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Sample records for cancer hela cells

  1. Amygdalin induces apoptosis in human cervical cancer cell line HeLa cells.

    Chen, Yu; Ma, Jinshu; Wang, Fang; Hu, Jie; Cui, Ai; Wei, Chengguo; Yang, Qing; Li, Fan

    2013-02-01

    Amygdalin, a naturally occurring substance, has been suggested to be efficacious as an anticancer substance. The effect of amygdalin on cervical cancer cells has never been studied. In this study, we found that the viability of human cervical cancer HeLa cell line was significantly inhibited by amygdalin. 4,6-Diamino-2-phenyl indole (DAPI) staining showed that amygdalin-treated HeLa cells developed typical apoptotic changes. The development of apoptosis in the amygdalin-treated HeLa cells were confirmed by double staining of amygdalin-treated HeLa cells with annexin V-FITC and propidium iodide (PI) along with increase in caspase-3 activity in these cells. Further studies indicated that antiapoptotic protein Bcl-2 was downregulated whereas proapoptotic Bax protein was upregulated in the amygdalin-treated HeLa cells implying involvement of the intrinsic pathway of apoptosis. In vivo, amygdalin administration inhibited the growth of HeLa cell xenografts through a mechanism of apoptosis. The results in the present study suggest that amygdalin may offer a new therapeutic option for patients with cervical cancer.

  2. Molecular mechanism of Skp2 in promoting cervical cancer HeLa cell proliferation

    2008-01-01

    Objective: To explore the impact of s-phase kinase-associated protein 2 (Skp2) on cervical cancer cell proliferation and the relationship between Skp2 and expression of cell regulation factors and transcription factors. Methods: RNAi technology was used to silence Skp2 gene in HeLa cells. After interference, RT-PCR was used for detection of Skp-2 mRNA, and Western blotting and flow cytometry were used for protein expression analysis. Results: siRNA significantly inhibited HeLa cell proliferation (P<0.05) and increased HeLa apoptosis, and G1/G0 phase cells were increased significantly (P<0.01). Skp2 siRNA transfected HeLa cells effectively reduced Skp2 protein levels, while p27 and p-p53 protein levels were increased significantly. RT-PCR results showed that after interference Skp2 mRNA, c-myc mRNA and cyclin A mRNA expressions decreased significantly compared with those in control group (P<0.01), and p27mRNA expression level was significantly higher (P<0.01). Conclusion: The change of Skp2 expression affects the expression of the cell cycle protein, thus affecting proliferation and apoptosis of HeLa cells. Skp2 protein plays an important role in the progression of cervical cancer; yet the specific mechanism still needs further study.

  3. Baicalein induces apoptosis of human cervical cancer HeLa cells in vitro.

    Peng, Yong; Guo, Congshan; Yang, Yanhong; Li, Fenglin; Zhang, Yanxia; Jiang, Bin; Li, Qingwang

    2015-03-01

    A number of studies have shown that baicalein shows high antitumor activity in vitro and in vivo. In this study, the inhibitory effect of baicalein on human cervical cancer HeLa cells was studied in vitro. HeLa cells were treated with high (100 µg/ml) and low (50 µg/ml) doses of baicalein, and cell growth inhibition rates were examined by the MTT assay. The morphological changes of apoptotic cells were observed under the light and electron microscope, while the rate of cell apoptosis was examined by flow cytometry. The expression of apoptosis-related proteins was analyzed by western blot, and caspase-3 activation was examined by a caspase-3 activity assay and spectrophotometry. The results demonstrated that baicalein inhibits the proliferation of HeLa cells and induces apoptosis in a caspase-3-dependent pathway, through downregulation of the B-cell lymphoma 2 (Bcl-2) protein and upregulation of the Bcl-2-associated X protein (Bax), Fas, Fas ligand (FasL) and caspase-8. Thus, we conclude that baicalein induces apoptosis of HeLa cells via the mitochondrial and the death receptor pathways. Cell apoptosis in HeLa cells was most likely promoted by the activation of the proteolytic enzyme caspase-3 in both pathways.

  4. Effects of TGF-β1 on the Proliferation and Apoptosis of Human Cervical Cancer Hela Cells In Vitro.

    Tao, Ming-Zhu; Gao, Xia; Zhou, Tie-Jun; Guo, Qing-Xi; Zhang, Qiang; Yang, Cheng-Wan

    2015-12-01

    To investigate the effects of TGF-β1 on the proliferation and apoptosis of cervical cancer Hela cells in vitro. Human cervical cancer Hela cells were cultured in vitro and divided into the experimental and control groups. In the experimental groups, Hela cells were stimulated with different concentrations of TGF-β1 (0.01, 0.1, 1, and 10 ng/mL), while Hela cells cultured in serum-free medium without TGF-β1 were used as controls. The CCK8 method was adopted to detect the effect of TGF-β1 on Hela cell proliferation, and flow cytometry was used to determine cell apoptosis 72 h after TGF-β1 treatment. Compared with the control group, the CCK-8 tests showed that different concentrations of TGF-β1 had no obvious effect on Hela cell proliferation 24 h after treatment (P > 0.05). However, upon 48 or 72 h of treatment, TGF-β1 significantly inhibited the proliferation of Hela cells in a time- and dose-dependent manner (P Hela cells in a dose-dependent manner after 72 h of treatment (P Hela cells in vitro.

  5. Kelussia odoratissima potentiates cytotoxic effects of radiation in HeLa cancer cell line

    Azar Hosseini

    2017-02-01

    Full Text Available Objective: Cervical cancer is the second most common cause of death from cancer in women throughout the world. The aim of this study was to evaluate the cytotoxic activity of Kelussia odoratissima (K. odoratissima extract associated with radiotherapy in cervical cancer cells (HeLa cell line.Materials and Methods: Different concentration of the extract (25-500µg/ml was tested in HeLa cell lines. Cell cytotoxicity of the extract and the effects of the extract on radiation (2Gy/min-induced damages were assessed by MTT assay. Apoptosis was assessed using flow cytometric analysis.Result: K. odoratissima decreased cell viability in HeLa cell line in a concentration and time-dependent manner. When compared to the control,K. odoratissima induced a sub-G1 peak in the flow cytometry histogram of treated cells, indicating that apoptotic cell death is involved in K. odoratissima-induced toxicity. It was also shown that K. odoratissima sensitizes cells to radiation-induced toxicity.Conclusion: Our result showed the extract increased the radiation effect. This observation may be related to the presence of active compounds such as phthalides and ferulic acid.

  6. Kelussia odoratissima potentiates cytotoxic effects of radiation in HeLa cancer cell line

    Hosseini, Azar; Saeidi Javadi, Shima; Fani-Pakdel, Azar; Mousavi, Seyed Hadi

    2017-01-01

    Objective: Cervical cancer is the second most common cause of death from cancer in women throughout the world. The aim of this study was to evaluate the cytotoxic activity of Kelussia odoratissima (K. odoratissima) extract associated with radiotherapy in cervical cancer cells (HeLa cell line). Materials and Methods: Different concentration of the extract (25-500µg/ml) was tested in HeLa cell lines. Cell cytotoxicity of the extract and the effects of the extract on radiation (2Gy/min)-induced damages were assessed by MTT assay. Apoptosis was assessed using flow cytometric analysis. Result: K. odoratissima decreased cell viability in HeLa cell line in a concentration and time-dependent manner. When compared to the control, K. odoratissima induced a sub-G1 peak in the flow cytometry histogram of treated cells, indicating that apoptotic cell death is involved in K. odoratissima-induced toxicity. It was also shown that K. odoratissima sensitizes cells to radiation-induced toxicity. Conclusion: Our result showed the extract increased the radiation effect. This observation may be related to the presence of active compounds such as phthalides and ferulic acid.

  7. Proteomic investigation into betulinic acid-induced apoptosis of human cervical cancer HeLa cells.

    Xu, Tao; Pang, Qiuying; Zhou, Dong; Zhang, Aiqin; Luo, Shaman; Wang, Yang; Yan, Xiufeng

    2014-01-01

    Betulinic acid is a pentacyclic triterpenoid that exhibits anticancer functions in human cancer cells. This study provides evidence that betulinic acid is highly effective against the human cervical cancer cell line HeLa by inducing dose- and time-dependent apoptosis. The apoptotic process was further investigated using a proteomics approach to reveal protein expression changes in HeLa cells following betulinic acid treatment. Proteomic analysis revealed that there were six up- and thirty down-regulated proteins in betulinic acid-induced HeLa cells, and these proteins were then subjected to functional pathway analysis using multiple analysis software. UDP-glucose 6-dehydrogenase, 6-phosphogluconate dehydrogenase decarboxylating, chain A Horf6-a novel human peroxidase enzyme that involved in redox process, was found to be down-regulated during the apoptosis process of the oxidative stress response pathway. Consistent with our results at the protein level, an increase in intracellular reactive oxygen species was observed in betulinic acid-treated cells. The proteins glucose-regulated protein and cargo-selection protein TIP47, which are involved in the endoplasmic reticulum pathway, were up-regulated by betulinic acid treatment. Meanwhile, 14-3-3 family proteins, including 14-3-3β and 14-3-3ε, were down-regulated in response to betulinic acid treatment, which is consistent with the decrease in expression of the target genes 14-3-3β and 14-3-3ε. Furthermore, it was found that the antiapoptotic bcl-2 gene was down-regulated while the proapoptotic bax gene was up-regulated after betulinic acid treatment in HeLa cells. These results suggest that betulinic acid induces apoptosis of HeLa cells by triggering both the endoplasmic reticulum pathway and the ROS-mediated mitochondrial pathway.

  8. HeLa human cervical cancer cell migration is inhibited by treatment with dibutyryl-cAMP.

    Lee, Jae-Wook; Lee, Jiyoung; Moon, Eun-Yi

    2014-07-01

    Cyclic AMP (cAMP) activates both protein kinase A (PKA) and guanine-nucleotide exchange factor exchange protein directly activated by CAMP (EPAC)-mediated Ras-related Protein1 (RAP1) GTPase that regulates various cellular functions including cell migration. Herein, we investigated whether cAMP-mediated PKA and EPAC1/RAP1 pathways differentially control HeLa cervical cancer cell migration. Although HeLa cell migration was reduced by dibutyryl-cAMP, we observed an increase in cAMP/PKA, cAMP/EPAC1/RAP1-GTPase, and RAC1-GTPase. HeLa cell migration and RAC1-GTPase were increased by treatment with 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cAMP analogue to activate EPAC-specific signaling pathways. When HeLa cells were treated with H-89, a PKA inhibitor, cell migration was enhanced but RAC1-GTPase was inhibited. In addition, cell migration induced by dibutyryl-cAMP was reversed but the activity of Rac1-GTPase was inhibited by H-89 treatment. Taken together, these data demonstrate that cAMP/PKA and cAMP/EPAC1/RAP1-GTPase might inversely control cervical cancer cell migration, although both signaling pathways may up-regulate RAC1-GTPase. It also suggests that cAMP-mediated cancer cell migration was independent of RAC1-GTPase activation.

  9. Growth inhibition and induction of apoptosis in human cancerous HeLa cells by Maytenus procumbens.

    Momtaz, S; Hussein, A A; Ostad, S N; Abdollahi, M; Lall, N

    2013-01-01

    The possible biochemical activities of the acetonic/ethanolic extract of the leaves of Maytenus procumbens (L.M.P), and its isolated compounds were investigated in the present study. In cytotoxicity assay, L.M.P showed IC(50) of 68.79, 51.22, 78.49, 76.59, and 76.64μg/ml on Caco-2, HeLa, HT29, NIH3T3, and T47D cells, respectively. Bioassay guided fractionation led to the isolation and identification of a new triterpene: '30-hydroxy-11α-methoxy-18β-olean-12-en-3-one' (HMO) in addition to a known terpenoid: 'asiatic acid' (AA). HMO exhibited the most cytotoxicity against HeLa cells and was further investigated for its ability to induce apoptosis in HeLa cells. HMO induced apoptosis up to 20.41% in HeLa cells versus control group (0.40%). Antioxidant/oxidative properties of L.M.P and HMO were investigated using extracellular (DPPH), and intracellular (ROS) assays. Experimental samples represented a time and concentration-dependent formation of ROS in Hela cells. Generation of ROS seems one of the mechanisms by which HMO induces apoptosis in Hela cells. Conclusion is that the active components in L.M.P might serve as a mediator of the ROS scavenging system and have the potential to act as prooxidant or antioxidant depending on the biological environment of the cells.

  10. Validation of a Strategy for Cancer Therapy: Delivering Aminoglycoside Drugs to Mitochondria in HeLa Cells.

    Abe, Jiro; Yamada, Yuma; Harashima, Hideyoshi

    2016-02-01

    Mitochondria in human cancer cells have been implicated in cancer cell proliferation, invasion, metastasis, and even drug-resistance mechanisms, making them a potential target organelle for the treatment of human malignancies. Gentamicin (GM), an aminoglycoside drug (AG), is a small molecule that functions as an antibiotic and has ototoxic and nephrotoxic characteristics. Thus, the delivery of GM to mitochondria in cancer cells would be an innovative anticancer therapeutic strategy. In this study, we attempted mitochondrial delivery of GM in HeLa cells derived from a human cervical cancer. For the mitochondrial delivery, we used MITO-Porter, a liposomal nanocarrier for mitochondrial delivery via membrane fusion. We first encapsulated GM in the aqueous phase of the carrier to construct GM-MITO-Porter. Flow cytometry analysis and fluorescent microscopy observations permitted us to confirm that the GM-MITO-Porter was efficiently taken up by HeLa cells and accumulated in mitochondria, whereas naked GM was not taken up by the cells. Moreover, cell viability assays using HeLa cells showed that the GM-MITO-Porter induced strong cytotoxic effects related to mitochondrial disorder. This finding is the first report of the mitochondrial delivery of an AG to cancer cells for cancer therapeutic strategy.

  11. 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

  12. Curcumin-mediated decrease in the expression of nucleolar organizer regions in cervical cancer (HeLa) cells.

    Lewinska, Anna; Adamczyk, Jagoda; Pajak, Justyna; Stoklosa, Sylwia; Kubis, Barbara; Pastuszek, Paulina; Slota, Ewa; Wnuk, Maciej

    2014-09-01

    Curcumin, the major yellow-orange pigment of turmeric derived from the rhizome of Curcuma longa, is a highly pleiotropic molecule with the potential to modulate inflammation, oxidative stress, cell survival, cell secretion, homeostasis and proliferation. Curcumin, at relatively high concentrations, was repeatedly reported to be a potent inducer of apoptosis in cancer cells and thus considered a promising anticancer agent. In the present paper, the effects of low concentrations of curcumin on human cervical cancer (HeLa) cells were studied. We found curcumin-mediated decrease in the cell number and viability, and increase in apoptotic events and superoxide level. In contrast to previously shown curcumin cytotoxicity toward different cervical cancer lines, we observed toxic effects when even as low as 1 μM concentration of curcumin was used. Curcumin was not genotoxic to HeLa cells. Because argyrophilic nucleolar protein (AgNOR protein) expression is elevated in malignant cells compared to normal cells reflecting the rapidity of cancer cell proliferation, we evaluated curcumin-associated changes in size (area) and number of silver deposits. We showed curcumin-induced decrease in AgNOR protein pools, which may be mediated by global DNA hypermethylation observed after low concentration curcumin treatment. In summary, we have shown for the first time that curcumin at low micromolar range may be effective against HeLa cells, which may have implications for curcumin-based treatment of cervical cancer in humans.

  13. 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.

  14. Kaempferol increases apoptosis in human cervical cancer HeLa cells via PI3K/AKT and telomerase pathways.

    Kashafi, Elham; Moradzadeh, Maliheh; Mohamadkhani, Ashraf; Erfanian, Saiedeh

    2017-02-28

    Cervical cancer is one of the most frequent cancers in women worldwide. Defects in the apoptotic pathways are responsible for both the disease pathogenesis and its therapy resistance. It is thus a good candidate for treatment by pro-apoptotic agents. Kaempferol as a flavonoid has antioxidant and anti-tumor properties. Kaempferol has been shown to induce apoptosis and cell death in cancer cells. However, due to the problems in the treatment of cervical cancer, this study is designed to investigate the molecular mechanism by which kaempferol suppresses the growth of cervical cancer HeLa cell as compared with HFF cells (normal cells). Cells treated with kaempferol (12-100μM) and 5-FU (1-10μM), as the positive control, up to 72h. Cell viability was determined by MTT assay and real time PCR was used to investigate apoptosis and telomerase genes expression. The results showed that kaempferol decreased cell viability as concentration- and time-dependently. IC50 values were 10.48μM for HeLa and 707.00μM for HFF cells, as compared with 1.40μM and 16.38μM for 5-FU after 72h treatment, respectively. Also, kaempferol induced cellular apoptosis and aging through down-regulating the PI3K/AKT and hTERT pathways. This study suggests that kaempferol may be a useful adjuvant therapeutic agent in the treatment of cervical cancer.

  15. 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.

  16. Experimental Studies on Cyclooxygenase-2 Inhibitor Induced Cervical Cancer Hela Cell Apoptosis and Its Molecular Mechanism

    Ling YIN; Li-bei WEI; Qiu-hong QU; Xiao-peng GUO

    2007-01-01

    Objective To investigate the Hela cells growth inhibition and apoptosis possible molecular mechanisms.Methods Hela cells were treated with various concentrations(100 μmol/L,200 μmol/L,300μmol/L,400 μmol/L) ofNS-398 (selective for COX-2 inhibition). Cell growth was measured by MTT (Thiazolyl blue).Apoptosis was detected by double staining flow cytomezry (FCM).Levels of PGE2 were measured by radioimmunoassay.The expressions of COX-2 protein were also examined by Western blot analysis.Results After treated with different concentrations ofNS-398,the growth of Hela cells was suppressed significantly in a dose-and time-dependent manner (P<0. 01).The NS-398 can induce apoptosis with the apoptosis rates at 8.53%-43.46% by FCM in a dose-dependent manner.The release of PGE2 was reduced in Hela cells with the values of 69.26 ±2.13, 47.46 ±2.18,28.15 ± 1.64 and 17.01 ± 1.12,respectively,there was significant difference compared with control group (83.78 ± 1.11)(P<0. 01).The NS-398 could inhibit the activity and expression of COX-2 in a dosedependent manner and down-regulated the expression of COX-2 protein greatly.Conclusion NS-398 could inhibit the proliferation and increase apoptosis in human Hela cells.These effects may be depended on the inhibition of the expression of COX-2 and PGE2 by NS-398.

  17. Effects of Geldanamycin on Expression of Bcl-2 in Human Cervical Cancer HeLa Cells

    Xue Du; Ruoran Mi; Quanxin Qu; Ye Qu; Tianfu Yue

    2008-01-01

    OBJECTIVE Geldanamycin, a natural product of Streptomyces geldanus, binds the heat shock protein 90 (Hsp90), a cell chaperone protein that interacts with Bcl-2. In this study, we investigated whether geldanamycin (GA) inhibits proliferation of HeLa cells through induction of apoptosis by decreasing the level of Bcl-2expression.METHODS HeLa cells, a human cervical cell line, were cultured in vitro and treated with different concentrations of GA (0, 0.02, 0.2,2, 10 μmol/L) for 24 h. Or were treated for different lengths of time at a GA concentration of 10 μmol/L. Proliferation of the cells was analyzed by an MTT assay, and cell apoptosis was determined by staining the cells with annexin V. In addition, cellular mRNA levels for Bcl-2 and Hsp90 were determined by the semi-quantitative polymerase chain reaction (PCR), and the levels of Bcl-2 and Hsp90 protein expression were determined by Western blots.RESULTS Treatment of cells with GA was found to inhibit HeLa cell proliferation in a concentration and time-dependent manner. The inhibition was a result of increased cellular apoptotic levels. Further analyses showed that while the mRNA and protein expression levels of Hsp90 were-not affected, GA treatment significantly reduced the level of Bcl-2 mRNA and protein expression in a concentration-dependent manner that correlated with the observed inhibition of cell proliferation.CONCLUSION GA can inhibit proliferation and increase apoptosis of HeLa cells by decreasing the transcription and expression of an anti-apoptotic gene bcl-2, probably through interaction and functional inhibition of Hsp90.

  18. Forced expression of Nanog with mRNA synthesized in vitro to evaluate the malignancy of HeLa cells through acquiring cancer stem cell phenotypes.

    Ding, Yan; Yu, Ai Qing; Wang, Xiao Li; Guo, Xing Rong; Yuan, Ya Hong; Li, Dong Sheng

    2016-05-01

    Nanog is a pluripotency-related factor. It was also found to play an important role in tumorigenesis. To date, the mechanisms underlying cervical tumorigenesis still need to be elucidated. In the present study, Nanog mRNA was synthesized in vitro and transfected into HeLa cells. After mRNA transfection, the forced expressed of Nanog in HeLa cells led to markedly increased invasion, migration, resistance to chemotherapeutic agents and dedifferentiation. In a subcutaneous xenograft assay, these cells had significantly increased tumorigenic capacity. Real-time PCR indicated that Nanog‑induced dedifferentiation was associated with increased expression of endogenous Oct4, Sox2 and FoxD3. In addition, the dedifferentiated HeLa cells acquired features associated with cancer stem cells (CSCs), such as multipotent differentiation capacity, and expression of CSC markers such as CD133. These data imply that Nanog is a positive regulator of cervical cancer dedifferentiation.

  19. Anticancer activity of synthetic bis(indolyl)methane-ortho-biaryls against human cervical cancer (HeLa) cells.

    Jamsheena, Vellekkatt; Shilpa, Ganesan; Saranya, Jayaram; Harry, Nissy Ann; Lankalapalli, Ravi Shankar; Priya, Sulochana

    2016-03-05

    Bis(indolyl)methane appended biaryls were designed, synthesized and evaluated in human cervical cancer cell lines (HeLa) for their anticancer activities and compared against normal rat cardiac myoblasts (H9C2) cells. Compounds 1-12 were synthesized, with variations in one of the phenyl unit, in a single step by condensation of biaryl-2-carbaldehydes with indole in the presence of para-toluenesulfonic acid. Compound 1 exhibited a GI50 value of 11.00 ± 0.707 μM and the derivatives, compounds 4 and 11 showed a GI50 value of 8.33 ± 0.416 μM and 9.13 ± 0.177 μM respectively in HeLa cells and was found to be non-toxic to H9C2 cells up to 20 μM. Furthermore, compounds 1, 4 and 11 induced caspase dependent cellular apoptosis in a concentration-dependent manner, reduced mitochondrial membrane potential, inhibited the cell migration and downregulated the production of MMP-2 and MMP-9 in HeLa cells.

  20. Securinine from Phyllanthus glaucus Induces Cell Cycle Arrest and Apoptosis in Human Cervical Cancer HeLa Cells

    Krauze-Baranowska, Mirosława; Ochocka, J. Renata

    2016-01-01

    Background The Securinega-type alkaloids occur in plants belonging to Euphorbiaceae family. One of the most widely distributed alkaloid of this group is securinine, which was identified next to allosecurinine in Phyllanthus glaucus (leafflower). Recently, some Securinega-type alkaloids have paid attention to its antiproliferative potency towards different cancer cells. However, the cytotoxic properties of allosecurinine have not yet been evaluated. Methods The cytotoxicity of the extract, alkaloid fraction obtained from P. glaucus, isolated securinine and allosecurinine against HeLa cells was evaluated by real-time xCELLigence system and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was detected by annexin V and 7-amino-actinomycin (7-AAD) staining and confirmed with fluorescent Hoechst 33342 dye. The assessment of mitochondrial membrane potential (MMP), reactive oxygen species (ROS) generation, the level of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), caspase-3/7 activity and cell cycle analysis were measured by flow cytometry. The enzymatic activity of caspase-9 was assessed by a luminometric assay. The expression of apoptosis associated genes was analyzed by real-time PCR. Results The experimental data revealed that securinine and the alkaloid fraction were significantly potent on HeLa cells growth inhibition with IC50 values of 7.02 ± 0.52 μg/ml (32.3 μM) and 25.46 ± 1.79 μg/ml, respectively. The activity of allosecurinine and Phyllanthus extract were much lower. Furthermore, our study showed that the most active securinine induced apoptosis in a dose-dependent manner in the tested cells, increased the percentage of ROS positive cells and depolarized cells as well as stimulated the activity of ERK1/2, caspase-9 and -3/7. Securinine also induced cell cycle arrest in S phase. Real-time PCR analysis showed high expression of TNFRSF genes in the cells stimulated with securinine. Conclusions Securinine

  1. Synergistic combination of fluoro chalcone and doxorubicin on HeLa cervical cancer cells by inducing apoptosis

    Arianingrum, Retno; Arty, Indyah Sulistyo; Atun, Sri

    2017-03-01

    Doxorubicin (Dox), a primary chemotherapeutic agent used for cancer treatment is known to have various side effect included multidrug resistance (MDR) phenomenon. Combination chemotherapy is one of some approaches to reduce Dox side effect. Chalcones have been reported to reduce the proliferation of many cancer cells. The research were conducted to investigate the cytotoxic activity and apoptosis induction of a chalcone derivate which is containing fluoro substituent [1 - (4" - fluorophenyl) -3 - (4' - hydroxy - 3' - methoxyphenyl) - 2 - propene - 1 -on] (FHM) and its combination with Dox on HeLa cells line. The observation of the cytotoxic activity was conducted using MTT [3 - (4, 5 - dimethyl thiazol - 2 - y1) - 2.5 - diphenyltetrazolium bromide] assay. Apoptosis induction was determined by flow cytometric. The changes of cell morphology were observed using phase contrast microscopy. The combination index (CI) was used to determine the effect of the combination. The study showed that FHM inhibited the HeLa cell growth with IC50 of 34 μM, while the IC50 of Dox was 1 μM. The combination had a higher inhibitory effect on cell growth compare to the single treatment of FHM and Dox. All of the combination doses under IC50 of FHM and Dox gave synergistic (CI: - 0.7) up to strong synergistic effect (CI: 0.l - 0.3). The synergistic effects of the combination were due to their ability to induce apoptosis in the HeLa cells. According to the result, FHM was potential to be developed as a co-chemotherapeutic agent with Dox for cervical cancer.

  2. Effects of Smac gene over-expression on the radiotherapeutic sensitivities of cervical cancer cell line HeLa

    ZHENG Li-duan; XIONG Zhou-fang; ZHU Jian-wen; WANG Ze-hua

    2005-01-01

    Background The second mitochondria-derived activator of caspases (Smac) is a novel proapoptotic gene, which plays an important role in the apoptosis-inducing effects of irradiation on tumor cells. The purpose of this study was to investigate the effects of extrinsic Smac gene transfer and its over-expression in radiotherapeutic sensitivities of cervical cancer cells. Methods After the Smac gene was transferred into the cervical cancer cell line HeLa, subcloned cells were obtained by persistent G418 selection. Cellular Smac gene expression was detected by RT-PCR and Western blot, while in vitro cell viabilities were detected by trypan blue staining assay. After treatment with X-ray irradiation, cellular radiotherapeutic sensitivities were investigated by tetrazolium bromide colorimetry. Cellular apoptosis and its rate were determined by electronic microscopy, annexin V-FITC and propidium iodide staining flow cytometry. The expression and activities of cellular caspase-3 were assayed by Western blot and colorimetry. Results Smac mRNA and protein levels in HeLa/Smac cells and the selected subclone cell line of cervical cancer were significantly higher than those of HeLa (P0.05). However, after irradiation with 8 Gy X-ray, growth activities of HeLa/Smac were reduced by 22.42% (P<0.01). When compared with those of HeLa, partial HeLa/Smac cells presented characteristic morphological changes of apoptosis under electronic microscope, with higher apoptosis rates (16.4% vs. 6.2%, P<0.01); the caspase-3 expression levels in HeLa/Smac cells were improved significantly (P<0.01), while its activities were increased by 3.42 times (P<0.01).Conclusions Stable transfer of the extrinsic Smac gene and its over-expression in cervical cancer cell line could significantly enhance the expression and activities of cellular caspase-3 and ameliorate apoptosis-inducing effects of irradiation on cancer cells, which was a novel strategy to improve radiotherapeutic effects on cervical cancer.

  3. Analysis of energy metabolism of HeLa cancer cells in vitro and in vivo using fluorescence lifetime microscopy

    Lukina, Maria; Shirmanova, Marina; Dudenkova, Varvara; Druzhkova, Irina; Shumilova, Anastasia; Zagaynova, Elena

    2016-04-01

    The aim of the present work was to study energy metabolism in human cervical carcinoma (HeLa) cells in vitro and in vivo using two-photon FLIM. Cellular metabolism was examined by monitoring of the fluorescence lifetimes of free and protein-bound forms of NAD(P)H and FAD and their relative contributions. Two-photon fluorescence and second harmonic generation microscopy as well as standard histopathology with hematoxylin and eosin were used to characterize tissue structure. Cellular metabolism was analyzed in cancer cells co-cultured with human fibroblasts and in tumor xenografts transplanted to nude mice. In the HeLa-huFB co-culture we observed a metabolic shift from OXPHOS toward glycolysis in cancer cells, and from glycolysis to OXPHOS in fibroblasts, starting from Day 2 of co-culturing. In the tumor tissue we detected metabolic heterogeneity with more glycolytic metabolism of cancer cells in the stroma-rich zones. The results of the study are of a great importance for understanding metabolic behavior of tumors and for development of anticancer drugs targeted to metabolic pathways.

  4. Myricetin and methyl eugenol combination enhances the anticancer activity, cell cycle arrest and apoptosis induction of cis-platin against HeLa cervical cancer cell lines.

    Yi, Jin-Ling; Shi, Song; Shen, Yan-Li; Wang, Ling; Chen, Hai-Yan; Zhu, Jun; Ding, Yan

    2015-01-01

    Drug combination therapies are common practice in the treatment of cancer. In this study, we evaluated the anticancer effects of myricetin (MYR), methyl eugenol (MEG) and cisplatin (CP) both separately as well as in combination against cervical cancer (HeLa) cells. To demonstrate whether MYR and MEG enhance the anticancer activity of CP against cervical cancer cells, we treated HeLa cells with MYR and MEG alone or in combination with cisplatin and evaluated cell growth and apoptosis using MTT (3 (4, 5 dimethyl thiazol 2yl) 2, 5 diphenyltetrazolium bromide) assay, LDH release assay, flow cytometry and fluorescence microscopy. The results revealed that, as compared to single drug treatment, the combination of MYR or MEG with CP resulted in greater effect in inhibiting cancer cell growth and inducing apoptosis. Cell apoptosis induction, Caspase-3 activity, cell cycle arrest and mitochondrial membrane potential loss were systematically studied to reveal the mechanisms of synergy between MYR, MEG and CP. Combination of MYR or MEG with CP resulted in more potent apoptosis induction as revealed by fluorescence microscopy using Hoechst 33258 and AO-ETBR staining. The combination treatment also increased the number of cells in G0/G1 phase dramatically as compared to single drug treatment. Mitochondrial membrane potential loss (ΛΨm) as well as Caspase-3 activity was much higher in combination treatment as compared to single drug treatment. Findings of this investigation suggest that MYR and MEG combined with cisplatin is a potential clinical chemotherapeutic approach in human cervical cancer.

  5. 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.

  6. 放疗耐受性宫颈癌Hela细胞的生物学特性%Biological Characteristics in Cervical Cancer Cell Line Hela Tolerated to Radiation

    黄靖然; 彭永排; 周晖; 冯敏清; 姚婷婷; 饶群仙; 王丽娟; 林仲秋

    2012-01-01

    [目的]研究耐放疗的宫颈癌Hela细胞生物学特性的改变,并探讨其与宫颈癌肿瘤干细胞间的关系.[方法]采用多次分割剂量照射技术建立宫颈癌Hela细胞的耐放疗模型(Hela-R),实验分4组:Hela-R1组,Hela-R2组,Hela-R3组和对照组.四甲基偶氮唑蓝法检测细胞生长情况,克隆形成实验测定放射敏感性和克隆能力,流式细胞术检测细胞周期分布和增殖能力,球囊培养法检测细胞自我更新能力.[结果]Hela、Hela-R细胞接受照射后均呈现先加速增殖后出现生长抑制的现象.Hela-R1、Hela-R2、Hela-R3的细胞倍增时间分别为(43.4±1.0)h、(49.2±2.0)h和(48.7±3.3)h,克隆形成率分别为(20.3±4.0)%、(49.3±11.6)%和(6.3±5.9)%,S期细胞比例分别为(9.9±0.4)%、(13.0±0.9)%和(9.6±0.7)%,增殖指数(PI)分别为(27.3±2.6)%、(31.8±4.9)%和(37.4±8.0)%.与对照组比较,Hela-R3组的放射抗拒性增强.非粘附性球囊培养法培养Hela及Hela-R细胞可得到肿瘤细胞球,四组的球囊形成率分别为(9.9±0.4)%、(13.0±0.9)%、(9.6±0.7)%和(5.0±0.3)%.[结论]多次分割剂量照射可在体外建立宫颈癌Hela细胞的耐放疗模型,并可富集肿瘤干细胞;多次分割照射后,Hela细胞生长速度减慢,增殖能力有升高趋势,自我更新能力、克隆能力增强,细胞周期无明显变化.%[Object] The aim of the study was to investigate biological behaviors of radioresistant cervical cancer cells. The relationship between radioresistant cancer cells and cancer stem cells would be discussed. [Methods] Hela cells were treated with fractionated irradiation, yielding Hela-R, the radioresistant model. There were four groups; Hela-Rl, Hela-R2, Hela-R3 and control. Biological characteristics, including cell growth, clone-generating capability, cell cycle distribution and proliferation, and tumor sphere-forming rate were detected by MTT assay, clone formation assay, flow cytometry assay and

  7. Melatonin sensitizes human cervical cancer HeLa cells to cisplatin-induced cytotoxicity and apoptosis: effects on oxidative stress and DNA fragmentation.

    Pariente, Roberto; Pariente, José A; Rodríguez, Ana B; Espino, Javier

    2016-01-01

    Melatonin has antitumor activity via several mechanisms including its antiproliferative and pro-apoptotic effects as well as its potent antioxidant actions, although recent evidence has indicated that melatonin may perform pro-oxidant actions in tumor cells. Therefore, melatonin may be useful in the treatment of tumors in association with chemotherapy drugs. This study was intended to evaluate the in vitro effect of melatonin on the cytotoxic and pro-apoptotic actions of various chemotherapeutic agents in cervical cancer HeLa cells. Herein, we found that both melatonin and three of the chemotherapeutic drugs tested, namely cisplatin (CIS), 5-fluorouracil (5-FU), and doxorubicin, induced a decrease in HeLa cell viability. Furthermore, melatonin significantly increased the cytotoxic effect of such chemotherapeutic agents. Consistently, costimulation of HeLa cells with any chemotherapeutic agent in the presence of melatonin further increased caspase-3 activation, particularly in CIS- and 5-FU-challenged cells. Likewise, concomitant treatments with melatonin and CIS significantly enhanced the ratio of cells entering mitochondrial apoptosis due to reactive oxygen species (ROS) overproduction, substantially augmented the population of apoptotic cells, and markedly enlarged DNA fragmentation compared to the treatments with CIS alone. Nonetheless, melatonin only displayed moderate chemosensitizing effects in 5-FU-stimulated HeLa cells, as suggested by slight increments in the percentage of cells stimulated for ROS production and in the proportion of early apoptotic cells compared to the treatments with 5-FU alone. In summary, our findings provided evidence that in vitro melatonin strongly enhances CIS-induced cytotoxicity and apoptosis in HeLa cells and, hence, the indoleamine could be potentially applied to cervical cancer treatment as a powerful synergistic agent.

  8. 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

    2015-07-21

    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.

  9. 1-(2-Hydroxy-5-methylphenyl)-3-phenyl-1,3-propanedione Induces G1 Cell Cycle Arrest and Autophagy in HeLa Cervical Cancer Cells

    Tsai, Jie-Heng; Hsu, Li-Sung; Huang, Hsiu-Chen; Lin, Chih-Li; Pan, Min-Hsiung; Hong, Hui-Mei; Chen, Wei-Jen

    2016-01-01

    The natural agent, 1-(2-hydroxy-5-methylphenyl)-3-phenyl-1,3-propanedione (HMDB), has been reported to have growth inhibitory effects on several human cancer cells. However, the role of HMDB in cervical cancer remains unclear. Herein, we found that HMDB dose- and time-dependently inhibited growth of HeLa cervical cancer cells, accompanied with G1 cell cycle arrest. HMDB decreased protein expression of cyclins D1/D3/E and cyclin-dependent kinases (CDKs) 2/4/6 and reciprocally increased mRNA and protein levels of CDK inhibitors (p15, p16, p21, and p27), thereby leading to the accumulation of hypophosphorylated retinoblastoma (Rb) protein. HMDB also triggered the accumulation of acidic vesicles and formation of microtubule-associated protein-light chain 3 (LC3), followed by increased expression of LC3 and Beclin-1 and decreased expression of p62, suggesting that HMDB triggered autophagy in HeLa cells. Meanwhile, suppression of the expression of survivin and Bcl-2 implied that HMDB-induced autophagy is tightly linked to apoptosis. Exploring the action mechanism, HMDB induced autophagy via the modulation of AMP-activated protein kinase (AMPK) and mTOR signaling pathway rather than the class III phosphatidylinositol 3-kinase pathway. These results suggest that HMDB inhibits HeLa cell growth by eliciting a G1 arrest through modulation of G1 cell cycle regulators and by concomitantly inducing autophagy through the mediation of AMPK-mTOR and Akt-mTOR pathways, and may be a promising antitumor agent against cervical cancer. PMID:27527160

  10. 1-(2-Hydroxy-5-methylphenyl-3-phenyl-1,3-propanedione Induces G1 Cell Cycle Arrest and Autophagy in HeLa Cervical Cancer Cells

    Jie-Heng Tsai

    2016-08-01

    Full Text Available The natural agent, 1-(2-hydroxy-5-methylphenyl-3-phenyl-1,3-propanedione (HMDB, has been reported to have growth inhibitory effects on several human cancer cells. However, the role of HMDB in cervical cancer remains unclear. Herein, we found that HMDB dose- and time-dependently inhibited growth of HeLa cervical cancer cells, accompanied with G1 cell cycle arrest. HMDB decreased protein expression of cyclins D1/D3/E and cyclin-dependent kinases (CDKs 2/4/6 and reciprocally increased mRNA and protein levels of CDK inhibitors (p15, p16, p21, and p27, thereby leading to the accumulation of hypophosphorylated retinoblastoma (Rb protein. HMDB also triggered the accumulation of acidic vesicles and formation of microtubule-associated protein-light chain 3 (LC3, followed by increased expression of LC3 and Beclin-1 and decreased expression of p62, suggesting that HMDB triggered autophagy in HeLa cells. Meanwhile, suppression of the expression of survivin and Bcl-2 implied that HMDB-induced autophagy is tightly linked to apoptosis. Exploring the action mechanism, HMDB induced autophagy via the modulation of AMP-activated protein kinase (AMPK and mTOR signaling pathway rather than the class III phosphatidylinositol 3-kinase pathway. These results suggest that HMDB inhibits HeLa cell growth by eliciting a G1 arrest through modulation of G1 cell cycle regulators and by concomitantly inducing autophagy through the mediation of AMPK-mTOR and Akt-mTOR pathways, and may be a promising antitumor agent against cervical cancer.

  11. 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, pHeLa cells was detected after treatment and the apoptosis rate with the concentration and longer incubation time (r=1.0, pHeLa 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.

  12. 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.

  13. 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.

  14. Assessment of Cytotoxic Activity of Rosemary (Rosmarinus officinalis L.), Turmeric (Curcuma longa L.), and Ginger (Zingiber officinale R.) Essential Oils in Cervical Cancer Cells (HeLa)

    Santos, P. A. S. R.; Avanço, G. B.; Nerilo, S. B.; Marcelino, R. I. A.; Janeiro, V.; Valadares, M. C.

    2016-01-01

    The objective of this study was to evaluate the cytotoxic activity of rosemary (REO, Rosmarinus officinalis L.), turmeric (CEO, Curcuma longa L.), and ginger (GEO, Zingiber officinale R.) essential oils in HeLa cells. Cytotoxicity tests were performed in vitro, using tetrazolium (MTT) and neutral red assays for evaluation of antiproliferative activity by different mechanisms, trypan blue assay to assess cell viability and evaluation of cell morphology for Giemsa to observe the cell damage, and Annexin V to evaluate cell death by apoptosis. CEO and GEO exhibited potent cytotoxic activity against HeLa cells. IC50 obtained was 36.6 μg/mL for CEO and 129.9 μg/mL for GEO. The morphology of HeLa cells showed condensation of chromatin, loss of cell membrane integrity with protrusions (blebs), and cell content leakage for cells treated with CEO and GEO, from the lowest concentrations studied, 32.81 μg/mL of CEO and 32.12 μg/mL of GEO. The Annexin V assay revealed a profile of cell death by apoptosis for both CEO and GEO. The results indicate cytotoxic activity in vitro for CEO and GEO, suggesting potential use as anticancer agents for cervical cancer cells. PMID:28042599

  15. Assessment of Cytotoxic Activity of Rosemary (Rosmarinus officinalis L., Turmeric (Curcuma longa L., and Ginger (Zingiber officinale R. Essential Oils in Cervical Cancer Cells (HeLa

    P. A. S. R. Santos

    2016-01-01

    Full Text Available The objective of this study was to evaluate the cytotoxic activity of rosemary (REO, Rosmarinus officinalis L., turmeric (CEO, Curcuma longa L., and ginger (GEO, Zingiber officinale R. essential oils in HeLa cells. Cytotoxicity tests were performed in vitro, using tetrazolium (MTT and neutral red assays for evaluation of antiproliferative activity by different mechanisms, trypan blue assay to assess cell viability and evaluation of cell morphology for Giemsa to observe the cell damage, and Annexin V to evaluate cell death by apoptosis. CEO and GEO exhibited potent cytotoxic activity against HeLa cells. IC50 obtained was 36.6 μg/mL for CEO and 129.9 μg/mL for GEO. The morphology of HeLa cells showed condensation of chromatin, loss of cell membrane integrity with protrusions (blebs, and cell content leakage for cells treated with CEO and GEO, from the lowest concentrations studied, 32.81 μg/mL of CEO and 32.12 μg/mL of GEO. The Annexin V assay revealed a profile of cell death by apoptosis for both CEO and GEO. The results indicate cytotoxic activity in vitro for CEO and GEO, suggesting potential use as anticancer agents for cervical cancer cells.

  16. Effects of Livin Gene RNA Interference on Apoptosis of Cervical Cancer Hela Cells and Enhanced Sensitivity to Cisplatin

    Lili YU; Zehua WANG

    2009-01-01

    The recombinant plasmids pGenesii-1-BIRC71 and pGenesil-1-BIRC72 were transfected into Hela cells and cisplatin was added with different concentrations in order to study the inhibitory ef-fects of Livin gene, increase the apoptosis induced by cisplatin, and detect the expression of Bcl-2, Bax,caspase-3, and survivin genes. The pGenesil-1-BIRC71 and pGenesil-1-BIRC72 were transfected into Hela cells, and the expression levels of Livin, Bcl-2, Bax, caspase-3, and survivin genes were detected by using fluorescence quantitative real-time PCR. Then cisplatin at different concentrations (3.0, 6.0 and 9.9 μg/mL) was added into the transfected Hela cells, and 24, and 48 h later, the apoptosis rate was measured by flow cytometry. After transfection of pGenesil-1-BIRC71 and pGenesil-1-BIRC72 into Hela cells, the expression level of Livin gene was obviously reduced, and the apoptosis rate was sig-nificantly increased in transfection group as compared with control group (P<0.05). Cisplatin could in-crease the apoptosis rate in a dose- and time-dependent manner. After cisplatin was added, the expres-sion levels of Bcl-2 mRNA were reduced, and those of Bax, caspase-3, and survivin mRNA were in-creased in transfection group as compared with those in control group (P<0.05). It was concluded that shRNA expression vector targeting Livin gene could inhibit the expression of Livin gene in Hela cells and enhance the apoptosis induced by cisplatin, which was related to the decreased expression of Bcl-2and activation of Bax and caspase-3. Survivin might play an important role as an antagonist in the proc-ess of apoptosis induction.

  17. 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.

  18. Cytotoxic and apoptogenic effects of Bryonia aspera root extract against Hela and HN-5 cancer cell lines

    Pourgonabadi, Solmaz; Amiri, Mohammad Sadegh; Mousavi, Seyed Hadi

    2017-01-01

    Objective: Bryonia aspera (Stev. ex Ledeb) is a plant that grows in northeast of Iran. In the present study, cytotoxic and apoptogenic properties of B. aspera root extract was determined against HN-5(head and neck squamous cell carcinoma) and Hela (cervix adenocarcinoma) cell lines. Materials and Methods: HN-5 and Hela cell lines were cultured in DMEM medium and incubated with different concentrations of B. aspera root extract. Cell viability was quantitated by MTT assay and the optical absorbance was measured at 570 nm (620 nm as the reference) by an ELISA reader, in each experiment. Apoptotic cells were assessed using PI staining of DNA fragmentation by flow cytometry (sub-G1 peak). The B. aspera inhibited 50% growth (IC50) of Hela and HN-5 cell lines at 100±28 μg/ml and 12.5±4 μg/ml, respectively after 48 hr of incubation. Results: Cell viability assay showed that inhibitory effects of B. aspera were time and dose-dependent in both cell lines, which were consistent with morphological changes, observed under light microscope. Apoptosis was investigated by flow cytometry in which percentage of apoptotic cells increased in a dose and time-dependent manner. Conclusion: Based on our data, B. aspera has cytotoxic effects in which apoptosis played an important role. Further evaluations are needed to assess the possible anti-tumor properties of this plant. PMID:28265548

  19. Properties of Surfactin C-15 Nanopeptide and Its Cytotoxic Effect on Human Cervix Cancer (HeLa Cell Line

    Zahra Nozhat

    2012-01-01

    Full Text Available Surfactin is one of the most powerful biosurfactants that has been known so far. It is an acidic cyclic nonribosomal lipoheptapeptide that is produced by Bacillus subtilis. In this presentation we investigated different properties of surfactin C-15. The nanomicelle forming ability of surfactin C-15 in different aqueous environments with various ionic strengths was studied by scanning electron microscope. Surfactin second structure was investigated by Far-UV CD spectrum. Its hemolytic activity and cytotoxicity were measured by hemolysis and MTT assays, respectively. Surfactin formed spherical nanomicelles in distilled water and amorphous nanomicelles in PBS buffer . The hemolysis assay results indicated that HC50 of surfactin was 47 μM. Surfactin C-15 arrested growth of human cervix cancer HeLa cell line in a time- and dosage-dependent method, so that its IC50 at 16, 24, and 48h were 86.9, 73.1, and 50.2 μM, respectively.

  20. 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.

  1. Effect of EBI3 on radiation-induced immunosuppression of cervical cancer HeLa cells by regulating Treg cells through PD-1/PD-L1 pathway.

    Zhang, Song-An; Niyazi, Hu-Er-Xi-Dan; Hong, Wen; Tuluwengjiang, Gu-Li-Xian; Zhang, Lei; Zhang, Yang; Su, Wei-Peng; Bao, Yong-Xing

    2017-03-01

    This study aimed to investigate the effect of EBI3 on radiation-induced immunosuppression of cervical cancer HeLa cells by regulating Treg cells through PD-1/PD-L1 signaling pathway. A total of 43 adult female Wistar rats were selected and injected with HeLa cells in the caudal vein to construct a rat model of cervical cancer. All model rats were randomly divided into the radiotherapy group ( n = 31) and the control group ( n = 12). The immunophenotype of Treg cells was detected by the flow cytometry. The protein expressions of EBI3, PD-1, and PD-L1 in cervical cancer tissues were tested by the streptavidin-peroxidase method. HeLa cells in the logarithmic growth phase were divided into four groups: the blank, the negative control group, the EBI3 mimics group, and the EBI3 inhibitors group. Western blotting was used to detect PD-1 and PD-L1 protein expressions. MTT assay was performed to measure the proliferation of Treg cells. Flow cytometry was used to detect cell cycle and apoptosis, and CD4(+)/CD8(+) T cell ratio in each group. Compared with before and 1 week after radiotherapy, the percentages of CD4(+)T cells and CD8(+)T cells were significantly decreased in the radiotherapy group at 1 month after radiotherapy. Furthermore, down-regulation of EBI3 and up-regulation of PD-1 and PD-L1 were observed in cervical cancer tissues at 1 month after radiotherapy. In comparison to the blank and negative control groups, increased expression of EBI3 and decreased expressions of PD-1 and PD-L1 were found in the EBI3 mimics group. However, the EBI3 inhibitors group had a lower expression of EBI3 and higher expressions of PD-1 and PD-L1 than those in the blank and negative control groups. The EBI3 mimics group showed an increase in the optical density value (0.43 ± 0.05), while a decrease in the optical density value (0.31 ± 0.02) was found in the EBI3 inhibitors group. Moreover, compared with the blank and negative control groups, the apoptosis rates

  2. Suppression of human cervical cancer cell lines Hela and DoTc2 4510 by a mixture of lysine, proline, ascorbic acid, and green tea extract.

    Roomi, M W; Ivanov, V; Kalinovsky, T; Niedzwiecki, A; Rath, M

    2006-01-01

    Cervical cancer, the second most common cancer in women, once metastasized, leads to poor prognosis. We investigated the antitumor effect of a nutrient mixture (NM) containing lysine, proline, arginine, ascorbic acid, and green tea extract on human cervical cancer cells Hela (CCL-2) and DoTc2 4510 by measuring cell proliferation (MTT assay), modulation of matrix metalloproteinases (MMP)-2 and MMP-9) expression (gelatinase zymography), and cancer cell invasive potential (Matrigel). NM showed significant antiproliferative effect on CCL-2 and DoTc2 4510 cancer cells. The NM inhibited CCL-2 expression of MMP-2 and MMP-9 in a dose-dependent fashion, with virtual total inhibition of MMP-2 at 1000 microg/mL and MMP-9 at 500 microg/mL NM. Untreated DoTc2 4510 cells showed MMP-9 expression, which was enhanced with phorbol 12-myristate 13-acetate treatment. NM inhibited MMP-9 expression in a dose-dependent fashion, with virtual inhibition at 500 microg/mL. Invasion of human cervical cancer cells CCL-2 and DoTc2 4510 through Matrigel decreased in a dose-dependent fashion, with 100% inhibition at 500 microg/mL NM (P cervical cancer by inhibiting critical steps in cancer development and spread.

  3. Inotodiol对宫颈癌HeLa细胞增殖的影响%effect of Inotodiol on proliferation of cervical cancer HeLa cells

    赵丽微; 钟秀宏; 杨淑艳; 杨宁江

    2013-01-01

    目的:研究桦褐孔菌单体(Inotodiol)对宫颈癌HeLa细胞增殖的影响.方法:采用MTT法检测Inotodiol对HeLa细胞的增殖抑制率;通过倒置显微镜、HE染色观察HeLa细胞形态学改变;通过免疫细胞化学法检测核增殖抗原Ki-67表达.结果:随着Inotodiol浓度增加及作用时间延长,抑制率明显增强,差异有统计学意义(P<0.05).Inotodiol作用于Hela细胞后,形态学观察示细胞凋亡.Inotodiol组Ki-67表达明显降低,与对照组相比差异有统计学意义(P<0.01).结论:Inoto-diol对宫颈癌HeLa细胞有明显的抑制增殖和诱导凋亡作用.%Objective: To research the effect of Inotodiol on proliferation of cervical cancer HeLa cells.Methods: MTT assay was used to detect the inhibition rate of Inotodiol on proliferation of HeLa cells.The morphological changes of HeLa cells were observed by invert microscope and HE staining.The expression of Ki - 67 was detected by immunocytochemical method.Results: The inhibition rate increased significantly with the increase of Inotodiol concentration and the time, there was statistically significant difference (P < 0.05) .Apoptosis of HeLa cells was observed after treated with Inotodiol.The expression level of Ki - 67 in Inotodiol group decreased significantly, compared with control group, there was statistically significant difference (P <0.01 ) .Conclusion: Inotodiol can inhabit proliferation and induce apoptosis of cervical cancer HeLa cells significantly.

  4. 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.

  5. The Effect of Coatings on the Affinity of Lanthanide Nanoparticles to MKN45 and HeLa Cancer Cells and Improvement in Photodynamic Therapy Efficiency

    Takashi Sawamura

    2015-09-01

    Full Text Available An improvement in photodynamic therapy (PDT efficiency against a human gastric cancer cell line (MKN45 with 5-aminolevulinic acid (ALA and lanthanide nanoparticles (LNPs is described. An endogenous photosensitizer, protoporphyrin IX, biosynthesized from ALA and selectively accumulated in cancer cells, is sensitizable by the visible lights emitted from up-conversion LNPs, which can be excited by a near-infrared light. Ten kinds of surface modifications were performed on LNPs, NaYF4(Sc/Yb/Er and NaYF4(Yb/Tm, in an aim to distribute these irradiation light sources near cancer cells. Among these LNPs, only the amino-functionalized LNPs showed affinity to MKN45 and HeLa cancer cells. A PDT assay with MKN45 demonstrated that amino-modified NaYF4(Sc/Yb/Er gave rise to a dramatically enhanced PDT effect, reaching almost perfect lethality, whereas NaYF4(Yb/Tm-based systems caused little improvement in PDT efficiency. The improvement of PDT effect with the amino-modified NaYF4(Sc/Yb/Er is promising for a practical PDT against deep cancer cells that are reachable only by near-infrared lights.

  6. The Effect of Coatings on the Affinity of Lanthanide Nanoparticles to MKN45 and HeLa Cancer Cells and Improvement in Photodynamic Therapy Efficiency.

    Sawamura, Takashi; Tanaka, Tatsumi; Ishige, Hiroyuki; Iizuka, Masayuki; Murayama, Yasutoshi; Otsuji, Eigo; Ohkubo, Akihiro; Ogura, Shun-Ichiro; Yuasa, Hideya

    2015-09-16

    An improvement in photodynamic therapy (PDT) efficiency against a human gastric cancer cell line (MKN45) with 5-aminolevulinic acid (ALA) and lanthanide nanoparticles (LNPs) is described. An endogenous photosensitizer, protoporphyrin IX, biosynthesized from ALA and selectively accumulated in cancer cells, is sensitizable by the visible lights emitted from up-conversion LNPs, which can be excited by a near-infrared light. Ten kinds of surface modifications were performed on LNPs, NaYF₄(Sc/Yb/Er) and NaYF₄(Yb/Tm), in an aim to distribute these irradiation light sources near cancer cells. Among these LNPs, only the amino-functionalized LNPs showed affinity to MKN45 and HeLa cancer cells. A PDT assay with MKN45 demonstrated that amino-modified NaYF₄(Sc/Yb/Er) gave rise to a dramatically enhanced PDT effect, reaching almost perfect lethality, whereas NaYF₄(Yb/Tm)-based systems caused little improvement in PDT efficiency. The improvement of PDT effect with the amino-modified NaYF₄(Sc/Yb/Er) is promising for a practical PDT against deep cancer cells that are reachable only by near-infrared lights.

