A dual inhibitor against prolyl isomerase Pin1 and cyclophilin discovered by a novel real-time fluorescence detection method

International Nuclear Information System (INIS)

Mori, Tadashi; Hidaka, Masafumi; Lin, Yi-Chin; Yoshizawa, Ibuki; Okabe, Takayoshi; Egashira, Shinichiro; Kojima, Hirotatsu; Nagano, Tetsuo; Koketsu, Mamoru; Takamiya, Mari; Uchida, Takafumi

2011-01-01

Research highlights: → A Pin1 (prolyl isomerase) inhibitor, TME-001, has been discovered by using a new established high-throughput screening method. → The TME-001 showed a cell-active inhibition with lower cytotoxic effect than known Pin1 inhibitors. → Kinetic analyses revealed that the TME-001 is the first compound that exhibits dual inhibition of Pin1 and another type of prolyl isomerase, cyclophilin. → Thus, similarities of structure and reaction mechanism between Pin1 and cyclophilin are proposed. -- Abstract: Pin1, a peptidyl prolyl cis/trans isomerase (PPIase), is a potential target molecule for cancer, infectious disease, and Alzheimer's disease. We established a high-throughput screening method for Pin1 inhibitors, which employs a real-time fluorescence detector. This screening method identified 66 compounds that inhibit Pin1 out of 9756 compounds from structurally diverse chemical libraries. Further evaluations of surface plasmon resonance methods and a cell proliferation assay were performed. We discovered a cell-active inhibitor, TME-001 (2-(3-chloro-4-fluoro-phenyl)-isothiazol-3-one). Surprisingly, kinetic analyses revealed that TME-001 is the first compound that exhibits dual inhibition of Pin1 (IC 50 = 6.1 μM) and cyclophilin, another type of PPIase, (IC 50 = 13.7 μM). This compound does not inhibit FKBP. This finding suggests the existence of similarities of structure and reaction mechanism between Pin1 and cyclophilin, and may lead to a more complete understanding of the active sites of PPIases.

Molecular cloning and characterization of Aspergillus nidulans cyclophilin B.

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Joseph, J D; Heitman, J; Means, A R

1999-06-01

Cyclophilins are an evolutionarily conserved family of proteins which serve as the intracellular receptors for the immunosuppressive drug cyclosporin A. Here we report the characterization of the first cyclophilin cloned from the filamentous fungus Aspergillus nidulans (CYPB). Sequence analysis of the cypB gene predicts an encoded protein with highest homology to the murine cyclophilin B protein. The sequence similarity includes an N-terminal sequence predicted to target the protein to the endoplasmic reticulum (ER) as well as a C-terminal sequence predicted to retain the mature protein in the ER. The bacterially expressed hexa-histidine tagged protein displays peptidyl-prolyl isomerase activity which is inhibited by cyclosporin A. In the presence of cyclosporin A, the expressed protein also inhibits purified calcineurin. When the endogenous cypB gene was disrupted and placed under the control of the regulatable alcohol dehydrogenase promoter, the strain demonstrated no detectable growth phenotype under conditions which induce or repress cypB transcription. Induction or repression of the cypB gene also did not effect sensitivity of A. nidulans to cyclosporin A. cypB mRNA levels were significantly elevated under severe heat shock conditions, indicating a possible role for the A. nidulans cyclophilin B protein during growth in high stress environments. Copyright 1999 Academic Press.

From Drosophila to humans: Reflections on the roles of the prolyl-isomerases and chaperones, cyclophilins, in cell function and disease

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Ferreira, Paulo A.; Orry, Andrew

2013-01-01

Despite remarkable advances in human genetics and other genetic model systems, the fruit fly, Drosophila melanogaster, remains a powerful experimental tool to probe with ease the inner workings of a myriad of biological and pathological processes, even when evolutionary forces impart apparent divergences to some of such processes. The understanding of such evolutionary differences provides mechanistic insights into genotype-phenotype correlations underpinning biological processes across metazoans. The pioneering work developed by the William Pak laboratory for the past four decades, and the work of others, epitomize the notion of how the Drosophila system breaks new fertile ground or complements research fields of high scientific and medical relevance. Among the three major genetic complementation groups produced by the Pak's laboratory and impairing distinct facets of photoreceptor neuronal function, the nina group (ninaA….J) selectively affects the biogenesis of G protein-coupled receptors (GPCR) mediating the photoconversion and transduction of light-stimuli. Among the nina genes identified, ninaA arguably assumes heightened significance for several reasons. First, it presents unique physiological selectivity toward the biogenesis of a subset of GPCRs, a standalone biological manifestation yet to be discerned for most mammalian homologues of NinaA. Second, NinaA belongs to a family of proteins, immunophilins, which are the primary targets for immunosuppressive drugs at the therapeutic forefront of a multitude of medical conditions. Third, NinaA closest homologue, cyclophilin-B (CyPB/PPIB), is an immunophilin whose loss-of-function was found recently to cause osteogenesis imperfecta in the human. This report highlights advances made by studies on some members of immunophilins, the cyclophilins. Finally, it re-examines critically data and dogmas derived from past and recent genetic, structural, biological and pathological studies on NinaA and few other

Ultra-violet B (UVB)-induced skin cell death occurs through a cyclophilin D intrinsic signaling pathway

International Nuclear Information System (INIS)

Ji, Chao; Yang, Bo; Yang, Zhi; Tu, Ying; Yang, Yan-li; He, Li; Bi, Zhi-Gang

2012-01-01

Highlights: ► UVB radiated skin keratinocytes show cyclophilin D (Cyp-D) upregulation. ► NAC inhibits UVB induced Cyp-D expression, while H 2 O 2 facilitates it. ► Cyp-D-deficient cells are significantly less susceptible to UVB induced cell death. ► Over-expression of Cyp-D causes spontaneous keratinocytes cell death. -- Abstract: UVB-induced skin cell damage involves the opening of mitochondrial permeability transition pore (mPTP), which leads to both apoptotic and necrotic cell death. Cyclophilin D (Cyp-D) translocation to the inner membrane of mitochondrion acts as a key component to open the mPTP. Our Western-Blot results in primary cultured human skin keratinocytes and in HaCaT cell line demonstrated that UVB radiation and hydrogen peroxide (H 2 O 2 ) induced Cyp-D expression, which was inhibited by anti-oxidant N-acetyl cysteine (NAC). We created a stable Cyp-D deficiency skin keratinocytes by expressing Cyp-D-shRNA through lentiviral infection. Cyp-D-deficient cells were significantly less susceptible than their counterparts to UVB- or H 2 O 2 -induced cell death. Further, cyclosporine A (Cs-A), a Cyp-D inhibitor, inhibited UVB- or H 2 O 2 -induced keratinocytes cell death. Reversely, over-expression of Cyp-D in primary keratinocytes caused spontaneous keratinocytes cell death. These results suggest Cyp-D’s critical role in UVB/oxidative stress-induced skin cell death.

CD147 is a signaling receptor for cyclophilin B.

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Yurchenko, V; O'Connor, M; Dai, W W; Guo, H; Toole, B; Sherry, B; Bukrinsky, M

2001-11-09

Cyclophilins A and B (CyPA and CyPB) are cyclosporin A binding proteins that can be secreted in response to inflammatory stimuli. We recently identified CD147 as a cell-surface receptor for CyPA and demonstrated that CD147 is an essential component in the CyPA-initiated signaling cascade that culminates in ERK activation and chemotaxis. Here we demonstrate that CD147 also serves as a receptor for CyPB. CyPB induced Ca(2+) flux and chemotaxis of CD147-transfected, but not control, CHO cells, and the chemotactic response of primary human neutrophils to CyPB was blocked by antibodies to CD147. These results suggest that CD147 serves as a receptor for extracellular cyclophilins. Copyright 2001 Academic Press.

Structural and Biochemical Characterization of the Human Cyclophilin Family of Peptidyl-Prolyl Isomerases

Energy Technology Data Exchange (ETDEWEB)

Davis, Tara L.; Walker, John R.; Campagna-Slater, Valérie; Finerty, Jr., Patrick J.; Paramanathan, Ragika; Bernstein, Galina; MacKenzie, Farrell; Tempel, Wolfram; Ouyang, Hui; Lee, Wen Hwa; Eisenmesser, Elan Z.; Dhe-Paganon, Sirano (Toronto); (Colorado)

2011-12-14

Peptidyl-prolyl isomerases catalyze the conversion between cis and trans isomers of proline. The cyclophilin family of peptidyl-prolyl isomerases is well known for being the target of the immunosuppressive drug cyclosporin, used to combat organ transplant rejection. There is great interest in both the substrate specificity of these enzymes and the design of isoform-selective ligands for them. However, the dearth of available data for individual family members inhibits attempts to design drug specificity; additionally, in order to define physiological functions for the cyclophilins, definitive isoform characterization is required. In the current study, enzymatic activity was assayed for 15 of the 17 human cyclophilin isomerase domains, and binding to the cyclosporin scaffold was tested. In order to rationalize the observed isoform diversity, the high-resolution crystallographic structures of seven cyclophilin domains were determined. These models, combined with seven previously solved cyclophilin isoforms, provide the basis for a family-wide structure:function analysis. Detailed structural analysis of the human cyclophilin isomerase explains why cyclophilin activity against short peptides is correlated with an ability to ligate cyclosporin and why certain isoforms are not competent for either activity. In addition, we find that regions of the isomerase domain outside the proline-binding surface impart isoform specificity for both in vivo substrates and drug design. We hypothesize that there is a well-defined molecular surface corresponding to the substrate-binding S2 position that is a site of diversity in the cyclophilin family. Computational simulations of substrate binding in this region support our observations. Our data indicate that unique isoform determinants exist that may be exploited for development of selective ligands and suggest that the currently available small-molecule and peptide-based ligands for this class of enzyme are insufficient for isoform

Intracellularly Induced Cyclophilins Play an Important Role in Stress Adaptation and Virulence of Brucella abortus

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García Fernández, Lucía; DelVecchio, Vito G.; Briones, Gabriel

2013-01-01

Brucella is an intracellular bacterial pathogen that causes the worldwide zoonotic disease brucellosis. Brucella virulence relies on its ability to transition to an intracellular lifestyle within host cells. Thus, this pathogen must sense its intracellular localization and then reprogram gene expression for survival within the host cell. A comparative proteomic investigation was performed to identify differentially expressed proteins potentially relevant for Brucella intracellular adaptation. Two proteins identified as cyclophilins (CypA and CypB) were overexpressed in the intracellular environment of the host cell in comparison to laboratory-grown Brucella. To define the potential role of cyclophilins in Brucella virulence, a double-deletion mutant was constructed and its resulting phenotype was characterized. The Brucella abortus ΔcypAB mutant displayed increased sensitivity to environmental stressors, such as oxidative stress, pH, and detergents. In addition, the B. abortus ΔcypAB mutant strain had a reduced growth rate at lower temperature, a phenotype associated with defective expression of cyclophilins in other microorganisms. The B. abortus ΔcypAB mutant also displays reduced virulence in BALB/c mice and defective intracellular survival in HeLa cells. These findings suggest that cyclophilins are important for Brucella virulence and survival in the host cells. PMID:23230297

The Activation of Phytophthora Effector Avr3b by Plant Cyclophilin is Required for the Nudix Hydrolase Activity of Avr3b.

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Kong, Guanghui; Zhao, Yao; Jing, Maofeng; Huang, Jie; Yang, Jin; Xia, Yeqiang; Kong, Liang; Ye, Wenwu; Xiong, Qin; Qiao, Yongli; Dong, Suomeng; Ma, Wenbo; Wang, Yuanchao

2015-08-01

Plant pathogens secrete an arsenal of effector proteins to impair host immunity. Some effectors possess enzymatic activities that can modify their host targets. Previously, we demonstrated that a Phytophthora sojae RXLR effector Avr3b acts as a Nudix hydrolase when expressed in planta; and this enzymatic activity is required for full virulence of P. sojae strain P6497 in soybean (Glycine max). Interestingly, recombinant Avr3b produced by E. coli does not have the hydrolase activity unless it was incubated with plant protein extracts. Here, we report the activation of Avr3b by a prolyl-peptidyl isomerase (PPIase), cyclophilin, in plant cells. Avr3b directly interacts with soybean cyclophilin GmCYP1, which activates the hydrolase activity of Avr3b in a PPIase activity-dependent manner. Avr3b contains a putative Glycine-Proline (GP) motif; which is known to confer cyclophilin-binding in other protein substrates. Substitution of the Proline (P132) in the putative GP motif impaired the interaction of Avr3b with GmCYP1; as a result, the mutant Avr3bP132A can no longer be activated by GmCYP1, and is also unable to promote Phytophthora infection. Avr3b elicits hypersensitive response (HR) in soybean cultivars producing the resistance protein Rps3b, but Avr3bP132A lost its ability to trigger HR. Furthermore, silencing of GmCYP1 rendered reduced cell death triggered by Avr3b, suggesting that GmCYP1-mediated Avr3b maturation is also required for Rps3b recognition. Finally, cyclophilins of Nicotiana benthamiana can also interact with Avr3b and activate its enzymatic activity. Overall, our results demonstrate that cyclophilin is a "helper" that activates the enzymatic activity of Avr3b after it is delivered into plant cells; as such, cyclophilin is required for the avirulence and virulence functions of Avr3b.

The SARS-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors.

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Susanne Pfefferle

2011-10-01

Full Text Available Coronaviruses (CoVs are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS in 2002/2003 has demonstrated human vulnerability to (Coronavirus CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B as interaction partners of the CoV non-structural protein 1 (Nsp1. These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock.

Ultra-violet B (UVB)-induced skin cell death occurs through a cyclophilin D intrinsic signaling pathway

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Ji, Chao [Department of Dermatology, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210024, Jiangsu (China); Yang, Bo [Department of Dermatology, Huashan Hospital, Fudan University, Shanghai 200040 (China); Yang, Zhi; Tu, Ying [Department of Dermatology, The First Affiliated Hospital of Kunming Medical University, Yunnan Provincial Institute of Dermatology, Kunming 650032, Yunnan (China); Yang, Yan-li [Department of Otorhinolaryngology, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210024, Jiangsu (China); He, Li, E-mail: heli2662@yahoo.com.cn [Department of Otorhinolaryngology, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210024, Jiangsu (China); Bi, Zhi-Gang, E-mail: eltonbibenqhospital@yahoo.com.cn [Department of Dermatology, BenQ Medical Center, Nanjing Medical University, Nanjing 210019, Jiangsu (China)

2012-09-07

Highlights: Black-Right-Pointing-Pointer UVB radiated skin keratinocytes show cyclophilin D (Cyp-D) upregulation. Black-Right-Pointing-Pointer NAC inhibits UVB induced Cyp-D expression, while H{sub 2}O{sub 2} facilitates it. Black-Right-Pointing-Pointer Cyp-D-deficient cells are significantly less susceptible to UVB induced cell death. Black-Right-Pointing-Pointer Over-expression of Cyp-D causes spontaneous keratinocytes cell death. -- Abstract: UVB-induced skin cell damage involves the opening of mitochondrial permeability transition pore (mPTP), which leads to both apoptotic and necrotic cell death. Cyclophilin D (Cyp-D) translocation to the inner membrane of mitochondrion acts as a key component to open the mPTP. Our Western-Blot results in primary cultured human skin keratinocytes and in HaCaT cell line demonstrated that UVB radiation and hydrogen peroxide (H{sub 2}O{sub 2}) induced Cyp-D expression, which was inhibited by anti-oxidant N-acetyl cysteine (NAC). We created a stable Cyp-D deficiency skin keratinocytes by expressing Cyp-D-shRNA through lentiviral infection. Cyp-D-deficient cells were significantly less susceptible than their counterparts to UVB- or H{sub 2}O{sub 2}-induced cell death. Further, cyclosporine A (Cs-A), a Cyp-D inhibitor, inhibited UVB- or H{sub 2}O{sub 2}-induced keratinocytes cell death. Reversely, over-expression of Cyp-D in primary keratinocytes caused spontaneous keratinocytes cell death. These results suggest Cyp-D's critical role in UVB/oxidative stress-induced skin cell death.

The Roles of CD147 and/or Cyclophilin A in Kidney Diseases

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Wang, Chunting; Zhang, Jicheng; Qie, Guoqiang

2014-01-01

CD147 is a widely expressed integral plasma membrane glycoprotein and has been involved in a variety of physiological and pathological activities in combination with different partners, including cyclophilins, caveolin-1, monocarboxylate transporters, and integrins. Recent data demonstrate that both CyPA and CD147 significantly contribute to renal inflammation, acute kidney injury, renal fibrosis, and renal cell carcinoma. Here we review the current understanding of cyclophilin A and CD147 expression and functions in kidney diseases and potential implications for treatment of kidney diseases. PMID:25580061

Cyclophilin B protects SH-SY5Y human neuroblastoma cells against MPP(+)-induced neurotoxicity via JNK pathway.

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Oh, Yoojung; Jeong, Kwon; Kim, Kiyoon; Lee, Young-Seok; Jeong, Suyun; Kim, Sung Soo; Yoon, Kyung-Sik; Ha, Joohun; Kang, Insug; Choe, Wonchae

2016-09-23

Parkinson's disease (PD) is the second most common neurodegenerative disorder of aging. PD involves a progressive loss of dopaminergic neurons in the substantia nigra pars compacta. 1-Methyl-4-phenyl-1, 2, 3, 6-tetrahydropyidine (MPTP) and its toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) inhibit the complex I of the mitochondrial electron transport chain, and have been widely used to construct PD models. Cyclophilin B (CypB) is an endoplasmic reticulum protein that binds to cyclosporine A as a cyclophilin family member. CypB has peptidyl-prolyl cis-trans isomerase (PPIase) activity. We investigated the protective effects of overexpressed CypB on MPP+-induced neurocytotoxicity in SH-SY5Y human neuroblastoma cells. Overexpressed CypB decreased MPP(+)-induced oxidative stress through the modulation of antioxidant enzymes including manganese superoxide dismutase and catalase, and prevented neurocytotoxicity via mitogen-activated protein kinase, especially the c-Jun N-terminal kinase pathway. In addition, CypB inhibited the activation of MPP(+)-induced the pro-apoptotic molecules poly (ADP-ribose) polymerase, Bax, and Bcl-2, and attenuated MPP(+)-induced mitochondrial dysfunction. The data suggest that overexpressed CypB protects neuronal cells from MPP+-induced dopaminergic neuronal cell death. Copyright © 2016 Elsevier Inc. All rights reserved.

Hypoxia induces cyclophilin B through the activation of transcription factor 6 in gastric adenocarcinoma cells

OpenAIRE

JEONG, KWON; KIM, KIYOON; KIM, HUNSUNG; OH, YOOJUNG; KIM, SEONG-JIN; JO, YUNHEE; CHOE, WONCHAE

2015-01-01

Hypoxia is an important form of physiological stress that induces cell death, due to the resulting endoplasmic reticulum (ER) stress, particularly in solid tumors. Although previous studies have indicated that cyclophilin B (CypB) plays a role in ER stress, there is currently no direct information supporting the mechanism of CypB involvement under hypoxic conditions. However, it has previously been demonstrated that ER stress positively regulates the expression of CypB. In the present study, ...

Cloning and Expression of Cyclophilin from Platanus orientalis Pollens in Escherichia coli

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Mojtaba Sankian

2012-10-01

Full Text Available Background: Allergy is a clinical disorder affecting the human population with wide geographical distribution. Platanus orientalis (P. orientalis trees are planted in many countries and their pollen causes allergic reactions. Cyclophilin has recently been identified as one of the most important allergens of P. orientalis pollen. We aimed to clone and purify this allergen in Escherichia coli for further studies and therapeutic and diagnostic purposes for allergy to P. orientalis. Methods: RNA was extracted from P. orientalis. A full-length fragment encoding cyclophilin was prepared by polymerase chain reaction amplification of the first-strand cDNA synthesized from P. orientalis RNA. The cDNA was inserted into the pET32b (+ vector, and the construct transformed into E. coli Top10 and BL21 cells. The expressed protein was purified by the CuSO4 method. Results: The cDNA for the cyclophilin of P. orientalis pollen was cloned, and a specific reactivity of recombinant cyclophin was confirmed by immunoblotting using sera from patients allergic to P. orientalis pollen. Conclusion: The recombinant cyclophilin has a potential for immunologic assays for evaluation of allergy to P. orientalis pollen.

Cyclophilin B as a co-regulator of prolactin-induced gene expression and function in breast cancer cells

OpenAIRE

Fang, Feng; Zheng, Jiamao; Galbaugh, Traci L; Fiorillo, Alyson A; Hjort, Elizabeth E; Zeng, Xianke; Clevenger, Charles V

2010-01-01

The effects of prolactin (PRL) during the pathogenesis of breast cancer are mediated in part though Stat5 activity enhanced by its interaction with its transcriptional inducer, the prolyl isomerase cyclophilin B (CypB). We have demonstrated that knockdown of CypB decreases cell growth, proliferation, and migration, and CypB expression is associated with malignant progression of breast cancer. In this study, we examined the effect of CypB knockdown on PRL signaling in breast cancer cells. CypB...

The Roles of CD147 and/or Cyclophilin A in Kidney Diseases

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Xin Qu

2014-01-01

Full Text Available CD147 is a widely expressed integral plasma membrane glycoprotein and has been involved in a variety of physiological and pathological activities in combination with different partners, including cyclophilins, caveolin-1, monocarboxylate transporters, and integrins. Recent data demonstrate that both CyPA and CD147 significantly contribute to renal inflammation, acute kidney injury, renal fibrosis, and renal cell carcinoma. Here we review the current understanding of cyclophilin A and CD147 expression and functions in kidney diseases and potential implications for treatment of kidney diseases.

Cyclosporine A-sensitive, cyclophilin B-dependent endoplasmic reticulum-associated degradation.

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Riccardo Bernasconi

2010-09-01

Full Text Available Peptidyl-prolyl cis/trans isomerases (PPIs catalyze cis/trans isomerization of peptide bonds preceding proline residues. The involvement of PPI family members in protein refolding has been established in test tube experiments. Surprisingly, however, no data is available on the involvement of endoplasmic reticulum (ER-resident members of the PPI family in protein folding, quality control or disposal in the living cell. Here we report that the immunosuppressive drug cyclosporine A (CsA selectively inhibits the degradation of a subset of misfolded proteins generated in the ER. We identify cyclophilin B (CyPB as the ER-resident target of CsA that catalytically enhances disposal from the ER of ERAD-L(S substrates containing cis proline residues. Our manuscript presents the first evidence for enzymatic involvement of a PPI in protein quality control in the ER of living cells.

Cyclosporine A-Sensitive, Cyclophilin B-Dependent Endoplasmic Reticulum-Associated Degradation

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Luban, Jeremy; Molinari, Maurizio

2010-01-01

Peptidyl-prolyl cis/trans isomerases (PPIs) catalyze cis/trans isomerization of peptide bonds preceding proline residues. The involvement of PPI family members in protein refolding has been established in test tube experiments. Surprisingly, however, no data is available on the involvement of endoplasmic reticulum (ER)-resident members of the PPI family in protein folding, quality control or disposal in the living cell. Here we report that the immunosuppressive drug cyclosporine A (CsA) selectively inhibits the degradation of a subset of misfolded proteins generated in the ER. We identify cyclophilin B (CyPB) as the ER-resident target of CsA that catalytically enhances disposal from the ER of ERAD-LS substrates containing cis proline residues. Our manuscript presents the first evidence for enzymatic involvement of a PPI in protein quality control in the ER of living cells. PMID:20927389

Cyclophilin C Participates in the US2-Mediated Degradation of Major Histocompatibility Complex Class I Molecules.

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Chapman, Daniel C; Stocki, Pawel; Williams, David B

2015-01-01

Human cytomegalovirus uses a variety of mechanisms to evade immune recognition through major histocompatibility complex class I molecules. One mechanism mediated by the immunoevasin protein US2 causes rapid disposal of newly synthesized class I molecules by the endoplasmic reticulum-associated degradation pathway. Although several components of this degradation pathway have been identified, there are still questions concerning how US2 targets class I molecules for degradation. In this study we identify cyclophilin C, a peptidyl prolyl isomerase of the endoplasmic reticulum, as a component of US2-mediated immune evasion. Cyclophilin C could be co-isolated with US2 and with the class I molecule HLA-A2. Furthermore, it was required at a particular expression level since depletion or overexpression of cyclophilin C impaired the degradation of class I molecules. To better characterize the involvement of cyclophilin C in class I degradation, we used LC-MS/MS to detect US2-interacting proteins that were influenced by cyclophilin C expression levels. We identified malectin, PDIA6, and TMEM33 as proteins that increased in association with US2 upon cyclophilin C knockdown. In subsequent validation all were shown to play a functional role in US2 degradation of class I molecules. This was specific to US2 rather than general ER-associated degradation since depletion of these proteins did not impede the degradation of a misfolded substrate, the null Hong Kong variant of α1-antitrypsin.

Evidence that human milk isolated cyclophilin B corresponds to a truncated form.

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Mariller, C; Allain, F; Kouach, M; Spik, G

1996-03-07

Cyclophilin B (CyPB) is a member of the cyclophilin family (cyclosporin A-binding proteins) with specific N- and C-terminal extensions. In contrast to cyclophilin A, CyPB owns a signal sequence leading to its translocation in the endoplasmic reticulum. CyPB was reported to be present in human blood and milk, suggesting it is secreted. For this purpose, CyPB was purified to homogeneity from human milk and compared to recombinant CyPB expressed in E. coli. Ion spray mass spectrometry revealed that CyPB secreted in human milk exhibits a lower molecular mass than the one expected. Identification of phenylalanine as the C-terminus amino-acid residue of human milk CyPB indicates that the difference in molecular mass may be explained by the absence of the five C-terminal amino-acid residues AIAKE. These results suggest that in the sequence VEKPFAIAKE known to be responsible for retention of CyPB in the endoplasmic reticulum, the sequence AIAKE is more particularly necessary. Our findings raise the possibility that the CyPB may be processed to promote its release. As recombinant CyPB was shown to bind specifically to Jurkat cells, a lymphoblastic T-cell line, we then wanted to investigate the binding of human milk CyPB to these cells. Despite lacking the five C-terminal amino-acid residues, human milk CyPB is able to inhibit the binding of recombinant CyPB to Jurkat T cells.

Expression and Significance of Cyclophilin J in Primary Gastric Adenocarcinoma.

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Gong, Zhaohua; Mu, Yuling; Chen, Jian; Chu, Hongjin; Lian, Peiwen; Wang, Congcong; Wang, Jiahui; Jiang, Lixin

2017-08-01

Biomarkers are essential in early diagnosis and understanding of the molecular mechanism of human cancer. The expression of cyclophilin J, a novel member of the cyclophilin family, was investigated in primary gastric adenocarcinoma. Western blot analysis was carried out on 36 paired tumor and normal tissue samples; immunohistochemical analysis was carried out on 120 gastric carcinoma tissues and normal adjacent tissue. Cyclophilin J protein was overexpressed in 72.2% of gastric carcinoma tissues compared to adjacent normal tissues. Immunohistochemical analysis revealed that cyclophilin J was overexpressed in 49.2% (59/120) and 23.3% (28/120) of gastric carcinoma tissues and adjacent tissues, respectively (pJ was associated with the degree of differentiation, but not with lymph node metastasis, gender or depth of tumor infiltration. The overall survival of patients showed no association with the overexpression of cyclophilin J protein. Cyclophilin J expression was up-regulated in gastric carcinoma compared to normal gastric tissues. However, in order to confirm its association with the survival of patients with gastric cancer, more cases need to be studied. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

The ability of multimerized cyclophilin A to restrict retrovirus infection

International Nuclear Information System (INIS)

Javanbakht, Hassan; Diaz-Griffero, Felipe; Yuan Wen; Yeung, Darwin F.; Li Xing; Song Byeongwoon; Sodroski, Joseph

2007-01-01

In owl monkeys, the typical retroviral restriction factor of primates, TRIM5α, is replaced by TRIMCyp. TRIMCyp consists of the TRIM5 RING, B-box 2 and coiled-coil domains, as well as the intervening linker regions, fused with cyclophilin A. TRIMCyp restricts infection of retroviruses, such as human immunodeficiency virus (HIV-1) and feline immunodeficiency virus (FIV), with capsids that can bind cyclophilin A. The TRIM5 coiled coil promotes the trimerization of TRIMCyp. Here we show that cyclophilin A that is oligomeric as a result of fusion with a heterologous multimer exhibits substantial antiretroviral activity. The addition of the TRIM5 RING, B-box 2 and Linker 2 to oligomeric cyclophilin A generated a protein with antiretroviral activity approaching that of wild-type TRIMCyp. Multimerization increased the binding of cyclophilin A to the HIV-1 capsid, promoting accelerated uncoating of the capsid and restriction of infection

The cyclophilin D/Drp1 axis regulates mitochondrial fission contributing to oxidative stress-induced mitochondrial dysfunctions in SH-SY5Y cells

International Nuclear Information System (INIS)

Xiao, Anqi; Gan, Xueqi; Chen, Ruiqi; Ren, Yanming; Yu, Haiyang; You, Chao

2017-01-01

Oxidative stress plays a central role in the pathogenesis of various neurodegenerative diseases. Increasing evidences have demonstrated that structural abnormalities in mitochondria are involved in oxidative stress related nerve cell damage. And Drp1 plays a critical role in mitochondrial dynamic imbalance insulted by oxidative stress-derived mitochondria. However, the status of mitochondrial fusion and fission pathway and its relationship with mitochondrial properties such as mitochondrial membrane permeability transition pore (mPTP) have not been fully elucidated. Here, we demonstrated for the first time the role of Cyclophilin D (CypD), a crucial component for mPTP formation, in the regulation of mitochondrial dynamics in oxidative stress treated nerve cell. We observed that CypD-mediated phosphorylation of Drp1 and subsequently augmented Drp1 recruitment to mitochondria and shifts mitochondrial dynamics toward excessive fission, which contributes to the mitochondrial structural and functional dysfunctions in oxidative stress-treated nerve cells. CypD depletion or over expression accompanies mitochondrial dynamics/functions recovery or aggravation separately. We also demonstrated first time the link between the CypD to mitochondrial dynamics. Our data offer new insights into the mechanism of mitochondrial dynamics which contribute to the mitochondrial dysfunctions, specifically the role of CypD in Drp1-mediated mitochondrial fission. The protective effect of CsA, or other molecules affecting the function of CypD hold promise as a potential novel therapeutic strategy for governing oxidative stress pathology via mitochondrial pathways. - Highlights: • Demonstrated first time the link between the mPTP to mitochondrial dynamics. • The role of Cyclophilin D in the regulation of Drp1-mediated mitochondrial fission. • CsA as a potential target for governing oxidative stress related neuropathology.

Cyclophilin A enhances cell proliferation and tumor growth of liver fluke-associated cholangiocarcinoma

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Sawanyawisuth Kanlayanee

2011-08-01

Full Text Available Abstract Background Cyclophilin A (CypA expression is associated with malignant phenotypes in many cancers. However, the role and mechanisms of CypA in liver fluke-associated cholangiocarcinoma (CCA are not presently known. In this study, we investigated the expression of CypA in CCA tumor tissues and CCA cell lines as well as regulation mechanisms of CypA in tumor growth using CCA cell lines. Methods CypA expression was determined by real time RT-PCR, Western blot or immunohistochemistry. CypA silence or overexpression in CCA cells was achieved using gene delivery techniques. Cell proliferation was assessed using MTS assay or Ki-67 staining. The effect of silencing CypA on CCA tumor growth was determined in nude mice. The effect of CypA knockdown on ERK1/2 activation was assessed by Western blot. Results CypA was upregulated in 68% of CCA tumor tissues. Silencing CypA significantly suppressed cell proliferation in several CCA cell lines. Likewise, inhibition of CypA peptidyl-prolyl cis-trans isomerase (PPIase activity using cyclosporin A (CsA decreased cell proliferation. In contrast, overexpression of CypA resulted in 30% to 35% increases in proliferation of CCA cell lines. Interestingly, neither silence nor overexpression of CypA affected cell proliferation of a non-tumor human cholangiocyte cell line, MMNK1. Suppression of CypA expression attenuated ERK1/2 activity in CCA M139 cells by using both transient and stable knockdown methods. In the in vivo study, there was a 43% reduction in weight of tumors derived from CypA-silenced CCA cell lines compared with control vector CCA tumors in mice; these tumors with stable CypA silencing showed a reduced cell proliferation. Conclusions CypA is upregulated in majority of CCA patients' tissues and confers a significant growth advantage in CCA cells. Suppression of CypA expression decreases proliferation of CCA cell lines in vitro and reduces tumor growth in the nude mouse model. Inhibition of Cyp

Effect of cyclophilin A on gene expression in human pancreatic cancer cells.

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Li, Min; Wang, Hao; Li, Fei; Fisher, William E; Chen, Changyi; Yao, Qizhi

2005-11-01

We previously found that cyclophilin A (CypA) is overexpressed in human pancreatic cancer cells and stimulates cell proliferation through CD147. In this study, we further investigated the effect of CypA on gene expression of several key molecules that are involved in pancreatic cancer cell proliferation. Human pancreatic cancer cell lines (Panc-1, MIA PaCa-2, and BxPC-3) and human pancreatic ductal epithelial (HPDE) cells were used. The messenger RNA (mRNA) levels of CypA, CypB, CD147, neuropilins (NRPs), vascular endothelial growth factor (VEGF), and VEGF receptors upon the treatment of exogenous recombinant human CypA were determined by real-time reverse-transcription polymerase chain reaction. Exogenous human recombinant CypA reduced the mRNA levels of NRP-1 and VEGF, but not endogenous CypA, CypB, and CD147, in Panc-1, MIA PaCa-2, and BxPC-3 cells. In contrast, HPDE cells showed a decrease of endogenous CypA and CD147 mRNA, but not detectable changes of CypB, NRPs, and VEGF mRNA levels upon exogenous CypA treatment. These data show that exogenous CypA downregulates NRP-1 and VEGF expression in pancreatic cancer cells. This effect is different in normal HPDE cells. Thus, soluble CypA may affect cell growth of pancreatic cancer.

Essential role of cyclophilin A for hepatitis C virus replication and virus production and possible link to polyprotein cleavage kinetics.

