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Sample records for cancer hela cells

  1. Radiation sensitization by CAPE on human HeLa cells of cervical cancer

    Objective: To study the radiosensitizing effect of caffic acid phenethyl ester (CAPE) on human cervical cancer HeLa cells. Methods: MTT assay was used to measure the relation between the inhibition effect and CAPE concentrations by CAPE with different concentrations on HeLa cells for 24 hours. HeLa cells were divided into the control and experimental groups, both of which were given 0, 2, 4, 6 and 8 Gy of 60Co γ-irradiation, respectively. The cell clones were counted. Meanwhile HeLa cells were divided into the control, CAPE, irradiation and combination groups. Flow cytometric analysis was adopted to detect the changes of cell cycle distribution induced by CAPE. Results: The inhibition rate of CAPE acting on Hela cells increased with concentrations (F=126. 49 ∼ 3654.88, P0) (1.45 and 1.82 Gy) and the quasi-threshold dose (Dq) (1.89 and 3.21 Gy) of HeLa cells in experimental group decreased comparing with control group, SER was 1.26. Compared with the sole irradiation group, cells in G2/M phase of the CAPE group and the sole irradiation group increased (P2/M arrest and may be related to the inhibition of the sub-lethal damage repair. (authors)

  2. HIF-1 and NDRG2 contribute to hypoxia-induced radioresistance of cervical cancer Hela cells

    Hypoxia inducible factor 1 (HIF-1), the key mediator of hypoxia signaling pathways, has been shown involved in hypoxia-induced radioresistance. However, the underlying mechanisms are unclear. The present study demonstrated that both hypoxia and hypoxia mimetic cobalt chloride could increase the radioresistance of human cervical cancer Hela cells. Meanwhile, ectopic expression of HIF-1 could enhance the resistance of Hela cells to radiation, whereas knocking-down of HIF-1 could increase the sensitivity of Hela cells to radiation in the presence of hypoxia. N-Myc downstream-regulated gene 2 (NDRG2), a new HIF-1 target gene identified in our lab, was found to be upregulated by hypoxia and radiation in a HIF-1-dependent manner. Overexpression of NDRG2 resulted in decreased sensitivity of Hela cells to radiation while silencing NDRG2 led to radiosensitization. Moreover, NDRG2 was proved to protect Hela cells from radiation-induced apoptosis and abolish radiation-induced upregulation of Bax. Taken together, these data suggest that both HIF-1 and NDRG2 contribute to hypoxia-induced tumor radioresistance and that NDRG2 acts downstream of HIF-1 to promote radioresistance through suppressing radiation-induced Bax expression. It would be meaningful to further explore the clinical application potential of HIF-1 and NDRG2 blockade as radiosensitizer for tumor therapy.

  3. Effects of Tatariside G Isolated from Fagopyrum tataricum Roots on Apoptosis in Human Cervical Cancer HeLa Cells

    Yuan Li; Su-Juan Wang; Wei Xia; Khalid Rahman; Yan Zhang; Hao Peng; Hong Zhang; Lu-Ping Qin

    2014-01-01

    Cervical cancer is the second most common female carcinoma. Current therapies are often unsatisfactory, especially for advanced stage patients. The aim of this study was to explore the effects of tatariside G (TG) on apoptosis in human cervical cancer HeLa cells and the possible mechanism of action involved. An MTT assay was employed to evaluate cell viability. Hoechst 33258 staining and flow cytometry (FCM) assays were used to detect cell apoptosis. The protein expression of phosphorylated J...

  4. Mechanisms of arsenic trioxide induced apoptosis of human cervical cancer HeLa cells and protection by Bcl-2

    邓友平; 林晨; 郑杰; 梁萧; 陈洁平; 付明; 肖培根; 吴旻

    1999-01-01

    It was recently reported that arsenic trioxide (As2O3) can induce complete remission in patients with acute promyelocytic leukemia (APL). In this present article, the biological effect of As2O3 on human cervical cancer HeLa cells and HeLa cells overexpressing Bcl-2 is studied. By MTT and colony forming ability assays, morphology alteration, flow cytometric analysis, DNA gel electrephoresis and in situ cell death detection (TUNEL), it was found that As2O3 inhibited the growth of HeLa cells and induced G2/M arrest and apoptosis of the cells. RT-PCR, Northern blot, Western blot analysis revealed that As2O3 induced HeLa cell apoptosis possibly via decreasing the expression of c-myc and viral genes. HeLa cells overexpressing Bcl-2 partly resist As2O3 induced apoptosis, which might be relative to preventing the cells from As2O3 caused G2/M block, downregulation of c-myc gene expression and inhibition of viral gene expression was also noted, However, it was found that As2O3 at a high concentratio

  5. The Expression of Cyclooxygenase-2 in Cervical Cancers and Hela Cells Was Regulated by Estrogen/Progestogen

    LI Yunguang; PU Demin; LI Yanli

    2007-01-01

    To investigate the relationship between the expression of cyclooxygenase-2 (COX-2) and menstrual cycle, the regulatory effects of 17-β-estradiol (E2) and medroxyprogesterone acetate (MPA) on the expression of COX-2 in cervical cancer Hela cells were examined. Cervical cancer specimens were obtained from 47 pre-menopausal patients. The phase of menstrual cycle was determined by case history and HE staining of uterine endometrium. COX-2 was immunohistochemically stained by SABC staining and the staining intensity was determined with computerized image analysis system.Hela cells were incubated with alcohol, E2, E2+MPA, MPA for 12, 24 and 48 h respectively. The expression of COX-2 in Hela cells was detected by Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). Our results showed that the expression of COX-2 was significantly higher during proliferative phase than secretory phase (P<0.05), but there was no difference in the positive rate between proliferative phase and secretory phase (P>0.05). Incubation with E2 could significantly enhance the expression of COX-2 continually. On the contrary, E2+MPA and MPA alone could decrease the expression of COX-2 as compared with the control and E2 group (P<0.05 and P<0.01 respectively). It is concluded that the expression of COX-2 in cervical cancer of pre-menopausal patients and Hela cells was regulated by estrogen/progestogen.

  6. Serum ferritin in patients with cancer: determination with antibodies to HeLa cell and spleen ferritin

    Some malignant tissues and cell lines contain acidic isoferritins and it has been suggested that the assay of such isoferritins in serum may be of value in the diagnosis of malignancy. This paper describes a radioimmunoassay for acidic ferritin purified from HeLa cells. Examination of purified heart, kidney, liver and spleen ferritin showed that the assay was highly specific for acidic isoferritins. Ferritin concentrations have been measured with antibodies to HeLa cell and spleen ferritin in extracts of normal and tumour tissue. Although the tumours contained more HeLa type ferritin than the corresponding normal tissue the HeLa/spleen type ferritin ratio was low. HeLa-type ferritin concentrations have been compared with values obtained with anti-spleen ferritin in over 1000 sera from normal subjects and patients with cancer and leukaemia. HeLa-type ferritin was not detected (<2 μg/l) in most normal sera. Concentrations of up to 53 μg/l were found in sera from patients with malignant disease but the HeLa/spleen type ferritin ratio was always very low. There appears to be little application for antibodies to HeLa cell or heart ferritin in the diagnosis or monitoring of cancer. (Auth.)

  7. Cytotoxic Effects of Native and Recombinant Frutalin, a Plant Galactose-Binding Lectin, on HeLa Cervical Cancer Cells

    Carla Oliveira

    2011-01-01

    Full Text Available Frutalin is the α-D-galactose-binding lectin isolated from breadfruit seeds. Frutalin was obtained from two different sources: native frutalin was purified from its natural origin, and recombinant frutalin was produced and purified from Pichia pastoris. This work aimed to study and compare the effect of native and recombinant frutalin on HeLa cervical cancer cells proliferation and apoptosis. Furthermore, the interaction between frutalin and the HeLa cells was investigated by confocal microscopy. Despite having different carbohydrate-binding affinities, native and recombinant frutalin showed an identical magnitude of cytotoxicity on HeLa cells growth (IC50~100 μg/mL and equally induced cell apoptosis. The interaction studies showed that both lectins were rapidly internalised and targeted to HeLa cell's nucleus. Altogether, these results indicate that frutalin action is not dependent on its sugar-binding properties. This study provides important information about the bioactivity of frutalin and contributes to the understanding of the plant lectins cytotoxic activity.

  8. Apoptosis induced by dioscin in Hela cells.

    Cai, Jing; Liu, Mingjie; Wang, Zhao; Ju, Yong

    2002-02-01

    Dioscin, a saponin extracted from the root of Polygonatum Zanlanscianense Pamp, markedly inhibited proliferation of Hela cells. The results indicated that Hela cells underwent apoptosis in dose- and time-dependent manners when treated with Dioscin. Caspase-3, -8 and -9 activities were also detected. The low enzymatic activity of caspase-8 and high activity of caspase-9 showed that the mitochondrial pathway was activated in apoptosis. The reduced expression of the survival protein Bcl-2 also confirmed this result. These studies may be significant in finding a new drug to treat human cervical cancer. PMID:11853164

  9. The effects of ionizing radiation combined with autophagy inducers or inhibitors or inhibitors on human cervical cancer hela cells

    Objective: To detect the effects of ionizing radiation combined with autophagy inhibitors and inducers on the proliferation of human cervical cancer cell line. Methods: MTT and flowcytometry (FCM) were used to detect the surviving and proliferation of human cervical cancer cells,and analysis of the relationship of dose-effect and time-effect was made. Results: With the increase of irradiation doses (2, 4, 6, 8 and 10 Gy) and the elongation of irradiation time (24, 48 and 72 h), the inhibiting effect of ionizing radiation on the proliferation of human cervical cancer cells increased (P< 0.05 or P< 0.01). The inhibiting effect of 6 Gy combined with autophagy inducer rapamycin on the proliferation of Hela cells weakened (P< 0.05). The inhibiting effects of 6 Gy combined with autophagy inhibitor 3-MA on the cell proliferation were higher than those in 6 Gy group (P< 0.05). Conclusion: Ionizing radiation combined with autophagy inducers can inhibit apoptosis in Hela cells, while the ionizing radiation combined with autophagy inhibitors can promote their apoptosis. (authors)

  10. The Enhanced Inhibitory Effect of Different Antitumor Agents in Self-Microemulsifying Drug Delivery Systems on Human Cervical Cancer HeLa Cells

    Zoltán Ujhelyi; Azin Kalantari; Miklós Vecsernyés; Eszter Róka; Ferenc Fenyvesi; Róbert Póka; Bence Kozma; Ildikó Bácskay

    2015-01-01

    The aim of this study was to develop topical self-microemulsifying drug delivery systems (SMEDDS) containing antitumor agents (bleomycin, cisplatin and ifosfamide) and to investigate their inhibitory potential in SMEDDS on human cervical cancer HeLa cells. The physicochemical properties of cytostatic drug loaded SMEDDS were characterized. The cytotoxicity of main components of SMEDDS was also investigated. Their IC50 values were determined. HeLa cells were treated by different concentrations ...

  11. The Ability to Survive Mitosis in the Presence of Microtubule Poisons Differs Significantly Between Human Nontransformed (RPE-1) and Cancer (U2OS, HeLa) Cells

    Brito, Daniela A; Rieder, Conly L.

    2009-01-01

    We used live cell imaging to compare the fate of human nontransformed (RPE-1) and cancer (HeLa, U2OS) cells as they entered mitosis in nocodazole or taxol. In the same field, and in either drug, a cell in all lines could die in mitosis, exit mitosis and die within 10 h, or exit mitosis and survive ≥10 h. Relative to RPE-1 cells, significantly fewer HeLa or U2OS cells survived mitosis or remained viable after mitosis: in nocodazole concentrations that inhibit spindle microtubule assembly, or i...

  12. Mechanical trapping of the nucleus on micropillared surfaces inhibits the proliferation of vascular smooth muscle cells but not cervical cancer HeLa cells.

    Nagayama, Kazuaki; Hamaji, Yumi; Sato, Yuji; Matsumoto, Takeo

    2015-07-16

    The interaction between cells and the extracellular matrix on a topographically patterned surface can result in changes in cell shape and many cellular functions. In the present study, we demonstrated the mechanical deformation and trapping of the intracellular nucleus using polydimethylsiloxane (PDMS)-based microfabricated substrates with an array of micropillars. We investigated the differential effects of nuclear deformation on the proliferation of healthy vascular smooth muscle cells (SMCs) and cervical cancer HeLa cells. Both types of cell spread normally in the space between micropillars and completely invaded the extracellular microstructures, including parts of their cytoplasm and their nuclei. We found that the proliferation of SMCs but not HeLa cells was dramatically inhibited by cultivation on the micropillar substrates, even though remarkable deformation of nuclei was observed in both types of cells. Mechanical testing with an atomic force microscope and a detailed image analysis with confocal microscopy revealed that SMC nuclei had a thicker nuclear lamina and greater expression of lamin A/C than those of HeLa cells, which consequently increased the elastic modulus of the SMC nuclei and their nuclear mechanical resistance against extracellular microstructures. These results indicate that the inhibition of cell proliferation resulted from deformation of the mature lamin structures, which might be exposed to higher internal stress during nuclear deformation. This nuclear stress-induced inhibition of cell proliferation occurred rarely in cancer cells with deformable nuclei. PMID:26054426

  13. Inhibitory effect of 13 taxane diterpenoids from Chinese yew (Taxus chinensis var. mairei) on the proliferation of HeLa cervical cancer cells.

    Liu, Hai-Sheng; Gao, Yu-Huan; Liu, Li-Hong; Liu, Wei; Shi, Qing-Wen; Dong, Mei; Suzuki, Toshikazu; Kiyota, Hiromasa

    2016-10-01

    The inhibitory effect of 13 taxanes isolated from the Chinese yew (Taxus chinensis var. mairei) on the proliferation of human cervical cancer HeLa cells were examined using an MTT assay. Four compounds having a hydrophobic cinnamate side chain showed antiproliferative activity, which may be due to increased cell permeability. PMID:27296359

  14. Effects of tatariside G isolated from Fagopyrum tataricum roots on apoptosis in human cervical cancer HeLa cells.

    Li, Yuan; Wang, Su-Juan; Xia, Wei; Rahman, Khalid; Zhang, Yan; Peng, Hao; Zhang, Hong; Qin, Lu-Ping

    2014-01-01

    Cervical cancer is the second most common female carcinoma. Current therapies are often unsatisfactory, especially for advanced stage patients. The aim of this study was to explore the effects of tatariside G (TG) on apoptosis in human cervical cancer HeLa cells and the possible mechanism of action involved. An MTT assay was employed to evaluate cell viability. Hoechst 33258 staining and flow cytometry (FCM) assays were used to detect cell apoptosis. The protein expression of phosphorylated JNK, P38, ERK and Akt and cleaved caspase-3 and caspase-9 was evaluated by western blot analysis. Additionally, the mRNA expression of caspase-3 and caspase-9 was measured by fluorescent quantitative reverse transcription-PCR (FQ-RT-PCR). TG notably inhibited cell viability, enhanced the percentage of apoptotic cells, facilitated the phosphorylation of p38 MAPK and JNK proteins and caspase-3 and caspase-9 cracking, downregulated the phosphorylation level of Akt, and increased the loss of MMP and the mRNA expression of caspase-3 and caspase-9. TG-induced apoptosis is associated with activation of the mitochondrial death pathway. TG may be an effective candidate for chemotherapy against cervical cancer. PMID:25076146

  15. Effects of Tatariside G Isolated from Fagopyrum tataricum Roots on Apoptosis in Human Cervical Cancer HeLa Cells

    Yuan Li

    2014-07-01

    Full Text Available Cervical cancer is the second most common female carcinoma. Current therapies are often unsatisfactory, especially for advanced stage patients. The aim of this study was to explore the effects of tatariside G (TG on apoptosis in human cervical cancer HeLa cells and the possible mechanism of action involved. An MTT assay was employed to evaluate cell viability. Hoechst 33258 staining and flow cytometry (FCM assays were used to detect cell apoptosis. The protein expression of phosphorylated JNK, P38, ERK and Akt and cleaved caspase-3 and caspase-9 was evaluated by western blot analysis. Additionally, the mRNA expression of caspase-3 and caspase-9 was measured by fluorescent quantitative reverse transcription-PCR (FQ-RT-PCR. TG notably inhibited cell viability, enhanced the percentage of apoptotic cells, facilitated the phosphorylation of p38 MAPK and JNK proteins and caspase-3 and caspase-9 cracking, downregulated the phosphorylation level of Akt, and increased the loss of MMP and the mRNA expression of caspase-3 and caspase-9. TG-induced apoptosis is associated with activation of the mitochondrial death pathway. TG may be an effective candidate for chemotherapy against cervical cancer.

  16. Inotodiol inhabits proliferation and induces apoptosis through modulating expression of cyclinE, p27, bcl-2, and bax in human cervical cancer HeLa cells.

    Zhao, Li-Wei; Zhong, Xiu-Hong; Yang, Shu-Yan; Zhang, Yi-Zhong; Yang, Ning-Jiang

    2014-01-01

    Inonotus obliquus is a medicinal mushroom that has been used as an effective agent to treat various diseases such as diabetes, tuberculosis and cancer. Inotodiol, an included triterpenoid shows significant anti-tumor effect. However, the mechanisms have not been well documented. In this study, we aimed to explore the effect of inotodiol on proliferation and apoptosis in human cervical cancer HeLa cells and investigated the underlying molecular mechanisms. HeLa cells were treated with different concentrations of inotodiol. The MTT assay was used to evaluate cell proliferating ability, flow cytometry (FCM) was employed for cell cycle analysis and cell apoptosis, while expression of cyclinE, p27, bcl-2 and bax was detected by immunocytochemistry. Proliferation of HeLa cells was inhibited by inotodiolin a dose-dependent manner at 24h (r=0.9999, pInonotus obliquus inhibited the proliferation of HeLa cells and induced apoptosis in vitro. The mechanisms may be related to promoting apoptosis through increasing the expression of bax and cutting bcl-2 and affecting the cell cycle by down-regulation the expression of cyclin E and up-regulation of p27. The results further indicate the potential value of inotodiol for treatment of human cervical cancer. PMID:24815470

  17. Inhibition of clathrin by pitstop 2 activates the spindle assembly checkpoint and induces cell death in dividing HeLa cancer cells

    Smith Charlotte M

    2013-01-01

    Full Text Available Abstract Background During metaphase clathrin stabilises the mitotic spindle kinetochore(K-fibres. Many anti-mitotic compounds target microtubule dynamics. Pitstop 2™ is the first small molecule inhibitor of clathrin terminal domain and inhibits clathrin-mediated endocytosis. We investigated its effects on a second function for clathrin in mitosis. Results Pitstop 2 did not impair clathrin recruitment to the spindle but disrupted its function once stationed there. Pitstop 2 trapped HeLa cells in metaphase through loss of mitotic spindle integrity and activation of the spindle assembly checkpoint, phenocopying clathrin depletion and aurora A kinase inhibition. Conclusions Pitstop 2 is therefore a new tool for investigating clathrin spindle dynamics. Pitstop 2 reduced viability in dividing HeLa cells, without affecting dividing non-cancerous NIH3T3 cells, suggesting that clathrin is a possible novel anti-mitotic drug target.

  18. The Enhanced Inhibitory Effect of Different Antitumor Agents in Self-Microemulsifying Drug Delivery Systems on Human Cervical Cancer HeLa Cells

    Zoltán Ujhelyi

    2015-07-01

    Full Text Available The aim of this study was to develop topical self-microemulsifying drug delivery systems (SMEDDS containing antitumor agents (bleomycin, cisplatin and ifosfamide and to investigate their inhibitory potential in SMEDDS on human cervical cancer HeLa cells. The physicochemical properties of cytostatic drug loaded SMEDDS were characterized. The cytotoxicity of main components of SMEDDS was also investigated. Their IC50 values were determined. HeLa cells were treated by different concentrations of cisplatin, bleomycin and ifosfamide alone and in various SMEDDS. The inhibitory effect on cell growth was analyzed by MTT cell viability assay. Inflammation is a driving force that accelerates cancer development. The inhibitory effect of these antitumor agents has also been tested on HeLa cells in the presence of inflammatory mediators (IL-1-β, TNF-α as an in vitro model of inflamed human cervix. Significant differences in the cytotoxicity of cytostatic drugs alone and in SMEDDS have been found in a concentration-dependent manner. The self-micro emulsifying system may potentiate the effectiveness of bleomycin, cisplatin and ifosfamide topically. The effect of SMEDDS containing antitumor agents was decreased significantly in the presence of inflammatory mediators. According to our experiments, the optimal SMEDDS formulation is 1:1:2:6:2 ratios of Isopropyl myristate, Capryol 90, Kolliphor RH 40, Cremophor RH40, Transcutol HP and Labrasol. It can be concluded that SMEDDS may increase the inhibitory effect of bleomycin, ifosfamide and cisplatin on human cervical cancer HeLa cells. Inflammation on HeLa cells hinders the effectiveness of SMEDDS containing antitumor agents. Our results might ensure useful data for development of optimal antitumor formulations.

  19. The enhanced inhibitory effect of different antitumor agents in self-microemulsifying drug delivery systems on human cervical cancer HeLa cells.

    Ujhelyi, Zoltán; Kalantari, Azin; Vecsernyés, Miklós; Róka, Eszter; Fenyvesi, Ferenc; Póka, Róbert; Kozma, Bence; Bácskay, Ildikó

    2015-01-01

    The aim of this study was to develop topical self-microemulsifying drug delivery systems (SMEDDS) containing antitumor agents (bleomycin, cisplatin and ifosfamide) and to investigate their inhibitory potential in SMEDDS on human cervical cancer HeLa cells. The physicochemical properties of cytostatic drug loaded SMEDDS were characterized. The cytotoxicity of main components of SMEDDS was also investigated. Their IC50 values were determined. HeLa cells were treated by different concentrations of cisplatin, bleomycin and ifosfamide alone and in various SMEDDS. The inhibitory effect on cell growth was analyzed by MTT cell viability assay. Inflammation is a driving force that accelerates cancer development. The inhibitory effect of these antitumor agents has also been tested on HeLa cells in the presence of inflammatory mediators (IL-1-β, TNF-α) as an in vitro model of inflamed human cervix. Significant differences in the cytotoxicity of cytostatic drugs alone and in SMEDDS have been found in a concentration-dependent manner. The self-micro emulsifying system may potentiate the effectiveness of bleomycin, cisplatin and ifosfamide topically. The effect of SMEDDS containing antitumor agents was decreased significantly in the presence of inflammatory mediators. According to our experiments, the optimal SMEDDS formulation is 1:1:2:6:2 ratios of Isopropyl myristate, Capryol 90, Kolliphor RH 40, Cremophor RH40, Transcutol HP and Labrasol. It can be concluded that SMEDDS may increase the inhibitory effect of bleomycin, ifosfamide and cisplatin on human cervical cancer HeLa cells. Inflammation on HeLa cells hinders the effectiveness of SMEDDS containing antitumor agents. Our results might ensure useful data for development of optimal antitumor formulations. PMID:26197311

  20. Requirement of T-lymphokine-activated killer cell-originated protein kinase for TRAIL resistance of human HeLa cervical cancer cells

    T-lymphokine-activated killer cell-originated protein kinase (TOPK) appears to be highly expressed in various cancer cells and to play an important role in maintaining proliferation of cancer cells. However, the underlying mechanism by which TOPK regulates growth of cancer cells remains elusive. Here we report that upregulated endogenous TOPK augments resistance of cancer cells to apoptosis induced by tumor necrosis factor-related apoptosis inducing ligand (TRAIL). Stable knocking down of TOPK markedly increased TRAIL-mediated apoptosis of human HeLa cervical cancer cells, as compared with control cells. Caspase 8 or caspase 3 activities in response to TRAIL were greatly incremented in TOPK-depleted cells. Ablation of TOPK negatively regulated TRAIL-mediated NF-κB activity. Furthermore, expression of NF-κB-dependent genes, FLICE-inhibitory protein (FLIP), inhibitor of apoptosis protein 1 (c-IAP1), or X-linked inhibitor of apoptosis protein (XIAP) was reduced in TOPK-depleted cells. Collectively, these findings demonstrated that TOPK contributed to TRAIL resistance of cancer cells via NF-κB activity, suggesting that TOPK might be a potential molecular target for successful cancer therapy using TRAIL.

  1. Induction of Apoptotic Effects of Antiproliferative Protein from the Seeds of Borreria hispida on Lung Cancer (A549 and Cervical Cancer (HeLa Cell Lines

    S. Rupachandra

    2014-01-01

    Full Text Available A 35 KDa protein referred to as F3 was purified from the seeds of Borreria hispida by precipitation with 80% ammonium sulphate and gel filtration on Sephadex G-100 column. RP-HPLC analysis of protein fraction (F3 on an analytical C-18 column produced a single peak, detected at 220 nm. F3 showed an apparent molecular weight of 35 KDa by SDS PAGE and MALDI-TOF-MS analyses. Peptide mass fingerprinting analysis of F3 showed the closest homology with the sequence of 1-aminocyclopropane-1-carboxylate deaminase of Pyrococcus horikoshii. The protein (F3 exhibited significant cytotoxic activity against lung (A549 and cervical (HeLa cancer cells in a dose-dependent manner at concentrations ranging from 10 µg to 1000 µg/mL, as revealed by the MTT assay. Cell cycle analysis revealed the increased growth of sub-G0 population in both cell lines exposed to a concentration of 1000 µg/mL of protein fraction F3 as examined from flow cytometry. This is the first report of a protein from the seeds of Borreria hispida with antiproliferative and apoptotic activity in lung (A549 and cervical (HeLa cancer cells.

  2. Non-thermal plasma inhibits human cervical cancer HeLa cells invasiveness by suppressing the MAPK pathway and decreasing matrix metalloproteinase-9 expression

    Li, Wei; Yu, K. N.; Bao, Lingzhi; Shen, Jie; Cheng, Cheng; Han, Wei

    2016-01-01

    Non-thermal plasma (NTP) has been proposed as a novel therapeutic method for anticancer treatment. However, the mechanism underlying its biological effects remains unclear. In this study, we investigated the inhibitory effect of NTP on the invasion of HeLa cells, and explored the possible mechanism. Our results showed that NTP exposure for 20 or 40 s significantly suppressed the migration and invasion of HeLa cells on the basis of matrigel invasion assay and wound healing assay, respectively. Moreover, NTP reduced the activity and protein expression of the matrix metalloproteinase (MMP)-9 enzyme. Western blot analysis indicated that NTP exposure effectively decreased phosphorylation level of both ERK1/2 and JNK, but not p38 MAPK. Furthermore, treatment with MAPK signal pathway inhibitors or NTP all exhibited significant depression of HeLa cells migration and MMP-9 expression. The result showed that NTP synergistically suppressed migration and MMP-9 expression in the presence of ERK1/2 inhibitor and JNK inhibitor, but not p38 MAPK inhibitor. Taken together, these findings suggested that NTP exposure inhibited the migration and invasion of HeLa cells via down-regulating MMP-9 expression in ERK1/2 and JNK signaling pathways dependent manner. These findings provide hints to the potential clinical research and therapy of NTP on cervical cancer metastasis.

  3. Involvement of mitochondria and caspase pathways in N-demethyl-clarithromycin-induced apoptosis in human cervical cancer HeLa cell

    Ai-min QIAO; Takashi IKEJIMA; Shini-chi TASHIRO; Satoshi ONODERA; Wei-ge ZHANG; Ying-liang WU

    2006-01-01

    Aim: To study the mechanisms by which N-demethyl-clarithromycin (NDC) induces human cervical cancer HeLa cell apoptosis in vitro. Methods: The viability of N-demethyl-clarithromycin-induced HeLa cells was measured by MTT assay. Apoptotic cells with condensed nuclei were visualized by phase contrast microscopy. Nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis. Measurement of mitochondrial transmembrane potential was analyzed by a FACScan flowcytometer. Caspase-3, poly-(ADP-ribose) polymerase (PARP), caspase-activated DNase (ICAD), Bcl-2, Bax, p53, and SIRT1 protein expression and the release of cytochrome c were detected by Western blot analysis. Results: N-demethyl-clarithromycin, an anti-inflammatory substance, inhibited HeLa cell growth in a dose- and time-dependent manner.N-demethyl-clarithromycin induced HeLa cell death through the apoptotic pathways. The pan-caspase inhibitor (z-VAD-fmk), caspase-3 inhibitor (z-DEVD-fmk) and the caspase-9 inhibitor (z-LEHD-fmk) partially enhanced cell viability induced by N-demethyl-clarithromycin, but the caspase-8 inhibitor (z-IETD-fmk) had almost no effect. Caspase-3 was activated then followed by the degradation of caspase-3 substrates, the inhibitor of ICAD and PARP. Simultaneously, mitochondrial transmembrane potential was markedly reduced and the release of cytochrome c in the cytosol was increased.N-demethyl-clarithromycin upregulated the expression ratio of mitochondrial Bax/Bcl-2, and significantly increased the expression of the p53 protein. It also downregulated anti-apoptotic protein SIRT1 expression. Conclusion: N-demethyl-clarithromycin induced apoptosis in HeLa cells via the mitochondrial pathway.

  4. Isolation of Melittin from Iranian Honey Bee Venom and Investigation of Its Effect on Proliferation of Cervical Cancer- HeLa Cell Line

    K Pooshang Bagheri

    2013-06-01

    Full Text Available Introduction: Cervical cancer is the second prevalent cancer in developing countries and the sixth prevalent cancer in USA. Since conventional treatment methods are associated with detrimental side effects, searching for new drugs using natural ingredients is very important. Previous studies have shown that melittin (main component of honey bee venom has anticancer properties along with the effect on cell membrane and activation of apoptosis. In this study, inhibitory effects of melittin on the viability and proliferation of cervical cancer cell line (HeLa was investigated. Methods: Melittin was purified from honeybee venom using reversed-phase HPLC method. Then, biological activity of melittin was examined by hemolytic activity analysis on the red blood cells. In order to investigate whether melittin inhibits proliferation of HeLa cell, MTT assay was performed. HeLa cells were plated in a 96-well plate and treated with serially diluted concentrations of melittin for 12 and 24 hours. The viability of the cells was measured via MTT assay at 540nm. Results: Melittin showed a strong hemolytic activity (HD50=0.5 µg/ml which can be reduced by FBS(HD50=2 µg/ml. Results of MTT assay indicated that melittin shows cytotoxic effect on cervical cancer cells with IC50 = 1.2 ug/ml at 12h incubation period. Conclusion: In this study, biological activity of melittin and inhibitory effect of FBS on hemolysis were determined via hemolytic activity analysis. MTT assay indicated that melittin induced cytotoxic effects in a dose dependent manner on cervical cancer cells and it also revealed dependence on incubation time as well.

  5. 紫花牡荆素体外抑制人宫颈癌HeLa细胞增殖的研究%Proliferation inhibition of human cervical cancer HeLa cells by Casticin in vitro

    Jing Xie; Jun Bai; Xifeng Sheng; Jianguo Cao; Wanyu Xie

    2011-01-01

    Objective: The aim of the study was to investigate the effect of Casticin on proliferation inhibition of human cervical cancer HeLa cells in vitro and to unravel the associated mechanisms. Methods: Human cervical HeLa cells were cultured in vitro. The inhibitory effect of Casticin on the viability of human cervical cancer HeLa cells was evaluated by the MTT assay.The colony formation ability was detected by plate colony formation assay. Distribution of cell cycle was analyzed by flow cytometry. The protein expression levels were analyzed by Western blot. Results: Casticin significantly inhibited the growth of human cervical cancer HeLa cells in a dose- and time-dependent manner, and the IC50 was 2.82 μg/mL. The colony-forming rate was reduced drastically compared with control group (P < 0.05). The cells were markedly arrested at G2/M phase after the treatment of Casticin for 48 h. Western blot showed that the expression of p21 protein was up-regulated and protein level of Cyclin B1 was depressed by Casticin in a concentration dependent manner. Conclusion: Casticin could inhibit the cell growth and lead to cell arrest in human cervical cancer HeLa cells, and the down-regulation of Cyclin B1 protein expression and activation of p21 protein might contribute to Casticin induced cell arrest in human cervical cancer HeLa cells.

  6. Adenovirus-mediated expression of UHRF1 reduces the radiosensitivity of cervical cancer HeLa cells to γ-irradiation

    Xin-li LI; Qing-hui MENG; Sai-jun FAN

    2009-01-01

    Aim:An in vitro study was carried out to determine the effect of UHRF1 overexpression on radiosensitivity in human cervical cancer HeLa ceUs using adenovirus-mediated UHRF1 gene transfer (Ad5-UHRF1). Methods: Cell survival was evaluated using the clonogenic survival assay and the MTT assay; apoptosis and cell cycle distribution were monitored by flow cytometry. Protein levels were measured by Western blotting. Silencing XRCC4 expression was performed by transfection of small interfering RNA (siRNA).Results: Increased expression of UHRF1 by AdS-UHRF1 significantly reduced the radiosensitivity of HeLa cells. The UHRF1-mediated radioresistance was correlated with increased DNA repair capability and increased expression of the DNA damage repair protein, XRCC4. Knocking down XRCC4 expression in the cells using XRCC4 siRNA markedly reduced the UHRFl-mediated radioresistance. Conclusion: These results provide the first evidence for revealing a functional role of UHRF1 in human cervical cancer cells as a negative regulator of radiosensitivity.

  7. Outcome of treatment of human HeLa cervical cancer cells with roscovitine strongly depends on the dosage and cell cycle status prior to the treatment.

    Wesierska-Gadek, Józefa; Borza, Andreea; Walzi, Eva; Krystof, Vladimir; Maurer, Margarita; Komina, Oxana; Wandl, Stefanie

    2009-04-01

    Exposure of asynchronously growing human HeLa cervical carcinoma cells to roscovitine (ROSC), a selective cyclin-dependent kinases (CDKs) inhibitor, arrests their progression at the transition between G(2)/M and/or induces apoptosis. The outcome depends on the ROSC concentration. At higher dose ROSC represses HPV-encoded E7 oncoprotein and initiates caspase-dependent apoptosis. Inhibition of the site-specific phosphorylation of survivin and Bad, occurring at high-dose ROSC treatment, precedes the onset of apoptosis and seems to be a prerequisite for cell death. Considering the fact that in HeLa cells the G(1)/S restriction checkpoint is abolished by E7, we addressed the question whether the inhibition of CDKs by pharmacological inhibitors in synchronized cells would be able to block the cell-cycle in G(1) phase. For this purpose, we attempted to synchronize cells by serum withdrawal or by blocking of the mitotic apparatus using nocodazole. Unlike human MCF-7 cells, HeLa cells do not undergo G(1) block after serum starvation, but respond with a slight increase of the ratio of G(1) population. Exposure of G(1)-enriched HeLa cells to ROSC after re-feeding does not block their cell-cycle progression at G(1)-phase, but increases the ratio of S- and G(2)-phase, thereby mimicking the effect on asynchronously growing cells. A quite different impact is observed after treatment of HeLa cells released from mitotic block. ROSC prevents their cell cycle progression and cells transiently accumulate in G(1)-phase. These results show that inhibition of CDKs by ROSC in cells lacking the G(1)/S restriction checkpoint has different outcomes depending on the cell-cycle status prior to the onset of treatment. PMID:19180585

  8. The aqueous extract of Ficus religiosa induces cell cycle arrest in human cervical cancer cell lines SiHa (HPV-16 Positive and apoptosis in HeLa (HPV-18 positive.

    Amit S Choudhari

    Full Text Available Natural products are being extensively explored for their potential to prevent as well as treat cancer due to their ability to target multiple molecular pathways. Ficus religiosa has been shown to exert diverse biological activities including apoptosis in breast cancer cell lines. In the present study, we report the anti-neoplastic potential of aqueous extract of F. religiosa (FRaq bark in human cervical cancer cell lines, SiHa and HeLa. FRaq altered the growth kinetics of SiHa (HPV-16 positive and HeLa (HPV-18 positive cells in a dose-dependent manner. It blocked the cell cycle progression at G1/S phase in SiHa that was characterized by an increase in the expression of p53, p21 and pRb proteins with a simultaneous decrease in the expression of phospho Rb (ppRb protein. On the other hand, in HeLa, FRaq induced apoptosis through an increase in intracellular Ca(2+ leading to loss of mitochondrial membrane potential, release of cytochrome-c and increase in the expression of caspase-3. Moreover, FRaq reduced the migration as well as invasion capability of both the cervical cancer cell lines accompanied with downregulation of MMP-2 and Her-2 expression. Interestingly, FRaq reduced the expression of viral oncoproteins E6 and E7 in both the cervical cancer cell lines. All these data suggest that F. religiosa could be explored for its chemopreventive potential in cervical cancer.

  9. The aqueous extract of Ficus religiosa induces cell cycle arrest in human cervical cancer cell lines SiHa (HPV-16 Positive) and apoptosis in HeLa (HPV-18 positive).

    Choudhari, Amit S; Suryavanshi, Snehal A; Kaul-Ghanekar, Ruchika

    2013-01-01

    Natural products are being extensively explored for their potential to prevent as well as treat cancer due to their ability to target multiple molecular pathways. Ficus religiosa has been shown to exert diverse biological activities including apoptosis in breast cancer cell lines. In the present study, we report the anti-neoplastic potential of aqueous extract of F. religiosa (FRaq) bark in human cervical cancer cell lines, SiHa and HeLa. FRaq altered the growth kinetics of SiHa (HPV-16 positive) and HeLa (HPV-18 positive) cells in a dose-dependent manner. It blocked the cell cycle progression at G1/S phase in SiHa that was characterized by an increase in the expression of p53, p21 and pRb proteins with a simultaneous decrease in the expression of phospho Rb (ppRb) protein. On the other hand, in HeLa, FRaq induced apoptosis through an increase in intracellular Ca(2+) leading to loss of mitochondrial membrane potential, release of cytochrome-c and increase in the expression of caspase-3. Moreover, FRaq reduced the migration as well as invasion capability of both the cervical cancer cell lines accompanied with downregulation of MMP-2 and Her-2 expression. Interestingly, FRaq reduced the expression of viral oncoproteins E6 and E7 in both the cervical cancer cell lines. All these data suggest that F. religiosa could be explored for its chemopreventive potential in cervical cancer. PMID:23922932

  10. Thymoquinone-Loaded Nanostructured Lipid Carrier Exhibited Cytotoxicity towards Breast Cancer Cell Lines (MDA-MB-231 and MCF-7) and Cervical Cancer Cell Lines (HeLa and SiHa)

    Wei Keat Ng; Latifah Saiful Yazan; Li Hua Yap; Wan Abd Ghani Wan Nor Hafiza; Chee Wun How; Rasedee Abdullah

    2015-01-01

    Thymoquinone (TQ) has been shown to exhibit antitumor properties. Thymoquinone-loaded nanostructured lipid carrier (TQ-NLC) was developed to improve the bioavailability and cytotoxicity of TQ. This study was conducted to determine the cytotoxic effects of TQ-NLC on breast cancer (MDA-MB-231 and MCF-7) and cervical cancer cell lines (HeLa and SiHa). TQ-NLC was prepared by applying the hot high pressure homogenization technique. The mean particle size of TQ-NLC was 35.66 ± 0.1235 nm with a narr...

  11. TRICHOSTATIN A INHIBITS PROLIFERATION, INDUCES APOPTOSIS AND CELL CYCLE ARREST IN HELA CELLS

    XU Zhou-min; WANG Yi-qun; MEI Qi; CHEN Jian; DU Jia; WEI Yan; XU Ying-chun

    2006-01-01

    Objective: The histone deacetylase inhibitors (HDACIS) have been shown to inhibit cancer cell proliferation, stimulate apoptosis, an induce cell cycle arrest. Our purpose was to investigate the antiproliferative effects of a HDACI, trichostatin A (TSA), against human cervical cancer cells (HeLa). Methods: HeLa cells were treated in vitro with various concentrations of TSA. The inhibitory effect of TSA on the growth of HeLa cells was measured by MTT assay. To detect the characteristic of apoptosis chromatin condensation, HeLa cells were stained with Hoechst 33258 in the presence of TSA. Induction of cell cycle arrest was studied by flow cytometry. Changes in gene expression of p53, p21Waf1 and p27Kip1 were studied by semiquantitative RT-PCR. Results: TSA inhibited cell growth in a time- and dose-dependent manner. Hoechst 33258 staining assay showed that TSA induced apoptosis. Cell cycle analysis indicated that treatment with TSA decreased the proportion of cells in S phase and increased the proportion of cells in G0/G1 and/or G2/M phases of the cell cycle. This was concomitant with overexpression of genes related to malignant phenotype, including an increase in p53, p21Waf1 and p27Kip1. Conclusion: These results suggest that TSA is effective in inhibiting growth of HeLa cells in vitro. The findings raise the possibility that TSA may prove particularly effective in treatment of cervical cancers.

  12. Targeting pro-apoptotic trail receptors sensitizes HeLa cervical cancer cells to irradiation-induced apoptosis

    Maduro, John H.; de Vries, Elisabeth G. E.; Meersma, Gert-Jan; Hougardy, Brigitte M. T.; van der Zee, Ate G. J.; De Jong, Steven

    2008-01-01

    Purpose: To investigate the potential of irradiation in combination with drugs targeting the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor (DR)4 and DR5 and their mechanism of action in a cervical cancer cell line. Methods and Materials: Recombinant human TRAIL (rhTR

  13. UJI AKTIVITAS SITOTOKSIK EKSTRAK KULIT JERUK PURUT (Citrus hystrix) PADA SEL HeLa CERVICAL CANCER CELL LINE

    Nathanael, Joshua

    2015-01-01

    Citrus hystrix or know as kefir lime in Indonesia were not used as medicine. Kefir lime were more used as skin and body treatment rather as food composition. The research of cytotoxic effect on HeLa from ethanolic extract of kefir lime peels is never been done before, ethanolic extract of kefir lime peels were used on this research. The point of this research is to know the main compound that built ethanolic extract of kefir lime peels and to know the cytotoxic effect of ethanolic extract ...

  14. Biofabrication of Ag nanoparticles using Sterculia foetida L. seed extract and their toxic potential against mosquito vectors and HeLa cancer cells

    A one-step and eco-friendly process for the synthesis of silver-(protein-lipid) nanoparticles (Ag-PL NPs) (core–shell) has been developed using the seed extract from wild Indian Almond tree, Sterculia foetida (L.) (Sterculiaceae). The reaction temperature played a major role in controlling the size and shell formation of NPs. The amount of NPs synthesized and qualitative characterization was done by UV–vis spectroscopy and transmission electron microscopy (TEM), respectively. TEM studies exhibited controlled dispersity of spherical shaped NPs with an average size of 6.9 ± 0.2 nm. Selected area electron diffraction (SAED) and X-ray diffraction (XRD) revealed ‘fcc’ phase and crystallinity of the particles. X-ray photoelectron spectroscopy (XPS) was used to identify the protein–lipid (PL) bilayer that appears as a shell around the Ag core particles. The thermal stability of the Ag-PL NPs was examined using thermogravimetric analysis (TGA). Further analysis was carried out by using Fourier transform infrared spectroscopy (FTIR), where the spectra provided evidence for the presence of proteins and lipid moieties ((2n-octylcycloprop-1-enyl)-octanoic acid (I)), and their role in synthesis and stabilization of Ag NPs. This is the first report of plant seed assisted synthesis of PL conjugated Ag NPs. These formed Ag-PL NPs showed potential mosquito larvicidal activity against Aedes aegypti (L.), Anopheles stephensi Liston and Culex quinquefasciatus Say. These Ag-PL NPs can also act as promising agents in cancer therapy. They exhibited anti-proliferative activity against HeLa cancer cell lines and a promising toxicity was observed in a dose dependent manner. Toxicity studies were further supported by the cellular DNA fragmentation in the Ag-PL NPs treated HeLa cells. - Highlights: • Green synthesis of protein-lipid conjugated Ag NPs using S. foetida L. seed extract. • S. foetida seed extract acted as good reducing and stabilizing agent for Ag NPs. • XPS and

  15. Biofabrication of Ag nanoparticles using Sterculia foetida L. seed extract and their toxic potential against mosquito vectors and HeLa cancer cells

    Rajasekharreddy, Pala; Rani, Pathipati Usha, E-mail: usharani65@yahoo.com

    2014-06-01

    A one-step and eco-friendly process for the synthesis of silver-(protein-lipid) nanoparticles (Ag-PL NPs) (core–shell) has been developed using the seed extract from wild Indian Almond tree, Sterculia foetida (L.) (Sterculiaceae). The reaction temperature played a major role in controlling the size and shell formation of NPs. The amount of NPs synthesized and qualitative characterization was done by UV–vis spectroscopy and transmission electron microscopy (TEM), respectively. TEM studies exhibited controlled dispersity of spherical shaped NPs with an average size of 6.9 ± 0.2 nm. Selected area electron diffraction (SAED) and X-ray diffraction (XRD) revealed ‘fcc’ phase and crystallinity of the particles. X-ray photoelectron spectroscopy (XPS) was used to identify the protein–lipid (PL) bilayer that appears as a shell around the Ag core particles. The thermal stability of the Ag-PL NPs was examined using thermogravimetric analysis (TGA). Further analysis was carried out by using Fourier transform infrared spectroscopy (FTIR), where the spectra provided evidence for the presence of proteins and lipid moieties ((2n-octylcycloprop-1-enyl)-octanoic acid (I)), and their role in synthesis and stabilization of Ag NPs. This is the first report of plant seed assisted synthesis of PL conjugated Ag NPs. These formed Ag-PL NPs showed potential mosquito larvicidal activity against Aedes aegypti (L.), Anopheles stephensi Liston and Culex quinquefasciatus Say. These Ag-PL NPs can also act as promising agents in cancer therapy. They exhibited anti-proliferative activity against HeLa cancer cell lines and a promising toxicity was observed in a dose dependent manner. Toxicity studies were further supported by the cellular DNA fragmentation in the Ag-PL NPs treated HeLa cells. - Highlights: • Green synthesis of protein-lipid conjugated Ag NPs using S. foetida L. seed extract. • S. foetida seed extract acted as good reducing and stabilizing agent for Ag NPs. • XPS and

  16. Thymoquinone-Loaded Nanostructured Lipid Carrier Exhibited Cytotoxicity towards Breast Cancer Cell Lines (MDA-MB-231 and MCF-7 and Cervical Cancer Cell Lines (HeLa and SiHa

    Wei Keat Ng

    2015-01-01

    Full Text Available Thymoquinone (TQ has been shown to exhibit antitumor properties. Thymoquinone-loaded nanostructured lipid carrier (TQ-NLC was developed to improve the bioavailability and cytotoxicity of TQ. This study was conducted to determine the cytotoxic effects of TQ-NLC on breast cancer (MDA-MB-231 and MCF-7 and cervical cancer cell lines (HeLa and SiHa. TQ-NLC was prepared by applying the hot high pressure homogenization technique. The mean particle size of TQ-NLC was 35.66 ± 0.1235 nm with a narrow polydispersity index (PDI lower than 0.25. The zeta potential of TQ-NLC was greater than −30 mV. Polysorbate 80 helps to increase the stability of TQ-NLC. Differential scanning calorimetry showed that TQ-NLC has a melting point of 56.73°C, which is lower than that of the bulk material. The encapsulation efficiency of TQ in TQ-NLC was 97.63 ± 0.1798% as determined by HPLC analysis. TQ-NLC exhibited antiproliferative activity towards all the cell lines in a dose-dependent manner which was most cytotoxic towards MDA-MB-231 cells. Cell shrinkage was noted following treatment of MDA-MB-231 cells with TQ-NLC with an increase of apoptotic cell population (P<0.05. TQ-NLC also induced cell cycle arrest. TQ-NLC was most cytotoxic towards MDA-MB-231 cells. It induced apoptosis and cell cycle arrest in the cells.

  17. Adeno-associated virus sensitizes HeLa cell tumors to gamma rays.

    Walz, C; Schlehofer, J R; Flentje, M; Rudat, V; zur Hausen, H

    1992-01-01

    Infection with the helper virus-dependent human parvovirus adeno-associated virus (AAV) is known to interfere with cellular transformation in vitro and oncogenesis in vivo. Here we report on sensitization to gamma irradiation by AAV infection of cells in culture and of tumors established from HeLa cells grafted into immunodeficient (nude) mice: infection of HeLa cells with AAV type 2 enhanced cell killing and reduced plating efficiency after irradiation compared with uninfected cells. Similarly, HeLa cell tumors in nude mice displayed a reduced growth rate and were more sensitive to gamma irradiation when the animals were infected with AAV type 2 prior to or after tumor cell inoculation. Since no pathogenicity is known for AAV, the ability of this virus to render radiotherapy of human tumor cells more efficient may up open novel approaches in cancer treatment. Images PMID:1323717

  18. Immune Killing Activity of Lymphocytes on Hela Cells Expressing Interleukin-12 In Vitro

    Huiyan WANG; Suhua CHEN

    2008-01-01

    The killing effects of lymphocytes on Hela cells expressing intedeukin-12 (IL-12) in vitro were explored. By using gene transfection technique, full length IL-12 gene was transfected into Hela cells. The expression of IL-12 in Hela cells was detected quantitatively by ELISA; Changes in killing effects of lymphocytes on Hela cells expressing IL-12 were observed by MTT. It was found that Hela cells could express IL-12 between 24h and 72h after transfection. Killing activity of lymphocytes on Hela cells expressing IL-12 was significantly enhanced. It was concluded by cell transfection technique, Hela cells could express IL-12 and were more easily killed by lymphocytes.

  19. Antiproliferative effects of some medicinal plants on HeLa cells

    Cenić-Milošević Desanka

    2013-01-01

    Full Text Available Medicinal plants maintain the health and vitality of individuals, and also have potential curative effect on various diseases, including cancer. In this study were investigated the antiproliferative effects of water extracts of previously obtained ethanolic dry extracts of three different medicinal plants (Echinacea angustifolia, Salvia officinalis and Melissa officinalis on cell lines derived from human cervix adenocarcinoma (HeLa cells. The best cytotoxic activity (IC50 = 43.52 μg/ml on HeLa cell lines was exhibited by Echinacea angustifolia. The extract of Salvia officinalis also showed a good cytotoxic activity against HeLa cell lines; the IC50 value was 70.41 μg/ml. Melissa officinalis manifested a slightly weaker cytotoxic activity and an IC50 value of 122.22 μg/ml. [Projekat Ministarstva nauke Republike Srbije, br. 34021 i br. 175011

  20. Condurango (Gonolobus condurango Extract Activates Fas Receptor and Depolarizes Mitochondrial Membrane Potential to Induce ROS-dependent Apoptosis in Cancer Cells in vitro CE-treatment on HeLa: a ROS-dependent mechanism

    Kausik Bishayee

    2015-09-01

    Full Text Available Objectives: Condurango (Gonolobus condurango extract is used by complementary and alternative medicine (CAM practitioners as a traditional medicine, including homeopathy, mainly for the treatment of syphilis. Condurango bark extract is also known to reduce tumor volume, but the underlying molecular mechanisms still remain unclear. Methods: Using a cervical cancer cell line (HeLa as our model, the molecular events behind condurango extract’s (CE’s anticancer effect were investigated by using flow cytometry, immunoblotting and reverse transcriptase-polymerase chain reaction (RT-PCR. Other included cell types were prostate cancer cells (PC3, transformed liver cells (WRL-68, and peripheral blood mononuclear cells (PBMCs. Results: Condurango extract (CE was found to be cytotoxic against target cells, and this was significantly deactivated in the presence of N-acetyl cysteine (NAC, a scavenger of reactive oxygen species (ROS, suggesting that its action could be mediated through ROS generation. CE caused an increase in the HeLa cell population containing deoxyribonucleic acid (DNA damage at the G zero/Growth 1 (G0/G1 stage. Further, CE increased the tumor necrosis factor alpha (TNF-α and the fas receptor (FasR levels both at the ribonucleic acid (RNA and the protein levels, indicating that CE might have a cytotoxic mechanism of action. CE also triggered a sharp decrease in the expression of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB both at the RNA and the protein levels, a possible route to attenuation of B-cell lymphoma 2 (Bcl-2, and caused an opening of the mitochondrial membrane’s permeability transition (MPT pores, thus enhancing caspase activities. Conclusion: Overall, our results suggest possible pathways for CE mediated cytotoxicity in model cancer cells.

  1. Tumoricidal effects of nanomaterials in HeLa cell line

    Fakhar-E-Alam, M.; Kishwar, S.; Khan, Y.; Siddique, M.; Atif, M.; Nur, O.; Willander, M.

    2011-11-01

    The current study exhibits the cellular response of HeLa (cervical cancer) cells to metal oxides ultrafine nanomaterials e.g. manganese dioxide nanowires (MnO2 NRs), iron oxide nanoparticles (Fe2O3 NPs) and zinc oxide nanorods (ZnO NRs) as bare and as conjugated with photosensitizers. For cytotoxic evaluations, the cellular morphology, (MTT) assay, reactive oxygen species (ROS) production were used for cases with and without photo sensitizer as well illuminated with UV-visible laser exposed conditions. Three different photosensitizers were tested. These are 5-aminolevulinic acid (5-ALA), Photofrin® and protopor phyrin dimethyl ester (PPDME). Significant loss in cell viability was noted with 100-500 μg/ml in bare and conjugated forms of the metal oxides used. The effect was insignificant with lower concentrations (0.05-50 μg/ml). While notable anticancer effect of 5-ALA under 30 J/cm2 of diode laser irradiation was noted as compared to other photo sensitizer. By increasing the UV irradiation time of labeled cells, generation of ROS was observed, indicating the possibility of achieving efficient photodynamic therapy (PDT).

  2. Inhibition of 3-bromopyruvate on proliferation of human cervical cancer HeLa cells%3-溴丙酮酸对人宫颈癌HeLa细胞增殖的抑制作用

    黄小艳; 余进进; 潘敏; 杨文霞; 任峰

    2012-01-01

    Objective: To observe the inhibitory effect of 3 - bromopyruvate (3 - BrPA) on the cell proliferation of HeLa human cervical cancer cell line. Methods ;The HeLa cells were exposed to 3-bromopyruvate environment with different concentrations and different times, then cell growth was measured by MTT, the cell cycle distribution and apoptotic or necrotic cell death were detected with flow cytometry. The morphologic changes of HeLa cells were observed with convert light microscopy and fluorescent staining. Results:3 - bromopyruvate( >50nmol/L) significantly inhibited the proliferation of HeLa cells in a does - dependent manner, induced G2/M phase arrest(P < 0.01), cell necrosis and apoptosis (P <0. 01). The HeLa cells were treated with 3 - bromopyruvate for 24h, convert light microscopy showed cell growth was sparse, clarity cytoplasm decreased and dead cell increased. Conclusion: 3 - bro-mopyruvate can efficiently inhibit the proliferation of Hela cells.%目的:研究3-溴丙酮酸(3-BrPA)对宫颈癌HeLa细胞生长及增殖的影响.方法:HeLa细胞经不同浓度3-BrPA作用后,用四甲基偶氮唑蓝(MTT)检测HeLa细胞的增殖情况,倒置显微镜及荧光显微镜观察细胞形态,流式细胞仪检测细胞周期分布及坏死或凋亡情况.结果:经3-BrPA作用一段时间后:MTT检测发现在一定浓度范围内( 50-200) μmol/L对HeLa细胞增殖的抑制作用呈浓度依赖性,与对照组比较,各浓度组差异均有统计学意义(P<0.01).倒置显微镜下可见HeLa细胞生长稀疏,细胞透亮度下降,细胞皱缩、破碎.Hoechst染色后荧光显微镜下呈现典型的凋亡核固缩表现.细胞周期分析结果表明,可诱导HeLa细胞G2/M期阻滞(P<0.01).流式细胞仪及荧光显微镜观察表明3-BrPA可诱导细胞坏死和凋亡(P<0.01).结论:3-BrPA对宫颈癌细胞增殖有显著抑制作用.

  3. ANTICANCER AND CYTOTOXIC POTENTIAL OF TRITICUM AESTIVUM EXTRACT ON HELA CELL LINE

    Patel Janki B.; Patel Piyush M.

    2013-01-01

    The objective of the study was to analyze the anticancer property of the leaves of Triticum aestivum on HeLa cells. The Indian medicinal plant Triticum aestivum that is used in traditional medicine for cancer and non cancerous diseases was collected. The crude methanolic extract was prepared by using standard protocols. The antiproliferative effect the methanolic extract was evaluated in vitro by employing MTT assay. The potency of each plant extract concentration was calculated in terms of p...

  4. Isolation of Melittin from Iranian Honey Bee Venom and Investigation of Its Effect on Proliferation of Cervical Cancer- HeLa Cell Line

    K Pooshang Bagheri; A Mahmoodzadeh; H Zarinnahad; M. Mahdavi; Shahbazzadeh, D.; A Moradi

    2013-01-01

    Introduction: Cervical cancer is the second prevalent cancer in developing countries and the sixth prevalent cancer in USA. Since conventional treatment methods are associated with detrimental side effects, searching for new drugs using natural ingredients is very important. Previous studies have shown that melittin (main component of honey bee venom) has anticancer properties along with the effect on cell membrane and activation of apoptosis. In this study, inhibitory effects of melittin on ...

  5. In vitro studies on radiosensitization effect of glucose capped gold nanoparticles in photon and ion irradiation of HeLa cells

    Kaur, Harminder; Pujari, Geetanjali; Semwal, Manoj K.; Sarma, Asitikantha; Avasthi, Devesh Kumar

    2013-04-01

    Noble metal nanoparticles are of great interest due to their potential applications in diagnostics and therapeutics. In the present work, we synthesized glucose capped gold nanoparticle (Glu-AuNP) for internalization in the HeLa cell line (human cervix cancer cells). The capping of glucose on Au nanoparticle was confirmed by Raman spectroscopy. The Glu-AuNP did not show any toxicity to the HeLa cell. The γ-radiation and carbon ion irradiation of HeLa cell with and without Glu-AuNP were performed to evaluate radiosensitization effects. The study revealed a significant reduction in radiation dose for killing the HeLa cells with internalized Glu-AuNPs as compared to the HeLa cells without Glu-AuNP. The Glu-AuNP treatment resulted in enhancement of radiation effect as evident from increase in relative biological effectiveness (RBE) values for carbon ion irradiated HeLa cells.

  6. Phosphatidylinositol anchor of HeLa cell alkaline phosphatase

    Alkaline phosphatase from cancer cells, HeLa TCRC-1, was biosynthetically labeled with either 3H-fatty acids or [3H]ethanolamine as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitated material. Phosphatidylinositol-specific phospholipase C (PI-PLC) released a substantial proportion of the 3H-fatty acid label from immunoaffinity-purified alkaline phosphatase but had no effect on the radioactivity of [3H]ethanolamine-labeled material. PI-PLC also liberated catalytically active alkaline phosphatase from viable cells, and this could be selectively blocked by monoclonal antibodies to alkaline phosphatase. However, the alkaline phosphatase released from 3H-fatty acid labeled cells by PI-PLC was not radioactive. By contrast, treatment with bromelain removed both the 3H-fatty acid and the [3H]ethanolamine label from purified alkaline phosphatase. Subtilisin was also able to remove the [3H]ethanolamine label from the purified alkaline phosphatase. The 3H radioactivity in alkaline phosphatase purified from [3H]ethanolamine-labeled cells comigrated with authentic [3H]ethanolamine by anion-exchange chromatography after acid hydrolysis. The data suggest that the 3H-fatty acid and [3H]ethanolamine are covalently attached to the carboxyl-terminal segment since bromelain and subtilisin both release alkaline phosphatase from the membrane by cleavage at that end of the polypeptide chain. The data are consistent with findings for other proteins recently shown to be anchored in the membrane through a glycosylphosphatidylinositol structure and indicate that a similar structure contributes to the membrane anchoring of alkaline phosphatase

  7. Phosphatidylinositol anchor of HeLa cell alkaline phosphatase

    Jemmerson, R.; Low, M.G.

    1987-09-08

    Alkaline phosphatase from cancer cells, HeLa TCRC-1, was biosynthetically labeled with either /sup 3/H-fatty acids or (/sup 3/H)ethanolamine as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitated material. Phosphatidylinositol-specific phospholipase C (PI-PLC) released a substantial proportion of the /sup 3/H-fatty acid label from immunoaffinity-purified alkaline phosphatase but had no effect on the radioactivity of (/sup 3/H)ethanolamine-labeled material. PI-PLC also liberated catalytically active alkaline phosphatase from viable cells, and this could be selectively blocked by monoclonal antibodies to alkaline phosphatase. However, the alkaline phosphatase released from /sup 3/H-fatty acid labeled cells by PI-PLC was not radioactive. By contrast, treatment with bromelain removed both the /sup 3/H-fatty acid and the (/sup 3/H)ethanolamine label from purified alkaline phosphatase. Subtilisin was also able to remove the (/sup 3/H)ethanolamine label from the purified alkaline phosphatase. The /sup 3/H radioactivity in alkaline phosphatase purified from (/sup 3/H)ethanolamine-labeled cells comigrated with authentic (/sup 3/H)ethanolamine by anion-exchange chromatography after acid hydrolysis. The data suggest that the /sup 3/H-fatty acid and (/sup 3/H)ethanolamine are covalently attached to the carboxyl-terminal segment since bromelain and subtilisin both release alkaline phosphatase from the membrane by cleavage at that end of the polypeptide chain. The data are consistent with findings for other proteins recently shown to be anchored in the membrane through a glycosylphosphatidylinositol structure and indicate that a similar structure contributes to the membrane anchoring of alkaline phosphatase.

  8. Dosimetry study on photodynamic effect of PSD-007 on human cervical cancer cell line Hela%PSD-007对宫颈癌Hela细胞光动力杀伤效应的剂量学研究

    叶绪英; 阴慧娟; 王宏; 张洪梅; 顾立超; 刘天军

    2014-01-01

    Objective To investigate the photochemotherapeutic effect and the main affecting factors of PSD-007 on human cervical cancer Hela in vitro.Methods Hela cells were treated with different concentrations of PSD-007 (0,3.125,6.25,12.5,25,50,100 μg/ml) for 2 h under the influence of low-level laser (635 nm) therapy at different doses (0,0.6,1.2,2.4,4.8,9.6 J/cm2).Then the OD values and survival rates of Hela cells were measured by MTT assay compared with breast cancer cells MCF-7 in same treatment.Hela cells were treated with 12.5 μg/ml of PSD-007 for 2 h and were treated with different intensities of laser (1.2,2.4,4.8 J/cm2).The cellular apoptosis rate and cell cycle phase distribution of Hela were measured by a flow cytometry (FCM).Results Survival rates of Hela cells declined with more than 25 μg/ml of PSD-007 only,and significant difference in the inhibitory between the PDT group and control group was observed (P<0.05).The survival rates of Hela after PDT was decreased by the concentration of sensitizer and dose of laser.There were no significant differences of cell survival rates among the groups with concentrations more than 12.5 μg/ml and laser energy density more than 4.8 J/cm2.The FCM assay showed a G0/G1 cell cycle arrest in a time-dependent manner.Conclusions PSD-007 has a photodynamic effect on Hela in vitro.Photodynamic effect of PSD-007 was more significant in Hela than MCF-7.Less photosensitizer and laser energy density were needed.%目的 探讨癌光啉(PSD-007)对人宫颈癌Hela细胞体外光动力杀伤效应及主要影响因素.方法 不同质量浓度(0、3.125、6.25、12.5、25、50、100 μg/ml)的PSD-007与Hela细胞共同孵育2h后,予以不同能量(0、0.6、1.2、2.4、4.8、9.6 J/cm2)635 nm波长的激光照射,以相同剂量光照和光敏剂剂量的人乳腺癌细胞系MCF-7光动力杀伤作用做对比,通过噻唑蓝(MTT)比色法测定细胞的光密度(OD)值及存活率;质量浓度为12.5 μg/ml的PSD-007

  9. Energy metabolism in hela and walker-256 cells

    Attempts to measure ATP content in terms of amino acid incorporation into proteins in the presence of oxamate with and without glucose seem to indicate that uptake of labelled amino acid is independent of ATP level. This was quite in contrast to actual growth measurements where growth inhibition was observed only in the presence of glucose. Probably oxamate interfered with the transport of amino acid into HeLa cells and this was readily releived by adding pyruvate. In another system using Walker 256 carcinoma cells, oxamate effect to interfere with the uptake of label either through incorporation or transport was not observed suggesting a difference between HeLa and Walker cells in energy potential. (author)

  10. Dynamic friction measurements on living HeLa cells

    Goulet, Marc-Antoni; Colbert, Marie-Josée; Dalnoki-Veress, Kari

    2008-03-01

    The interaction of cells with various interfaces, and especially man-made surfaces, is an active field of research. In our experiment we use a micropipette to measure both the friction and normal force as a cell slides across a surface. A thin substrate, coated with Poly-L-Lysine is brought into contact with a HeLa cell. The adjustable substrate motion is used to study the response of the cell at various normal forces and speeds. Analysis of the micropipette provides dynamic measurements of both the friction and normal force. With our novel setup we are able to probe the attachment/detachment process of living cells.

  11. ANTICANCER AND CYTOTOXIC POTENTIAL OF TRITICUM AESTIVUM EXTRACT ON HELA CELL LINE

    Patel Janki B.

    2013-01-01

    Full Text Available The objective of the study was to analyze the anticancer property of the leaves of Triticum aestivum on HeLa cells. The Indian medicinal plant Triticum aestivum that is used in traditional medicine for cancer and non cancerous diseases was collected. The crude methanolic extract was prepared by using standard protocols. The antiproliferative effect the methanolic extract was evaluated in vitro by employing MTT assay. The potency of each plant extract concentration was calculated in terms of percent decrease in viable HeLa cells as compared to the control value. The extract showed dose dependent antitumor activity. The MTT assay showed an anti proliferative activity (IC50 at 156 μg/ml of crude extract.

  12. Reduced BCL2 and CCND1 mRNA expression in human cervical cancer HeLa cells treated with a combination of everolimus and paclitaxel

    Yilmaz, Akin; Alp, Ebru; Onen, H. Ilke; MENEVSE, SEVDA

    2016-01-01

    Aim of the study Cervical cancer is the second most common malignancy in women worldwide. Everolimus displays direct effects on growth and proliferation of cancer cells via inhibition of mammalian target of rapamycin (mTOR) protein, which is known to be associated with drug resistance. In this study, we aimed to investigate the effects of everolimus, gemcitabine, and paclitaxel in terms of cell viability and mRNA expression levels of GRP78, CCND1, CASP2, and BCL2 genes. Material and methods H...

  13. Harmonizing HeLa cell cytoskeleton behavior by multi-Ti oxide phased nanostructure synthesized through ultrashort pulsed laser

    Chinnakkannu Vijayakumar, Chandramouli; Venkatakrishnan, Krishnan; Tan, Bo

    2015-10-01

    Knowledge about cancer cell behavior on heterogeneous nanostructures is relevant for developing a distinct biomaterial that can actuate cancer cells. In this manuscript, we have demonstrated a harmonized approach of forming multi Ti-oxide phases in a nanostructure (MTOP nanostructure) for its unique cancer cell controlling behavior.Conventionally, single phases of TiO2 are used for targeted therapy and as drug carrier systems.In this research, we have shown a biomaterial that can control HeLa cells diligently using a combination of TiO, Ti3O and TiO2 phases when compared to fibroblast (NIH3T3) cells.MTOP-nanostructures are generated by varying the ionization energy in the vapor plume of the ultrashort pulse laser; this interaction with the material allows accurate tuning and composition of phases within the nanostructure. In addition, the lattice spacing of MTOP-nanostructures was analyzed as shown by HR-TEM investigations. An FESEM investigation of MTOP-nanostructures revealed a greater reduction of HeLa cells relative to fibroblast cells. Altered cell adhesion was followed by modulation of HeLa cell architecture with a significant reduction of actin stress fibers.The intricate combination of MTOP-nanostructures renders a biomaterial that can precisely alter HeLa cell but not fibroblast cell behavior, filling a void in the research for a biomaterial to modulate cancer cell behavior.

  14. Effects of antisense oligonucleotides targeting VEGF on radio sensitivity of uterine cervix cancer Hela cells%血管内皮生长因子反义核酸对宫颈癌Hela细胞的放射增敏作用

    Lina Xing; Li Qi

    2009-01-01

    Objective: To determine the impact of antisense oligonucleotides targeting vascular endothelial growth factor (VEGF) on radiosensitivity of uterine cervix cancer Hela cells. Methods: VEGF antisense oligodeoxynucleotides (ASODN) was transfected into Hela cells by liposome-mediated method. Cells transfected with the oligodeoxynuclecotide and saline were used as control groups. Cells were irradiated by 6 MV X ray at the dose of 0 Gy, 2 Gy, 4 Gy and 6 Gy respectively. The expression of VEGF mRNA was determined by RT-PCR. Apoptosis were evaluated using FCM. Cloning efficiency was deter-mined by colony formation assay. Results: The expression of VEGF mRNA was inhibited by ASODN (P < 0.01) in Hela cells. The inhibited activation which was influenced by radiation resulted in increasing apoptosis (P < 0.01) and inhibiting plating efficiency (P < 0.01). Conclusion: The expression of VEGF induced by Ⅹ irradiation in Hela cells can be blocked by VEGF ASODN. Treatment with VEGF might increase apoptosis in HeLa cells and enhance radiosensitivity.

  15. STUDY OF ENHANCED IMMUNOGENECITY OF B7-1 GENE TRANSFECTED HUMAN HELA CELL LINE

    He Xi; Qin Huilian; Xiang Rong; Zhang Yuejian; Ye Wenfei; He Qiuzao

    1998-01-01

    Although cervical carcinoma cells may express the human papillomavirus protein E6 and E7, they fail to induce an effective specific cytotoxic T lymphocyte response. Recent studies suggest that expression of CD-80(B7-1) on tumor cells is effective to induce anfitumor immune responses.1,2 In our study, CD-80 gene was transfected into human Hela cell line with a CD-80expression plasmid (B7-1+pcDNA3) by electroporation,then the immunogenecity of the modified Hela cell was tested in TLMC (tumor lymphocyte mixed culture)system. [3H]thymidine lymphocyte proliferation assays showed that the response of human peripheral blood lymphocytes (PBLS) to CD-80 positive Hela cells demonstrated a substantial increase in cell proliferation compared to the response to control cells. Cocultivation of allogeneic PBLs with CD-80 positive tumor cells for three days can induce an increased secretion of IL-2. Our results demonstrate an immunostimulatory effect of CD-80 expression on cervical cancer cells, which provides a basis for the development of a therapeutic tumor vaccine.

  16. Investigation of role of aspartame on apoptosis process in HeLa cells -->.

    Pandurangan, Muthuraman; Enkhtaivan, Gansukh; Mistry, Bhupendra; Chandrasekaran, Murugesan; Noorzai, Rafi; Kim, Doo Hwan

    2016-07-01

    Aspartame is an artificial sweetener used as an alternate for sugar in several foods and beverages. The study reports that consumption of aspartame containing product could lead to cancer. However, the effect of aspartame on apoptosis process in cancer is not yet understood clearly. HeLa cells were exposed to different concentrations (0.01-0.05 mg/ml) of aspartame for 48 h. Cytotoxicity of aspartame on cancer cells was determined by SRB assay. The result indicates no significant changes on cell viability. Aspartame suppresses apoptosis process in cancer cells by down-regulation of mRNA expression of tumor suppressor gene p53, and pro-apoptotic gene bax. It up-regulates anti-apoptotic gene bcl-2 mRNA expression. In addition, Ki 67 and PCNA mRNA, and protein expressions were determined. Taking all these together, we conclude that aspartame may be a potent substance to slow-down the apoptosis process in HeLa cells. Further works are ongoing to understand the biochemical and molecular mechanism of aspartame in cancer cells. PMID:27298583

  17. Investigation of role of aspartame on apoptosis process in HeLa cells

    Muthuraman Pandurangan

    2016-07-01

    Full Text Available Aspartame is an artificial sweetener used as an alternate for sugar in several foods and beverages. The study reports that consumption of aspartame containing product could lead to cancer. However, the effect of aspartame on apoptosis process in cancer is not yet understood clearly. HeLa cells were exposed to different concentrations (0.01–0.05 mg/ml of aspartame for 48 h. Cytotoxicity of aspartame on cancer cells was determined by SRB assay. The result indicates no significant changes on cell viability. Aspartame suppresses apoptosis process in cancer cells by down-regulation of mRNA expression of tumor suppressor gene p53, and pro-apoptotic gene bax. It up-regulates anti-apoptotic gene bcl-2 mRNA expression. In addition, Ki 67 and PCNA mRNA, and protein expressions were determined. Taking all these together, we conclude that aspartame may be a potent substance to slow-down the apoptosis process in HeLa cells. Further works are ongoing to understand the biochemical and molecular mechanism of aspartame in cancer cells.

  18. Three-dimensional printing of Hela cells for cervical tumor model in vitro

    Advances in three-dimensional (3D) printing have enabled the direct assembly of cells and extracellular matrix materials to form in vitro cellular models for 3D biology, the study of disease pathogenesis and new drug discovery. In this study, we report a method of 3D printing for Hela cells and gelatin/alginate/fibrinogen hydrogels to construct in vitro cervical tumor models. Cell proliferation, matrix metalloproteinase (MMP) protein expression and chemoresistance were measured in the printed 3D cervical tumor models and compared with conventional 2D planar culture models. Over 90% cell viability was observed using the defined printing process. Comparisons of 3D and 2D results revealed that Hela cells showed a higher proliferation rate in the printed 3D environment and tended to form cellular spheroids, but formed monolayer cell sheets in 2D culture. Hela cells in 3D printed models also showed higher MMP protein expression and higher chemoresistance than those in 2D culture. These new biological characteristics from the printed 3D tumor models in vitro as well as the novel 3D cell printing technology may help the evolution of 3D cancer study. (paper)

  19. Research of the relationship of intracellular acidification and apoptosis in Hela cells based on pH nanosensors

    HE XiaoXiao; WANG Yan; WANG KeMin; PENG JiaoFeng; LIU Fang; TAN WeiHong

    2007-01-01

    In this paper, the relationship of intracellular acidification and apoptosis in Hela cells induced by vincristine sulfate has been studied by use of the ratiometric pH nanosensors that have been developed by our group, employing fluorescein isothiocyanate (FITC) doped as the pH-sensitive dye and Tris(2,2'-bipyidyl) dichlororuthenium(Ⅱ) hexahydrate (RuBpy) doped as reference dye. The pH change of the Hela cells induced by vincristine sulfate has been monitored in vivo, in situ and real time by use of the ratiometric pH nanosensors. The experimental results show that the pH of the apoptotic Hela cells induced by vincristine sulfate has been acidified from 7.11 to 6.51, and the percentage of intracellular acidification is correlated with the induced concentration and incubation time of the vincristine sulfate. The further study of the percentage of intracellular acidification and the percentage of apoptosis of Hela cells at the same time reveals that apoptosis of Hela cells induced by vincristine sulfate is preceded by intracellular acidification. These results would provide theoretical foundation for the therapy of cancer through interfering the pH of cells by use of vincristine sulfate or other anti-cancer drugs.

  20. In vitro studies on radiosensitization effect of glucose capped gold nanoparticles in photon and ion irradiation of HeLa cells

    Kaur, Harminder; Pujari, Geetanjali [Radiation Biology Group, Inter University Accelerator Centre, Post Box 10502, New Delhi 110067 (India); Semwal, Manoj K. [Army Hospital (R and R), Delhi Cantonment, New Delhi 110010 (India); Sarma, Asitikantha [Radiation Biology Group, Inter University Accelerator Centre, Post Box 10502, New Delhi 110067 (India); Avasthi, Devesh Kumar, E-mail: dka@iuac.res.in [Radiation Biology Group, Inter University Accelerator Centre, Post Box 10502, New Delhi 110067 (India)

    2013-04-15

    Highlights: ► Glucose capped gold nanoparticles (Glu-AuNPs) are synthesized for internalization in HeLa cells (cervical cancer cells). ► Internalization of Glu-AuNPs in HeLa cells is confirmed by cross section TEM of cells. ► Irradiation (by C ion or γ-rays) of HeLa cells with internalized Glu-AuNPs results in enhanced radiosensitization. ► There is about 30% reduction in radiation dose for 90% cell killing of HeLa cells, when internalized by Glu-AuNPs. ► The enhanced radiosensitization due to Glu-AuNPs is of interest for researchers in nanobiotechnology and radiation biology. -- Abstract: Noble metal nanoparticles are of great interest due to their potential applications in diagnostics and therapeutics. In the present work, we synthesized glucose capped gold nanoparticle (Glu-AuNP) for internalization in the HeLa cell line (human cervix cancer cells). The capping of glucose on Au nanoparticle was confirmed by Raman spectroscopy. The Glu-AuNP did not show any toxicity to the HeLa cell. The γ-radiation and carbon ion irradiation of HeLa cell with and without Glu-AuNP were performed to evaluate radiosensitization effects. The study revealed a significant reduction in radiation dose for killing the HeLa cells with internalized Glu-AuNPs as compared to the HeLa cells without Glu-AuNP. The Glu-AuNP treatment resulted in enhancement of radiation effect as evident from increase in relative biological effectiveness (RBE) values for carbon ion irradiated HeLa cells.

  1. Berberine alters epigenetic modifications, disrupts microtubule network, and modulates HPV-18 E6-E7 oncoproteins by targeting p53 in cervical cancer cell HeLa: a mechanistic study including molecular docking.