  7. Apoptotic Effects of Hypocrellin A on HeLa Cells

    2006-01-01

    Hypocrellin A(HA), a photosensitive perylenequinone compound of Hypocrella bambusae , inhibited the proliferation of several tumor cell lines. Human cervical cancer cells, HeLa cells, were used as a model to elucidate the molecular mechanisms of HA-induced tumor cell death. The results show that HA can induce the oligonucleosomal fragmentation of DNA in HeLa cells and also can increase the expression of apoptosis inducer Bax mRNA and that it decreases the expression of apoptosis suppressor, Bcl-2 mRNA, in mitochondria. It can be concluded from the data that HA-induced apoptosis is related to the balance between Bcl-2 and Bax gene expressions.

  8. Juglans mandshurica Maxim extracts exhibit antitumor activity on HeLa cells in vitro.

    Xin, Nian; Hasan, Murtaza; Li, Wei; Li, Yan

    2014-04-01

    The present study examined the potential application of Juglans mandshurica Maxim extracts (HT) for cancer therapy by assessing their anti‑proliferative activity, reduction of telomerase activity, induction of apoptosis and cell cycle arrest in S phase in HeLa cells. From the perspective of using HT as a herbal medicine, photomicroscopy and florescent microscopy techniques were utilized to characterize the effect of the extracts on telomerase activity and cell morphology. Flow cytometry was employed to study apoptosis and cell cycle of HeLa cells, and DNA laddering was performed. The results showed that HT inhibited cell proliferation and telomerase activity, induced apoptosis and caused S phase arrest of HeLa cells in vitro. HT inhibited HeLa cell proliferation significantly, and the highest inhibition rate was 83.7%. A trap‑silver staining assay showed that HT was capable of markedly decreasing telomerase activity of HeLa cells and this inhibition was enhanced in a time‑ and dose‑dependent manner. Results of a Hoechst 33258 staining assay showed that HeLa cells treated by HT induced cell death. Through DNA agarose gel electrophoresis, DNA ladders of HeLa cells treated with HT were observed, indicating apoptosis. In conclusion, the present study demonstrated that HT exhibited anti‑tumor effects comprising the inhibition of growth and telomerase activity as well as apoptosis and cell cycle arrest in HeLa cells.

  9. Synergistic effect of fisetin combined with sorafenib in human cervical cancer HeLa cells through activation of death receptor-5 mediated caspase-8/caspase-3 and the mitochondria-dependent apoptotic pathway.

    Lin, Ming-Te; Lin, Chia-Liang; Lin, Tzu-Yu; Cheng, Chun-Wen; Yang, Shun-Fa; Lin, Chu-Liang; Wu, Chih-Chien; Hsieh, Yi-Hsien; Tsai, Jen-Pi

    2016-05-01

    Combining antitumor agents with bioactive compounds is a potential strategy for improving the effect of chemotherapy on cancer cells. The goal of this study was to elucidate the antitumor effect of the flavonoid, fisetin, combined with the multikinase inhibitor, sorafenib, against human cervical cancer cells in vitro and in vivo. The combination of fisetin and sorafenib synergistically induced apoptosis in HeLa cells, which is accompanied by a marked increase in loss of mitochondrial membrane potential. Apoptosis induction was achieved by caspase-3 and caspase-8 activation which increased the ratio of Bax/Bcl-2 and caused the subsequent cleavage of PARP level while disrupting the mitochondrial membrane potential in HeLa cells. Decreased Bax/Bcl-2 ratio level and mitochondrial membrane potential were also observed in siDR5-treated HeLa cells. In addition, in vivo studies revealed that the combined fisetin and sorafenib treatment was clearly superior to sorafenib treatment alone using a HeLa xenograft model. Our study showed that the combination of fisetin and sorafenib exerted better synergistic effects in vitro and in vivo than either agent used alone against human cervical cancer, and this synergism was based on apoptotic potential through a mitochondrial- and DR5-dependent caspase-8/caspase-3 signaling pathway. This combined fisetin and sorafenib treatment represents a novel therapeutic strategy for further clinical developments in advanced cervical cancer.

  10. Synthesis of 7-hydroxy-4'-methoxyflavanone and 7-hydroxy-4'-methoxyflavone as a candidate anticancer against cervical (HeLa) cancer cell and colon (WiDr) cancer cell by in vitro

    Matsjeh, Sabirin; Anwar, Chairil; Solikhah, Eti Nurwening; Farah, Harra Ismi; Nurfitria, Kurnia

    2017-03-01

    The compound 7-hydroxy-4'-methoxyflavanone and 7-hydroxy-4'-methoxyflavone have been synthesized through cyclization reaction of 2 ', 4'-dihydroxy-4-methoxychalcone (1,3-diphenyl-2-propene-1-one). The 2 ', 4'-dihydroxy-4-methoxychalcone were synthesized through Claisen-Schmidt condensation from 2,4-dihydroxyacetophenone and 4-methoxybenzaldehyde (anisaldehyde) in aqueous KOH as a catalyst in ethanol. The 7-hydroxy-4'-methoxyflavanone has been synthesized through cyclization reaction of 2 ', 4'-dihydroxy-4-methoxychalcone by Oxa-Michael addition reaction with sulfuric acid as a catalyst in ethanol. The 7-hydroxy-4'-methoxyflavone has been synthesized through oxidative cyclization reaction of 2 ', 4'-dihydroxy-4-methoxychalcone using I2 in DMSO as a catalyst with a mole ratio (1: 1) mol. All these producets were characterized by FT-IR, GC-MS, and 1H-NMR and 13C-NMR spectrometer. Both of these compounds were tested citotoxycity activity as an anticancer against cervical and colon cancer cells (HeLa and WiDr cell lines) using MTT assay in vitro. Dose series given test solution concentration on HeLa and WiDr cells starting from 0,78; 1,56; 3,12; 6,25; 12,50; 25; 50 and 100 µg/mL with a long incubation treatment for 24 hours. The results study showed that the 7-hydroxy-4'-methoxyflavanone as bright yellow crystals with a melting point 172-174 ° C and a yield of 56.67% and the 7-hydroxy-4'-methoxyflavone as bright yellow crystals with a yield of 88, 31%, and a melting point of 263-265 ° C. The test results cytotoxic 7-hydroxy-4-methoxyflavone showed active against HeLa cells with IC50 value of 25.73 µg/mL and was quite active in the WiDr cells with IC50 value of 83.75 µg/mL. The result of the activity of 7-hydroxy-4-methoxyflavanone show active cytotoxic activity against HeLa and WiDr cell growth with IC50 value of 40.13 µg/mL and 37.85 µg/mL. IC50 value indicated that 7-hydroxy-4'-methoxyflavone and 7-hydroxy-4'-methoxyflavanone potential as inhibitors in HeLa and

  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. 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

    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±0.2nm. 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.

  13. Auranofin induces apoptosis and necrosis in HeLa cells via oxidative stress and glutathione depletion.

    You, Bo Ra; Shin, Hye Rim; Han, Bo Ram; Kim, Suhn Hee; Park, Woo Hyun

    2015-02-01

    Auranofin (Au), an inhibitor of thioredoxin reductase, is a known anti‑cancer drug. In the present study, the anti‑growth effect of Au on HeLa cervical cancer cells was examined in association with levels of reactive oxygen species (ROS) and glutathione (GSH). Au inhibited the growth of HeLa cells with an IC50 of ~2 µM at 24 h. This agent induced apoptosis and necrosis, accompanied by the cleavage of poly (ADP‑ribose) polymerase and loss of mitochondrial membrane potential. The pan‑caspase inhibitor, benzyloxycarbonyl‑Val‑Ala‑Asp‑fluoromethylketone, prevented apoptotic cell death and each of the assessed caspase inhibitors inhibited necrotic cell death induced by Au. With respect to the levels of ROS and GSH, Au increased intracellular O2•- in the HeLa cells and induced GSH depletion. The pan‑caspase inhibitor reduced the levels of O2•- and GSH depletion in Au‑treated HeLa cells. The antioxidant, N‑acetyl cysteine, not only attenuated apoptosis and necrosis in the Au‑treated HeLa cells, but also decreased the levels of O2•- and GSH depletion in the cells. By contrast, L‑buthionine sulfoximine, a GSH synthesis inhibitor, intensified cell death O2•- and GSH depletion in the Au‑treated HeLa cells. In conclusion, Au induced apoptosis and necrosis in HeLa cells via the induction of oxidative stress and the depletion of GSH.

  14. The chemosensitivity of cisplatin on cervical cancer Hela cells influenced by PTEN%PTEN影响宫颈癌Hela细胞对顺铂的化疗敏感性

    范幸; 王比男; 李建军

    2012-01-01

    Objective:To investigate whether the chemosensitivity of cisplatin on cervical cancer Hela cell could be influenced by enhanced PTEN(phosphatase and tensin homology deletion on chromosome ten)expression from transfected PTEN vector.Methods: Three groups of Hela cell were cultured: blank control (non-trans-fection),plasmid control (Hela cells transfected with blank plasmid)and PTEN transfected cells (Hela cells transfected with plasmid contains PTEN gene).RT-PCR and Western blotting were used to detect and assess the expression level of the PTEN mRNA and protein in three groups of Hela cells respectively.Three groups of Hela cells were treated with different concentrations of cisplatin.MTT assay was used to assess chemosensitivity in three groups of Hela cells treated with cisplatin.RT-PCR was used to detect the expression level of phosphatidylinositol 3-kinase (PI3K)in three groups of Hela cell.Results:PTEN expression in PTEN transfected Hela cells was significantly higher than in other two groups(P<0.01).After treated with cisplatin,the growth inhibition ratio of PTEN transfected Hela cells was higher than other groups of cells (P<0.01).PI3K expression was significantly inhibited in PTEN transfected cells than the other two groups (P<0.01).Conclusion:PTEN could increase chemosensitivity of cisplatin on cervical cancer Hela cells,which could be induced by reducing drug resistance from inhibition of PI3K in Hela cells by PTEN.%目的:通过转染PTEN基因表达载体,提高宫颈癌Hela细胞中PTEN的表达水平,观察能否影响Hela细胞对顺铂的敏感性,并探讨其分子学机制.方法:将培养的宫颈癌Hela细胞分为空白对照(未转染质粒的Hela细胞)、质粒对照(转染空质粒的Hela细胞)和PTEN转染(转染PTEN表达载体的Hela细胞)3组.分别用RT-PCR和Western检测各组Hela细胞中PTEN的mRNA和蛋白的表达水平;MTT法检测各组Hela细胞对顺铂的化学敏感性;RT-PCR检测各组Hela细胞中磷脂酰肌醇3-

  15. Progress on the study of effective constituent of Chinese medicinals inducing cervical cancer Hela cell apoptosis%中药有效成分影响宫颈癌 Hela 细胞凋亡机制的研究进展

    史海敏; 丁库克; 张新

    2015-01-01

    With the increasing of cervical cancer incidence , the effects of Chinese medicinals in treating cervical cancer radiotherapy and chemotherapy side reaction and postoperative complications were gradually attached importance to .Influencing Bax/Bcl-2 family, Caspase family , Fas/Fasl system and the expression of some related genes could induce cervical cancer Hela cells apoptosis .This article focuses on several kinds of Chinese medicinals , summarizes the faction of the mechanism of effective constituent of these Chinese medicinals in cervical cancer Hela cells apoptosis .For example , the effective constituent of curcuma zedoaria promotes Hela cell apoptosis by down-regulated expression of Bcl-2.The procedure of promoting He-la cell apoptosis of Trichosanthin has relationship with Caspase famlily .The purposes of researching Chinese medicine effective constituent influnes cervical cancer Hela cell apoptosis are providing the scientific basis for treating cervical cancer with Chinese medicine and ideas about filtering new medicines for the cancer .%目的:随着宫颈癌发病率的逐年上升,中药在治疗宫颈癌放化疗副反应及术后并发症中的作用逐渐被重视。大量研究发现诱导宫颈癌Hela细胞凋亡可以通过影响Bcl-2基因家族、Caspase蛋白酶家族、Fas/Fasl基因系统、某些相关基因的表达等方式来实现。文章从目前国内外研究比较热门的一些中药入手,综述这些中药的有效成分在宫颈癌Hela细胞凋亡中的作用。例如文中提到中药温莪术有效成分β-榄香烯是通过下调Bcl-2基因表达来促进Hela凋亡;栝楼根的有效活性成分天花粉蛋白促凋亡过程与Caspase家族有关;薏苡仁酯诱导Fas表达上调、Fasl表达下调来促进宫颈癌Hela细胞凋亡。研究中药有效成分影响宫颈癌Hela细胞凋亡机制的目的是为临床应用中药治疗宫颈癌提供科学依据和筛选抗宫颈癌新药提供思路。

  16. 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

  17. Enhanced Cancer Cell (HeLa Killing Efficacy of Mixed Αlpha and Gamma Iron Oxide Superparamagnetic Nanoparticles under Combined AC (Alternating Current Magnetic-Field and Photoexcitation

    Md. Shariful Islam, Yoshihumi Kusumoto, Md. Abdulla Al-Mamun and Yuji Horie

    2011-12-01

    Full Text Available We synthesized mixed α and γ-Fe2O3 nanoparticles and investigated their toxic effects against HeLa cells under induced AC (alternating current magnetic-fields and photoexcited conditions at room temperature. The findings revealed that the cell-killing percentage was increased with increasing dose for all types of treatments. Finally, 99% cancer cells were destructed at 1.2 mL dose when exposed to combined AC magnetic-field and photoexcited conditions (T3 whereas 89 and 83 % of HeLa cells were killed under only AC magnetic-field induced (T1 or only photoexcited (T2 condition at the same dose.ABSTRAK: Campuran α dan zarah γ-Fe2O3 bersaiz nano disintesiskan dan kesan toksidnya terhadap sel HeLa dikaji dibawah aruhan medan magnet arus ulang-alik (alternating current (AC dan keadaan photoexcited (proses ransangan atom atau molekul suatu bahan dengan penyerapan tenaga sinaran pada suhu bilik. Penemuan mendedahkan bahawa peratusan sel yang musnah bertambah dengan pertambahan dos untuk semua jenis rawatan. Akhirnya, 99% sel kanser dimusnahkan pada kadar dos 1.2mL setelah didedahkan terhadap kombinasi medan magnet AC dan keadaan photoexcited (T3 dimana 89% dan 83% sel HeLa dimusnahkan dengan hanya di bawah aruhan medan magnet AC (T1 atau hanya pada keadaan photoexcited (T2 pada kadar dos yang sama.KEY WORDS : Cancer, Hyperthermia, Iron oxide nanoparticles, Heat dissipation,    Cytotoxicity, HeLa cell.

  18. Quercetin suppresses HeLa cells by blocking PI3K/Akt pathway.

    Xiang, Tao; Fang, Yong; Wang, Shi-Xuan

    2014-10-01

    To explore the effect of quercetin on the proliferation and apoptosis of HeLa cells, HeLa cells were incubated with quercetin at different concentrations. Cell viability was evaluated by MTT assay, cell apoptosis was detected by Annexin-V/PI double labeled cytometry and DNA ladder assay. Cell cycle was flow cytometrically determined and the morphological changes of the cells were observed under a fluorescence microscope after Hoechst 33258 staining and the apoptosis-related proteins in the HeLa cells were assessed by Western blotting. The results showed that quercetin significantly inhibited the growth of HeLa cells and induced obvious apoptosis in vitro in a time- and dose-dependent manner. Moreover, quercetin induced apoptosis of HeLa cells in cell cycle-dependent manner because quercetin could induce arrest of HeLa cells at G0/G1 phase. Quercetin treatment down-regulated the expression of the PI3K and p-Akt. In addition, quercetin could down-regulate expression of bcl-2, up-regulate Bax, but exerted no effect on the overall expression of Akt. We are led to conclude that quercetin induces apoptosis via PI3k/Akt pathways, and quercetin has potential to be used as an anti-tumor agent against human cervix cancer.

  19. 那可丁对宫颈癌HeLa细胞的抑制作用及机制探讨%Inhibitory Effects of Noscapine on Human Cervical Cancer Cell Line HeLa and Its Mechanism

    苏文敬; 黄磊; 敖启林; 张庆华; 田训; 方勇; 卢运萍

    2011-01-01

    Objective To investigate the effects of noscapine on human cervical cancer cell line HeLa and its mechanism. Methods HeLa cells were cultured in vitro and treated with gradient concentrations of noscapine. MTT assay was used to detect the proliferation of HeLa cells. Soft-agar colony-forming assay was used to measure tumorforming ability. Flow cytometry(FCM) was used to measure apoptosis and cell cycle distribution of HeLa cells. Western blot was used to detect protein levels of Caspase-3 and poly ADP-ribose polymerase(PARP). Results MTT assay showed that noscapine decreased the survival rate of HeLa cells in a time and dosedependent manner(P<0. 05). Softagar colonyforming assay displayed that clone number of HeLa cells treated with 10,20.30 or 40 μmol/L noscapine was (19±3) ,(13±2) ,(4±1) and (3±1) respectively, that was (62±8) in group control, which indicated a significantly decreased tumorforming ability of HeLa cells(P<0. 01). FCM analysis revealed that noscapine increased apoptosis rate of HeLa cells in a time and dose-dependent manner(P<0. 05). Western blot analysis demonstrated that noscapine increased protein levels of Caspase-3 (17 kD) and PARP ( 85 kD) in HelLa cells. Noscapine arrested HeLa cells in G2/M phase ( P < 0. 05 ) . Conclusion Noscapine significantly inhibits HeLa cells. Noscapine-induced G2/M phase arrest may play an important role in the effects.%目的 探讨那可丁对宫颈癌HeLa细胞株的抑制作用及其机制.方法 体外培养人宫颈癌HeLa细胞株,梯度浓度那可丁干预后,MTT测定细胞存活率,软琼脂克隆形成实验检测细胞成瘤能力,Western blot检测Caspase-3、PARP(多聚ADP-核糖聚合酶)蛋白水平变化,流式细胞术检测细胞凋亡率及细胞周期分布.结果 MTT检测发现那可丁显著降低HeLa细胞存活率(P<0.05),该效应具有浓度依赖性和时间依赖性;软琼脂克隆形成实验发现,10、20、30、40 μmol/L那可丁组的细胞克隆计数依次为(19

  20. 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.

  1. Phosphofructokinase-P Modulates P44/42 MAPK Levels in HeLa Cells.

    Cardim Pires, Thyago Rubens; Albanese, Jamille Mansur; Schwab, Michael; Marette, André; Carvalho, Renato Sampaio; Sola-Penna, Mauro; Zancan, Patricia

    2017-05-01

    It is known that interfering with glycolysis leads to profound modification of cancer cell proliferation. However, energy production is not the major reason for this correlation. Here, using HeLa cells as a model for cancer, we demonstrate that phosphofructokinase-P (PFK-P), which is overexpressed in diverse types of cancer including HeLa cells, modulates expression of P44/42 mitogen-activated protein kinase (MAPK). Silencing of PFK-P did not alter HeLa cell viability or energy production, including the glycolytic rate. On the other hand, silencing of PFK-P induced the downregulation of p44/42 MAPK, augmenting the sensitivity of HeLa cells to different drugs. Conversely, overexpression of PFK-P promotes the upregulation of p44/42 MAPK, making the cells more resistant to the drugs. These results indicate that overexpression of PFK-P by cancer cells is related to activation of survival pathways via upregulation of MAPK and suggest PFK-P as a promising target for cancer therapy. J. Cell. Biochem. 118: 1216-1226, 2017. © 2016 Wiley Periodicals, Inc.

  2. The critical role of quercetin in autophagy and apoptosis in HeLa cells.

    Wang, Yijun; Zhang, Wei; Lv, Qiongying; Zhang, Juan; Zhu, Dingjun

    2016-01-01

    In recent years, the effects of quercetin on autophagy and apoptosis of cancer cells have been widely reported, while effects on HeLa cells are still unclear. Here, HeLa cells were subjected to quercetin treatment, and then proliferation, apoptosis, and autophagy were evaluated using MTT, flow cytometry, and MDC staining, respectively. The LC3-I/II, Beclin 1, active caspase-3, and S6K1 phosphorylation were detected using Western blot assay. The ultrastructure of HeLa was observed via transmission electron microscope (TEM). Our findings showed that quercetin can dose-dependently inhibit the growth of HeLa cells. The MDC fluorescence was enhanced with increased concentration of quercetin and hit a plateau at 50 μmol/l. Western blot assay revealed that LC3-I/II ratio, Beclin 1, and active caspase-3 protein were enforced in a dose-dependent method. However, the phosphorylation of S6K1 gradually decreased, concomitant with an increase of autophagy. In addition, TEM revealed that the number of autophagic vacuoles was peaked at 50 μmol/l of quercetin. Besides, interference of autophagy with 3-MA led to proliferation inhibition and increased apoptosis in HeLa cells, accompanied by the decreased LC3-I/II conversion and the increased active caspase-3. In conclusion, quercetin can inhibit HeLa cell proliferation and induce protective autophagy at low concentrations; thus, 3-MA plus quercetin would suppress autophagy and effectively increased apoptosis.

  3. Synthesis of 2',4-dihydroxy-3-methoxychalcone and 2',4',4-trihydroxy-3-methoxychalcone as a candidate anticancer against cervical (WiDr), colon (HeLa), and breast (T47d) cancer cell lines in vitro

    Matsjeh, Sabirin; Swasono, Respati Tri; Anwar, Chairil; Solikhah, Eti Nurwening; Lestari, Endang

    2017-03-01

    The compound 2',4-dihydroxy-3-methoxychalcone and 2',4',4-trihydroxy-3-methoxychalcone have been synthesized through Claisen-Schmidt reaction from 2-hydroxyacetophenone and 2,4-dihydroxyacetophenone with 4-hydroxy-3-methoxy benzaldehida (vanillin) in aqueous KOH 40% and KSF montmorillonite as catalyst in methanol. All these products were characterized by FT-IR, TLC Scanner, GC-MS, MS-Direct, and 1H-NMR and 13C-NMR spectrometer. Both of these compounds were tested citotoxycity activity as an anticancer against cervical, colon, and breast cancer cells (Hela, WiDr, and T47D cell lines) using MTT assay in vitro. Dose series given test solution concentration on Hela, WiDr, and T47D cells started from 6,25; 25; 50 and 100 µg/mL with incubation treatment for 24 hours. The result of study showed that the 2',4-dihydroxy-3-methoxychalcone as bright yellow crystal with the melting point of 114-115 °C and the yield of 13.77% and the 2',4',4-trihydroxy-3-methoxychalcone as bright yellow crystals with the melting point of 195-197 °C and the yield of 6%. Other 2',4-dihydroxy-3-methoxychalcone and 2',4',4-trihydroxy-3-methoxychalcone also exhibited cytotoxic activity against the cancer cell lines, with the 2',4',4-trihydroxy-3-methoxychalcone showed greater activities than the 2',4-dihydroxy-3-methoxychalcone in WiDr cell lines. The 2',4-dihydroxy-3-methoxychalcone and 2',4',4-trihydroxy-3-methoxychalcone exhibited strong anticancer activities with IC50 value below 20 µg/mL. The activity of 2',4',4-trihydroxy-3-methoxychalcone showed the most active against Hela and WiDr cell lines with IC50 value 8.53 and 2.66 µg/mL respectively, than T47D cell lines with IC50 value 24.61 µg/mL. The test results cytotoxic of 2',4-dihydroxy-3-methoxychalcone showed the most active against Hela and WiDr cell lines with IC50 value 12.80, 19.57 µg/mL than T47D cell lines with IC50 value of 20.73 µg/mL. IC50 value indicated that 2',4-dihydroxy-3-methoxychalcone and 2',4',4-trihydroxy-3

  4. The enhancement of propyl gallate-induced apoptosis in HeLa cells by a proteasome inhibitor MG132.

    You, Bo Ra; Park, Woo Hyun

    2011-03-01

    Propyl gallate (PG) used in processed food and medicinal preparations has been shown to induce cell death in normal and cancer cells. The inhibition of proteasome function has emerged as a useful strategy to maneuver apoptosis. Here, we investigated the combined effects of PG and MG132 (a proteasome inhibitor) on HeLa cells in relation to cell growth, cell death, reactive oxygen species (ROS) and glutathione (GSH). PG induced growth inhibition and apoptosis in HeLa cells, accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨm), activation of caspase 3 and PARP cleavage. The levels of ROS and GSH depletion were increased in PG-treated HeLa cells. MG132 intensified apoptosis and PARP cleavage in PG-treated HeLa cells. MG132 also increased ROS levels including mitochondrial O2•-, MMP (ΔΨm) loss and GSH depletion in PG-treated HeLa cells. PG induced a G1 phase arrest of the cell cycle in HeLa cells, which was significantly prevented by MG132. MG132 alone inhibited HeLa cell growth via inducing the cell cycle arrests and triggering apoptosis. Conclusively, the inhibition of proteasome by MG132 plays a role as an enhancement factor in PG-induced apoptosis of HeLa cells via increasing ROS levels and GSH depletion.

  5. Apoptosis of Hela cell induced by Celastrus orbiculatus Thunb extract and primary mechanisms

    Weimin Wang; Yanqing Liu; Xiaojun Dai

    2011-01-01

    Objective: The apoptosis of Hela cells induced by ethyl acetate extract and n-butanol extract of Celastrus or-biculatus Thunb was studied in order to assess its antitumor effect. Methods: Hela cells were cultured in vitro and treated by a series of concentrations of ethyl acetate extract and n-butanol extract of Celastrus orbiculatus Thunb. Cell proliferation was detected based on MTT assay. Quantity of apoptosis were observed and analyzed by flow cytometry with Annexin V and propidium iodide double staining. P53 gene expression was detected by flow cytometry. Results: The proliferation of Hela cells was obviously inhibited by 15, 30, 60 and 120 μg/mL extract of Celastrus orbiculatus Thunb and apoptosis of Hela was induced by dosed dependent manner. P53 gene showed increasing tendency when treated by 60-480 μg/mL extract of Celas-trus orbiculatus Thunb. Conclusion: The ethyl acetate extract and n-butanol extract of Celastrus orbiculatus Thunb could induce apoptosis of Hela gastric cancer cells by dose dependent manner, which maybe one of the important mechanisms of Celastrus orbiculatus Thunb's anticancer effects. P53 protein expression in Hela was up-regulated by Celastrus orbiculatus Thunb, which maybe one of the molecular mechanisms involved in the anticancer and proapoptotic effect of Celastrus or-biculatus Thunb.

  6. PSD-007光动力对人子宫颈癌Hela细胞体外杀伤效应研究%STUDY ON PHOTODYNAMIC EFFECT OF PSD-007 ON HUMAN CERVICAL CANCER CELL LINE HE-LA

    张洪梅; 关毅; 王永恒; 白璐; 王宏; 秦雪元; 赵继华; 王婧瑶

    2016-01-01

    Objective To investigate the killing effects and the main affecting factors of the photosensizer with 635nm laser based photodynamic therapy on human cervical cancer cell line Hela cell in vitro .Methods MTT assay were performed to examine human cervical cancer cell line Hela cells treated with different concentrations of PSD - 007 and different intensities of laser ,to explore cell proliferation in different concentrations of PSD - 007 and different intensities of laser .Different intensities of laser (1 .2 ,2 .4 ,4 .8J/ cm2 ) treated Hela cells with 6 .25μg/mL of PSD - 007 .The morphological changes of Hela cells were observed by DAPI staining by microscope .Results Significant differences in the inhibitory was observed in PSD - 007 mediated photodynamic therapy(P< 0 .05) .Survival rates of Hela cells declined with more than 25μg/ml of PSD - 007 only .The survival rates of Hela after decreased by the concentration of sensitizer and dose of laser .There were no significant diffierences of cell survival rates among the groups with concentra ‐ tion more than 12 .5μg/ml and laser energy density more than 4 .8J/cm2 .The morphological study found that the multinucleate giant cells existed in the apoptotic cells after PSD - 007 mediated photodynamic therapy .Conclusion Photosensitizer PSD - 007 has low toxicity and efficient features .DTPP mediated photodynamic therapy can kill human breast cancer cell line Hela in vitro effectively and show a dose - time - dependent effect .PSD - 007 - PDT can induce typical apoptotic cells .%目的:探讨不同剂量癌光啉(PSD -007)及不同激光照射能量对人宫颈癌 Hela细胞增殖和凋亡的影响。方法通过采用不同质量浓度的PSD-007、不同激光照射能量介导的光动力作用于Hela细胞,采用噻唑蓝(M T T )比色法测定细胞的光密度(OD )值,计算细胞存活率;6.25μg/ml的 PSD -007作用于Hela细胞,以不同能量的激光照射,DAPI染色观察

  7. 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.

  8. Phorbol Esters from Jatropha Meal Triggered Apoptosis, Activated PKC-δ, Caspase-3 Proteins and Down-Regulated the Proto-Oncogenes in MCF-7 and HeLa Cancer Cell Lines

    Syahida Ahmad

    2012-09-01

    Full Text Available Jatropha meal produced from the kernel of Jatropha curcas Linn. grown in Malaysia contains phorbol esters (PEs. The potential benefits of PEs present in the meal as anticancer agent are still not well understood. Hence, this study was conducted to evaluate the cytotoxic effects and mode of actions of PEs isolated from Jatropha meal against breast (MCF-7 and cervical (HeLa cancer cell lines. Isolated PEs inhibited cells proliferation in a dose-dependent manner of both MCF-7 and HeLa cell lines with the IC50 of 128.6 ± 2.51 and 133.0 ± 1.96 µg PMA equivalents/mL respectively, while the values for the phorbol 12-myristate 13-acetate (PMA as positive control were 114.7 ± 1.73 and 119.6 ± 3.73 µg/mL, respectively. Microscopic examination showed significant morphological changes that resemble apoptosis in both cell lines when treated with PEs and PMA at IC50 concentration after 24 h. Flow cytometry analysis and DNA fragmentation results confirmed the apoptosis induction of PEs and PMA in both cell lines. The PEs isolated from Jatropha meal activated the PKC-δ and down-regulated the proto-oncogenes (c-Myc, c-Fos and c-Jun. These changes probably led to the activation of Caspase-3 protein and apoptosis cell death occurred in MCF-7 and HeLa cell lines upon 24 h treatment with PEs and PMA. Phorbol esters of Jatropha meal were found to be promising as an alternative to replace the chemotherapeutic drugs for cancer therapy.

  9. Phorbol esters from Jatropha meal triggered apoptosis, activated PKC-δ, caspase-3 proteins and down-regulated the proto-oncogenes in MCF-7 and HeLa cancer cell lines.

    Oskoueian, Ehsan; Abdullah, Norhani; Ahmad, Syahida

    2012-09-10

    Jatropha meal produced from the kernel of Jatropha curcas Linn. grown in Malaysia contains phorbol esters (PEs). The potential benefits of PEs present in the meal as anticancer agent are still not well understood. Hence, this study was conducted to evaluate the cytotoxic effects and mode of actions of PEs isolated from Jatropha meal against breast (MCF-7) and cervical (HeLa) cancer cell lines. Isolated PEs inhibited cells proliferation in a dose-dependent manner of both MCF-7 and HeLa cell lines with the IC₅₀ of 128.6 ± 2.51 and 133.0 ± 1.96 µg PMA equivalents/mL respectively, while the values for the phorbol 12-myristate 13-acetate (PMA) as positive control were 114.7 ± 1.73 and 119.6 ± 3.73 µg/mL, respectively. Microscopic examination showed significant morphological changes that resemble apoptosis in both cell lines when treated with PEs and PMA at IC₅₀ concentration after 24 h. Flow cytometry analysis and DNA fragmentation results confirmed the apoptosis induction of PEs and PMA in both cell lines. The PEs isolated from Jatropha meal activated the PKC-δ and down-regulated the proto-oncogenes (c-Myc, c-Fos and c-Jun). These changes probably led to the activation of Caspase-3 protein and apoptosis cell death occurred in MCF-7 and HeLa cell lines upon 24 h treatment with PEs and PMA. Phorbol esters of Jatropha meal were found to be promising as an alternative to replace the chemotherapeutic drugs for cancer therapy.

  10. Photodynamic Effects of Pterin on HeLa Cells

    Denofrio, M. Paula; Lorente, Carolina; Breitenbach, Thomas

    2011-01-01

    cells (HeLa) and that these cells die upon UV-A irradiation of Ptr. Cell death was assessed using two tests: (1) the Rhodamine 123 fluorescence assay for mitochondrial viability and (2) the Trypan Blue assay for membrane integrity. The data suggest that, for Ptr-dependent photoinitiated cell death...

  11. 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

  12. 宫颈癌 HeLa 细胞抗顺铂作用的 Notch1 机制研究%Notch1 Mediates the Anti-cisplatin Character of Cervical Cancer LeLa Cell Line

    杨晶; 张军红; 马慧娟; 王志华; 王琼; 王川

    2015-01-01

    目的 利用宫颈癌HeLa细胞系探讨宫颈癌的抗化疗机制. 方法 利用球形成试验( sphere forming assay )和Western blot法检测顺铂对癌症干细胞形成的影响以及 Notch1在HeLa细胞系的表达. 基因沉默试验分析Notch1基因是否在宫颈癌HeLa癌症干细胞中发挥关键作用. 结果 顺铂能促进球结构(意味着癌症干细胞形成)的形成. 5μmol/L顺铂较1μmol/L顺铂能促进更多球结构形成(P<0.05). 10μmol/L顺铂会抑制球结构形成. 5μmol/L顺铂还能提高HeLa中Notch1的表达(P<0.05),而沉默HeLa细胞的Notch1后,HeLa细胞中形成的球数量降低1.7倍(P<0.05). 结论 宫颈癌HeLa细胞系具有抗顺铂治疗的特性,尤其是5μmol/L的顺铂,该抗化疗作用与宫颈癌HeLa细胞中的Notch1表达升高,最终促进癌症干细胞自我更新有关.%Objective To explore the anti -chemotherapy mechanism of cervical cancer using the cervical cancer HeLa cell line . Methods Sphere forming assay and Western blotting were used to measure the potential effects of Cisplatin on cancer stem cell formation and Notch1 expression in HeLa cells .Gene silencing assay was performed to analyze whether Notch 1 played a key role in the proliferation of HeLa cancer stem cells .Results Cispaltin basically promoted the formation of ball structures ( an indicator of cancer stem cells ) ex-cept at larger concentration (10μmol/L).Cispaltin at 5μmol/L exerted greater promoting effect on the ball structures compared with that at 1μmol/L (P<0.05).Cispaltin at 10μmol/L inhibited the formation of ball structures .Cispaltin at 5μmol/L also promoted Notch1 ex-pression in HeLa cells (P<0.05), while silencing Notch1 decreased ball structure formation by 1.7 folds (P<0.05).Conclusion Cervical cancer HeLa cell line behaves an character of anti -Cispaltin therapy especially at 5μmol/L, and this tendency is likely associat-ed the promoting effect of Cisplatin on the expression of the self -renewal factor

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

    Fariba Samani; Ali Shabestani Monfared; Ebrahim Zabihi; Soraya Khafri; Maesoumeh Karimi; Haleh Akhavan Niaki

    2014-01-01

    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 m...

  14. Suberoyl bishydroxamic acid-induced apoptosis in HeLa cells via ROS-independent, GSH-dependent manner.

    You, Bo Ra; Park, Woo Hyun

    2013-05-01

    Suberoyl bishydroxamic acid (SBHA) is a HDAC inhibitor that can regulate many biological functions including apoptosis and proliferation in various cancer cells. Here, we evaluated the effect of SBHA on the growth of HeLa cervical cancer cells in relation to apoptosis, reactive oxygen species (ROS) and glutathione (GSH) levels. Dose-dependent inhibition of cell growth was observed in HeLa cells with an IC50 of approximately 15 μM at 72 h. SBHA also induced apoptosis in HeLa cells, as evidenced by sub-G1 cells, annexin V-FITC staining cells, activations of caspase 3 and 8, and the loss of mitochondrial membrane potential (ΔΨm). In addition, all of the tested caspase inhibitors rescued some cells from SBHA-induced HeLa cell death. SBHA increased ROS levels including O2(•-) and induced GSH depletion in HeLa cells. Generally, caspase inhibitors did not affect ROS levels in SBHA-treated HeLa cells, but they significantly prevented GSH depletion in these cells. Furthermore, while the well-known antioxidants, N-acetyl cysteine and vitamin C, did not affect cell death, ROS level or GSH depletion in SBHA-treated HeLa cells, L-buthionine sulfoximine, a GSH synthesis inhibitor, enhanced cell death and GSH depletion in these cells. In conclusion, SBHA inhibits the growth of HeLa cervical cancer cells via caspase-dependent apoptosis, and the inhibition is independent of ROS level changes, but dependent on GSH level changes.

  15. 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.

  16. Autophagy facilitates Salmonella replication in HeLa cells.

    Yu, Hong B; Croxen, Matthew A; Marchiando, Amanda M; Ferreira, Rosana B R; Cadwell, Ken; Foster, Leonard J; Finlay, B Brett

    2014-03-11

    Autophagy is a process whereby a double-membrane structure (autophagosome) engulfs unnecessary cytosolic proteins, organelles, and invading pathogens and delivers them to the lysosome for degradation. We examined the fate of cytosolic Salmonella targeted by autophagy and found that autophagy-targeted Salmonella present in the cytosol of HeLa cells correlates with intracellular bacterial replication. Real-time analyses revealed that a subset of cytosolic Salmonella extensively associates with autophagy components p62 and/or LC3 and replicates quickly, whereas intravacuolar Salmonella shows no or very limited association with p62 or LC3 and replicates much more slowly. Replication of cytosolic Salmonella in HeLa cells is significantly decreased when autophagy components are depleted. Eventually, hyperreplication of cytosolic Salmonella potentiates cell detachment, facilitating the dissemination of Salmonella to neighboring cells. We propose that Salmonella benefits from autophagy for its cytosolic replication in HeLa cells. IMPORTANCE As a host defense system, autophagy is known to target a population of Salmonella for degradation and hence restricting Salmonella replication. In contrast to this concept, a recent report showed that knockdown of Rab1, a GTPase required for autophagy of Salmonella, decreases Salmonella replication in HeLa cells. Here, we have reexamined the fate of Salmonella targeted by autophagy by various cell biology-based assays. We found that the association of autophagy components with cytosolic Salmonella increases shortly after initiation of intracellular bacterial replication. Furthermore, through a live-cell imaging method, a subset of cytosolic Salmonella was found to be extensively associated with autophagy components p62 and/or LC3, and they replicated quickly. Most importantly, depletion of autophagy components significantly reduced the replication of cytosolic Salmonella in HeLa cells. Hence, in contrast to previous reports, we propose

  17. Trichostatin A induces apoptotic cell death of HeLa cells in a Bcl-2 and oxidative stress-dependent manner.

    You, Bo Ra; Park, Woo Hyun

    2013-01-01

    Trichostatin A (TSA) as a HDAC inhibitor can regulate many biological properties including apoptosis and cell proliferation in various cancer cells. Here, we evaluated the effect of TSA on the growth and death of HeLa cervical cancer cells in relation to reactive oxygen species (ROS) and glutathione (GSH) levels. Dose- and time-dependent growth inhibition was observed in HeLa cells with an IC50 of approximately 20 nM at 72 h. This agent also induced apoptotic cell death, as evidenced by annexin V-FITC staining cells, caspase-3 activation and the loss of mitochondrial membrane potential (MMP; ∆ψm). In addition, the administration of Bcl-2 siRNA intensified TSA-induced HeLa cell death. All of the tested caspase inhibitors significantly rescued some cells from TSA-induced HeLa cell death. TSA increased O2•- level and induced GSH depletion in HeLa cells. Caspase inhibitors significantly attenuated O2•- level and GSH depletion in TSA-treated HeLa cells. In addition, N-acetyl cysteine (NAC; a well known antioxidant) significantly prevented cell death and GSH depletion in TSA-treated HeLa cells via decreasing O2•- level. In conclusion, TSA inhibited the growth of HeLa cells via Bcl-2-mediated apoptosis, which was closely related to O2•- and GSH content levels.

  18. 曲古霉素A对宫颈癌HeLa细胞的毒性及放射增敏作用%Effect of Trichostatina A on Cytotoxicity and Cell Radiosensitivity of Human Cervical Cancer Cell Line HeLa

    田晓予; 余娟娟; 米建强; 王爱红; 刘华

    2011-01-01

    Objective To investigate the effects of trichostatin A(TSA) on cytotoxicity and radiosensitivity of human cervical cancer cell line HeLa, Methods HeLa cells were treated with different concentrations of TSA for 12, 24, 48 and 72 hours. Cell proliferation and radiosensitizing effect of IC10 TSA combining different low dose of fractionated radiotherapy was detected by methylthiazolyl tetrazolium (MTT) assay. Cloniesformation was used to determine the radiosensitization effect induced by 10% inhibition concentration of TSA. Results TSA siginificantly inhibited the proliferation of HeLa cells in a dose-and time-dependent manner,the 50% inhibition concentration and 10% inhibition concentration of co-treatment with TSA for 24 hours were 0. 0312 and 0. 5865 μmol/L, respectively; and when concentration over 0. 6 μmol/L and treatment time longer than 48 hours,TSA showed no more cytotoxicity. When exposed to TSA for 24 h, the SER of HeLa cells was 1. 6858,SERs of lC10 TSA combining 2. 0 Gy/time, 1. 0 Gy/time( × 2) ,0. 5 Gy/time ( × 4) fractionated radiotherapy groups were 1. 4496,1. 6410 and 1. 9554,respectively. Conclusion TSA can significantly enhance radiosensitivity of HeLa cells,especially combining with low dose fractionated radiotherapy.%目的 探讨曲古霉素A(TSA)对宫颈癌HeLa细胞的毒性及放射增敏作用.方法MTT法检测TSA处理宫颈癌HeLa细胞12、24、48、72 h的细胞毒性及TSA作用24 h的10%细胞抑制浓度(10%inhibition concentration,IC10)和半数抑制率IC50对HeLa细胞不同分割放疗方式的增敏效果;克隆形成法分析IC10的TSA对HeLa细胞放射增敏作用的影响,单击多靶模型拟合细胞存活曲线,计算放射增敏比(sensitive enhencement ratio,SER).结果0.05~0.8 μmol/L TSA对HeLa细胞具有增殖抑制作用,且呈一定的时间-剂量依赖性,TSA作用24 h的IC10和IC50,分别为0.0312和0.5865.0.6μmol/L以上浓度及药物作用时间超过48 h的TSA对HeLa细胞的增殖抑

  19. Glucose capped silver nanoparticles induce cell cycle arrest in HeLa cells.

    Panzarini, Elisa; Mariano, Stefania; Vergallo, Cristian; Carata, Elisabetta; Fimia, Gian Maria; Mura, Francesco; Rossi, Marco; Vergaro, Viviana; Ciccarella, Giuseppe; Corazzari, Marco; Dini, Luciana

    2017-02-20

    This study aims to determine the interaction (uptake and biological effects on cell viability and cell cycle progression) of glucose capped silver nanoparticles (AgNPs-G) on human epithelioid cervix carcinoma (HeLa) cells, in relation to amount, 2×10(3) or 2×10(4) NPs/cell, and exposure time, up to 48h. The spherical and well dispersed AgNPs (30±5nm) were obtained by using glucose as reducing agent in a green synthesis method that ensures to stabilize AgNPs avoiding cytotoxic soluble silver ions Ag(+) release. HeLa cells take up abundantly and rapidly AgNPs-G resulting toxic to cells in amount and incubation time dependent manner. HeLa cells were arrested at S and G2/M phases of the cell cycle and subG1 population increased when incubated with 2×10(4) AgNPs-G/cell. Mitotic index decreased accordingly. The dissolution experiments demonstrated that the observed effects were due only to AgNPs-G since glucose capping prevents Ag(+) release. The AgNPs-G influence on HeLa cells viability and cell cycle progression suggest that AgNPs-G, alone or in combination with chemotherapeutics, may be exploited for the development of novel antiproliferative treatment in cancer therapy. However, the possible influence of the cell cycle on cellular uptake of AgNPs-G and the mechanism of AgNPs entry in cells need further investigation.

  20. New cancer-treatment model of photodynamic therapy combined with a type I topoisomerase inhibitor, CPT-11, against HeLa cell tumors in nude mice used by OPO parametric tunable laser

    Yoshida, Takato O.; Matsuzawa, Eiji; Matsuo, Tetsumichi; Koide, Yukio; Terakawa, Susumu; Yokokura, Teruo; Hirano, Toru

    1995-03-01

    A new cancer-treatment model, photodynamic therapy (PDT) combined with a type I topoisomerase inhibitor, camptothecin derivative (CPT-11), against HeLa cell tumors in BALB/c nude mice has been developed using a wide-band tunable coherent light source operated on optical parametric oscillation (OPO parametric tunable laser). The Photosan-3 PDT and CPT-11 combined therapy was remarkably effective, that is the inhibition rate (I.R.) 40 - 80%, as compared to PDT only in vivo. The analysis of HpD (Photosan-3) and CPT-11 effects on cultured HeLa cells in vitro has been studied by a video-enhanced contrast differential interference contrast microscope (VEC-DIC). Photosan-3 with 600 nm light killed cells by mitochondrial damage within 50 min, but not with 700 nm light. CPT-11 with 700 - 400 nm light killed cells within 50 min after nucleolus damage appeared after around 30 min. The localization of CPT-11 in cells was observed as fluorescence images in the nucleus, particularly the nucleoral area produced clear images using an Argus 100.

  1. 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.

  2. X染色体连锁的凋亡抑制蛋白对米非司酮诱导宫颈癌HeLa细胞凋亡的影响%The Influence of Mifepristone on Induction of Apoptosis and Effectiveness of XIAP in Uterine Cervix Cancer HeLa Cells

    汤飒爽

    2011-01-01

    目的 观察X染色体连锁的凋亡抑制蛋白(XIAP蛋白)对米非司酮诱导宫颈癌HeLa细胞凋亡的影响.方法 XIAF重组质粒转染HeLa细胞,共聚焦显微镜观察其在细胞内的分布;流式细胞仪检测米非司酮处理和未处理的各组细胞凋亡率.结果 XIAP在HeLa细胞中弥漫分布于细胞核和细胞浆,米非司酮可诱导HeLA细胞凋亡,XIAP能抑制由米非司酮诱导的HeLa细胞凋亡.结论 米非司酮能促进HeLa细胞凋亡,XIAP能抑制由米非司酮诱导的HeLa细胞凋亡.%Objective To investigate the effects of mifepristone -induced apoptosis in uterine cervix cancer cell line HeLa and the influence of mifepristone on the effectiveness of XIAP in uterine cervix cancer cells. Methods Human uterine cervix cancer cells lines HeLa were used for the current project. Expression,intracellular localization and function of the XIAP in HeLa cells were observed by Laser scanning confocal microscope after transfection. HeLa cells were induced by mifepristone. At the same time,the apoptosis were analyzed for different groups by FCM. Results Expression of DsRed2 - XIAP fusion proteins in HeLa cells were observed in both nucleus and cytoplasm of HeLa cells. HeLa cells which were induced by mifepristone showed partly apoptosis. The DsRed2 - XIAP fusion protein can inhibit apoptosis of HeLa cells by inducing of mifepristone. Conclusion The mifepristone can promote apoptosis of HeLa cells. The DsRed2 - XIAP fusion protein can inhibit apoptosis of HeLa cells by inducing of mifepristone.

  3. Effect of a bispidinone analog on mitochondria‑mediated apoptosis in HeLa cells.

    Yi, Myeongjin; Parthiban, Paramasivam; Hwang, Jiyoung; Zhang, Xin; Jeong, Hyunjin; Park, Dong Ho; Kim, Dong-Kyoo

    2014-01-01

    The present study was carried out to investigate the effect of 2,4,6,8-tetraaryl-3,7-diazabicyclo[3.3.1]nonan-9-one (bispidinone) analogs on the in vitro growth of human cervical carcinoma (HeLa) cells. A series of 11 bispidinone analogs was synthesized with substituents, e.g., fluoro/methyl/ethyl/isopropyl/thiomethyl/methoxy groups, at various positions. These compounds were synthesized to identify which substituent and position induced the strongest cytotoxic effect in cancer cells. Among these synthetics, analog 9, which contains methoxy groups, had the most significant cytotoxic effect on HeLa cells, and its IC50 value was less than 13 µM. A WST-8 assay also showed that analog 9 inhibited the proliferation of HeLa cells. By using DNA content analysis, we found that analog 9 induced sub-G1 and G1 phase arrest in a time-dependent manner. A [3H]-thymidine incorporation assay suggested that analog 9 inhibited DNA replication in HeLa cells. On performing light microscopy, morphological changes such as cellular shrinkage and disruption, which are apoptotic features, were observed in HeLa cells. Annexin V/propidium iodide double staining and rhodamine-123 staining showed that analog 9 induced apoptosis and disrupted the intracellular mitochondrial membrane potential in HeLa cells. The western blot analysis results suggested that analog 9 induced mitochondria-mediated apoptosis. In addition, we have shown that analog 9 may play a role in the Fas signaling apoptotic pathway.

  4. 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.

  5. Immuno-detection of OCTN1 (SLC22A4) in HeLa cells and characterization of transport function.

    Pochini, Lorena; Scalise, Mariafrancesca; Indiveri, Cesare

    2015-11-01

    OCTN1 was immuno-detected in the cervical cancer cell HeLa, in which the complete pattern of acetylcholine metabolizing enzymes is expressed. Comparison of immuno-staining intensity of HeLa OCTN1 with the purified recombinant human OCTN1 allowed measuring the specific OCTN1 concentration in the HeLa cell extract and, hence calculating the HeLa OCTN1 specific transport activity that was about 10 nmol×min(-1)×mg protein(-1), measured as uptake of [(3)H]acetylcholine in proteoliposomes reconstituted with HeLa extract. This value was very similar to the specific activity of the recombinant protein. Acetylcholine transport was suppressed by incubation of the protein or proteoliposomes with the anti-OCTN1 antibody and was strongly inhibited by PLP and MTSEA, known inhibitors of OCTN1. The absence of ATP in the internal side of proteoliposomes strongly impaired transport function of both the HeLa and, as expected, the recombinant OCTN1. HeLa OCTN1 was inhibited by spermine, NaCl (Na(+)), TEA, γ-butyrobetaine, choline, acetylcarnitine and ipratropium but not by neostigmine. Besides acetylcholine, choline was taken up by HeLa OCTN1 proteoliposomes. The transporter catalyzed also acetylcholine and choline efflux which, differently from uptake, was not inhibited by MTSEA. Time course of [(3)H]acetylcholine uptake in intact HeLa cells was measured. As in proteoliposomes, acetylcholine transport in intact cells was inhibited by TEA and NaCl. Efflux of [(3)H]acetylcholine occurred in intact cells, as well. The experimental data concur in demonstrating a role of OCTN1 in transporting acetylcholine and choline in HeLa cells.

  6. Effect of Rhizoma Curcumae on proliferation and immune function of Hela cells of cervical cancer%莪术对人宫颈癌Hela细胞增殖及免疫功能的影响

    任艳珍; 索玉平; 李晓旭; 郭敏红

    2015-01-01

    目的:探讨莪术对人宫颈癌细胞的增殖及免疫功能的影响。方法将50只裸鼠移植瘤模型简单随机分成5组,分别给予不同的处理,观察小鼠的免疫功能及癌组织的病理结果变化;用 M TT 方法检测莪术水煎液对宫颈癌 Hela 细胞的抑制作用。结果莪术抑制宫颈癌细胞的生长,最高达(71.2±3.4)%,呈时间、浓度依赖性;不同剂量莪术组与顺铂组相比较,莪术对小鼠免疫功能影响不大,白细胞降低不明显,差异有统计学意义;病理切片可见莪术治疗组的癌组织可出现不同程度的坏死。结论莪术抑制宫颈癌细胞的生长,促进癌细胞凋亡,对机体的不良反应小,较安全。%Objective Effect of Rhizoma Curcumae proliferation and immune function of cervical cancer cell . Methods The model of transplanted tumor of 50 mice were randomly divided into 5 groups ,were given different treatment ,pathological results observation of immune function of mice and cancer tissue changes ;inhibitory effect on Hela cells of cervical cancer by M T T method for detection of zedoary Decoction• Results Rhizoma Curcumae inhibit the growth of cervical cancer cells ,up to (71 .2 ± 3 .4)% ,which shows the dependent of time and concen‐tration ;Compare cisplatin group with different doses of Rhizoma Curcumae group ,little effect of Rhizoma Cur‐cumae on immune function in mice ,the decrease of white blood cell is not obvious ,the difference is statistically significant ;pathological section shows cancer tissue of Rhizoma Curcuma treatment group can appear different de‐gree of necrosis .Conclusion Conclusion Rhizoma Curcumae inhibit the growth of cervical cancer cells ,and pro‐mote apoptosis of cancer cells ,small side effects on the body ,relatively safe.