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Artur Kaul

2009-08-01

Full Text Available Viruses are obligate intracellular parasites and therefore their replication completely depends on host cell factors. In case of the hepatitis C virus (HCV, a positive-strand RNA virus that in the majority of infections establishes persistence, cyclophilins are considered to play an important role in RNA replication. Subsequent to the observation that cyclosporines, known to sequester cyclophilins by direct binding, profoundly block HCV replication in cultured human hepatoma cells, conflicting results were obtained as to the particular cyclophilin (Cyp required for viral RNA replication and the underlying possible mode of action. By using a set of cell lines with stable knock-down of CypA or CypB, we demonstrate in the present work that replication of subgenomic HCV replicons of different genotypes is reduced by CypA depletion up to 1,000-fold whereas knock-down of CypB had no effect. Inhibition of replication was rescued by over-expression of wild type CypA, but not by a mutant lacking isomerase activity. Replication of JFH1-derived full length genomes was even more sensitive to CypA depletion as compared to subgenomic replicons and virus production was completely blocked. These results argue that CypA may target an additional viral factor outside of the minimal replicase contributing to RNA amplification and assembly, presumably nonstructural protein 2. By selecting for resistance against the cyclosporine analogue DEBIO-025 that targets CypA in a dose-dependent manner, we identified two mutations (V2440A and V2440L close to the cleavage site between nonstructural protein 5A and the RNA-dependent RNA polymerase in nonstructural protein 5B that slow down cleavage kinetics at this site and reduce CypA dependence of viral replication. Further amino acid substitutions at the same cleavage site accelerating processing increase CypA dependence. Our results thus identify an unexpected correlation between HCV polyprotein processing and CypA dependence

Essential role of cyclophilin A for hepatitis C virus replication and virus production and possible link to polyprotein cleavage kinetics.

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Kaul, Artur; Stauffer, Sarah; Berger, Carola; Pertel, Thomas; Schmitt, Jennifer; Kallis, Stephanie; Zayas, Margarita; Lopez, Margarita Zayas; Lohmann, Volker; Luban, Jeremy; Bartenschlager, Ralf

2009-08-01

Viruses are obligate intracellular parasites and therefore their replication completely depends on host cell factors. In case of the hepatitis C virus (HCV), a positive-strand RNA virus that in the majority of infections establishes persistence, cyclophilins are considered to play an important role in RNA replication. Subsequent to the observation that cyclosporines, known to sequester cyclophilins by direct binding, profoundly block HCV replication in cultured human hepatoma cells, conflicting results were obtained as to the particular cyclophilin (Cyp) required for viral RNA replication and the underlying possible mode of action. By using a set of cell lines with stable knock-down of CypA or CypB, we demonstrate in the present work that replication of subgenomic HCV replicons of different genotypes is reduced by CypA depletion up to 1,000-fold whereas knock-down of CypB had no effect. Inhibition of replication was rescued by over-expression of wild type CypA, but not by a mutant lacking isomerase activity. Replication of JFH1-derived full length genomes was even more sensitive to CypA depletion as compared to subgenomic replicons and virus production was completely blocked. These results argue that CypA may target an additional viral factor outside of the minimal replicase contributing to RNA amplification and assembly, presumably nonstructural protein 2. By selecting for resistance against the cyclosporine analogue DEBIO-025 that targets CypA in a dose-dependent manner, we identified two mutations (V2440A and V2440L) close to the cleavage site between nonstructural protein 5A and the RNA-dependent RNA polymerase in nonstructural protein 5B that slow down cleavage kinetics at this site and reduce CypA dependence of viral replication. Further amino acid substitutions at the same cleavage site accelerating processing increase CypA dependence. Our results thus identify an unexpected correlation between HCV polyprotein processing and CypA dependence of HCV

A proteome study of secreted prostatic factors affecting osteoblastic activity, identification and characterisation of cyclophilin A

DEFF Research Database (Denmark)

Andersen, H.; Jensen, O.N.; Eriksen, E.F.

2003-01-01

ranging from 5 to 30 kDa were analysed by matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS). One of these spots was identified as cyclophilin A. We examined whether cyclophilin A alone or in combination with insulin-like growth factor-I (IGF-I) had any effects...... was to characterise the protein profile of conditioned medium (CM) from PC3 cells in the molecular weight range of 5-30 kDa using proteome analysis. A protein profile of the CM from PC3 cells was performed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Thirty protein spots with molecular weights...

Feline coronavirus replication is affected by both cyclophilin A and cyclophilin B.

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Tanaka, Yoshikazu; Sato, Yuka; Sasaki, Takashi

2017-02-01

Feline coronavirus (FCoV) causes the fatal disease feline infectious peritonitis, which is currently incurable by drug treatment, and no effective vaccines are available. Cyclosporin A (CsA), a cyclophilin (Cyp) inhibitor, inhibits the replication of FCoV in vitro and in vivo as well as the replication of human and animal coronaviruses. However, the mechanism underlying the regulation of coronavirus replication by CsA is unknown. In this study, we analysed the role of Cyps in FCoV replication using knockdown and knockout cells specific to Cyps. Inhibition of CypA and CypB reduced FCoV replication, with replication in knockout cells being much less than that in knockdown cells. Furthermore, the proteins expressed by CypA and CypB harbouring mutations in their respective predicted peptidyl-prolyl cis-transisomerase active sites, which also alter the affinities between Cyps and CsA, inhibited FCoV replication. These findings indicate that the peptidyl-prolyl cis-transisomerase active sites of Cyps might be required for FCoV replication.

Cyclophilins contribute to Stat3 signaling and survival of multiple myeloma cells.

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Bauer, K; Kretzschmar, A K; Cvijic, H; Blumert, C; Löffler, D; Brocke-Heidrich, K; Schiene-Fischer, C; Fischer, G; Sinz, A; Clevenger, C V; Horn, F

2009-08-06

Signal transducer and activator of transcription 3 (Stat3) is the major mediator of interleukin-6 (IL-6) family cytokines. In addition, Stat3 is known to be involved in the pathophysiology of many malignancies. Here, we show that the cis-trans peptidyl-prolyl isomerase cyclophilin (Cyp) B specifically interacts with Stat3, whereas the highly related CypA does not. CypB knockdown inhibited the IL-6-induced transactivation potential but not the tyrosine phosphorylation of Stat3. Binding of CypB to Stat3 target promoters and alteration of the intranuclear localization of Stat3 on CypB depletion suggested a nuclear function of Stat3/CypB interaction. By contrast, CypA knockdown inhibited Stat3 IL-6-induced tyrosine phosphorylation and nuclear translocation. The Cyp inhibitor cyclosporine A (CsA) caused similar effects. However, Stat1 activation in response to IL-6 or interferon-gamma was not affected by Cyp silencing or CsA treatment. As a result, Cyp knockdown shifted IL-6 signaling to a Stat1-dominated pathway. Furthermore, Cyp depletion or treatment with CsA induced apoptosis in IL-6-dependent multiple myeloma cells, whereas an IL-6-independent line was not affected. Thus, Cyps support the anti-apoptotic action of Stat3. Taken together, CypA and CypB both play pivotal roles, yet at different signaling levels, for Stat3 activation and function. These data also suggest a novel mechanism of CsA action.

Involvement of the N-terminal part of cyclophilin B in the interaction with specific Jurkat T-cell binding sites.

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Mariller, C; Haendler, B; Allain, F; Denys, A; Spik, G

1996-07-15

Cyclophilin B (CyPB) is secreted in biological fluids such as blood or milk and binds to a specific receptor present on the human lymphoblastic cell line Jurkat and on human peripheral blood lymphocytes. This study was intended to specify the areas of CyPB that are involved in the interaction with the receptor. A synthetic peptide corresponding to the first 24 N-terminal amino acid residues of CyPB was shown to specifically recognize the receptor. Moreover, modification of Arg18 of CyPB by p-hydroxyphenlglyoxal led to a dramatic loss of affinity for the receptor. However, when this residue was replaced by an alanine residue using site-directed mutagenesis, no modification of the binding properties was found, suggesting that Arg18 is not directly involved but is sufficiently close to the interaction site to interfere with the binding when modified. Competitive binding experiments using a chimaeric protein made up of the 24 N-terminal amino acid residues of CyPB fused to the cyclophilin A core sequence confirmed the involvement of this region of CyPB in receptor binding.

Overexpressed cyclophilin B suppresses aldosterone-induced proximal tubular cell injury both in vitro and in vivo.

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Wang, Bin; Lin, Lilu; Wang, Haidong; Guo, Honglei; Gu, Yong; Ding, Wei

2016-10-25

The renin-angiotensin-aldosterone system (RAAS) is overactivated in patients with chronic kidney disease. Oxidative stress and endoplasmic reticulum stress (ERS) are two major mechanisms responsible for aldosterone-induced kidney injury. Cyclophilin (CYP) B is a chaperone protein that accelerates the rate of protein folding through its peptidyl-prolyl cis-trans isomerase (PPIase) activity. We report that overexpression of wild-type CYPB attenuated aldosterone-induced oxidative stress (evidenced by reduced production of reactive oxygen species and improved mitochondrial dysfunction), ERS (indicated by reduced expression of the ERS markers glucose-regulated protein 78 [GRP78] and C/-EBP homologous protein [CHOP]), and tubular cell apoptosis in comparison with aldosterone-induced human kidney-2 (HK-2) cells. The in vivo study also yielded similar results. Hence, CYPB performs a crucial function in protecting cells against aldosterone-induced oxidative stress, ERS, and tubular cell injury via its PPIase activity.

Overexpressed cyclophilin B suppresses apoptosis associated with ROS and Ca2+ homeostasis after ER stress.

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Kim, Jinhwan; Choi, Tae Gyu; Ding, Yan; Kim, Yeonghwan; Ha, Kwon Soo; Lee, Kyung Ho; Kang, Insug; Ha, Joohun; Kaufman, Randal J; Lee, Jinhwa; Choe, Wonchae; Kim, Sung Soo

2008-11-01

Prolonged accumulation of misfolded proteins in the endoplasmic reticulum (ER) results in ER stress-mediated apoptosis. Cyclophilins are protein chaperones that accelerate the rate of protein folding through their peptidyl-prolyl cis-trans isomerase (PPIase) activity. In this study, we demonstrated that ER stress activates the expression of the ER-localized cyclophilin B (CypB) gene through a novel ER stress response element. Overexpression of wild-type CypB attenuated ER stress-induced cell death, whereas overexpression of an isomerase activity-defective mutant, CypB/R62A, not only increased Ca(2+) leakage from the ER and ROS generation, but also decreased mitochondrial membrane potential, resulting in cell death following exposure to ER stress-inducing agents. siRNA-mediated inhibition of CypB expression rendered cells more vulnerable to ER stress. Finally, CypB interacted with the ER stress-related chaperones, Bip and Grp94. Taken together, we concluded that CypB performs a crucial function in protecting cells against ER stress via its PPIase activity.

Activation of CD147 with Cyclophilin A Induces the Expression of IFITM1 through ERK and PI3K in THP-1 Cells

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Ju-Young Kim

2010-01-01

Full Text Available CD147, as a receptor for Cyclophilins, is a multifunctional transmembrane glycoprotein. In order to identify genes that are induced by activation of CD147, THP-1 cells were stimulated with Cyclophilin A and differentially expressed genes were detected using PCR-based analysis. Interferon-induced transmembrane 1 (IFITM1 was detected to be induced and it was confirmed by RT-PCR and Western blot analysis. CD147-induced expression of IFITM1 was blocked by inhibitors of ERK, PI3K, or NF-κB, but not by inhibitors of p38, JNK, or PKC. IFITM1 appears to mediate inflammatory activation of THP-1 cells since cross-linking of IFITM1 with specific monoclonal antibody against it induced the expression of proinflammatory mediators such as IL-8 and MMP-9. These data indicate that IFITM1 is one of the pro-inflammatory mediators that are induced by signaling initiated by the activation of CD147 in macrophages and activation of ERK, PI3K, and NF-κB is required for the expression of IFITM1.

Ultra-violet B (UVB)-induced skin cell death occurs through a cyclophilin D intrinsic signaling pathway.

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Ji, Chao; Yang, Bo; Yang, Zhi; Tu, Ying; Yang, Yan-li; He, Li; Bi, Zhi-Gang

2012-09-07

UVB-induced skin cell damage involves the opening of mitochondrial permeability transition pore (mPTP), which leads to both apoptotic and necrotic cell death. Cyclophilin D (Cyp-D) translocation to the inner membrane of mitochondrion acts as a key component to open the mPTP. Our Western-Blot results in primary cultured human skin keratinocytes and in HaCaT cell line demonstrated that UVB radiation and hydrogen peroxide (H(2)O(2)) induced Cyp-D expression, which was inhibited by anti-oxidant N-acetyl cysteine (NAC). We created a stable Cyp-D deficiency skin keratinocytes by expressing Cyp-D-shRNA through lentiviral infection. Cyp-D-deficient cells were significantly less susceptible than their counterparts to UVB- or H(2)O(2)-induced cell death. Further, cyclosporine A (Cs-A), a Cyp-D inhibitor, inhibited UVB- or H(2)O(2)-induced keratinocytes cell death. Reversely, over-expression of Cyp-D in primary keratinocytes caused spontaneous keratinocytes cell death. These results suggest Cyp-D's critical role in UVB/oxidative stress-induced skin cell death. Copyright © 2012 Elsevier Inc. All rights reserved.

Synthesis of Heparan Sulfate with Cyclophilin B-binding Properties Is Determined by Cell Type-specific Expression of Sulfotransferases*

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Deligny, Audrey; Denys, Agnès; Marcant, Adeline; Melchior, Aurélie; Mazurier, Joël; van Kuppevelt, Toin H.; Allain, Fabrice

2010-01-01

Cyclophilin B (CyPB) induces migration and adhesion of T lymphocytes via a mechanism that requires interaction with 3-O-sulfated heparan sulfate (HS). HS biosynthesis is a complex process with many sulfotransferases involved. N-Deacetylases/N-sulfotransferases are responsible for N-sulfation, which is essential for subsequent modification steps, whereas 3-O-sulfotransferases (3-OSTs) catalyze the least abundant modification. These enzymes are represented by several isoforms, which differ in term of distribution pattern, suggesting their involvement in making tissue-specific HS. To elucidate how the specificity of CyPB binding is determined, we explored the relationships between the expression of these sulfotransferases and the generation of HS motifs with CyPB-binding properties. We demonstrated that high N-sulfate density and the presence of 2-O- and 3-O-sulfates determine binding of CyPB, as evidenced by competitive experiments with heparin derivatives, soluble HS, and anti-HS antibodies. We then showed that target cells, i.e. CD4+ lymphocyte subsets, monocytes/macrophages, and related cell lines, specifically expressed high levels of NDST2 and 3-OST3 isoforms. Silencing the expression of NDST1, NDST2, 2-OST, and 3-OST3 by RNA interference efficiently decreased binding and activity of CyPB, thus confirming their involvement in the biosynthesis of binding sequences for CyPB. Moreover, we demonstrated that NDST1 was able to partially sulfate exogenous substrate in the absence of NDST2 but not vice versa, suggesting that both isoenzymes do not have redundant activities but do have rather complementary activities in making N-sulfated sequences with CyPB-binding properties. Altogether, these results suggest a regulatory mechanism in which cell type-specific expression of certain HS sulfotransferases determines the specific binding of CyPB to target cells. PMID:19940140

Synthesis of heparan sulfate with cyclophilin B-binding properties is determined by cell type-specific expression of sulfotransferases.

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Deligny, Audrey; Denys, Agnès; Marcant, Adeline; Melchior, Aurélie; Mazurier, Joël; van Kuppevelt, Toin H; Allain, Fabrice

2010-01-15

Cyclophilin B (CyPB) induces migration and adhesion of T lymphocytes via a mechanism that requires interaction with 3-O-sulfated heparan sulfate (HS). HS biosynthesis is a complex process with many sulfotransferases involved. N-Deacetylases/N-sulfotransferases are responsible for N-sulfation, which is essential for subsequent modification steps, whereas 3-O-sulfotransferases (3-OSTs) catalyze the least abundant modification. These enzymes are represented by several isoforms, which differ in term of distribution pattern, suggesting their involvement in making tissue-specific HS. To elucidate how the specificity of CyPB binding is determined, we explored the relationships between the expression of these sulfotransferases and the generation of HS motifs with CyPB-binding properties. We demonstrated that high N-sulfate density and the presence of 2-O- and 3-O-sulfates determine binding of CyPB, as evidenced by competitive experiments with heparin derivatives, soluble HS, and anti-HS antibodies. We then showed that target cells, i.e. CD4+ lymphocyte subsets, monocytes/macrophages, and related cell lines, specifically expressed high levels of NDST2 and 3-OST3 isoforms. Silencing the expression of NDST1, NDST2, 2-OST, and 3-OST3 by RNA interference efficiently decreased binding and activity of CyPB, thus confirming their involvement in the biosynthesis of binding sequences for CyPB. Moreover, we demonstrated that NDST1 was able to partially sulfate exogenous substrate in the absence of NDST2 but not vice versa, suggesting that both isoenzymes do not have redundant activities but do have rather complementary activities in making N-sulfated sequences with CyPB-binding properties. Altogether, these results suggest a regulatory mechanism in which cell type-specific expression of certain HS sulfotransferases determines the specific binding of CyPB to target cells.

Gastric Cancer Cell Proliferation and Survival Is Enabled by a Cyclophilin B/STAT3/miR-520d-5p Signaling Feedback Loop.

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Li, Ting; Guo, Hanqing; Zhao, Xiaodi; Jin, Jiang; Zhang, Lifeng; Li, Hong; Lu, Yuanyuan; Nie, Yongzhan; Wu, Kaichun; Shi, Yongquan; Fan, Daiming

2017-03-01

Molecular links between inflammation and cancer remain obscure despite their great pathogenic significance. The JAK2/STAT3 pathway activated by IL6 and other proinflammatory cytokines has garnered attention as a pivotal link in cancer pathogenesis, but the basis for its activation in cancer cells is not understood. Here we report that an IL6-triggered feedback loop involving STAT3-mediated suppression of miR-520d-5p and upregulation of its downstream target cyclophilin B (CypB) regulate the growth and survival of gastric cancer cells. In clinical specimens of gastric cancer, we documented increased expression of CypB and activation of STAT3. Mechanistic investigations identified miR-520d-5p as a regulator of CypB mRNA levels. This signaling axis regulated gastric cancer growth by modulating phosphorylation of STAT3. Furthermore, miR-520d-5p was identified as a direct STAT3 target and IL6-mediated inhibition of miR-520d-5p relied upon STAT3 activity. Our findings define a positive feedback loop that drives gastric carcinogenesis as influenced by H. pylori infections that involve proinflammatory IL6 stimulation. Cancer Res; 77(5); 1227-40. ©2016 AACR . ©2016 American Association for Cancer Research.

Comparing human pancreatic cell secretomes by in vitro aptamer selection identifies cyclophilin B as a candidate pancreatic cancer biomarker.

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Ray, Partha; Rialon-Guevara, Kristy L; Veras, Emanuela; Sullenger, Bruce A; White, Rebekah R

2012-05-01

Most cases of pancreatic cancer are not diagnosed until they are no longer curable with surgery. Therefore, it is critical to develop a sensitive, preferably noninvasive, method for detecting the disease at an earlier stage. In order to identify biomarkers for pancreatic cancer, we devised an in vitro positive/negative selection strategy to identify RNA ligands (aptamers) that could detect structural differences between the secretomes of pancreatic cancer and non-cancerous cells. Using this molecular recognition approach, we identified an aptamer (M9-5) that differentially bound conditioned media from cancerous and non-cancerous human pancreatic cell lines. This aptamer further discriminated between the sera of pancreatic cancer patients and healthy volunteers with high sensitivity and specificity. We utilized biochemical purification methods and mass-spectrometric analysis to identify the M9-5 target as cyclophilin B (CypB). This molecular recognition-based strategy simultaneously identified CypB as a serum biomarker and generated a new reagent to recognize it in body fluids. Moreover, this approach should be generalizable to other diseases and complementary to traditional approaches that focus on differences in expression level between samples. Finally, we suggest that the aptamer we identified has the potential to serve as a tool for the early detection of pancreatic cancer.

Pharmacological Cyclophilin Inhibitors Prevent Intoxication of Mammalian Cells with Bordetella pertussis Toxin.

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Ernst, Katharina; Eberhardt, Nina; Mittler, Ann-Katrin; Sonnabend, Michael; Anastasia, Anna; Freisinger, Simon; Schiene-Fischer, Cordelia; Malešević, Miroslav; Barth, Holger

2018-05-01

The Bordetella pertussis toxin (PT) is one important virulence factor causing the severe childhood disease whooping cough which still accounted for approximately 63,000 deaths worldwide in children in 2013. PT consists of PTS1, the enzymatically active (A) subunit and a non-covalently linked pentameric binding/transport (B) subunit. After endocytosis, PT takes a retrograde route to the endoplasmic reticulum (ER), where PTS1 is released into the cytosol. In the cytosol, PTS1 ADP-ribosylates inhibitory alpha subunits of trimeric GTP-binding proteins (Giα) leading to increased cAMP levels and disturbed signalling. Here, we show that the cyclophilin (Cyp) isoforms CypA and Cyp40 directly interact with PTS1 in vitro and that Cyp inhibitors cyclosporine A (CsA) and its tailored non-immunosuppressive derivative VK112 both inhibit intoxication of CHO-K1 cells with PT, as analysed in a morphology-based assay. Moreover, in cells treated with PT in the presence of CsA, the amount of ADP-ribosylated Giα was significantly reduced and less PTS1 was detected in the cytosol compared to cells treated with PT only. The results suggest that the uptake of PTS1 into the cytosol requires Cyps. Therefore, CsA/VK112 represent promising candidates for novel therapeutic strategies acting on the toxin level to prevent the severe, life-threatening symptoms caused by PT.

Cyclophilin B mediates cyclosporin A incorporation in human blood T-lymphocytes through the specific binding of complexed drug to the cell surface.

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Allain, F; Denys, A; Spik, G

1996-07-15

Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein located within intracellular vesicles and released in biological fluids. We recently reported the specific binding of this protein to T-cell surface receptor which is internalized even in the presence of CsA. These results suggest that CyPB might target the drug to lymphocytes and consequently modify its activity. To verify this hypothesis, we have first investigated the binding capacity and internalization of the CsA-CyPB complex in human peripheral blood T-lymphocytes and secondly compared the inhibitory effect of both free and CyPB-complexed CsA on the CD3-induced activation and proliferation of T-cells. Here, we present evidence that both the CsA-CyPB complex and free CyPB bind to the T-lymphocyte surface, with similar values of Kd and number of sites. At 37 degrees C, the complex is internalized but, in contrast to the protein, the drug is accumulated within the cell. Moreover, CyPB receptors are internalized together with the ligand and rapidly recycled to the cell surface. Finally, we demonstrate that CyPB-complexed CsA remains as efficient as uncomplexed CsA and that CyPB enhances the immunosuppressive activity of the drug. Taken together, our results support the hypothesis that surface CyPB receptors may be related to the selective and variable action of CsA, through specific binding and targeting of the CyPB-CsA complex to peripheral blood T-lymphocytes.

Plasma level of cyclophilin A is increased in patients with type 2 diabetes mellitus and suggests presence of vascular disease.

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Ramachandran, Surya; Venugopal, Anila; Kutty, V Raman; A, Vinitha; G, Divya; Chitrasree, V; Mullassari, Ajit; Pratapchandran, N S; Santosh, K R; Pillai, M Radhakrishna; Kartha, C C

2014-02-07

Cyclophilin A, an immunophilin is secreted from human monocytes activated by high glucose. Given its role as an inflammatory mediator of vascular tissue damage associated with inflammation and oxidative stress, we examined plasma levels of cyclophilin A in normal healthy volunteers and patients with type 2 diabetes (DM), with or without coronary artery disease (CAD). Study subjects comprised of 212 patients with DM and CAD,101 patients with diabetes, 122 patients with CAD and 121 normal healthy volunteers. Diabetes was assessed by HbA1c levels while coronary artery disease was established by a positive treadmill test and/or coronary angiography. Plasma cyclophilin A was measured using a cyclophilin A ELISA Kit. Relationship of plasma cyclophilin A levels with blood markers of type 2 diabetes, blood lipid levels and medication for diabetes and coronary artery disease were also explored. Plasma Cyclophilin levels were higher in diabetes patients with or without CAD compared to normal subjects (P levels and HbA1C levels were positively associated with increased plasma cyclophilin. Patients using metformin had reduced levels of plasma cyclophilin (p levels of total cholesterol, LDL cholesterol and triglycerides had no significant association with plasma cyclophilin levels. In patients with increased serum CRP levels, plasma cyclophilin A was also elevated (p = 0.016). Prevalence odds for DM, DM + CAD and CAD are higher in those with high cyclophilin values, compared to those with lower values, after adjusting for age and sex, indicating strong association of high cyclophilin values with diabetes and vascular disease. Our study demonstrates that patients with type 2 diabetes have higher circulating levels of cyclophilin A than the normal population. Plasma cyclophilin levels were increased in patients with diabetes and coronary artery disease suggesting a role of this protein in accelerating vascular disease in type 2 diabetes. Considering the evidence that

Neuroprotective effects of overexpressed cyclophilin B against Aβ-induced neurotoxicity in PC12 cells.

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Oh, Yoojung; Kim, Eun Young; Kim, Yeonghwan; Jin, Jizi; Jin, Byung Kwan; Jahng, Geon-Ho; Jung, Min Hyung; Park, Chan; Kang, Insug; Ha, Joohun; Choe, Wonchae

2011-08-15

Accumulated amyloid-β (Aβ) is a well-known cause of neuronal apoptosis in Alzheimer disease and functions in part by generating oxidative stress. Our previous work suggested that cyclophilin B (CypB) protects against endoplasmic reticulum (ER) stress. Therefore, in this study we examined the ability of CypB to protect against Aβ toxicity. CypB is present in the neurons of rat and mouse brains, and treating neural cells with Aβ(25-35) mediates apoptotic cell death. Aβ(25-35)-induced neuronal toxicity was inhibited by the overexpression of CypB as measured by cell viability, apoptotic morphology, sub-G1 cell population, intracellular reactive oxygen species accumulation, activated caspase-3, PARP cleavage, Bcl-2 proteins, mitogen-activated protein kinase (MAPK) activation, and phosphoinositide 3-kinase (PI-3-K) activation. CypB/R95A PPIase mutants did not reduce Aβ(25-35) toxicity. We showed that Aβ(25-35)-induced apoptosis is more severe in a CypB knockdown model, confirming that CypB protects against Aβ(25-35)-induced toxicity. Consequently, these findings suggest that CypB may protect against Aβ toxicity by its antioxidant properties, by regulating MAPK and PI-3-K signaling, and through the ER stress pathway. Copyright © 2011 Elsevier Inc. All rights reserved.

The level of CD147 expression correlates with cyclophilin-induced signalling and chemotaxis

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Constant Stephanie

2011-10-01

Full Text Available Abstract Background Previous studies identified CD147 as the chemotactic receptor on inflammatory leukocytes for extracellular cyclophilins (eCyp. However, CD147 is not known to associate with signal transducing molecules, so other transmembrane proteins, such as proteoglycans, integrins, and CD98, were suggested as receptor or co-receptor for eCyp. CD147 is ubiquitously expressed on many cell types, but relationship between the level of CD147 expression and cellular responses to eCyp has never been analyzed. Given the role of eCyp in pathogenesis of many diseases, it is important to know whether cellular responses to eCyp are regulated at the level of CD147 expression. Results Here, we manipulated CD147 expression levels on HeLa cells using RNAi and investigated the signalling and chemotactic responses to eCypA. Both Erk activation and chemotaxis correlated with the level of CD147 expression, with cells exhibiting low level expression being practically unresponsive to eCypA. Conclusions Our results provide the first demonstration of a chemotactic response of HeLa cells to eCypA, establish a correlation between the level of CD147 expression and the magnitude of cellular responses to eCypA, and indicate that CD147 may be a limiting factor in the receptor complex determining cyclophilin-induced Erk activation and cell migration.

Interaction with glycosaminoglycans is required for cyclophilin B to trigger integrin-mediated adhesion of peripheral blood T lymphocytes to extracellular matrix

OpenAIRE

Allain, Fabrice; Vanpouille, Christophe; Carpentier, Mathieu; Slomianny, Marie-Christine; Durieux, Sandrine; Spik, Geneviève

2002-01-01

Cyclophilins A and B (CyPA and CyPB) are cyclosporin A-binding proteins that are involved in inflammatory events. We have reported that CyPB interacts with two types of cell-surface-binding sites. The first site corresponds to a functional receptor and requires interaction with the central core of CyPB. This region is highly conserved in cyclophilins, suggesting that CyPA and CyPB might share biological activities mediated by interaction with this receptor. The second site is identified with ...

Discovery of non-peptidic small molecule inhibitors of cyclophilin D as neuroprotective agents in Aβ-induced mitochondrial dysfunction

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Park, Insun; Londhe, Ashwini M.; Lim, Ji Woong; Park, Beoung-Geon; Jung, Seo Yun; Lee, Jae Yeol; Lim, Sang Min; No, Kyoung Tai; Lee, Jiyoun; Pae, Ae Nim

2017-10-01

Cyclophilin D (CypD) is a mitochondria-specific cyclophilin that is known to play a pivotal role in the formation of the mitochondrial permeability transition pore (mPTP).The formation and opening of the mPTP disrupt mitochondrial homeostasis, cause mitochondrial dysfunction and eventually lead to cell death. Several recent studies have found that CypD promotes the formation of the mPTP upon binding to β amyloid (Aβ) peptides inside brain mitochondria, suggesting that neuronal CypD has a potential to be a promising therapeutic target for Alzheimer's disease (AD). In this study, we generated an energy-based pharmacophore model by using the crystal structure of CypD—cyclosporine A (CsA) complex and performed virtual screening of ChemDiv database, which yielded forty-five potential hit compounds with novel scaffolds. We further tested those compounds using mitochondrial functional assays in neuronal cells and identified fifteen compounds with excellent protective effects against Aβ-induced mitochondrial dysfunction. To validate whether these effects derived from binding to CypD, we performed surface plasmon resonance (SPR)—based direct binding assays with selected compounds and discovered compound 29 was found to have the equilibrium dissociation constants (KD) value of 88.2 nM. This binding affinity value and biological activity correspond well with our predicted binding mode. We believe that this study offers new insights into the rational design of small molecule CypD inhibitors, and provides a promising lead for future therapeutic development.

Cyclophilin 40 facilitates HSP90-mediated RISC assembly in plants.

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Iki, Taichiro; Yoshikawa, Manabu; Meshi, Tetsuo; Ishikawa, Masayuki

2012-01-18

Posttranscriptional gene silencing is mediated by RNA-induced silencing complexes (RISCs) that contain AGO proteins and single-stranded small RNAs. The assembly of plant AGO1-containing RISCs depends on the molecular chaperone HSP90. Here, we demonstrate that cyclophilin 40 (CYP40), protein phosphatase 5 (PP5), and several other proteins with the tetratricopeptide repeat (TPR) domain associates with AGO1 in an HSP90-dependent manner in extracts of evacuolated tobacco protoplasts (BYL). Intriguingly, CYP40, but not the other TPR proteins, could form a complex with small RNA duplex-bound AGO1. Moreover, CYP40 that was synthesized by in-vitro translation using BYL uniquely facilitated binding of small RNA duplexes to AGO1, and as a result, increased the amount of mature RISCs that could cleave target RNAs. CYP40 was not contained in mature RISCs, indicating that the association is transient. Addition of PP5 or cyclophilin-binding drug cyclosporine A prevented the association of endogenous CYP40 with HSP90-AGO1 complex and inhibited RISC assembly. These results suggest that a complex of AGO1, HSP90, CYP40, and a small RNA duplex is a key intermediate of RISC assembly in plants.

Pancreatic acinar cells-derived cyclophilin A promotes pancreatic damage by activating NF-κB pathway in experimental pancreatitis

International Nuclear Information System (INIS)

Yu, Ge; Wan, Rong; Hu, Yanling; Ni, Jianbo; Yin, Guojian; Xing, Miao; Shen, Jie; Tang, Maochun; Chen, Congying; Fan, Yuting; Xiao, Wenqin; Zhao, Yan; Wang, Xingpeng

2014-01-01

Highlights: • CypA is upregulated in experimental pancreatitis. • CCK induces expression and release of CypA in acinar cell in vitro. • rCypA aggravates CCK-induced acinar cell death and inflammatory cytokine production. • rCypA activates the NF-κB pathway in acinar cells in vitro. - Abstract: Inflammation triggered by necrotic acinar cells contributes to the pathophysiology of acute pancreatitis (AP), but its precise mechanism remains unclear. Recent studies have shown that Cyclophilin A (CypA) released from necrotic cells is involved in the pathogenesis of several inflammatory diseases. We therefore investigated the role of CypA in experimental AP induced by administration of sodium taurocholate (STC). CypA was markedly upregulated and widely expressed in disrupted acinar cells, infiltrated inflammatory cells, and tubular complexes. In vitro, it was released from damaged acinar cells by cholecystokinin (CCK) induction. rCypA (recombinant CypA) aggravated CCK-induced acinar cell necrosis, promoted nuclear factor (NF)-κB p65 activation, and increased cytokine production. In conclusion, CypA promotes pancreatic damage by upregulating expression of inflammatory cytokines of acinar cells via the NF-κB pathway

Pancreatic acinar cells-derived cyclophilin A promotes pancreatic damage by activating NF-κB pathway in experimental pancreatitis

Energy Technology Data Exchange (ETDEWEB)

Yu, Ge [Department of Gastroenterology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai (China); Wan, Rong [Department of Gastroenterology, Shanghai First People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai (China); Hu, Yanling [Department of Gastroenterology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai (China); Ni, Jianbo [Department of Gastroenterology, Shanghai First People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai (China); Yin, Guojian; Xing, Miao [Department of Gastroenterology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai (China); Shen, Jie [Department of Gastroenterology, Shanghai First People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai (China); Tang, Maochun [Department of Gastroenterology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai (China); Chen, Congying [Department of Gastroenterology, Shanghai First People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai (China); Fan, Yuting; Xiao, Wenqin; Zhao, Yan [Department of Gastroenterology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai (China); Wang, Xingpeng, E-mail: wangxingpeng@hotmail.com [Department of Gastroenterology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai (China); Department of Gastroenterology, Shanghai First People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai (China); and others

2014-01-31

Highlights: • CypA is upregulated in experimental pancreatitis. • CCK induces expression and release of CypA in acinar cell in vitro. • rCypA aggravates CCK-induced acinar cell death and inflammatory cytokine production. • rCypA activates the NF-κB pathway in acinar cells in vitro. - Abstract: Inflammation triggered by necrotic acinar cells contributes to the pathophysiology of acute pancreatitis (AP), but its precise mechanism remains unclear. Recent studies have shown that Cyclophilin A (CypA) released from necrotic cells is involved in the pathogenesis of several inflammatory diseases. We therefore investigated the role of CypA in experimental AP induced by administration of sodium taurocholate (STC). CypA was markedly upregulated and widely expressed in disrupted acinar cells, infiltrated inflammatory cells, and tubular complexes. In vitro, it was released from damaged acinar cells by cholecystokinin (CCK) induction. rCypA (recombinant CypA) aggravated CCK-induced acinar cell necrosis, promoted nuclear factor (NF)-κB p65 activation, and increased cytokine production. In conclusion, CypA promotes pancreatic damage by upregulating expression of inflammatory cytokines of acinar cells via the NF-κB pathway.