    Saha, Santu Kumar; Khuda-Bukhsh, Anisur Rahman

    2014-12-01

    Increased evidence of chemo-resistance, toxicity and carcinogenicity necessitates search for alternative approaches for determining next generation cancer therapeutics and targets. We therefore tested the efficacy of plant alkaloid berberine on human papilloma virus (HPV) -18 positive cervical cancer cell HeLa systematically-involving certain cellular, viral and epigenetic factors. We observed disruptions of microtubule network and changes in membrane topology due to berberine influx through confocal and atomic force microscopies (AFM). We examined nuclear uptake, internucleosomal DNA damages, mitochondrial membrane potential (MMP) alterations and cell migration assays to validate possible mode of cell death events. Analytical data on interactions of berberine with pBR322 through fourier transform infrared (FTIR) and gel migration assay strengthen berberine׳s biologically significant DNA binding abilities. We measured cellular uptake, DNA ploidy and DNA strand-breaks through fluorescence activated cell sorting (FACS). To elucidate epigenetic modifications, in support of DNA binding associated processes, if any, we conducted methylation-specific restriction enzyme (RE) assay, methylation specific-PCR (MSP) and expression studies of histone proteins. We also analyzed differential interactions and localization of cellular tumor suppressor p53 and viral oncoproteins HPV-18 E6-E7 through siRNA approach. We further made in-silico approaches to determine possible binding sites of berberine on histone proteins. Overall results indicated cellular uptake of berberine through cell membrane depolarization causing disruption of microtubule networks and its biological DNA binding abilities that probably contributed to epigenetic modifications. Results of modulation in p53 and viral oncoproteins HPV-18 E6-E7 by berberine further proved its potential as a promising chemotherapeutic agent in cervical cancer. PMID:25448308

  2. Metabolic switch in uterine cervix cancer: in vitro study of adenocarcinoma (HeLa) and squamous cell carcinoma (SiHa) cell lines

    Silva, Lídia Jorge Santos, 1989-

    2012-01-01

    Tese de mestrado. Biologia (Biologia Humana e Ambiente). Universidade de Lisboa, Faculdade de Ciências, 2012 O cancro é uma doença complexa que envolve numerosas alterações na fisiologia da célula que conduzem, em última instância, a tumores malignos. Os processos biológicos através dos quais as células normais são transformadas em células cancerígenas malignas têm sido alvo de vasta investigação durante várias décadas (Seyfried & Shelton, 2010). Existem seis alterações essenciais na fisio...

  3. Cloning of smac gene and its overexpression effects on radiosensitivity of HeLa cells to γ-rays

    Objective: To clone smac gene and construct eukaryocytic expression vector pcDNA3.1/ smac. The smac gene was transfected into HeLa cells to explore the effects of over-expression of extrinsic smac gene on radiosensitivity to γ-rays of HeLa cells. Methods: The full-length smac gene was amplified from total RNA of HeLa cells by RTPCR. The RTPCR product was ligated with the vector pcDNA3.1 and sequenced. The correct pcDNA3.1/smac was transfected into HeLa cells. The expression of smac gene was tested by RTPCR and Western blot. The cellular growth inhibition rates were evaluated by MTT 48 horns after irradiation with different doses of γ-rays. Results: Recombinant eukaryocytic expression vector pcDNA3.1/smac was successfully constructed. RTPCR and Western blot results indicated that the expression of smac gene of HeLa/smac cells was significantly enhanced compared with the expression of smac gene of HeLa/pcDNA3.1 and HeLa cells. 48 hours after different doses of γ-ray irradiation was significantly higher in pcDNA3.1/smac transfected HeLa/smac cells than those of non-transfected HeLa cells or pcDNA3.1 transfected HeLa/pcDNA3.1 cells, inhabitation rates were 38.85%, 17.64% and 20.32%, respectively. Conclusions: smac gene was successfully cloned. Extrinsic smac gene over-expression could significantly enhance radiosensitivity to γ-ray of HeLa cells, which would herald a new approach to improve radiosensitivity of cervical cancer. (authors)

  4. Outcome of Treatment of Human HeLa Cervical Cancer Cells With Roscovitine Strongly Depends on the Dosage and Cell Cycle Status Prior to the Treatment

    Wesierska-Gadek, J.; Borza, A.; Walzi, E.; Kryštof, Vladimír; Maurer, M.; Komina, O.; Wandl, S.

    2009-01-01

    Roč. 106, č. 5 (2009), s. 937-955. ISSN 0730-2312 Institutional research plan: CEZ:AV0Z50380511 Keywords : APOPTOSIS * CELL CYCLE ARREST * CYCLIN-DEPENDENT KINASES Subject RIV: ED - Physiology Impact factor: 2.935, year: 2009

  5. From HeLa cell division to infectious diarrhoea

    Stephen, J.; Osborne, M.P.; Spencer, A.J.; Warley, A. (Univ. of Birmingham (England))

    1990-09-01

    Hela S3 cells were grown in suspension both randomly and, synchronously using hydroxyurea which blocks cells at the G1/S interface. Cryosections were prepared, freeze-dried and analyzed by X-ray microanalysis. As cells moved into S and through M phases (Na) and (Cl) increased; both returned to normal levels upon re-entering G1 phase. The Na/K ratio was 1:1 in G1 phase. Infection of HeLa S3 cells in G1 phase with vaccinia virus resulted in no change in intracellular (Na). Infection of neonatal mice with murine rotavirus was localized to villus tip enterocytes and gave rise to diarrhoea which was maximal at 72h post-infection (p.i.). Diarrhoea was preceded by ischemia of villi (18-42h p.i.) and villus shortening (maximal at 42h p.i.), and was also coincident with a dramatic regrowth of villi. At 48h p.i. a proliferative zone of electron lucent cells was observed in villus base regions. Cryosections of infected gut, taken before, during, and after infection, together with corresponding age-matched controls, were freeze-dried and analysed by X-ray microanalysis. At 48h p.i. electron lucent villus base cells were shown to be more hydrated, and, to contain higher levels of both Na and Cl and lower levels of P, S, K and Mg than corresponding control cells. These studies increase confidence in the use of X-ray microanalysis in studying biological systems, provide some insight into the process of cell division, and constitute the basis of a new concept of diarrhoeal secretion.27 references.

  6. Effects of depsidones from Hypogymnia physodes on HeLa cell viability and growth.

    Stojanović, I Z; Najman, S; Jovanović, O; Petrović, G; Najdanović, J; Vasiljević, P; Smelcerović, A

    2014-01-01

    The anti-proliferative activitiy of Hypogymnia physodes methanol extracts (ME) and its main constituents, physodalic acid (P1), physodic acid (P2), and 3-hydroxy physodic acid (P3), was tested on human cancer HeLa cell lines. Three lichen depsidones, P1, P2 and P3, were isolated from H. physodes ME using column chromatography and their structures were determined by UV, ESI TOF MS, 1H and 13C NMR. The content of P1, P2 and P3 in ME was determined using reversed-phase highperformance liquid chromatography with photodiode array detection. P1-3 represented even 70 % of the studied extract. The HeLa cells were incubated during 24 and 72 h in the presence of ME and depsidones P1, P2 and P3, at concentrations of 10-1000 μg/ml. Compounds P2 and P3 showed higher activity than compound P1. Half maximal inhibitory concentrations (IC50, μg/ml) of P1, P2, P3 and ME for 24-h incubation were 964, 171, 97 and 254 μg/ml, respectively, while for 72-h incubation they were 283, 66, 63 and 68 μg/ml. As far as we know, this is the first report on the effect of H. physodes ME and their depsidones on HeLa cells. PMID:24785112

  7. UDP-glucuronosyltransferase (UGT) 1A9-overexpressing HeLa cells is an appropriate tool to delineate the kinetic interplay between breast cancer resistance protein (BRCP) and UGT and to rapidly identify the glucuronide substrates of BCRP.

    Jiang, Wen; Xu, Beibei; Wu, Baojian; Yu, Rong; Hu, Ming

    2012-02-01

    The interplay between phase II enzymes and efflux transporters leads to extensive metabolism and low bioavailability for flavonoids. To investigate the simplest interplay between one UDP-glucuronosyltransferase isoform and one efflux transporter in flavonoid disposition, engineered HeLa cells stably overexpressing UGT1A9 were developed, characterized, and further applied to investigate the metabolism of two model flavonoids (genistein and apigenin) and excretion of their glucuronides. The results indicated that the engineered HeLa cells overexpressing UGT1A9 rapidly excreted the glucuronides of genistein and apigenin. The kinetic characteristics of genistein or apigenin glucuronidation were similar with the use of UGT1A9 overexpressed in HeLa cells or the commercially available UGT1A9. Small interfering (siRNA)-mediated UGT1A9 silencing resulted in a substantial decrease in glucuronide excretion (>75%, p MRP) 2 and MRP3 did not affect excretion of flavonoid glucuronides. In conclusion, the engineered HeLa cells overexpressing UGT1A9 is an appropriate model to study the kinetic interplay between UGT1A9 and BCRP in the phase II disposition of flavonoids. This simple cell model should also be very useful to rapidly identify whether a phase II metabolite is the substrate of BCRP. PMID:22071170

  8. Radiosensitizing effect of Chitosan on HeLa and LN 18 brain tumor cells exposed to electron beam radiation

    Chitosan has been widely used for multiple applications because it is a non-toxic biocompatible, biodegradable, and adsorptive material. A previous study has shown that low-molecular-weight chitosan (LMWC) exerts a cytotoxic effect on oral cancer cells. Although a higher concentration of LMWC in comparison to cisplatin was needed in order to kill cancer cells, it was relatively less cytotoxic to non-cancer cells. Some of the well known anticancer drugs have the property of sensitizing the cell to radiation, which will be more applicable during combination therapy of cancer. The present study was undertaken to find the radiosensitizing effect of chitosan on Hela and Brain tumor (LN18) cells against electron beam radiation (EBR). Both the cancer cell lines, Hela and LN 18 were treated with different concentration of chitosan (50 and 100 μg/ml) pre and post exposure to 4 Gy EBR. The percentage of cell viability, percentage of apoptosis and ssDNA damage in the treated cells were assessed by MTT assay, DNA diffusion assay and comet assay respectively. The obtained results showed 62.1315.08 and 65.2412.45 percent Hela and LN 18 viable cells at 24 hour after the exposure to 4 Gy EBR. The percentage of viability was found to be decreased in cells exposed to EBR in the presence of chitosan. Supporting to this, percentage of apoptotic cells was found to be more in treated groups (28.1314.34 and 25.1313.76) when compared with control (23.1911.07 and 20.7914.86). Treatment of HeLa and LN18 before and after the exposure of EBR showed significantly (P<0.05) more frequency of micronucleus and % of DNA damage than the 4 Gy EBR control group. These results conclude the sensitizing effect of chitosan on cancer cell line against EBR exposure. (author)

  9. Photodynamic Effect of Ni Nanotubes on an HeLa Cell Line

    Hammad Aziz, Muhammad; Fakhar-e-Alam, M.; Fatima, Mahvish; Shaheen, Fozia; Iqbal, Seemab; Atif, M.; Talha, Muhammad; Mansoor Ali, Syed; Afzal, Muhammad; Majid, Abdul; Shelih Al.Harbi, Thamir; Ismail, Muhammad; Wang, Zhiming M.; AlSalhi, M. S.; Alahmed, Z. A.

    2016-01-01

    Nickel nanomaterials are promising in the biomedical field, especially in cancer diagnostics and targeted therapy, due to their distinctive chemical and physical properties. In this experiment, the toxicity of nickel nanotubes (Ni NTs) were tested in an in vitro cervical cancer model (HeLa cell line) to optimize the parameters of photodynamic therapy (PDT) for their greatest effectiveness. Ni NTs were synthesized by electrodeposition. Morphological analysis and magnetic behavior were examined using a Scanning electron microscope (SEM), an energy dispersive X-ray analysis (EDAX) and a vibrating sample magnetometer (VSM) analysis. Phototoxic and cytotoxic effects of nanomaterials were studied using the Ni NTs alone as well as in conjugation with aminolevulinic acid (5-ALA); this was performed both in the dark and under laser exposure. Toxic effects on the HeLa cell model were evaluated by a neutral red assay (NRA) and by detection of intracellular reactive oxygen species (ROS) production. Furthermore, 10–200 nM of Ni NTs was prepared in solution form and applied to HeLa cells in 96-well plates. Maximum toxicity of Ni NTs complexed with 5-ALA was observed at 100 J/cm2 and 200 nM. Up to 65–68% loss in cell viability was observed. Statistical analysis was performed on the experimental results to confirm the worth and clarity of results, with p-values = 0.003 and 0.000, respectively. Current results pave the way for a more rational strategy to overcome the problem of drug bioavailability in nanoparticulate targeted cancer therapy, which plays a dynamic role in clinical practice. PMID:26990435

  10. Photodynamic Effect of Ni Nanotubes on an HeLa Cell Line.

    Muhammad Hammad Aziz

    Full Text Available Nickel nanomaterials are promising in the biomedical field, especially in cancer diagnostics and targeted therapy, due to their distinctive chemical and physical properties. In this experiment, the toxicity of nickel nanotubes (Ni NTs were tested in an in vitro cervical cancer model (HeLa cell line to optimize the parameters of photodynamic therapy (PDT for their greatest effectiveness. Ni NTs were synthesized by electrodeposition. Morphological analysis and magnetic behavior were examined using a Scanning electron microscope (SEM, an energy dispersive X-ray analysis (EDAX and a vibrating sample magnetometer (VSM analysis. Phototoxic and cytotoxic effects of nanomaterials were studied using the Ni NTs alone as well as in conjugation with aminolevulinic acid (5-ALA; this was performed both in the dark and under laser exposure. Toxic effects on the HeLa cell model were evaluated by a neutral red assay (NRA and by detection of intracellular reactive oxygen species (ROS production. Furthermore, 10-200 nM of Ni NTs was prepared in solution form and applied to HeLa cells in 96-well plates. Maximum toxicity of Ni NTs complexed with 5-ALA was observed at 100 J/cm2 and 200 nM. Up to 65-68% loss in cell viability was observed. Statistical analysis was performed on the experimental results to confirm the worth and clarity of results, with p-values = 0.003 and 0.000, respectively. Current results pave the way for a more rational strategy to overcome the problem of drug bioavailability in nanoparticulate targeted cancer therapy, which plays a dynamic role in clinical practice.

  11. Evaluation of Radiosensitivity of HeLa Cells Infected with Polio Virus Irradiated by Co 60

    F Seif

    2008-04-01

    Full Text Available ABSTRACT: Introduction & Objective: The main purpose of radiotherapy is exposing enough doses of radiation to tumor tissue and protecting the normal tissues around it. Tumor dose for each session in radiotherapy will be considered based on radiosensitivity of the tissues. The presence of viral diseases in tumoral area can affect the radiosensitivity of cells. This study aimed to evaluate the radiosensitivity of Hela cells infected with poliomyelitis virus irradiated by Co 60. Materials & Methods: In this study, the radiosensitivity of HeLa cells, with or without the viral infection, after gamma radiation of cobalt 60, was assessed. Results: Results of comparison of the radisensitivity of infected and uninfected cells indicates that after 2 Gy irradiation by Co 60, polio infection in low, moderate and high virus load, increases the cell death by 20-30%, 30-40% and 70-90% respectively. Conclusion : Radiosensitivity of tumoral cells increase when they are infected with viral agents. Results of this study showed that non cancer diseases should be considered when prescribing dose fraction in radiotherapy of cancers.

  12. Anti-Tumor Effect of Curcumin on Human Cervical Carcinoma HeLa Cells In Vitro and In Vivo

    ZHAO Jing; ZHAO Yong; ZHANG Yan; CHEN Wei

    2007-01-01

    Objective: To investigate the anti-tumor effect of curcumin on human cervical carcinoma HeLa cells in vitro and in vivo. Methods: (1) Human cervical carcinoma cell line HeLa was cultured in vitro. HeLa cells were treated with 5-50μmol/L curcumin for 24. 48, 72 h and the growth inhibition rates of HeLa cells were measured by MTT method. Cell apoptosis was inspected by electron microscopy and flow cytometry (FCM). (2) A transplanted tumor model by injecting HeLa cells into subcutaneous tissue of BABL/C mice was established and its growth curve was measured. 30 BABL/C mice with tumors were divided into 2 groups at random and 0.2 ml saline or 0.2 ml 250 μmol/L curcumin was injected into abdominal cavity respectively once everyday and lasted for ten days. The changes of tumor volume were measured continuously and tumor inhibition rate was calculated. At last the expressions of caspase-3 and bax protein in transplanted tumors were detected by immunohistochemistry. Results: (1) Curcumin inhibited the proliferation of Lela cells on a dose-depending manner. Apoptosis of cells could be observed by FCM. Partial cells presented the characteristic morphological changes of apoptosis under electron microseope. (2) When 1×107 HeLa cells were inoculated for each mouse, 100% of the mice developed growing tumors after seven days. An inhibition effect was observed in treatment group, and the inhibition rate of curcumin was 74.33%. The expressions of caspase-3 and bax in the transplanted tumors were increased in curcumin group. Conclusion: Curcumin is effective as an anti-cancer drug not only in vitro but also in vivo.

  13. Bioactive compounds from crocodile (Crocodylus siamensis) white blood cells induced apoptotic cell death in hela cells.

    Patathananone, Supawadee; Thammasirirak, Sompong; Daduang, Jureerut; Chung, Jing Gung; Temsiripong, Yosapong; Daduang, Sakda

    2016-08-01

    Crocodile (Crocodylus siamensis) white blood cell extracts (WBCex) were examined for anticancer activity in HeLa cell lines using the MTT assay. The percentage viability of HeLa cells significantly deceased after treatment with WBCex in a dose- and time-dependent manner. The IC50 dose was suggested to be approximately 225 μg/mL protein. Apoptotic cell death occurred in a time-dependent manner based on investigation by flow cytometry using annexin V-FITC and PI staining. DAPI nucleic acid staining indicated increased chromatin condensation. Caspase-3, -8 and -9 activities also increased, suggesting the induction of the caspase-dependent apoptotic pathway. Furthermore, the mitochondrial membrane potential (ΔΨm ) of HeLa cells was lost as a result of increasing levels of Bax and reduced levels of Bcl-2, Bcl-XL, Bcl-Xs, and XIAP. The decreased ΔΨm led to the release of cytochrome c and the activation of caspase-9 and -3. Apoptosis-inducing factor translocated into the nuclei, and endonuclease G (Endo G) was released from the mitochondria. These results suggest that anticancer agents in WBCex can induce apoptosis in HeLa cells via both caspase-dependent and -independent pathways. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 986-997, 2016. PMID:25691005

  14. MicroRNA-21 promotes cell proliferation and down-regulates the expression of programmed cell death 4 (PDCD4) in HeLa cervical carcinoma cells

    MicroRNAs are involved in cancer-related processes. The microRNA-21(miR-21) has been identified as the only miRNA over-expressed in a wide variety of cancers, including cervical cancer. However, the function of miR-21 is unknown in cervical carcinomas. In this study, we found that the inhibition of miR-21 in HeLa cervical cancer cells caused profound suppression of cell proliferation, and up-regulated the expression of the tumor suppressor gene PDCD4. We also provide direct evidence that PDCD4-3'UTR is a functional target of miR-21 and that the 18 bp putative target site can function as the sole regulatory element in HeLa cells. These results suggest that miR-21 may play an oncogenic role in the cellular processes of cervical cancer and may serve as a target for effective therapies.

  15. Evaluation of the effects of paederus beetle extract and gamma irradiation on HeLa cells

    Fariba Samani

    2014-04-01

    Full Text Available Objective(s:Cervical cancer is a malignancy that is the second most common cause of death from cancer in women throughout the world. Paederus beetle (Paederus fuscipes extract (PBE, contains bioactive compounds such as pederine which has cytotoxic properties and blocks DNA and protein synthesis at very low concentrations. In this investigation we tried to determine the effects co-treatment with PBE and gamma irradiation on HeLa cells. Materials and Methods: The viability of the cells was measured by two methods: MTT and Colony assay. Results: We found that supplementing gamma irradiation therapy with PBE does not increase cell death and it might even interfere with its cytotoxicty at the concentrations below 0.1 ng/ml and the viability for irradiation vs irradiation + PBE was 37%: 60%.   Conclusion: This finding might be due to radioprotective effects of the very low doses of PBE against gamma radiation.

  16. Iron metabolism and cell membranes. III. Iron-induced alterations in HeLa cells.

    Jauregui, H. O.; Bradford, W. D.; Arstila, A. U.; Kinney, T. D.; Trump, B. F.

    1975-01-01

    The morphologic characteristics of acute iron loading were studied in HeLa cells incubated in an iron-enriched Eagle's medium containing 500 mug/ml of iron. Chemical studies showed that ferritin synthesis was rapidly induced and the concentration of intracellular ferritin increased up to 72 hours. Closely coupled with an increase in HeLa cell ferritin was a marked decrease in the rate of cell multiplication. The significant ultrastructural findings of iron-induced HeLa cell injury are characterized by the appearance of both autophagic multivesicular and residual bodies over the first 72 hours of iron incubation. The prominence of multivesicular bodies was noted after only 4 hours' incubation, with iron and myelin figures first appearing after 6 hours. Thus, the partial arrest of cell multiplication was associated with an increase in cytoplasmic residual bodies containing iron and other debris. The distribution of intracellular ferritin within HeLa cells differs significantly from the distribution described previously in hepatic parenchymal cells. In HeLa cells, ferritin particles were confined to lysosomal vesicles and were not identified in cell sap, endoplasmic reticulum, or Golgi apparatus. Images Figure 8 Figure 1 Figure 9 Figure 10 Figure 11 Figure 12 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:1155583

  17. Terbium doped SnO2 nanoparticles as white emitters and SnO2:5Tb/Fe3O4 magnetic luminescent nanohybrids for hyperthermia application and biocompatibility with HeLa cancer cells.

    Singh, Laishram Priyobarta; Singh, Ningthoujam Premananda; Srivastava, Sri Krishna

    2015-04-14

    SnO2:5Tb (SnO2 doped with 5 at% Tb(3+)) nanoparticles were synthesised by a polyol method and their luminescence properties at different annealing temperatures were studied. Characterization of nanomaterials was done by X-ray diffraction (XRD), Fourier transformation infrared spectroscopy (FTIR), transmission electron microscopy (TEM) and vibrating sample magnetometry (VSM). XRD studies indicate that the prepared nanoparticles were of tetragonal structures. Upon Tb(3+) ion incorporation into SnO2, Sn(4+) changes to Sn(2+) and, on annealing again at higher temperature, Sn(2+) changes to Sn(4+). The prepared nanoparticles were spherical in shape. Sn-O vibrations were found from the FTIR studies. In photoluminescence studies, the intensity of the emission peaks of Tb(3+) ions increases with the increase of annealing temperature, and emission spectra lie in the region of white emission in the CIE diagram. CCT calculations show that the SnO2:5Tb emission lies in cold white emission. Quantum yields up to 38% can be obtained for 900 °C annealed samples. SnO2:5Tb nanoparticles were well incorporated into the PVA polymer and such a material incorporated into the polymer can be used for display devices. The SnO2:5Tb/Fe3O4 nanohybrid was prepared and investigated for hyperthermia applications at different concentrations of the nanohybrid. This achieves a hyperthermia temperature (42 °C) under an AC magnetic field. The hybrid nanomaterial SnO2:5Tb/Fe3O4 was found to exhibit biocompatibility with HeLa cells (human cervical cancer cells) at concentrations up to 74% for 100 μg L(-1). Also, this nanohybrid shows green emission and thus it will be helpful in tracing magnetic nanoparticles through optical imaging in vivo and in vitro application. PMID:25747103

  18. Campylobacter jejuni cell lysates differently target mitochondria and lysosomes on HeLa cells.

    Canonico, B; Campana, R; Luchetti, F; Arcangeletti, M; Betti, M; Cesarini, E; Ciacci, C; Vittoria, E; Galli, L; Papa, S; Baffone, W

    2014-08-01

    Campylobacter jejuni is the most common cause of bacterial gastroenteritis in humans. The synthesis of cytolethal distending toxin appears essential in the infection process. In this work we evaluated the sequence of lethal events in HeLa cells exposed to cell lysates of two distinct strains, C. jejuni ATCC 33291 and C. jejuni ISS3. C. jejuni cell lysates (CCLys) were added to HeLa cell monolayers which were analysed to detect DNA content, death features, bcl-2 and p53 status, mitochondria/lysosomes network and finally, CD54 and CD59 alterations, compared to cell lysates of C. jejuni 11168H cdtA mutant. We found mitochondria and lysosomes differently targeted by these bacterial lysates. Death, consistent with apoptosis for C. jejuni ATCC 33291 lysate, occurred in a slow way (>48 h); concomitantly HeLa cells increase their endolysosomal compartment, as a consequence of toxin internalization besides a simultaneous and partial lysosomal destabilization. C. jejuni CCLys induces death in HeLa cells mainly via a caspase-dependent mechanism although a p53 lysosomal pathway (also caspase-independent) seems to appear in addition. In C. jejuni ISS3-treated cells, the p53-mediated oxidative degradation of mitochondrial components seems to be lost, inducing the deepest lysosomal alterations. Furthermore, CD59 considerably decreases, suggesting both a degradation or internalisation pathway. CCLys-treated HeLa cells increase CD54 expression on their surface, because of the action of lysate as its double feature of toxin and bacterial peptide. In conclusion, we revealed that C. jejuni CCLys-treated HeLa cells displayed different features, depending on the particular strain. PMID:24880782

  19. Comparative proteomics analysis of lanthanum citrate complex-induced apoptosis in HeLa cells

    2009-01-01

    In a previous study,the lanthanum citrate complex([LaCit2]3-) has been found to induce apoptosis in the human HeLa cervical cancer cell line.To clarify the mechanism,we carried out comparative proteomics analysis between treated and control cells.Differentially expressed proteins were separated electrophoretically and identified by MALDI-TOF/TOF tandem mass spectrometry.There were profound changes in 14 proteins related to mitochondrial function and oxidative stress,suggesting that mitochondrial dysfunction plays a key role in [LaCit2]3--induced apoptosis.This was confirmed by a decrease in the mitochondrial transmembrane potential,and increases in cytochrome c release and reactive oxygen species generation in [LaCit2]3--treated cells.Western blotting analyses show that [LaCit2]3--induced apoptosis was accompanied by the activation of caspase-9 and the specific proteolytic cleavage of PARP,leading to an increase in the proapoptotic protein Bax and a decrease in the antiapoptotic protein Bcl-2.These results suggest that [LaCit2]3-induced the apoptosis of HeLa cells through oxidative stress mediated pathway involving MT participation.

  20. Induction of apoptosis in HeLa cells by chloroform fraction of seed extracts of Nigella sativa

    Alshatwi Ali A

    2009-11-01

    Full Text Available Abstract Background Cancer remains one of the most dreaded diseases causing an astonishingly high death rate, second only to cardiac arrest. The fact that conventional and newly emerging treatment procedures like chemotherapy, catalytic therapy, photodynamic therapy and radiotherapy have not succeeded in reverting the outcome of the disease to any drastic extent, has made researchers investigate alternative treatment options. The extensive repertoire of traditional medicinal knowledge systems from various parts of the world are being re-investigated for their healing properties. This study progresses in the direction of identifying component(s from Nigella sativa with anti cancer acitivity. In the present study we investigated the efficacy of Organic extracts of Nigella sativa seed powder for its clonogenic inhibition and induction of apoptosis in HeLa cancer cell. Results Methanolic, n-Hexane and chloroform extracts of Nigella sativa seedz effectively killed HeLa cells. The IC50 values of methanolic, n-hexane, and chloroform extracts of Nigella sativa were 2.28 μg/ml, 2.20 μg/ml and 0.41 ng/ml, respectively. All three extracts induced apoptosis in HeLa cells. Apoptosis was confirmed by DNA fragmentation, western blot and terminal transferase-mediated dUTP-digoxigenin-end labeling (TUNEL assay. Conclusion Western Blot and TUNEL results suggested that Nigella sativa seed extracts regulated the expression of pro- and anti- apoptotic genes, indicating its possible development as a potential therapeutic agent for cervical cancer upon further investigation.

  1. Methanolic extract of Pterocarpus santalinus induces apoptosis in HeLa cells.

    Kwon, H J; Hong, Y K; Kim, K H; Han, C H; Cho, S H; Choi, J S; Kim, Byung-Woo

    2006-04-21

    Ptercarpus santalinus (Fabaceae) has been used as a folk remedy in Korea, and it has been shown to exhibit antiinflammations, antiulcers and anticancer effects. In this study, therefore, we report the cytotoxic activity and the mechanism of cell death exhibited by the methanol extract of Ptercarpus santalinus (MEPS) against human cervical adenocarcinoma cell line, HeLa. Treatment of HeLa cells with various concentrations of MEPS resulted in growth inhibition and induction of apoptosis in a dose-dependent manner as determined by cell viability, chromatin condensation, DNA fragmentation and sub-G1 phase accumulation. In Western blot analysis, apoptosis in the HeLa cells was associated with the release of cytochrome C from mitochondria into the cytosol, activation of caspases-3, -8, -9 and proteolytic cleavage of PARP. These results suggest that MEPS exhibits antiproliferative effect on HeLa cells via apoptosis, and it may be a potential candidate in field of anticancer drug discovery. PMID:16326057

  2. Factors influencing the accumulation of tetraphenylphosphonium cation in HeLa cells.

    Hiller, R.; Schaefer, A; Zibirre, R; Kaback, H R; Koch, G

    1984-01-01

    Exposure of HeLa cells to tetraphenylphosphonium cation (TPP+) results in a rapid accumulation intracellularly, and a steady-state level is reached within 10 min. Accumulation of [3H]TPP+ in HeLa cells is reduced under the following conditions: (i) after preincubation of cells in buffered saline or in medium containing two- to fourfold higher concentrations of amino acids, (ii) exposure to the alkylating agent L-1-tosylamido-2-phenyl-ethylchloromethyl ketone, (iii) ouabain-mediated inhibition...

  3. The Sensitivity of Hela Kyoto Cell Line Transfected with Sensor HyPer2 to Cisplatin

    Belova A.S.

    2014-12-01

    Full Text Available The aim of the investigation is to compare by means of MTT assay cytotoxic effect of cisplatin on the cells of HeLa Kyoto line and HeLa Kyoto line containing genetically-encoded sensor of hydrogen peroxide HyPer2 (HeLa Kyoto–HyPer2 line, and using staining by trypan blue to identify the doses of cisplatin causing cell death at different exposure time. Materials and Methods. A HeLa Kyoto cell line of human cervical carcinoma and HeLa Kyota line transfected with the cytoplasmic sensor of hydrogen peroxide (HeLa Kyoto–HyPer2 were used in the study. The analysis of cytotoxic and antiproliferative action of cisplatin in relation to the given cells was performed using MTT assay. Cell viability was determined after 24 h of incubation with the preparation at concentrations from 0 to 50 μmol/L, then within the period from 0 to 24 h with an interval of 2 h at concentration of IC50; and also after 2, 4, 6, 8 h at concentrations from 9.3 to 833.3 μmol/L a quantity of live and destructed cells was counted using staining by trypan blue. Results. After cisplatin expose the dose-response curves for cell viability of Hela Kyoto and HeLa Kyoto–HyPer2 cell lines were built according to MTT assay data. It was established that concentration of IC50 corresponding to the dose causing a loss of viability of 50% of cells is 1.3 times lower for HeLa Kyoto–HyPer2 compared to HeLa Kyoto. The results of staining by a vital agent trypan blue showed that inhibiting effects of cisplatin in concentration of IC50 by 24 h are mainly linked with the delay of cell division but not with their death. At concentrations up to 52 μmol/L damage of the membranes does not occur during 8 h, and at superhigh concentrations — 416.7 μmol/L — the damage is possible already 4 h after the exposure. Conclusion. Comparison of sensibility of the two cell lines to the effect of cisplatin showed that transfection of the cells with the fluorescent protein results in the increase of the

  4. INHIBITORY ROLE OF TRANSCRIPTION FACTOR COUP-TFⅡ IN EXPRESSION OF HTERT IN HELA CELLS

    Qiang Wang; Zeng-liang Bai; Li Xuan; Lin Hou; Bo Zhang

    2004-01-01

    Objective To clone and identify the proteins involved in regulating the transcription of hTERT and study the role of genes in both hTERT transcription and telomerase activity.Methods The full cDNA of COUP-TFⅡ was cloned from HeLa cDNA library by hTERT promoter-based yeast one-hybrid assay and then in-frame inserted into His-tag fusion expression vector pEK318. The His-tag COUP-TFⅡ fusion proteins were purified by Ni-NTA chromatography. The interaction of COUP-TFⅡ with hTERT promoter in vitro was identified by lectrophoretic mobility shift assay and Footprint. The role of COUP-TFⅡ in both hTERT transcription and telomerase activity were probed through Luciferase reporter assay, Northern blot, and TRAP-PCR ELISA.Results COUP-TFⅡ could firmly bind to the downstream E-box and the other two binding sites in hTERT promoter.Luciferase reporter assay indicated COUP-TFⅡ could suppress hTERT promoter activity and stable introduction of COUP TFⅡ into HeLa cells also decreased both endogenous hTERT transcription and telomerase activity.Conclusion The human COUP-TFⅡ can firmly bind to hTERT promoter, and inhibit telomerase activity through decreasing hTERT transcription. It will greatly facilitate understanding of telomerase regulation in normal and cancer cells.

  5. Antiproliferative activity of methanolic extracts from two green algae, Enteromorpha intestinalis and Rizoclonium riparium on HeLa cells

    Paul, Subhabrata; Kundu, Rita

    2013-01-01

    Background Natural compounds can be alternative sources for finding new lead anti-cancer molecules. Marine algae have been a traditional source for bioactive compounds. Enteromorpha intestinalis and Rhizoclonium riparium are two well distributed saline/brackish water algae from Sundarbans. There’s no previous report of these two for their anti-proliferative activities. Methods Cytotoxicity of the algal methanolic extracts (AMEs) on HeLa cells were assayed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5...

  6. Inhibitory Activity of Synthesized Acetylated Procyanidin B1 Analogs against HeLa S3 Cells Proliferation

    Syuhei Okamoto

    2014-02-01

    Full Text Available Proanthocyanidins, also known as condensed tannins and/or oligomeric flavonoids, occur in many edible plants and have various interesting biological activities. Previously, we reported a synthetic method for the preparation of various procyanidins in pure form and described their biological activities. Here, we describe the synthesis of procyanidin B1 acetylated analogs and discuss their inhibition activities against HeLa S3 cell proliferation. Surprisingly, the lower-unit acetylated procyanidin B1 strongly inhibited the proliferation of HeLa S3 cells. This molecule showed much stronger inhibitory activity than did epigallocatechin-3-O-gallate (EGCG, green tea polyphenol, and dimeric compounds that included EGCG as a unit. This result suggests that the phenolic hydroxyl groups of the upper-units in flavan-3-ols are important for their inhibitory activity against cancer cell proliferation and that a hydrophobic lower unit dimer enhances this activity.

  7. Heterofucan from Sargassum filipendula Induces Apoptosis in HeLa Cells

    Hugo Alexandre Oliveira Rocha

    2011-04-01

    Full Text Available Fucan is a term used to denominate a family of sulfated polysaccharides rich in sulfated L-fucose. Heterofucan SF-1.5v was extracted from the brown seaweed Sargassum filipendula by proteolytic digestion followed by sequential acetone precipitation. This fucan showed antiproliferative activity on Hela cells and induced apoptosis. However, SF-1.5v was not able to activate caspases. Moreover, SF-1.5v induced glycogen synthase kinase (GSK activation, but this protein is not involved in the heterofucan SF-1.5v induced apoptosis mechanism. In addition, ERK, p38, p53, pAKT and NFκB were not affected by the presence of SF-1.5v. We determined that SF-1.5v induces apoptosis in HeLa mainly by mitochondrial release of apoptosis-inducing factor (AIF into cytosol. In addition, SF-1.5v decreases the expression of anti-apoptotic protein Bcl-2 and increased expression of apoptogenic protein Bax. These results are significant in that they provide a mechanistic framework for further exploring the use of SF-1.5v as a novel chemotherapeutics against human cervical cancer.

  8. Effect of Twist short hairpin RNA on adhesion, spreading and migration of human cervical cancer HeLa cells in vitro%Twist shRNA对人宫颈癌细胞体外黏附、铺展及迁移能力的影响

    周奇; 李婷; 王国增; 赵婧; 刘岚; 吴乾渝

    2011-01-01

    目的 通过RNA干扰抑制Twist基因在人宫颈癌HeLa细胞中的表达,观察Twist基因沉默对HeLa细胞体外黏附、铺展及迁移能力的影响.方法 根据shRNA设计原则,构建两种靶向Twist基因的短发夹RNA(shRNA)干扰质粒,稳定转染HeLa细胞.通过荧光定量PCR及Western印迹法检测HeLa细胞中Twist基因mRNA和蛋白的表达水平,利用黏附实验、铺展实验和划痕实验检测其对细胞黏附力和迁移力的影响.结果 成功构建的Twist shRNA真核表达载体稳定转染HeLa细胞后可显著降低Twist基因的表达.与对照组相比,Twist基因沉默组细胞的黏附、铺展及迁移能力明显下降(P<0.05).结论 成功构建的Twist shRNA真核表达载体,能有效抑制HeLa细胞黏附、铺展及迁移能力.%Objective To investigate the effect of shRNA of Twist gene on adhesion, spreading and migration of human cervical cancer HeLa cells. Methods Two kinds of shRNA vectors targeting to Twist gene were constructed and stably transfected into HeLa cells. The expressions of Twist gene and protein were detected by fluorescent quantitative PCR and Western blotting. The cell adhesion assay, cell spreading assay and cell migration assay were performed. Results The recombinant plasmids were successfully constructed and stably transfected into HeLa cells. The expression of Twist gene in HeLa cells was depressed significantly. The cell adhesion, spreading and migration activity were inhibited (P < 0.05). Conclusion The shRNA expressing plasmids targeting to Twist gene can inhibit the adhesion, spreading and migration activity of HeLa cells in vitro.

  9. Adjuvant antiproliferative and cytotoxic effect of aloin in irradiated HeLaS3 cells

    Nićiforović, A.; Adžić, M.; Zarić, B.; Radojčić, M. B.

    2007-09-01

    Naturally occurring phytoanthracycline, aloin, was used to radiosensitize HeLaS3 human cervix carcinoma cells. The results indicated that the cytotoxic adjuvant effect of aloin was synergistic with gammaionizing radiation at all drug concentrations and comparable to the cytotoxicity of 5-10 Gy ionizing radiation alone. Radiosensitization of HeLaS3 cells was achieved by 60 μM aloin, which reduced the IC50 dose of ionizing radiation from 3.4 to 2 Gy. Ionizing radiation and aloin alone or in combination are shown to cause perturbation of the HeLaS3 cell-cycle and increase the percentage of cells in the DNA synthesis (S) phase of the cell cycle. While either of the agents applied alone causes programmed cell death by apoptosis, the simultaneous cell damage by both agents through the altered redox balance compromised cell capacity to conduct this program and led to synergic cytotoxic cell death by necrosis.

  10. Methanolic Extracts from Brown Seaweeds Dictyota cilliolata and Dictyota menstrualis Induce Apoptosis in Human Cervical Adenocarcinoma HeLa Cells

    Dayanne Lopes Gomes

    2015-04-01

    Full Text Available Carcinoma of the uterine cervix is the second most common female tumor worldwide, surpassed only by breast cancer. Natural products from seaweeds evidencing apoptotic activity have attracted a great deal of attention as new leads for alternative and complementary preventive or therapeutic anticancer agents. Here, methanol extracts from 13 species of tropical seaweeds (Rhodophytas, Phaeophyta and Chlorophyta collected from the Northeast of Brazil were assessed as apoptosis-inducing agents on human cervical adenocarcinoma (HeLa. All extracts showed different levels of cytotoxicity against HeLa cells; the most potent were obtained from the brown alga Dictyota cilliolata (MEDC and Dictyota menstrualis (MEDM. In addition, MEDC and MEDM also inhibits SiHa (cervix carcinoma cell proliferation. Studies with these two extracts using flow cytometry and fluorescence microscopy showed that HeLa cells exposed to MEDM and MEDC exhibit morphological and biochemical changes that characterize apoptosis as shown by loss of cell viability, chromatin condensation, phosphatidylserine externalization, and sub-G1 cell cycle phase accumulation, also MEDC induces cell cycle arrest in cell cycle phase S. Moreover, the activation of caspases 3 and 9 by these extracts suggests a mitochondria-dependent apoptosis route. However, other routes cannot be ruled out. Together, these results point out the methanol extracts of the brown algae D. mentrualis and D. cilliolata as potential sources of molecules with antitumor activity.

  11. Hyperthermia HeLa cell treatment with silica coated manganese oxide nanoparticles

    Villanueva, A; Alonso, JM; Rueda, T; Martínez, A; Crespo, P; Morales, MP; Fernandez, MA Gonzalez; Valdes, J; Rivero, G

    2009-01-01

    HeLa tumour cells incubated with ferromagnetic nanoparticles of manganese oxide perovskite La0.56(SrCa)0.22MnO3 were treated with a high frequency alternating magnetic field. The particles were previously coated with silica to improve their biocompatibility. The control assays made with HeLa tumour cells showed that cell survival and growth rate were not affected by the particle internalization in cells, or by the electromagnetic field on cells without nanoparticles. The application of an alternating electromagnetic field to cells incubated with this silica coated manganese oxide induced a significant cellular damage that finally lead to cell death by an apoptotic mechanism.

  12. Proteasome Inhibition Contributed to the Cytotoxicity of Arenobufagin after Its Binding with Na, K-ATPase in Human Cervical Carcinoma HeLa Cells.

    Qingxi Yue

    Full Text Available Although the possibility of developing cardiac steroids/cardiac glycosides as novel cancer therapeutic agents has been recognized, the mechanism of their anticancer activity is still not clear enough. Toad venom extract containing bufadienolides, which belong to cardiac steroids, has actually long been used as traditional Chinese medicine in clinic for cancer therapy in China. The cytotoxicity of arenobufagin, a bufadienolide isolated from toad venom, on human cervical carcinoma HeLa cells was checked. And, the protein expression profile of control HeLa cells and HeLa cells treated with arenobufagin for 48 h was analyzed using two-dimensional electrophoresis, respectively. Differently expressed proteins in HeLa cells treated with arenobufagin were identified and the pathways related to these proteins were mapped from KEGG database. Computational molecular docking was performed to verify the binding of arenobufagin and Na, K-ATPase. The effects of arenobufagin on Na, K-ATPase activity and proteasome activity of HeLa cells were checked. The protein-protein interaction network between Na, K-ATPase and proteasome was constructed and the expression of possible intermediate proteins ataxin-1 and translationally-controlled tumor protein in HeLa cells treated with arenobufagin was then checked. Arenobufagin induced apoptosis and G2/M cell cycle arrest in HeLa cells. The cytotoxic effect of arenobufagin was associated with 25 differently expressed proteins including proteasome-related proteins, calcium ion binding-related proteins, oxidative stress-related proteins, metabolism-related enzymes and others. The results of computational molecular docking revealed that arenobufagin was bound in the cavity formed by the transmembrane alpha subunits of Na, K-ATPase, which blocked the pathway of extracellular Na+/K+ cation exchange and inhibited the function of ion exchange. Arenobufagin inhibited the activity of Na, K-ATPase and proteasome, decreased the

  13. FRAKSINASI PROTEIN KAPANG LAUT Xylaria psidii KT30 DAN SITOTOKSISITASNYA TERHADAP SEL HeLa [Fractionation of Proteins of Marine Fungus Xylaria psidii KT30 and their Cytotoxicity against HeLa Cells

    Mita Gebriella Inthe

    2014-06-01

    Full Text Available Cervical cancer is the most common cause of death for Indonesian women after human breast cancer. One of the efforts of cancer treatment is the utilization of natural compounds. One of the microorganisms having the potential as anticancer agent is endophytic fungi. Endophytic fungi from the marine habitat can be isolated from sea weeds, sea grasses, sponges, and mangroves. Xylaria psidii KT30, a marine fungus used in this study was isolated from red seaweed Kappaphycus alvarezii. Xylaria psidii KT30 was cultivated in potato dextrose broth medium for nine days at room temperature 27-29°C in shaking condition. This study aimed to obtain protein fractions from X. psidii KT30 and determine their toxicity againt Chang and HeLa cells. The fractionation process was conducted using DEAE Sephadex A-50 column chromatography and the toxicity was determined by Brine Shrimp Lethality Test (BSLT. The metabolites excreted in the culture broth was extracted using 90% of ammonium sulphate. The extract was then tested for their toxicity against HeLa and Chang cells by Microculture Tetrazolium Technique (MTT assay.The results revealed that LC50 of the protein extract of X. psidii KT30 was 104.95 ppm and IC50 was 69.9 ppm. Based on the National Cancer Institute (NCI, this value showed moderate cytotoxicity against HeLa cells.

  14. Adjuvant antiproliferative and cytotoxic effect of aloin in irradiated HeLaS3 cells

    Naturally occurring phytoanthracycline, aloin, was used to radiosensitize HeLaS3 human cervix carcinoma cells. The results indicated that the cytotoxic adjuvant effect of aloin was synergistic with IR at all drug concentrations and comparable to the cytotoxicity of 5-10Gy IR alone. Radiosensitization of HeLaS3 cells was achieved by 60μM aloin which reduced IC50 dose of IR from 3.4- to 2Gy. The cell damage by both agents compromised cell capacity to conduct programmed cell death by apoptosis, and led to the synergic cytotoxic cell death by necrosis. (author)

  15. Low voltage irreversible electroporation induced apoptosis in HeLa cells

    Wei Zhou

    2012-01-01

    Full Text Available Background: High-voltage electric field pulses can make cell membrane electroporated irreversibly and eliminate malignant cells via necrosis. However, low-voltage is not efficient as that. Aims: This study determined the differential effects of high- and low-voltage electric field pulses on HeLa cells, when the power of low-voltage was enhanced by increasing quantity of pulses. Materials and Methods: Pulses electric fields with permanent frequency (1 Hz and pulse length (100 μs were performed on HeLa cells. Voltage and pulse sets (8 pulses/set were various during treatment. CCK-8 assay was used to detect cell viability. The quantitative determination of apoptosis and necrosis were performed by flow cytometry with Annexin V and PI staining. Transmission electron microscopy was used to observe the ultrastructure of HeLa cells. Caspase-3 and caspase-8, the enzymes in apoptotic pathway, were determined by western blot. Results: The data showed that low-voltage electric field pulses also could make cell irreversible electroporation (IRE and ablate HeLa cells effectively by induction of apoptosis. The ablating effect due to low-voltage treatments delivered with a greater number of pulses may be as satisfactory as high-voltage, or even preferable because it causes less necrosis and more apoptosis. Conclusions: IRE induced by low voltage with more pulses could ablate HeLa cells effectively as high voltage, and it was preferable that less necrosis and more apoptosis occurred under such condition.

  16. Lipid Peroxidation-Mediated Telomere Shortening in Hydroxyl Radical-Induced Apoptosis in HeLa Cells

    任建国; 陈晶; 戴尧仁

    2001-01-01

    Many anti-cancer drugs have been found to trigger apoptosis in tumor cells through the production of reactive oxygen species (ROS) including hydroxyl radicals (@ OH) regardless of chemical types. At the same time, telomerase is found to be associated with malignancy and reduced apoptosis. However, little is known about the linkage between ROS (such as @ OH) and telomerase/telomere. The focus of this investigation was to examine the possible pathway of the apoptosis induced by @ OH production via Fe2+ and H2O2. Results of the present study demonstrated that after exposure of HeLa cells to Fe2+-H2O2 system, an increase in lipid peroxidation and reduction of GSH was observed. These events proceeded and triggered apoptosis, resulting in DNA fragmentation. More interestingly, we did not observe any changes of telomerase activity. However, the telomere length in apoptotic cells shortened significantly. We also found that GSH rescued @ OH-induced HeLa cell death and prevened telomere shortening, and that 3,3'-diethyoxadicarbocyanine (DODCB), a telomerase inhibitor, increased susceptibility of HeLa cells to @ OH-induced apoptosis. Our results suggest that @ OH-induced telomere shortening is not through telomerase inhibition but possibly a direct effect of @ OH on telomeres themselves via lipid peroxidation.

  17. Study of Paclitaxel-Treated HeLa Cells by Differential Electrical Impedance Flow Cytometry

    Kirkegaard, Julie; Clausen, Casper Hyttel; Rodriguez-Trujíllo, Romén;

    2014-01-01

    This work describes the electrical investigation of paclitaxel-treated HeLa cells using a custom-made microfluidic biosensor for whole cell analysis in continuous flow. We apply the method of differential electrical impedance spectroscopy to treated HeLa cells in order to elucidate the changes in...... on investigating the changes in the electrical properties of the cell membrane caused by the effect of paclitaxel. We observe good agreement between the model and the obtained results. This establishes the proof-of-concept for the application in cell drug therapy....

  18. Inhibitory action of docetaxel on the proliferation of HeLa and SiHa cells

    2010-01-01

    Objective To study the inhibitory action of docetaxel(DOC)on the proliferation of HeLa and SiHa cells.Methods Cell morphological changes were observed with inverted phase contrast microscope.MTT was adopted to test and calculate the cell inhibition ratio.Flow cytometry was used to detect cell cycle.Results DOC had an obvious concentration-dependent inhibitory effect on the proliferation of both HeLa and SiHa cells.The inhibition ratio of DOC on SiHa was significantly higher than that on HeLa(P<0.05).DOC blo...

  19. ALG-2 knockdown in HeLa cells results in G2/M cell cycle phase accumulation and cell death

    Høj, Berit Rahbek; la Cour, Peter Jonas Marstrand; Mollerup, Jens;

    2009-01-01

    ALG-2 (apoptosis-linked gene-2 encoded protein) has been shown to be upregulated in a variety of human tumors questioning its previously assumed pro-apoptotic function. The aim of the present study was to obtain insights into the role of ALG-2 in human cancer cells. We show that ALG-2 downregulat......ALG-2 (apoptosis-linked gene-2 encoded protein) has been shown to be upregulated in a variety of human tumors questioning its previously assumed pro-apoptotic function. The aim of the present study was to obtain insights into the role of ALG-2 in human cancer cells. We show that ALG-2...... downregulation induces accumulation of HeLa cells in the G2/M cell cycle phase and increases the amount of early apoptotic and dead cells. Caspase inhibition by the pan-caspase inhibitor zVAD-fmk attenuated the increase in the amount of dead cells following ALG-2 downregulation. Thus, our results indicate that...

  20. Effects of spider Macrothele raven venom on cell proliferation and cytotoxicity in HeLa cells

    Li GAO; Bao-en SHAN; Jing CHEN; Jiang-hui LIU; Da-xiang SONG; Bao-cheng ZHU

    2005-01-01

    Aim: To examine the effect of venom from the spider Macrothele raven on cell proliferation and cytotoxicity in human cervical carcinoma, HeLa cells. Methods:Morphological and biochemical signs of apoptosis appeared using acridine orange-ethidium bromide (AO/EB) staining. Marked morphological changes in HeLa cells after treatment with spider venom were observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Cell proliferation and cytotoxicity were determined by [methyl-3H] thymidine assay ([3H]TdR) and lactate dehydrogenase (LDH) release, respectively. DNA fragmentation and cell cycle distribution were monitored using flow cytometry. In addition, Western blot analysis was used to evaluate the level of caspase-3 expression. In vivo examination of the inhibition of the size of tumors in nude mice treated with spider venom was measured. Results: Marked morphological changes were observed using AO/EB staining, SEM and TEM assay. Spider venom at concentrations of 10-40 mg/L caused dose- and time-dependent inhibition of HeLa cell proliferation.The ratio of apoptosis and necrosis increased. The activity of caspase-3 was upregulated after spider venom treatment. In vivo study of tumor size revealed that tumors significantly decreased in size from controls to tumors treated for 3 weeks with spider venom (P<0.05). Conclusion: The inhibition of HeLa cells by the venom of the spider Macrothele raveni was carried out in three ways: induction of apoptosis, necrosis of toxicity damage and direct lysis. Spider venom is a novel anti-tumor material both in vitro and in vivo.

  1. Effect of bortezomib on migration and invasion in cervical carcinoma HeLa cell

    Chong Shi; Guo-Bin Zhang; Shu-Wang Yin

    2015-01-01

    Objective: To explore the effect of bortezomib on migration and invasion of cervical carcinoma HeLa cell and specific molecular mechanism. Methods:The effect of bortezomib on the viability of HeLa cell was measured by MTT assay. The effect of bortezomib on cell migration and invasion was measured by Transwell assay and invasion experiment respectively. The activation of Akt/mTOR signaling pathway and expression level of MMP2, MMP9 were assayed by western blot. Results:MTT assay indicated bortezomib (2.5μM, 5μM, 10μM) could inhibit HeLa cell viability, and the inhibitory rate was highest at 48 h. Transwell assay and invasion experiment results showed that bortezomib inhibited HeLa cell migration and invasion. Western blotting assays presented bortezomib could suppress the phosphorylation of Akt and mTOR, and down-regulate the expression of MMP2 and MMP9. Conclusions:These results suggested bortezomib could inhibit migration and invasion in cervical carcinoma HeLa cell, which might be related to Akt/mTOR signal pathway.

  2. Effect of bortezomib on migration and invasion in cervical carcinoma HeLa cell

    Chong; Shi; Guo-Bin; Zhang; Shu-Wang; Yin

    2015-01-01

    Objective:To explore the effect of bortezomib on migration and invasion of cervical carcinoma HeLa cell and specific molecular mechanism.Methods:The effect of bortezomib on the viability of HeLa cell was measured by MTT assay.The effect of bortezomib on cell migration and invasion was measured by Transwell assay and invasion experiment respectively.The activation of Akt/mTOR signaling pathway and expression level of MMP2,MMP9 were assayed by western blot.Results:MTT assay indicated bortezomib(2.5 μM.5 μM,10 μM)could inhibit HeLa cell viability,and the inhibitory rate was highest at 48 h.Transwell assay and invasion experiment results showed that bortezomib inhibited HeLa cell migration and invasion.Western blotting assays presented bortezomib could suppress the phosphorylation of Akt and mTOR.and down-regulate the expression of MMP2 and MMP9.Conclusions:These results suggested bortezomib could inhibit migration and invasion in cervical carcinoma HeLa cell,which might be related to Akt/mTOR signal pathway.

  3. Evaluation of Antiproliferative Potential of Cerium Oxide Nanoparticles on HeLa Human Cervical Tumor Cell

    Zoriţa Diaconeasa

    2015-05-01

    Full Text Available Cerium oxide nanoparticles (CeO2 nanoparticles as nanomaterials have promising biomedical applications. In this paper, the cytotoxicity induced by CONPs human cervical tumor cells was investigated. Cerium oxide nanoparticles were synthesized using the precipitation method. The nanoparticles were found to inhibit the proliferation of HeLa human cervical tumor cells in a dose dependent manner but did not showed to be cytotoxic as analyzed by MTT assay. The administrated treatment decreased the HeLa cell viability cells from 100% to 65% at the dose of 100 μg/mL.

  4. Dynamic behavior of histone H1 microinjected into HeLa cells

    Histone H1 was purified from bovine thymus and radiolabeled with tritium by reductive methylation or with 125I using chloramine-T. Red blood cell-mediated microinjection was then used to introduce the labeled H1 molecules into HeLa cells synchronized in S phase. The injected H1 molecules rapidly entered HeLa nuclei, and a number of tests indicate that their association with chromatin was equivalent to that of endogenous histone H1. The injected molecules copurified with HeLa cell nucleosomes, exhibited a half-life of ∼100h, and were hyperphosphorylated at mitosis. When injected HeLa cells were fused with mouse 3T3 fibroblasts < 10% of the labeled H1 molecules migrated to mouse nuclei during the next 48 h. Despite their slow rate of migration between nuclei, the injected H1 molecules were evenly distributed on mouse and human genomes soon after mitosis of HeLa-3T3 heterokaryons. These results suggest that although most histone H1 molecules are stably associated with interphase chromatin, they undergo extensive redistribution after mitosis

  5. Effect of PKC pathway on G1/S progression control in HeLa cells

    2000-01-01

    The effect of PKC activity on G1/S progression in HeLa cells has been studied.The result shows that (ⅰ) PKC activity alteration in G1 phase affects G1/S progression in HeLa cells.It has been observed that G1/S progression is stimulated by PKC agonist TPA and inhibited by PKC inhibitor GF-109203X.(ⅱ) The expression of c-myc and c-jun is stimulated by TPA and inhibited by GF-109203X treatment in early G1 phase.(ⅲ) During G1/S progression,the expression of CyclinD1 is stimulated by TPA treatment and inhibited by GF-109203X treatment.There is no effect on the expression of CDK4.It is likely that PKC pathway regulates G1/S progression through regulating the expression of some early response genes and engine molecules in HeLa cells.

  6. p150 ADAR1 isoform involved in maintenance of HeLa cell proliferation

    RNA-specific adenosine deaminase ADAR1 is ubiquitously expressed in a variety of mammalian cells and tissues. Although its physiological importance in non-nervous tissues has been confirmed by analysis of null mutation phenotypes, few endogenous editing substrates have been identified in numerous peripheral tissues and biological function of ADAR1 has not been fully understood. A conditional site-specific, ribozyme-based gene knock-down strategy was utilized to study the function of full-length isoform of ADAR1 (p150 protein) in HeLa cell. Double-stable HeLa cell lines were developed by transfecting HeLa Tet-On cells with a pTRE-derived plasmid that can express a hammerhead ribozyme against mRNA of p150 ADAR1 isoform under induction condition. Semi-quantitative RT-PCR and Western blotting were performed to measure the expression of p150 in selected cell clones. Cell proliferation was evaluated by means of MTT assay and growth curve analysis. Cellular morphological changes were observed under light microscope. Flow Cytometry was used for cell cycle analysis. Growth rate of cell transplants in BALB/c nude mice was also investigated. Both HeLa cell proliferation in vitro and the growth rate of transplanted HeLa cell-derived tumors in nude mice in vivo were significantly inhibited due to reduced expression of ADAR1 p150. Additionally, cell cycle analysis showed that cell progression from G1 phase to S phase was retarded in the ADAR1 p150 suppressed cells. Our results suggest that normal expression and functioning of p150 ADAR1 is essential for the maintenance of proper cell growth. The mechanisms underlying ADAR1's action might include both editing of currently unknown double-stranded RNAs and interacting with other cellular dsRNA-related processes

  7. tRNA modifying enzymes, NSUN2 and METTL1, determine sensitivity to 5-fluorouracil in HeLa cells.

    Mayumi Okamoto

    2014-09-01

    Full Text Available Nonessential tRNA modifications by methyltransferases are evolutionarily conserved and have been reported to stabilize mature tRNA molecules and prevent rapid tRNA decay (RTD. The tRNA modifying enzymes, NSUN2 and METTL1, are mammalian orthologs of yeast Trm4 and Trm8, which are required for protecting tRNA against RTD. A simultaneous overexpression of NSUN2 and METTL1 is widely observed among human cancers suggesting that targeting of both proteins provides a novel powerful strategy for cancer chemotherapy. Here, we show that combined knockdown of NSUN2 and METTL1 in HeLa cells drastically potentiate sensitivity of cells to 5-fluorouracil (5-FU whereas heat stress of cells revealed no effects. Since NSUN2 and METTL1 are phosphorylated by Aurora-B and Akt, respectively, and their tRNA modifying activities are suppressed by phosphorylation, overexpression of constitutively dephosphorylated forms of both methyltransferases is able to suppress 5-FU sensitivity. Thus, NSUN2 and METTL1 are implicated in 5-FU sensitivity in HeLa cells. Interfering with methylation of tRNAs might provide a promising rationale to improve 5-FU chemotherapy of cancer.

  8. Caveolin-1 and CDC42 mediated endocytosis of silica-coated iron oxide nanoparticles in HeLa cells

    Nils Bohmer

    2015-01-01

    Full Text Available Nanomedicine is a rapidly growing field in nanotechnology, which has great potential in the development of new therapies for numerous diseases. For example iron oxide nanoparticles are in clinical use already in the thermotherapy of brain cancer. Although it has been shown, that tumor cells take up these particles in vitro, little is known about the internalization routes. Understanding of the underlying uptake mechanisms would be very useful for faster and precise development of nanoparticles for clinical applications. This study aims at the identification of key proteins, which are crucial for the active uptake of iron oxide nanoparticles by HeLa cells (human cervical cancer as a model cell line. Cells were transfected with specific siRNAs against Caveolin-1, Dynamin 2, Flotillin-1, Clathrin, PIP5Kα and CDC42. Knockdown of Caveolin-1 reduces endocytosis of superparamagnetic iron oxide nanoparticles (SPIONs and silica-coated iron oxide nanoparticles (SCIONs between 23 and 41%, depending on the surface characteristics of the nanoparticles and the experimental design. Knockdown of CDC42 showed a 46% decrease of the internalization of PEGylated SPIONs within 24 h incubation time. Knockdown of Dynamin 2, Flotillin-1, Clathrin and PIP5Kα caused no or only minor effects. Hence endocytosis in HeLa cells of iron oxide nanoparticles, used in this study, is mainly mediated by Caveolin-1 and CDC42. It is shown here for the first time, which proteins of the endocytotic pathway mediate the endocytosis of silica-coated iron oxide nanoparticles in HeLa cells in vitro. In future studies more experiments should be carried out with different cell lines and other well-defined nanoparticle species to elucidate possible general principles.

  9. In vitro studies of the toxic effects of silver nanoparticles on HeLa and U937 cells

    Kaba SI

    2015-03-01

    Full Text Available Said I Kaba, Elena M Egorova Institute of General Pathology and Pathophysiology, Moscow, Russia Abstract: In the last decade, much attention has been paid to studies of the effect of silver nanoparticles (Ag NPs on tumor cells. Apart from elucidation of the mechanism of NPs’ interaction with mammalian cells, these studies are aimed at discovering new effective antitumor drugs. In this work, we report about the toxic effects of Ag NPs observed on two types of tumor cells: HeLa (adhesive cells and U937 (suspension cells. The Ag NPs were obtained by an original method of biochemical synthesis. Particle size was 13.2±4.72 nm, and zeta potential was -61.9±3.2 mV. The toxicity of Ag NPs in the concentration range 0.5–8.0 µg Ag/mL was determined by means of 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay and cytofluorometry after 4 and 24 hours' incubation. It was found that Ag NPs had high toxicity toward both cell types. The minimal concentrations where a toxicity effect was registered (toxicity thresholds lied in the range 0.5–2.0 µg Ag/mL. In parallel with the Ag NP solution, cells were incubated with water solutions of the NP stabilizer (aerosol-OT and Ag+ ions (as silver nitrate. It was shown that aerosol-OT had no effect on the viability on HeLa cells, but was moderately toxic toward U937, though less dangerous for these cells than Ag NPs. With Ag+ ions, for HeLa no toxic effect was observed, while for U937 they were as toxic as the Ag NPs. The data obtained indicate that Ag NPs as used in this study may prove to be useful for the creation of medicines for cancer therapy. Keywords: silver nanoparticles, cell viability, apoptosis, tumor cells

  10. Co-encapsulation of chrysophsin-1 and epirubicin in PEGylated liposomes circumvents multidrug resistance in HeLa cells.

    Lo, Yu-Li; Tu, Wei-Chen

    2015-12-01

    Chrysophsin-1, an amphipathic alpha-helical antimicrobial peptide, is isolated from the gills of the red sea bream and possesses different structure and mechanism(s) in comparison with traditional multidrug resistance (MDR) modulators. For the purpose of reducing off-target normal cell toxicity, it is rational to incorporate chrysophsin-1 and epirubicin in a PEGylated liposomal formulation. In the present study, we report a multifunctional liposomes with epirubicin as an antineoplastic agent and an apoptosis inducer, as well as chrysophsin-1 as a MDR transporter inhibitor and an apoptosis modulator in human cervical cancer HeLa cells. Co-incubation of HeLa cells with PEGylated liposomal formulation of epirubicin and chrysophsin-1 resulted in a significant increase in the cytotoxicity of epirubicin. The liposomal formulations of epirubicin and/or chrysophsin-1 were shown to considerably improve the intracellular H2O2 and O2(-) levels of HeLa cells. Furthermore, these treatments were found to extensively reduce mRNA expression levels of MDR1, MRP1, and MRP2. The addition of chrysophsin-1 in liposomes was demonstrated to substantially enhance the intracellular accumulation of epirubicin in HeLa cells. Moreover, the PEGylated liposomes of epirubicin and chrysophsin-1 were also found to significantly increase the mRNA expressions of p53, Bax, and Bcl-2. The ratio of Bax to Bcl-2 was noticeably amplified in the presence of these formulations. Apoptosis induction was also validated by chromatin condensation, a reduction in mitochondrial membrane potential, the increased sub-G1 phase of cell cycle, and more populations of apoptosis using annexin V/PI assay. These formulations were verified to increase the activity and mRNA expression levels of caspase-9 and caspases-3. Collectively, our findings provide the first evidence that cotreatment with free or liposomal chrysophsin-1 and epirubicin leads to cell death in human cervical cancer cells through the ROS

  11. The space of enzyme regulation in HeLa cells can be inferred from its intracellular metabolome

    Diener, Christian; Muñoz-Gonzalez, Felipe; Encarnación, Sergio; Resendis-Antonio, Osbaldo

    2016-01-01

    During the transition from a healthy state to a cancerous one, cells alter their metabolism to increase proliferation. The underlying metabolic alterations may be caused by a variety of different regulatory events on the transcriptional or post-transcriptional level whose identification contributes to the rational design of therapeutic targets. We present a mechanistic strategy capable of inferring enzymatic regulation from intracellular metabolome measurements that is independent of the actual mechanism of regulation. Here, enzyme activities are expressed by the space of all feasible kinetic constants (k-cone) such that the alteration between two phenotypes is given by their corresponding kinetic spaces. Deriving an expression for the transformation of the healthy to the cancer k-cone we identified putative regulated enzymes between the HeLa and HaCaT cell lines. We show that only a few enzymatic activities change between those two cell lines and that this regulation does not depend on gene transcription but is instead post-transcriptional. Here, we identify phosphofructokinase as the major driver of proliferation in HeLa cells and suggest an optional regulatory program, associated with oxidative stress, that affects the activity of the pentose phosphate pathway. PMID:27335086

  12. Vacuolization and apoptosis induced by nano-selenium in HeLa cell line

    2010-01-01

    Selenium(Se),a potential drug candidate for cancer prevention,has a special property:Its nutritional dosage and tolerable upper intake level appear in a narrow range,while the therapeutic use of this mineral may depend on a higher body intake level.Nano-selenium(nano-Se) particles,however,preserve the selenium element’s low toxicity characteristic but give a high biochemical activity effect of selenium compounds.In the present study different morphologies of synthesized nano-Se were evaluated concerning its anti-proliferation and apoptosis-inducing effect.Then nano-Se(sphere) were picked out to investigate its influence on two significant events involved in apoptosis,cell cycle arrest and mitochondrial membrane potential disruption.Furthermore,massive vacuolization of HeLa cells treated by nano-Se(sphere) was observed and more methods were used to measure the level of vacuolization.Such vacuolization needs energy supply and has been demonstrated to be related to Se endocytosis.These results suggest a possible mechanism to trigger apoptosis initiation.

  13. A novel metabolite from aspergillus ochraceus JGI 25 showing cytotoxicity to hela cells

    Varalakshmi K Nadumane

    2013-01-01

    Full Text Available This study aims at the isolation of filamentous fungi, extraction of metabolites, and evaluation of the cytotoxic properties on HeLa cells and normal human lymphocytes. We isolated fungi from the soil by serial dilution method. One of the isolates was chosen and identified as Aspergillus ochraceus Wilhelm (Trichocomaceae by standard techniques. The metabolites were extracted using methanol. Different concentrations of the extract were evaluated for their potential anticancer activity on HeLa cells by 3-(4,5-dimethylthiazol-2yl-2,5-diphenyl tetrazolium bromide assay and the safety of the extract was checked on normal human lymphocytes. The extract was purified by chromatographic techniques like thin-layer chromatography and high-performance liquid chromatography, and subjected to mass spectrometric analysis. The extract showed significant cytotoxic potential on HeLa cells at low concentrations with a half maximal inhibitory concentration value of <50 ΅g/ml. The extract gave 10 fractions by thin layer chromatography, and fraction B had higher toxicity than the rest. This fraction gave a single peak by high-performance liquid chromatography and had a mass-to-charge ratio of 905.65, which did not match any of the earlier known fungal metabolites or metabolites from other strains of A. ochraceus. The metabolite from A. ochraceus is alkaloid in nature, cytotoxic to HeLa cells, and appears to be a novel with anticancer potentials, which could be explored further for characterization of the active component.

  14. Visualizing the molecular sociology at the HeLa cell nuclear periphery

    Mahamid, Julia; Pfeffer, Stefan; Schaffer, Miroslava; Villa, Elizabeth; Danev, Radostin; Cuellar, Luis Kuhn; Förster, Friedrich; Hyman, Anthony A; Plitzko, Jürgen M; Baumeister, Wolfgang

    2016-01-01

    The molecular organization of eukaryotic nuclear volumes remains largely unexplored. Here we combined recent developments in cryo-electron tomography (cryo-ET) to produce three-dimensional snapshots of the HeLa cell nuclear periphery. Subtomogram averaging and classification of ribosomes revealed th

  15. Adenovirus DNA replication in vitro is stimulated by RNA from uninfected HeLa cells

    Vliet, P.C. van der; Dam, D. van; Kwant, M.M.

    1984-01-01

    Adenovirus DNA replication was studied in a partially reconstituted system consisting of purified viral proteins (DNA-binding protein, precursor terminal protein and Ad DNA polymerase) and a nuclear extract from uninfected HeLa cells. Optimal DNA replication required the presence of a heat-stable, r

  16. Inhibition of thioredoxin reductase by alantolactone prompts oxidative stress-mediated apoptosis of HeLa cells.

    Zhang, Junmin; Li, Ya; Duan, Dongzhu; Yao, Juan; Gao, Kun; Fang, Jianguo

    2016-02-15

    The mammalian thioredoxin reductase (TrxR) isoenzymes, TrxR1 in cytosol or nucleus, TrxR2 in mitochondria, and TrxR3 in testis, are essential seleno-flavoenzymes with a conserved penultimate selenocysteine (Sec) residue at the C-terminus, and have attracted increasing interests as potential targets for development of cancer chemotherapeutic agents. The sesquiterpene lactone alantolactone (ATL), an active component from the traditional folk medicine Inula helenium, has been documented possessing multiple pharmacological functions, especially the anticancer activity. However, the underlying mechanism has not been well defined. We reported that ATL inhibits both the recombinant TrxR and the enzyme in the cellular environment. The alpha-methylene-gamma-lactone moiety in ATL and the Sec residue in TrxR are critical for targeting TrxR by ATL. By employing our newly developed pull down assay, we demonstrated the remarkable elevation of the oxidized thioredoxin in HeLa cells after ATL treatment. In addition, ATL elicits accumulation of reactive oxygen species, and eventually induces apoptosis of HeLa cells. Importantly, overexpression of the functional TrxR attenuates the cytotoxicity of ATL, while knockdown of the enzyme sensitizes the cells to ATL treatment. Targeting TrxR thus discloses a novel molecular mechanism underlying the cellular action of ATL, and sheds light in considering the usage of ATL as a potential cancer chemotherapeutic agent. PMID:26686580

  17. Polypeptide Fraction from Arca subcrenata Induces Apoptosis and G2/M Phase Arrest in HeLa Cells via ROS-Mediated MAPKs Pathways

    Xianjing Hu

    2015-01-01

    Full Text Available Arca subcrenata is documented in the literature of marine Traditional Chinese Medicine. Polypeptide fraction from A. subcrenata, coded as P2, was demonstrated to possess significant antitumor activity in our previous study. However, the underlying mechanism remains undefined. The present study was carried out to investigate the underlying antitumor mechanism of P2 in human cervical cancer HeLa cells by MTT, FCM, LSCM, and western blot assays. The results revealed that P2 significantly induced apoptosis of HeLa cells in a concentration- and time-dependent manner. High level of ROS was provoked by P2, which was in turn responsible for induction of apoptosis through activation of intrinsic mitochondrial pathway and JNK1/2, p38 MAPK pathways, as well as inhibition of ERK1/2 pathway, as evidenced by the abrogation of P2’s effect on HeLa cells preincubated with the ROS scavenger NAC. P2 also was observed to display significant effect on G2/M phase arrest by downregulating the expression of cyclin B1/cdc2 complex and upregulating the expression of p21. These findings demonstrate that P2 induces apoptosis and G2/M phase arrest in HeLa cells through ROS-mediated MAPKs pathways, suggesting that P2 would be worth investigating as a promising agent within the scope of marine drugs for treatment of cervical cancer.

  18. Nanosecond pulsed electric fields induce poly(ADP-ribose) formation and non-apoptotic cell death in HeLa S3 cells

    Morotomi-Yano, Keiko; Akiyama, Hidenori [Institute of Pulsed Power Science, Kumamoto University, Kumamoto 860-8555 (Japan); Yano, Ken-ichi, E-mail: yanoken@kumamoto-u.ac.jp [Priority Organization for Innovation and Excellence, Kumamoto University, Kumamoto 860-8555 (Japan)

    2013-08-30

    Highlights: •Nanosecond pulsed electric field (nsPEF) is a new and unique means for life sciences. •Apoptosis was induced by nsPEF exposure in Jurkat cells. •No signs of apoptosis were detected in HeLa S3 cells exposed to nsPEFs. •Formation of poly(ADP-ribose) was induced in nsPEF-exposed HeLa S3 cells. •Two distinct modes of cell death were activated by nsPEF in a cell-dependent manner. -- Abstract: Nanosecond pulsed electric fields (nsPEFs) have recently gained attention as effective cancer therapy owing to their potency for cell death induction. Previous studies have shown that apoptosis is a predominant mode of nsPEF-induced cell death in several cell lines, such as Jurkat cells. In this study, we analyzed molecular mechanisms for cell death induced by nsPEFs. When nsPEFs were applied to Jurkat cells, apoptosis was readily induced. Next, we used HeLa S3 cells and analyzed apoptotic events. Contrary to our expectation, nsPEF-exposed HeLa S3 cells exhibited no molecular signs of apoptosis execution. Instead, nsPEFs induced the formation of poly(ADP-ribose) (PAR), a hallmark of necrosis. PAR formation occurred concurrently with a decrease in cell viability, supporting implications of nsPEF-induced PAR formation for cell death. Necrotic PAR formation is known to be catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1), and PARP-1 in apoptotic cells is inactivated by caspase-mediated proteolysis. Consistently, we observed intact and cleaved forms of PARP-1 in nsPEF-exposed and UV-irradiated cells, respectively. Taken together, nsPEFs induce two distinct modes of cell death in a cell type-specific manner, and HeLa S3 cells show PAR-associated non-apoptotic cell death in response to nsPEFs.

  19. Glycans coated silver nanoparticles induces autophagy and necrosis in HeLa cells

    Panzarini, Elisa; Mariano, Stefania; Dini, Luciana

    2015-06-01

    This study reports the induction of autophagy by two concentrations (2×103 or 2×104 NPs/cell) of 30 nm sized β-D-Glucose- and β-D-Glucose/Sucrose-coated silver NanoParticles (AgNPs-G and AgNPs-GS respectively) in HeLa cells treated for 6, 12, 24 and 48 hrs. Cell viability was assessed by Neutral Red (NR) test and morphological evaluation. In addition ROS generation (NBT test) and induction of apoptosis/necrosis (Annexin V/Propidium Iodide-Annexin V/PI staining) and autophagy (Monodansylcadaverine-MDC staining) were evaluated. Cytotoxicity, ROS generation and morphology changes depend on NPs type and amount, and incubation time. As a general result, AgNPs-G are more toxic than AgNPs-GS. Moreover, the lowest AgNPs-GS concentration is ineffective on cell viability and ROS generation. Only 10% and 25% of viable HeLa cells were found at the end of incubation time in the presence of higher amount of AgNPs - G and AgNPs-GS respectively and in parallel ROS generation is induced. To elucidate the type of cell death, Annexin V/PI and MDC staining was performed. Interestingly, irrespective of coating type and NPs amount the percentage of apoptotic cells (Annexin V+/PI-) is similar to viable HeLa cells. At contrary, we observed a NPs amount dependent autophagy and necrosis induction. In fact, the lower amount of NPs induces autophagy (MDC+/PI- cells) whereas the higher one induces necrosis (Annexin V+/PI+ cells). Our findings suggest that AgNPs-induced cytotoxicity depends on AgNPs amount and type and provide preliminary evidence of induction of autophagy in HeLa cells cultured in the presence of AgNPs.