  7. Effects of garcinol on the radiosensitivity of human cervical cancer Hela cells%garcinol对宫颈癌Hela细胞放射敏感性影响的研究

    孙玲娣; 王侠; 张艳; 章龙珍

    2014-01-01

    目的:研究多聚异戊二烯基苯甲酮( garcinol )对宫颈癌Hela细胞株放射敏感性的影响。方法不同浓度garcinol作用于人宫颈癌Hela细胞12、24、48 h,CCK-8法检测garcinol对Hela细胞增殖活性的影响,并计算出24 h时的20%和50%的药物抑制浓度( IC20和IC50)值;克隆形成实验检测20μmol/L garcinol对Hela 细胞放射敏感性的影响;反转录PCR法和Western blot法检测Hela细胞内环氧化酶-2(cyclooxygenase -2, COX-2)在mRNA水平和蛋白表达水平的变化。结果 garcinol抑制宫颈癌Hela细胞的生长,并且有明显的时间-剂量依赖性;通过单击多靶模型计算放射生物学参数,经20μmol/L garcinol处理24 h的 Hela细胞,与单纯照射组相比,放射敏感性增加(t=6.69,P<0.05),放射增敏比(SER)为1.26;经20μmol/L garcinol 处理24 h的Hela细胞,COX-2的蛋白表达水平下降(P<0.05),而COX-2 mRNA水平无明显变化(P>0.05)。结论 garcinol 可以增加宫颈癌Hela细胞的放射敏感性,这可能与其降低Hela细胞中COX-2的蛋白表达水平有关。%Objective To investigate the effects of polyisoprenylated benzophenone ( garcino ) on the radiosensitivity of human cervical cancer Hela cells .Methods Hela cells were exposed to different concentrations of garcinol for 12 , 24 and 48 hours.The proliferative rate of the cells was detected by CCK -8 assay, while the values of IC20 and IC50 were calculated after 24 hours of incubation .Clonogenic survival assay was adopted to evaluate the effects of 20μmol/L garci-nol on the radiosensitivity of Hela cells .The levels of COX -2 mRNA and protein were measured by RT -PCR and Western blotting , respectively .Results Garcinol could inhibit the growth of Hela cells in time -and dose -dependent ways.Incubation with 20μmol/L garcinol for 24 house stimulated the radiosensitivity of the cells (t=6.69, P0.05 ) .Conclusion Garcinol can enhance

  8. Loss of Selenium-Binding Protein 1 Decreases Sensitivity to Clastogens and Intracellular Selenium Content in HeLa Cells

    Zhao, Changhui; Zeng, Huawei; Wu, Ryan T. Y.; Cheng, Wen-Hsing

    2016-01-01

    Selenium-binding protein 1 (SBP1) is not a selenoprotein but structurally binds selenium. Loss of SBP1 during carcinogenesis usually predicts poor prognosis. Because genome instability is a hallmark of cancer, we hypothesize that SBP1 sequesters cellular selenium and sensitizes cancer cells to DNA-damaging agents. To test this hypothesis, we knocked down SBP1 expression in HeLa cervical cancer cells by employing a short hairpin RNA (shRNA) approach. Reduced sensitivity to hydrogen peroxide, paraquat and camptothecin, reactive oxygen species content, and intracellular retention of selenium after selenomethionine treatment were observed in SBP1 shRNA HeLa cells. Results from Western analyses showed that treatment of HeLa cells with selenomethionine resulted in increased SBP1 protein expression in a dose-dependent manner. Knockdown of SBP1 rendered HeLa cells increased expression of glutathione peroxidase-1 but not glutathione peroxidase-4 protein levels and accelerated migration from a wound. Altogether, SBP1 retains supplemental selenium and sensitizes HeLa cancer cells to clastogens, suggesting a new cancer treatment strategy by sequestering selenium through SBP1. PMID:27404728

  9. APOPTOSIS INDUCTION BY THE RECOMBINANT FUSION APOPTOSIS INDUCING FACTOR ON HELA CELLS

    于翠娟; 孟艳玲; 桂俊豪; 赵晶; 金明; 王智; 王成济; 杨安钢

    2003-01-01

    Objective: To obtain the recombinant fusion AIF genes inserted into the eukaryotic expression vector Pires2-EGFP, to observe the expression and location of the fusion AIF genes (3NE: PE(280-358)-AIFΔ1-120, and 4NE: PE(280-364)-AIFΔ1-120), and to detect and compare their apoptosis inducing effects on the transfected HeLa cells. Methods: Full-length human AIF gene was cloned by RT-PCR, and its N-terminal mitochondrial localization sequence (MLS) was replaced by part sequence of Psuedomonas exotoxin A (PE) translocation domain (PEII(280-358/364)), then the recombinant fusion genes were inserted into the Pires2-EGFP eukaryotic expression vector. After these genes were transiently transfected into HeLa cells with LipofectAmine, the expression of the recombinant fusion AIF genes and their effects on HeLa cells were detected by fluorescent microscopy, laser confocal microscopy and electron microscopy. Results: The eukaryotic expression vectors containing the recombinant fusion AIF genes (Pires2-EGFP-PEII(280-358/364)- AIFΔ1- 120) were constructed successfully. It was demonstrated that the fusion AIF protein genes were expressed effectively in the transfected cells, with the GFP comco-expressed in cells by indirect immunofluorescence staining analysis. After transfection, expression of the genes could induce HeLa cells to exhibit the typical apoptosis features: such as plasma membrane blebbing and peripheral chromatin condensation. As compared with control groups, the untreated cells and the void vector transfected cells, the living cell number of the AIF gene transfected cells reduced distinctly. Conclusion: Our data prove that the expression of the recombinant human AIF fusion genes could induce apoptosis in transfected HeLa cells, which provides new strategy for cancer killing.

  10. 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

  11. Antiproliferative effects of Tanaceti partheni, Hypericum perforatum and propolis on HeLa cells

    Cenić-Milošević Desanka

    2014-01-01

    Full Text Available Tanaceti partheni, Hypericum perforatum and propolis have been widely used for centuries and are well-documented medicinal plants and natural product. In this study, we investigated the antiproliferative effects of water extracts of ethanolic dry extracts of two different medicinal plants (Tanaceti partheni and Hypericum perforatum and propolis on HeLa cells. The Tanaceti partheni extract exhibited mild cytotoxic activity. The IC50 was 153.71 μg/mL. The extract of Hypericum perforatum did not show active cytotoxic activity against HeLa cells (IC50 >200 μg/mL. Regarding the antiproliferative effects of Hypericum perforatum, our results are not in correlation with the results of other authors, probably because different Hypericum species and different human cancer cell lines were used. The extract of propolis did not show active cytotoxic activity against HeLa cells (IC50 = 1.08 ± 0.01 mg/mL. The weak antiproliferative effect of propolis on HeLa cells is either due to the use of a low concentration of propolis extracted in weakly polar solvents, or the use of propolis collected in the autumn. [Projekat Ministarstva nauke Republike Srbije, br. 34021 i br. 175011

  12. Cesium reversibly suppresses HeLa cell proliferation by inhibiting cellular metabolism.

    Kobayashi, Daisuke; Kakinouchi, Kei; Nagae, Tomoki; Nagai, Toshihiko; Shimura, Kiyohito; Hazama, Akihiro

    2017-03-01

    The aim of the present study was to investigate the influence of Cs(+) on cultured human cells. We find that HeLa cell growth is suppressed by the addition of 10 mm CsCl into the culture media. In the Cs(+) -treated cells, the intracellular Cs(+) and K(+) concentrations are increased and decreased, respectively. This leads to a decrease in activity of the glycolytic enzyme pyruvate kinase, which uses K(+) as a cofactor. Cs(+) -treated cells show an intracellular pH shift towards alkalization. Based on these results, CsCl presumably suppresses HeLa cell proliferation by inducing an intracellular cation imbalance that affects cell metabolism. Our findings may have implications for the use of Cs(+) in cancer therapy.

  13. 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.

  14. 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

    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 ALG-2 has an anti-apoptotic function in HeLa cells by facilitating the passage through checkpoints in the G2/M cell cycle phase.......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...

  15. Inhibitory effects and underlying mechanism of 7-hydroxyflavone phosphate ester in HeLa cells.

    Zhang, Ting; Du, Jiang; Liu, Liguo; Chen, Xiaolan; Yang, Fang; Jin, Qi

    2012-01-01

    Chrysin and its phosphate ester have previously been shown to inhibit cell proliferation and induce apoptosis in Hela cells; however, the underlying mechanism remains to be characterized. In the present study, we therefore synthesized diethyl flavon-7-yl phosphate (FP, C(19)H(19)O(6)P) by a simplified Atheron-Todd reaction, and explored its anti-tumor characteristics and mechanisms. Cell proliferation, cell cycle progression and apoptosis were measured by MTS, flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling techniques, respectively in human cervical cancer HeLa cells treated with 7-hydroxyflavone (HF) and FP. p21, proliferating cell nuclear antigen (PCNA) and cAMP levels in Hela cells were analyzed by western blot and radioimmunoassay. Both HF and FP inhibited proliferation and induced apoptosis in HeLa cells via induction of PCNA/p21 expression, cleaved caspase-3/poly (ADP-ribose) polymerase (PARP)-1, elevation of cAMP levels, and cell cycle arrest with accumulation of cells in the G0/G1 fraction. The effects of FP were more potent than those of HF. The interactions of FP with Ca(2+)-calmodulin (CaM) and Ca(2+)-CaM-phosphodiesterase (PDE)1 were explored by electrospray ionization-mass spectrometry and fluorescence spectra. FP, but not HF, formed non-covalent complexes with Ca(2+)-CaM-PDE1, indicating that FP is an inhibitor of PDE1, and resulting in elevated cellular cAMP levels. It is possible that the elevated cAMP levels inhibit growth and induce apoptosis in Hela cells through induction of p21 and cleaved caspase-3/PARP-1 expression, and causing down-regulation of PCNA and cell cycle arrest with accumulation of cells in the G0/G1 and G2/M fractions. In conclusion, FP was shown to be a Ca(2+)-CaM-PDE inhibitor, which might account for its underlying anti-cancer mechanism in HeLa cells. These observations clearly demonstrate the special roles of phosphorylated flavonoids in biological processes, and suggest that FP might represent

  16. Screening and identification of the peptides specifically binding to cervical cancer HeLa cell%宫颈癌HeLa细胞特异性结合肽的筛选及鉴定

    何晓娟; 郭彩霞; 杨陈; 梁晓秋

    2012-01-01

    Background and purpose: Molecular targeted therapy was paid much attention for its good therapeutic effects on cervical cancer and deeply decreased the injuries of the human body. This study aimed to screen and identify the peptides specifically binding to cervical cancer by using phage display in vivo. It may be the targeting vector of the chemotherapeutics and be the basic of the targeted drugs of cervical cancer. Methods: Human cervical cancer HeLa cell was cultured in vivo and the tumor animal model was made in nude mice. After phage peptide library was injected in mouse by intravenous injection and heart perfusion was carried out after 15 minutes, cervical cancer was harvested. Cervical cancer specific peptides that used in the next screening were obtained after amplification and purification in vivo. After 3 circulations, the affinity and specificity of phage clones to cervical cancer cells were preliminary indentified by enzyme linked immunosorbent assay (ELISA) and immunohistochemistry. The positive phage clone was amplified and the sequences were analyzed to determine the collective sequence. Results: The 10 phage clones were identified by ELISA, 8 of them were specially bound to the HeLa cell line which had the same amino acid sequence that is LLRSTGF. Conclusion: The peptides specifically binding to cervical cancer HeLa cell are screened and identified. All the results lay the foundation for the development of diagnostic reagents and drugs of cervical cancer.%背景与目的:宫颈癌的分子靶向治疗具有很好的疗效,同时可以显著减少抗癌药物对人体自身的损伤,因此备受关注.本研究利用噬菌体体内展示技术筛选及鉴定宫颈癌特异性结合肽,将有可能成为化疗药物的靶向载体,为宫颈癌靶向药物治疗奠定基础.方法:体外培养宫颈癌HeLa细胞接种裸鼠,建立肿瘤动物模型.将随机肽库尾静脉注入裸鼠体内,循环15 min,心脏灌注后回收肿瘤组织噬菌体扩增

  17. The cellular internalization of liposome encapsulated protoporphyrin IX by HeLa cells.

    Przybylo, Magdalena; Glogocka, Daria; Dobrucki, Jerzy W; Fraczkowska, Kaja; Podbielska, Halina; Kopaczynska, Marta; Borowik, Tomasz; Langner, Marek

    2016-03-31

    The proper lipid composition of liposomes designed to carry drugs determines their surface properties ensuring their accumulation within selected tissue. The electrostatic potential and surface topology of liposomes affect the internalization by single cells. The high-resolution imaging of cancer cells and the distribution of protoporphyrin-loaded liposomes within the cytoplasm and its dependence on the liposome surface properties are presented. In the paper, HeLa cells were used to investigate the uptake of porphyrin-loaded liposomes and liposomes alone by means of confocal and differential interference contrast microscopies. The effect of liposomes surface electrostatic potential and surface topology on their intracellular distribution was evaluated. The time evolution of the intracellular distribution of liposomes labelled with Rhodamine-PE was examined on HeLa cells. These studies allow for the identification of the liposome lipid composition so the efficient delivery of the active substance to cancer cells will be achieved. The obtained results showed that neutral PC-liposomes are the most efficiently internalized by HeLa cells. Moreover, results showed that properties of liposomes affect not only the internalization efficiency of the photosensitizer but also its distribution within the cells, as revealed by colocalization measurements.

  18. 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.

  19. 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.

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

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

    2015-10-15

    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.

  1. Pulse electric fields effect on invasion and metastasis of Hela cells in human cervical cancer%脉冲电场对人宫颈癌Hela细胞侵袭转移能力的影响

    张琴; 熊正爱; 周玮; 李成祥; 姚陈果

    2011-01-01

    cervical cancer Hela cells. It is probably one of its molecular mechanisms to inhibit the expression of MMP-2 and RhoC.

  2. Lysosome dysfunction enhances oxidative stress-induced apoptosis through ubiquitinated protein accumulation in Hela cells.

    Yu, Chunyan; Huang, Xiaowei; Xu, Ye; Li, Hongyan; Su, Jing; Zhong, Jiateng; Kang, Jinsong; Liu, Yuhe; Sun, Liankun

    2013-01-01

    The role of lysosomal system in oxidative stress-induced apoptosis in cancer cells is not fully understood. Menadione is frequently used as oxidative stress model. It is indicated that menadione could induce autophagy in Hela cells. In the present study, we examined whether the lysosomal inhibitor, ammonium chloride (NH(4)Cl) could prevent the autophagy flux by inhibiting the fusion of autophagosomes with lysosomes and enhance apoptosis induced by menadione via mitochondrial pathway. The results demonstrated generation and accumulation of reactive oxygen species and increased levels of ubiquitinated proteins and GRP78 in cells treated with both menadione and NH(4)Cl. Our data indicates that lysosomal system through autophagy plays an important role in preventing menadione-induced apoptosis in Hela cells by clearing misfolded proteins, which alleviates endoplasmic reticulum stress.

  3. 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.

  4. 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.

  5. 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.

  6. Inactivation of Hela cancer cells by an atmospheric pressure cold plasma jet∗%大气压冷等离子体射流灭活子宫颈癌Hela细胞

    黄骏†; 陈维; 李辉; 王鹏业; 杨思泽

    2013-01-01

    An inactivation mechanism study on Hela cancer cells by means of an atmospheric pressure cold plasma jet is presented. Cell morphology is observed under an inverted microscope after plasma treatment. The neutral red uptake assay provides quantitative evaluations of cell viability under different conditions. The effect of the inactivation efficiency of Hela cancer cells in the argon (900 mL/min) with addition of different amount of oxygen (1%, 2%, 4%, 8%) into atmospheric pressure cold plasma jet is discussed under the fixed power 18 W. Results show that 2% O2 addition provides the best inactivation efficiency, and the survival rate can be reduced to 7%after 180 s treatment. When the oxygen addition exceeds 2%, the inactivation efficiency gradually weakens. The effect is not so good as that in pure argon plasma when the oxygen addition arrives at 8%. According to the emission spectrum of the plasmum, it is concluded that the reactive oxygen species in the plasma play a key role in cancer cell inactivation process.%  研究了大气压冷等离子体射流对子宫颈癌Hela细胞的灭活机制。在倒置显微镜下观察不同等离子体处理条件下的细胞形态,并通过中性红吸收测试定量测定各个条件下的细胞存活率。将功率维持在18 W,在900 mL/min氩等离子体中添入氧气的百分含量分别为1%,2%,4%和8%的条件下处理Hela细胞,探讨活性气体氧气在惰性气体氩气中的百分含量对Hela癌细胞灭活效率的影响,发现添加2%氧气时,氩/氧等离子体灭活效果最佳,处理180 s后细胞存活率可降至7%。当继续添加氧超过2%时,灭活效果逐渐减弱,直至8%时,其效果反而不如单纯氩等离子体。通过测量等离子体发射光谱,结果表明活性氧自由基在癌细胞灭活过程中可能起关键作用。

  7. Insulin on cisplatin sensitivity of human cervical cancer cell HeLa line%胰岛素对人宫颈癌Hela细胞化疗敏感性的影响

    赵婷; 张蓓

    2013-01-01

    目的 探讨胰岛素能否增强人宫颈癌Hela细胞对顺铂(DDP)的化疗敏感性及其相关机制.方法体外培养人宫颈癌Hela细胞株,MTT法检测 5 U/L胰岛素作用3、6、9、12、15、18、21 h后,联合DDP(7.26 mg/L)对人宫颈癌Hela细胞增殖抑制率的影响.流式细胞仪检测对照组、DDP组、胰岛素组及联合组(胰岛素化疗前作用6 h)的细胞周期时相变化情况.结果 DDP组、胰岛素化疗前作用不同时间组与对照组相比,Hela细胞的增殖抑制率明显上升(P<0.01);且胰岛素于化疗前提前作用3、6、9、12、15 h后加入 DDP,宫颈癌Hela细胞的增殖抑制率明显高于DDP组(P<0.01).流式分析显示DDP组及胰岛素组的S期细胞比例较对照组高(P均<0.01),联合组S期细胞比例较DDP组高(P<0.01).结论 胰岛素具有较显著的DDP化疗增敏作用,胰岛素作用时机选择是胰岛素对宫颈癌细胞化疗增敏作用的关键.%Objective To investigate the ability of insulin to enhance sensitivity to chemotherapy of human cervical cancer HeLa cells to cisplatin and its associated mechanisms . Methods The inhibition rate of giving insulin at 5 U/L at different times before combining with cisplatin at 7. 26 mg/L was calculated by MTT assay. Flow cytometry was used to study the cell cycle distribution of the Hela cells of control group,DDP group,insulin group and the combination group. Results After being cultured with 5 U/L insulin for 3 h,6 h,9h,12,15 h,respectively,the inhibition rate was significantly higher than the DDP group ( P < 0.01 ). Flow cytometry showed insulin could increase the S phase arrest induced by DDP. Conclusion The timing of the action of insulin is the key of enhancing the sensitivity to chemotherapy of cervi-cal cancer cell line. The mechanism may be as follows : the toxic effects of cisplatin on cervical cancer HeLa cells are as-sociated with S phase arrest,and insulin can also cause cervical cancer cells in S phase arrest,there is

  8. 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.

  9. 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.

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

    2007-01-01

    In this paper,the relationship of intracellular acidification and apoptosis in Hela cells induced by vin-cristine 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 intra-cellular 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 apop-tosis 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.

  11. INHIBITION EFFECT AND ITS MECHANISM OF MATRINE ON HeLa CELLS IN CERVICAL CANCER%苦参碱抑制宫颈癌HeLa细胞增殖作用及机制

    付婷婷; 邹存华; 赵淑萍; 徐样; 鲁强; 张志磊

    2012-01-01

    目的 探讨苦参碱对人宫颈癌HeLa细胞体外增殖的抑制作用及其分子机制.方法 以不同浓度(0.5、1.0、1.5、2.0 g/L)的苦参碱分别处理HeLa细胞24、48、72 h后,应用MTT法检测细胞增殖抑制率,Western Blot方法检测培养48 h细胞中真核细胞翻译起始因子4E(eIF4E)、4E结合蛋白1(4E-BP1)蛋白表达情况:1.0 g/L的苦参碱作用HeLa细胞1、3、6、12 h后,Western Blot方法测其eIF4E、4E-BP1蛋白磷酸化水平.结果 苦参碱明显抑制HeLa细胞增殖,且呈时间和剂量依赖性:在相同时间内,随着苦参碱浓度的增加,HeLa细胞的增殖抑制率明显升高(F=235.035,P<0.05);在同一浓度时,随着苦参碱作用时间的延长,HeLa细胞的增殖抑制率也明显升高(F=320.207,P<0.05);2.0 g/L苦参碱作用72 h对HeLa细胞增殖抑制率最大,为(65.30±2.17)%.不同浓度苦参碱处理HeLa细胞48 h后,eIF4E、4E-BP1蛋白的表达均无明显变化(F=0.171、0.932,P>0.05).1.0 g/L苦参碱作用于HeLa细胞不同时间后,细胞中eIF4E、4E-BP1蛋白磷酸化水平均降低,且呈时间依赖性(F=43.48、107.23,P<0.01).结论 苦参碱可以明显抑制体外培养宫颈癌HeLa细胞的增殖,其作用可能是通过抑制eIF4E以及4E-BP1的磷酸化,从而抑制蛋白翻译来实现的.%Objective To study the in-vitro inhibition and its mechanism of matrine on HeLa cells of cervical cancer. Methods HeLa cells were treated with different concentrations (0. 5, 1.0, 1.5, and 2. 0 g/L) of matrine for 24, 48 and 72 hours, the cytostasis rate was measured by MTT method. Using Western Blot analysis, the expressions of eIF4E and 4F.-BP1 were detected after cultured for 48 hours, and phosphorylation levels of p-eIF4E and p-4E-BPl in HeLa cells after treated with 1. 0 g/L matrine for 1,3,6, and 12 hours. Results HeLa cells were significantly inhibited by matrine with dose- and time- dependent: In a same duration, the cytostasis rate of HeLa cells increased along with

  12. 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

    2014-12-05

    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.

  13. Study on the Mechanism of Autophagy of HeLa Cells in Human Cervical Cancer Induced by Fucoxanthin%褐藻素诱导人宫颈癌HeLa细胞的自噬作用机制研究

    廖政邦; 于五辈; 赵辉

    2015-01-01

    目的:研究褐藻素诱导人宫颈癌HeLa细胞的自噬作用机制。方法:1、10、20、40、80μmol/L褐藻素培养HeLa细胞48 h后,MTT法测定细胞活力并计算抑制率。0(空白对照)、10、20、40μmol/L褐藻素培养HeLa细胞48 h后,流式细胞仪测定细胞周期与凋亡率;通过吖啶橙染色与Lyso-Tracker Red染色、HeLa-GFP-LC3细胞测试法在荧光显微镜下观察细胞自噬状态;Western blot法检测细胞自噬流量与相关蛋白的表达。结果:10、20、40、80μmol/L褐藻素培养细胞48 h后对细胞生长有明显抑制作用。10、20、40μmol/L褐藻素培养细胞48 h后可将细胞阻滞于G0/G1期,但对细胞凋亡无明显影响;40μmol/L褐藻素培养细胞48 h后细胞自噬程度加重;与空白对照比较,40μmol/L褐藻素可促进LC3Ⅰ转化为LC3Ⅱ以及Beclin-1、PTEN、p21的表达,但抑制p-Akt、p-p70S6K、p-mTOR、Cyclin D1、CDKZ表达。自噬抑制剂3-甲基腺嘌呤(3-MA,5 mmol/L)预处理可逆转褐藻素诱导的HeLa细胞自噬作用;U0126可部分逆转褐藻素诱导的HeLa细胞自噬作用。结论:褐藻素可通过抑制Akt信号通路以及激活MEK/ERK信号通路诱导HeLa细胞自噬。%OBJECTIVE:To study the mechanism of autophagy of HeLa cell in human cervical cancer induced by fucoxanthin. METHODS:MTT was adopted to determine the cell activity and calculate the inhibition rate after HeLa cells were cultured by 1, 10,20,40 and 80 μmol/L of fucoxanthin for 48 h. Flow cytometry was used to determine the cell cycle and apoptosis rate after HeLa cells were cultured by 0(blank control),10,20 and 40 μmol/L of fucoxanthin for 48 h;acridine orange staining,LysoTrack-er Red staining,HeLa-GFP-LC3 method and fluorescence microscope were used to observe the autophagy state;Western blot was used to determine the expressions of proteins related to autophagy. RESULTS:The cells had obvious inhibition effect on the cell growth after being

  14. Apoptosis of HeLa Cells Induced by Cisplatin and Its Mechanism

    Youqing LIU; Hui XING; Xiaobing HAN; Xiaoyan SHI; Fengqi LIANG; Gang CHENG; Yunping LU; Ding MA

    2008-01-01

    To study the apoptosis induced by cisplatin in cervical cancer cell line HeLa and its mechanism, cell growth inhibition of cisplatin on HeLa cells was analyzed by MTT assay. Cell apoptosis was examined by cytometry and Hoechst33258 staining after treatment with cisplatin. The ef- fects of cisplatin on transcription of E6 were analyzed by RT-PCR. The protein expressions of E6, p53, p21, Bax and Bcl-2 were studied by Western blotting. Cisplatin inhibited proliferation in a time- and dose-dependant manner. Cytometically, sub-G1 peak showed higher apoptosis rates in the ex- perimental group than those in the control. Hoechst33258staining exhibited apoptosis induced by cis- platin. RT-PCR revealed that cisplatin decreased transcription of E6. Western blotting showed that cisplatin decreased protein expression of E6 and increased protein expression of p53, p21and Bax. It had no effect on protein expression of Bcl-2. It is concluded that cisplatin can induce apoptosis in HeLa cells by suppressing HPV E6 and thereby restoring the function of p53.

  15. The Mechanism of Cisplatin-induced Apoptosis in HeLa Cells

    Youqing Liu; Hui Xing; Xiaobing Han; Xiaoyan Shi; Fengqi Liang; Gang Chen; Ding Ma

    2005-01-01

    OBJECTIVE To study the mechanism of apoptosis induced by cisplatin in vitro in HeLa cells cervical cancer cell line.METHODS The inhibitory effect of cisplatin on HeLa cell growth was analyzed by the MTT assay. Cell apoptosis was measured with flow cytometry and Hoechst 33258 staining following cisplatin treatment. The effect of cisplatin on transcription of HPV E6 was analyzed by RT-PCR and protein expression of E6, P53, p21, Bax and Bcl-2 was studied by Western blots.RESULTS Cisplatin inhibited cellular proliferation in a time and dosedependant manner. The sub-G1 peak by flow cytometry showed a higher apoptotic rate in the experimental group compared to the controls and Hoechst 33258 staining indicated that apoptosis was induced by cisplatin. Results of RT-PCR demonstrated that cisplatin decreased transcription of E6. Western Blots showed that cisplatin decreased protein expression of E6 and increased protein expression of P53, p21 and Bax but had no effect on protein expression of Bcl-2.CONCLUSION Cisplatin induces apoptosis and death of HeLa cells through the suppression of HPV E6 and restoration of p53 function.

  16. LAPTM4B Down Regulation Inhibits the Proliferation, Invasion and Angiogenesis of HeLa Cells In Vitro

    Fanling Meng

    2015-09-01

    Full Text Available Background/Aims: LAPTM4B (lysosome-associated protein transmembrane 4 beta is a novel oncogene with important functions in aggressive human carcinomas, including cervical cancer. However, the specific functions and internal molecular mechanisms associated with this gene in the context of cervical cancer remain unclear. Methods: In this study, we explored the effects and mechanisms of LAPTM4B on tumor growth, metastasis and angiogenesis in vitro by depletion of LAPTM4B in Hela cell. RNA interference was used to induce down regulation of LAPTM4B gene expression in Hela cells. The motility, migration potential, and proliferation of the Hela cells were measured by flow cytometry, Transwell migration assays, wound healing assays, and Cell Counting Kit-8 assays. In addition, the cell cycle analysis utilized fluorescence-activated cell sorting. Results: In this study, RNAi-mediated LAPTM4B knockdown inhibited cell growth and angiogenesis. In vitro, HeLa cells with down regulated LAPTM4B also exhibited decreased migration and invasion activity as well as significantly reduced CDK12, HIF-1α, MMP-2, MMP-9 and VEGF expression. LAPTM4B blockade significantly decreased cord lengths and branch points in a tube formation assay. Conclusions: These results suggested that LAPTM4B inactivation could be a novel therapeutic target for cervical cancer.

  17. Akt is translocated to the mitochondria during etoposide-induced apoptosis of HeLa cells.

    Park, Byoungduck; Je, Young-Tae; Chun, Kwang-Hoon

    2015-11-01

    Akt, or protein kinase B, is a key serine-threonine kinase, which exerts anti-apoptotic effects and promotes cell proliferation in response to various stimuli. Recently, however, it was demonstrated that Akt exhibits a proapoptotic role in certain contexts. During etoposide‑induced apoptosis of HeLa cells, Akt enhances the interaction of second mitochondria‑derived activator of caspases/direct IAP binding protein with low pI (Smac/DIABLO) and X‑linked inhibitor of apoptosis protein by phosphorylating Smac at serine 67, and thus promotes apoptosis. However, the detailed mechanisms underlying Akt regulation in etoposide‑mediated apoptosis remain to be determined. The present study investigated whether etoposide triggers the translocation of Akt into the mitochondria. It was found that Akt activity was increased and sustained during apoptosis triggered by etoposide in HeLa cells. During apoptosis, Akt was translocated from the cytoplasm into the mitochondria in a phosphoinositide 3‑kinase-dependent manner at the early and late stages of apoptosis. Concomitantly, the depletion of Akt in the nuclear fraction was observed after etoposide treatment from analysis of confocal microscopy. The results suggest that etoposide‑stimulated Akt is translocated into the mitochondria, thereby possibly enhancing its interaction with Smac and promoting apoptosis in HeLa cells. These results indicate that Akt may be a promising candidate for a pro-apoptotic approach in cancer treatment.

  18. Intense picosecond pulsed electric fields inhibit proliferation and induce apoptosis of HeLa cells.

    Zhang, Min; Xiong, Zheng-Ai; Chen, Wen-Juan; Yao, Cheng-Guo; Zhao, Zhong-Yong; Hua, Yuan-Yuan

    2013-06-01

    A picosecond pulsed electric field (psPEF) is a localized physical therapy for tumors that has been developed in recent years, and that may in the future be utilized as a targeted non‑invasive treatment. However, there are limited studies regarding the biological effects of psPEF on cells. Electric field amplitude and pulse number are the main parameters of psPEF that influence its biological effects. In this study, we exposed HeLa cells to a psPEF with a variety of electric field amplitudes, from 100 to 600 kV/cm, and various pulse numbers, from 1,000 to 3,000. An MTT assay was used to detect the growth inhibition, while flow cytometry was used to determine the occurrence of apoptosis and the cell cycle of the HeLa cells following treatment. The morphological changes during cell apoptosis were observed using transmission electron microscopy (TEM). The results demonstrated that the cell growth inhibition rate gradually increased, in correlation with the increasing electric field amplitude and pulse number, and achieved a plateau of maximum cell inhibition 12 h following the pulses. In addition, typical characteristics of HeLa cell apoptosis in the experimental groups were observed by TEM. The results demonstrated that the rate of apoptosis in the experimental groups was significantly elevated in comparison with the untreated group. In the treatment groups, the rate of apoptosis was greater in the higher amplitude groups than in the lower amplitude groups. The same results were obtained when the variable was the pulse number. Flow cytometric analysis indicated that the cell cycle of the HeLa cells was arrested at the G2/M phase following psPEF treatment. Overall, our results indicated that psPEF inhibited cell proliferation and induced cell apoptosis, and that these effects occurred in a dose-dependent manner. In addition, the results demonstrated that the growth of the HeLa cells was arrested at the G2/M phase following treatment. This study may provide a

  19. Julibroside J8-induced HeLa cell apoptosis through caspase pathway.

    Zheng, L; Zheng, J; Wu, L-J; Zhao, Y-Y

    2006-01-01

    The julibroside J8 was isolated from the Albizia julibrissin and evaluated for antiproliferatived on six cancer cell lines (BGC-823, Bel-7402, HeLa, PC-3MIE8, MDA-MB-435 and LH-60) in vitro. Julibroside J8 at 100 microg mL- 1 (46.08 micromol.L- 1) significantly inhibited growth in the first three cell lines. In addition, in HeLa cells typical apoptotic changes in morphology were observed, and further, nuclear damage was observed by Giemsa staining and DNA fragmentation was exhibited. Effects of julibrosideJ8 on induction of DNA fragmentation, caspase-3 activation and downregulation of ICAD expression were effectively inhibited by a caspase-3 inhibitor, z-DEVD-fmk. In addition, apoptosis induced with julibroside J8 was associated with an increase in expression of the apoptosis inducer Bax, and a significant reduction in expression of the apoptosis suppressor Bcl-2 in mitochondria. These results suggest that julibroside J8 induces HeLa death through caspase pathway.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

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

    Yao, Qing; Xu, Hui; Zhang, Qian-Qian; Zhou, Hui [Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory for Biocontrol, Sun Yat-Sen University, Guangzhou 510275 (China); Qu, Liang-Hu, E-mail: lssqlh@mail.sysu.edu.cn [Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory for Biocontrol, Sun Yat-Sen University, Guangzhou 510275 (China)

    2009-10-23

    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.

  5. 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...

  6. The effect of MAPK inhibitors and ROS modulators on cell growth and death of H₂O₂-treated HeLa cells.

    Park, Woo Hyun

    2013-08-01

    Reactive oxygen species (ROS) influence the signaling of mitogen‑activated protein kinases (MAPKs) involved in cell survival and death. In the present study, the toxicological effect of hydrogen peroxide (H2O2) on HeLa cervical cancer cells was evaluated following treatment with MAPK inhibitors [MAP kinase or ERK kinase (MEK), c‑Jun N‑terminal kinase (JNK) or p38], N‑acetyl cysteine (NAC) and propyl gallate (PG) (well‑known antioxidants), or L‑buthionine sulfoximine [BSO; an inhibitor of glutathione (GSH) synthesis]. Treatment with 100 µM H2O2 inhibited the growth of HeLa cells and induced cell death, which was accompanied by loss of the mitochondrial membrane potential (MMP; ΔΨm). H2O2 did not induce any specific phase arrests of the cell cycle. ROS levels increased, while GSH levels decreased in H2O2‑treated HeLa cells after 1 and 24 h of treatment. The MAPK inhibitors enhanced H2O2‑induced HeLa cell death, while only p38 inhibitor increased ROS levels. Both NAC and PG attenuated H2O2‑induced HeLa cell growth inhibition and death together with the suppression of ROS levels. BSO increased ROS levels in H2O2‑treated HeLa cells without increasing cell death. The levels of MMP (ΔΨm) loss and GSH depletion were not closely associated with the levels of apoptosis in HeLa cells treated with the MAPK inhibitors, NAC, PG or BSO, in the presence of H2O2. In conclusion, H2O2 induced HeLa cell growth inhibition and death. MAPK inhibitors generally enhanced H2O2‑induced HeLa cell death. In particular, p38 inhibitor increased ROS levels in H2O2‑treated HeLa cells, while NAC and PG attenuated H2O2‑induced HeLa cell death by suppressing ROS levels.

  7. 透骨草提取物诱导人宫颈癌Hela细胞凋亡及其相关机制研究%The study on apoptosis of human cervical cancer Hela cell induced by Phryma Leptostachya L.var.asiatica Hara extract and related molecular mechanism

    于秀艳; 曾常茜; 高松; 王文龙; 高海燕; 刘晓峰

    2012-01-01

    Objective:To investigate the apoptosis of human cervical cancer Hela cell induced by Phryma Leptostachya L. var. asiatica Hara extract and related molecular mechanism. Methods:The morphology of Hela cells was observed with fluorescence microscope. The ultrastructure of Hela cells was observed with transmission electronic microscope. Flow cytometry was used to detect the apoptosis rate of Hela cells. Western blot was used to determine the expressions of Caspase-3 and Bcl-2 proteins of Hela cells. Results; After Hela cells were treated with 100μg/ml Phryma Leptostachya L. var. asiatica Hara Extract, fluorescence microscope showed that the Hela cell shrinkage, nucleus concentrate beside theca like crescent. The transmission electronic microscope showed that the surface processes and microvillus decreased the Hela cells, nucleus fragmented,chromatin condensated and marginalized, "budding phenomenon" appeared. The flow cytometry indicated that the apoptotic rate of Hela cell was (25. 9 ± 1. 13)% and increased obviously than control group (P <0. 01) . The Western blot showed that the expression of Caspase-3 and Bcl-2 proteins of Hela cells decreased than control group (P<0.05). Conclusion; Phryma Leptostachya L. var. asiatica Hara extract could induce the apoptosis of Hela cells and the mechanism related to downregulating the expression of Bcl-2 and Caspase-3 protein.%目的:探讨透骨草提取物诱导人宫颈癌Hela细胞凋亡及其相关机制.方法:荧光显微镜观察Hela细胞形态,透射电镜观察Hela细胞超微结构,流式细胞术检测Hela细胞凋亡率,Western blot检测Bcl-2蛋白和Caspase-3蛋白表达水平.结果:100 μg/ml透骨草提取物处理的Hela细胞后,荧光显微镜显示Hela细胞皱缩,细胞核浓缩呈新月状边集.透射电镜可见Hella细胞表面突起和微绒毛减少,核断裂、染色质凝聚且边缘化,有"出芽"现象.流式细胞术显示透骨草提取物处理的Hela细胞凋亡率为(25.90±1.13)%,

  8. 氯化镧对子宫颈癌HeLa细胞增殖和迁移能力的影响%Effects of lanthanum chloride on proliferation and migration of human cervical cancer cell line HeLa cells

    刘丝荪; 陆丹; 缪丽芳; 熊秋迎; 陈新萍; 汪泱; 郭菲

    2010-01-01

    ,抑制宫颈癌细胞的增殖和迁移并诱导其凋亡.%Objective To investigate the effects of lanthanum chloride on proliferation and migration activity of human cervical cancer cells in vitro which may be a new anti-cervical cancer drug and provide experimental data for cervical cancer treatment. Methods HeLa cells cultured in vitro were divided into two groups: experimental group and control group. In experimental group, the cells were respectively treated with lanthanum chloride at different concentrations, 5, 50 and 100 μmol/L, while the cells in the control group were not treated with lanthanum chloride. The cell growth was observed by inverted microscope and the morphology changes of the cells were observed by the laser scanning confocal microscope (LSCM).Proliferation of HeLa cells in the two groups was detected by methyl thiazolyl tetrazolium (MTT) test;apoptosis rate was analyzed by flow cytometry (FCM). Cell migration test was applied to observe the effect of lanthanum chloride on migration. Reverse transcription (RT)-PCR was employed to evaluate the effects of lanthanum chloride on proliferation gene (cyclinD1), anti-apoptosis gene (zinc finger protein A20) and migration-related gene (matrix metalloproteinase 9, MMP-9). Results The status of cell growth was observed under the inverted microscope: with the increased of the lanthanum chloride concentrations, the cell density of reduced, the granule in cytoplasm increased, color intensifying and intercellular space enlarged; some cells became rounding and dead, floating in the culture media; the exfoliated cells increased gradually in the experimental groups. While In the control group, the cells grew adherently, with clear morphology and plump cytoplasm, and adjacent cell grew in lamellar. Observed with LSCM: the nuclear chromatin condensated and marginated with the volume of nuclear decreased in experimental groups. With the increase of the lanthanum chloride concentrations, nuclei in the

  9. CRISPR-Cas9 Mediated NOX4 Knockout Inhibits Cell Proliferation and Invasion in HeLa Cells

    Park, Rackhyun; Li, Liqing; Jang, Minsu; Morris, Andrew J.; Huang, Cai

    2017-01-01

    Increased expression of NOX4 protein is associated with cancer progression and metastasis but the role of NOX4 in cell proliferation and invasion is not fully understood. We generated NOX4 knockout HeLa cell lines using the CRISPR-Cas9 gene editing system to explore the cellular functions of NOX4. After transfection of CRISPR-Cas9 construct, we performed T7 endonuclease 1 assays and DNA sequencing to generate and identify insertion and deletion of the NOX4 locus. We confirmed the knockout of NOX4 by Western blotting. NOX4 knockout cell lines showed reduced cell proliferation with an increase of sub-G1 cell population and the decrease of S/G2/M population. Moreover, NOX4 deficiency resulted in a dramatic decrease in invadopodium formation and the invasive activity. In addition, NOX4 deficiency also caused a decrease in focal adhesions and cell migration in HeLa cells. These results suggest that NOX4 is required for both efficient proliferation and invasion of HeLa cells. PMID:28099519

  10. CRISPR-Cas9 Mediated NOX4 Knockout Inhibits Cell Proliferation and Invasion in HeLa Cells.

    Jafari, Naser; Kim, Hyunju; Park, Rackhyun; Li, Liqing; Jang, Minsu; Morris, Andrew J; Park, Junsoo; Huang, Cai

    2017-01-01

    Increased expression of NOX4 protein is associated with cancer progression and metastasis but the role of NOX4 in cell proliferation and invasion is not fully understood. We generated NOX4 knockout HeLa cell lines using the CRISPR-Cas9 gene editing system to explore the cellular functions of NOX4. After transfection of CRISPR-Cas9 construct, we performed T7 endonuclease 1 assays and DNA sequencing to generate and identify insertion and deletion of the NOX4 locus. We confirmed the knockout of NOX4 by Western blotting. NOX4 knockout cell lines showed reduced cell proliferation with an increase of sub-G1 cell population and the decrease of S/G2/M population. Moreover, NOX4 deficiency resulted in a dramatic decrease in invadopodium formation and the invasive activity. In addition, NOX4 deficiency also caused a decrease in focal adhesions and cell migration in HeLa cells. These results suggest that NOX4 is required for both efficient proliferation and invasion of HeLa cells.

  11. 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.

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

    Samani, Fariba; Monfared, Ali Shabestani; Zabihi, Ebrahim; Khafri, Soraya; Karimi, Maesoumeh; Akhavan Niaki, Haleh

    2014-01-01

    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. PMID:24904724

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

    Abbas Jafarain

    2014-01-01

    Full Text Available 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. The amount of phenolic compounds of the extracts was determined by Folin Ciocalteu method. The cytotoxicity of the extracts against Hela tumor cells was carried out using MTT assay. Briefly, cells were seeded in microplates and different concentrations of the extract were added. Cells were incubated for 48 h and their viability was evaluated by addition of tetrazolium salt solution. After 3 h medium was aspirated, dimethyl sulfoxide was added and absorbance was determined at 540 nm with an ELISA plate reader. Cytotoxicity was considered when more than 50% reduction on cell survival was observed. Results: Callus and leaf extracts of M. oleifera significantly decreased the viability of Hela cells in a concentration-dependent manner. However, leaf extract of M. oleifera were more potent than that of callus extract. Conclusion: As the content of phenolic compounds of leaf extract was higher than that of callus extract, it can be concluded that phenolic compounds are involved in the cytotoxicity of M. oleifera.

  14. 3-甲基腺嘌呤对喜树碱诱导的宫颈癌Hela细胞凋亡的影响%Effect of 3-MA on camptothecin-induced cervical cancer Hela cell apoptosis

    王晓娜; 任来峰; 赵安江; 杨万霞; 任云青

    2016-01-01

    Objective:To explore the effect of autophagy inhibitor 3-methyladenine(3-MA) on camptothecin(CPT)-induced Hela cell apoptosis.Methods:MTT assays were carried out to determine the optimal concentration and time of CPT on Hela cells and the effect of different drugs on Hela cell proliferation activity .After Hela cells were treated with different drugs ,the changes of autophagy marker protein( microtubule-associated protein 1 light chain 3,LC3),p62 and apoptosis-related protein were detected using Western blot and immunofluorescence ( IF) .DAPI ( nuclear ) staining was used to observe cell apoptosis rate .Results: In CPC-treated Hela cells,Hela cell proliferation activity declined dramatically ,and autophagy could be induced to occur .Compared with CPT group ,the cell proliferation activity was lower in CPT combined with 3-MA group,the level of autophagy decreased ,but the apoptosis rate significantly increased.Conclusion:CPT can induce autophagy while inducing Hela cell death .Hela cells chemosensitivity to CPT treatment can be enhanced by 3-MA inhibiting autophagy .%目的:本研究探讨自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)对喜树碱(Camptothecin,CPT)诱导的Hela细胞凋亡的影响。方法:用四甲基偶氮唑盐比色法( MTT方法)检测CPT对Hela细胞作用的最佳药物浓度和时间,以及不同药物对Hela细胞增殖活性的影响;用免疫印迹及免疫荧光检测不同药物作用于Hela细胞后,Hela细胞自噬标志蛋白微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)、p62及凋亡相关蛋白的变化;DAPI核染色观察细胞凋亡。结果:CPT作用于Hela细胞后,Hela细胞增殖活性明显下降,并且可诱导自噬现象的发生。 CPT和3-MA联合作用较单独CPT作用Hela细胞的增殖活性降低,细胞自噬水平下降,凋亡率明显升高。结论:CPT在诱导Hela细胞凋亡的同时可诱导自噬,通过3-MA

  15. Effect of histone deacetylase inhibitor trichostatin A on radiosensitivity of human cervical cancer cell line HeLa under hypoxia condition%组蛋白去乙酞化酶抑制剂对乏氧宫颈癌HeLa细胞放射敏感度的影响

    余娟娟; 王爱红; 戚世芳; 田晓予

    2011-01-01

    Objective: To investigate the effects of histone deacetylase inhibitor trichostatin A (TSA) on radiosensitivity of human cervical cancer cell line HeLa under hypoxia condition. Methods: Under hypoxia condition, the HeLa cells were treated with different concentrations of TSA for 12, 24, 48 and 72 h, respectively. The survival rate of HeLa cells was detected by MTT assay. The half inhibitory concentration (IC50) and IC10 values for TSA were calculated. The radiosensitivity in HeLa cells treated with TSA (IC10) for 24 h under hypoxia condition was detected by colony formation assay. The expressions of hypoxia-inducible factor-1 alpha (HIF-1 α ) and vascular endothelial growth factor (VEGF) proteins were measured by immunocytochemistry. Results: The survival rate of HeLa cells induced by TSA and hypoxia was decreased in a concentration and time-dependent manner. The radiosensitivity of HeLa cells under hypoxia condition wasincreased by treatment with TSA (IC10) for 24 h (P<0.05). The expressions of HIF-1 α and VEGF proteins were higher in HeLa cells exposed to hypoxia than that in HeLa cells exposed to normoxia. The expressions of HIF-1α and VEGF proteins in HeLa cells under hypoxia condition were decreased by treatment with TSA (IC10) for 24 h. Conclusion: TSA can significantly enhance the radiosentivity of HeLa cells under hypoxia condition. This effect may be related with the down-regulation of HIF-1 α and VEGF protein expression.%目的:观察组蛋白去乙酸化酶抑制剂曲古霉素A (trichostatin A,TSA)对乏氧状态下人宫颈癌HeLa细胞放射敏感度的影响.方法:应用不同浓度TSA作用经乏氧预处理的人宫颈癌HeLa细胞12、24、48和72 h,MTT法检测HeLa细胞的增殖率,计算半数抑制浓度(half inhibitory concentration,IC(50))和IC(10)值;克隆形成实验检测TSA (IC(10))作用24 h对乏氧HeLa细胞的放射增敏效应;免疫细胞化学法检测TSA (IC(10))对HeLa细胞中乏氧诱导因子-1α (hypoxia

  16. A lipidomics investigation of the induced hypoxia stress on HeLa cells by using MS and NMR techniques.

    Yu, Yang; Vidalino, Laura; Anesi, Andrea; Macchi, Paolo; Guella, Graziano

    2014-04-01

    Induced hypoxia stress on cervical cancer derived cells (HeLa cells) leads to significant changes in their membrane lipid profiles. The lipidome of HeLa cells was characterized by a joint approach wherein liquid chromatography-mass spectrometry (LC-MS) analysis was followed by high resolution NMR measurements. Multivariate data analysis showed apparent separation between control and hypoxia-treated HeLa cells and thus demonstrated hypoxia effects on lipid metabolism. The most striking finding was that hypoxia stimulation significantly reduced the total amount of cellular phosphoinositols (PI) but caused a prominent increase in the amount of lyso phosphocholines (lyso-PC) and lyso phosphoethanolamines (lyso-PE). The observed decrease of PI amount under hypoxic conditions is probably due to the accumulation of cellular myo-inositol, which is known to play a critical role in de novo synthesis of PI. Moreover, our study suggests that polyunsaturated phospholipid species are stronger biomarkers for discriminating the effect of hypoxia treatment. The evaluation of changes in the average unsaturation index (UI) of the membrane lipids acyl chains reveals that UI slightly increases in several lipid classes, thus affecting membrane fluidity and further membrane-dependent functions. The plausible mechanisms by which HeLa cells adapt to hypoxia conditions are also briefly reported.

  17. 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.

  18. 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.

  19. 槲皮素对宫颈癌HeLa细胞放射增敏的体外实验研究∗%Radiosensitization effect of quercetin on cervical cancer HeLa cell line cultured in vitro

    佐志刚; 陈萍; 邓守恒; 张莉; 曹凤军

    2016-01-01

    Objective To investigate the radiosensitization effect of quercetin on cervical cancer HeLa cells and to explore its possible mechanism. Methods Clone forming assay was used to observe the radiosensitizing effect of quercetin on HeLa cells after ra⁃diation with drug by various schedules and different drug concentrations(20% IC50 and IC50). According to the experimental protocol, the experiments were carried out in radiation alone group, 20% IC50 quercetin+radiation group, radiation+20% IC50 quercetin group, IC50 quercetin+radiation group and radiation+IC50 quercetin group. The surviving fraction( SF) of above all groups after X⁃ray radiation of 12, 8, 6, 4, 2 and 0 Gy were calculated and the optimum schedule was selected. The below experiments were carried out in negative control group, quercetin group(20% IC50 and IC50), radiation group and quercetin+irradiation group(20% IC50 and IC50), and the dose of radiation was 4 Gy. Flow cytometry, DAPI staining, Western blotting(20%IC50 quercetin only) were explored to detect the cell cycle distribution, apoptotic changes and expression of Bcl⁃2 and Bax protein at 24 h after treatment. Results Compared with radiation alone group, the SF were reduced by the treatment of different concentration of radiation, quercetin in different sequence(P<0�05). The SF were inversely proportional to the concentration of quercetin, while the radiosensitization of quercetin was in concentration de⁃pended manner. Under the same concentration of quercetin and dose of X⁃ray, the SF in radiation+quercetin groups were lower than those in quercetin+radiation groups. DAPI staining showed that nucleus shrinkage were observed with the treatment of quercetin or radi⁃ation, and the combination of them could cause nucleus shrinkage and fragmentation more marked. Flow cytometry demonstrated that compare with other groups, quercetin+radiation groups could arrest cell cycle in G2/M phase( P<0�05) . Western blotting showed that the levels

  20. 塞来昔布对宫颈癌HeLa细胞的放射增敏作用及影响因素的研究%Study on radiosensitization effect of celecoxib on human cervical cancer HeLa cells and the effect factors

    田晓予; 王爱红; 余娟娟; 刘华

    2011-01-01

    目的:研究塞来昔布(Celecoxib)对人宫颈癌HeLa细胞放射增敏作用以及给药顺序对放射增敏作用的影响.方法:采用MTT法测定塞来昔布作用于HeLa细胞的半数抑制浓度(IC50),应用克隆形成和单击多靶数字模型检测塞来昔布的放射增敏作用及不同给药顺序的细胞存活率及放射增敏比(SER).结果:塞来昔布对宫颈癌Hela细胞株增殖有抑制作用,其作用呈现剂量-效应和时间-效应依赖性,塞来昔布对Hela细胞的IC50为44 μmol/L,取40 μmol/L塞来昔布联合X射线照射作用于Hela细胞,先给药后照射组SER为2.46,给药中照射组SER为1.91,先照射后给药组SER为1.44;免疫化学实验证实塞 来昔布可下调Hela细胞VEGF的表达.结论:塞来昔布对Hela细胞具有放射增敏作用,其增敏作用有顺序依从性;其机制可能是通过下调人宫颈癌Hela细胞的VEGF 表达而实现.%Objective: To study the radiosensitization effect of celecoxib on human cervical cancer HeLa cells and the effect of administration sequence on radiosensitization. Methods: MTT method was used to detect the inhibitory concentration 50 ( IC 50) of Hela cells after treated with celecoxib, clone formation and single - hit multitarget model detection were used to detect the radiosensitization effect of celecoxib, cell survival rate and sensitive enhancement ratio ( SER) of different administration sequences. Results: Celecoxib inhibited the proliferation of cervical cancer Hela cell strains, showing a dose - and time - dependent manner, IC 50 of Hela cells after treated with celecoxib was 44 μmol/L; after treating Hela cells with 40 μmol/L celecoxib combined with X - ray, SERs in administration prior to radiation group, administration combined with radiation group and radiation prior to administration group were 2. 46, 1.91 and 1.44, respectively; immunochemistry confirmed that celecoxib could down -regulate the expression of VEGF in Hela cells. Conclusion

  1. 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.

  2. Polyketide Derivatives from Annona muricata Linn Leaves as Potencial Anticancer Material by Combination Treatment With Doxorubicin on Hela Cell Line

    Artanti, A. N.; Astirin, O. P.; Prayito, A.; Widiyaningsih, R. F.; Prihapsara, F.

    2017-02-01

    One of the compounds found effication as an anticancer agent on cervical cancer is acetogenin, a polyketide compound that is abundant in Annona muricata L. leaves. This study has been done to examine polyketide derivatives was isolated from Annona muricata L. which has potency to induce apoptosis by p53 expression on hela cell line. An approach recently develop to overcome side effect of chemoterapeutic agent is used of combined chemoterapeutic agent, i.e doxorubicin. The determination of cytotoxic combination activity from polyketide derivative and doxorubicin was evaluated using MTT assay to obtain the value of CI (combination index). The expression of p53 profile was evaluated by immunohistochemistry on hela cell line. Data analysis showed that combination of polyketide derivative from Annona muricata L. (38,5 µg/ml) and doxorubicin with all of concentration performed synergistic effect on hela cell line with CI value from 0,33 – 0,65. The analysis on immucytochemistry showed that polyketide derivative from Annona muricata L. leaves could enhance p53 pathway significantly on hela cell line.

  3. THE INFLUENCE OF TIME ON THE DEATH OF HELA CELLS AT ELEVATED TEMPERATURES

    The time required to kill HeLa cells in suspension was determined for the range of temperatures from 48 to 65 C. The cells were placed between 6...elevated temperature episode and distinguished live from dead cells. The time (t) to cause death of the HeLa cells was found to range from 1 second at 65 C to 1900 seconds at 48 C. (Author)

  4. Antioxidant, anticancer, and apoptosis-inducing effects of Piper extracts in HeLa cells

    Wahyu Widowati

    2013-06-01

    Full Text Available Objective: Cervical cancer is the second most common cancer as well as one of leading cause of cancer-related death for women worldwide. In regards to that issue, focus of this paper will be on popularly used Piperaceae members including Piper betle L, Piper cf fragile Benth, Piper umbellatum L, Piper aduncum L, Piper pellucidum L. This research was conducted to elucidate the antioxidant, anticancer and apoptosis inducing activities of Piperaceae extracts on cervical cancer cells, namely HeLa cell line. Methods: The anticancer activity was determined by inhibiting the proliferation of cells. Apoptosis inducing was determined by inhibiting proliferation cells and by SubG1 flow cytometry. The antioxidant activity is determined by using superoxide dismutase value and 2,2-diphenyl-1-picrylhydrazyl (DPPH radical scavenging activity. Results: The highest anticancer activity at 24 h incubation was found for P.pellucidum extract (IC50: 2.85 µg/ml; The anticancer activity at 48 h incubation was more than at 24 h for all extracts. The highest apoptotic activity was found for P.betle (12.5 µg/ml at both 24 and 48 h incubatio. The highest antioxidant activity was also represented by P.betle extract. Conclusions: All Piperaceae extracts have high anticancer activity; longer incubation increase anticancer activity. P.betle extract has the highest antioxidant property. [J Exp Integr Med 2013; 3(3.000: 225-230

  5. 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.

  6. Anti-apoptotic effect of caspase inhibitors on H₂O₂-treated HeLa cells through early suppression of its oxidative stress.

    Park, Woo Hyun

    2014-05-01

    Oxidative stress-induced cytotoxicity in cervical cancer cells may be of toxicological interest. In the present study, the effects of exogenous H2O2 on cell growth and death in HeLa cervical cancer cells were investigated, and the anti-apoptotic effects of various caspase (pan-caspase, caspase-3, -8 or -9) inhibitors on H2O2-treated HeLa cells were also evaluated with regard to reactive oxygen species (ROS) and glutathione (GSH) levels. Based on MTT assays, H2O2 inhibited the growth of HeLa cells with an IC50 value of ~75 µM at 24 h. H2O2 increased the number of dead cells and Annexin V-FITC-positive cells in the HeLa cells, which was accompanied by the activation of caspase-3 and the loss of mitochondrial membrane potential (MMP; ΔΨm). However, relatively higher doses of H2O2 induced necrosis in HeLa cells. Caspase inhibitors significantly prevented H2O2-induced HeLa cell death. H2O2 increased ROS including O2•- at 24 h and increased the activity of catalase in HeLa cells. H2O2 also increased the ROS level at 1 h, and several caspase inhibitors attenuated the increased level at 1 h but not at 6, 12 and 24 h. H2O2 decreased the GSH level in HeLa cells at 1 h, and several caspase inhibitors attenuated the decreased level of GSH at this time. H2O2 induced GSH depletion at 24 h. In conclusion, H2O2 inhibited the growth of HeLa cells via apoptosis and/or necrosis, which was accompanied by intracellular increases in ROS levels and GSH depletion. Caspase inhibitors are suggested to suppress H2O2-induced oxidative stress to rescue HeLa cells at the early time point of 1 h.