Cyclophilin B as a co-regulator of prolactin-induced gene expression and function in breast cancer cells.

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Fang, Feng; Zheng, Jiamao; Galbaugh, Traci L; Fiorillo, Alyson A; Hjort, Elizabeth E; Zeng, Xianke; Clevenger, Charles V

2010-06-01

The effects of prolactin (PRL) during the pathogenesis of breast cancer are mediated in part though Stat5 activity enhanced by its interaction with its transcriptional inducer, the prolyl isomerase cyclophilin B (CypB). We have demonstrated that knockdown of CypB decreases cell growth, proliferation, and migration, and CypB expression is associated with malignant progression of breast cancer. In this study, we examined the effect of CypB knockdown on PRL signaling in breast cancer cells. CypB knockdown with two independent siRNAs was shown to impair PRL-induced reporter expression in breast cancer cell line. cDNA microarray analysis was performed on these cells to assess the effect of CypB reduction, and revealed a significant decrease in PRL-induced endogenous gene expression in two breast cancer cell lines. Parallel functional assays revealed corresponding alterations of both anchorage-independent cell growth and cell motility of breast cancer cells. Our results demonstrate that CypB expression levels significantly modulate PRL-induced function in breast cancer cells ultimately resulting in enhanced levels of PRL-responsive gene expression, cell growth, and migration. Given the increasingly appreciated role of PRL in the pathogenesis of breast cancer, the actions of CypB detailed here are of biological significance.

Targeting colorectal cancer cells by a novel sphingosine kinase 1 inhibitor PF-543

International Nuclear Information System (INIS)

Ju, TongFa; Gao, DaQuan; Fang, Zheng-yu

2016-01-01

In this study, we showed that PF-543, a novel sphingosine kinase 1 (SphK1) inhibitor, exerted potent anti-proliferative and cytotoxic effects against a panel of established (HCT-116, HT-29 and DLD-1) and primary human colorectal cancer (CRC) cells. Its sensitivity was negatively associated with SphK1 expression level in the CRC cells. Surprisingly, PF-543 mainly induced programmed necrosis, but not apoptosis, in the CRC cells. CRC cell necrotic death was detected by lactate dehydrogenase (LDH) release, mitochondrial membrane potential (MMP) collapse and mitochondrial P53-cyclophilin-D (Cyp-D) complexation. Correspondingly, the necrosis inhibitor necrostatin-1 largely attenuated PF-543-induced cytotoxicity against CRC cells. Meanwhile, the Cyp-D inhibitors (sanglifehrin A and cyclosporin A), or shRNA-mediated knockdown of Cyp-D, remarkably alleviated PF-543-induced CRC cell necrotic death. Reversely, over-expression of wild-type Cyp-D in HCT-116 cells significantly increased PF-543's sensitivity. In vivo, PF-543 intravenous injection significantly suppressed HCT-116 xenograft growth in severe combined immunodeficient (SCID) mice, whiling remarkably improving the mice survival. The in vivo activity by PF-543 was largely attenuated when combined with the Cyp-D inhibitor cyclosporin A. Collectively, our results demonstrate that PF-543 exerts potent anti-CRC activity in vitro and in vivo. Mitochondrial programmed necrosis pathway is likely the key mechanism responsible for PF-543's actions in CRC cells. - Highlights: • PF-543 is anti-proliferative and cytotoxic to established and primary CRC cells. • PF-543 induces programmed necrosis, but not apoptosis, in CRC cells. • Modulation of mitochondrial protein cyclophilin-D alters PF-543's sensitivity. • PF-543 inhibits HCT-116 xenograft growth in SCID mice, improving mice survival. • Co-administration of cyclophilin-D inhibitor CsA inhibits PF-543's activity in vivo.

Targeting colorectal cancer cells by a novel sphingosine kinase 1 inhibitor PF-543

Energy Technology Data Exchange (ETDEWEB)

Ju, TongFa [Department of Anal-colorectal Surgery, HangZhou First People' s Hospital, HangZhou (China); Gao, DaQuan [Hematological Department, HangZhou First People' s Hospital, HangZhou (China); Fang, Zheng-yu, E-mail: fangzhengyu158@sina.com [Department of Anal-colorectal Surgery, The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou (China)

2016-02-12

In this study, we showed that PF-543, a novel sphingosine kinase 1 (SphK1) inhibitor, exerted potent anti-proliferative and cytotoxic effects against a panel of established (HCT-116, HT-29 and DLD-1) and primary human colorectal cancer (CRC) cells. Its sensitivity was negatively associated with SphK1 expression level in the CRC cells. Surprisingly, PF-543 mainly induced programmed necrosis, but not apoptosis, in the CRC cells. CRC cell necrotic death was detected by lactate dehydrogenase (LDH) release, mitochondrial membrane potential (MMP) collapse and mitochondrial P53-cyclophilin-D (Cyp-D) complexation. Correspondingly, the necrosis inhibitor necrostatin-1 largely attenuated PF-543-induced cytotoxicity against CRC cells. Meanwhile, the Cyp-D inhibitors (sanglifehrin A and cyclosporin A), or shRNA-mediated knockdown of Cyp-D, remarkably alleviated PF-543-induced CRC cell necrotic death. Reversely, over-expression of wild-type Cyp-D in HCT-116 cells significantly increased PF-543's sensitivity. In vivo, PF-543 intravenous injection significantly suppressed HCT-116 xenograft growth in severe combined immunodeficient (SCID) mice, whiling remarkably improving the mice survival. The in vivo activity by PF-543 was largely attenuated when combined with the Cyp-D inhibitor cyclosporin A. Collectively, our results demonstrate that PF-543 exerts potent anti-CRC activity in vitro and in vivo. Mitochondrial programmed necrosis pathway is likely the key mechanism responsible for PF-543's actions in CRC cells. - Highlights: • PF-543 is anti-proliferative and cytotoxic to established and primary CRC cells. • PF-543 induces programmed necrosis, but not apoptosis, in CRC cells. • Modulation of mitochondrial protein cyclophilin-D alters PF-543's sensitivity. • PF-543 inhibits HCT-116 xenograft growth in SCID mice, improving mice survival. • Co-administration of cyclophilin-D inhibitor CsA inhibits PF-543's activity in vivo.

Computational insight into small molecule inhibition of cyclophilins.

Science.gov (United States)

Sambasivarao, Somisetti V; Acevedo, Orlando

2011-02-28

Cyclophilins (Cyp) are a family of cellular enzymes possessing peptidyl-prolyl isomerase activity, which catalyze the cis-trans interconversion of proline-containing peptide bonds. The two most abundant family members, CypA and CypB, have been identified as valid drug targets for a wide range of diseases, including HCV, HIV, and multiple cancers. However, the development of small molecule inhibitors that possess nM potency and high specificity for a particular Cyp is difficult given the complete conservation of all active site residues between the enzymes. Monte Carlo statistical sampling coupled to free energy perturbation theory (MC/FEP) calculations have been carried out to elucidate the origin of the experimentally observed nM inhibition of CypA by acylurea-based derivatives and the >200-fold in vitro selectivity between CypA and CypB from aryl 1-indanylketone-based μM inhibitors. The computed free-energies of binding were in close accord with those derived from experiments. Binding affinity values for the inhibitors were determined to be dependent upon the stabilization strength of the nonbonded interactions provided toward two catalytic residues: Arg55 and Asn102 in CypA and the analogous Arg63 and Asn110 residues in CypB. Fine-tuning of the hydrophobic interactions allowed for enhanced potency among derivatives. The aryl 1-indanylketones are predicted to differentiate between the cyclophilins by using distinct binding motifs that exploit subtle differences in the active site arrangements. Ideas for the development of new selective compounds with the potential for advancement to low-nanomolar inhibition are presented.

Cyclophilin B Expression Is Associated with In Vitro Radioresistance and Clinical Outcome after Radiotherapy

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Paul D. Williams

2011-12-01

Full Text Available The tools for predicting clinical outcome after radiotherapy are not yet optimal. To improve on this, we applied the COXEN informatics approach to in vitro radiation sensitivity data of transcriptionally profiled human cells and gene expression data from untreated head and neck squamous cell carcinoma (HNSCC and bladder tumors to generate a multigene predictive model that is independent of histologic findings and reports on tumor radiosensitivity. The predictive ability of this 41-gene model was evaluated in patients with HNSCC and was found to stratify clinical outcome after radiotherapy. In contrast, this model was not useful in stratifying similar patients not treated with radiation. This led us to hypothesize that expression of some of the 41 genes contributes to tumor radioresistance and clinical recurrence. Hence, we evaluated the expression the 41 genes as a function of in vitro radioresistance in the NCI-60 cancer cell line panel and found cyclophilin B (PPIB, a peptidylprolyl isomerase and target of cyclosporine A (CsA, had the strongest direct correlation. Functional inhibition of PPIB by small interfering RNA depletion or CsA treatment leads to radiosensitization in cancer cells and reduced cellular DNA repair. Immunohistochemical evaluation of PPIB expression in patients with HNSCC was found to be associated with outcome after radiotherapy. This work demonstrates that a novel 41-gene expression model of radiation sensitivity developed in bladder cancer cell lines and human skin fibroblasts predicts clinical outcome after radiotherapy in head and neck cancer patients and identifies PPIB as a potential target for clinical radiosensitization.

Cyclophilin B interacts with sodium-potassium ATPase and is required for pump activity in proximal tubule cells of the kidney.

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Suñé, Guillermo; Sarró, Eduard; Puigmulé, Marta; López-Hellín, Joan; Zufferey, Madeleine; Pertel, Thomas; Luban, Jeremy; Meseguer, Anna

2010-11-10

Cyclophilins (Cyps), the intracellular receptors for Cyclosporine A (CsA), are responsible for peptidyl-prolyl cis-trans isomerisation and for chaperoning several membrane proteins. Those functions are inhibited upon CsA binding. Albeit its great benefits as immunosuppressant, the use of CsA has been limited by undesirable nephrotoxic effects, including sodium retention, hypertension, hyperkalemia, interstial fibrosis and progressive renal failure in transplant recipients. In this report, we focused on the identification of novel CypB-interacting proteins to understand the role of CypB in kidney function and, in turn, to gain further insight into the molecular mechanisms of CsA-induced toxicity. By means of yeast two-hybrid screens with human kidney cDNA, we discovered a novel interaction between CypB and the membrane Na/K-ATPase β1 subunit protein (Na/K-β1) that was confirmed by pull-down, co-immunoprecipitation and confocal microscopy, in proximal tubule-derived HK-2 cells. The Na/K-ATPase pump, a key plasma membrane transporter, is responsible for maintenance of electrical Na+ and K+ gradients across the membrane. We showed that CypB silencing produced similar effects on Na/K-ATPase activity than CsA treatment in HK-2 cells. It was also observed an enrichment of both alpha and beta subunits in the ER, what suggested a possible failure on the maturation and routing of the pump from this compartment towards the plasma membrane. These data indicate that CypB through its interaction with Na/K-β1 might regulate maturation and trafficking of the pump through the secretory pathway, offering new insights into the relationship between cyclophilins and the nephrotoxic effects of CsA.

Mitochondrial Cyclophilin D in Vascular Oxidative Stress and Hypertension.

Science.gov (United States)

Itani, Hana A; Dikalova, Anna E; McMaster, William G; Nazarewicz, Rafal R; Bikineyeva, Alfiya T; Harrison, David G; Dikalov, Sergey I

2016-06-01

Vascular superoxide (O˙2 (-)) and inflammation contribute to hypertension. The mitochondria are an important source of O˙2 (-); however, the regulation of mitochondrial O˙2 (-) and the antihypertensive potential of targeting the mitochondria remain poorly defined. Angiotensin II and inflammatory cytokines, such as interleukin 17A and tumor necrosis factor-α (TNFα) significantly contribute to hypertension. We hypothesized that angiotensin II and cytokines co-operatively induce cyclophilin D (CypD)-dependent mitochondrial O˙2 (-) production in hypertension. We tested whether CypD inhibition attenuates endothelial oxidative stress and reduces hypertension. CypD depletion in CypD(-/-) mice prevents overproduction of mitochondrial O˙2 (-) in angiotensin II-infused mice, attenuates hypertension by 20 mm Hg, and improves vascular relaxation compared with wild-type C57Bl/6J mice. Treatment of hypertensive mice with the specific CypD inhibitor Sanglifehrin A reduces blood pressure by 28 mm Hg, inhibits production of mitochondrial O˙2 (-) by 40%, and improves vascular relaxation. Angiotensin II-induced hypertension was associated with CypD redox activation by S-glutathionylation, and expression of the mitochondria-targeted H2O2 scavenger, catalase, abolished CypD S-glutathionylation, prevented stimulation mitochondrial O˙2 (-), and attenuated hypertension. The functional role of cytokine-angiotensin II interplay was confirmed by co-operative stimulation of mitochondrial O˙2 (-) by 3-fold in cultured endothelial cells and impairment of aortic relaxation incubated with combination of angiotensin II, interleukin 17A, and tumor necrosis factor-α which was prevented by CypD depletion or expression of mitochondria-targeted SOD2 and catalase. These data support a novel role of CypD in hypertension and demonstrate that targeting CypD decreases mitochondrial O˙2 (-), improves vascular relaxation, and reduces hypertension. © 2016 American Heart Association, Inc.

Role of cyclophilin B in tumorigenesis and cisplatin resistance in hepatocellular carcinoma in humans.

Science.gov (United States)

Kim, Yeonghwan; Jang, Miran; Lim, Sangbin; Won, Hyeran; Yoon, Kyung-Sik; Park, Jae-Hoon; Kim, Hyo Jong; Kim, Byung-Ho; Park, Won-Sang; Ha, Joohun; Kim, Sung-Soo

2011-11-01

Cyclophilin B (CypB) performs diverse roles in living cells, but its role in hepatocellular carcinoma (HCC) is largely unclear. To reveal its role in HCC, we investigated the induction of CypB under hypoxia and its functions in tumor cells in vitro and in vivo. Here, we demonstrated that hypoxia-inducible factor 1α (HIF-1α) induces CypB under hypoxia. Interestingly, CypB protected tumor cells, even p53-defective HCC cells, against hypoxia- and cisplatin-induced apoptosis. Furthermore, it regulated the effects of HIF-1α, including those in angiogenesis and glucose metabolism, via a positive feedback loop with HIF-1α. The tumorigenic and chemoresistant effects of CypB were confirmed in vivo using a xenograft model. Finally, we showed that CypB is overexpressed in 78% and 91% of the human HCC and colon cancer tissues, respectively, and its overexpression in these cancers reduced patient survival. These results indicate that CypB induced by hypoxia stimulates the survival of HCC via a positive feedback loop with HIF-1α, indicating that CypB is a novel candidate target for developing chemotherapeutic agents against HCC and colon cancer. Copyright © 2011 American Association for the Study of Liver Diseases.

Hypoxia induces cyclophilin B through the activation of transcription factor 6 in gastric adenocarcinoma cells.

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Jeong, Kwon; Kim, Kiyoon; Kim, Hunsung; Oh, Yoojung; Kim, Seong-Jin; Jo, Yunhee; Choe, Wonchae

2015-06-01

Hypoxia is an important form of physiological stress that induces cell death, due to the resulting endoplasmic reticulum (ER) stress, particularly in solid tumors. Although previous studies have indicated that cyclophilin B (CypB) plays a role in ER stress, there is currently no direct information supporting the mechanism of CypB involvement under hypoxic conditions. However, it has previously been demonstrated that ER stress positively regulates the expression of CypB. In the present study, it was demonstrated that CypB is transcriptionally regulated by hypoxia-mediated activation of transcription factor 6 (ATF6), an ER stress transcription factor. Subsequently, the effects of ATF6 on CypB promoter activity were investigated and an ATF6-responsive region in the promoter was identified. Hypoxia and ATF6 expression each increased CypB promoter activity. Collectively, these results demonstrate that ATF6 positively regulates the expression of CypB by binding to an ATF6-responsive region in the promoter, which may play an important role in the attenuation of apoptosis in the adaption to hypoxia. These results suggest that CypB may be a key molecule in the adaptation of cells to hypoxic conditions.

Distribution of cyclophilin B-binding sites in the subsets of human peripheral blood lymphocytes.

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Denys, A; Allain, F; Foxwell, B; Spik, G

1997-08-01

Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein, mainly associated with the secretory pathway and released in biological fluids. We have recently demonstrated that both free CyPB and CyPB-CsA complex specifically bind to peripheral blood T lymphocytes and are internalized. These results suggest that CyPB might promote the targeting of the drug into sensitive cells. Peripheral blood lymphocytes are subdivided in several populations according to their biological functions and sensitivity to CsA. We have investigated the binding of CyPB to these different subsets using a CyPB derivatized by fluorescein through its single cysteine which retains its binding properties. We have confirmed that only T cells were involved in the interaction with CyPB. The ligand binding was found to be heterogeneously distributed on the different T-cell subsets and surface-bound CyPB was mainly associated with the CD4-positive cells. No significant difference was noted between the CD45RA and CD45RO subsets, demonstrating that CyPB-binding sites were equally distributed between native and memory T cells. CD3 stimulation of T lymphocytes led to a decrease in the CyPB-binding capacity, that may be explained by a down-regulation of the CyPB-receptor expression upon T-cell activation. Finally, we demonstrated that CyPB-receptor-positive cells, isolated on CyPB sulphydryl-coupled affinity matrices, are more sensitive to CyPB-complexed CsA than mixed peripheral blood lymphocytes, suggesting that CyPB potentiates CsA activity through the binding of the complex. Taken together, our results demonstrate that CyPB-binding sites are mainly associated with resting cells of the helper T lymphocyte, and that CyPB might modulate the distribution of CsA through the drug targeting to sensitive cells.

Cellular growth kinetics distinguish a cyclophilin inhibitor from an HSP90 inhibitor as a selective inhibitor of hepatitis C virus.

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Rudolf K F Beran

Full Text Available During antiviral drug discovery, it is critical to distinguish molecules that selectively interrupt viral replication from those that reduce virus replication by adversely affecting host cell viability. In this report we investigate the selectivity of inhibitors of the host chaperone proteins cyclophilin A (CypA and heat-shock protein 90 (HSP90 which have each been reported to inhibit replication of hepatitis C virus (HCV. By comparing the toxicity of the HSP90 inhibitor, 17-(Allylamino-17-demethoxygeldanamycin (17-AAG to two known cytostatic compounds, colchicine and gemcitabine, we provide evidence that 17-AAG exerts its antiviral effects indirectly through slowing cell growth. In contrast, a cyclophilin inhibitor, cyclosporin A (CsA, exhibited selective antiviral activity without slowing cell proliferation. Furthermore, we observed that 17-AAG had little antiviral effect in a non-dividing cell-culture model of HCV replication, while CsA reduced HCV titer by more than two orders of magnitude in the same model. The assays we describe here are useful for discriminating selective antivirals from compounds that indirectly affect virus replication by reducing host cell viability or slowing cell growth.

Synthesis of heparan sulfate with cyclophilin B-binding properties is determined by cell type-specific expression of sulfotransferases.

NARCIS (Netherlands)

Deligny, A.; Denys, A.; Marcant, A.; Melchior, A.; Mazurier, J.; Kuppevelt, A.H.M.S.M. van; Allain, F.

2010-01-01

Cyclophilin B (CyPB) induces migration and adhesion of T lymphocytes via a mechanism that requires interaction with 3-O-sulfated heparan sulfate (HS). HS biosynthesis is a complex process with many sulfotransferases involved. N-Deacetylases/N-sulfotransferases are responsible for N-sulfation, which

Downregulation of Cyclophilin A by siRNA diminishes non-small cell lung cancer cell growth and metastasis via the regulation of matrix metallopeptidase 9

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Qian Zhe

2012-10-01

Full Text Available Abstract Background Cyclophilin A (CypA is a cytosolic protein possessing peptidyl-prolyl isomerase activity that was recently reported to be overexpressed in several cancers. Here, we explored the biology and molecular mechanism of CypA in non-small cell lung cancer (NSCLC. Methods The expression of CypA in human NSCLC cell lines was detected by real-time reverse transcription PCR. The RNA interference-mediated knockdown of CypA was established in two NSCLC cell lines (95C and A549. 239836 CypA inhibitor was also used to suppress CypA activity. Tumorigenesis was assessed based on cellular proliferation, colony formation assays, and anchorage-independent growth assays; metastasis was assessed based on wound healing and transwell assays. Results Suppression of CypA expression inhibited the cell growth and colony formation of A549 and 95C cells. CypA knockdown resulted in the inhibition of cell motility and invasion. Significantly, we show for the first time that CypA increased NSCLC cell invasion by regulating the activity of secreted matrix metallopeptidase 9 (MMP9. Likewise, suppression of CypA with 239836 CypA inhibitor decreased cell proliferation and MMP9 activity. Conclusions The suppression of CypA expression was correlated with decreased NSCLC cell tumorigenesis and metastasis.

Cyclophilin B interacts with sodium-potassium ATPase and is required for pump activity in proximal tubule cells of the kidney.

Directory of Open Access Journals (Sweden)

Guillermo Suñé

Full Text Available Cyclophilins (Cyps, the intracellular receptors for Cyclosporine A (CsA, are responsible for peptidyl-prolyl cis-trans isomerisation and for chaperoning several membrane proteins. Those functions are inhibited upon CsA binding. Albeit its great benefits as immunosuppressant, the use of CsA has been limited by undesirable nephrotoxic effects, including sodium retention, hypertension, hyperkalemia, interstial fibrosis and progressive renal failure in transplant recipients. In this report, we focused on the identification of novel CypB-interacting proteins to understand the role of CypB in kidney function and, in turn, to gain further insight into the molecular mechanisms of CsA-induced toxicity. By means of yeast two-hybrid screens with human kidney cDNA, we discovered a novel interaction between CypB and the membrane Na/K-ATPase β1 subunit protein (Na/K-β1 that was confirmed by pull-down, co-immunoprecipitation and confocal microscopy, in proximal tubule-derived HK-2 cells. The Na/K-ATPase pump, a key plasma membrane transporter, is responsible for maintenance of electrical Na+ and K+ gradients across the membrane. We showed that CypB silencing produced similar effects on Na/K-ATPase activity than CsA treatment in HK-2 cells. It was also observed an enrichment of both alpha and beta subunits in the ER, what suggested a possible failure on the maturation and routing of the pump from this compartment towards the plasma membrane. These data indicate that CypB through its interaction with Na/K-β1 might regulate maturation and trafficking of the pump through the secretory pathway, offering new insights into the relationship between cyclophilins and the nephrotoxic effects of CsA.

Uncaria rhynchophylla upregulates the expression of MIF and cyclophilin A in kainic acid-induced epilepsy rats: A proteomic analysis.

Science.gov (United States)

Lo, Wan-Yu; Tsai, Fuu-Jen; Liu, Chung-Hsiang; Tang, Nou-Ying; Su, Shan-Yu; Lin, Shinn-Zong; Chen, Chun-Chung; Shyu, Woei-Cherng; Hsieh, Ching-Liang

2010-01-01

Uncaria rhynchophylla (Miq) Jack (UR) is a traditional Chinese herb and is used for the treatment of convulsive disorders, including epilepsy. Our previous study has shown that UR, as well as its major component rhynchophylline (RH), has an anticonvulsive effect and this effect is closely related to its scavenging activities of oxygen free radicals. The purpose of the present study was to investigate the effect of (UR) on the expression of proteins using a proteomics analysis in Sprague-Dawley (SD) rats with kainic acid (KA)-induced epileptic seizures. We profiled the differentially expressed proteins on two-dimensional electrophoresis (2-DE) maps derived from the frontal cortex and hippocampus of rat brain tissue 24 hours after KA-induced epileptic seizures. The results indicated that macrophage migration inhibitory factor (MIF) and cyclophilin A were under expressed in frontal cortex by an average of 0.19- and 0.23-fold, respectively. In the frontal cortex, MIF and cyclophilin A were significantly decreased in the KA group and these decreases were confirmed by the Western blots. However, in the hippocampus, only cyclophilin A was significantly decreased in the KA group. In addition, in real-time quantitative PCR (Q-PCR), MIF and cyclophilin A gene expressions were also significantly under expressed in the frontal cortex, and only the cyclophilin A gene was also significantly under expressed in the hippocampus in the KA group. These under expressions of MIF and cyclophilin A could be overcome by the treatment of UR and RH. In conclusion, the under expressions of MIF and cyclophilin A in the frontal cortex and hippocampus in KA-treated rats, which were overcome by both UR and UH treatment, suggesting that both MIF and cyclophilin A at least partly participate in the anticonvulsive effect of UR.

Mutation in cyclophilin B that causes hyperelastosis cutis in American Quarter Horse does not affect peptidylprolyl cis-trans isomerase activity but shows altered cyclophilin B-protein interactions and affects collagen folding.

Science.gov (United States)

Ishikawa, Yoshihiro; Vranka, Janice A; Boudko, Sergei P; Pokidysheva, Elena; Mizuno, Kazunori; Zientek, Keith; Keene, Douglas R; Rashmir-Raven, Ann M; Nagata, Kazuhiro; Winand, Nena J; Bächinger, Hans Peter

2012-06-22

The rate-limiting step of folding of the collagen triple helix is catalyzed by cyclophilin B (CypB). The G6R mutation in cyclophilin B found in the American Quarter Horse leads to autosomal recessive hyperelastosis cutis, also known as hereditary equine regional dermal asthenia. The mutant protein shows small structural changes in the region of the mutation at the side opposite the catalytic domain of CypB. The peptidylprolyl cis-trans isomerase activity of the mutant CypB is normal when analyzed in vitro. However, the biosynthesis of type I collagen in affected horse fibroblasts shows a delay in folding and secretion and a decrease in hydroxylysine and glucosyl-galactosyl hydroxylysine. This leads to changes in the structure of collagen fibrils in tendon, similar to those observed in P3H1 null mice. In contrast to cyclophilin B null mice, where little 3-hydroxylation was found in type I collagen, 3-hydroxylation of type I collagen in affected horses is normal. The mutation disrupts the interaction of cyclophilin B with the P-domain of calreticulin, with lysyl hydroxylase 1, and probably other proteins, such as the formation of the P3H1·CypB·cartilage-associated protein complex, resulting in less effective catalysis of the rate-limiting step in collagen folding in the rough endoplasmic reticulum.

Mutation in Cyclophilin B That Causes Hyperelastosis Cutis in American Quarter Horse Does Not Affect Peptidylprolyl cis-trans Isomerase Activity but Shows Altered Cyclophilin B-Protein Interactions and Affects Collagen Folding*

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Ishikawa, Yoshihiro; Vranka, Janice A.; Boudko, Sergei P.; Pokidysheva, Elena; Mizuno, Kazunori; Zientek, Keith; Keene, Douglas R.; Rashmir-Raven, Ann M.; Nagata, Kazuhiro; Winand, Nena J.; Bächinger, Hans Peter

2012-01-01

The rate-limiting step of folding of the collagen triple helix is catalyzed by cyclophilin B (CypB). The G6R mutation in cyclophilin B found in the American Quarter Horse leads to autosomal recessive hyperelastosis cutis, also known as hereditary equine regional dermal asthenia. The mutant protein shows small structural changes in the region of the mutation at the side opposite the catalytic domain of CypB. The peptidylprolyl cis-trans isomerase activity of the mutant CypB is normal when analyzed in vitro. However, the biosynthesis of type I collagen in affected horse fibroblasts shows a delay in folding and secretion and a decrease in hydroxylysine and glucosyl-galactosyl hydroxylysine. This leads to changes in the structure of collagen fibrils in tendon, similar to those observed in P3H1 null mice. In contrast to cyclophilin B null mice, where little 3-hydroxylation was found in type I collagen, 3-hydroxylation of type I collagen in affected horses is normal. The mutation disrupts the interaction of cyclophilin B with the P-domain of calreticulin, with lysyl hydroxylase 1, and probably other proteins, such as the formation of the P3H1·CypB·cartilage-associated protein complex, resulting in less effective catalysis of the rate-limiting step in collagen folding in the rough endoplasmic reticulum. PMID:22556420

Cyclosporin A treatment of Leishmania donovani reveals stage-specific functions of cyclophilins in parasite proliferation and viability.

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Wai-Lok Yau

Full Text Available BACKGROUND: Cyclosporin A (CsA has important anti-microbial activity against parasites of the genus Leishmania, suggesting CsA-binding cyclophilins (CyPs as potential drug targets. However, no information is available on the genetic diversity of this important protein family, and the mechanisms underlying the cytotoxic effects of CsA on intracellular amastigotes are only poorly understood. Here, we performed a first genome-wide analysis of Leishmania CyPs and investigated the effects of CsA on host-free L. donovani amastigotes in order to elucidate the relevance of these parasite proteins for drug development. METHODOLOGY/PRINCIPAL FINDINGS: Multiple sequence alignment and cluster analysis identified 17 Leishmania CyPs with significant sequence differences to human CyPs, but with highly conserved functional residues implicated in PPIase function and CsA binding. CsA treatment of promastigotes resulted in a dose-dependent inhibition of cell growth with an IC50 between 15 and 20 microM as demonstrated by proliferation assay and cell cycle analysis. Scanning electron microscopy revealed striking morphological changes in CsA treated promastigotes reminiscent to developing amastigotes, suggesting a role for parasite CyPs in Leishmania differentiation. In contrast to promastigotes, CsA was highly toxic to amastigotes with an IC50 between 5 and 10 microM, revealing for the first time a direct lethal effect of CsA on the pathogenic mammalian stage linked to parasite thermotolerance, independent from host CyPs. Structural modeling, enrichment of CsA-binding proteins from parasite extracts by FPLC, and PPIase activity assays revealed direct interaction of the inhibitor with LmaCyP40, a bifunctional cyclophilin with potential co-chaperone function. CONCLUSIONS/SIGNIFICANCE: The evolutionary expansion of the Leishmania CyP protein family and the toxicity of CsA on host-free amastigotes suggest important roles of PPIases in parasite biology and implicate

Cyclophilin B enhances HIV-1 infection

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DeBoer, Jason; Madson, Christian J. [Department of Medical Microbiology and Immunology, Creighton University, Omaha, NE (United States); Belshan, Michael, E-mail: michaelbelshan@creighton.edu [Department of Medical Microbiology and Immunology, Creighton University, Omaha, NE (United States); The Nebraska Center for Virology, University of Nebraska, Lincoln, NE (United States)

2016-02-15

Cyclophilin B (CypB) is a member of the immunophilin family and intracellular chaperone. It predominantly localizes to the ER, but also contains a nuclear localization signal and is secreted from cells. CypB has been shown to interact with the Gag protein of human immunodeficiency type 1 (HIV-1). Several proteomic and genetic studies identified it as a potential factor involved in HIV replication. Herein, we show that over-expression of CypB enhances HIV infection by increasing nuclear import of viral DNA. This enhancement was unaffected by cyclosporine treatment and requires the N-terminus of the protein. The N-terminus contains an ER leader sequence, putative nuclear localization signal, and is required for secretion. Deletion of the N-terminus resulted in mislocalization from the ER and suppression of HIV infection. Passive transfer experiments showed that secreted CypB did not impact HIV infection. Combined, these experiments show that intracellular CypB modulates a pathway of HIV nuclear import. - Highlights: • CypB has been identified in several proteomic studies of HIV-1 infection. • CypB expression is upregulated in activated and infected T-cells. • Over-expression of CypB enhances HIV nuclear import and infection. • The N-terminus of CypB is necessary for these effects.

Cyclophilin B enhances HIV-1 infection

International Nuclear Information System (INIS)

DeBoer, Jason; Madson, Christian J.; Belshan, Michael

2016-01-01

Cyclophilin B (CypB) is a member of the immunophilin family and intracellular chaperone. It predominantly localizes to the ER, but also contains a nuclear localization signal and is secreted from cells. CypB has been shown to interact with the Gag protein of human immunodeficiency type 1 (HIV-1). Several proteomic and genetic studies identified it as a potential factor involved in HIV replication. Herein, we show that over-expression of CypB enhances HIV infection by increasing nuclear import of viral DNA. This enhancement was unaffected by cyclosporine treatment and requires the N-terminus of the protein. The N-terminus contains an ER leader sequence, putative nuclear localization signal, and is required for secretion. Deletion of the N-terminus resulted in mislocalization from the ER and suppression of HIV infection. Passive transfer experiments showed that secreted CypB did not impact HIV infection. Combined, these experiments show that intracellular CypB modulates a pathway of HIV nuclear import. - Highlights: • CypB has been identified in several proteomic studies of HIV-1 infection. • CypB expression is upregulated in activated and infected T-cells. • Over-expression of CypB enhances HIV nuclear import and infection. • The N-terminus of CypB is necessary for these effects.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of Arabidopsis thaliana cyclophilin 38 (AtCyp38)

International Nuclear Information System (INIS)

Vasudevan, Dileep; Gopalan, Gayathri; He, Zengyong; Luan, Sheng; Swaminathan, Kunchithapadam

2005-01-01

Crystallization of Arabidopsis thaliana cyclophilin 38. The crystal diffracts X-rays to 2.5 Å resolution. AtCyp38 is one of the highly divergent multidomain cyclophilins from Arabidopsis thaliana. A recombinant form of AtCyp38 (residues 83–437) was expressed in Escherichia coli and purified to homogeneity. The protein was crystallized using the vapour-batch technique with PEG 6000 and t-butanol as precipitants. Crystals of recombinant AtCyp38 diffracted X-rays to better than 2.5 Å resolution at 95 K using a synchrotron-radiation source. The crystal belongs to the C-centred orthorhombic space group C222 1 , with unit-cell parameters a = 58.2, b = 95.9, c = 167.5 Å, and contains one molecule in the asymmetric unit. The selenomethionine derivative of the AtCyp38 protein was overexpressed, purified and crystallized in the same space group and data were collected to 3.5 Å at the NSLS synchrotron. The structure is being solved by the MAD method

Cyclophilin A interacts with diverse lentiviral capsids

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Emerman Michael

2006-10-01

Full Text Available Abstract Background The capsid (CA protein of HIV-1 binds with high affinity to the host protein cyclophilin A (CypA. This binding positively affects some early stage of the viral life-cycle because prevention of binding either by drugs that occupy that active site of cyclophilin A, by mutation in HIV-1 CA, or RNAi that knocks down intracellular CypA level diminishes viral infectivity. The closely related lentivirus, SIVcpz also binds CypA, but it was thought that this interaction was limited to the HIV-1/SIVcpz lineage because other retroviruses failed to interact with CypA in a yeast two-hybrid assay. Results We find that diverse lentiviruses, FIV and SIVagmTAN also bind to CypA. Mutagenesis of FIV CA showed that an amino acid that is in a homologous position to the proline at amino acid 90 of HIV-1 CA is essential for FIV interactions with CypA. Conclusion These results demonstrate that CypA binding to lentiviruses is more widespread than previously thought and suggest that this interaction is evolutionarily important for lentiviral infection.