  20. TSPY potentiates cell proliferation and tumorigenesis by promoting cell cycle progression in HeLa and NIH3T3 cells

    TSPY is a repeated gene mapped to the critical region harboring the gonadoblastoma locus on the Y chromosome (GBY), the only oncogenic locus on this male-specific chromosome. Elevated levels of TSPY have been observed in gonadoblastoma specimens and a variety of other tumor tissues, including testicular germ cell tumors, prostate cancer, melanoma, and liver cancer. TSPY contains a SET/NAP domain that is present in a family of cyclin B and/or histone binding proteins represented by the oncoprotein SET and the nucleosome assembly protein 1 (NAP1), involved in cell cycle regulation and replication. To determine a possible cellular function for TSPY, we manipulated the TSPY expression in HeLa and NIH3T3 cells using the Tet-off system. Cell proliferation, colony formation assays and tumor growth in nude mice were utilized to determine the TSPY effects on cell growth and tumorigenesis. Cell cycle analysis and cell synchronization techniques were used to determine cell cycle profiles. Microarray and RT-PCR were used to investigate gene expression in TSPY expressing cells. Our findings suggest that TSPY expression increases cell proliferation in vitro and tumorigenesis in vivo. Ectopic expression of TSPY results in a smaller population of the host cells in the G2/M phase of the cell cycle. Using cell synchronization techniques, we show that TSPY is capable of mediating a rapid transition of the cells through the G2/M phase. Microarray analysis demonstrates that numerous genes involved in the cell cycle and apoptosis are affected by TSPY expression in the HeLa cells. These data, taken together, have provided important insights on the probable functions of TSPY in cell cycle progression, cell proliferation, and tumorigenesis

  1. Curcumin targeting the thioredoxin system elevates oxidative stress in HeLa cells

    Cai, Wenqing; Zhang, Baoxin; Duan, Dongzhu [State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou, Gansu 730000 (China); Wu, Jincai [College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou, Gansu 730000 (China); Fang, Jianguo, E-mail: fangjg@lzu.edu.cn [State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou, Gansu 730000 (China); College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou, Gansu 730000 (China)

    2012-08-01

    The thioredoxin system, composed of thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH, is ubiquitous in all cells and involved in many redox-dependent signaling pathways. Curcumin, a naturally occurring pigment that gives a specific yellow color in curry food, is consumed in normal diet up to 100 mg per day. This molecule has also been used in traditional medicine for the treatment of a variety of diseases. Curcumin has numerous biological functions, and many of these functions are related to induction of oxidative stress. However, how curcumin elicits oxidative stress in cells is unclear. Our previous work has demonstrated the way by which curcumin interacts with recombinant TrxR1 and alters the antioxidant enzyme into a reactive oxygen species (ROS) generator in vitro. Herein we reported that curcumin can target the cytosolic/nuclear thioredoxin system to eventually elevate oxidative stress in HeLa cells. Curcumin-modified TrxR1 dose-dependently and quantitatively transfers electrons from NADPH to oxygen with the production of ROS. Also, curcumin can drastically down-regulate Trx1 protein level as well as its enzyme activity in HeLa cells, which in turn remarkably decreases intracellular free thiols, shifting the intracellular redox balance to a more oxidative state, and subsequently induces DNA oxidative damage. Furthermore, curcumin-pretreated HeLa cells are more sensitive to oxidative stress. Knockdown of TrxR1 sensitizes HeLa cells to curcumin cytotoxicity, highlighting the physiological significance of targeting TrxR1 by curcumin. Taken together, our data disclose a previously unrecognized prooxidant mechanism of curcumin in cells, and provide a deep insight in understanding how curcumin works in vivo. -- Highlights: ► Curcumin induces oxidative stress by targeting the thioredoxin system. ► Curcumin-modified TrxR quantitatively oxidizes NADPH to generate ROS. ► Knockdown of TrxR1 augments curcumin's cytotoxicity in HeLa cells.

  2. Curcumin targeting the thioredoxin system elevates oxidative stress in HeLa cells

    The thioredoxin system, composed of thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH, is ubiquitous in all cells and involved in many redox-dependent signaling pathways. Curcumin, a naturally occurring pigment that gives a specific yellow color in curry food, is consumed in normal diet up to 100 mg per day. This molecule has also been used in traditional medicine for the treatment of a variety of diseases. Curcumin has numerous biological functions, and many of these functions are related to induction of oxidative stress. However, how curcumin elicits oxidative stress in cells is unclear. Our previous work has demonstrated the way by which curcumin interacts with recombinant TrxR1 and alters the antioxidant enzyme into a reactive oxygen species (ROS) generator in vitro. Herein we reported that curcumin can target the cytosolic/nuclear thioredoxin system to eventually elevate oxidative stress in HeLa cells. Curcumin-modified TrxR1 dose-dependently and quantitatively transfers electrons from NADPH to oxygen with the production of ROS. Also, curcumin can drastically down-regulate Trx1 protein level as well as its enzyme activity in HeLa cells, which in turn remarkably decreases intracellular free thiols, shifting the intracellular redox balance to a more oxidative state, and subsequently induces DNA oxidative damage. Furthermore, curcumin-pretreated HeLa cells are more sensitive to oxidative stress. Knockdown of TrxR1 sensitizes HeLa cells to curcumin cytotoxicity, highlighting the physiological significance of targeting TrxR1 by curcumin. Taken together, our data disclose a previously unrecognized prooxidant mechanism of curcumin in cells, and provide a deep insight in understanding how curcumin works in vivo. -- Highlights: ► Curcumin induces oxidative stress by targeting the thioredoxin system. ► Curcumin-modified TrxR quantitatively oxidizes NADPH to generate ROS. ► Knockdown of TrxR1 augments curcumin's cytotoxicity in HeLa cells. ► Curcumin

  3. Mitochondria-targeted superoxide dismutase (SOD2) regulates radiation resistance and radiation stress response in HeLa cells

    Reactive oxygen species (ROS) act as a mediator of ionizing radiation-induced cellular damage. Previous studies have indicated that MnSOD (SOD2) plays a critical role in protection against ionizing radiation in mammalian cells. In this study, we constructed two types of stable HeLa cell lines overexpressing SOD2, HeLa S3/SOD2 and T-REx HeLa/SOD2, to elucidate the mechanisms underlying the protection against radiation by SOD2. SOD2 overexpression in mitochondria enhanced the survival of HeLa S3 and T-REx HeLa cells following γ-irradiation. The levels of γH2AX significantly decreased in HeLa S3/SOD2 and T-REx HeLa/SOD2 cells compared with those in the control cells. MitoSoxTM Red assays showed that both lines of SOD2-expressing cells showed suppression of the superoxide generation in mitochondria. Furthermore, flow cytometry with a fluorescent probe (2',7'-dichlorofluorescein) revealed that the cellular levels of ROS increased in HeLa S3 cells during post-irradiation incubation, but the increase was markedly attenuated in HeLa S3/SOD2 cells. DNA microarray analysis revealed that, of 47,000 probe sets analyzed, 117 and 166 probes showed more than 2-fold changes after 5.5 Gy of γ-irradiation in control and HeLa S3/SOD2 cells, respectively. Pathway analysis revealed different expression profiles in irradiated control cells and irradiated SOD2-overexpressing cells. These results indicate that SOD2 protects HeLa cells against cellular effects of γ-rays through suppressing oxidative stress in irradiated cells caused by ROS generated in the mitochondria and through regulating the expression of genes which play a critical role in protection against ionizing radiation. (author)

  4. A class of DNA-binding peptides from wheat bud causes growth inhibition, G2 cell cycle arrest and apoptosis induction in HeLa cells

    Elgjo Kjell

    2009-07-01

    Full Text Available Abstract Background Deproteinized DNA from eukaryotic and prokaryotic cells still contains a low-molecular weight peptidic fraction which can be dissociated by alkalinization of the medium. This fraction inhibits RNA transcription and tumor cell growth. Removal from DNA of normal cells causes amplification of DNA template activity. This effect is lower or absent in several cancer cell lines. Likewise, the amount of active peptides in cancer cell DNA extracts is lower than in DNA preparation of the corresponding normal cells. Such evidence, and their ubiquitous presence, suggests that they are a regulatory, conserved factor involved in the control of normal cell growth and gene expression. Results We report that peptides extracted from wheat bud chromatin induce growth inhibition, G2 arrest and caspase-dependent apoptosis in HeLa cells. The growth rate is decreased in cells treated during the S phase only and it is accompanied by DNA damage and DNA synthesis inhibition. In G2 cells, this treatment induces inactivation of the CDK1-cyclin B1 complex and an increase of active chk1 kinase expression. Conclusion The data indicate that the chromatin peptidic pool inhibits HeLa cell growth by causing defective DNA replication which, in turn, arrests cell cycle progression to mitosis via G2 checkpoint pathway activation.

  5. EFFECTS OF CURCUMIN ON PROLIFERATION AND APOPTOSIS OF HUMAN CERVICAL CARCINOMA HeLa CELLS IN VITRO

    赵敬; 赵涌

    2004-01-01

    Objective: To investigate the regulatory effect of curcumin on proliferation and apoptosis in human cervical carcinoma cell line HeLa in vitro. Methods: Human cervical carcinoma cell line Hela was cultured in vitro. HeLa cells were treated with 10(50 (mol/L curcumin for 24(72 h and the growth inhibition rates of HeLa cells were measured by MTT method. Cell apoptosis was inspected by electron microscopy. In addition, the expression of bcl-2, bcl-xl and caspase-3 protein in HeLa cell were observed by SP immunohistochemistry. Results: Curcumin inhibited the proliferation of HeLa cells on a dose-depending manner. Peak of subG1 appeared on DNA histogram in FCM. A portion of the cells presented the characteristic morphological changes of apoptosis under the electron microscope. The bcl-2, bcl-xl expression was decreased while Caspase-3 expression was increased. Conclusion: Curcumin could significantly inhibit the growth of HeLa cells; inducing apoptosis through up-regulating Caspase-3 and down-regulating expression of bcl-2 and bcl-xl was probably one of its molecular mechanisms.

  6. A phthalide derivative isolated from endophytic fungi Pestalotiopsis photiniae induces G1 cell cycle arrest and apoptosis in human HeLa cells

    C. Chen

    2013-08-01

    Full Text Available MP [4-(3′,3′-dimethylallyloxy-5-methyl-6-methoxyphthalide] was obtained from liquid culture of Pestalotiopsis photiniae isolated from the Chinese Podocarpaceae plant Podocarpus macrophyllus. MP significantly inhibited the proliferation of HeLa tumor cell lines. After treatment with MP, characteristic apoptotic features such as DNA fragmentation and chromatin condensation were observed in DAPI-stained HeLa cells. Flow cytometry showed that MP induced G1 cell cycle arrest and apoptosis in a dose-dependent manner. Western blotting and real-time reverse transcription-polymerase chain reaction were used to investigate protein and mRNA expression. MP caused significant cell cycle arrest by upregulating the cyclin-dependent kinase inhibitor p27KIP1 protein and p21CIP1 mRNA levels in HeLa cells. The expression of p73 protein was increased after treatment with various MP concentrations. mRNA expression of the cell cycle-related genes, p21CIP1 , p16INK4a and Gadd45α, was significantly upregulated and mRNA levels demonstrated significantly increased translation of p73, JunB, FKHR, and Bim. The results indicate that MP may be a potential treatment for cervical cancer.

  7. A phthalide derivative isolated from endophytic fungi Pestalotiopsis photiniae induces G1 cell cycle arrest and apoptosis in human HeLa cells

    Chen, C. [College of Life Science, Hebei University, Baoding (China); Yang, R.L. [Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Baoding, China, Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Baoding (China)

    2013-07-30

    MP [4-(3′,3′-dimethylallyloxy)-5-methyl-6-methoxyphthalide] was obtained from liquid culture of Pestalotiopsis photiniae isolated from the Chinese Podocarpaceae plant Podocarpus macrophyllus. MP significantly inhibited the proliferation of HeLa tumor cell lines. After treatment with MP, characteristic apoptotic features such as DNA fragmentation and chromatin condensation were observed in DAPI-stained HeLa cells. Flow cytometry showed that MP induced G1 cell cycle arrest and apoptosis in a dose-dependent manner. Western blotting and real-time reverse transcription-polymerase chain reaction were used to investigate protein and mRNA expression. MP caused significant cell cycle arrest by upregulating the cyclin-dependent kinase inhibitor p27{sup KIP1} protein and p21{sup CIP1} mRNA levels in HeLa cells. The expression of p73 protein was increased after treatment with various MP concentrations. mRNA expression of the cell cycle-related genes, p21{sup CIP1}, p16{sup INK4a} and Gadd45α, was significantly upregulated and mRNA levels demonstrated significantly increased translation of p73, JunB, FKHR, and Bim. The results indicate that MP may be a potential treatment for cervical cancer.

  8. A phthalide derivative isolated from endophytic fungi Pestalotiopsis photiniae induces G1 cell cycle arrest and apoptosis in human HeLa cells

    MP [4-(3′,3′-dimethylallyloxy)-5-methyl-6-methoxyphthalide] was obtained from liquid culture of Pestalotiopsis photiniae isolated from the Chinese Podocarpaceae plant Podocarpus macrophyllus. MP significantly inhibited the proliferation of HeLa tumor cell lines. After treatment with MP, characteristic apoptotic features such as DNA fragmentation and chromatin condensation were observed in DAPI-stained HeLa cells. Flow cytometry showed that MP induced G1 cell cycle arrest and apoptosis in a dose-dependent manner. Western blotting and real-time reverse transcription-polymerase chain reaction were used to investigate protein and mRNA expression. MP caused significant cell cycle arrest by upregulating the cyclin-dependent kinase inhibitor p27KIP1 protein and p21CIP1 mRNA levels in HeLa cells. The expression of p73 protein was increased after treatment with various MP concentrations. mRNA expression of the cell cycle-related genes, p21CIP1, p16INK4a and Gadd45α, was significantly upregulated and mRNA levels demonstrated significantly increased translation of p73, JunB, FKHR, and Bim. The results indicate that MP may be a potential treatment for cervical cancer

  9. Microscopic Detection of Thermogenesis in a Single HeLa Cell

    Suzuki, Madoka; Tseeb, Vadim; Oyama, Kotaro; Ishiwata, Shin'ichi

    2007-01-01

    We report here the technique for detection and measurement of the temperature changes in single cells using a recently devised microthermometer (a glass micropipette filled with the thermosensitive fluorescent dye Europium (III) thenoyltrifluoroacetonate trihydrate). We found that the heat production in a single HeLa cell occurred with some time delay after the ionomycin-induced Ca2+ influx from the extracellular space. The time delay inversely depended on extracellular [Ca2+], and the increa...

  10. Analysis of lysosomal membrane proteins exposed to melanin in HeLa cells

    Bang, Seung Hyuck; Park, Dong Jun; Kim, Yang-Hoon; Min, Jiho

    2016-01-01

    Objectives There have been developed to use targeting ability for antimicrobial, anticancerous, gene therapy and cosmetics through analysis of various membrane proteins isolated from cell organelles. Methods It was examined about the lysosomal membrane protein extracted from lysosome isolated from HeLa cell treated by 100 ppm melanin for 24 hours in order to find associated with targeting ability to melanin using by 2-dimensional electrophoresis. Results The result showed 14 up-regulated (1.5...

  11. Inhibition of protein synthesis in intact HeLa cells by Shigella dysenteriae 1 toxin.

    Brown, J.E.; Rothman, S W; Doctor, B P

    1980-01-01

    Shiga toxin purified to near homogeneity from cell lysates of Shigella dysenteriae 1 inhibited protein and deoxyribonucle acid syntheses in intact HeLa cells. Inhibition was dependent on toxin concentration and time of incubation. A minimal latent period of 30 min was observed with saturating doses of toxin. Ribonucleic acid synthesis, uptake of alpha-aminoisobutyric acid, and maintenance of intracellular K+ concentrations were not affected until well after maximal inhibition of protein and d...

  12. Transport of NaYF4:Er3+, Yb3+ up-converting nanoparticles into HeLa cells

    An effective, simple and practically useful method to incorporate fluorescent nanoparticles inside live biological cells was developed. The internalization time and concentration dependence of a frequently used liposomal transfection factor (Lipofectamine 2000) was studied. A user friendly, one-step technique to obtain water and organic solvent soluble Er3+ and Yb3+ doped NaYF4 nanoparticles coated with polyvinylpyrrolidone was obtained. Structural analysis of the nanoparticles confirmed the formation of nanocrystals of the desired sizes and spectral properties. The internalization of NaYF4 nanoparticles in HeLa cervical cancer cells was determined at different nanoparticle concentrations and for incubation periods from 3 to 24 h. The images revealed a redistribution of nanoparticles inside the cell, which increases with incubation time and concentration levels, and depends on the presence of the transfection factor. The study identifies, for the first time, factors responsible for an effective endocytosis of the up-converting nanoparticles to HeLa cells. Thus, the method could be applied to investigate a wide range of future ‘smart’ theranostic agents. Nanoparticles incorporated into the liposomes appear to be very promising fluorescent probes for imaging real-time cellular dynamics. (paper)

  13. Transport of NaYF4:Er3+, Yb3+ up-converting nanoparticles into HeLa cells

    Sikora, Bożena; Fronc, Krzysztof; Kamińska, Izabela; Koper, Kamil; Szewczyk, Sebastian; Paterczyk, Bohdan; Wojciechowski, Tomasz; Sobczak, Kamil; Minikayev, Roman; Paszkowicz, Wojciech; Stępień, Piotr; Elbaum, Danek

    2013-06-01

    An effective, simple and practically useful method to incorporate fluorescent nanoparticles inside live biological cells was developed. The internalization time and concentration dependence of a frequently used liposomal transfection factor (Lipofectamine 2000) was studied. A user friendly, one-step technique to obtain water and organic solvent soluble Er3+ and Yb3+ doped NaYF4 nanoparticles coated with polyvinylpyrrolidone was obtained. Structural analysis of the nanoparticles confirmed the formation of nanocrystals of the desired sizes and spectral properties. The internalization of NaYF4 nanoparticles in HeLa cervical cancer cells was determined at different nanoparticle concentrations and for incubation periods from 3 to 24 h. The images revealed a redistribution of nanoparticles inside the cell, which increases with incubation time and concentration levels, and depends on the presence of the transfection factor. The study identifies, for the first time, factors responsible for an effective endocytosis of the up-converting nanoparticles to HeLa cells. Thus, the method could be applied to investigate a wide range of future ‘smart’ theranostic agents. Nanoparticles incorporated into the liposomes appear to be very promising fluorescent probes for imaging real-time cellular dynamics.

  14. Cytotoxicity and apoptotic effects of nickel oxide nanoparticles in cultured HeLa cells

    The aim of this study was to observe the cytotoxicity and apoptotic effects of nickel oxide nanoparticles on human cervix epithelioid carcinoma cell line (HeLa). Nickel oxide precursors were synthesized by an nickel sulphate-excess urea reaction in boiling aqueous solution. The synthesized NiO nanoparticles (< 200 nm) were investigated by X-ray diffraction analysis and transmission electron microscopy techniques. For cytotoxicity experiments, HeLa cells were incubated in 50-500 micro g/ml NiO for 2, 6, 12 and 16 hours. The viable cells were counted with a haemacytometer using light microscopy. The cytotoxicity was observed low in 50-200 micro g/ml concentration for 16 h, but high in 400-500 micro g/ml concentration for 2-6 h. HeLa cells cytoplasm membrane was lysed and detached from the well surface in 400 micro g/ml concentration NiO nanoparticles. Double staining and M30 immunostaining were performed to quantify the number of apoptotic cells in culture on the basis of apoptotic cell nuclei scores. The apoptotic effect was observed 20% for 16 h incubation. (authors)

  15. Cytotoxicity and apoptotic effects of nickel oxide nanoparticles in cultured HeLa cells

    Kezban Ada

    2010-04-01

    Full Text Available The aim of this study was to observe the cytotoxicity and apoptotic effects of nickel oxide nanoparticles on humancervix epithelioid carcinoma cell line (HeLa. Nickel oxide precursors were synthesized by an nickel sulphate-excess ureareaction in boiling aqueous solution. The synthesized NiO nanoparticles (<200 nm were investigated by X-ray diffractionanalysis and transmission electron microscopy techniques. For cytotoxicity experiments, HeLa cells were incubated in50-500 μg/mL NiO for 2, 6, 12 and 16 hours. The viable cells were counted with a haemacytometer using light microscopy.The cytotoxicity was observed low in 50-200 μg/mL concentration for 16 h, but high in 400-500 μg/mL concentration for2-6 h. HeLa cells' cytoplasm membrane was lysed and detached from the well surface in 400 μg/mL concentration NiOnanoparticles. Double staining and M30 immunostaining were performed to quantify the number of apoptotic cells in cultureon the basis of apoptotic cell nuclei scores. The apoptotic effect was observed 20% for 16 h incubation.

  16. Curcumin targeting the thioredoxin system elevates oxidative stress in HeLa cells.

    Cai, Wenqing; Zhang, Baoxin; Duan, Dongzhu; Wu, Jincai; Fang, Jianguo

    2012-08-01

    The thioredoxin system, composed of thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH, is ubiquitous in all cells and involved in many redox-dependent signaling pathways. Curcumin, a naturally occurring pigment that gives a specific yellow color in curry food, is consumed in normal diet up to 100mg per day. This molecule has also been used in traditional medicine for the treatment of a variety of diseases. Curcumin has numerous biological functions, and many of these functions are related to induction of oxidative stress. However, how curcumin elicits oxidative stress in cells is unclear. Our previous work has demonstrated the way by which curcumin interacts with recombinant TrxR1 and alters the antioxidant enzyme into a reactive oxygen species (ROS) generator in vitro. Herein we reported that curcumin can target the cytosolic/nuclear thioredoxin system to eventually elevate oxidative stress in HeLa cells. Curcumin-modified TrxR1 dose-dependently and quantitatively transfers electrons from NADPH to oxygen with the production of ROS. Also, curcumin can drastically down-regulate Trx1 protein level as well as its enzyme activity in HeLa cells, which in turn remarkably decreases intracellular free thiols, shifting the intracellular redox balance to a more oxidative state, and subsequently induces DNA oxidative damage. Furthermore, curcumin-pretreated HeLa cells are more sensitive to oxidative stress. Knockdown of TrxR1 sensitizes HeLa cells to curcumin cytotoxicity, highlighting the physiological significance of targeting TrxR1 by curcumin. Taken together, our data disclose a previously unrecognized prooxidant mechanism of curcumin in cells, and provide a deep insight in understanding how curcumin works in vivo. PMID:22634334

  17. Substitued (E-b-(benzoylacrylic acids suppressed survival of neoplastic human HeLa cells

    I. JURANIC

    1999-09-01

    Full Text Available The bacteriostatic activity of some of alkyl substituted (E-b-(benzoylacrylic acids was shown earlier. The aim of this study was to investigate the antiproliferative action of 19 alkyl-, or halogeno-, or methoxy-, or acetamido- substituted (E-b-(benzoylacrylic acids, against human cervix carcinoma, HeLa, cells. Target HeLa cells were continuously treated with increasing concentrations of substituted (E-b-(benzoylacrylic acids during two days. The MTT test was used for assessment of the antiproliferative action of this group of compounds. Treatment of HeLa cells with 4-methyl-, 4-fluoro-, 4-chloro-, 4-bromo- and 4-methoxy- derivatives of (E-b-(benzoyl acrylic acid leads to the expression of cytostatic activity against HeLa cells (IC50 were in the range from 31-40 µM. Their antiproliferative action was less than that of the basic compound (E-b-(benzoylacrylic acid whose IC50 was 28.5 µM. The 3,4-dimethyl-, 2,4-dimethyl- and 2,5-dimethyl- derivatives as well as the 4-ethyl- and 3,4-dichloro- and 2,4-dichloro-derivatives, have stronger cytostatic activity than the correspoding monosubstituted and parent compound. Their IC50 were 18.5 µM; 17.5 µM; 17.0 mM; 17.5 µM; 22.0 µM and 18 µM, respectively. The 4-iso-propyl- and 4-n-butyl-derivatives exerted higher cytostatic activity than the compounds with a lower number of methylene -CH2- groups in the substitutent. Their IC50 were 14.5 µM and 6.5 µM respectively. The 2,5-di-iso-propyl- and 4-tert-butyl-derivatives expressed the most strong antiproliferative action against the investigated HeLa cells, IC50 being 4.5 µM and 5.5 µM, respectively. The investigated compounds affected the survival of HeLa cells, expressing a strong structure-activity relationship of the Hansch type.

  18. Biocompatibility of various ferrite nanoparticles evaluated by in vitro cytotoxicity assays using HeLa cells

    Tomitaka, Asahi [Department of Electrical and Computer Engineering, Yokohama National University, Tokiwadai 79-5, Yokohama, Kanagawa 240-8501 (Japan)], E-mail: d07gd158@ynu.ac.jp; Hirukawa, Atsuo; Yamada, Tsutomu [Department of Electrical and Computer Engineering, Yokohama National University, Tokiwadai 79-5, Yokohama, Kanagawa 240-8501 (Japan); Morishita, Shin [Department of Mechanical Engineering and Materials Science, Yokohama National University, Tokiwadai 79-5, Yokohama, Kanagawa 240-8501 (Japan); Takemura, Yasushi [Department of Electrical and Computer Engineering, Yokohama National University, Tokiwadai 79-5, Yokohama, Kanagawa 240-8501 (Japan)

    2009-05-15

    Magnetic nanoparticles for thermotherapy must be biocompatible and possess high thermal efficiency as heating elements. The biocompatibility of Fe{sub 3}O{sub 4} (20-30 nm), ZnFe{sub 2}O{sub 4} (15-30 nm) and NiFe{sub 2}O{sub 4} (20-30 nm) nanoparticles was studied using a cytotoxicity colony formation assay and a cell viability assay. The Fe{sub 3}O{sub 4} sample was found to be biocompatible on HeLa cells. While ZnFe{sub 2}O{sub 4} and NiFe{sub 2}O{sub 4} were non-toxic at low concentrations, HeLa cells exhibited cytotoxic effects when exposed to concentrations of 100 {mu}g/ml nanoparticles.

  19. Coxsackievirus B5 induced apoptosis of HeLa cells: Effects on p53 and SUMO

    Coxsackievirus B5 (CVB5), a human enterovirus of the family Picornaviridae, is a frequent cause of acute and chronic human diseases. The pathogenesis of enteroviral infections is not completely understood, and the fate of the CVB5-infected cell has a pivotal role in this process. We have investigated the CVB5-induced apoptosis of HeLa cells and found that it happens by the intrinsic pathway by a mechanism dependent on the ubiquitin-proteasome system, associated with nuclear aggregation of p53. Striking redistribution of both SUMO and UBC9 was noted at 4 h post-infection, simultaneously with a reduction in the levels of the ubiquitin-ligase HDM2. Taken together, these results suggest that CVB5 infection of HeLa cells elicit the intrinsic pathway of apoptosis by MDM2 degradation and p53 activation, destabilizing protein sumoylation, by a mechanism that is dependent on a functional ubiquitin-proteasome system.

  20. A key inactivation factor of HeLa cell viability by a plasma flow

    Sato, Takehiko; Yokoyama, Mayo [Institute of Fluid Science, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577 (Japan); Johkura, Kohei, E-mail: sato@ifs.tohoku.ac.jp [Department of Histology and Embryology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621 (Japan)

    2011-09-21

    Recently, a plasma flow has been applied to medical treatment using effects of various kinds of stimuli such as chemical species, charged particles, heat, light, shock wave and electric fields. Among them, the chemical species are known to cause an inactivation of cell viability. However, the mechanisms and key factors of this event are not yet clear. In this study, we focused on the effect of H{sub 2}O{sub 2} in plasma-treated culture medium because it is generated in the culture medium and it is also chemically stable compared with free radicals generated by the plasma flow. To elucidate the significance of H{sub 2}O{sub 2}, we assessed the differences in the effects of plasma-treated medium and H{sub 2}O{sub 2}-added medium against inactivation of HeLa cell viability. These two media showed comparable effects on HeLa cells in terms of the survival ratios, morphological features of damage processes, permeations of H{sub 2}O{sub 2} into the cells, response to H{sub 2}O{sub 2} decomposition by catalase and comprehensive gene expression. The results supported that among chemical species generated in a plasma-treated culture medium, H{sub 2}O{sub 2} is one of the main factors responsible for inactivation of HeLa cell viability. (fast track communication)

  1. Regulation of the cell cycle via mitochondrial gene expression and energy metabolism in HeLa cells

    Wei Xiong; Yang Jiao; Weiwei Huang; Mingxing Ma; Min Yu; Qinghua Cui; Deyong Tan

    2012-01-01

    Human cervical cancer HeLa cells have functional mitochondria.Recent studies have suggested that mitochondrial metabolism plays an essential role in tumor cell proliferation.Nevertheless,how cells coordinate mitochondrial dynamics and cell cycle progression remains to be clarified.To investigate the relationship between mitochondrial function and cell cycle regulation,the mitochondrial gene expression profile and cellular ATP levels were determined by cell cycle progress analysis in the present study.HeLa cells were synchronized in the G0/G1 phase by serum starvation,and re-entered cell cycle by restoring serum culture,time course experiment was performed to analyze the expression of mitochondrial transcription regulators and mitochondrial genes,mitochondrial membrane potential (MMP),cellular ATP levels,and cell cycle progression.The results showed that when arrested G0/G1 cells were stimulated in serum-containing medium,the amount of DNA and the expression levels of both mRNA and proteins in mitochondria started to increase at 2 h time point,whereas the MMP and ATP level elevated at 4 h.Furthermore,the cyclin D1 expression began to increase at 4 h after serum triggered cell cycle.ATP synthesis inhibitor-oligomycintreatment suppressed the cyclin D1 and cyclin B1 expression levels and blocked cell cycle progression.Taken together,our results suggested that increased mitochondrial gene expression levels,oxidative phosphorylation activation,and cellular ATP content increase are important events for triggering cell cycle.Finally,we demonstrated that mitochondrial gene expression levels and cellular ATP content are tightly regulated and might play a central role in regulating cell proliferation.

  2. Novel microtubule-targeted agent 6-chloro-4-(methoxyphenyl) coumarin induces G2-M arrest andapoptosis in HeLa cells

    Yi-ming MA; Yu-bo ZHOU; Chuan-ming XIE; Dong-mei CHEN; Jia LI

    2012-01-01

    To identify a novel coumarin analogue with the highest anticancer activity and to further investigate its anticancer mechanisms.Methods:The viability of cancer cells was investigated using the MTT assay.The cell cycle progression was evaluated using both flow cytometric and Western blotting analysis.Microtubule depolymerization was observed with immunocytochemistry in vivo and a tubu-lin depolymerization assay in vitro.Apoptosis was demonstrated using Annexin V/Propidium Iodide (PI) double-staining and sub-G1analysis.Results:Among 36 analogues of coumarin,6-chloro-4-(methoxyphenyl) coumarin showed the best anticancer activity (IC50 value about 200 nmol/L) in HCT116 cells.The compound had a broad spectrum of anticancer activity against 9 cancer cell lines derived from colon cancer,breast cancer,liver cancer,cervical cancer,leukemia,epidermoid cancer with IC5o value of 75 nmol/L-1.57 μmol/L but with low cytotocitity against WI-38 human lung fibroblasts (IC50 value of 12.128 μmol/L).The compound (0.04-10 μmol/L) induced G2-M phase arrest in HeLa cells in a dose-dependent manner,which was reversible after the compound was removed.The compound (10-300 μmol/L) induced the depolymerization of purified porcine tubulin in vitro.Finally,the compound (0.04-2.5 μmol/L) induced apoptosis of HeLa cells in dose- and time-dependent manners.Conclusion:6-Chloro-4-(methoxyphenyl) coumarin is a novel microtubule-targeting agent that induces G2-M arrest and apoptosis in HeLa cells.

  3. Radioprotective effect of calorie restriction in Hela cells and SD rats

    Objective: To explore the effect of low calorie metabolism on the survival of HeLa cells exposed to X-rays, and the influence of starvation on the antioxidative factors in the blood of rats after irradiation. Methods: MTT method was used to evaluate the impact of different concentration glucose on the proliferation of HeLa cells. Colony formation assay was employed to detect the influence of glucose (1, 5, 10 and 25 mmol/L) on radiosensitivity of HeLa cells. Flow cytometry assay was used to analyze distribution of cell cycle and apoptosis. 60 male SD rats were randomly divided into 6 groups with 10 rats each. Rats in every two groups were fed ad libitum, fasted for 24 h and fasted for 48 h, respectively. Rats in one group of each approach were respectively exposed to whole-body X-rays at 11 Gy. At 2 h after irradiation,all of rats were sacrificed and their venous blood was collected. Elisa kits were used to detect superoxide dismutase (SOD) and total antioxidant capacity (T-AOC). Results: An increased viability was observed in HeLa cells treated with the glucose at low concentration (<25 mmol/L), while HeLa cell growth was inhibited by glucose at doses of >25 mmol/L. Relevant to cells treated with 1 mmoL/L glucose, SERs (sensitive enhancement ratio) in cells exposed to 5, 10 and 25 mmol/L glucose were 1.07, 1.10 and 1.23,respectively. A reduction of G2/M and S arrests and apoptosis caused by 6 Gy X-ray irradiation were observed [(49.68 ±1.88)% and (35.54±1.45)% at G2/M phase, (16.88 ±1.22)% and (10.23 ±1.65)% at S phase, t=10.42, 5.61, P<0.05] and in the cells treated with 1 mmol/L glucose compared with cells treated with 25 mmol/L glucose [(25.50 ± 0.95)% and (7.56 ± 1.07)%, t=21.72, P<0.05].Without irradiation, calorie restriction exhibited a negligible influence on SOD and T-AOC in rats. However, after 11 Gy irradiation, compared with rats fed ad libitum, the levels of SOD and T-AOC were significantly increased in rats with calorie restriction (t=40.32, 42

  4. Laser stimulation can activate autophagy in HeLa cells

    Wang, Yisen; Hu, Minglie; Wang, Chingyue [Ultrafast Laser Laboratory, Key Laboratory of Optoelectronic Information Technology (Ministry of Education), College of Precision Instrument and Optoelectronics Engineering, Tianjin University, Tianjin (China); Lan, Bei; Cao, Youjia [Key Laboratory of Microbial Functional Genomics of Ministry of Education, College of Life Sciences, Nankai University, Tianjin (China); He, Hao, E-mail: haohe@tju.edu.cn [Ultrafast Laser Laboratory, Key Laboratory of Optoelectronic Information Technology (Ministry of Education), College of Precision Instrument and Optoelectronics Engineering, Tianjin University, Tianjin (China); Med-X Research Institute, School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai (China)

    2014-10-27

    For decades, lasers have been a daily tool in most biological research for fluorescent excitation by confocal or multiphoton microscopy. More than 20 years ago, cell photodamage caused by intense laser stimulation was noticed by generating reactive oxygen species, which was then thought as the main damage effect by photons. In this study, we show that laser stimulation can induce autophagy, an important cell lysosomal pathway responding to immune stimulation and starvation, without any biochemical treatment. Two different types of laser stimulations are found to be capable of activating autophagy: continuous scanning by continuous-wave visible lasers and a short-time flash of femtosecond laser irradiation. The autophagy generation is independent from wavelength, power, and scanning duration of the visible lasers. In contrast, the power of femtosecond laser is very critical to autophagy because the multiphoton excited Ca{sup 2+} dominates autophagy signaling. In general, we show here the different mechanisms of autophagy generation by such laser stimulation, which correspond to confocal microscopy and cell surgery, respectively. Those results can help further understanding of photodamage and autophagy signaling.

  5. Laser stimulation can activate autophagy in HeLa cells

    Wang, Yisen; Lan, Bei; He, Hao; Hu, Minglie; Cao, Youjia; Wang, Chingyue

    2014-10-01

    For decades, lasers have been a daily tool in most biological research for fluorescent excitation by confocal or multiphoton microscopy. More than 20 years ago, cell photodamage caused by intense laser stimulation was noticed by generating reactive oxygen species, which was then thought as the main damage effect by photons. In this study, we show that laser stimulation can induce autophagy, an important cell lysosomal pathway responding to immune stimulation and starvation, without any biochemical treatment. Two different types of laser stimulations are found to be capable of activating autophagy: continuous scanning by continuous-wave visible lasers and a short-time flash of femtosecond laser irradiation. The autophagy generation is independent from wavelength, power, and scanning duration of the visible lasers. In contrast, the power of femtosecond laser is very critical to autophagy because the multiphoton excited Ca2+ dominates autophagy signaling. In general, we show here the different mechanisms of autophagy generation by such laser stimulation, which correspond to confocal microscopy and cell surgery, respectively. Those results can help further understanding of photodamage and autophagy signaling.

  6. Laser stimulation can activate autophagy in HeLa cells

    For decades, lasers have been a daily tool in most biological research for fluorescent excitation by confocal or multiphoton microscopy. More than 20 years ago, cell photodamage caused by intense laser stimulation was noticed by generating reactive oxygen species, which was then thought as the main damage effect by photons. In this study, we show that laser stimulation can induce autophagy, an important cell lysosomal pathway responding to immune stimulation and starvation, without any biochemical treatment. Two different types of laser stimulations are found to be capable of activating autophagy: continuous scanning by continuous-wave visible lasers and a short-time flash of femtosecond laser irradiation. The autophagy generation is independent from wavelength, power, and scanning duration of the visible lasers. In contrast, the power of femtosecond laser is very critical to autophagy because the multiphoton excited Ca2+ dominates autophagy signaling. In general, we show here the different mechanisms of autophagy generation by such laser stimulation, which correspond to confocal microscopy and cell surgery, respectively. Those results can help further understanding of photodamage and autophagy signaling.

  7. Curcumin and Ellagic acid synergistically induce ROS generation, DNA damage, p53 accumulation and apoptosis in HeLa cervical carcinoma cells.

    Kumar, Devbrat; Basu, Soumya; Parija, Lucy; Rout, Deeptimayee; Manna, Sanjeet; Dandapat, Jagneshwar; Debata, Priya Ranjan

    2016-07-01

    Cervical cancer and precancerous lesions of the cervix continue to be a global health issue, and the medication for the treatment for chronic HPV infection so far has not been effective. Potential anticancer and anti HPV activities of two known phytochemicals, Curcumin and Ellagic acid were evaluated in HeLa cervical cancer cells. Curcumin is a natural compound found in the root of Curcuma longa plant and Ellagic acid a polyphenol found in fruits of strawberries, raspberries and walnuts. The combination of Curcumin and Ellagic acid at various concentrations showed better anticancer properties than either of the drug when used alone as evidenced by MTT assay. Besides this, Curcumin and Ellagic acid also restore p53, induce ROS formation and DNA damage. Mechanistic study further indicated that Curcumin and Ellagic acid show anti-HPV activity as evidenced by decrease in the HPV E6 oncoprotein on HeLa cells. PMID:27261574

  8. Effect of 5-fluorodeoxyuridine on DNA replication in ultraviolet-irradiated HeLa cells

    In HeLa cells precultivated for 6 hours with 5-fluorodeoxyuridine (FUdR) and for 18 hours in FUdR-free medium, DNA synthesis was much more resistant to UV irradiation than that of untreated cells. DNA synthesized in FUdR-pretreated and UV irradiated cells represents a semiconservative DNA replication and shows more rapid shift of the pulse-labelled chased DNA to high molecular weight. This DNA synthesis is not induced by synchronization of the cell cycle. It is assumed that either the changes of chromatine structure, or an enhanced level of some enzymes might be involved in the replication of the damaged template. (author)

  9. Dioscin Induces Apoptosis in Human Cervical Carcinoma HeLa and SiHa Cells through ROS-Mediated DNA Damage and the Mitochondrial Signaling Pathway

    Xinwei Zhao

    2016-06-01

    Full Text Available Dioscin, a natural product, has activity against glioblastoma multiforme, lung cancer and colon cancer. In this study, the effects of dioscin against human cervical carcinoma HeLa and SiHa cells were further confirmed, and the possible mechanism(s were investigated. A transmission electron microscopy (TEM assay and DAPI staining were used to detect the cellular morphology. Flow cytometry was used to assay cell apoptosis, ROS and Ca2+ levels. Single cell gel electrophoresis and immunofluorescence assays were used to test DNA damage and cytochrome C release. The results showed that dioscin significantly inhibited cell proliferation and caused DNA damage in HeLa and SiHa cells. The mechanistic investigation showed that dioscin caused the release of cytochrome C from mitochondria into the cytosol. In addition, dioscin significantly up-regulated the protein levels of Bak, Bax, Bid, p53, caspase-3, caspase-9, and down-regulated the protein levels of Bcl-2 and Bcl-xl. Our work thus demonstrated that dioscin notably induces apoptosis in HeLa and SiHa cells through adjusting ROS-mediated DNA damage and the mitochondrial signaling pathway.

  10. Dioscin Induces Apoptosis in Human Cervical Carcinoma HeLa and SiHa Cells through ROS-Mediated DNA Damage and the Mitochondrial Signaling Pathway.

    Zhao, Xinwei; Tao, Xufeng; Xu, Lina; Yin, Lianhong; Qi, Yan; Xu, Youwei; Han, Xu; Peng, Jinyong

    2016-01-01

    Dioscin, a natural product, has activity against glioblastoma multiforme, lung cancer and colon cancer. In this study, the effects of dioscin against human cervical carcinoma HeLa and SiHa cells were further confirmed, and the possible mechanism(s) were investigated. A transmission electron microscopy (TEM) assay and DAPI staining were used to detect the cellular morphology. Flow cytometry was used to assay cell apoptosis, ROS and Ca(2+) levels. Single cell gel electrophoresis and immunofluorescence assays were used to test DNA damage and cytochrome C release. The results showed that dioscin significantly inhibited cell proliferation and caused DNA damage in HeLa and SiHa cells. The mechanistic investigation showed that dioscin caused the release of cytochrome C from mitochondria into the cytosol. In addition, dioscin significantly up-regulated the protein levels of Bak, Bax, Bid, p53, caspase-3, caspase-9, and down-regulated the protein levels of Bcl-2 and Bcl-xl. Our work thus demonstrated that dioscin notably induces apoptosis in HeLa and SiHa cells through adjusting ROS-mediated DNA damage and the mitochondrial signaling pathway. PMID:27271587

  11. Real-time observation of irradiated Hela-cell Modified by Fluorescent ubiquitination-based Cell Cycle Indicator Using Synchrotron X-Ray Microbeam

    Fluorescent ubiquitination-based cell-cycle indicator (FUCCI) human cancer (HeLa) cells (red indicates G1; green, S/G2) were exposed to a synchrotron X-ray microbeam. Cells in either G1 or S/G2 were irradiated selectively according to their colour in the same microscopic field. Time-lapse micrographs of the irradiated cells were acquired for 24 h after irradiation. For fluorescent immunostaining, phosphorylated histone proteins (γ-H2AX) indicated the induction of DNA double-strand breaks. The cell cycle was arrested by irradiation at S/G2. In contrast, cells irradiated at G1 progressed to S/G2. The foci were induced in cells irradiated at both G1 and S/G2, suggesting that the G1-S (or S) checkpoint pathway does not function in HeLa cells due to the fact that the cells are functionally p53 deficient, even though X-ray microbeam irradiation significantly induces double-strand breaks. These results demonstrate that single FUCCI cell exposure and live cell imaging are powerful methods for studying the effects of radiation on the cell cycle. (authors)

  12. PMA synergistically enhances apicularen A-induced cytotoxicity by disrupting microtubule networks in HeLa cells

    Combination therapy is key to improving cancer treatment efficacy. Phorbol 12-myristate 13-acetate (PMA), a well-known PKC activator, increases the cytotoxicity of several anticancer drugs. Apicularen A induces cytotoxicity in tumor cells through disrupting microtubule networks by tubulin down-regulation. In this study, we examined whether PMA increases apicularen A-induced cytotoxicity in HeLa cells. Cell viability was examined by thiazolyl blue tetrazolium (MTT) assays. To investigate apoptotic potential of apicularen A, DNA fragmentation assays were performed followed by extracting genomic DNA, and caspase-3 activity assays were performed by fluorescence assays using fluorogenic substrate. The cell cycle distribution induced by combination with PMA and apicularen A was examined by flow cytometry after staining with propidium iodide (PI). The expression levels of target proteins were measured by Western blotting analysis using specific antibodies, and α-tubulin mRNA levels were assessed by reverse transcription polymerase chain reaction (RT-PCR). To examine the effect of combination of PMA and apicularen A on the microtubule architecture, α-tubulin protein and nuclei were visualized by immunofluorescence staining using an anti-α-tubulin antibody and PI, respectively. We found that apicularen A induced caspase-dependent apoptosis in HeLa cells. PMA synergistically increased cytotoxicity and apoptotic sub-G1 population induced by apicularen A. These effects were completely blocked by the PKC inhibitors Ro31-8220 and Go6983, while caspase inhibition by Z-VAD-fmk did not prevent cytotoxicity. RNA interference using siRNA against PKCα, but not PKCβ and PKCγ, inhibited cytotoxicity induced by combination PMA and apicularen A. PMA increased the apicularen A-induced disruption of microtubule networks by further decreasing α- and β-tubulin protein levels in a PKC-dependent manner. These results suggest that the synergy between PMA and apicularen A is involved by

  13. Combination Effect of Ethylacetate Extract of Plectranthus amboinicus (Lour.) Spreng. with Doxorubicin Againts HeLa Cell Lines

    Hasibuan, Poppy Anjelisa Z.; Rosidah

    2016-01-01

    The aims of the study were to investigate the growth-inhibiting mediating effect of Plectranthus amboinicus (Lour.) Spreng. ethylacetate extract (PAE) in combination therapy with doxorubicin againts HeLa cell lines, to analyzed the apoptotic induction and expression of cyclin D1, Bcl2 and COX-2 (cyclooxigenase-2) proteins of HeLa cell lines after treatment of PAE. The cytotoxicity effects were determined by using MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromi...

  14. Suppression in vivo of human papillomavirus type 18 E6-E7 gene expression in nontumorigenic HeLa X fibroblast hybrid cells.

    Bosch, F X; Schwarz, E; Boukamp, P; Fusenig, N E; Bartsch, D; zur Hausen, H

    1990-01-01

    The E6 and E7 genes of the cancer-associated human papillomavirus (HPV) types 16 (HPV16) and 18 (HPV18) can induce cell immortalization in vitro in normal human keratinocytes. This, however, is not associated with tumorigenicity in vivo. On the other hand, tumorigenicity of HPV18-positive HeLa cervical carcinoma cells can be suppressed by fusion of HeLa cells with normal human keratinocytes or fibroblasts. We have addressed the question of whether suppression of tumorigenicity in HeLa x fibroblast hybrid cells might be due to a reduced ability of these cells to express the HPV18 E6-E7 genes in vivo. Nontumorigenic hybrid cells and tumorigenic hybrid segregants were transplanted as organotypical cultures or injected subcutaneously into immunocompromised mice and were analyzed for HPV18 E6-E7 gene expression by RNA-RNA in situ hybridization. The tumorigenic hybrid cells showed a continuous and invasive growth that was associated with high levels of HPV18 E6-E7 mRNAs at all time points examined. In contrast, the nontumorigenic hybrid cells stopped cell proliferation approximately 3 days after transplantation. At this time they expressed the E6-E7 genes at low levels, whereas at day 2 high expression levels were observed. However, the mRNA levels of the cytoskeletal genes beta-actin and vimentin remained high for at least 14 days, demonstrating that inhibition of growth and of HPV18 E6-E7 gene expression was not due to cell death. These results suggest that growth inhibition of the nontumorigenic HeLa x fibroblast hybrid cells in vivo might be caused by suppression of HPV18 E6-E7 gene expression and are compatible with the idea of an intracellular surveillance mechanism for HPV gene expression existing in nontumorigenic cells. Images PMID:2168962

  15. Apoptosis of HeLa cells induced by a new targeting photosensitizer-based PDT via a mitochondrial pathway and ER stress

    Li D

    2015-04-01

    Full Text Available Donghong Li,1 Lei Li,2 Pengxi Li,1 Yi Li,3 Xiangyun Chen1 1State Key Laboratory of Trauma, Burn and Combined Injury, The Second Department of Research Institute of Surgery, 2The First Department of Research Institute of Surgery, 3Cancer Center, Daping Hospital, Third Military Medical University, Chongqing, People’s Republic of China Abstract: Photodynamic therapy (PDT is emerging as a viable treatment for many cancers. To decrease the cutaneous photosensitivity induced by PDT, many attempts have been made to search for a targeting photosensitizer; however, few reports describe the molecular mechanism of PDT mediated by this type of targeting photosensitizer. The present study aimed to investigate the molecular mechanism of PDT induced by a new targeting photosensitizer (PS I, reported previously by us, on HeLa cells. Apoptosis is the primary mode of HeLa cell death in our system, and apoptosis occurs in a manner dependent on concentration, irradiation dose, and drug–light intervals. After endocytosis mediated by the folate receptor, PS I was primarily localized to the mitochondria and the endoplasmic reticulum (ER of HeLa cells. PS I PDT resulted in rapid increases in intracellular reactive oxygen species (ROS production and Ca2+ concentration, both of which reached a peak nearly simultaneously at 15 minutes, followed by the loss of mitochondrial membrane potential at 30 minutes, release of cytochrome c from mitochondria into the cytoplasm, downregulation of Bcl-2 expression, and upregulation of Bax expression. Meanwhile, activation of caspase-3, -9, and -12, as well as induction of C/EBP homologous protein (CHOP and glucose-regulated protein (GRP78, in HeLa cells after PS I PDT was also detected. These results suggest that apoptosis of HeLa cells induced by PS I PDT is not only triggered by ROS but is also regulated by Ca2+ overload. Mitochondria and the ER serve as the subcellular targets of PS I PDT, the effective activation of which

  16. siRNA-mediated silencing of Cockayne Cyndrome group B gene potentiates radiation-induced apoptosis and antiproliferative effect in HeLa cells

    LIU Feng; YU Zi-jian; SUI Jian-li; BAI Bei; ZHOU Ping-kun

    2006-01-01

    Background Cockayne syndrome (CS) is a rare human genetic disorder characterized by increased UV sensitivity, developmental abnormalities and premature aging. Cells isolated from individuals with CS have a defect in transcription-coupled DNA repair. Despite the repair defect, there is no any increased risk of spontaneous or UV-induced cancer for CS individuals. The strategy of RNA interfering was used here to explore the potential radiosensitizing and anticancer activity of targeting CS group B (CSB) gene.Methods The vectors encoding CSB-specific siRNAs were constructed by inserting duplex siRNA encoding oligonucleotides into the plasmid psilencer TM 3.1. The cell lines expressing the CSB-siRNA were generated from HeLa cells transfected with the above vectors. Colony-forming ability was used to assay cell survival. Cell cycle was analyzed by FACScan flow cytometry. The apoptosis was measured by detecting the accumulation of sub-G1 population as well as by fluorescence staining assay. Reverse transcriptase polymerase chain reaction (RT-PCR)was used to semi-quantify mRNA expression. Protein level was detected by Western blotting analysis.Results Two constructs encoding CSB-specific siRNA were generated, both of them resulted in remarkable suppression on CSB expression in HeLa cells, and led to an increased sensitivity to γ-ray and UV light.siRNA-mediated silencing of CSB decreased cell proliferation rate, increased spontaneous apoptosis as well as the occurrence of UV- or cisplatin-induced apoptosis by 2 to 3.5 fold. A significant S phase blockage and a remarkable reduction of G1 population were induced in control HeLa cells at 18 hours after being exposed to 10J/m2 of UV light. The S phase blockage was also observed in UV-irradiated CSB-siRNA transfected HeLa cells,but the extent of increased S phase population was lower than that in the UV-irradiated control cells. No or a relative weak reduction on G1 phase population was observed in UV-irradiated CSB

  17. Immunotherapy: rAAV2 expressing interleukin-15 inhibits HeLa cell tumor growth in mice

    Hung Yu-Ting

    2009-05-01

    Full Text Available Abstract Human interleukin-15 (hIL15 has anti-tumor activities, but it is not convenient for tumor treatment because of its short half-life. A gene therapy for mouse lung cancer using an adenovirus vector expressing IL15 has been reported. However, adenovirus vector-mediated gene therapy can provoke cellular toxicity and inflammatory reactions. The recombinant adenovirus-associated vector 2 (rAAV2 is safer due to minimal cellular toxicity and immune response. In order to demonstrate that gene therapy can be used safely and successfully for human cancer treatment, the rAAV2 expressing hIL15 gene (rAAV2-hIL15 is applied for human cervical cancer, HeLa cell, in this study. This study successfully demonstrates that rAAV2-hIL15 can express IL15 with bioactivities in vitro and in vivo. In conclusion, our studies show that human cervical cancers are inhibited on animal model with rAAV2-hIL15 treatment and provide a safer and important reference for human cancer gene therapy.

  18. Spontaneous and radiation induced cell death in HeLa S3 human carcinoma

    Radiation biologists have classified radiation-induced cell death based on cell proliferative capacity to either mitotic or interphase death. Cytologists have revealed two morphologically and biochemically diverse forms of cell death, apoptosis and necrosis. While the knowledge of the former is already well exploited by radiologists, cell susceptibility to apoptosis and necrosis is still under investigation. We studied characteristics of spontaneous cell death, and dose dependence and time course of radiation-induced cell death of human uterine cervix epitheloid carcinoma HeLaS3 in culture. Cells were irradiated with 2-40 Gy of γ-rays. The effect on growth, viability, morphology and genomic DNA structure were followed 24-72 h after irradiation. Cell viability was evaluated by trypan-blue exclusion assay and cell morphology by in situ DNA staining with propidium iodide. Cell genomic DNA fragmentation pattern was determined by electrophoresis on 2% agarose gels. At all cell densities 25-35% cells were PI positive and their DNA was fragmented to a high molecular size (≥20 kbp), but the internucleosomal ladder was not observed. A significant decrease in viability to 33% was observed 72 h post 40 Gy irradiation. It corresponded to 55% of PI positive cells. A smear of smaller DNA fragments (0.1-1 kbp), 24 h after 10-20 Gy irradiation was considered as proof that the dominant form of radiation-induced cell death was necrosis. It was concluded that the dominant form of radiation-induced cell death in HeLaS3 population was necrosis and the radiation dose which caused 50% of cell death after 72 h (termed ND50) was between 30-40 Gy. (author)

  19. Electroporation of micro-droplet encapsulated HeLa cells in oil phase

    Xiao, Kang

    2010-08-27

    Electroporation (EP) is a method widely used to introduce foreign genes, drugs or dyes into cells by permeabilizing the plasma membrane with an external electric field. A variety of microfluidic EP devices have been reported so far. However, further integration of prior and posterior EP processes turns out to be very complicated, mainly due to the difficulty of developing an efficient method for precise manipulation of cells in microfluidics. In this study, by means of a T-junction structure within a delicate microfluidic device, we encapsulated HeLa cells in micro-droplet of poration medium in oil phase before EP, which has two advantages: (i) precise control of cell-encapsulating droplets in oil phase is much easier than the control of cell populations or individuals in aqueous buffers; (ii) this can minimize the electrochemical reactions on the electrodes. Finally, we successfully introduced fluorescent dyes into the micro-droplet encapsulated HeLa cells in oil phase. Our results reflected a novel way to realize the integrated biomicrofluidic system for EP. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.

  20. FePt nanoparticles as a potential X-ray activated chemotherapy agent for HeLa cells

    Zheng Y

    2015-10-01

    Full Text Available Yanhong Zheng,1 Yunlan Tang,2 Zhirong Bao,1 Hui Wang,1 Feng Ren,1 Mingxiong Guo,2 Hong Quan,1 Changzhong Jiang11Key Laboratory of Artificial Micro- and Nano-structures of the Ministry of Education and Center for Electronic Microscopy and Department of Physics, Wuhan University; 2College of Life Sciences, Wuhan University, Wuhan, People’s Republic of ChinaAbstract: Nanomaterials have an advantage in “personalized” therapy, which is the ultimate goal of tumor treatment. In order to investigate the potential ability of FePt nanoparticles (NPs in the diagnosis and chemoradiotherapy treatment of malignant tumors, superparamagnetic, monodispersed FePt (~3 nm alloy NPs were synthesized, using cysteamine as a capping agent. The NPs were characterized by means of X-ray diffraction; transmission electron microscopy, Physical Property Measurement System, and Fourier transform infrared spectroscopy. The cytotoxicity of FePt NPs on Vero cells was assessed using an MTT assay, and tumor cell proliferation inhibited by individual FePt NPs and FePt NPs combined with X-ray beams were also collected using MTT assays; HeLa human cancer cell lines were used as in vitro models. Further confirmation of the combined effect of FePt NPs and X-rays was verified using HeLa cells, after which, the cellular uptake of FePt NPs was captured by transmission electron microscopy. The results indicated that the growth of HeLa cells was significantly inhibited by FePt NPs in a concentration-dependent manner, and the growth was significantly more inhibited by FePt NPs combined with a series of X-ray beam doses; the individual NPs did not display any remarkable cytotoxicity on Vero cells at a concentration <250 µg/mL. Meanwhile, the FePt NPs showed negative/positive contrast enhancement for MRI/CT molecule imaging at the end of the study. Therefore, the combined results implied that FePt NPs might potentially serve as a promising nanoprobe for the integration of tumor

  1. Apoptosis and necrosis of HeLa cells in response to low-energy ion radiation

    2006-01-01

    The aim of this study was to investigate the damage of low-energy ions to HeLa cells and to particularly examine the relationship between apoptotic and necrotic effects and the low-energy ion radiation. In this study, HeLa cells were irradiated by low-energy ions (30keV N+) at different doses. The level of apoptosis and necrosis was evaluated using flow cytometry. Since vacuum is required for experimental low-energy ion generation and irradiation, the cells must be placed in vacuum. Mineral oil was used to prevent dehydration of cells. The results show that the apoptotic rate reached 7.09% when the ion implantation dose was 1 × 1015 ions/cm2; and when the cells were exposed to and implanted at 2 × 1015 ions/cm2 dose, the apoptotic rate was higher than that at 1 × 1015 ions/cm2, and the necrotic rate was 15.63%. In addition, the survival fraction gradually decreased with the increase in implantation dose. Some relationships have been found between the radiation-induced apoptosis and the incubated time after irradiation.

  2. Study on the characteristics of cell-cycle perturbation in hela cell exposed to continuous β irradiation of 32P

    In an attempt to understand radiobiological basis for targeted radiotherapy in oncology, the cell cycle perturbations have studied in Hela cell lines after exposed to different doses and dose-rate of 32P radiation. Asynchronous Hela cells, cultured in vitro, were exposed to β radiation from radioactive filter papers (absorbed 32P) which were put close under culture plate of growing monolayer of Hela cells. The characteristic radiation response to different dose, dose-rate and radiation time was evaluated through cell cycle perturbation studied by flow cytometry. Cell cycle status showed G2 phase blockage in a way of dose dependence, a plateau of G2 block can be recognized at about 24h. Interestingly, the G2 phase declined even though the accumulated doses increased as the time of radiation prolonged. This result suggested that the cell cycle progress could not be inhibited completely when exposed to continuous radiation, rather it seems to be controlled somehow by the nature of cell cycle itself for a certain cell line. G2 blockage, one of the major changes caused by β radiation, is dose-dependent, but the time reaching the plateau of G2 phase blockage is most likely related with the intrinsic nature of cell cycle

  3. Structures of nuclear phosphoproteins characteristic of rapidly growing HeLa cells

    To study characteristic events of phosphorylation in cell growth, phosphoproteins were labeled with [32P]-phosphate at mid-logarithmic phase of HeLa cell proliferation. Among a number of nuclear phosphoproteins isolated, three characteristic classes of most highly labeled phosphoproteins were identified by DEAE-column chromatography (0.2-0.25 M NaCl gradient, pH 6.0), followed by 7.5% SDS polyacrylamide gel electrophoresis. Chemical characterization of their structures showed that they contained three different forms of post-translational modifications: Class I with phosphoserine, Class II with phosphoserine and oligonulceotides (5-10 nucleotides long), and Class III with phosphoserine, 5'-GMP and poly(ADP-ribose). Class I is represented by nucleolar C-23. Class II is represented by nucleolar 125 kDa and nucleoplasmic 50 kDa with GC rich sequences (G = 30%, C = 40%) and 5'-linking pCp. Class III is represented by nucleoplasmic poly(ADP-ribose) proteins (18 different species, MW ranges 30 kDa-200 kDa) with branched poly(ADP-ribose) longer than tRNA. When HeLa cells were labeled at non-mid-logarithmic phase, labeling of these classes were 4 fold less efficient, indicating their functional importance in cell proliferation

  4. Exposure to TiO2 nanoparticles increases Staphylococcusaureusinfection of HeLa cells

    Xu, Yan; Wei, Ming-Tzo; Walker, Stephen. G.; Wang, Hong Zhan; Gondon, Chris; Brink, Peter; Guterman, Shoshana; Zawacki, Emma; Applebaum, Eliana; Rafailovich, Miriam; Ou-Yang, H. Daniel; Mironava, Tatsiana

    TiO2 is one of the most common nanoparticles in industry from food additives to energy generation. Even though TiO2 is also used as an anti-bacterial agent in combination with UV, we found that, in the absence of UV, exposure of HeLa cells to TiO2 nanoparticles largely increased their risk of bacterial invasion. HeLa cells cultured with low dosage rutile and anatase TiO2 nanoparticles (0.1 mg/ml) for 24 hrs prior to exposure to bacteria had 350% and 250% respectively more bacteria infected per cell. The increase was attributed to increased LDH leakage, and changes in the mechanical response of the cell membrane. On the other hand, macrophages exposed to TiO2 particles ingested 40% fewer bacteria, further increasing the risk of infection. In combination, these two factors raise serious concerns regarding the impact of exposure to TiO2 nanoparticles on the ability of organisms to resist bacterial infection.

  5. Fractionation of HeLa cell nuclear extracts reveals minor small nuclear ribonucleoprotein particles.

    Krämer, A

    1987-01-01

    Upon chromatographic fractionation of HeLa cell nuclear extracts, small RNAs of 145 and 66/65 nucleotides, respectively, were detected that are distinct from the abundant small RNAs present in the extract. These RNAs are precipitated by antibodies directed against the trimethylguanosine cap structure, characteristic for small nuclear RNAs (snRNAs) of the U type. The RNAs of 145 and 66/65 nucleotides appear to be associated with at least one of the proteins common to the major small nuclear ri...

  6. Expression of the papillomavirus E2 protein in HeLa cells leads to apoptosis.

    Desaintes, C.; Demeret, C; Goyat, S; Yaniv, M; Thierry, F

    1997-01-01

    The papillomavirus E2 protein plays a central role in the viral life cycle as it regulates both transcription and replication of the viral genome. In this study, we showed that transient expression of bovine papillomavirus type 1 or human papillomavirus type 18 (HPV18) E2 proteins in HeLa cells activated the transcriptional activity of p53 through at least two pathways. The first one involved the binding of E2 to its recognition elements located in the integrated viral P105 promoter. E2 bindi...

  7. Flexible synthesis of isomeric pyranoindolones and evaluation of cytotoxicity towards HeLa cells

    J C JEYAVEERAN; CHANDRASEKAR PRAVEEN; Y ARUN; A A M PRINCE; P T PERUMAL

    2016-05-01

    A hybrid pharmacophore approach for the synthesis of isomeric pyranoindolones was achievedby employing gold(III) chloride-catalyzed cycloisomerization of alkyne-tethered indole carboxylic acids ingood to excellent yield. All the synthesized compounds were evaluated for their tumor cell growth inhibitoryactivity against human cervix adenocarcinoma (HeLa) which revealed that three compounds exhibited activitycomparable with the standard cis-platin $(IC_{50} = 0.μM)$. Molecular docking of all the compounds in Vaccinia H1-Related (VHR) Phosphatase receptor also supported that compound 7d as the most active with a free energyof binding as - 8.27 kcal/mol.

  8. Increase of UV-resistance in xeroderma pigmentosum cells by human HeLaS3 DNA transfection

    The DNA-mediated gene transfer had been carried out by both calcium phosphate coprecipitation and electroporation method. The cellular DNA and DNA fragments from human cervical carcinoma HeLaS3 cells were introduced with PSV2Neo DNA into XP20S (SV40) cells. The transfectants were picked up after twice selections by G418 and 3 J/m2 UV-irradiation. The results showed that cellular DNA and Bg1 I, Xho I digested DNA fragments from HeLaS3 could correct the deficiency of excision repair gene in XP cells, and cause the recipient cells resistant to UV irradiation. The second transfection experiment confirmed that HeLaS3 DNA were really integrated into XP cell chromosome and stably expressed within the cell genome

  9. Effect of the interaction between M-CSF and MCM7 on DNA replication in HeLa cells

    ZHANG Meng-xia; WU Hai-yan; TU Jian; ZHANG Xiao-hong; LE Xiao-yong; TANG Sheng-song

    2008-01-01

    Objective To explore the effect of the interaction between microphage colony-stimulating factor (M-CSF) and minichromosome maintenance protein-7(Mcm7) on DNA replication in HeLa cells. Methods pCMV/nuc/mye, pCMV/nuc/GFP and pCMV/nuc/M-CSF vector were stably transfected into HeLa cells by Lipofectarnine, respectively. After screening with G418, the expression and localization of M-CSF in HeLa cells were verified by RT-PCR, Western blot and immunofluoreseence staining. The statue and interaction between intracellular M-CSF and Mcm7 in HeLa cells was analyzed by co-immunoprecipitation. The effect of the interaction between M-CSF and Mcm7 on DNA replication was analyzed by a mammalian cell or cell-free DNA replication system in vitro. Results The results indicated that the M-CSF-transfected HeLa cells stably express both M-CSF mRNA and protein, and that M-CSF protein is located to the nuclei of HeLa cells mentioned above. To further analyze the status and interaction between intracellular M-CSF and Mcm7, the Mcm7 from HeLa cells was precipitated with anti-Mcm7 antibody and followed by Protein A/G PLUS agarose. The precipitation was blotted with anti-M-CSF monoclonal antibody. The results show that M-CSF was coprecipitated with Mcm7, so intracellular M-CSF existed in Mcm7-bound state. The DNA replication experiments reveal that a higher percentage of the replicating nuclei is present either in unsyn-chronized or in both synchronized G1 and S phase M-CSF-transfected HeLa cells, compared with both pCMV/nuc-transfeeted and un-transfected HeLa cells, which suggests that interaction between M-CSF and Mcm7 promote both the initiation and elongation of DNA replication. Conclusions M-CSF directly interacts with McmT. The interaction between M-CSF and Mcm7 promotes both the initiation and elongation of DNA replication.

  10. Yeast CUP1 protects HeLa cells against copper-induced stress

    As an essential trace element, copper can be toxic in mammalian cells when present in excess. Metallothioneins (MTs) are small, cysteine-rich proteins that avidly bind copper and thus play an important role in detoxification. YeastCUP1 is a member of the MT gene family. The aim of this study was to determine whether yeast CUP1 could bind copper effectively and protect cells against copper stress. In this study,CUP1 expression was determined by quantitative real-time PCR, and copper content was detected by inductively coupled plasma mass spectrometry. Production of intracellular reactive oxygen species (ROS) was evaluated using the 2',7'-dichlorofluorescein-diacetate (DCFH-DA) assay. Cellular viability was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and the cell cycle distribution of CUP1 was analyzed by fluorescence-activated cell sorting. The data indicated that overexpression of yeast CUP1 in HeLa cells played a protective role against copper-induced stress, leading to increased cellular viability (P<0.05) and decreased ROS production (P<0.05). It was also observed that overexpression of yeast CUP1 reduced the percentage of G1 cells and increased the percentage of S cells, which suggested that it contributed to cell viability. We found that overexpression of yeast CUP1 protected HeLa cells against copper stress. These results offer useful data to elucidate the mechanism of the MT gene on copper metabolism in mammalian cells

  11. Yeast CUP1 protects HeLa cells against copper-induced stress

    Xie, X.X. [Department of Animal Sciences, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai (China); Shanghai Key Laboratory of Veterinary Biotechnology, Shanghai (China); College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou (China); Ma, Y.F.; Wang, Q.S.; Chen, Z.L.; Liao, R.R.; Pan, Y.C. [Department of Animal Sciences, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai (China); Shanghai Key Laboratory of Veterinary Biotechnology, Shanghai (China)

    2015-06-12

    As an essential trace element, copper can be toxic in mammalian cells when present in excess. Metallothioneins (MTs) are small, cysteine-rich proteins that avidly bind copper and thus play an important role in detoxification. YeastCUP1 is a member of the MT gene family. The aim of this study was to determine whether yeast CUP1 could bind copper effectively and protect cells against copper stress. In this study,CUP1 expression was determined by quantitative real-time PCR, and copper content was detected by inductively coupled plasma mass spectrometry. Production of intracellular reactive oxygen species (ROS) was evaluated using the 2',7'-dichlorofluorescein-diacetate (DCFH-DA) assay. Cellular viability was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and the cell cycle distribution of CUP1 was analyzed by fluorescence-activated cell sorting. The data indicated that overexpression of yeast CUP1 in HeLa cells played a protective role against copper-induced stress, leading to increased cellular viability (P<0.05) and decreased ROS production (P<0.05). It was also observed that overexpression of yeast CUP1 reduced the percentage of G1 cells and increased the percentage of S cells, which suggested that it contributed to cell viability. We found that overexpression of yeast CUP1 protected HeLa cells against copper stress. These results offer useful data to elucidate the mechanism of the MT gene on copper metabolism in mammalian cells.

  12. Role of Endonuclease G in Exogenous DNA Stability in HeLa Cells.

    Misic, V; El-Mogy, M; Haj-Ahmad, Y

    2016-02-01

    Endonuclease G (EndoG) is a well-conserved mitochondrial-nuclear nuclease with dual lethal and vital roles in the cell. The aim of our study was to examine whether EndoG exerts its nuclease activity on exogenous DNA substrates such as plasmid DNA (pDNA), considering their importance in gene therapy applications. The effects of EndoG knockdown on pDNA stability and levels of encoded reporter gene expression were evaluated in the cervical carcinoma HeLa cells. Transfection of pDNA vectors encoding short-hairpin RNAs (shRNAs) reduced levels of EndoG mRNA in HeLa cells. In physiological circumstances, EndoG knockdown did not have an effect on the stability of pDNA or the levels of encoded transgene expression as measured over a four-day time course. However, when endogenous expression of EndoG was induced by an extrinsic stimulus, targeting of EndoG by shRNA improved the perceived stability and transgene expression of pDNA vectors. Therefore, EndoG is not a mediator of exogenous DNA clearance, but in non-physiological circumstances, it may nonspecifically cleave intracellular DNA regardless of its origin. These findings make it unlikely that targeting of EndoG is a viable strategy for improving the duration and level of transgene expression from nonviral DNA vectors in gene therapy efforts. PMID:27260396

  13. Suppressive effect on HeLa cells proliferation by phenothiazine derivatives alone and combining with ionizing radiation

    Objective: To examine the antiproliferative effects of phenothiazine derivatives (PTZDs) alone on HeLa cells and in combination with ionizing radiation. Methods: MTT and colony-forming method were used to evaluate the proliferation activity and cellular radiosensitivity of HeLa cells. Results: We compared the antiproliferative effects of six phenothiazine derivatives, and found that the derivatives α-chloro-N-dimethylamine phenothiazine (PTZD2), α-triflumethyl-N-α(dimethylamine ethyl) phenothiazine (PTZD3) and α-chloro-N-(dimethylamine ethyl) phenothiazine (PTZD5) showed a significant antiproliferative effect at concentration of 10 μmol/L. HeLa cells proliferation was completely suppressed when treated with PTZDs of 40-50 μmol/L. PTZD2/PTZD3 and cobalt-60 gamma-irradiation showed synergistic suppressive effect on proliferation of HeLa cells. The enhancement ratios of 10 μmol/L PTZD3 combination with 2 Gy and 4 Gy irradiations were 3.5 and 1.8, respectively. The maximum synergistic suppressive effect was observed when cells administered with PTZD3 at 18 h before being irradiated. Conclusion: Phenothiazine derivatives show antiproliferations on HeLa cells, and differ in degrees. The synergistic anticancer effect could be obtained by combining phenothiazine derivatives with radiotherapy. (authors)

  14. The changes in telomerase activity and telomere length in HeLa cells undergoing apop- tosis induced by sodium butyrate

    2001-01-01

    The changes in telomerase activity and telomere length during apoptosis in HeLa cells as induced by sodium butyrate (SB) have been studied. After a 48 h SB treatment, HeLa cells demonstrated characteristic apoptotic hallmarks including chromatin condensation, formation of apoptotic bodies and DNA Laddering which were caused by the cleavage and degradation of DNA between nucleosomes. There were no significant changes in telomerase activity of apoptotic cells, while the telomere length shortened markedly. In the meanwhile, cells became more susceptible to apoptotic stimuli and telomere became more vulnerable to degradation after telomerase activity was inhibited. All the results suggest that the apoptosis induced by SB is closely related to telomere shortening, while telomerase enhances resistance of HeLa cells to apoptotic stimuli by protecting telomere.

  15. Newly synthesized bis-benzimidazole compound 8 induces apoptosis, autophagy and reactive oxygen species generation in HeLa cells.

    Chu, Naying; Yao, Guodong; Liu, Yuan; Cheng, Maosheng; Ikejima, Takashi

    2016-09-01

    Compound 8 (C8) is a newly synthesized bis-benzimidazole derivative and exerts significant anti-tumor activity in vitro. Previous studies demonstrated that C8 induced apoptosis and autophagy in human promyelocytic leukemia HL60 cells. However, cytotoxicity study on human peripheral blood mononuclear cells (hPBMC) showed that C8 exhibited less toxicity in normal cells. In this study, the molecular mechanism of C8 on human cervical carcinoma HeLa cells was investigated. The results showed that C8 inhibited the growth of HeLa cells and triggered both apoptotic and autophagic cell death. Subsequent experiment also indicated that reactive oxygen species (ROS) generation was induced in C8-treated HeLa cells. Since ROS scavenger decreased the ratio of apoptotic and autophagic cells, ROS generation contributed to C8-induced apoptosis and autophagy. Furthermore, inhibitors of apoptosis and autophagy also reduced ROS generation, respectively. Autophagy inhibition increased cell growth compared to C8-treated group and attenuated apoptotic cell death, indicating that C8-induced autophagy promoted apoptosis for cell death. However, the percentage of autophagic cells was enhanced when limiting apoptosis process. Taken together, C8 induced ROS-mediated apoptosis and autophagy in HeLa cells, autophagy promoted apoptosis but the former was antagonized by the latter. The data also gave us a new perspective on the anti-tumor effect of C8. PMID:27497983

  16. MicroRNA-mediated NBS1 Gene Silence and Its Effects on Telomerase Activation in Hela Cells

    CAO Sun-qiong; REN Chang-shan

    2008-01-01

    Objective:To research the silence of NBS1 after transfection microRNA expressing eukaryotic recombinants and the changes of telomerase activation in teiomerase-positive cell line Hela.Methods:According to the sequence of NBS1 mRNA,the NBS1 pre-microRNA was designed and synthesized,then cloned into the GFP reporter pcDNA6.2-GW/EmGFP-miR vector and transfected into Hela cells.The integrity of the insert fragment was verified through colony PCR and sequencing analysis.The NBS1 gene expression of NBS1 microRNA recombinants was detected by Real-Time PCR and western blot.Telomerase activity in Hela cells was assayed by TRAP-PCR-EB.Results:Sequences of insert fragment in microRNA expressing recombinants were correct.The NBS1 gene expression was decreased,and the telomerase activation of Hela cell reduced.Conclusion:NBS1 microRNA inhibits NBS1 gene expression,and depresses telomerase activation of Hela cells.This confirms that there is relevance between NBS1 gene and telomerase activity.

  17. PVA engineered microcapsules for targeted delivery of camptothecin to HeLa cells

    Galbiati, Alice; Rocca, Blasco Morozzo della; Tabolacci, Claudio; Beninati, Simone; Desideri, Alessandro [Dipartimento di Biologia, Universita di Roma Tor Vergata, Via della Ricerca Scientifica, 00133 Rome (Italy); Paradossi, Gaio, E-mail: paradossi@stc.uniroma2.it [Dipartimento di Scienze e Tecnologie Chimiche, Universita di Roma Tor Vergata, Via della Ricerca Scientifica, 00133 Rome (Italy)

    2011-12-01

    Capsular microvectors are an important tool in the recent research field of nanomedicine to address a drug cargo for the therapeutic treatment of several pathologies. In this study we describe how the product of the conjugation of the polysaccharide chitosan with folate can be used as a coating of poly (vinyl alcohol), PVA, based microcapsules for an efficient targeting of HeLa cells. The influence of the coating on the bioadhesive properties of the vector and on its cargo capacity was also considered using camptothecin as an anticancer drug model. The coating strategy was finalized to exploit the good chemical versatility of PVA, used to form the shell of the vector. This study is a follow up of an investigation activity aiming to show the potentialities of PVA-shelled microcapsules or microbubbles as injectable microdevices supporting a theranostic approach for different types of tumour. Highlights: {yields}Coating of PVA-shelled microcapsules with chitosan-folate. {yields} Selective bioadhesion of microcapsules to HeLa Cells. {yields} Effective loading and release of camptothecin. {yields} In vitro anti-proliferative action of camptothecin loaded microcapsules.

  18. Apoptosis induced by (di-isopropyloxyphoryl-Trp)2-Lys-OCH3 in K562 and HeLa cells

    Feng Liu; Shi-Ying Liu; Ping Xu; Zhen-Hua Xie; Guo-Ping Cai; Yu-Yang Jiang

    2008-03-01

    According to the method used in our laboratory, our group synthesized (DIPP-Trp)2-Lys-OCH3. It inhibited the proliferation of K562 and HeLa cells in a dose- and time-dependent manner with an IC50 of 15.12 and 42.23 M, respectively. (DIPP-Trp)2-Lys-OCH3 induced a dose-dependent increase of the G2/M cell population in K562 cells, and S cell population in HeLa cells; the sub-G0 population increased dramatically in both cell lines as seen by PI staining experiments using a FACS Calibur Flow cytometer (BeckmanCoulter, USA). Phosphatidylserine could significantly translocate to the surface of the membrane in (DIPP-Trp)2-Lys-OCH3-treated K562 and HeLa cells. The increase of an early apoptotic population was observed in a dose-dependent manner by both annexin-FITC and PI staining. It was concluded that (DIPP-Trp)2-Lys-OCH3 not only induced cells to enter into apoptosis, but also affected the progress of the cell cycle. It may have arrested the K562 and HeLa cells in the G2/M, S phases, respectively. The apoptotic pathway was pulsed at this point, resulting in the treated cells entering into programmed cell death. (DIPP-Trp)2-Lys-OCH3 is a potential anticancer drug that intervenes in the signalling pathway.