  7. 人宫颈癌获得性放射抗拒细胞株(Hela-R)DNA修复能力的观察%Observation of the DNA repair capability of acquired radioresistant cell line from human cervical carcinoma cells

    刘晨; 庞学利

    2013-01-01

    目的 观察人宫颈癌获得性放射抗拒细胞株(Heal-R)与其亲本细胞(Hela)DNA修复能力的差异.方法 采用克隆形成实验测定细胞的放射敏感性;CCK-8细胞增殖实验检测增殖情况,流式细胞术检测凋亡及周期变化,彗星实验及5-乙基-2′-脱氧尿苷(EdU)掺入检测DNA的修复能力.结果 Hela-R细胞株的平均致死剂量显著高于Hela细胞株.X射线照射后,Hela-R细胞株早、晚期凋亡率均低于 Hela细胞.24、48、72 h Hela和Hela-R细胞株的光密度值(OD)值分别为1.13±0.12、1.46±0.13(P<0.05);1.34±0.07、1.20±0.07(P<0.05); 1.58±0.07、1.48±0.07(P<0.05).Hela-R细胞株放疗后12~48 h G2期细胞显著增多.Hela-R细胞株放疗后24 h与48 h,彗星尾长均短于Hela细胞株.X射线照射1 h后,Hela-R细胞株EdU荧光强度为121.32±39.67(P<0.01),高于Hela细胞株.结论 Hela-R较Hela细胞株具有显著的放射抗拒性,DNA损伤修复能力的增强是其产生的重要机制之一.%Objective To observe the difference of DNA repair capability between the radioresistant cell (Hela R) line and its parents cell(Hela) line. Methods The radiosensitivity was measured with colony formation. CCK 8 cell proliferation assay was used to detect proliferation after radiation. Flow cytometry was used to detect the change of apoptosis and cell cycle after radiation. Comet assay and incorporation of ethynyl deoxyuridine were used to detect DNA repair capability. Results The mean lethal dose of Hela R was significantly superior to its parental cell (Hela). After irradiation,Hela R cell line's early apoptotic rate and late apop totic rate were significantly less than the Hela cells. CCK 8 proliferation experiment showed that Hela and Hela R cell's OD value, respectively,in 24,48 and 72 hours,were 1. 13 ± 0. 12 and 1. 46 ± 0. 13 (P<0. 05);1.34±0. 07 and 1. 20 ± 0. 07 (P<0. 05) ; 1. 58 ± 0. 07 and 1. 48±0. 07(P<0. 05). Hela R's G2 phase rate was significantly higher than Hela

  8. 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.

  9. 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

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

    Yue, Qingxi; Zhen, Hong; Huang, Ming; Zheng, Xi; Feng, Lixing; Jiang, Baohong; Yang, Min; Wu, Wanying; Liu, Xuan; Guo, Dean

    2016-01-01

    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 expression of Na, K

  11. Heterofucan from Sargassum filipendula induces apoptosis in HeLa cells.

    Costa, Leandro Silva; Telles, Cinthia Beatrice Silva; Oliveira, Ruth Medeiros; Nobre, Leonardo Thiago Duarte Barreto; Dantas-Santos, Nednaldo; Camara, Rafael Barros Gomes; Costa, Mariana Santana Santos Pereira; Almeida-Lima, Jailma; Melo-Silveira, Raniere Fagundes; Albuquerque, Ivan Rui Lopes; Leite, Edda Lisboa; Rocha, Hugo Alexandre Oliveira

    2011-01-01

    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.

  12. 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.

  13. Rheological properties of mammalian cell culture suspensions: Hybridoma and HeLa cell lines.

    Shi, Y; Ryu, D D; Ballica, R

    1993-03-25

    Data on viscous (eta') and elastic (eta'') components of the complex viscosity versus oscillatory angular frequency (0.01 to 4.0 rad/s) with increasing strains were obtained for hybridoma cell (62'D3) and HeLa cell (S3) suspensions in PBS at 0.9 (mL/mL) cell volume fraction using a Weissenberg rheogoniometer equipped with two parallel plate geometry at ambient temperature. Both cell suspensions exhibited shear thinning behavior. From the measured viscoelastic properties, the yield stress was calculated. Hybridoma cell suspension (15 microm as the mean diameter of cells) showed the yield stress at 550 dyne/cm(2) that was 1.8 times higher than the value of HeLa cell suspension (22 microm mean diameter) as measured at the oscillatory angular frequency, 4.0 rad/s. The apparent viscosities of HeLa cell suspension at four concentrations and varying steady shear rate were also determined using the Brookfield rotational viscometer. The yield stress to steady shear test was about 130 dyne/cm(2) for HeLa cell suspension at 0.9 (mL/mL) cell volume fraction. The apparent viscosity was in the range about 1 approximately 1000 Poise depending on the cell concentration and shear rate applied. A modified semiempirical Mooney equation, eta = eta(0) exp[K gamma(.)(-beta)phi(c)(1 - K'' sigmaphi(c) /D)] was derived based on the cell concentration, the cell morphology, and the steady shear rate. The beta, shear rate index, was estimated as 0.159 in the range of shear rate, 0.16 to 22.1 s(-1), for the cell volume fractions from 0.6 to 0.9 (mL/mL). In this study, the methods of determining the shear sensitivity and the viscous and the elastic components of mammalian cell suspensions are described under the steady shear field.

  14. Photodynamic damage study of HeLa cell line using ALA

    AlSalhi, M. S.; Atif, M.; AlObiadi, A. A.; Aldwayyan, A. S.

    2011-04-01

    The present study evaluates the photodynamic damage with 5-aminolevulinic acid (5-ALA) using HeLa as experimental model. HeLa cell line was irradiated with red light (He-Ne laser, λ = 632.8 CW nm). The influence of different incubation times and concentrations of 5-ALA, different irradiation doses and various combinations of photosensitizer and light doses on the cellular viability of HeLa cells were studied. The optimal uptake of photosensitizer ALA in HeLa cells was investigated by means of PpIX fluorescence intensity by exciting the HeLa cell suspension at 450 nm and a detection wavelength set at 690 nm. Cells viability was determined by means of trypan blue solution. The spectrometric measurements showed that the maximal cellular uptake of 5-ALA occurred after 4 h in vitro incubation. We found that the combination with 5-ALA and laser irradiation leads to time/concentration-dependent increase of cells death and also energy doses-dependent enlarge the cells death. The fluorescence intensity after PDD of carcinoma cells reduce when compared with the control group. The fluorescence emission spectral profiles after PDD of carcinoma cells showed a dip around 425-525 nm when compared with the control group. This may be due to the damage of mitochondria component of cells. The percentage of HeLa cells after PDD shows that the percentage of cells survival rate as function of laser dose (power). Hence it is clear that at 200 μg/ml ALA and 20 mW laser irradiation, more than 70% of HeLa cells were dead after 15 min.

  15. 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.

  16. 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.

  17. Apoptosis and necrosis in vaccinia virus-infected HeLa G and BSC-40 cells.

    Liskova, Jana; Knitlova, Jarmila; Honner, Richard; Melkova, Zora

    2011-09-01

    In most cells, vaccinia virus (VACV) infection is considered to cause a lytic cell death, an equivalent of necrosis. However, upon infection of the epithelial cell lines HeLa G and BSC-40 with VACV strain Western Reserve (WR), we have previously observed an increased activation of and activity attributable to caspases, a typical sign of apoptosis. In this paper, we have further analyzed the type of cell death in VACV-infected cells HeLa G and BSC-40. In a cell-based flow cytometric assay, we showed a specific activation of caspase-2 and 4 in HeLa G and BSC-40 cells infected with VACV, strain WR, while we did not find any effects of inhibitors of calpain and cathepsin D and E. The actual activity of the two caspases, but also of caspase-3, was then confirmed in lysates of infected HeLa G, but not in BSC-40 cells. Accordingly, poly(ADP)-ribose polymerase (PARP) cleavage was found increased only in infected HeLa G cells. Consequently, we have determined morphological features of apoptosis and/or activity of the executioner caspase-3 in infected HeLa G cells in situ, while only a background apoptosis was observed in infected BSC-40 cells. Finally, vaccination strains Dryvax and Praha were found to induce apoptosis in both HeLa G and BSC-40 cells, as characterized morphologically and by PARP cleavage. These findings may be important for understanding the differences in VACV-host interactions and post-vaccination complications in different individuals.

  18. Taxifolin synergizes Andrographolide-induced cell death by attenuation of autophagy and augmentation of caspase dependent and independent cell death in HeLa cells.

    Alzaharna, Mazen; Alqouqa, Iyad; Cheung, Hon-Yeung

    2017-01-01

    Andrographolide (Andro) has emerged recently as a potential and effective anticancer agent with induction of apoptosis in some cancer cell lines while induction of G2/M arrest with weak apoptosis in others. Few studies have proved that Andro is also effective in combination therapy. The flavonoid Taxifolin (Taxi) has showed anti-oxidant and antiproliferative effects against different cancer cells. Therefore, the present study investigated the cytotoxic effects of Andro alone or in combination with Taxi on HeLa cells. The combination of Andro with Taxi was synergistic at all tested concentrations and combination ratios. Andro alone induced caspase-dependent apoptosis which was enhanced by the combination with Taxi and attenuated partly by using Z-Vad-Fmk. Andro induced a protective reactive oxygen species (ROS)-dependent autophagy which was attenuated by Taxi. The activation of p53 was involved in Andro-induced autophagy where the use of Taxi or pifithrin-α (PFT-α) decreased it while the activation of JNK was involved in the cell death of HeLa cells but not in the induction of autophagy. The mitochondrial outer-membrane permeabilization (MOMP) plays an important role in Andro-induced cell death in HeLa cells. Andro alone increased the MOMP which was further increased in the case of combination. This led to the increase in AIF and cytochrome c release from mitochondria which consequently increased caspase-dependent and independent cell death. In conclusion, Andro induced a protective autophagy in HeLa cells which was reduced by Taxi and the cell death was increased by increasing the MOMP and subsequently the caspase-dependent and independent cell death.

  19. Effect of human mesenchymal stem cells on the growth of HepG2 and Hela cells.

    Long, Xiaohui; Matsumoto, Rena; Yang, Pengyuan; Uemura, Toshimasa

    2013-01-01

    Human mesenchymal stem cells (hMSCs) accumulate at carcinomas and have a great impact on cancer cell's behavior. Here we demonstrated that hMSCs could display both the promotional and inhibitive effects on growth of HepG2 and Hela cells by using the conditioned media, indirect co-culture, and cell-to-cell co-culture. Cell growth was increased following the addition of lower proportion of hMSCs while decreased by treatment of higher proportion of hMSCs. We also established a novel noninvasive label way by using internalizing quantum dots (i-QDs) for study of cell-cell contact in the co-culture, which was effective and sensitive for both tracking and distinguishing different cells population without the disturbance of cells. Furthermore, we investigated the role of hMSCs in regulation of cell growth and showed that mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways were involved in hMSC-mediated cell inhibition and proliferation. Our findings suggested that hMSCs regulated cancer cell function by providing a suitable environment, and the discovery from the study would provide some clues for development of effective strategy for hMSC-based cancer therapies.

  20. Metabolomic profiles delineate potential roles for gadolinium chloride in the proliferation or inhibition of Hela cells.

    Long, Xiao-Hui; Yang, Peng-Yuan; Liu, Qiong; Yao, Jun; Wang, Yi; He, Guo-Hua; Hong, Guang-Yan; Ni, Jia-Zuan

    2011-08-01

    Lanthanides (Lns) compounds have been reported to possess contrary effects on cell activity, i.e., promoting cell cycle progression and cell growth by lower concentration treatment, but suppressing cell proliferation and inducing cell apoptosis at higher dosing. However, the cellular processes during the intervention and the possible underlying mechanisms are still not well clarified. Using a combination of high-throughput liquid chromatography (LC) with mass spectrometry (MS), we have investigated the metabolomic profiles of Hela cells following gadolinium chloride (GdCl(3)) treatment in time- and concentration- dependent manners. A total of 48 metabolites released by Hela cells are identified to be differentially expressed (P strategy for the first time, disclose that different cell signaling pathways are activated by GdCl(3) treatment with different concentrations, leading to inhibitory or promotional effect on Hela cells.

  1. 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.

  2. Toxicity of cadmium sulfide (CdS) nanoparticles against Escherichia coli and HeLa cells.

    Hossain, Sk Tofajjen; Mukherjee, Samir Kumar

    2013-09-15

    The present study endeavours to assess the toxic effect of synthesized CdS nanoparticles (NPs) on Escherichia coli and HeLa cells. The CdS NPs were characterized by DLS, XRD, TEM and AFM studies and the average size of NPs was revealed as ∼3 nm. On CdS NPs exposure bacterial cells changed morphological features to filamentous form and damage of the cell surface was found by AFM study. The expression of two conserved cell division components namely ftsZ and ftsQ in E. coli was decreased both at transcriptional and translational levels upon CdS NPs exposure. CdS NPs inhibited proper cell septum formation without affecting the nucleoid segregation. Viability of HeLa cells declined with increasing concentration of CdS NPs and the IC₅₀ value was found to be 4 μg/mL. NPs treated HeLa cells showed changed morphology with condensed and fragmented nuclei. Increased level of reactive oxygen species (ROS) was found both in E. coli and HeLa cells on CdS NPs exposure. The inverse correlation between declined cell viabilities and elevated ROS level suggested that oxidative stress seems to be the key event by which NPs induce toxicity both in E. coli and HeLa cells.

  3. Discovery of HeLa Cell Contamination in HES Cells: Call for Cell Line Authentication in Reproductive Biology Research.

    Kniss, Douglas A; Summerfield, Taryn L

    2014-08-01

    Continuous cell lines are used frequently in reproductive biology research to study problems in early pregnancy events and parturition. It has been recognized for 50 years that many mammalian cell lines contain inter- or intraspecies contaminations with other cells. However, most investigators do not routinely test their culture systems for cross-contamination. The most frequent contributor to cross-contamination of cell lines is the HeLa cell isolated from an aggressive cervical adenocarcinoma. We report on the discovery of HeLa cell contamination of the human endometrial epithelial cell line HES isolated in our laboratory. Short tandem repeat analysis of 9 unique genetic loci demonstrated molecular identity between HES and HeLa cells. In addition, we verified that WISH cells, isolated originally from human amnion epithelium, were also contaminated with HeLa cells. Inasmuch as our laboratory did not culture HeLa cells at the time of HES cell derivations, the source of contamination was the WISH cell line. These data highlight the need for continued diligence in authenticating cell lines used in reproductive biology research.

  4. Je-Chun-Jun induced apoptosis of human cervical carcinoma HeLa cells

    Han-jung CHAE; Kyung-mi PARK; Geun-youn LEE; Gi-seup JEONG; Hyung-rae PARK; Hyung-min KIM; Soo-wan CHAE; Shim-keun YOO; Hyung-ryong KIM

    2004-01-01

    AIM: To study the mechanism of Je-Chun-Jun (JCJ)-inducing the apoptosis of the human cervical carcinoma,HeLa cells. METHODS: The cell viability was assessed using MTT assay. The optical density was measured at 570 nm. The caspase activity was measured using 50 mmol/L of fluorogenic substrate, AC-DEVD-AMC (caspase3), AC-VEID-AMC (caspase-8) or AC-LEHD-AFC (caspase-9). To confirm the expression of proteins, Western blotting was performed. To detect the characteristic of apoptosis chromatin condensation, HeLa cells were stained with Hoechst 33258 in the presence of JCJ. For the cell cycle analysis, HeLa cells were incubated with Propidium iodide (PI) solution. Fluorescence intensity of cell cycle was measured using flow cytometry system. RESULTS:The loss of viability occurred following the exposure of 10 g/L JCJ. Cells treated with 10 g/L JCJ for 3 d exhibited the apoptotic morphology (brightly blue-fluorescent condensed nuclei by Hoechst 33258-staining) and the reduction of cell volume. Cells incubated with JCJ for 48 h were arrested at the G1 phase of cell cycle and their G1 checkpoint related gene products such as cyclin D1 were transiently decreased. We showed that JCJ induced the p38 MAPK activation in HeLa cells. The p38 MAPK inhibitor, SB203580 protected Hela cells from the JCJ-induced death as well as intervened the JCJ-induced accumulation of cells at the G1 phase. In contrast, MEK1 (-ERK upstream) inhibitor, PD98059 had no effect on HeLa cells. CONCLUSION: JCJ induced cell cycle arrest and apoptosis of HeLa cells through p38 MAPK pathway.

  5. 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.

  6. REST is up-regulated by epidermal growth factor in HeLa cells and inhibits apoptosis by influencing histone H3 acetylation.

    Baiula, Monica; Carbonari, Gioia; Dattoli, Samantha D; Calienni, Maria; Bedini, Andrea; Spampinato, Santi

    2012-08-01

    REST (repressor element 1-silencing transcription factor) is a transcription factor that recruits histone deacetylases to silence gene transcription. REST appears to play a paradoxical role in cancer cells: it exhibits tumor suppressor activity or promotes tumorigenesis, depending upon the setting. The extracellular signaling molecules that control REST gene expression in cancer cells remain poorly understood. In this study, we report that REST expression in HeLa cells is elevated in cells exposed to epidermal growth factor or serum, whereas the rate of cell apoptosis is low. Apoptosis induced by serum withdrawal is significantly increased in HeLa cells treated with an antisense phosphorothioate oligodeoxynucleotide (AS ODN) capable of down-regulating REST expression, whereas in HeLa cells transfected with a REST expressing plasmid, REST overexpression reduces the marked apoptosis caused, in absence of serum, by exposure to an anti-Fas receptor antibody imitating the Fas ligand activity plus PD 98059, a blocker of extracellular signal-regulated kinase 1/2 activation. REST knockdown also reduces mRNA levels of the antiapoptotic protein Bcl-X(L) whereas in HeLa cells overexpressing REST, the reduction of Bcl-X(L) mRNA caused by the anti-Fas receptor antibody plus PD 98059 is significantly decreased. Finally, we report that acetylation of histone H3 is increased in HeLa cells exposed to AS ODN or anti-Fas receptor antibody, whereas it is reduced in cells transfected with the REST expressing plasmid. Our findings indicate that REST is a novel gene regulated by EGF in HeLa cells that potentially contributes to the modulation of apoptosis via epigenetic mechanisms.

  7. Toxicity of cadmium sulfide (CdS) nanoparticles against Escherichia coli and HeLa cells

    Hossain, Sk Tofajjen; Mukherjee, Samir Kumar, E-mail: dr.samirmukherjee@gmail.com

    2013-09-15

    Highlights: • Toxic effect of CdS NPs on the growth and cell division in E. coli was studied. • CdS NPs affected cell surface topology and cell division. • Downregulation of both FtsZ and FtsQ was observed due to NPs exposure. • CdS NPs affected HeLa cell morphology with fragmented nuclei. • All such effects might be due to elevated oxidative stress. -- Abstract: The present study endeavours to assess the toxic effect of synthesized CdS nanoparticles (NPs) on Escherichia coli and HeLa cells. The CdS NPs were characterized by DLS, XRD, TEM and AFM studies and the average size of NPs was revealed as ∼3 nm. On CdS NPs exposure bacterial cells changed morphological features to filamentous form and damage of the cell surface was found by AFM study. The expression of two conserved cell division components namely ftsZ and ftsQ in E. coli was decreased both at transcriptional and translational levels upon CdS NPs exposure. CdS NPs inhibited proper cell septum formation without affecting the nucleoid segregation. Viability of HeLa cells declined with increasing concentration of CdS NPs and the IC{sub 50} value was found to be 4 μg/mL. NPs treated HeLa cells showed changed morphology with condensed and fragmented nuclei. Increased level of reactive oxygen species (ROS) was found both in E. coli and HeLa cells on CdS NPs exposure. The inverse correlation between declined cell viabilities and elevated ROS level suggested that oxidative stress seems to be the key event by which NPs induce toxicity both in E. coli and HeLa cells.

  8. 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...

  9. Diosgenin induces apoptosis in HeLa cells via activation of caspase pathway

    Rui HUO; Qiu-li ZHOU; Ben-xiang WANG; Shin-ichi TASHIRO; Satoshi ONODERA; Takashi IKEJIMA

    2004-01-01

    AIM: To investigate the mechanism of diosgenin-induced HeLa cell apoptosis. METHODS: HeLa cell growth was measured by MTr method. Apoptosis was detected by electron microscopy and agarose gel electrophoresis. Ratio of apoptotic cells was measured by APO-BRDU kit. Cell cycle distribution and changes of mitochondrial membrane potential were monitored by flow cytometry. Caspase activities were assayed by caspase apoptosis detection kit.Western blot analysis was used to evaluate the level of mitochondrial Bcl-2 expression. RESULTS: Diosgenin inhibited HeLa cell growth. HeLa cells treated with diosgenin showed typical characteristics of apoptosis including the morphological changes and DNA fragmentation. Caspase family inhibitor (z-VAD-fmk), caspase-9 inhibitor (Ac-AAVALPAVLLALLAPLEHD-CHO), and caspase-3 inhibitor (z-DEVD-fmk) partially prevented diosgenin-induced apoptosis, but not caspase-8 inhibitor (z-IETD-fmk) and caspase-10 inhibitor (z-AEVD-fmk). Diosgenin caused reduction of mitochondrial membrane potential and down-regulated Bcl-2 expression. CONCLUSION:Diosgenin induced HeLa cell apoptosis through caspase pathway.

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

    Morotomi-Yano, Keiko; Akiyama, Hidenori; Yano, Ken-ichi

    2013-08-30

    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.

  11. Parkin induces G2/M cell cycle arrest in TNF-α-treated HeLa cells.

    Lee, Min Ho; Cho, Yoonjung; Jung, Byung Chul; Kim, Sung Hoon; Kang, Yeo Wool; Pan, Cheol-Ho; Rhee, Ki-Jong; Kim, Yoon Suk

    2015-08-14

    Parkin is a known tumor suppressor. However, the mechanism by which parkin acts as a tumor suppressor remains to be fully elucidated. Previously, we reported that parkin expression induces caspase-dependent apoptotic cell death in TNF-α-treated HeLa cells. However, at that time, we did not consider the involvement of parkin in cell cycle control. In the current study, we investigated whether parkin is involved in cell cycle regulation and suppression of cancer cell growth. In our cell cycle analyses, parkin expression induced G2/M cell cycle arrest in TNF-α-treated HeLa cells. To elucidate the mechanism(s) by which parkin induces this G2/M arrest, we analyzed cell cycle regulatory molecules involved in the G2/M transition. Parkin expression induced CDC2 phosphorylation which is known to inhibit CDC2 activity and cause G2/M arrest. Cyclin B1, which is degraded during the mitotic transition, accumulated in response to parkin expression, thereby indicating parkin-induced G2/M arrest. Next, we established that Myt1, which is known to phosphorylate and inhibit CDC2, increased following parkin expression. In addition, we found that parkin also induces increased Myt1 expression, G2/M arrest, and reduced cell viability in TNF-α-treated HCT15 cells. Furthermore, knockdown of parkin expression by parkin-specific siRNA decreased Myt1 expression and phosphorylation of CDC2 and resulted in recovered cell viability. These results suggest that parkin acts as a crucial molecule causing cell cycle arrest in G2/M, thereby suppressing tumor cell growth.

  12. 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.

  13. Effect of tunicamycin on cisplatin induced apoptosis of HeLa cells

    Ye XU

    2013-04-01

    Full Text Available Objective  To investigate the effect of endoplasmic reticulum stress (ER stress in cisplatin-induced apoptosis of human cervical cancer HeLa cells. Methods  HeLa cells were used as the study object which were divided into four groups: TUNI (5mg/L group, cisplatin (6mg/L group, TUNI(5mg/L+cisplatin(6mg/L group, and negative control group (no drug treatment. MTT assay was employed to examine the growth status of the cells. Hoechst staining was used to observe the morphological change in the nucleus. Immunoblotting was used to detect the activation of apoptotic proteins, caspase-3 and caspase-4. Indirect immunofluorescence was used to assess the expression of the protein disulfide isomerase (PDI and phosphorylated histone H2AX (γ-H2AX. Results  MTT assay showed that the growth inhibition rates were 2.65%±2.71%, 19.60%±4.34%, 44.69%±7.07% and 0% in TUNI group, cisplatin group, TUNI+cisplatin group and control group, respectively (P<0.05. Cisplatin showed a significant inhibitory effect on the growth of HeLa cells, and TUNI enhanced the effect of cisplatin. Statistical significance was found between TUNI+cisplatin group and cisplatin group (P<0.05. Hoechst staining showed that the fluorescence of the nucleus in control group was weak and well-distributed. At 12h after treatment, the nuclei in some HeLa cells in cisplatin group and TUNI+cisplatin group diminished in size, thus showing dense hyperfluorescence, and some of them were broken. The proportion of karyorrhexis cells in TUNI+cisplatin group (44.5%±5.1% was significantly higher than that in cisplatin group (22.7%±3.9%, P<0.05. Immunoblotting showed the expressions of activated caspase-3 and caspase-4 were up-regulated obviously in cisplatin group. Compared to cisplatin group, the expressions of those proteins significantly increased in TUNI+cisplatin group (P<0.05. Indirect immunofluorescence staining showed no PDI expression and weak fluorescence was found in control group. PDI

  14. 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.

  15. 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.

  16. Effects of trichostatin A (TSA) on growth and gene expression in HeLa cells%曲古菌素A对HeLa细胞增殖及其基因表达的影响研究

    Fengqiu Zhang; Huasheng Fang; Yuxiao Li; Guangyong Qin

    2008-01-01

    Objective:To investigate the expressions of p53,RB1,Fas,c-fos,Ras,EGFR mRNA in human cervical cancer (HeLa) cell in response to the trichostatin A (TSA).Methods:We took count of HeLa cells in different incubation times with TSA (0.2 IJm/L).The result indicated that HeLa cells changed evidently when HeLa cells were incubated for 36 h.Then,we investigated the genes expression (mRNA levels) of HeLa cells after treatment for 36 h using SYBR green real-time PCR.Results:We demonstrated that trichostatin A (TSA) could make human cervical cancer (HeLa) cell morphological change and induce HeLa cell apoptosis.Furthermore,the data suggest that TSA-induced down-regulation of p53,RB1,Fas,but upregulated c-fos gene expression after treatment for 36 h,and Ras,EGFR did not show obvious response to TSA treatments.Conclusion:TSA has different effects on gene expression.

  17. 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.

  18. Bid and calpains cooperate to trigger oxaliplatin-induced apoptosis of cervical carcinoma HeLa cells.

    Anguissola, Sergio; Köhler, Barbara; O'Byrne, Robert; Düssmann, Heiko; Cannon, Mary D; Murray, Frank E; Concannon, Caoimhin G; Rehm, Markus; Kögel, Donat; Prehn, Jochen H M

    2009-11-01

    The Bcl-2 homology 3-only protein Bid is an important mediator of death receptor-induced apoptosis. Recent reports and this study suggest that Bid may also mediate genotoxic drug-induced apoptosis of various human cancer cells. Here, we characterized the role of Bid and the mechanism of Bid activation during oxaliplatin-induced apoptosis of HeLa cervical cancer cells. Small hairpin RNA-mediated silencing of Bid protected HeLa cells against both death receptor- and oxaliplatin-induced apoptosis. Expression of a Bid mutant in which caspase-8 cleavage site was mutated (D59A) reactivated oxaliplatin-induced apoptosis in Bid-deficient cells but failed to reactivate death receptor-induced apoptosis, suggesting that caspase-8-mediated Bid cleavage did not contribute to oxaliplatin-induced apoptosis. Overexpression of bcl-2 or treatment with the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone abolished caspase-2, -8, -9, and -3 activation as well as Bid cleavage in response to oxaliplatin, suggesting that Bid cleavage occurred downstream of mitochondrial permeabilization and was predominantly mediated by caspases. We also detected an early activation of calpains in response to oxaliplatin. Calpain inhibition reduced Bid cleavage, mitochondrial depolarization, and activation of caspase-9, -3, -2, and -8 in response to oxaliplatin. Further experiments, however, suggested that Bid cleavage by calpains was not a prerequisite for oxaliplatin-induced apoptosis: single-cell imaging experiments using a yellow fluorescent protein-Bid-cyan fluorescent protein probe demonstrated translocation of full-length Bid to mitochondria that was insensitive to calpain or caspase inhibition. Moreover, calpain inhibition showed a potent protective effect in Bid-silenced cells. In conclusion, our data suggest that calpains and Bid act in a cooperative, but mutually independent, manner to mediate oxaliplatin-induced apoptosis of HeLa cells.

  19. 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.

  20. Norcantharidin induces apoptosis in HeLa cells through caspase, MAPK, and mitochondrial pathways

    Wei-weiAN; Xian-fengGONG; Min-weiWANG; Shin-ichiTASHIRO; SatoshiONODERA; TakashiIKEJIMA

    2004-01-01

    AIM: To investigate the mechanism of norcantharidin (NCTD)-induced HeLa cell apoptosis. METHODS: HeLa cell growth inhibition was measured by MTT method. Apoptosis was detected by Hoechst 33258 staining and agarose gel electrophoresis. Caspase activities were assayed using caspase apoptosis detection kit. Western blot analysis was used to evaluate the level of ICAD, ERK/p-ERK, JNK/p-JNK, and Bcl-X.L/Bax expression. RESULTS: Norcantharidin inhibited HeLa cell growth in a time- and dose-dependent manner. HeLa cells treated with norcantharidin showed typical characteristics of apoptosis including the morphological changes and DNA fragmentation. Caspase family inhibitor (z-VAD-fmk), caspase-8, -9 inhibitor (z-IETD-fmk, Ac-LEHD-CHO, respectively) and caspase-3 inhibitor (z-DEVD-fmk) partially prevent norcantharidin-induced apoptosis, but initiator caspase-1 inhibitor (Ac-YVAD-fmk) did not. The activities of caspase-3, -8, and -9 were up-regulated after norcantharidin treatment. Furthermore, NCTD-induced activation of caspase-3 resulted in the degradation of the inhibitor of caspase-activated DNase (ICAD). Up-regulation of mitochondrial Bax expression and down-regulation of Bcl-xLexpression also participated in the apoptosis induced by NCTD. Although p38 MAPK inhibitor (SB203580) failed to block cell death, ERK MAPK inhibitor (PD98059) and JNK MAPK inhibitor (SP600125) had marked inhibitory effects on norcantharidin-induced apoptosis. Moreover, the phosphorylation of JNK were up-regulated followed by delayed ERK phosphorylation after treatment with NCTD, suggesting that ERK and JNK were both responsible for NCTD-induced apoptosis in HeLa cells and worked at different stages. CONCLUSION: The cytotoxic effect of NCTD on HeLa cells was mainly due to apoptosis. The anti-tumor mechanism of NCTD might involve caspses, mitochondrial, and MAPKs pathways.

  1. Norcantharidin induces apoptosis in HeLa cells through caspase,MAPK,and mitochondrial pathways

    Wei-wei AN; Xian-feng GONG; Min-wei WANG; Shin-ichi TASHIRO; Satoshi ONODERA; Takashi IKEJIMA

    2004-01-01

    AIM: To investigate the mechanism of norcantharidin (NCTD)-induced HeLa cell apoptosis. METHODS: HeLa cell growth inhibition was measured by MTT method. Apoptosis was detected by Hoechst 33258 staining and agarose gel electrophoresis. Caspase activities were assayed using caspase apoptosis detection kit. Western blot analysis was used to evaluate the level of ICAD, ERK/p-ERK, JNK/p-JNK, and Bcl-XL/Bax expression. RESULTS:Norcantharidin inhibited HeLa cell growth in a time- and dose-dependent manner. HeLa cells treated with norcantharidin showed typical characteristics of apoptosis including the morphological changes and DNA fragmentation. Caspase family inhibitor (z-VAD-fmk), caspase-8, -9 inhibitor (z-IETD-fmk, Ac-LEHD-CHO,respectively) and caspase-3 inhibitor (z-DEVD-fmk) partially prevent norcantharidin-induced apoptosis, but initiator caspase-1 inhibitor (Ac-YVAD-fmk) did not. The activities of caspase-3, -8, and -9 were up-regulated after norcantharidin treatment. Furthermore, NCTD-induced activation of caspase-3 resulted in the degradation of the inhibitor of caspase-activated DNase (ICAD). Up-regulation of mitochondrial Bax expression and down-regulation of Bcl-xL expression also participated in the apoptosis induced by NCTD. Although p38 MAPK inhibitor (SB203580)failed to block cell death, ERK MAPK inhibitor (PD98059) and JNK MAPK inhibitor (SP600125) had marked inhibitory effects on norcantharidin-induced apoptosis. Moreover, the phosphorylation of JNK were up-regulated followed by delayed ERK phosphorylation after treatment with NCTD, suggesting that ERK and JNK were both responsible for NCTD-induced apoptosis in HeLa cells and worked at different stages. CONCLUSION: The cytotoxic effect of NCTD on HeLa cells was mainly due to apoptosis. The anti-tumor mechanism of NCTD might involve caspses, mitochondrial, and MAPKs pathways.

  2. 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

  3. 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.

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

    Lo, Yu-Li; Tu, Wei-Chen

    2015-12-05

    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

  5. The comparison of radiation responses in MCF-7 and HeLa cells

    Kang, Mi Young; Jang, Eun Yeong; Ryu, Tae Ho; Chung, Dong-Min; Kim, Jin Hong; Kim, Jin Kyu [Advanced Radiation Technology Institute, Jeongeup (Korea, Republic of)

    2014-11-15

    Activation of this pathway temporarily arrests cells at the G1 or G2 checkpoints of cell cycle, or terminates DNA replication and cell division. The present study was carried out to identify the fate of cells to cope with DNA damage stress. Cellular responses following IR treatment were different depending on the characteristics (origin, organism and genes expressed etc.) of cell line used and extent of genomic injury. p53 expression level was increased in a dose-dependent manner in both cells. IR induced a drastic increase in expression of p21 in MCF-7 compared to that in HeLa cells. Cell cycle analysis using flow cytometry showed a significant accumulation in G2/M phase after treatment of MCF-7 with IR. This study identified that IR-induced cell fates were determined through p53-dependent activation of p21, which resulted in senescence of MCF-7 cells and autophagy of HeLa cells.

  6. IR-induced autophagy plays a role in survival of HeLa cells

    Kang, Mi Young; Jang, Eun Yeong; Ryu, Tae Ho; Chung, Dong Min; Kim, Jin Hong; Kim, Jin Kyu [Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of)

    2014-04-15

    Cells respond to stress with repair, or are diverted into irreversible cell cycle exit (senescence) or are eliminated through programmed cell death. There are two major morphologically distinctive forms of programmed cell death, apoptosis and autophagic cell death. Apoptosis contribute to cell death, whereas autophagy can play a dual role in mediating either cell survival or death in response to various stress stimuli. Here we analysed cellular responses induced by IR. The understanding of an appropriate cellular stress response is of crucial importance in foreseeing the cell fate. Apoptotic feagures were not detected in HeLa under our experimental irradiation condition. Autophagic cell death in HeLa may play an important role in cell protection and can result in cell survival.

  7. Cholesterol esters are detected by Raman microspectroscopy in HeLa cells

    Manen, van Henk-Jan; Otto, Cees

    2008-01-01

    The detection of trans-unsaturated lipids in single HeLa cells by Raman microspectroscopy was recently reported in this journal by Onogi et al. Based on our previously published Raman microspectroscopy data of individual macrophage foam cells, a detailed comparison between our spectra and spectrum r

  8. MCPIP1 contributes to the toxicity of proteasome inhibitor MG-132 in HeLa cells by the inhibition of NF-κB.

    Skalniak, Lukasz; Dziendziel, Monika; Jura, Jolanta

    2014-10-01

    Recently, we have shown that the treatment of cells with proteasome inhibitor MG-132 results in the induction of expression of monocyte chemotactic protein-1 induced protein 1 (MCPIP1). MCPIP1 is a ribonuclease, responsible for the degradation of transcripts encoding certain pro-inflammatory cytokines. The protein is also known as an inhibitor of NF-κB transcription factor. Thanks to its molecular properties, MCPIP1 is considered as a regulator of inflammation, differentiation, and survival. Using siRNA technology, we show here that MCPIP1 expression contributes to the toxic properties of MG-132 in HeLa cells. The inhibition of proteasome by MG-132 and epoxomicin markedly increased MCPIP1 expression. While MG-132 induces HeLa cell death, down-regulation of MCPIP1 expression by siRNA partially protects HeLa cells from MG-132 toxicity and restores Nuclear factor-κB (NF-κB) activity, inhibited by MG-132 treatment. Inversely, overexpression of MCPIP1 decreased constitutive activity of NF-κB and limited the survival of HeLa cells, as we have shown in the previous study. Interestingly, although MG-132 decreased the expression of IκBα and increased p65 phosphorylation, the inhibition of constitutive NF-κB activity was observed in MG-132-treated cells. Since the elevated constitutive activity of NF-κB is one of the mechanisms providing increased survival of cancer cells, including HeLa cells, we propose that death-promoting properties of MCPIP1 in MG-132-treated HeLa cells may, at least partially, derive from the negative effect on the constitutive NF-κB activity.

  9. Inhibition of Cell Proliferation of Active-protein Fraction from Bactrian Camel Whey on Cervical Cancer HeLa Cells in vitro%新疆双峰驼乳清蛋白组分对人宫颈癌HeLa细胞增殖的抑制作用∗

    杨洁; 王红娟; 豆智华; 李冠

    2015-01-01

    目的:探讨新疆双峰驼乳清蛋白组分对人宫颈癌HeLa细胞增殖的抑制作用.方法:采用四甲基偶氮唑盐(MTT)法测定新疆双峰驼乳蛋白组分对HeLa细胞增殖的抑制作用;荧光染色检测HeLa细胞凋亡的形态变化;倒置显微镜、扫描电镜和透射电镜观察HeLa细胞的形态变化;用流式细胞术检测HeLa细胞凋亡率、线粒体膜电位的变化.结果表明:新疆双峰驼乳清蛋白组分TR35可抑制人宫颈癌HeLa细胞的增殖并诱导其凋亡,并呈时间和剂量依赖性(P <0.01);倒置显微镜、扫描电镜和透射电镜观察到典型的凋亡特征,荧光显微观察到凋亡小体;Annexin-Ⅴ/PI双染检测出现早期凋亡细胞;线粒体膜电位去极化,并将细胞周期阻滞在S期.结论:新疆双峰驼乳清蛋白组分TR35可抑制HeLa细胞的增殖和诱导细胞凋亡,TR35组分诱导细胞凋亡的途径可能与线粒体通路有关.%To explore the Xinjiang bactrian camel milk suppressor component that inhibit tumor cell pro-liferation in vitro and its mechanism, we adopt MTT to screen the whey protein components with anticancer activity. Hela cell morphological changes were studied by inverted microscope, scanning electron microscopy (sem) and transmission electron microscopy and fluorescence microscopy. Flow cytometry was used to detect apoptosis rate, the change of mitochondrial membrane potential in the Hela cells after 24 h treatment with camel whey tumor-suppressor component. Results show that the TR35 fractionation from camel whey have a certain inhibitory effect on cancer cell, and had a certain amount of time-dose dependent manner. TR35 components can induce the apoptosis of Hela cells, the apoptotic body, mitochondrial membrane potential depolarization, and cell cycle arrest in S phase. Under the light microscope and electron microscope showed typical apoptosis characteristics. Annexin-Ⅴ/PI double dye detection appeared early

  10. Molecular mechanism of ophiopogonin B induced cellular autophagy of human cervical cancer HeLa cells%麦冬皂苷B诱导人宫颈癌HeLa细胞自噬的机制

    许秋菊; 侯莉莉; 胡国强; 谢松强

    2013-01-01

    This study is to investigate the antitumor activity of ophiopogonin B (OP-B).MTT assay,flow cytometric analysis,acridine orange staining,Lyso-Tracker Red staining and HeLa-GFP-LC3 transfect cells assay were used to detect the proliferation activity,apoptosis and autophagy of HeLa cells.The results showed that OP-B exerted potent antiproliferative activity on HeLa cells,the cell growth inhibition effect of OP-B was not due to apoptosis and OP-B could induce autophagy of HeLa cells.OP-B also induced the protein expression up-regulation of Beclin-1 and promoted LC3 Ⅰ transformation LC3 Ⅱ,which were representative proteins of autophagy.Furthermore,3-MA,an inhibitor of autophagy,not only inhibited OP-B-mediated autophagy but also almost completely reversed the antiproliferative effect of OP-B,suggesting that the growth inhibition effect of OP-B was autophagy dependent.Western blotting demonstrated that OP-B inhibited the phosphorylation of Akt and its' downstream vital protein,such as mTOR and p70S6K.In addition,OP-B also induced the protein expression up-regulation of PTEN,which is a negative regulation protein for Akt/mTOR signaling pathway.However,OP-B did not affect the protein expression of total Akt.Collectively,the antitumor effects of OP-B were autophagy-dependent via repression Akt/mTOR signaling pathway.Therefore,OP-B is a prospective inhibitor of Akt/mTOR and may be used as an alternative compound to treat cervical carcinoma.%以人宫颈癌HeLa细胞为研究对象,探讨麦冬皂苷B的抗肿瘤作用及其分子机制.采用MTT检测、流式细胞仪分析、吖啶橙染色、Lyso-Tracker Red染色及HeLa-GFP-LC3转染细胞实验,分别检测HeLa细胞的增殖、凋亡及自噬.结果表明,麦冬皂苷B可抑制细胞增殖,但并不诱导细胞凋亡,可诱导细胞自噬,并引起自噬标志性蛋白Beclin-1表达增加及LC3 Ⅰ转变为LC3 Ⅱ;自噬抑制剂3-MA不但可以抑制该自噬作用而且几乎完全逆转其抗增殖作用,提

  11. Establishment of HeLa cell mutants deficient in sphingolipid-related genes using TALENs.

    Toshiyuki Yamaji

    Full Text Available Sphingolipids are essential components in eukaryotes and have various cellular functions. Recent developments in genome-editing technologies have facilitated gene disruption in various organisms and cell lines. We here show the disruption of various sphingolipid metabolic genes in human cervical carcinoma HeLa cells by using transcription activator-like effector nucleases (TALENs. A TALEN pair targeting the human CERT gene (alternative name COL4A3BP encoding a ceramide transport protein induced a loss-of-function phenotype in more than 60% of HeLa cells even though the cell line has a pseudo-triploid karyotype. We have isolated several loss-of-function mutant clones for CERT, UGCG (encoding glucosylceramide synthase, and B4GalT5 (encoding the major lactosylceramide synthase, and also a CERT/UGCG double-deficient clone. Characterization of these clones supported previous proposals that CERT primarily contributes to the synthesis of SM but not GlcCer, and that B4GalT5 is the major LacCer synthase. These newly established sphingolipid-deficient HeLa cell mutants together with our previously established stable transfectants provide a 'sphingolipid-modified HeLa cell panel,' which will be useful to elucidate the functions of various sphingolipid species against essentially the same genomic background.

  12. Effect of beta-adrenergic stimulants on cytotoxicity of mitomycin C in HeLa cells.

    Miyamoto, K; Sanae, F; Iwasaki, M; Koshiura, R

    1982-12-01

    Effects of several autonomic agents on the cytotoxicity of mitomycin C in HeLa cells were studied. When beta-adrenergic stimulants such as isoproterenol, epinephrine, terbutaline and turobuterol were added at concentrations over 10(-14) M 15 to 60 min before mitomycin C, the colony-forming ability of HeLa cells was significantly inhibited more than by mitomycin C alone. The action of isoproterenol and epinephrine on the colony-forming ability of the cells was abolished by propranolol. The intracellular cyclic AMP level of HeLa cells reached the peak of about two-fold the basal level at 30 min after the addition of 10(-8) M isoproterenol. In combination with mitomycin C, the high level of intracellular cyclic AMP induced by isoproterenol was maintained for a significantly longer period in comparison with that by isoproterenol alone, while mitomycin C alone caused essentially no change in the cyclic AMP level. The pretreatment with dibutyryl cyclic AMP also enhanced the effect of mitomycin C. From these findings, it is strongly suggested that the synergistic effect of beta-adrenergic stimulants on the cytotoxicity of mitomycin C is mediated via stimulation of the beta-adrenoceptors of HeLa cells which elevates the intracellular cyclic AMP for a long time in combination with mitomycin C.

  13. Rose Bengal acetate photodynamic therapy (RBAc-PDT) induces exposure and release of Damage-Associated Molecular Patterns (DAMPs) in human HeLa cells.

    Panzarini, Elisa; Inguscio, Valentina; Fimia, Gian Maria; Dini, Luciana

    2014-01-01

    The new concept of Immunogenic Cell Death (ICD), associated with Damage Associated Molecular Patterns (DAMPs) exposure and/or release, is recently becoming very appealing in cancer treatment. In this context, PhotoDynamic Therapy (PDT) can give rise to ICD and to immune response upon dead cells removal. The list of PhotoSensitizers (PSs) able to induce ICD is still short and includes Photofrin, Hypericin, Foscan and 5-ALA. The goal of the present work was to investigate if Rose Bengal Acetate (RBAc), a powerful PS able to trigger apoptosis and autophagy, enables photosensitized HeLa cells to expose and/or release pivotal DAMPs, i.e. ATP, HSP70, HSP90, HMGB1, and calreticulin (CRT), that characterize ICD. We found that apoptotic HeLa cells after RBAc-PDT exposed and released, early after the treatment, high amount of ATP, HSP70, HSP90 and CRT; the latter was distributed on the cell surface as uneven patches and co-exposed with ERp57. Conversely, autophagic HeLa cells after RBAc-PDT exposed and released HSP70, HSP90 but not CRT and ATP. Exposure and release of HSP70 and HSP90 were always higher on apoptotic than on autophagic cells. HMGB1 was released concomitantly to secondary necrosis (24 h after RBAc-PDT). Phagocytosis assay suggests that CRT is involved in removal of RBAc-PDT generated apoptotic HeLa cells. Altogether, our data suggest that RBAc has all the prerequisites (i.e. exposure and/or release of ATP, CRT, HSP70 and HSP90), that must be verified in future vaccination experiments, to be considered a good PS candidate to ignite ICD. We also showed tha CRT is involved in the clearance of RBAc photokilled HeLa cells. Interestingly, RBAc-PDT is the first cancer PDT protocol able to induce the translocation of HSP90 and plasma membrane co-exposure of CRT with ERp57.

  14. NHE1 mediates migration and invasion of HeLa cells via regulating the expression and localization of MT1-MMP.

    Lin, Yani; Wang, Jian; Jin, Weina; Wang, Lihong; Li, Huawen; Ma, Li; Li, Qinghua; Pang, Tianxiang

    2012-01-01

    Na(+)/H(+) exchanger 1 (NHE1), acting as an important regulator of intracellular pH (pH(i)) and extracellular pH (pH(e)), has been known to play a key role in the metastasis of many solid tumours. However, the exact mechanism underlying these processes, especially in cervical cancer, is still poorly understood. In the current study, we first showed that the inhibition of NHE1 activity by the specific inhibitor cariporide could suppress migration and invasion of HeLa cells in vitro. Moreover, cariporide also reversed the enhanced migration and invasion in HeLa cells by overexpressed membrane-type 1 matrix metalloproteinase (MT1-MMP). Subsequently, our results showed that NHE1 regulated the expression of MT1-MMP at both messenger RNA and protein levels as well as its localization. Meanwhile, we observed slight modification in the morphology of HeLa cell after treating with cariporide. The present work indicates that NHE1 mediates HeLa cell metastasis via regulating the expression and localization of MT1-MMP and provides a theoretical basis for the development of novel therapeutic strategies targeting cervical cancer.

  15. 塞来昔布对宫颈癌HeLa细胞的放射增敏作用观察及机制探讨%Effect and mechanism of celecoxib on radiosensitivity enhancement in cervical cancer HeLa cells

    王爱红; 余娟娟; 戚世芳; 田晓予

    2011-01-01

    目的 观察环氧合酶2(COX-2)抑制剂塞来昔布对宫颈癌HeLa细胞的放射增敏作用,并探讨其机制.方法 将对数生长期的HeLa细胞分为3组,阴性对照组单纯培养液培养,单纯放射组采用6 MV的X线照射治疗,放射加药组先用20、40 μmol/L塞来昔布作用72 h,再行X线照射.采用克隆形成实验测算细胞放射敏感性参数平均致死剂量(Do)、准域剂量(Dq)、2 Gy照射存活分数(SF2)及放射增敏比(SERDo),采用免疫细胞化学法检测HeLa细胞中的COX-2、VEGF-C.结果 放射加药组 Do、Dq和SF2值小于单纯放射组(P均<0.05),其SERDo由2.01±0.06增至2.41±0.08;与阴性对照组、单纯放射组比较,放射加药组HeLa细胞的COX-2、VEGF-C表达显著降低(P均<0.05).结论 塞来昔布能增强宫颈癌HeLa细胞的放射敏感性,可能与下调宫颈癌细胞的COX-2、VEGF-C表达有关.%Objective To observe the effect of celecoxib on radiosensitivity enhancement in cervical cancer HeLa cells and explore its mechanism. Methods Logarithmic phase HeLa cells were divided into 3 groups, the negative control group were culture medium, radiotherapy alone group was treated with 6 MV X ray radiation therapy, radiation plus drug group was treated with 20,40 μmol / L of celecoxib, for 72 h before radiation therapy. The average lethal dose ( Do), Quasi-field dose (Dq), 2 Gy irradiation of the surviving fraction (SF2) and radiosensitization ratio (SER) was measured by colony formation assay cells radiosensitivity parameters, the expression of cyclooxygenase-2、VEGF-C、antigen were detected by immunohistochemistry. Results Do 、 Dq and SF2 in radiation plus drug group were less than the control group (P all <0.05), its SERDo increased from 2.01 ±0.06 to 2.41 ± 0.08. The expreasion of COX-2, VEGF-C was significantly decreased in radiotherapy plus drug group HeLa cells ( P all < 0. 05 ), compared with the negative control group and radiotherapy alone. Conclusion Celecoxib

  16. 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.

  17. Effects of AuNPs@PEG-AS1411 nanoparticles on radiosensitization of HeLa cancer cells%聚乙二醇和核酸适配体AS1411修饰的金纳米粒子对人宫颈癌HeLa细胞放射敏感性的影响

    马洪鸽; 林温文; 史盼影; 张保国

    2015-01-01

    Objective To study the effects of AuNPs@PEG-AS1411 nanoparticles on radiosensitization of human uterine cervix cancer HeLa cells.Methods AuNPs were synthesized by citrate reduction method and then functioned with PEG and PEG-AS1411, respectively.CCK-8 assay and colon forming assay were used to detect the acute and chronic toxicity effects of AuNPs on HeLa cells, respectively.At the same time, clonogenic survival assay was applied to measure the cell survival rate of HeLa cells after exposure to AuNPs@PEG and AuNPs@PEG-AS1411 combined with X-ray radiation.The intracellular uptake of AuNPs@PEG and AuNPs@PEG-AS1411 in HeLa cells were detected by ICP-MS.Results The CCK-8 assay showed that AuNPs@PEG and AuNPs@PEG-AS1411 were not toxical on HeLa cells(P >0.05).But the clonogenic survival assay showed that AuNPs@PEG and AuNPs@PEG-AS1411 had toxicity on HeLa cells significantly after 10 d(t =4.38-11.60, P < 0.05).AuNPs functioned with AS1411 could increase the cellular uptake of AuNPs.AuNPs@PEG and AuNPs@PEG-AS1411 both had significant radiosensitive effect on HeLa cells (F =7.90,48.23, P < 0.05).The values of SERDo for AuNPs@PEG and AuNPs@PEG-AS1411 were 1.12 and 1.20, respectively, when the concentration of Au was 10 mg/L.Conclusions AuNPs@PEG and AuNPs@PEG-AS1411 could cause chronic toxicity on HeLa cells instead of acute effect.PEGylated AuNPs functioned with AS1411 could enhance the radiosensitivity of HeLa cells in vitro.%目的 研究聚乙二醇(PEG)和核酸适配体AS1411修饰的金纳米粒子(AuNPs)对人宫颈癌HeLa细胞辐射敏感性的影响.方法 用PEG和PEG-AS1411分别修饰经柠檬酸钠还原法制备的AuNPs,制备纳米粒子AuNPs@PEG和AuNPs@PEG-AS1411.分别用CCK-8法和克隆形成法检测纳米粒子的细胞毒性.用电感耦合等离子体质谱仪(ICP-MS)检测HeLa细胞对纳米粒子的吸收量.用克隆形成法检测纳米粒子联合X射线照射对HeLa细胞存活率的影响.结果 CCK-8实验结果

  18. Chlamydia trachomatis serovar L2 induces protein tyrosine phosphorylation during uptake by HeLa cells

    Birkelund, Svend; Johnsen, H; Christiansen, Gunna

    1994-01-01

    . By use of a monoclonal antibody against phosphotyrosine, we showed that three classes of proteins are tyrosine phosphorylated: a triple band of 68, 66, and 64 kDa, a 97-kDa band, and a 140-kDa band. The phosphorylation could be detected by immunoblotting from 15 min after infection of HeLa cells. We...

  19. 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

  20. 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.

  1. 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

  2. Induction of apoptosis in human cervical carcinoma Hela cells with active components of Menispermum dauricum.

    Wang, J Y; Sun, S; Liu, L; Yang, W S

    2014-02-13

    Menispermum dauricum DC possesses a wide range of pharmacological effects. In this study, the mechanism of apoptosis induced by active components of M. dauricum was investigated in the human cervical carcinoma HeLa cell line. HeLa cells were treated with different M. dauricum concentrations over different time periods. The proliferation-inhibitory rate and cytotoxic effect of HeLa cells were measured by using the methyl thiazolyl tetrazolium (MTT) assay, and the apoptotic rate was detected by flow cytometry. Expressions of caspase-9, caspase-8, caspase-3, Bcl-2, and Fas proteins, in the apoptotic pathway, and the expression of nuclear factor-kappa B (NF-κB) were detected by SP immunocytochemistry. The MTT assay showed that active components of M. dauricum could significantly inhibit the growth of HeLa cells in a dose- and time-dependent manner (Pdauricum, the expressions of caspase-8, caspase-9, caspase-3, Fas protein, and NF-κB all increased, and the expression of the Bcl-2 protein decreased, with significant differences relative to the control group (Pdauricum through the NF-κB signal transduction pathway and the caspase pathway, which was related to the downregulation of Bcl-2 expression and the upregulation of Fas expression.

  3. 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.