Cyclophilin B is involved in p300-mediated degradation of CHOP in tumor cell adaptation to hypoxia.

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Jeong, K; Kim, H; Kim, K; Kim, S-J; Hahn, B-S; Jahng, G-H; Yoon, K-S; Kim, S S; Ha, J; Kang, I; Choe, W

2014-03-01

The regulation of CCAAT/enhancer-binding protein-homologous protein (CHOP), an endoplasmic reticulum (ER) stress-response factor, is key to cellular survival. Hypoxia is a physiologically important stress that induces cell death in the context of the ER, especially in solid tumors. Although our previous studies have suggested that Cyclophilin B (CypB), a molecular chaperone, has a role in ER stress, currently, there is no direct information supporting its mechanism under hypoxia. Here, we demonstrate for the first time that CypB is associated with p300 E4 ligase, induces ubiquitination and regulates the proteasomal turnover of CHOP, one of the well-known pro-apoptotic molecules under hypoxia. Our findings show that CypB physically interacts with the N-terminal α-helix domain of CHOP under hypoxia and cooperates with p300 to modulate the ubiquitination of CHOP. We also show that CypB is transcriptionally induced through ATF6 under hypoxia. Collectively, these findings demonstrate that CypB prevents hypoxia-induced cell death through modulation of ubiquitin-mediated CHOP protein degradation, suggesting that CypB may have an important role in the tight regulation of CHOP under hypoxia.

Cyclophilin B enhances HIV-1 infection.

Science.gov (United States)

DeBoer, Jason; Madson, Christian J; Belshan, Michael

2016-02-01

Cyclophilin B (CypB) is a member of the immunophilin family and intracellular chaperone. It predominantly localizes to the ER, but also contains a nuclear localization signal and is secreted from cells. CypB has been shown to interact with the Gag protein of human immunodeficiency type 1 (HIV-1). Several proteomic and genetic studies identified it as a potential factor involved in HIV replication. Herein, we show that over-expression of CypB enhances HIV infection by increasing nuclear import of viral DNA. This enhancement was unaffected by cyclosporine treatment and requires the N-terminus of the protein. The N-terminus contains an ER leader sequence, putative nuclear localization signal, and is required for secretion. Deletion of the N-terminus resulted in mislocalization from the ER and suppression of HIV infection. Passive transfer experiments showed that secreted CypB did not impact HIV infection. Combined, these experiments show that intracellular CypB modulates a pathway of HIV nuclear import. Copyright © 2015 Elsevier Inc. All rights reserved.

Enhancing the effect of secreted cyclophilin B on immunosuppressive activity of cyclosporine.

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Denys, A; Allain, F; Masy, E; Dessaint, J P; Spik, G

1998-04-27

Cyclophilin B (CyPB) is a cyclosporine (CsA)-binding protein, located within intracellular vesicles and secreted in biological fluids. In previous works, we reported that CyPB specifically interacts with the T-cell membrane and potentiates the ability of CsA to inhibit CD3-induced proliferation of T lymphocytes. CyPB levels were measured in plasma from healthy donors and transplant patients. The role of extracellular CyPB on the distribution and activity of CsA was investigated first by studies on the uptake of free and CyPB-complexed drug by blood cells, and second by studies on the inhibitory effects of these two compounds on the CD3-induced proliferation of peripheral blood mononuclear cells. A significant increase in plasma CyPB level was observed for CsA-treated patients (13+/-6.4 nM, n=42) in comparison with untreated donors (4.3+/-2.1 nM, n=34). In vitro, extracellular CyPB dose dependently modified CsA distribution between plasma, erythrocyte, and lymphocyte contents, by both retaining the complexed drug extracellularly and promoting its specific accumulation within peripheral blood mononuclear cells. Moreover, the enhanced ability of CyPB-complexed CsA to suppress CD3-induced T-cell proliferation was preserved in the presence of other blood cells, implying specific targeting of the drug to sensitive cells. Furthermore, although a large interindividual variability of sensitivity to the drug was confirmed for 18 individuals, we found that CyPB potentiated the activity of CsA in restoring a high sensitivity to the immunosuppressant. These results suggest that plasma CyPB may contribute to the acceptance and the good maintenance of organ transplantation by enhancing the immunosuppressive activity of CsA through a receptor-mediated incorporation of CyPB-complexed CsA within peripheral blood lymphocytes.

Preclinical characterization of naturally occurring polyketide cyclophilin inhibitors from the sanglifehrin family.

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Gregory, Matthew A; Bobardt, Michael; Obeid, Susan; Chatterji, Udayan; Coates, Nigel J; Foster, Teresa; Gallay, Philippe; Leyssen, Pieter; Moss, Steven J; Neyts, Johan; Nur-e-Alam, Mohammad; Paeshuyse, Jan; Piraee, Mahmood; Suthar, Dipen; Warneck, Tony; Zhang, Ming-Qiang; Wilkinson, Barrie

2011-05-01

Cyclophilin inhibitors currently in clinical trials for hepatitis C virus (HCV) are all analogues of cyclosporine (CsA). Sanglifehrins are a group of naturally occurring cyclophilin binding polyketides that are structurally distinct from the cyclosporines and are produced by a microorganism amenable to biosynthetic engineering for lead optimization and large-scale production by fermentation. Preclinical characterization of the potential utility of this class of compounds for the treatment of HCV revealed that the natural sanglifehrins A to D are all more potent than CsA at disrupting formation of the NS5A-CypA, -CypB, and -CypD complexes and at inhibition of CypA, CypB, and CypD isomerase activity. In particular, sanglifehrin B (SfB) was 30- to 50-fold more potent at inhibiting the isomerase activity of all Cyps tested than CsA and was also shown to be a more potent inhibitor of the 1b subgenomic replicon (50% effective concentrations [EC50s] of 0.070 μM and 0.16 μM in Huh 5-2 and Huh 9-13 cells, respectively). Physicochemical and mouse pharmacokinetic analyses revealed low oral bioavailability (F5 h). These data demonstrate that naturally occurring sanglifehrins are suitable lead compounds for the development of novel analogues that are less immunosuppressive and that have improved metabolism and pharmacokinetic properties.

Crystal structures of wild-type and mutated cyclophilin B that causes hyperelastosis cutis in the American quarter horse

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Boudko Sergei P

2012-11-01

Full Text Available Abstract Background Hyperelastosis cutis is an inherited autosomal recessive connective tissue disorder. Affected horses are characterized by hyperextensible skin, scarring, and severe lesions along the back. The disorder is caused by a mutation in cyclophilin B. Results The crystal structures of both wild-type and mutated (Gly6->Arg horse cyclophilin B are presented. The mutation neither affects the overall fold of the enzyme nor impairs the catalytic site structure. Instead, it locally rearranges the flexible N-terminal end of the polypeptide chain and also makes it more rigid. Conclusions Interactions of the mutated cyclophilin B with a set of endoplasmic reticulum-resident proteins must be affected.

Cyclosporine A suppresses immunoglobulin G biosynthesis via inhibition of cyclophilin B in murine hybridomas and B cells.

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Lee, Jisun; Choi, Tae Gyu; Ha, Joohun; Kim, Sung Soo

2012-01-01

Immunoglubulin G (IgG) is a major isotype of antibody, which is predominantly involved in immune response. The complete tetramer is needed to fold and assemble in endoplasmic reticulum (ER) prior to secretion from cells. Protein quality control guided by ER chaperons is most essential for full biological activity. Cyclophilin B (CypB) was initially identified as a high-affinity binding protein for the immunosuppressive drug Cyclosporine A (CsA). CsA suppresses organ rejection by halting productions of pro-inflammatory molecules in T cell and abolishes the enzymatic property of CypB that accelerates the folding of proteins by catalysing the isomerization of peptidyl-proline bonds in ER. Here, we reported that CsA significantly inhibited IgG biosynthesis at posttranslational level in antibody secreting cells. Moreover, CsA stimulated the extracellular secretion of CypB and induced ROS generation, leading to expressions of ER stress markers. In addition, the absence of intracellular CypB impaired the formation of ER multiprotein complex, which is most important for resisting ER stress. Interestingly, CsA interrupted IgG folding via occupying the PPIase domain of CypB in ER. Eventually, unfolded IgG is degraded via Herp-dependent ERAD pathway. Furthermore, IgG biosynthesis was really abrogated by inhibition of CypB in primary B cells. We established for the first time the immunosuppressive effect of CsA on B cells. Conclusively, the combined results of the current study suggest that CypB is a pivotal molecule for IgG biosynthesis in ER quality control. Copyright © 2011 Elsevier B.V. All rights reserved.

Cyclophilin B is a functional regulator of hepatitis C virus RNA polymerase.

Science.gov (United States)

Watashi, Koichi; Ishii, Naoto; Hijikata, Makoto; Inoue, Daisuke; Murata, Takayuki; Miyanari, Yusuke; Shimotohno, Kunitada

2005-07-01

Viruses depend on host-derived factors for their efficient genome replication. Here, we demonstrate that a cellular peptidyl-prolyl cis-trans isomerase (PPIase), cyclophilin B (CyPB), is critical for the efficient replication of the hepatitis C virus (HCV) genome. CyPB interacted with the HCV RNA polymerase NS5B to directly stimulate its RNA binding activity. Both the RNA interference (RNAi)-mediated reduction of endogenous CyPB expression and the induced loss of NS5B binding to CyPB decreased the levels of HCV replication. Thus, CyPB functions as a stimulatory regulator of NS5B in HCV replication machinery. This regulation mechanism for viral replication identifies CyPB as a target for antiviral therapeutic strategies.

Syndecan-1/CD147 association is essential for cyclophilin B-induced activation of p44/42 mitogen-activated protein kinases and promotion of cell adhesion and chemotaxis.

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Pakula, Rachel; Melchior, Aurélie; Denys, Agnès; Vanpouille, Christophe; Mazurier, Joël; Allain, Fabrice

2007-05-01

Many of the biological functions attributed to cell surface proteoglycans are dependent on the interaction with extracellular mediators through their heparan sulphate (HS) moieties and the participation of their core proteins in signaling events. A class of recently identified inflammatory mediators is secreted cyclophilins, which are mostly known as cyclosporin A-binding proteins. We previously demonstrated that cyclophilin B (CyPB) triggers chemotaxis and integrin-mediated adhesion of T lymphocytes mainly of the CD4+/CD45RO+ phenotype. These activities are related to interactions with two types of binding sites, CD147 and cell surface HS. Here, we demonstrate that CyPB-mediated adhesion of CD4+/CD45RO+ T cells is related to p44/42 mitogen-activated protein kinase (MAPK) activation by a mechanism involving CD147 and HS proteoglycans (HSPG). Although HSPG core proteins are represented by syndecan-1, -2, -4, CD44v3 and betaglycan in CD4+/CD45RO+ T cells, we found that only syndecan-1 is physically associated with CD147. The intensity of the heterocomplex increased in response to CyPB, suggesting a transient enhancement and/or stabilization in the association of CD147 to syndecan-1. Pretreatment with anti-syndecan-1 antibodies or knockdown of syndecan-1 expression by RNA interference dramatically reduced CyPB-induced p44/p42 MAPK activation and consequent migration and adhesion, supporting the model in which syndecan-1 serves as a binding subunit to form the fully active receptor of CyPB. Altogether, our findings provide a novel example of a soluble mediator in which a member of the syndecan family plays a critical role in efficient interaction with signaling receptors and initiation of cellular responses.

Human cellular restriction factors that target HIV-1 replication

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Jeang Kuan-Teh

2009-09-01

Full Text Available Abstract Recent findings have highlighted roles played by innate cellular factors in restricting intracellular viral replication. In this review, we discuss in brief the activities of apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G, bone marrow stromal cell antigen 2 (BST-2, cyclophilin A, tripartite motif protein 5 alpha (Trim5α, and cellular microRNAs as examples of host restriction factors that target HIV-1. We point to countermeasures encoded by HIV-1 for moderating the potency of these cellular restriction functions.

A BAX/BAK and cyclophilin D-independent intrinsic apoptosis pathway.

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Sebastián Zamorano

Full Text Available Most intrinsic death signals converge into the activation of pro-apoptotic BCL-2 family members BAX and BAK at the mitochondria, resulting in the release of cytochrome c and apoptosome activation. Chronic endoplasmic reticulum (ER stress leads to apoptosis through the upregulation of a subset of pro-apoptotic BH3-only proteins, activating BAX and BAK at the mitochondria. Here we provide evidence indicating that the full resistance of BAX and BAK double deficient (DKO cells to ER stress is reverted by stimulation in combination with mild serum withdrawal. Cell death under these conditions was characterized by the appearance of classical apoptosis markers, caspase-9 activation, release of cytochrome c, and was inhibited by knocking down caspase-9, but insensitive to BCL-X(L overexpression. Similarly, the resistance of BIM and PUMA double deficient cells to ER stress was reverted by mild serum withdrawal. Surprisingly, BAX/BAK-independent cell death did not require Cyclophilin D (CypD expression, an important regulator of the mitochondrial permeability transition pore. Our results suggest the existence of an alternative intrinsic apoptosis pathway emerging from a cross talk between the ER and the mitochondria.

Preclinical Characterization of Naturally Occurring Polyketide Cyclophilin Inhibitors from the Sanglifehrin Family▿†

Science.gov (United States)

Gregory, Matthew A.; Bobardt, Michael; Obeid, Susan; Chatterji, Udayan; Coates, Nigel J.; Foster, Teresa; Gallay, Philippe; Leyssen, Pieter; Moss, Steven J.; Neyts, Johan; Nur-e-Alam, Mohammad; Paeshuyse, Jan; Piraee, Mahmood; Suthar, Dipen; Warneck, Tony; Zhang, Ming-Qiang; Wilkinson, Barrie

2011-01-01

Cyclophilin inhibitors currently in clinical trials for hepatitis C virus (HCV) are all analogues of cyclosporine (CsA). Sanglifehrins are a group of naturally occurring cyclophilin binding polyketides that are structurally distinct from the cyclosporines and are produced by a microorganism amenable to biosynthetic engineering for lead optimization and large-scale production by fermentation. Preclinical characterization of the potential utility of this class of compounds for the treatment of HCV revealed that the natural sanglifehrins A to D are all more potent than CsA at disrupting formation of the NS5A-CypA, -CypB, and -CypD complexes and at inhibition of CypA, CypB, and CypD isomerase activity. In particular, sanglifehrin B (SfB) was 30- to 50-fold more potent at inhibiting the isomerase activity of all Cyps tested than CsA and was also shown to be a more potent inhibitor of the 1b subgenomic replicon (50% effective concentrations [EC50s] of 0.070 μM and 0.16 μM in Huh 5-2 and Huh 9-13 cells, respectively). Physicochemical and mouse pharmacokinetic analyses revealed low oral bioavailability (F 5 h). These data demonstrate that naturally occurring sanglifehrins are suitable lead compounds for the development of novel analogues that are less immunosuppressive and that have improved metabolism and pharmacokinetic properties. PMID:21383094

Cloning, purification, crystallization and preliminary X-ray crystallographic analysis of a cyclophilin A-like protein from Piriformospora indica

International Nuclear Information System (INIS)

Bhatt, Harshesh; Trivedi, Dipesh Kumar; Pal, Ravi Kant; Johri, Atul Kumar; Tuteja, Narendra; Bhavesh, Neel Sarovar

2012-01-01

Cyclophin A like protein from Piriformospora indica involved in salt-stress tolerance was cloned, overexpressed, purified and crystallized. The crystals obtained diffracted X-rays to 2 Å resolution and belonged to space group C222 1 . Cyclophilins are widely distributed both in eukaryotes and prokaryotes and have a primary role as peptidyl-prolyl cis–trans isomerases (PPIases). This study focuses on the cloning, expression, purification and crystallization of a salinity-stress-induced cyclophilin A (CypA) homologue from the symbiotic fungus Piriformospora indica. Crystallization experiments in the presence of 56 mM sodium phosphate monobasic monohydrate, 1.34 M potassium phosphate dibasic pH 8.2 yielded crystals that were suitable for X-ray diffraction analysis. The crystals belonged to the orthorhombic space group C222 1 , with unit-cell parameters a = 121.15, b = 144.12, c = 110.63 Å. The crystals diffracted to a resolution limit of 2.0 Å. Analysis of the diffraction data indicated the presence of three molecules of the protein per asymmetric unit (V M = 4.48 Å 3 Da −1 , 72.6% solvent content)

Expression of Cyclophilin B is Associated with Malignant Progression and Regulation of Genes Implicated in the Pathogenesis of Breast Cancer

OpenAIRE

Fang, Feng; Flegler, Ayanna J.; Du, Pan; Lin, Simon; Clevenger, Charles V.

2009-01-01

Cyclophilin B (CypB) is a 21-kDa protein with peptidyl-prolyl cis-trans isomerase activity that functions as a transcriptional inducer for Stat5 and as a ligand for CD147. To better understand the global function of CypB in breast cancer, T47D cells with a small interfering RNA-mediated knockdown of CypB were generated. Subsequent expression profiling analysis showed that 663 transcripts were regulated by CypB knockdown, and that many of these gene products contributed to cell proliferation, ...

The cyclophilin inhibitor Debio-025 shows potent anti-hepatitis C effect in patients coinfected with hepatitis C and human immunodeficiency virus.

Science.gov (United States)

Flisiak, Robert; Horban, Andrzej; Gallay, Philippe; Bobardt, Michael; Selvarajah, Suganya; Wiercinska-Drapalo, Alicja; Siwak, Ewa; Cielniak, Iwona; Higersberger, Jozef; Kierkus, Jarek; Aeschlimann, Christian; Grosgurin, Pierre; Nicolas-Métral, Valérie; Dumont, Jean-Maurice; Porchet, Hervé; Crabbé, Raf; Scalfaro, Pietro

2008-03-01

Debio-025 is an oral cyclophilin (Cyp) inhibitor with potent anti-hepatitis C virus activity in vitro. Its effect on viral load as well as its influence on intracellular Cyp levels was investigated in a randomized, double-blind, placebo-controlled study. Mean hepatitis C viral load decreased significantly by 3.6 log(10) after a 14-day oral treatment with 1200 mg twice daily (P CypB) levels in peripheral blood mononuclear cells decreased from 67 +/- 6 (standard error) ng/mg protein (baseline) to 5 +/- 1 ng/mg protein at day 15 (P CypB levels, coinciding with the decrease in hepatitis C viral load. These are the first preliminary human data supporting the hypothesis that CypB may play an important role in hepatitis C virus replication and that Cyp inhibition is a valid target for the development of anti-hepatitis C drugs.

Cyclophilin B facilitates the replication of Orf virus

OpenAIRE

Zhao, Kui; Li, Jida; He, Wenqi; Song, Deguang; Zhang, Ximu; Zhang, Di; Zhou, Yanlong; Gao, Feng

2017-01-01

Background Viruses interact with host cellular factors to construct a more favourable environment for their efficient replication. Expression of cyclophilin B (CypB), a cellular peptidyl-prolyl cis-trans isomerase (PPIase), was found to be significantly up-regulated. Recently, a number of studies have shown that CypB is important in the replication of several viruses, including Japanese encephalitis virus (JEV), hepatitis C virus (HCV) and human papillomavirus type 16 (HPV 16). However, the f...

Cyclophilin B expression in renal proximal tubules of hypertensive rats.

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Kainer, D B; Doris, P A

2000-04-01

Rat cyclophilin-like protein (Cy-LP) is a candidate hypertension gene initially identified by differential hybridization and implicated in renal mechanisms of salt retention and high blood pressure. We report the molecular characterization of rat cyclophilin B (CypB) and demonstrate, through sequence analysis and an allele-specific polymerase chain reaction primer assay, that CypB but not Cy-LP is expressed in rat kidney. CypB is an endoplasmic reticulum-localized prolyl-isomerase that interacts with elongation initiation factor 2-beta, an important regulator of protein translation and a central component of the endoplasmic reticulum stress response to hypoxia or ATP depletion. Active renal transport of sodium is increased in the spontaneously hypertensive rat (SHR), and there is evidence that this coincides with hypoxia and ATP depletion in the renal cortex. In the present studies we have examined expression of CypB in rat proximal tubules, which contributes to the increased renal sodium reabsorption in this model of hypertension. We report that CypB transcript abundance is significantly elevated in proximal convoluted tubules from SHR compared with the control Wistar-Kyoto strain. This upregulation occurs in weanling animals and precedes the development of hypertension, indicating that it is not a simple response to hypertension in SHR. Further, CypB expression is also higher in a proximal tubule cell line derived from SHR compared with a similar line derived from Wistar-Kyoto rats, indicating that this difference is genetically determined. No sequence differences were observed in the CypB cDNA from these 2 strains. These observations suggest that a genetically determined alteration in proximal tubules from SHR occurs that leads to increased expression of CypB. In view of evidence linking CypB to the regulation of elongation initiation factor-2, the upregulation of CypB may result from metabolic stress.

Cyclosporin A associated helicase-like protein facilitates the association of hepatitis C virus RNA polymerase with its cellular cyclophilin B.

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Kengo Morohashi

Full Text Available BACKGROUND: Cyclosporin A (CsA is well known as an immunosuppressive drug useful for allogeneic transplantation. It has been reported that CsA inhibits hepatitis C virus (HCV genome replication, which indicates that cellular targets of CsA regulate the viral replication. However, the regulation mechanisms of HCV replication governed by CsA target proteins have not been fully understood. PRINCIPAL FINDINGS: Here we show a chemical biology approach that elucidates a novel mechanism of HCV replication. We developed a phage display screening to investigate compound-peptide interaction and identified a novel cellular target molecule of CsA. This protein, named CsA associated helicase-like protein (CAHL, possessed RNA-dependent ATPase activity that was negated by treatment with CsA. The downregulation of CAHL in the cells resulted in a decrease of HCV genome replication. CAHL formed a complex with HCV-derived RNA polymerase NS5B and host-derived cyclophilin B (CyPB, known as a cellular cofactor for HCV replication, to regulate NS5B-CyPB interaction. CONCLUSIONS: We found a cellular factor, CAHL, as CsA associated helicase-like protein, which would form trimer complex with CyPB and NS5B of HCV. The strategy using a chemical compound and identifying its target molecule by our phage display analysis is useful to reveal a novel mechanism underlying cellular and viral physiology.

Cyclosporin A associated helicase-like protein facilitates the association of hepatitis C virus RNA polymerase with its cellular cyclophilin B.

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Morohashi, Kengo; Sahara, Hiroeki; Watashi, Koichi; Iwabata, Kazuki; Sunoki, Takashi; Kuramochi, Kouji; Takakusagi, Kaori; Miyashita, Hiroki; Sato, Noriyuki; Tanabe, Atsushi; Shimotohno, Kunitada; Kobayashi, Susumu; Sakaguchi, Kengo; Sugawara, Fumio

2011-04-29

Cyclosporin A (CsA) is well known as an immunosuppressive drug useful for allogeneic transplantation. It has been reported that CsA inhibits hepatitis C virus (HCV) genome replication, which indicates that cellular targets of CsA regulate the viral replication. However, the regulation mechanisms of HCV replication governed by CsA target proteins have not been fully understood. Here we show a chemical biology approach that elucidates a novel mechanism of HCV replication. We developed a phage display screening to investigate compound-peptide interaction and identified a novel cellular target molecule of CsA. This protein, named CsA associated helicase-like protein (CAHL), possessed RNA-dependent ATPase activity that was negated by treatment with CsA. The downregulation of CAHL in the cells resulted in a decrease of HCV genome replication. CAHL formed a complex with HCV-derived RNA polymerase NS5B and host-derived cyclophilin B (CyPB), known as a cellular cofactor for HCV replication, to regulate NS5B-CyPB interaction. We found a cellular factor, CAHL, as CsA associated helicase-like protein, which would form trimer complex with CyPB and NS5B of HCV. The strategy using a chemical compound and identifying its target molecule by our phage display analysis is useful to reveal a novel mechanism underlying cellular and viral physiology.

Implication of the Whitefly Bemisia tabaci Cyclophilin B Protein in the Transmission of Tomato yellow leaf curl virus.

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Kanakala, Surapathrudu; Ghanim, Murad

2016-01-01

Tomato yellow leaf curl virus (TYLCV) is a single-stranded (ssDNA) begomoviruses that causes severe damage to tomato and several other crops worldwide. TYLCV is exclusively transmitted by the sweetpotato whitefly, Bemisia tabaci in a persistent circulative and propagative manner. Previous studies have shown that the transmission, retention, and circulation of TYLCV in its vector involves interaction with insect and endosymbiont proteins, which aid in the transmission of the virus, or have a protective role in response to the presence of the virus in the insect body. However, only a low number of such proteins have been identified. Here, the role of B. tabaci Cyclophilin B (CypB) in the transmission of TYLCV protein was investigated. Cyclophilins are a large family of cellular prolyl isomerases that have many molecular roles including facilitating protein-protein interactions in the cell. One cyclophilin protein has been implicated in aphid-luteovirus interactions. We demonstrate that the expression of CypB from B. tabaci is altered upon TYLCV acquisition and retention. Further experiments used immunocapture-PCR and co-immunolocalization and demonstrated a specific interaction and colocalization between CypB and TYLCV in the the midgut, eggs, and salivary glands. Membrane feeding of anti-CypB antibodies and TYLCV-infected plants showed a decrease in TYLCV transmission, suggesting a critical role that CypB plays in TYLCV transmission. Further experiments, which used membrane feeding with the CypB inhibitor Cyclosporin A showed decrease in CypB-TYLCV colocalization in the midgut and virus transmission. Altogether, our results indicate that CypB plays an important role in TYLCV transmission by B. tabaci .

Implication of the whitefly Bemisia tabaci Cyclophilin B protein in the transmission of Tomato yellow leaf curl virus

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Surapathrudu Kanakala

2016-11-01

Full Text Available Tomato yellow leaf curl virus (TYLCV is a single-stranded (ssDNA begomoviruses that causes severe damage to tomato and several other crops worldwide. TYLCV is exclusively transmitted by the sweetpotato whitefly, Bemisia tabaci in a persistent circulative and propagative manner. Previous studies have shown that the transmission, retention and circulation of TYLCV in its vector involves interaction with insect and endosymbiont proteins, which aid in the transmission of the virus, or have a protective role in response to the presence of the virus in the insect body. However, only a low number of such proteins have been identified. Here, the role of B. tabaci Cyclophilin B (CypB in the transmission of TYLCV protein was investigated. Cyclophilins (Cyps are a large family of cellular prolyl isomerases that have many molecular roles including facilitating protein-protein interactions in the cell. One cyclophilin protein has been implicated in aphid-luteovirus interactions. We demonstrate that the expression of CypB from B. tabaci is altered upon TYLCV acquisition and retention. Further experiments used immunocapture-PCR and co-immunolocalization and demonstrated a specific interaction and colocalization between CypB and TYLCV in the the midgut, eggs and salivary glands. Membrane feeding of anti-CypB antibodies and TYLCV infected plants showed a decrease in TYLCV transmission, suggesting a critical role that CypB plays in TYLCV transmission. Further experiments, which used membrane feeding with the CypB inhibitor Cyclosporin A (CsA showed decrease in CypB-TYLCV colocalization in the midgut and virus transmission. Altogether, our results indicate that CypB plays an important role in TYLCV transmission by B. tabaci.

Molecular dynamics simulations of site point mutations in the TPR domain of cyclophilin 40 identify conformational states with distinct dynamic and enzymatic properties

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Gur, Mert; Blackburn, Elizabeth A.; Ning, Jia; Narayan, Vikram; Ball, Kathryn L.; Walkinshaw, Malcolm D.; Erman, Burak

2018-04-01

Cyclophilin 40 (Cyp40) is a member of the immunophilin family that acts as a peptidyl-prolyl-isomerase enzyme and binds to the heat shock protein 90 (Hsp90). Its structure comprises an N-terminal cyclophilin domain and a C-terminal tetratricopeptide (TPR) domain. Cyp40 is overexpressed in prostate cancer and certain T-cell lymphomas. The groove for Hsp90 binding on the TPR domain includes residues Lys227 and Lys308, referred to as the carboxylate clamp, and is essential for Cyp40-Hsp90 binding. In this study, the effect of two mutations, K227A and K308A, and their combinative mutant was investigated by performing a total of 5.76 μs of all-atom molecular dynamics (MD) simulations in explicit solvent. All simulations, except the K308A mutant, were found to adopt two distinct (extended or compact) conformers defined by different cyclophilin-TPR interdomain distances. The K308A mutant was only observed in the extended form which is observed in the Cyp40 X-ray structure. The wild-type, K227A, and combined mutant also showed bimodal distributions. The experimental melting temperature, Tm, values of the mutants correlate with the degree of compactness with the K308A extended mutant having a marginally lower melting temperature. Another novel measure of compactness determined from the MD data, the "coordination shell volume," also shows a direct correlation with Tm. In addition, the MD simulations show an allosteric effect with the mutations in the remote TPR domain having a pronounced effect on the molecular motions of the enzymatic cyclophilin domain which helps rationalise the experimentally observed increase in enzyme activity measured for all three mutations.

Cyclophilin40 isomerase activity is regulated by a temperature-dependent allosteric interaction with Hsp90.

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Blackburn, Elizabeth A; Wear, Martin A; Landré, Vivian; Narayan, Vikram; Ning, Jia; Erman, Burak; Ball, Kathryn L; Walkinshaw, Malcolm D

2015-09-01

Cyclophilin 40 (Cyp40) comprises an N-terminal cyclophilin domain with peptidyl-prolyl isomerase (PPIase) activity and a C-terminal tetratricopeptide repeat (TPR) domain that binds to the C-terminal-EEVD sequence common to both heat shock protein 70 (Hsp70) and Hsp90. We show in the present study that binding of peptides containing the MEEVD motif reduces the PPIase activity by ∼30%. CD and fluorescence assays show that the TPR domain is less stable than the cyclophilin domain and is stabilized by peptide binding. Isothermal titration calorimetry (ITC) shows that the affinity for the-MEEVD peptide is temperature sensitive in the physiological temperature range. Results from these biophysical studies fit with the MD simulations of the apo and holo (peptide-bound) structures which show a significant reduction in root mean square (RMS) fluctuation in both TPR and cyclophilin domains when-MEEVD is bound. The MD simulations of the apo-protein also highlight strong anti-correlated motions between residues around the PPIase-active site and a band of residues running across four of the seven helices in the TPR domain. Peptide binding leads to a distortion in the shape of the active site and a significant reduction in these strongly anti-correlated motions, providing an explanation for the allosteric effect of ligand binding and loss of PPIase activity. Together the experimental and MD results suggest that on heat shock, dissociation of Cyp40 from complexes mediated by the TPR domain leads to an increased pool of free Cyp40 capable of acting as an isomerase/chaperone in conditions of cellular stress. © 2015 Authors.

Extracellular cyclophilin levels associate with parameters of asthma in phenotypic clusters.

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Stemmy, Erik J; Benton, Angela S; Lerner, Jennifer; Alcala, Sarah; Constant, Stephanie L; Freishtat, Robert J

2011-12-01

Leukocyte persistence during chronic (quiescent) phases of asthma is a major hallmark of the disease. The mechanisms regulating these persistent leukocyte populations are not clearly understood. An alternative family of chemoattracting proteins, cyclophilins (Cyps), has recently been shown to contribute to leukocyte recruitment in animal models of allergic asthma. The goals of this study were to determine whether Cyps are present in asthma patients during the chronic phase of the disease and to investigate whether levels of Cyps associate with clinical parameters of disease severity. Nasal wash samples from an urban cohort of 137 patients of age 6-20 years with physician-diagnosed asthma were examined for the presence of cyclophilin A (CypA), cyclophilin B (CypB), as well as several other classical chemokines. Linear, logistic, or ordinal regressions were performed to identify associations between Cyps, chemokines, and clinical parameters of asthma. The asthma cohort was further divided into previously established phenotypic clusters (cluster 1: n = 55; cluster 2: n = 31; and cluster 3: n = 51) and examined for associations. Levels of CypB in the asthma group were highly elevated compared to nonasthmatic controls, while a slight increase in Monocyte Chemotactic Protein-1 (MCP-1) was also observed. CypA and MCP-1 were associated with levels of eosinophil cationic protein (ECP; a marker of eosinophil activation). Cluster-specific associations were found for CypA and CypB and clinical asthma parameters [e.g. forced expiratory volume in 1 second (FEV(1)) and ECP]. Cyps are present in nasal wash samples of asthma patients and may be a novel biomarker for clinical parameters of asthma severity.

Evaluation of Porin Interaction with Adenine Nucleotide Translocase and Cyclophilin-D Proteins after Brain Ischemia and Reperfusion

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Mohammad Ali Atlasi

2011-01-01

Full Text Available Objective (s Porin is a mitochondrial outer membrane channel, which usually functions as the pathway for the movement of various substances in and out of the mitochondria and is considered to be a component of the permeability transition (PT pore complex that plays a role in the PT. We addressed the hypothesis that porin interacts with other mitochondrial proteins after ischemic injury.Materials and MethodsFor this purpose, we used in vivo 4-vessel occlusion model of rat brain and porin purification method by hydroxyapatite column. After SDS gel electrophoresis and silver nitrate staining, Western blotting was done for porin, adenine nucleotide translocase and cyclophilin-D proteins.Results Porin was purified from mitochondrial mixture in ischemic brain and control groups. Investigation of interaction of adenine nucleotide transposes (ANT and cyclophilin-D with porin by Western blotting showed no proteins co-purified with porin from injured tissues.Conclusion The present study implies that there may not be interaction between porin, and ANT or cyclophilin-D, and if there is any, it is not maintained during the purification procedure.

Biochemical characterization of the prolyl 3-hydroxylase 1.cartilage-associated protein.cyclophilin B complex.

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Ishikawa, Yoshihiro; Wirz, Jackie; Vranka, Janice A; Nagata, Kazuhiro; Bächinger, Hans Peter

2009-06-26

The rough endoplasmic reticulum-resident protein complex consisting of prolyl 3-hydroxylase 1 (P3H1), cartilage-associated protein (CRTAP), and cyclophilin B (CypB) can be isolated from chick embryos on a gelatin-Sepharose column, indicating some involvement in the biosynthesis of procollagens. Prolyl 3-hydroxylase 1 modifies a single proline residue in the alpha chains of type I, II, and III collagens to (3S)-hydroxyproline. The peptidyl-prolyl cis-trans isomerase activity of cyclophilin B was shown previously to catalyze the rate of triple helix formation. Here we show that cyclophilin B in the complex shows peptidyl-prolyl cis-trans isomerase activity and that the P3H1.CRTAP.CypB complex has another important function: it acts as a chaperone molecule when tested with two classical chaperone assays. The P3H1.CRTAP.CypB complex inhibited the thermal aggregation of citrate synthase and was active in the denatured rhodanese refolding and aggregation assay. The chaperone activity of the complex was higher than that of protein-disulfide isomerase, a well characterized chaperone. The P3H1.CRTAP.CypB complex also delayed the in vitro fibril formation of type I collagen, indicating that this complex is also able to interact with triple helical collagen and acts as a collagen chaperone.