  19. Changes in the protein-synthesizing system of HeLa cells in culture in the presence of trace elements

    This paper studies the state of the protein-synthesizing system of HeLa cells in culture in the presence of certain trace elements. The cytopathic action of zinc, nickel, cobalt, cadmium, and fluorine was studied in the presence of maximal allowable concentrations adopted for liquid media. Thirty minutes before the end of incubation with the elements to be studied, (H 3)-uridine or (H 3)-leucine was added to the cultures of HeLa cells. The autoradiographic data showed that variation in the integral parameters of cell function as the level of synthesis of total fast-labeled RNA and total protein in fact do take place during incubation of the HeLa cell culture with trace elements

  20. Frequency domain analysis of membrane capacitance of cultured cells (HeLa and myeloma) using the micropipette technique.

    Asami, K; Takahashi, Y.; Takashima, S.

    1990-01-01

    The membrane capacitance and conductance of cultured cells (HeLa and mouse myeloma) are investigated using the micropipette method. Mean values of the membrane capacities were found to be 1.9 microF/cm2 for HeLa cells and 1.0 microF/cm2 for myeloma cells. These values are in agreement with those obtained using the suspension method. Whereas the suspension method is unable to provide the information on membrane conductance, the micropipette method is able to measure even an extremely small mem...

  1. Combination Effect Of N-Hexane Extract Of Plectranthus Amboinicus (LOUR.) Spreng. With Doxorubicin Againts HeLa Cell Lines

    Rosidah; Hasibuan, Poppy Anjelisa Z.

    2016-01-01

    The study was aimed to investigate the growth inhibiting effect of Plectranthus amboinicus, (Lour.) Spreng n-hexane extract (PAN) in combination with doxorubicin againts HeLa cell Lines, to observe the apoptotic induction and immunocytochemistry of HeLa cell Lines on cyclin D1, Bcl-2, and Cox-2 after treatment of PAN. The percentage viability of the cell were carried out by using MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide] assay. The effect of apoptosis...

  2. Cytotoxic Activity of Three South Sulawesi Medicinal Plant Extracts Used in the Treatment of HeLa Cell Line: Jati Putih (Gmelina arborea Roxb., Jati Belanda (Guazuma ulmifolia Lamk. and Lakkalakka (Curculigo orchioides Gaerth

    LUKMAN M

    2014-09-01

    Full Text Available Gmelina arborea Roxb, Guazuma ulmifolia Lamk and Curculigo orchioides Gaerth, the three plants frequently used in South Sulawesi for the treatment of cancerous diseases, have been selected to examine their action in cervical epithelial carcinoma. These extracts were assessed using HeLa cell cancer (Human cervix cancer and doxorubicin was used as the positive control. Data are presented as the dose that inhibited 50% control growth (IC50. Cytotoxic activity was measured using MTT colorimetric assay. Dose-dependent studies revealed IC50 of 113.61±0.12 μg/mL, 174.90±1.22 μg/mL and 126.05±2.43 μg/mL for eGA, eGU and eCO on HeLa cell cancer, respectively and correlated with treatment of cancer.

  3. The role of glutathione in the radiosensitive effect induced by treating HeLa cells with sanazole

    Objective: To investigate radiosensitive effect of sanazole on HeLa cells and its relationship with glutathione (GSH). Methods: The anoxia model was made by inflow of nitrogen gas. The survival rate of HeLa cells was observed with method of colony formation after treatment with sanazole and 60Co γ irradiation. Radiosensitive effect was evaluated through measurement of sensitizing enhancement ratio (SER) resulted from single-target multi-hit model. The GSH content in these HeLa cells was determined by the tetra-oxypyrimidine UV-spectrophotometer method to explore the mechanism of radiosensitive effect. Results: SER was more than 1.4. The concentration of GSH decreased significantly with increasing concentration of sensitizer and dose of radiation, especially under anoxia condition. Conclusions: Sanazole has significant radiosensitive effect and decrease in GSH content resulted from combination with 60Co γ irradiation may be one of its radiosensitive mechanisms

  4. A Novel Photosensitizer 3¹,13¹-phenylhydrazine -Mppa (BPHM) and Its in Vitro Photodynamic Therapy against HeLa Cells.

    Li, Wenting; Tan, Guanghui; Cheng, Jianjun; Zhao, Lishuang; Wang, Zhiqiang; Jin, Yingxue

    2016-01-01

    Photodynamic therapy (PDT) has attracted widespread attention due to its potential in the treatment of various cancers. Porphyrinic pyropheophorbide-a (PPa) has been shown to be a potent photosensitizer in PDT experiments. In this paper, a C-3¹,13¹ bisphenylhydrazone modified methyl pyropheophorbide-a (BPHM) was designed and synthesized with the consideration that phenylhydrazone structure may extend absorption wavelength of methyl pyro-pheophorbide-a (Mppa), and make the photosensitizer potential in deep tumor treatment. The synthesis, spectral properties and in vitro photodynamic therapy (PDT) against human HeLa cervical cancer cell line was studied. Methyl thiazolyl tetrazolium (MTT) assay showed the title compound could achieve strong inhibition of cervical cancer cell viability under visible light (675 nm, 25 J/cm²). Cell uptake experiments were performed on HeLa cells. Morphological changes were examined and analyzed by fluorescent inverted microscope. In addition, the mechanism of the photochemical processes of PDT was investigated, which showed that the formation of singlet oxygen after treatment with PDT played a moderate important role. PMID:27136527

  5. Suppression of postmitochondrial signaling and delayed response to UV-induced nuclear apoptosis in HeLa cells

    Sasai, Kaori; Yajima, Hirohiko; Suzuki, Fumio [Hiroshima Univ., (Japan). Research Inst. for Radiation Biology and Medicine

    2002-03-01

    Activation of postmitochondrial pathways by UV irradiation was examined using mouse lymphoma 3SB and human leukemic Jurkat cells and two human carcinoma cell lines (HeLa and MCF-7). Exposure of 3SB and Jurkat cells resulted in large amounts of cytochrome c and apoptosis-inducing factor (AIF) being released into the cytosol, and a clear laddering pattern of DNA fragments was observed within 3 h of incubation after irradiation. Simultaneously, activation of caspase-9 and its downstream caspases was detected. HeLa and MCF-7 cells also showed extensive release of mitochondrial factors and caspase-9 activation at 4 to 6 h after exposure, but apoptotic nuclear changes appeared much later. Compared with 3SB and Jurkat cells, these carcinoma cell lines exhibited reduced activation of caspase-9-like proteolytic activity by UV radiation, and levels of caspase-3-like activity in HeLa cells were extremely low, similar to those in caspase-3-deficient MCF-7 cells. These results suggest that the delayed response to UV-induced nuclear apoptosis in HeLa cells is due to a reduced activation of the caspase cascade downstream of cytochrome c release and suppression of caspase-3 activity. (author)

  6. Treatment of HeLa cells with Giloe (Tinospora cordifolia meirs) increases the radiosensitivity by increasing DNA damage

    Radiotherapy is an important treatment modality and screening of phytoceuticals may enhance the clinical outcome of radiotherapy, therefore radiosensitizing activity of various guduchi (Tinospora cordifolia) extracts was studied in HeLa cells. Chromosomal aberrations were scored in HeLa cells treated with 10 μg/ml of aqueous, methanol, or methylene chloride guduchi extracts or doxorubicin before exposure to 0, 0.5, 1, 2 or 3 Gy of γ-radiation at 12, 24, 36 or 48 h post-irradiation. Irradiation of HeLa cells caused a dose dependent rise in the chromatid breaks, chromosome breaks, dicentric, centric rings, acentric fragments and total aberrations at all post-irradiation times and the dose response was linear quadratic for all types of aberrations scored. Chromatid breaks increased up to 12 h post-irradiation and declined steadily up to 48 h post-irradiation, whereas chromosome breaks, dicentric, acentric fragments and total aberrations elevated up to 24 h post-irradiation and declined thereafter. However, centric rings continued to rise steadily up to 48 h post-irradiation. Treatment of HeLa cells with aqueous, methanol or methylene chloride guduchi extract or doxorubicin before irradiation significantly enhanced various types of chromosomal aberrations and a maximum rise in the chromosome aberrations was observed in the HeLa cells treated with methylene chloride extract before irradiation when compared to other groups. Various guduchi extracts enhanced the effect of radiation in HeLa cells by increasing the molecular damage to cellular genome and their effect was similar to or even greater than doxorubicin (positive control) pretreatment, depending on the type of guduchi extract used. (author)

  7. Effects of a tumor promoter on phospholipid metabolism in HeLa cells

    The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) caused a marked stimulation of inorganic [32P]orthophosphate incorporation into HeLa-cell phosphatidylcholine (PC), phosphatidylethanolamine (PE), and lysophosphatidylethanolamine. The increased incorporation of inorganic [32P]orthophosphate into PE and lysophosphatidylethanolamine in the presence of TPA was not associated with an increase in PE synthesis as detected by the incorporation of [3H]serine or [3H]ethanolamine. The PC-specific exchange protein from beef liver was used to insert PC labeled with [3H]choline, inorganic [32P]orthophosphate, or [14C]arachidonic acid plus [3H]palmitic acid into the outer monolayer of intact HeLa cell membranes. Radioactivity from the latter two compounds was rapidly incorporated into PE and lysophosphatidylethanolamine; the incorporation was stimulated by TPA. It was concluded that TPA stimulated the formation of PE by base exchange between ethanolamine and PC

  8. Heat Inactivation of Garlic (Allium sativum) Extract Abrogates Growth Inhibition of HeLa Cells.

    Chintapalli, Renuka; Murray, Matthew J J; Murray, James T

    2016-07-01

    The potential anticancer properties of garlic (Allium sativum) may depend on the method of preparation and its storage. Storage of garlic has not been thoroughly investigated to determine whether anticancer properties are retained. Garlic was prepared and processed to mimic normal options for storage and preparation for consumption. Cytotoxicity was determined by crystal violet assay and mechanisms of cytotoxicity were established by microscopy, SDS-PAGE, and Western immunoblotting. Significant (P garlic. Depending on the method of storage, garlic extract induced either type I or type II programmed cell death, detectable by caspase 9 cleavage, or Poly (adenosine diphosphate-ribose) polymerase (PARP) cleavage and LC3-II accumulation, respectively. The conflicting literature on the anticancer properties of garlic may be explained by differences in processing and storage. This study has highlighted that the potency of the antiproliferative properties of cooked garlic, compared to the uncooked form, is diminished in HeLa cells. PMID:27176674

  9. Evaluation of hela cell lineage response to β radiation from Holmium-166 embedded in ceramic seeds

    Eduardo Sarmento Valente

    2011-10-01

    Full Text Available This work studied the effects of β radiation of Ho-166 embedded in ceramic seeds on HeLa cells. Methodology consisted in the production of ceramic seeds with holmium-165 by sol-gel route. Chemical and physical characterizations of the seeds were performed. Subsequently, nuclear characterization was performed by gamma spectrometry. Experimental and theoretical activities were defined and initial dose rate were evaluated by MIRD (Medical Internal Radiation Dose Committee methodology. The seeds were placed in confluent culture flasks and remained for six radionuclide half-lives. Biological results were represented by a clean 6 mm diameter area around the seed where the tumour cells were killed. The initial dose rate was 15.5 Gy. h-1. The maximum absorbed dose was 591.3 Gy. The features of the Ho-166 seeds suggested that such ceramic seeds were suitable for high dose rate brachytherapy.

  10. The influence of γ-irradiation in different modes on the isolated and united HeLa cells in culture

    It was shown that isolated and united HeLa cells in culture responded to single irradiation and repeated low dose irradiation by different ways. The effect on radiosensitivity was revealed in united cells only. 7 refs., 2 figs., 1 tab

  11. Cell Cycle Inhibition from Ethylacetate Extracts of Plectranthus amboinicus, (Lour.) Spreng.) Leaves on HeLa Cells Lines

    Rosidah; Hasibuan, Poppy Anjelisa Z.; Satria, Denny

    2016-01-01

    Objective: To evaluate the effects of ethylacetate extract (EAE) of Plectranthus amboinicus (Lour.) Spreng.) leaves on cell cyle on HeLa cell lines. Methods: Analysis of cell cycle distribution was performed using flowcytometer and the data was analyzed using ModFit LT 3.0 program. Results: The EAE changes the accumulation of cell cycle phase from G0 -G1 phase (54.61%) to sub-G1 phase (69.73%). Conclusions: Based on the results, EAE is potential to be developed as co-chemoth...

  12. Inhibition of Neisseria gonorrhoeae attachment to HeLa cells with monoclonal antibody directed against a protein II.

    Sugasawara, R J; Cannon, J G; Black, W J; Nachamkin, I; Sweet, R L; Brooks, G F

    1983-01-01

    This study showed that a protein II (PII) of Neisseria gonorrhoeae FA1090 appeared to act as a mediator of attachment to HeLa cells. Two colony variants of FA1090 were selected. Both gonococcal variants were nonpiliated, but one contained a PII and the other did not. A monoclonal antibody (1090-10.1), which was directed against the PII, inhibited the apparent PII-mediated attachment to HeLa cells. Antibodies produced from clone 1035-4, which had no PII specificity, did not inhibit the attachm...

  13. Overexpression of IGF-I receptor in HeLa cells enhances in vivo radioresponse

    Insulin-like growth factor I receptor (IGF-IR) is a transmembrane receptor tyrosine kinase whose activation strongly promotes cell growth and survival. We previously reported that IGF-IR activity confers intrinsic radioresistance in mouse embryo fibroblasts in vitro. However, it is still unclear whether tumor cells overexpressing IGF-IR exhibit radioresistance in vivo. For this purpose, we established HeLa cells that overexpress IGF-IR (HeLa-R), subcutaneously transplanted these cells into nude mice, and examined radioresponse in the resulting solid tumors. HeLa-R cells exhibited typical in vitro phenotypes generally observed in IGF-IR-overexpressing cells, as well as significant intrinsic radioresistance in vitro compared with parent cells. As expected, the transplanted HeLa-R tumors grew at a remarkably higher rate than parent tumors. Histological analysis revealed that HeLa-R tumors expressed more VEGF and had a higher density of tumor vessels. Unexpectedly, a marked growth delay was observed in HeLa-R tumors following 10 Gy of X-irradiation. Immunostaining of HeLa-R tumors for the hypoxia marker pimonidazole revealed a significantly lower level of hypoxic cells. Moreover, clamp hypoxia significantly increased radioresistance in HeLa-R tumors. Tumor microenvironments in vivo generated by the IGF-IR expression thus could be a major factor in determining the tumor radioresponse in vivo

  14. Invitro and Invivo anticancer activity of Ethanolic extract of Canthium Parviflorum Lam on DLA and Hela cell lines

    Purushoth Prabhu.T

    2011-12-01

    Full Text Available Background: Wild Jessamine, Canthium Parviflorum Lam, ( fam: Rubiaceae is traditionally used for snake bite in some villages in shimoga district of Karnataka. Canthium Species are used in the treatment of tumor, cough, astringent and anthlementic. Objective: In this study, invitro and invivo anticancer activity of crude ethanolic extracts from the leaves of Canthium Parviflorum Lam was investigated Method: The invitro anticancer activity was measured by MTT assay and Exclusion method. The invivo studies was determined in mice using Dalton’s lymphoma ascetic (DLA cells. Results: The ethanolic extacts of C.Parviflorum greatly inhibited DLA and Hela cell growth with IC50 Of 61.24μg/ml and 43.15μg/ml respectively. A significant increase in the life span and a decrease in the cancer cell number & tumour weight were noted in the tumor induced mice after treatment with Canthium Parviflorum Lam. Conclusion: Anticancer activity of Canthium Parviflorum was may be due to flavonoid present in the plant . Further studies are also in process to evaluate the most potent fraction of the plant and to isolate the constituents of the fraction

  15. Total Alkaloids of Sophora alopecuroides Inhibit Growth and Induce Apoptosis in Human Cervical Tumor HeLa Cells In vitro

    Li, Jian-Guang; Yang, Xiao-Yi; Huang, Wei

    2016-01-01

    Background: Uygur females of Xinjiang have the higher incidence of cervical tumor in the country. Alkaloids are the major active ingredients in Sophora alopecuroides, and its antitumor effect was recognized by the medical profession. Xinjiang is the main site of S. alopecuroides production in China so these plants are abundant in the region. Studies on the antitumor properties of total alkaloids of S. alopecuroides (TASA) can take full use of the traditional folk medicine in antitumor unique utility. Objectives: To explore the effects of TASA on proliferation and apoptosis of human cervical tumor HeLa cells in vitro. Materials and Methods: TASA was extracted, purified, and each monomer component was analyzed by high-performance liquid chromatography. The effect of TASA at different concentrations on the survival of HeLa cells was determined after 24 h using the Cell Counting Kit-8. In addition, cells were photographed using an inverted microscope to document morphological changes. The effect of TASA on apoptotic rate of HeLa cells was assessed by flow cytometry. Results: Monomers of TASA were found to be sophoridine, matrine, and sophocarpine. On treatment with 8.75 mg/ml of TASA, more than 50% of HeLa cells died, and cell death rate increased further with longer incubation. The apoptotic rates of HeLa cells in the experimental groups were 16.0% and 33.3% at concentrations of 6.25 mg/ml and 12.50 mg/ml, respectively. Conclusion: TASA can induce apoptosis in cervical tumor HeLa cells, and it has obvious inhibitory effects on cell growth. SUMMARY Total alkaloids of Sophora alopecuroides (TASA) exhibits anti-human cervical tumor propertiesMonomer component of TASA was analyzed by high-performance liquid chromatography, and its main effect component are sophoridine, matrine, and sophocarpineTASA inhibits growth and induces apoptosis in HeLa cells. Abbreviations used: TASA: Total alkaloids of S. alopecuroides, CCK-8: Cell Counting Kit-8, FBS: Fetal bovine serum, PBS

  16. Dynamic distribution of TTK in HeLa cells: insights from an ultrastructural study

    ZHEN DOU; AKIRA SAWAGECHI; JIE ZHANG; HONG LUO; LAWRENCE BRAKO; XUE BIAO YAO

    2003-01-01

    Entry into mitosis is driven by signaling cascades of mitotic kinases.Our recent studies show that TTK,a kinetochore-associated protein kinase,interacts with CENP-E,a mitotic kinesin located to corona fiber ofkinetochore.Using immunoelectron microscopy,here we show that TTK is present at the nuclear pore adjacent complex of interphase HeLa cells.Upon nuclear envelope fragmentation,TTK targets to the outermostregion of the developing kinetochores ofmonoorient chromosome as well as to spindle poles.After stable attachment,throughout chromosome congression,TTK is a constituent of the corona fibers,extending up to 90 nm away from the kinetochore outer plate.Upon metaphase alignment,TTK departs from the kinetochore and migrates toward the centrosomes.Taken together,this evidence strongly supports a model in which TTK functions in spindle checkpoint signaling cascades at both kinetochore and centrosome.

  17. Caveolae-mediated endocytosis of biocompatible gold nanoparticles in living Hela cells

    Efficient intracellular delivery of gold nanoparticles (AuNPs) and unraveling the mechanism underlying the intracellular delivery are essential for advancing the applications of AuNPs toward in vivo imaging and therapeutic interventions. We employed fluorescence microscopy to investigate the internalization mechanism of small-size AuNPs by living Hela cells. Herein, we found that the caveolae-mediated endocytosis was the dominant pathway for the intracellular delivery of small-size AuNPs. The intracellular delivery was suppressed when we depleted the cholesterol with methyl-β-cyclodextrin (MβCD); in contrast, the sucrose that disrupts the formation of clathrin-mediated endocytosis did not block the endocytosis of AuNPs. Meanwhile, we examined the intracellular localization of AuNPs in endocytic vesicles by fluorescent colocalization. This work would provide a potential technique to study the intracellular delivery of small-size nanoparticles for biomedical applications. (paper)

  18. The acquired radioresistance in HeLa cells under conditions mimicking hypoxia was attenuated by a decreased expression of HIF subunit genes induced by RNA interference

    The cancer cells residing in the hypoxic layer are resistant to radiation and these are ones responsible for cancer recurrence after radiation therapy. One of the reasons why hypoxic cancer cells acquire radioresistance may be attributable to changes in the gene expression profile by the activation of hypoxia inducible factors (HIFs). However, the details underlying this process remain unknown. In this study, we investigated the effects of knockdown of HIF subunit genes to elucidate how HIF subunit genes may be involved in the radioresistance acquired by HeLa cells following exposure to a hypoxia mimic. Interestingly, HIF-1α and HIF-2α seemed mutually complementary for each other when either of them was suppressed. We thus suppressed the expression of both genes simultaneously. To do this, we developed a short hairpin RNA (shRNA) targeting a high homology region between HIF-1α and HIF-2α. It was shown that the expression of the shRNA effectively suppressed the acquisition of radioresistance following the hypoxia mimic. Moreover, it was confirmed that suppression of both subunits resulted in the downregulation of stem cell markers and the suppression of spheroid formation during the hypoxia mimicking-conditions. This shRNA-mediated knockdown method targeting a common region shared by a family of genes may offer a new candidate cancer treatment. - Highlights: • Incubation with CoCl2 confers radioresistance to HeLa cells. • Both HIF-1α and HIF-2α are involved in the acquisition of radioresistance. • An shRNA to a homology region of HIF-1α and HIF-2α suppressed the radioresistance. • The shRNA decreased cells with stem cell markers and a stem cell phenotype

  19. The acquired radioresistance in HeLa cells under conditions mimicking hypoxia was attenuated by a decreased expression of HIF subunit genes induced by RNA interference

    Doi, Nobutaka [Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194 (Japan); New Products Research & Development, Gene Engineering Division, NIPPON GENE Co., Ltd. (Japan); Ogawa, Ryohei, E-mail: ogawa@med.u-toyama.ac.jp [Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194 (Japan); Cui, Zheng-Guo [Department of Public Health, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama (Japan); Morii, Akihiro; Watanabe, Akihiko [Department of Urology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama (Japan); Kanayama, Shinji; Yoneda, Yuko [New Products Research & Development, Gene Engineering Division, NIPPON GENE Co., Ltd. (Japan); Kondo, Takashi [Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194 (Japan)

    2015-05-01

    The cancer cells residing in the hypoxic layer are resistant to radiation and these are ones responsible for cancer recurrence after radiation therapy. One of the reasons why hypoxic cancer cells acquire radioresistance may be attributable to changes in the gene expression profile by the activation of hypoxia inducible factors (HIFs). However, the details underlying this process remain unknown. In this study, we investigated the effects of knockdown of HIF subunit genes to elucidate how HIF subunit genes may be involved in the radioresistance acquired by HeLa cells following exposure to a hypoxia mimic. Interestingly, HIF-1α and HIF-2α seemed mutually complementary for each other when either of them was suppressed. We thus suppressed the expression of both genes simultaneously. To do this, we developed a short hairpin RNA (shRNA) targeting a high homology region between HIF-1α and HIF-2α. It was shown that the expression of the shRNA effectively suppressed the acquisition of radioresistance following the hypoxia mimic. Moreover, it was confirmed that suppression of both subunits resulted in the downregulation of stem cell markers and the suppression of spheroid formation during the hypoxia mimicking-conditions. This shRNA-mediated knockdown method targeting a common region shared by a family of genes may offer a new candidate cancer treatment. - Highlights: • Incubation with CoCl{sub 2} confers radioresistance to HeLa cells. • Both HIF-1α and HIF-2α are involved in the acquisition of radioresistance. • An shRNA to a homology region of HIF-1α and HIF-2α suppressed the radioresistance. • The shRNA decreased cells with stem cell markers and a stem cell phenotype.

  20. Do altered activities of superoxide dismutases and the level of NF-kB modulate the effects of gamma radiation in HeLaS3 cells?

    ANA NICIFOROVIC

    2007-10-01

    Full Text Available Most experimental models, including cell culture studies, have demon­strated that over-expression of manganese superoxide dismutase (MnSOD in cells bearing a carcinoma phenotype has anti-proliferative and tumour suppression chara­cteristics. In contrast, when cervical carcinoma biopsies express MnSOD, there is a poor prognosis and resistance to radiation therapy. The results herein indicate that human cervical adenocarcinoma (HeLaS3 cells have increased MnSOD activity (up to 50 % of the total SOD activity due to low expression of its repressor p53 and a high level of oxidative stress arising from the cell culture conditions. High MnSOD activity may be related to HeLaS3 cell radioresistance, illustrated by a high IC50 of 3.4 Gy and by a relatively high level of cell viability after gamma irradiation. In contrast to MnSOD activity, cytosolic CuZnSOD activity decreased after ionising radiation. The catalase (Cat activity was unchanged. IR also increa­sed the nitric oxide synthase (NOS activity. Such conditions lead to increased con­centrations of the superoxide radical, hydrogen peroxide and NO., which together may be responsible for the decreased expression of NF-kB and unaltered Cat ac­tivity. Therefore, the disturbed redox balance within HeLaS3 cells may be respon­sible for the cytotoxicity observed at higher irradiation doses. It could be concluded that inhibition of the CuZnSOD activity may be an important target for the selective killing of radioresistant cancer cells.

  1. Oridonin induces apoptosis via PI3K/Akt pathway in cervical carcinoma HeLa cell line

    Hong-zhen HU; Yue-bo YANG; Xiang-dong XU; Hong-wei SHEN; Yi-min SHU; Zi REN; Xiao-mao LI; Hui-ming SHEN; Hai-tao ZENG

    2007-01-01

    Aim:To investigate the apoptosis-inducing effect of oridonin,a diterpenoid isolated from Rabdosia rubescens,in the human cervical carcinoma HeLa cell line.Methods:A morphological analysis,nuclear condensation,and fragmentation of chromatin were monitored using Hoechst 33342 staining. Cell viability was assessed using the 3-(4,5-dimethylthiazol-(2)-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cell apoptosis and the apoptosis-related activation in the HeLa cell line were evaluated by flow cytometry and Western blotting. Results:Oridonin suppressed the proliferation of the HeLa cell line in a dose- and time-dependent fashion. Oridonin treatment downregulated the activation of protein kinase B (Akt),the expression of forkhead box class O (FOXO) transcription factor,and glycogen synthase kinase 3 (GSK3). Oridonin also induced the release of cytochrome c accompanied by the activation of caspase-3 and poly-adenosine diphosphate-ribose polymerase cleavage. In addition,Z-D(OMe)-E(OMe)-V-D(OMe)FMK (z-DEVD-fmk),an inhibitor of caspases,prevented caspase-3 activation and abrogated oridonin-induced cell death. Finally,oridonin treatment of the HeLa cell line downregulated the expression of the inhibitor of the apoptosis protein.Conclusion:Our results showed that oridonin-induced apoptosis involved several molecular pathways. Oridonin may suppress constitutively activated targets of phosphatidylinositol 3-kinase (Akt,FOXO,and GSK3) in the HeLa cell line,inhibiting the proliferation and induction of caspase-dependent apoptosis.

  2. Photodynamic therapy of HeLa cell cultures by using LED or laser sources.

    Etcheverry, María E; Pasquale, Miguel A; Garavaglia, Mario

    2016-07-01

    The photodynamic therapy (PDT) on HeLa cell cultures was performed utilizing a 637nm LED lamp with 1.06W power and m-tetrahydroxyphenyl chlorin (m-THPC) as photosensitizer and compared to a laser source emitting at 654nm with the same power. Intracellular placement of the photosensitizer and the effect of its concentration (CP), its absorption time (TA) and the illumination time (TI) were evaluated. It was observed that for CP>40μg/ml and TA>24h, m-THPC had toxicity on cells in culture, even in the absence of illumination. For the other tested concentrations, the cells remained viable if not subjected to illumination doses. No effect on cells was observed for CPlaser source. Results demonstrate the feasibility of using a LED lamp as alternative to laser source. Here the main characteristic is not the light coherence but achieving a certain light fluence of the appropriate wavelength on cell cultures. We conclude that the efficacy was achieved satisfactorily and is essential for convenience, accessibility and safety. PMID:27152675

  3. Chk1 prevents abnormal mitosis of S-phase HeLa cells containing DNA damage

    LI XiaoFang; WARD Tarsha; YAO XueBiao; WU JiaRui

    2009-01-01

    To explore effects of DNA damage on cell-cycle progression in p53-deficient tumor cells,synchronized HeLa cells at G1,S and G2/M phases were treated with methyl methanesulfnate (MMS).The results showed that the MMS treatment resulted in the cell-cycle arrest or delay in all 3 phases,while the S-phase cells were the most sensitive to MMS.Further studies demonstrated that ATM-Chk2 and p38 MAPK signaling pathways were activated in all 3 phases when the cells were treated with MMS;whereas Chk1 was activated only in S phase under the drug treatment,indicating that Chk1 specifically participated in S-phase checkpoints.To analyze the role of Chk1 in S-phase checkpoints,we administered a specific Chk1 inhibitor,UCN-01,to the S-phase cells.The results showed that the S-phase cells treated with MMS+UCN-01 could enter aberrant mitosis without finishing DNA replication,indicating that Chk1 mainly functions in the DNA damage checkpoint rather than in the replication checkpoint.In addition,MMS treatment alone inhibited the accumulation of cyclin B1,a key component of M-phase CDK-cyclin complex,in the S-phase cells,whereas the inhibition of Chk1 activation resulted in the accumulation of cyclin B1 in the MMS-treated S-phase cells.This observation further supports the view that DNA-damaged S-phase cells enter abnormal mitosis when Chk1 activation is inhibited.Our results demonstrate that Chk1 is a specific kinase that plays an important role in the MMS-induced S-phase DNA damage checkpoint.As p53 is not involved in this process,Chk1 may be a potential target for p53-deficient tumor therapy.

  4. Binucleated HeLa cells are formed by cytokinesis failure in starvation and keep the potential of proliferation.

    Nishimura, Kazunori; Watanabe, Sumiko; Hayashida, Ryo; Sugishima, Setsuo; Iwasaka, Tsuyoshi; Kaku, Tsunehisa

    2016-08-01

    Many cytological studies have reported that the numbers of binucleated cells were elevated in various tumors. However, binucleated cells are observed in not only malignant tumors but also normal tissues. Thus, the clinical significance of binucleated cells is controversial. Here we attempted to elucidate the characteristics of binucleated HeLa cells using time-lapse microscopy. To examine the frequency, viability, proliferation, and formation mechanism of binucleated cells, we grew HeLa cells on chamber slides and tissue culture dishes in DMEM supplemented with (10, 3, 1 and 0.5 % media) and without fetal bovine serum (0 % medium). The proliferation was evaluated by the medium improvement examination (cultured for 2 more days in 10% medium after culturing in 0% medium; starvation). In the 0 % medium, 150 binucleated cells were formed by cytokinesis failure. There were significantly more binucleated cells in the 0 % medium than in the 10, 3, 1 and 0.5 % media. About twice the number of binucleated cells underwent mitosis in the improvement examinations than in the serum-free examination. We found here that starvation induced the binucleation of HeLa cells and that some binucleated cells can reproduce. These findings might be helpful for understanding binucleated cells in tumors. PMID:25894790

  5. Hormesis and adaptive response of survival in Hela cells induced by low dose X-ray irradiation

    The survival fraction in HeLa cells irradiated by low dose X-rays was observed using clone method. The results showed that the survival fraction in the cells irradiated by less than 0.5 Gy X-rays was higher than control, 'hormesis' of HeLa cell survival was obtained and was significant at doses near 0.25 Gy; also, the damage degree of cells induced by the following irradiation was reduced because of pre-treating the cells with low dose D1 of 0.05, 0.75 Gy; it was found from above that 'adaptive response' of cell survival was induced by the low dose irradiation

  6. Multiple origins of spontaneously arising micronuclei in HeLa cells: Direct evidence from long-term live cell imaging

    Although micronuclei (MNi) are extensively used to evaluate genotoxic effects and chromosome instability, the most basic issue regarding their origins has not been completely addressed due to limitations of traditional methods. Recently, long-term live cell imaging was developed to monitor the dynamics of single cell in a real-time and high-throughput manner. In the present study, this state-of-the-art technique was employed to examine spontaneous micronucleus (MN) formation in untreated HeLa cells. We demonstrate that spontaneous MNi are derived from incorrectly aligned chromosomes in metaphase (displaced chromosomes, DCs), lagging chromosomes (LCs) and broken chromosome bridges (CBs) in later mitotic stages, but not nuclear buds in S phase. However, most of bipolar mitoses with DCs (91.29%), LCs (73.11%) and broken CBs (88.93%) did not give rise to MNi. Our data also show directly, for the first time, that MNi could originate spontaneously from (1) MNi already presented in the mother cells; (2) nuclear fragments that appeared during mitosis with CB; and (3) chromosomes being extruded into a minicell which fused with one of the daughter cells later. Quantitatively, most of MNi originated from LCs (63.66%), DCs (10.97%) and broken CBs (9.25%). Taken together, these direct evidences show that there are multiple origins for spontaneously arising MNi in HeLa cells and each mechanism contributes to overall MN formation to different extents

  7. DNA repair in HeLa Zh-63 cells after irradiation and action of chemical carcinogens

    In HeLa Zh-63 cells prelabelled with 3H-thymidine the repair of γ-ray-induced single-strand breaks in the nutrient medium and in the buffer proceeds with the same intensity. The process is inhibited with acriflavine, quinacrine and 2,4-dinitrophenol but not with caffeine. The repair of double-strand breaks of DNA in the nutrient medium is more complete than in the buffer. It is inhibited by acriflavine but not by caffeine. In cells pulse-labelled with 3H-thymidine after U.V.-irradiation and 7-bromomethylbenz-(α)anthracene (BMBA) treatment the nascent DNA has a low molecular weight (Msub(w)). In U.V.-irradiated cells during incubation Msub(w) restores to the control level. In BMBA-treated cells Msub(w) is not restored within the first 6 h, but 18-21 h later DNA of a normal Msub(w) is synthesized. After the action of γ-rays and 2-aminofluorene (AF) nascent DNA with a normal Msub(w) is synthesized. When a pulse label is introduced simultaneously with caffeine after γ-ray-irradiation and BMBA (but not U.V.-light or AF) treatment the Msub(w) of nascent DNA is less than in the case without caffeine. It is assumed to be due to fast post-replication repair (during pulse labelling) which is sensitive to caffeine. (author)

  8. The epimer of kaurenoic acid from Croton antisyphiliticus is cytotoxic toward B-16 and HeLa tumor cells through apoptosis induction.

    Fernandes, V C; Pereira, S I V; Coppede, J; Martins, J S; Rizo, W F; Beleboni, R O; Marins, M; Pereira, P S; Pereira, A M S; Fachin, A L

    2013-01-01

    Cancer has become the leading cause of death in developing countries due to increased life expectancy of the population and changes in lifestyle. Studies on active principles of plant have motivated researchers to develop new antitumor agents that are specific and effective for treatment of neoplasms. Kaurane diterpenes are considered important compounds in the development of new and highly effective anticancer chemotherapeutic agents due to their cytotoxic properties in the induction of apoptosis. We evaluated the cytotoxic and apoptotic activity of the epimer of kaurenoic acid (EKA) isolated from the medicinal plant Croton antisyphiliticus (Euphorbiaceae) toward tumor cell lines HeLa and B-16 and normal fibroblasts 3T3. Based on analyses with the MTT test, EKA showed cytotoxic activity, with half maximal inhibitory concentration values of 59.41, 68.18 and 60.30 µg/mL for the B-16, HeLa and 3T3 cell lines, respectively. The assay for necrotic or apoptotic cells by differential staining showed induction of apoptosis in all three cell lines. We conclude that EKA is not selective between tumor and normal cell lines; the mechanism of action of EKA is induction of apoptosis, which is part of the innate mechanism of cell defense against neoplasia. PMID:23613246

  9. Identification and characterization of a DNA primase activity present in herpes simplex virus type 1-infected HeLa cells

    A novel DNA primase activity has been identified in HeLa cells infected with herpes simplex virus type 1 (HSV-1). Such an activity has not been detected in mock-infected cells. The primase activity coeluted with a portion of HSV-1 DNA polymerase from single-stranded DNA agarose columns loaded with high-salt extracts derived from infected cells. This DNA primase activity could be distinguished from host HeLa cell DNA primase by several criteria. First, the pH optimum of the HSV primase was relatively broad and peaked at 8.2 to 8.7 pH units. Second, freshly isolated HSV DNA primase was less salt sensitive than the HeLa primase. Third, antibodies raised against individual peptides of the calf thymus DNA polymerase:primase complex cross-reacted with the HeLa primase but did not react with the HSV DNA primase. Fourth, freshly prepared HSV DNA primase appeared to be associated with the HSV polymerase, but after storage at 4 degree C for several weeks, the DNA primase separated from the viral DNA polymerase. This free DNA primase had an apparent molecular size of approximately 40 kilodaltons, whereas free HeLa DNA primase had an apparent molecular size of approximately 110 kilodaltons. On the basis of these data, the authors believe that the novel DNA primase activity in HSV-infected cells may be virus coded and that this enzyme represents a new and important function involved in the replication of HSV DNA

  10. NF-κB plays a key role in microcystin-RR-induced HeLa cell proliferation and apoptosis.

    Chen, Liang; Zhang, Xin; Chen, Jun; Zhang, Xuezhen; Fan, Huihui; Li, Shangchun; Xie, Ping

    2014-09-01

    Microcystins (MCs) are well-known cyanobacterial toxins produced in eutrophic waters and can act as potential carcinogens and have caused serious risk to human health. However, pleiotropic even paradoxical actions of cells exposure to MCs have been reported, and the mechanisms of MC-induced tumorigenesis and apoptosis are still unknown. In this study, we performed the first comprehensive in vitro investigation on carcinogenesis associated with nuclear factor kappa B (NF-κB) and its downstream genes in HeLa cells (Human cervix adenocarcinoma cell line from epithelial cells) exposure to MC-RR. HeLa cells were treated with 0, 20, 40, 60, and 80 µg/mL MC-RR for 4, 8, 12, and 24 h. HeLa cells presented dualistic responses to different doses of MCs. CCK8 assay showed that MC-RR exposure evidently enhanced cell viability of HeLa cells at lower MCs doses. Cell cycle and apoptosis analysis revealed that lower MCs doses promoted G1/S transition and cell proliferation while higher doses of MCs induced apoptosis, with a dose-dependent manner. Electrophoretic mobility shift assay (EMSA) revealed that MC-RR could increase/decrease NF-κB activity at lower/higher MC-RR doses, respectively. Furthermore, the expression of NF-κB downstream target genes including c-FLIP, cyclinD1, c-myc, and c-IAP2 showed the same variation trend as NF-κB activity both at mRNA and protein levels, which were induced by lower doses of MC-RR and suppressed by higher doses. Our data verified for the first time that NF-κB pathway may mediate MC-induced cell proliferation and apoptosis and provided a better understanding of the molecular mechanism for potential carcinogenicity of MC-RR. PMID:24932741

  11. High LET radiation enhances nocodazole induced cell death in HeLa cells through mitotic catastrophe and apoptosis

    To understand how human tumor cells respond to the combined treatment with nocodazole and high linear energy transfer (LET) radiation, alterations in cell cycle, mitotic disturbances and cell death were investigated in the present study. Human cervix carcinoma HeLa cells were exposed to nocodazole for 18 h immediately followed by high LET iron ion irradiation and displayed a sequence of events leading to DNA damages, mitotic aberrations, interphase restitution and endocycle as well as cell death. A prolonged mitotic arrest more than 10 h was observed following nocodazole exposure, no matter the irradiation was present or not. The occurrence of mitotic slippage following the mitotic arrest was only drug-dependent and the irradiation did not accelerate it. The amount of polyploidy cells was increased following mitotic slippage. No detectable G2 or G1 arrest was observed in cells upon the combined treatment and the cells reentered the cell cycle still harboring unrepaired cellular damages. This premature entry caused an increase of multipolar mitotic spindles and amplification of centrosomes, which gave rise to lagging chromosomal material, failure of cytokinesis and polyploidization. These mitotic disturbances and their outcomes confirmed the incidence of mitotic catastrophe and delayed apoptotic features displayed by terminal-transferased UTP- nick end-labeling (TUNEL) method after the combined treatment. These results suggest that the addition of high-LET iron ion irradiation to nocodazole enhanced mitotic catastrophe and delayed apoptosis in HeLa cells. These might be important cell death mechanisms involved in tumor cells in response to the treatment of antimitotic drug combined with high LET radiation. (author)

  12. Depletion of cellular poly (A) binding protein prevents protein synthesis and leads to apoptosis in HeLa cells

    Highlights: → Depletion of cellular PABP level arrests mRNA translation in HeLa cells. → PABP knock down leads to apoptotic cell death. → PABP depletion does not affect transcription. → PABP depletion does not lead to nuclear accumulation of mRNA. -- Abstract: The cytoplasmic poly (A) binding protein (PABP) is important in mRNA translation and stability. In yeast, depletion of PABP leads to translation arrest. Similarly, the PABP gene in Drosophila is important for proper development. It is however uncertain, whether mammalian PABP is essential for mRNA translation. Here we showed the effect of PABP depletion on mRNA metabolism in HeLa cells by using a small interfering RNA. Our results suggest that depletion of PABP prevents protein synthesis and consequently leads to cell death through apoptosis. Interestingly, no detectable effect of PABP depletion on transcription, transport and stability of mRNA was observed.

  13. Depletion of cellular poly (A) binding protein prevents protein synthesis and leads to apoptosis in HeLa cells

    Thangima Zannat, Mst.; Bhattacharjee, Rumpa B. [Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G2W1 (Canada); Bag, Jnanankur, E-mail: jbag@uoguelph.ca [Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G2W1 (Canada)

    2011-05-13

    Highlights: {yields} Depletion of cellular PABP level arrests mRNA translation in HeLa cells. {yields} PABP knock down leads to apoptotic cell death. {yields} PABP depletion does not affect transcription. {yields} PABP depletion does not lead to nuclear accumulation of mRNA. -- Abstract: The cytoplasmic poly (A) binding protein (PABP) is important in mRNA translation and stability. In yeast, depletion of PABP leads to translation arrest. Similarly, the PABP gene in Drosophila is important for proper development. It is however uncertain, whether mammalian PABP is essential for mRNA translation. Here we showed the effect of PABP depletion on mRNA metabolism in HeLa cells by using a small interfering RNA. Our results suggest that depletion of PABP prevents protein synthesis and consequently leads to cell death through apoptosis. Interestingly, no detectable effect of PABP depletion on transcription, transport and stability of mRNA was observed.

  14. A New Diterpene from Litsea cubeba Fruits: Structure Elucidation and Capability to Induce Apoptosis in HeLa Cells

    Piyapat Trisonthi

    2014-05-01

    Full Text Available A new diterpene, identified as (+-6-(4-hydroxy-4-methyl-2-pentenoyl-4,6-dimethyl-5-(3-methyl-2-butenyl-1,3-cyclohexadienecarbaldehyde (1, cubelin, was isolated from a methanol extract of Litsea cubeba fruits by normal phase column chromatography and purified by preparative HPLC. The structure elucidation was conducted by spectroscopic methods (UV, IR, ESI-TOF-MS, 1-D and 2-D NMR. Cubelin exhibited activity against HeLa cell viability and proliferation. The cells also exhibited changes in nuclear morphology which are hallmarks of apoptotic cell death. The presence of cleaved caspase-3/-7, caspase-8 and caspase-9 in the cubelin treated population indicated the potential of the compound to induce apoptosis in HeLa cells via both intrinsic and extrinsic pathways.

  15. Axin is expressed in mitochondria and suppresses mitochondrial ATP synthesis in HeLa cells.

    Shin, Jee-Hye; Kim, Hyun-Wook; Rhyu, Im Joo; Kee, Sun-Ho

    2016-01-01

    Many recent studies have revealed that axin is involved in numerous cellular functions beyond the negative regulation of β-catenin-dependent Wnt signaling. Previously, an association of ectopic axin with mitochondria was observed. In an effort to investigate the relationship between axin and mitochondria, we found that axin expression suppressed cellular ATP production, which was more apparent as axin expression levels increased. Also, mitochondrial expression of axin was observed using two axin-expressing HeLa cell models: doxycycline-inducible ectopic axin expression (HeLa-axin) and axin expression enhanced by long-term treatment with XAV939 (HeLa-XAV). In biochemical analysis, axin is associated with oxidative phosphorylation (OXPHOS) complex IV and is involved in defects in the assembly of complex IV-containing supercomplexes. Functionally, axin expression reduced the activity of OXPHOS complex IV and the oxygen consumption rate (OCR), suggesting axin-mediated mitochondrial dysfunction. Subsequent studies using various inhibitors of Wnt signaling showed that the reduction in cellular ATP levels was weaker in cases of ICAT protein expression and treatment with iCRT3 or NSC668036 compared with XAV939 treatment, suggesting that XAV939 treatment affects ATP synthesis in addition to suppressing Wnt signaling activity. Axin-mediated regulation of mitochondrial function may be an additional mechanism to Wnt signaling for regulation of cell growth. PMID:26704260

  16. Inhibition of Bcl-2 expression by a novel tumor-specific RNA interference system increases chemosensitivity to 5-fluorouracil in Hela cells

    Sheng-lin HUANG; Yi WU; Hai YU; Ping ZHANG; Xing-qian ZHANG; Lei YING; Han-fang ZHAO

    2006-01-01

    Aim: RNA interference (RNAi) has been proposed as a potential treatment for cancer, but the lack of cellular targets limits its use in cancer gene therapy. No current technology has achieved direct tumor-specific gene silencing using RNAi.In the present study we attempt to develop a tumor-specific RNAi system using the human telomerase reverse transcriptase (hTERT) promoter; furthermore, we analyzed its inhibitive effect on Bcl-2 expression. Methods: The vectors containing a small hairpin RNA (shRNA) to target exogenous reporters [firefly luciferase and enhanced green fluorescent protein (EGFP)] and endogenous gene (Bcl-2)were constructed. Luciferase expression was determined by dual luciferase assay.Reverse transcription-polymerase chain reaction (RT-PCR), fluorescence microscopy and fluorescence-activated cell sorting (FACS) were used to measure EGFP expression. Inhibition of Bcl-2 was evaluated by RT-PCR and Western blotting.Cell proliferation and viability were measured by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. FACS was used to analyze the cell cycle distribution profile. Results: We showed that with the hTERT promoter directly driving shRNA transcription, expression of the exogenous reporters (LUC and EGFP) in tumor cells, but not normal cells, was specifically inhibited in vitro. The hTERT promoter-driven shRNA also depressed the expression of Bcl-2. Inhibition of Bcl-2 did not affect cell proliferation, but increased the chemosensitivity of HeLa cells to 5-fluorouracil. Conclusion: The present study describes an efficient RNAi system for gene silencing that is specific to tumor cells using the hTERT promoter. Suppression of Bcl-2 by using this system sensitized HeLa cells to 5-fluorouracil. This system may be useful for RNAi therapy.

  17. Detection of Chlamydia trachomatis inclusions in McCoy and HeLa-229 cells: an alternative staining technique using toluidine blue.

    Mohammed, N R; Hillary, I B

    1984-01-01

    Toluidine blue staining was used to detect Chlamydia trachomatis inclusions in both McCoy and HeLa-229 cells from clinical specimens. This method was more sensitive than iodine staining for detecting C trachomatis inclusions in both McCoy and HeLa-229 cells and also more sensitive than Giemsa staining for detecting chlamydial inclusions in HeLa-229 cells. While its sensitivity for detection of chlamydial inclusions in McCoy cells is equal to that of Giemsa staining, toluidine blue staining is...

  18. A novel dithiocarbamate derivative induces cell apoptosis through p53-dependent intrinsic pathway and suppresses the expression of the E6 oncogene of human papillomavirus 18 in HeLa cells.

    Li, Yanhong; Qi, Hongxue; Li, Xiaobo; Hou, Xueling; Lu, Xueying; Xiao, Xiangwen

    2015-06-01

    Dithiocarbamates (DTCs) exhibit a broad spectrum of antitumor activities, however, their molecular mechanisms of antitumor have not yet been elucidated. Previously, we have synthesized a series of novel dithiocarbamate derivatives. These DTCs were examined for cytotoxic activities against five human cancer cell lines. In this study, one of dithiocarbamate (DTC1) with higher potential for HeLa cells was chosen to investigate molecular mechanisms for its anti-tumor activities. DTC1 could inhibit proliferation, and highly induce apoptosis in HeLa cells by activating caspase-3, -6 and -9; moreover, activities of caspase-3, -6 and -9 were inhibited by pan-caspase inhibitor, Z-VAD-FMK. Furthermore, DTC1 decreased the levels of Bcl-2 and Bcl-xL, and increased expression of cytosol cytochrome c, Bak, Bax and p53 in a time-dependent manner but had no effect on the level of Rb. It was shown that DTC1 induced HeLa cells apoptosis through a p53-dependent pathway as tested by the wild type p53 inhibitor, pifithrin-α. Additionally, the relative expression of E6 and E7 were evaluated in HPV18-positive (HeLa cells) by real-time PCR and western blotting. The results firstly demonstrated that DTC1 suppressed both expression of E6 mRNA and E6 oncoprotein, but had no effect on the expression of E7 mRNA and protein in HPV18. Our results suggested that DTC1 may serve as novel chemotherapeutic agents in the treatment of cervical cancer and potential anti-HPV virus candidates that merit further studies. PMID:25772545

  19. Acetylcholinesterase inhibition, antioxidant activity and toxicity of Peumus boldus water extracts on HeLa and Caco-2 cell lines.

    Falé, P L; Amaral, F; Amorim Madeira, P J; Sousa Silva, M; Florêncio, M H; Frazão, F N; Serralheiro, M L M

    2012-08-01

    This work aimed to study the inhibition on acetylcholinesterase activity (AChE), the antioxidant activity and the toxicity towards Caco-2 and HeLa cells of aqueous extracts of Peumus Boldus. An IC(50) value of 0.93 mg/mL, for AChE inhibition, and EC(50) of 18.7 μg/mL, for the antioxidant activity, was determined. This activity can be attributed to glycosylated flavonoid derivatives detected, which were the main compounds, although boldine and other aporphine derivatives were also present. No changes in the chemical composition or the biochemical activities were found after gastrointestinal digestion. Toxicity of P. boldus decoction gave an IC(50) value 0.66 mg/mL for HeLa cells, which caused significant changes in the cell proteome profile. PMID:22617353

  20. Constitutive expression of human coagulating factor IX in HeLa cells by homologous recombination of the promoter

    2001-01-01

    Constitutive expression of hFIX protein in nonhepatocytes wasstudied. The gene targeting vector was constructed and transferred into HeLa cells. With the detection system of PCR, we demonstrated that the endogenous hFIX promoter was replaced with an hCMV promoter when targeted insertion of the constructor was directed by the sequence homology. The expression of hFIX in the modified HeLa cells, 11.2 ng/106 cell/24 h, strongly suggested that hFIX gene could be activated by a powerful promoter in nonhepatocytes. The results would make it possible to examine the feasibility of re-regulate gene expression by promoter replacement.

  1. HeLa cell tumor response to 60Co, Cs-137, Cf-252 radiations and cisplatin chemotherapy in nude mice

    HeLa cells were implanted into athymic nude mice from tissue culture and solid tumors established (HeLa cell tumor or HCT). Large cell numbers of 1 X 107 were required to obtain consistent and progressive growth, and tumor growth followed a Gompertzian mode. Irradiation studies were carried out using acute Cobalt-60 (60Co), low-dose-rate (LDR) Cs-137 and LDR Cf-252. Cf-252, a neutron-emitting radioisotope, produced an immediate tumor shrinkage and regression response after a dose of 279 cGy. Acute 60Co or LDR Cs-137 irradiation with 1000 cGy had little effect on the HCT. After a dose of 2000 cGy of 60Co radiation tumor shrinkage followed a latent period of approximately 5 days. Cisplatin had no effect on the HCT in nude mice in stationary or late exponential growth

  2. Organellar proteome analyses of ricin toxin-treated HeLa cells.

    Liao, Peng; Li, Yunhu; Li, Hongyang; Liu, Wensen

    2016-07-01

    Apoptosis triggered by ricin toxin (RT) has previously been associated with certain cellular organellar compartments, but the diversity in the composition of the organellar proteins remains unclear. Here, we applied a shotgun proteomics strategy to examine the differential expression of proteins in the mitochondria, nuclei, and cytoplasm of HeLa cells treated and not treated with RT. Data were combined with a global bioinformatics analysis and experimental confirmations. A total of 3107 proteins were identified. Bioinformatics predictors (Proteome Analyst, WoLF PSORT, TargetP, MitoPred, Nucleo, MultiLoc, and k-nearest neighbor) and a Bayesian model that integrated these predictors were used to predict the locations of 1349 distinct organellar proteins. Our data indicate that the Bayesian model was more efficient than the individual implementation of these predictors. Additionally, a Biomolecular Interaction Network (BIN) analysis was used to identify 149 BIN subnetworks. Our experimental confirmations indicate that certain apoptosis-related proteins (e.g. cytochrome c, enolase, lamin B, Bax, and Drp1) were found to be translocated and had variable expression levels. These results provide new insights for the systematic understanding of RT-induced apoptosis responses. PMID:25227225

  3. Effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on phosphatidylethanolamine metabolism in HeLa cells

    The potent tumor promoter, TPA, exerts its earliest effects at the plasma membrane. Recent findings have shown that TPA stimulates a phospholipase C-mediated turnover of phosphatidyl-choline in several different cell types. The present study was undertaken to investigate whether TPA elicits a similar effect on the phosphatidylethanolamine (PE) pool of HeLa cells. Three different series of experiments were performed. First, in HeLa cells pulse-labeled with [3H]ethanolamine, TPA stimulated a 5-fold release of aqueous radiolabeled products into the extra-cellular medium after a 1-hour incubation. Second, when [3H]ethanolamine and TPA were added simultaneously to the cells, TPA stimulated a 2-fold incorporation of radiolabel into the cellular PE pool. In both the release and incorporation of [3H]ethanolamine, TPA had no significant effect on PE mass. Finally, when HeLa cells were incubated with exogenous 1-radyl-2-acyl-sn-glycero-3-phospho-[3H]ethanolamine, TPA stimulated the formation of an aqueous radiolabeled product in the medium, which was identified as phosphoethanolamine. These results provide evidence that TPA stimulates a phospholipase C-mediated turnover of PE

  4. Interaction of translationally controlled tumor protein with Apaf-1 is involved in the development of chemoresistance in HeLa cells

    Translationally controlled tumor protein (TCTP), alternatively called fortilin, is believed to be involved in the development of the chemoresistance of tumor cells against anticancer drugs such as etoposide, taxol, and oxaliplatin, the underlying mechanisms of which still remain elusive. Cell death analysis of TCTP-overexpressing HeLa cells was performed following etoposide treatment to assess the mitochondria-dependent apoptosis. Apoptotic pathway was analyzed through measuring the cleavage of epidermal growth factor receptor (EGFR) and phospholipase C-γ (PLC-γ), caspase activation, mitochondrial membrane perturbation, and cytochrome c release by flow cytometry and western blotting. To clarify the role of TCTP in the inhibition of apoptosome, in vitro apoptosome reconstitution and immunoprecipitation was used. Pull-down assay and silver staining using the variants of Apaf-1 protein was applied to identify the domain that is responsible for its interaction with TCTP. In the present study, we confirmed that adenoviral overexpression of TCTP protects HeLa cells from cell death induced by cytotoxic drugs such as taxol and etoposide. TCTP antagonized the mitochondria-dependent apoptotic pathway following etoposide treatment, including mitochondrial membrane damage and resultant cytochrome c release, activation of caspase-9, and -3, and eventually, the cleavage of EGFR and PLC-γ. More importantly, TCTP interacts with the caspase recruitment domain (CARD) of Apaf-1 and is incorporated into the heptameric Apaf-1 complex, and that C-terminal cleaved TCTP specifically associates with Apaf-1 of apoptosome in apoptosome-forming condition thereby inhibiting the amplification of caspase cascade. TCTP protects the cancer cells from etoposide-induced cell death by inhibiting the mitochondria-mediated apoptotic pathway. Interaction of TCTP with Apaf-1 in apoptosome is involved in the molecular mechanism of TCTP-induced chemoresistance. These findings suggest that TCTP may serve

  5. Human papillomavirus 18 E6 inhibits phosphorylation of p53 expressed in HeLa cells

    Ajay Amrendra K

    2012-01-01

    Full Text Available Abstract Background In HPV infected cells p53 function is abrogated by E6 and even ectopically expressed p53 is unable to perform tumor suppressor functions. In addition to facilitating its degradation, E6 may also inhibit p53 transactivity, though the mechanisms are still poorly understood. It has been reported that inhibition of p300, an acetyltransferase responsible for p53 acetylation is inactivated by E6. Activation of overexpressed p53 to cause cell growth inhibition is facilitated by its phosphorylation. Previously, we reported that non-genotoxically overexpressed p53 in HeLa cells needs to be phosphorylated to perform its cell growth inhibitory functions. Since over expressed p53 by itself was not activated, we hypothesized an inhibitory role for E6. Results Majority of reports proposes E6 mediated degradation of p53 as a possible reason for its inactivation. However, results presented here for the first time demonstrate that overexpressed p53 is not directly associated with E6 and therefore free, yet it is not functionally active in HPV positive cells. Also, the stability of overexpressed p53 does not seem to be an issue because inhibition of proteasomal degradation did not increase the half-life of overexpressed p53, which is more than endogenous p53. However, inhibition of proteasomal degradation prevents the degradation of endogenous p53. These findings suggest that overexpressed p53 and endogenous p53 are differentially subjected to proteasomal degradation and the reasons for this discrepancy remain unclear. Our studies demonstrate that p53 over expression has no effect on anchorage independent cell-growth and E6 nullifies its cell growth inhibitory effect. E6 overexpression abrogates OA induced p53 occupancy on the p21 promoter and cell death as well. E6 did not decrease p53 protein but phospho-p53 level was significantly reduced. Conclusion We report for the first time that E6 de-activates p53 by inhibiting its phosphorylation

  6. Investigation of siRNA Nanoparticle Formation Using Mono-Cationic Detergents and Its Use in Gene Silencing in Human HeLa Cells

    Yamada, Yuma; Suzuki, Ryosuke; Harashima, Hideyoshi, E-mail: harashima@pharm.hokudai.ac.jp [Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812 (Japan)

    2013-11-01

    The focus of recent research has been on the development of siRNA vectors to achieve an innovative gene therapy. Most of the conventional vectors are siRNA nanoparticles complexed with cationic polymers and liposomes, making it difficult to release siRNA. In this study, we report on the use of MCD, a quaternary ammonium salt detergent containing a long aliphatic chain (L-chain) as an siRNA complexation agent using human HeLa cells (a model cancer cell). We prepared siRNA nanoparticles using various MCDs, and measured the diameters and zeta-potentials of the particles. The use of an MCD with a long L-chain resulted in the formation of a positively charged nanoparticle. In contrast, a negatively charged nanoparticle was formed when a MCD with a short L-chain was used. We next evaluated the gene silencing efficiency of the nanoparticles using HeLa cells expressing the luciferase protein. The results showed that the siRNA/MCD nanoparticles showed a higher gene silencing efficiency than Lipofectamine 2000. We also found that the efficiency of gene silencing is a function of the length of the alkyl chain in MCD and zeta-potential of the siRNA/MCD nanoparticles. Such information provides another viewpoint for designing siRNA vectors.

  7. Investigation of siRNA Nanoparticle Formation Using Mono-Cationic Detergents and Its Use in Gene Silencing in Human HeLa Cells

    The focus of recent research has been on the development of siRNA vectors to achieve an innovative gene therapy. Most of the conventional vectors are siRNA nanoparticles complexed with cationic polymers and liposomes, making it difficult to release siRNA. In this study, we report on the use of MCD, a quaternary ammonium salt detergent containing a long aliphatic chain (L-chain) as an siRNA complexation agent using human HeLa cells (a model cancer cell). We prepared siRNA nanoparticles using various MCDs, and measured the diameters and zeta-potentials of the particles. The use of an MCD with a long L-chain resulted in the formation of a positively charged nanoparticle. In contrast, a negatively charged nanoparticle was formed when a MCD with a short L-chain was used. We next evaluated the gene silencing efficiency of the nanoparticles using HeLa cells expressing the luciferase protein. The results showed that the siRNA/MCD nanoparticles showed a higher gene silencing efficiency than Lipofectamine 2000. We also found that the efficiency of gene silencing is a function of the length of the alkyl chain in MCD and zeta-potential of the siRNA/MCD nanoparticles. Such information provides another viewpoint for designing siRNA vectors

  8. Nuclear proteome analysis of benzo(a)pyrene-treated HeLa cells

    Previously, we employed a proteomics-based 2-D gel electrophoresis assay to show that exposure to 10 μM benzo(a)pyrene (BaP) during a 24 h frame can lead to changes in nuclear protein expression and alternative splicing. To further expand our knowledge about the DNA damage response (DDR) induced by BaP, we investigated the nuclear protein expression profiles in HeLa cells treated with different concentrations of BaP (0.1, 1, and 10 μM) using this proteomics-based 2-D gel electrophoresis assay. We found 125 differentially expressed proteins in BaP-treated cells compared to control cells. Among them, 79 (63.2%) were down-regulated, 46 (36.8%) were up-regulated; 8 showed changes in the 1 μM and 10 μM BaP-treated groups, 2 in the 0.1 μM and 10 μM BaP-treated groups, 4 in the 0.1 μM and 1 μM BaP-treated groups, and only one showed changes in all three groups. Fifty protein spots were chosen for liquid chromatography–tandem mass spectrometry (LC–MS/MS) identification, and of these, 39 were identified, including subunits of the 26S proteasome and Annexin A1. The functions of some identified proteins were further examined and the results showed that they might be involved in BaP-induced DDR. Taken together, these data indicate that proteomics is a valuable approach in the study of environmental chemical–host interactions, and the identified proteins could provide new leads for better understanding BaP-induced mutagenesis and carcinogenesis.

  9. Mechanism of Induction of Apoptosis by siRNA Targeting hTERT in HeLa Cells

    WANG Jian; REN Chang-shan

    2008-01-01

    Objective:To investigate the molecular mechanism of induction of apoptosis by siRNA targeting human telomerase reverse transcriptase(hTERT)in HeLa Cells.Methods:HeLa cells were transfected with siRNAs by liposome method.RT-PCR was used to examine mRNA levels of hTERT in HeLa cells.Microarray assay was adopted to explore the transcriptional profiling of apoptosis associated genes.The protein levels of hTERT,TRAIL,Bcl-2,and cytoplasm Cyt C were detected by Western Blotting.The apoptosis rate was determined by flow cytometry using PI staining.Relative activity of Caspase-3 and Caspase-8 was measured by colorimetric assay.Results:The siRNA targeting hTERT suppressed the expression of hTERT gene significantly.Forty-eight hours after transfection,the expression level of TRAIL was increased,the expression level of Bcl-2 was decreased,the releasing of Cyt C was enhanced,the activation of Caspase-3 was increased and the apoptosis rate was increased.Conclusion:hTERT-siRNA induces apoptosis of HeLa cells via activating mitochondrial signal transduction pathway.

  10. Radiosensitizing effect of gold nanoparticles in carbon ion irradiation of human cervical cancer cells

    Noble metal nanoparticles have received considerable attention in biotechnology for their role in bio sensing due to surface plasmon resonance, medical diagnostics due to better imaging contrast and therapy. The radiosensitization effect of gold nanoparticles (AuNP) has been gaining popularity in radiation therapy of cancer cells. The better depth dose profile of energetic ion beam proves its superiority over gamma radiation for fighting against cancer. In the present work, the glucose capped gold nanoparticles (Glu-AuNP) were synthesised and internalized in the HeLa cells. Transmission electron microscopic analysis of ultrathin sections of Glu-AuNP treated HeLa cells confirmed the internalization of Glu-AuNPs. Control HeLa cells and Glu-AuNp treated HeLa cells were irradiated at different doses of 62 MeV 12C ion beam (LET – 290keV/μm) at BIO beam line of using 15UD Pelletron accelerator at Inter University Accelerator Centre, New Delhi, India. The survival fraction was assessed by colony forming assay which revealed that the dose of carbon ion for 90% cell killing in Glu-AuNP treated HeLa cells and control HeLa cells are 2.3 and 3.2 Gy respectively. This observation shows ∼ 28% reduction of 12C6+ ion dose for Glu-AuNP treated HeLa cells as compared to control HeLa cells

  11. Characterisation of Ca(2+)-dependent inwardly rectifying K+ currents in HeLa cells.

    Díaz, M; Sepúlveda, F V

    1995-06-01

    The whole-cell configuration of the patch-clamp technique was used to examine K+ currents in HeLa cells. Under quasi-physiological ionic gradients, using an intracellular solution containing 10(-7) mol/l free Ca2+, mainly outward currents were observed. Large inwardly rectifying currents were elicited in symmetrical 145 mmol/l KCl. Replacement of all extracellular K+ by isomolar Na+, greatly decreased inward currents and shifted the reversal potential as expected for K+ selectivity. The inwardly rectifying K+ currents exhibited little or no apparent voltage dependence within the range of from -120 mV to 120 mV. A square-root relationship between chord conductance and [K+] at negative potentials could be established. The inwardly rectifying nature of the currents was unaltered after removal of intracellular Mg2+ and chelation with ATP and ethylenediaminetetraacetic acid (EDTA). Permeability ratios for other monovalent cations relative to K+ were: K+ (1.0) > Rb+ (0.86) > Cs+ (0.12) > Li (0.08) > Na+ (0.03). Slope conductance ratios measured at -100 mV were: Rb+ (1.66) > K+ (1.0) > Na+ (0.09) > Li (0.08) > Cs+ (0.06). K+ conductance was highly sensitive to intracellular free Ca2+ concentration. The relationship between conductance at 0 mV and Ca2+ concentration was well described by a Hill expression with a dissociation constant, KD, of 70 nmol/l and a Hill coefficient, n, of 1.81. Extracellular Ba2+ blocked the currents in a concentration- and voltage-dependent manner. The dependence of the KD for the blockade was analysed using a Woodhull-type treatment, locating the ion interaction site at 19% of the distance across the electrical field of the membrane and a KD (0 mV) of 7 mmol/l. Tetraethylammonium and 4-aminopyridine were without effect whilst quinine and quinidine blocked the currents with concentrations for half-maximum effects equal to 7 mumol/l and 3.5 mumol/l, respectively. The unfractionated venom of the scorpion Leiurus quinquestriatus (LQV) blocked the K

  12. Some structural differentiations in the HeLa cell: heavy bodies, annulate lamellae and cotte de maillet endoplasmic reticulum

    Franke, Werner W.; Scheer, Ulrich

    2010-01-01

    A small fraction of HeLa cells within an exponentially growing culture showed cisternal differentiations, such as cytoplasmic as well as intranuclear annulate lamellae and special smooth surfaced endoplasmic reticulum aggregates with a typical "Cotte de maillet" appearance. Additionally, clusters of dense granules were observed in the cytoplasm which were often associated with polysomes and strongly resembled the so-called "heavy bodies" known in particular in diverse oocytes. The functional ...

  13. Evaluation of biological activities of Physalis peruviana ethanol extracts and expression of Bcl-2 genes in HeLa cells

    Özgür Çakir; Murat Pekmez; Elif Çepni; Bilgin Candar; Kerem Fidan

    2014-01-01

    Physalis species are used in folk medicine for phytotherapeutic properties. The extracts of medicinal plants are known to possess cytotoxic and chemopreventative compounds. In this study we investigated antibacterial, antioxidant, DNA damage preventative properties of Physalis peruviana (golden berry) on leaf and shoot ethanol extracts and their effects on cytotoxicity of HeLa cells and expression of apoptotic pathway genes. Among the tested bacteria for antibacterial activity, maximum inhibi...

  14. Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells

    1995-01-01

    DNAs coding for seven murine connexins (Cx) (Cx26, Cx31, Cx32, Cx37, Cx40, Cx43, and Cx45) are functionally expressed in human HeLa cells that were deficient in gap junctional communication. We compare the permeabilities of gap junctions comprised of different connexins to iontophoretically injected tracer molecules. Our results show that Lucifer yellow can pass through all connexin channels analyzed. On the other hand, propidium iodide and ethidium bromide penetrate very poorly or not at all...

  15. Comparative experimental studies into radioimmunoscintigraphy using radioactive antibodies in animals with HeLa cell carcinomas and Yoshida sarcomas

    TPA-positive and TPA-negative tumour-bearing animal systems (HeLa cell carcinomas in RNU rats and Yoshida sarcomas in Wistar rats) were examined to show that the method of scanning can well be used to visualise tumour tissue. In this connection, further attempts were made to shed light on the specifity of immunoscintigraphy in the search for tumour tissue. 125-Iodine-anti-TPA was found to be a specific carcinoma-seeking substance. The amount of antibodies accumulating in the tumour was multiplied by previous intravenous treatment of test animals with unspecific immunoglobulin. In control studies using 125-iodine-immunoglobulin the site of the carcinomatous tissue could not be determined with sufficient diagnostic accuracy. It was found that the discriminating power of radioimmunoscintigraphy using 125-iodine-anti-TPA is quite unrelated to an increased circulation in the proliferating carcinomatous tissue. For the detection of TPA in HeLa cell carcinomas anti-TPA PAP stains were prepared. Radionuclide studies using 125-iodine-anti-TPA were also useful in the visualisation of the Yoshida sarcoma, even though this scores negative on TPA. Here, the amounts of radioactivity accumulating in the tumour were smaller than with the HeLa cell carcinoma. Moreover, peak levels were measured after no less than one day, as compared to the five days required for HeLa cell tumours to reach maximum levels. This finding would appear to provide presumptive evidence that there are other, unspecific mechanisms of tumour selectivity. (orig/MG)

  16. Ctotoxic and apoptogenic effects of Perovskia abrotanoides flower extract on MCF-7 and HeLa cell lines

    Geryani, Mohamad Ali; Mahdian, Davood; Mousavi, Seyed Hadi; Hosseini, Azar

    2016-01-01

    Objective: Perovskia abrotanoides Karel, belongs to the family Lamiaceae and grows wild alongside the mountainous roads inarid and cold climate of Northern Iran. The anti-tumor activity of P. abrotanoides root extract has been shown previously. This study was designed to examine in vitro anti-proliferative and pro-apoptotic effects of flower extract of P. abrotanoides on MCF-7 and Hela cell lines. Materials and Methods: Cells were cultured in DMEM medium with 10% fetal bovine serum, 100 units/ml penicillin and 100 µg/ml streptomycin and incubated with different concentrations of plant extracts. Cell viability was quantified by MTT assay. Apoptotic cells were determined using propidium iodide (PI) staining of DNA fragmentation by flow cytometry (sub-G1 peak). Results: P. abrotanoides extract inhibited the growth of malignant cells in a time and dose-dependent manner and 1000 µg/ml of extract following 48h of incubation was the most cytotoxic dose against Hela cell in comparison with other doses; however, in MCF-7 cells,1000 and 500 µg/ml PA induced toxicity at all time points but with different features.. Analysis of flowcytometry histogram of treated cells compared with control cells indicated that the cytotoxic effect is partly due toapoptosis induction. Conclusion: Hydro-alcoholic extract of P. abrotanoides flowers inhibits the growth of MCF-7 and HeLa cell lines, partly via inducing apoptosis. Their inhibitory effect was increased in a time and dose-dependent manner, especially in MCF7 cells. However, further studies are needed to reveal the mechanisms of P. abrotanoides extract-induced cell death.

  17. Ctotoxic and apoptogenic effects of Perovskia abrotanoides flower extract on MCF-7 and HeLa cell lines

    Mohamad Ali Geryani

    2016-06-01

    Full Text Available Objective: Perovskia abrotanoides Karel, belongs to the family Lamiaceae and grows wild alongside the mountainous roads inarid and cold climate of Northern Iran. The anti-tumor activity of P. abrotanoides root extract has been shown previously. This study was designed to examine in vitro anti-proliferative and pro-apoptotic effects of flower extract of P. abrotanoides on MCF-7 and Hela cell lines. Materials and Methods: Cells were cultured in DMEM medium with 10% fetal bovine serum, 100 units/ml penicillin and 100 µg/ml streptomycin and incubated with different concentrations of plant extracts. Cell viability was quantified by MTT assay. Apoptotic cells were determined using propidium iodide (PI staining of DNA fragmentation by flow cytometry (sub-G1 peak. Results: P. abrotanoides extract inhibited the growth of malignant cells in a time and dose-dependent manner and 1000 µg/ml of extract following 48h of incubation was the most cytotoxic dose against Hela cell in comparison with other doses; however, in MCF-7 cells,1000 and 500 µg/ml PA induced toxicity at all time points but with different features.. Analysis of flowcytometry histogram of treated cells compared with control cells indicated that the cytotoxic effect is partly due toapoptosis induction. Conclusion: Hydro-alcoholic extract of P. abrotanoides flowers inhibits the growth of MCF-7 and HeLa cell lines, partly via inducing apoptosis. Their inhibitory effect was increased in a time and dose-dependent manner, especially in MCF7 cells. However, further studies are needed to reveal the mechanisms of P. abrotanoides extract-induced cell death.

  18. In vitro activity of nonoxynol 9 on HeLa 229 cells and primary monkey cervical epithelial cells infected with Chlamydia trachomatis.

    Patton, D L; Wang, S. K.; Kuo, C C

    1992-01-01

    Nonoxynol 9 (non-9) is the active ingredient in a wide variety of vaginal contraceptive preparations. The manufacturer recommendation for optimal contraceptive practice is repeated application every 6 h. We studied the in vitro activity of non-9 against Chlamydia trachomatis (E/UW-5/Cx) and its toxicity against HeLa 229 cells and monkey cervical epithelial cells. With a contact time of 6 h, non-9 was toxic to HeLa cells at concentrations of 50 micrograms/ml or greater and to monkey cervical c...

  19. Application of a FRET probe for Caspase-3 activation in living HeLa cells by sequentially treated cisplatin and TRAIL

    Lin, Juqiang; Zhang, Zhihong; Yi, Qiushi; Zeng, Shaoqun; Luo, Qingming

    2006-02-01

    Caspase-3 is a kind of cysteine proteases that plays an important role in cell apoptosis. We have constructed a FRET (fluorescence resonance energy transfer) probe fused with ECFP (enhanced cyan fluorescence protein) and DsRed (Discosoma red fluorescent protein) with a linker containing a caspase-3 cleavage sequence (CCS, DEVD).It could be observed much change in fluorescence emission ratio when the probe was cleaved by caspase-3. Therefore, application of this probe we can real-time detected the activation of caspase-3. It was already confirmed that caspase-3 was activated in HeLa cells treated by cisplatin or TRAIL (Tumor necrosis factor (TNF)-related apoptosis-inducing ligand). In the present study, we detected the activation of caspase-3 during cisplatin or TRAIL induced apoptosis in living HeLa cells, and also observed the activation of caspase-3 caused by both cisplatin and TRAIL combined treatment. Our results demonstrated a synergistic effect between cisplatin and TRAIL. Cisplatin is one of the most broadly used drugs in the Clinical applications of cancer chemotherapy, and TRAIL, which belongs to the TNF family proteins, can selectively induce apoptosis in many transformed cells but not in normal cells. Therefore, TRAIL is a very valuably prospective utility as its potential tumor-specific cancer therapeutic. Most of anticancer drugs can induce apoptosis which mediated by the activation of caspase pathway. We can select the best synergistic effect group by our FRET probe. This finding would be useful in the design of treatment modalities for patients.

  20. Effects of 3-aminobenzamide on poly(ADP-ribose)polymerase expression,apoptosis and cell cycle progression of HeLa cells after X-ray irradiation

    2008-01-01

    The aim of this paper is to study the changes of apoptosis and cell cycle progression in HeLa cells after the poly(ADP-ribose)polymerase(PARP)was inhibited by its inhibitor 3-aminobenzamide(3-AB)and the mechanisms of PARP action on HeLa cells damaged by irradiation.Flow cytometry(FCM)was used to examine the PARP expression and the percentage of apoptotic cells and cell cycle progression.The percentage of HeLa cells with positive expression of PARP protein 2,4,8 and 12 h after administrated with 3-AB was significantly lower than that of the control(P<0.01).The percentages of apoptotic cells in the 3-AB plus irradiation group at the time points of 2,8,12 and 24 h after 2 Gy irradiation were higher than that in the irradiation group(P<0.01 or P<0.05)and the percentage of G2 cells decreased significantly(P<0.01 or P<0.05).It indicates that 3-AB can rapidly inhibit PARP expression of HeLa cells,promote cell apoptosis and block G2 arrest induced by irradiation.

  1. In vitro study of 5-aminolevulinic acid-based photodynamic therapy for apoptosis in human cervical HeLa cell line

    5-aminolevulanic acid (ALA), belonging among the promising second generation of sensitizers, was evaluated as an inducer of photodamage on HeLa (human cervical adenocarcinoma) cell line. A diode laser (635 nm) was used as a source for initiation of the photodynamic effect. We studied the influence of different incubation times, various concentrations of sensitizer, different irradiation doses and various combinations of sensitizer and light doses on the photodamage of HeLa cells. Viability of cells was determined by means of neutral red assay. The quantitative cellular uptake of ALA sensitizer was done by spectrophotometric measurements. No prominent cytotoxic or phototoxic effects on HeLa were observed due to sensitizer or light doses when studied independently of each other. However phototoxicity evoked by laser irradiated sensitizer was detected in HeLa cell line

  2. Effects of serum containing Suberect spatholobus on Hela cells%鸡血藤含药血清对Hela细胞的影响

    梁宁; 张雯艳; 杨焕琪; 陆惠燕

    2011-01-01

    目的 观察鸡血藤提取物(MHD)的毒性及体外的抗肿瘤作用.方法 以Hela细胞株为研究对象,以昆明小鼠含药血清为药物,用MTT法观察MHD含药血清对Hela细胞增殖的抑制作用.结果 MHD含药血清对Hela细胞有抑制作用,结果具有统计学意义.结论 MHD含药血清具有一定抑制Hela肿瘤细胞增殖作用.%OBJECTIVE To observe the effects of serum containing Suberect spatholobus(MHD) on Hela Cells. METHODS MTT method was used to detect the proliferation of Hela cells. RESULTS Hela cells were obviously inhibited by the serum containing MHD. CONCLUSION MHD had the inhibitory effects on Hela cells.