  4. As4S4 Induced Apoptosis in HeLa Cells and Its Molecular Mechanism

    YIN Ling; PU De-min; CHENG Yan-xiang; LIU Rong; LI Tian

    2007-01-01

    Objective: To investigate the As4S4 induced growth inhibition and apoptosis in HeLa cells and its possible relationship with cyclooxygenase-2 (COX-2). Methods: HeLa cells were treated with various concentrations (7.5, 15, 30, 60 mg/L) of As4S4 at different times (12, 24, 36, 48, 60 h). Cell growth was measured by MTT. Apoptosis was detected by double staining flow cytometry (FCM). Levels of PGE2 were measured by radioimmunoassay. The expression of COX-2 protein was examined by Western blot analysis. Results: After treated with different concentrations of As4S4, the growth of HeLa cells was suppressed significantly in a dose-and time-dependent manner. The IC50 of 24 h was 30 mg/L (P<0.01). As4S4 induced apoptosis with apoptosis rates at 8.13%-62.36% by flow cytometry (FCM) in a dose-dependent manners. The release of PGE2 was reduced in HeLa cells with the values being (70.56±2.03), (48.58±2.28), (29.25±1.57) and (18.02±1.04) respectively, significantly different compared with control group (3.15±0.01) (P<0.01). As4S4 also inhibited the activity and expression of COX-2 in a dose dependent manner and down-regulated the expression of COX-2 protein greatly. Conclusion: As4S4 could inhibit the proliferation and increase apoptosis in human HeLa cells. These effects may depend on the inhibition of the expression of COX-2 and PGE2 by As4S4.

  5. Imaging of apoptotic HeLa cells by using scanning near-field optical microscopy

    2002-01-01

    By using scanning near-field optical microscopy (SNOM), HeLa cells in apoptosis process are imaged with a higher optical resolution beyond the diffraction limit. Since SNOM provides both topographic and transmitted light intensity information of a cell, it can correlate the structural characteristics and optical properties with the spatial position of the apoptotic cells. Wavelength imaging by using near-field spectroscopy shows that there is a great difference in light propagation and absorption in the cell. This unique technique can be applied to the super high resolution imaging of different components in the cell. The observations by near-field optical imaging and near-field spectroscopy indicate an inhomogeneous aggregation of the inner structure in the apoptotic HeLa cells and the change of transmission intensity of light with the apoptosis status.

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

    Chan Wai-Yee

    2006-06-01

    Full Text Available Abstract Background 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. Methods 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. Results 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. Conclusion These data, taken together, have provided important insights on the probable functions of TSPY in cell cycle progression, cell proliferation, and tumorigenesis.

  7. Anticancer Activity of Certain Herbs and Spices on the Cervical Epithelial Carcinoma (HeLa Cell Line

    Danielle Berrington

    2012-01-01

    Full Text Available Acetone extracts of selected plant species were evaluated for their in vitro cytotoxicity against a noncancerous African green monkey kidney (Vero cell line and an adenocarcinoma cervical cancer (HeLa cell line. The plants studied were Origanum vulgare L. (Oregano, Rosmarinus officinalis L. (Upright and ground cove rosemary, Lavandula spica L. (Lavender, Laurus nobilis L. (Bay leaf, Thymus vulgaris L. (Thyme, Lavandula x intermedia L. (Margaret Roberts Lavender, Petroselinum crispum Mill. (Curly leaved parsley, Foeniculum vulgare Mill. (Fennel, and Capsicum annuum L. (Paprika. Antioxidant activity was determined using a quantitative DPPH (1,1-diphenyl-2-picryl hydrazyl assay. The rosemary species exhibited effective radical scavenging capacity with 50% inhibitory concentration (IC50 of 3.48±0.218 μg/mL and 10.84±0.125 μg/mL and vitamin C equivalents of 0.351 g and 1.09 g for McConnell’s Blue and Tuscan Blue, respectively. Cytotoxicity was measured using XTT (Sodium 3′-[1-(phenyl amino-carbonyl-3,4-tetrazolium]-bis-[4-methoxy-6-nitro] benzene sulfonic acid hydrate colorimetric assay. Only L. nobilis and O. vulgare exhibited pronounced effects on the HeLa cell line. Dose-dependent studies revealed IC50 of 34.46±0.48 μg/mL and 126.3±1.00 μg/mL on the HeLa cells and on the Vero cells 124.1 μg/mL ± 18.26 and 163.8 μg/mL ± 2.95 for L. nobilis and O. vulgare, respectively. Light (eosin and haematoxylin staining and confocal microscopy (Hoechst 33342, acridine orange, and propidium iodide staining were used to evaluate the cytotoxic mechanism of action for L. nobilis and O. vulgare.

  8. Cytotoxic effects of Pinus eldarica essential oil and extracts on HeLa and MCF-7 cell lines

    Sarvmeili, Najmeh; Jafarian-Dehkordi, Abbas; Zolfaghari, Behzad

    2016-01-01

    Several attempts have so far been made in the search of new anticancer agents of plant origin. Some studies have reported that different species of Pine genus possess cytotoxic activities against various cancer cell lines. In the present study, we evaluated the cytotoxic effects of Pinus eldarica bark and leaf extracts or leaf essential oil on HeLa and MCF-7 tumor cell lines. Hydroalcoholic and phenolic extracts and the essential oil of plant were prepared. Total phenolic contents of the extracts were measured using Folin-Ciocalteu reagent. Essential oil components were determined by gas chromatography-mass spectroscopy (GC-MS). Cytotoxic activity of the extracts and essential oil against HeLa and MCF-7 tumor cell lines were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The polyphenolic content of hydroalcoholic and phenolic extracts of the bark and hydroalcoholic extract of the leaf were 48.31%, 47.2%, and 8.47%, respectively. According to the GC-MS analysis, the major components of the leaf oil of P. eldarica were: β -caryophyllene (14.8%), germacrene D (12.9%), α–terpinenyl acetate (8.15%), α –pinene (5.7%), and –α humulene (5.9%). Bark extracts and leaf essential oil of P. eldarica significantly reduced the viability of both HeLa and MCF-7 cells in a concentration dependent manner. However, leaf extract showed less inhibitory effects against both cell lines. The essential oil of P. eldarica was more cytotoxic than its hydroalcoholic and phenolic extracts. The terpenes and phenolic compounds were probably responsible for cytotoxicity of P. eldarica. Therefore, P. eldarica might have a good potential for active anticancer agents. PMID:28003841

  9. HeLa Cells Containing a Truncated Form of DNA Polymerase Beta are More Sensitized to Alkylating Agents than to Agents Inducing Oxidative Stress.

    Khanra, Kalyani; Chakraborty, Anindita; Bhattacharyya, Nandan

    2015-01-01

    The present study was aimed at determining the effects of alkylating and oxidative stress inducing agents on a newly identified variant of DNA polymerase beta (polβ Δ208-304) specific for ovarian cancer. Pol β Δ208-304 has a deletion of exons 11-13 which lie in the catalytic part of enzyme. We compared the effect of these chemicals on HeLa cells and HeLa cells stably transfected with this variant cloned into in pcDNAI/neo vector by MTT, colony forming and apoptosis assays. Polβ Δ208-304 cells exhibited greater sensitivity to an alkylating agent and less sensitivity towards H2O2 and UV when compared with HeLa cells alone. It has been shown that cell death in Pol β Δ208-304 transfected HeLa cells is mediated by the caspase 9 cascade. Exon 11 has nucleotidyl selection activity, while exons 12 and 13 have dNTP selection activity. Hence deletion of this part may affect polymerizing activity although single strand binding and double strand binding activity may remain same. The lack of this part may adversely affect catalytic activity of DNA polymerase beta so that the variant may act as a dominant negative mutant. This would represent clinical significance if translated into a clinical setting because resistance to radiation or chemotherapy during the relapse of the disease could be potentially overcome by this approach.

  10. Phosphorylated extracellular signal-regulated kinase up-regulated p53 expression in shikonin-induced HeLa cell apoptosis

    WU Zhen; WU Li-jun; TASHIRO Shinichi; ONODERA Satoshi; IKEJIMA Takashi

    2005-01-01

    Background The role of extracellular signal-regulated kinase 1/2 (ERK1/2) in shikonin-induced HeLa cells apoptosis remains vague. This study was to investigate the activation of caspase pathways and the role of ERK1/2 in human cervical cancer cells, HeLa, by shikonin.Methods The inhibitory effect of shikonin on the growth of HeLa cells was measured by MTT assay. Fluorescent microscopic analysis of apoptotic cells stained with 4’,6’-oliiamiclino-2-phenylindole C (DAPI) and Hoechst 33258 was carried out. Caspase-3 and -8 activities were detected using caspase-3 substrate and caspase-8 substrate as substrates, respectively. The protein levels of ERK, p53 and p-ERK were determined by Western blot analysis.Results Shikonin inhibited cell growth in a time- and dose-dependent manner. Caspase-3 and caspase-8 were activated in the apoptotic process and caspase inhibitors effectively reversed shikonin-induced apoptosis. Phosphorylation of ERK resulted in up-regulation of p53 expression, which was blocked by mitogen-activated protein kinase (MEK), inhibitor PD 98059.Conclusion Shikonin induces HeLa cell apoptosis through the ERK, p53 and caspase pathways.

  11. Mathematical Modeling of the Heat-Shock Response in HeLa Cells

    2015-07-01

    Journal 109(2) 182–193 186 Scheff et al. phase of the heat -stress response. The behavior of the prior models showing no free homeostatic HSF, and... chemical reactions (15,43). Because cells respond differently to different types of heat -shock-response-inducing stimuli and different cell types have...Article Mathematical Modeling of the Heat -Shock Response in HeLa Cells Jeremy D. Scheff,1 Jonathan D. Stallings,2 Jaques Reifman,1,* and Vineet

  12. 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.

  13. 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.

  14. 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.

  15. Growth and apoptosis of HeLa cells induced by intense picosecond pulsed electric field

    Yuan-yuan HUA

    2011-07-01

    Full Text Available Objective To investigate the growth and apoptosis of HeLa cells induced by intense picosecond pulsed electric field(PEF in vitro.Methods HeLa cells cultured in vitro were divided into experimental group and control group(with or without intense picosecond PEF.With constant pulse width,frequency and voltage,the cells in experimental group were divided into 6 sub-groups according to the number of pulse(100,200,500,1000,1500,2000,the growth inhibition of HeLa cells by PEF and the dose-effect relationship were analyzed by MTT.Caspase 3 protein activity was detected in the cells in 500,1000 and 2000 sub-groups.Mitochondrial transmembrane potential was detected by rhodamine 123 staining with the cells in 2000 sub-groups.Results MTT assay demonstrated that intense picosecond PEF significantly inhibited the proliferation of HeLa cells in dose-dependent manner.The survival rates of cells declined along with the increase in pulse number,and were 96.23%±0.76%,94.11%±2.42%,90.31%±1.77%,64.59%±1.59%,32.95%±0.73%,23.85%±2.38% and 100%,respectively,in 100,200,500,1000,1500,2000 sub-groups and control group(P < 0.01.The Caspase 3 protein activity was significantly enhanced by intense picosecond PEF,and the absorbancy indexes(A were 0.174±0.012,0.232±0.017,0.365±0.016 and 0.122±0.011,respectively,in 500,1000,2000 sub-groups and control group(P < 0.05.The mitochondrial transmembrane potential of HeLa cells was significantly inhibited by intense picosecond PEF,and the fluorescence intensity in 2000 sub-group(76.66±13.38 was much lower than that in control group(155.81±2.33,P < 0.05.Conclusion Intense picosecond PEF may significantly inhibit the growth of HeLa cells,and induce cell apoptosis via mitochondrial pathway.

  16. 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.

  17. Oxygen depletion speeds and simplifies diffusion in HeLa cells.

    Edwald, Elin; Stone, Matthew B; Gray, Erin M; Wu, Jing; Veatch, Sarah L

    2014-10-21

    Many cell types undergo a hypoxic response in the presence of low oxygen, which can lead to transcriptional, metabolic, and structural changes within the cell. Many biophysical studies to probe the localization and dynamics of single fluorescently labeled molecules in live cells either require or benefit from low-oxygen conditions. In this study, we examine how low-oxygen conditions alter the mobility of a series of plasma membrane proteins with a range of anchoring motifs in HeLa cells at 37°C. Under high-oxygen conditions, diffusion of all proteins is heterogeneous and confined. When oxygen is reduced with an enzymatic oxygen-scavenging system for ≥ 15 min, diffusion rates increase by > 2-fold, motion becomes unconfined on the timescales and distance scales investigated, and distributions of diffusion coefficients are remarkably consistent with those expected from Brownian motion. More subtle changes in protein mobility are observed in several other laboratory cell lines examined under both high- and low-oxygen conditions. Morphological changes and actin remodeling are observed in HeLa cells placed in a low-oxygen environment for 30 min, but changes are less apparent in the other cell types investigated. This suggests that changes in actin structure are responsible for increased diffusion in hypoxic HeLa cells, although superresolution localization measurements in chemically fixed cells indicate that membrane proteins do not colocalize with F-actin under either experimental condition. These studies emphasize the importance of controls in single-molecule imaging measurements, and indicate that acute response to low oxygen in HeLa cells leads to dramatic changes in plasma membrane structure. It is possible that these changes are either a cause or consequence of phenotypic changes in solid tumor cells associated with increased drug resistance and malignancy.

  18. 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.

  19. IBTK Differently Modulates Gene Expression and RNA Splicing in HeLa and K562 Cells

    Giuseppe Fiume

    2016-11-01

    Full Text Available The IBTK gene encodes the major protein isoform IBTKα that was recently characterized as substrate receptor of Cul3-dependent E3 ligase, regulating ubiquitination coupled to proteasomal degradation of Pdcd4, an inhibitor of translation. Due to the presence of Ankyrin-BTB-RCC1 domains that mediate several protein-protein interactions, IBTKα could exert expanded regulatory roles, including interaction with transcription regulators. To verify the effects of IBTKα on gene expression, we analyzed HeLa and K562 cell transcriptomes by RNA-Sequencing before and after IBTK knock-down by shRNA transduction. In HeLa cells, 1285 (2.03% of 63,128 mapped transcripts were differentially expressed in IBTK-shRNA-transduced cells, as compared to cells treated with control-shRNA, with 587 upregulated (45.7% and 698 downregulated (54.3% RNAs. In K562 cells, 1959 (3.1% of 63128 mapped RNAs were differentially expressed in IBTK-shRNA-transduced cells, including 1053 upregulated (53.7% and 906 downregulated (46.3%. Only 137 transcripts (0.22% were commonly deregulated by IBTK silencing in both HeLa and K562 cells, indicating that most IBTKα effects on gene expression are cell type-specific. Based on gene ontology classification, the genes responsive to IBTK are involved in different biological processes, including in particular chromatin and nucleosomal organization, gene expression regulation, and cellular traffic and migration. In addition, IBTK RNA interference affected RNA maturation in both cell lines, as shown by the evidence of alternative 3′- and 5′-splicing, mutually exclusive exons, retained introns, and skipped exons. Altogether, these results indicate that IBTK differently modulates gene expression and RNA splicing in HeLa and K562 cells, demonstrating a novel biological role of this protein.

  20. Effect of extracts of trichosanthes root tubers on HepA-H cells and HeLa cells

    Chang-Ming Dou; Ji-Cheng Li

    2004-01-01

    AIM: To investigate the cytotoxic activity of extracts of trichosanthes root tubers (EOT) on HepA-H cells and HeLa cells compared with trichosanthin (TCS), and to explore the possible mechanism of growth inhibitory effect of EOT on HeLa cells.METHODS: Tumor cells were culturedin vitro, and then microculture tetrzoalium assay (MTT) was used to investigate drugs' cytotoxic activity. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to observe ultrastructural changes of cells, and electrophoresis was performed to detect changes of biochemical characteristics of intercellular DNA.RESULTS: TCS and EOT had no obvious effects on HepAH cells (P>0.05), but had remarkable effects on HeLa cells in a time and dose dependent manner (r>0.864, P<0.05or P<0.01). The inhibitory rate of EOT was much higher than that of TCS (P<0.01). Median inhibitory rates (IC50)of TCS and EOT on HeLa cells were 610.9 mg/L and 115.6 mg/L for 36 h, and 130.7 mg/L and 33.4 mg/L for 48 h respectively. Marked morphologic changes were observed including microvillus disappearance or reduction, cell membrane bledding, cell shrinkage, condensation of chromosomes and apoptotic bodies with complete membranes. Meanwhile, apoptosis of HeLa cells was confirmed by DNA ladder formation on gel electrophoresis.CONCLUSION: TCS and EOT have no obvious effects on HepA-H cells, but have significant inhibitory effects on HeLa cells, indicating that EOT is superior to TCS in anti-tumor activity.

  1. Attenuated vesicular stomatitis virus induces apoptosis in cervical cancer HeLa cells and its mechanism%减毒水泡性口炎病毒诱导宫颈癌HeLa细胞的凋亡及其作用机制

    连海; 张静敏; 夏志平; 汤晶; 张爽; 于玲

    2011-01-01

    Objective: To investigate the apoptosis-inducing effect of an attenuated vesicular stomatitis virus in cervical cancer HeLa cells, and to explore the possible mechanism. Methods: HeLa cells were infected with VSV ( MOI = 1.0) and the cell proliferation was determined by MTT assay at 6, 12, 18, 24, and 30 h after infection. Morphological changes of apoptosis were observed by acridine orange (AO)/ethidium bromide (EB) staining. Annexin V/PI double-staining was performed to detect early apoptosis rate of HeLa cells and the sub-G1 apoptotic peak was examined by flow cytometry. The mitochondrial membrane potential of HeLa cells was measured by the JC-1 staining. The activities of caspase-9, -8 and -3 were measured by caspase assay kit. Results: After HeLa cells were exposed to attenuated VSV for 12 h and 24 h, the viabilities of cells were reduced to (78.4 ± 1.9 ) % and (63.1 ± 5.6 ) % ( P < 0.01 ); the early apoptosis rates were 16.88±2.48)% and (31.9 ±4.24)% (P<0.01); the Sub-G1 apoptotic peaks were (14.85 ±1.48)% and 21.05 ± 2. 28) % (P < 0.01 ), respectively. Attenuated VSV significantly decreased mitochondrial membrane potential in HeLa cells with the increase of infection time ( P < 0.05 ). The activities of caspase-9 and caspase-3 of HeLa cells were significantly increased after VSV infection ( all P < 0.05 ). Conclusion: The attenuated VSV can inhibit the proliferation of HeLa cells and trigger apoptosis via caspase-9 and caspase-3-dependent pathway.%目的:研究减毒水泡性口炎病毒(vesicular stomatitis virus,VSV)对宫颈癌HeLa细胞的凋亡诱导作用,并探讨其可能的机制.方法:将减毒VSV以1.0 MOl的接种密度感染HeLa细胞,在6、12、18、24和30 h后收集细胞,MTT法检测HeLa细胞的增殖;AO/EB染色观察HeLa细胞凋亡形态学变化,Annexin V-FITC/PI双染法检测HeLa细胞早期凋亡率,流式细胞术分析HeLa细胞sub-G1凋亡峰,JC-1染色法测定细胞线粒体跨膜电位水

  2. Killing of glucose-deprived hypoxic cells with moderate hyperthermia. [HeLa cells

    Kim, J.H.; Kim, S.H.; Hahn, E.W.

    1978-08-01

    Investigations were carried out to determine the importance of glucose as a modifying factor for hyperthermic cellular damage. HeLa S-3 cells were heated under oxic and hypoxic conditions, in the presence and absence of D-glucose. Temperatures as low as 41/sup 0/C selectively enhanced killing of the glucose deprived hypoxic cell. Glucose deprivation (37/sup 0/C, 2 hr) or heat alone (41/sup 0/C for 2 hr in regular medium with glucose) produced minimal cell kill under oxic or hypoxic conditions. No enhancement was seen with hyperthermia under oxic condition in the absence of glucose. The result suggests that the interplay between glucose concentration is important for hyperthermic cell kill.

  3. Cytocompatibility of HeLa Cells to Nano-Sized Ceramics Particles.

    Seitoku, Eri; Abe, Shigeaki; Kusaka, Teruo; Nakamura, Mariko; Inoue, Satoshi; Yoshida, Yasuhiro; Sano, Hidehiko

    2016-04-01

    In this study, we investigated the behaviors and cytocompatibility response of human cervical carcinoma (HeLa) cells expose to nano-sized particles. Cultivated cells exposed to titanium oxide and indium oxide nanoparticles remained highly viable. In the presence of copper oxide (CuO); however, the cells became seriously inflamed. To understand the mechanism by which CuO causes cell death, we evaluated cell death and apoptosis cytometry. CuO induced cells apoptosis more strongly than exposure to titania nanoparticles. Confocal fluorescence microscopy revealed that the nano-sized particles penetrate the cells.

  4. Erythropoietin Receptor Antagonist Suppressed Ectopic Hemoglobin Synthesis in Xenografts of HeLa Cells to Promote Their Destruction.

    Yoshiko Yasuda

    Full Text Available The aim of this study is to explore a cause-oriented therapy for patients with uterine cervical cancer that expresses erythropoietin (Epo and its receptor (EpoR. Epo, by binding to EpoR, stimulates the proliferation and differentiation of erythroid progenitor cells into hemoglobin-containing red blood cells. In this study, we report that the HeLa cells in the xenografts expressed ε, γ, and α globins as well as myoglobin (Mb to produce tetrameric α2ε2 and α2γ2 and monomeric Mb, most of which were significantly suppressed with an EpoR antagonist EMP9. Western blotting revealed that the EMP9 treatment inhibited the AKT-pAKT, MAPKs-pMAPKs, and STAT5-pSTAT5 signaling pathways. Moreover, the treatment induced apoptosis and suppression of the growth and inhibited the survival through disruption of the harmonized hemoprotein syntheses in the tumor cells concomitant with destruction of vascular nets in the xenografts. Furthermore, macrophages and natural killer (NK cells with intense HIF-1α expression recruited significantly more in the degenerating foci of the xenografts. These findings were associated with the enhanced expressions of nNOS in the tumor cells and iNOS in macrophages and NK cells in the tumor sites. The treated tumor cells exhibited a substantial number of perforations on the cell surface, which indicates that the tumors were damaged by both the nNOS-induced nitric oxide (NO production in the tumor cells as well as the iNOS-induced NO production in the innate immune cells. Taken together, these data suggest that HeLa cells constitutively acquire ε, γ and Mb synthetic capacity for their survival. Therefore, EMP9 treatment might be a cause-oriented and effective therapy for patients with squamous cell carcinoma of the uterine cervix.

  5. Antibody ligation of CM1 on cisplatin-exposed HeLa cells induces apoptosis through reactive oxygen species-dependent Fas ligand expression.

    Park, Ga Bin; Kim, Daejin; Yoon, Hoi Soo; Kim, Yeong-Seok; Lee, Hyun-Kyung; Kim, Ki Tae; Jeong, Dae Hoon; Hur, Dae Young

    2014-06-01

    Centrocyte/centroblast marker 1 (CM1) has been identified as a pro-apoptosis molecule on B-cell lymphoma cells as well as several types of cancer cells. In this study, we investigated its signaling mechanism in HeLa cells after treatment with cisplatin in order to potentially identify a new therapeutic target. The CM1 molecule was induced on the surface of cisplatin-exposed HeLa cells. In these cells, ligation of CM1 with anti-CM1 monoclonal antibodies inhibited cell proliferation and produced reactive oxygen species. Fas ligand (FasL) expression was upregulated without upregulating Fas in cisplatin-exposed HeLa cells after CM1 stimulation. Pretreatment with N-acetylcysteine, a pan-capase inhibitor, and ZB4, an antagonistic anti-Fas antibody, effectively inhibited the apoptotic effect triggered by CM1. CM1 ligation induced apoptosis through disruption of the mitochondrial membrane potential, decreased Bcl-2 and phosphorylated ERK expression. These findings identify CM1 as a potential new therapeutic target related to cisplatin-exposed cervical cancer.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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)

  13. INHIBITION AND RADIATION SENSITIZING EFFECT OF INDOLEACETIC ACID COMBINED WITH HORSERADISH PEROXIDASE ON HELA CELLS

    宋丽萍; 黄辰; 邱曙东; 王月英; 张健; 陈顺昌; 马军; 王全丽

    2004-01-01

    Objective To observe the inhibition and radiation-sensitizing effect of Indoleacetic acid (IAA) combined with horseradish peroxidase(HRP)on Hela cells. Methods Hela cells were cultured in vitro and classified into control group, drug group incubated with different doses of IAA(30, 60, 90μmol·L-1) plus 1.2μg·mL-1 HRP, radiation group (6MV-X, 4Gy ) and group of radiation plus IAA plus HRP(same dose as above). All the above were treated for 24-96 hours.The growth inhibition, radiation-sensitizing effect were observed with methyl thiazolyl tetrazolium (MTT) photocolorimetric assay and trypan blue dye assay. The-effect on cell proliferation cycle was determined by flow cytometry. Results The antiproliferation activities showed a significant time-effect and dose-effect relationship to some extent after Hela cells were treated with IAA combined with HRP. The group of radiation plus 60μmol·L-1 IAA plus 1.2μg·mL-1 HRP and radiation plus 90μmol·L-1 IAA plus 1.2μg·mL-1 HRP showed an obvious radiation sensitizing effect. After treatment with 90μmol·L-1 IAA plus 1.2μg·mL-1 HRP for 72 hours, the determination of cell cycle showed that the percentages of the cells on stages G2-M and S were all higher than those of the control group. For the group of radiation plus IAA combined with HRP, the percentages of the cells on stages G2-M were higher than those of the radiation group. Conclusion The above findings suggest that IAA combined with HRP has an inhibitive and killing effect on Hela cells. The effect was stronger during the cell cycles of G2-M and S. It also has a radiation sensitizing effect. Its mechanism might be that Hela cells were blocked on stages G2-M, and it presents a collaborative killing effect during miototic time.

  14. A molecular understanding of D-homoestrone-induced G2/M cell cycle arrest in HeLa human cervical carcinoma cells.

    Minorics, Renáta; Bózsity, Noémi; Molnár, Judit; Wölfling, János; Mernyák, Erzsébet; Schneider, Gyula; Ocsovszki, Imre; Zupkó, István

    2015-10-01

    2-Methoxyestradiol (ME), one of the most widely investigated A-ring-modified metabolites of estrone, exerts significant anticancer activity on numerous cancer cell lines. Its pharmacological actions, including cell cycle arrest, microtubule disruption and pro-apoptotic activity, have already been described in detail. The currently tested D-ring-modified analogue of estrone, D-homoestrone, selectively inhibits cervical cancer cell proliferation and induces a G2/M phase cell cycle blockade, resulting in the development of apoptosis. The question arose of whether the difference in the chemical structures of these analogues can influence the mechanism of anticancer action. The aim of the present study was therefore to elucidate the molecular contributors of intracellular processes induced by D-homoestrone in HeLa cells. Apoptosis triggered by D-homoestrone develops through activation of the intrinsic pathway, as demonstrated by determination of the activities of caspase-8 and -9. It was revealed that D-homoestrone-treated HeLa cells are not able to enter mitosis because the cyclin-dependent kinase 1-cyclin B complex loses its activity, resulting in the decreased inactivation of stathmin and a concomitant disturbance of microtubule formation. However, unlike 2-ME, D-homoestrone does not exert a direct effect on tubulin polymerization. These results led to the conclusion that the D-homoestrone-triggered intracellular processes resulting in a cell cycle arrest and apoptosis in HeLa cells differ from those in the case of 2-ME. This may be regarded as an alternative mechanism of action among steroidal anticancer compounds.

  15. Biosynthesis of gold nanoparticles using Sargassum swartzii and its cytotoxicity effect on HeLa cells.

    Dhas, T Stalin; Kumar, V Ganesh; Karthick, V; Govindaraju, K; Shankara Narayana, T

    2014-12-10

    In this investigation, biological synthesis of gold nanoparticles (AuNPs) using Sargassum swartzii and its cytotoxicity against human cervical carcinoma (HeLa) cells is reported. The biological synthesis involved the reduction of chloroauric acid led to the formation of AuNPs within 5min at 60°C and the formation of AuNPs was confirmed using UV-vis spectrophotometer. The AuNPs were stable; spherical in shape with well-defined dimensions, and the average size of the particle is 35nm. A zeta potential value of -27.6mV revealed synthesized AuNPs were highly stable. The synthesized AuNPs exhibited a dose-dependent cytotoxicity against human cervical carcinoma (HeLa) cells. Furthermore, induction of apoptosis was measured by DAPI (4',6-Diamidino-2-phenylindole dihydrochloride) staining.

  16. Receptor ganglioside content of three hosts for Sendai virus. MDBK, HeLa, and MDCK cells.

    Markwell, M A; Fredman, P; Svennerholm, L

    1984-08-01

    Specific gangliosides GD1a, GT1b and GQ1b isolated from brain have been shown to function as receptors for Sendai virus by conferring susceptibility to infection when they are incorporated into receptor-deficient cells (Markwell, M.A.K., Svennerholm, L. and Paulson, J.C. (1981) Proc. Natl. Acad. Sci. USA 78, 5406-5410). The endogenous gangliosides of three commonly used hosts for Sendai virus: MDBK, HeLa, and MDCK cells were analyzed to determine the amount and type of receptor gangliosides present. In all three cell lines, GM3 was the major ganglioside component. The presence of GM1, GD1a and the more complex homologs of the gangliotetraose series was also established. In cell lines derived from normal tissue, MDBK and MDCK cells, gangliosides contributed 47-65% of the total sialic acid. In HeLa cells, gangliosides contributed substantially less (17% of the total sialic acid). The ganglioside content of each cell line was shown not to be immutable but instead to depend on the state of differentiation, passage number, and surface the cells were grown on. Thus, the ganglioside concentration of undifferentiated MDCK cells was found to be substantially greater than that of MDBK or HeLa cells, but decreased as the MDCK cells underwent differentiation. Changes in culture conditions that were shown to decrease the receptor ganglioside content of the cells resulted in a corresponding decrease in susceptibility to infection. The endogenous oligosialogangliosides present in susceptible host cells were shown to function as receptors for Sendai virus.

  17. Polyimidazole conjugated oligonucleotides reach the nucleus of HeLa cells.

    Morvan, F; Castex, C; Vivès, E; Imbach, J L

    2001-01-01

    Oligonucleotide models bearing 6, 12 or 18 histamine residues were synthesized on solid support and labeled with fluorescein. Only the oligo with 6 histamine residues showed a high uptake in HeLa cells with a nuclear localization. Experiment a 4 degrees C or with bafilomicyn A1 suggest that uptake proceeded by an endocytosis mechanism followed by a destabilization of the membrane. Once in the cytoplasm the oligo reached rapidly the nucleus.

  18. The nonstructural protein NP1 of human bocavirus 1 induces cell cycle arrest and apoptosis in Hela cells

    Sun, Bin; Cai, Yingyue; Li, Yongshu [College of Life Science, Central China Normal University, Wuhan 430079, Hubei (China); Li, Jingjing [College of Life Science, Hubei Normal University, Huangshi 435002, Hubei (China); Liu, Kaiyu [College of Life Science, Central China Normal University, Wuhan 430079, Hubei (China); Li, Yi, E-mail: johnli2668@hotmail.com [College of Life Science, Central China Normal University, Wuhan 430079, Hubei (China); Bioengineering Department, Wuhan Bioengineering Institute, Wuhan 430415, Hubei (China); Yang, Yongbo, E-mail: yongboyang@mail.ccnu.edu.cn [College of Life Science, Central China Normal University, Wuhan 430079, Hubei (China)

    2013-05-25

    Human bocavirus type 1 (HBoV1) is a newly identified pathogen associated with human respiratory tract illnesses. Previous studies demonstrated that proteins of HBoV1 failed to cause cell death, which is considered as a possible common feature of bocaviruses. However, our work showed that the NP1 of HBoV1 induced apoptotic cell death in Hela cells in the absence of viral genome replication and expression of other viral proteins. Mitochondria apoptotic pathway was involved in the NP1-induced apoptosis that was confirmed by apoptotic characteristics including morphological changes, DNA fragmentation and caspase activation. We also demonstrated that the cell cycle of NP1-transfected Hela cells was transiently arrested at G2/M phase followed by rapid appearance of apoptosis and that the N terminal domain of NP1 was critical to its nuclear localization and function in apoptosis induction in Hela cells. These findings might provide alternative information for further study of mechanism of HBoV1 pathogenesis. - Highlights: ► NP1 protein of HBoV1 induced apoptosis in Hela cells was first reported. ► NP1 induced-apoptosis followed the cell cycle arrest at G2/M phase. ► The NP1 induced-apoptosis was mediated by mitochondrion apoptotic pathway. ► N terminal of NP1 was critical for apoptosis induction and nuclear localization.

  19. Automatic segmentation of HeLa cell images

    Urban, Jan

    2011-01-01

    In this work, the possibilities for segmentation of cells from their background and each other in digital image were tested, combined and improoved. Lot of images with young, adult and mixture cells were able to prove the quality of described algorithms. Proper segmentation is one of the main task of image analysis and steps order differ from work to work, depending on input images. Reply for biologicaly given question was looking for in this work, including filtration, details emphasizing, segmentation and sphericity computing. Order of algorithms and way to searching for them was also described. Some questions and ideas for further work were mentioned in the conclusion part.

  20. Monitoring the elasticity changes of HeLa cells during mitosis by atomic force microscopy

    Jiang, Ningcheng; Wang, Yuhua; Zeng, Jinshu; Ding, Xuemei; Xie, Shusen; Yang, Hongqin

    2016-10-01

    Cell mitosis plays a crucial role in cell life activity, which is one of the important phases in cell division cycle. During the mitosis, the cytoskeleton micro-structure of the cell changed and the biomechanical properties of the cell may vary depending upon different mitosis stages. In this study, the elasticity property of HeLa cells during mitosis was monitored by atomic force microscopy. Also, the actin filaments in different mitosis stages of the cells were observed by confocal imaging. Our results show that the cell in anaphase is stiffer than that in metaphase and telophase. Furthermore, lots of actin filaments gathered in cells' center area in anaphase, which contributes to the rigidity of the cell in this phase. Our findings demonstrate that the nano-biomechanics of living cells could provide a new index for characterizing cell physiological states.

  1. 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.

  2. 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.

  3. 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

  4. Ethanolic Neem (Azadirachta indica Leaf Extract Prevents Growth of MCF-7 and HeLa Cells and Potentiates the Therapeutic Index of Cisplatin

    Chhavi Sharma

    2014-01-01

    Full Text Available The present study was designed to gain insight into the antiproliferative activity of ethanolic neem leaves extract (ENLE alone or in combination with cisplatin by cell viability assay on human breast (MCF-7 and cervical (HeLa cancer cells. Nuclear morphological examination and cell cycle analysis were performed to determine the mode of cell death. Further, to identify its molecular targets, the expression of genes involved in apoptosis, cell cycle progression, and drug metabolism was analyzed by RT-PCR. Treatment of MCF-7, HeLa, and normal cells with ENLE differentially suppressed the growth of cancer cells in a dose- and time-dependent manner through apoptosis. Additionally, lower dose combinations of ENLE with cisplatin resulted in synergistic growth inhibition of these cells compared to the individual drugs (combination index <1. ENLE significantly modulated the expression of bax, cyclin D1, and cytochrome P450 monooxygenases (CYP 1A1 and CYP 1A2 in a time-dependent manner in these cells. Conclusively, these results emphasize the chemopreventive ability of neem alone or in combination with chemotherapeutic treatment to reduce the cytotoxic effects on normal cells, while potentiating their efficacy at lower doses. Thus, neem may be a prospective therapeutic agent to combat gynecological cancers.

  5. 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

  6. Effects of matrine on apoptosis hnduction in Cervi Calcarcinoma hela cells%苦参碱诱导宫颈癌Hela细胞的凋亡作用

    王丽; 苏宝山; 吴静

    2012-01-01

    目的:研究不同浓度的苦参碱(Mat)对宫颈癌Hela细胞株的体外增殖抑制作用.方法:采用体外细胞培养技术,以不同浓度的Mat作用于宫颈癌Hela细胞,相同条件下培养24h、48h、72h后,倒置显微镜观察细胞形态变化;MTT法测定Mat对 Hela细胞的增殖抑制率;流式细胞仪(FCM)检测细胞凋亡率.结果:不同浓度Mat在体外均能不同程度抑制宫颈癌Hela细胞增殖,并且作用呈现时间-剂量依赖性;苦参碱诱导Hela细胞凋亡率增加.结论:Mat有抑制宫颈癌Hela细胞的增殖,诱导其凋亡作用.%Objective:To study the inhibitory effects of matrine at different concentrations on cervical carcinoma Hela cells in vitro. Methods:Matrine at different concentrations (0. 5,1. 0,1. 5 and 2. 0mg/ml) was re-spectively used to cultivate cervical cancer Hela cells in vitro. After Hela cells were cultivated for 24, 48 and 72 hours respectively, the proliferation of Hela cells treated with matrine was observed by discrepancy microscope; the effect of matrine on cervical cancer cells was assayed by MTT and flowcytometry(FCM). Results: Matrine of differ-ent concentration could significantly inhibit proliferation of Hela cells, and its inhibitory effects increased as the con-centration higher. It was shown that the inhibitory efficacy of matrine in vitro depended on the reaction time and dos-age. The effect on apoptosis rate was also increased. Conclusions: Matrine can significantly inhibit proliferation of cervical carcinoma Hela cells, and induce apoptosis of them.

  7. Red-luminescent europium (III) doped silica nanoshells: synthesis, characterization, and their interaction with HeLa cells.

    Yang, Jian; Sandoval, Sergio; Alfaro, Jesus G; Aschemeyer, Sharraya; Liberman, Alex; Martin, David T; Makale, Milan; Kummel, Andrew C; Trogler, William C

    2011-06-01

    A simple method to fabricate Eu(3+) doped silica nanoshells particles with 100 and 200 nm diameters is reported. Amino polystyrene beads were used as templates, and an 8 to 10 nm thick silica gel coating was formed by the sol-gel reaction. After removing the template by calcination, porous dehydrated silica gel nanoshells of uniform size were obtained. The Eu(3+) doped silica nanoshells exhibited a red emission at 615 nm on UV excitation. The porous structure of the silica shell wall was characterized by transmission electron microscopy measurements, while particle size and zeta potentials of the particles suspended in aqueous solution were characterized by dynamic light scattering. Two-photon microscopy was used to image the nanoshells after assimilation by HeLa cancer cells.

  8. Enhancement of transfection efficiency for HeLa cells via incorporating arginine moiety into chitosan

    2007-01-01

    Arginine-rich peptides have attracted considerable attention due to their distinct internalization mechanism. It was reported that arginine and guanidino moieties were able to translocate through cell membranes and played a critical role in the process of membrane permeation. In this work, arginine was conjugated to the backbone of chitosan to form a novel chitosan derivative, arginine modified chitosan (Arg-CS). Arg-CS/DNA complexes were prepared according to the method of coacervation process. The physicochemical properties of Arg-CS and Arg-CS/DNA complexes were characterized and the transfection activity and efficiency mediated by Arg-CS/DNA complexes were investigated taking HeLa cells as target cells. Arg-CS was characterized by FTIR and 13C NMR. Arg-CS/DNA polyelectrolyte complexes were investigated by agarose gel retardation, dynamic light scattering (DLS) and atomic force microscopy (AFM). The results revealed that the Arg-CS/DNA complexes started to form at N/P ratio of 2:1, and the size of particles varied from 100 to 180 nm. The cytotoxicity of Arg-CS and their complexes with plasmid DNA were determined by MTT assay for HeLa cells, and the results suggested that Arg-CS/DNA complexes were slightly less toxic than Arg-CS. Moreover, the derivative alone and their complexes showed significantly lower toxicity than PEI and PEI/DNA complexes, respectively. Taking HeLa cells as target cells and using pGL3-control as reporter gene, the luciferase expression mediated by Arg-CS was greatly enhanced to about 100 folds compared with the luciferase expression mediated by chitosan at different pH media. These results suggest that Arg-CS is a promising candidate as a safe and efficient vector for gene delivery and transfection.

  9. Ginsenoside Rh2 Showing Ability to Induce Apoptosis in HeLa Cells

    2003-01-01

    This paper deals with the inhibitory mechanisms of ginsenoside G-Rh2 on the growth of tumor cells. G-Rh2 significantly inhibited the proliferation of human cervical adenocarcinoma HeLa cells in a time- and dose-dependent manner. G-Rh2 induced apoptotic manifestations in HeLa cells as evidenced by the changes in the cell morphology, the DNA fragmentation and the activation of caspases. Caspase inhibitors, caspase family inhibitor, z-Val-Ala-Asp-fmk(z-VAD-fmk); caspase-1 inhibitor, Ac-Tyr-Val-Ala-Asp-chloromethyl-ketone(Ac-YVAD-cmk); caspase-3 inhibitor, z-Asp-Glu-Val-Asp-fmk(z-DEVE-fmk) and caspase-8 inhibitor, z-Ile-Glu-Asp-fmk(z-IETD-fmk) effectively attenuated G-Rh2-induced cell death. The activities of caspase-1 and caspase-3 were increased in the G-Rh2-induced apoptotic process. However, caspase inhibitors can not inhibit G-Rh-2 induced cell death completely. These results suggest that G-Rh2-induced cell death is mediated by the activation of caspase cascade, but there might be some other pathways for induction of this apoptosis.

  10. The pro-survival function of p53 in HeLa cells

    Kim, Jin Kyu; Kang, Mi Young; Jang, Eun Yeong; Kim, Jin Hong [Korea Atomic Energy Research Institute, Advanced Radiation Technology Institute, Jeongeup (Korea, Republic of)

    2014-11-15

    The rate of apoptosis and autophagy was variable with different p53 status after IR treatment of cells. The influence of p53 status on cell fate suggests a role of p53 in two fundamentally important cell biological pathways: autophagy and apoptosis. p53 coordinates cell cycle arrest and apoptosis to govern cell fate. This study was done to identify p53-mediated regulation of cell's fate. Autophagy induced by IR may prevent cells from undergoing apoptosis, implying an interlink modulation between autophagy and apoptosis. The rate of apoptosis and autophagy was determined with different p53 status after IR treatment of HeLa cells in this study. Our research on IR-induced cellular responses may provide new information about fate decision between the processes of apoptosis and autophagy.

  11. Concomitant Use of Sea Cucumber Organic Extract and Radiotherapy on Proliferation and Apoptosis of Cervical (HeLa Cell Line

    Javad Baharara

    2016-04-01

    Full Text Available Background Cervical carcinoma is gynecologic malignancy with conventional treatment modality but drug resistance interferes with current therapeutic methods. Therefore, identification of novel new modality with low toxicity has uniquely favorable strategy. Objectives The aim of this study is evaluation of concomitant use of sea cucumber organic extract and radiotherapy on ovarian cancer. Materials and Methods In this in vitro experimental study, HeLa cancer cells were cultured and suspended in RPMI (Roswell Park Memorial Institute 1640 medium supplemented with 10 % FBS (Fetal Bovine Serum, 1 % antibiotic. Cells were treated with extract at different concentrations (0 to 100 μg/mL for 24 hours. After determination of suitable concentration, the cells were exposed to 2 Gray gamma radiation in presence of extract for 192 seconds and 66 hours were kept in incubator till anti-proliferative assay were evaluated. To assess apoptosis, flow cytometry with PI (Propodium Iodide and acridine orange staining were performed. Results Morphological analysis and results from cytotoxicity assay exhibited that 50 µg/mL of sea cucumber extract alone is considered IC50 and combination of gamma radiation became more valuable in growth inhibition. Also, flow cytometry histogram of treated cells indicated sub-G1 peak demonstrating disturbance in membrane integrity and apoptosis cell death. Fluorescence images have been confirmed apoptosis cell death in treatment groups. Conclusions These data indicate that sea cucumber extract as novel resource of aquatic natural products significantly can inhibit cervical cancer cell growth and synergistic effect of natural extract along with radiation therapy was more effective in anti-cervical cancer therapy.

  12. 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.

  13. Cell-based chip for the detection of anticancer effect on HeLa cells using cyclic voltammetry.

    El-Said, Waleed Ahmed; Yea, Cheol-Heon; Kim, Hyunhee; Oh, Byung-Keun; Choi, Jeong-Woo

    2009-01-01

    HeLa cells directly immobilized on gold-patterned silicon substrate were used to assess the biological toxicity of anticancer drugs (hydroxyurea and cyclophosphamide). Immobilization of HeLa cells was confirmed by optical microscopy, and cell growth, viability and drug-related toxicity were examined by cyclic voltammetry and potentiometric stripping analysis. The voltammetric behaviors of HeLa cells displayed a quasi-reversible pattern with the peak current exhibiting a linear relationship with cell number. The attached living cells were exposed to different concentrations of hydroxyurea and cyclophosphamide as anticancer drugs, which induced the change of cyclic voltammetry current peak. As the exposed concentration of anticancer drugs was increased, the change of current peak was increased, which indicates the decrease of cell viability. Trypan Blue dyeing was performed to confirm the results of the effect of anticancer drugs on the cell viability which was obtained from cyclic voltammetry assay. The proposed direct cell immobilization method technique can be applied to the fabrication of cell chip for diagnosis, drug detection, and on-site monitoring.

  14. Cyclic RGD peptide incorporation on phage major coat proteins for improved internalization by HeLa cells.

    Choi, Dong Shin; Jin, Hyo-Eon; Yoo, So Young; Lee, Seung-Wuk

    2014-02-19

    Delivering therapeutic materials or imaging reagents into specific tumor tissues is critically important for development of novel cancer therapeutics and diagnostics. Genetically engineered phages possess promising structural features to develop cancer therapeutic materials. For cancer targeting purposes, we developed a novel engineered phage that expressed cyclic RGD (cRGD) peptides on the pVIII major coat protein using recombinant DNA technology. Using a type 88 phage engineering approach, which inserts a new gene to express additional major coat protein in the noncoding region of the phage genome, we incorporated an additional pVIII major coat protein with relatively bulky cRGD and assembled heterogeneous major coat proteins on the F88.4 phage surfaces. With IPTG control, we could tune different numbers of cRGD peptide displayed on the phage particles up to 140 copies. The resulting phage with cRGD on the recombinant pVIII protein exhibited enhanced internalization efficiency into HeLa cells in a ligand density and conformational structure dependent manner when comparing with the M13 phages modified with either linear RGD on pVIII or cRGD on pIII. Our cRGD peptide engineered phage could be useful for cancer therapy or diagnostic purposes after further modifying the phage with drug molecules or contrast reagents in the future.

  15. Metabolism of HeLa cells revealed through autofluorescence lifetime upon infection with enterohemorrhagic Escherichia coli

    Buryakina, Tatyana Yu.; Su, Pin-Tzu; Syu, Wan-Jr; Allen Chang, C.; Fan, Hsiu-Fang; Kao, Fu-Jen

    2012-10-01

    Fluorescence lifetime imaging microscopy (FLIM) is a sensitive technique in monitoring functional and conformational states of nicotinamide adenine dinucleotide reduced (NADH) and flavin adenine dinucleotide (FAD),main compounds participating in oxidative phosphorylation in cells. In this study, we have applied FLIM to characterize the metabolic changes in HeLa cells upon bacterial infection and made comparison with the results from the cells treated with staurosporine (STS), a well-known apoptosis inducer. The evolving of NADH's average autofluorescence lifetime during the 3 h after infection with enterohemorragic Escherichia coli (EHEC) or STS treatment has been observed. The ratio of the short and the long lifetime components' relative contributions of NADH increases with time, a fact indicating cellular metabolic activity, such as a decrease of oxidative phosphorylation over the course of infection, while opposite dynamics is observed in FAD. Being associated with mitochondria, FAD lifetimes and redox ratio could indicate heterogeneous mitochondrial function, microenvironment with bacterial infection, and further pathway to cell death. The redox ratios for both EHEC-infected and STS-treated HeLa cells have been observed and these observations also indicate possible apoptosis induced by bacterial infection.

  16. Nanomolar ouabain elicits apoptosis through a direct action on HeLa cell mitochondria.

    Alonso, Elba; Cano-Abad, María F; Moreno-Ortega, Ana J; Novalbos, Jesús; Milla, Juan; García, Antonio G; Ruiz-Nuño, Ana

    2013-11-01

    The steroid Na(+)/K(+) ATPase (NKA) blocker ouabain has been shown to exhibit pro-apoptotic effects in various cell systems; however, the mechanism involved in those effects is unclear. Here, we have demonstrated that incubation of HeLa cells during 24h with nanomolar concentrations of ouabain or digoxin causes apoptotic death of 30-50% of the cells. Ouabain caused the activation of caspases-3/7 and -9; however, caspase-8 was unaffected. The fact that compound Z-LEHD-FMK reduced both apoptosis and caspase-9 activation elicited by ouabain, suggest a mitochondrially-mediated pathway. This was strengthened by the fact that ouabain caused ATP depletion and the release of mitochondrial cytochrome c into the cytosol. Furthermore, upon ouabain treatment mitochondrial disruption and redistribution into the cytosol were observed. A mitochondrial site of action for ouabain was further corroborated by tight co-localisation of fluorescent ouabain with mitochondria. Finally, in ouabain-treated cells the histamine-elicited elevation of cytosolic Ca(2+) concentration ([Ca(2+)]c) suggests an additional effect on the endoplasmic reticulum (ER) leading to Ca(2+) store depletion. We conclude that fluorescent ouabain is taken up and tightly co-localises with mitochondria of HeLa cells. This indicates that apoptosis may be triggered by a direct action of ouabain on mitochondria.

  17. 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.

  18. Visualizing cell-cycle kinetics after hypoxia/reoxygenation in HeLa cells expressing fluorescent ubiquitination-based cell cycle indicator (Fucci).

    Goto, Tatsuaki; Kaida, Atsushi; Miura, Masahiko

    2015-12-10

    Hypoxia induces G1 arrest in many cancer cell types. Tumor cells are often exposed to hypoxia/reoxygenation, especially under acute hypoxic conditions in vivo. In this study, we investigated cell-cycle kinetics and clonogenic survival after hypoxia/reoxygenation in HeLa cells expressing fluorescent ubiquitination-based cell cycle indicator (Fucci). Hypoxic treatment halted cell-cycle progression during mid-S to G2 phase, as determined by the cell cycle-regulated E3 ligase activities of SCF(Skp2) and APC/C(Cdh1), which are regulators of the Fucci probes; however, the DNA content of the arrested cells was equivalent to that in G1 phase. After reoxygenation, time-lapse imaging and DNA content analysis revealed that all cells reached G2 phase, and that Fucci fluorescence was distinctly separated into two fractions 24h after reoxygenation: red cells that released from G2 arrest after repairing DNA double-strand breaks (DSBs) exhibited higher clonogenic survival, whereas most cells that stayed green contained many DSBs and exhibited lower survival. We conclude that hypoxia disrupts coordination of DNA synthesis and E3 ligase activities associated with cell-cycle progression, and that DSB repair could greatly influence cell-cycle kinetics and clonogenic survival after hypoxia/reoxygenation.

  19. Hyperthermia Differently Affects Connexin43 Expression and Gap Junction Permeability in Skeletal Myoblasts and HeLa Cells

    Ieva Antanavičiūtė

    2014-01-01

    Full Text Available Stress kinases can be activated by hyperthermia and modify the expression level and properties of membranous and intercellular channels. We examined the role of c-Jun NH2-terminal kinase (JNK in hyperthermia-induced changes of connexin43 (Cx43 expression and permeability of Cx43 gap junctions (GJs in the rabbit skeletal myoblasts (SkMs and Cx43-EGFP transfected HeLa cells. Hyperthermia (42°C for 6 h enhanced the activity of JNK and its target, the transcription factor c-Jun, in both SkMs and HeLa cells. In SkMs, hyperthermia caused a 3.2-fold increase in the total Cx43 protein level and enhanced the efficacy of GJ intercellular communication (GJIC. In striking contrast, hyperthermia reduced the total amount of Cx43 protein, the number of Cx43 channels in GJ plaques, the density of hemichannels in the cell membranes, and the efficiency of GJIC in HeLa cells. Both in SkMs and HeLa cells, these changes could be prevented by XG-102, a JNK inhibitor. In HeLa cells, the changes in Cx43 expression and GJIC under hyperthermic conditions were accompanied by JNK-dependent disorganization of actin cytoskeleton stress fibers while in SkMs, the actin cytoskeleton remained intact. These findings provide an attractive model to identify the regulatory players within signalosomes, which determine the cell-dependent outcomes of hyperthermia.

  20. Hyperthermia differently affects connexin43 expression and gap junction permeability in skeletal myoblasts and HeLa cells.

    Antanavičiūtė, Ieva; Mildažienė, Vida; Stankevičius, Edgaras; Herdegen, Thomas; Skeberdis, Vytenis Arvydas

    2014-01-01

    Stress kinases can be activated by hyperthermia and modify the expression level and properties of membranous and intercellular channels. We examined the role of c-Jun NH2-terminal kinase (JNK) in hyperthermia-induced changes of connexin43 (Cx43) expression and permeability of Cx43 gap junctions (GJs) in the rabbit skeletal myoblasts (SkMs) and Cx43-EGFP transfected HeLa cells. Hyperthermia (42°C for 6 h) enhanced the activity of JNK and its target, the transcription factor c-Jun, in both SkMs and HeLa cells. In SkMs, hyperthermia caused a 3.2-fold increase in the total Cx43 protein level and enhanced the efficacy of GJ intercellular communication (GJIC). In striking contrast, hyperthermia reduced the total amount of Cx43 protein, the number of Cx43 channels in GJ plaques, the density of hemichannels in the cell membranes, and the efficiency of GJIC in HeLa cells. Both in SkMs and HeLa cells, these changes could be prevented by XG-102, a JNK inhibitor. In HeLa cells, the changes in Cx43 expression and GJIC under hyperthermic conditions were accompanied by JNK-dependent disorganization of actin cytoskeleton stress fibers while in SkMs, the actin cytoskeleton remained intact. These findings provide an attractive model to identify the regulatory players within signalosomes, which determine the cell-dependent outcomes of hyperthermia.

  1. Temporal proteomic profiling of Chlamydia trachomatis-infected HeLa-229 human cervical epithelial cells.

    Tan, Grace Min Yi; Lim, Hui Jing; Yeow, Tee Cian; Movahed, Elaheh; Looi, Chung Yeng; Gupta, Rishein; Arulanandam, Bernard P; Abu Bakar, Sazaly; Sabet, Negar Shafiei; Chang, Li-Yen; Wong, Won Fen

    2016-05-01

    Chlamydia trachomatis is the leading causative agent of bacterial sexually transmitted infections worldwide which can lead to female pelvic inflammatory disease and infertility. A greater understanding of host response during chlamydial infection is essential to design intervention technique to reduce the increasing incidence rate of genital chlamydial infection. In this study, we investigated proteome changes in epithelial cells during C. trachomatis infection by using an isobaric tags for relative and absolute quantitation (iTRAQ) labeling technique coupled with a liquid chromatography-tandem mass spectrometry (LC-MS(3) ) analysis. C. trachomatis (serovar D, MOI 1)-infected HeLa-229 human cervical carcinoma epithelial cells (at 2, 4 and 8 h) showed profound modifications of proteome profile which involved 606 host proteins. MGST1, SUGP2 and ATXN10 were among the top in the list of the differentially upregulated protein. Through pathway analysis, we suggested the involvement of eukaryotic initiation factor 2 (eIF2) and mammalian target of rapamycin (mTOR) in host cells upon C. trachomatis infection. Network analysis underscored the participation of DNA repair mechanism during C. trachomatis infection. In summary, intense modifications of proteome profile in C. trachomatis-infected HeLa-229 cells indicate complex host-pathogen interactions at early phase of chlamydial infection.