Cyclophilin B facilitates the replication of Orf virus.

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Zhao, Kui; Li, Jida; He, Wenqi; Song, Deguang; Zhang, Ximu; Zhang, Di; Zhou, Yanlong; Gao, Feng

2017-06-15

Viruses interact with host cellular factors to construct a more favourable environment for their efficient replication. Expression of cyclophilin B (CypB), a cellular peptidyl-prolyl cis-trans isomerase (PPIase), was found to be significantly up-regulated. Recently, a number of studies have shown that CypB is important in the replication of several viruses, including Japanese encephalitis virus (JEV), hepatitis C virus (HCV) and human papillomavirus type 16 (HPV 16). However, the function of cellular CypB in ORFV replication has not yet been explored. Suppression subtractive hybridization (SSH) technique was applied to identify genes differentially expressed in the ORFV-infected MDBK cells at an early phase of infection. Cellular CypB was confirmed to be significantly up-regulated by quantitative reverse transcription-PCR (qRT-PCR) analysis and Western blotting. The role of CypB in ORFV infection was further determined using Cyclosporin A (CsA) and RNA interference (RNAi). Effect of CypB gene silencing on ORFV replication by 50% tissue culture infectious dose (TCID 50 ) assay and qRT-PCR detection. In the present study, CypB was found to be significantly up-regulated in the ORFV-infected MDBK cells at an early phase of infection. Cyclosporin A (CsA) exhibited suppressive effects on ORFV replication through the inhibition of CypB. Silencing of CypB gene inhibited the replication of ORFV in MDBK cells. In conclusion, these data suggest that CypB is critical for the efficient replication of the ORFV genome. Cellular CypB was confirmed to be significantly up-regulated in the ORFV-infected MDBK cells at an early phase of infection, which could effectively facilitate the replication of ORFV.

The science of direct-acting antiviral and host-targeted agent therapy.

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Pawlotsky, Jean-Michel

2012-01-01

Direct-acting antiviral drugs targeting two major steps of the HCV life cycle, polyprotein processing and replication, and cyclophilin inhibitors, that target a host cell protein required to interact with the replication complex, have reached clinical development. In order to achieve a sustained virological response, that is, a cure of the HCV infection, it is necessary to shut down virus production, to maintain viral inhibition throughout treatment and to induce a significant, slower second-phase decline in HCV RNA levels that leads to definitive clearance of infected cells. Recent findings suggest that the interferon era is coming to an end in hepatitis C therapy and HCV infection can be cured by all-oral interferon-free treatment regimens within 12 to 24 weeks. Further results are awaited that will allow the establishment of an ideal first-line all-oral, interferon-free treatment regimen for patients with chronic HCV infection.

Spatiotemporal behavior of nuclear cyclophilin B indicates a role in RNA transcription.

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Dieriks, Birger; Van Oostveldt, Patrick

2012-06-01

Cyclophilin B (CypB) is an ubiquitously expressed protein, which performs several intra- and extracellular functions. Despite its abundant use as a household protein, little is known about its exact cellular localization and dynamics. In the present study we show that endogenous CypB localizes in one of two distinct compartments, either within the endoplasmic reticulum (ER) or inside the nucleus, accumulating in the fibrillar centers of the nucleoli. By means of a genetic deletion screen, we identified a minimal nucleolar localization signal for efficient relocation to the nucleoli. Within the fibrillar centers, CypB colocalized with RNA polymerase, upstream binding factor-1 (UBF), fibrillarin and dyskerin (DCK1). Even after chemical disruption of the nucleoli, a strong interaction with these proteins remained. Using live cell imaging, we showed a persistent colocalization of CypB with proteins involved in the ribosome biogenesis during the transcriptionally more active phases of the cell cycle. Supported by in silico data, our observations suggest that CypB interacts with these proteins and is involved in ribosome biogenesis and RNA transcription.

Role of cyclophilins in somatolactogenic action.

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Rycyzyn, M A; Clevenger, C V

2000-01-01

Prolactin (PRL) and growth hormone (GH) are members of the somatolactogenic hormone family, the pleiotropic actions of which are necessary for vertebrate growth and mammary differentiation. The basis for the specific function of these hormones has remained uncertain; however, their action is associated with internalization and translocation into the nucleus. A yeast two-hybrid screen identified an interaction between PRL and cyclophilin B (CypB), a peptidyl prolyl isomerase (PPI) found in the endoplasmic reticulum (ER), extracellular space, and nucleus. The interaction between CypB and PRL/GH was confirmed in vitro and in vivo through the use of recombinant proteins and coimmunoprecipitation studies. The exogenous addition of CypB potentiated the proliferation of PRL- and GH-dependent cell lines 18- and 40-fold, respectively. The potentiation of PRL action by CypB was accompanied by a dramatic increase in the nuclear retrotranslocation of PRL. Immunogold electron microscopy has revealed this retrotransport to occur via a vesicular pathway. A CypB mutant, termed CypB-NT, was generated that lacked the putative wild-type N-terminal nuclear localization sequence. Although CypB-NT demonstrated levels of PRL binding and PPI activity equivalent to wild-type CypB, it was incapable of mediating the nuclear retrotranslocation of PRL or enhancing PRL-driven proliferation. These studies reveal CypB as an important chaperone facilitating the nuclear retrotransport and action of the somatolactogenic hormone family.

Biochemical Characterization of the Prolyl 3-Hydroxylase 1·Cartilage-associated Protein·Cyclophilin B Complex*

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Ishikawa, Yoshihiro; Wirz, Jackie; Vranka, Janice A.; Nagata, Kazuhiro; Bächinger, Hans Peter

2009-01-01

The rough endoplasmic reticulum-resident protein complex consisting of prolyl 3-hydroxylase 1 (P3H1), cartilage-associated protein (CRTAP), and cyclophilin B (CypB) can be isolated from chick embryos on a gelatin-Sepharose column, indicating some involvement in the biosynthesis of procollagens. Prolyl 3-hydroxylase 1 modifies a single proline residue in the α chains of type I, II, and III collagens to (3S)-hydroxyproline. The peptidyl-prolyl cis-trans isomerase activity of cyclophilin B was shown previously to catalyze the rate of triple helix formation. Here we show that cyclophilin B in the complex shows peptidyl-prolyl cis-trans isomerase activity and that the P3H1·CRTAP·CypB complex has another important function: it acts as a chaperone molecule when tested with two classical chaperone assays. The P3H1·CRTAP·CypB complex inhibited the thermal aggregation of citrate synthase and was active in the denatured rhodanese refolding and aggregation assay. The chaperone activity of the complex was higher than that of protein-disulfide isomerase, a well characterized chaperone. The P3H1·CRTAP·CypB complex also delayed the in vitro fibril formation of type I collagen, indicating that this complex is also able to interact with triple helical collagen and acts as a collagen chaperone. PMID:19419969

A novel interaction between calcium-modulating cyclophilin ligand and Basigin regulates calcium signaling and matrix metalloproteinase activities in human melanoma cells.

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Long, Tingting; Su, Juan; Tang, Wen; Luo, Zhongling; Liu, Shuang; Liu, Zhaoqian; Zhou, Honghao; Qi, Min; Zeng, Weiqi; Zhang, Jianglin; Chen, Xiang

2013-10-01

Intracellular free calcium is a ubiquitous second messenger regulating a multitude of normal and pathogenic cellular responses, including the development of melanoma. Upstream signaling pathways regulating the intracellular free calcium concentration ([Ca2+]i) may therefore have a significant impact on melanoma growth and metastasis. In this study, we demonstrate that the endoplasmic reticulum (ER)-associated protein calcium-modulating cyclophilin ligand (CAML) is bound to Basigin, a widely expressed integral plasma membrane glycoprotein and extracellular matrix metalloproteinase inducer (EMMPRIN, or CD147) implicated in melanoma proliferation, invasiveness, and metastasis. This interaction between CAML and Basigin was first identified using yeast two-hybrid screening and further confirmed by co-immunoprecipitation. In human A375 melanoma cells, CAML and Basigin were co-localized to the ER. Knockdown of Basigin in melanoma cells by siRNA significantly decreased resting [Ca2+]i and the [Ca2+]i increase induced by the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor thapsigargin (TG), indicating that the interaction between CAML and Basigin regulates ER-dependent [Ca2+]i signaling. Meanwhile upregulating the [Ca2+]i either by TG or phorbol myristate acetate (PMA) could stimulate the production of MMP-9 in A375 cells with the expression of Basigin. Our study has revealed a previously uncharacterized [Ca2+]i signaling pathway that may control melanoma invasion, and metastasis. Disruption of this pathway may be a novel therapeutic strategy for melanoma treatment. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Mitochondrial cyclophilin-D as a critical mediator of ischaemic preconditioning

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Hausenloy, Derek J.; Lim, Shiang Y.; Ong, Sang-Ging; Davidson, Sean M.; Yellon, Derek M.

2010-01-01

Aims It has been suggested that mitochondrial reactive oxygen species (ROS), Akt and Erk1/2 and more recently the mitochondrial permeability transition pore (mPTP) may act as mediators of ischaemic preconditioning (IPC), although the actual interplay between these mediators is unclear. The aim of the present study is to determine whether the cyclophilin-D (CYPD) component of the mPTP is required by IPC to generate mitochondrial ROS and subsequently activate Akt and Erk1/2. Methods and results...

Cyclophilin A Levels Dictate Infection Efficiency of Human Immunodeficiency Virus Type 1 Capsid Escape Mutants A92E and G94D ▿

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Ylinen, Laura M. J.; Schaller, Torsten; Price, Amanda; Fletcher, Adam J.; Noursadeghi, Mahdad; James, Leo C.; Towers, Greg J.

2009-01-01

Cyclophilin A (CypA) is an important human immunodeficiency virus type 1 (HIV-1) cofactor in human cells. HIV-1 A92E and G94D capsid escape mutants arise during CypA inhibition and in certain cell lines are dependent on CypA inhibition. Here we show that dependence on CypA inhibition is due to high CypA levels. Restricted HIV-1 is stable, and remarkably, restriction is augmented by arresting cell division. Nuclear entry is not inhibited. We propose that high CypA levels and capsid mutations combine to disturb uncoating, leading to poor infectivity, particularly in arrested cells. Our data suggest a role for CypA in uncoating the core of HIV-1 to facilitate integration. PMID:19073742

Keratinocyte secretion of cyclophilin B via the constitutive pathway is regulated through its cyclosporin-binding site.

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Fearon, Paula; Lonsdale-Eccles, Ann A; Ross, O Kehinde; Todd, Carole; Sinha, Aparna; Allain, Fabrice; Reynolds, Nick J

2011-05-01

Cyclophilin B (CypB) is an endoplasmic reticulum (ER)-resident member of the cyclophilin family of proteins that bind cyclosporin A (CsA). We report that as in other cell types, CypB trafficked from the ER and was secreted by keratinocytes into the media in response to CsA. Concentrations as low as 1 pM of CsA induced secretion of CypB. Using brefeldin A, we showed that CypB is secreted from keratinocytes via the constitutive secretory pathway. We defined that substitution of tryptophan residue 128 in the CsA-binding site of CypB with alanine resulted in dissociation of CypB(W128A)-green fluorescent protein (GFP) from the ER. Photobleaching studies revealed a significant reduction in the diffusible mobility of CypB(W128A)-GFP compared with CypB(WT)-GFP, consistent with redistribution of CypB(W128A)-GFP into secretory vesicles disconnected from the ER/Golgi network. Furthermore, CsA significantly decreased the mobility of CypB(WT)-GFP but not CypB(W128A)-GFP. These studies demonstrate that therapeutically relevant concentrations of CsA regulate secretion of CypB by keratinocytes, and that a key residue within the CsA-binding site of CypB controls retention of CypB within the ER and regulates entry into the secretory pathway. As keratinocytes express CypB receptors (CD147) and CypB exhibits chemotactic properties, these data have implications for the therapeutic effects of CsA in inflammatory skin disease.

Octasaccharide is the minimal length unit required for efficient binding of cyclophilin B to heparin and cell surface heparan sulphate.

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Vanpouille, Christophe; Denys, Agnès; Carpentier, Mathieu; Pakula, Rachel; Mazurier, Joël; Allain, Fabrice

2004-09-01

Cyclophilin B (CyPB) is a heparin-binding protein first identified as a receptor for cyclosporin A. In previous studies, we reported that CyPB triggers chemotaxis and integrin-mediated adhesion of T-lymphocytes by way of interaction with two types of binding sites. The first site corresponds to a signalling receptor; the second site has been identified as heparan sulphate (HS) and appears crucial to induce cell adhesion. Characterization of the HS-binding unit is critical to understand the requirement of HS in pro-adhesive activity of CyPB. By using a strategy based on gel mobility shift assays with fluorophore-labelled oligosaccharides, we demonstrated that the minimal heparin unit required for efficient binding of CyPB is an octasaccharide. The mutants CyPB(KKK-) [where KKK- refers to the substitutions K3A(Lys3-->Ala)/K4A/K5A] and CyPB(DeltaYFD) (where Tyr14-Phe-Asp16 has been deleted) failed to interact with octasaccharides, confirming that the Y14FD16 and K3KK5 clusters are required for CyPB binding. Molecular modelling revealed that both clusters are spatially arranged so that they may act synergistically to form a binding site for the octasaccharide. We then demonstrated that heparin-derived octasaccharides and higher degree of polymerization oligosaccharides inhibited the interaction between CyPB and fluorophore-labelled HS chains purified from T-lymphocytes, and strongly reduced the HS-dependent pro-adhesive activity of CyPB. However, oligosaccharides or heparin were unable to restore adhesion of heparinase-treated T-lymphocytes, indicating that HS has to be present on the cell membrane to support the pro-adhesive activity of CyPB. Altogether, these results demonstrate that the octasaccharide is likely to be the minimal length unit required for efficient binding of CyPB to cell surface HS and consequent HS-dependent cell responses.

Ricinus communis cyclophilin: functional characterisation of a sieve tube protein involved in protein folding.

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Gottschalk, Maren; Dolgener, Elmar; Xoconostle-Cázares, Beatriz; Lucas, William J; Komor, Ewald; Schobert, Christian

2008-09-01

The phloem translocation stream of the angiosperms contains a special population of proteins and RNA molecules which appear to be produced in the companion cells prior to being transported into the sieve tube system through the interconnecting plasmodesmata. During this process, these non-cell-autonomous proteins are thought to undergo partial unfolding. Recent mass spectroscopy studies identified peptidyl-prolyl cis-trans isomerase (PPIases) as potential molecular chaperones functioning in the phloem translocation stream (Giavalisco et al. 2006). In the present study, we describe the cloning and characterisation of a castor bean phloem cyclophilin, RcCYP1 that has high peptidyl-prolyl cis-trans isomerase activity. Equivalent enzymatic activity was detected with phloem sap or purified recombinant (His)(6)-tagged RcCYP1. Mass spectrometry analysis of proteolytic peptides, derived from a 22 kDa band in HPLC-fractionated phloem sap, immunolocalisation studies and Western analysis of proteins extracted from castor bean tissues/organs indicated that RcCYP1 is an abundant protein in the companion cell-sieve element complex. Microinjection experiments established that purified recombinant (His)(6)-RcCYP1 can interact with plasmodesmata to both induce an increase in size exclusion limit and mediate its own cell-to-cell trafficking. Collectively, these findings support the hypothesis that RcCYP1 plays a role in the refolding of non-cell-autonomous proteins after their entry into the phloem translocation stream.

First characterization of three cyclophilin family proteins in the oyster, Crassostrea ariakensis Gould.

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Xu, Ting; Xie, Jiasong; Yang, Shoubao; Ye, Shigen; Luo, Ming; Wu, Xinzhong

2016-08-01

Cyclophilins (CyPs) are a family of proteins that bind the immunosuppressive agent cyclosporin A (CsA) with high-affinity and belong to one of the three superfamilies of peptidyl-prolyl cis-trans isomerases (PPIase). In this report, three cyclophilin genes (Ca-CyPs), including Ca-CyPA, Ca-CyPB and Ca-PPIL3, were identified from oyster, Crassostrea ariakensis Gould in which Ca-CyPA encodes a protein with 165 amino acid sequences, Ca-CyPB encodes a protein with 217 amino acid sequences and Ca-PPIL3 encodes a protein with 162 amino acid sequences. All of the three Ca-CyPs genes contain a typical CyP-PPIase domain with its signature sequences and Ca-CyPB contains an N-signal peptide sequences. Tissue distribution study revealed that Ca-CyPs were ubiquitously expressed in all examined tissues and the highest levels were observed in hemocytes. RLO incubation upregulated the mRNA expression levels of Ca-CyPs, indicating that three Ca-CyPs might be involved in oyster immune response against RLO infection. Copyright © 2016 Elsevier Ltd. All rights reserved.

Involvement of two classes of binding sites in the interactions of cyclophilin B with peripheral blood T-lymphocytes.

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Denys, A; Allain, F; Carpentier, M; Spik, G

1998-12-15

Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein, mainly associated with the secretory pathway, and is released in biological fluids. We recently reported that CyPB specifically binds to T-lymphocytes and promotes enhanced incorporation of CsA. The interactions with cellular binding sites involved, at least in part, the specific N-terminal extension of the protein. In this study, we intended to specify further the nature of the CyPB-binding sites on peripheral blood T-lymphocytes. We first provide evidence that the CyPB binding to heparin-Sepharose is prevented by soluble sulphated glycosaminoglycans (GAG), raising the interesting possibility that such interactions may occur on the T-cell surface. We then characterized CyPB binding to T-cell surface GAG and found that these interactions involved the N-terminal extension of CyPB, but not its conserved CsA-binding domain. In addition, we determined the presence of a second CyPB binding site, which we termed a type I site, in contrast with type II for GAG interactions. The two binding sites exhibit a similar affinity but the expression of the type I site was 3-fold lower. The conclusion that CyPB binding to the type I site is distinct from the interactions with GAG was based on the findings that it was (1) resistant to NaCl wash and GAG-degrading enzyme treatments, (2) reduced in the presence of CsA or cyclophilin C, and (3) unmodified in the presence of either the N-terminal peptide of CyPB or protamine. Finally, we showed that the type I binding sites were involved in an endocytosis process, supporting the hypothesis that they may correspond to a functional receptor for CyPB.

Interaction with glycosaminoglycans is required for cyclophilin B to trigger integrin-mediated adhesion of peripheral blood T lymphocytes to extracellular matrix.

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Allain, Fabrice; Vanpouille, Christophe; Carpentier, Mathieu; Slomianny, Marie-Christine; Durieux, Sandrine; Spik, Geneviève

2002-03-05

Cyclophilins A and B (CyPA and CyPB) are cyclosporin A-binding proteins that are involved in inflammatory events. We have reported that CyPB interacts with two types of cell-surface-binding sites. The first site corresponds to a functional receptor and requires interaction with the central core of CyPB. This region is highly conserved in cyclophilins, suggesting that CyPA and CyPB might share biological activities mediated by interaction with this receptor. The second site is identified with glycosaminoglycans (GAGs), the binding region located in the N terminus of CyPB. The difference in the N-terminal extensions of CyPA and CyPB suggests that a unique interaction with GAGs might account for selective activity of CyPB. To explore this hypothesis, we analyzed the lymphocyte responses triggered by CyPA, CyPB, and CyPB(KKK-), a mutant unable to interact with GAGs. The three ligands seemed capable enough to elicit calcium signal and chemotaxis by binding to the same signaling receptor. In contrast, only CyPB enhanced firm adhesion of T cells to the extracellular matrix. This activity depended on the interactions with GAGs and signaling receptor. CyPB-mediated adhesion required CD147 presumably because it was a costimulatory molecule and was related to an activation of alpha4beta1 and alpha4beta7 integrins. Finally, we showed that CyPB was capable mainly to enhance T cell adhesion of the CD4+CD45RO+ subset. The present data indicate that CyPB rather than CyPA is a proinflammatory factor for T lymphocytes and highlight the crucial role of CyPB-GAG interaction in the chemokine-like activity of this protein.

Cyclophilin B binding to platelets supports calcium-dependent adhesion to collagen.

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Allain, F; Durieux, S; Denys, A; Carpentier, M; Spik, G

1999-08-01

We have recently reported that cyclophilin B (CyPB), a secreted cyclosporine-binding protein, could bind to T lymphocytes through interactions with two types of binding sites. The first ones, referred to as type I, involve interactions with the conserved domain of CyPB and promote the endocytosis of surface-bound ligand, while the second type of binding sites, termed type II, are represented by glycosaminoglycans (GAG). Here, we further investigated the interactions of CyPB with blood cell populations. In addition to lymphocytes, CyPB was found to interact mainly with platelets. The binding is specific, with a dissociation constant (kd) of 9 +/- 3 nmol/L and the number of sites estimated at 960 +/- 60 per cell. Platelet glycosaminoglycans are not required for the interactions, but the binding is dramatically reduced by active cyclosporine derivatives. We then analyzed the biologic effects of CyPB and found a significant increase in platelet adhesion to collagen. Concurrently, CyPB initiates a transmembranous influx of Ca(2+) and induces the phosphorylation of the P-20 light chains of myosin. Taken together, the present results demonstrate for the first time that extracellular CyPB specifically interacts with platelets through a functional receptor related to the lymphocyte type I binding sites and might act by regulating the activity of a receptor-operated membrane Ca(2+) channel.

Cyclophilin B attenuates the expression of TNF-α in lipopolysaccharide-stimulated macrophages through the induction of B cell lymphoma-3.

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Marcant, Adeline; Denys, Agnès; Melchior, Aurélie; Martinez, Pierre; Deligny, Audrey; Carpentier, Mathieu; Allain, Fabrice

2012-08-15

Extracellular cyclophilin A (CyPA) and CyPB have been well described as chemotactic factors for various leukocyte subsets, suggesting their contribution to inflammatory responses. Unlike CyPA, CyPB accumulates in extracellular matrixes, from which it is released by inflammatory proteases. Hence, we hypothesized that it could participate in tissue inflammation by regulating the activity of macrophages. In the current study, we confirmed that CyPB initiated in vitro migration of macrophages, but it did not induce production of proinflammatory cytokines. In contrast, pretreatment of macrophages with CyPB attenuated the expression of inflammatory mediators induced by LPS stimulation. The expression of TNF-α mRNA was strongly reduced after exposure to CyPB, but it was not accompanied by significant modification in LPS-induced activation of MAPK and NF-κB pathways. LPS activation of a reporter gene under the control of TNF-α gene promoter was also markedly decreased in cells treated with CyPB, suggesting a transcriptional mechanism of inhibition. Consistent with this hypothesis, we found that CyPB induced the expression of B cell lymphoma-3 (Bcl-3), which was accompanied by a decrease in the binding of NF-κB p65 to the TNF-α promoter. As expected, interfering with the expression of Bcl-3 restored cell responsiveness to LPS, thus confirming that CyPB acted by inhibiting initiation of TNF-α gene transcription. Finally, we found that CyPA was not efficient in attenuating the production of TNF-α from LPS-stimulated macrophages, which seemed to be due to a modest induction of Bcl-3 expression. Collectively, these findings suggest an unexpected role for CyPB in attenuation of the responses of proinflammatory macrophages.

Hepatitis C virus NS5A protein is a substrate for the peptidyl-prolyl cis/trans isomerase activity of cyclophilins A and B.

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Hanoulle, Xavier; Badillo, Aurélie; Wieruszeski, Jean-Michel; Verdegem, Dries; Landrieu, Isabelle; Bartenschlager, Ralf; Penin, François; Lippens, Guy

2009-05-15

We report here a biochemical and structural characterization of domain 2 of the nonstructural 5A protein (NS5A) from the JFH1 Hepatitis C virus strain and its interactions with cyclophilins A and B (CypA and CypB). Gel filtration chromatography, circular dichroism spectroscopy, and finally NMR spectroscopy all indicate the natively unfolded nature of this NS5A-D2 domain. Because mutations in this domain have been linked to cyclosporin A resistance, we used NMR spectroscopy to investigate potential interactions between NS5A-D2 and cellular CypA and CypB. We observed a direct molecular interaction between NS5A-D2 and both cyclophilins. The interaction surface on the cyclophilins corresponds to their active site, whereas on NS5A-D2, it proved to be distributed over the many proline residues of the domain. NMR heteronuclear exchange spectroscopy yielded direct evidence that many proline residues in NS5A-D2 form a valid substrate for the enzymatic peptidyl-prolyl cis/trans isomerase (PPIase) activity of CypA and CypB.

Target cells in internal dosimetry

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Goessner, W

2003-07-01

Data related to radium induced bone sarcomas in humans are used as a model for defining target cells on bone surfaces and in the bone marrow. The differential distribution of radiation induced bone sarcoma types with a high ratio of non-bone producing, mainly fibroblastic tumours, challenges the ICRP concept that the bone lining cells are target cells. Multipotential mesenchymal stem cells are located within the range of alpha particles, and are the most likely target cells for the fibroblastic type of bone sarcoma. The histogenesis of bone sarcomas after irradiation with alpha emitters shows that their final histopathology is not dependent on a single target cell. Each target cell has a microenvironment, which has to be regarded as a synergistic morpho-functional tissue unit. For this the concept of 'histion', a term used in general pathology, is proposed. Interactions between target cells that have been hit by alpha-particles, leading to lethal, mutational or transformation events with all components of a 'histion', will prove critical to understanding the pathogenesis of both deterministic and stochastic late effects. (author)

Target cells in internal dosimetry

International Nuclear Information System (INIS)

Goessner, W.

2003-01-01

Data related to radium induced bone sarcomas in humans are used as a model for defining target cells on bone surfaces and in the bone marrow. The differential distribution of radiation induced bone sarcoma types with a high ratio of non-bone producing, mainly fibroblastic tumours, challenges the ICRP concept that the bone lining cells are target cells. Multipotential mesenchymal stem cells are located within the range of alpha particles, and are the most likely target cells for the fibroblastic type of bone sarcoma. The histogenesis of bone sarcomas after irradiation with alpha emitters shows that their final histopathology is not dependent on a single target cell. Each target cell has a microenvironment, which has to be regarded as a synergistic morpho-functional tissue unit. For this the concept of 'histion', a term used in general pathology, is proposed. Interactions between target cells that have been hit by alpha-particles, leading to lethal, mutational or transformation events with all components of a 'histion', will prove critical to understanding the pathogenesis of both deterministic and stochastic late effects. (author)

High level expression and characterization of the cyclophilin B gene from the anaerobic fungus Orpinomyces sp. strain PC-2.

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Chen, Huizhong; Li, Xin-Liang; Xu, Haiyan; Ljungdahl, Lars G; Cerniglia, Carl E

2006-01-01

Cyclophilins are an evolutionarily conserved family of peptidyl-prolyl cis-trans isomerases (PPIases). A cyclophilin B (cypB) gene from the anaerobic fungus Orpinomyces sp. strain PC-2 was cloned and overexpressed in Escherichia coli. It was expressed as an amino-terminal 6 x His-tagged recombinant protein to facilitate purification. Highly purified protein (26.5 kDa) was isolated by two chromatographic steps involving affinity and gel filtration for biochemical studies of the enzyme. The recombinant CypB displayed PPIase activity with a k(cat)/K(m) of 8.9 x 10(6) M(-1) s(-1) at 10 degrees C and pH 7.8. It was inhibited by cyclosporin A (CsA) with an IC(50) of 23.5 nM, similar to those of the native protein and other cyclophilin B enzymes from animals. Genomic DNA analysis of cypB revealed that it was present as a single copy in Orpinomyces PC-2 and contained two introns, indicating it has a eukaryotic origin. It is one of the most heavily interrupted genes with intron sequences found in anaerobic fungi. The three-dimensional model of Orpinomyces PC-2 CypB was predicted with a homology modeling approach using the Swiss-Model Protein Modeling Server and three dimensional structure of human CypB as a template. The overall architecture of the CypB molecule is very similar to that of human CypB.

Genomic and proteomic analysis of Schizaphis graminum reveals cyclophilin proteins are involved in the transmission of cereal yellow dwarf virus.

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Cecilia Tamborindeguy

Full Text Available Yellow dwarf viruses cause the most economically important virus diseases of cereal crops worldwide and are transmitted by aphid vectors. The identification of aphid genes and proteins mediating virus transmission is critical to develop agriculturally sustainable virus management practices and to understand viral strategies for circulative movement in all insect vectors. Two cyclophilin B proteins, S28 and S29, were identified previously in populations of Schizaphisgraminum that differed in their ability to transmit the RPV strain of Cereal yellow dwarf virus (CYDV-RPV. The presence of S29 was correlated with F2 genotypes that were efficient virus transmitters. The present study revealed the two proteins were isoforms, and a single amino acid change distinguished S28 and S29. The distribution of the two alleles was determined in 12 F2 genotypes segregating for CYDV-RPV transmission capacity and in 11 genetically independent, field-collected S. graminum biotypes. Transmission efficiency for CYDV-RPV was determined in all genotypes and biotypes. The S29 isoform was present in all genotypes or biotypes that efficiently transmit CYDV-RPV and more specifically in genotypes that efficiently transport virus across the hindgut. We confirmed a direct interaction between CYDV-RPV and both S28 and S29 using purified virus and bacterially expressed, his-tagged S28 and S29 proteins. Importantly, S29 failed to interact with a closely related virus that is transported across the aphid midgut. We tested for in vivo interactions using an aphid-virus co-immunoprecipitation strategy coupled with a bottom-up LC-MS/MS analysis using a Q Exactive mass spectrometer. This analysis enabled us to identify a third cyclophilin protein, cyclophilin A, interacting directly or in complex with purified CYDV-RPV. Taken together, these data provide evidence that both cyclophilin A and B interact with CYDV-RPV, and these interactions may be important but not sufficient to mediate

Menadione triggers cell death through ROS-dependent mechanisms involving PARP activation without requiring apoptosis.

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Loor, Gabriel; Kondapalli, Jyothisri; Schriewer, Jacqueline M; Chandel, Navdeep S; Vanden Hoek, Terry L; Schumacker, Paul T

2010-12-15

Low levels of reactive oxygen species (ROS) can function as redox-active signaling messengers, whereas high levels of ROS induce cellular damage. Menadione generates ROS through redox cycling, and high concentrations trigger cell death. Previous work suggests that menadione triggers cytochrome c release from mitochondria, whereas other studies implicate the activation of the mitochondrial permeability transition pore as the mediator of cell death. We investigated menadione-induced cell death in genetically modified cells lacking specific death-associated proteins. In cardiomyocytes, oxidant stress was assessed using the redox sensor RoGFP, expressed in the cytosol or the mitochondrial matrix. Menadione elicited rapid oxidation in both compartments, whereas it decreased mitochondrial potential and triggered cytochrome c redistribution to the cytosol. Cell death was attenuated by N-acetylcysteine and exogenous glutathione or by overexpression of cytosolic or mitochondria-targeted catalase. By contrast, no protection was observed in cells overexpressing Cu,Zn-SOD or Mn-SOD. Overexpression of antiapoptotic Bcl-X(L) protected against staurosporine-induced cell death, but it failed to confer protection against menadione. Genetic deletion of Bax and Bak, cytochrome c, cyclophilin D, or caspase-9 conferred no protection against menadione-induced cell death. However, cells lacking PARP-1 showed a significant decrease in menadione-induced cell death. Thus, menadione induces cell death through the generation of oxidant stress in multiple subcellular compartments, yet cytochrome c, Bax/Bak, caspase-9, and cyclophilin D are dispensable for cell death in this model. These studies suggest that multiple redundant cell death pathways are activated by menadione, but that PARP plays an essential role in mediating each of them. Copyright © 2010 Elsevier Inc. All rights reserved.

Identification of cellular and viral factors related to anti-hepatitis C virus activity of cyclophilin inhibitor.

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Goto, Kaku; Watashi, Koichi; Inoue, Daisuke; Hijikata, Makoto; Shimotohno, Kunitada

2009-10-01

We have so far reported that an immunosuppressant cyclosporin A (CsA), a well-known cyclophilin (CyP) inhibitor (CPI), strongly suppressed hepatitis C virus (HCV) replication in cell culture, and that CyPB was a cellular cofactor for viral replication. To further investigate antiviral mechanisms of CPI, we here developed cells carrying CsA-resistant HCV replicons, by culturing the HCV subgenomic replicon cells for 4 weeks in the presence of CsA with G418. Transfection of total RNA from the isolated CsA-resistant cells to naïve Huh7 cells conferred CsA resistance, suggesting that the replicon RNA itself was responsible for the resistant phenotype. Of the identified amino acid mutations, D320E in NS5A conferred the CsA resistance. The replicon carrying the D320E mutation was sensitive to interferon-alpha, but was resistant to CsA and other CPIs including NIM811 and sanglifehrin A. Knockdown of individual CyP subtypes revealed CyP40, in addition to CyPA and CyPB, contributed to viral replication, and CsA-resistant replicons acquired independence from CyPA for efficient replication. These data provide important evidence on the mechanisms underlying the regulation of HCV replication by CyP and for designing novel and specific anti-HCV strategies with CPIs.

Extracellular and Intracellular Cyclophilin A, Native and Post-Translationally Modified, Show Diverse and Specific Pathological Roles in Diseases.

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Xue, Chao; Sowden, Mark P; Berk, Bradford C

2018-05-01

CypA (cyclophilin A) is a ubiquitous and highly conserved protein with peptidyl prolyl isomerase activity. Because of its highly abundant level in the cytoplasm, most studies have focused on the roles of CypA as an intracellular protein. However, emerging evidence suggests an important role for extracellular CypA in the pathogenesis of several diseases through receptor (CD147 or other)-mediated autocrine and paracrine signaling pathways. In this review, we will discuss the shared and unique pathological roles of extracellular and intracellular CypA in human cardiovascular diseases. In addition, the evolving role of post-translational modifications of CypA in the pathogenesis of disease is discussed. Finally, recent studies with drugs specific for extracellular CypA show its importance in disease pathogenesis in several animal models and make extracellular CypA a new therapeutic target. © 2018 American Heart Association, Inc.

Receptor-mediated transcytosis of cyclophilin B through the blood-brain barrier.

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Carpentier, M; Descamps, L; Allain, F; Denys, A; Durieux, S; Fenart, L; Kieda, C; Cecchelli, R; Spik, G

1999-07-01

Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein mainly located in intracellular vesicles and secreted in biological fluids. In previous works, we demonstrated that CyPB interacts with T lymphocytes and enhances in vitro cellular incorporation and activity of CsA. In addition to its immunosuppressive activity, CsA is able to promote regeneration of damaged peripheral nerves. However, the crossing of the drug from plasma to neural tissue is restricted by the relative impermeability of the blood-brain barrier. To know whether CyPB might also participate in the delivery of CsA into the brain, we have analyzed the interactions of CyPB with brain capillary endothelial cells. First, we demonstrated that CyPB binds to two types of binding sites present at the surface of capillary endothelial cells from various species of tissues. The first type of binding sites (K(D) = 300 nM; number of sites = 3 x 10(6)) is related to interactions with negatively charged compounds such as proteoglycans. The second type of binding sites, approximately 50,000 per cell, exhibits a higher affinity for CyPB (K(D) = 15 nM) and is involved in an endocytosis process, indicating it might correspond to a functional receptor. Finally, the use of an in vitro model of blood-brain barrier allowed us to demonstrate that CyPB is transcytosed by a receptor-mediated pathway (flux = 16.5 fmol/cm2/h). In these conditions, CyPB did not significantly modify the passage of CsA, indicating that it is unlikely to provide a pathway for CsA brain delivery.