  3. Radiosensitizing effect of 2,4-dinitroimidazole-1-ethanol and its cytotoxicity in HeLa S3 cells

    Using cultured HeLa S3 cells, the radiosensitizing and cytotoxic effects of newly synthesized derivatives of dinitroimidazole were investigated and compared with those of misonidazole. 2,4-dinitroimidazole-1-ethanol radiosensitized hypoxic cells selectively. At 5 mM misonidazole, the enhancement ratio was 1.95; with 0.5 mM 2,4-dinitroimidazole-1-ethanol, almost the same enhancement could be obtained. This indicates that the radiosensitizing effect of the latter agent was about 10 times greater than that of misonidazole. However, its cytotoxicity was twice that of misonidazole under hypoxic conditions and there was no apparent differential cytotoxicity to hypoxic and aerobic cells. (orig.)

  4. Effect on invasion ability of cervical cancer cells after silence heparanase gene expression in Hela cells%RNAi技术沉默子宫颈癌HeLa细胞中HPA基因的表达对细胞侵袭力的影响

    吕琼莹; 张蔚; 程静; 张文婷; 钟亚娟

    2013-01-01

    Objective Design and synthesize short hairpin RNA (shRNA) expression vector of RNA for specific silencing of heparanase (HPA) gene,screened plasmid which silence effects is the best.Observe the function of ceil invasion after inhibiting the expression of HPA in cervical carcinoma cell lines (HeLa).Methods The genomic sequence of HPA gene was retrieved from GenBank database.Designed four pairs of specific oligonucleotide sequences and a negative control according to the shRNA design principles.They were inserted into the vector pYr-1.1,vectors,and transfected into HeLa cells via lipofectamine.Reverse transcription (RT)-PCR and immunofluorescence were employed to detect the expression of HPA gene in the transfected cells at the mRNA and protein levels,respectively.The plasmid were screened and transfected into HeLa cells,then transwell small room stromal invasion experiment were employed to observe the cervical carcinoma cell invasion.Results RT-PCR results of transfected HeLa cells shown that the mRNA amplification multiples were 0.54 ±0.05 in the HPA-592 group,0.89 ±0.18 in HPA-995 group,0.82 ±0.22 in the HPA-1351 group,0.91 ±0.47 in HPA-1658 group.While,they were 1.31 ±0.72 and 1.09 ±0.16 in negative control and blank control group,respectively.Green fluorescence was visible in the cytoplasm,which indicated that the HPA protein was expressed in the cytoplasm,of them the weakest green fluorescence in the HPA-592 group.The relative numbers of invasive cells among the HeLa cells were as follows:182 ±6 in the blank control group,258 ± 17 in the negative control group,and 44 ± 4 in the HPA-592-specific interference group(P < 0.01).Conclusion Successfully screened shRNA vector targeting human HPA,efficiently inhibit expression of HPA gene when transfected into HeLa cells,and significantly reduced the invasion capacity of cervical carcinoma cells.%目的 利用RNA干扰(RNAi)技术沉默宫颈癌细胞系HeLa细胞中乙酰肝素酶(HPA)基因的表达,并探

  5. An evidence on G2/M arrest, DNA damage and caspase mediated apoptotic effect of biosynthesized gold nanoparticles on human cervical carcinoma cells (HeLa)

    Jeyaraj, M. [Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu (India); Arun, R. [Department of Biomedical Sciences, Bharathidasan University, Tiruchirappalli 620024 (India); Sathishkumar, G. [Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu (India); MubarakAli, D. [Central Inter-Disciplinary Research Facility, Mahatma Gandhi Medical College and Research Institute Campus, Pondicherry 607402 (India); Rajesh, M.; Sivanandhan, G.; Kapildev, G.; Manickavasagam, M. [Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu (India); Thajuddin, N. [Department of Microbiology, Bharathidasan University, Tiruchirappalli 620024 (India); Ganapathi, A., E-mail: aganapathi2007@gmail.com [Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu (India)

    2014-04-01

    Highlights: • Gold nanoparticles (AuNPs) have been synthesized using Podophyllum hexandrum L. • AuNPs induces the oxidative stress to cell death in human cervical carcinoma cells. • It activates the caspase-cascade to cellular death. • It is actively blocks G2/M phase of cell cycle. - Abstract: Current prospect of nanobiotechnology involves in the greener synthesis of nanostructured materials particularly noble metal nanoparticles for various biomedical applications. In this study, biologically (Podophyllum hexandrum L.) synthesized crystalline gold nanoparticles (AuNPs) with the size range between 5 and 35 nm were screened for its anticancereous potential against human cervical carcinoma cells (HeLa). Stoichiometric proportion of the reaction mixture and conditions were optimized to attain stable nanoparticles with narrow size range. Different high throughput techniques like transmission electron microscope (TEM), X-ray diffraction (XRD) and UV–vis spectroscopy were adopted for the physio-chemical characterization of AuNPs. Additionally, Fourier transform infrared spectroscopy (FTIR) study revealed that the water soluble fractions present in the plant extract solely influences the reduction of AuNPs. Sublimely, synthesized AuNPs exhibits an effective in vitro anticancer activity against HeLa cells via induction of cell cycle arrest and DNA damage. Furthermore, it was evidenced that AuNPs treated cells are undergone apoptosis through the activation of caspase cascade which subsequently leads to mitochondrial dysfunction. Thereby, this study proves that biogenic colloidal AuNPs can be developed as a promising drug candidature for human cervical cancer therapy.

  6. Control of placental alkaline phosphatase gene expression in HeLa cells: induction of synthesis by prednisolone and sodium butyrate

    HeLa S3 cells produce an alkaline phosphatase indistinguishable from the enzyme from human term placenta. The phosphatase activity in these cells was induced by both prednisolone and sodium butyrate. Both agents stimulated de novo synthesis of the enzyme. The increase in phosphatase activity paralleled the increase in immunoactivity and biosynthesis of placental alkaline phosphatase. The fully processed phosphatase monomer in control, prednisolone-treated or butyrate-treated cells was a 64.5 K polypeptide, measured by both incorporation of L-[35S]methionine into enzyme protein and active-site labeling. The 64.5K polypeptide was formed by the incorporation of additional N-acetylneuraminic acid moieties to a precursor polypeptide of 61.5K. However, this biosynthetic pathway was identified only in butyrate-treated cells. In prednisolone-treated cells, the processing of 61.5K to 64.5K monomer was accelerated, and the presence of the 61.5 precursor could only be detected by either neuraminidase or monensin treatment. Phosphatase mRNA which comigrated with the term placental alkaline phosphatase mRNA of 2.7 kilobases was induced in the presence of either prednisolone or butyrate. Alkaline phosphatase mRNA is untreated HeLa S3 cells migrated slightly faster than the term placental alkaline phosphatase mRNA. Butyrate also induced a second still faster migrating alkaline phosphatase mRNA. Both prednisolone and butyrate increased the steady-state levels of placental alkaline phosphatase mRNA. The data indicate that the increase in phosphatase mRNA by prednisolone and butyrate resulted in the induction of alkaline phosphatase activity and biosynthesis in HeLa S3 cells. Furthermore, both agents induced the expression of different alkaline phosphatase gene transcripts without altering its protein product

  7. Inhibition of autophagic flux by ROS promotes apoptosis during DTT-induced ER/oxidative stress in HeLa cells.

    Xiang, Xi-Yan; Yang, Xiao-Chun; Su, Jin; Kang, Jing-Song; Wu, Yao; Xue, Ya-Nan; Dong, Yu-Tong; Sun, Lian-Kun

    2016-06-01

    As targets for cancer therapy, endoplasmic reticulum (ER) stress and autophagy are closely linked. However, the signaling pathways responsible for induction of autophagy in response to ER stress and its cellular consequences appear to vary with cell type and stimulus. In the present study, we showed that dithiothreitol (DTT) induced ER stress in HeLa cells in a time- and dose-dependent fashion. With increased ER stress, reactive oxygen species (ROS) production increased and autophagy flux, assessed by intracellular accumulation of LC3B-II and p62, was inhibited. N-acetyl-L-cysteine (NAC), a classic antioxidant, exacerbated cell death induced by 3.2 mM of DTT, but attenuated that induced by 6.4 mM DTT. Low cytotoxic doses of DTT transiently activated c-JNU N-terminal kinase (JNK) and p38, whereas high dose of DTT persistently activated JNK and p38 and simultaneously reduced extracellular signal-regulated kinase (ERK) activity. Combined treatment with DTT and U0126, an inhibitor of ERK upstream activators mitogen-activated protein kinase (MAPK) kinase 1 and 2 (MEK1/2), blocked autophagy flux in HeLa cells. This effect was similar to that caused by a combination of DTT and chloroquine (CQ). These data suggested that insufficient autophagy was accompanied by increased ROS production during DTT-induced ER stress. ROS appeared to regulate MAPK signaling, switching from a pro-survival to a pro-apoptotic signal as ER stress increased. ERK inhibition by ROS during severe ER stress blocked autophagic flux. Impaired autophagic flux, in turn, aggravated ER stress, ultimately leading to cell death. Taken together, our data provide the first reported evidence that ROS may control cell fate through regulating the MAPK pathways and autophagic flux during DTT-induced ER/oxidative stress. PMID:27035858

  8. Development of electrochemical reporter assay using HeLa cells transfected with vector plasmids encoding various responsive elements

    Electrochemical assay using HeLa cell lines transfected with various plasmid vectors encoding SEAP (secreted alkaline phosphatase) as the reporter has been performed by using SECM (scanning electrochemical microscopy). The plasmid vector contains different responsive elements that include GRE (glucocorticoid response elements), CRE (cAMP responsive elements), or κB (binding site for NFκB (nuclear factor kappa B)) upstream of the SEAP sequence. The transfected HeLa cells were patterned on a culture dish in a 4 x 4 array of circles of diameter 300 μm by using the PDMS (poly(dimethylsiloxane)) stencil technique. The cellular array was first exposed to 100 ng mL-1 dexamethasone, 10 ng mL-1 forskolin, or 100 ng mL-1 TNF-α (tumor necrosis factor α) after which it was further cultured in an RPMI culture medium for 6 h. After incubation, the cellular array was soaked in a measuring solution containing 4.7 mM PAPP (p-aminophenylphosphate) at pH 9.5, following which electrochemical measurements were performed immediately within 40 min. The SECM method allows parallel evaluation of different cell lines transfected with pGRE-SEAP, pCRE-SEAP, and pNFκB-SEAP patterned on the same solid support for detection of the oxidation current of PAP (p-aminophenol) flux produced from only 300 HeLa cells in each stencil pattern. The results of the SECM method were highly sensitive as compared to those obtained from the conventional CL (chemiluminescence) protocol with at least 5 x 104 cells per well.

  9. Development of electrochemical reporter assay using HeLa cells transfected with vector plasmids encoding various responsive elements

    Shiku, Hitoshi, E-mail: shiku@bioinfo.che.tohoku.ac.jp [Graduate School of Environmental Studies, Tohoku University, 6-6-11-604 Aramaki-Aoba, Sendai 980-8579 (Japan); Takeda, Michiaki; Murata, Tatsuya [Graduate School of Environmental Studies, Tohoku University, 6-6-11-604 Aramaki-Aoba, Sendai 980-8579 (Japan); Akiba, Uichi; Hamada, Fumio [Graduate School of Engineering and Resource Science, Akita University, 1-1 Tegata gakuen-machi, Akita 010-8502 (Japan); Matsue, Tomokazu, E-mail: matsue@bioinfo.che.tohoku.ac.jp [Graduate School of Environmental Studies, Tohoku University, 6-6-11-604 Aramaki-Aoba, Sendai 980-8579 (Japan)

    2009-04-27

    Electrochemical assay using HeLa cell lines transfected with various plasmid vectors encoding SEAP (secreted alkaline phosphatase) as the reporter has been performed by using SECM (scanning electrochemical microscopy). The plasmid vector contains different responsive elements that include GRE (glucocorticoid response elements), CRE (cAMP responsive elements), or {kappa}B (binding site for NF{kappa}B (nuclear factor kappa B)) upstream of the SEAP sequence. The transfected HeLa cells were patterned on a culture dish in a 4 x 4 array of circles of diameter 300 {mu}m by using the PDMS (poly(dimethylsiloxane)) stencil technique. The cellular array was first exposed to 100 ng mL{sup -1} dexamethasone, 10 ng mL{sup -1} forskolin, or 100 ng mL{sup -1} TNF-{alpha} (tumor necrosis factor {alpha}) after which it was further cultured in an RPMI culture medium for 6 h. After incubation, the cellular array was soaked in a measuring solution containing 4.7 mM PAPP (p-aminophenylphosphate) at pH 9.5, following which electrochemical measurements were performed immediately within 40 min. The SECM method allows parallel evaluation of different cell lines transfected with pGRE-SEAP, pCRE-SEAP, and pNF{kappa}B-SEAP patterned on the same solid support for detection of the oxidation current of PAP (p-aminophenol) flux produced from only 300 HeLa cells in each stencil pattern. The results of the SECM method were highly sensitive as compared to those obtained from the conventional CL (chemiluminescence) protocol with at least 5 x 10{sup 4} cells per well.

  10. Interaction of hyperthermia and radiation in tolerant and nontolerant HeLa S3 cells: role of DNA polymerase inactivation

    The activities of DNA polymerase α and β were measured in tolerant and nontolerant HeLa S3 suspension cells. The heat-inactivation of the enzymes and their recovery when cells were incubated at 370C after the heat challenge was compared to the synergistic action of heat and x-radiation and its disappearance at the level of cell survival. Thermotolerant cells were radiosensitized by heat similarly to nontolerant cells, but the sensitization decreased more rapidly in the tolerant cells when time at 370C was allowed between the two treatments. For polymerase activities the extent of inactivation, as well as the kinetics of recovery, were similar in tolerant and nontolerant cells. (author)

  11. Evaluation of cytotoxicity of Moringa oleifera Lam. callus and leaf extracts on Hela cells

    Abbas Jafarain; Gholamreza Asghari; Erfaneh Ghassami

    2014-01-01

    Background: There are considerable attempts worldwide on herbal and traditional compounds to validate their use as anti-cancer drugs. Plants from Moringaceae family including Moringa oleifera possess several activities such as antitumor effect on tumor cell lines. In this study we sought to determine if callus and leaf extracts of M. oleifera possess any cytotoxicity. Materials and Methods: Ethanol-water (70-30) extracts of callus and leaf of M. oleifera were prepared by maceration method...

  12. Genistein Increase Intracellular Distribution of the High Motility Group Box - 1 through p38 Pathway in HeLa culture cells induced by Tumor Necrosis Factor - α

    Merlita Herbani

    2014-05-01

    Full Text Available Cervical cancer is one kind of many cancers that cause death to women around the world. Many studies had support the statement that inflammation has a strong linkage with cancer development. Several factors like proinflammatory factor can influence tumor cell microenvironment, and induce a faster proliferation. TNF-α is suspected can induce proliferation. While cancer itself can induce inflammation, which is marked by several marker. One of them is HMGB1, released from the cell as active secretory lysosomes or passive diffusion. Genistein has demonstrated growth inhibitory effects of various types of cancer cells. It inhibits tyrosine kinase pathway, which can be activated by TNF-α. One of those pathways that have the link with proliferation is p38. This study tries to reveal about inhibitory effect of genistein toward p38 pathway that had been activated by TNF-α. This research was conducted by exposing cultured HeLa cells with various doses of genistein for 90 minutes, and then exposed to TNF-α 10 ng / mL for 20 minutes. Observations were made with a confocal microscope, by staining the cells with pp38-TRITC and HMGB1 antibody. The intensity was measured and analyzed by Fluoview software. The results suggest that there be significant differences between pp38 intranuclear intensity and HMGB1 extranuclear intensity of each dose of genistein (p = 0.000, ANOVA. pp38 and HMGB1 intensity were increased along with increasing genistein dose, but at high dose there were noted decreasing of pp38 and HMGB1 intensity. At apoptotic dose, pp38 and HMGB1 intensity were increased markedly, showing the effect of apoptosis. In general, increasing doses of genistein increase intranuclear p38 activation and HMGB1 extranuclear translocation. So there were a strong linkage between p38 activation and HMGB1 translocation in this study.

  13. Gene expression responses of HeLa cells to chemical species generated by an atmospheric plasma flow

    Highlights: • Response of HeLa cells to a plasma-irradiated medium was revealed by DNA microarray. • Gene expression pattern was basically different from that in a H2O2-added medium. • Prominently up-/down-regulated genes were partly shared by the two media. • Gene ontology analysis showed both similar and different responses in the two media. • Candidate genes involved in response to ROS were detected in each medium. - Abstract: Plasma irradiation generates many factors able to affect the cellular condition, and this feature has been studied for its application in the field of medicine. We previously reported that hydrogen peroxide (H2O2) was the major cause of HeLa cell death among the chemical species generated by high level irradiation of a culture medium by atmospheric plasma. To assess the effect of plasma-induced factors on the response of live cells, HeLa cells were exposed to a medium irradiated by a non-lethal plasma flow level, and their gene expression was broadly analyzed by DNA microarray in comparison with that in a corresponding concentration of 51 μM H2O2. As a result, though the cell viability was sufficiently maintained at more than 90% in both cases, the plasma-medium had a greater impact on it than the H2O2-medium. Hierarchical clustering analysis revealed fundamentally different cellular responses between these two media. A larger population of genes was upregulated in the plasma-medium, whereas genes were downregulated in the H2O2-medium. However, a part of the genes that showed prominent differential expression was shared by them, including an immediate early gene ID2. In gene ontology analysis of upregulated genes, the plasma-medium showed more diverse ontologies than the H2O2-medium, whereas ontologies such as “response to stimulus” were common, and several genes corresponded to “response to reactive oxygen species.” Genes of AP-1 proteins, e.g., JUN and FOS, were detected and notably elevated in the plasma-medium. These

  14. Sulfated fucan from marine alga inhibits HeLa cells infection by HTLV-1 free particles: semi-quantitative analysis

    Maria T. V. Romanos

    2011-04-01

    Full Text Available A sulfated fucan from Laminaria abyssalis marine alga prevented the interaction of HTLV-1 particles, purified from the MT-2 cell line, with HeLa cells. The infection obtained using a concentrated virus suspension was detected only by amplification of the newly synthesized HTLV-1 proviral cDNA by the nested-polymerase chain reaction (PCR. The sulfated polysaccharide was not toxic to the cells at a concentration of 100 µg/mL and prevented infection by the viral particles when added to the cell monolayers. The proviral cDNA was only detected when the sulfated polysaccharide was added to the cells three hours post-infection, indicating that the inhibitory activity occurred in the initial stages of virus-cell interaction. Our results demonstrate, for the first time, the ability of a sulfated fucan from marine algae to inhibit virus transmission through free virus particles.

  15. Mechanism of the radiosensitizing effect of low dose ionizing radiation in the HeLa cell culture

    The sensitizing effect of preliminary irradiation (10 R) 2-3 min prior to applying the main dose of 490 R was studied on a HeLa cell culture. The overall dose, divided in this manner, was found to initially have the same stimulative effect as 10 R. The inhibitory action of the main dose showed only after 1.5-2.0 hr. The results on the radiosensitizing effect of preliminary irradiation with 10 R might be explained by the delayed action of a complex of adaptive responses. An examination of the dosage curve showed preliminary irradiation with 10 R to cause a decline in reparation processes

  16. Low-level laser therapy: Effects on human face aged skin and cell viability of HeLa cells exposed to UV radiation

    Mezghani Sana

    2015-01-01

    Full Text Available Chronic and excessive exposure to UV radiation leads to photoaging and photocarcinogenesis. Adequate protection of the skin against the deleterious effects of UV irradiation is essential. Low-level laser therapy (LLLT is a light source in the red to near-infrared range that has been accepted in a variety of medical applications. In this study, we explored the effect of LLLT in human face aged skin and the cell viability of HeLa cells exposed to UV radiation. We found that LLLT significantly reduced visible wrinkles and the loss of firmness of facial skin in aging subjects. Additionally, treatment of cultured HeLa cells with LLLT prior to or post UVA or UVB exposure significantly protected cells from UV-mediated cell death. All results showed the beneficial effects of LLLT on relieving signs of skin aging and its prevention and protection of the cell viability against UV-induced damage.

  17. The fibrate decreases radiation sensitivity via peroxisome proliferator-activated receptor {alpha}-mediated superoxide dismutase induction in HeLa cells

    Liu, Xianguang; An, Zhengzhe; Song, Hye Jin; Kim, Won Dong; Park, Woo Yoon [Chungbuk National University College of Medicine, Cheongju (Korea, Republic of); Jang, Seong Soon [The Catholic University of Korea College of Medicine, Seoul (Korea, Republic of); Yu, Jae Ran [Konkuk University College of Medicine, Chungju (Korea, Republic of)

    2012-06-15

    The fibrates are ligands for peroxisome proliferator-activated receptor (PPAR) {alpha} and used clinically as hypolipidemic drugs. The fibrates are known to cause peroxisome proliferation, enhance superoxide dismutase (SOD) expression and catalase activity. The antioxidant actions of the fibrates may modify radiation sensitivity. Here, we investigated the change of the radiation sensitivity in two cervix cancer cell lines in combination with fenofi brate (FF). Activity and protein expression of SOD were measured according to the concentration of FF. The mRNA expressions were measured by using real time reverse-transcription polymerase chain reaction. Combined cytotoxic effect of FF and radiation was measured by using clonogenic assay. In HeLa cells total SOD activity was increased with increasing FF doses up to 30 {mu}M. In the other hand, the catalase activity was increased a little. As with activity the protein expression of SOD1 and SOD2 was increased with increasing doses of FF. The mRNAs of SOD1, SOD2, PPAR{alpha} and PPAR{gamma} were increased with increasing doses of FF. The reactive oxygen species (ROS) produced by radiation was decreased by preincubation with FF. The surviving fractions (SF) by combining FF and radiation was higher than those of radiation alone. In Me180 cells SOD and catalase activity were not increased with FF. Also, the mRNAs of SOD1, SOD2, and PPAR{alpha} were not increased with FF. However, the mRNA of PPAR{gamma} was increased with FF. FF can reduce radiation sensitivity by ROS scavenging via SOD induction in HeLa. SOD induction by FF is related with PPAR{alpha}.

  18. Evaluation of biological activities of Physalis peruviana ethanol extracts and expression of Bcl-2 genes in HeLa cells

    Özgür Çakir

    2014-06-01

    Full Text Available Physalis species are used in folk medicine for phytotherapeutic properties. The extracts of medicinal plants are known to possess cytotoxic and chemopreventative compounds. In this study we investigated antibacterial, antioxidant, DNA damage preventative properties of Physalis peruviana (golden berry on leaf and shoot ethanol extracts and their effects on cytotoxicity of HeLa cells and expression of apoptotic pathway genes. Among the tested bacteria for antibacterial activity, maximum inhibition zone was determined in Lactococcus lactis. The phenolic content was found higher in leaf extracts than shoot extracts. The antioxidant activity showed the highest TEAC values of the leaf (2 mg/mL and the shoot (0.5 mg/mL extracts as 0.291±0.04 and 0.192±0.015, respectively. In DNA damage prevention assay both leaf and shoot extracts, especially 30 and 20 µg/mL concentrations, exhibited significant protection against DNA damage-induced by hydroxyl radical generated by Fenton reaction. Our results suggest that leaf and shoot extracts possess cytotoxic effect on HeLa cells when applied as 100 µg/mL concentration. Also mRNA expression analysis showed the alteration of antiapoptotic genes, so the results suggest that P. peruviana ethanol extracts induce apoptotic cell death and should be investigated for identification of active compounds and their mechanisms of action.

  19. Oridonin induces apoptosis of HeLa cells via altering expres sion of Bcl-2/Bax and activating caspase-3/ICAD pathway

    Chun-ling ZHANG; Li-jun WU; Shin-ichi TASHIRO; Satoshi ONODERA; Takashi IKEJIMA

    2004-01-01

    AIM: To study the mechanisms by which oridonin inhibited HeLa cell growth in vitro. METHODS: Viability of oridonin-induced HeLa cells was measured by MTT assay. Apoptotic cells with condensed nuclei were visualized by phase contrast microscopy. Nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis.Caspase activity was assayed using fiuorometric protease assay. ICAD, Bcl-2, and Bax proteins expression were detected by Western blot analysis. RESULTS: Oridonin induced oligonucleosomal fragmentation of DNA and increased caspase-3 activity, on the other hand, reduced the expression of inhibitor of caspase-3-activated DNase (ICAD), a caspase-3 substrate, at 12 h in HeLa cells. Oridonin-induced DNA fragmentation, caspase-3 activation and down-regulation of ICAD expression were effectively inhibited by a caspase-3 inhibitor, z-DEVD-fmk (z-AspGlu-Val-Asp-fmk). However, pretreatment with an inhibitor of poly (ADP-ribose) polymerase (PARP), 3, 4-dihydro5-[4-(1-piperidinyl)butoxy]-1 (2H)-isoquinolinone (DPQ), did not suppress oridonin-induced HeLa cell death. In addition, oridonin-induced apoptosis was associated with an increase in the expression of the apoptosis inducer Bax, and a significant reduction in expression of the apoptosis suppressor Bcl-2 in mitochondria. CONCLUSION:Oridonin induces HeLa cells apoptosis by altering balance of Bcl-2 and Bax protein expression and activation of caspase-3/ICAD pathway.

  20. Radioadaptive response to the medium-mediated bystander induction of DNA strand breaks in HeLa cells

    Full text: Numerous investigators have reported two cellular responses of importance at low doses that have a potential impact on the risk estimation of ionizing radiation. The radioadaptive response confers resistance to a subsequent dose by a low priming dose, while the bystander effect exaggerates the effect of small doses. The present study was conducted to examine the interaction of the radioadaptive response with the bystander effect in HeLa cells. The culture was irradiated with 0.5 to 8 Gy of 140 kVp X-rays and one hour later, the medium was taken, passed through a filter and transferred to the parallel culture of non-irradiated HeLa cells as non-targeted cells. After incubation for 30 min, the induced DNA damage was analyzed by the single cell gel-electrophoresis assay under alkaline or neutral conditions. The treatments resulted in a dose-dependent increase in tail moment under either conditions, indicating the induction of DNA single- and double-strand breaks. The clonogenic survival of non-irradiated cells was also reduced after they were cultured in the medium that was taken from irradiated cultures. Any change was not observed when the medium alone was irradiated. These results give the disputed evidence that certain genotoxic factor(s) released from irradiated cells into the culture medium can induce DNA strand breaks leading to cell death. It is also suggested that physical contact between irradiated and non-irradiated cells may not be required for the bystander effect. In adapted cells that were pre-exposed to 5 cGy of X-rays and cultured for 4 h beforehand, the yield of DNA strand breaks induced by X-rayed medium was reduced by about 50 %. The results, in conjunction with our early finding (Ikushima et al., 1996) suggest that the radioadaptive response resulting from such a low dose may diminish the bystander effect through an enhanced DNA repair function

  1. Cell-Cycle-Dependent Variations in the FTIR Spectroscopy of HeLa Cells Treated with Trichostatin A

    ZHANG Feng-qiu; QI Jian; YANG Zhan-guo

    2011-01-01

    It is quite complex to evaluate the mechanism of action for antitumor drugs on cancer cells.Studies have pointed out that there is an unique advantage of Fourier transform infrared spectrum to obtain a fingerprint of all molecules present in the cells when cancer cells were exposed to anti-cancer drugs.Trichostatin A (TSA) is a most potent reversible inhibitor of mammalian histone deacetylases.It can inhibit cancer cell growth in vitro and in vivo.In the present study,HeLa cells were exposed to 0,50,100,200,300 and 400 nmol · L-1 TSA,and FTIR spectra were applied to evaluate the effect of TSA on cancer cells.Results show that there is some significant relationship between the changes in FTIR absorption and cell cycle arresting.On the other hand,this investigation shows that the concentration of TSA had to be more than 200 nmol · L-1 in order to ensure A1080 cm-1/A1540cm-1 ≥1 for inhibiting cell proliferation.

  2. Lung cancer - small cell

    Cancer - lung - small cell; Small cell lung cancer; SCLC ... About 15% of all lung cancer cases are SCLC. Small cell lung cancer is slightly more common in men than women. Almost all cases of SCLC ...

  3. Lung cancer - small cell

    Cancer - lung - small cell; Small cell lung cancer; SCLC ... About 15% of all lung cancer cases are SCLC. Small cell lung cancer is slightly more common in men than women. Almost all cases of SCLC are ...

  4. Apoptosis in HeLa cell exposed to different dose, dose-rate of 32P β-irradiation and the correlation with cell-killing efficacy

    In an attempt to elucidate some aspects of the radiobiological basis of targeted radiotherapy in oncology, the apoptosis occurred have been studied in Hela cell lines after exposing to different doses and dose-rate radiation of 32P and the relationship between apoptosis occurred and the capacity of cell proliferation, which might be of help to the understanding of targeted radiotherapy. Asynchronous Hela cells were exposed to β radiation from 32P absorbed in filter papers which were put closely under culture dishes of growing monolayer of Hela cell. The radiation response characteristics to different dose, dose-rate and radiation time were evaluated through cell-proliferation assessed by the colony-forming assay, cell cycle perturbation studied by flow cytometry and quantity analysis of apoptosis analyzed by flow cytometry and fluorescence microscopy. Morphological and flow cytometry analysis showed a delayed apoptosis. The programmed cell death approached a plateau between 48-72h post-irradiation. Electron and fluorescence microscopic studies showed the presence of morphologically apoptotic cells. Single dose radiation showed a higher apoptosis ratio than multiple low dose radiation, which did not correlate with clonal-forming assay, suggesting apoptosis ratio at a near time point post-irradiation is not a convincing indicator of radiation efficacy in the current experimental setting

  5. Purification and characterization of the glycoprotein hormone α-subunit-like material secreted by HeLa cells

    The protein secreted by HeLa cells that cross-reacts with antiserum developed against the α-subunit of human chorionic gonadotropin (hCG) has been purified approximately 30,000-fold from concentrated culture medium by organic solvent fractionation followed by ion exchange, gel filtration, and lectin affinity chromatography. The final preparation had a specific activity (by RIA) of 6.8 x 105 ng of α/mg of protein and appeared homogeneous by electrophoresis on reducing/denaturing polyacrylamide gels (SDS-PAGE). Amino acid analysis indicated that HeLa-α had a composition very similar to that of the urinary hCG α-subunit. However, comparison of hCG-α and HeLa-α demonstrated that the tumor-associated subunit was not identical with its normal counterpart. The purified tumor protein had an apparent molecular weight greater than that of the urinary α-subunit when analyzed by SDS-PAGE, and this difference was even greater when a partially purified preparation was examined by an immunoblot technique (Western). Isoelectric focusing of the HeLa and hCG subunits demonstrated that the tumor protein had a lower pI. Immunoprecipitation and electrophoresis of α-subunit from HeLa cultures labeled with [3H]fucose indicated that the tumor subunit was fucosylated, whereas analysis of hCG-α hydrosylates by HPLC confirmed previous reports that the placental subunit does not contain fucose. The results indicate that, regardless of whether or not a single α-subunit gene is being expressed in both normal and neoplastic tissues, posttranslational modifications lead to a highly altered subunit in the tumor. The differences observed may be useful in diagnosing neoplastic vs hyperplastic conditions and may lend insight into the mechanism of ectopic hormone production by tumors

  6. Time-course proteomics dataset monitoring HeLa cells subjected to DTT induced endoplasmic reticulum stress.

    Cheng, Zhe; Rendleman, Justin; Vogel, Christine

    2016-09-01

    The data described here provide an analysis of the dynamic response of HeLa cell proteome to dithiothreitol (DTT) inducing stress of the endoplasmic reticulum (ER). During ER stress, accumulation of misfolded and unfolded proteins in the lumen of the ER initiates the Unfolded Protein Response (UPR), resulting in a large-scale redistribution of proteins. We used label-free mass spectrometry to monitor the proteomic changes of HeLa cells during a 30-h time course, monitoring eight time points (0, 0.5, 1, 2, 8, 16, 24, and 30 h). The data are associated with the research article "Differential dynamics of the mammalian mRNA and protein expression response to misfolding stress" [1], which discusses a core dataset of 1237 proteins. Here, we present the extended dataset of 2131 proteins. The raw mass spectrometry data and the analysis results have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PRIDE: PXD002039. PMID:27547793

  7. Reactive oxygen species contribute to oridonininduced apoptosis and autophagy in human cervical carcinoma HeLa cells

    Ya-hong ZHANG; Ying-liang WU; Shin-ichi TASHIRO; Satoshi ONODERA; Takashi IKF lIMA

    2011-01-01

    Aim:To investigate the role of reactive oxygen species (ROS) in oridonin-induced apoptosis and autophagy in HeLa cells.Methods:The cell viability was measured using MTr assay.Morphological changes of apoptosis and autophagy were examined using Hoechst 33258 staining and monodansylcadaverine (MDC) staining,respectively.The mitochondrial membrane potential (△ψm) was measured using fluorescent dye rhodamine 123.DCF-induced fluorescence was used to measure the intraceliular ROS level.Protein expression was examined using Western blot.Results:Treatment of HeLa cells with oridonin (20-160 μmol/L) inhibited the cell growth in time- and concentration-dependent manners.The cells treated with oridonin (80 μmol/L) for 24 h displayed marked DNA fragmentation and MDC-positive autophagosomes.In the presence of the specific autophagy inhibitor 3-MA (2 mmol/L),the oridonin-induced apoptosis was significantly enhanced.Treatment of HeLa cells with oridonin (20-120 μmol/L) induced intracellular ROS generation in a concentration-dependent manner.In the presence of the ROS scavenger NAC (5 mmol/L),the oridinin-induced ROS generation was markedly reduced.NAC (5 mmol/L) or non-thiol antioxidant catalase (1000 U/mL) significantly reduced the oridonin-induced inhibition of cell growth and apoptosis.Furthermore,oridonin significantly reduced △ψm,which was blocked by NAC.Oridonin markedly increased Bax expression in mitochondria,and decreased Bcl-2 expression in both the cytosol and mitochondria.Oridonin also markedly increased the phosphorylation of Bcl-2 in the cytosol.All the effects were blocked by NAC.Oridonin increased the levels of caspase-3 and caspase-8,and decreased the expression of pro-caspase 3 and pro-caspase 9,which were blocked by NAC.Conclusion:ROS plays a critical role in oridonin-induced apoptosis and autophagy.

  8. Low-level laser therapy: Effects on human face aged skin and cell viability of HeLa cells exposed to UV radiation

    Mezghani Sana; Hammami Amira; Amri Mohamed

    2015-01-01

    Chronic and excessive exposure to UV radiation leads to photoaging and photocarcinogenesis. Adequate protection of the skin against the deleterious effects of UV irradiation is essential. Low-level laser therapy (LLLT) is a light source in the red to near-infrared range that has been accepted in a variety of medical applications. In this study, we explored the effect of LLLT in human face aged skin and the cell viability of HeLa cells exposed to UV radiatio...

  9. Combination of aloe-emodin with radiation enhances radiation effects and improves differentiation in human cervical cancer cells.

    Luo, Jinghua; Yuan, Yong; Chang, Pengyu; Li, Dawei; Liu, Zhiqiang; Qu, Yaqin

    2014-08-01

    The aim of the present study was to investigate the effects of aloe-emodin (AE) on the radiosensitivity and differentiation of HeLa human cervical cancer cells. Cell proliferation was assessed in the HeLa cervical cancer cell line by a methylthiazolyldiphenyl-tetrazolium bromide assay. Radiosensitivity was determined by a colony‑forming assay. Flow cytometry was used for analysis of cell cycle distribution and apoptosis. The expression of γ-H2AX and cyclin B was assessed by western blotting. Alkaline phosphatase (ALP) activity was measured by an ALP activity kit. It was demonstrated that AE inhibited the proliferation of HeLa cells in a concentration- and time-dependent manner, induced G2/M and S phase cell cycle arrest and enhanced the radiosensitivity of HeLa cells. The combination of AE and radiation induced apoptosis, upregulated cyclin B and γ-H2AX expression and further improved ALP activity compared with treatment with AE or radiation alone. AE enhanced the radiosensitivity of HeLa human cervical cancer cells in vitro, inhibited the proliferation of HeLa cells, induced G2/M phase cell cycle arrest and, in combination with radiation, induced the apoptosis and improved the differentiation of HeLa cells. PMID:24920336

  10. Inhibition of Proliferation of Human Hela Cells by Small Interference RNA against Pokemon Gene

    DENG Yi-jing; NI Bing; JIANG Man; YANG Di; LI Fan; WU Yu-zhang

    2008-01-01

    Objective:The transcriptional repressor Pokemon(encoded by the Zbtb7 gene)is a critical factor in oncogenesis.Pokemon overexpression leads to overt oncogenic transformation both in vitro and in vivo in transgenic mice. The objective of this study was to investigate the effect of retrovirus expressing the siRNA targeting Pokemon in human cervical cancer cells. Methods:We constructed and identified the recombinant retrovirus particle expressing siRNA of Pokemon gene,and then testified the suppression of recombinant plasmid and evaluated the gene-silencing effect. Results:We got the positive evaluation from colony forming experiment we found that the retrovirus expressing siRNA targeting Pokemon had repressing effect. Conclusion:Our work provides basis for the study of suppression effect of retrovirus in vivo and the design of the target-complex.

  11. An in-cell NMR study of monitoring stress-induced increase of cytosolic Ca2+ concentration in HeLa cells.

    Hembram, Dambarudhar Shiba Sankar; Haremaki, Takahiro; Hamatsu, Jumpei; Inoue, Jin; Kamoshida, Hajime; Ikeya, Teppei; Mishima, Masaki; Mikawa, Tsutomu; Hayashi, Nobuhiro; Shirakawa, Masahiro; Ito, Yutaka

    2013-09-01

    Recent developments in in-cell NMR techniques have allowed us to study proteins in detail inside living eukaryotic cells. The lifetime of in-cell NMR samples is however much shorter than that in culture media, presumably because of various stresses as well as the nutrient depletion in the anaerobic environment within the NMR tube. It is well known that Ca(2+)-bursts occur in HeLa cells under various stresses, hence the cytosolic Ca(2+) concentration can be regarded as a good indicator of the healthiness of cells in NMR tubes. In this study, aiming at monitoring the states of proteins resulting from the change of cytosolic Ca(2+) concentration during experiments, human calbindin D9k (P47M+C80) was used as the model protein and cultured HeLa cells as host cells. Time-resolved measurements of 2D (1)H-(15)N SOFAST-HMQC experiments of calbindin D9k (P47M+C80) in HeLa cells showed time-dependent changes in the cross-peak patterns in the spectra. Comparison with in vitro assignments revealed that calbindin D9k (P47M+C80) is initially in the Mg(2+)-bound state, and then gradually converted to the Ca(2+)-bound state. This conversion process initiates after NMR sample preparation. These results showed, for the first time, that cells inside the NMR tube were stressed, presumably because of cell precipitation, the lack of oxygen and nutrients, etc., thereby releasing Ca(2+) into cytosol during the measurements. The results demonstrated that in-cell NMR can monitor the state transitions of stimulated cells through the observation of proteins involved in the intracellular signalling systems. Our method provides a very useful tool for in situ monitoring of the "healthiness" of the cells in various in-cell NMR studies. PMID:23933251

  12. Design and Synthesis of New Chacones Substituted with Azide/Triazole Groups and Analysis of Their Cytotoxicity Towards HeLa Cells

    José A. F. P. Villar

    2012-08-01

    Full Text Available A series of new chalcones substituted with azide/triazole groups were designed and synthesized, and their cytotoxic activity was evaluated in vitro against the HeLa cell line. O-Alkylation, Claisen-Schmidt condensation and Cu(I-catalyzed cycloaddition of azides with terminal alkynes were applied in key steps. Fifteen compounds were tested against HeLa cells. Compound 8c was the most active molecule, with an IC50 value of 13.03 µM, similar to the value of cisplatin (7.37 µM.

  13. Confocal Raman imaging for cancer cell classification

    Mathieu, Evelien; Van Dorpe, Pol; Stakenborg, Tim; Liu, Chengxun; Lagae, Liesbet

    2014-05-01

    We propose confocal Raman imaging as a label-free single cell characterization method that can be used as an alternative for conventional cell identification techniques that typically require labels, long incubation times and complex sample preparation. In this study it is investigated whether cancer and blood cells can be distinguished based on their Raman spectra. 2D Raman scans are recorded of 114 single cells, i.e. 60 breast (MCF-7), 5 cervix (HeLa) and 39 prostate (LNCaP) cancer cells and 10 monocytes (from healthy donors). For each cell an average spectrum is calculated and principal component analysis is performed on all average cell spectra. The main features of these principal components indicate that the information for cell identification based on Raman spectra mainly comes from the fatty acid composition in the cell. Based on the second and third principal component, blood cells could be distinguished from cancer cells; and prostate cancer cells could be distinguished from breast and cervix cancer cells. However, it was not possible to distinguish breast and cervix cancer cells. The results obtained in this study, demonstrate the potential of confocal Raman imaging for cell type classification and identification purposes.

  14. In vitro Evaluation of Cytotoxic Activities of Essential Oil from Moringa oleifera Seeds on HeLa, HepG2, MCF-7, CACO-2 and L929 Cell Lines.

    Elsayed, Elsayed Ahmed; Sharaf-Eldin, Mahmoud A; Wadaan, Mohammad

    2015-01-01

    Moringa oleifera Lam. (Moringaceae) is widely consumed in tropical and subtropical regions for their valuable nutritional and medicinal characteristics. Recently, extensive research has been conducted on leaf extracts of M. oleifera to evaluate their potential cytotoxic effects. However, with the exception of antimicrobial and antioxidant activities, little information is present on the cytotoxic activity of the essential oil obtained from M. oleifera seeds. Therefore, the present investigation was designed to investigate the potential cytotoxic activity of seed essential oil obtained from M. oleifera on HeLa, HepG2, MCF-7, CACO-2 and L929 cell lines. The different cell lines were subjected to increasing oil concentrations ranging from 0.15 to 1 mg/mL for 24h, and the cytotoxicity was assessed using MTT assay. All treated cell lines showed a significant reduction in cell viability in response to the increasing oil concentration. Moreover, the reduction depended on the cell line as well as the oil concentration applied. Additionally, HeLa cells were the most affected cells followed by HepG2, MCF-7, L929 and CACO-2, where the percentages of cell toxicity recorded were 76.1, 65.1, 59.5, 57.0 and 49.7%, respectively. Furthermore, the IC50 values obtained for MCF-7, HeLa and HepG2 cells were 226.1, 422.8 and 751.9 μg/mL, respectively. Conclusively, the present investigation provides preliminary results which suggest that seed essential oil from M. oleifera has potent cytotoxic activities against cancer cell lines. PMID:26107222

  15. Gene expression responses of HeLa cells to chemical species generated by an atmospheric plasma flow

    Yokoyama, Mayo, E-mail: yokoyama@plasma.ifs.tohoku.ac.jp [Institute of Fluid Science, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577 (Japan); Johkura, Kohei, E-mail: kohei@shinshu-u.ac.jp [Department of Histology and Embryology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621 (Japan); Sato, Takehiko, E-mail: sato@ifs.tohoku.ac.jp [Institute of Fluid Science, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577 (Japan)

    2014-08-08

    Highlights: • Response of HeLa cells to a plasma-irradiated medium was revealed by DNA microarray. • Gene expression pattern was basically different from that in a H{sub 2}O{sub 2}-added medium. • Prominently up-/down-regulated genes were partly shared by the two media. • Gene ontology analysis showed both similar and different responses in the two media. • Candidate genes involved in response to ROS were detected in each medium. - Abstract: Plasma irradiation generates many factors able to affect the cellular condition, and this feature has been studied for its application in the field of medicine. We previously reported that hydrogen peroxide (H{sub 2}O{sub 2}) was the major cause of HeLa cell death among the chemical species generated by high level irradiation of a culture medium by atmospheric plasma. To assess the effect of plasma-induced factors on the response of live cells, HeLa cells were exposed to a medium irradiated by a non-lethal plasma flow level, and their gene expression was broadly analyzed by DNA microarray in comparison with that in a corresponding concentration of 51 μM H{sub 2}O{sub 2}. As a result, though the cell viability was sufficiently maintained at more than 90% in both cases, the plasma-medium had a greater impact on it than the H{sub 2}O{sub 2}-medium. Hierarchical clustering analysis revealed fundamentally different cellular responses between these two media. A larger population of genes was upregulated in the plasma-medium, whereas genes were downregulated in the H{sub 2}O{sub 2}-medium. However, a part of the genes that showed prominent differential expression was shared by them, including an immediate early gene ID2. In gene ontology analysis of upregulated genes, the plasma-medium showed more diverse ontologies than the H{sub 2}O{sub 2}-medium, whereas ontologies such as “response to stimulus” were common, and several genes corresponded to “response to reactive oxygen species.” Genes of AP-1 proteins, e.g., JUN

  16. The effects and mechanisms of cytoplasmic Macrophage colony-stimulating factor (M-CSF) on the proliferation, migration and invasion of HeLa cells

    ZHANG Meng-xia; WU Hai-yan; TU Jian; ZHANG Xiao-hong; LE Xiao-yong; TANG Sheng-song

    2008-01-01

    Objective To explore the effects and mechanisms of cytoplasmic M-CSF on the proliferation, migration and invasion of HeLa cells. Methods Both pCMV/cyto/myc vector and pCMV/cyto/myc-M-CSF vector was transfected into HeLa-cell by transfectaimine. After screening by G418, the positive clones were amplified and confirmed by RT-PCR, Western blot and immunocytochemistry. The effect of cytoplasmic MCSF on the proliferation of HeLa cells were analyzed by cell conuting and antisense oligonucleotides. The migration and invasion of cell was measured by in vitro Transwell assay and Matrigel-coated polycarbonate filters. The expression of cyclinE, cyclinD1/2/3, CDK2/4/6, Rac1, and matrix metalloproteinase 2 and 9 (MMP2/9) were assayed by semiquantitative RT-PCR. And expression of both α-tubulin and cdc42 were displayed by immunofluorescence. The activity of MMP2 was detected by gelatin zymography. Results Results A cell line (referred as to HeLa-M cell) that highly expresses cytoplasmic M-CSF was successfully established in the test. Our result indicated that HeLa-M cell had a larger volume, faster growth rate and shorter doubling time than either pCMV/cyto/myc transfected HeLa cells (referred as to HeLa-C cell) or untransfected HeLa cells (referred as to HeLa cell). M-CSF-specific antisense oligonucleoside significantly inhibited HeLa-M cell proliferation and had little effect on either HeLa-C cell or HeLa-C cell growth. Cytoplasmic M-CSF up-regulated both the expression of cyclinE, cyclinD1 and cyclinD3, CDK2, CDK 4 and CDK6,a Rho GTPase ralative protein (Rac1), cdc42 and MMP2, but had little effect on expression of MMP9 and cyclin D2. Furthermore, cytoplasmic M-CSF induced the rearrangement of the α-tubulin in HeLa cells and significantly promoted the migration and invasion of HeLa cells in vitro. Conclusions Cytoplasmic M-CSFs up-regulate the expression of cyclinE, cyclinD1 and cyclinD3, CDK2, CDK 4 and CDK6 and induces the proliferation of HeLa cells. Cytoplasmic M

  17. An in-cell NMR study of monitoring stress-induced increase of cytosolic Ca2+ concentration in HeLa cells

    Highlights: •We performed time-resolved NMR observations of calbindin D9k in HeLa cells. •Stress-induced increase of cytosolic Ca2+ concentration was observed by in-cell NMR. •Calbindin D9k showed the state-transition from Mg2+- to Ca2+-bound state in cells. •We provide a useful tool for in situ monitoring of the healthiness of the cells. -- Abstract: Recent developments in in-cell NMR techniques have allowed us to study proteins in detail inside living eukaryotic cells. The lifetime of in-cell NMR samples is however much shorter than that in culture media, presumably because of various stresses as well as the nutrient depletion in the anaerobic environment within the NMR tube. It is well known that Ca2+-bursts occur in HeLa cells under various stresses, hence the cytosolic Ca2+ concentration can be regarded as a good indicator of the healthiness of cells in NMR tubes. In this study, aiming at monitoring the states of proteins resulting from the change of cytosolic Ca2+ concentration during experiments, human calbindin D9k (P47M + C80) was used as the model protein and cultured HeLa cells as host cells. Time-resolved measurements of 2D 1H–15N SOFAST–HMQC experiments of calbindin D9k (P47M + C80) in HeLa cells showed time-dependent changes in the cross-peak patterns in the spectra. Comparison with in vitro assignments revealed that calbindin D9k (P47M + C80) is initially in the Mg2+-bound state, and then gradually converted to the Ca2+-bound state. This conversion process initiates after NMR sample preparation. These results showed, for the first time, that cells inside the NMR tube were stressed, presumably because of cell precipitation, the lack of oxygen and nutrients, etc., thereby releasing Ca2+ into cytosol during the measurements. The results demonstrated that in-cell NMR can monitor the state transitions of stimulated cells through the observation of proteins involved in the intracellular signalling systems. Our method provides a very useful tool

  18. An in-cell NMR study of monitoring stress-induced increase of cytosolic Ca{sup 2+} concentration in HeLa cells

    Hembram, Dambarudhar Shiba Sankar; Haremaki, Takahiro; Hamatsu, Jumpei; Inoue, Jin; Kamoshida, Hajime; Ikeya, Teppei; Mishima, Masaki [Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, 1-1 Minami-Osawa, Hachioji-shi, Tokyo 192-0373 (Japan); Mikawa, Tsutomu [Cellular and Molecular Biology Unit, RIKEN Advanced Science Institute, Wako-shi, Saitama 351-0198 (Japan); Hayashi, Nobuhiro [Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 B-1, Nagatsuda-chou, Midori-ku, Yokohama, Kanagawa 226-8501 (Japan); Shirakawa, Masahiro [Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Nishikyo-ku, Kyoto 615-8510 (Japan); Ito, Yutaka, E-mail: ito-yutaka@tmu.ac.jp [Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, 1-1 Minami-Osawa, Hachioji-shi, Tokyo 192-0373 (Japan)

    2013-09-06

    Highlights: •We performed time-resolved NMR observations of calbindin D{sub 9k} in HeLa cells. •Stress-induced increase of cytosolic Ca{sup 2+} concentration was observed by in-cell NMR. •Calbindin D{sub 9k} showed the state-transition from Mg{sup 2+}- to Ca{sup 2+}-bound state in cells. •We provide a useful tool for in situ monitoring of the healthiness of the cells. -- Abstract: Recent developments in in-cell NMR techniques have allowed us to study proteins in detail inside living eukaryotic cells. The lifetime of in-cell NMR samples is however much shorter than that in culture media, presumably because of various stresses as well as the nutrient depletion in the anaerobic environment within the NMR tube. It is well known that Ca{sup 2+}-bursts occur in HeLa cells under various stresses, hence the cytosolic Ca{sup 2+} concentration can be regarded as a good indicator of the healthiness of cells in NMR tubes. In this study, aiming at monitoring the states of proteins resulting from the change of cytosolic Ca{sup 2+} concentration during experiments, human calbindin D{sub 9k} (P47M + C80) was used as the model protein and cultured HeLa cells as host cells. Time-resolved measurements of 2D {sup 1}H–{sup 15}N SOFAST–HMQC experiments of calbindin D{sub 9k} (P47M + C80) in HeLa cells showed time-dependent changes in the cross-peak patterns in the spectra. Comparison with in vitro assignments revealed that calbindin D{sub 9k} (P47M + C80) is initially in the Mg{sup 2+}-bound state, and then gradually converted to the Ca{sup 2+}-bound state. This conversion process initiates after NMR sample preparation. These results showed, for the first time, that cells inside the NMR tube were stressed, presumably because of cell precipitation, the lack of oxygen and nutrients, etc., thereby releasing Ca{sup 2+} into cytosol during the measurements. The results demonstrated that in-cell NMR can monitor the state transitions of stimulated cells through the observation of

  19. Single-cell lineage tracking analysis reveals that an established cell line comprises putative cancer stem cells and their heterogeneous progeny

    Sato, Sachiko; Rancourt, Ann; Sato, Yukiko; Satoh, Masahiko S.

    2016-01-01

    Mammalian cell culture has been used in many biological studies on the assumption that a cell line comprises putatively homogeneous clonal cells, thereby sharing similar phenotypic features. This fundamental assumption has not yet been fully tested; therefore, we developed a method for the chronological analysis of individual HeLa cells. The analysis was performed by live cell imaging, tracking of every single cell recorded on imaging videos, and determining the fates of individual cells. We found that cell fate varied significantly, indicating that, in contrast to the assumption, the HeLa cell line is composed of highly heterogeneous cells. Furthermore, our results reveal that only a limited number of cells are immortal and renew themselves, giving rise to the remaining cells. These cells have reduced reproductive ability, creating a functionally heterogeneous cell population. Hence, the HeLa cell line is maintained by the limited number of immortal cells, which could be putative cancer stem cells. PMID:27003384

  20. A simple naphthalene-based fluorescent probe for high selective detection of formaldehyde in toffees and HeLa cells via aza-Cope reaction.

    Xu, Junchao; Zhang, Yue; Zeng, Lintao; Liu, Jinbiao; Kinsella, Joseph M; Sheng, Ruilong

    2016-11-01

    A simple naphthalene-based fluorescent probe (AENO) for formaldehyde (FA) was successfully synthesized, which exhibited a significant fluorescence turn-on response towards FA in aqueous solution. The probe could quantitatively determine the concentration of FA (0-1.0mM) with excellent selectivity, high sensitivity and low limit of detection (0.57µM). The sensing mechanism was proposed as 2-aza-Cope rearrangement for AENO after reaction with FA, which was confirmed by (1)H NMR, HR-MS, FT-IR, UV-vis and fluorescence spectra. The probe has been employed to determine the FA contents in several commercially available toffee samples with satisfactory performance. Thus, AENO might be used as a promising tool for quantitative detection of FA in food. Furthermore, fluorescence imaging of HeLa cells indicated that the probe was cell membrane permeable and could be used for visualizing/imaging the FA trace/transportation in cancer cells. PMID:27591661

  1. Inducible HSP70 Antagonizes IL-1β Cytocidal Effects through Inhibiting NF-kB Activation via Destabilizing TAK1 in HeLa Cells

    Cao, Xiang; Yue, Ling; Song, JiYun; Wu, Qiuyue; Li, Na; Luo, Lan; Lan, Lei; Yin, Zhimin

    2012-01-01

    Background Despite several reports describing the HSP70-mediated cytoprotection against IL-1, the precise mechanism for this phenomenon remains to be determined. Methods/Principal Findings Here we used HeLa cells, a human epithelial carcinoma cell line, to evaluate the role of inducible HSP70 in response of IL-1β stimulation. We found that inducible HSP70 antagonized the cytotoxicity of IL-1β and improved the survival of HeLa cells. Further investigation demonstrated that increased expression level of inducible HSP70 reduced the complex of TAK1 and HSP90, and promoted the degradation of TAK1 protein via proteasome pathway. By overexpression and RNAi knockdown, we showed that inducible HSP70 modulated the NF-kB but not MAPKs signalings through influencing the stability of TAK1 protein in HeLa cells. Moreover, overexpression of HSP70 attenuated the production of iNOS upon IL-1β stimulation, validating that inducible HSP70 serves as a cytopretective factor to antagonize the cytocidal effects of IL-1β in HeLa cells. Conclusions/Significance Our observations provide evidence for a novel signaling mechanism involving HSP70, TAK1, and NF-κB in the response of IL-1β cytocidal effects. This research also provides insight into mechanisms by which HSP70 exerts its cytoprotective action upon toxic stimuli in tumor cells. PMID:23185533

  2. Squamous cell skin cancer

    ... earliest form of squamous cell cancer is called Bowen disease (or squamous cell carcinoma in situ). This type ... cancer; Squamous cell carcinoma of the skin Images Bowen's disease on the hand Keratoacanthoma Keratoacanthoma Skin cancer, squamous ...

  3. Effect of inhibitors of poly(ADP-ribose) polymerase on the heat response of HeLa S3 cells.

    Burgman, P; Konings, A W

    1988-12-01

    The purpose of this study was to investigate a possible involvement of poly(ADP-ribosyl)ation reactions in hyperthermic cell killing and hyperthermic DNA strand-break induction and repair in HeLa S3 cells. The inhibitors of poly(ADP-ribose) polymerase, 3-aminobenzamide (3AB) and 4-aminobenzamide (4AB), were used as tools in this study. Both inhibitors could sensitize the cells for hyperthermic cell killing equally well, although 3AB is known to be a more effective enzyme inhibitor. The heat sensitization at the level of cell killing could be reversed when the compounds were still present during a 4-h postincubation at 37 degrees C. More heat-induced DNA strand breaks were formed in the presence of 3AB and 4AB. Repair of strand breaks was inhibited during the postincubation at 37 degrees C. Thus the effect of 3AB and 4AB on DNA strand-break repair was different from the cited effect on cell survival. It is concluded that the sensitizing effect of 3AB and 4AB on hyperthermic cell killing is not caused by inhibition of poly(ADP-ribose) polymerase and is also not related to repair of DNA strand breaks. PMID:3144718

  4. Synergistic interactions of saponins and monoterpenes in HeLa cells, Cos7 cells and in erythrocytes.

    Herrmann, Florian; Wink, Michael

    2011-10-15

    In phytomedicine complex extracts consisting of phenolics, monoterpenes or saponins are traditionally used. It is often impossible to attribute the biological activity of an extract to one or few compounds. As an explanation of the superior activity of extracts, a synergistic effect of combinations of active compounds has been suggested. Since lipophilic monoterpenes or saponins targeting the biomembrane usually accompany polar polyphenols in phytomedical preparations, we decided to investigate their effect as single substances and in combination to gain further insight into potential synergistic effects of herbal medicine. Combinations of the monoterpenes α-pinene, thymol and menthol with the monodesmosidic saponins digitonin, aescin, glycyrrhizic acid and Quillaja saponin demonstrated strong synergistic activity. The IC(50) of haemolysis was lowered by a factor of 10-100 from 316μg/ml to 2μg/ml for aescin, 157μg/ml to 11μg/ml for Quillaja saponins and 20μg/ml to 3μg/ml for digitonin when combined with thymol. A similar significant synergistic cytotoxicity occurred both in HeLa and Cos7 cells by combining the α-pinene, thymol and menthol with the saponins. The IC(50) of glycyrrhizic acid was lowered by a factor 100 from around 300μg/ml to around 1-10μg/ml and the IC(50) of aescin, digitonin and Quillaja saponins about the factor 10. Monoterpenes and monodesmosidic saponins have a common target, the biomembrane, which is present in all animal, fungal and bacterial cells. Disturbance of membrane fluidity and permeability is the mode of action. This activity is non-specific which makes it extremely difficult for bacteria and fungi to develop resistance. This explains the overall success of these molecules as defence chemicals in the plant kingdom. The synergistic effect of combinations of saponins with monoterpenes opens a complete new field of possible applications in medicine to overcome resistance in multidrug resistant microbial and human cell. PMID

  5. Phenol-soluble modulin α induces G2/M phase transition delay in eukaryotic HeLa cells.

    Deplanche, Martine; Filho, Rachid Aref El-Aouar; Alekseeva, Ludmila; Ladier, Emilie; Jardin, Julien; Henry, Gwénaële; Azevedo, Vasco; Miyoshi, Anderson; Beraud, Laetitia; Laurent, Frederic; Lina, Gerard; Vandenesch, François; Steghens, Jean-Paul; Le Loir, Yves; Otto, Michael; Götz, Friedrich; Berkova, Nadia

    2015-05-01

    Staphylococcus aureus is a gram-positive bacterium responsible for a wide range of infections. Host cell cycle alteration is a sophisticated mechanism used by pathogens to hijack the defense functions of host cells. We previously demonstrated that S. aureus MW2 (USA400) bacteria induced a G2/M phase transition delay in HeLa cells. We demonstrate here that this activity is triggered by culture supernatant compounds. Using size exclusion chromatography of the MW2 supernatant, followed by mass spectroscopy analysis of corresponding peaks, we identified phenol-soluble modulin α (PSMα) peptides as the likely candidates for this effect. Indeed, synthetic PSMα1 and PSMα3 caused a G2/M phase transition delay. The implication of PSMα in cell cycle alteration was confirmed by comparison of S. aureus Los Angeles County clone (LAC) wild-type with the isogenic mutant LAC∆psmα, which lacks the psmα operon encoding PSMα1-4. PSMα-induced G2/M transition delay correlated with a decrease in the defensin genes expression suggesting a diminution of antibacterial functions of epithelial cells. By testing the supernatant of S. aureus human clinical isolates, we found that the degree of G2/M phase transition delay correlated with PSMα1 production. We show that PSMs secreted by S. aureus alter the host cell cycle, revealing a newly identified mechanism for fostering an infection. PMID:25648996

  6. Silencing Bcl-2 Expression in Epithelial Cancer Cells Using “Smart” Particles

    Yen-Ling Lin; Guohua Jiang; Zhaocheng Zhang; Jacques E Nör; Mohamed E H ElSayed

    2014-01-01

    Short interfering RNA (siRNA) targeted against anti-apoptotic Bcl-2 protein proved to knockdown its expression and trigger cancer cell death. We used degradable, pH-sensitive, comb-like [P(EAA-co-BMA)-b-PNASI-g-P(HMA-co-TMAEMA)] polymer to condense anti-Bcl-2 siRNA into “smart” particles, which proved to shuttle their cargo past the endosomal membrane and into the cytoplasm of HeLa and UM-SCC-17B cancer cells. HeLa and UM-SCC-17B cancer cells were treated with anti-Bcl-2 particles followed by...

  7. In Vitro Ultramorphological Assessment of Apoptosis Induced by Zerumbone on (HeLa

    Siddig Ibrahim Abdel Wahab

    2009-01-01

    Full Text Available Zerumbone (ZER, a potential anticancer compound, isolated from the fresh rhizomes of Zingiber zerumbet. In this investigation, the cytotoxic properties of ZER were evaluated, on cancer cells of human cervix (HeLa, breast and ovary, and normal cells of Chinese Hamster ovary, using MTT assay. Apoptogenic effects of ZER on HeLa were studied using fluorescence microscopy (AO/PI double staining, scanning and transmission electron microscopy (SEM and TEM, and colorimetric assay of the apoptosis promoter enzyme, caspase-3. The results of MTT assay showed that ZER has less effect on normal cells compared to cancer cells. The lowest IC50 of ZER was observed on HeLa cells. Cytological observations showed nuclear and chromatin condensation, cell shrinkage, multinucleation, abnormalities of mitochondrial cristae, membrane blebbing, holes, cytoplasmic extrusions and formation of apoptotic bodies as confirmed collectively by double staining of AO/PI, SEM and TEM. Statistical analysis (two-tailed t-test of differential counting of 200 cells under fluorescence microscope revealed significant difference in apoptotic cells populations between treated and untreated HeLa cells. In addition, ZER has increased the cellular level of caspase-3 on the treated HeLa cells. It could be concluded that ZER was able to produce distinctive morphological features of cell death that corresponds to apoptosis.

  8. Effect of inhibitors of poly(ADP-ribose)polymerase on the radiation response of HeLa S3 cells

    The purpose of this study was to investigate possible involvement of poly(ADP-ribosyl)ation reactions in X-ray-induced cell killing, repair of potentially lethal damage (PLD), and formation and repair of radiation-induced DNA damage. As tools we used the inhibitors of poly(ADP-ribose)polymerase, 3-aminobenzamide (3AB), and 4-aminobenzamide (4AB). Both drugs inhibited PLD repair equally well but did not increase radiation-induced cell killing when cells were plated immediately after irradiation. 3AB affected repair of radiation-induced DNA damage, while 4AB had no effect. When 3AB was combined with aphidicolin (APC), it was found that the amount of DNA damage increased during the postirradiation incubation period. This means that the presence of 3AB stimulates the formation of DNA damage after X-irradiation. It is concluded that 3AB and 4AB sensitize HeLaS3 cells for radiation-induced cell killing by inhibiting repair of PLD. Because of the different effects of both inhibitors on repair of PLD and repair of radiation-induced DNA damage (a process known to be affected by inhibition of poly(ADP-ribosyl)ation), it is concluded that the observed inhibition of PLD repair is not caused by inhibition of poly(ADP-ribose)polymerase, and that the inhibitors affect repair of PLD and repair of DNA damage through independent mechanisms

  9. Effect of inhibitors of poly(ADP-ribose)polymerase on the radiation response of HeLa S3 cells

    Burgman, P.; Konings, A.W. (State Univ. Groningen (Netherlands))

    1989-08-01

    The purpose of this study was to investigate possible involvement of poly(ADP-ribosyl)ation reactions in X-ray-induced cell killing, repair of potentially lethal damage (PLD), and formation and repair of radiation-induced DNA damage. As tools we used the inhibitors of poly(ADP-ribose)polymerase, 3-aminobenzamide (3AB), and 4-aminobenzamide (4AB). Both drugs inhibited PLD repair equally well but did not increase radiation-induced cell killing when cells were plated immediately after irradiation. 3AB affected repair of radiation-induced DNA damage, while 4AB had no effect. When 3AB was combined with aphidicolin (APC), it was found that the amount of DNA damage increased during the postirradiation incubation period. This means that the presence of 3AB stimulates the formation of DNA damage after X-irradiation. It is concluded that 3AB and 4AB sensitize HeLaS3 cells for radiation-induced cell killing by inhibiting repair of PLD. Because of the different effects of both inhibitors on repair of PLD and repair of radiation-induced DNA damage (a process known to be affected by inhibition of poly(ADP-ribosyl)ation), it is concluded that the observed inhibition of PLD repair is not caused by inhibition of poly(ADP-ribose)polymerase, and that the inhibitors affect repair of PLD and repair of DNA damage through independent mechanisms.

  10. Effect of inhibitors of poly(ADP-ribose)polymerase on the radiation response of HeLa S3 cells.

    Burgman, P; Konings, A W

    1989-08-01

    The purpose of this study was to investigate possible involvement of poly(ADP-ribosyl)ation reactions in X-ray-induced cell killing, repair of potentially lethal damage (PLD), and formation and repair of radiation-induced DNA damage. As tools we used the inhibitors of poly(ADP-ribose)polymerase, 3-aminobenzamide (3AB), and 4-aminobenzamide (4AB). Both drugs inhibited PLD repair equally well but did not increase radiation-induced cell killing when cells were plated immediately after irradiation. 3AB affected repair of radiation-induced DNA damage, while 4AB had no effect. When 3AB was combined with aphidicolin (APC), it was found that the amount of DNA damage increased during the postirradiation incubation period. This means that the presence of 3AB stimulates the formation of DNA damage after X-irradiation. It is concluded that 3AB and 4AB sensitize HeLaS3 cells for radiation-induced cell killing by inhibiting repair of PLD. Because of the different effects of both inhibitors on repair of PLD and repair of radiation-induced DNA damage (a process known to be affected by inhibition of poly(ADP-ribosyl)ation), it is concluded that the observed inhibition of PLD repair is not caused by inhibition of poly(ADP-ribose)polymerase, and that the inhibitors affect repair of PLD and repair of DNA damage through independent mechanisms. PMID:2502817

  11. Mapping and identification of interferon gamma-regulated HeLa cell proteins separated by immobilized pH gradient two-dimensional gel electrophoresis

    Shaw, AC; Rossel Larsen, M; Roepstorff, P;

    1999-01-01

    magnitude of IFN-gamma responsive genes has been reported previously. Our goal is to identify and map IFN-gamma-regulated HeLa cell proteins to the two-dimensional polyacrylamide gel electrophoresis with the immobilized pH gradient (IPG) two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) system...

  12. Oxidative stress-mediated cytotoxicity and apoptosis induction by TiO2 nanofibers in HeLa cells

    Ramkumar, Kunga Mohan; Manjula, Chinnasamy; GnanaKumar, Georgepeter;

    2012-01-01

    attracted a lot of attention due to their possible negative health effects as suggested by their morphological similarities with asbestos. In the present study, therefore, toxicity of TiO2NFs was evaluated in human cervical adenocarcinoma HeLa cells. The TEM and XRD analyses showed that TiO2NFs used in this...

  13. Increased expression of cyclin B1 mRNA coincides with diminished G2-phase arrest in irradiated HeLa cells treated with staurosporine or caffeine

    The irradiation of cells results in delayed progression through the G2 phase of the cell cycle. Treatment of irradiated HeLa cells with caffeine greatly reduces the G2-phase delay, while caffeine does not alter progression of cells through the cell cycle in unirradiated cells. In this report we demonstrate that treatment of HeLa cells with the kinase inhibitor staurosporine, but not with the inhibitor H7, also results in a reduction of the G2-phase arrest after irradiation. Cell cycle progression in unirradiated cells is unaffected by 4.4 nM (2ng/ml) staurosporine, which releases the radiation-induced G2-phase arrest. In HeLa cells, the G2-phase delay after irradiation in S phase is accompanied by decreased expression of cyclin B1 mRNA. Coincident with the reduction in G2-phase delay, we observed an increase in cyclin B1 mRNA accumulation in irradiated, staurosporine-treated cells compared to cells treated with irradiation alone. Caffeine treatment of irradiated HeLa cells also resulted in an elevation in the levels of cyclin B1 message. These results support the hypothesis that diminished cyclin B1 mRNA levels influence G2-phase arrest to some degree. The findings that both staurosporine and caffeine treatments reverse the depression in cyclin B1 expression suggest that these two compounds may act on a common pathway of cell cycle control in response to radiation injury. 33 refs., 6 figs

  14. RGDS-functionalized polyethylene glycol hydrogel-coated magnetic iron oxide nanoparticles enhance specific intracellular uptake by HeLa cells

    Nazli C

    2012-04-01

    Full Text Available Caner Nazli1, Tugba Ipek Ergenc2, Yasemin Yar1, Havva Yagci Acar1,3, Seda Kizilel1,21Graduate School of Sciences and Engineering, Koç University, 2Department of Chemical and Biological Engineering, College of Engineering, Koç University, 3Department of Chemistry, Faculty of Arts and Sciences, Koç University, Istanbul, TurkeyAbstract: The objective of this study was to develop thin, biocompatible, and biofunctional hydrogel-coated small-sized nanoparticles that exhibit favorable stability, viability, and specific cellular uptake. This article reports the coating of magnetic iron oxide nanoparticles (MIONPs with covalently cross-linked biofunctional polyethylene glycol (PEG hydrogel. Silanized MIONPs were derivatized with eosin Y, and the covalently cross-linked biofunctional PEG hydrogel coating was achieved via surface-initiated photopolymerization of PEG diacrylate in aqueous solution. The thickness of the PEG hydrogel coating, between 23 and 126 nm, was tuned with laser exposure time. PEG hydrogel-coated MIONPs were further functionalized with the fibronectin-derived arginine-glycine-aspartic acid-serine (RGDS sequence, in order to achieve a biofunctional PEG hydrogel layer around the nanoparticles. RGDS-bound PEG hydrogel-coated MIONPs showed a 17-fold higher uptake by the human cervical cancer HeLa cell line than that of amine-coated MIONPs. This novel method allows for the coating of MIONPs with nano-thin biofunctional hydrogel layers that may prevent undesirable cell and protein adhesion and may allow for cellular uptake in target tissues in a specific manner. These findings indicate that the further biofunctional PEG hydrogel coating of MIONPs is a promising platform for enhanced specific cell targeting in biomedical imaging and cancer therapy.Keywords: PEG hydrogel, surface-initiated photopolymerization, nanoparticle encapsulation, agglomeration

  15. Action of caffeine on x-irradiated HeLa cells. I. Delayed inhibition of DNA synthesis

    Treatment of HeLa S3 cells with 1 mM caffeine delays progression through G1 by 1.5 hours but causes no other detectable inhibition of cell progression; it sometimes results in a large stimulation of thymidine incorporation. When this concentration is applied to cells that have been irradiated with 1-krad doses of 220-kV x rays, there is a marked suppression of both the inhibition of DNA synthesis and G2 arrest induced by the radiation. Larger doses require higher concentrations of caffeine to suppress the inhibition of DNA synthesis. Delaying addition until the rate of synthesis is at its minimum (1.5 hours after irradiation with 1 krad) results in a slightly accelerated recovery of the rate. Treatment before or during irradiation is without effect on the inhibition. Removal of the caffeine as late as 6 hours after its addition at the time of irradiation results in a prompt inhibition in DNA synthesis that mimics that observed immediately after irradiation in the absence of caffeine. These findings raise the possibility that the depression in rate of DNA systhesis might not result from radiation damage introduced into the replicon initiation system, but rather may be an indirect consequence of damage residing elsewhere in the irradiated cell

  16. Modulation of intracellular calcium homeostasis by trimethyltin chloride in human tumour cells: Neuroblastoma SY5Y and cervix adenocarcinoma HeLa S3

    Physiological modifications of intracellular Ca2+ ([Ca2+]i) levels trigger and/or regulate a diversity of cellular activities (e.g. neurotransmitter release, synaptic plasticity, muscular contraction, cell proliferation), while calcium overloads could result in cytotoxicity. Previously, we have shown that trimethyltin chloride (Me3SnCl; TMT) modulates calcium homeostasis in cervix adenocarcinoma (HeLa S3) cells [Florea, A.-M., Dopp, E., Buesselberg, D., 2005. TMT induces elevated calcium transients in HeLa cells: types and levels of response. Cell Calcium 37, 252-258]. Here we compare [Ca2+]i-changes induced by trimethyltin chloride in neuroblastoma SY5Y and HeLa S3 cells using calcium-sensitive dyes (fluo-4/AM (fluo-4) and rhod-2/AM (rhod-2)) and laser scanning microscopy (LSM). TMT-induced calcium elevations in neuroblastoma SY5Y as well as in HeLa S3 cells. [Ca2+]i rose to a sustained plateau or to transient spikes. Overall, the detected averaged increase of the maximum calcium elevation were: 0.5 μM ∼125.6%; 5 μM ∼130.1%; 500 μM ∼145% in HeLa S3 cells and 0.5 μM ∼133.3%; 5 μM ∼136.1%; 500 μM ∼147.1% in neuroblastoma SY5Y cells. The calcium rise derived from internal stores did not significantly depend on the presence of calcium in the external solution: ∼109% (no calcium added) versus ∼117% (2 mM calcium; 5 μM TMT) in HeLa cells. This difference was similar in neuroblastoma SY5Y cells, were ∼127% versus ∼136% increase (5 μM TMT) were measured. Staining of calcium stores with rhod-2 showed a TMT-induced [Ca2+]i-decrease in the stores followed by an increase of the calcium concentration in the nuclei of the two cell lines tested. Our results suggest that toxic effects in human tumour cells after exposure to trimethyltin compounds might be due to an elevation of [Ca2+]i

  17. Non-biased enrichment does not improve quantitative proteomic delineation of reovirus T3D-infected HeLa cell protein alterations

    KevinM.Coombs

    2012-01-01

    Mass spectrometry-based methods have allowed elucidation of alterations in complex proteomes, such as eukaryotic cells. Such studies have identified and measured relative abundances of thousands of host proteins after cells are infected with a virus. One of the potential limitations in such studies is that generally only the most abundant proteins are identified, leaving the deep richness of the cellular proteome largely unexplored. We differentially labeled HeLa cells with light and heavy st...

  18. Delay of HeLa cell cleavage into interphase using dihydrocytochalasin B: retention of a postmitotic spindle and telophase disc correlates with synchronous cleavage recovery

    1995-01-01

    The molecular signals that determine the position and timing of the cleavage furrow during mammalian cell cytokinesis are presently unknown. We have studied in detail the effect of dihydrocytochalasin B (DCB), a drug that interferes with actin assembly, on specific late mitotic events in synchronous HeLa cells. When cleavage furrow formation is blocked at 10 microM DCB, cells return to interphase by the criteria of reformation of nuclei with lamin borders, degradation of the cyclin B componen...

  19. Loss of FADS2 Function Severely Impairs the Use of HeLa Cells as an In Vitro Model for Host Response Studies Involving Fatty Acid Effects

    Jaudszus, Anke; Degen, Christian; Barth, Stephan W.; Klempt, Martin; Schlörmann, Wiebke; Roth, Alexander; Rohrer, Carsten; Sauerwein, Helga; Sachse, Konrad; Jahreis, Gerhard

    2014-01-01

    Scope Established epithelial cell lines equipped with pattern recognition receptors such as the Toll-like receptor (TLR)-2 are common tools for immune response studies on invading pathogens, e.g. the obligate intracellular species of Chlamydia. Moreover, such models are widely used to elucidate fatty acid-mediated immune effects. In several transformed cell lines, however, unusual loss of metabolic functions was described. The cell lines A549 and HeLa are poorly characterized in this respect....

  20. Growth-dependent modulation of casein kinase II and its substrate nucleolin in primary human cell cultures and HeLa cells

    Schneider, H R; Issinger, O G

    1989-01-01

    We have previously provided evidence that casein kinase II (CKII) and its substrate nucleolin increase concomitantly during certain development stages during embryogenesis (Schneider et al., Eur. J. Biochem. 161, 733-738). We now show that during normal growth of primary cell cultures and He......La cells CKII activity is increased concomitant with cellular growth and that the activity declines when confluency is reached. Parallel to the CKII activity increase, nucleolin, which has been shown to be a potential substrate of CKII changes its phosphorylation status, reaching a maximum at the time when...