  2. 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.

  3. 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.

  4. 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.

  5. Association of Chromosomal Telomere DNA With Nuclear Matrix in HeLa Cell

    汪国顺; 罗文捷; 潘惟钧; 丁明孝; 翟中和

    1994-01-01

    Using Electron Spectroscopic Imaging (ESI), we visualized the in situ binding of nucleic acids to nuclear matrix and 3H-thymidine incorporation which indicates that a small partial DNA bound to nuclear matrix tightly. Furthermore we found that chromosomal telomere DNA could bind to nuclear matrix specifically by the dot and Southern hybridization. The result of the Southwestern blot suggests that telomere DNA has high affinity to lamin B, vimentin and some nuclear matrix proteins. Therefore, the nuclear matrix and lamina of HeLa cell are possibly associated with spatial organization and action of chromosome.

  6. Comparative multiparametric analysis of HeLa and RD cell culture reactions to solcoseryl.

    Magakian, Yu A; Karalyan, Z A; Karalova, E M; Abroyan, L O; Akopyan, L A; Gasparyan, M H; Jaghacpanyan, N G; Semerjyan, Z B; Ter-Pogossyan, Z R

    2009-10-01

    Reactions of continuous HeLa and RD cell cultures and their nuclear and nucleolar apparatus to addition of solcoseryl into the medium were studied. The monolayer density, proliferation activity, percentage of dead cells, RNA and DNA content in the nuclei and nucleoli, number of nucleoli in the nuclei, cell distribution in the population by the number of nucleoli in the nuclei, volume and complete surface area of the nuclei and nucleoli, and the nucleolar/nuclear ratio were evaluated. The cultures differently reacted to solcoseryl in the medium at the population and cellular levels of their organization. By the results of multiparametric analysis of the reactions of cells and their nuclear and nucleolar apparatus, solcoseryl can be referred to bioactive substances with characteristics of a factor regulating cell population growth.

  7. Hela细胞端粒DNA断裂损伤%Strand breaks in telomeres in Hela cells

    阳芳; 杨洁萍; 李清焕; 邵兰; 谭铮

    2003-01-01

    Telomeres are the repetitive G-rich DNA sequences at the end of chromosomes and shorten at each round of cell division.Besides the incomplete DNA synthesis,single and double DNA strand breaks,if not repaired, also contribute to the telomere shortening.To assess the frequency of strand breaks in proliferating Hela cells,telomere fragments were released by alkaline denaturing and electrophoresis from cells embedded in agarose,blotted onto membrane,and detected by probe specific to telomere sequence.The quantity of telomere fragments released was estimated to be less than 0.4% of the total telomere content,which corresponded to less than one break per cell.Since the mean length of the terminal restriction fragments of the cells was about 7 kbp,the fragments detected would lead to less than 19 bp in mean telomere shortening [Acta Zoologica Sinica 49(6):873-877,2003].

  8. BLCAP arrests G₁/S checkpoint and induces apoptosis through downregulation of pRb1 in HeLa cells.

    Zhao, Min; Zhang, Li; Qiu, Xiaoping; Zeng, Fanyu; Chen, Wen; An, Yuehui; Hu, Bicheng; Wu, Xufeng; Wu, Xinxing

    2016-05-01

    BLCAP (bladder cancer-associated protein) gene exhibited tumor suppressor function in different tumors and is regarded as a candidate tumor suppressor gene; however, the mechanism by which BLCAP exerts its function remains elusive. This study investigated the functional association between BLCAP and proliferation or apoptosis in cervical cancer cells, to identify the functional motifs of BLCAP. The BLCAP-shRNA expression vector based on pRNA-U6.1/Hygro plasmid was used to specifically inhibit BLCAP activity in HeLa cells. The optimal shRNA plasmid was selected to knock down BLCAP expression and the biological effects were investigated. The effects on cell cycle and apoptosis were detected by flow cytometric or Annexin V-FITC staining analysis. The gene expression profiles of HeLa cells transfected with blcap-wt and BLCAP-shRNA were analyzed using human signal pathway gene Oligochips. The levels of protein expression and interaction of BLCAP with Rb1 proteins were determined by western blotting and Co-IP assays. The site-specific mutagenesis assay was used to identify amino acid residues important for BLCAP. Significantly differentially expressed genes were found by gene Oligo chips analysis. These genes were all correlated with proliferation, cell cycle and apoptosis. The results of western blotting and Co-IP assays confirmed that overexpression of BLCAP could interact with Rb1 and inhibit Rb1 phosphorylation. Further investigation revealed that SAXX mutation in the key regions of BLCAP suppressed the function of BLCAP and significantly increased the level of phosphorylated Rb1 protein. Here our findings suggested that the functional association of BLCAP and Rb1 might play important roles in proliferation and apoptosis of HeLa cells. It suggested that BLCAP could be a novel therapeutic target for cervical cancer.

  9. 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.

  10. 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.

  11. Usefulness of HeLa cells to evaluate inverse agonistic activity of antihistamines.

    Mizuguchi, Hiroyuki; Ono, Shohei; Hattori, Masashi; Sasaki, Yohei; Fukui, Hiroyuki

    2013-03-01

    Antihistamines are thought to antagonize histamine and prevent it from binding to the histamine H1 receptor (H1R). However, recent studies indicate that antihistamines are classified into two groups, i.e., inverse agonists and neutral antagonists on the basis of their ability to down-regulate the constitutive activity of H1R. As H1R is an allergy-sensitive gene whose expression influences the severity of allergic symptoms, inverse agonists should more potently alleviate allergic symptoms than neutral antagonists by inhibiting H1R constitutive activity. Therefore, it is important to assess inverse agonistic activity of antihistamines. Here we report a novel assay method using HeLa cells expressing H1R endogenously for evaluation of inverse agonistic activity of antihistamines. Pretreatment with inverse agonists down-regulated H1R gene expression below to its basal level. On the other hand, basal H1R mRNA expression was unchanged by neutral antagonist pretreatment. Both inverse agonists and neutral antagonists suppressed histamine-induced H1R mRNA elevation. Classification of antihistamines on the basis of their suppressive activity of basal H1R gene expression was consistent with that of inositol phosphate accumulation in H1R-overexpressed cells. Our data suggest that the assay method using HeLa cells is more convenient and useful than the existing methods and may contribute to develop new antihistamines with inverse agonistic activity.

  12. 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.

  13. 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.

  14. Passive permeability and effective pore size of HeLa cell nuclear membranes.

    Samudram, Arunkarthick; Mangalassery, Bijeesh M; Kowshik, Meenal; Patincharath, Nandakumar; Varier, Geetha K

    2016-09-01

    Nuclear pore complexes in the nuclear membrane act as the sole gateway of transport of molecules from the cytoplasm to the nucleus and vice versa. Studies on biomolecular transport through nuclear membranes provide vital data on the nuclear pore complexes. In this work, we use fluorescein isothiocyanate-labeled dextran molecules as a model system and study the passive nuclear import of biomolecules through nuclear pore complexes in digitonin-permeabilized HeLa cells. Experiments are carried out under transient conditions in the time lapse imaging scheme using an in-house constructed confocal laser scanning microscope. Transport rates of dextran molecules having molecular weights of 4-70 kDa corresponding to Stokes radius of 1.4-6 nm are determined. Analyzing the permeability of the nuclear membrane for different sizes the effective pore radius of HeLa cell nuclear membrane is determined to be 5.3 nm, much larger than the value reported earlier using proteins as probe molecules. The range of values reported for the nuclear pore radius suggest that they may not be rigid structures and it is quite probable that the effective pore size of nuclear pore complexes is critically dependent on the probe molecules and on the environmental factors.

  15. Rapid increase of inositol 1,4,5-trisphosphate in the HeLa cells after hypergravity exposure

    Kumei, Yasuhiro; Whitson, Peggy A.; Cintron, Nitza M.; Sato, Atsushige

    1990-01-01

    The IP3 level in HeLa cells has been elevated through the application in hypergravity in a time-dependent manner. The data obtained for the hydrolytic products of PIP2, IP3, and DG are noted to modulate c-myc gene expression. It is also established that the cAMP accumulation by the IBMX in hypergravity-exposed cells was suppressed relative to the control. In light of IP3 increase and cAMP decrease results, a single GTP-binding protein may play a role in the hypergravity signal transduction of HeLa cells by stimulating PLC while inhibiting adenylate cyclase.

  16. 利用RNA干扰技术构建IER5-SiRNA-Hela 细胞系%Recombination IER5-siRNA-Hela cells line using RNAi technology

    李莉; 赵焕英; 杨川杰; 鲁苇媛; 郝淳; 乔茶; 周平坤; 徐勤芝; 丁库克

    2011-01-01

    Objective To investigate the effect of RNA interference targeting IER5 gene on Hela cells in vitro and screen highly efficient small interfering RNA (siRNA ) .Methods RNAi technology was used to make the IER5 RNAi expression vector to transfect to Hela cells and established the stable transfected cell lines .Results Comparing the IER5-siRNA-Hela to the normal Hela cells ,the IER5 knock down Hela cells (IER5-siRNA-Hela) has high number as well as with a larger cell size using real-time PCR and Westem blot analysis .Conclusion We confinned the IER5 RNAi could effectively knock down the endogenous IER 5 from the Hela cells ,and established the stable transfected cell lines .%目的 观察小干扰RNA(siRNA)转染Hela细胞后,对IER5基因抑制效果,筛选出高效的siRNA转染质粒.方法 利用siRNA设计软件,将设计好的RNA片断插入到siRNA表达质粒载体PsilencerTM 3.1-H1 hygro中,测序正确后转染至Hela细胞中.利用Real-time PCR的方法,测定siRNA对Hela细胞中IRE5的抑制效果.结果 在mRNA水平及Western blot测试,IER5-siRNA-Hela细胞的IER5基因mRNA表达抑制效果明显.从细胞形态上讲,IER5-siRNA-Hela细胞与正常Hela细胞相比,IER5-siRNA-Hela细胞比Hela细胞体积大.结论 本实验成功的获得了特异性抑制IER5基因的IER5-siRNA-Hela细胞系.

  17. The influence of COX - 2 inhibitor on the expression of HPV18 - E6 and COX - 2 in Hela cells%环氧化酶2抑制剂对宫颈癌 Hela 细胞中 HPV18- E6和 COX -2表达的影响

    吴丽; 陶光实; 陈亦乐; 唐真姿; 杨俊

    2016-01-01

    Objective:To explore the influence of COX - 2 inhibitor Celecoxib on the expression of HPV18 - E6 and COX - 2 in Hela cells and its viability and apoptosis. Methods:Human uterine cervix cancer Hela cells were treated with different concentrations of Celecoxib. The expression of HPV18 - E6 and COX - 2 were detected by im-munocytochemical SP method and reverse transcription - polymerase chain reaction. Cell survival viability was tested by using the MTT assay. The change of cell apoptosis was detected by flow cytometry and its morphology was observed under microscope after Hoechst staining. Results:The expression of HPV18 - E6 and COX - 2 protein was inhibited by COX - 2 inhibitor,with time/ dose - dependent manner and they were positively related in Hela cells. The growth was inhibited and the apoptosis of Hela cells was induced by COX - 2 inhibitor. Conclusion:COX - 2 inhibitor can lower HPV18 - E6 and COX - 2 expression at the same time inhibit Hela cells viability and enhance its apoptosis.%目的:探讨环氧化酶2抑制剂 Celecoxib 对宫颈癌细胞 Hela 中 HPV18- E6、COX -2表达及其细胞增殖和凋亡的影响。方法:用不同浓度 Celecoxib 处理 Hela 细胞株后,免疫细胞化学 SP 法检测 HPV18- E6和COX -2蛋白的表达变化。RT - PCR 检测 HPV18- E6及 COX -2 mRNA 表达的改变。MTT 法检测其对细胞增殖的影响。流式细胞仪测定细胞凋亡情况。Hoechst33258染色观察细胞的形态学变化。结果:Celecoxib 作用 Hela 细胞后 HPV18- E6和 COX -2基因 mRNA 及蛋白表达量明显低于对照组,且随着药物浓度增加表达呈梯度下降。各组之间差异具有显著性(P <0.05),且两者之间具有显著相关性(P <0.01)。Celecoxib 抑制Hela 细胞增殖,作用呈量-效关系(P <0.05)。流式细胞仪显示各实验组凋亡率随浓度升高而增加,与对照组之间有显著差异(P <0.05)。Hoechst33258荧光染色显示经20μmol/ L 药物作用 Hela

  18. Isolation of chromatin DNA tightly bound to the nuclear envelope of HeLa cells.

    Kuvichkin, Vasily Vladimirovich

    2012-11-01

    Recent discovery of the role of nuclear pores in transcription, predicted by our early DNA-membrane complex (DMC) model, makes membrane-bound DNA (MBD) isolation from the cell nucleus and analysis of the MBD actual. The method of MBD isolation proposed by us retains DMC integrity during isolation. We used HeLa cells for DMC extraction. Changing the ionic composition of the isolation medium and replacing DNase I, used commonly for chromatin destruction, with a set of restriction enzymes allowed us to isolate the MBD. Treatment of a nuclear membrane with proteinase K and ultrasound has been used to increase the yield of MBD. Electron microscopic analysis of the purified fraction of isolated DMC supports our previous model of nuclear envelope lipid-chromatin interaction in the nuclear pore assembly.

  19. Kinetic analysis of RNA interference for lamin A/C in HeLa cells

    Sung-Ju You; Sang-Hoon Lee; Jae-Seung Lee

    2010-01-01

    We kinetically analyzed RNA interference(RNAi)for lamin A/C in HeLa cells,assuming suppression and recovery phases of gene expression,and dilution of transfected small interfering RNA(siRNA)by cell divisions.We observed the inhibitory effect of RNAi over a period of 6 days using various siRNA concentrations,and the maximum gene silencing efficiency occurred at Day 2 or 3.The gene silencing efficiency as a function of time and siRNA concentration was further quantitatively evaluated using a kinetic analysis method,demonstrating that RNAi for lamin A/C can be understood as a conventional drug response system.This work provides potentially important guidelines for future applications using nanomater ials as delivery vehicles of siRNA in RNAi for lamin A/C.

  20. Downregulation of FoxM1 inhibits proliferation, invasion and angiogenesis of HeLa cells in vitro and in vivo.

    Chen, Hong; Zou, Yang; Yang, Hong; Wang, Jingjing; Pan, Hong

    2014-12-01

    FoxM1 is a specific transcription factor that has an important function in aggressive human carcinomas, including cervical cancer. However, the specific function and internal molecular mechanism in cervical cancer remain unclear. In this study, RNAi-mediated FoxM1 knockdown inhibited cell growth. This process also decreased the migration and invasion activities of HeLa cells in vitro. Downregulation of FoxM1 inhibited tumor growth and angiogenesis in vivo. In addition, the expressions of uPA, matrix metalloproteinase (MMP)-2, MMP-9 and VEGF were significantly decreased in vitro and in vivo. These results suggested that the inactivation of FoxM1 could be a novel therapeutic target for cervical cancer treatment.

  1. Differential Expression of miR-21, miR-126, miR-143, miR-373 in Normal Cervical Tissue, Cervical Cancer Tissue and Hela Cell%miR-21、miR-126、miR-143和miR-373在正常宫颈组织、宫颈癌组织及Hela细胞中的表达差异

    刘琳; 王月玲; 王江芬

    2012-01-01

    目的 研究miR-21、miR-126、miR-143和miR-373在正常宫颈组织、宫颈癌组织及Hela细胞中的表达差异,探讨microRNAs(miRNAs)对宫颈癌发生的调控作用.方法 荧光实时定量检测20例良性肿瘤患者的正常宫颈组织,20例宫颈癌组织及Hela细胞中miR-21、miR-126、miR-143和miR-373的表达.结果 m iR-21在宫颈癌组织及Hela细胞系中高表达,在正常宫颈标本中低表达,在宫颈癌组织中相对定量为正常宫颈组织的11.3196倍(P<0.05);miR-143、miR-373在宫颈癌组织及Hela细胞中均低表达,在正常宫颈标本中高表达,miR-143、miR-373在宫颈癌组织中相对定量分别为正常宫颈组织的0.1553倍和0.4907倍(P<0.05);miR-126的表达无明显变化.结论 miRNAs与宫颈癌的发生和调控密切相关.在宫颈癌组织和Hela细胞中,miR-21表达上调,可能在宫颈癌的发生过程中发挥癌基因的作用;miR-143、miR-373表达下调可能发挥抑癌基因的作用;miR-126表达无差异,与宫颈癌无明显关系.%Objective To investigate the differential expression of miR-21 ,miR-126 ,miR-143 and miR-373 in normal cervical tissue, cervical cancer tissue and Hela cell.Methods The expressions of miR-21, miR-126, miR-143 and miR-373 were detected by real-time PCR in cervical cancer tissue,cervical tissue of benign uterine tumor and Hela cell. Results High expression of miR-21 was observed in cervical cancer and Hela cell, while low expression was observed in normal cervical tissue. The relative quantification of miR-21 in cerveical cancer was 11.3196 times that of miR-21 in normal cervical tissue (P0. 05). Conclusion miRNAs are closely related to the occurrence and regulation of cervical cancer. The high expression of miR-21 in cervical cancer and Hela cell indicate that it may play a possible role of oncogenes, while miR-143 and miR-373 with low expression may play the role of tumor suppressor genes.

  2. Effects of all-trans-retinoic acid on the expression and tyrosine phosphorylation of gap junction connexin 43 in HeLa cell line and its significance

    CHEN Bi-liang; MA Xiang-dong; XIN Xiao-yan; WANG De-tang; WANG Chun-mei

    2001-01-01

    Objective: To investigate the signal transduction mechanism of gap junctional genes connexin43 in human cervical carcinogenesis. Methods: Human cervical carcinoma cell line HeLa was cultured and treated by all-trans-retinoic acid (ATRA). Flow cytometer (FCM) was employed to detect expression of Cx43 protein in HeLa cells. Fluo-3 AM loading and laser scanning confocal microscope (LSCM) were used to measure the concentrations of intracellular calcium ([Ca2+]i) in HeLa cells. Phosphorylation on tyrosine of connexin43 protein was examined by immunoblot. Results: The positive rate of Cx43 protein increased from 1.9% in untreated HeLa cells to 26.3% in RA-treated HeLa cells as shown by FCM. [Ca2+]i was 35.73 nmol/L in untreated HeLa cells which was increased to 58.16 nmol/L in ATRA-treated cells.Immunoblot showed that ATRA-treated HeLa cells had phosphorylation on tyrosine in Cx43 protein whereas untreated cells had not. Conclusions: Carcinogenesis of human cervical carcinoma is related with the abnormal expression of cx43gene and disorder of signal transduction manifested as the decrease of [Ca2+]i and post-translation phosphorylation on tyrosine of Cx43 protein. The anti-tumor effect of ATRA in HeLa cells might be due to the up-regulation of cx43 gene and its signal transduction pathway.

  3. Staphylococcus aureus Lpl Lipoproteins Delay G2/M Phase Transition in HeLa Cells

    Nguyen, Minh-Thu; Deplanche, Martine; Nega, Mulugeta; Le Loir, Yves; Peisl, Loulou; Götz, Friedrich; Berkova, Nadia

    2016-01-01

    The cell cycle is an ordered set of events, leading to cell growth and division into two daughter cells. The eukaryotic cell cycle consists of interphase (G1, S, and G2 phases), followed by the mitotic phase and G0 phase. Many bacterial pathogens secrete cyclomodulins that interfere with the host cell cycle. In Staphylococcus aureus four cyclomodulins have been described so far that all represent toxins and are secreted into the culture supernatant. Here we show that the membrane-anchored lipoprotein-like proteins (Lpl), encoded on a genomic island called νSaα, interact with the cell cycle of HeLa cells. By comparing wild type and lpl deletion mutant it turned out that the lpl cluster is causative for the G2/M phase transition delay and also contributes to increased invasion frequency. The lipoprotein Lpl1, a representative of the lpl cluster, also caused G2/M phase transition delay. Interestingly, the lipid modification, which is essential for TLR2 signaling and activation of the immune system, is not necessary for cyclomodulin activity. Unlike the other staphylococcal cyclomodulins Lpl1 shows no cytotoxicity even at high concentrations. As all Lpl proteins are highly conserved there might be a common function that is accentuated by their multiplicity in a tandem gene cluster. The cell surface localized Lpls' suggests a correlation between G2/M phase transition delay and host cell invasion. PMID:28083519

  4. IL-6 Antibody and RGD Peptide Conjugated Poly(amidoamine) Dendrimer for Targeted Drug Delivery of HeLa Cells.

    Mekuria, Shewaye Lakew; Debele, Tilahun Ayane; Chou, Hsiao-Ying; Tsai, Hsieh-Chih

    2016-01-14

    In this study, PAMAM dendrimer (G4.5) was conjugated with two targeting moieties, IL-6 antibody and RGD peptide (G4.5-IL6 and G4.5-RGD conjugates). Doxorubicin anticancer drug was physically loaded onto G4.5-IL6 and G4.5-RGD with the encapsulation efficiency of 51.3 and 30.1% respectively. The cellular internalization and uptake efficiency of G4.5-IL6/DOX and G4.5-RGD/DOX complexes was observed and compared by confocal microscopy and flow cytometry using HeLa cells, respectively. The lower IC50 value of G4.5-IL6/DOX in comparison to G4.5-RGD/DOX is indication that higher drug loading and faster drug release rate corresponded with greater cytotoxicity. The cytotoxic effect was further verified by increment in late apoptotic/necrotic cells due to delivery of drug through receptor-mediated endocytosis. On the basis of these results, G4.5-IL6 is a better suited carrier for targeted drug delivery of DOX to cervical cancer cells.

  5. Hypoxia Strongly Affects Mitochondrial Ribosomal Proteins and Translocases, as Shown by Quantitative Proteomics of HeLa Cells.

    Bousquet, Paula A; Sandvik, Joe Alexander; Arntzen, Magnus Ø; Jeppesen Edin, Nina F; Christoffersen, Stine; Krengel, Ute; Pettersen, Erik O; Thiede, Bernd

    2015-01-01

    Hypoxia is an important and common characteristic of many human tumors. It is a challenge clinically due to the correlation with poor prognosis and resistance to radiation and chemotherapy. Understanding the biochemical response to hypoxia would facilitate the development of novel therapeutics for cancer treatment. Here, we investigate alterations in gene expression in response to hypoxia by quantitative proteome analysis using stable isotope labeling with amino acids in cell culture (SILAC) in conjunction with LCMS/MS. Human HeLa cells were kept either in a hypoxic environment or under normoxic conditions. 125 proteins were found to be regulated, with maximum alteration of 18-fold. In particular, three clusters of differentially regulated proteins were identified, showing significant upregulation of glycolysis and downregulation of mitochondrial ribosomal proteins and translocases. This interaction is likely orchestrated by HIF-1. We also investigated the effect of hypoxia on the cell cycle, which shows accumulation in G1 and a prolonged S phase under these conditions. Implications. This work not only improves our understanding of the response to hypoxia, but also reveals proteins important for malignant progression, which may be targeted in future therapies.

  6. 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.

  7. Analysis and prediction of pathways in HeLa cells by integrating biological levels of organization with systems-biology approaches.

    Juan Carlos Higareda-Almaraz

    Full Text Available It has recently begun to be considered that cancer is a systemic disease and that it must be studied at every level of complexity using many of the currently available approaches, including high-throughput technologies and bioinformatics. To achieve such understanding in cervical cancer, we collected information on gene, protein and phosphoprotein expression of the HeLa cell line and performed a comprehensive analysis of the different signaling pathways, transcription networks and metabolic events in which they participate. A total expression analysis by RNA-Seq of the HeLa cell line showed that 19,974 genes were transcribed. Of these, 3,360 were over-expressed, and 2,129 under-expressed when compared to the NHEK cell line. A protein-protein interaction network was derived from the over-expressed genes and used to identify central elements and, together with the analysis of over-represented transcription factor motifs, to predict active signaling and regulatory pathways. This was further validated by Metal-Oxide Affinity Chromatography (MOAC and Tandem Mass Spectrometry (MS/MS assays which retrieved phosphorylated proteins. The 14-3-3 family members emerge as important regulators in carcinogenesis and as possible clinical targets. We observed that the different over- and under-regulated pathways in cervical cancer could be interrelated through elements that participate in crosstalks, therefore belong to what we term "meta-pathways". Additionally, we highlighted the relations of each one of the differentially represented pathways to one or more of the ten hallmarks of cancer. These features could be maintained in many other types of cancer, regardless of mutations or genomic rearrangements, and favor their robustness, adaptations and the evasion of tissue control. Probably, this could explain why cancer cells are not eliminated by selective pressure and why therapy trials directed against molecular targets are not as effective as expected.

  8. 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

  9. 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.

  10. 雷帕霉素对宫颈癌HeLa细胞侵袭转移的影响及机制研究%Study on Effects and Mechanism of Rapamycin on Invasion and Metastasis of Cervical Cancer HeLa Cells

    贾利刚; 田菲; 张媛

    2016-01-01

    目的:研究雷帕霉素对宫颈癌HeLa细胞侵袭转移的影响及其机制。方法:将HeLa细胞分为对照组和雷帕霉素低、中、高剂量(10、30、100 nmol/L)组,药物作用48 h后,MTT法检测细胞活力,计算抑制率;Transwell法检测细胞迁移和侵袭能力;Western blot法检测细胞中蛋白激酶B(Akt)、雷帕霉素靶蛋白(mTOR)磷酸化水平,以及基质金属蛋白酶2(MMP-2)、MMP-9、波形蛋白(Vimentin)、钙黏蛋白(E-cadherin)表达水平。结果:与对照组比较,雷帕霉素各剂量组细胞的抑制率增加(P<0.01),迁移细胞数目和侵袭细胞数目减少(P<0.01),MMP-2、MMP-9、Vimentin表达水平降低(P<0.01或P<0.05),E-cadherin表达水平增强(P<0.01或P<0.05),Akt及mTOR磷酸化水平降低(P<0.01)。结论:雷帕霉素可通过Akt/mTOR信号通路抑制HeLa细胞的侵袭转移。%OBJECTIVE:To study the effects and mechanism of rapamycin on invasion and metastasis of cervical cancer HeLa cell. METHODS:HeLa cells were divided into control group and rapamycin low-dose,medium-dose and high-dose groups (10, 30,100 nmol/L). After treated for 48 h,cell viability was measured by MTT assay,and inhibitory rate was calculated;migration and invasion of cell was tested by Transwell assay. The expression of matrix metalloproteinase 2(MMP-2),MMP-9,Vimentin and E-cadherin,and phosphorylation of protein kinase B (Akt),mammalian target of rapamycin (mTOR) were detected by Western blot. RESULTS:Compared with control group,the inhibition rate of cell viability was increased in rapamycin groups(P<0.01);the number of invasion and metastasis cells decreased(P<0.01);the expression of MMP-2,MMP-9 and Vimentin were decreased (P<0.01 or P<0.05);the expression of E-cadherin was enhanced(P<0.01 or P<0.05);the phosphorylation of Akt and mTOR were reduced (P<0.01). CONCLUSIONS:Rapamycin could inhibit invasion and metastasis of HeLa cell via

  11. Differential susceptibility of human trophoblastic (BeWo) and uterine cervical (HeLa) cells to Neospora caninum infection.

    Carvalho, Julianne V; Alves, Celene M O S; Cardoso, Mariana R D; Mota, Caroline M; Barbosa, Bellisa F; Ferro, Eloísa A V; Silva, Neide M; Mineo, Tiago W P; Mineo, José R; Silva, Deise A O

    2010-12-01

    Neospora caninum is an apicomplexan parasite, closely related to Toxoplasma gondii, and causes abortion and congenital neosporosis in cattle worldwide. Trophoblast cells act in mechanisms of innate immune defense at the fetal-maternal interface and no data are available about the interaction of Neospora with human trophoblasts. Thus, this study aimed to verify the susceptibility of human trophoblastic (BeWo) compared with uterine cervical (HeLa) cell lines to N. caninum. BeWo and HeLa cells were infected with different parasite:cell ratios of N. caninum tachyzoites and analyzed at different times after infection for cell viability using thiazolyl blue tetrazole and lactate dehydrogenase assays. Both cell lines were also evaluated for cytokine production and parasite infection/replication assays when pre-treated or not with Neospora lysate antigen (NLA) or human recombinant IFN-γ. Cell viability was increased up to 48 h of infection in both types of cells, suggesting that infection could inhibit early cell death and/or induce cell proliferation. Neospora infection induced up-regulation of the macrophage migration inhibitory factor (MIF), mainly in HeLa cells, which was enhanced by cell pre-treatment by NLA or IFN-γ. Conversely, parasite infection induced down-regulation of the transforming growth factor (TGF-β), mostly in BeWo cells, which was decreased with NLA or IFN-γ pre-treatment. HeLa cells were more susceptible to Neospora infection than BeWo cells and IFN-γ pre-treatment resulted in reduced infection indices in both cell lines. Control of parasite growth was mediated by IFN-γ through an indoleamine-2,3-dioxygenase-dependent mechanism in HeLa cells alone. Based on these results, we concluded that BeWo and HeLa cells are readily infected by N. caninum, although presenting differences in susceptibility to infection, cytokine production and cell viability. Thus, these host cells can be considered in comparative approaches to understand strategies used by N

  12. Structural and physicochemical aspects of silica encapsulated ZnO quantum dots with high quantum yield and their natural uptake in HeLa cells.

    Depan, D; Misra, R D K

    2014-09-01

    Photoluminescent semiconductor quantum dots (QDs) are of significant interest for bioimaging and fluorescence labeling. In this regard, we describe here the design of high sensitivity and high specificity non-toxic ZnO QDs (∼5 nm) with long-term stability of up to 12 months. The embedding of ZnO QDs on silica nanospheres led to significant increase in photoluminescence intensity rendering them highly bright QD-based probes. The QDs were characterized in vitro with respect to cancer cells (HeLa) and evaluated in terms of viability, fluorescence and cytoskeletal organization. The immobilization of ZnO QDs on silica nanospheres promoted the internalization and enhanced fluorescence emission of HeLa cells. The fluorescence emission from QDs was stable for 3 days, indicating excellent stability toward photobleaching. Cytoskeletal reorganization was observed after internalization of QDs such that the ZnO QDS on silica nanospheres resulted in broadening of the actin cytoskeleton. The study underscores that ZnO QDs immobilized on Si nanospheres are promising for tracking cancer cells in cell therapy.

  13. Involvement of transcription factor activator protein-2α in doxazosin-induced HeLa cell apoptosis

    Lu GAN; Dong-xing ZHU; Li-ping YANG; Ru-shi LIU; Feng YAN; Jian ZHANG

    2008-01-01

    Aim:To investigate the pro-apoptotic effects of α- 1-adrenergic inhibitor doxazosin in HeLa cells and the potential involvement of transcription factor activator pro-tein-2α (AP-2α) in doxazosin-indueed apoptosis. Methods:The HeLa cells were exposed to various concentrations of doxazosin for 16 h. Apoptosis was detected using a DNA fragmentation assay, Hoechst 33258 staining, and flow cytometric analysis. The expression of AP-2α and caspase-3 was detected by relative quan-titative RT-PCR and Western blot assays, respectively. After the respective trans-fections of the HeLa cells with AP-2α overexpressing constructs and an antisense oligonucleotide against AP-2α, apoptosis was assessed by flow cytometric analysis, and the expression of AP-2α and easpase-3 was detected by relative quantitative RT-PCR and Western blot assays. The colorimetric assay was per-formed to detect the caspase-3 activity. Results:Treatment with various concen-trations of doxazosin for 16 h increased the apoptotic rate and total cell death rate of the HeLa cells in a dose-dependent manner and upregulated the expression of AP-2α and caspase-3 in a dose-dependent manner. A dose-dependent increase was observed in the caspase-3 activity. Overexpressing AP-2α led to the in-creased rate of doxazosin-induced apoptosis and the total cell death, whereas doxazosin-induced apoptosis and the total cell death in HeLa cells decreased by antisense AP-2α. Furthermore, overexpressing AP-2α increased the expression and activity of caspase-3, whereas antisense AP-2α in part abolished the increased effects of doxazosin on caspase-3 expression and activity. Conclusion:Doxazosin induces apoptosis in HeLa cells in a dose-dependent manner, and transcription factor AP-2α is functionally involved in doxazosin-induced HeLa cell apoptosis.

  14. Efficacy of Proliferation of HeLa Cells under Three Different Low-Intensity Red Lasers Irradiation

    H. Q. Yang

    2012-01-01

    Full Text Available This study was intended to compare the efficacy of proliferation of HeLa cells under three different low-intensity laser irradiation (LIL, that is, 633 nm, 658 nm, and 785 nm. The time-dependent responses of proliferation of HeLa cells after the red laser irradiation and the influence of fetal bovine serum (FBS at 1%, 2%, 5%, or 10% on the proliferation of cells were also investigated. The results indicated that the proliferation of HeLa cells in 10% FBS was in proliferation-specific homeostasis (PSH so that it was not modulated with LIL; the proliferation in FBS at 1%, 2%, or 5% was far from PSH so that it may be wavelength dependently modulated with LIL, and the maximum proliferation promotion was conducted with LIL at 633 nm amongst the three different LIL. It was concluded the wavelength-dependent photobiomodulation of LIL on proliferation of HeLa cells may be homeostatic.

  15. 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.

  16. Single hepatitis-B virus core capsid binding to individual nuclear pore complexes in Hela cells.

    Lill, Yoriko; Lill, Markus A; Fahrenkrog, Birthe; Schwarz-Herion, Kyrill; Paulillo, Sara; Aebi, Ueli; Hecht, Bert

    2006-10-15

    We investigate the interaction of hepatitis B virus capsids lacking a nuclear localization signal with nuclear pore complexes (NPCs) in permeabilized HeLa cells. Confocal and wide-field optical images of the nuclear envelope show well-spaced individual NPCs. Specific interactions of capsids with single NPCs are characterized by extended residence times of capsids in the focal volume which are characterized by fluorescence correlation spectroscopy. In addition, single-capsid-tracking experiments using fast wide-field fluorescence microscopy at 50 frames/s allow us to directly observe specific binding via a dual-color colocalization of capsids and NPCs. We find that binding occurs with high probability on the nuclear-pore ring moiety, at 44 +/- 9 nm radial distance from the central axis.

  17. Mesoporous silica nanoparticles enhance MTT formazan exocytosis in HeLa cells and astrocytes.

    Fisichella, Matthieu; Dabboue, Hinda; Bhattacharyya, Sanjib; Saboungi, Marie-Louise; Salvetat, Jean-Paul; Hevor, Tobias; Guerin, Martine

    2009-06-01

    We report on the observation that mesoporous silica nanoparticles (MSNs), after being endocytosed, interfere with the MTT test in HeLa cells and astrocytes by accelerating the exocytosis of formazan crystals. The stimulation of MTT formazan exocytosis is probably related to perturbation of intracellular vesicle trafficking by MSN uptake as revealed by experiments in presence of chloroquine and genistein. Similar effect has been previously observed with a number of chemicals, especially with neurotoxic beta amyloid peptides, but not with nanoparticles. We showed also that MTT reduction test gives an overestimation of the cytotoxicity of mesoporous silica nanoparticles compared to other tests such as LDH activity, WST-1 test and flow cytometry. These findings show that MTT assay should not be used for the study of MSN toxicity, and that perturbation of intracellular trafficking has to be taken into account in evaluating biocompatibility of MSNs.

  18. 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.

  19. The invasion of tobacco mosaic virus RNA induces endoplasmic reticulum stress-related autophagy in HeLa cells.

    Li, Li; Wang, Li; Xiao, Ruijing; Zhu, Guoguo; Li, Yan; Liu, Changxuan; Yang, Ru; Tang, Zhiqing; Li, Jie; Huang, Wei; Chen, Lang; Zheng, Xiaoling; He, Yuling; Tan, Jinquan

    2012-04-01

    The ability of human cells to defend against viruses originating from distant species has long been ignored. Owing to the pressure of natural evolution and human exploration, some of these viruses may be able to invade human beings. If their 'fresh' host had no defences, the viruses could cause a serious pandemic, as seen with HIV, SARS (severe acute respiratory syndrome) and avian influenza virus that originated from chimpanzees, the common palm civet and birds, respectively. It is unknown whether the human immune system could tolerate invasion with a plant virus. To model such an alien virus invasion, we chose TMV (tobacco mosaic virus) and used human epithelial carcinoma cells (HeLa cells) as its 'fresh' host. We established a reliable system for transfecting TMV-RNA into HeLa cells and found that TMV-RNA triggered autophagy in HeLa cells as shown by the appearance of autophagic vacuoles, the conversion of LC3-I (light chain protein 3-I) to LC3-II, the up-regulated expression of Beclin1 and the accumulation of TMV protein on autophagosomal membranes. We observed suspected TMV virions in HeLa cells by TEM (transmission electron microscopy). Furthermore, we found that TMV-RNA was translated into CP (coat protein) in the ER (endoplasmic reticulum) and that TMV-positive RNA translocated from the cytoplasm to the nucleolus. Finally, we detected greatly increased expression of GRP78 (78 kDa glucose-regulated protein), a typical marker of ERS (ER stress) and found that the formation of autophagosomes was closely related to the expanded ER membrane. Taken together, our data indicate that HeLa cells used ERS and ERS-related autophagy to defend against TMV-RNA.

  20. Effect of Lon protease knockdown on mitochondrial function in HeLa cells.

    Bayot, Aurélien; Gareil, Monique; Chavatte, Laurent; Hamon, Marie-Paule; L'Hermitte-Stead, Caroline; Beaumatin, Florian; Priault, Muriel; Rustin, Pierre; Lombès, Anne; Friguet, Bertrand; Bulteau, Anne-Laure

    2014-05-01

    ATP-dependent proteases are currently emerging as key regulators of mitochondrial functions. Among these proteolytic systems, Lon protease is involved in the control of selective protein turnover in the mitochondrial matrix. In the absence of Lon, yeast cells have been shown to accumulate electron-dense inclusion bodies in the matrix space, to loose integrity of mitochondrial genome and to be respiratory deficient. In order to address the role of Lon in mitochondrial functionality in human cells, we have set up a HeLa cell line stably transfected with a vector expressing a shRNA under the control of a promoter which is inducible with doxycycline. We have demonstrated that reduction of Lon protease results in a mild phenotype in this cell line in contrast with what have been observed in other cell types such as WI-38 fibroblasts. Nevertheless, deficiency in Lon protease led to an increase in ROS production and to an accumulation of carbonylated protein in the mitochondria. Our study suggests that Lon protease has a wide variety of targets and is likely to play different roles depending of the cell type.

  1. 1.5-NM PROJECTION STRUCTURE OF HELA-CELL PROSOME-MCP (PROTEASOME) PROVIDED BY 2-DIMENSIONAL CRYSTALS

    PERKINS, GA; BERGSMASCHUTTER, W; KEEGSTRA, W; ARNBERG, AC; COUX, O; SCHERRER, K

    1994-01-01

    We grew two-dimensional crystals of HeLa cell prosomes, also called multicatalytic proteinases (MCP) and proteasomes, for a structure determination by electron microscopy. The molecules were arranged in side views in these crystals. The crystals have p21 plane group symmetry with one particle per un

  2. Subnanomolar antisense activity of phosphonate-peptide nucleic acid (PNA) conjugates delivered by cationic lipids to HeLa cells

    Shiraishi, Takehiko; Hamzavi, Ramin; Nielsen, Peter E

    2008-01-01

    oligomer. This modification of the PNA does not interfere with the nucleic acid target binding affinity based on thermal stability of the PNA/RNA duplexes. When delivered to cultured HeLa pLuc705 cells by Lipofectamine, the PNAs showed dose-dependent nuclear antisense activity in the nanomolar range...

  3. 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.

  4. 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.

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

    Rao Xiaotang; Zhang Yingyin; Yi Qiyi; Hou Heli; Xu Bo; Chu Liang; Huang Yun; Zhang Wenrui [Laboratory of Molecular and Cell Genetics, Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027 (China); Fenech, Michael [CSIRO Human Nutrition, PO Box 10041, Adelaide BC, Adelaide, SA 5000 (Australia); Shi Qinghua [Laboratory of Molecular and Cell Genetics, Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027 (China)], E-mail: qshi@ustc.edu.cn

    2008-11-10

    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.

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

    Kaur, Harminder; Avasthi, D. K.; Pujari, Geetanjali; Sarma, Asitikantha

    2013-07-01

    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.

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

    Kaur, Harminder; Avasthi, D. K.; Pujari, Geetanjali; Sarma, Asitikantha [Inter University Accelerator Centre, Aruna Asaf Ali Marg, Post box-10502, New Delhi-110067 (India)

    2013-07-18

    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/{mu}m) 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 {approx} 28% reduction of {sup 12}C{sup 6+} ion dose for Glu-AuNP treated HeLa cells as compared to control HeLa cells.

  8. 槲皮素对Hela 细胞细胞周期和RNA聚合酶活性的影响%Effects of quercetin on cell cycle and RNA polymerase activity in Hela cells

    侯敢; 黄迪南; 祝其锋

    2001-01-01

    Objective:To study the effects of quercetin on cell cycle and RNA polymerase in Hela cells.Methods:Hela cells were incubated with 4、20 and 100 μ mol/L quercetin for 3 days,respectively.Cell cycle was analyzed with flow cytometry,and the activities of total RNA polymerase and RNA polymerase Ⅱ were measured in isolated Hela cell nuclei by nuclear transcription in vitro.Results:After incubation with quercetin,the percentage of Hela cells at G1 phase increased in a dose-dependent manner,and the activities of total RNA polymerase and RNA polymerase Ⅱ were inhibited in the same way.Conclusion:Quercetin is able to arrest cell cycle progression of Hela cells at G1 phase,which may be related to the inhibition of the activities of total RNA polymerase and RNA polymerase Ⅱ.%目的:观察槲皮素对Hela 细胞细胞周期和RNA聚合酶活性的影响及其相互关系。方法:分别用4、20和100 μ mol/L槲皮素处理培养的Hela细胞3 d后,应用流式细胞仪进行细胞周期分析,并通过体外核转录实验测定总RNA聚合酶活性和RNA聚合酶Ⅱ活性。结果:Hela细胞经槲皮素处理后,细胞周期中G1期细胞增加并呈现量-效关系,显示槲皮素有阻断Hela细胞周期于G1期作用;总RNA聚合酶活性和RNA聚合酶Ⅱ活性均受抑制,并呈现量-效关系。结论:槲皮素阻断Hela细胞周期于G1期与抑制RNA聚合酶活性有关。

  9. Measurement of interaction force between RGD-peptide and Hela cell surface by optical tweezers

    Mincheng Zhong; Guosheng Xue; Jinhua Zhou; Ziqiang Wang; Yinmei Li

    2012-01-01

    Since RGD peptides (R:arginine; G:glycine; D:aspartic acid) are found to promote cell adhesion,they are modified at numerous materials surface for medical applications such as drug delivery and regenerative medicine.Peptide-cell surface interactions play a key role in the above applications.In this letter,we study the adhesion force between the RGD-coated bead and Hela cell surface by optical tweezes.The adhesion is dominated by the binding of α5β1 and RGD-peptide with higher adhesion probability and stronger adhesion strength compared with the adhesion of bare bead and cell surface.The binding force for a single α5β1-GRGDSP pair is determined to be 16.8 pN at a loading rate of 1.5 nN/s.The unstressed off-rate is 1.65 × 10-2 s-1 and the distance of transition state for the rigid binding model is 3.0 nm.

  10. 3D printing of biomimetic microstructures for cancer cell migration

    Huang, Tina Qing; Qu, Xin; Liu, Justin; Chen, Shaochen

    2013-01-01

    To understand the physical behavior and migration of cancer cells, a 3D in vitro micro-chip in hydrogel was created using 3D projection printing. The micro-chip has a honeycomb branched structure, aiming to mimic 3D vascular morphology to test, monitor, and analyze differences in the behavior of cancer cells (i.e. HeLa) vs. non-cancerous cell lines (i.e. 10T1/2). The 3D Projection Printing system can fabricate complex structures in seconds from user-created designs. The fabricated microstructures have three different channel widths of 25, 45, and 120 microns wide to reflect a range of blood vessel diameters. HeLa and 10T1/2 cells seeded within the micro-chip were then analyzed for morphology and cell migration speed. 10T1/2 cells exhibited greater changes in morphology due to channel size width than HeLa cells; however, channel width had a limited effect on 10T1/2 cell migration while HeLa cancer cell migration increased as channel width decreased. This physiologically relevant 3D cancer tissue model has the potential to be a powerful tool for future drug discoveries and cancer migration studies PMID:24150602

  11. 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.

  12. A study on inhibition of docetaxel combined with wild type p53 gene on proliferation of Hela cells%多西他赛联合野生型p53对Hela细胞的抑制作用

    宋巧丽; 章根琴; 张军琴

    2009-01-01

    Objective To investigate inhibitory effects of docetaxel alone and docetaxel combined with wild type p53(wt p53) on proliferation of human cervical cancer cells-Hela cells. Methods The Hela cells were divided into two groups: wt p53 transfected Hela cells group(p53-Hela group, obtained by using lipofection-mediated gene transfection method) and blank control group (not transfected with wt p53). The expression of wt p53 was deteced by using reverse transcription polymerase chain reaction(RT-PCR) method. Docetaxel acted on Hela cells and p53-Hela cells. After 24 hours and 48 hours,the morphology of the cells in the two groups were observed respectively and OD450 were tested by using cell counting kit-8(CCK-8) to caculate the inhibition rate. Results After transfection with wt p53, the expression of p53mRNA in p53-Hela group was increased, and there was a significant difference as compared with that in Hela group (t=-17.504, P<0.01). Docetaxel exerted an inhibitary effect on the Hela cell line in the two groups at all action times (Fp53-Hela=53.500,P<0.01;FHela=430.225,P<0.01). This cellular inhibitory effects showed dose and time dependent manners. Conclusion Docetaxel alone exerts an inhibitory effect on Hela cells and this inhibitary effect could be increased when docetaxel is combined with wt p53.%目的 探讨多西他赛单用及与野生型p53基因联合应用对宫颈癌Hela细胞的抑制作用.方法 Hela细胞分两组培养,p53转染实验组(p53-Hela组)和空白对照组(Hela组),野生型p53基因表达用逆转录-多聚酶链反应鉴定.多西他赛作用于p53-Hela细胞及Hela细胞,24小时与48小时后形态学观察并使用活细胞计数试剂盒检测吸光度值OD450,计算细胞抑制率.结果 用逆转录-多聚酶链反应法检测到p53-Hela细胞的p53 mRNA表达,与对照组Hela细胞的p53 mRNA表达相比,差异有统计学意义(t=-17.504,P<0.01).多西他赛对p53-Hela及Hela细胞作用时,不同时间都有抑制作用(Fp53

  13. 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.

  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. Reovirus type 3 synthesizes proteins in interferon-treated HeLa cells without reversing the antiviral state.

    Feduchi, E; Esteban, M; Carrasco, L

    1988-06-01

    Treatment of HeLa cells with human lymphoblastoid interferon (IFN-alpha) does not inhibit reovirus type 3 protein synthesis during virus infection. In contrast, reovirus translation is blocked by treatment of L cells with mouse IFN-alpha. The (2'-5')A synthetase activity is induced in HeLa cells by IFN-alpha treatment and is activated after reovirus infection, since cell lysates from these cells synthesize in vitro (2'-5')A oligonucleotides. The IFN-induced protein kinase activity is also triggered in those lysates upon dsRNA addition. Thus, contrary to DNA-containing viruses, such as vaccinia virus or adenovirus, reovirus infection does not destroy or reverse the IFN-induced antiviral state. In support of this conclusion, superinfection with poliovirus or vesicular stomatitis virus of reovirus-infected HeLa cells treated with IFN leads only to a blockade of translation of the former viruses. These results provide a remarkable example where in the same cells doubly infected with two different viruses, the antiviral state induced by IFN-alpha is manifested by selectively inhibiting translation of one kind of virus (poliovirus or vesicular stomatitis virus) without affecting the translation of reovirus type 3. In addition, these results indicate that the resistance of reovirus translation to inhibition by IFN is different from the mechanism of resistance induced by DNA-containing viruses.

  16. 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.

  17. Studies on the control of ribosomal RNA synthesis in HeLa cells.

    Chesterton, C J; Coupar, B E; Butterworth, P H; Green, M H

    1975-09-01

    In many eucaryotic systems protein synthesis is coupled to ribosomal RNA synthesis such that shut-down of the former causes inhibition of the latter. We have investigated this stringency phenomenon in HeLa cells. The protein synthesis inhibitors cycloheximide and puromycin cause inactivation of both processes but valine starvation totally inhibits only the processing of 45-S RNA. DNA-dependent RNA polymerases from A, B and C (or I, II and III respectively) were extracted, separated partially by DEAE-cellulose chromatography and their activity levels determined. These do not decrease significantly during inhibition of protein synthesis. To find out whether or not form A is bound to its template under these conditions, proteins were removed from chromatin with the detergent sarkosyl. This does not affect bound RNA polymerase. Inhibition of protein synthesis caused up to 50% reduction in endogenous alpha-amanitin-insensitive chromatin-RNA-synthesising activity. This reduced level of activity was not affected by sarkosyl treatment. Levels in normal cells were stimulated. This result indicates that the form A RNA polymerase is not bound to its template when protein synthesis is inhibited.

  18. 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.

  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.

  20. Carboxymethyl chitosan nanoparticles coupled with CD59-specific ligand peptide for targeted delivery of C-phycocyanin to HeLa cells.

    Yang, Peng; Li, Bing; Yin, Qi-Feng; Wang, Yu-Juan

    2017-03-01

    The combination of nanotechnology and medicine will be the next generation of vehicles for targeted drug delivery. Carboxymethyl chitosan loaded with the anticancer drug C-phycocyanin and the CD59-specific ligand peptide for cancer cell targeting were used to create C-phycocyanin/carboxymethyl chitosan-CD59-specific ligand peptide nanoparticles using the ionic-gelation method. Optimal synthesis conditions, selected by response surface methodology, comprised the ratio carboxymethyl chitosan:C-phycocyanin = 3:1, and carboxymethyl chitosan and CaCl2 concentrations of 2.0 and 1.0 mg/mL, respectively. The resulting nanoparticles were spherical, with diameters of approximately 200 nm; the entrapment efficient was about 65%; and the drug loading was about 20%. The release of C-phycocyanin from C-phycocyanin/carboxymethyl chitosan nanoparticles was pH sensitive and had a sustainable effect in vitro. Guided by the CD59-specific ligand peptide, the nanoparticles efficiently targeted the surface of HeLa cells and had an obvious inhibitory effect on HeLa cell proliferation as determined by methyl thiazolyl tetrazolium assays. The nanoparticles were hemocompatible and induced apoptosis by upregulation of cleaved caspase-3 and cleaved polyADP-ribose polymerase proteins, and downregulation of Bcl-2 proteins. Our study provides a novel approach to the research and development of marine drugs, and support for targeted therapy using anticancer drugs.

  1. 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.

  2. 18 SrRNA Degradation is Not Accompanied by Altered rRNA Transport at Early Times Following Irradiation of HeLa Cells

    1990-01-01

    Altered rRNA Transport at Early Times following Irradiation of HeLa Cells PINHAS Ft CHS.* JOHN M. KROLAKt DAVID MCCLAINt AND KENNETH W. MINTONt "I...ratio of 2:1 that is ob- high as 1.6:1 during the interval 5 to 20 h following irradiation of HeLa cells at _7.5 Gy. Alterations in 28 S:18 S ratio were...m 2 5 15 20 GY findings that the movement of the newly formed 40 S ribo- 10 somal subunits from the nuclei in HeLa cells proceeds more 2swiftly

  3. Overexpressed active Notch1 induces cell growth arrest of HeLa cervical carcinoma cells.

    Wang, L; Qin, H; Chen, B; Xin, X; Li, J; Han, H

    2007-01-01

    Human cervical carcinoma is one of the most common malignant tumors, but the mechanisms that orchestrate the multiple oncogenic insults required for initiation and progression are not clear. Notch signaling plays a critical role in maintaining the balance between cell proliferation, differentiation, and apoptosis, but perturbed Notch signaling may contribute to tumorigenesis. We now show that Notch1 is detected in all cervical cancer, including advanced diseases. We also constitutively overexpressed active Notch1 in human cervical carcinoma to explore the effects of Notch1 signaling on human cervical carcinoma cell growth and to investigate the underlying molecular mechanisms. The signaling may participate in the development of human cervical carcinoma cells, but overexpressed active Notch1 inhibits their growth through induction of cell cycle arrest. Increased Notch1 signaling induced a downmodulation of human papillomavirus transcription through suppression of activator protein (AP)-1 activity by upregulation of c-Jun and the decreased expression of c-Fos. Thus, Notch1 signaling plays a key role and exerts dual effects, functioning in context-specific manner.

  4. [Effect of endonuclease G depletion on plasmid DNA uptake and levels of homologous recombination in hela cells].

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

    2016-01-01

    Endonuclease G (EndoG) is a mitochondrial apoptosis regulator that also has roles outside of programmed cell death. It has been implicated as a defence DNase involved in the degradation of exogenous DNA after transfection of mammalian cells and in homologous recombination of viral and endogenous DNA. In this study, we looked at the effect of EndoG depletion on plasmid DNA uptake and the levels of homologous recombination in HeLa cells. We show that the proposed defence role of EndoG against uptake of non-viral DNA vectors does not extend to the cervical carcinoma HeLa cells, as targeting of EndoG expression by RNA interference failed to increase intracellular plasmid DNA levels. However, reducing EndoG levels in HeLa cells resulted in a statistically significant reduction of homologous recombination between two plasmid DNA substrates. These findings suggest that non-viral DNA vectors are also substrates for EndoG in its role in homologous recombination.

  5. Triphala, a formulation of traditional Ayurvedic medicine, shows protective effect against X-radiation in HeLa cells.

    Takauji, Yuki; Miki, Kensuke; Mita, Juma; Hossain, Mohammad Nazir; Yamauchi, Masatake; Kioi, Mitomu; Ayusawa, Dai; Fujii, Michihiko

    2016-12-01

    Ayurveda is a holistic medical system of traditional medicine, and Triphala is one of the most popular formulations in Ayurveda. Triphala is composed of three kinds of herb, Terminalia chebula, Terminalia bellirica, and Emblica officinalis. Since Triphala is shown to exhibit a protective activity against ionizing radiation in mice, we investigated its activity in HeLa cells. We found that Triphala showed the protective effects against X-radiation and bleomycin, both of which generate DNA strand breaks, in HeLa cells. Further, Triphala efficiently eliminated reactive oxygen species (ROS) in HeLa cells. Thus, the antioxidant activity of Triphala would likely play a role in its protective actions against X-radiation and bleomycin because both agents damage DNA through the generation of ROS. These observations suggested that the radioprotective activity of Triphala can be, at least partly, studied with the cells cultured in vitro. The simple bioassay system with human cultured cells would facilitate the understanding of the molecular basis for the beneficial effects of Triphala.