Phase 1 clinical study of cyclophilin B peptide vaccine for patients with lung cancer.

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Gohara, Rumi; Imai, Nobue; Rikimaru, Toru; Yamada, Akira; Hida, Naoya; Ichiki, Masao; Kawamoto, Mayumi; Matsunaga, Kazuko; Ashihara, Junko; Yano, Sayoko; Tamura, Mayumi; Ohkouchi, Shinya; Yamana, Hideaki; Oizumi, Kotaro; Itoh, Kyogo

2002-01-01

Cyclophilin B (CypB) possesses two antigenic epitopes (CypB(84-92) and CypB(91-99) ) recognized by HLA-A24-restricted and tumor-specific cytotoxic T lymphocytes (CTLs). To determine the safety of CypB-derived peptides and its ability to generate antitumor immune responses, patients with advanced lung cancer received subcutaneous vaccinations of these peptides or their modified peptides. All 16 patients were vaccinated with CypB(91-99) or its modified peptide, whereas only two patients were vaccinated with the modified CypB(84-92), as immediate-type hypersensitivity to CypB(84-92) or its modified peptide was observed in the remaining patients. No severe adverse events were associated with the vaccination. No significant increase in cellular responses to either peptides or tumor cells was observed in the postvaccination PBMCs by the conventional CTL assays in any patients tested. These results suggest that the vaccination of CypB(91-99) peptide was safe, but failed to induce objective immune responses at this regimen.

Severe osteogenesis imperfecta in cyclophilin B-deficient mice.

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Jae Won Choi

2009-12-01

Full Text Available Osteogenesis Imperfecta (OI is a human syndrome characterized by exquisitely fragile bones due to osteoporosis. The majority of autosomal dominant OI cases result from point or splice site mutations in the type I collagen genes, which are thought to lead to aberrant osteoid within developing bones. OI also occurs in humans with homozygous mutations in Prolyl-3-Hydroxylase-1 (LEPRE1. Although P3H1 is known to hydroxylate a single residue (pro-986 in type I collagen chains, it is unclear how this modification acts to facilitate collagen fibril formation. P3H1 exists in a complex with CRTAP and the peptidyl-prolyl isomerase cyclophilin B (CypB, encoded by the Ppib gene. Mutations in CRTAP cause OI in mice and humans, through an unknown mechanism, while the role of CypB in this complex has been a complete mystery. To study the role of mammalian CypB, we generated mice lacking this protein. Early in life, Ppib-/- mice developed kyphosis and severe osteoporosis. Collagen fibrils in Ppib-/- mice had abnormal morphology, further consistent with an OI phenotype. In vitro studies revealed that in CypB-deficient fibroblasts, procollagen did not localize properly to the golgi. We found that levels of P3H1 were substantially reduced in Ppib-/- cells, while CRTAP was unaffected by loss of CypB. Conversely, knockdown of either P3H1 or CRTAP did not affect cellular levels of CypB, but prevented its interaction with collagen in vitro. Furthermore, knockdown of CRTAP also caused depletion of cellular P3H1. Consistent with these changes, post translational prolyl-3-hydroxylation of type I collagen by P3H1 was essentially absent in CypB-deficient cells and tissues from CypB-knockout mice. These data provide significant new mechanistic insight into the pathophysiology of OI and reveal how the members of the P3H1/CRTAP/CypB complex interact to direct proper formation of collagen and bone.

Severe Osteogenesis Imperfecta in Cyclophilin B–Deficient Mice

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Choi, Jae Won; Sutor, Shari L.; Lindquist, Lonn; Evans, Glenda L.; Madden, Benjamin J.; Bergen, H. Robert; Hefferan, Theresa E.; Yaszemski, Michael J.; Bram, Richard J.

2009-01-01

Osteogenesis Imperfecta (OI) is a human syndrome characterized by exquisitely fragile bones due to osteoporosis. The majority of autosomal dominant OI cases result from point or splice site mutations in the type I collagen genes, which are thought to lead to aberrant osteoid within developing bones. OI also occurs in humans with homozygous mutations in Prolyl-3-Hydroxylase-1 (LEPRE1). Although P3H1 is known to hydroxylate a single residue (pro-986) in type I collagen chains, it is unclear how this modification acts to facilitate collagen fibril formation. P3H1 exists in a complex with CRTAP and the peptidyl-prolyl isomerase cyclophilin B (CypB), encoded by the Ppib gene. Mutations in CRTAP cause OI in mice and humans, through an unknown mechanism, while the role of CypB in this complex has been a complete mystery. To study the role of mammalian CypB, we generated mice lacking this protein. Early in life, Ppib-/- mice developed kyphosis and severe osteoporosis. Collagen fibrils in Ppib-/- mice had abnormal morphology, further consistent with an OI phenotype. In vitro studies revealed that in CypB–deficient fibroblasts, procollagen did not localize properly to the golgi. We found that levels of P3H1 were substantially reduced in Ppib-/- cells, while CRTAP was unaffected by loss of CypB. Conversely, knockdown of either P3H1 or CRTAP did not affect cellular levels of CypB, but prevented its interaction with collagen in vitro. Furthermore, knockdown of CRTAP also caused depletion of cellular P3H1. Consistent with these changes, post translational prolyl-3-hydroxylation of type I collagen by P3H1 was essentially absent in CypB–deficient cells and tissues from CypB–knockout mice. These data provide significant new mechanistic insight into the pathophysiology of OI and reveal how the members of the P3H1/CRTAP/CypB complex interact to direct proper formation of collagen and bone. PMID:19997487

Severe osteogenesis imperfecta in cyclophilin B-deficient mice.

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Choi, Jae Won; Sutor, Shari L; Lindquist, Lonn; Evans, Glenda L; Madden, Benjamin J; Bergen, H Robert; Hefferan, Theresa E; Yaszemski, Michael J; Bram, Richard J

2009-12-01

Osteogenesis Imperfecta (OI) is a human syndrome characterized by exquisitely fragile bones due to osteoporosis. The majority of autosomal dominant OI cases result from point or splice site mutations in the type I collagen genes, which are thought to lead to aberrant osteoid within developing bones. OI also occurs in humans with homozygous mutations in Prolyl-3-Hydroxylase-1 (LEPRE1). Although P3H1 is known to hydroxylate a single residue (pro-986) in type I collagen chains, it is unclear how this modification acts to facilitate collagen fibril formation. P3H1 exists in a complex with CRTAP and the peptidyl-prolyl isomerase cyclophilin B (CypB), encoded by the Ppib gene. Mutations in CRTAP cause OI in mice and humans, through an unknown mechanism, while the role of CypB in this complex has been a complete mystery. To study the role of mammalian CypB, we generated mice lacking this protein. Early in life, Ppib-/- mice developed kyphosis and severe osteoporosis. Collagen fibrils in Ppib-/- mice had abnormal morphology, further consistent with an OI phenotype. In vitro studies revealed that in CypB-deficient fibroblasts, procollagen did not localize properly to the golgi. We found that levels of P3H1 were substantially reduced in Ppib-/- cells, while CRTAP was unaffected by loss of CypB. Conversely, knockdown of either P3H1 or CRTAP did not affect cellular levels of CypB, but prevented its interaction with collagen in vitro. Furthermore, knockdown of CRTAP also caused depletion of cellular P3H1. Consistent with these changes, post translational prolyl-3-hydroxylation of type I collagen by P3H1 was essentially absent in CypB-deficient cells and tissues from CypB-knockout mice. These data provide significant new mechanistic insight into the pathophysiology of OI and reveal how the members of the P3H1/CRTAP/CypB complex interact to direct proper formation of collagen and bone.

Cyclophilin B induces integrin-mediated cell adhesion by a mechanism involving CD98-dependent activation of protein kinase C-delta and p44/42 mitogen-activated protein kinases.

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Melchior, Aurélie; Denys, Agnès; Deligny, Audrey; Mazurier, Joël; Allain, Fabrice

2008-02-01

Initially identified as a cyclosporin-A binding protein, cyclophilin B (CyPB) is an inflammatory mediator that induces adhesion of T lymphocytes to fibronectin, by a mechanism dependent on CD147 and alpha 4 beta 1 integrins. Recent findings have suggested that another cell membrane protein, CD98, may cooperate with CD147 to regulate beta1 integrin functions. Based on these functional relationships, we examined the contribution of CD98 in the pro-adhesive activity of CyPB, by utilizing the responsive promonocyte cell line THP-1. We demonstrated that cross-linking CD98 with CD98-AHN-18 antibody mimicked the responses induced by CyPB, i.e. homotypic aggregation, integrin-mediated adhesion to fibronectin and activation of p44/42 MAPK. Consistent with previous data, immunoprecipitation confirmed the existence of a heterocomplex wherein CD147, CD98 and beta1 integrins were associated. We then demonstrated that CyPB-induced cell adhesion and p44/42 MAPK activation were dependent on the participation of phosphoinositide 3-kinase and subsequent activation of protein kinase C-delta. Finally, silencing the expression of CD98 by RNA interference potently reduced CyPB-induced cell responses, thus confirming the role of CD98 in the pro-adhesive activity of CyPB. Altogether, our results support a model whereby CyPB induces integrin-mediated adhesion via interaction with a multimolecular unit formed by the association between CD147, CD98 and beta1 integrins.

Cooperative tumour cell membrane targeted phototherapy

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Kim, Heegon; Lee, Junsung; Oh, Chanhee; Park, Ji-Ho

2017-06-01

The targeted delivery of therapeutics using antibodies or nanomaterials has improved the precision and safety of cancer therapy. However, the paucity and heterogeneity of identified molecular targets within tumours have resulted in poor and uneven distribution of targeted agents, thus compromising treatment outcomes. Here, we construct a cooperative targeting system in which synthetic and biological nanocomponents participate together in the tumour cell membrane-selective localization of synthetic receptor-lipid conjugates (SR-lipids) to amplify the subsequent targeting of therapeutics. The SR-lipids are first delivered selectively to tumour cell membranes in the perivascular region using fusogenic liposomes. By hitchhiking with extracellular vesicles secreted by the cells, the SR-lipids are transferred to neighbouring cells and further spread throughout the tumour tissues where the molecular targets are limited. We show that this tumour cell membrane-targeted delivery of SR-lipids leads to uniform distribution and enhanced phototherapeutic efficacy of the targeted photosensitizer.

PpiA, a surface PPIase of the cyclophilin family in Lactococcus lactis.

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Nicolas Trémillon

Full Text Available BACKGROUND: Protein folding in the envelope is a crucial limiting step of protein export and secretion. In order to better understand this process in Lactococcus lactis, a lactic acid bacterium, genes encoding putative exported folding factors like Peptidyl Prolyl Isomerases (PPIases were searched for in lactococcal genomes. RESULTS: In L. lactis, a new putative membrane PPIase of the cyclophilin subfamily, PpiA, was identified and characterized. ppiA gene was found to be constitutively expressed under normal and stress (heat shock, H(2O(2 conditions. Under normal conditions, PpiA protein was synthesized and released from intact cells by an exogenously added protease, showing that it was exposed at the cell surface. No obvious phenotype could be associated to a ppiA mutant strain under several laboratory conditions including stress conditions, except a very low sensitivity to H(2O(2. Induction of a ppiA copy provided in trans had no effect i on the thermosensitivity of an mutant strain deficient for the lactococcal surface protease HtrA and ii on the secretion and stability on four exported proteins (a highly degraded hybrid protein and three heterologous secreted proteins in an otherwise wild-type strain background. However, a recombinant soluble form of PpiA that had been produced and secreted in L. lactis and purified from a culture supernatant displayed both PPIase and chaperone activities. CONCLUSIONS: Although L. lactis PpiA, a protein produced and exposed at the cell surface under normal conditions, displayed a very moderate role in vivo, it was found, as a recombinant soluble form, to be endowed with folding activities in vitro.

Targeting of Mesenchymal Stromal Cells by Cre-Recombinase Transgenes Commonly Used to Target Osteoblast Lineage Cells.

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Zhang, Jingzhu; Link, Daniel C

2016-11-01

The targeting specificity of tissue-specific Cre-recombinase transgenes is a key to interpreting phenotypes associated with their use. The Ocn-Cre and Dmp1-Cre transgenes are widely used to target osteoblasts and osteocytes, respectively. Here, we used high-resolution microscopy of bone sections and flow cytometry to carefully define the targeting specificity of these transgenes. These transgenes were crossed with Cxcl12 gfp mice to identify Cxcl12-abundant reticular (CAR) cells, which are a perivascular mesenchymal stromal population implicated in hematopoietic stem/progenitor cell maintenance. We show that in addition to osteoblasts, Ocn-Cre targets a majority of CAR cells and arteriolar pericytes. Surprisingly, Dmp1-Cre also targets a subset of CAR cells, in which expression of osteoblast-lineage genes is enriched. Finally, we introduce a new tissue-specific Cre-recombinase, Tagln-Cre, which efficiently targets osteoblasts, a majority of CAR cells, and both venous sinusoidal and arteriolar pericytes. These data show that Ocn-Cre and Dmp1-Cre target broader stromal cell populations than previously appreciated and may aid in the design of future studies. Moreover, these data highlight the heterogeneity of mesenchymal stromal cells in the bone marrow and provide tools to interrogate this heterogeneity. © 2016 American Society for Bone and Mineral Research. © 2016 American Society for Bone and Mineral Research.

Chemotactic Activity of Cyclophilin A in the Skin Mucus of Yellow Catfish (Pelteobagrus fulvidraco) and Its Active Site for Chemotaxis

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Dawar, Farman Ullah; Tu, Jiagang; Xiong, Yang; Lan, Jiangfeng; Dong, Xing Xing; Liu, Xiaoling; Khattak, Muhammad Nasir Khan; Mei, Jie; Lin, Li

2016-01-01

Fish skin mucus is a dynamic barrier for invading pathogens with a variety of anti-microbial enzymes, including cyclophilin A (CypA), a multi-functional protein with peptidyl-prolyl cis/trans isomerase (PPIase) activity. Beside various other immunological functions, CypA induces leucocytes migration in vitro in teleost. In the current study, we have discovered several novel immune-relevant proteins in yellow catfish skin mucus by mass spectrometry (MS). The CypA present among them was further detected by Western blot. Moreover, the CypA present in the skin mucus displayed strong chemotactic activity for yellow catfish leucocytes. Interestingly, asparagine (like arginine in mammals) at position 69 was the critical site in yellow catfish CypA involved in leucocyte attraction. These novel efforts do not only highlight the enzymatic texture of skin mucus, but signify CypA to be targeted for anti-inflammatory therapeutics. PMID:27589721

Chemotactic Activity of Cyclophilin A in the Skin Mucus of Yellow Catfish (Pelteobagrus fulvidraco and Its Active Site for Chemotaxis

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Farman Ullah Dawar

2016-08-01

Full Text Available Fish skin mucus is a dynamic barrier for invading pathogens with a variety of anti-microbial enzymes, including cyclophilin A (CypA, a multi-functional protein with peptidyl-prolyl cis/trans isomerase (PPIase activity. Beside various other immunological functions, CypA induces leucocytes migration in vitro in teleost. In the current study, we have discovered several novel immune-relevant proteins in yellow catfish skin mucus by mass spectrometry (MS. The CypA present among them was further detected by Western blot. Moreover, the CypA present in the skin mucus displayed strong chemotactic activity for yellow catfish leucocytes. Interestingly, asparagine (like arginine in mammals at position 69 was the critical site in yellow catfish CypA involved in leucocyte attraction. These novel efforts do not only highlight the enzymatic texture of skin mucus, but signify CypA to be targeted for anti-inflammatory therapeutics.

Delineation of the calcineurin-interacting region of cyclophilin B.

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Carpentier, M; Allain, F; Haendler, B; Slomianny, M C; Spik, G

2000-12-01

The immunosuppressant drug cyclosporin A (CsA) inhibits T-cell function by blocking the phosphatase activity of calcineurin. This effect is mediated by formation of a complex between the drug and cyclophilin (CyP), which creates a composite surface able to make high-affinity contacts with calcineurin. In vitro, the CyPB/CsA complex is more effective in inhibiting calcineurin than the CyPA/CsA and CyPC/CsA complexes, pointing to fine structural differences in the calcineurin-binding region. To delineate the calcineurin-binding region of CyPB, we mutated several amino acids, located in two loops corresponding to CyPA regions known to be involved, as follows: R76A, G77H, D155R, and D158R. Compared to wild-type CyPB, the G77H, D155R, and D158R mutants had intact isomerase and CsA-binding activities, indicating that no major conformational changes had taken place. When complexed to CsA, they all displayed only reduced affinity for calcineurin and much decreased inhibition of calcineurin phosphatase activity. These results strongly suggest that the three amino acids G77, D155, and D158 are directly involved in the interaction of CyPB/CsA with calcineurin, in agreement with their exposed position. The G77, D155, and D158 residues are not maintained in CyPA and might therefore account for the higher affinity of the CyPB/CsA complex for calcineurin.

Role of cyclophilin B in prolactin signal transduction and nuclear retrotranslocation.

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Rycyzyn, M A; Reilly, S C; O'Malley, K; Clevenger, C V

2000-08-01

The pleiotropic actions of PRL are necessary for mammary growth and differentiation and in vitro lymphoid proliferation. The proximal action of this ligand is mediated by its cell surface receptor via associated networks. PRL action, however, is also associated with the internalization and translocation of this hormone into the nucleus. To delineate the mechanism of this retrotranslocation, a yeast two-hybrid screen was performed and revealed an interaction between PRL and cyclophilin B (CypB). CypB is a peptidyl prolyl isomerase (PPI) found in the endoplasmic reticulum, extracellular space, and nucleus. The interaction between CypB and PRL was subsequently confirmed in vitro and in vivo through the use of recombinant proteins and coimmunoprecipitation studies. The exogenous addition of CypB potentiated the 3H-thymidine incorporation of PRL-dependent cell lines up to 18-fold. CypB by itself was nonmitogenic and did not potentiate the action of GH or other interleukins. CypB did not alter the affinity of the PRL receptor (PRLr) for its ligand, or increase the phosphorylation of PRLr-associated Jak2 or Stat5a. The potentiation of PRL-action by CypB, however, was accompanied by a dramatic increase in the nuclear retrotranslocation of PRL. A CypB mutant, termed CypB-NT, was generated that lacked the wild-type N-terminal nuclear localization sequence. Although CypB-NT demonstrated levels of PRL binding and PPI activity equivalent to wild-type CypB, it was incapable of mediating the nuclear retrotranslocation of PRL or enhancing PRL-driven proliferation. These studies reveal CypB as an important chaperone facilitating the nuclear retrotransport and action of the lactogenic hormones.

Ebselen induces reactive oxygen species (ROS-mediated cytotoxicity in Saccharomyces cerevisiae with inhibition of glutamate dehydrogenase being a target

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Gajendra Kumar Azad

2014-01-01

Full Text Available Ebselen is a synthetic, lipid-soluble seleno-organic compound. The high electrophilicity of ebselen enables it to react with multiple cysteine residues of various proteins. Despite extensive research on ebselen, its target molecules and mechanism of action remains less understood. We performed biochemical as well as in vivo experiments employing budding yeast as a model organism to understand the mode of action of ebselen. The growth curve analysis and FACS (florescence activated cell sorting assays revealed that ebselen exerts growth inhibitory effects on yeast cells by causing a delay in cell cycle progression. We observed that ebselen exposure causes an increase in intracellular ROS levels and mitochondrial membrane potential, and that these effects were reversed by addition of antioxidants such as reduced glutathione (GSH or N-acetyl-l-cysteine (NAC. Interestingly, a significant increase in ROS levels was noticed in gdh3-deleted cells compared to wild-type cells. Furthermore, we showed that ebselen inhibits GDH function by interacting with its cysteine residues, leading to the formation of inactive hexameric GDH. Two-dimensional gel electrophoresis revealed protein targets of ebselen including CPR1, the yeast homolog of Cyclophilin A. Additionally, ebselen treatment leads to the inhibition of yeast sporulation. These results indicate a novel direct connection between ebselen and redox homeostasis.

Ebselen induces reactive oxygen species (ROS)-mediated cytotoxicity in Saccharomyces cerevisiae with inhibition of glutamate dehydrogenase being a target.

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Azad, Gajendra Kumar; Singh, Vikash; Mandal, Papita; Singh, Prabhat; Golla, Upendarrao; Baranwal, Shivani; Chauhan, Sakshi; Tomar, Raghuvir S

2014-01-01

Ebselen is a synthetic, lipid-soluble seleno-organic compound. The high electrophilicity of ebselen enables it to react with multiple cysteine residues of various proteins. Despite extensive research on ebselen, its target molecules and mechanism of action remains less understood. We performed biochemical as well as in vivo experiments employing budding yeast as a model organism to understand the mode of action of ebselen. The growth curve analysis and FACS (florescence activated cell sorting) assays revealed that ebselen exerts growth inhibitory effects on yeast cells by causing a delay in cell cycle progression. We observed that ebselen exposure causes an increase in intracellular ROS levels and mitochondrial membrane potential, and that these effects were reversed by addition of antioxidants such as reduced glutathione (GSH) or N-acetyl-l-cysteine (NAC). Interestingly, a significant increase in ROS levels was noticed in gdh3-deleted cells compared to wild-type cells. Furthermore, we showed that ebselen inhibits GDH function by interacting with its cysteine residues, leading to the formation of inactive hexameric GDH. Two-dimensional gel electrophoresis revealed protein targets of ebselen including CPR1, the yeast homolog of Cyclophilin A. Additionally, ebselen treatment leads to the inhibition of yeast sporulation. These results indicate a novel direct connection between ebselen and redox homeostasis.

Elevated Serum Cyclophilin B Levels Are Associated with the Prevalence and Severity of Metabolic Syndrome

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Zhang, Hang; Fan, Qin; Xie, Hongyang; Lu, Lin; Tao, Rong; Wang, Fang; Xi, Rui; Hu, Jian; Chen, Qiujing; Shen, Weifeng; Zhang, Ruiyan; Yan, Xiaoxiang

2017-01-01

Objective Inflammation plays a central role in the pathogenesis of metabolic syndrome (MetS). Cyclophilin B (CypB) can be constitutively secreted in response to inflammatory stimuli and oxidative stress, participating in tissue or systemic inflammation. We investigated the relationship between CypB and MetS in both humans and mice. Methods Serum CypB levels were determined in 211 subjects with MetS and 292 subjects without MetS (non-MetS) (133 healthy controls and 159 high-risk subjects with ...

Elevated Serum Cyclophilin B Levels Are Associated with the Prevalence and Severity of Metabolic Syndrome

OpenAIRE

Hang Zhang; Hang Zhang; Qin Fan; Qin Fan; Hongyang Xie; Hongyang Xie; Lin Lu; Lin Lu; Rong Tao; Fang Wang; Rui Xi; Jian Hu; Qiujing Chen; Weifeng Shen; Ruiyan Zhang

2017-01-01

ObjectiveInflammation plays a central role in the pathogenesis of metabolic syndrome (MetS). Cyclophilin B (CypB) can be constitutively secreted in response to inflammatory stimuli and oxidative stress, participating in tissue or systemic inflammation. We investigated the relationship between CypB and MetS in both humans and mice.MethodsSerum CypB levels were determined in 211 subjects with MetS and 292 subjects without MetS (non-MetS) (133 healthy controls and 159 high-risk subjects with one...

Activation of mPTP-dependent mitochondrial apoptosis pathway by a novel pan HDAC inhibitor resminostat in hepatocellular carcinoma cells

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Fu, Meili [Department of Infectious Disease, Linyi People’s Hospital, Linyi (China); Shi, Wenhong [Department of Radiotherapy, Linyi Tumor Hospital, Linyi (China); Li, Zhengling [Department of Nursing, Tengzhou Central People’s Hospital, Tengzhou (China); Liu, Haiyan, E-mail: liuhaiyanlinyi5@sina.com [Department of Nursing, Linyi People’s Hospital, No. 27 Jiefang Road, Linyi 276000, Shandong (China)

2016-09-02

Over-expression and aberrant activation of histone deacetylases (HDACs) are often associated with poor prognosis of hepatocellular carcinoma (HCC). Here, we evaluated the potential anti-hepatocellular carcinoma (HCC) cell activity by resminostat, a novel pan HDAC inhibitor (HDACi). We demonstrated that resminostat induced potent cytotoxic and anti-proliferative activity against established HCC cell lines (HepG2, HepB3, SMMC-7721) and patient-derived primary HCC cells. Further, resminostat treatment in HCC cells activated mitochondrial permeability transition pore (mPTP)-dependent apoptosis pathway, which was evidenced by physical association of cyclophilin-D and adenine nucleotide translocator 1 (ANT-1), mitochondrial depolarization, cytochrome C release and caspase-9 activation. Intriguingly, the mPTP blockers (sanglifehrin A and cyclosporine A), shRNA knockdown of cyclophilin-D or the caspase-9 inhibitor dramatically attenuated resminostat-induced HCC cell apoptosis and cytotoxicity. Reversely, HCC cells with exogenous cyclophilin-D over-expression were hyper-sensitive to resminostat. Intriguingly, a low concentration of resminostat remarkably potentiated sorafenib-induced mitochondrial apoptosis pathway activation, leading to a profound cytotoxicity in HCC cells. The results of this preclinical study indicate that resminostat (or plus sorafenib) could be further investigated as a valuable anti-HCC strategy. - Highlights: • Resminostat inhibits human HCC cell survival and proliferation. • Resminostat activates mPTP-dependent mitochondrial apoptosis pathway in HCC cells. • Resminostat potentiates sorafenib-induced mitochondrial apoptosis pathway activation. • mPTP or caspase-9 inhibition attenuates apoptosis by resminostat or plus sorafenib.

Cyclophilin A potentiates TRIM5α inhibition of HIV-1 nuclear import without promoting TRIM5α binding to the viral capsid.

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Mallori Burse

Full Text Available The host immunophilin cyclophilin A (CypA binds to the capsid protein (CA of HIV-1 and regulates its infectivity. Depending on the target cell type, CypA can either promote or inhibit HIV-1 infection. The ability of CypA to promote HIV-1 infection has been extensively studied and linked to several steps in early replication including uncoating, reverse transcription and nuclear import. By contrast, the mechanism by which CypA inhibits infection is less well understood. We investigated the mechanism by which CypA potentiates restriction of HIV-1 by the tripartite motif-containing protein 5 (TRIM5α. Depletion of TRIM5α in the African green monkey cell line Vero, resulted in a loss of inhibition of infection by CypA, demonstrating that inhibition by CypA is mediated by TRIM5α. Complementary genetic and biochemical assays failed to demonstrate an ability of CypA to promote binding of TRIM5α to the viral capsid. TRIM5α inhibits HIV-1 reverse transcription in a proteasome-dependent manner; however, we observed that inhibition of proteasome activity did not reduce the ability of CypA to inhibit infection, suggesting that CypA acts at a step after reverse transcription. Accordingly, we observed a CypA-dependent reduction in the accumulation of nuclear HIV-1 DNA, indicating that CypA specifically promotes TRIM5α inhibition of HIV-1 nuclear import. We also observed that the ability of CypA to inhibit HIV-1 infection is abolished by amino acid substitutions within the conserved CPSF6-binding surface in CA. Our results indicate that CypA inhibits HIV-1 infection in Vero cells not by promoting TRIM5α binding to the capsid but by blocking nuclear import of the HIV-1 preintegration complex.

Release of overexpressed CypB activates ERK signaling through CD147 binding for hepatoma cell resistance to oxidative stress.

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Kim, Kiyoon; Kim, Hunsung; Jeong, Kwon; Jung, Min Hyung; Hahn, Bum-Soo; Yoon, Kyung-Sik; Jin, Byung Kwan; Jahng, Geon-Ho; Kang, Insug; Ha, Joohun; Choe, Wonchae

2012-08-01

Cyclophilin, a cytosolic receptor for the immunosuppressive drug cyclosporin A, plays a role in diverse pathophysiologies along with its receptor, CD147. Although the interaction between cyclophilin A and CD147 is well established in inflammatory disease, that of cyclophilin B (CypB) with CD147 has not been fully explored, especially in cancer cell biology, and the exact molecular mechanism underlying such an association is poorly understood. In this study, we first identified high expression levels of CypB in 54 % of hepatocellular carcinoma patient tissues but in only 12.5 % of normal liver tissues. Then, we demonstrated that CypB overexpression protects human hepatoma cells against oxidative stress through its binding to CD147; this protective effect depends on the peptidyl prolyl isomerase activity of CypB. siRNA-mediated knockdown of CypB expression rendered hepatoma cells more vulnerable to ROS-mediated apoptosis. Furthermore, we also determined that a direct interaction between secreted CypB and CD147 regulates the extracellular signal-regulated kinase intracellular signaling pathway and is indispensible for the protective functions of CypB. For the first time, we demonstrated that CypB has an essential function in protecting hepatoma cells against oxidative stress through binding to CD147 and regulating the ERK pathway.

Vital role for cyclophilin B (CypB) in asexual development, dimorphic transition and virulence of Beauveria bassiana.

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Chu, Zhen-Jian; Sun, Huan-Huan; Ying, Sheng-Hua; Feng, Ming-Guang

2017-08-01

Cyclophilin B (CypB) was previously revealed as one of many putative secretory proteins in the transcriptome of Beauveria bassiana infection to a lepidopteran pest. Here we show a main localization of CypB in hyphal cell walls and septa and its essential role in the in vitro and in vivo asexual cycles of the fungal insect pathogen. Deletion of cypB reduced colony growth by 16-42% on two rich media and 30 scant media with different carbon or nitrogen sources. The deletion mutant suffered a delayed conidiation on a standard medium and a final 47% reduction in conidial yield, accompanied with drastic transcript depression of several key genes required for conidiation and conidial maturation. The mutant conidia required 10h longer to germinate 50% at optimal 25°C than wild-type conidia. Intriguingly, cultivation of the mutant conidia in a trehalose-peptone broth mimic to insect hemolymph resulted in 83% reduction in blastospore yield but only slight decrease in biomass level, indicating severe defects in transition of hyphae to blastospores. LT 50 for the deletion mutant against Galleria mellonella larvae through normal cuticle infection was prolonged to 7.4d from a wild-type estimate of 4.7d. During colony growth, additionally, the deletion mutant displayed hypersensitivity to Congo red, menadione, H 2 O 2 and heat shock but increased tolerance to cyclosporine A and rapamycin. All of changes were restored by targeted gene complementation. Altogether, CypB takes part in sustaining normal growth, aerial conidiation, conidial germination, dimorphic transition, stress tolerance and pathogenicity in B. bassiana. Copyright © 2017 Elsevier Inc. All rights reserved.

Pharmacologic suppression of target cell recognition by engineered T cells expressing chimeric T-cell receptors.

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Alvarez-Vallina, L; Yañez, R; Blanco, B; Gil, M; Russell, S J

2000-04-01

Adoptive therapy with autologous T cells expressing chimeric T-cell receptors (chTCRs) is of potential interest for the treatment of malignancy. To limit possible T-cell-mediated damage to normal tissues that weakly express the targeted tumor antigen (Ag), we have tested a strategy for the suppression of target cell recognition by engineered T cells. Jurkat T cells were transduced with an anti-hapten chTCR tinder the control of a tetracycline-suppressible promoter and were shown to respond to Ag-positive (hapten-coated) but not to Ag-negative target cells. The engineered T cells were then reacted with hapten-coated target cells at different effector to target cell ratios before and after exposure to tetracycline. When the engineered T cells were treated with tetracycline, expression of the chTCR was greatly decreased and recognition of the hapten-coated target cells was completely suppressed. Tetracycline-mediated suppression of target cell recognition by engineered T cells may be a useful strategy to limit the toxicity of the approach to cancer gene therapy.

Lung Squamous Cell Carcinoma Stem Cells as Immunotherapy Targets

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2017-08-01

AWARD NUMBER: W81XWH-16-1-0260 TITLE: Lung Squamous Cell Carcinoma Stem Cells as Immunotherapy Targets PRINCIPAL INVESTIGATOR: Carla Kim... Cell Carcinoma Stem Cells as Immunotherapy Targets 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-16-1-0260 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S...SUPPLEMENTARY NOTES 14. ABSTRACT Lung squamous cell carcinoma (SCC) is the second most common type of lung cancer, and immunotherapy is a promising new

Structural and functional characterization of the interaction between cyclophilin B and a heparin-derived oligosaccharide.

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Hanoulle, Xavier; Melchior, Aurélie; Sibille, Nathalie; Parent, Benjamin; Denys, Agnès; Wieruszeski, Jean-Michel; Horvath, Dragos; Allain, Fabrice; Lippens, Guy; Landrieu, Isabelle

2007-11-23

The chemotaxis and integrin-mediated adhesion of T lymphocytes triggered by secreted cyclophilin B (CypB) depend on interactions with both cell surface heparan sulfate proteoglycans (HSPG) and the extracellular domain of the CD147 membrane receptor. Here, we use NMR spectroscopy to characterize the interaction of CypB with heparin-derived oligosaccharides. Chemical shift perturbation experiments allowed the precise definition of the heparan sulfate (HS) binding site of CypB. The N-terminal extremity of CypB, which contains a consensus sequence for heparin-binding proteins was modeled on the basis of our experimental NMR data. Because the HS binding site extends toward the CypB catalytic pocket, we measured its peptidyl-prolyl cis-trans isomerase (PPIase) activity in the absence or presence of a HS oligosaccharide toward a CD147-derived peptide. We report the first direct evidence that CypB is enzymatically active on CD147, as it is able to accelerate the cis/trans isomerization of the Asp(179)-Pro(180) bond in a CD147-derived peptide. However, HS binding has no significant influence on this PPIase activity. We thus conclude that the glycanic moiety of HSPG serves as anchor for CypB at the cell surface, and that the signal could be transduced by CypB via its PPIase activity toward CD147.

Regulatory polymorphisms in the cyclophilin A gene, PPIA, accelerate progression to AIDS.