  1. A comparison of the growth of selected mycobacteria in HeLa, monkey kidney, and human amnion cells in tissue culture.

    SHEPARD, C C

    1958-02-01

    HeLa, monkey kidney, and human amnion cells in tissue cultures were compared as sites for the multiplication of strains of tubercle bacilli or original and reduced pathogenicity, and for several other species of mycobacteria capable of causing disease in humans. The arrangement of the pathogenic species inorder of their growth rates in HeLa cells was Mycobacterium fortuitum, Mycobacterium balnei, and the "yellow bacillus," followed closely by the tubercle bacillus. This order was also correct for these species in monkey kidney and human amnion cells, and is the same as that seen in bacteriological media. The arrangement of the strains of tubercle bacilli in order of their growth rates in all three types of cells was: H37Rv, then R1Rv, and lastly H37Ra, which multiplied about as slowly as BCG. An INH-resistant strain grew about as rapidly as H37Rv. Growth of the pathogenic species occurred at about the same rates in HeLa and monkey kidney cells, but was distinctly slower in human amnion cells, which are less active metabolically. Irradiation of the cells in doses up to 5000 r did not affect the subsequent growth of mycobacteria in them. Preliminary experiments with human leprosy bacilli indicate that they can be introduced into these cells in high numbers and that the bacilli then persist for the life of the cells. PMID:13491759

  2. Analysis of Relevant Parameters for Autophagic Flux Using HeLa Cells Expressing EGFP-LC3.

    Muñoz-Braceras, Sandra; Escalante, Ricardo

    2016-01-01

    Macroautophagy (called just autophagy hereafter) is an intracellular degradation machinery essential for cell survival under stress conditions and for the maintenance of cellular homeostasis. The hallmark of autophagy is the formation of double membrane vesicles that engulf cytoplasmic material. These vesicles, called autophagosomes, mature by fusion with endosomes and lysosomes that allows the degradation of the cargo. Autophagy is a dynamic process regulated at multiple steps. Assessment of autophagy is not trivial because the number autophagosomes might not necessarily reflect the real level of autophagic degradation, the so-called autophagic flux. Here, we describe an optimized protocol for the analysis of relevant parameters of autophagic flux using HeLa cells stably expressing EGFP-LC3. These cells are a convenient tool to determine the influence of the downregulation or overexpression of specific proteins in the autophagic flux as well as the analysis of autophagy-modulating compounds. Western blot analysis of relevant parameters, such as the levels of EGFP-LC3, free EGFP generated by autophagic degradation and endogenous LC3·I-II are analyzed in the presence and absence of the autophagic inhibitor chloroquine. PMID:27613046

  3. Thymidine 5'-O-monophosphorothioate induces HeLa cell migration by activation of the P2Y6 receptor.

    Gendaszewska-Darmach, Edyta; Szustak, Marcin

    2016-06-01

    ATP, ADP, UTP, and UDP acting as ligands of specific P2Y receptors activate intracellular signaling cascades to regulate a variety of cellular processes, including proliferation, migration, differentiation, and cell death. Contrary to a widely held opinion, we show here that nucleoside 5'-O-monophosphorothioate analogs, containing a sulfur atom in a place of one nonbridging oxygen atom in a phosphate group, act as ligands for selected P2Y subtypes. We pay particular attention to the unique activity of thymidine 5'-O-monophosphorothioate (TMPS) which acts as a specific partial agonist of the P2Y6 receptor (P2Y6R). We also collected evidence for the involvement of the P2Y6 receptor in human epithelial adenocarcinoma cell line (HeLa) cell migration induced by thymidine 5'-O-monophosphorothioate analog. The stimulatory effect of TMPS was abolished by siRNA-mediated P2Y6 knockdown and diisothiocyanate derivative MRS 2578, a selective antagonist of the P2Y6R. Our results indicate for the first time that increased stability of thymidine 5'-O-monophosphorothioate as well as its affinity toward the P2Y6R may be responsible for some long-term effects mediated by this receptor. PMID:26746211

  4. Development of Microelectrode Arrays Using Electroless Plating for CMOS-Based Direct Counting of Bacterial and HeLa Cells.

    Niitsu, Kiichi; Ota, Shoko; Gamo, Kohei; Kondo, Hiroki; Hori, Masaru; Nakazato, Kazuo

    2015-10-01

    The development of two new types of high-density, electroless plated microelectrode arrays for CMOS-based high-sensitivity direct bacteria and HeLa cell counting are presented. For emerging high-sensitivity direct pathogen counting, two technical challenges must be addressed. One is the formation of a bacteria-sized microelectrode, and the other is the development of a high-sensitivity and high-speed amperometry circuit. The requirement for microelectrode formation is that the gold microelectrodes are required to be as small as the target cell. By improving a self-aligned electroless plating technique, the dimensions of the microelectrodes on a CMOS sensor chip in this work were successfully reduced to 1.2 μm × 2.05 μm. This is 1/20th of the smallest size reported in the literature. Since a bacteria-sized microelectrode has a severe limitation on the current flow, the amperometry circuit has to have a high sensitivity and high speed with low noise. In this work, a current buffer was inserted to mitigate the potential fluctuation. Three test chips were fabricated using a 0.6- μm CMOS process: two with 1.2 μm × 2.05 μm (1024 × 1024 and 4 × 4) sensor arrays and one with 6- μm square (16 × 16) sensor arrays; and the microelectrodes were formed on them using electroless plating. The uniformity among the 1024 × 1024 electrodes arranged with a pitch of 3.6 μm × 4.45 μm was optically verified. For improving sensitivity, the trenches on each microelectrode were developed and verified optically and electrochemically for the first time. Higher sensitivity can be achieved by introducing a trench structure than by using a conventional microelectrode formed by contact photolithography. Cyclic voltammetry (CV) measurements obtained using the 1.2 μm × 2.05 μm 4 × 4 and 6- μm square 16 × 16 sensor array with electroless-plated microelectrodes successfully demonstrated direct counting of the bacteria-sized microbeads and HeLa cells. PMID:26561481

  5. [Investigation of antitumorigenic effects of food-borne non-pathogenic and pathogenic Salmonella enterica strains on MEF, DU145 and HeLa cell lines].

    Altıntaş Kazar, Gamze; Şen, Ece

    2016-07-01

    Basic applications in cancer therapy may fail to eradicate cancer cells completely, they can show toxic affects to healthy cells and development of resistance to antitumor agents may increase tendency to metastasis. Bacterial therapies have the advantage of specific targetting of tumors by selective toxicity, responsiveness to external signals, self-propelling capacity, and the sense of microenvironment. The most interest on the bacterial cancer therapy is about Salmonella spp. with a special emphasis of S.Typhimurium. The aim of this study was to investigate the antitumorigenic effects of food-borne non-pathogenic and pathogenic Salmonella enterica strains on different cell cultures. Non-pathogenic Salmonella Enteriditis (A17) and pathogenic Salmonella Telaviv (A22) strains isolated from chicken carcasses which were put on the market in Edirne province (located at Thrace region of Turkey), and Salmonella Typhimurium ATCC 14028 strain were used in the study. ATCC-derived MEF (mouse embryonic fibroblasts), DU145 (human prostate cancer cells), and HeLa (human cervical cancer cells) cell lines were cocultivated with Salmonella strains of MOI (Multiplicity of infection; number of bacteria:number of cell) of 1000:1, 100:1, 10:1, 1:1, 0.1:1. The cell viability was measured by colorimetric MTT cytotoxicity assay, the percentage of apoptosis was assessed by Tali® Apoptosis Assay-Annexin V Alexa Fluor® 488 kit (Invitrogen, Molecular Probes, Life Technologies, USA), and the caspase-3 activity was determined by colorimetric protease ApoTarget™ kit (Invitrogen, BioSource International, USA). It was shown that non-pathogenic S.Enteriditis (A17) decreased cell viability approximately to 70%, wheras patogenic S.Telaviv (A22) and standart S.Typhimurium ATCC 14028 strains reduced cell viability approximately to 80%. Adversely, it was also observed that pathogenic S.Telaviv (A22) strain induces apoptosis more effectively than non-pathogenic S.Enteriditis (A17) and S

  6. Synchronization of HeLa cell cultures by inhibition of DNA polymerase alpha with aphidicolin.

    Pedrali-Noy, G; Spadari, S; Miller-Faurès, A; Miller, A O; Kruppa, J; Koch, G

    1980-01-01

    Both the inhibitory effect of aphidicolin on the replicative alpha-polymerase and the reversibility of its action in vivo (Pedrali-Noy & Spadari, 1979, Biochem. Biophys. Res. Commun. 88, 1194-2002) allow the synchronization of cells in culture. Aphidicolin prevents G1 cells from entering the DNA synthetic period, blocks cells in "S" phase, allows G2, M and G1 cells to continue the cell cycle and to accumulate at the G1/S border. Aphidicolin is a more useful reagent than hydroxyurea and thymid...

  7. hMTERF4 knockdown in HeLa cells results in sub-G1 cell accumulation and cell death

    Min Yu; Jie Dai; Weiwei Huang; Yang Jiao; Liang Liu; Min Wu; Deyong Tan

    2011-01-01

    Mitochondrial activity and cell energy status play important roles in the regulation of cell cycle and cell proliferation. Regulation of mitochondrial gene expression is crucial for mitochondrial activity regulation. The mitochondrial transcription termination factor (MTERF)family is a group of important mitochondrial transcription regulatory factors. It has been demonstrated that MTERF1-3 are involved in the regulation of mitochondrial gene transcription and oxidative phosphorylation However, the function of the newest member MTERF4 has not been characterized. In this study, human MTERF4 full-length open reading frame was cloned, and the protein structure prediction revealed that hMTERF4 protein contained leucine-zipper motifs, wbich is similar to human MTERFI-3. The expressed pMTERF4-green fluorescence fusion protein in HeLa cells localized the mitochondria. (3(4,5)dimethylthiahiazo(zy1)3,5diphenytetrazoliumromide) (MTT) proliferation assay and flow cytometry analysis showed that hMTERF4 knockdown induced sub-G1 phase cells accumulation, whereas its overexpression promoted cell proliferation. Furthermore,double staining with Annexin V and PI revealed that hMTERF4 knockdown increased necrosis but not apoptosis. In conclusion, our data suggested that hMTERF4 is an essential factor for cell proliferation, which is probably modulated by mitochondrial transcription to promote cell proliferation.

  8. Brucella abortus Cyclic β-1,2-Glucan Mutants Have Reduced Virulence in Mice and Are Defective in Intracellular Replication in HeLa Cells

    Briones, Gabriel; Iñón de Iannino, Nora; Roset, Mara; VIGLIOCCO, ANA; Paulo, Patricia Silva; Ugalde, Rodolfo A.

    2001-01-01

    Null cyclic β-1,2-glucan synthetase mutants (cgs mutants) were obtained from Brucella abortus virulent strain 2308 and from B. abortus attenuated vaccinal strain S19. Both mutants show greater sensitivity to surfactants like deoxycholic acid, sodium dodecyl sulfate, and Zwittergent than the parental strains, suggesting cell surface alterations. Although not to the same extent, both mutants display reduced virulence in mice and defective intracellular multiplication in HeLa cells. The B. abort...

  9. Quantitation of RNA Polymerase II and Its Transcription Factors in an HeLa Cell: Little Soluble Holoenzyme but Significant Amounts of Polymerases Attached to the Nuclear Substructure

    Kimura, Hiroshi; Tao, Yong; Roeder, Robert G.; Cook, Peter R.

    1999-01-01

    Various complexes that contain the core subunits of RNA polymerase II associated with different transcription factors have been isolated from eukaryotes; their precise molecular constitution depends on the purification procedure. We estimated the numbers of various components of such complexes in an HeLa cell by quantitative immunoblotting. The cells were lysed with saponin in a physiological buffer; ∼140,000 unengaged polymerases (mainly of form IIA) were released. Only ∼4,000 of these solub...

  10. Association to HeLa cells and surface behavior of exogenous gangliosides studied with a fluorescent derivative of GM1

    Cultured HeLa cells were incubated with pyrene-GM1/3H-radiolabeled GM1 ganglioside (1:4 M/M) mixtures for various times. The process of association of pyrene-GM1 with cells was qualitatively and quantitatively the same as that of 3H-GM1. The pyrene-GM1 and 3H-GM1 proportions in the various forms of association with cells were similar to that of the starting ganglioside mixture. After 2-h incubation, the association of ganglioside with cells was well established whereas almost no metabolic processing had occurred. During a 24-h incubation, pyrene- and 3H-GM1 underwent similar metabolic processing and gave rise to catabolic (GM2 and GM3) and anabolic (GDla) derivatives. Fluorescence spectroscopy experiments carried out with the excimer formation technique on subcellular fractions containing plasma membranes showed that exogenous ganglioside was, in part, associated with the cells in a micellar form removable by trypsin treatment, and in part inserted in a seemingly molecular dispersion. Addition of Ca2+ salts caused aggregation of the ganglioside, as indicated by the increase of the excimer:monomer fluorescence ratio. The phenomenon was Ca2+ concentration dependent (maximum at 10 mM), and subsequent addition of EDTA has no effect. The saccharide portion of exogenously incorporated pyrene-GM1 was available to interact with external ligands, as shown by its ability to bind cholera toxin whose addition reduced the collision rate among the ganglioside lipid moieties

  11. HN protein of Newcastle disease virus sensitizes HeLa cells to TNF-α-induced apoptosis by downregulating NF-κB expression.

    Rajmani, R S; Gupta, Shishir Kumar; Singh, Prafull Kumar; Gandham, Ravi Kumar; Sahoo, A P; Chaturvedi, Uttara; Tiwari, Ashok K

    2016-09-01

    Hemagglutinin neuraminidase (HN) is a membrane protein of Newcastle disease virus (NDV) with the ability to induce apoptosis in many transformed cell lines. TNF-α is a multi-factorial protein that regulates cell survival, differentiation and apoptosis. In a previous study, we reported that HN protein induces apoptosis by downregulating NF-κB expression. Further, we speculated that downregulation of NF-κB expression might sensitize HeLa cells to TNF-α-mediated apoptosis. Therefore, the present study was undertaken to investigate if HN protein could sensitize HeLa cells to TNF-α and to examine the apoptotic potential of the HN protein and TNF-α in combination. The results revealed that the pro-apoptotic effects were more pronounced with the combination of HN and TNF-α than with HN or TNF-α alone, which indicates that the HN protein indeed sensitized the HeLa cells to TNF-α-induced cell death. The results of the study provide a mechanistic insight into the apoptotic action of HN protein along with TNF-α, which could be valuable in treating tumor types that are naturally resistant to TNF-α. PMID:27294845

  12. Analysis of Replication Foci and Replication Domains in HeLa Cells

    Ligasová, Anna; Malínský, Jan; Raška, Ivan; Koberna, Karel

    Lisboa : organizer, 2005. PB-09. [XLth Meeting of the Portuguese Society for Electron Microscopy and Cell Biology. 08.12.2005-09.12.2005, Lisboa] R&D Projects: GA ČR GA304/03/1121 Institutional research plan: CEZ:AV0Z50390512 Keywords : cells Subject RIV: EB - Genetics ; Molecular Biology

  13. Heat induced protein denaturation in the particulate fraction of HeLa S3 cells: effect of thermotolerance.

    Burgman, P W; Konings, A W

    1992-10-01

    In this study we investigated the effect of heat on the proteins of the particulate fraction (PF) of HeLa S3 cells using electron spin resonance (ESR) and thermal gel analysis (TGA). ESR detects overall conformational changes in proteins, while TGA detects denaturation (aggregation due to formation of disulfide bonds) in specific proteins. For ESR measurements the -SH groups of the proteins were labelled with a maleimido bound spin label (4-maleimido-tempo). The sample was heated inside the ESR spectrometer at a rate of 1 degree C/min. ESR spectra were made every 2-3 degrees C between 20 degrees C and 70 degrees C. In the PF of untreated cells conformational changes in proteins were observed in three temperature stretches: between 38 and 44 degrees C (transition A, TA); between 47 and 53 degrees C (transition B, TB); and above 58 degrees C (transition C, TC). With TGA, using the same heating rate, we identified three proteins (55, 70, and 90 kD) which denatured during TB. No protein denaturation was observed during TA, while during TC denaturation of all remaining proteins in the PF occurred. When the ESR and TGA measurements were done with the PF of (heat-induced) thermotolerant cells, TA was unchanged while TB and TC started at higher temperatures. The temperature shift for the onset of these transitions correlated with the degree of thermotolerance that was induced in the cells. These results suggest that protection against heat-induced denaturation of proteins in the PF is involved in heat induced thermotolerance. PMID:1325981

  14. HeLa cell response proteome alterations induced by mammalian reovirus T3D infection

    Coombs, Kevin M.

    2013-01-01

    Background Cells are exposed to multiple stressors that induce significant alterations in signaling pathways and in the cellular state. As obligate parasites, all viruses require host cell material and machinery for replication. Virus infection is a major stressor leading to numerous induced modifications. Previous gene array studies have measured infected cellular transcriptomes. More recently, mass spectrometry-based quantitative and comparative assays have been used to complement such stud...

  15. [Construction of ADAMTS13-pEGFP-N1 vector and its expression in HeLa cells].

    Ling, Jing; Ma, Zhen-Ni; Su, Jian; Ruan, Chang-Geng

    2013-02-01

    This study was aimed to construct a pEGFP-N1 vector of von Willebrand factor cleaving protease (ADAMTS13, a disintegrin and metalloprotease with a thrombospondin type 1 motifs 13) so as to pave the way for further studying its synthesis and secretion. Human full-length cDNA sequence of ADAMTS13 was acquired by polymerase chain reaction (PCR) with Phusion(®) High-Fidelity (NEB), then the PCR product was double digested with EcoRI and XhoI. After digestion, the ADAMTS13 cDNA sequence was purified and recombined with the pEGFP-N1 vector. The DNA sequence analysis showed that ADAMTS13 was ligated to the pEGFP-N1 vector correctly. After transient expression in HeLa cells, the expression of EGFP could be detected by fluorescent microscopy, and the expression of ADAMTS13 protein could be detected by SDS-PAGE and Western blot. It is concluded that the ADAMTS13-pEGFP-N1 vector is successfully constructed, and it can be widely used in further research on the mechanism of the synthesis and secretion of ADAMTS13. PMID:23484705

  16. A novel chemosensor with visible light excitability for sensing Zn2+ in physiological medium and in HeLa cells.

    Datta, Barun Kumar; Thiyagarajan, Durairaj; Samanta, Soham; Ramesh, Aiyagari; Das, Gopal

    2014-07-21

    In the present study a novel imine-hydrazone based fluorescent chemosensor () for efficient and selective sensing of Zn(2+) over other biologically important metal ions under physiological conditions is reported. An enhancement in fluorescence emission intensity of the developed probe with a red shift of ∼25 nm was observed for Zn(2+), whereas other metal ions failed to reveal any significant change in the emission spectra. Interestingly, the receptor functioned under completely physiological conditions (99.7% HEPES buffer) and has visible light excitability. Sensing of Zn(2+) was investigated in detail by absorption spectroscopy, emission spectroscopy, DFT calculation, (1)H-NMR titration experiment and ESI-MS experiment. The association constant between and Zn(2+) was found to be 5.58 × 10(5) M(-1). The receptor could detect as low as 69 ppb Zn(2+). Sensing of Zn(2+) is proposed through switch-on of intramolecular charge transfer (ICT) and chelation enhanced fluorescence (CHEF) processes after the introduction of Zn(2+) into the free ligand. The developed receptor was non-toxic and rendered intracellular sensing of Zn(2+) in HeLa cells through fluorescence imaging studies. PMID:24879606

  17. Effect of Regulation of HSV-tk Gene Expression and Tumor Killed Activity with a Single Tetracycline-regulatable Plasmid Vector on HeLa Cells

    WANG Qian; DU Zhen-wu; MA Qing-shan; ZHANG Yu-cheng; WU Xiao-dong; YANG Shao-juan; WANG Ya-li; ZHANG Gui-zhen

    2009-01-01

    To construct a single tetracycline-regulatable plasmid vector based on the double tetracycline-regulatable plasmid vector system for regulating HSV-tk gene expression so as to effectively kill HeLa cells. Two tetracycline operator(TetO2) was cloned into pcDNA3.1 and a cassette was made for a cytomegalovirus-type 2 tetracycline oper-ator(CMV-TetO2) promoter, and the obtained vector was named pcDNA3.1-CMV-TetO2. Herpes simplex virus thy-midine kinase(HSV-tk) gene and tetracycline repressor(TR) gene were cloned into pcDNA3.1-CMV-TetO2 and the two genes were linked with internal ribosome entry site(IRES) to gain a vector named pcDNA3.1-CMV-TetO2-HSV-tk-IRES-TR. The HeLa cells were stablly transfected with pcDNA3.1-CMV-TetO2-HSV-tk-IRES-TR plasmid. The expression of HSV-tk and TR were detected by RT-PCR, the tumorcidal activity of HSV-tk/GCV was determined by MTT assay. In Hela cells transfected with the above plasmid vector, HSV-tk gene and TR gene can be expressed lowly and the concentration of GCV producing a 50% decrease in cell viability was about 50 ug/mL without adding deoxycycline; in contrast, the expessions of HSV-tk gene and TR gene increased significantly and the concentration of GCV producing a 50% decrease in cell viability was about 5 u,g/mL with adding deoxycycline. Therefore tetracycline can regulate the expression and tumorcidal activity of HSV-tk gene in HeLa cells with this single plasmid vector.

  18. Loss of a putative tumor suppressor locus after gamma-ray-induced neoplastic transformation of HeLa x Skin fibroblast human cell hybrids

    The nontumorigenic HeLa x skin fibroblast hybrid cell line, CGL1, can be induced to re-express HeLa tumor-associated cell surface antigen, p75-IAP (intestinal alkaline phosphatase), with resulting neoplastic transformation, by exposure to γ radiation. This has allowed the human hybrid system to be developed into a quantitative in vitro model for radiation-induced neoplastic transformation of human cells. Recently, several γ-ray-induced IAP-expression mutants (GIMs) of the nontumorigenic HeLa x skin fibroblast hybrid CGL1 were isolated and all were tumorigenic when injected subcutaneously into nude mice. Control cell lines which were negative for p75-IAP (CONs) were also isolated from irradiated populations, and none were found to be tumorigenic. We have now begun to investigate the molecular basis of radiation-induced neoplastic transformation in this system by studying the potential genetic linkage between p75/IAP expression, tumorigenicity and damage to a putative tumor suppressor locus on fibroblast chromosome 11. Previous analysis of rare spontaneous segregants has indicated that this locus is involved in the regulation of tumorigenicity and in the expression of the HeLa tumor-associated cell surface marker intestinal alkaline phosphatase (p75-IAP) in this system. Therefore, analysis by restriction fragment length polymorphism and chromosome painting have been performed for chromosome 11, and for chromosome 13 as a control, for the p75/IAP-positive GIM and p75/IAP-negative CON cell lines. We report that in five of eight of the GIMs large-scale damage to the fibroblast chromosome 11's is evident (four GIMs have lost one complete copy of a fibroblast chromosome 11 heavily damaged). None of the CONs, however (0/5), have lost a complete copy of either fibroblast chromosome 11. No large-scale damage to the control chromosome 13's was detected in the GIMs or CONs. 49 refs., 3 figs., 2 tabs

  19. Hsp105 family proteins suppress staurosporine-induced apoptosis by inhibiting the translocation of Bax to mitochondria in HeLa cells

    Hsp105 (Hsp105α and Hsp105β), major heat shock proteins in mammalian cells, belong to a subgroup of the HSP70 family, HSP105/110. Previously, we have shown that Hsp105α has completely different effects on stress-induced apoptosis depending on cell type. However, the molecular mechanisms by which Hsp105α regulates stress-induced apoptosis are not fully understood. Here, we established HeLa cells that overexpress either Hsp105α or Hsp105β by removing doxycycline and examined how Hsp105 modifies staurosporine (STS)-induced apoptosis in HeLa cells. Apoptotic features such as the externalization of phosphatidylserine on the plasma membrane and nuclear morphological changes were induced by the treatment with STS, and the STS-induced apoptosis was suppressed by overexpression of Hsp105α or Hsp105β. In addition, we found that overexpression of Hsp105α or Hsp105β suppressed the activation of caspase-3 and caspase-9 by preventing the release of cytochrome c from mitochondria. Furthermore, the translocation of Bax to mitochondria, which results in the release of cytochrome c from the mitochondria, was also suppressed by the overexpression of Hsp105α or Hsp105β. Thus, it is suggested that Hsp105 suppresses the stress-induced apoptosis at its initial step, the translocation of Bax to mitochondria in HeLa cells

  20. Retinoic acid-mediated repression of human papillomavirus 18 transcription and different ligand regulation of the retinoic acid receptor beta gene in non-tumorigenic and tumorigenic HeLa hybrid cells.

    Bartsch, D; Boye, B; Baust, C; zur Hausen, H; Schwarz, E

    1992-01-01

    Human papillomavirus type 18 (HPV18) belongs to the group of genital papillomaviruses involved in the development of cervical carcinomas. Since retinoic acid (RA) is a key regulator of epithelial cell differentiation and a growth inhibitor in vitro of HPV18-positive HeLa cervical carcinoma cells, we have used HeLa and HeLa hybrid cells in order to analyse the effects of RA on expression of the HPV18 E6 and E7 oncogenes and of the cellular RA receptor genes RAR-beta and -gamma. We show here that RA down-regulates HPV18 mRNA levels apparently due to transcriptional repression. Transient cotransfection assays indicated that RARs negatively regulate the HPV18 upstream regulatory region and that the central enhancer can confer RA-dependent repression on a heterologous promoter. RA treatment resulted in induction of RAR-beta mRNA levels in non-tumorigenic HeLa hybrid cells, but not in tumorigenic hybrid segregants nor in HeLa cells. No alterations of the RAR-beta gene or of the HeLa RAR-beta promoter could be revealed by Southern and DNA sequence analysis, respectively. As determined by transient transfection assays, however, the RAR-beta control region was activated by RA more strongly in non-tumorigenic hybrid cells than in HeLa cells, thus indicating differences in trans-acting regulatory factors. Our data suggest that the RARs are potential negative regulators of HPV18 E6 and E7 gene expression, and that dysregulation of the RAR-beta gene either causatively contributes to or is an indicator of tumorigenicity in HeLa and HeLa hybrid cells. Images PMID:1318198

  1. Extracellular gentamicin reduces the activity of connexin hemichannels and interferes with purinergic Ca2+ signaling in HeLa cells

    Figueroa, Vania A.; Retamal, Mauricio A.; Cea, Luis A.; Salas, José D.; Vargas, Aníbal A.; Verdugo, Christian A.; Jara, Oscar; Martínez, Agustín D.; Sáez, Juan C.

    2014-01-01

    Gap junction channels (GJCs) and hemichannels (HCs) are composed of protein subunits termed connexins (Cxs) and are permeable to ions and small molecules. In most organs, GJCs communicate the cytoplasm of adjacent cells, while HCs communicate the intra and extracellular compartments. In this way, both channel types coordinate physiological responses of cell communities. Cx mutations explain several genetic diseases, including about 50% of autosomal recessive non-syndromic hearing loss. However, the possible involvement of Cxs in the etiology of acquired hearing loss remains virtually unknown. Factors that induce post-lingual hearing loss are diverse, exposure to gentamicin an aminoglycoside antibiotic, being the most common. Gentamicin has been proposed to block GJCs, but its effect on HCs remains unknown. In this work, the effect of gentamicin on the functional state of HCs was studied and its effect on GJCs was reevaluated in HeLa cells stably transfected with Cxs. We focused on Cx26 because it is the main Cx expressed in the cochlea of mammals where it participates in purinergic signaling pathways. We found that gentamicin applied extracellularly reduces the activity of HCs, while dye transfer across GJCs was not affected. HCs were also blocked by streptomycin, another aminoglycoside antibiotic. Gentamicin also reduced the adenosine triphosphate release and the HC-dependent oscillations of cytosolic free-Ca2+ signal. Moreover, gentamicin drastically reduced the Cx26 HC-mediated membrane currents in Xenopus laevis oocytes. Therefore, the extracellular gentamicin-induced inhibition of Cx HCs may adversely affect autocrine and paracrine signaling, including the purinergic one, which might partially explain its ototoxic effects. PMID:25237294

  2. Herpes Simplex Virus (HSV) Modulation of Staphylococcus aureus and Candida albicans Initiation of HeLa 299 Cell-Associated Biofilm.

    Plotkin, Balbina J; Sigar, Ira M; Tiwari, Vaibhav; Halkyard, Scott

    2016-05-01

    Although herpes simplex virus type-1 (HSV-1), and type-2 (HSV-2), Staphylococcus aureus and Candida albicans co-habit the oral and genital mucosa, their interaction is poorly understood. We determined the effect HSV has on bacterial and/or fungal adherence, the initial step in biofilm formation. HeLa229 cells were infected with HSV-1 (KOS) gL86 or HSV-2 (KOS) 333gJ (-) at a multiplicity of infection (MOI) of 50 and 10. S. aureus (ATCC 25923) and/or C. albicans (yeast forms or germ tube forms) were co-incubated for 30 min (37 °C; 5 % CO2; 5:1 organism: HeLa cell ratio; n = 16) with virus-infected HeLa cells or uninfected HeLa cell controls. Post-incubation, the monolayers were washed (3x; PBS), lysed (RIPA), and the lysate plated onto Fungisel and/or mannitol salts agar for standard colony count. The level of HeLa-associated S. aureus was significantly decreased (P albicans yeast forms and germ tube approximately two-fold, respectively. The effect of S. aureus on germ tube and yeast form adherence to HSV-1- and HSV-2-infected cells was specific for the Candida phenotype tested. Our study suggests that HSV, while antagonist towards S. aureus adherence enhances Candida adherence. Furthermore, the combination of the three pathogens results in S. aureus adherence that is either unaffected, or partially restored depending on both the herpes viral species and the fungal phenotype present. PMID:26758707

  3. Transporter Molecules influence the Gene Expression in HeLa Cells

    Waldeck, Waldemar; Pipkorn, Ruediger; Korn, Bernhard; Mueller, Gabriele; Schick, Matthias; Tóth, Katalin; Wiessler, Manfred; Didinger, Bernd; Braun, Klaus

    2008-01-01

    Progresses in biology and pharmacology led to highly specific bioactive substances, but their poor bioavailability at the site of action is a result of their physico-chemical properties. Various design approaches for transport carrier molecules facilitating the cellular entry of bioactive substances could help to reach their molecular target in cells and tissues. The transfer efficacy and the subsequent pharmacological effects of the cargo molecules are well investigated, but the investigatio...

  4. Transporter Molecules influence the Gene Expression in HeLa Cells

    Waldemar Waldeck, Ruediger Pipkorn, Bernhard Korn, Gabriele Mueller, Matthias Schick, Katalin Tóth, Manfred Wiessler, Bernd Didinger, Klaus Braun

    2009-01-01

    Progresses in biology and pharmacology led to highly specific bioactive substances, but their poor bioavailability at the site of action is a result of their physico-chemical properties. Various design approaches for transport carrier molecules facilitating the cellular entry of bioactive substances could help to reach their molecular target in cells and tissues. The transfer efficacy and the subsequent pharmacological effects of the cargo molecules are well investigated, but the investigatio...

  5. Transporter Molecules influence the Gene Expression in HeLa Cells

    Waldemar Waldeck, Ruediger Pipkorn, Bernhard Korn, Gabriele Mueller, Matthias Schick, Katalin Tóth, Manfred Wiessler, Bernd Didinger, Klaus Braun

    2009-01-01

    Full Text Available Progresses in biology and pharmacology led to highly specific bioactive substances, but their poor bioavailability at the site of action is a result of their physico-chemical properties. Various design approaches for transport carrier molecules facilitating the cellular entry of bioactive substances could help to reach their molecular target in cells and tissues. The transfer efficacy and the subsequent pharmacological effects of the cargo molecules are well investigated, but the investigations of effects of the carrier molecules themselves on the target cells or tissues remain necessary. A special attention should be paid to the differential gene expression, particularly in the interpretation of the data achieved by highly specific active pharmaceutical products. After application of transmembrane transport peptides, particularly the pAnt and also the HIV-1 Tat, cells respond with a conspicuous altered gene expression of at least three genes. The PKN1 gene was induced and two genes (ZCD1 and BSG were slightly repressed. The genes and the chromosomes are described, the moderate differential gene expression graphed, and the ontology is listed.

  6. Transporter Molecules influence the Gene Expression in HeLa Cells

    Waldeck, Waldemar; Pipkorn, Ruediger; Korn, Bernhard; Mueller, Gabriele; Schick, Matthias; Tóth, Katalin; Wiessler, Manfred; Didinger, Bernd; Braun, Klaus

    2009-01-01

    Progresses in biology and pharmacology led to highly specific bioactive substances, but their poor bioavailability at the site of action is a result of their physico-chemical properties. Various design approaches for transport carrier molecules facilitating the cellular entry of bioactive substances could help to reach their molecular target in cells and tissues. The transfer efficacy and the subsequent pharmacological effects of the cargo molecules are well investigated, but the investigations of effects of the carrier molecules themselves on the target cells or tissues remain necessary. A special attention should be paid to the differential gene expression, particularly in the interpretation of the data achieved by highly specific active pharmaceutical products. After application of transmembrane transport peptides, particularly the pAnt and also the HIV-1 Tat, cells respond with a conspicuous altered gene expression of at least three genes. The PKN1 gene was induced and two genes (ZCD1 and BSG) were slightly repressed. The genes and the chromosomes are described, the moderate differential gene expression graphed, and the ontology is listed. PMID:19214198

  7. mRNAs containing the unstructured 5' leader sequence of alfalfa mosaic virus RNA 4 translate inefficiently in lysates from poliovirus-infected HeLa cells.

    Hann, L E; Gehrke, L

    1995-01-01

    Poliovirus infection is accompanied by translational control that precludes translation of 5'-capped mRNAs and facilitates translation of the uncapped poliovirus RNA by an internal initiation mechanism. Previous reports have suggested that the capped alfalfa mosaic virus coat protein mRNA (AIMV CP RNA), which contains an unstructured 5' leader sequence, is unusual in being functionally active in extracts prepared from poliovirus-infected HeLa cells (PI-extracts). To identify the cis-acting nu...

  8. A new assay for invasion of HeLa 229 cells by Bordetella pertussis: effects of inhibitors, phenotypic modulation, and genetic alterations.

    Lee, C. K.; Roberts, A. L.; Finn, T M; Knapp, S; Mekalanos, J J

    1990-01-01

    Invasion and intracellular survival of Bordetella pertussis in HeLa 229 cells was studied by a new assay that utilizes polymyxin B instead of gentamicin to rapidly kill extracellular organisms. Invasion measured by this assay was time and temperature dependent and was inhibited by the microfilament drug cytochalasin D. The invasion process was also dependent on a functional vir locus (also known as bvg), the positive regulator of virulence gene expression in B. pertussis. Four spontaneous Vir...

  9. Evaluation of Cytotoxic Effects of Dichloromethane Extract of Guduchi (Tinospora cordifolia Miers ex Hook F & THOMS) on Cultured HeLa Cells

    Shaival Kamalaksha Rao; Ganesh Chandra Jagetia

    2006-01-01

    Extracts of Tinospora cordifolia (TCE) have been shown to possess anti-tumor properties, but the mechanism of the anti-tumor function of TCE is poorly understood. This investigation elucidates the possible mechanism underlying the cytotoxic effects of dichlormethane extracts of TCE, after selecting optimal duration and concentration for treatment. HeLa cells were exposed to various concentrations of TCE, which has resulted in a concentration-dependent decline in the clonogenicity, glutathione...

  10. Functional homology between the sequence-specific DNA-binding proteins nuclear factor I from HeLa cells and the TGGCA protein from chicken liver.

    Leegwater, P.A.; van der Vliet, P C; Rupp, R A; Nowock, J; Sippel, A E

    1986-01-01

    Nuclear factor I from HeLa cells, a protein with enhancing function in adenovirus DNA replication, and the chicken TGGCA protein are specific DNA-binding proteins that were first detected by independent methods and that appeared to have similar DNA sequence specificity. To test whether they are homologous proteins from different species we have compared (i) their DNA binding properties and (ii) their function in reconstituted adenovirus DNA replication systems. Using deletion and substitution...

  11. RGDS-functionalized polyethylene glycol hydrogel-coated magnetic iron oxide nanoparticles enhance specific intracellular uptake by HeLa cells

    Nazli C; Ergenc TI; Yar Y; Acar HY; Kizilel S

    2012-01-01

    © 2012 Nazli et al, publisher and licensee Dove Medical Press Ltd. This is an Open Access article which permits unrestricted noncommercial use, provided the original work is properly cited. International Journal of Nanomedicine 2012:7 1903–1920 International Journal of Nanomedicine RGDS-functionalized polyethylene glycol hydrogel-coated magnetic iron oxide nanoparticles enhance specific intracellular uptake by HeLa cells Caner Nazli1 Tugba Ipek Ergenc2 Yasemin Ya...

  12. The HeLa Documentary Film: An Engaging Writing and Culturally Relevant Assignment on Cell Division and Ethics for Nonscience Majors†

    Diann Jordan; Timetria Bonds

    2015-01-01

    Historically black institutions play a pivotal role in educating the next generation of scientists and engineers as well as promoting scientific literacy among all of its students. Students would like to have more culturally relevant assignments that reflect their life experiences as it relates to course content.  We used the HeLa documentary film, "The Way of All Flesh Film," as an effective teaching tool in the first survey course of general biology to supplement our discussion on the cell ...

  13. Synthetic time series resembling human (HeLa) cell-cycle gene expression data and application to gene regulatory network discovery

    Tam, GHF; Hung, YS; Chang, C.

    2013-01-01

    Evaluation of gene regulatory network (GRN) discovery methods relies heavily on synthetic time series. However, synthetic data generated by traditional method deviate a lot from real data, making such evaluation questionable. Guiding by decaying sinusoids, we propose a new method that generates synthetic data resembling human (HeLa) cell-cycle gene expression data. Using the new synthetic data, a simple comparison between four GRN discovery methods reveals that Granger causality (GC) methods ...

  14. Thermal neutron-induced killing effect on HeLa cells modified by a new nucleic acid precursor with boron-10

    A new 10B-containing nucleic acid precursor for neutron capture therapy, 5-carboranyluridine was used to increase the killing effect of thermal neutron beam of Kyoto University Reactor on HeLa S3 cells in vitro. The increase was calculated with four parameters of a linear-quadratic model on dose-survival curve, α, β, a mean inactivation dose (D) and a surviving fraction at 2 Gy irradiation (SF(2)). The results showed that this compound was taken in cells and probably accumulated on cell surface during the incubation of cells and increased the thermal neutron induced killing effect. (author)

  15. Arsenic trioxide inhibits cell proliferation and human papillomavirus oncogene expression in cervical cancer cells

    Highlights: • As2O3 inhibits growth of cervical cancer cells and expression of HPV oncogenes in these cells. • HPV-negative cervical cancer cells are more sensitive to As2O3 than HPV-positive cervical cancer cells. • HPV-18 positive cervical cancer cells are more sensitive to As2O3 than HPV-16 positive cancer cells. • Down-regulation of HPV oncogenes by As2O3 is partially due to the diminished AP-1 binding. - Abstract: Arsenic trioxide (As2O3) has shown therapeutic effects in some leukemias and solid cancers. However, the molecular mechanisms of its anticancer efficacy have not been clearly elucidated, particularly in solid cancers. Our previous data showed that As2O3 induced apoptosis of human papillomavirus (HPV) 16 DNA-immortalized human cervical epithelial cells and cervical cancer cells and inhibited the expression of HPV oncogenes in these cells. In the present study, we systemically examined the effects of As2O3 on five human cervical cancer cell lines and explored the possible molecular mechanisms. MTT assay showed that HPV-negative C33A cells were more sensitive to growth inhibition induced by As2O3 than HPV-positive cervical cancer cells, and HPV 18-positive HeLa and C4-I cells were more sensitive to As2O3 than HPV 16-positive CaSki and SiHa cells. After As2O3 treatment, both mRNA and protein levels of HPV E6 and E7 obviously decreased in all HPV positive cell lines. In contrast, p53 and Rb protein levels increased in all tested cell lines. Transcription factor AP-1 protein expression decreased significantly in HeLa, CaSki and C33A cells with ELISA method. These results suggest that As2O3 is a potential anticancer drug for cervical cancer

  16. Lung Cancer Stem Cells

    Sharon R. Pine

    2008-01-01

    Full Text Available Lung cancer remains a major cause of cancer-related lethality because of high incidence and recurrence in spite of significant advances in staging and therapies. Recent data indicates that stem cells situated throughout the airways may initiate cancer formation. These putative stem cells maintain protumorigenic characteristics including high proliferative capacity, multipotent differentiation, drug resistance and long lifespan relative to other cells. Stem cell signaling and differentiation pathways are maintained within distinct cancer types, and destabilization of this machinery may participate in maintenance of cancer stem cells. Characterization of lung cancer stem cells is an area of active research and is critical for developing novel therapies. This review summarizes the current knowledge on stem cell signaling pathways and cell markers used to identify the lung cancer stem cells.

  17. Cell phones and cancer

    Cancer and cell phones; Do cell phones cause cancer? ... Several major studies show no link between cell phones and cancer at this time. However, since the information available is based on short-term studies, the impact of many years of ...

  18. Phospholipase C-β1 and β4 contribute to non-genetic cell-to-cell variability in histamine-induced calcium signals in HeLa cells.

    Sachiko Ishida

    Full Text Available A uniform extracellular stimulus triggers cell-specific patterns of Ca(2+ signals, even in genetically identical cell populations. However, the underlying mechanism that generates the cell-to-cell variability remains unknown. We monitored cytosolic inositol 1,4,5-trisphosphate (IP3 concentration changes using a fluorescent IP3 sensor in single HeLa cells showing different patterns of histamine-induced Ca(2+ oscillations in terms of the time constant of Ca(2+ spike amplitude decay and the Ca(2+ oscillation frequency. HeLa cells stimulated with histamine exhibited a considerable variation in the temporal pattern of Ca(2+ signals and we found that there were cell-specific IP3 dynamics depending on the patterns of Ca(2+ signals. RT-PCR and western blot analyses showed that phospholipase C (PLC-β1, -β3, -β4, -γ1, -δ3 and -ε were expressed at relatively high levels in HeLa cells. Small interfering RNA-mediated silencing of PLC isozymes revealed that PLC-β1 and PLC-β4 were specifically involved in the histamine-induced IP3 increases in HeLa cells. Modulation of IP3 dynamics by knockdown or overexpression of the isozymes PLC-β1 and PLC-β4 resulted in specific changes in the characteristics of Ca(2+ oscillations, such as the time constant of the temporal changes in the Ca(2+ spike amplitude and the Ca(2+ oscillation frequency, within the range of the cell-to-cell variability found in wild-type cell populations. These findings indicate that the heterogeneity in the process of IP3 production, rather than IP3-induced Ca(2+ release, can cause cell-to-cell variability in the patterns of Ca(2+ signals and that PLC-β1 and PLC-β4 contribute to generate cell-specific Ca(2+ signals evoked by G protein-coupled receptor stimulation.

  19. Evaluation and comparison of Hela, Hep2C and Vero cell lines sensitivity to polio vaccinal virus using micro and macro vaccine potency tests

    Soleimani, S.,

    2012-11-01

    Full Text Available Poliomyelitis, an acute viral infectious disease caused by poliovirus, still remains a public health problem in developing countries. Despite the global effort to eradicate polio, continuing the polio immunization with a potent and safe vaccine is essential. For accurate vaccine evaluation, three types of cell lines including Hela, Hep2C and Vero were evaluated and compared using two methods of polio vaccine potency tests (micro & macro. For cells comparison, five different batches from polio vaccines were tested and to develop the test, five variables including viruses, cells, serum, media and Co2 were studied. For validation, the titer of which has been well established as a working reference preparation (WRP was applied to control the accuracy and reproducibility of the testing system. Multiple comparisons were performed by analysis of variance (ANOVA followed by Tokey HDS and LSD. No significant differences were found between the potency of vaccine batches and between macro and micro methods. Reduction in cells sensitivity and potency of vaccines was found with increasing passage number. Significant differences were found between the sensitivity of the cell lines. The highest potency of polio vaccines was obtained using Hela cells (GMT in macro and micro test = 10 6.35; Hep2C cells were afterwards (GMT in macro= 10 6.01 and in micro test= 10 5.94; Vero cells were lowest (GMT in macro= 10 5.78 and in micro test= 10 5.72. So, the sensitivity and accuracy of the potency test for evaluation of the polio vaccine in immunization program in Iran will be assured using the Hela cell line with low passage number in macro and micro methods.

  20. Study of cancer cell lines with Fourier transform infrared (FTIR)/vibrational absorption (VA) spectroscopy

    Uceda Otero, E. P.; Eliel, G. S. N.; Fonseca, E. J. S.;

    2013-01-01

    In this work we have used Fourier transform infrared (FTIR) / vibrational absorption (VA) spectroscopy to study two cancer cell lines: the Henrietta Lacks (HeLa) human cervix carcinoma and 5637 human bladder carcinoma cell lines. Our goal is to experimentally investigate biochemical changes and d...

  1. Expression of arthritis-causing HLA-B27 on Hela cells promotes induction of c-fos in response to in vitro invasion by Salmonella typhimurium.

    Ikawa, T; Ikeda, M; Yamaguchi, A; Tsai, W.C.; Tamura, N; Seta, N; Trucksess, M; Raybourne, R B; Yu, D T

    1998-01-01

    HLA-B27 confers a very strong genetic predisposition to development of a reactive arthritis after infection by bacteria such as Salmonella typhimurium. This study examines the role of HLA-B27 in the initiation of the earliest host activities after exposure to Salmonella, namely activation of the immediate early genes in the epithelial cells. Our major finding is that in Hela cells, the expression of c-fos was induced by Salmonella invasion only when the cells expressed the transfected HLA-B27...

  2. 4-Amino-2-arylamino-5-indoloyl/cinnamoythiazoles, analogs of topsentin-class of marine alkaloids, induce apoptosis in HeLa cells.

    Juneja, Manisha; Vanam, Uma; Paranthaman, Sripriya; Bharathan, Asha; Keerthi, Venugopal S; Reena, Justus K; Rajaram, Rama; Rajasekharan, Kallikat N; Karunagaran, Devarajan

    2013-05-01

    Marine organisms provide several biologically active compounds that include alkaloids with high cytotoxic activity but only a few of them have so far reached clinical stage, due partly to their limited supply and complex structural features. In an attempt to develop novel anticancer compounds, we have now synthesized diaminoindoloylthiazoles (4a-c; DIT1-3) and diaminocinnamoylthiazoles (5a,b; DCT1-2) as analogs based on a topsentin scaffold and investigated the cytotoxic and apoptotic activities of these compounds in HeLa cells. The results suggest that diaminoindoloylthiazoles (DIT1-3) inhibit cell growth and among these, DIT3 is the most cytotoxic against HeLa cells (IC50 1 μM). The diaminocinnamoylthiazoles DCT1 and DCT2, which can be viewed as curcumin-diaminothiazole hybrids, also inhibited cell growth but at relatively higher concentrations with IC50 values of 60 and 30 μM, respectively. These compounds induced apoptosis through the intrinsic pathway by reducing the mitochondrial membrane potential and activating caspases, 9 and 3, but not caspase 8. Among the marine alkaloid analogs tested in this study, DIT1-3 are very effective in inducing apoptosis of HeLa cells followed by DCT2 and DCT1. The treated cells were arrested in G2/M phase followed by accumulation of the cells in the Sub G0 phase. The curcumin-diaminothiazole hybrid DCT1 had the maximum effect in downregulating TNF-induced NF-κB activation among the compounds tested in this study. Thus, we demonstrate that diaminoindoloylthiazoles and diaminocinnamoylthiazoles induce apoptosis, regulate cell cycle and NF-κB signaling and thus show promising anticancer effects that warrant further investigation. PMID:23524113

  3. Artemisinin derivative artesunate induces radiosensitivity in cervical cancer cells in vitro and in vivo

    Cervical cancer is the third most common type of cancer in women worldwide and radiotherapy remains its predominant therapeutic treatment. Artesunate (ART), a derivative of artemisinin, has shown radiosensitization effect in previous studies. However, such effects of ART have not yet been revealed for cervical cancer cells. The effect of ART on radiosensitivity of human cervical cancer cell lines HeLa and SiHa was assessed using the clonogenic assay. Cell cycle progression and apoptosis alterations were analyzed by flow cytometry. For in vivo study, HeLa or SiHa cells were inoculated into nude mice to establish tumors. Tissues from xenografts were obtained to detect the changes of microvessel density, apoptosis and cell cycle distribution. Microarray was used to analyze differentially expressed genes. ART increased the radiosensitivity of HeLa cells (SER = 1.43, P < 0.001) but not of SiHa cells. Apoptosis and the G2-M phase transition induced by X-ray irradiation (IR) were enhanced by ART via increased Cyclin B1 expression in HeLa cells. Tumor growth of xenografts from HeLa but not SiHa cells was significantly inhibited by irradiation combined with ART (tumor volume reduction of 72.34% in IR + ART group vs. 41.22% in IR group in HeLa cells and 48.79% in IR + ART group vs. 44.03% in IR alone group in SiHa cells). Compared with the irradiated group, cell apoptosis was increased and the G2/M cell cycle arrest was enhanced in the group receiving irradiation combined with ART. Furthermore, compared with radiation alone, X-ray irradiation plus ART affected the expression of 203 genes that function in multiple pathways including RNA transport, the spliceosome, RNA degradation and p53 signaling. ART potently abrogates the G2 checkpoint control in HeLa cells. ART can induce radiosensitivity of HeLa cells in vitro and in vivo

  4. Cancer Stem Cells

    Katarzyna Wieczorek; Jolanta Niewiarowska

    2008-01-01

    Cancer stem cell theory gains increasingly greater significance in the world of medicine. Numerous findings of scientific research in vivo and in vitro indicate that it is the population of undifferentiated, self-renewing cells which is responsible for recurrence of cancer and metastasis. Similarly to normal stem cells, cancer stem cells (CSC) function in the environment of the other cells of the organism, called the niche, where they receive signals for differentiation and proliferation proc...

  5. Lethal Sectoring, genomic instability and cell cycle variation of HeLa S3 cells survived after alpha- and X-ray irradiation

    When cells with infinite growth ability are irradiated by radiation, survived cells occasionally yield cells without colony forming ability after several generations (lethal sectoring). The sectoring is the process to exclude cells with latent lethal damage, which results in the inhibition of cell cycle progression. Further, clonogen still having the infinite growth ability appears during the sectoring process and has genomic instability. The present study was conducted to compare these processes after irradiation of α- and X-ray. A time-lapse motion camera (1 picture/15 min) was used for microscopic recording of HeLa S3-9IV cells within a single scope (about 50 cells). They were irradiated by 0.45 Gy of α-ray (point source of 241Am, fluence rate 1.5 x 106/cm2/min, 0.3 Gy/min, LET=125 keV/μm) or 3 Gy of X-ray (200 kVp, 0.7 Gy/min), of which doses were equivalent to the survival rate of 0.2. The film recording was done for 30 and 150 hr before and after irradiation, respectively, from which the pedigree of individual cells was figured out by selection of surviving clonogen. Analysis of the pedigree of the survived cells revealed that there was an essential difference in the three processes, which was possibly based on the LET difference. (N.I.)

  6. Effects of natural flavones on membrane properties and citotoxicity of HeLa cells Efeitos de flavonas naturais em propriedades de membranas e em citotoxicidade de células HeLa

    Tatiana Herrerias

    2010-07-01

    Full Text Available The aim of this study was to determine whether eupafolin and hispidulin, flavones extracted from Eupatorium littorale Cabrera, Asteraceae, have the ability to change properties of biological membranes and promote cytotoxic effects. Eupafolin (50-200 µM decreased approximately 30% the rate and total amplitude of valinomycin induced swelling and 60-100% the energy-dependent mitochondrial swelling. Moreover, eupafolin (200 µM reduced 35% the mitochondrial permeability transition, and hispidulin did not change this parameter in any of the doses tested. The evaluation of phase transition of DMPC liposomes with the probe DPH demonstrated that hispidulin and eupafolin affect gel and fluid phase. With mitochondrial membrane as model, hispidulin increased the polarization of fluorescence when used DPH-PA probe. Eupafolin and hispidulin (100 µM promoted a reduction of 40% in cellular viability of HeLa cells in 24 h. Our results suggest that eupafolin and hispidulin have cytotoxic effects that can be explained, in part, by alterations promoted on biological membranes properties and mitochondrial bioenergetics.O objetivo deste estudo foi avaliar se eupafolina e hispidulina, flavonas extraídas do Eupatorium littorale Cabrera, Asteraceae, possuíam a capacidade de alterar propriedades das membranas biológicas e promover efeitos citotóxicos. Eupafolina (50-200 µM reduziu em aproximadamente 30% a velocidade e amplitude do inchamento mitocondrial induzido por valinomicina e 60-100% o inchamento mitocondrial dependente de substrato. Além disso, eupafolina na dose de 200 µM reduziu a transição de permeabilidade mitocondrial em 35% entretanto, a hispidulina não alterou este parâmetro em todas as doses testadas. A avaliação da transição de fase dos lipossomas de DMPC com a sonda DPH demonstrou que ambas as flavonas afetam a fase gel e fluida. Quando lipossomas de membranas mitocondriais e a sonda DPH-PA foram utilizados, houve aumento da polariza

  7. ANTICANCER ACTIVITY OF PONGAMIA GLABRA V. SEED OIL EXTRACT AGAINST SELECTED HUMAN CANCER CELL LINES

    Chinnasamy Arulvasu

    2012-08-01

    Full Text Available Screening of the seed oil extract from Pongamia glabra V. (Fabaceae has been carried out for antiproliferative activity of cancer cells. The seed oil was extracted with methanol and then persuasive activity was tested on human cancer cell lines MCF-7 and HeLa. The cell growth inhibitory effects of seed oil extract was observed. The cell viability was assessed using trypan blue dye exclusion method and 3-(4, 5- Dimethyl thiazol-2yl-2, 5-dimethyltetrazolium bromide (MTT assay. The IC50 value of the methanolic seed oil extract against MCF-7 and HeLa was found to be 6 mg/ml and 6 mg/ml respectively after 48 hours of incubation. The P.glabra seed oil extract increased the proportion of DNA fragmentation in MCF-7 and HeLa cancer cell lines. Moreover, the inhibitory effect is correlated with DNA fragmentation. These results suggest that the P.glabra seed oil extract has an inhibitory effect on human cancer cell lines MCF-7 and HeLa.

  8. Lung Cancer Stem Cells

    Pine, Sharon R.; Blair Marshall; Lyuba Varticovski

    2008-01-01

    Lung cancer remains a major cause of cancer-related lethality because of high incidence and recurrence in spite of significant advances in staging and therapies. Recent data indicates that stem cells situated throughout the airways may initiate cancer formation. These putative stem cells maintain protumorigenic characteristics including high proliferative capacity, multipotent differentiation, drug resistance and long lifespan relative to other cells. Stem cell signaling and differentiation p...

  9. Low power ultrasound inhibits cell proliferation and invasion of human cancer cells in vitro

    Etienne Mfoumou

    2012-01-01

    Full Text Available Background: Applications of ultrasound in medicine for therapeutic purposes have been accepted, and they have several beneficial uses for many years. However, the outcome of low power ultrasound waves on cell proliferation, especially cell cycle progression and invasion as well as their associated genes on human breast and cervical cancer cells has not been investigated yet. Therefore, we examined the effect of low power ultrasound on BT20, BT20-E6/E7 and HeLa cell lines. Materials and Methods: BT20, BT20-E6/E7 and HeLa cell lines were used in this study. On the other hand, cell proliferation, cell cycle, and invasion assays were applied to study the effect of low ultrasound irradiation on these cell lines. Meanwhile, western blot was performed to study the expression patterns of some selected genes associated with this effect. Results: We found that low power ultrasound inhibits cell proliferation and provokes G0-G1 cell cycle arrest and reduction of S as well as an increase in the G2-M phase of HeLa cells in comparison with the untreated cells. This is accompanied by a down-regulation of Cdk-6 (cyclin dependent kinase which is a major control switch for the cell cycle. Moreover, low power ultrasound inhibits cell invasion and consequently down-regulates the expression of Id-1, caveolin, and EGF-R which are widely considered as main regulators of cell invasion and metastasis of human cancer. Conclusion: These results suggest that application of low power ultrasound on human breast and cervical cancer could be an effective method to reduce cell proliferation and invasion of these cancers.

  10. Compatibility of cancer cells with nanostructured oxidized porous silicon substrates

    Zeidman, Tal; Parush, Ran; Massad, Na' ama [Department of Biotechnology and Food Engineering, Technion - Israel Institute of Technology, Haifa 32000 (Israel); Segal, Ester [Department of Biotechnology and Food Engineering, Technion - Israel Institute of Technology, Haifa 32000 (Israel); Russell Berrie Nanotechnology Institute, Technion - Israel Institute of Technology, Haifa 32000 (Israel)

    2011-06-15

    The attachment and long-term viability of three types of human cancer cell lines (glioma U87, breast cancer MDA-MB-231, and cervical cancer HeLa) onto nanostructured oxidized porous Si substrates is investigated. The porous layers are fabricated to give cylindrically-shaped structures with pore diameters in the tunable range of 10 to 150 nm by anodizing a heavily-doped p-type Si. The Alamar Blue viability assay and optical microscopy are employed to assess the attachment, viability and the morphology of the cells. The results show that cells remain viable and proliferate on all surfaces. The nano-architecture of the studied scaffolds does not exert a deleterious effect on cancer cells. Cell coverage levels comparable to standard culture preparations on tissue culture polystyrene are observed (copyright 2011 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  11. Study on the Inhibition of Fermented Soybean to Cancer Cells

    LU Yan; WANG Wei; SHAN Yi; E Zhiqiang; WANG Liqun

    2009-01-01

    In the experiment, the inhibition of isoflavones extracted from soybean and tempe to SP2/0 and Hela cells was studied,and the inhibition rate of each unit for cancer cells was also studied. The results showed that the inhibition rate of tempe isoflavones to SP2/0 was 96.9% and to Hela cells was 69.5% when the concentration was 20 μg·mL-1. In the same condition, the inhibition rate of soybean isoflavones was 83.16% and 60.5%. With the decline of concentration, the inhibition rate decreased. The inhibition of isoflavones to SP2/0 did not exist when the concentration was 5-1.25 μg·mL-1.

  12. Action of caffeine on x-irradiated HeLa cells. IV. Progression delays and enhanced cell killing at high caffeine concentrations

    The response of x-irradiated and unirradiated HeLa S3 cells to treatment with caffeine at concentrations between 1 and 10 nM has been examined with respect to both delay in progression through the cell generation cycle and enhancement of the expression of potentially lethal x-ray damage. Progression is delayed in a concentration-dependent fashion: the generation time is doubled at about 4 mM. The duration of G1 is lengthened, and the rate of DNA synthesis is reduced, although the kinetics are different in the two phases; the rate of DNA synthesis is usually unaffected at 1 or 2 mM, while there is no concentration threshold for the slowing of progression through G1. Progression through G2 appears to be unaffected by concentrations up to at least 10 mM. Killing of irradiated cells in G2 is somewhat greater after treatment with the higher caffeine concentrations than reported previously for 1 mM. Moreover, an additional mode of killing is observed in irradiated G1 cells which had been found previously to be only slightly affected by 1 mM caffeine; they suffer extensive killing at concentrations above 5 mM. The time-survival curves for irradiated, caffeine-treated G1 and G2 cells have characteristically different shapes. The dose-survival curves for cells treated with the higher caffeine concentrations display steeper terminal slopes and narrower shoulders

  13. Breast cancer stem cells

    Owens, Thomas W.; Naylor, Matthew J.

    2013-01-01

    Cancer metastasis, resistance to therapies and disease recurrence are significant hurdles to successful treatment of breast cancer. Identifying mechanisms by which cancer spreads, survives treatment regimes and regenerates more aggressive tumors are critical to improving patient survival. Substantial evidence gathered over the last 10 years suggests that breast cancer progression and recurrence is supported by cancer stem cells (CSCs). Understanding how CSCs form and how they contribute to th...

  14. CYB5D2 requires heme-binding to regulate HeLa cell growth and confer survival from chemotherapeutic agents.

    Anthony Bruce

    Full Text Available The cytochrome b5 domain containing 2 (CYB5D2; Neuferricin protein has been reported to bind heme, however, the critical residues responsible for heme-binding are undefined. Furthermore, the relationship between heme-binding and CYB5D2-mediated intracellular functions remains unknown. Previous studies examining heme-binding in two cytochrome b5 heme-binding domain-containing proteins, damage-associated protein 1 (Dap1; Saccharomyces cerevisiae and human progesterone receptor membrane component 1 (PGRMC1, have revealed that conserved tyrosine (Y 73, Y79, aspartic acid (D 86, and Y127 residues present in human CYB5D2 may be involved in heme-binding. CYB5D2 binds to type b heme, however, only the substitution of glycine (G at D86 (D86G within its cytochrome b5 heme-binding (cyt-b5 domain abolished its heme-binding ability. Both CYB5D2 and CYB5D2(D86G localize to the endoplasmic reticulum. Ectopic CYB5D2 expression inhibited cell proliferation and anchorage-independent colony growth of HeLa cells. Conversely, CYB5D2 knockdown and ectopic CYB5D2(D86G expression increased cell proliferation and colony growth. As PGRMC1 has been reported to regulate the expression and activities of cytochrome P450 proteins (CYPs, we examined the role of CYB5D2 in regulating the activities of CYPs involved in sterol synthesis (CYP51A1 and drug metabolism (CYP3A4. CYB5D2 co-localizes with cytochrome P450 reductase (CYPOR, while CYB5D2 knockdown reduced lanosterol demethylase (CYP51A1 levels and rendered HeLa cells sensitive to mevalonate. Additionally, knockdown of CYB5D2 reduced CYP3A4 activity. Lastly, CYB5D2 expression conferred HeLa cell survival from chemotherapeutic agents (paclitaxel, cisplatin and doxorubicin, with its ability to promote survival being dependent on its heme-binding ability. Taken together, this study provides evidence that heme-binding is critical for CYB5D2 in regulating HeLa cell growth and survival, with endogenous CYB5D2 being required to

  15. Global quantitative proteomics reveal up-regulation of endoplasmic reticulum stress response proteins upon depletion of eIF5A in HeLa cells.

    Mandal, Ajeet; Mandal, Swati; Park, Myung Hee

    2016-01-01

    The eukaryotic translation factor, eIF5A, is a translation factor essential for protein synthesis, cell growth and animal development. By use of a adenoviral eIF5A shRNA, we have achieved an effective depletion of eIF5A in HeLa cells and undertook in vivo comprehensive proteomic analyses to examine the effects of eIF5A depletion on the total proteome and to identify cellular pathways influenced by eIF5A. The proteome of HeLa cells transduced with eIF5A shRNA was compared with that of scramble shRNA-transduced counterpart by the iTRAQ method. We identified 972 proteins consistently detected in three iTRAQ experiments and 104 proteins with significantly altered levels (protein ratio ≥1.5 or ≤0.66, p-value ≤0.05) at 72 h and/or 96 h of Ad-eIF5A-shRNA transduction. The altered expression levels of key pathway proteins were validated by western blotting. Integration of functional ontology with expression data of the 104 proteins revealed specific biological processes that are prominently up- or down-regulated. Heatmap analysis and Cytoscape visualization of biological networks identified protein folding as the major cellular process affected by depletion of eIF5A. Our unbiased, quantitative, proteomic data demonstrate that the depletion of eIF5A leads to endoplasmic reticulum stress, an unfolded protein response and up-regulation of chaperone expression in HeLa cells. PMID:27180817

  16. Evidence that a triplex-forming oligodeoxyribonucleotide binds to the c-myc promoter in HeLa cells, thereby reducing c-myc mRNA levels

    Postel, E.H.; Flint, S.J. (Princeton Univ., NJ (United States)); Kessler, D.J.; Hogan, M.E. (Baylor College of Medicine, The Woodlands, TX (United States))

    1991-09-15

    A synthetic 27-base-long oligodeoxyribonucleotide, termed PU1, has been shown to bind to duplex DNA to form a triplex at a single site within the human c-myc P1 promoter. PU1 has been administered to HeLa cells in culture to examine the feasibility of influencing transcription of the c-myc gene in vivo. It is shown that uptake of PU1 into the nucleus of HeLa cells is efficient and that the compound remains intact for at least 4 hours. In nuclei extracted from PU1-treated cells, inhibition of DNase I cleavage is detected within the c-myc P1 promoter at the target site for triplex formation. The inhibition is shown to be both site and oligodeoxyribonucleotide specific. After cellular uptake of PU1, it is shown that steady-state mRNA arising from the c-myc P2 initiation site, and relative to mRNA derived form the {beta}-actin promoter. Significant mRNA repression is not seen upon treating cells with oligodeoxyuribonucleotides that fail to bind to the P1 promoter target. Taken together, these data suggest that triplex formation can occur between an exogenous oligodeoxy-ribonucleotide and duplex DNA in the nucleus of treated cells.

  17. Gecko Proteins Exert Anti-Tumor Effect against Cervical Cancer Cells Via PI3-Kinase/Akt Pathway.

    Jeong, Ae-Jin; Chung, Chung-Nam; Kim, Hye-Jin; Bae, Kil Soo; Choi, Song; Jun, Woo Jin; Shim, Sang In; Kang, Tae-Hong; Leem, Sun-Hee; Chung, Jin Woong

    2012-10-01

    Anti-tumor activity of the proteins from Gecko (GP) on cervical cancer cells, and its signaling mechanisms were assessed by viable cell counting, propidium iodide (PI) staining, and Western blot analysis. GP induced the cell death of HeLa cells in a dose-dependent manner while it did not affect the viability of normal cells. Western blot analysis showed that GP decreased the activation of Akt, and co-administration of GP and Akt inhibitors synergistically exerted anti-tumor activities on HeLa cells, suggesting the involvement of PI3-kinase/Akt pathway in GP-induced cell death of the cancer cells. Indeed, the cytotoxic effect of GP against HeLa cells was inhibited by overexpression of constituvely active form of Akt in HeLa cells. The candidates of the functional proteins in GP were analyzed by Mass-spectrum. Taken together, our results suggest that GP elicits anti-tumor activity against HeLa cells by inhibition of PI3-kinase/Akt pathway. PMID:23118562

  18. Stages of Renal Cell Cancer

    ... cell cancer is a disease in which malignant (cancer) cells form in tubules of the kidney. Renal cell ... diagnosed, tests are done to find out if cancer cells have spread within the kidney or to other ...

  19. Ubiquitin B in cervical cancer: critical for the maintenance of cancer stem-like cell characters.

    Yuan Tian

    Full Text Available Cervical cancer cells exhibit an increased requirement for ubiquitin-dependent protein degradation associated with an elevated metabolic turnover rate. Ubiquitin, which is a small, highly conserved protein expressed in all eukaryotic cells, can be covalently linked to certain target proteins to mark them for degradation by the ubiquitin-proteasome system. Previous studies highlight the essential role of Ubiquitin B (UbB and UbB-dependent proteasomal protein degradation in histone deacetylase inhibitor (HDACi -induced tumor selectivity. We hypothesized that UbB plays a critical role in the function of cervical cancer stem cells. We measured endogenous UbB levels in mammospheres in vitro by real-time PCR and Western blotting. The function of UbB in cancer stem-like cells was assessed after knockdown of UbB expression in prolonged Trichostatin A-selected HeLa cells (HeLa/TSA by measuring in vitro cell proliferation, cell apoptosis, invasion, and chemotherapy resistance as well as by measuring in vivo growth in an orthotopic model of cervical cancer. We also assessed the cancer stem cell frequency, tumorsphere formation, and in vivo growth of human cervical cancer xenografts after UbB silencing. We found that HeLa/TSA were resistant to chemotherapy, highly expressed the UbB gene and the stem cell markers Sox2, Oct4 and Nanog. These cells also displayed induced differentiation abilities, including enhanced migration/invasion/malignancy capabilities in vitro and in vivo. Furthermore, an elevated expression of UbB was shown in the tumor samples of chemotherapy patients. Silencing of UbB inhibited tumorsphere formation, lowered the expression of stem cell markers and decreased cervical xenograft growth. Our results demonstrate that UbB was significantly increased in prolonged Trichostatin A-selected HeLa cells and it played a key role in the maintenance of cervical cancer stem-like cells.

  20. Gastric Cancer Stem Cells

    Takaishi, Shigeo; Okumura, Tomoyuki; Timothy C Wang

    2008-01-01

    Cancer stem cells are defined as the unique subpopulation in the tumors that possess the ability to initiate tumor growth and sustain self-renewal as well as metastatic potential. Accumulating evidence in recent years strongly indicate the existence of cancer stem cells in solid tumors of a wide variety of organs. In this review, we will discuss the possible existence of a gastric cancer stem cell. Our recent data suggest that a subpopulation with a defined marker shows spheroid colony format...

  1. Cancer stem cell metabolism

    Peiris-Pagès, Maria; Martinez-Outschoorn, Ubaldo E.; Pestell, Richard G.; Sotgia, Federica; Lisanti, Michael P

    2016-01-01

    Cancer is now viewed as a stem cell disease. There is still no consensus on the metabolic characteristics of cancer stem cells, with several studies indicating that they are mainly glycolytic and others pointing instead to mitochondrial metabolism as their principal source of energy. Cancer stem cells also seem to adapt their metabolism to microenvironmental changes by conveniently shifting energy production from one pathway to another, or by acquiring intermediate metabolic phenotypes. Deter...

  2. Liver Cancer Stem Cells

    Sameh Mikhail; Aiwu Ruth He

    2011-01-01

    Hepatocellular carcinoma is the most common primary malignancy of the liver in adults. It is also the fifth most common solid cancer worldwide and the third leading cause of cancer-related death. Recent research supports that liver cancer is a disease of adult stem cells. From the models of experimental hepatocarcinogenesis, there may be at least three distinct cell lineages with progenitor properties susceptible to neoplastic transformation. Identification of specific cell surface markers fo...

  3. In Vitro Investigations on the Toxicity and Cell Death Induced by Tamoxifen on Two Non-Breast Cancer Cell Types

    Majumdar, S. K.; Valdellon, J. A.; Brown, K A

    2001-01-01

    Tamoxifen, a potent anticancer agent known to interrupt the enhanced estrogen activity of malignant mammary gland cells, was recently approved by the Food and Drug Administration (FDA) for the treatment of breast cancer. In this investigation, the toxic effects of tamoxifen were evaluated through cell multiplication, and cytological, surface ultrastructural, and biochemical studies on human cervical carcinoma cells (HeLa) and/or murine erythroleukemic (MEL) cells (BB-88). Tamoxifen treatment ...

  4. Effect of troglitazone on radiation sensitivity in cervix cancer cells

    An, Zheng Zhe; Liu, Xian Guang; Song, Hye Jin; Choi, Chi Hwan; Kim, Won Dong; Park, Woo Yoon [Chungbuk National University College of Medicine, Cheongju (Korea, Republic of); Yu, Jae Ran [Konkuk University College of Medicine, Chungju (Korea, Republic of)

    2012-06-15

    Troglitazone (TRO) is a peroxisome proliferator-activated receptor {gamma} (PPAR{gamma} ) agonist. TRO has antiproliferative activity on many kinds of cancer cells via G1 arrest. TRO also increases Cu{sup 2+}/Zn{sup 2+} -superoxide dismutase (CuZnSOD) and catalase. Cell cycle, and SOD and catalase may affect on radiation sensitivity. We investigated the effect of TRO on radiation sensitivity in cancer cells in vitro. Three human cervix cancer cell lines (HeLa, Me180, and SiHa) were used. The protein expressions of SOD and catalase, and catalase activities were measured at 2-10 {mu}M of TRO for 24 hours. Cell cycle was evaluated with flow cytometry. Reactive oxygen species (ROS) was measured using 2',7'-dichlorofluorescin diacetate. Cell survival by radiation was measured with clonogenic assay. By 5 {mu}M TRO for 24 hours, the mRNA, protein expression and activity of catalase were increased in all three cell lines. G0- G1 phase cells were increased in HeLa and Me180 by 5 {mu}M TRO for 24 hours, but those were not increased in SiHa. By pretreatment with 5 {mu}M TRO radiation sensitivity was increased in HeLa and Me180, but it was decreased in SiHa. In Me180, with 2 {mu}M TRO which increased catalase but not increased G0-G1 cells, radiosensitization was not observed. ROS produced by radiation was decreased with TRO. TRO increases radiation sensitivity through G0-G1 arrest or decreases radiation sensitivity through catalasemediated ROS scavenging according to TRO dose or cell types. The change of radiation sensitivity by combined with TRO is not dependent on the PPAR {gamma} expression level.

  5. Azithromycin Synergistically Enhances Anti-Proliferative Activity of Vincristine in Cervical and Gastric Cancer Cells

    Zhou, Xuezhang; Zhang, Yuyan; Li, Yong; Hao, Xiujing; Liu, Xiaoming, E-mail: erc1080@gmail.com; Wang, Yujiong, E-mail: erc1080@gmail.com [Key Laboratory of the Ministry of Education for the Conservation and Utilization of Special Biological Resources of Western China, Yinchuan 750021, Ningxia (China); College of Life Science, Ningxia University, Yinchuan 750021, Ningxia (China)

    2012-12-04

    In this study, the anti-proliferative and anticancer activity of azithromycin (AZM) was examined. In the presence of AZM, cell growth was inhibited more effectively in Hela and SGC-7901 cancer cells, relative to transformed BHK-21 cells. The respective 50% inhibition of cell growth (IC{sub 50}) values for Hela, SGC-7901 and BHK-21 were 15.66, 26.05 and 91.00 µg/mL at 72 h post incubation, indicative of a selective cytotoxicity against cancer cells. Cell apoptosis analysis using Hoechst nuclear staining and annexin V-FITC binding assay further demonstrated that AZM was capable of inducing apoptosis in both cancer cells and transformed cells. The apoptosis induced by AZM was partly through a caspase-dependent mechanism with an up-regulation of apoptotic protein cleavage PARP and caspase-3 products, as well as a down-regulation of anti-apoptotic proteins, Mcl-1, bcl-2 and bcl-X1. More importantly, a combination of AZM and a low dose of the common anti-cancer chemotherapeutic agent vincristine (VCR), produced a selectively synergistic effect on apoptosis of Hela and SGC-7901 cells, but not BHK-21 cells. In the presence of 12.50 μg/mL of VCR, the respective IC{sub 50} values of Hela, SGC-7901 and BHK-21 cells to AZM were reduced to 9.47 µg/mL, 8.43 µg/mL and 40.15 µg/mL at 72 h after the incubation, suggesting that the cytotoxicity of AZM had a selective anti-cancer effect on cancer over transformed cells in vitro. These results imply that AZM may be a potential anticancer agent for use in chemotherapy regimens, and it may minimize side effects via reduction of dosage and enhancing the effectiveness common chemotherapeutic drugs.

  6. Synergic effect of human IL-21 gene transfer combined with γ-ray irradiation on the growth of cervical carcinoma HeLa cells

    Objective: To study the combined effect of interleukin-21 gene transfer and ionizing radiation on the growth of cervical carcinoma HeLa cells. Methods: Previously constructed Ad-IL-21 gene was amplified by infecting 293A cells and the titer was measured by TCID50 method. HeLa cells were transfected with Ad-IL-21 and then irradiated with 6 Gy 137Cs γ-rays. The cells were divided into 5 groups, including blank control, Ad-LacZ group, Ad-IL-21 group,radiation group and Ad-IL-21 combined with radiation group (combination group). The cell growth, cell cycle, apoptosis, and the expressions of IL-21 gene and protein in HeLa cells were detected. Results: Ad-IL-21 was successfully amplified and the titer of Ad-11.-21 was 9 × 1010 pfu/ml. Compared with Ad-IL-21 group and radiation group,the cell growth of combination group was significantly inhibited at 96 h after transfection (F=85.26, 72.98, P<0.05). The cells in combination group were arrested in G1 phase and decreased at S phase (F=36.69, 34.83, P<0.05), while the cellular apoptosis increased markedly (F=28.23, 25.57, Pcell growth. (authors)

  7. Green synthesis of Se/Ru alloy nanoparticles using gallic acid and evaluation of theiranti-invasive effects in HeLa cells.