  6. Inhibiting PI3K/Akt Pathway Increases DNA Damage of Cervical Carcinoma HeLa Cells by Drug Radiosensitization

    夏曙; 于世英; 付强; 刘飞; 郑微; 付秀根; 赵茵

    2010-01-01

    This study examined the role of PI3K/Akt pathway in radiosensitization of DNA damage of cervical carcinoma cells.The 50% inhibition concentration(IC50) of cisplatin and docetaxel in HeLa cells was detected by Mono-nuclear cell direct cytotoxicity assay(MTT) in vitro.HeLa cells were treated by cisplatin/docetaxel of 10 percent of IC20 alone or combined with LY294002 for 24 h,and then radiated by different doses of X-ray.The cell survival ratio was obtained by means of clone formation.One-hit multi-target mod...

  7. Panax notoginseng saponins inhibiting the proliferation of Hela cell%三七总皂苷抑制Hela细胞增殖的实验研究

    邹存华; 付婷婷; 鲁强; 赵淑萍; 徐祥

    2012-01-01

    OBJECTIVE: To study the possible mechanism of Panax notoginseng saponins in inhibiting the proliferation of cervical cancer Hela cells. METHODS* Using MTT to test the proliferation rate after Hela cells were exposured to different density of PNS for 24,48,72 hours. Adopt the technology of luciferase reporter gene to detect the variance of lu-ciferase after PNS stimulate Hela cells for 24 h, The expression of VEGF was assessed by ELISA; the change of 4E-BP1, S6Kl,mTOR and the levels of their phosphorylation were detected by Western-Blot. RESULTS; PNS could significantly inhibit the growth of Hela cells, positively correlated with higher concentration and increasing time- The relative light unit of LUC was significantly lower than control group. In comparison with blank control group, 4E-BP1, S6K1 , mTOR and the level of their phosphorylation and VEGF of treatment group decrease significantlly after be treated with 200 μg/mL PNS for 24 hours (all F<0, 01). CONCLUSIONS: The growth of Hela cells are significantly inhibited by PNS, which mechanism may be block mTOR signaling pathway. At the same time,it decreases the efficiency of mRNA translation and represses the formation of translation initiation complex, which inhibits the proliferation of Hela cells.%目的:研究三七总皂苷(PNS)抑制宫颈癌Hela细胞增殖的可能机制.方法:MTT法检测不同浓度PNS作用于Hela细胞24、48和72 h Hela细胞增殖率;检测PNS作用于Hela细胞24 h荧光素酶报告基因(LUC)的变化;蛋白质印迹法检测200 μg/mL PNS作用于Hela细胞4E-BP1、S6K1、mTOR及其磷酸化水平变化;ELISA法检测PNS作用于Hela细胞24h VEGF的变化.结果:PNS可以明显抑制Helá细胞生长,随时间延长及PNS剂量增加抑制率明显增高;LUC相对光单位值(RLU值)明显低于对照组,其S6K1、4E-BP1、mTOR蛋白表达及其磷酸化水平均明显降低,200 μg/mL PNS作用于Hela细胞24 h后其VEGF表达明显降低,差异

  8. 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.

  9. Rapid Biosynthesis of Silver Nanoparticles Using Pepino (Solanum muricatum Leaf Extract and Their Cytotoxicity on HeLa Cells

    Mónica Gorbe

    2016-04-01

    Full Text Available Within nanotechnology, gold and silver nanostructures have unique physical, chemical, and electronic properties [1,2], which make them suitable for a number of applications. Moreover, biosynthetic methods are considered to be a safer alternative to conventional physicochemical procedures for both the environmental and biomedical applications, due to their eco-friendly nature and the avoidance of toxic chemicals in the synthesis. For this reason, employing bio routes in the synthesis of functionalized silver nanoparticles (FAgNP have gained importance recently in this field. In the present study, we report the rapid synthesis of FAgNP through the extract of pepino (Solanum muricatum leaves and employing microwave oven irradiation. The core-shell globular morphology and characterization of the different shaped and sized FAgNP, with a core of 20–50 nm of diameter is established using the UV-Visible spectroscopy (UV-vis, field emission scanning electron microscopy (FESEM, transmission electron microscopy (TEM and Zeta potential and dynamic light scanning (DLS studies. Moreover, cytotoxic studies employing HeLa (human cervix carcinoma cells were undertaken to understand FAgNP interactions with cells. HeLa cells showed significant dose dependent antiproliferative activity in the presence of FAgNP at relatively low concentrations. The calculated IC50 value was 37.5 µg/mL, similar to others obtained for FAgNPs against HeLa cells.

  10. Triphala, a formulation of traditional Ayurvedic medicine, shows protective effect against X-radiation in HeLa cells

    YUKI TAKAUJI; KENSUKE MIKI; JUMA MITA; MOHAMMAD NAZIR HOSSAIN; MASATAKE YAMAUCHI; MITOMU KIOI; DAI AYUSAWA; MICHIHIKO FUJII

    2016-12-01

    Ayurveda is a holistic medical system of traditional medicine, and Triphala is one of the most popular formulations inAyurveda. Triphala is composed of three kinds of herb, Terminalia chebula, Terminalia bellirica, and Emblicaofficinalis. Since Triphala is shown to exhibit a protective activity against ionizing radiation in mice, we investigatedits activity in HeLa cells. We found that Triphala showed the protective effects against X-radiation and bleomycin,both of which generate DNA strand breaks, in HeLa cells. Further, Triphala efficiently eliminated reactive oxygenspecies (ROS) in HeLa cells. Thus, the antioxidant activity of Triphala would likely play a role in its protective actionsagainst X-radiation and bleomycin because both agents damage DNA through the generation of ROS. Theseobservations suggested that the radioprotective activity of Triphala can be, at least partly, studied with the cellscultured in vitro. The simple bioassay system with human cultured cells would facilitate the understanding of themolecular basis for the beneficial effects of Triphala.

  11. Toxicity of Aluminum Silicates Used in Hemostatic Dressings Toward Human Umbilical Veins Endothelial Cells, HeLa Cells, and RAW267.4 Mouse Macrophages

    2011-09-01

    HUVEC, HeLa cells derived from a cervical carcinoma, and RAW264.7 mouse macrophage-like cells (RAW), a cell line often used for in vitro screening of...nanopore size and enzyme immobilization . Langmuir. 2008;24:14254–14260. 7. Dexter LA. The pharmaceutical uses of bentonite. Can Pharm J. 1948; 81:316–318

  12. Gallic acid reduces cell viability, proliferation, invasion and angiogenesis in human cervical cancer cells

    Zhao, Bing; HU, MENGCAI

    2013-01-01

    Gallic acid is a trihydroxybenzoic acid, also known as 3,4,5-trihydroxybenzoic acid, which is present in plants worldwide, including Chinese medicinal herbs. Gallic acid has been shown to have cytotoxic effects in certain cancer cells, without damaging normal cells. The objective of the present study was to determine whether gallic acid is able to inhibit human cervical cancer cell viability, proliferation and invasion and suppress cervical cancer cell-mediated angiogenesis. Treatment of HeLa...

  13. ASSOCIATION OF PSL (p55),A S-PHASE-RELATED NUCLEAR ANTIGEN,WITH CHROMATIN IN HeLa CELLS

    袁耀宗; Barque JP; Dellavalle V; Larsen CJ

    1993-01-01

    PSL is a S-phase-related 55 kDa antigen which has been previously located inchromatin areas of the nucleus by electron microscopic studies. In the present report, weshow that it is released from purified HeLa cell nuclei by micrococcal nuclease treatment,which relieves chromatin under the form of nucleosome. However, the antigen is not associ-ated with nucleosomes but is part of a structure that sediments through 5-20% sucrosegradient. Moreover, PSL is completely retained on single-stranded DNA (ss-DNA) affini-ty columns and binds more efficiently HeLa cell ss-DNA than double stranded DNA.Although no direct evidence could be obtained, these data suggest that PSL might bea ss-DNA-binding or a RNA-binding protein.

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

    Yan Chunlan; Chen Zhaojun; Li Huanrong; Zhang Guanglin [The First Affiliated Hospital, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003 (China); Department of Toxicology, Zhejiang University School of Public Health, Hangzhou, Zhejiang 310058 (China); Li Feng [The First Renmin Hospital, Houma, Shanxi 043000 (China); Duerksen-Hughes, Penelope J. [Department of Basic Sciences, Loma Linda University School of Medicine, Loma Linda, CA 92354 (United States); Zhu Xinqiang, E-mail: zhuxq@zju.edu.cn [Department of Toxicology, Zhejiang University School of Public Health, Hangzhou, Zhejiang 310058 (China); Yang Jun, E-mail: gastate@zju.edu.cn [The First Affiliated Hospital, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003 (China); Department of Toxicology, Hangzhou Normal University School of Public Health, Hangzhou, Zhejiang 310036 (China)

    2012-03-01

    Previously, we employed a proteomics-based 2-D gel electrophoresis assay to show that exposure to 10 {mu}M 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 {mu}M) 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 {mu}M and 10 {mu}M BaP-treated groups, 2 in the 0.1 {mu}M and 10 {mu}M BaP-treated groups, 4 in the 0.1 {mu}M and 1 {mu}M 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.

  15. Quantification of functionalised gold nanoparticle-targeted knockdown of gene expression in HeLa cells.

    Meesbah Jiwaji

    Full Text Available Gene therapy continues to grow as an important area of research, primarily because of its potential in the treatment of disease. One significant area where there is a need for better understanding is in improving the efficiency of oligonucleotide delivery to the cell and indeed, following delivery, the characterization of the effects on the cell.In this report, we compare different transfection reagents as delivery vehicles for gold nanoparticles functionalized with DNA oligonucleotides, and quantify their relative transfection efficiencies. The inhibitory properties of small interfering RNA (siRNA, single-stranded RNA (ssRNA and single-stranded DNA (ssDNA sequences targeted to human metallothionein hMT-IIa are also quantified in HeLa cells. Techniques used in this study include fluorescence and confocal microscopy, qPCR and Western analysis.We show that the use of transfection reagents does significantly increase nanoparticle transfection efficiencies. Furthermore, siRNA, ssRNA and ssDNA sequences all have comparable inhibitory properties to ssDNA sequences immobilized onto gold nanoparticles. We also show that functionalized gold nanoparticles can co-localize with autophagosomes and illustrate other factors that can affect data collection and interpretation when performing studies with functionalized nanoparticles.The desired outcome for biological knockdown studies is the efficient reduction of a specific target; which we demonstrate by using ssDNA inhibitory sequences targeted to human metallothionein IIa gene transcripts that result in the knockdown of both the mRNA transcript and the target protein.

  16. The activation of TLR7 regulates the expression of VEGF, TIMP1, MMP2, IL-6, and IL-15 in Hela cells.

    Li, Lei; Cheng, Feng-Wei; Wang, Fang; Jia, Bo; Luo, Xin; Zhang, Sheng-Quan

    2014-04-01

    Toll-like receptors (TLRs) play important roles in activation of immunoreaction and tumor development. Toll-like receptor 7 (TLR7), one of the TLRs binding with single-stranded RNA, activates intracellular pathways and stimulates the release of proinflammatory cytokines, chemokines. In this study, we investigated the impact of the TLR7-signaling pathway on the expression of vascular endothelial growth factor (VEGF), matrix metalloproteinase 2 (MMP2), tissue inhibitor of metalloproteinase 1 (TIMP1), interleukin 6 (IL-6), and interleukin 15 (IL-15), which have been testified to refer to the immunomodulating and tumor progression. We confirmed that the TLR7 was expressed by Hela cells, despite the abundance was weak. Gardiquimod, one of the TLR7 ligands, can promote these five genes expression in varying degrees. After stimulating with gardiquimod, the expression of the IL-15V1, 3 increased about 4.5 times on RNA level, the other expression was only up-regulated about 2 times. We also discovered that gardiquimod could activate the MAPK/ERK- and PI3K/AKT-signaling pathways, and the specific inhibitors studies indicate that, the effect of gardiquimod on these genes expression is mainly or partially dependent on the activation of these two signaling pathways. To sum up, the activation of TLR7 signaling pathway may modulate some genes expression in Hela cells and may contribute to the pathogenesis of the cervical cancer.

  17. Cytotoxic isolates of Helicobacter pylori from Peptic Ulcer Diseases decrease K+-dependent ATPase Activity in HeLa cells

    Archana Ayyagari

    2003-11-01

    Full Text Available Abstract Background Helicobacter pylori is a Gram negative bacterium that plays a central role in the etiology of chronic gastritis and peptic ulcer diseases. However, not all H. pylori positive cases develop advanced disease. This discriminatory behavior has been attributed to the difference in virulence of the bacteria. Among all virulence factors, cytotoxin released by H. pylori is the most important factor. In this work, we studied variation in H. pylori isolates from Indian dyspeptic patients on the basis of cytotoxin production and associated changes in K+-dependent ATPase (one of its targets enzyme activity in HeLa cells. Methods The patients were retrospectively grouped on the basis of endoscopic and histopathological observation as having gastritis or peptic ulcer. The HeLa cells were incubated with the broth culture filtrates (BCFs of H. pylori isolates from patients of both groups and observed for the cytopathic effects: morphological changes and viability. In addition, the K+-dependent ATPase activity was measured in HeLa cells extracts. Results The cytotoxin production was observed in 3/7 (gastritis and 4/4 (peptic ulcer H. pylori isolates. The BCFs of cytotoxin producing H. pylori strains reduced the ATPase activity of HeLa cells to 40% of that measured with non-cytotoxin producing H. pylori strains (1.33 μmole Pi/mg protein and 3.36 μmole Pi/mg protein, respectively, p Conclusions Our results suggest that the isolation of cytotoxic H. pylori is more common in severe form of acid peptic diseases (peptic ulcer than in gastritis patients from India. Also the cytotoxin released by H. pylori impairs the ion-transporting ATPase and is a measure of cytotoxicity.

  18. 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.

  19. Cytotoxic activity of proteins isolated from extracts of Corydalis cava tubers in human cervical carcinoma HeLa cells

    Balcerkiewicz Stanislaw

    2010-12-01

    Full Text Available Abstract Background Corydalis cava Schweigg. & Koerte, the plant of numerous pharmacological activities, together with the studied earlier by our group Chelidonium majus L. (Greater Celandine, belong to the family Papaveraceae. The plant grows in Central and South Europe and produces the sizeable subterraneous tubers, empty inside, which are extremely resistant to various pathogen attacks. The Corydalis sp. tubers are a rich source of many biologically active substances, with the extensive use in European and Asian folk medicine. They have analgetic, sedating, narcotic, anti-inflammatory, anti-allergic and anti-tumour activities. On the other hand, there is no information about possible biological activities of proteins contained in Corydalis cava tubers. Methods Nucleolytic proteins were isolated from the tubers of C. cava by separation on a heparin column and tested for DNase activity. Protein fractions showing nucleolytic activity were tested for cytotoxic activity in human cervical carcinoma HeLa cells. Cultures of HeLa cells were conducted in the presence of three protein concentrations: 42, 83 and 167 ng/ml during 48 h. Viability of cell cultures was appraised using XTT colorimetric test. Protein fractions were separated and protein bands were excised and sent for identification by mass spectrometry (LC-ESI-MS/MS. Results The studied protein fractions showed an inhibiting effect on mitochondrial activity of HeLa cells, depending on the administered dose of proteins. The most pronounced effect was obtained with the highest concentration of the protein (167 ng/ml - 43.45 ± 3% mitochondrial activity of HeLa cells were inhibited. Mass spectrometry results for the proteins of applied fractions showed that they contained plant defense- and pathogenesis-related (PR proteins. Conclusions The cytotoxic effect of studied proteins toward HeLa cell line cells has been evident and dependent on increasing dose of the protein. The present study, most

  20. 越橘花色苷对宫颈癌Hela细胞凋亡及抗氧化能力的影响%Effects of Anthocyanin from Vaccinium uliginosum on cell apoptosis and antioxidation in cervical Hela cells

    姜艳霞; 芦晓静; 徐俊杰; 吕士杰

    2011-01-01

    Objective To research the mechanisms of Anthocyanin from Vaccinium uliginosum on proliferation of cervical cancer Hela cells. Methods Different concentrations of Anthocyanin from Vaccinium uliginosum on Hela cells was done for 48 hours to observe its inhibiting cervical cancer Hela cells and the change of cell tnorpholoy by Gimsa dying was used to observe cell apoptosis by flow cytometry ( Annexin V FITC/PI) and to detect P53 expression by RT-PCR, and detect SOD, GSH and MDA in Hela cells. Results Increasing concentration of anthocyanin from Vaccinium made Hela cell counts lower and rare; cells became small and pyknosis nucleolus by Gimsa staining; with Anthocyanin concentration increasing, apoptosis rate increased by fluorescent dye and P53 expression increase was of significant difference in comparison with control group( P <0.05). 30mg/ml and 40mg/ml of anthocyanin could increase SOD and GSH in the Hela cells, with a significant difference in comparison with control group ( P < 0.05). Conclusion Anthocyanin from Vaccinium uliginosum can inhibit the growth of cervical cancer Hela cells by promoting cell apoptosis and increasing P53 expression and antioxidation.%目的 探讨越橘花色苷对宫颈癌Hela细胞的作用机制.方法 以不同浓度的越橘花色苷作用于宫颈癌Hela细胞48h后,通过Gimsa染色观察越橘花色苷抑制Hela细胞的形态学改变,通过流式双染观察Hela细胞凋亡情况,通过RT-PCR观察Hela内P53的表达,并测定Hela细胞内的SOD、GSH、MDA值.结果 Gimsa染色可见越橘花色苷可使Hela细胞光泽度下降,细胞数减少,稀疏,细胞变小、核固缩.流式双染可见,随着橘花色苷浓度升高,凋亡率升高.与对照组比较,越橘花色苷浓度达到30mg/ml时,P53的表达量最高,差异有统计学意义(P<0.05).与对照组比较,30mg/ml和40mg/ml的越橘花色苷组SOD和GSH含量升高,差异有统计学意义(P<0.05).结论越橘花色苷可通过上调抑癌基因P53的表

  1. Antibodies against recombinant catalytic domain of lethal toxin of Clostridium sordellii neutralize lethal toxin toxicity in HeLa cells.

    Arya, Preetika; Ponmariappan, S; Singh, Lokendra; Prasad, G B K S

    2013-02-01

    Lethal toxin of Clostridium sordellii (MLD 150 ng/kg) is one of the most potent Clostridial toxins and is responsible for most of the diseases including sudden death syndrome in cattle, sheep and toxic shock syndrome, necrotizing faciitis, neonatal omphalitis and gangrene in humans. Lethal toxin (TcsL) is a single chain protein of about 270 kDa. In the present study, 1.6 kb DNA fragment encoding for the catalytic domain of TcsL was PCR amplified, cloned in pQE30 UA vector and expressed in E. coli SG 13009. The expression of recombinant lethal toxin protein (rTcsL) was optimized and it was purified under native conditions using a single step Ni-NTA affinity chromatography. The purified recombinant protein was used for the production of polyclonal antibodies in mice and rabbit. The raised antibodies reacted specifically with the purified rTcsL and intact native lethal toxin on Western blot. The biological activity of the recombinant protein was tested in HeLa cells where it showed the cytotoxicity. Further, the polyclonal antibodies were used for in-vitro neutralization of purified rTcsL, acid precipitated C. sordellii and C. difficile native toxins in HeLa cells. Mice and rabbit anti-rTcsL sera effectively neutralized the cytotoxicity of rTcsL and C. sordellii native toxin but it did not neutralize the cytotoxicity of C. difficile toxin in HeLa cells.

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

    Romanos,Maria T. V.; Maria J. Andrada-Serpa; Mourão, Paulo A. S.; Yocie Yoneshigue-Valentin; Pereira,Mariana S.; Norma Santos; Marcia D. Wigg

    2011-01-01

    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...

  3. 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.

  4. Highly sensitive determination of copper in HeLa cell using capillary electrophoresis combined with a simple cell extraction treatment.

    Meng, Lingchen; Fang, Ziyuan; Lin, Jian; Li, Meixian; Zhu, Zhiwei

    2014-04-01

    A new separation system of capillary electrophoresis (CE1) for the highly sensitive determination of copper was established by using ethylenediaminetetraacetic acid (EDTA) as a complexing agent and employing cetyltrimethylammonium chloride (CTAC) as a capillary inner wall modifier. Benefitted from the combination of field-enhanced sample injection (FESI) method, a limit of detection (LOD) of 2.7 nM was obtained, which was much lower than that of the conventional methods. This made it possible to determine trace copper in HeLa cell only by a simple cell extraction (CE2) treatment. Two copper-extraction methods-acid-hydrolysis and freeze-thaw-were compared. Limited by the requirement of low ion strength in FESI, only the extract using freeze-thaw could be successfully applied in the determination. The effectiveness assessment of this CE(2)-FESI method was adopted by inductively coupled plasma-atomic emission spectrometry (ICP-AES) as a gold standard.

  5. 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.

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

    Atif, M.; Firdous, S.; Khurshid, A.; Noreen, L.; Zaidi, S. S. Z.; Ikram, M.

    2009-12-01

    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.

  7. Ethanol Metabolism by HeLa Cells Transduced with Human Alcohol Dehydrogenase Isoenzymes: Control of the Pathway by Acetaldehyde Concentration†

    Matsumoto, Michinaga; Cyganek, Izabela; Sanghani, Paresh C.; Cho, Won Kyoo; Liangpunsakul, Suthat; Crabb, David W.

    2010-01-01

    Background Human class I alcohol dehydrogenase 2 isoenzymes (encoded by the ADH1B locus) have large differences in kinetic properties; however, individuals inheriting the alleles for the different isoenzymes exhibit only small differences in alcohol elimination rates. This suggests that other cellular factors must regulate the activity of the isoenzymes. Methods The activity of the isoenzymes expressed from ADH1B*1, ADH1B*2, and ADH1B*3 cDNAs was examined in stably transduced HeLa cell lines, including lines which expressed human low Km aldehyde dehydrogenase (ALDH2). The ability of the cells to metabolize ethanol was compared with that of HeLa cells expressing rat class I ADH (HeLa-rat ADH cells), rat hepatoma (H4IIEC3) cells, and rat hepatocytes. Results The isoenzymes had similar protein half-lives in the HeLa cells. Rat hepatocytes, H4IIEC3 cells, and HeLa-rat ADH cells oxidized ethanol much faster than the cells expressing the ADH1B isoenzymes. This was not explained by high cellular NADH levels or endogenous inhibitors; but rather because the activity of the β1 and β2 ADHs were constrained by the accumulation of acetaldehyde, as shown by the increased rate of ethanol oxidation by cell lines expressing β2 ADH plus ALDH2. Conclusion The activity of the human β2 ADH isoenzyme is sensitive to inhibition by acetaldehyde, which likely limits its activity in vivo. This study emphasizes the importance of maintaining a low steady–state acetaldehyde concentration in hepatocytes during ethanol metabolism. PMID:21166830

  8. Changes of the cell cycle regulators and cell cycle arrest in cervical cancer cells after cisplatin therapy

    Ke-xiu Zhu; Ya-li Cao; Bin Li; Jia Wang; Xiao-bing Han

    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 eisplatin of different concentrations were measured by MTT assays. The mRNA and protein expressions of ATM, Chk2 and p53 of HeLa cells with and withont cisplatin were detected by RT-PCR and Western blot, respectively. The cell cycle analysis was conducted by flow cytometric analysis. Results Cisplatin inhibited the proliferation of HeLa cells in a dose- and time-dependent manner. The mRNA and protein expressions of ATM, Chk2 and p53 were increased in HeLa cells treated with cisplatin. The cell cycle was arrested in G2/M phase in HeLa cells treated with cisplatin. Conclusion Activation of ATM, Chk2 and p53 might be critical in determining whether cells survive or undergo apoptesis. Targeting ATM, Chk2 and p53 pathway might he a promising strategy for reversing chemoresistance to clsplatin in cervical cancer.

  9. In vitro transcription of a cloned vaccinia virus gene by a soluble extract prepared from vaccinia virus-infected HeLa cells.

    Foglesong, P D

    1985-01-01

    Faithful transcription of a vaccinia virus gene was accomplished in vitro by using a soluble extract prepared from vaccinia virus-infected HeLa cells. Specific transcription of the cloned vaccinia virus gene was detected by using template DNA restricted within the transcribed region. The vaccinia virus gene was not transcribed by extracts prepared from uninfected HeLa cells even with supplementation by purified vaccinia virus RNA polymerase, nor was a clone of adenovirus 2 DNA bearing the maj...

  10. Cloning of rat glutamine synthetase gene and its expression in Hela-G cells%大鼠谷氨酰胺合成酶基因的克隆及其在Hela-G细胞中的表达

    孙伟峰; 刘春兴; 邹健

    2011-01-01

    Objective To clone rat glutamine synthetase(GS) gene and to express it in mammalian cells. Methods Rat GS cD NA was amplified by RT-PCR from RNA of rat cerebral cortex tissue. GS cDNA was inserted into eukaryotic expression vector pEGFP-N3. The recombinant expression vector was transiently transfected into Hela-G cells by LipofectamineTM 2000 reagent. The expression of GS in Hela-G cells was detected by immunocytochemistry. Results The sequence of the cloncd GS was confirmed by DNA sequencing. The Hela-G cells transfected with pEGFP-N3-GS could efficicntly express GS protein. Conclusion The cloning of rat GS gene and the construction of its eukaryotic expression vector are successful,which lavs the foundation for further investiga ting the role of GS in astrocytes.%目的 克隆大鼠谷氨酰胺合成酶(GS)基因,构建其真核表达载体,并观察其在Hela-G细胞中的表达. 方法 采用RT-PCR方法,以大鼠大脑皮层总RNA为模板,扩增GS基因,定向克隆到pEGFP-N3载体中.以LipofectamineTM2000试剂转染pEGFP-N3-GS表达载体至Hela-G细胞中进行瞬时真核表达.以免疫细胞化学方法鉴定GS的表达.结果 从大鼠大脑皮层组织中克隆到序列正确的GS全长编码序列.所构建的pEGFP-N3-GS质粒在Hela-G细胞中获得高效表达.结论 大鼠GS基因的克隆、真核表达载体的构建及在Hela-G中的表达获得成功.

  11. 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.

  12. 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

  13. Paclitaxel-resistant HeLa cells have up-regulated levels of reactive oxygen species and increased expression of taxol resistance gene 1.

    Bi, Wenxiang; Wang, Yuxia; Sun, Gaoying; Zhang, Xiaojin; Wei, Yongqing; Li, Lu; Wang, Xiaoyuan

    2014-07-01

    This study is to establish a paclitaxel (PTX)-resistant human cervical carcinoma HeLa cell line (HeLa/PTX) and to investigate its redox characteristics and the expression of taxol resistance gene 1 (Txr1). HeLa cells were treated with PTX and effects of PTX on cell proliferation were detected through cell counting and the MTT assay. Levels of cellular reactive oxygen species (ROS), reduced glutathione (GSH), and oxidized glutathione (GSSG) as well as the ratio of GSH to GSSG were measured by the 2,7-difluorescein diacetate (DCFH-DA) method and the 5,5'dithiobis(2-nitrobenzoic acid) (DTNB) method. Activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were determined by the nitrite formation method, the molybdate colorimetric method, and the DTNB colorimetric method, respectively. The level of Txr1 mRNA was determined by real-time PCR. Compared with the regular HeLa cells, HeLa/PTX cells were larger in size and had more cytoplasmic granules. The population doubling time for HeLa/PTX cells was 1.32 times of that of HeLa cells (PHeLa cells with a resistance index of 122.69. HeLa/PTX cells had higher levels of ROS (PHeLa cells. HeLa/PTX cells, with higher levels of ROS and Txr1 mRNA expression, are more resistant to PTX than HeLa cells.

  14. Pravastatin and simvastatin inhibit the adhesion, replication and proliferation of Toxoplasma gondii (RH strain) in HeLa cells.

    Sanfelice, Raquel Arruda; da Silva, Suelen Santos; Bosqui, Larissa Rodrigues; Miranda-Sapla, Milena Menegazzo; Barbosa, Bellisa Freitas; Silva, Rafaela José; Ferro, Eloísa A Vieira; Panagio, Luciano Aparecido; Navarro, Italmar Teodorico; Bordignon, Juliano; Conchon-Costa, Ivete; Pavanelli, Wander Rogerio; Almeida, Ricardo Sergio; Costa, Idessania Nazareth

    2017-03-01

    The conventional treatment for toxoplasmosis with pyrimethamine and sulfadiazine shows toxic effects to the host, and it is therefore necessary to search for new drugs. Some studies suggest the use of statins, which inhibit cholesterol synthesis in humans and also the initial processes of isoprenoid biosynthesis in the parasite. Thus, the objective of this study was to evaluate the activity of the statins pravastatin and simvastatin in HeLa cells infected in vitro with the RH strain of T. gondii. HeLa cells (1×10(5)) were infected with T. gondii tachyzoites (5×10(5)) following two different treatment protocols. In the first protocol, T. gondii tachyzoites were pretreated with pravastatin (50 and 100μg/mL) and simvastatin (1.56 and 3.125μg/mL) for 30min prior to infection. In the second, HeLa cells were first infected (5×10(5)) with tachyzoites and subsequently treated with pravastatin and simvastatin for 24h at the concentrations noted above. Initially, we evaluated the cytotoxicity of drugs by the MTT assay, number of tachyzoites adhered to cells, number of infected cells, and viability of tachyzoites by trypan blue exclusion. The supernatant of the cell cultures was collected post-treatment for determination of the pattern of Th1/Th2/Th17 cytokines by cytometric bead array. There was no cytotoxicity to HeLa cells with 50 and 100μg/mL pravastatin and 1.56 and 3.125μg/mL simvastatin. There was no change in the viability of tachyzoites that received pretreatment. Regarding the pre- and post-treatment of the cells with pravastatin and simvastatin alone, there was a reduction in adhesion, invasion and proliferation of cells to T. gondii. As for the production of cytokines, we found that IL-6 and IL-17 were significantly reduced in cells infected with T. gondii and treated with pravastatin and simvastatin, when compared to control. Based on these results, we can infer that pravastatin and simvastatin alone possess antiproliferative effects on tachyzoites forms

  15. 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 ...

  16. Inhibition ef ect of WBG on Hela cel s%扇贝多肽(WBG)对宫颈癌Hela细胞的抑制作用

    张震宇

    2014-01-01

    Objective To investigate the WBG in cervical cancer Hela cellline.Methods To investigate WBG on the cytotoxicity of Hela cells by MTT method.Results WBG inhibit Hela cells.Conclusion WBG inhibit Hela cervical cancer cellgrowth in a dose dependent manner.Cervical cancer Hela cells treatment with 20 -200 μMWBG in vitro slow growth,cellmorphology obviously change,and the inhibition rate reached 55.74% at 200 μMWBG.%目的:探讨扇贝多肽(WBG)对宫颈癌 Hela 细胞的抑制作用。方法:MTT 法测定扇贝多肽(WBG)对 Hela 细胞的细胞抑制作用.结果:WBG 对宫颈癌 Hela 细胞有明显的抑制效应。结论:WBG 对宫颈癌 Hela 细胞生长的抑制作用呈剂量依赖性,经20-200μMWBG 处理,体外培养宫颈癌 Hela 细胞生长缓慢,细胞形态发生明显改变,200μMWBG时抑制率达到55.74%。

  17. Proteomic Analysis Revealed the Important Role of Vimentin in Human Cervical Carcinoma HeLa Cells Treated With Gambogic Acid.

    Yue, Qingxi; Feng, Lixing; Cao, Biyin; Liu, Miao; Zhang, Dongmei; Wu, Wanying; Jiang, Baohong; Yang, Min; Liu, Xuan; Guo, Dean

    2016-01-01

    Gambogic acid (GA) is an anticancer agent in phase IIb clinical trial in China. In HeLa cells, GA inhibited cell proliferation, induced cell cycle arrest at G2/M phase and apoptosis, as showed by results of MTT assay and flow cytometric analysis. Possible target-related proteins of GA were searched using comparative proteomic analysis (2-DE) and nine proteins at early (3 h) stage together with nine proteins at late (24 h) stage were found. Vimentin was the only target-related protein found at both early and late stage. Results of both 2-DE analysis and Western blotting assay suggested cleavage of vimentin induced by GA. MS/MS analysis of cleaved vimentin peptides indicated possible cleavage sites of vimentin at or near ser51 and glu425. Results of targeted proteomic analysis showed that GA induced change in phosphorylation state of the vimentin head domain (aa51-64). Caspase inhibitors could not abrogate GA-induced cleavage of vimentin. Over-expression of vimentin ameliorated cytotoxicity of GA in HeLa cells. The GA-activated signal transduction, from p38 MAPK, heat shock protein 27 (HSP27), vimentin, dysfunction of cytoskeleton, to cell death, was predicted and then confirmed. Results of animal study showed that GA treatment inhibited tumor growth in HeLa tumor-bearing mice and cleavage of vimentin could be observed in tumor xenografts of GA-treated animals. Results of immunohistochemical staining also showed down-regulated vimentin level in tumor xenografts of GA-treated animals. Furthermore, compared with cytotoxicity of GA in HeLa cells, cytotoxicity of GA in MCF-7 cells with low level of vimentin was weaker whereas cytotoxicity of GA in MG-63 cells with high level of vimentin was stronger. These results indicated the important role of vimentin in the cytotoxicity of GA. The effects of GA on vimentin and other epithelial-to-mesenchymal transition (EMT) markers provided suggestion for better usage of GA in clinic.

  18. Quaternized chitosan-coated nanofibrous materials containing gossypol: preparation by electrospinning, characterization and antiproliferative activity towards HeLa cells.

    Ignatova, Milena; Manolova, Nevena; Toshkova, Reneta; Rashkov, Iliya; Gardeva, Elena; Yossifova, Liliya; Alexandrov, Marin

    2012-10-15

    Nanofibrous polylactide-based materials loaded with a natural polyphenolic compound gossypol (GOS) with antitumor properties were prepared by electrospinning. The nanofibrous materials were coated with a thin film of crosslinked quaternized chitosan (QCh). GOS incorporated in the nanofibrous mats was in the amorphous state. GOS release was diffusion-controlled and its in vitro release profiles depended on the mat composition. The nanofibrous materials exhibited high cytotoxicity towards HeLa tumor cells. Interestingly, it was particularly pronounced in the case of fibrous materials, which contain both QCh and GOS. The observed strong antiproliferative effect of the nanofibrous mats was mainly due to induction of cell apoptosis.

  19. Visualisation of cell cycle modifications by X-ray irradiation of single HeLa cells using fluorescent ubiquitination-based cell cycle indicators.

    Kaminaga, K; Noguchi, M; Narita, A; Sakamoto, Y; Kanari, Y; Yokoya, A

    2015-09-01

    To explore the effects of X-ray irradiation on mammalian cell cycle dynamics, single cells using the fluorescent ubiquitination-based cell cycle indicator (Fucci) technique were tracked. HeLa cells expressing Fucci were used to visualise cell cycle modifications induced by irradiation. After cultured HeLa-Fucci cells were exposed to 5 Gy X-rays, fluorescent cell images were captured every 20 min for 48 h using a fluorescent microscope. Time dependence of the fluorescence intensity of S/G2 cells was analysed to examine the cell cycle dynamics of irradiated and non-irradiated control cells. The results showed that irradiated cells could be divided into two populations: one with similar cell cycle dynamics to that of non-irradiated cells, and another displaying a prolonged G2 phase. Based on these findings, it is proposed in this article that an underlying switch mechanism is involved in cell cycle regulation and the G2/M checkpoint of HeLa cells.

  20. Photodynamic effects induced by meso-tris(pentafluorophenyl)corrole and its cyclodextrin conjugates on cytoskeletal components of HeLa cells.

    Barata, Joana F B; Zamarrón, Alicia; Neves, M Graça P M S; Faustino, M Amparo F; Tomé, Augusto C; Cavaleiro, José A S; Röder, Beate; Juarranz, Ángeles; Sanz-Rodríguez, Francisco

    2015-03-06

    The aim of this work was to synthesize new corrole β-cyclodextrin conjugates βCD1 (with one β-cyclodextrin moiety) and βCD2 (with two β-cyclodextrin moieties) from 5,10,15-tris(pentafluorophenyl)corrole (TPFC) and to test in vitro the efficacy of these compounds towards tumoral HeLa cells. No dark cytotoxicity was observed for TPFC and βCD1 at the concentration used for PDT cell treatment, even during long incubation periods (24 h). Fluorescence microscopy showed that TPFC and βCD1 accumulate in HeLa cells at lysosomes and in the Golgi apparatus, respectively. The cell survival after the PDT treatment with visible light was dependent on light exposure level and compound concentration. βCD1 was able to penetrate efficiently in the cytoplasm of the HeLa cells. In particular, we have analyzed the photodynamic effect of the corrole derivatives on the microtubules of HeLa cells and the morphological alterations on the mitotic spindle. TPFC and βCD1 caused photocytotoxicity in tumoral HeLa cells and induced a rapid metaphase blockage of cells that also showed clearly altered configurations of the mitotic spindle. The results showed that TPFC has the highest photosensitizing efficiency on tumoral cells.

  1. Anticancer Drug Resistance of HeLa Cells Transfected With Rat Glutathione S-transferase pi Gene

    WEI CAO; JIN ZUO; YAN MENG; QIANG WEI; ZHAO-HU SHI; LI-MEI JU; FU-DE FANG

    2003-01-01

    Objective To establish a cytologic expressing system of rat glutathione S-transferase pi(GST-pi) cDNA for detecting the resistance of HeLa cells to anticancer drugs. Methods Theassessment was made with various anticancer drugs (adriamycin, mitomycin, cisplatinum andvincristine) that showed different cytotoxicities in transfectant HeLa cells with pSV-GT containing ratGST-pi cDNA (HeLa/pSV-GT) or control pSV-neo (HeLa/pSV-neo). Expression levels of GST-pimRNA in HeLa/pSV-GT and HeLa/pSV-neo were measured by in situ hybridization usingDigoxin-labelled cDNA probe. Results HeLa/pSV-GT expressed significantly high degree ofGST-pi mRNA, whereas both HeLa/pSV-neo and HeLa cells had very low expression. Cytotoxicitiesof HeLa/pSV-GT and HeLa/pSV-neo with 4 anticancer drugs were measured by MTT assay. Drugconcentrations for yielding 50% inhibition (IC50) in HeLa/pSV-GT by adriamycin, mitomycin andcisplatinum were 70.13 μg/mL, 10.95 μg/mL and 16.52 μg/mL, respectively. In contrast, IC50 inHeLa/pSV-neo was 10.34 μg/mL, 7.48 μg/mL and 13.70 μg/mL, respectively. The cytotoxicities ofvincristine on both HeLa/pSV-GT and HeLa/pSV-neo were not significantly different. ConclusionsOur findings suggest that HeLa/pSV-GT containing rat GST-pi cDNA is resistant to some anticancerdrugs due to overexpression of GST-pi. Also, HeLa/pSV-GT cell line could serve as a usefulcytogenetic model for further research.

  2. 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 HeLa...

  3. Metallothionein isoform 2A expression is inducible and protects against ROS-mediated cell death in rotenone-treated HeLa cells.

    Reinecke, F.; Levanets, O.; Olivier, Y.; Louw, R.; Semete, B.; Grobler, A.; Hidalgo, J.; Smeitink, J.A.M.; Olckers, A.; Westhuizen, F.H. van der

    2006-01-01

    The role of MT (metallothionein) gene expression was investigated in rotenone-treated HeLa cells to induce a deficiency of NADH:ubiquinone oxidoreductase (complex I). Complex I deficiency leads to a diversity of cellular consequences, including production of ROS (reactive oxygen species) and apoptos

  4. Inhibition of antiviral drug cidofovir on proliferation of human papillomavirus-infected cervical cancer cells

    Yang, Jing; Dai, Lv-Xia; Chen, Ming; Li, Bei; Ding, Nana; Li, Gang; Liu, Yan-Qing; Li, Ming-Yuan; Wang, Bao-Ning; Shi, Xin-Li; Tan, Hua-Bing

    2016-01-01

    In order to evaluate the potential application value of cidofovir (CDV) in the prevention of human papillomavirus (HPV) infection and treatment of cervical cancer, the inhibitory effect of CDV on the proliferation of HPV 18-positive HeLa cells in cervical cancer was preliminarily investigated, using cisplatin (DDP) as a positive control. An MTT assay was used to analyze the effects of CDV and DDP on HeLa cell proliferation. In addition, clone formation assay and Giemsa staining were used to examine the extent of HeLa cell apoptosis caused by CDV and DDP. Flow cytometry was also used to detect the shape and size of apoptotic cells following propidium iodide staining, while western blot analysis identified the expression levels of of E6 and p53 proteins in HeLa cells. A cell climbing immunofluorescence technique was used to locate the subcellular position of p53 in HeLa cells. The results demonstrated that CDV and DDP inhibited the proliferation of HeLa cells in a concentration- and time-dependent manner. Flow cytometry showed that CDV and DDP treatments resulted in cell arrest in the S-phase, and triggered programmed cell death. Furthermore, western blot analysis revealed that CDV and DDP inhibited E6 protein expression and activated p53 expression in HeLa cells. Finally, the immunofluorescence results indicated that CDV and DDP inhibited the nuclear export of p53 by E6 protein, which is required for degradation of endogenous p53 by MDM2 and human papilloma virus E6. In conclusion, CDV and DDP inhibited HeLa cell proliferation in a concentration- and time-dependent manner, reduced the expression of E6 protein, and reinstated p53 protein activity. Thus, CDV regulates cell cycle arrest and apoptosis, and may be a potential cervical cancer therapeutic strategy. PMID:27882102

  5. Inhibition of antiviral drug cidofovir on proliferation of human papillomavirus-infected cervical cancer cells.

    Yang, Jing; Dai, Lv-Xia; Chen, Ming; Li, Bei; Ding, Nana; Li, Gang; Liu, Yan-Qing; Li, Ming-Yuan; Wang, Bao-Ning; Shi, Xin-Li; Tan, Hua-Bing

    2016-11-01

    In order to evaluate the potential application value of cidofovir (CDV) in the prevention of human papillomavirus (HPV) infection and treatment of cervical cancer, the inhibitory effect of CDV on the proliferation of HPV 18-positive HeLa cells in cervical cancer was preliminarily investigated, using cisplatin (DDP) as a positive control. An MTT assay was used to analyze the effects of CDV and DDP on HeLa cell proliferation. In addition, clone formation assay and Giemsa staining were used to examine the extent of HeLa cell apoptosis caused by CDV and DDP. Flow cytometry was also used to detect the shape and size of apoptotic cells following propidium iodide staining, while western blot analysis identified the expression levels of of E6 and p53 proteins in HeLa cells. A cell climbing immunofluorescence technique was used to locate the subcellular position of p53 in HeLa cells. The results demonstrated that CDV and DDP inhibited the proliferation of HeLa cells in a concentration- and time-dependent manner. Flow cytometry showed that CDV and DDP treatments resulted in cell arrest in the S-phase, and triggered programmed cell death. Furthermore, western blot analysis revealed that CDV and DDP inhibited E6 protein expression and activated p53 expression in HeLa cells. Finally, the immunofluorescence results indicated that CDV and DDP inhibited the nuclear export of p53 by E6 protein, which is required for degradation of endogenous p53 by MDM2 and human papilloma virus E6. In conclusion, CDV and DDP inhibited HeLa cell proliferation in a concentration- and time-dependent manner, reduced the expression of E6 protein, and reinstated p53 protein activity. Thus, CDV regulates cell cycle arrest and apoptosis, and may be a potential cervical cancer therapeutic strategy.

  6. 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...

  7. 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.

  8. [Effects of the expression of mouse metallothionein-I gene in human HeLa cell line on drug resistance].

    Li, X; Lü, W; Yin, S; Li, L

    2000-07-01

    Metallothionein-I (MT-I) gene was inserted into EcoRI site by using pSV2-neo plasmid vector. Recombiant plasmid was transfected into HeLa cells by DNA-calcium phosphate precipitation technique. MT-I expression colones were growing in medium including G418. The amount of MT-I expression in transfected cells was found 2.6 times higher than that of non-transfected ones. In order to observe the relationship between the expression of MT-I gene in cells and drug resistance, cells were treated with different concentrations of cisplatin and adriamycin respectively. The results indicated that cisplatin (0.1 mumol/ml) inhibited the growth of both transfected and non-transfected cells. The inhibitory rates were 34% and 82% respectively(P 0.05). The results indicated that MT was related to drug resistance of tumor cells.

  9. 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.

  10. Gallic acid reduces cell viability, proliferation, invasion and angiogenesis in human cervical cancer cells.

    Zhao, Bing; Hu, Mengcai

    2013-12-01

    Gallic acid is a trihydroxybenzoic acid, also known as 3,4,5-trihydroxybenzoic acid, which is present in plants worldwide, including Chinese medicinal herbs. Gallic acid has been shown to have cytotoxic effects in certain cancer cells, without damaging normal cells. The objective of the present study was to determine whether gallic acid is able to inhibit human cervical cancer cell viability, proliferation and invasion and suppress cervical cancer cell-mediated angiogenesis. Treatment of HeLa and HTB-35 human cancer cells with gallic acid decreased cell viability in a dose-dependent manner. BrdU proliferation and tube formation assays indicated that gallic acid significantly decreased human cervical cancer cell proliferation and tube formation in human umbilical vein endothelial cells, respectively. Additionally, gallic acid decreased HeLa and HTB-35 cell invasion in vitro. Western blot analysis demonstrated that the expression of ADAM17, EGFR, p-Akt and p-Erk was suppressed by gallic acid in the HeLa and HTB-35 cell lines. These data indicate that the suppression of ADAM17 and the downregulation of the EGFR, Akt/p-Akt and Erk/p-Erk signaling pathways may contribute to the suppression of cancer progression by Gallic acid. Gallic acid may be a valuable candidate for the treatment of cervical cancer.

  11. 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.

  12. Gene expression alteration during redox-dependent enhancement of arsenic cytotoxicity by emodin in HeLa cells

    Xiao Jing WANG; Jie YANG; Hui CANG; Yan Qiong ZOU; Jing YI

    2005-01-01

    Emodin (1,3,8-trihydroxy-6-methylanthraquinone) could enhance the sensitivity of tumor cells to arsenic trioxide (As2O3)-induced apoptosis via generation of ROS,but the molecular mechanism has not been elucidated.Here,we carried out cDNA microarray-based global transcription profiling of HeLa cells in response to As2O3/emodin cotreatment,comparing with As2O3-only treatment.The results showed that the expression of a number of genes was substantially altered at two time points.These genes are involved in different aspects of cell function.In addition to redox regulation and apoptosis,ROS affect genes encoding proteins associated with cell signaling,organelle functions,cell cycle,cytoskeleton,etc.These data suggest that based on the cytotoxicity of As2O3,emodin mobilize every genomic resource through which the As2O3-induced apoptosis is facilitated.

  13. 热休克蛋白70反义脱氧寡核苷酸对宫颈癌HeLa细胞生长和凋亡及化学治疗敏感性的影响%Effect of heat shock protein 70 antisense oligodeoxynucleotides on growth, apoptosis and chemosensitivity for human cervical cancer HeLa cells

    刘春梅; 丁依玲

    2013-01-01

    Objective:To explore the effect of heat shock protein 70 (HSP70) antisense oligodeoxynucleotides on proliferation, apoptosis and chemosensitivity for HeLa cells induced by cisplatin. Methods:l)HeLa cells were transfected by antisense, sense or random oligonucleotides. The cells were divided into a control group (Ctrl group, n=S), a antisense oligodeoxynucleotides treatment group (AS group, n=5), a sense oligodeoxynucleotides treatment group (S group, n=5) and a random oligonucleotides treatment group (R group, n=5). The expression of heat shock proteins 70 (HSP70) in the cells from each group was detected. 2) The HeLa cells were treated by cisplatin and were divided into a control group (control group, n=8), a cisplatin treatment group (Cis group, n=8), a antisense oligodeoxynucleotides + cisplatin treatment group (AS +Cis group, n=8), a sense oligodeoxynucleotides + cisplatin treatment group (S +Cis group, n=8) and a random oligonucleotides + cisplatin treatment group (R +Cis group, n=8). The growth inhibitory rate of HeLa cells was determined by methyl thiazolyl tetrazolium (MTT) assay. The apoptosis rate was detected by flow cytometry (FCM). Results:l)The optical density of HSP70 band in the Ctrl group, the AS group, the S group, and the Rgroup were (1.365 ±0.187) , (0.379 ±0.134), (1.403 ±0.163) and (l.410±0.158), respectively, which was significantly decreased in the AS group (P<0.0l). 2) Compared with the control group, the growth inhibitory rate and the apoptosis rate of HeLa cells were significantly increased only in AS+Cis group (P<0.05). Conclusion:HSP70 antisense oligodeoxynucleotides can effectively inhibit the proliferation of HeLa cells and induce its apoptosis through down-regulation of HSP70 expression. HSP70 antisense oligodeoxynucleotides can increase the chemosensitivity of tumor to cisplatin treatment.%目的:探讨反义脱氧寡核苷酸封闭HSP70基因对体外宫颈癌HeLa细胞增殖、凋亡及化疗敏感性的影响.方法:1)将体

  14. Investigation on the effect of static magnetic field up to 30 mT on viability percent, proliferation rate and IC50 of HeLa and fibroblast cells.

    Zafari, Jaber; Javani Jouni, Fatemeh; Abdolmaleki, Parviz; Jalali, Amir; Khodayar, Mohammad Javad

    2015-09-01

    We have investigated the effects of static magnetic field (SMF) on the viability of the human cervical cancer (HeLa) cell line and fibroblast cells. The cells were cultured in DMEM medium and treated several times (24, 48,72 and 96 h) and at several intensities (5, 10, 20 and 30 mT) of magnetic field (MF). The cytotoxicity and cell viability percent in treated cells were performed using MTT assay by evaluating mitochondrial dehydrogenase activity. The MF ability on inducing cell death or inhibiting biochemical function was reported as cell death percent. The results showed that the increase of MF intensity and the time that cells were exposed to this treatment increased sharply cell death percent and proliferation rate in HeLa cell compare to fibroblast cells. Our data suggest that SMF biological effects on cell death were different in our selected targets. Cell type and time of exposure have been therefore found to be significant factors. These findings could be used to improve new effective method using SMF in conjunction with the common therapeutic approaches.

  15. On-Line Monitoring the Growth of E. coli or HeLa Cells Using an Annular Microelectrode Piezoelectric Biosensor

    Tong, Feifei; Lian, Yan; Han, Junliang

    2016-01-01

    Biological information is obtained from the interaction between the series detection electrode and the organism or the physical field of biological cultures in the non-mass responsive piezoelectric biosensor. Therefore, electric parameter of the electrode will affect the biosensor signal. The electric field distribution of the microelectrode used in this study was simulated using the COMSOL Multiphysics analytical tool. This process showed that the electric field spatial distribution is affected by the width of the electrode finger or the space between the electrodes. In addition, the characteristic response of the piezoelectric sensor constructed serially with an annular microelectrode was tested and applied for the continuous detection of Escherichia coli culture or HeLa cell culture. Results indicated that the piezoelectric biosensor with an annular microelectrode meets the requirements for the real-time detection of E. coli or HeLa cells in culture. Moreover, this kind of piezoelectric biosensor is more sensitive than the sensor with an interdigital microelectrode. Thus, the piezoelectric biosensor acts as an effective analysis tool for acquiring online cell or microbial culture information. PMID:27999343

  16. Electrospun nanofibrous mats containing quaternized chitosan and polylactide with in vitro antitumor activity against HeLa cells.

    Ignatova, Milena G; Manolova, Nevena E; Toshkova, Reneta A; Rashkov, Iliya B; Gardeva, Elena G; Yossifova, Lilia S; Alexandrov, Marin T

    2010-06-14

    Nanofibrous materials containing the antitumor drug doxorubicin hydrochloride (DOX) were easily prepared using a one-step method by electrospinning of DOX/poly(L-lactide-co-D,L-lactide) (coPLA) and DOX/quaternized chitosan (QCh)/coPLA solutions. The pristine and DOX-containing mats were characterized by ATR-FTIR and X-ray photoelectron spectroscopy (XPS). The release rate of DOX from the prepared fibers increased with the increase in DOX content. The DOX release process was diffusion-controlled. MTT cell viability studies revealed that incorporation of DOX and QCh in the nanofibrous mats led to a significant reduction in the HeLa cells viability. It was found, that the antitumor efficacy of the DOX-containing mats at 6 h was higher than that of the free DOX. SEM, TEM, and fluorescence microscopic observations confirmed that the antitumor effect of QCh-based and DOX-containing fibrous mats was mainly due to induction of apoptosis in the HeLa cells.

  17. Yatein from Chamaecyparis obtusa suppresses herpes simplex virus type 1 replication in HeLa cells by interruption the immediate-early gene expression.

    Kuo, Yuh-Chi; Kuo, Yueh-Hsiung; Lin, Yuang-Lian; Tsai, Wei-Jern

    2006-07-01

    Inhibitory effects of methanolic extracts from nine Chinese herbs on herpes simplex virus type 1 (HSV-1) replication were studied. By a bioassay-guided fractionation procedure, yatein (C(22)H(23)O(7); M.W.399) was isolated from Chamaecyparis obtusa; yatein significantly suppressed HSV-1 multiplication in HeLa cells without apparent cytotoxicity. To further localize the point in the HSV-1 replication cycle where arrest occurred, a set of key regulatory events leading to the viral multiplication was examined, including viral immediate-early (alpha) and late (gamma) gene expression and DNA replication. Results indicated that levels of glycoprotein B (gB) and gC mRNA expression in HeLa cells were impeded by yatein. Data from polymerase chain reaction showed that replication of HSV-1 DNA in HeLa cells was arrested by yatein. Furthermore, yatein decreased ICP0 and ICP4 gene expression in HeLa cells. Results of an electrophoretic mobility shift assay demonstrated that yatein interrupted the formation of alpha-trans-induction factor/C1/Oct-1/GARAT multiprotein complex. The mechanisms of antiviral action of yatein seem to be mediated, by inhibiting HSV-1 alpha gene expression, including expression of the ICP0 and ICP4 genes, and by arresting HSV-1 DNA synthesis and structural protein expression in HeLa cells. These results suggest that yatein is an antiviral agent against HSV-1 replication.

  18. Isolation of an arabinogalactan from Endopleura uchi bark decoction and its effect on HeLa cells.

    Bento, João Francisco; Noleto, Guilhermina Rodrigues; de Oliveira Petkowicz, Carmen Lúcia

    2014-01-30

    Endopleura uchi is a native plant from the Amazon used in popular medicine to treat myomas. A crude polysaccharide (AGb) obtained from E. uchi bark decoction was purified, yielding a type II arabinogalactan (AG) that was characterized by chemical and spectroscopic methods. AG was evaluated for its cytotoxic effects on HeLa cells. AG (5-500 μg/ml) reduced cell viability at 48 and 72 h (approximately 20%) but not in a dose-dependent manner. Cell proliferation was also reduced by AG, with a 25% inhibition (100 μg/ml) at 72 h. The results suggest that the cytotoxicity exhibited by AG does not involve pathways related to the cell cycle.