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Ping An

2007-06-01

Full Text Available Human cyclophilin A, or CypA, encoded by the gene peptidyl prolyl isomerase A (PPIA, is incorporated into the HIV type 1 (HIV-1 virion and promotes HIV-1 infectivity by facilitating virus uncoating. We examined the effect of single nucleotide polymorphisms (SNPs and haplotypes within the PPIA gene on HIV-1 infection and disease progression in five HIV-1 longitudinal history cohorts. Kaplan-Meier survival statistics and Cox proportional hazards model were used to assess time to AIDS outcomes. Among eight SNPs tested, two promoter SNPs (SNP3 and SNP4 in perfect linkage disequilibrium were associated with more rapid CD4(+ T-cell loss (relative hazard = 3.7, p = 0.003 in African Americans. Among European Americans, these alleles were also associated with a significant trend to more rapid progression to AIDS in a multi-point categorical analysis (p = 0.005. Both SNPs showed differential nuclear protein-binding efficiencies in a gel shift assay. In addition, one SNP (SNP5 located in the 5' UTR previously shown to be associated with higher ex vivo HIV-1 replication was found to be more frequent in HIV-1-positive individuals than in those highly exposed uninfected individuals. These results implicate regulatory PPIA polymorphisms as a component of genetic susceptibility to HIV-1 infection or disease progression, affirming the important role of PPIA in HIV-1 pathogenesis.

Different mechanisms of hepatitis C virus RNA polymerase activation by cyclophilin A and B in vitro.

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Weng, Leiyun; Tian, Xiao; Gao, Yayi; Watashi, Koichi; Shimotohno, Kunitada; Wakita, Takaji; Kohara, Michinori; Toyoda, Tetsuya

2012-12-01

Cyclophilins (CyPs) are cellular proteins that are essential to hepatitis C virus (HCV) replication. Since cyclosporine A was discovered to inhibit HCV infection, the CyP pathway contributing to HCV replication is a potential attractive stratagem for controlling HCV infection. Among them, CyPA is accepted to interact with HCV nonstructural protein (NS) 5A, although interaction of CyPB and NS5B, an RNA-dependent RNA polymerase (RdRp), was proposed first. CyPA, CyPB, and HCV RdRp were expressed in bacteria and purified using combination column chromatography. HCV RdRp activity was analyzed in vitro with purified CyPA and CyPB. CyPA at a high concentration (50× higher than that of RdRp) but not at low concentration activated HCV RdRp. CyPB had an allosteric effect on genotype 1b RdRp activation. CyPB showed genotype specificity and activated genotype 1b and J6CF (2a) RdRps but not genotype 1a or JFH1 (2a) RdRps. CyPA activated RdRps of genotypes 1a, 1b, and 2a. CyPB may also support HCV genotype 1b replication within the infected cells, although its knockdown effect on HCV 1b replicon activity was controversial in earlier reports. CyPA activated HCV RdRp at the early stages of transcription, including template RNA binding. CyPB also activated genotype 1b RdRp. However, their activation mechanisms are different. These data suggest that both CyPA and CyPB are excellent targets for the treatment of HCV 1b, which shows the greatest resistance to interferon and ribavirin combination therapy. Copyright © 2012 Elsevier B.V. All rights reserved.

Human immune cell targeting of protein nanoparticles - caveospheres

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Glass, Joshua J.; Yuen, Daniel; Rae, James; Johnston, Angus P. R.; Parton, Robert G.; Kent, Stephen J.; de Rose, Robert

2016-04-01

Nanotechnology has the power to transform vaccine and drug delivery through protection of payloads from both metabolism and off-target effects, while facilitating specific delivery of cargo to immune cells. However, evaluation of immune cell nanoparticle targeting is conventionally restricted to monocultured cell line models. We generated human caveolin-1 nanoparticles, termed caveospheres, which were efficiently functionalized with monoclonal antibodies. Using this platform, we investigated CD4+ T cell and CD20+ B cell targeting within physiological mixtures of primary human blood immune cells using flow cytometry, imaging flow cytometry and confocal microscopy. Antibody-functionalization enhanced caveosphere binding to targeted immune cells (6.6 to 43.9-fold) within mixed populations and in the presence of protein-containing fluids. Moreover, targeting caveospheres to CCR5 enabled caveosphere internalization by non-phagocytic CD4+ T cells--an important therapeutic target for HIV treatment. This efficient and flexible system of immune cell-targeted caveosphere nanoparticles holds promise for the development of advanced immunotherapeutics and vaccines.

Effects of intracellular iron overload on cell death and identification of potent cell death inhibitors.

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Fang, Shenglin; Yu, Xiaonan; Ding, Haoxuan; Han, Jianan; Feng, Jie

2018-06-11

Iron overload causes many diseases, while the underlying etiologies of these diseases are unclear. Cell death processes including apoptosis, necroptosis, cyclophilin D-(CypD)-dependent necrosis and a recently described additional form of regulated cell death called ferroptosis, are dependent on iron or iron-dependent reactive oxygen species (ROS). However, whether the accumulation of intracellular iron itself induces ferroptosis or other forms of cell death is largely elusive. In present study, we study the role of intracellular iron overload itself-induced cell death mechanisms by using ferric ammonium citrate (FAC) and a membrane-permeable Ferric 8-hydroxyquinoline complex (Fe-8HQ) respectively. We show that FAC-induced intracellular iron overload causes ferroptosis. We also identify 3-phosphoinositide-dependent kinase 1 (PDK1) inhibitor GSK2334470 as a potent ferroptosis inhibitor. Whereas, Fe-8HQ-induced intracellular iron overload causes unregulated necrosis, but partially activates PARP-1 dependent parthanatos. Interestingly, we identify many phenolic compounds as potent inhibitors of Fe-8HQ-induced cell death. In conclusion, intracellular iron overload-induced cell death form might be dependent on the intracellular iron accumulation rate, newly identified cell death inhibitors in our study that target ferroptosis and unregulated oxidative cell death represent potential therapeutic strategies against iron overload related diseases. Copyright © 2018 Elsevier Inc. All rights reserved.

Targeting Cell Polarity Machinery to Exhaust Breast Cancer Stem Cells

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2017-10-01

AWARD NUMBER: W81XWH-15-1-0644 TITLE: Targeting Cell Polarity Machinery to Exhaust Breast Cancer Stem Cells PRINCIPAL INVESTIGATOR: Chun-Ju...Targeting Cell Polarity Machinery to Exhaust Breast Cancer Stem Cells 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-15-1-0644 5c. PROGRAM ELEMENT...Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Cancer stem cells (CSCs), a cell population with acquired perpetuating self-renewal properties which

Cell-specific targeting by heterobivalent ligands.

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Josan, Jatinder S; Handl, Heather L; Sankaranarayanan, Rajesh; Xu, Liping; Lynch, Ronald M; Vagner, Josef; Mash, Eugene A; Hruby, Victor J; Gillies, Robert J

2011-07-20

Current cancer therapies exploit either differential metabolism or targeting to specific individual gene products that are overexpressed in aberrant cells. The work described herein proposes an alternative approach--to specifically target combinations of cell-surface receptors using heteromultivalent ligands ("receptor combination approach"). As a proof-of-concept that functionally unrelated receptors can be noncovalently cross-linked with high avidity and specificity, a series of heterobivalent ligands (htBVLs) were constructed from analogues of the melanocortin peptide ligand ([Nle(4), dPhe(7)]-α-MSH) and the cholecystokinin peptide ligand (CCK-8). Binding of these ligands to cells expressing the human Melanocortin-4 receptor and the Cholecystokinin-2 receptor was analyzed. The MSH(7) and CCK(6) were tethered with linkers of varying rigidity and length, constructed from natural and/or synthetic building blocks. Modeling data suggest that a linker length of 20-50 Å is needed to simultaneously bind these two different G-protein coupled receptors (GPCRs). These ligands exhibited up to 24-fold enhancement in binding affinity to cells that expressed both (bivalent binding), compared to cells with only one (monovalent binding) of the cognate receptors. The htBVLs had up to 50-fold higher affinity than that of a monomeric CCK ligand, i.e., Ac-CCK(6)-NH(2). Cell-surface targeting of these two cell types with labeled heteromultivalent ligand demonstrated high avidity and specificity, thereby validating the receptor combination approach. This ability to noncovalently cross-link heterologous receptors and target individual cells using a receptor combination approach opens up new possibilities for specific cell targeting in vivo for therapy or imaging.

Expression of cyclophilin B is associated with malignant progression and regulation of genes implicated in the pathogenesis of breast cancer.

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Fang, Feng; Flegler, Ayanna J; Du, Pan; Lin, Simon; Clevenger, Charles V

2009-01-01

Cyclophilin B (CypB) is a 21-kDa protein with peptidyl-prolyl cis-trans isomerase activity that functions as a transcriptional inducer for Stat5 and as a ligand for CD147. To better understand the global function of CypB in breast cancer, T47D cells with a small interfering RNA-mediated knockdown of CypB were generated. Subsequent expression profiling analysis showed that 663 transcripts were regulated by CypB knockdown, and that many of these gene products contributed to cell proliferation, cell motility, and tumorigenesis. Real-time PCR confirmed that STMN3, S100A4, S100A6, c-Myb, estrogen receptor alpha, growth hormone receptor, and progesterone receptor were all down-regulated in si-CypB cells. A linkage analysis of these array data to protein networks resulted in the identification of 27 different protein networks that were impacted by CypB knockdown. Functional assays demonstrated that CypB knockdown also decreased cell growth, proliferation, and motility. Immunohistochemical and immunofluorescent analyses of a matched breast cancer progression tissue microarray that was labeled with an anti-CypB antibody demonstrated a highly significant increase in CypB protein levels as a function of breast cancer progression. Taken together, these results suggest that the enhanced expression of CypB in malignant breast epithelium may contribute to the pathogenesis of this disease through its regulation of the expression of hormone receptors and gene products that are involved in cell proliferation and motility.

mTOR inhibition sensitizes human hepatocellular carcinoma cells to resminostat

Energy Technology Data Exchange (ETDEWEB)

Peng, Xingang, E-mail: pengxinggang26@sina.com [Department of Emergency General Surgery, The Affiliated Hospital of Qingdao University, Qingdao (China); Zhang, Donghui, E-mail: zhangdonghuiyx@sina.com [Department of Infectious Disease, Linyi People’s Hospital, Linyi (China); Li, Zhengling, E-mail: lizhenglingzz@sina.com [Department of Nursing, Tengzhou Central People’s Hospital, Tengzhou (China); Fu, Meili, E-mail: fumeilidrlinyi@tom.com [Department of Infectious Disease, Linyi People’s Hospital, Linyi (China); Liu, Haiyan, E-mail: liuhaiyanlinyi5@sina.com [Department of Nursing, Linyi People’s Hospital, Linyi (China)

2016-09-02

Histone deacetylases (HDACs) hyper-activity in hepatocellular carcinoma (HCC) is often associated with patients’ poor prognosis. Our previous study has shown that resminostat, a novel HDAC inhibitor (HDACi), activated mitochondrial permeability transition pore (mPTP)-dependent apoptosis pathway in HCC cells. Here we explored the potential resminostat resistance factor by focusing on mammalian target of rapamycin (mTOR). We showed that AZD-2014, a novel mTOR kinase inhibitor, potentiated resminostat-induced cytotoxicity and proliferation inhibition in HCC cells. Molecularly, AZD-2014 enhanced resminostat-induced mPTP apoptosis pathway activation in HCC cells. Inhibition of this apoptosis pathway, by the caspase-9 specific inhibitor Ac-LEHD-CHO, the mPTP blockers (sanglifehrin A/cyclosporine A), or by shRNA-mediated knockdown of mPTP component cyclophilin-D (Cyp-D), significantly attenuated resminostat plus AZD-2014-induced cytotoxicity and apoptosis in HCC cells. Significantly, mTOR shRNA knockdown or kinase-dead mutation (Asp-2338-Ala) also sensitized HCC cells to resminostat, causing profound cytotoxicity and apoptosis induction. Together, these results suggest that mTOR could be a primary resistance factor of resminostat. Targeted inhibition of mTOR may thus significantly sensitize HCC cells to resminostat. - Highlights: • AZD-2014 potentiates resminostat’s cytotoxicity against HCC cells. • AZD-2014 facilitates resminostat-induced HCC cell apoptosis. • AZD-2014 augments resminostat-induced mitochondrial apoptosis pathway activation. • mTOR shRNA or kinase-dead mutation significantly sensitizes HCC cells to resminostat.

The clinical implication of serum cyclophilin A in patients with chronic obstructive pulmonary disease

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Zhang M

2018-01-01

Full Text Available Ming Zhang,1 Jingjing Tang,1 Jiafeng Yin,2 Xiaoying Wang,3 Xiangli Feng,1 Xia Yang,1 Hu Shan,1 Qiuhong Zhang,1 Jie Zhang,1 Yali Li1 1Department of Respiratory and Critical Care Medicine, The Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an, 2Department of Laboratory Examination, The Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an, 3Health Examination Center, The Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an, People’s Republic of China Background: Cyclophilin A (CyPA is a secreted molecule that is regulated by inflammatory stimuli. Although inflammation has an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD, little is known regarding the relationship between serum CyPA and COPD. Methods: Ninety-three COPD patients with acute exacerbation were enrolled in the study and were reassessed during the convalescence phase. Eighty-eight controls were matched for age, gender, body mass index, smoking index and comorbidity. The basic clinical information and pulmonary function of all participants were collected. Serum levels of CyPA and other inflammation indexes were further measured. Results: Serum CyPA was significantly increased in convalescent COPD patients compared to healthy controls, and further elevated in COPD patients with acute exacerbation. Serum CyPA positively correlated with serum interleukin-6, matrix metalloproteinase-9 and high-sensitivity C-reactive protein in both the exacerbation and convalescence phases of COPD. Furthermore, it negatively correlated with percent value of forced expiratory volume in 1 second (FEV1% predicted and FEV1/forced vital capacity in convalescent COPD patients. Conclusion: These results suggest that serum CyPA can be used as a potential inflammatory biomarker for COPD and assessment of serum CyPA may reflect the severity of inflammation in COPD. Keywords: cyclophilin A, chronic obstructive pulmonary disease

[Cloning and expressing of cyclophilin B gene from Schistosoma japonnicum and the analysis of immunoprotective effect].

Science.gov (United States)

Peng, Jinbiao; Han, Hongxiao; Hong, Yang; Wang, Yan; Guo, Fanji; Shi, Yaojun; Fu, Zhiqiang; Liu, Jinming; Cheng, Guofeng; Lin, Jiaojiao

2010-03-01

The present study was intend to clone and express the cDNA encoding Cyclophilin B (CyPB) of Schistosoma japonicum, its preliminary biological function and further immunoprotective effect against schistosome infection in mice. RT-PCR technique was applied to amplify a full-length cDNA encoding protein Cyclophilin B (Sj CyPB) from schistosomula cDNA. The expression profiles of Sj CyPB were determined by Real-time PCR using the template cDNAs isolated from 7, 13, 18, 23, 32 and 42 days parasites. The cDNA containing the Open Reading Frame of CyPB was then subcloned into a pGEX-6P-1 vector and transformed into competent Escherichia coli BL21 for expressing. The recombinant protein was renaturated, purified and its antigenicity were detected by Western blotting, and the immunoprotective effect induced by recombinant Sj CyPB was evaluated in Balb/C mice. The cDNA containing the ORF of Sj CyPB was cloned with the length of 672 base pairs, encoding 223 amino acids. Real-time PCR analysis revealed that the gene had the highest expression in 18-day schistosomula, suggesting that Sj CyPB was schistosomula differentially expressed gene. The recombinant protein showed a good antigenicity detected by Western blotting. Animal experiment indicated that the vaccination of recombinant CyPB protein in mice led to 31.5% worm and 41.01% liver egg burden reduction, respectively, compared with those of the control. A full-length cDNA differentially expressed in schistosomula was obtained. The recombinant Sj CyPB protein could induce partial protection against schistosome infection.

Involvement of two classes of binding sites in the interactions of cyclophilin B with peripheral blood T-lymphocytes.

OpenAIRE

Denys, A; Allain, F; Carpentier, M; Spik, G

1998-01-01

Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein, mainly associated with the secretory pathway, and is released in biological fluids. We recently reported that CyPB specifically binds to T-lymphocytes and promotes enhanced incorporation of CsA. The interactions with cellular binding sites involved, at least in part, the specific N-terminal extension of the protein. In this study, we intended to specify further the nature of the CyPB-binding sites on peripheral blood T-lymphocytes...

Radiation responses of stem cells: targeted and non-targeted effects

International Nuclear Information System (INIS)

Kavanagh, J.N.; Waring, E.J.; Prise, K.M.

2015-01-01

Stem cells are fundamental to the development of any tissue or organism via their ability to self-renew, which is aided by their unlimited proliferative capacity and their ability to produce fully differentiated offspring, often from multiple lineages. Stems cells are long lived and have the potential to accumulate mutations, including in response to radiation exposure. It is thought that stem cells have the potential to be induced into a cancer stem cell phenotype and that these may play an important role in resistance to radiotherapy. For radiation-induced carcinogenesis, the role of targeted and non-targeted effects is unclear with tissue or origin being important. Studies of genomic instability and bystander responses have shown consistent effects in haematopoietic models. Several models of radiation have predicted that stem cells play an important role in tumour initiation and that bystander responses could play a role in proliferation and self-renewal. (authors)

Proliferation of NS0 cells in protein-free medium: the role of cell-derived proteins, known growth factors and cellular receptors.

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Spens, Erika; Häggström, Lena

2009-05-20

NS0 cells proliferate without external supply of growth factors in protein-free media. We hypothesize that the cells produce their own factors to support proliferation. Understanding the mechanisms behind this autocrine regulation of proliferation may open for the novel approaches to improve animal cell processes. The following proteins were identified in NS0 conditioned medium (CM): cyclophilin A, cyclophilin B (CypB), cystatin C, D-dopachrome tautomerase, IL-25, isopentenyl-diphosphate delta-isomerase, macrophage migration inhibitory factor (MIF), beta(2)-microglobulin, Niemann pick type C2, secretory leukocyte protease inhibitor, thioredoxin-1, TNF-alpha, tumour protein translationally controlled 1 and ubiquitin. Further, cDNA microarray analysis indicated that the genes for IL-11, TNF receptor 6, TGF-beta receptor 1 and the IFN-gamma receptor were transcribed. CypB, IFN-alpha/beta/gamma, IL-11, IL-25, MIF, TGF-beta and TNF-alpha as well as the known growth factors EGF, IGF-I/II, IL-6, leukaemia inhibitory factor and oncostatin M (OSM) were excluded as involved in autocrine regulation of NS0 cell proliferation. The receptors for TGF-beta, IGF and OSM are however present in NS0 cell membranes since TGF-beta(1) caused cell death, and IGF-I/II and OSM improved cell growth. Even though no ligand was found, the receptor subunit gp130, active in signal transduction of the IL-6 like proteins, was shown to be essential for NS0 cells as demonstrated by siRNA gene silencing.

Hsp47 and cyclophilin B traverse the endoplasmic reticulum with procollagen into pre-Golgi intermediate vesicles. A role for Hsp47 and cyclophilin B in the export of procollagen from the endoplasmic reticulum.

Science.gov (United States)

Smith, T; Ferreira, L R; Hebert, C; Norris, K; Sauk, J J

1995-08-04

Hsp47 and cyclophilin B (CyPB) are residents of the endoplasmic reticulum (ER). Both of these proteins are closely associated with polysome-associated alpha 1(I) procollagen chains. Hsp47 possesses chaperone properties early during the translation of procollagen while the cis/trans-isomerase properties of CyPB facilitate procollagen folding. In this report, we further investigate the interaction of these proteins with procollagen I during export from the ER. To inhibit vesicular budding and retain procollagen within the ER, cells were treated with the heterotrimeric G protein inhibitor mastoparan or calphostin C, a specific inhibitor of diacylglycerol/phorbol ester binding proteins. To arrest procollagen in pre-Golgi intermediate vesicles, cells were treated with guanosine 5'-3-O-(thio)triphosphate. Pulse-chase experiments of cells labeled with [35S]methionine followed by immunoprecipitation during the chase period with anti-procollagen, anti-Hsp47, and anti-CyPB antibodies were performed to reveal the relationship between Hsp47/CyPB/procollagen I. The distribution of procollagen, Hsp47, and CyPB to the ER and/or pre-Golgi vesicles was verified by immunofluorescence. Hsp47 and CyPB remained associated with procollagen retained within the ER. Hsp47 and CyPB were also associated with procollagen exported from the ER into pre-Golgi intermediate vesicles. Treatment of cells with cyclosporin A diminished the levels of CyPB bound to procollagen and diminished the rate of Hsp47 released from procollagen and the rate of procollagen secretion, suggesting that Hsp47 release from procollagen may be driven by helix formation. Also, these studies suggest that Hsp47 may resemble protein disulfide isomerase and possess both chaperone and anti-chaperone properties. During translation, high levels of Hsp47 are seen to limit protein aggregation and facilitate chain registration. Later, Hsp47 and/or CyPB and protein disulfide isomerase act as anti-chaperones and provide the basis for

Lack of cyclophilin B in osteogenesis imperfecta with normal collagen folding.

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Barnes, Aileen M; Carter, Erin M; Cabral, Wayne A; Weis, MaryAnn; Chang, Weizhong; Makareeva, Elena; Leikin, Sergey; Rotimi, Charles N; Eyre, David R; Raggio, Cathleen L; Marini, Joan C

2010-02-11

Osteogenesis imperfecta is a heritable disorder that causes bone fragility. Mutations in type I collagen result in autosomal dominant osteogenesis imperfecta, whereas mutations in either of two components of the collagen prolyl 3-hydroxylation complex (cartilage-associated protein [CRTAP] and prolyl 3-hydroxylase 1 [P3H1]) cause autosomal recessive osteogenesis imperfecta with rhizomelia (shortening of proximal segments of upper and lower limbs) and delayed collagen folding. We identified two siblings who had recessive osteogenesis imperfecta without rhizomelia. They had a homozygous start-codon mutation in the peptidyl-prolyl isomerase B gene (PPIB), which results in a lack of cyclophilin B (CyPB), the third component of the complex. The proband's collagen had normal collagen folding and normal prolyl 3-hydroxylation, suggesting that CyPB is not the exclusive peptidyl-prolyl cis-trans isomerase that catalyzes the rate-limiting step in collagen folding, as is currently thought. 2010 Massachusetts Medical Society

Buoyancy-activated cell sorting using targeted biotinylated albumin microbubbles.

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Yu-Ren Liou

Full Text Available Cell analysis often requires the isolation of certain cell types. Various isolation methods have been applied to cell sorting, including fluorescence-activated cell sorting and magnetic-activated cell sorting. However, these conventional approaches involve exerting mechanical forces on the cells, thus risking cell damage. In this study we applied a novel isolation method called buoyancy-activated cell sorting, which involves using biotinylated albumin microbubbles (biotin-MBs conjugated with antibodies (i.e., targeted biotin-MBs. Albumin MBs are widely used as contrast agents in ultrasound imaging due to their good biocompatibility and stability. For conjugating antibodies, biotin is conjugated onto the albumin MB shell via covalent bonds and the biotinylated antibodies are conjugated using an avidin-biotin system. The albumin microbubbles had a mean diameter of 2 μm with a polydispersity index of 0.16. For cell separation, the MDA-MB-231 cells are incubated with the targeted biotin-MBs conjugated with anti-CD44 for 10 min, centrifuged at 10 g for 1 min, and then allowed 1 hour at 4 °C for separation. The results indicate that targeted biotin-MBs conjugated with anti-CD44 antibodies can be used to separate MDA-MB-231 breast cancer cells; more than 90% of the cells were collected in the MB layer when the ratio of the MBs to cells was higher than 70:1. Furthermore, we found that the separating efficiency was higher for targeted biotin-MBs than for targeted avidin-incorporated albumin MBs (avidin-MBs, which is the most common way to make targeted albumin MBs. We also demonstrated that the recovery rate of targeted biotin-MBs was up to 88% and the sorting purity was higher than 84% for a a heterogenous cell population containing MDA-MB-231 cells (CD44(+ and MDA-MB-453 cells (CD44-, which are classified as basal-like breast cancer cells and luminal breast cancer cells, respectively. Knowing that the CD44(+ is a commonly used cancer-stem-cell

Manipulating the mitochondria activity in human hepatic cell line Huh7 by low-power laser irradiation

Science.gov (United States)

Lynnyk, Anna; Lunova, Mariia; Jirsa, Milan; Egorova, Daria; Kulikov, Andrei; Kubinová, Šárka; Lunov, Oleg; Dejneka, Alexandr

2018-01-01

Low-power laser irradiation of red light has been recognized as a promising tool across a vast variety of biomedical applications. However, deep understanding of the molecular mechanisms behind laser-induced cellular effects remains a significant challenge. Here, we investigated mechanisms involved in the death process in human hepatic cell line Huh7 at a laser irradiation. We decoupled distinct cell death pathways targeted by laser irradiations of different powers. Our data demonstrate that high dose laser irradiation exhibited the highest levels of total reactive oxygen species production, leading to cyclophilin D-related necrosis via the mitochondrial permeability transition. On the contrary, low dose laser irradiation resulted in the nuclear accumulation of superoxide and apoptosis execution. Our findings offer a novel insight into laser-induced cellular responses, and reveal distinct cell death pathways triggered by laser irradiation. The observed link between mitochondria depolarization and triggering ROS could be a fundamental phenomenon in laser-induced cellular responses. PMID:29541521

Cyclophilin B Deficiency Causes Abnormal Dentin Collagen Matrix.

Science.gov (United States)

Terajima, Masahiko; Taga, Yuki; Cabral, Wayne A; Nagasawa, Masako; Sumida, Noriko; Hattori, Shunji; Marini, Joan C; Yamauchi, Mitsuo

2017-08-04

Cyclophilin B (CypB) is an endoplasmic reticulum-resident protein that regulates collagen folding, and also contributes to prolyl 3-hydroxylation (P3H) and lysine (Lys) hydroxylation of collagen. In this study, we characterized dentin type I collagen in CypB null (KO) mice, a model of recessive osteogenesis imperfecta type IX, and compared to those of wild-type (WT) and heterozygous (Het) mice. Mass spectrometric analysis demonstrated that the extent of P3H in KO collagen was significantly diminished compared to WT/Het. Lys hydroxylation in KO was significantly diminished at the helical cross-linking sites, α1/α2(I) Lys-87 and α1(I) Lys-930, leading to a significant increase in the under-hydroxylated cross-links and a decrease in fully hydroxylated cross-links. The extent of glycosylation of hydroxylysine residues was, except α1(I) Lys-87, generally higher in KO than WT/Het. Some of these molecular phenotypes were distinct from other KO tissues reported previously, indicating the dentin-specific control mechanism through CypB. Histological analysis revealed that the width of predentin was greater and irregular, and collagen fibrils were sparse and significantly smaller in KO than WT/Het. These results indicate a critical role of CypB in dentin matrix formation, suggesting a possible association between recessive osteogenesis imperfecta and dentin defects that have not been clinically detected.

Radioprotection of targeted and bystander cells by methylproamine

International Nuclear Information System (INIS)

Burdak-Rothkamm, Susanne; Smith, Andrea; Lobachevsky, Pavel; Martin, Roger; Prise, Kevin M.

2015-01-01

Radioprotective agents are of interest for application in radiotherapy for cancer and in public health medicine in the context of accidental radiation exposure. Methylproamine is the lead compound of a class of radioprotectors which act as DNA binding anti-oxidants, enabling the repair of transient radiation-induced oxidative DNA lesions. This study tested methylproamine for the radioprotection of both directly targeted and bystander cells. T98G glioma cells were treated with 15 μM methylproamine and exposed to 137 Cs γ-ray/X-ray irradiation and He 2+ microbeam irradiation. Radioprotection of directly targeted cells and bystander cells was measured by clonogenic survival or γH2AX assay. Radioprotection of directly targeted T98G cells by methylproamine was observed for 137 Cs γ-rays and X-rays but not for He 2+ charged particle irradiation. The effect of methylproamine on the bystander cell population was tested for both X-ray irradiation and He 2+ ion microbeam irradiation. The X-ray bystander experiments were carried out by medium transfer from irradiated to non-irradiated cultures and three experimental designs were tested. Radioprotection was only observed when recipient cells were pretreated with the drug prior to exposure to the conditioned medium. In microbeam bystander experiments targeted and nontargeted cells were co-cultured with continuous methylproamine treatment during irradiation and postradiation incubation; radioprotection of bystander cells was observed. Methylproamine protected targeted cells from DNA damage caused by γ-ray or X-ray radiation but not He 2+ ion radiation. Protection of bystander cells was independent of the type of radiation which the donor population received. (orig.) [de

The heat shock protein-90 co-chaperone, Cyclophilin 40, promotes ALK-positive, anaplastic large cell lymphoma viability and its expression is regulated by the NPM-ALK oncoprotein

International Nuclear Information System (INIS)

Pearson, Joel D; Mohammed, Zubair; Bacani, Julinor T C; Lai, Raymond; Ingham, Robert J

2012-01-01

Anaplastic lymphoma kinase-positive, anaplastic large cell lymphoma (ALK+ ALCL) is a T cell lymphoma defined by the presence of chromosomal translocations involving the ALK tyrosine kinase gene. These translocations generate fusion proteins (e.g. NPM-ALK) with constitutive tyrosine kinase activity, which activate numerous signalling pathways important for ALK+ ALCL pathogenesis. The molecular chaperone heat shock protein-90 (Hsp90) plays a critical role in allowing NPM-ALK and other signalling proteins to function in this lymphoma. Co-chaperone proteins are important for helping Hsp90 fold proteins and for directing Hsp90 to specific clients; however the importance of co-chaperone proteins in ALK+ ALCL has not been investigated. Our preliminary findings suggested that expression of the immunophilin co-chaperone, Cyclophilin 40 (Cyp40), is up-regulated in ALK+ ALCL by JunB, a transcription factor activated by NPM-ALK signalling. In this study we examined the regulation of the immunophilin family of co-chaperones by NPM-ALK and JunB, and investigated whether the immunophilin co-chaperones promote the viability of ALK+ ALCL cell lines. NPM-ALK and JunB were knocked-down in ALK+ ALCL cell lines with siRNA, and the effect on the expression of the three immunophilin co-chaperones: Cyp40, FK506-binding protein (FKBP) 51, and FKBP52 examined. Furthermore, the effect of knock-down of the immunophilin co-chaperones, either individually or in combination, on the viability of ALK+ ALCL cell lines and NPM-ALK levels and activity was also examined. We found that NPM-ALK promoted the transcription of Cyp40 and FKBP52, but only Cyp40 transcription was promoted by JunB. We also observed reduced viability of ALK+ ALCL cell lines treated with Cyp40 siRNA, but not with siRNAs directed against FKBP52 or FKBP51. Finally, we demonstrate that the decrease in the viability of ALK+ ALCL cell lines treated with Cyp40 siRNA does not appear to be due to a decrease in NPM-ALK levels or the

Osteosarcoma: Cells-of-Origin, Cancer Stem Cells, and Targeted Therapies

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Ander Abarrategi

2016-01-01

Full Text Available Osteosarcoma (OS is the most common type of primary solid tumor that develops in bone. Although standard chemotherapy has significantly improved long-term survival over the past few decades, the outcome for those patients with metastatic or recurrent OS remains dismally poor and, therefore, novel agents and treatment regimens are urgently required. A hypothesis to explain the resistance of OS to chemotherapy is the existence of drug resistant CSCs with progenitor properties that are responsible of tumor relapses and metastasis. These subpopulations of CSCs commonly emerge during tumor evolution from the cell-of-origin, which are the normal cells that acquire the first cancer-promoting mutations to initiate tumor formation. In OS, several cell types along the osteogenic lineage have been proposed as cell-of-origin. Both the cell-of-origin and their derived CSC subpopulations are highly influenced by environmental and epigenetic factors and, therefore, targeting the OS-CSC environment and niche is the rationale for many recently postulated therapies. Likewise, some strategies for targeting CSC-associated signaling pathways have already been tested in both preclinical and clinical settings. This review recapitulates current OS cell-of-origin models, the properties of the OS-CSC and its niche, and potential new therapies able to target OS-CSCs.

Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells.

Science.gov (United States)

Herce, Henry D; Schumacher, Dominik; Schneider, Anselm F L; Ludwig, Anne K; Mann, Florian A; Fillies, Marion; Kasper, Marc-André; Reinke, Stefan; Krause, Eberhard; Leonhardt, Heinrich; Cardoso, M Cristina; Hackenberger, Christian P R

2017-08-01

Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain VHH antibody fragments. We used this strategy for the non-endocytic delivery of two recombinant nanobodies into living cells, which enabled the relocalization of the polymerase clamp PCNA (proliferating cell nuclear antigen) and tumour suppressor p53 to the nucleolus, and thereby allowed the detection of protein-protein interactions that involve these two proteins in living cells. Furthermore, cell-permeable nanobodies permitted the co-transport of therapeutically relevant proteins, such as Mecp2, into the cells. This technology constitutes a major step in the labelling, delivery and targeted manipulation of intracellular antigens. Ultimately, this approach opens the door towards immunostaining in living cells and the expansion of immunotherapies to intracellular antigen targets.

Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells

Science.gov (United States)

Herce, Henry D.; Schumacher, Dominik; Schneider, Anselm F. L.; Ludwig, Anne K.; Mann, Florian A.; Fillies, Marion; Kasper, Marc-André; Reinke, Stefan; Krause, Eberhard; Leonhardt, Heinrich; Cardoso, M. Cristina; Hackenberger, Christian P. R.

2017-08-01

Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain VHH antibody fragments. We used this strategy for the non-endocytic delivery of two recombinant nanobodies into living cells, which enabled the relocalization of the polymerase clamp PCNA (proliferating cell nuclear antigen) and tumour suppressor p53 to the nucleolus, and thereby allowed the detection of protein-protein interactions that involve these two proteins in living cells. Furthermore, cell-permeable nanobodies permitted the co-transport of therapeutically relevant proteins, such as Mecp2, into the cells. This technology constitutes a major step in the labelling, delivery and targeted manipulation of intracellular antigens. Ultimately, this approach opens the door towards immunostaining in living cells and the expansion of immunotherapies to intracellular antigen targets.