    Zhou, Yanhui; Xu, Meng; Liu, Yanan; Bai, Yan; Deng, Yuqian; Liu, Jie; Chen, Lanmei

    2016-08-01

    Methods for the synthesis of nanoparticles (NPs) for biomedical applications ideally involve the use of nontoxic reducing and capping agents, and more importantly, enable control over the shape and size of the particles. As such, we used gallic acid (GA) as both a reducing and a capping agent in a simple and "green" synthesis of stable Se/Rualloy NPs (GA-Se/RuNPs). The diameter and morphology of the Se/Ru alloy NPs were regulated by GA concentration, and the presence of Ru was found to be a key factor in regulating and controlling the size of GA-Se/RuNPs. Moreover, GA-Se/RuNPs suppressed HeLa cell proliferation through the induction of apoptosis at concentrations that were nontoxic in normal cells. Furthermore, GA-Se/RuNPs effectively inhibited migration and invasion in HeLa cells via the inhibition of MMP-2 and MMP-9 proteins. Our findings confirm that bimetallic (Se/Ru) NPs prepared via GA-mediated synthesis exhibit enhanced anticancer effects. PMID:27085043

  8. Construction of eukaryotic expression vector encoding ATP synthase lipid-binding protein-like protein gene of Sj and its expression in HeLa cells

    Ouyang Danming; Hu Yongxuan; Li Mulan; Zeng Xiaojun; He Zhixiong; Yuan Caijia

    2008-01-01

    Objective: To clone and construct the recombinant plasmid containing ATP synthase lipid-binding protein-like protein gene of Schistosoma japonicum,(SjAslp) and transfer it into mammalian cells to express the objective protein. Methods: By polymerase chain reaction (PCR) technique, SjAslp was amplified from the constructed recombinant plasmid pBCSK+/SjAslp, and inserted into cloning vector pUCm-T. Then, SjAslp was subcloned into an eukaryotic expression vector pcDNA3.1(+). After identifying it by PCR, restrictive enzymes digestion and DNA sequencing, the recombinant plasmid was transfected into HeLa cells using electroporation, and the expression of the recombinant protein was analyzed by immunocytochemical assay. Resnlts: The specific gene fragment of 558 bp was successfully amplified. The DNA vaccine of SjAslp was successfully constructed. Immunocytochemical assay showed that SjAslp was expressed in the cytoplasm of HeLa cells. Conclusion: SjAslp gene can be expressed in eukaryotic system, which lays the foundation for development of the SjAslp DNA vaccine against schitosomiasis.

  9. Calcitriol Inhibits Cervical Cancer Cell Proliferation Through Downregulation of HCCR1 Expression.

    Wang, Guoqing; Lei, Lei; Zhao, Xixia; Zhang, Jun; Zhou, Min; Nan, Kejun

    2014-01-01

    Calcitriol (1α,25-dihydroxyvitamin D3) has demonstrated anticancer activity against several tumors. However, the underlying mechanism for this activity is not yet fully understood. Our experiment was designed and performed to address one aspect of this issue in cervical cancer. HeLa S3 cells were cultured in media with various concentrations of calcitriol. Cell proliferation and cell cycle were assessed by spectrophotometry and flow cytometry, respectively. The mRNA and protein expression levels of human cervical cancer oncogene (HCCR-1) and p21 were determined by RT-PCR and Western blot, respectively. Results indicated that calcitriol inhibited HeLa S3 cell proliferation and induced cell cycle arrest at the G1 phase. Calcitriol decreased HCCR-1 protein expression in a dose- and time-dependent manner. Furthermore, promoter activity analyses revealed that transcriptional regulation was involved in the inhibition of HCCR-1 expression. Overexpression of HCCR-1 in HeLa S3 cells reversed the inhibition of cell proliferation and G1 phase arrest that resulted from calcitriol treatment. In addition, calcitriol increased p21 expression and promoter activity. HCCR-1 overexpression decreased p21 expression and promoter activity. Thus, our results suggested that calcitriol inhibited HeLa S3 cell proliferation by decreasing HCCR-1 expression and increasing p21 expression. PMID:26629942

  10. In vitro antiproliferative activity of Annona reticulata roots on human cancer cell lines

    H M Suresh

    2011-01-01

    Full Text Available Background: The phytochemical and pharmacological activities of Annona reticulata components suggest a wide range of clinical application in lieu of cancer chemotherapy. Materials and Methods: Ethanol and aqueous extracts of roots of Annona reticulata Linn were studied for their in vitro antiproliferative activity on A-549 (human lung carcinoma, K-562 (human chronic myelogenous leukemia bone marrow, HeLa (human cervix and MDA-MB (human adenocarcinoma mammary gland cancer cell lines by MTT [3-(4,5-dimethyl thiazol-2-yl-2,5-diphenyl tetrazolium bromide] colorimetric assay. Results: The ethanol extract exhibited a prominent inhibitory effect against A-549, K-562, HeLa and MDA-MB human cancer cell lines at a concentration range between 10 and 40 μg/ml, whereas the aqueous extract showed a lower activity at the same concentration. Simultaneously, the effect of the ethanol extract toward the inhibition of Vero cell line proliferation was lower in comparison with the cancer cell lines. Conclusion: The significant antiproliferative activity of the ethanol extract of Annona reticulata roots against A-549, K-562, HeLa and MDA-MB human cancer cell lines may be attributed toward the collective presence of acetogenins, alkaloids and lower inhibitory effect on Vero cell line, which suggests Annona reticulata be used as a chemopreventive agent in cancer therapy.

  11. Gecko Proteins Exert Anti-Tumor Effect against Cervical Cancer Cells Via PI3-Kinase/Akt Pathway

    Jeong, Ae-Jin; Chung, Chung-Nam; Kim, Hye-Jin; Bae, Kil Soo; Choi, Song; Jun, Woo Jin; Shim, Sang In; Kang, Tae-Hong; Leem, Sun-Hee; Chung, Jin Woong

    2012-01-01

    Anti-tumor activity of the proteins from Gecko (GP) on cervical cancer cells, and its signaling mechanisms were assessed by viable cell counting, propidium iodide (PI) staining, and Western blot analysis. GP induced the cell death of HeLa cells in a dose-dependent manner while it did not affect the viability of normal cells. Western blot analysis showed that GP decreased the activation of Akt, and co-administration of GP and Akt inhibitors synergistically exerted anti-tumor activities on HeLa...

  12. Design and fabrication of a microplatform for the proximity effect study of localized ELF-EMF on the growth of in vitro HeLa and PC-12 cells

    Chen, Y. C.; Chen, C. C.; Tu, W.; Cheng, Y. T.; Tseng, F. G.

    2010-12-01

    This paper presents a platform technology with experimental results that show the scientists and biologists a way to rapidly investigate and analyze the biological effects of localized extremely low frequency (ELF) electromagnetic field (EMF) on living cells. The proximity effect of the localized ELF-EMF on living cells is revealed using the bio-compatible microplatform on which an on-glass inductive coil array, the source of the localized ELF-EMF in micro scale, is designed, fabricated and operated with a field strength of 1.2 ± 0.1 mT at 60 Hz for cell culturing study. After a 72 h ELF-EMF exposure, HeLa (human cervical cancer) and PC-12 (rat pheochromocytoma) cells exhibit about 18.4% and 12.9% cell proliferation rate reduction, respectively. Furthermore, according to the presented dynamic model, the reduction of the proliferation can be attributed to the interference of signal transduction processes due to the tangential currents induced around the cells.

  13. Mapping and identification of HeLa cell proteins separated by immobilized pH-gradient two-dimensional gel electrophoresis and construction of a two-dimensional polyacrylamide gel electrophoresis database

    Shaw, AC; Rossel Larsen, M; Roepstorff, P; Holm, A; Christiansen, Gunna; Birkelund, Svend

    1999-01-01

    The HeLa cell line, a human adenocarcinoma, is used in many research fields, since it can be infected with a wide range of viruses and intracellular bacteria. Therefore, the mapping of HeLa cell proteins is useful for the investigation of parasite host cell interactions. Because of the recent imp...... and future data accessible for interlaboratory comparison, we constructed a 2-D PAGE database on the World Wide Web....... mapping of [35S]methionine/cysteine-labeled HeLa cell proteins with the 2-D PAGE (IPG)-system, using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and N-terminal sequencing for protein identification. To date 21 proteins have been identified and mapped. In order to make these...

  14. Estrogenic Activity of Coumestrol, DDT, and TCDD in Human Cervical Cancer Cells

    Kenneth Ndebele

    2010-05-01

    Full Text Available Endogenous estrogens have dramatic and differential effects on classical endocrine organ and proliferation. Xenoestrogens are environmental estrogens that have endocrine impact, acting as both estrogen agonists and antagonists, but whose effects are not well characterized. In this investigation we sought to delineate effects of xenoestrogens. Using human cervical cancer cells (HeLa cells as a model, the effects of representative xenoestrogens (Coumestrol-a phytoestrogen, tetrachlorodioxin (TCDD-a herbicide and DDT-a pesticide on proliferation, cell cycle, and apoptosis were examined. These xenoestrogens and estrogen inhibited the proliferation of Hela cells in a dose dependent manner from 20 to 120 nM suggesting, that 17-β-estrtadiol and xenoestrogens induced cytotoxic effects. Coumestrol produced accumulation of HeLa cells in G2/M phase, and subsequently induced apoptosis. Similar effects were observed in estrogen treated cells. These changes were associated with suppressed bcl-2 protein and augmented Cyclins A and D proteins. DDT and TCDD exposure did not induce apoptosis. These preliminary data taken together, suggest that xenoestrogens have direct, compound-specific effects on HeLa cells. This study further enhances our understanding of environmental modulation of cervical cancer.

  15. Inhibitors of cysteine cathepsin and calpain do not prevent ultraviolet-B-induced apoptosis in human keratinocytes and HeLa cells

    Bang, Bo; Baadsgaard, Ole; Skov, Lone;

    2004-01-01

    Caspases, members of the cysteine protease family, execute UVB-induced apoptosis in several cell lines and keratinocytes. Several researchers investigating UVB-induced apoptosis have demonstrated a dose-dependent protective effect of the synthetic peptide caspase inhibitor zVAD-fmk. However, z......VAD-fmk displays a dose-dependent protective effect against UVB-induced apoptosis, even at doses higher than those required to block all known proapoptotic caspases. In addition, it is known that zVAD-fmk also inhibits other cysteine proteases including cathepsins and calpains, and these proteases have recently...... been demonstrated to play a role in the execution of programmed cell death induced by other stimuli, e.g. TNF-alpha. The purpose of the present study was therefore to investigate whether inhibitors of cysteine cathepsins and calpains could prevent UVB-induced apoptosis in HeLa cells and keratinocytes...

  16. Functional interaction between hMYH and hTRADD in the TNF-α-mediated survival and death pathways of HeLa cells

    Vy Tran, An Hue; Hahm, Soo-Hyun; Han, Se Hee [Department of Advanced Technology Fusion, Konkuk University, Hwayang-dong, Gwangjin-gu, Seoul 143-701 (Korea, Republic of); Chung, Ji Hyung [Department of Applied Bioscience, College of Life Science, CHA University, Gyeonggi-do 463-836 (Korea, Republic of); Park, Geon Tae [Cornell University, Ithaca, NY 14850 (United States); Han, Ye Sun, E-mail: yshan@konkuk.ac.kr [College of Global Integrated Studies, Division of Interdisciplinary Studies, Konkuk University, Hwayang-dong, Gwangjin-gu, Seoul 143-701 (Korea, Republic of)

    2015-07-15

    Highlights: • We determine the interaction between hMYH and hTRADD. • We examine changes in the level of hMYH–hTRADD interaction under TNF-α treatment. • hTRADD–hMYH association is involved in the nuclear translocation of NFκB. • hTRADD–hMYH complex influences the TNFR1–TRADD association. - Abstract: The tumor necrosis factor (TNF) signaling pathway is a classical immune system pathway that plays a key role in regulating cell survival and apoptosis. The TNF receptor-associated death domain (TRADD) protein is recruited to the death domain of TNF receptor 1 (TNFR1), where it interacts with TNF receptor-associated factor 2 (TRAF2) and receptor-interacting protein (RIP) for the induction of apoptosis, necrosis, nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), and mitogen-activated protein (MAP) kinase activation. In this study, we found that the human MutY homolog (hMYH) interacted with human TRADD (hTRADD) via the C-terminal domain of hMYH. Moreover, under conditions promoting TNF-α-induced cell death or survival in HeLa cells, this interaction was weakened or enhanced, respectively. The interaction between hMYH and hTRADD was important for signaling pathways mediated by TNF-α. Our results also suggested that the hTRADD–hMYH association was involved in the nuclear translocation of NFκB and formation of the TNFR1–TRADD complex. Thus, this study identified a novel mechanism through which the hMYH–hTRADD interaction may affect the TNF-α signaling pathway. Implications: In HeLa cells, the hTRADD–hMYH interaction functioned in both cell survival and apoptosis pathways following TNF-α stimulation.

  17. Functional interaction between hMYH and hTRADD in the TNF-α-mediated survival and death pathways of HeLa cells

    Highlights: • We determine the interaction between hMYH and hTRADD. • We examine changes in the level of hMYH–hTRADD interaction under TNF-α treatment. • hTRADD–hMYH association is involved in the nuclear translocation of NFκB. • hTRADD–hMYH complex influences the TNFR1–TRADD association. - Abstract: The tumor necrosis factor (TNF) signaling pathway is a classical immune system pathway that plays a key role in regulating cell survival and apoptosis. The TNF receptor-associated death domain (TRADD) protein is recruited to the death domain of TNF receptor 1 (TNFR1), where it interacts with TNF receptor-associated factor 2 (TRAF2) and receptor-interacting protein (RIP) for the induction of apoptosis, necrosis, nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), and mitogen-activated protein (MAP) kinase activation. In this study, we found that the human MutY homolog (hMYH) interacted with human TRADD (hTRADD) via the C-terminal domain of hMYH. Moreover, under conditions promoting TNF-α-induced cell death or survival in HeLa cells, this interaction was weakened or enhanced, respectively. The interaction between hMYH and hTRADD was important for signaling pathways mediated by TNF-α. Our results also suggested that the hTRADD–hMYH association was involved in the nuclear translocation of NFκB and formation of the TNFR1–TRADD complex. Thus, this study identified a novel mechanism through which the hMYH–hTRADD interaction may affect the TNF-α signaling pathway. Implications: In HeLa cells, the hTRADD–hMYH interaction functioned in both cell survival and apoptosis pathways following TNF-α stimulation

  18. Regulated Necrosis in HeLa Cells Induced by ZnPc Photodynamic Treatment: A New Nuclear Morphology

    Jorge Soriano

    2014-12-01

    Full Text Available Photodynamic therapy (PDT is a cancer treatment modality based on the administration of a photosensitizer (PS, which accumulates preferentially in tumor cells. Subsequent irradiation of the neoplastic area triggers a cascade of photochemical reactions that leads to the formation of highly reactive oxygen species responsible for cell inactivation. Photodynamic treatments in vitro are performed with the PS, zinc-phthalocyanine (ZnPc. The PS is near the plasma membrane during uptake and internalization. Inactivation clearly occurs by a necrotic process, manifested by nuclear pyknosis, negative TUNEL and Annexin V assays and non-relocation of cytochrome c. In contrast, by increasing the incubation time, ZnPc is accumulated in the Golgi apparatus and produces cell inactivation with characteristics of apoptosis and necrosis: TUNEL positive, relocated cytochrome c and negative Annexin V assay. This type of death produces a still undescribed granulated nuclear morphology, which is different from that of necrosis or apoptosis. This morphology is inhibited by necrostatin-1, a specific inhibitor of regulated necrosis.

  19. Photothermal therapy of cancer cells using novel hollow gold nanoflowers

    Han J

    2014-01-01

    Full Text Available Jing Han,1 Jinru Li,1 Wenfeng Jia,1 Liangming Yao,2 Xiaoqin Li,1 Long Jiang,1 Yong Tian21Beijing National Laboratory for Molecular Sciences, Institute of Chemistry, 2Beijing Key Laboratory of Noncoding RNA, Institute of Biophysics, Chinese Academy of Sciences, Beijing, People's Republic of ChinaAbstract: This article presents a new strategy for fabricating large gold nanoflowers (AuNFs that exhibit high biological safety under visible light and very strong photothermal cytotoxicity to HeLa cells under irradiation with near-infrared (NIR light. This particular type of AuNF was constructed using vesicles produced from a multiamine head surfactant as a template followed by depositing gold nanoparticles (AuNPs and growing their crystallites on the surface of vesicles. The localized surface plasmon-resonance spectrum of this type of AuNF can be easily modulated to the NIR region by controlling the size of the AuNFs. When the size of the AuNFs increased, biosafety under visible light improved and cytotoxicity increased under NIR irradiation. Experiments in vitro with HeLa cells and in vivo with small mice have been carried out, with promising results. The mechanism for this phenomenon is based on the hypothesis that it is difficult for larger AuNFs to enter the cell without NIR irradiation, but they enter the cell easily at the higher temperatures caused by NIR irradiation. We believe that these effects will exist in other types of noble metallic NPs and cancer cells. In addition, the affinity between AuNPs and functional biomolecules, such as aptamers and biomarkers, will make this type of AuNF a good recognition device in cancer diagnosis and therapy.Keywords: HeLa cells, endocytosis, cytotoxicity, AuNFs, NIR, cancer therapy

  20. Cancer Stem Cells, Cancer Cell Plasticity and Radiation Therapy

    Vlashi, Erina; Pajonk, Frank

    2014-01-01

    Since the first prospective identification of cancer stem cells in solid cancers the cancer stem cell hypothesis has reemerged as a research topic of increasing interest. It postulates that solid cancers are organized hierarchically with a small number of cancer stem cells driving tumor growth, repopulation after injury and metastasis. They give rise to differentiated progeny, which lack these features. The model predicts that for any therapy to provide cure, all cancer stem cells have to be ...

  1. A systematic High-Content Screening microscopy approach reveals key roles for Rab33b, OATL1 and Myo6 in nanoparticle trafficking in HeLa cells

    Panarella, Angela; Bexiga, Mariana G.; Galea, George; O’ Neill, Elaine D.; Salvati, Anna; Dawson, Kenneth A.; Simpson, Jeremy C.

    2016-01-01

    Synthetic nanoparticles are promising tools for imaging and drug delivery; however the molecular details of cellular internalization and trafficking await full characterization. Current knowledge suggests that following endocytosis most nanoparticles pass from endosomes to lysosomes. In order to design effective drug delivery strategies that can use the endocytic pathway, or by-pass lysosomal accumulation, a comprehensive understanding of nanoparticle uptake and trafficking mechanisms is therefore fundamental. Here we describe and apply an RNA interference-based high-content screening microscopy strategy to assess the intracellular trafficking of fluorescently-labeled polystyrene nanoparticles in HeLa cells. We screened a total of 408 genes involved in cytoskeleton and membrane function, revealing roles for myosin VI, Rab33b and OATL1 in this process. This work provides the first systematic large-scale quantitative assessment of the proteins responsible for nanoparticle trafficking in cells, paving the way for subsequent genome-wide studies. PMID:27374232

  2. Breast cancer stem cells

    MatthewJNaylor

    2013-08-01

    Full Text Available Cancer metastasis, resistance to therapies and disease recurrence are significant hurdles to successful treatment of breast cancer. Identifying mechanisms by which cancer spreads, survives treatment regimes and regenerates more aggressive tumours are critical to improving patient survival. Substantial evidence gathered over the last 10 years suggests that breast cancer progression and recurrence is supported by cancer stem cells (CSCs. Understanding how CSCs form and how they contribute to the pathology of breast cancer will greatly aid the pursuit of novel therapies targeted at eliminating these cells. This review will summarise what is currently known about the origins of breast CSCs, their role in disease progression and ways in which they may be targeted therapeutically.

  3. In vitro antiproliferative activity of Annona reticulata roots on human cancer cell lines

    Suresh, H. M.; B Shivakumar; K.Hemalatha; S S Heroor; Hugar, D. S.; Sambasiva Rao, K. R. S.

    2011-01-01

    Background: The phytochemical and pharmacological activities of Annona reticulata components suggest a wide range of clinical application in lieu of cancer chemotherapy. Materials and Methods: Ethanol and aqueous extracts of roots of Annona reticulata Linn were studied for their in vitro antiproliferative activity on A-549 (human lung carcinoma), K-562 (human chronic myelogenous leukemia bone marrow), HeLa (human cervix) and MDA-MB (human adenocarcinoma mammary gland) cancer cell lines by MTT...

  4. Silencing Bcl-2 Expression in Epithelial Cancer Cells Using “Smart” Particles

    Yen-Ling Lin

    2014-09-01

    Full Text Available Short interfering RNA (siRNA targeted against anti-apoptotic Bcl-2 protein proved to knockdown its expression and trigger cancer cell death. We used degradable, pH-sensitive, comb-like [P(EAA-co-BMA-b-PNASI-g-P(HMA-co-TMAEMA] polymer to condense anti-Bcl-2 siRNA into “smart” particles, which proved to shuttle their cargo past the endosomal membrane and into the cytoplasm of HeLa and UM-SCC-17B cancer cells. HeLa and UM-SCC-17B cancer cells were treated with anti-Bcl-2 particles followed by quantifying Bcl-2 mRNA and protein levels using qRT-PCR and western blotting, respectively. “Smart” anti-Bcl-2 particles selectively suppress Bcl-2 mRNA and protein levels in HeLa cells by 50%–60% and 79%–81%, respectively. Similarly, “smart” anti-Bcl-2 particles inhibited Bcl-2 mRNA levels by 30%, 40%, and 20% upon incubation with UM-SCC-17B cancer cells for 48, 72, and 96 h, respectively. Bcl-2 protein expression in UM-SCC-17B cancer cells was inhibited by 30% after treatment for 72 h. Results show that pH-sensitive comb-like polymer complex anti-Bcl-2 siRNA forming “smart” nanoparticles that deliver their cargo into the cytoplasm of HeLa and UM-SCC-17B cancer cells causing Bcl-2 knockdown at the mRNA and protein levels.

  5. Inhibitory effects of 405 nm irradiation on Chlamydia trachomatis growth and characterization of the ensuing inflammatory response in HeLa cells

    Wasson Cassandra J

    2012-08-01

    Full Text Available Abstract Background Chlamydia trachomatis is an intracellular bacterium that resides in the conjunctival and reproductive tract mucosae and is responsible for an array of acute and chronic diseases. A percentage of these infections persist even after use of antibiotics, suggesting the need for alternative treatments. Previous studies have demonstrated anti-bacterial effects using different wavelengths of visible light at varying energy densities, though only against extracellular bacteria. We investigated the effects of visible light (405 and 670 nm irradiation via light emitting diode (LEDs on chlamydial growth in endocervical epithelial cells, HeLa, during active and penicillin-induced persistent infections. Furthermore, we analyzed the effect of this photo treatment on the ensuing secretion of IL-6 and CCL2, two pro-inflammatory cytokines that have previously been identified as immunopathologic components associated with trichiasis in vivo. Results C. trachomatis-infected HeLa cells were treated with 405 or 670 nm irradiation at varying energy densities (0 – 20 J/cm2. Bacterial growth was assessed by quantitative real-time PCR analyzing the 16S: GAPDH ratio, while cell-free supernatants were examined for IL-6 and monocyte chemoattractant protein-1 (CCL2 production. Our results demonstrated a significant dose-dependent inhibitory effect on chlamydial growth during both active and persistent infections following 405 nm irradiation. Diminished bacterial load corresponded to lower IL-6 concentrations, but was not related to CCL2 levels. In vitro modeling of a persistent C. trachomatis infection induced by penicillin demonstrated significantly elevated IL-6 levels compared to C. trachomatis infection alone, though 405 nm irradiation had a minimal effect on this production. Conclusion Together these results identify novel inhibitory effects of 405 nm violet light on the bacterial growth of intracellular bacterium C. trachomatis in

  6. AKT–THE MAMMALIAN TARGET OF RAPAMYCIN (MTOR PATHWAY INHIBITION INCREASES CERVICAL CANCER CELL CHEMOSENSITIVITY TO ACTIVE FORM OF IRINOTECAN (SN-38

    Leri Septiani

    2015-07-01

    Full Text Available Objective: To investigate the molecular pathway of the cytotoxic effect of SN-38 in human cervical cancer cell lines. Methods: Two human cervical cancer cell lines were treated with various concentrations of irinotecan for 24–72 hours and the sensitivity was analysed using the MTT assay. Apoptosis was further observed through microscopic examinations. The protein expression was determined using Western blot analysis. Results: CaSki cells demonstrated the highest sensitivity to SN-38, whereas HeLa cells showed the lowest. In cervical cancer cells, SN-38 induced apoptosis through an intrinsic- and extrinsic-pathways. In addition, we showed that SN-38 downregulated the phosphorylation of Akt-mTOR pathways in CaSki cells, but not in HeLa cells. Interestingly, in HeLa cells, which were more suggestive of a resistant phenotype, pre-treatment with LY294002 and rapamycin inhibited activation of Akt-mTOR signaling and significantly enhanced the sensitivity of HeLa cells to SN-38. Conclusions: Irinotecan exerts its anti-neoplastic effects on cervical cancer cells by inducing apoptosis through caspase-cascade. Inhibition of Akt-mTOR, LY294002 and rapamycin, which is targeted to Akt-mTOR pathways, may sensitize irinotecan-resistant cervical cancer cells.

  7. Tailoring folic acid and methotrexate-attributed quantum dots for integrated cancer cell imaging and therapy

    Fahmi, Mochamad Zakki; Chang, Jia-Yaw

    2016-03-01

    Potential application of folic acid and methotrexate-attributed AgInS2-ZnS quantum dots on both detection and therapeutic of cancer cell were intensively investigated on this study. In the initial step, the bright luminescent of QDs, with % QY up to 55.3, were synthesized with one-pot two-step process resulting narrow particle distribution and successfully transferred to water phase without significant effect on optical properties. The water-soluble AgInS2-ZnS quantum dots (QDs) encapsulated with oleylamine have been successfully prepared by ultrasonication assisting. Several aspect including QDs characterization, pH stability, ionic strength, and bonding properties were investigated to reach desired condition of water-soluble AgInS2-ZnS QDs. Folic acid was further conjugated to QDs for HeLa and MCF7 cancer cell imaging to performs the targeting capability. Moreover, folic acid is efficiently internalized into cell through the receptor-mediated endocytosis even when conjugated with a wide variety of molecules. Confocal imaging characterization further informs folic acid-conjugated AgInS2-ZnS QDs could most specific targeted to the human cervical (HeLa) cells. The therapeutic feature of QDs on HeLa cancer cell was conjugated by attributing methotrexate on the QDs, instead of folic acid, and the design could improve on inhibiting the cancer cell viability as well as its fluorescent intensity.

  8. Fingerprints in cancer cells

    Gene research has shown that factors causing cancer, or carcinogens, may leave marks typical of each particular carcinogen (fingerprints) in the genotype of the cell. Radiation, for instance, may leave such fingerprints in a cancer cell. In particular, the discovery of a gene called p53 has yielded much new information on fingerprints. It has been discovered, for example, that toxic fungus and UV-radiation each leave fingerprints in the p53 gene. Based on the detection of fingerprints, it may be possible in the future to tell a cancer patient what factor had trigged the maglinancy

  9. Evaluation of Cytotoxic Effects of Dichloromethane Extract of Guduchi (Tinospora cordifolia Miers ex Hook F & THOMS on Cultured HeLa Cells

    Ganesh Chandra Jagetia

    2006-01-01

    Full Text Available Extracts of Tinospora cordifolia (TCE have been shown to possess anti-tumor properties, but the mechanism of the anti-tumor function of TCE is poorly understood. This investigation elucidates the possible mechanism underlying the cytotoxic effects of dichlormethane extracts of TCE, after selecting optimal duration and concentration for treatment. HeLa cells were exposed to various concentrations of TCE, which has resulted in a concentration-dependent decline in the clonogenicity, glutathione-S-transferase (GST activity and a concentration-dependent increase in lipid peroxidation (TBARS with a peak at 4 h and lactate dehydrogenase (LDH release with a peak at 2 h. Our results suggest that the cytotoxic effect of TCE may be due to lipid peroxidation and release of LDH and decline in GST.

  10. Dose-Survival Curves for HeLa Cell Cultures using Thermal Neutrons and the B10(n, α) Li7 Reaction

    The effects of 250-kVp X-ray, thermal neutron irradiation and the thermal neutron capture reaction of boron-10, B10(n, α)Li7, on the ability of HeLa cells to proliferate have been evaluated with multiple event dose-survival curves. The dose-survival data following thermal neutron irradiation for cultures containing 10 μg of boron-10 per ml can be expressed by a single-event curve. Dose survival data having an inflection point are expressed as a composite curve consisting of a single-event and multiple-event fraction. The dose-survival data demonstrate a decreased radiosensitivity following dose fractionation or following irradiation in a nitrogen-carbon dioxide atmosphere. (author)

  11. The HeLa Documentary Film: An Engaging Writing and Culturally Relevant Assignment on Cell Division and Ethics for Nonscience Majors

    Diann Jordan

    2015-02-01

    Full Text Available Historically black institutions play a pivotal role in educating the next generation of scientists and engineers as well as promoting scientific literacy among all of its students. Students would like to have more culturally relevant assignments that reflect their life experiences as it relates to course content.  We used the HeLa documentary film, "The Way of All Flesh Film," as an effective teaching tool in the first survey course of general biology to supplement our discussion on the cell cycle and ethics in scientific studies.  Over 90% of our students preferred this additional teaching method compared to a traditional lecture only.  Furthermore, the exercise enhanced the students' writing, research, and critical thinking skills through the ethical implications of the film.

  12. 以端粒酶为靶标抗癌药物筛选模型建立及端粒酶抑制剂筛选%Determination of Telomerase from HeLa Cells as a Target for Screening Antitumor Agents

    郑晓飞; 王升启; 孙志贤

    2002-01-01

    Telomerase, a ribonucleoprotein enzyme, has been found in immortalized but not in most sonatic adult human tissues, and thus emerged as a novel target for cancer chemotherapy. Recently it has been found that telomerase is a fruitful target for oncologic drug development. A new method for screening antitumor agents by using telomerase as a target has been established according to the phenomena that the enzyme activity ean be affected bv some types of antitumor agents or chemicals. The telomerase was extracted from HeLa cells. The telomeric repeat amplification protocol(TRAP) was used to measure enzyme activity. Telomerase activity can be inhibited by 4 kinds of chemical compounds.

  13. MOLECULAR CLONING OF hTRT CATALYTIC DOMAIN FROM HeLa CELLS AND ITS EXPRESSION IN E. Coli AND PURIFICATION

    2000-01-01

    Objective. To investigate the expression of telomerase gene hTRT mRNA in HeLa cells and to obtain hTRT pro-tein for futher study. Methods. The gene for encoding hTRT catalytic domain was cloned based on RT-PCR amplification from HeLa cells and sequenced. The cloned hTRTcDNA was in-frame inserted into His-tag fusion expression vector pEK318. The His-tag hTRT fusion proteins were purified by Ni-NTA chromatography and stained by westerm blotting. Results. An approximately 620bp fragment was generated and cloned into pBluescript SK + between Sail and BamHI sites. DNA sequencing showed the isolated fragment was consistem to those reported. SDS-PAGE present that a 17kDa protein was expressed stably in E. coli JM109 harboring pEKTRTM4 containing 6 × His-tag and hTRT 150aa, and the expression level of the protein was about 26% of the total bacterial proteins, while the expression of pEKTRT containing 6 × His-tag and hTRT 243aa was only detectable as 27 kDa band in western blotting. Both of fu-sion proteins were purified by Ni-NTA chromatography and showed single band( > 95% purifity) in Coomassie Bril-liant staining. Westem-blotting confirmed that two proteins could be recognized by the Ni-NTA AP conjugate. Conclusions. The hTRT catalytic domain was highly conserved. The expressed hTRT protein contained recogniz-able His-tag, telomerase-specific and strong antigenic epitops, which may be convenient for further investigation.

  14. Prostate cancer stem cells

    Tu, Shi-Ming; Lin, Sue-Hwa

    2011-01-01

    Stem cells have long been implicated in prostate glandular formation. The prostate undergoes regression after androgen deprivation and regeneration after testosterone replacement. Regenerative studies suggest that these cells are found in the proximal ducts and basal layer of the prostate. Many characteristics of prostate cancer indicate that it originates from stem cells. For example, the putative AR− status of prostate stem cells renders them inherently insensitive to androgen blockade ther...

  15. Crude aqueous extracts of Pluchea indica (L. Less. inhibit proliferation and migration of cancer cells through induction of p53-dependent cell death

    Cho Jonathan J

    2012-12-01

    Full Text Available Abstract Background Pluchea indica (L. Less. (Asteraceae is a perennial shrub plant with anti-inflammatory and antioxidant medicinal properties. However, the anti-cancer properties of its aqueous extracts have not been studied. The aim of this study was to investigate the anti-proliferation, anti-migration, and pro-apoptotic properties of crude aqueous extracts of P. indica leaf and root on human malignant glioma cancer cells and human cervical cancer cells, and the underlying molecular mechanism. Methods GBM8401 human glioma cells and HeLa cervical carcinoma cells were treated with various concentrations of crude aqueous extracts of P. indica leaf and root and cancer cell proliferation and viability were measured by cell growth curves, trypan blue exclusions, and the tetrazolium reduction assay. Effects of the crude aqueous extracts on focus formation, migration, and apoptosis of cancer cells were studied as well. The molecular mechanism that contributed to the anti-cancer activities of crude aqueous extracts of P. indica root was also examined using Western blotting analysis. Results Crude aqueous extracts of P. indica leaf and root suppressed proliferation, viability, and migration of GBM8401 and HeLa cells. Treatment with crude aqueous extracts of P. indica leaf and root for 48 hours resulted in a significant 75% and 70% inhibition on proliferation and viability of GBM8401 and HeLa cancer cells, respectively. Crude aqueous extracts of P. indica root inhibited focus formation and promoted apoptosis of HeLa cells. It was found that phosphorylated-p53 and p21 were induced in GBM8401 and HeLa cells treated with crude aqueous extracts of P. indica root. Expression of phosphorylated-AKT was decreased in HeLa cells treated with crude aqueous extracts of P. indica root. Conclusion The in vitro anti-cancer effects of crude aqueous extracts of P. indica leaf and root indicate that it has sufficient potential to warrant further examination and

  16. Action of caffeine on x-irradiated HeLa cells. VII. Evidence that caffeine enhances expression of potentially lethal radiation damage

    HeLa cells irradiated with 2 Gy of 220-kV X rays suffer a 60-70% loss of colony-forming ability which is increased to 90% by postirradiation treatment with 10 mM caffeine for 6 hr. The detailed postirradiation patterns of cell death and sister-cell fusion in such cultures and in cultures in which the colony-forming ability was brought to about the same level by treatment with a larger (4 Gy) X-ray dose alone or by longer (48 hr) treatment with 10 mM caffeine alone were recorded by time-lapse cinemicrography. Because the patterns of cell death and fusion differ radically in irradiated and in caffeine-treated cultures, the response of the additional cells killed by the combined treatment can be identified as X-ray induced rather than caffeine induced. The appearance of cultures after several days of incubation confirms the similarity of the post-treatment patterns of proliferation in cultures suffering enhanced killing to those occurring in cultures treated with larger doses of X rays alone. It is concluded that x rays do not sensitize cells to caffeine, but rather that caffeine enhanced the expression of potentially lethal radiation-induced damage

  17. Connexin30.2: in vitro interaction with connexin36 in HeLa cells and expression in AII amacrine cells and intrinsically photosensitive ganglion cells in the mouse retina

    Arndt eMeyer

    2016-05-01

    Full Text Available Electrical coupling via gap junctions is an abundant phenomenon in the mammalian retina and occurs in all major cell types. Gap junction channels are assembled from different connexin subunits, and the connexin composition of the channel confers specific properties to the electrical synapse. In the mouse retina, gap junctions were demonstrated between intrinsically photosensitive ganglion cells and displaced amacrine cells but the underlying connexin remained undetermined. In the primary rod pathway, gap junctions play a crucial role, coupling AII amacrine cells among each other and to ON cone bipolar cells. Although it has long been known that connexin36 and connexin45 are necessary for the proper functioning of this most sensitive rod pathway, differences between homocellular AII/AII gap junctions and AII/ON bipolar cell gap junctions suggested the presence of an additional connexin in AII amacrine cells. Here, we used a connexin30.2-lacZ mouse line to study the expression of connexin30.2 in the retina. We show that connexin30.2 is expressed in intrinsically photosensitive ganglion cells and AII amacrine cells. Moreover, we tested whether connexin30.2 and connexin36 – both expressed in AII amacrine cells – are able to interact with each other and are deposited in the same gap junctional plaques. Using newly generated anti-connexin30.2 antibodies, we show in HeLa cells that both connexins are indeed able to interact and may form heteromeric channels: both connexins were co-immunoprecipitated from transiently transfected HeLa cells and connexin30.2 gap junction plaques became significantly larger when co-expressed with connexin36. These data suggest that connexin36 is able to form heteromeric gap junctions with another connexin. We hypothesize that co-expression of connexin30.2 and connexin36 may endow AII amacrine cells with the means to differentially regulate its electrical coupling to different synaptic partners.

  18. Arsenic trioxide inhibits cell proliferation and human papillomavirus oncogene expression in cervical cancer cells

    Wang, Hongtao [Department of Pathology, School of Medicine, Southeast University, Nanjing 210009 (China); Gao, Peng [Department of Internal Medicine, University of Iowa, Iowa City, IA 52242 (United States); Zheng, Jie, E-mail: jiezheng54@126.com [Department of Pathology, School of Medicine, Southeast University, Nanjing 210009 (China)

    2014-09-05

    Highlights: • As{sub 2}O{sub 3} inhibits growth of cervical cancer cells and expression of HPV oncogenes in these cells. • HPV-negative cervical cancer cells are more sensitive to As{sub 2}O{sub 3} than HPV-positive cervical cancer cells. • HPV-18 positive cervical cancer cells are more sensitive to As{sub 2}O{sub 3} than HPV-16 positive cancer cells. • Down-regulation of HPV oncogenes by As{sub 2}O{sub 3} is partially due to the diminished AP-1 binding. - Abstract: Arsenic trioxide (As{sub 2}O{sub 3}) has shown therapeutic effects in some leukemias and solid cancers. However, the molecular mechanisms of its anticancer efficacy have not been clearly elucidated, particularly in solid cancers. Our previous data showed that As{sub 2}O{sub 3} induced apoptosis of human papillomavirus (HPV) 16 DNA-immortalized human cervical epithelial cells and cervical cancer cells and inhibited the expression of HPV oncogenes in these cells. In the present study, we systemically examined the effects of As{sub 2}O{sub 3} on five human cervical cancer cell lines and explored the possible molecular mechanisms. MTT assay showed that HPV-negative C33A cells were more sensitive to growth inhibition induced by As{sub 2}O{sub 3} than HPV-positive cervical cancer cells, and HPV 18-positive HeLa and C4-I cells were more sensitive to As{sub 2}O{sub 3} than HPV 16-positive CaSki and SiHa cells. After As{sub 2}O{sub 3} treatment, both mRNA and protein levels of HPV E6 and E7 obviously decreased in all HPV positive cell lines. In contrast, p53 and Rb protein levels increased in all tested cell lines. Transcription factor AP-1 protein expression decreased significantly in HeLa, CaSki and C33A cells with ELISA method. These results suggest that As{sub 2}O{sub 3} is a potential anticancer drug for cervical cancer.

  19. Connexin30.2: In Vitro Interaction with Connexin36 in HeLa Cells and Expression in AII Amacrine Cells and Intrinsically Photosensitive Ganglion Cells in the Mouse Retina

    Meyer, Arndt; Tetenborg, Stephan; Greb, Helena; Segelken, Jasmin; Dorgau, Birthe; Weiler, Reto; Hormuzdi, Sheriar G.; Janssen-Bienhold, Ulrike; Dedek, Karin

    2016-01-01

    Electrical coupling via gap junctions is an abundant phenomenon in the mammalian retina and occurs in all major cell types. Gap junction channels are assembled from different connexin subunits, and the connexin composition of the channel confers specific properties to the electrical synapse. In the mouse retina, gap junctions were demonstrated between intrinsically photosensitive ganglion cells and displaced amacrine cells but the underlying connexin remained undetermined. In the primary rod pathway, gap junctions play a crucial role, coupling AII amacrine cells among each other and to ON cone bipolar cells. Although it has long been known that connexin36 and connexin45 are necessary for the proper functioning of this most sensitive rod pathway, differences between homocellular AII/AII gap junctions and AII/ON bipolar cell gap junctions suggested the presence of an additional connexin in AII amacrine cells. Here, we used a connexin30.2-lacZ mouse line to study the expression of connexin30.2 in the retina. We show that connexin30.2 is expressed in intrinsically photosensitive ganglion cells and AII amacrine cells. Moreover, we tested whether connexin30.2 and connexin36—both expressed in AII amacrine cells—are able to interact with each other and are deposited in the same gap junctional plaques. Using newly generated anti-connexin30.2 antibodies, we show in HeLa cells that both connexins are indeed able to interact and may form heteromeric channels: both connexins were co-immunoprecipitated from transiently transfected HeLa cells and connexin30.2 gap junction plaques became significantly larger when co-expressed with connexin36. These data suggest that connexin36 is able to form heteromeric gap junctions with another connexin. We hypothesize that co-expression of connexin30.2 and connexin36 may endow AII amacrine cells with the means to differentially regulate its electrical coupling to different synaptic partners. PMID:27303262

  20. Stem Cells and Cancer

    Stem cell research has thrived over the last years due to their therapeutic and regenerative potential. Scientific breakthroughs in the field are immediately translated from the scientific journals to the mass media, which is not surprising as the characterisation of the molecular mechanisms that regulate the biology of stem cells is crucial for the treatment of degenerative and cardiovascular diseases, as well as cancer. In the Molecular Oncology Unit at Ciemat we work to unravel the role of cancer stem cells in tumour development, and to find new antitumor therapies. (Author)

  1. Detection of Auger enhancement induced in HeLa cells labeled with iododeoxyuridine and irradiated with 150 kV X-rays. Effects of cysteamine and dimethylsulfoxide

    We examined the effects of cysteamine and dimethylsulfoxide on the lethality of iododeoxyuridine-labeled HeLa cells irradiated with 150 kV X-rays (effective energy was 48 keV) and compared with those irradiated with 60Co γ-rays. The protectable fraction was estimated form the regression lines for the reciprocal plots of the concentration of protectors, versus the degree of protection. The protectable fraction by cysteamine was 9.4% lower in iododeoxyuridine-labeled cells than in control cells when they were irradiated with X-rays, whereas no such difference was observed with dimethylsulfoxide. The slopes of the regression lines were steeper (i.e., more difficult to protect) for both protectors in iododeoxyuridine-labeled cells irradiated with X-rays. No such differences were observed in γ-irradiated cells. The cause for these differences are attributable to Auger enhancement. The results suggest that at least a portion of Auger enhancement is not protectable by cysteamine and is hard to be protected by both protectors cysteamine and dimethylsulfoxide. (orig.)

  2. Cancer Stem Cells in Pancreatic Cancer

    Pancreatic cancer is an aggressive malignant solid tumor well-known by early metastasis, local invasion, resistance to standard chemo- and radiotherapy and poor prognosis. Increasing evidence indicates that pancreatic cancer is initiated and propagated by cancer stem cells (CSCs). Here we review the current research results regarding CSCs in pancreatic cancer and discuss the different markers identifying pancreatic CSCs. This review will focus on metastasis, microRNA regulation and anti-CSC therapy in pancreatic cancer

  3. Action of caffeine on x-irradiated HeLa cells. III. enhancement of x-ray-induced killing during G2 arrest

    The ability of caffeine to enhance the expression of potentially lethal x-ray damage in HeLa S3 cells was examined as a function of the age of the cells in the generation cycle. Synchronous populations were irradiated at different times after mitotic collection and treated for various intervals with 1 mM caffeiene, which causes negligible killing of unirradiated cells. The response was thereby determined as a function of cell age at both the time of irradiation and the time of exposure to caffeine. The amount of cell killing depends strongly on when in the cycle caffeine is present and only weakly on when the cells are irradiated. If cells are irradiated in early G1, caffeine treatment enhances killing for 2 to 3 hr. No additional enhancement is observed until 16 to 17 hr postcollection, corresponding to G2; here they enter a second period of much greater sensitivity. Similarly, fluorodeoxyuridine resynchronized cells irradiated during S and treated with caffeine suffer no enhanced killing until they pass into this sensitive phase in G2, approximately 7 hr after release from the fluorodeoxyuridine block. The sensitive period appears to coincide with G2 arrest. The rate and extent of killing during this period are dependent upon the x-ray dose and the caffeine concentration. In the absence of caffeine, cells irradiated in G1 lose sensitivity to caffeine in about 9 hr; they do so faster in G2. It is concluded that the potentially lethal x-ray damage expressed on treatment with caffeine is retained for many hours in the presence of caffeine and is maximally manifested by G2-arrested cells

  4. Evaluation of the Anti-proliferative Effects of Ophiocoma erinaceus Methanol Extract Against Human Cervical Cancer Cells

    Baharara, Javad; Amini, Elaheh; Namvar, Farideh

    2016-01-01

    Background: Marine organisms provide appreciable source of novel bioactive compounds with pharmacological potential. There is little information in correlation with anti-cancer activities of brittle star. In the present study, anti-neoplastic efficacy of Ophiocoma erinaceus methanol extract against human cervical cancer cells was investigated. Methods: The HeLa cells were cultured and exposed to brittle star methanol extract for 24 and 48 hr. The anti-proliferative properties were examined by...

  5. Cancer Stem Cells

    Aurelio Lorico; Eric Deutsch; Bo Lu; Shih-Hwa Chiou

    2011-01-01

    Cancer Stem Cells (CSCs) are a small subpopulation of cells within tumors with capabilities of self-renewal, differentiation, and tumorigenicity when transplanted into an animal host. A number of cell surface markers such as CD44, CD24, and CD133 are often used to identify and enrich CSCs. A regulatory network consisting of microRNAs and Wnt/β-catenin, Notch, and Hedgehog signaling pathways controls the CSC properties. The clinical relevance of CSCs has been strengthened by emerging evidence,...

  6. Effects of NHERF expression on drug resistance in cervical cancer cells%NHERF表达对宫颈癌细胞耐药性的影响

    施文; 陶涛; 杨晓梅; 贺俊崎

    2016-01-01

    Objective The effects of NHERF expression on drug resistance in hela cells were investigated. Methods Expression of NHERF were detected in hela cells dealed with different concentrations of cisplatin. The effects of NHERF knock down on the viability of cisplatin resistant hela cells were detected. Therefore,the effects of cisplatin,taxol and methotrexate on apoptosis of NHERF knocked down HeLa cells were determined. Results Down - regulation of NHERF was detected in hela cells dealed with high concentrations of cisplatin. The cell viability was increased in NHERF knocked down hela cells dealed with cisplatin. The apoptosis of NHERF knocked down hela cells dealed with cisplatin and taxol were decreased. Conclusion Down - regulation of NHERF can increase the multiple drug resistance of cervical cancer cells.%目的:探讨钠氢交换调控因子(NHERF)表达对宫颈癌细胞耐药性的影响。方法检测顺铂处理下 HeLa细胞 NHERF 表达量的差异。干扰 HeLa 细胞中 NHERF 的表达,检测 HeLa 细胞在顺铂处理下细胞活力的变化。并在NHERF 表达减少的情况下,检测顺铂、紫杉醇及甲氨喋呤处理对 HeLa 细胞凋亡的影响。结果顺铂处理下 HeLa 细胞中 NHERF 表达量下降。干扰 NHERF 表达的 HeLa 细胞耐顺铂活力增加。顺铂及紫杉醇处理组中,NHERF 表达下降可以减少细胞凋亡。结论 NHERF 表达下降可以增加宫颈癌细胞的多药耐药性。

  7. Non-biased enrichment does not improve quantitative proteomic delineation of reovirus T3D-infected HeLa cell protein alterations

    Jieyuan eJiang

    2012-09-01

    Full Text Available Mass spectrometry-based methods have allowed elucidation of alterations in complex proteomes, such as eukaryotic cells. Such studies have identified and measured relative abundances of thousands of host proteins after cells are infected with a virus. One of the potential limitations in such studies is that generally only the most abundant proteins are identified, leaving the deep richness of the cellular proteome largely unexplored. We differentially labeled HeLa cells with light and heavy stable isotopic forms of lysine and arginine (SILAC and infected cells with reovirus strain T3D. Cells were harvested at 24 hours post-infection. Heavy-labeled infected and light-labeled mock-infected cells were mixed together 1:1. Cells were then divided into cytosol and nuclear fractions and each fraction analyzed, both by standard 2D-HPLC/MS, and also after each fraction had been reacted with a random hexapeptide library (Proteominer® beads to attempt to enrich for low-abundance cellular proteins. A total of 2736 proteins were identified by 2 or more peptides at >99% confidence, of which 66 were significantly up-regulated and 67 were significantly down-regulated. Up-regulated proteins included those involved in antimicrobial and antiviral responses, GTPase activity, nucleotide binding, interferon signaling, and enzymes associated with energy generation. Down-regulated proteins included those involved in cell and biological adhesion, regulation of cell proliferation, structural molecule activity, and numerous molecular binding activities. Comparisons of the r2 correlations, degree of dataset overlap, and numbers of peptides detected suggest that non-biased enrichment approaches may not provide additional data to allow deeper quantitative and comparative mining of complex proteomes.

  8. Binding ability of LHRH-PE40 to LHRH receptors on cancer cell line

    Objective: To evaluate the binding ability of LHRH-PE40, a fusion protein, to the LHRH receptors on cancer cell line. Methods: The radioligand binding assay of receptors was used to calculate the Kd and Bmax. Results: Hela cell line: Kd=(0.36 +- 0.12) nmol, Bmax=(0.23+-0.15) μmol·g-1; Hep2 cell line: Kd=(0.33 +- 0.11) nmol, Bmax=(0.46 +- 0.12)μmol·g-1. Conclusion: LHRH-PE40 has a high binding affinity to the LHRH receptors on cancer cell line, which is the same as the natural LHRH

  9. ANTICANCER ACTIVITY OF PONGAMIA GLABRA V. SEED OIL EXTRACT AGAINST SELECTED HUMAN CANCER CELL LINES

    Chinnasamy Arulvasu; Subramanian Vasantha suppriya; Gajendran Babu

    2012-01-01

    Screening of the seed oil extract from Pongamia glabra V. (Fabaceae) has been carried out for antiproliferative activity of cancer cells. The seed oil was extracted with methanol and then persuasive activity was tested on human cancer cell lines MCF-7 and HeLa. The cell growth inhibitory effects of seed oil extract was observed. The cell viability was assessed using trypan blue dye exclusion method and 3-(4, 5- Dimethyl thiazol-2yl)-2, 5-dimethyltetrazolium bromide (MTT) assay. The IC50 value...

  10. Cancer Stem Cells in Breast Cancer

    Fumitaka Takeshita; Tomohiro Fujiwara; Takahiro Ochiya; Makiko Ono; Ryou-u Takahashi

    2011-01-01

    The cancer stem cell (CSC) theory is generally acknowledged as an important field of cancer research, not only as an academic matter but also as a crucial aspect of clinical practice. CSCs share a variety of biological properties with normal somatic stem cells in self-renewal, the propagation of differentiated progeny, the expression of specific cell markers and stem cell genes, and the utilization of common signaling pathways and the stem cell niche. However, CSCs differ from normal stem cel...

  11. Biomedical applications of SPION@APTES@PEG-folic acid@carboxylated quercetin nanodrug on various cancer cells

    Akal, Z. Ü.; Alpsoy, L.; Baykal, A.

    2016-08-01

    In this study, carboxylated quercetin (CQ) was conjugated to superparamagnetic iron oxide nanoparticles (SPIONs) which were modified by (3-aminopropyl) triethoxysilane (APTES), Folic acid (FA) and carboxylated Polyethylene glycol (PEG); (SPION@APTES@FA-PEG@CQ), nanodrug has been synthesized via polyol and accompanying by various chemical synthesis routes. The characterization of the final product was done via X-ray powder diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Thermal gravimetric analysis (TGA), Transmission electron spectroscopy (TEM) and Vibrating sample magnetometer (VSM). Its cytotoxic and apoptotic activities on over expressed folic acid receptor (FR +) (MCF-7, HeLa) and none expressed folic acid receptor (FR-) (A549) cancer cell lines were determined by using MTT assay, Real-Time Cell Analysis, TUNEL assay, Annexin assay and RT-PCR analysis for Caspase3/7 respectively. SPION@APTES@FA-PEG@CQ nanodrug showed higher cytotoxicity against HeLa and MCF-7 cell lines as compared with A549 cell line. Moreover, SPION@APTES@FA-PEG@CQ nanodrug also caused higher apoptotic and necrotic effects in 100 μg/mL HeLa and MCF-7 cells than A549 cells. The findings showed that SPION@APTES@FA-PEG@CQ nanodrug has cytotoxic, apoptotic and necrotic effects on HeLa and MCF-7 which are FR over expressed cell lines and can be potentially used for the delivery of quercetin to cervical and breast cancer cells.

  12. Inhibition of proliferation of cervical and leukemic cancer cells by penicillin G.

    Banerjee, Aditya; Dahiya, Meetu; Anand, M T; Kumar, Sudhir

    2013-01-01

    Cancer, despite all the efforts, still causes one in five deaths worldwide. Surgery, chemotherapy and radiotherapy provide inadequate protection and instead affect normal cells along with cancer cells. The search for cancer cures from natural products (plants and animals) has been practice for over a decade and the use of purified chemical to treat cancer still continues. Several studies have been undertaken during last three decades to find the anti-cancerous property of various plant extract and toxins secreted by animals and micro-organism. These lead to the discovery of several promising molecule having anticancer activity, some of which are in clinical trial and may emerged to be a potential future drug in cancer therapy. In this study we have used penicillin to evaluate its anti-cancer activity. It shown significant effects at cellular and molecular levels against growth of HeLa and K562 cell lines. PMID:23679330

  13. Extragonadal Germ Cell Cancer (EGC)

    ... Testicular Cancer Resource Center Extragonadal Germ Cell Cancer (EGC) 95% of all testicular tumors are germ cell ... seen in young adults. Patients with mediastinal nonseminomatous EGC are typically classed as poor risk patients because ...

  14. Repression of the integrated papillomavirus E6/E7 promoter is required for growth suppression of cervical cancer cells.

    Francis, D A; Schmid, S I; Howley, P M

    2000-03-01

    The human papillomavirus (HPV) E2 protein is an important regulator of viral E6 and E7 gene expression. E2 can repress the viral promoter for E6 and E7 expression as well as block progression of the cell cycle in cancer cells harboring the DNA of "high-risk" HPV types. Although the phenomenon of E2-mediated growth arrest of HeLa cells and other HPV-positive cancer cells has been well documented, the specific mechanism by which E2 affects cellular proliferation has not yet been elucidated. Here, we show that bovine papillomavirus (BPV) E2-induced growth arrest of HeLa cells requires the repression of the E6 and E7 promoter. This repression is specific for E2TA and not E2TR, a BPV E2 variant that lacks the N-terminal transactivation domain. We demonstrate that expression of HPV16 E6 and E7 from a heterologous promoter that is not regulated by E2 rescues HeLa cells from E2-mediated growth arrest. Our data indicate that the pathway of E2-mediated growth arrest of HeLa cells requires repression of E6 and E7 expression through an activity specified by the transactivation domain of E2TA. PMID:10684283

  15. Cancer stem cells in prostate cancer

    Moltzahn, Felix; Thalmann, George N

    2013-01-01

    Prostate cancer (P-Ca) remains a leading cause of cancer-related death in men. Lately, increasing evidence for a hierarchically organized cancer stem cell (CSC) model emerged for different tumors entities, including P-Ca. CSCs are defined by several characteristics including self-renewal, pluripotency and tumorigenicity and are thought to be responsible for tumor recurrence, metastasis and cancer related death. In this review we discuss the recent research in the field of CSCs, its limitation...

  16. Cancer Stem Cells in Pancreatic Cancer

    Karl-Walter Jauch; Hendrik Seeliger; Hanno Niess; Qi Bao; Andrea Renner; Yue Zhao; Bruns, Christiane J.

    2010-01-01

    Pancreatic cancer is an aggressive malignant solid tumor well-known by early metastasis, local invasion, resistance to standard chemo- and radiotherapy and poor prognosis. Increasing evidence indicates that pancreatic cancer is initiated and propagated by cancer stem cells (CSCs). Here we review the current research results regarding CSCs in pancreatic cancer and discuss the different markers identifying pancreatic CSCs. This review will focus on metastasis, microRNA regulation and anti-CSC t...

  17. The dynamin chemical inhibitor dynasore impairs cholesterol trafficking and sterol-sensitive genes transcription in human HeLa cells and macrophages.

    Emmanuelle Girard

    Full Text Available Intracellular transport of cholesterol contributes to the regulation of cellular cholesterol homeostasis by mechanisms that are yet poorly defined. In this study, we characterized the impact of dynasore, a recently described drug that specifically inhibits the enzymatic activity of dynamin, a GTPase regulating receptor endocytosis and cholesterol trafficking. Dynasore strongly inhibited the uptake of low-density lipoprotein (LDL in HeLa cells, and to a lower extent in human macrophages. In both cell types, dynasore treatment led to the abnormal accumulation of LDL and free cholesterol (FC within the endolysosomal network. The measure of cholesterol esters (CE further showed that the delivery of regulatory cholesterol to the endoplasmic reticulum (ER was deficient. This resulted in the inhibition of the transcriptional control of the three major sterol-sensitive genes, sterol-regulatory element binding protein 2 (SREBP-2, 3-hydroxy-3-methyl-coenzymeA reductase (HMGCoAR, and low-density lipoprotein receptor (LDLR. The sequestration of cholesterol in the endolysosomal compartment impaired both the active and passive cholesterol efflux in HMDM. Our data further illustrate the importance of membrane trafficking in cholesterol homeostasis and validate dynasore as a new pharmacological tool to study the intracellular transport of cholesterol.

  18. JWA is required for arsenic trioxide induced apoptosis in HeLa and MCF-7 cells via reactive oxygen species and mitochondria linked signal pathway

    Arsenic trioxide, emerging as a standard therapy for refractory acute promyelocytic leukemia, induces apoptosis in a variety of malignant cell lines. JWA, a novel retinoic acid-inducible gene, is known to be involved in apoptosis induced by various agents, for example, 12-O-tetradecanoylphorbol 13-acetate, N-4-hydroxy-phenyl-retinamide and arsenic trioxide. However, the molecular mechanisms underlying how JWA gene is functionally involved in apoptosis remain largely unknown. Herein, our studies demonstrated that treatment of arsenic trioxide produced apoptosis in HeLa and MCF-7 cells in a dose-dependent manner and paralleled with increased JWA expression. JWA expression was dependent upon generation of intracellular reactive oxygen species induced by arsenic trioxide. Knockdown of JWA attenuated arsenic trioxide induced apoptosis, and was accompanied by significantly reduced activity of caspase-9, enhanced Bad phosphorylation and inhibited MEK1/2, ERK1/2 and JNK phosphorylations. Arsenic trioxide induced loss of mitochondrial transmembrane potential was JWA-dependent. These findings suggest that JWA may serve as a pro-apoptotic molecule to mediate arsenic trioxide triggered apoptosis via a reactive oxygen species and mitochondria-associated signal pathway

  19. Highly Luminescent Heterostructured Copper-Doped Zinc Sulfide Nanocrystals for Application in Cancer Cell Labeling.

    Ang, Huixiang; Bosman, Michel; Thamankar, Ramesh; Zulkifli, Muhammad Faizal B; Yen, Swee Kuan; Hariharan, Anushya; Sudhaharan, Thankiah; Selvan, Subramanian Tamil

    2016-08-18

    The structural characteristics of the seed-mediated synthesis of heterostructured CuS-ZnS nanocrystals (NCs) and Cu-doped ZnS (ZnS:Cu) NCs synthesized by two different protocols are compared and analyzed. At high Cu dopant concentrations, segregated subclusters of ZnS and CuS are observed. The photoluminescence quantum yield of ZnS:Cu NCs is about 50-80 %; a value much higher than that of ZnS NCs (6 %). Finally, these NCs are coated with a thin silica shell by using (3-mercaptopropyl)triethoxysilane in a reverse microemulsion to make them water soluble. Cytotoxicity experiments show that these silica-coated NCs have greatly reduced toxicity on both cancerous HeLa and noncancerous Chinese hamster ovary cells. The labeling of cancerous HeLa cells is also demonstrated. PMID:27146419

  20. Analysis of pre-rRNAs in heat-shocked HeLa cells allows identification of the upstream termination site of human polymerase I transcription

    Human rRNA precursors from normal or stressed HeLa cells were studied by S1 nuclease mapping of unlabeled RNA and by antisense RNase mapping of RNA from cells that had been labeled in vivo with [32P]PO4. Heating cells to 430C decreased the amount of newly synthesized rRNA to less than 5% of the control level and led to greater than 95% inhibition of transcription termination at a region 355 to 362 nucleotides downstream of the 3' end of 28S rRNA, with readthrough continuing into the next transcription unit. Heating of cells to 420C led to 60% inhibition of termination at this site; 50% of transcripts that extended into the nontranscribed spacer ended in a region 200 to 210 nucleotides upstream of the polymerase I (Pol I) initiation site. This is presumed to be the human upstream transcription termination site because of the absence of RNAs with a 5' end corresponding to this region, the location relative to the Pol 1 initiation site (which is similar to the location of upstream terminators in other species), and the fact that it is 15 to 25 nucleotides upstream of the sequence GGGTTGACC, which has an 8-of-9 base identify with the sequence 3' of the downstream termination site. Surprisingly, treatment of cells with sodium arsenite, which also leads to the induction of a stress response, did not inhibit termination. Pol I initiation was decreased to the same extent as termination, which lends support to the hypothesis that termination and initiation are coupled. Although termination was almost completely inhibited at 430C, the majority of the recently synthesized rRNAs were processed to have the correct 3' end of 28S. This finding suggests that 3'-end formation can involve an endonucleolytic cut and is not solely dependent on exonucleolytic trimming of correctly terminated rRNAs

  1. Characterization of a novel Dp71 dystrophin-associated protein complex (DAPC) present in the nucleus of HeLa cells: Members of the nuclear DAPC associate with the nuclear matrix

    Dystrophin is an essential component in the assembly and maintenance of the dystrophin-associated protein complex (DAPC), which includes members of the dystroglycan, syntrophin, sarcoglycan and dystrobrevin protein families. Distinctive complexes have been described in the cell membrane of different tissues and cultured cells. In this work, we report the identification and characterization of a novel DAPC present in the nuclei of HeLa cells, which contains dystrophin Dp71 as a key component. Using confocal microscopy and cell fractionation analyses, we found the presence of Dp71, β-sarcoglycan, β-dystroglycan, α- and β-syntrophin, α1- and β-dystrobrevin and nNOS in the nuclei of HeLa cells. Furthermore, we demonstrated by co-immunoprecipitation experiments that most of these proteins form a complex in the nuclear compartment. Next, we analyze the possible association of the nuclear DAPC with the nuclear matrix. We found the presence of Dp71, β-dystroglycan, nNOS, β-sarcoglycan, α/β syntrophin, α1-dystrobrevin and β-dystrobrevin in the nuclear matrix protein fractions and in situ nuclear matrix preparations from HeLa cells. Moreover, we found that Dp71, β-dystroglycan and β-dystrobrevin co-immunoprecipitated with the nuclear matrix proteins lamin B1 and actin. The association of members of the nuclear DAPC with the nuclear matrix indicates that they may work as scaffolding proteins involved in nuclear architecture

  2. Cancer stem cell subsets and their relationships

    Pan Yi-Fei; Yang Han; Chen Chong; Liu Hai-Guang; Zhang Xiao-Hua

    2011-01-01

    Abstract Emerging evidence suggests that cancer stem cells account for the initiation and progression of cancer. While many types of cancer stem cells with specific markers have been isolated and identified, a variety of differences among them began to be appreciated. Cancer stem cells are hierarchical populations that consist of precancerous stem cells, primary cancer stem cells, migrating cancer stem cells and chemoradioresistant cancer stem cells, playing different roles in cancer initiati...

  3. Unusual expression of red fluorescence at M phase induced by anti-microtubule agents in HeLa cells expressing the fluorescent ubiquitination-based cell cycle indicator (Fucci)

    Highlights: ► Fucci visualizes cell cycle by green and red fluorescence. ► Plinabulin, induced unusual red fluorescence at M-phase in HeLa-Fucci cells. ► The unusual pattern was followed by mitotic catastrophe. ► The unusual pattern may be an early indicator of cell death in HeLa cells. -- Abstract: Plinabulin (NPI-2358) is a novel microtubule-depolymerizing agent. In HeLa cells, plinabulin arrests the cell-cycle at M phase and subsequently induces mitotic catastrophe. To better understand the effects on this compound on the cell-cycle, we used the fluorescent ubiquitination-based cell cycle indicator (Fucci), which normally enables G1 and S/G2/M cells to emit red and green fluorescence, respectively. When HeLa-Fucci cells were treated with 50 nM plinabulin, cells began to fluoresce both green and red in an unusual pattern; most cells exhibited the new pattern after 24 h of treatment. X-irradiation efficiently induced G2 arrest in plinabulin-treated cells and significantly retarded the emergence of the unusual pattern, suggesting that entering M phase is essential for induction of the pattern. By simultaneously visualizing chromosomes with GFP-histone H2B, we established that the pattern emerges after nuclear envelope breakdown but before metaphase. Pedigree assay revealed a significant relationship between the unusual expression and mitotic catastrophe. Nocodazole, KPU-133 (a more potent derivative of plinabulin), and paclitaxel also exerted similar effects. From these data, we conclude that the unusual pattern may be associated with dysregulation of late M phase-specific E3 ligase activity and mitotic catastrophe following treatment with anti-microtubule agents.

  4. Unusual expression of red fluorescence at M phase induced by anti-microtubule agents in HeLa cells expressing the fluorescent ubiquitination-based cell cycle indicator (Fucci)

    Honda-Uezono, Asumi [Section of Oral Radiation Oncology, Department of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan); Section of Maxillofacial Surgery, Department of Maxillofacial and Neck Reconstruction, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan); Kaida, Atsushi [Section of Oral Radiation Oncology, Department of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan); Michi, Yasuyuki; Harada, Kiyoshi [Section of Maxillofacial Surgery, Department of Maxillofacial and Neck Reconstruction, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan); Hayashi, Yoshiki; Hayashi, Yoshio [Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392 (Japan); Miura, Masahiko, E-mail: masa.mdth@tmd.ac.jp [Section of Oral Radiation Oncology, Department of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan)

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer Fucci visualizes cell cycle by green and red fluorescence. Black-Right-Pointing-Pointer Plinabulin, induced unusual red fluorescence at M-phase in HeLa-Fucci cells. Black-Right-Pointing-Pointer The unusual pattern was followed by mitotic catastrophe. Black-Right-Pointing-Pointer The unusual pattern may be an early indicator of cell death in HeLa cells. -- Abstract: Plinabulin (NPI-2358) is a novel microtubule-depolymerizing agent. In HeLa cells, plinabulin arrests the cell-cycle at M phase and subsequently induces mitotic catastrophe. To better understand the effects on this compound on the cell-cycle, we used the fluorescent ubiquitination-based cell cycle indicator (Fucci), which normally enables G1 and S/G2/M cells to emit red and green fluorescence, respectively. When HeLa-Fucci cells were treated with 50 nM plinabulin, cells began to fluoresce both green and red in an unusual pattern; most cells exhibited the new pattern after 24 h of treatment. X-irradiation efficiently induced G2 arrest in plinabulin-treated cells and significantly retarded the emergence of the unusual pattern, suggesting that entering M phase is essential for induction of the pattern. By simultaneously visualizing chromosomes with GFP-histone H2B, we established that the pattern emerges after nuclear envelope breakdown but before metaphase. Pedigree assay revealed a significant relationship between the unusual expression and mitotic catastrophe. Nocodazole, KPU-133 (a more potent derivative of plinabulin), and paclitaxel also exerted similar effects. From these data, we conclude that the unusual pattern may be associated with dysregulation of late M phase-specific E3 ligase activity and mitotic catastrophe following treatment with anti-microtubule agents.

  5. Resolution of the diadenosine 5',5'''-P1,P4-tetraphosphate binding subunit from a multiprotein form of HeLa cell DNA polymerase α

    A diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) binding subunit has been resolved from a high molecular weight (640,000) multiprotein form of DNA polymerase α [deoxy-nucleoside triphosphate:DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7] from HeLa cells. The Ap4A binding activity copurifies with the DNA polymerizing activity during the course of purification. Hydrophobic chromatograpy on butylagarose resolves the Ap4A binding activity from the DNA polymerase. The Ap4A binding activity is protein in nature since the binding of Ap4A is abolished by treatment of the isolated binding activity with proteinase K but is insensitive to treatment with DNase or RNase. The molecular weight of the Ap4A binding protein, as determined by polyacrylamide gel electrophoresis under nondenaturing conditions or by NaDodSO4/polyacrylamide gel electrophoresis after photoaffinity labeling of the protein with [32P]Ap4A is 92,000 or 47,000. The binding activity of this protein is highly specific for Ap4A

  6. Urothelial Cancer Stem Cells

    Irena Dimov

    2010-01-01

    Full Text Available There is mounting evidence supporting the idea that tumors, similar to normal adult tissues, arise from a specific stem-like cell population, the cancer stem cells (CSCs, which are considered as the real driving force behind tumor growth, the ability to metastasize, as well as resistance to conventional antitumor therapy. The concept that cancer growth recapitulates normal proliferative and/or regenerative processes, even though in very dysfunctional ways, has tremendous implications for cancer therapy. The rapid development of the CSC field, shoulder to shoulder with powerful genome-wide screening techniques, has provided cause for optimism for the development of more reliable therapies in the future. However, several important issues still lie ahead. Recent identification of a highly tumorigenic stem-like compartment and existence of urothelial differentiation programs in urothelial cell carcinomas (UCCs raised important questions about UCC initiation and development. This review examines the present knowledge on CSCs in UCCs regarding the similarities between CSCs and the adult urothelial stem cells, potential origin of urothelial CSCs, main regulatory pathways, surface markers expression, and the current state of CSC-targeting therapeutic strategies.

  7. Doxorubicin loaded 17β-estradiol based SWNT dispersions for target specific killing of cancer cells.

    Ghosh, Moumita; Das, Prasanta Kumar

    2016-06-01

    The present work reports the synthesis of a 17β-estradiol based amphiphiles comprising of polyethylene glycol (PEG) moiety linked through succinic acid that non-covalently dispersed (76%) the single walled carbon nanotubes (SWNTs) in water. The superior exfoliation of carbon nanotubes was characterized by microscopic and spectroscopic studies. Significant stability of these SWNT dispersions was observed in the presence of protein in cell culture media and the nanohybrids were highly biocompatible toward mammalian cells. Anticancer drug doxorubicin loaded on these nanohybrids was selectively delivered within estrogen receptor rich cancer cells, MCF7 (breast cancer cell) and A549 (lung cancer cell). Microscopic studies showed the localization of doxorubicin within the cancer cell nucleus whereas no such localization was observed in ER negative cells. Both these ER positive cancer cells were killed by ∼3 fold higher efficiency than that of ER negative MDA-MB-231 (advanced breast cancer cell) and HeLa cells that are deprived of estrogen receptors. Thus, judiciously designed estradiol based nanohybrids proved to be excellent tool for SWNT dispersion and also for selectively killing of ER positive cancer cells. To the best of our knowledge, for the first time non-covalently modified SWNTs by estradiol based amphiphilic dispersing agent have been used for selective killing of ER positive cancer cells by doxorubicin loaded on dispersed SWNTs. It holds immense promise to be exploited as a cancer therapeutic agent. PMID:26970825

  8. Effects of irreversible electroporation on cervical cancer cell lines in vitro.

    Qin, Qin; Xiong, Zheng-Ai; Liu, Ying; Yao, Chen-Guo; Zhou, Wei; Hua, Yuan-Yuan; Wang, Zhi-Liang

    2016-09-01

    The effects of irreversible electroporation (IRE) on the proliferation, migration, invasion and adhesion of human cervical cancer cell lines HeLa and SiHa were investigated in the present study. HeLa and SiHa cells were divided into a treatment group and control group. The treatment group cells were exposed to electric pulses at 16 pulses, 1 Hz frequency for 100 µsec with 1,000 V/cm strength. Cellular proliferation was determined 24 h after treatment using a Cell Counting Kit‑8 (CCK‑8) assay and carboxyfluorescein diacetate‑succinimidyl ester (CFDA‑SE) labeling assay. The different phases of the cell cycle were detected using flow cytometry. Wound healing, Transwell invasion and Matrigel adhesion assays were performed to evaluate the migration, invasion and adhesion abilities of HeLa and SiHa cells. The expression levels of metastasis‑associated proteins were determined by western blot analysis. CCK‑8 and CFSE labeling assays indicated that the inhibition of cellular proliferation occurs in cells treated with IRE. Additionally, cell cycle progression was arrested at the G1/S phase. A western blot analysis indicated that the expression levels of p53 and p21 proteins were increased, whilst those of cyclin‑dependent kinase 2 (CDK2) and proliferating cell nuclear antigen (PCNA) proteins were decreased. However, wound healing, invasion and adhesion assays indicated that cellular migration, invasion and adhesion abilities were not significantly altered following exposure to IRE. IRE was not observed to promote the migration, invasion or adhesion capacity of HeLa and SiHa cells. However, IRE may inhibit the capacity of cells to proliferate and their progression through the cell cycle in vitro. Preliminary evidence suggests that the underlying mechanism involves increased expression levels of p53 and p21 and decreased expression levels of CDK2 and PCNA. PMID:27431825

  9. Curcumin and emodin down-regulate TGF-β signaling pathway in human cervical cancer cells.

    Pooja Chandrakant Thacker

    Full Text Available Cervical cancer is the major cause of cancer related deaths in women, especially in developing countries and Human Papilloma Virus infection in conjunction with multiple deregulated signaling pathways leads to cervical carcinogenesis. TGF-β signaling in later stages of cancer is known to induce epithelial to mesenchymal transition promoting tumor growth. Phytochemicals, curcumin and emodin, are effective as chemopreventive and chemotherapeutic compounds against several cancers including cervical cancer. The main objective of this work was to study the effect of curcumin and emodin on TGF-β signaling pathway and its functional relevance to growth, migration and invasion in two cervical cancer cell lines, SiHa and HeLa. Since TGF-β and Wnt/β-catenin signaling pathways are known to cross talk having common downstream targets, we analyzed the effect of TGF-β on β-catenin (an important player in Wnt/β-catenin signaling and also studied whether curcumin and emodin modulate them. We observed that curcumin and emodin effectively down regulate TGF-β signaling pathway by decreasing the expression of TGF-β Receptor II, P-Smad3 and Smad4, and also counterbalance the tumorigenic effects of TGF-β by inhibiting the TGF-β-induced migration and invasion. Expression of downstream effectors of TGF-β signaling pathway, cyclinD1, p21 and Pin1, was inhibited along with the down regulation of key mesenchymal markers (Snail and Slug upon curcumin and emodin treatment. Curcumin and emodin were also found to synergistically inhibit cell population and migration in SiHa and HeLa cells. Moreover, we found that TGF-β activates Wnt/β-catenin signaling pathway in HeLa cells, and curcumin and emodin down regulate the pathway by inhibiting β-catenin. Taken together our data provide a mechanistic basis for the use of curcumin and emodin in the treatment of cervical cancer.

  10. The effect of lance geometry and carbon coating of silicon lances on propidium iodide uptake in lance array nanoinjection of HeLa 229 cells

    Sessions, John W.; Lindstrom, Dallin L.; Hanks, Brad W.; Hope, Sandra; Jensen, Brian D.