  19. 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.

  20. Optical parameters measurement for diagnostic and photodynamic therapy of human cervical adenocarcinoma (HeLa) cell line

    Rehman, A.; Firdous, S.; Nawaz, M.; Ahmad, M.

    2011-11-01

    The purpose of this study was to investigate the optical properties, absorption coefficient (μ a ) scattering coefficient (μ s ) and refractive indices, (n) of HeLa cell line in a suspension of 2% minimum essential medium (MEM) at two different (632.8 and 532.0 nm) wave lengths of laser light. Optical properties were determined with Kubelka Munk Model (KMM) and refractive index measurement was made through minimum angle of deviation method (MAD). We reported μ a = 8.643 ± 0.187 and 2.348 ± 0.249 cm-1 and μ s = 5.609 ± 0.287 and 88.166 ± 2.833 cm-1 at 632.8 and 532.0 nm, respectively. Refractive index was found to be 1.332 and 1.312 at 632.8 nm and 532.0 nm, respectively. The discussed results provide a route of information for clinical diagnosis, therapeutic application and dosimetry studies in HeLa and other cell lines.

  1. NADH oxidase activity (NOX) and enlargement of HeLa cells oscillate with two different temperature-compensated period lengths of 22 and 24 minutes corresponding to different NOX forms

    Wang, S.; Pogue, R.; Morre, D. M.; Morre, D. J.

    2001-01-01

    NOX proteins are cell surface-associated and growth-related hydroquinone (NADH) oxidases with protein disulfide-thiol interchange activity. A defining characteristic of NOX proteins is that the two enzymatic activities alternate to generate a regular period length of about 24 min. HeLa cells exhibit at least two forms of NOX. One is tumor-associated (tNOX) and is inhibited by putative quinone site inhibitors (e.g., capsaicin or the antitumor sulfonylurea, LY181984). Another is constitutive (CNOX) and refractory to inhibition. The periodic alternation of activities and drug sensitivity of the NADH oxidase activity observed with intact HeLa cells was retained in isolated plasma membranes and with the solubilized and partially purified enzyme. At least two activities were present. One had a period length of 24 min and the other had a period length of 22 min. The lengths of both the 22 and the 24 min periods were temperature compensated (approximately the same when measured at 17, 27 or 37 degrees C) whereas the rate of NADH oxidation approximately doubled with each 10 degrees C rise in temperature. The rate of increase in cell area of HeLa cells when measured by video-enhanced light microscopy also exhibited a complex period of oscillations reflective of both 22 and 24 min period lengths. The findings demonstrate the presence of a novel oscillating NOX activity at the surface of cancer cells with a period length of 22 min in addition to the constitutive NOX of non-cancer cells and tissues with a period length of 24 min.

  2. 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.

  3. Squamous cell skin cancer

    ... that reflect light more, such as water, sand, concrete, and areas that are painted white. The higher ... - skin - squamous cell; Skin cancer - squamous cell; Nonmelanoma skin cancer - squamous ...

  4. Enhanced cellular uptake of albumin-based lyophilisomes when functionalized with cell-penetrating peptide TAT in HeLa cells.

    Etienne van Bracht

    Full Text Available Lyophilisomes are a novel class of biodegradable proteinaceous nano/micrometer capsules with potential use as drug delivery carrier. Cell-penetrating peptides (CPPs including the TAT peptide have been successfully implemented for intracellular delivery of a broad variety of cargos including various nanoparticulate pharmaceutical carriers. In the present study, lyophilisomes were modified using CPPs in order to achieve enhanced cellular uptake. Lyophilisomes were prepared by a freezing, annealing, and lyophilization method and a cystein-elongated TAT peptide was conjugated to the lyophilisomes using a heterobifunctional linker. Fluorescent-activated cell sorting (FACS was utilized to acquire a lyophilisome population with a particle diameter smaller than 1000 nm. Cultured HeLa, OVCAR-3, Caco-2 and SKOV-3 cells were exposed to unmodified lyophilisomes and TAT-conjugated lyophilisomes and examined with FACS. HeLa cells were investigated in more detail using a trypan blue quenching assay, confocal microscopy, and transmission electron microscopy. TAT-conjugation strongly increased binding and cellular uptake of lyophilisomes in a time-dependent manner in vitro, as assessed by FACS. These results were confirmed by confocal microscopy. Transmission electron microscopy indicated rapid cellular uptake of TAT-conjugated lyophilisomes via phagocytosis and/or macropinocytosis. In conclusion, TAT-peptides conjugated to albumin-based lyophilisomes are able to enhance cellular uptake of lyophilisomes in HeLa cells.

  5. 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.

  6. Correlation between slowly repairable double-strand breaks and thermal radiosensitization in the human HeLa S3 cell line

    Kampinga, HH; Hiemstra, YS; Konings, AWT; Dikomey, E

    1997-01-01

    The effect of heat on double-strand breaks (dsb) repair was compared with thermal radiosensitization using HeLa S3 cells. Cells were exposed to a combined treatment of X-irradiation followed by heat (44 degrees C, 0.5 h) separated by time intervals up to 8h. DNA dsb were measured by PFGE and surviva

  7. 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

    2013-09-06

    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.

  8. 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.

  9. 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

  10. Molecular mechanism of inhibitory effects of C-phycocyanin combined with all-trans-retinoic acid on the growth of HeLa cells in vitro.

    Yang, Fan; Li, Bing; Chu, Xian-Ming; Lv, Cong-Yi; Xu, Ying-Jie; Yang, Peng

    2014-06-01

    We studied the effects of all-trans-retinoic acid (ATRA), C-phycocyanin (C-PC), or ATRA+C-PC on the growth of cervical cells (HeLa cells), cell cycle distribution, and apoptosis. The anticancer mechanism of the drug combination was revealed. MTT assay was adopted to determine the effects of C-PC and ATRA on the growth of HeLa cells. The expression quantities of cyclin-dependent kinase (CDK) 4, cyclin D1, Bcl-2, caspase-3, and CD59 were determined by in situ hybridization, immunofluorescence, immunohistochemistry staining, Western blot, and RT-PCR. TUNEL assay was adopted to determine the cellular apoptosis levels. Both C-PC and ATRA could inhibit the growth of HeLa cells, and the combination of ATRA+C-PC functioned cooperatively to induce apoptosis in HeLa cells. The dosage of ATRA was reduced when it cooperated with C-PC to reduce the toxicity. ATRA treated with C-PC could induce more cell cycle arrests than the single drug used by decrease in cyclin D1 and CDK4 expression. The combination of the two drugs could upregulate caspase-3 and downregulate the Bcl-2 gene and induce cell apoptosis. Moreover, the combination therapy has an important immunological significance in decreased expression of the CD59 protein. Singly, C-PC or ATRA could inhibit the growth of HeLa cells, and the effects of treatment were further enhanced in the combination group. In combination with C-PC, the dosage of ATRA was effectively reduced. The C-PC + ATRA combination might take effect by inhibiting the progress of the cell cycle, inducing cell apoptosis and promoting complement-mediated cytolysis.

  11. [Comparison of the indirect immunofluorescence assay performance of Bartonella henselae antigens obtained by co-cultivation in Vero and HeLa cells].

    Ergin, Cağrı; Akkaya, Yüksel; Kiriş Satılmış, Ozgün; Yılmaz, Cansev

    2011-07-01

    The laboratory diagnosis of Bartonella henselae infection is mainly based on serological testing by indirect immunofluorescence assay (IFA). Cell line co-cultivation with B.henselae and agar derivated antigens are the two major procedures used for evaluation of anti-Bartonella antibodies. Vero and Hep-2 cell lines are the most commonly used media for co-cultivation both in-house and commercial diagnostic kits production. However, HeLa cells which are easily supplied and grown, also can easily be infected by B.henselae. The aim of this study was to compare the performances of antigens obtained by co-cultivation of B.henselae ATCC 49882 (Houston-1) in Vero and HeLa Cells in IFA serology. Out of 381 sera samples, 127 (33.3%) were found positive and 195 (51.2%) were found negative by IFA performed by both cell line co-cultivations. The total agreement between the methods were found as 84.5% (322/381), and Cohen kappa value was calculated as 0.68 (strong, coherent). As a result, He-La cells were found to be useful for the preparation of B.henselae antigens to be used in IFA for the serologic diagnosis of B.henselae infections. However different genotype strains and cross reactions with other infectious agents should be investigated by further studies before routine applications of HeLa cell co-cultivations procedure is established.

  12. 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

  13. 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

  14. Anticancer-cytotoxic activity of saponins isolated from the leaves of Gymnema sylvestre and Eclipta prostrata on HeLa cells

    Khanna Venkatesan

    2009-01-01

    Full Text Available The anticancer-cytotoxic activities of isolated saponins, gymnemagenol (C 30 H 50 O 4 from Gymnema sylvestre and dasyscyphin C (C 28 H 40 O 8 from Eclipta prostrata leaves were tested under in vitro conditions in HeLa cells. The gymnemagenol and dayscyphin C at 50 μg/ml showed a good cytotoxic activity (63% and 52%, respectively in HeLa cells at 48 hours with the IC50 value of 37 and 50 μg/ml, respectively. 5-Fluorouracil (5-FU, a positive control, showed 57.5 % cell death with the IC50 value of 36 μg/ml. The percentage of HeLa cell death was maximum (73% after 96 hours with gymnemagenol, whereas dasyscyphin C showed only 53%. The isolated saponins were not toxic to Vero cells. From this study, it can be concluded that the saponins, gymnemagenol, and dayscyphin C have significant anticancer-cytotoxic activity on HeLa cells under in vitro conditions.

  15. Apoptosis induction and mitochondria alteration in human HeLa tumour cells by photoproducts of Rose Bengal acetate.

    Panzarini, Elisa; Tenuzzo, Bernadette; Palazzo, Fabio; Chionna, Alfonsina; Dini, Luciana

    2006-04-01

    The aim of this work was to investigate the apoptosis induction and mitochondria alteration after photodamage exerted by incubation of HeLa cells with Rose Bengal acetate-derivative (RBAc) followed by irradiation for a total dose of 1.6 J/cm2. This treatment was previously demonstrated to reduce cell viability under mild treatment conditions, suggesting the restoration of the photoactive molecule in particularly sensitive cell sites. Indeed, Rose Bengal (RB) is a very efficient photosensitizer, whose photophysical properties are inactivated by addition of the quencher group acetate. The RBAc behaves as a fluorogenic substrate by entering easily the cells where the original, photoactive molecule is restored by specific esterases. Different intracellular sites of photodamage of RB are present. In particular, fluorescence imaging of Rodamine 123 and JC-1 labelled cells showed altered morphology and loss of potential membrane of mitochondria. MTT and NR assays gave indications of alteration of mitochondrial and lysosomal enzyme activities. These damaged sites were likely responsible for triggering apoptosis. Significant amount of apoptotic cell death (about 40%) was induced after light irradiation followed RBAc incubation as revealed by morphological (modification of cell shape and blebs formation), cytochemical (FITC-Annexin-V positive cells) and nuclear fragmentation assays.

  16. Up-regulation of Raf kinase inhibitor protein enhances chemosensitivity of cervical cancer cell

    Xiao Chu; Xinqiang Ji; Mingcui Wang; Wenqing Zhang; Hui Ou; Chong Li

    2014-01-01

    Objective:The purpose of the study is to investigate the ef ects of up-regulation of Raf kinase inhibitor protein (RKlP) on the chemosensitivity of cervical cancer Hela cells. Methods:Eukaryotic expression plasmid pcDNA3.1(+)-ssRKIP containing human overal length RKIPcDNA was transfected into cervical cancer Hela cellby lipofectin assay, establishing a stable cellline containing a target gene by G418. Expression of RKIP in Hela cells was measured by Western blot analysis. After treatment with cisplatin of dif erent concentrations and intervals of time, the ef ect of RKIP on the proliferation of Hela cells was evaluated by MTT method. The flow cytometry was used to investigate whether the RKIP could inhibit apoptosis in Hela cells induced by cisplatin. Results:The expression of RKIP in Hela cells transfected with pcDNA3.1-ssRKIP was increased obviously. After dif erent concentrations of cisplatin treatment cells for 24, 48 and 72 h, the growth inhibition rate in Hela cells transfected with pcDNA3.1-ssRKIP was significantly higher than in control cells (P<0.05). With 5μg/mL cisplatin treatment for 24 h, pcDNA3.1-ssRKIP-transfected Hela cells had an obviously higher percentage of apoptosis (23.2 ± 0.24)%than non-transfected cells (12.4 ± 0.31)%and empty vector-transfected cells (13.4 ± 0.47)%. Without treatment of cisplatin, the percentage of apoptosis for Hela cells transfected with pcDNA3.1-ssRKIP was (5.7 ± 0.12)%, which was stil higher than those of the non-transfected cells (2.9 ± 0.21)%and empty vector-transfected cells (3 ± 0.08)%. Conclusion:Higher expres-sion of RKIP gene can improve chemosensitivitv of cervical cancer Hela cells to cisplatin.

  17. Picosecond pulsed electric fields induce apoptosis in HeLa cells via the endoplasmic reticulum stress and caspase-dependent signaling pathways.

    Chen, Wen-Juan; Xiong, Zheng-Ai; Zhang, Min; Yao, Chen-Guo; Zhao, Zhong-Yong; Hua, Yuan-Yuan; Zhou, Wei

    2013-03-01

    The non-invasive treatment of tumors with preserved fertility holds great promise. The application of pulsed electric field (PEF) is a new biomedical engineering technique for tumor therapy. Picosecond pulsed electric fields (psPEF) can be transferred to target deep tissue non-invasively and precisely; however, research of the biological effects of psPEF on cells is limited. Electric theory predicts that when the pulse duration decreases to nanoseconds and picoseconds, it will mainly affect organelles and lead to intracellular electromanipulations. Previous studies have shown that psPEF targets the mitochondria and induces apoptosis through a mitochondrial-mediated pathway in HeLa cells. The endoplasmic reticulum is also involved in the intrinsic pathways of apoptosis. In the present study, HeLa cells were exposed to psPEF to investigate the underlying mechanisms of apoptosis. MTT assay demonstrated that psPEF displayed strong growth inhibitory effects on HeLa cells. Treatment with psPEF led to marked cell apoptosis and cell cycle arrest at the G2/M phase. In addition, psPEF affected the phosphorylation levels of endoplasmic reticulum sensors and upregulated the expression of glucose-regulated protein 78 (GRP78), glucose-regulated protein 94 (GRP94) and CCAAT enhancer-binding protein (C/EBP) homologous protein (CHOP). These changes were accompanied by the elevation of intracellular Ca2+ concentrations. Furthermore, the activation of caspase-12, -9 and -3, led to the release of cytochrome c, as well as the upregulation of Bax and the downregulation of Bcl-2, as observed in the HeLa cells. Taken together, these data suggest that psPEF is an efficient apoptosis-inducing agent for HeLa cells, which exerts its effects, at least partially, via the endoplasmic reticulum stress and caspase-dependent signaling pathways.

  18. Accurate identification of UDP-glucuronosyltransferase 1A1 (UGT1A1) inhibitors using UGT1A1-overexpressing HeLa cells.

    Sun, Hua; Zhou, Xiaotong; Wu, Baojian

    2015-01-01

    1. UDP-glucuronosyltransferase 1A1 (UGT1A1) plays an irreplaceable role in detoxification of bilirubin and many drugs (e.g., SN-38). Here we aimed to explore the potential of UGT1A1-overexpressing HeLa cells (or HeLa1A1 cells) as a tool to accurately identify UGT1A1 inhibitors. 2. Determination of glucuronidation rates (β-estradiol and SN-38 as the substrates) was performed using HeLa1A1 cells and uridine diphosphoglucuronic acid (UDPGA)-supplemented cDNA expressed UGT1A1 enzyme (or microsomes). The inhibitory effects (IC50 values) of 20 structurally diverse compounds on the UGT1A1 activity were determined using HeLa1A1 cells and microsomal incubations. 3. In HeLa1A1 cells, the IC50 values for inhibition of β-estradiol glucuronidation by the tested compounds ranged from 0.33 to 94.6 µM. In the microsomal incubations, the IC50 values ranged from 0.47 to 155 µM. It was found that the IC50 values of all test compounds derived from the cells were well consistent with those from the microsomes (deviated by less than two-fold). Further, the IC50 values from the cells were strongly correlated with those from microsomes (r = 0.944, p HeLa cells were an appropriate tool to accurately depict the inhibition profiles of chemicals against UGT1A1.

  19. SENSITIZATION OF ACNU KILLING EFFECTS ON HeLa S3 CELLS BY MGMT ANTISENSE RNA TRANSFECTION

    季守平; 由英; 吴英; 陈建敏; 杨军; 章扬培

    1998-01-01

    O’-methylguanlne-DNA-msthybransferase(MGMT)plays a very important role in the ceUular resistsnce to uitrosoureas drugs. Inhibition of MGMT might be a useful approach in tumor chemotherapy. In this study, the depletlon vii MGMT activity hy retroviral-mediated antisense RNA transfectkm were reported. Three retroviral vectors expressing MGMT antisense RNA were constructed and transfected into HeLa S3 cells. The difference of MGMT mRNA, MGMT activity as well as cellular resistance to ACNU before and after transtecfion were ohserved. It was found that antisense RNA targeting 5''region and whole length of MGMT mRNA could partially deplete MGMT activity and enhance killing effects of ACNU.However, 3'' region antisense RNA had no effect on MGMT modulation.

  20. 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 HSV-1- and HSV-2-infected cells, as compared to virus-free HeLa cell controls (38 and 59 % of control, respectively). In contrast, HSV-1 and HSV-2 significantly (P 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.

  1. Thermotolerance induced at a mild temperature of 40°C alleviates heat shock-induced ER stress and apoptosis in HeLa cells.

    Bettaieb, Ahmed; Averill-Bates, Diana A

    2015-01-01

    Hyperthermia (39-45°C) has emerged as an alternate prospect for cancer therapy in combination with radiation and chemotherapy. Despite promising progress in the clinic, molecular mechanisms involved in hyperthermia-induced cell death are not clear. Hyperthermia causes protein denaturation/aggregation, which results in cell death by apoptosis and/or necrosis. Hyperthermia also induces thermotolerance, which renders cells resistant to subsequent exposure to lethal heat shock. This study investigates the role of both lethal (42-43°C) and mild (40°C) hyperthermia in regulating ER stress and ER stress-induced apoptosis in HeLa cells. The ability of mild thermotolerance induced at 40°C to alleviate either or both of these processes is also determined. Hyperthermia (42-43°C) induced ER stress, revealed by phosphorylation of PERK, eIF2α and IRE1α, cleavage of ATF6 and increased expression of BiP and sXBP1. Real-time PCR revealed that mRNA levels of ATF6, ATF4, BiP, sXBP1 and CHOP increased in cells exposed to hyperthermia. Moreover, hyperthermia caused disruption of calcium homeostasis and activated the calpain-calpastatin proteolytic system and ER resident caspase 4. Pre-exposure to mild hyperthermia (40°C) alleviated the induction of cytotoxicity and ER stress by hyperthermia (42-43°C) and protected cells against ER stress-induced apoptosis. ShRNA-mediated depletion of Hsp72 abrogated protective effects of mild thermotolerance (40°C) against heat-shock induced ER stress and sensitized cells to ER stress-mediated apoptosis. Our findings show that Hsp72 contributes to the protective effects of mild hyperthermia (40°C) against hyperthermia-induced ER stress and apoptosis.

  2. 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...

  3. Inhibition of nucleoside diphosphate kinase activity by in vitro phosphorylation by protein kinase CK2. Differential phosphorylation of NDP kinases in HeLa cells in culture

    Biondi, R M; Engel, M; Sauane, M

    1996-01-01

    that in vitro protein kinase CK2 catalyzed phosphorylation of human NDPK A inhibits its enzymatic activity by inhibiting the first step of its ping-pong mechanism of catalysis: its autophosphorylation. Upon in vivo 32P labeling of HeLa cells, we observed that both human NDPKs, A and B, were autophosphorylated...

  4. Effect of Ureaplasma parvum co-incubation on Chlamydia trachomatis maturation in human epithelial HeLa cells treated with interferon-γ.

    Yamazaki, Tomohiro; Matsuo, Junji; Nakamura, Shinji; Oguri, Satoshi; Yamaguchi, Hiroyuki

    2014-08-01

    Chlamydia trachomatis is an obligate intracellular bacterium that causes a sexually transmitted disease. Ureaplasma parvum is commensal in the human genital tract, with a minimal contribution to urogenital infection. We have recently found that U. parvum has a significant effect on the presence of C. trachomatis in the genital tract of healthy women. We therefore assessed the effect of U. parvum co-incubation on C. trachomatis maturation from reticulate bodies (RBs) to elementary bodies (EBs) in HeLa cells in the absence or presence of interferon (IFN)-γ, which is a critical host defense factor. IFN-γ stimulation of viable U. parvum significantly prompted chlamydial growth with an increase in infectious particles, EBs, in HeLa cells. IFN-γ treatment of killed U. parvum had a similar effect on C. trachomatis maturation in HeLa cells. There was no change in expression of indoleamine 2,3-dioxygenase (IDO) in cultures of viable or killed U. parvum. We concluded that U. parvum co-incubation by IFN-γ helped C. trachomatis to mature from RBs to EBs in HeLa cells, independent of IDO expression. This suggests a novel survival strategy of C. trachomatis against IFN-γ exposure, prompting secondary infection of the genital mucosa, with possible clinical implications.

  5. 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...

  6. A systems biology analysis of the changes in gene expression via silencing of HPV-18 E1 expression in HeLa cells.

    Castillo, Andres; Wang, Lu; Koriyama, Chihaya; Eizuru, Yoshito; Jordan, King; Akiba, Suminori

    2014-10-01

    Previous studies have reported the detection of a truncated E1 mRNA generated from HPV-18 in HeLa cells. Although it is unclear whether a truncated E1 protein could function as a replicative helicase for viral replication, it would still retain binding sites for potential interactions with different host cell proteins. Furthermore, in this study, we found evidence in support of expression of full-length HPV-18 E1 mRNA in HeLa cells. To determine whether interactions between E1 and cellular proteins play an important role in cellular processes other than viral replication, genome-wide expression profiles of HPV-18 positive HeLa cells were compared before and after the siRNA knockdown of E1 expression. Differential expression and gene set enrichment analysis uncovered four functionally related sets of genes implicated in host defence mechanisms against viral infection. These included the toll-like receptor, interferon and apoptosis pathways, along with the antiviral interferon-stimulated gene set. In addition, we found that the transcriptional coactivator E1A-binding protein p300 (EP300) was downregulated, which is interesting given that EP300 is thought to be required for the transcription of HPV-18 genes in HeLa cells. The observed changes in gene expression produced via the silencing of HPV-18 E1 expression in HeLa cells indicate that in addition to its well-known role in viral replication, the E1 protein may also play an important role in mitigating the host's ability to defend against viral infection.

  7. Novel localisation and possible function of LIN7 and IRSp53 in mitochondria of HeLa cells.

    Ferrari, Ilaria; Crespi, Arianna; Fornasari, Diego; Pietrini, Grazia

    2016-08-01

    By means of immunofluorescence and subcellular fractionation experiments, we here demonstrate mitochondrial distribution of LIN7 and IRSp53 in HeLa cells. These peripheral proteins displayed a tight association with mitochondria and coimmunoprecipitated from mitochondrial fractions. In line with a role for LIN7 in the regulation of IRSp53 activity on actin dynamics, the morphology of mitochondria was similarly altered by changing the expression levels of either each protein or both, whereas mitochondrial morphology was preserved in cells overexpressing IRSp53 deleted of its binding domains for LIN7 (IRSp53Δ5) or for actin polymerisation modulators (IRSp53ΔSH3). In particular, the overexpression of full length LIN7 and/or IRSp53 increased the percentage of cells with short mitochondria, while downregulation of the endogenous proteins by shRNAs increased the amount of cells with elongated and perinuclear clustered mitochondria. These mitochondria were only partially resistant to fragmentation induced by dissipation of the mitochondrial membrane potential (i.e. treatment with sodium azide), whereas mitochondria were fully protected by the fission defective mutant Drp1 K38A. Overexpression of LIN7 or IRSp53 did not prevent the formation of hyperfused mitochondria in cells coexpressing the Drp1 K38A mutant, thus suggesting that LIN7-IRSp53 complex requires functional Drp1 to regulate mitochondrial morphology.

  8. [EFFECT OF FUCOIDANS ISOLATED FROM SEAWEEDS LAMINARIA DIGITATA AND FUCUS VESICULOSUS ON CELL LINES HELA G-63, ECV 304 AND PC 12].

    Zhurishkina, E V; Lapina, I M; Ivanen, D R; Stepanov, S I; Shvetsova, S V; Shavarda, A L; Giliano, N Ya; Kulminskaya, A A

    2015-01-01

    The aim of the research was to investigate cytotoxicity of fucoidans on mammals cells. Three different samples of fucoidans were isolated from mechanically grounded brown algae Laminaria digitata and Fucus ve- siculosus. The sample F2 that differed from the others by higher sulfatation level and suppression of HeLa G-63 line culture growth was taken for further study in cell lines HeLa G-63, ECV 304 and PC 12. We have shown that fucoidan preparation F2 inhibits proliferation and induces cell death in a dose- and time-dependent manner for all investigated cell lines. Neuroendocrine tumor rat cell line PC 12 appeared to be the most sensitive to fucoidan treatment whereas endothelial human cells ECV 304 were the least sensitive.

  9. Queuosine biosynthesis is required for sinorhizobium meliloti-induced cytoskeletal modifications on HeLa Cells and symbiosis with Medicago truncatula

    Marta Marchetti; Delphine Capela; Renaud Poincloux; Nacer Benmeradi; Marie-Christine Auriac; Aurélie Le Ru; Isabelle Maridonneau-Parini; Jacques Batut; Catherine Masson-Boivin

    2013-01-01

    Rhizobia are symbiotic soil bacteria able to intracellularly colonize legume nodule cells and form nitrogen-fixing symbiosomes therein. How the plant cell cytoskeleton reorganizes in response to rhizobium colonization has remained poorly understood especially because of the lack of an in vitro infection assay. Here, we report on the use of the heterologous HeLa cell model to experimentally tackle this question. We observed that the model rhizobium Sinorhizobium meliloti, and other rhizobia as...

  10. Cellular Cultivation: Growing HeLa Cells Using Standard High School Laboratory Equipment.

    Woloschak, Gayle; And Others

    1995-01-01

    Describes experiments to culture cells in a laboratory that provide students with hands-on experience in manipulating cells and a chance to observe cell growth characteristics first hand. Exposes students to sterile technique, cell culture, cell growth concepts, and eukaryotic cell structure. (JRH)

  11. HeLa和SiHa细胞中Skp2、p27和C-myc的表达%Expressions of Skp2、p27 and C-myc in HeLa and SiHa Cells

    薛翔; 公丕军; 范引侠

    2012-01-01

    Objective: To test the expressions of S-phase kinase-associated protein 2 (Skp2), C-myc and p27 in HeLa and SiHa cells from the levels of RNA and protein and to investgate the meaning of those indexes in cervical cancerous cells. Methods:We tested the expressions of Skp2, p27 and C-myc in HeLa and SiHa cell lines by immunohistochemistry, western blotting as well as semiquantitative PCR . Results: Immunohistochemistry results showed that the expressions of Skp2 and C-myc were obviously stronger in HeLa cells than those in SiHa cells (P =0.032 and P = 0.026). While the expression of p27 was weaker in HeLa cells than that in SiHa cells (P=0.035). Western blot results showed that the expressions of endogenous Skp2 and C-myc protein in HeLa cells were as 2.8 and 1.5 times of SiHa cells respectively. However, the expression of p27 protein in SiHa cells was as 2.7 times of those in HeLa cells. RT-PCR results showed that the expression levels of endogenous Skp2 and C-myc mRNA in HeLa cells were higher than those in Si-Hha cells(P = 0.034 and P = 0.028, respectively), meanwhile, the expression of p27mRNA in SiHa cells was higher than that of HeLa cells (P=0.002). Conclusions: The expressions of Skp2 and C-myc in HeLa cells are obviously higher than those in SiHa cells. The expression of p27 in HeLa cells is lower than that in SiHa cells.%目的:检测S期激酶相关蛋白2( Skp2)、C-myc、p27在宫颈癌HeLa及SiHa细胞系中的表达,明确这些指标在宫颈癌细胞系表达的意义.方法:通过细胞免疫组化、Western blot、RT-PCR技术分析Skp2、p27和C-myc在蛋白和mRNA水平表达的差异.结果:免疫组化显示:HeLa细胞中Skp2和C-myc表达强度高于SiHa细胞(P=0.032和P=0.026),而HeLa细胞中p27蛋白表达弱于SiHa细胞(P=0.035).Western blot显示:在HeLa细胞中Skp2和C-myc蛋白表达分别是SiHa细胞表达量的2.8倍和1.5倍;而SiHa细胞中的p27蛋白的表达水平是He-La细胞中表达的2.7倍.RT-PCR结果

  12. 5-氮杂-2'-脱氧胞苷对Hela细胞增殖及CDH1、HPV18E6mRNA表达的影响%Effect of 5-Aza-CdR on cell growth and mRNA expressions of CDH1 and HPV18E6 in Hela cells

    林颖; 陶光实

    2012-01-01

    目的:探讨DNA去甲基化试剂5-氮杂-2'-脱氧胞苷(5-Aza-CdR)对宫颈癌Hela细胞株增殖及抑癌基因CDH1和HPV18E6 mRNA的表达水平的影响.方法:不同浓度5-Aza-CdR干预Hela细胞株24h以后,四唑氮蓝法(MTT)检测其对Hela细胞增殖的影响,半定量RT-PCR检测CDH1及HPV18E6 mRNA表达的改变.结果:与对照组(DMSO)相比,5-Aza-CdR能显著抑制Hela细胞增值,且呈量-效性.5-Aza-CdR能显著抑制Hela细胞CDH1 mRNA的表达水平(P<0.05),而对HPV18E6 mRNA表达水平无明显影响(P>0.05).结论:5-Aza-CdR能抑制Hela细胞增殖,其作用可能是通过上调宫颈癌抑癌基因CDH1的表达实现,有望为宫颈癌的治疗提供新的治疗思路.%Objective To investigate the effect of 5-Aza-2'-deoxycytidine (5-Aza-CdR) on cell growth and mRNA expressions of HPV E6 and CDH1 in cervical cancer Hela cells. Methods The cell proliferative vitality of Hela cells was estimated by methabenzthiazuron (MTT) assay after Hela cells were cultured and treated by 5-Aza-CdR with 5,10,20|xmol/L concentrations for 24 hours respectively. The mRNA expression of CDH1 and HPV18E6 in Hela cells after treated by 5-Aza-CdR with different dosage were examined by Semi- quantitative PT-PCR. Results As compared with that in the control group, 5-Aza-CdR could significantly inhibit the growth, CDH1 mRNA level of Hela cells in dosage-dependent manner (P0.05). Conclusion 5-Aza-CdR could effectively inhibit the proliferation of Hela cells in dosage-dependent by up-regulating the CDHlmRNA expression, which would be a novel strategy to improve effects of cervical cancer.

  13. 索拉非尼对Hela细胞增殖及VEGFR-3和PDGFR-β表达的影响%Effect of sorafenib on proliferation of Hela cells and the expression of VEGFR-3 and PDGFR-βin Hela cells

    梁玲霞; 田晓予; 王瑞芳; 王盈; 王建刚; 席守民

    2014-01-01

    目的:研究索拉非尼能否抑制宫颈癌Hela细胞增殖、抑制Hela细胞中VEGFR-3和PDGFR-β的表达。方法:四甲基偶氮唑蓝(MTT)快速比色法检测索拉非尼作用于Hela细胞24h、48h、72h后细胞增殖率, Hoechst荧光染色观察药物作用(1mg/L)48h后的凋亡细胞,细胞免疫荧光法和Western blot检测不同浓度的索拉非尼(1mg/L、0.1mg/L、0.01mg/L)作用Hela细胞VEGFR-3和PDGFR-β表达情况。结果:不同浓度的索拉非尼作用Hela细胞后,Hela 细胞的增殖率下降,呈浓度依赖性(P<0.05),随着药物浓度的增加Hela细胞的增殖率不断下降。药物作用后能观察到凋亡的Hela细胞,细胞核变小、固缩,荧光明显增强。正常Hela细胞中有VEGFR-3和PDGFR-β表达,索拉非尼作用后,Hela细胞绿色荧光明显减弱,VEGFR-3和PDGFR-β表达明显降低,呈浓度依赖性。结论:索拉非尼能够诱导Hela细胞的凋亡,抑制其增殖且呈浓度依赖性。索拉非尼能够抑制Hela细胞VEGFR-3和PDGFR-β表达,可能降低Hela细胞侵袭转移的能力。%Objective:To investigate whether sorafenib can inhabit the proliferation of Hela cells and inhibit the expression of VEGFR-3 and PDGFR-βin Hela cells.Methods:MTT assay was used to analyze cells proliferation rate after treatment with different concentration of sorafenib for 24h,48h,72h.Hoechst 33258 was used to stain nucle-us of Hela cells treatment with sorafenib (1.0mg/L),48h later,observed nucleus morphological changes.Immunoflu-orescence and Western blot was used to observe the expression of VEGFR-3 and PDGFR-βin Hela cells with the treatment of different concentration of sorafenib (1mg/L,0.1mg/L,0.01mg/L).Results:After treatment with differ-ent concentration of sorafenib ,the proliferation rate of Hela cells was down ,and it has dose-dependent effect (P<0.05).Sorafenib can make Hela cells apoptosis.The apoptotic cells,nuclear small

  14. TRPV4对HeLa细胞增殖的作用%The effect of TRPV4 regulation on HeLa cells proliferation in vitro

    李孝琼; 陈莉; 王昌梅

    2013-01-01

    Objective To observe the effect of transient receptor potential vanilloid 4 regulation on HeLa cells proliferation in vitro.Methods Expressed TRPV4 by HeLa and W12 cells were measured by western blot.Then HeLa cells were randomly divided into two groups:control group and RN1734 group.At different time points (0 h,12 h,24 h,48 h and 72 h),the changes of HeLa proliferation and cell cycle progression were detected by cell number counted assay,LDH release assay and flow cytometry.Results Compared with W12 cells,the expression of TRPV4 was increased in HeLa cells.The proliferation of HeLa cells was blocked by RN1734 (P < 0.05).At the mean time,the percentage of HeLa cells in S phase in RN1734 groups were significantly less (P <0.05) than control groups while that in G0/G1 phase were increased at these time points.Conclusions Transient receptor potential vanilloid 4 blocker RN1734 can remarkably inhibit HeLa cells prcliferationin vitro.Our results suggested that the transient receptor potential vanilloid 4 were involved in HeLa cells proliferation.%目的 观察瞬时感受器电位离子通道香草素受体4(TRPV4)与宫颈癌HeLa细胞增殖的关系.方法 采用宫颈癌HeLa细胞系,用Western blot技术检测HeLa细胞中TRPV4的表达量;并分别用TRPV4阻滞剂RN1734干预体外培养HeLa细胞,通过细胞计数法、LDH检测及流式细胞技术观察TRPV4通道调控对HeLa增殖和细胞周期进展的影响.结果 与对照组W12细胞系相比,宫颈癌HeLa细胞系中TRPV4呈高表达.TRPV4阻滞剂干预48和72 h后,HeLa细胞增殖较未干预组降低(P<0.05);而TRPV4阻滞剂干预24h和48 h时,处于S期细胞的百分率明显低于未干预组;处于G0/G1期细胞的百分率则明显高于未干预组(P<0.05).结论 TRPV4抑制可以阻滞体外培养宫颈癌HeLa细胞增殖及细胞周期进展.

  15. A Stable HeLa Cell Line That Inducibly Expresses Poliovirus 2Apro: Effects on Cellular and Viral Gene Expression

    Barco, Angel; Feduchi, Elena; Carrasco, Luis

    2000-01-01

    A HeLa cell clone (2A7d) that inducibly expresses the gene for poliovirus protease 2A (2Apro) under the control of tetracycline has been obtained. Synthesis of 2Apro induces severe morphological changes in 2A7d cells. One day after tetracycline removal, cells round up and a few hours later die. Poliovirus 2Apro cleaves both forms of initiation factor eIF4G, causing extensive inhibition of capped-mRNA translation a few hours after protease induction. Methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone, a selective inhibitor of 2Apro, prevents both eIF4G cleavage and inhibition of translation but not cellular death. Expression of 2Apro still allows both the replication of poliovirus and the translation of mRNAs containing a picornavirus leader sequence, while vaccinia virus replication is drastically inhibited. Translation of transfected capped mRNA is blocked in 2A7d-On cells, while luciferase synthesis from a mRNA bearing a picornavirus internal ribosome entry site (IRES) sequence is enhanced by the presence of 2Apro. Moreover, synthesis of 2Apro in 2A7d cells complements the translational defect of a poliovirus 2Apro-defective variant. These results show that poliovirus 2Apro expression mimics some phenotypical characteristics of poliovirus-infected cells, such as cell rounding, inhibition of protein synthesis and enhancement of IRES-driven translation. This cell line constitutes a useful tool to further analyze 2Apro functions, to complement poliovirus 2Apro mutants, and to test antiviral compounds. PMID:10666269

  16. Photodamage induced by Zinc(II)-phthalocyanine to microtubules, actin, alpha-actinin and keratin of HeLa cells.

    Juarranz, A; Espada, J; Stockert, J C; Villanueva, A; Polo, S; Domínguez, V; Cañete, M

    2001-03-01

    We have studied the photosensitizing effects of zinc(II)-phthalocyanine (ZnPc) on the cytoskeleton of HeLa cells using sublethal (10(-7) M, followed by 1 or 3 min of red light to induce 20%, LD20, or 60%, LD60, cell death, respectively) or lethal (5 x 10(-6) M and 15 min of irradiation, LD100) experimental conditions. The immunofluorescent analysis of the cytoskeleton showed a variable photodamage to microtubules (MT), actin microfilaments (AF) and intermediate filaments of keratin (KF), as well as on alpha-actinin, which was dependent on treatment conditions. Both sublethal treatments induced deep alterations on interphase and mitotic MT. The mitotic index increased with time with the maximum at 18 h (12%) or 24 h (14%) after LD20 or LD60, respectively. The alterations on AF and alpha-actinin were much more severe than those observed on KF at any evaluated time. With the exception of the KF, which remained partially organized, the MT and AF network was severely damaged by the lethal treatment. Western blot analysis for alpha-tubulin, G-actin and alpha-actinin from soluble and insoluble fractions confirmed the results observed by immunofluorescence, thus indicating that these cytoskeletal components are involved in cell damage and death by ZnPc photosensitization.

  17. 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.

  18. Oleifolioside A mediates caspase-independent human cervical carcinoma HeLa cell apoptosis involving nuclear relocation of mitochondrial apoptogenic factors AIF and EndoG.

    Yu, Hai Yang; Jin, Cheng-Yun; Kim, Kyoung-Sook; Lee, Young-Choon; Park, Shin-Hyung; Kim, Gi-Young; Kim, Wun-Jae; Moon, Hyung-In; Choi, Yung Hyun; Lee, Jai-Heon

    2012-05-30

    Apoptosis, the main type of programmed cell death, plays an essential role in a variety of biological events. Whereas "classical" apoptosis is dependent on caspase activation, caspase-independent death is increasingly recognized as an alternative pathway. To develop new anticancer agents, oleifolioside A was isolated from Dendropanax morbifera Leveille and the biochemical mechanisms of oleifolioside A-induced apoptosis in HeLa cells were investigated. Exposure to oleifolioside A resulted in caspase activation and typical features of apoptosis, although cell death was not prevented by caspase inhibition. Oleifolioside A treatment induced up-regulation of Bad, loss of mitochondrial membrane potential, nuclear relocation of mitochondrial factors, apoptosis-inducing factor (AIF), endonuclease G (EndoG), and apoptosis induction. This is the first report of anticancer activity of oleifolioside A, and nuclear translocation of AIF and EndoG in oleifolioside A-treated HeLa cells might represent an alternative death signaling pathway in the absence of caspase activity.

  19. The proliferation inhibition of Hela cells by histone deacetylases-1 siRNA%组蛋白去乙酰化酶1siRNA转染对Hela细胞的抑制

    Na Li; Shiying Yu

    2008-01-01

    Objective:To investigate the anti-proliferative effect of histone deacetylases-1(HDAC-1)knockdown in Hela cells.Methods:The HDAC-1 protein was knockdowned using siRNA.The expression of HDAC-1 was detected by Western blotting.Apoptosis was assessed by flow cytometry.The inhibition of cell growth was assesses by MTT assay.Result:HDAC-1 siRNA knockdowned the expression of HDAC-1 protein.HDAC siRNA inhibited lhe proliferation of Hela cells.HDAC-1 siRNA induced apoptosis.Conclusion:HDAC-1 siRNA may inhibit the growth of Hela cells by inducing apoptosis.

  20. TLR7激活对Hela细胞增殖的影响%The regulation of the proliferation on activation of TLR7 in Hela cells

    李磊; 程丰伟; 王芳; 金锐; 罗欣; 张胜权

    2014-01-01

    目的:探讨Toll样受体7(TLR7)在Hela细胞中的表达以及激活TLR7后对Hela细胞增殖和MAPKs-ERK1/2及PI3K-AKT两条信号通路的 ERK 和 AKT 磷酸化水平的影响。方法采用Real-time PCR分析TLR7在Hela细胞中的表达;使用不同浓度的TLR7激动剂 Gardiquimod经过不同的时间刺激Hela细胞,采用MTS比色法分析其对Hela细胞增殖的影响; Western blot 分析 Gardiquimod 对 Hela 细胞ERK1/2及 AKT 蛋白磷酸化水平的影响。结果 TLR7在Hela细胞中呈组成性弱表达;TLR7激动剂Gardiquimod可促进Hela细胞的增殖,且呈时间及剂量依赖性; Gardiquimod激活TLR7后可以显著增加Hela细胞中ERK1/2和AKT的蛋白磷酸化水平。结论 TLR7激动剂Gardiquimod能够活化MAPK-ERK1/2和PI3K-AKT信号通路的ERK1/2和AKT蛋白磷酸化水平,并促进Hela细胞的体外增殖。%Objective To explore the expression of Toll like receptor 7 ( TLR7 ) on Hela cells, and the effect of TLR7 agonist-Gardiquimod on the proliferation of activation of TLR7 in Hela cells and related probable mechanism. Methods Firstly, using Real-time PCR to analyze the expression of TLR7 on Hela cells. Then, the cells were treated with different concentration of Gardiquimod for different times. MTS were performed to detect the impact of Gardiquimod on the proliferation of Hela cells. Using Western blot to analyze the variation of phosphorylation of ERK1/2 and AKT when Hela cells were treated with Gardiquimod. Results The constitutive expression of TLR7 on Hela cells was weak. The activation of TLR7 by its agonist of Gardiquimod could promote the proliferation of He-la cells apparently, and existing dose and time dependence. The protein level of phosphorylation of ERK1/2 and AKT enhanced after provoking the TLR7 in Hela cells. Conclusion The TLR7 ligand, Gardiquimod can promote proliferation through excitation protein level of phosphorylation of ERK1/2 and AKT of the signal pathway, MAPK-ERK1/2 and PI3K-AKT in

  1. 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.

  2. siRNA转染浓度和时间对HeLa细胞ANXA5表达的抑制作用%INHIBITING EFFECTS OF SIRNA TRANSFECTION CONCENTRATION AND TIME ON ANXA5 EXPRESSION IN HELA CELLS

    王小杰; 王永为; 曲银娥; 李欣

    2014-01-01

    Objective:To optimize the conditions of siRNA targeting ANXA5 transfected into HeLa cells, so to provide bases for further studying effects of ANXA5 low expression on biological functions of cervical cancer cells. Methods:siRNAtargetingANXA5 was transfected into HeLa cells, with different ratios ofANXA5 siRNAand liposomes, and different transfection time. Western Blot was used to detect the ANXA5 protein expression in HeLa cells. Results:The ANXA5 protein expression in HeLa cells was the lowest (the inhibitory effects was the strongest) when the ratio of siRNA and liposomes was 0.4:1 and transfection time was 72h. Conclusions:The ratio of siRNA and liposomes and transfection timecaninfluencetheexpressionofANXA5inHeLa cells, and has the best ratio and transfection time.%目的:优化靶向ANXA5的siRNA转染HeLa细胞的条件,为进一步研究ANXA5低表达对宫颈癌细胞生物功能的影响奠定基础。方法:将靶向ANXA5的siRNA转染HeLa细胞,设置不同的siRNA与脂质体的比值和转染时间,Western Blot检测ANXA5蛋白的表达。结果:siRNA与脂质体的比值为0.4:1时,转染入HeLa细胞72h后,HeLa细胞ANXA5蛋白的表达水平最低,即抑制作用最强。结论:siRNA与脂质体的比值和转染时间可影响HeLa细胞ANXA5的表达,存在最佳比值和转染时间。

  3. Depletion of SMN by RNA interference in HeLa cells induces defects in Cajal body formation.

    Girard, Cyrille; Neel, Henry; Bertrand, Edouard; Bordonné, Rémy

    2006-01-01

    Neuronal degeneration in spinal muscular atrophy (SMA) is caused by reduced expression of the survival of motor neuron (SMN) protein. The SMN protein is ubiquitously expressed and is present both in the cytoplasm and in the nucleus where it localizes in Cajal bodies. The SMN complex plays an essential role for the biogenesis of spliceosomal U-snRNPs. In this article, we have used an RNA interference approach in order to analyse the effects of SMN depletion on snRNP assembly in HeLa cells. Although snRNP profiles are not perturbed in SMN-depleted cells, we found that SMN depletion gives rise to cytoplasmic accumulation of a GFP-SmB reporter protein. We also demonstrate that the SMN protein depletion induces defects in Cajal body formation with coilin being localized in multiple nuclear foci and in nucleolus instead of canonical Cajal bodies. Interestingly, the coilin containing foci do not contain snRNPs but appear to co-localize with U85 scaRNA. Because Cajal bodies represent the location in which snRNPs undergo 2'-O-methylation and pseudouridylation, our results raise the possibility that SMN depletion might give rise to a defect in the snRNA modification process.

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

    Wang, Hongtao; Gao, Peng; Zheng, Jie

    2014-09-05

    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.

  5. 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.

  6. 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 ...

  7. 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.

  8. Cardiac glycoside-induced cell death and Rho/Rho kinase pathway: Implication of different regulation in cancer cell lines.

    Özdemir, Aysun; Şimay, Yaprak Dilber; İbişoğlu, Burçin; Yaren, Biljana; Bülbül, Döne; Ark, Mustafa

    2016-05-01

    Previously, we demonstrated that the Rho/ROCK pathway is involved in ouabain-induced apoptosis in HUVEC. In the current work, we investigated whether the Rho/ROCK pathway is functional during cardiac glycosides-induced cytotoxic effects in cancer cell lines, as well as in non-tumor cells. For that purpose, we evaluated the role of ROCK activation in bleb formation and cell migration over upstream and downstream effectors in addition to ROCK cleavage after cardiac glycosides treatment. All three cardiac glycosides (ouabain, digoxin and bufalin) induced cell death in HeLa and HepG2 cells and increased the formation of blebbing in HeLa cells. In contrast to our previous study, ROCK inhibitor Y27632 did not prevent bleb formation. Observation of ROCK II cleavage after ouabain, digoxin and oxaliplatin treatments in HeLa and/or HepG2 cells suggested that cleavage is independent of cell type and cell death induction. While inhibiting cleavage of ROCK II by the caspase inhibitors z-VAD-fmk, z-VDVAD-fmk and z-DEVD-fmk, evaluation of caspase 2 siRNA ineffectiveness on this truncation indicated that caspase-dependent ROCK II cleavage is differentially regulated in cancer cell lines. In HeLa cells, ouabain induced the activation of ROCK, although it did not induce phosphorylation of ERM, an upstream effector. While Y27632 inhibited the migration of HeLa cells, 10nM ouabain had no effect on cell migration. In conclusion, these findings indicate that the Rho/ROCK pathway is regulated differently in cancer cell lines compared to normal cells during cardiac glycosides-induced cell death.

  9. Synthetic peptides from two Pf sporozoite invasion-associated proteins specifically interact with HeLa and HepG2 cells.

    Arévalo-Pinzón, Gabriela; Curtidor, Hernando; Muñoz, Marina; Patarroyo, Manuel A; Patarroyo, Manuel E

    2011-09-01

    Two recently described molecules have been associated with sporozoite traversal ability and hepatocyte entry: sporozoite invasion-associated proteins (SIAP)-1 and -2. The HeLa and HepG2 cell binding ability of synthetic peptides spanning the whole SIAP-1 and -2 sequences has been studied in the search for identifying these proteins' functionally active specific regions. Twelve HepG-2 and seventeen HeLa cell high-activity binding peptides (HABPs) have been identified in SIAP-1, 8 of them having high specific binding affinity for both cell lines. Four HepG2 HABPs and two HeLa HABPs have been identified in SIAP-2, one of them interacting with both HeLa and HepG2 cells. SIAP-1 and SIAP-2 HABPs bound specifically and saturably to heparin sulfate and chondroitin sulfate-type membrane receptors on host cells. Circular dichroism assays have shown high α-helix content in SIAP-1 and SIAP-2 HABP secondary structure. Immunofluorescence analysis has revealed that specific peptides against SIAP proteins are highly immunogenic in mice and that anti-SIAP-1 and -2 antibodies recognize the native protein in Plasmodium falciparum sporozoites. Polymorphism studies have shown that a most SIAP-1 and -2 HABPs are conserved among P. falciparum strains. Our results have suggested that SIAP-1 and -2 participate in host-pathogen interactions during cell-traversal and hepatocyte invasion and highlighted the relevance of the ongoing identification and study of potentially new molecules when designing a fully protective antimalarial vaccine.

  10. A nucleic-acid hydrolyzing single chain antibody confers resistance to DNA virus infection in hela cells and C57BL/6 mice.

    Lee, Gunsup; Yu, Jaelim; Cho, Seungchan; Byun, Sung-June; Kim, Dae Hyun; Lee, Taek-Kyun; Kwon, Myung-Hee; Lee, Sukchan

    2014-06-01

    Viral protein neutralizing antibodies have been developed but they are limited only to the targeted virus and are often susceptible to antigenic drift. Here, we present an alternative strategy for creating virus-resistant cells and animals by ectopic expression of a nucleic acid hydrolyzing catalytic 3D8 single chain variable fragment (scFv), which has both DNase and RNase activities. HeLa cells (SCH07072) [corrected] expressing 3D8 scFv acquired significant resistance to DNA viruses. Virus challenging with Herpes simplex virus (HSV) in 3D8 scFv transgenic cells and fluorescence resonance energy transfer (FRET) assay based on direct DNA cleavage analysis revealed that the induced resistance in HeLa cells was acquired by the nucleic acid hydrolyzing catalytic activity of 3D8 scFv. In addition, pseudorabies virus (PRV) infection in WT C57BL/6 mice was lethal, whereas transgenic mice (STG90) that expressed high levels of 3D8 scFv mRNA in liver, muscle, and brain showed a 56% survival rate 5 days after PRV intramuscular infection. The antiviral effects against DNA viruses conferred by 3D8 scFv expression in HeLa cells as well as an in vivo mouse system can be attributed to the nuclease activity that inhibits viral genome DNA replication in the nucleus and/or viral mRNA translation in the cytoplasm. Our results demonstrate that the nucleic-acid hydrolyzing activity of 3D8 scFv confers viral resistance to DNA viruses in vitro in HeLa cells and in an in vivo mouse system.

  11. Comparison of the killing effects between nitrogen-doped and pure TiO2 on HeLa cells with visible light irradiation

    Li, Zheng; Pan, Xiaobo; Wang, Tianlong; Wang, Pei-Nan; Chen, Ji-Yao; Mi, Lan

    2013-02-01

    The killing effect of nitrogen-doped titanium dioxide (N-TiO2) nanoparticles on human cervical carcinoma (HeLa) cells by visible light photodynamic therapy (PDT) was higher than that of TiO2 nanoparticles. To study the mechanism of the killing effect, the reactive oxygen species produced by the visible-light-activated N-TiO2 and pure-TiO2 were evaluated and compared. The changes of the cellular parameters, such as the mitochondrial membrane potential (MMP), intracellular Ca2+, and nitrogen monoxide (NO) concentrations after PDT were measured and compared for N-TiO2- and TiO2-treated HeLa cells. The N-TiO2 resulted in more loss of MMP and higher increase of Ca2+ and NO in HeLa cells than pure TiO2. The cell morphology changes with time were also examined by a confocal microscope. The cells incubated with N-TiO2 exhibited serious distortion and membrane breakage at 60 min after the PDT.

  12. Functional analysis of a recombinant PIII-SVMP, GST-acocostatin; an apoptotic inducer of HUVEC and HeLa, but not SK-Mel-28 cells.

    Teklemariam, Takele; Seoane, Agustin I; Ramos, Carla J; Sanchez, Elda E; Lucena, Sara E; Perez, John C; Mandal, Stephanie A; Soto, Julio G

    2011-04-01

    Disintegrins and disintegrin-like peptides interact with integrins and interfere with cell-cell and cell-matrix interactions. A disintegrin-like snake venom gene, Acocostatin was cloned from the venom gland mRNA of Agkistrodon contortrix contortrix. Acocostatin belongs to the PIII-SVMP subfamily of disintegrin-like peptides. The recombinant acocostatin peptide was produced and purified as GST-fusion. The GST-acocostatin peptide, at 44 μg/mL, inhibited platelet aggregation by 30% in PRP and 18% in whole blood. In addition GST-acocostatin, at 220 μg/mL, inhibited SK-Mel-28 cell migration by 48%, but did not inhibit T24 cell migration. The GST-acocostatin peptide ability to induce apoptosis on HUVEC, HeLa, and SK-Mel-28 cells was determined using Annexin V-FITC and chromatin fragmentation assays after 24 h of treatment. At 5 μM GST-acocostatin peptide, 19.68%+/- 3.09 of treated HUVEC, and 35.86% +/- 2.05 of treated HeLa cells were in early apoptosis. The GST-acocostatin peptide also caused chromatin fragmentation of HUVEC and HeLa cells as determined by fluorescent microscopy and Hoechst staining. The GST-acocostatin peptide failed to induce apoptosis of SK-Mel-28 cells. We characterized the HUVEC, HeLa, and T24 integrin expression by flow cytometry, as the first step in determining GST-acocostatin binding specificity. Our results indicate that HUVEC express αv, αvβ3, αvβ5, α6, β1, and β3 integrin receptors. HeLa cells express α1, α2, α6, αv, αvβ5, and β1 integrin receptors. T24 cells express α1, α3, α6, αv, αvβ3, αvβ5, β1, β3, and β6 integrin receptors.

  13. Cell-cyc