    2016-04-01

    Connecting technology to biologic discovery is a core focus of non-viral gene therapy biotechnologies. One approach that leverages both the physical and electrical function of microelectromechanical systems (MEMS) in cellular engineering is a technology previously described as lance array nanoinjection (LAN). In brief, LAN consists of a silicon chip measuring 2 cm by 2 cm that has been etched to contain an array of 10 μm tall, solid lances that are spaced every 10 μm in a grid pattern. This array of lances is used to physically penetrate hundreds of thousands of cells simultaneously and to then electrically deliver molecular loads into cells. In this present work, two variables related to the microfabrication of the silicon lances, namely lance geometry and coating, are investigated. The purpose of both experimental variables is to assess these parameters’ effect on propidium iodide (PI), a cell membrane impermeable dye, uptake to injected HeLa 229 cells. For the lance geometry experimentation, three different microfabricated lance geometries were used which include a flat/narrow (FN, 1 μm diameter), flat/wide (FW, 2-2.5 μm diameter), and pointed (P, 1 μm diameter) lance geometries. From these tests, it was shown that the FN lances had a slightly better cell viability rate of 91.73% and that the P lances had the best PI uptake rate of 75.08%. For the lance coating experimentation, two different lances were fabricated, both silicon etched lances with some being carbon coated (CC) in a  <100 nm layer of carbon and the other lances being non-coated (Si). Results from this experiment showed no significant difference between lance types at three different nanoinjection protocols (0V, +1.5V DC, and  +5V Pulsed) for both cell viability and PI uptake rates. One exception to this is the comparison of CC/5V Pul and Si/5V Pul samples, where the CC/5V Pul samples had a cell viability rate 5% higher. Both outcomes were unexpected and reveal how to better

  11. The effect of lance geometry and carbon coating of silicon lances on propidium iodide uptake in lance array nanoinjection of HeLa 229 cells

    Connecting technology to biologic discovery is a core focus of non-viral gene therapy biotechnologies. One approach that leverages both the physical and electrical function of microelectromechanical systems (MEMS) in cellular engineering is a technology previously described as lance array nanoinjection (LAN). In brief, LAN consists of a silicon chip measuring 2 cm by 2 cm that has been etched to contain an array of 10 μm tall, solid lances that are spaced every 10 μm in a grid pattern. This array of lances is used to physically penetrate hundreds of thousands of cells simultaneously and to then electrically deliver molecular loads into cells. In this present work, two variables related to the microfabrication of the silicon lances, namely lance geometry and coating, are investigated. The purpose of both experimental variables is to assess these parameters’ effect on propidium iodide (PI), a cell membrane impermeable dye, uptake to injected HeLa 229 cells. For the lance geometry experimentation, three different microfabricated lance geometries were used which include a flat/narrow (FN, 1 μm diameter), flat/wide (FW, 2–2.5 μm diameter), and pointed (P, 1 μm diameter) lance geometries. From these tests, it was shown that the FN lances had a slightly better cell viability rate of 91.73% and that the P lances had the best PI uptake rate of 75.08%. For the lance coating experimentation, two different lances were fabricated, both silicon etched lances with some being carbon coated (CC) in a  <100 nm layer of carbon and the other lances being non-coated (Si). Results from this experiment showed no significant difference between lance types at three different nanoinjection protocols (0V, +1.5V DC, and  +5V Pulsed) for both cell viability and PI uptake rates. One exception to this is the comparison of CC/5V Pul and Si/5V Pul samples, where the CC/5V Pul samples had a cell viability rate 5% higher. Both outcomes were unexpected and reveal how to better

  12. Cancer-initiating cells derived from established cervical cell lines exhibit stem-cell markers and increased radioresistance

    Cancer-initiating cells (CICs) are proposed to be responsible for the generation of metastasis and resistance to therapy. Accumulating evidences indicates CICs are found among different human cancers and cell lines derived from them. Few studies address the characteristics of CICs in cervical cancer. We identify biological features of CICs from four of the best-know human cell lines from uterine cervix tumors. (HeLa, SiHa, Ca Ski, C-4 I). Cells were cultured as spheres under stem-cell conditions. Flow cytometry was used to detect expression of CD34, CD49f and CD133 antigens and Hoechst 33342 staining to identify side population (SP). Magnetic and fluorescence-activated cell sorting was applied to enrich and purify populations used to evaluate tumorigenicity in nude mice. cDNA microarray analysis and in vitro radioresistance assay were carried out under standard conditions. CICs, enriched as spheroids, were capable to generate reproducible tumor phenotypes in nu-nu mice and serial propagation. Injection of 1 × 103 dissociated spheroid cells induced tumors in the majority of animals, whereas injection of 1 × 105 monolayer cells remained nontumorigenic. Sphere-derived CICs expressed CD49f surface marker. Gene profiling analysis of HeLa and SiHa spheroid cells showed up-regulation of CICs markers characteristic of the female reproductive system. Importantly, epithelial to mesenchymal (EMT) transition-associated markers were found highly expressed in spheroid cells. More importantly, gene expression analysis indicated that genes required for radioresistance were also up-regulated, including components of the double-strand break (DSB) DNA repair machinery and the metabolism of reactive oxygen species (ROS). Dose-dependent radiation assay indicated indeed that CICs-enriched populations exhibit an increased resistance to ionizing radiation (IR). We characterized a self-renewing subpopulation of CICs found among four well known human cancer-derived cell lines (HeLa, Si

  13. Coordinate turnover of nuclear and cytoplasmic histone messenger RNA following inhibition of DNA replication of HeLa S3 cells

    The authors have examined the metabolism of human H4 histone mRNA in the nucleus and cytoplasm of HeLa S3 cells following inhibition of DNA synthesis to address the extent to which histone mRNA stability in these cellular compartments is coupled to DNA replication. The nuclear and cytoplasmic levels of histone mRNAs encoded by the pF0108A human H4 histone gene were determined by S1 nuclease analysis using a 32P-labeled probe that could distinguish pF0108A transcripts from those of other members of the H4 histone multigene family. Hydroxyurea treatment resulted within 15 min in a 75% reduction in the level of histone H4 mRNA in the nucleus, which corresponds to the 85% decrease observed for H4 histone mRNA in the cytoplasm. The kinetics of nuclear and cytoplasmic H4 mRNA turnover following hydroxyurea treatment were also similar. Northern blot analysis using a 32P-labeled mitochondrial cytochrome b probe indicated that the association of cytoplasmic RNA with the nuclear fraction was less than 0.5%. Treatment of cells with a protein synthesis inhibitor resulted in a 1.3-fold increase in nuclear H4 histone mRNA levels and a 1.5-fold increase of H4 mRNA in the cytoplasm after 45 min. Together, these results indicate that nuclear and cytoplasmic H4 histone mRNAs respond similarly to metabolic perturbations that influence message stability and that mechanisms operative in the turnover of histone mRNAs in the nucleus and cytoplasm may be similar

  14. DNA-binding protein from HeLa cells that binds preferentially to supercoiled DNA damaged by ultraviolet light or N-acetoxy-N-acetyl-2-aminofluorene

    A DNA-binding protein was partially purified from extracts of HeLa cells by high-speed centrifugation and chromatography on DEAE-cellulose, phosphocellulose and ultraviolet light-irradiated DNA-cellulose columns. It eluted from the phosphocellulose column with 0.375 M potassium phosphate and from the ultraviolet light-irradiated DNA-cellulose column between 0.5 M and 1 M NaCl. The protein binds preferentially to supercoiled PM2 DNA treated with ultraviolet light or N-acetoxy-N-acetyl-2-aminofluorene, as compared to native supercoiled PM2 DNA. The binding is non-cooperative. Nicked or linear forms of PM2 DNA (damaged or untreated) are not efficient substrates, indicating a requirement of DNA supercoiling for DNA binding. The sedimentation coefficient of the protein estimated by glycerol gradient centrifugation is 2.0-2.5 S, corresponding to a molecular weight of about 20000-25000 if the protein is spherical. The binding to DNA irradiated with ultraviolet light or treated with acetoxyacetylaminofluorene is optimal at around 100-200 mM NaCl and is relatively independent of temperature and pH. MgCl2 and MnCl2 at concentrations between 1 and 5 mM do not markedly affect the binding, but it is inhibited by sucrose, ATP and caffeine. The biological significance of the DNA-binding protein remains to be determined. It does not possess significant glycosylase, endonuclease or exonuclease activities. The dissociation equilibrium constant for the binding reaction of the protein to the ultraviolet light or acetoxyacetylaminofluorene-induced binding sites on DNA is estimated to be 4x10-11 M. There are at least 1x105 DNA-binding protein molecules/HeLa cell. (Auth.)

  15. Rapid effect of heat shock on two heterogeneous nuclear ribonucleoprotein-associated antigens in HeLa cells

    1989-01-01

    During severe heat shock, which known to interrupt both splicing of RNA transcripts and nucleocytoplasmic transport, it is to be expected that the substructure of heterogeneous nuclear ribonucleoproteins (hnRNP) is altered in some way. Recently, we have shown that such a stress actually induces rapid alterations at the level of individual proteins (Lutz, Y., M. Jacob, and J.-P. Fuchs. 1988 Exp. Cell Res. 175:109-124). Here we report further investigations on two related 72.5-74-kD hnRNP prote...

  16. Proteasome-dependent degradation of cytochromes P450 2E1 and 2B1 expressed in tetracycline-regulated HeLa cells

    The degradation of ethanol-inducible cytochrome P450 2E1 (CYP2E1) and phenobarbital-inducible cytochrome P450 2B1 (CYP2B1) expressed in tetracycline (Tc)-inducible HeLa cell lines was characterized. A steady-state pulse-chase analysis was used to determine a half-life of 3.8 h for CYP2E1 while the half-life of CYP2B1 was 2.3-fold greater in the same cell line. In contrast, NADPH cytochrome P450 reductase which is constitutively expressed in Tc-HeLa cells had a half-life of about 30 h. Lactacystin and other selective proteasome inhibitors including N-benzyloxycarbonyl-leucyl-leucyl-leucinal (MG132) and N-benzyloxycarbonyl-L-leucyl-L-leucyl-L-norvalinal (MG115) significantly inhibited both CYP2E1 and CYP2B1 degradation. The turnover of CYP2E1 was slightly inhibited by calpain inhibitors while CYP2B1 turnover was not altered. Inhibitors of lysosomal proteolysis had no effect on the degradation of either protein. Treatment of cells with brefeldin A did not alter the degradation of either P450 which suggested the degradation occurred in the endoplasmic reticulum (ER). Even in the presence of proteasome inhibitors high molecular weight ubiquitin conjugates were not observed. Mutagenesis of two putative ubiquitination sites (Lys 317 and 324) did not alter the degradation of CYP2E1. The role of ubiquitination in the degradation of CYP2E1 was also examined in a Chinese hamster mutant cell line E36ts20 that contains a thermolabile ubiquitin-activating enzyme (E1). The turnover of CYP2E1 was not significantly different at the nonpermissive temperature in the ts20 when compared to the control E36 cells. Furthermore, the addition of the hsp90 inhibitors geldanamycin, herbimycin, and radicicol had no effect on the turnover of CYP2E1, differentiating the degradation of CYP2E1 from other substrates for proteasome-dependent degradation

  17. Targeting of a chimeric human histone fusion mRNA to membrane-bound polysomes in HeLa cells

    The subcellular location of histone mRNA-containing polysomes may play a key role in the posttranscriptional events that mediate histone mRNA turnover following inhibition of DNA synthesis. Previously, it has been shown that histone mRNA is found primarily on free polysomes that are associated with the cytoskeleton. The authors report here the construction of an Escherichia coli pBR322 β-lactamase signal peptide-human H3 histone fusion gene. The fusion transcript is targeted to membrane-bound polysomes and remains stable following interruption of DNA replication. Relocating mRNA within the cell may provide a procedure for studying the posttranscriptional regulation of gene expression

  18. Targeting of a chimeric human histone fusion mRNA to membrane-bound polysomes in HeLa cells

    Zambetti, G.; Stein, J.; Stein, G.

    1987-05-01

    The subcellular location of histone mRNA-containing polysomes may play a key role in the posttranscriptional events that mediate histone mRNA turnover following inhibition of DNA synthesis. Previously, it has been shown that histone mRNA is found primarily on free polysomes that are associated with the cytoskeleton. The authors report here the construction of an Escherichia coli pBR322 ..beta..-lactamase signal peptide-human H3 histone fusion gene. The fusion transcript is targeted to membrane-bound polysomes and remains stable following interruption of DNA replication. Relocating mRNA within the cell may provide a procedure for studying the posttranscriptional regulation of gene expression.

  19. Selectivity of compounds isolated from the leaves of Nerium indicum Mill. on various human cancer cell lines.

    Mae, S H W; Sofia, M; Bolhuis, R L H; Nooter, K; Oostrum, R G; Subagus, W; Ibnu, G G

    2008-07-01

    The leaves of Nerium indicum Mill. have been utilized traditionally to cure cancer. By Bioassay (BST) guided isolation method, six compounds were isolated from the CHCl3 extract of the leaves. Selectivity of these compounds (in 0.6-12,500 ng/ml) was tested on various human cancer (MCF7, EVSA-T, T47D, H226, IGROV, A498, WIDR, M19, HeLa) and normal (Vero) cells in vitro. Doxorubicin and cysplatin were used as positive controls. The result indicated that NiO2D (5alpha-oleandrin) possessed the best cytotoxic effect on HeLa cells (IC50, 8.38 x10(-6) mM) and NiO2C (16, 17-dehidrodeasetil-5alpha-oleandrin) on A498 cells (IC50, 1.43 x 10(-6) mM). Those two compounds were not cytotoxic to normal cell. PMID:19024965

  20. A new prospect in cancer therapy: targeting cancer stem cells to eradicate cancer

    Yi-Min Zhu; Li-Hua Yuan; Ke-Feng Pu; Bing Dong; An-Xin Wang; Li-Sha Chen

    2012-01-01

    According to the cancer stem cell theory, cancers can be initiated by cancer stem cells. This makes cancer stem cells prime targets for therapeutic intervention. Eradicating cancer stem cells by efficient targeting agents may have the potential to cure cancer. In this review, we summarize recent breakthroughs that have improved our understanding of cancer stem cells, and we discuss the therapeutic strategy of targeting cancer stem cells, a promising future direction for cancer stem cell resea...

  1. A new prospect in cancer therapy: targeting cancer stem cells to eradicate cancer

    Li-Sha Chen; An-Xin Wang; Bing Dong; Ke-Feng Pu; Li-Hua Yuan; Yi-Min Zhu

    2012-01-01

    According to the cancer stem cell theory,cancers can be initiated by cancer stem cells.This makes cancer stem cells prime targets for therapeutic intervention.Eradicating cancer stem cells by efficient targeting agents may have the potential to cure cancer.In this review,we summarize recent breakthroughs that have improved our understanding of cancer stem cells,and we discuss the therapeutic strategy of targeting cancer stem cells,a promising future direction for cancer stem cell research.

  2. Nanoscopic exclusion between Rad51 and 53BP1 after ion irradiation in human HeLa cells

    Reindl, Judith; Drexler, Guido A.; Girst, Stefanie; Greubel, Christoph; Siebenwirth, Christian; Drexler, Sophie E.; Dollinger, Günther; Friedl, Anna A.

    2015-12-01

    Many proteins involved in detection, signalling and repair of DNA double-strand breaks (DSB) accumulate in large number in the vicinity of DSB sites, forming so called foci. Emerging evidence suggests that these foci are sub-divided in structural or functional domains. We use stimulated emission depletion (STED) microscopy to investigate localization of mediator protein 53BP1 and recombination factor Rad51 after irradiation of cells with low linear energy transfer (LET) protons or high LET carbon ions. With a resolution better than 100 nm, STED microscopy and image analysis using a newly developed analyzing algorithm, the reduced product of the differences from the mean, allowed us to demonstrate that with both irradiation types Rad51 occupies spherical regions of about 200 nm diameter. These foci locate within larger 53BP1 accumulations in regions of local 53BP1 depletion, similar to what has been described for the localization of Brca1, CtIP and RPA. Furthermore, localization relative to 53BP1 and size of Rad51 foci was not different after irradiation with low and high LET radiation. As expected, 53BP1 foci induced by low LET irradiation mostly contained one Rad51 focal structure, while after high LET irradiation, most foci contained >1 Rad51 accumulation.

  3. NEU3 inhibitory effect of naringin suppresses cancer cell growth by attenuation of EGFR signaling through GM3 ganglioside accumulation.

    Yoshinaga, Ayana; Kajiya, Natsuki; Oishi, Kazuki; Kamada, Yuko; Ikeda, Asami; Chigwechokha, Petros Kingstone; Kibe, Toshiro; Kishida, Michiko; Kishida, Shosei; Komatsu, Masaharu; Shiozaki, Kazuhiro

    2016-07-01

    Naringin, which is one of the flavonoids contained in citrus fruits, is well known to possess various healthy functions to humans. It has been reported that naringin suppresses cancer cell growth in vitro and in vivo, although the underlying mechanisms are not fully understood. Recently, the roles of glycoconjugates, such as gangliosides, in cancer cells have been focused because of their regulatory effects of malignant phenotypes. Here, to clarify the roles of naringin in the negative-regulation of cancer cell growth, the alteration of glycoconjugates induced by naringin exposure and its significance on cell signaling were investigated. Human cancer cells, HeLa and A549, were exposed to various concentrations of naringin. Naringin treatment induced the suppression of cell growth toward HeLa and A549 cells accompanied with an increase of apoptotic cells. In naringin-exposed cells, GM3 ganglioside was drastically increased compared to the GM3 content prior to the treatment. Furthermore, naringin inhibited NEU3 sialidase, a GM3 degrading glycosidase. Similarly, NEU3 inhibition activities were also detected by other flavanone, such as hesperidin and neohesperidin dihydrocalcone, but their aglycones showed less inhibitions. Naringin-treated cancer cells showed suppressed EGFR and ERK phosphorylation levels. These results suggest a novel mechanism of naringin in the suppression of cancer cell growth through the alteration of glycolipids. NEU3 inhibitory effect of naringin induced GM3 accumulation in HeLa and A549 cells, leading the attenuation of EGFR/ERK signaling accompanied with a decrease in cell growth. PMID:27105818

  4. Immunotargeting of cancer stem cells

    Kwiatkowska-Borowczyk, Eliza P.; Gąbka-Buszek, Agnieszka; Jankowski, Jakub; Mackiewicz, Andrzej

    2015-01-01

    Cancer stem cells (CSCs) represent a distinctive population of tumour cells that control tumour initiation, progression, and maintenance. Their influence is great enough to risk the statement that successful therapeutic strategy must target CSCs in order to eradicate the disease. Because cancer stem cells are highly resistant to chemo- and radiotherapy, new tools to fight against cancer have to be developed. Expression of antigens such as ALDH, CD44, EpCAM, or CD133, which distinguish CSCs fr...

  5. Depletion of kinesin 5B affects lysosomal distribution and stability and induces peri-nuclear accumulation of autophagosomes in cancer cells

    Cardoso, Carla M P; Groth-Pedersen, Line; Høyer-Hansen, Maria;

    2009-01-01

    BACKGROUND: Enhanced lysosomal trafficking is associated with metastatic cancer. In an attempt to discover cancer relevant lysosomal motor proteins, we compared the lysosomal proteomes from parental MCF-7 breast cancer cells with those from highly invasive MCF-7 cells that express an active form of...... the ErbB2 (DeltaN-ErbB2). METHODOLOGY/PRINCIPAL FINDINGS: Mass spectrometry analysis identified kinesin heavy chain protein KIF5B as the only microtubule motor associated with the lysosomes in MCF-7 cells, and ectopic DeltaN-ErbB2 enhanced its lysosomal association. KIF5B associated with lysosomes...... also in HeLa cervix carcinoma cells as analyzed by subcellular fractionation. The depletion of KIF5B triggered peripheral aggregations of lysosomes followed by lysosomal destabilization, and cell death in HeLa cells. Lysosomal exocytosis in response to plasma membrane damage as well as fluid phase...

  6. Confocal fluorescence microscopy: An ultra-sensitive tool used to evaluate intracellular antiretroviral nano-drug delivery in HeLa cells

    Mandal, Subhra; Zhou, You; Shibata, Annemarie; Destache, Christopher J.

    2015-08-01

    In the last decade, confocal fluorescence microscopy has emerged as an ultra-sensitive tool for real-time study of nanoparticles (NPs) fate at the cellular-level. According to WHO 2007 report, Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS) is still one of the world's major health threats by claiming approximately 7,000 new infections daily worldwide. Although combination antiretroviral drugs (cARV) therapy has improved the life-expectancy of HIV-infected patients, routine use of high doses of cARV has serious health consequences and requires complete adherence to the regimen for success. Thus, our research goal is to fabricate long-acting novel cARV loaded poly(lactide-co-glycolic acid) (PLGA) nanoparticles (cARV-NPs) as drug delivery system. However, important aspects of cARV-NPs that require special emphasis are their cellular-uptake, potency, and sustained drug release efficiency over-time. In this article, ultra-sensitive confocal microscopy is been used to evaluate the uptake and sustained drug release kinetics of cARV-NPs in HeLa cells. To evaluate with the above goal, instead of cARV-drug, Rhodamine6G dye (fluorescent dye) loaded NPs (Rho6G NPs) have been formulated. To correlate the Rhodamin6G release kinetics with the ARV release from NPs, a parallel HPLC study was also performed. The results obtained indicate that Rho6G NPs were efficiently taken up at low concentration (delivery with the potential to reduce drug dosage as well as the number of drug administrations per month.

  7. Prostate stem cells and cancer

    Nikitin, Alexander Y.; Matoso, A; Roy-Burman, P

    2007-01-01

    Properties shared by neoplastic and stem cells indicate a possibility that somatic stem cells or transit-amplifying cells that have reacquired stem cell properties, particularly the ability for self-renewal, represent favorable targets for malignant transformation. In this review we discuss significance of the stem cell model for understanding prostate cancer pathogenesis and describe relevant studies in animals. It is proposed that dissemination of rare cancer stem ce...

  8. General Information about Small Cell Lung Cancer

    ... Cancer Prevention Lung Cancer Screening Research Small Cell Lung Cancer Treatment (PDQ®)–Patient Version General Information About Small Cell Lung Cancer Go to Health Professional Version Key Points Small ...

  9. Stages of Small Cell Lung Cancer

    ... Cancer Prevention Lung Cancer Screening Research Small Cell Lung Cancer Treatment (PDQ®)–Patient Version General Information About Small Cell Lung Cancer Go to Health Professional Version Key Points Small ...

  10. Treatment Option Overview (Small Cell Lung Cancer)

    ... Cancer Prevention Lung Cancer Screening Research Small Cell Lung Cancer Treatment (PDQ®)–Patient Version General Information About Small Cell Lung Cancer Go to Health Professional Version Key Points Small ...

  11. Alteration of cell cycle progression by Sindbis virus infection

    Yi, Ruirong; Saito, Kengo [Department of Molecular Virology, Graduate School of Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba 260-8670 (Japan); Isegawa, Naohisa [Laboratory Animal Center, Graduate School of Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba 260-8670 (Japan); Shirasawa, Hiroshi, E-mail: sirasawa@faculty.chiba-u.jp [Department of Molecular Virology, Graduate School of Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba 260-8670 (Japan)

    2015-07-10

    We examined the impact of Sindbis virus (SINV) infection on cell cycle progression in a cancer cell line, HeLa, and a non-cancerous cell line, Vero. Cell cycle analyses showed that SINV infection is able to alter the cell cycle progression in both HeLa and Vero cells, but differently, especially during the early stage of infection. SINV infection affected the expression of several cell cycle regulators (CDK4, CDK6, cyclin E, p21, cyclin A and cyclin B) in HeLa cells and caused HeLa cells to accumulate in S phase during the early stage of infection. Monitoring SINV replication in HeLa and Vero cells expressing cell cycle indicators revealed that SINV which infected HeLa cells during G{sub 1} phase preferred to proliferate during S/G{sub 2} phase, and the average time interval for viral replication was significantly shorter in both HeLa and Vero cells infected during G{sub 1} phase than in cells infected during S/G{sub 2} phase. - Highlights: • SINV infection was able to alter the cell cycle progression of infected cancer cells. • SINV infection can affect the expression of cell cycle regulators. • SINV infection exhibited a preference for the timing of viral replication among the cell cycle phases.

  12. Enhanced Antiproliferative Effect of Carboplatin in Cervical Cancer Cells Utilizing Folate-Grafted Polymeric Nanoparticles

    Ji, Jing; Zuo, Ping; Wang, Yue-Ling

    2015-11-01

    Carboplatin (CRB) possesses superior anticancer effect in cervical cancer cells with lower incidence of side effects compared to that of cisplatin. However, CRB suffers from severe side effects due to undesirable tissue distributions which contribute to the low therapeutic efficacy. Here, we report a unique folic acid-conjugated chitosan-coated poly( d- l-lactideco-glycolide) (PLGA) nanoparticles (FPCC) prepared for the selective delivery of carboplatin to the cervical cancer cells. The particles were nanosized and spherical shaped with size less than chitosan layer controlled the overall release rate of CRB from chitosan-coated PLGA nanoparticles (PCC) and FPCC. FPCC displayed a higher cellular uptake capacity in HeLa cells than compared to non-targeted nanoparticles. Selective uptake of FPCC was due to an interaction of folic acid (FA) with the folate receptors alpha (FRs-α) which is overexpressed on the HeLa and promoted active targeting. These results indicated that FPCC had a specific affinity for the cancerous, HeLa cells owing to ligand-receptor (FA-FR-α) recognition. Consistently, FPCC showed superior cytotoxic effect than any other formulations. The IC50 (concentration of the drug required to kill 50 % of the cells) value of FPCC was 0.65 μg/ml while it was 1.08, 1.56, and 2.35 μg/ml for PCC, PLGA NP, and free CRB, respectively. Consistent with the cytotoxicity assay, FPCC induced higher fraction of early as well as late apoptosis cells. Especially, FPCC induced nearly 45 % of early apoptosis cells and more than 35 % in late apoptosis. Therefore, we propose that folate-conjugated nanoparticles might have potential applications in cervical cancer therapy.

  13. REAL-TIME DETECTION OF SURVIVIN mRNA EXPRESSION IN CERVICAL CANCER CELL LINES USING MOLECULAR BEACON IMAGING

    An Ruifang; He Dalin; Xue Yan; Wang Shu; Xie Li; Zhao Jun; Wang Xinyang; Yang Lili

    2006-01-01

    Objective To detect the expression of survivin mRNA in cervical cancer cell lines using molecular beacon imaging technology. Methods Human cervical cancer cells (HeLa and SiHa) and human fetal lung fibroblast HFL-I were cultured in vitro. After adding 100 nmol/L survivin mRNA molecular beacon, the fluorescent signals were observed under fluorescent microscope. The expressions of survivin in cervical cancer cells and HFL-I cell were examined by immunocytochemical streptravidin-biothin peroxidase (SP) assay at the same time. Results Two kinds of survivin mRNA molecular beacon, with different color fluorescence, had strong fluorescent signal in cervical cancer cell lines, and the signal in SiHa cell line was stronger, but these signals were not found in HFL-I ; Immunocytochemical staining of positive survivin was located in the cytoplasm of cervical cancer cell lines HeLa and SiHa, whereas, no expression of survivin was detected in HFL-I cell line. Conclusion The technology of molecular beacon imaging can be used to detect the expression of survivin mRNA in viable cells successfully, and may provide a new approach to the diagnosis of early stage cervical cancer and the following-up in the clinic.

  14. Characterisation of ribosomal proteins from HeLa and Krebs II mouse ascites tumor cells by different two-dimensional polyacrylamide gel electrophoresis techniques

    Issinger, O G; Beier, H

    1978-01-01

    Electrophoresis of ribosomal proteins according to Kaltschmidt and Wittmann, 1970a, b (pH 8.6/pH 4.5 urea system) yielded 29 proteins for the small subunits and 35 and 37 proteins for the large subunits of Krebs II ascites and HeLa ribosomes, respectively. Analysis of the proteins according to a ...

  15. The mRNA decay factor tristetraprolin (TTP) induces senescence in human papillomavirus-transformed cervical cancer cells by targeting E6-AP ubiquitin ligase

    Sanduja, Sandhya; Kaza, Vimala; Dixon, Dan A.

    2009-01-01

    The RNA-binding protein tristetraprolin (TTP) regulates expression of many cancer-associated and proinflammatory factors through binding AU-rich elements (ARE) in the 3'-untranslated region (3'UTR) and facilitating rapid mRNA decay. Here we report on the ability of TTP to act in an anti-proliferative capacity in HPV18-positive HeLa cells by inducing senescence. HeLa cells maintain a dormant p53 pathway and elevated telomerase activity resulting from HPV-mediated transformation, whereas TTP ex...

  16. Ovarian cancer: emerging concept on cancer stem cells

    Ponnusamy Moorthy P; Batra Surinder K

    2008-01-01

    Abstract Emerging evidence suggests that the capacity of a tumor to grow and propagate is dependent on a small subset of cells within a tumor, termed cancer stem cells. In fact, cancer cells, like stem cells, can proliferate indefinitely through a dysregulated cellular self-renewal capacity. Cancer stem cells may originate due to the distribution into self-renewal and differentiation pathways occurring in multi-potential stem cells, tissue-specific stem cells, progenitor cells and cancer cell...

  17. Repression of the Integrated Papillomavirus E6/E7 Promoter Is Required for Growth Suppression of Cervical Cancer Cells

    Francis, Delicia A.; Schmid, Susanne I.; Howley, Peter M.

    2000-01-01

    The human papillomavirus (HPV) E2 protein is an important regulator of viral E6 and E7 gene expression. E2 can repress the viral promoter for E6 and E7 expression as well as block progression of the cell cycle in cancer cells harboring the DNA of “high-risk” HPV types. Although the phenomenon of E2-mediated growth arrest of HeLa cells and other HPV-positive cancer cells has been well documented, the specific mechanism by which E2 affects cellular proliferation has not yet been elucidated. Her...

  18. Changes of the cell cycle regulators and cell cycle arrest in cervical cancer cells after cisplatin therapy

    2009-01-01

    Objective To investigate the changes of the cell cycle regulators ATM,Chk2 and p53 and cell cycle arrest in HeLa cells after cisplatin therapy. Methods The proliferation-inhibiting rates of HeLa cells induced by cisplatin of different concentrations were measured by MTT assays. The mRNA and protein expressions of ATM,Chk2 and p53 of HeLa cells with and without cisplatin were detected by RT-PCR and Western blot,respectively. The cell cycle analysis was conducted by flow cytometric analysis. Results Cisplatin...

  19. Effect of 630-NM pulsed laser irradiation on the proliferation of HeLa cells in Photofrin®-mediated photodynamic therapy

    Miyamoto, Yuichi; Nishikiori, Daisuke; Hagino, Fumika; Wakita, Masayoshi; Tanabe, Ichiro; Toida, Masahiro

    2011-01-01

    Background and Aims: Red laser light of wavelength 630 nm is usually used for Photofrin®-mediated photodynamic therapy (PDT). The 630-nm light employed in PDT corresponds to the region of the wavelength used in low-level laser therapy (LLLT) may influence on the photodynamic effect required for killing cancer cells. The aim of this in vitro study was to investigate the changes in cell viability and degree of cell proliferation after Photofrin®-mediated PDT using 630-nm pulsed laser irradiatio...

  20. VEGF-C在宫颈癌抗凋亡分子机制中的研究%Effect of VEGF-C Gene Transfection on the Expression of VEGF-C in Human Cervical Carcinoma HeLa Cells and the Molecular Meehnisms of Its Anti-apoptosis Effect

    陈星; 王美芬; 吴朝阳; 任虹平; 郑灵芝; 郑曙民; 熊冬生; 杨纯正; 糜若然

    2009-01-01

    目的:探讨脂质体介导VEGF-C基因转染人宫颈癌HeLa细胞及其对宫颈癌抗凋亡分子机制的研究.方法:前期构建的真核表达载体pcDNA3.1(+)/VEGF-C,用脂质体介导转染人宫颈癌HeLa细胞,并加压筛选获得转染成功的细胞株,经半定量RT-PCR检测转染后VEGF-C表达水平,ELISA检测培养上清中VEGF-C的表达.对转染成功的细胞检测NF-κB、bcl-2基因的表达.结果:在mRNA水平,转染组VEGF-C明显高于空载体组和未转染组;ELISA检测转染组(678.73±38.92ng/mL),也明显高于空载体组(129.52±50.73ng/ml),和未转染组(123.05±55.83ng/mL),成功构建了高表达VEGF-C的宫颈癌细胞株HeLa/S1;在HeLa/S1组NF-κB的表达(2.06±0.09 vs 1.35±0.02 vs 1.38±0.02P<0.05),bcl-2的表达(2.02±0.67 vs 0.41±0.06 vs 0.37±0.06 P<0.05)明显高于空载体组和未转染组.结论:脂质体介导VEGF-C基因转染人宫颈癌HeLa可显著增加VEGF-C表达,推测高表达的VEGF-C可激活NF-κB,使抗凋亡基因bcl-2高表达,从而促进肿瘤细胞的生长.%Objective: TO explore the effect of VEGF-C gene transfection on the expression of VEGF-C in human cervical carcinoma HeLa cells and the mechanisms of its anti-apoptosis effect. Methods: The con-structed pcDNA3.1(+)NEGF-C vector was transformed into human cervical cancer HeLa cells and was select-ed by G418. The changes in the expression level of VEGF-C mRNA and protein were determined by semi-quantitive RT-PCR and ELISA. HeLa cells with overexpression of VEGF-C were named as HeLa/S1. The expression level of NF-KB and bcl-2 mRNA was determined by RT-PCR in transfected cells. Results: After transfection by liposome, the VEGF-C mRNA level and the expression of VEGF-C protein in transfected cells were higher than those in the control groups. HeLa/S1 cell line was successfully established. In HeLa/S1 cells, the expression of NF-κB (2.06±0.09 vs 1.35±0.02 vs 1.38±0.02 P<0.05) and bcl-2 gene mRNA (2.02± 0.67 vs 0.41±0.06 vs 0.37±0

  1. Lung cancer - non-small cell

    Cancer - lung - non-small cell; Non-small cell lung cancer; NSCLC; Adenocarcinoma - lung; Squamous cell carcinoma - lung ... Horn L, Eisenberg R, Gius D, et al. Cancer of the lung. In: Niederhuber JE, Armitage JO, Doroshow JH, Kastan ...

  2. Prostate cancer stem cell biology

    Yu, Chunyan; Yao, Zhi; Jiang, Yuan; Keller, Evan T.

    2012-01-01

    The cancer stem cell (CSC) model provides insights into pathophysiology of cancers and their therapeutic response. The CSC model has been both controversial, yet provides a foundation to explore cancer biology. In this review, we provide an overview of CSC concepts, biology and potential therapeutic avenues. We then focus on prostate CSC including (1) their purported origin as either basal-derived or luminal-derived cells; (2) markers used for prostate CSC identification; (3) alterations of s...

  3. Aspirin Has Antitumor Effects via Expression of Calpain Gene in Cervical Cancer Cells

    Sang Koo Lee

    2008-01-01

    Full Text Available Aspirin and other nonsteroidal anti-inflammatory drugs show efficacy in the prevention of cancers. It is known that they can inhibit cyclooxygenases, and some studies have shown that they can induce apoptosis. Our objective in this study was to investigate the mechanism by which aspirin exerts its apoptosis effects in human cervical cancer HeLa cells. The effect of aspirin on the gene expression was studied by differential mRNA display RT-PCR. Among the isolated genes, mu-type calpain gene was upregulated by aspirin treatment. To examine whether calpain mediates the antitumor effects, HeLa cells were stably transfected with the mammalian expression vector pCR3.1 containing mu-type calpain cDNA (pCRCAL/HeLa, and tumor formations were measured in nude mice. When tumor burden was measured by day 49, HeLa cells and pCR/HeLa cells (vector control produced tumors of 2126 mm3 and 1638 mm3, respectively, while pCRCAL/HeLa cells produced markedly smaller tumor of 434 mm3 in volume. The caspase-3 activity was markedly elevated in pCRCAL/HeLa cells. The increased activity levels of caspase-3 in pCRCAL/HeLa cells, in parallel with the decreased tumor formation, suggest a correlation between caspase-3 activity and calpain protein. Therefore, we conclude that aspirin-induced calpain mediates an antitumor effect via caspase-3 in cervical cancer cells.

  4. Folate Functionalized Mesoporous Carbon Nanospheres as Nanocarrier for Targetted Delivery and Controlled Release of Doxorubicin to HeLa Cells%叶酸功能化介孔碳纳米球负载阿霉素的细胞靶向传递及可控释放

    朱杰; 廖蕾; 朱丽娜; 孔继烈; 刘宝红

    2013-01-01

    Ordered mesoporous carbon is a kind of novel carrier for intracellular drug release. There are few reports on the use of mesoporous carbon nanospheres (MCNs) as the transmembrane deliverer in human cancer cells; on the other hand, the particle size of MCNs synthesized by hard templates is usually larger than 100 nm. It is accepted that the optimal size of a transmembrane delivery vehicle should be less than 100 nm in diameter and the surface should be hydrophilic to circumvent clearance by macrophages, to maximize circulation times and targeting ability. In this work, MCNs with a diameter of ca. 90 nm have been developed as a targeted drug delivery system of an anticancer drug, doxorubicin (DOX). The small MCNs were synthesized using triblock copolymer Pluronic F127 as a template. The MCNs were first treated by acid to improve its dispersion property in an aqueous solution, and then modified by folic acid through EDC-NHS. The structure of the MCNs was well characterized by transmission electron microscopy, small-angle X-ray scattering, nitrogen adsorption/desorption and dynamic light scattering. pH-dependent drug release is successfully achieved due to the supramolecular π-π stacking between DOX and the carbonaceous structures. By effective passive and active targeting, MCNs can be readily internalized into HeLa cells, where the carried DOX can be efficiently released in the acidic microenvironment of the tumors for further therapy. The results from confocal laser scanning microscope and flow cytometry demonstrated that the cellular uptake efficiency of MCNs toward HeLa cells was increased through the functionalization with folic acid, and the folate modified MCNs show much higher endocytosis properties toward HeLa cells (folate receptor positive) than toward MCF-7 cells (folate receptor negative). The cytotoxicities toward HeLa cells were studied by MTT method, which indicated that the cytotoxicities of DOX loaded mesoporous carbon nanoparticles was also

  5. One Step Quick Detection of Cancer Cell Surface Marker by Integrated NiFe-based Magnetic Biosensing Cell Cultural Chip

    Chenchen Bao; Lei Chen; Tao Wang; Chong Lei; Furong Tian; Daxiang Cui; Yong Zhou

    2013-01-01

    RGD peptides has been used to detect cell surface integrin and direct clinical effective therapeutic drug selection. Herein we report that a quick one step detection of cell surface marker that was realized by a specially designed NiFe-based magnetic biosensing cell chip combined with functionalized magnetic nanoparti-cles. Magnetic nanoparticles with 20-30 nm in diameter were prepared by coprecipitation and modified with RGD-4C, and the resultant RGD-functionalized magnetic nanoparticles were used for targeting cancer cells cul-tured on the NiFe-based magnetic biosensing chip and distinguish the amount of cell surface receptor-integrin. Cell lines such as Calu3, Hela, A549, CaFbr, HEK293 and HUVEC exhibiting different integrin expression were chosen as test samples. Calu3, Hela, HEK293 and HUVEC cells were successfully identified. This approach has advantages in the qualitative screening test. Compared with traditional method, it is fast, sensitive, low cost, easy-operative, and needs very little human intervention. The novel method has great potential in applications such as fast clinical cell surface marker detection, and diagnosis of early cancer, and can be easily extended to other biomedical applications based on molecular recognition.

  6. Detecção da citotoxicidade de materiais biocompatíveis nas linhagens celulares MRC-5, HeLa e RC-IAL MRC-5, HeLa and RC-IAL cell lines sensitivity for detection of cytotoxicity of biocompatible materials

    Aurea S. Cruz

    1992-04-01

    Full Text Available A sensibilidade de uma linhagem celular diplóide e duas heteroplóides, para a detecção de citotoxicidade através do método de difusão em camada de ágar sobre culturas celulares, foi avaliada experimentalmente com solução de ácido ascórbico em diferentes concentrações e, na prática, frente a 562 amostras de 21 diferentes materiais industriais enviados para análise na Seção de Culturas Celulares do Instituto Adolfo Lutz. A linhagem celular heteroplóide designada RC-IAL apresentou, em relação às linhagens MRC-5 e HeLa, maior sensibilidade porque revelou a presença de efeito citotóxico nas menores concentrações utilizadas (10 e 25 ug/ml do ácido ascórbico e apresentou maior diâmetro do halo citotóxico em 15 amostras e igual diâmetro em 16 das 43 amostras (7,6% que resultaram positivas. Nas 43 amostras positivas, a linhagem MRC-5 não revelou citotoxicidade em 3 amostras de espuma e 1 de resina acrílica. O polivinilcloreto (PVC e o polietileno, raramente revelaram positividade, enquanto plástico, algodão e resinas acrílicas revelaram citotoxicidade ao redor de 5%. Em vista dos resultados é discutida a proposta da utilização da linhagem RC-IAL e HeLa para a continuidade das futuras análises solicitadas ao Instituto Adolfo LutzThe sensitivity of diploid and heteroploid cell lines for detection of cytotoxicity using the agar diffusion method on cell culture, was tested with ascorbic acid solution of different concentrations. A total of 562 samples of 21 various materials were tested. The heteroploid cell line, RC-IAL, showed in relation to the MRC-5 and HeLa cell lines, greater sensitivity because it showed the presence of cytotoxic effect with the lowest concentration used (10 and 25ug/ml of ascorbic acid and showed greater diameter of cytotoxic halo in 15 samples and equal diameter in 16 of the 43 positive samples (7.6%. Out of 43 positive samples, the MRC-5 line did not show cytotoxicity in 3 sponge samples and

  7. The Effects of Zeolite X and Y on Cancer Cell Lines

    Noor Azhana Ghazi

    2012-07-01

    Full Text Available Zeolites are hydrated silicates of aluminium that have been very useful in many industry because of its microporous property, absorbance ability and ion exchange capacity. It is currently viewed as a potential adjuvant in cancer therapy due to its ability to inhibit the proliferation of cancer cells. Research on natural zeolite clinoptilolite application as anticancer agent has been proven by others. However, the effect of other types of zeolite on cancer cells is still uncertain. This study is performed to determine the effects of zeolite X and Y on cancer cell lines proliferation in vitro. Cancer cell lines HeLa, AsPC-1 and 911 cells were cultured in designated medium treated with zeolite X and zeolite Y at the concentration of 5 mg/ml and 50 mg/ml. Fetal Bovine Serum (FBS concentrations were modified to 5%, 10%, 15% and 20%. After 72 hours incubation, the efficacy of zeolite to treat cancer cell lines were measured by means of cell viability test via MTT assay. Overall results showed that cancer cell lines cultivated in the medium treated with 50 mg/ml of zeolite X and 5% FBS exhibited the highest inhibition of cell proliferation and decrease in cell viability. This finding provides preliminary information in the study of determining the potential use of zeolite as anticancer agent for alternative or complementary therapy.

  8. Antibacterial and Antimetastatic Potential of Diospyros lycioides Extract on Cervical Cancer Cells and Associated Pathogens

    Bagla, V. P.; Lubisi, V. Z.; Ndiitwani, T.; Mokgotho, M. P.; Mampuru, L.; Mbazima, V.

    2016-01-01

    Cervical cancer is among the most prevalent forms of cancer in women worldwide. Diospyros lycioides was extracted using hexane, ethyl acetate, acetone, and methanol and finger print profiles were determined. The leaf material was tested for the presence of flavonoids, tannins, saponins, terpenoids, and cardiac glycosides using standard chemical methods and the presence of flavonoids and phenolics using thin layer chromatography. The total phenolic content was determined using Folin-Ciocalteu procedure. The four extracts were tested for antibacterial activity using bioautography against Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, and Escherichia coli. The acetone extract with the highest number of antibacterial and antioxidant compounds was assessed for its cytotoxicity on BUD-8 cells using the real-time xCELLigence system and its potential effects on metastatic cervical cancer (HeLa) cell migration and invasion were assessed using wound healing migration and invasion assays. The leaf extract tested positive for flavonoids, tannins, and terpenoids while the four different extracts tested in the antimicrobial assay contained constituents active against one or more of the organisms tested, except E. coli. The cytotoxicity of the acetone extract in real-time was concentration-dependent with potent ability to suppress the migration and invasion of HeLa cells. The finding demonstrates the acetone extract to contain constituents with antibacterial and antimetastatic effects on cervical cancer cells. PMID:27239210

  9. Induction of mitochondrial-mediated apoptosis by Morinda citrifolia (Noni) in human cervical cancer cells.

    Gupta, Rakesh Kumar; Banerjee, Ayan; Pathak, Suajta; Sharma, Chandresh; Singh, Neeta

    2013-01-01

    Cervical cancer is the second most common cause of cancer in women and has a high mortality rate. Cisplatin, an antitumor agent, is generally used for its treatment. However, the administration of cisplatin is associated with side effects and intrinsic resistance. Morinda citrifolia (Noni), a natural plant product, has been shown to have anti-cancer properties. In this study, we used Noni, cisplatin, and the two in combination to study their cytotoxic and apoptosis-inducing effects in cervical cancer HeLa and SiHa cell lines. We demonstrate here, that Noni/Cisplatin by themselves and their combination were able to induce apoptosis in both these cell lines. Cisplatin showed slightly higher cell killing as compared to Noni and their combination showed additive effects. The observed apoptosis appeared to be mediated particularly through the up-regulation of p53 and pro-apoptotic Bax proteins, as well as down- regulation of the anti-apoptotic Bcl-2, Bcl-XL proteins and survivin. Augmentation in the activity of caspase-9 and -3 was also observed, suggesting the involvement of the intrinsic mitochondrial pathway of apoptosis for both Noni and Cisplatin in HeLa and SiHa cell lines. PMID:23534730

  10. Efflux Transport Characterization of Resveratrol Glucuronides in UDP-Glucuronosyltransferase 1A1 Transfected HeLa Cells: Application of a Cellular Pharmacokinetic Model to Decipher the Contribution of Multidrug Resistance-Associated Protein 4.

    Wang, Shuai; Li, Feng; Quan, Enxi; Dong, Dong; Wu, Baojian

    2016-04-01

    Resveratrol undergoes extensive metabolism to form biologically active glucuronides in humans. However, the transport mechanisms for resveratrol glucuronides are not fully established. Here, we aimed to characterize the efflux transport of resveratrol glucuronides using UGT1A1-overexpressing HeLa cells (HeLa1A1 cells), and to determine the contribution of multidrug resistance-associated protein (MRP) 4 to cellular excretion of the glucuronides. Two glucuronide isomers [i.e., resveratrol 3-O-glucuronide (R3G) and resveratrol 4'-O-glucuronide (R4'G)] were excreted into the extracellular compartment after incubation of resveratrol (1-100 μM) with HeLa1A1 cells. The excretion rate was linearly related to the level of intracellular glucuronide, indicating that glucuronide efflux was a nonsaturable process. MK-571 (a dual inhibitor of UGT1A1 and MRPs) significantly decreased the excretion rates of R3G and R4'G while increasing their intracellular levels. Likewise, short-hairpin RNA (shRNA)-mediated silencing of MRP4 caused a significant reduction in glucuronide excretion but an elevation in glucuronide accumulation. Furthermore, β-glucuronidase expressed in the cells catalyzed the hydrolysis of the glucuronides back to the parent compound. A cellular pharmacokinetic model integrating resveratrol transport/metabolism with glucuronide hydrolysis/excretion was well fitted to the experimental data, allowing derivation of the efflux rate constant values in the absence or presence of shRNA targeting MRP4. It was found that a large percentage of glucuronide excretion (43%-46%) was attributed to MRP4. In conclusion, MRP4 participated in cellular excretion of R3G and R4'G. Integration of mechanistic pharmacokinetic modeling with transporter knockdown was a useful method to derive the contribution percentage of an exporter to overall glucuronide excretion. PMID:26758854

  11. Mouse models for cancer stem cell research

    Cheng, Le; Ramesh, Anirudh V.; Flesken-Nikitin, Andrea; Choi, Jinhyang; Nikitin, Alexander Yu.

    2009-01-01

    Cancer stem cell concept assumes that cancers are mainly sustained by a small pool of neoplastic cells, known as cancer stem cells or tumor initiating cells, which are able to reproduce themselves and produce phenotypically heterogeneous cells with lesser tumorigenic potential. Cancer stem cells represent an appealing target for development of more selective and efficient therapies. However, direct testing of the cancer stem cell concept and assessment of its therapeutic implications in human...

  12. Investigating the consequences of eIF4E2 (4EHP interaction with 4E-transporter on its cellular distribution in HeLa cells.

    Dorota Kubacka

    Full Text Available In addition to the canonical eIF4E cap-binding protein, eukaryotes have evolved sequence-related variants with distinct features, some of which have been shown to negatively regulate translation of particular mRNAs, but which remain poorly characterised. Mammalian eIF4E proteins have been divided into three classes, with class I representing the canonical cap-binding protein eIF4E1. eIF4E1 binds eIF4G to initiate translation, and other eIF4E-binding proteins such as 4E-BPs and 4E-T prevent this interaction by binding eIF4E1 with the same consensus sequence YX 4Lϕ. We investigate here the interaction of human eIF4E2 (4EHP, a class II eIF4E protein, which binds the cap weakly, with eIF4E-transporter protein, 4E-T. We first show that ratios of eIF4E1:4E-T range from 50:1 to 15:1 in HeLa and HEK293 cells respectively, while those of eIF4E2:4E-T vary from 6:1 to 3:1. We next provide evidence that eIF4E2 binds 4E-T in the yeast two hybrid assay, as well as in pull-down assays and by recruitment to P-bodies in mammalian cells. We also show that while both eIF4E1 and eIF4E2 bind 4E-T via the canonical YX 4Lϕ sequence, nearby downstream sequences also influence eIF4E:4E-T interactions. Indirect immunofluorescence was used to demonstrate that eIF4E2, normally homogeneously localised in the cytoplasm, does not redistribute to stress granules in arsenite-treated cells, nor to P-bodies in Actinomycin D-treated cells, in contrast to eIF4E1. Moreover, eIF4E2 shuttles through nuclei in a Crm1-dependent manner, but in an 4E-T-independent manner, also unlike eIF4E1. Altogether we conclude that while both cap-binding proteins interact with 4E-T, and can be recruited by 4E-T to P-bodies, eIF4E2 functions are likely to be distinct from those of eIF4E1, both in the cytoplasm and nucleus, further extending our understanding of mammalian class I and II cap-binding proteins.

  13. Aloe vera inhibits proliferation of human breast and cervical cancer cells and acts synergistically with cisplatin.

    Hussain, Arif; Sharma, Chhavi; Khan, Saniyah; Shah, Kruti; Haque, Shafiul

    2015-01-01

    Many of the anti-cancer agents currently used have an origin in natural sources including plants. Aloe vera is one such plant being studied extensively for its diverse health benefits, including cancer prevention. In this study, the cytotoxic potential of Aloe vera crude extract (ACE) alone or in combination with cisplatin in human breast (MCF-7) and cervical (HeLa) cancer cells was studied by cell viability assay, nuclear morphological examination and cell cycle analysis. Effects were correlated with modulation of expression of genes involved in cell cycle regulation, apoptosis and drug metabolism by RT-PCR. Exposure of cells to ACE resulted in considerable loss of cell viability in a dose- and time-dependent fashion, which was found to be mediated by through the apoptotic pathway as evidenced by changes in the nuclear morphology and the distribution of cells in the different phases of the cell cycle. Interestingly, ACE did not have any significant cytotoxicity towards normal cells, thus placing it in the category of safe chemopreventive agent. Further, the effects were correlated with the downregulation of cyclin D1, CYP 1A1, CYP 1A2 and increased expression of bax and p21 in MCF-7 and HeLa cells. In addition, low dose combination of ACE and cisplatin showed a combination index less than 1, indicating synergistic growth inhibition compared to the agents applied individually. In conclusion, these results signify that Aloe vera may be an effective anti-neoplastic agent to inhibit cancer cell growth and increase the therapeutic efficacy of conventional drugs like cispolatin. Thus promoting the development of plant-derived therapeutic agents appears warranted for novel cancer treatment strategies. PMID:25854386

  14. Eradicating cancer cells: struggle with a chameleon

    Di, Jiabo; Boer, Tjitske Duiveman-de; Figdor, Carl G.; Torensma, Ruurd

    2011-01-01

    Eradication of cancer stem cells to abrogate tumor growth is a new treatment modality. However, like normal cells cancer cells show plasticity. Differentiated tumor stem cells can acquire stem cell properties when they gain access to the stem cell niche. This indicates that eradicating of stem cells (emptying of the niche) alone will not lead to eradication of the tumor. Treatment should be directed to cancer stem cells ànd more mature cancer cells.

  15. The experimental study of radionuclide imaging and treatment of cervical cancer mediated by hNIS gene transfection

    Objective: To explore the feasibility of imaging and treatment of cervical cancer xenograft model using 131I mediated by hNIS gene transfection. Methods: The cervical cancer xenograft models were established with Hela-NIS( +) cells and Hela cells, respectively. Five Hela-NIS(+) xenograft models and five Hela xenograft models were dynamically imaged at 0.5, 1, 2, 4, 8, 16 and 20 h postinjection of 131I (7.4 MBq). Five Hela-NIS(+) xenograft models were imaged at 0.5, 1, 2, 4, 8, 16, 20 and 25 h postinjection of 99TcmO4- (11.1 MBq). Twenty Hela-NIS(+) cervical cancer xenograft models were randomly divided into four groups: Three 131I treating groups and one control group. The therapeutic effects of 131I at three levels (74, 111, 148 MBq) were investigated following intraperitoneal injection. Results: Hela-NIS(+)human cervical cancer xenografts were established successfully in nude mice. The Hela-NIS(+) xenografts significantly accumulated radioactivity after intraperitoneal injection of 131I, and the radioactivity was persistently present until 20 h postinjection, but Hela xenografts had no radioactive accumulation. The T/B value of the Hela-NIS(+) xenografts reached 17.34 at 8 h postinjection. The imaging with 99TcmO4- showed that the radioactivity was persistently present in Hela-NIS(+) xenografts for almost 25 h. The Hela-NIS(+)xenografts shrinked after 131I treatment. The inhibition ratios of tumor growth in 111 MBq and 148 MBq groups were both significantly higher than that of 74 MBq group (t: 2.74-5.75, P131I and 99TcmO4- and could be treated successfully with 131I. 131I treatment mediated by hNIS gene transfection could be a promising cancer treatment method. (authors)

  16. Induction of Apoptosis by Green Synthesized Gold Nanoparticles Through Activation of Caspase-3 and 9 in Human Cervical Cancer Cells

    Baharara, Javad; Ramezani, Tayebe; Divsalar, Adeleh; Mousavi, Marzieh; Seyedarabi, Arefeh

    2016-01-01

    Background: Gold Nanoparticles (GNPs) are used in imaging and molecular diagnostic applications. As the development of a novel approach in the green synthesis of metal nanoparticles is of great importance and a necessity, a simple and safe method for the synthesis of GNPs using plant extracts of Zataria multiflora leaves was applied in this study and the results on GNPs’ anticancer activity against HeLa cells were reported. Methods: The GNPs were characterized by UV-visible spectroscopy, FTIR, TEM, DLS and Zeta-potential measurements. In addition, the cellular up-take of nanoparticles was investigated using Dark Field Microscopy (DFM). Induction of apoptosis by high dose of GNPs in HeLa cells was assessed by MTT assay, Acridin orange, DAPI staining, Annexin V/PI double-labeling flow cytometry and caspase activity assay. Results: UV-visible spectroscopy results showed a surface plasmon resonance band for GNPs at 530 nm. FTIR results demonstrated an interaction between plant extract and nanoparticles. TEM images revealed different shapes for GNPs and DLS results indicated that the GNPs range in size from 10 to 42 nm. The Zeta potential values of the synthesized GNPs were between 30 to 50 Mev, indicating the formation of stable particles. As evidenced by MTT assay, GNPs inhibit proliferation of HeLa cells in dose-dependent GNPs and cytotoxicity of GNPs in Bone Marrow Mesenchymal Stem Cell (BMSCs) was lower than cancerous cells. At nontoxic concentrations, the cellular up-take of the nanoparticles took place. Acridin orange and DAPI staining showed morphological changes in the cell’s nucleus due to apoptosis. Finally, caspase activity assay demonstrated HeLa cell’s apoptosis through caspase activation. Conclusion: The results showed that GNPs have the ability to induce apoptosis in HeLa cells. PMID:27141266

  17. Expression of aggregative adherence to hela cells by Escherichia coli strains isolated from sick horses Expressão de aderência agregativa em células HeLa por amostras de E. coli isoladas de eqüinos doentes

    Ana Maria Alvim Liberatore

    2007-03-01

    Full Text Available The virulence attributes of 56 Escherichia coli strains isolated from sick horses (secretions of uterine cervices; gastrointestinal and lung fragments of necropsy; diarrheic feces, and tracheal washings was examined by determining their adherence pattern to HeLa cells and searching for the presence of virulence genes of the various E. coli pathotypes. Two non-adherent strains presented astA, which encodes the enteroaggregative E. coli heat-stable toxin. Twenty-seven strains (48.2% adhered to HeLa cells, 21 (77.8% of which presented the aggregative adherence pattern (AA that characterize the Enteroaggregative E. coli pathotype (EAEC. Nine of the strains presenting AA were isolated from secretions of uterine cervix, including one carrying virulence genes of the EAEC pathotype (aggR,aap,irp2, and pic. This is the first description of the AA phenotype amongst E. coli strains from sick horses. Such strains should be further evaluated regarding their potential role in the pathogenesis of diverse equine diseases and as reservoirs of human infections.Características de virulência de 56 amostras de Escherichia coli isoladas de eqüinos doentes (secreção de colo uterino, fragmentos de necrópsia do trato gastrointestinal e de pulmões, fezes diarréicas e lavado traqueal foram examinadas para determinar o padrão de aderência em células HeLa e pesquisar a presença de genes de virulência de vários patotipos de E. coli. Duas amostras não aderentes apresentaram astA, gene que codifica a toxina termo-estável de E. coli enteroagregativa. Das vinte e sete amostras (48,2% que aderiram a células HeLa, 21 (77,8% apresentaram o padrão de aderência agregativa (AA que caracteriza o patotipo de E. coli Enteroagregativa (EAEC. Nove destas amostras que apresentaram AA foram isoladas de secreção de colo uterino, incluindo uma que apresentava genes de virulência de patotipos de EAEC (aggR,aap,irp2 e pic. Esta é a primeira descrição do fenótipo AA em

  18. Recombinant adeno-associated virus 2-mediated transfer of the human superoxide-dismutase gene does not confer radioresistance on HeLa cervical carcinoma cells

    Background and purpose: The success rate of any therapeutic approach depends on the therapeutic window, which can be increased by either raising the resistance of the normal tissue without protecting the tumor cells or by sensitizing the tumor cells but not the normal cells. Two promising candidate genes for normal tissue protection against radiation-induced damage may be the copper-zinc (CuZnSOD) and manganese superoxide-dismutase genes (MnSOD). The recombinant adeno-associated virus 2 (rAAV-2) offers attractive advantages over other vector systems: low immunogenicity, ability to infect dividing and non-dividing tissues and a low chance of insertional mutagenesis, due to extra-chromosomal localization. We report the production of novel rAAV-2-SOD vectors and the investigation of their modulating effects on HeLa-RC cells after irradiation. Material and methods: rAAV-2 vectors were cloned containing the human CuZnSOD or MnSOD as transgene and vector stocks were produced. In the initial experiments human cervix carcinoma (HeLa-RC) cells were chosen for their susceptibility to rAAV-2. On day 0, cells were seeded and transduced with the rAAV-2-SOD vectors. On day 3, cells were harvested, irradiated (0.5-8 Gy) and reseeded in different assays (FACS, SOD, MTT and colony assays). Results: Although >70% of all cells expressed SOD and significant amounts of functional SOD protein were detected, no radioprotective effect of SOD was observed after transduction of HeLa-RC cells. Conclusions: Novel rAAV-2-SOD vectors that could be produced at high titer, were able to efficiently infect cells and express the SOD genes. The absence of a radioprotective effect in HeLa-RC cancer cells indicates an additional safety feature and suggests that rAAV-mediated MnSOD overexpression might contribute to increasing the therapeutic index when applied for normal tissue protection

  19. Radiobiological characteristics of cancer stem cells from esophageal cancer cell lines

    Wang, Jian-Lin; Yu, Jing-Ping; Zhi-qiang SUN; Sun, Su-Ping

    2014-01-01

    AIM: To study the cancer stem cell population in esophageal cancer cell lines KYSE-150 and TE-1 and identify whether the resulting stem-like spheroid cells display cancer stem cells and radiation resistance characteristics.

  20. Chemotherapy targeting cancer stem cells

    Liu, Haiguang; Lv, Lin; Yang, Kai

    2015-01-01

    Conventional chemotherapy is the main treatment for cancer and benefits patients in the form of decreased relapse and metastasis and longer overall survival. However, as the target therapy drugs and delivery systems are not wholly precise, it also results in quite a few side effects, and is less efficient in many cancers due to the spared cancer stem cells, which are considered the reason for chemotherapy resistance, relapse, and metastasis. Conventional chemotherapy limitations and the cance...

  1. Single cancer cell analysis on a chip

    Yang, Yoonsun

    2016-01-01

    Cancer cells in blood may represent “a real time liquid biopsy” through the interrogation of single cancer cells thereby determining the outspread of their heterogeneity and guiding therapy. In this thesis, we focused on single cancer cell analysis downstream of the isolation of cancer cells from blood. We designed and developed various microfluidic devices for genetic and phenotypic characterization of single cancer cells. The limited DNA content in a single cell requires DNA amplification t...

  2. Cell of origin of lung cancer

    Hanna, Jennifer M.; Onaitis, Mark W.

    2013-01-01

    Lung cancer is the leading cause of cancer deaths worldwide, and current therapies are disappointing. Elucidation of the cell(s) of origin of lung cancer may lead to new therapeutics. In addition, the discovery of putative cancer-initiating cells with stem cell properties in solid tumors has emerged as an important area of cancer research that may explain the resistance of these tumors to currently available therapeutics. Progress in our understanding of normal tissue stem cells, tumor cell o...

  3. Head and Neck Cancer Stem Cells

    Krishnamurthy, S.; Nör, J.E.

    2012-01-01

    Most cancers contain a small sub-population of cells that are endowed with self-renewal, multipotency, and a unique potential for tumor initiation. These properties are considered hallmarks of cancer stem cells. Here, we provide an overview of the field of cancer stem cells with a focus on head and neck cancers. Cancer stem cells are located in the invasive fronts of head and neck squamous cell carcinomas (HNSCC) close to blood vessels (perivascular niche). Endothelial cell-initiated signalin...

  4. Proteasome inhibition mediates p53 reactivation and anti-cancer activity of 6-Gingerol in cervical cancer cells

    Rastogi, Namrata; Duggal, Shivali; Singh, Shailendra Kumar; Porwal, Konica; Srivastava, Vikas Kumar; Maurya, Rakesh; Bhatt, Madan L.B.; Mishra, Durga Prasad

    2015-01-01

    Human papilloma virus (HPV) expressing E6 and E7 oncoproteins, is known to inactivate the tumor suppressor p53 through proteasomal degradation in cervical cancers. Therefore, use of small molecules for inhibition of proteasome function and induction of p53 reactivation is a promising strategy for induction of apoptosis in cervical cancer cells. The polyphenolic alkanone, 6-Gingerol (6G), present in the pungent extracts of ginger (Zingiber officinale Roscoe) has shown potent anti-tumorigenic and pro-apoptotic activities against a variety of cancers. In this study we explored the molecular mechanism of action of 6G in human cervical cancer cells in vitro and in vivo. 6G potently inhibited proliferation of the HPV positive cervical cancer cells. 6G was found to: (i) inhibit the chymotrypsin activity of proteasomes, (ii) induce reactivation of p53, (iii) increase levels of p21, (iv) induce DNA damage and G2/M cell cycle arrest, (v) alter expression levels of p53-associated apoptotic markers like, cleaved caspase-3 and PARP, and (vi) potentiate the cytotoxicity of cisplatin. 6G treatment induced significant reduction of tumor volume, tumor weight, proteasome inhibition and p53 accumulation in HeLa xenograft tumor cells in vivo. The 6G treatment was devoid of toxic effects as it did not affect body weights, hematological and osteogenic parameters. Taken together, our data underscores the therapeutic and chemosensitizing effects of 6G in the management and treatment of cervical cancer. PMID:26621832

  5. Triplex forming oligonucleotide targeted to 3′UTR downregulates the expression of the bcl-2 proto-oncogene in HeLa cells

    Shen, Changxian; Buck, Andreas; Mehrke, Gerhard; Polat, Bülent; Gross, Hans-Jügen; Bachem, Max; Reske, Sven

    2001-01-01

    The bcl-2 proto-oncogene is overexpressed in a variety of human cancers and plays an important role in programmed cell death. Recent reports implied that the 3′-untranslated region (3′UTR) functions effectively in the regulation of gene expression. Here, we attempt to assay the ability of triplex forming oligonucleotides (TFOs) to inhibit expression of a target gene in vivo and to examine the potential of the 3′UTR of the bcl-2 proto-oncogene in the regulati...

  6. Proteomic analysis of cervical cancer cells treated with suberonylanilide hydroxamic acid

    Jianxiong He; Canhua Huang; Aiping Tong; Bin Chen; Zhi Zeng; Peng Zhang; Chunting Wang; Yuquan Wei

    2008-12-01

    Suberonylanilide hydroxamic acid (SAHA) is an orally administered histone deacetylase inhibitor (HDACI) that has shown significant antitumour activity in a variety of tumour cells. To identify proteins involved in its antitumour activity, we utilized a proteomic approach to reveal protein expression changes in the human cervical cancer cell line HeLa following SAHA treatment. Protein expression profiles were analysed by 2-dimensional polyacrylamide gel electrophoresis (2-DE) and protein identification was performed on a MALDI-Q-TOF MS/MS instrument. As a result, a total of nine differentially expressed proteins were visualized by 2-DE and Coomassie brilliant blue (CBB) staining. Further, all the changed proteins were positively identified via mass spectrometry (MS)/MS analysis. Of these, PGAM1 was significantly downregulated in HeLa cells after treatment with SAHA. Moreover, PGAM1 has been proven to be downregulated in another cervical cancer cell line (CaSki) by western blot analysis. Together, using proteomic tools, we identified several differentially expressed proteins that underwent SAHA-induced apoptosis. These changed proteins may provide some clues to a better understanding of the molecular mechanisms underlying SAHA-induced apoptosis in cervical cancer.

  7. Introduction of optical reporter gene into cancer and immune cells using lentiviral vector

    For some applications such as gene therapy or reporter gene imaging, a gene has to be introduced into the organism of interest. Adenoviral vectors are capable of transducing both replicating and non-dividing cells. The adenoviral vectors do not integrate their DNA into host DNA, but do lead to an immune response. Lentiviruses belong to the retrovirus family and are capable of infecting both dividing and non-dividing cells. The human immunodeficiency virus (HIV) is an example of a lentavirus. A disabled HIV virus has been developed and could be used for in vivo gene delivery. A portion of the viral genome which encodes for accessory proteins canbe deleted without affecting production of the vector and efficiency of infection. Lentiviral delivery into various rodent tissues shows sustained expression of the transgene of up to six months. Furthermore, there seems to be little or no immune response with these vectors. These lentiviral vectors hold significant promise for in vivo gene delivery. We constructed lentiviral vector encoding firefly luciferase (Fluc) and eGFP. Fluc-eGFP fusion gene was inserted into multiple cloning sites of pLentiM1.3 vector. Reporter gene (Fluc-eGFP) was designed to be driven by murine CMV promoter with enhanced efficacy of transgene expression as compared to human CMV promoter. We transfected pLenti1.3-Fluc into human cervix cancer cell line (HeLa) and murine T lymphocytes. We also constructed adenovirus encoding Fluc and transfected to HeLa and T cells. This LentiM1.3-Fluc was transfected into HeLa cells and murine T lymphocytes in vitro, showing consistent expression of eGFP under the fluorescence microscopy from the 2nd day of transfection. Firefly luciferase reporter gene was not expressed in immune cells when it is mediated by adenovirus. Lentivirus was validated as a useful vector for both immune and cancer cells

  8. Introduction of optical reporter gene into cancer and immune cells using lentiviral vector

    Min, Jung Joon; Le, Uyenchi N.; Moon, Sung Min; Heo, Young Jun; Song, Ho Chun; Bom, Hee Seung [School of Medicine, Chonnam National University, Gwangju (Korea, Republic of); Kim, Yeon Soo [Schoole of Medicine, Inje University, Seoul (Korea, Republic of)

    2004-07-01

    For some applications such as gene therapy or reporter gene imaging, a gene has to be introduced into the organism of interest. Adenoviral vectors are capable of transducing both replicating and non-dividing cells. The adenoviral vectors do not integrate their DNA into host DNA, but do lead to an immune response. Lentiviruses belong to the retrovirus family and are capable of infecting both dividing and non-dividing cells. The human immunodeficiency virus (HIV) is an example of a lentavirus. A disabled HIV virus has been developed and could be used for in vivo gene delivery. A portion of the viral genome which encodes for accessory proteins canbe deleted without affecting production of the vector and efficiency of infection. Lentiviral delivery into various rodent tissues shows sustained expression of the transgene of up to six months. Furthermore, there seems to be little or no immune response with these vectors. These lentiviral vectors hold significant promise for in vivo gene delivery. We constructed lentiviral vector encoding firefly luciferase (Fluc) and eGFP. Fluc-eGFP fusion gene was inserted into multiple cloning sites of pLentiM1.3 vector. Reporter gene (Fluc-eGFP) was designed to be driven by murine CMV promoter with enhanced efficacy of transgene expression as compared to human CMV promoter. We transfected pLenti1.3-Fluc into human cervix cancer cell line (HeLa) and murine T lymphocytes. We also constructed adenovirus encoding Fluc and transfected to HeLa and T cells. This LentiM1.3-Fluc was transfected into HeLa cells and murine T lymphocytes in vitro, showing consistent expression of eGFP under the fluorescence microscopy from the 2nd day of transfection. Firefly luciferase reporter gene was not expressed in immune cells when it is mediated by adenovirus. Lentivirus was validated as a useful vector for both immune and cancer cells.

  9. Ca2+-mediated potentiation of the swelling-induced taurine efflux from HeLa cells: On the role of calmodulin and novel protein kinase C isoforms

    Falktoft, Birgitte; Lambert, Ian H.

    2004-01-01

    The present work sets out to investigate how Ca2+ regulates the volume-sensitive taurine-release pathway in HeLa cells. Addition of Ca2+-mobilizing agonists at the time of exposure to hypotonic NaCl medium augments the swelling-induced taurine release and subsequently accelerates the inactivation...... of the release pathway. The accelerated inactivation is not observed in hypotonic Ca2+-free or high-K+ media. Addition of Ca2+-mobilizing agonists also accelerates the regulatory volume decrease, which probably reflects activation of Ca2+-activated K+ channels. The taurine release from control cells...... and cells exposed to Ca2+ agonists is equally affected by changes in cell volume, application of DIDS and arachidonic acid, indicating that the volume-sensitive taurine leak pathway mediates the Ca2+-augmented taurine release. Exposure to Ca2+-mobilizing agonists prior to a hypotonic challenge also...

  10. Cancer Stem Cell Hypothesis: Implication for Cancer Prevention and Treatment

    Anna Meiliana; Nurrani Mustika Dewi; Andi Wijaya

    2016-01-01

    BACKGROUND: Cancer is a disease of genomic instability, evasion of immune cells, and adaptation of the tumor cells to the changing environment. Genetic heterogeneity caused by tumors and tumor microenvironmental factors forms the basis of aggressive behavior of some cancer cell populations. CONTENT: Cancers arise in self-renewing cell populations and that the resulting cancers, like their normal organ counterparts, are composed of hierarchically organized cell populations. Self–renewing “...

  11. Growth dynamics of cancer cell colonies and their comparison with noncancerous cells

    Huergo, M. A. C.; Pasquale, M. A.; González, P. H.; Bolzán, A. E.; Arvia, A. J.

    2012-01-01

    The two-dimensional (2D) growth dynamics of HeLa (cervix cancer) cell colonies was studied following both their growth front and the pattern morphology evolutions utilizing large population colonies exhibiting linearly and radially spreading fronts. In both cases, the colony profile fractal dimension was df=1.20±0.05 and the growth fronts displaced at the constant velocity 0.90±0.05 μm min-1. Colonies showed changes in both cell morphology and average size. As time increased, the formation of large cells at the colony front was observed. Accordingly, the heterogeneity of the colony increased and local driving forces that set in began to influence the dynamics of the colony front. The dynamic scaling analysis of rough colony fronts resulted in a roughness exponent α = 0.50±0.05, a growth exponent β = 0.32±0.04, and a dynamic exponent z=1.5±0.2. The validity of this set of scaling exponents extended from a lower cutoff lc≈60 μm upward, and the exponents agreed with those predicted by the standard Kardar-Parisi-Zhang continuous equation. HeLa data were compared with those previously reported for Vero cell colonies. The value of df and the Kardar-Parisi-Zhang-type 2D front growth dynamics were similar for colonies of both cell lines. This indicates that the cell colony growth dynamics is independent of the genetic background and the tumorigenic nature of the cells. However, one can distinguish some differences between both cell lines during the growth of colonies that may result from specific cooperative effects and the nature of each biosystem.

  12. Prostate Cancer Stem Cells: Research Advances

    Dagmara Jaworska; Wojciech Król; Ewelina Szliszka

    2015-01-01

    Cancer stem cells have been defined as cells within a tumor that possesses the capacity to self-renew and to cause the heterogeneous lineages of cancer cells that comprise the tumor. Experimental evidence showed that these highly tumorigenic cells might be responsible for initiation and progression of cancer into invasive and metastatic disease. Eradicating prostate cancer stem cells, the root of the problem, has been considered as a promising target in prostate cancer treatment to improve th...

  13. A role for PP1/NIPP1 in steering migration of human cancer cells.

    Cristina Martin-Granados

    Full Text Available Electrical gradients are present in many developing and regenerating tissues and around tumours. Mimicking endogenous electric fields in vitro has profound effects on the behaviour of many cell types. Intriguingly, specific cell types migrate cathodally, others anodally and some polarise with their long axis perpendicular to the electric vector. These striking phenomena are likely to have in vivo relevance since one of the determining factors during cancer metastasis is the ability to switch between attractive and repulsive migration in response to extracellular guidance stimuli. We present evidence that the cervical cancer cell line HeLa migrates cathodally in a direct current electric field of physiological intensity, while the strongly metastatic prostate cancer cell line PC-3-M migrates anodally. Notably, genetic disruption of protein serine/threonine phosphatase-1 (PP1 and its regulator NIPP1 decrease directional migration in these cell lines. Conversely, the inducible expression of NIPP1 switched the directional response of HeLa cells from cathodal to slightly anodal in a PP1-dependent manner. Remarkably, induction of a hyperactive PP1/NIPP1 holoenzyme, further shifted directional migration towards the anode. We show that PP1 association with NIPP1 upregulates signalling by the GTPase Cdc42 and demonstrate that pharmacological inhibition of Cdc42 in cells overexpressing NIPP1 recovered cathodal migration. Taken together, we provide the first evidence for regulation of directional cell migration by NIPP1. In addition, we identify PP1/NIPP1 as a novel molecular compass that controls directed cell migration via upregulation of Cdc42 signalling and suggest a way by which PP1/NIPP1 may contribute to the migratory properties of cancer cells.

  14. Cytotoxicity and Antitumor Properties of a Marine Compound , HESA-A , on Cancer Cells.

    Hojjat Sadeghi- Aliabadi

    2003-09-01

    Full Text Available Majority of the currently available anticancer drugs are designed to have selective toxicity to rapidly dividing cells. Among these agents the focus of many studies are compounds obtained from natural products with high therapeutic index. In this study the cytotoxicity of HESA-A, a marine compound, on cancer and normal cells was evaluated. HESA-A was prepared in normal saline as a stock solution (0.8 mg/ml, pH=7.4, sterilized and further diluted to final concentrations of 0.4, 0.2, 0.1 and 0.05 mg/ml. Cells (MDA-MB-468, Hep-2, Hela as cancer cells; L929 and McCoy as normal cells were grown in completed RPMI 1640 and seeded in 96 well micro plates at a concentration of 1-5 ´ 104 cells/ml. After incubation for 24 h, different concentrations of HESA-A were added and cells were further incubated for 72 h. Using MTT assay, percent cell survival was determined by ELISA at 540 nm. Doxorubicin was used as a positive control (20 mg /ml. HESA-A (0.4 mg/ml reduced the number of viable MDA-MB-468 and Hela cells to less than 50%. For Hep-2 cells the IC50 was 0.8 mg/ml. In normal cells IC50 could not be obtained at any given concentrations. These results suggest that HESA-A in therapeutic doses and in a concentration dependent manner inhibits the growth of cancer cells more selectively than normal cells.

  15. Fluorescent Cy5 silica nanoparticles for cancer cell imaging

    O'Connell, Claire; Nooney, Robert I.; Glynn, MacDara; Ducree, Jens; McDonagh, Colette

    2015-08-01

    Cancer is a leading cause of death worldwide, with metastasis responsible for the majority of cancer-related deaths. Circulating tumour cells (CTCs) play a central role in metastasis. Fluorescent silica particles (NPs), of diameter ~50 nm which contain a large concentration of Cy5 dye molecules and are extremely bright, have been developed to detect these rare CTCs. Due to this brightness, the particles have superior performance compared to single Cy5 dye molecule labels, for detecting cancer cells. Fluorescence measurements show that the NPs are almost 100 times brighter than the free dye. They do not photo bleach as readily and, due to the biocompatible silica surface, they can be chemically modified, layer-by-layer, in order to bind to cells. The choice of these chemical layers, in particular the NP to antibody linker, along with the incubation period and type of media used in the incubation, has a strong influence on the specific binding abilities of the NPs. In this work, NPs have been shown to selectively bind to the MCF-7 cell line by targeting epithelial cellular adhesion molecule (EpCAM) present on the MCF-7 cell membrane by conjugating anti-EpCAM antibody to the NP surface. Results have shown a high signal to noise ratio for this cell line in comparison to a HeLa control line. NP attachment to cells was verified qualitatively with the use of fluorescence microscopy and quantitatively using image analysis methods. Once the system has been optimised, other dyes will be doped into the silica NPs and their use in multiplexing will be investigated.

  16. Stem cells in human breast cancer

    Roberto Oliveira, Lucinei; Jeffrey, Stefanie S; Ribeiro Silva, Alfredo

    2010-01-01

    Increasing data support cancer as a stem cell-based disease. Cancer stem cells (CSCs) have beenfound in different human cancers, and recent evidenceindicates that breast cancer originates from and ismaintained by its own CSCs, as well as the normalmammary gland. Mammary stem cells and breast CSCshave been identified and purified in in vitroculturesystems, transplantation assays and/or by cell surfaceantigen identification. Cell surface markers enable thefunctional isolation of stem cells that...

  17. Targeting SPARC by lentivirus-mediated RNA interference inhibits cervical cancer cell growth and metastasis

    Chen Jie

    2012-10-01

    Full Text Available Abstract Background Secreted protein acidic and rich in cysteine (SPARC, a calcium-binding matricellular glycoprotein, is implicated in the progressions of some cancers. However, no information has been available to date regarding the function of SPARC in cervical cancer cell growth and metastasis. Methods In this study, we isolated and established high invasive subclones and low invasive subclones from human cervical cancer cell lines HeLa and SiHa by the limited dilution method. Real-time q-RT-PCR, Western Blot and ICC were performed to investigate SPARC mRNA and protein expressions in high invasive subclones and low invasive subclones. Then lentivirus vector with SPARC shRNA was constructed and infected the highly invasive subclones. Real-time q-RT-PCR, Western Blot and ICC were also pe