The quest for targets executing MYC-dependent cell transformation

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Markus eHartl

2016-06-01

Full Text Available MYC represents a transcription factor with oncogenic potential converting multiple cellular signals into a broad transcriptional response, thereby controlling the expression of numerous protein-coding and non-coding RNAs important for cell proliferation, metabolism, differentiation, and apoptosis. Constitutive activation of MYC leads to neoplastic cell transformation, and deregulated MYC alleles are frequently observed in many human cancer cell types. Multiple approaches have been performed to isolate genes differentially expressed in cells containing aberrantly activated MYC proteins leading to the identification of thousands of putative targets. Functional analyses of genes differentially expressed in MYC-transformed cells had revealed that so far more than forty upregulated or downregulated MYC targets are actively involved in cell transformation or tumorigenesis. However, for determination which of the known, or yet unidentified targets are responsible for processing the oncogenic MYC program, further systematic and selective approaches are required. The search for critical targets in MYC-dependent tumor cells is exacerbated by the fact that during tumor development, cancer cells progressively evolve in a multistep process thereby acquiring their characteristic features in an additive manner. Functional expression cloning, combinatorial gene expression and appropriate in vivo tests could represent adequate tools for dissecting the complex scenario of MYC-specified cell transformation. In this context, the central goal is to identify a minimal set of targets that suffices to phenocopy oncogenic MYC. Recently developed genomic editing tools could be employed to confirm the requirement of crucial transformation-associated targets.Knowledge about essential MYC regulated genes is beneficial to expedite the development of specific inhibitors to interfere with growth and viability of human tumor cells in which MYC is aberrantly activated

Targeting therapy-resistant cancer stem cells by hyperthermia

DEFF Research Database (Denmark)

Oei, A L; Vriend, L E M; Krawczyk, P M

2017-01-01

Eradication of all malignant cells is the ultimate but challenging goal of anti-cancer treatment; most traditional clinically-available approaches fail because there are cells in a tumour that either escape therapy or become therapy-resistant. A subpopulation of cancer cells, the cancer stem cells...... are limited. Here, we argue that hyperthermia - a therapeutic approach based on local heating of a tumour - is potentially beneficial for targeting CSCs in solid tumours. First, hyperthermia has been described to target cells in hypoxic and nutrient-deprived tumour areas where CSCs reside and ionising...

Targeting human breast cancer cells by an oncolytic adenovirus using microRNA-targeting strategy.

Science.gov (United States)

Shayestehpour, Mohammad; Moghim, Sharareh; Salimi, Vahid; Jalilvand, Somayeh; Yavarian, Jila; Romani, Bizhan; Mokhtari-Azad, Talat

2017-08-15

MicroRNA-targeting strategy is a promising approach that enables oncolytic viruses to replicate in tumor cells but not in normal cells. In this study, we targeted adenoviral replication toward breast cancer cells by inserting ten complementary binding sites for miR-145-5p downstream of E1A gene. In addition, we evaluated the effect of increasing miR-145 binding sites on inhibition of virus replication. Ad5-control and adenoviruses carrying five or ten copies of miR145-5p target sites (Ad5-5miR145T, Ad5-10miR145T) were generated and inoculated into MDA-MB-453, BT-20, MCF-7 breast cancer cell lines and human mammary epithelial cells (HMEpC). Titer of Ad5-10miR145T in HMEpC was significantly lower than Ad5-control titer. Difference between the titer of these two viruses at 12, 24, 36, and 48h after infection was 1.25, 2.96, 3.06, and 3.77 log TCID 50 . No significant difference was observed between the titer of both adenoviruses in MDA-MB-453, BT-20 and MCF-7 cells. The infectious titer of adenovirus containing 10 miR-145 binding sites in HMEpC cells at 24, 36, and 48h post-infection was 1.7, 2.08, and 4-fold, respectively, lower than the titer of adenovirus carrying 5 miR-145 targets. Our results suggest that miR-145-targeting strategy provides selectivity for adenovirus replication in breast cancer cells. Increasing the number of miRNA binding sites within the adenoviral genome confers more selectivity for viral replication in cancer cells. Copyright © 2017. Published by Elsevier B.V.

Cell cycle-tailored targeting of metastatic melanoma: Challenges and opportunities.

Science.gov (United States)

Haass, Nikolas K; Gabrielli, Brian

2017-07-01

The advent of targeted therapies of metastatic melanoma, such as MAPK pathway inhibitors and immune checkpoint antagonists, has turned dermato-oncology from the "bad guy" to the "poster child" in oncology. Current targeted therapies are effective, although here is a clear need to develop combination therapies to delay the onset of resistance. Many antimelanoma drugs impact on the cell cycle but are also dependent on certain cell cycle phases resulting in cell cycle phase-specific drug insensitivity. Here, we raise the question: Have combination trials been abandoned prematurely as ineffective possibly only because drug scheduling was not optimized? Firstly, if both drugs of a combination hit targets in the same melanoma cell, cell cycle-mediated drug insensitivity should be taken into account when planning combination therapies, timing of dosing schedules and choice of drug therapies in solid tumors. Secondly, if the combination is designed to target different tumor cell subpopulations of a heterogeneous tumor, one drug effective in a particular subpopulation should not negatively impact on the other drug targeting another subpopulation. In addition to the role of cell cycle stage and progression on standard chemotherapeutics and targeted drugs, we discuss the utilization of cell cycle checkpoint control defects to enhance chemotherapeutic responses or as targets themselves. We propose that cell cycle-tailored targeting of metastatic melanoma could further improve therapy outcomes and that our real-time cell cycle imaging 3D melanoma spheroid model could be utilized as a tool to measure and design drug scheduling approaches. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Advanced cell therapies: targeting, tracking and actuation of cells with magnetic particles.

Science.gov (United States)

Connell, John J; Patrick, P Stephen; Yu, Yichao; Lythgoe, Mark F; Kalber, Tammy L

2015-01-01

Regenerative medicine would greatly benefit from a new platform technology that enabled measurable, controllable and targeting of stem cells to a site of disease or injury in the body. Superparamagnetic iron-oxide nanoparticles offer attractive possibilities in biomedicine and can be incorporated into cells, affording a safe and reliable means of tagging. This review describes three current and emerging methods to enhance regenerative medicine using magnetic particles to guide therapeutic cells to a target organ; track the cells using MRI and assess their spatial localization with high precision and influence the behavior of the cell using magnetic actuation. This approach is complementary to the systemic injection of cell therapies, thus expanding the horizon of stem cell therapeutics.

B cells as a target of immune modulation

Directory of Open Access Journals (Sweden)

Hawker Kathleen

2009-01-01

Full Text Available B cells have recently been identified as an integral component of the immune system; they play a part in autoimmunity through antigen presentation, antibody secretion, and complement activation. Animal models of multiple sclerosis (MS suggest that myelin destruction is partly mediated through B cell activation (and plasmablasts. MS patients with evidence of B cell involvement, as compared to those without, tend to have a worse prognosis. Finally, the significant decrease in new gadolinium-enhancing lesions, new T2 lesions, and relapses in MS patients treated with rituximab (a monoclonal antibody against CD20 on B cells leads us to the conclusion that B cells play an important role in MS and that immune modulation of these cells may ameliorate the disease. This article will explore the role of B cells in MS and the rationale for the development of B cell-targeted therapeutics. MS is an immune-mediated disease that affects over 2 million people worldwide and is the number one cause of disability in young patients. Most therapeutic targets have focused on T cells; however, recently, the focus has shifted to the role of B cells in the pathogenesis of MS and the potential of B cells as a therapeutic target.

Targeting Stromal Recruitment by Prostate Cancer Cells

Science.gov (United States)

2006-03-01

Ensinger, C., Tumer , Z., Tommerup, N. et al.: Hedgehog signaling in small-cell lung cancer : frequent in vivo but a rare event in vitro. Lung Cancer , 52...W81XWH-04-1-0157 TITLE: Targeting Stromal Recruitment by Prostate Cancer Cells PRINCIPAL INVESTIGATOR: Jingxian Zhang, Ph.D...DATES COVERED (From - To) 15 Feb 2004 – 14 Feb 2006 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Targeting Stromal Recruitment by Prostate Cancer

Broad target cell selectivity of Kaposi's sarcoma-associated herpesvirus glycoprotein-mediated cell fusion and virion entry

International Nuclear Information System (INIS)

Kaleeba, Johnan A.R.; Berger, Edward A.

2006-01-01

The molecular mechanism of Kaposi's sarcoma-associated herpesvirus (KSHV, human herpesvirus 8) entry is poorly understood. We tested a broad variety of cell types of diverse species and tissue origin for their ability to function as targets in a quantitative reporter gene assay for KSHV-glycoprotein-mediated cell fusion. Several human, non-human primate, and rabbit cell lines were efficient targets, whereas rodent and all human lymphoblastoid cell lines were weak targets. Parallel findings were obtained with a virion entry assay using a recombinant KSHV encoding a reporter gene. No correlation was observed between target cell activity and surface expression of α3β1 integrin, a proposed KSHV receptor. We hypothesize that target cell permissiveness in both the cell fusion and virion entry assays reflects the presence of a putative KSHV fusion-entry receptor

Magnetic stem cell targeting to the inner ear

Science.gov (United States)

Le, T. N.; Straatman, L.; Yanai, A.; Rahmanian, R.; Garnis, C.; Häfeli, U. O.; Poblete, T.; Westerberg, B. D.; Gregory-Evans, K.

2017-12-01

Severe sensorineural deafness is often accompanied by a loss of auditory neurons in addition to injury of the cochlear epithelium and hair cell loss. Cochlear implant function however depends on a healthy complement of neurons and their preservation is vital in achieving optimal results. We have developed a technique to target mesenchymal stem cells (MSCs) to a deafened rat cochlea. We then assessed the neuroprotective effect of systematically delivered MSCs on the survival and function of spiral ganglion neurons (SGNs). MSCs were labeled with superparamagnetic nanoparticles, injected via the systemic circulation, and targeted using a magnetized cochlea implant and external magnet. Neurotrophic factor concentrations, survival of SGNs, and auditory function were assessed at 1 week and 4 weeks after treatments and compared against multiple control groups. Significant numbers of magnetically targeted MSCs (>30 MSCs/section) were present in the cochlea with accompanied elevation of brain-derived neurotrophic factor and glial cell-derived neurotrophic factor levels (p < 0.001). In addition we saw improved survival of SGNs (approximately 80% survival at 4 weeks). Hearing threshold levels in magnetically targeted rats were found to be significantly better than those of control rats (p < 0.05). These results indicate that magnetic targeting of MSCs to the cochlea can be accomplished with a magnetized cochlear permalloy implant and an external magnet. The targeted stem cells release neurotrophic factors which results in improved SGN survival and hearing recovery. Combining magnetic cell-based therapy and cochlear implantation may improve cochlear implant function in treating deafness.

Surface-modified gold nanorods for specific cell targeting

Science.gov (United States)

Wang, Chan-Ung; Arai, Yoshie; Kim, Insun; Jang, Wonhee; Lee, Seonghyun; Hafner, Jason H.; Jeoung, Eunhee; Jung, Deokho; Kwon, Youngeun

2012-05-01

Gold nanoparticles (GNPs) have unique properties that make them highly attractive materials for developing functional reagents for various biomedical applications including photothermal therapy, targeted drug delivery, and molecular imaging. For in vivo applications, GNPs need to be prepared with very little or negligible cytotoxicitiy. Most GNPs are, however, prepared using growth-directing surfactants such as cetyl trimethylammonium bromide (CTAB), which are known to have considerable cytotoxicity. In this paper, we describe an approach to remove CTAB to a non-toxic concentration. We optimized the conditions for surface modification with methoxypolyethylene glycol thiol (mPEG), which replaced CTAB and formed a protective layer on the surface of gold nanorods (GNRs). The cytotoxicities of pristine and surface-modified GNRs were measured in primary human umbilical vein endothelial cells and human cell lines derived from hepatic carcinoma cells, embryonic kidney cells, and thyroid papillary carcinoma cells. Cytotoxicity assays revealed that treating cells with GNRs did not significantly affect cell viability except for thyroid papillary carcinoma cells. Thyroid cancer cells were more susceptible to residual CTAB, so CTAB had to be further removed by dialysis in order to use GNRs for thyroid cell targeting. PEGylated GNRs are further modified to present monoclonal antibodies that recognize a specific surface marker, Na-I symporter, for thyroid cells. Antibody-conjugated GNRs specifically targeted human thyroid cells in vitro.

Curcumin targets fibroblast–tumor cell interactions in oral squamous cell carcinoma

International Nuclear Information System (INIS)

Dudás, József; Fullár, Alexandra; Romani, Angela; Pritz, Christian; Kovalszky, Ilona; Hans Schartinger, Volker; Mathias Sprinzl, Georg; Riechelmann, Herbert

2013-01-01

Co-culture of periodontal ligament fibroblasts (PDLs) and SCC-25 oral squamous carcinoma cells (OSCC) results in conversion of PDLs into carcinoma-associated fibroblasts (CAFs) and induces epithelial-to mesenchymal transition (EMT) of OSCC tumor cells. We hypothesized that Curcumin targets this dynamic mutual interaction between CAFs and tumor cells. Normal and 2 μM Curcumin-treated co-culture were performed for 4 days, followed by analysis of tumor cell invasivity, mRNA/protein expression of EMT-markers and mediators, activity measure of matrix metalloproteinase 9 (MMP-9), and western blot analysis of signal transduction in tumor cells and fibroblasts. In Curcumin-treated co-culture, in tumor cells, the levels of nuclear factor κB (NFκBα) and early response kinase (ERK)—decreased, in fibroblasts, integrin αv protein synthesis decreased compared to corresponding cells in normal co-culture. The signal modulatory changes induced by Curcumin caused decreased release of EMT-mediators in CAFs and reversal of EMT in tumor cells, which was associated with decreased invasion. These data confirm the palliative potential of Curcumin in clinical application. - Graphical abstract: Co-culture of periodontal ligament fibroblasts (PDLs) and SCC-25 oral squamous carcinoma cells (OSCC) results in conversion of PDLs into carcinoma-associated fibroblasts (CAFs) and induces epithelial-to mesenchymal transition (EMT) of tumor cells. Curcumin targets this dynamic mutual interaction between CAFs and tumor cells by inhibiting the production of EMT mediators in CAFs and by modification of intracellular signaling in tumor cells. This causes less invasivity and reversal of EMT in tumor cells. Highlights: ► Curcumin targets tumor–fibroblast interaction in head and neck cancer. ► Curcumin suppresses mediators of epithelial–mesenchymal transition. ► Curcumin decreases the invasivity of tumor cells

Curcumin targets fibroblast–tumor cell interactions in oral squamous cell carcinoma

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Dudás, József, E-mail: jozsef.dudas@i-med.ac.at [Department of Otorhinolaryngology and Head and Neck Surgery, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Fullár, Alexandra, E-mail: fullarsz@gmail.com [Department of Otorhinolaryngology and Head and Neck Surgery, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Üllői út 26, 1085 Budapest (Hungary); Romani, Angela, E-mail: angela.romani@i-med.ac.at [Department of Otorhinolaryngology and Head and Neck Surgery, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Pritz, Christian, E-mail: christian.pritz@i-med.ac.at [Department of Otorhinolaryngology and Head and Neck Surgery, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Kovalszky, Ilona, E-mail: koval@korb1.sote.hu [1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Üllői út 26, 1085 Budapest (Hungary); Hans Schartinger, Volker, E-mail: volker.schartinger@i-med.ac.at [Department of Otorhinolaryngology and Head and Neck Surgery, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Mathias Sprinzl, Georg, E-mail: georg.sprinzl@i-med.ac.at [Department of Otorhinolaryngology and Head and Neck Surgery, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Riechelmann, Herbert, E-mail: herbert.riechelmann@i-med.ac.at [Department of Otorhinolaryngology and Head and Neck Surgery, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria)

2013-04-01

Co-culture of periodontal ligament fibroblasts (PDLs) and SCC-25 oral squamous carcinoma cells (OSCC) results in conversion of PDLs into carcinoma-associated fibroblasts (CAFs) and induces epithelial-to mesenchymal transition (EMT) of OSCC tumor cells. We hypothesized that Curcumin targets this dynamic mutual interaction between CAFs and tumor cells. Normal and 2 μM Curcumin-treated co-culture were performed for 4 days, followed by analysis of tumor cell invasivity, mRNA/protein expression of EMT-markers and mediators, activity measure of matrix metalloproteinase 9 (MMP-9), and western blot analysis of signal transduction in tumor cells and fibroblasts. In Curcumin-treated co-culture, in tumor cells, the levels of nuclear factor κB (NFκBα) and early response kinase (ERK)—decreased, in fibroblasts, integrin αv protein synthesis decreased compared to corresponding cells in normal co-culture. The signal modulatory changes induced by Curcumin caused decreased release of EMT-mediators in CAFs and reversal of EMT in tumor cells, which was associated with decreased invasion. These data confirm the palliative potential of Curcumin in clinical application. - Graphical abstract: Co-culture of periodontal ligament fibroblasts (PDLs) and SCC-25 oral squamous carcinoma cells (OSCC) results in conversion of PDLs into carcinoma-associated fibroblasts (CAFs) and induces epithelial-to mesenchymal transition (EMT) of tumor cells. Curcumin targets this dynamic mutual interaction between CAFs and tumor cells by inhibiting the production of EMT mediators in CAFs and by modification of intracellular signaling in tumor cells. This causes less invasivity and reversal of EMT in tumor cells. Highlights: ► Curcumin targets tumor–fibroblast interaction in head and neck cancer. ► Curcumin suppresses mediators of epithelial–mesenchymal transition. ► Curcumin decreases the invasivity of tumor cells.

Cyclophilin B induces chemoresistance by degrading wild type p53 via interaction with MDM2 in colorectal cancer.

Science.gov (United States)

Choi, Tae Gyu; Nguyen, Minh Nam; Kim, Jieun; Jo, Yong Hwa; Jang, Miran; Nguyen, Ngoc Ngo Yen; Yun, Hyeong Rok; Choe, Wonchae; Kang, Insug; Ha, Joohun; Tang, Dean G; Kim, Sung Soo

2018-06-06

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. Chemoresistance is a major problem for effective therapy in CRC. Here, we investigated the mechanism by which peptidylprolyl isomerase B (PPIB; cyclophilin B, CypB) regulates chemoresistance in CRC. We found that CypB is a novel wild type p53 (p53WT)-inducible gene but a negative regulator of p53WT in response to oxaliplatin treatment. Overexpression of CypB shortens the half-life of p53WT and inhibits oxaliplatin-induced apoptosis in CRC cells, whereas knockdown of CypB lengthens the half-life of p53WT and stimulates p53WT dependent apoptosis. CypB interacts directly with MDM2, and enhances MDM2-dependent p53WT ubiquitination and degradation. Furthermore, we firmly validated using bioinformatics analyses that overexpression of CypB is associated with poor prognosis in CRC progression and chemoresistance. Hence, we suggest a novel mechanism of chemoresistance caused by overexpressed CypB, which may help to develop new anti-cancer drugs. We also propose that CypB may be utilized as a predictive biomarker in CRC patients. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Selective tumor cell targeting by the disaccharide moiety of bleomycin.

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Yu, Zhiqiang; Schmaltz, Ryan M; Bozeman, Trevor C; Paul, Rakesh; Rishel, Michael J; Tsosie, Krystal S; Hecht, Sidney M

2013-02-27

In a recent study, the well-documented tumor targeting properties of the antitumor agent bleomycin (BLM) were studied in cell culture using microbubbles that had been derivatized with multiple copies of BLM. It was shown that BLM selectively targeted MCF-7 human breast carcinoma cells but not the "normal" breast cell line MCF-10A. Furthermore, it was found that the BLM analogue deglycobleomycin, which lacks the disaccharide moiety of BLM, did not target either cell line, indicating that the BLM disaccharide moiety is necessary for tumor selectivity. Not resolved in the earlier study were the issues of whether the BLM disaccharide moiety alone is sufficient for tumor cell targeting and the possible cellular uptake of the disaccharide. In the present study, we conjugated BLM, deglycoBLM, and BLM disaccharide to the cyanine dye Cy5**. It was found that the BLM and BLM disaccharide conjugates, but not the deglycoBLM conjugate, bound selectively to MCF-7 cells and were internalized. The same was also true for the prostate cancer cell line DU-145 (but not for normal PZ-HPV-7 prostate cells) and for the pancreatic cancer cell line BxPC-3 (but not for normal SVR A221a pancreas cells). The targeting efficiency of the disaccharide was only slightly less than that of BLM in MCF-7 and DU-145 cells and comparable to that of BLM in BxPC-3 cells. These results establish that the BLM disaccharide is both necessary and sufficient for tumor cell targeting, a finding with obvious implications for the design of novel tumor imaging and therapeutic agents.

The human TRPV6 channel protein is associated with cyclophilin B in human placenta.

Science.gov (United States)

Stumpf, Tobias; Zhang, Qi; Hirnet, Daniela; Lewandrowski, Urs; Sickmann, Albert; Wissenbach, Ulrich; Dörr, Janka; Lohr, Christian; Deitmer, Joachim W; Fecher-Trost, Claudia

2008-06-27

Transcellular calcium transport in the kidney, pancreas, small intestine, and placenta is partly mediated by transient receptor potential (TRP) channels. The highly selective TRPV6 calcium channel protein is most likely important for the calcium transfer in different specialized epithelial cells. In the human placenta the protein is expressed in trophoblast tissue, where it is implicated in the transepithelial calcium transfer from mother to the fetus. We enriched the TRPV6 channel protein endogenously expressed in placenta together with annexin A2 and cyclophilin B (CypB), which is a member of the huge immunophilin family. In the human placenta TRPV6 and CypB are mainly located intracellularly in the syncytiotrophoblast layer, but a small amount of the mature glycosylated TRPV6 channel protein and CypB is also expressed in microvilli apical membranes, the fetomaternal barrier. To understand the role of CypB on the TRPV6 channel function, we evaluated the effect of CypB co-expression on TRPV6-mediated calcium uptake into Xenopus laevis oocytes expressing TRPV6. A significant increase of TRPV6-mediated calcium uptake was observed after CypB/TRPV6 co-expression. This stimulatory effect of CypB was reversed by the immunosuppressive drug cyclosporin A, which inhibits the enzymatic activity of CypB. Cyclosporin A had no significant effect on TRPV6 and CypB protein expression levels in the oocytes. In summary, our results establish CypB as a new TRPV6 accessory protein with potential involvement in TRPV6 channel activation through its peptidyl-prolyl cis/trans isomerase activity.

An innovative pre-targeting strategy for tumor cell specific imaging and therapy.

Science.gov (United States)

Qin, Si-Yong; Peng, Meng-Yun; Rong, Lei; Jia, Hui-Zhen; Chen, Si; Cheng, Si-Xue; Feng, Jun; Zhang, Xian-Zheng

2015-09-21

A programmed pre-targeting system for tumor cell imaging and targeting therapy was established based on the "biotin-avidin" interaction. In this programmed functional system, transferrin-biotin can be actively captured by tumor cells with the overexpression of transferrin receptors, thus achieving the pre-targeting modality. Depending upon avidin-biotin recognition, the attachment of multivalent FITC-avidin to biotinylated tumor cells not only offered the rapid fluorescence labelling, but also endowed the pre-targeted cells with targeting sites for the specifically designed biotinylated peptide nano-drug. Owing to the successful pre-targeting, tumorous HepG2 and HeLa cells were effectively distinguished from the normal 3T3 cells via fluorescence imaging. In addition, the self-assembled peptide nano-drug resulted in enhanced cell apoptosis in the observed HepG2 cells. The tumor cell specific pre-targeting strategy is applicable for a variety of different imaging and therapeutic agents for tumor treatments.

Potential targets for lung squamous cell carcinoma

Science.gov (United States)

Researchers have identified potential therapeutic targets in lung squamous cell carcinoma, the second most common form of lung cancer. The Cancer Genome Atlas (TCGA) Research Network study comprehensively characterized the lung squamous cell carcinoma gen

The heparin/heparan sulfate sequence that interacts with cyclophilin B contains a 3-O-sulfated N-unsubstituted glucosamine residue.

Science.gov (United States)

Vanpouille, Christophe; Deligny, Audrey; Delehedde, Maryse; Denys, Agnès; Melchior, Aurélie; Liénard, Xavier; Lyon, Malcolm; Mazurier, Joël; Fernig, David G; Allain, Fabrice

2007-08-17

Many of the biological functions of heparan sulfate (HS) proteoglycans can be attributed to specialized structures within HS moieties, which are thought to modulate binding and function of various effector proteins. Cyclophilin B (CyPB), which was initially identified as a cyclosporin A-binding protein, triggers migration and integrin-mediated adhesion of peripheral blood T lymphocytes by a mechanism dependent on interaction with cell surface HS. Here we determined the structural features of HS that are responsible for the specific binding of CyPB. In addition to the involvement of 2-O,6-O, and N-sulfate groups, we also demonstrated that binding of CyPB was dependent on the presence of N-unsubstituted glucosamine residues (GlcNH2), which have been reported to be precursors for sulfation by 3-O-sulfotransferases-3 (3-OST-3). Interestingly, 3-OST-3B isoform was found to be the main 3-OST isoenzyme expressed in peripheral blood T lymphocytes and Jurkat T cells. Moreover, down-regulation of the expression of 3-OST-3 by RNA interference potently reduced CyPB binding and consequent activation of p44/42 mitogen-activated protein kinases. Altogether, our results strongly support the hypothesis that 3-O-sulfation of GlcNH2 residues could be a key modification that provides specialized HS structures for CyPB binding to responsive cells. Given that 3-O-sulfation of GlcNH2-containing HS by 3-OST-3 also provides binding sites for glycoprotein gD of herpes simplex virus type I, these findings suggest an intriguing structural linkage between the HS sequences involved in CyPB binding and viral infection.

Targeting the bone marrow: applications in stem cell transplantation

International Nuclear Information System (INIS)

Orchard, K.; Cooper, M.

2004-01-01

Therapeutic doses of radiation cab be selectively directed to the bone marrow either directly using vectors that bind to myeloid and/or lymphoid specific antigens or indirectly by targeting bone matrix. The combination of an accessible target tissue and relatively radiation sensitive malignant cells favours the use of targeted radiotherapy in the treatment of haematopoietic malignancies. Dose escalation of targeted radiation can increase tumour cell destruction and has led to the use of myelosuppressive and possibly myeloablative doses of targeted radiation. A natural development has been the use of targeted radiation in conditioning prior to haematopoietic stem cell transplantation (HSCT). Several groups are actively exploring the use of targeted radiotherapy in the context of HSCT as treatment for haematological malignancies. Although no randomised trials using targeted radiotherapy in HSCT have been published, phase I and II trials have shown very encouraging results stimulating further clinical research in this field. After more than a decade of translational research the optimal combination of therapeutic radioisotope and vector has not been determined. This review summarises the clinical experience of targeted radiotherapy in HSCT and discusses the problems that still need to be solved to maximise the potential of this new treatment modality in HSCT

Emmprin (basigin/CD147): matrix metalloproteinase modulator and multifunctional cell recognition molecule that plays a critical role in cancer progression.

Science.gov (United States)

Nabeshima, Kazuki; Iwasaki, Hiroshi; Koga, Kaori; Hojo, Hironobu; Suzumiya, Junji; Kikuchi, Masahiro

2006-07-01

Emmprin (basigin, CD147) is a cell surface glycoprotein that belongs to the immunoglobulin superfamily. It is highly expressed on the surface of tumor cells and stimulates adjacent fibroblasts or tumor cells to produce matrix metalloproteinases. Moreover, it has recently been shown that emmprin also stimulates expression of vascular endothelial growth factor and hyaluronan, which leads to angiogenesis and anchorage-independent growth/multidrug resistance, respectively. These findings have made emmprin an important molecule in tumor progression and, thus, more attractive as a target for antitumor treatment. However, other functions of emmprin, including as an activator of T cells, a chaperone for monocarboxylate transporters, a receptor for cyclophilin A and a neural recognition molecule, are also being identified in physiological and pathological conditions. Therefore, it is essential to develop specific means to control particular functions of emmprin, for which elucidation of each mechanism is crucial. This review will discuss the role of emmprin in tumor progression and recent advances in the molecular mechanisms of diverse phenomena regulated by emmprin.

Targeting dendritic cells in vivo for cancer therapy

Directory of Open Access Journals (Sweden)

Irina eCaminschi

2012-02-01

Full Text Available Monoclonal antibodies that recognise cell surface molecules have been used deliver antigenic cargo to dendritic cells (DC for induction of immune responses. The encouraging anti-tumour immunity elicited using this immunisation strategy suggests its suitability for clinical trials. This review discusses the complex network of DC, the functional specialisation of DC-subsets, the immunological outcomes of targeting different DC-subsets and their cell surface receptors, and the requirements for the induction of effective anti-tumour immunity. Finally, we review preclinical experiments and the progress towards targeting human DC in vivo.

Targeting cancer stem cells in hepatocellular carcinoma

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He AR

2014-12-01

Full Text Available Aiwu Ruth He,1 Daniel C Smith,1 Lopa Mishra2 1Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC, 2Department of Gastroenterology, Hepatology, and Nutrition, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Abstract: The poor outcome of patients with hepatocellular carcinoma (HCC is attributed to recurrence of the disease after curative treatment and the resistance of HCC cells to conventional chemotherapy, which may be explained partly by the function of liver cancer stem cells (CSCs. Liver CSCs have emerged as an important therapeutic target against HCC. Numerous surface markers for liver CSCs have been identified, and include CD133, CD90, CD44, CD13, and epithelial cell adhesion molecules. These surface markers serve not only as tools for identifying and isolating liver CSCs but also as therapeutic targets for eradicating these cells. In studies of animal models and large-scale genomic analyses of human HCC samples, many signaling pathways observed in normal stem cells have been found to be altered in liver CSCs, which accounts for the stemness and aggressive behavior of these cells. Antibodies and small molecule inhibitors targeting the signaling pathways have been evaluated at different levels of preclinical and clinical development. Another strategy is to promote the differentiation of liver CSCs to less aggressive HCC that is sensitive to conventional chemotherapy. Disruption of the tumor niche essential for liver CSC homeostasis has become a novel strategy in cancer treatment. To overcome the challenges in developing treatment for liver CSCs, more research into the genetic makeup of patient tumors that respond to treatment may lead to more effective therapy. Standardization of HCC CSC tumor markers would be helpful for measuring the CSC response to these agents. Herein, we review the current strategies for developing treatment to eradicate liver CSCs and to improve the outcome for patients with

Breast cancer stem cells, EMT and therapeutic targets

Energy Technology Data Exchange (ETDEWEB)

Kotiyal, Srishti; Bhattacharya, Susinjan, E-mail: s.bhattacharya@jiit.ac.in

2014-10-10

Highlights: • Therapeutic targeting or inhibition of the key molecules of signaling pathways can control growth of breast cancer stem cells (BCSCs). • Development of BCSCs also involves miRNA interactions. • Therapeutic achievement can be done by targeting identified targets in the BCSC pathways. - Abstract: A small heterogeneous population of breast cancer cells acts as seeds to induce new tumor growth. These seeds or breast cancer stem cells (BCSCs) exhibit great phenotypical plasticity which allows them to undergo “epithelial to mesenchymal transition” (EMT) at the site of primary tumor and a future reverse transition. Apart from metastasis they are also responsible for maintaining the tumor and conferring it with drug and radiation resistance and a tendency for post-treatment relapse. Many of the signaling pathways involved in induction of EMT are involved in CSC generation and regulation. Here we are briefly reviewing the mechanism of TGF-β, Wnt, Notch, TNF-α, NF-κB, RTK signalling pathways which are involved in EMT as well as BCSCs maintenance. Therapeutic targeting or inhibition of the key/accessory players of these pathways could control growth of BCSCs and hence malignant cancer. Additionally several miRNAs are dysregulated in cancer stem cells indicating their roles as oncogenes or tumor suppressors. This review also lists the miRNA interactions identified in BCSCs and discusses on some newly identified targets in the BCSC regulatory pathways like SHIP2, nicastrin, Pin 1, IGF-1R, pro-inflammatory cytokines and syndecan which can be targeted for therapeutic achievements.

Multimodal Nanomedicine Strategies for Targeting Cancer Cells as well as Cancer Stem Cell Signalling Mechanisms.

Science.gov (United States)

Kanwar, Jagat R; Samarasinghe, Rasika M; Kamalapuram, Sishir K; Kanwar, Rupinder K

2017-01-01

Increasing evidence suggests that stem cells, a small population of cells with unique selfrenewable and tumour regenerative capacity, are aiding tumour re-growth and multidrug resistance. Conventional therapies are highly ineffective at eliminating these cells leading to relapse of disease and formation of chemoresistance tumours. Cancer and stem cells targeted therapies that utilizes nanotherapeutics to delivery anti-cancer drugs to specific sites are continuously investigated. This review focuses on recent research using nanomedicine and targeting entities to eliminate cancer cells and cancer stem cells. Current nanotherapeutics in clinical trials along with more recent publications on targeted therapies are addressed. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Targeting myeloid cells using nanoparticles to improve cancer immunotherapy.

Science.gov (United States)

Amoozgar, Zohreh; Goldberg, Michael S

2015-08-30

While nanoparticles have traditionally been used to deliver cytotoxic drugs directly to tumors to induce cancer cell death, emerging data suggest that nanoparticles are likely to generate a larger impact on oncology through the delivery of agents that can stimulate antitumor immunity. Tumor-targeted nanocarriers have generally been used to localize chemotherapeutics to tumors and thus decrease off-target toxicity while enhancing efficacy. Challengingly, tumor heterogeneity and evolution render tumor-intrinsic approaches likely to succumb to relapse. The immune system offers exquisite specificity, cytocidal potency, and long-term activity that leverage an adaptive memory response. For this reason, the ability to manipulate immune cell specificity and function would be desirable, and nanoparticles represent an exciting means by which to perform such manipulation. Dendritic cells and tumor-associated macrophages are cells of the myeloid lineage that function as natural phagocytes, so they naturally take up nanoparticles. Dendritic cells direct the specificity and potency of cellular immune responses that can be targeted for cancer vaccines. Herein, we discuss the specific criteria needed for efficient vaccine design, including but not limited to the route of administration, size, morphology, surface charge, targeting ligands, and nanoparticle composition. In contrast, tumor-associated macrophages are critical mediators of immunosuppression whose trans-migratory abilities can be exploited to localize therapeutics to the tumor core and which can be directly targeted for elimination or for repolarization to a tumor suppressive phenotype. It is likely that a combination of targeting dendritic cells to stimulate antitumor immunity and tumor-associated macrophages to reduce immune suppression will impart significant benefits and result in durable antitumor responses. Copyright © 2014 Elsevier B.V. All rights reserved.

Liposomes to target peripheral neurons and Schwann cells.

Directory of Open Access Journals (Sweden)

Sooyeon Lee

Full Text Available While a wealth of literature for tissue-specific liposomes is emerging, optimal formulations to target the cells of the peripheral nervous system (PNS are lacking. In this study, we asked whether a novel formulation of phospholipid-based liposomes could be optimized for preferential uptake by microvascular endothelia, peripheral neurons and Schwann cells. Here, we report a unique formulation consisting of a phospholipid, a polymer surfactant and cholesterol that result in enhanced uptake by targeted cells. Using fluorescently labeled liposomes, we followed particle internalization and trafficking through a distinct route from dextran and escape from degradative compartments, such as lysosomes. In cultures of non-myelinating Schwann cells, liposomes associate with the lipid raft marker Cholera toxin, and their internalization is inhibited by disruption of lipid rafts or actin polymerization. In contrast, pharmacological inhibition of clathrin-mediated endocytosis does not significantly impact liposome entry. To evaluate the efficacy of liposome targeting in tissues, we utilized myelinating explant cultures of dorsal root ganglia and isolated diaphragm preparations, both of which contain peripheral neurons and myelinating Schwann cells. In these models, we detected preferential liposome uptake into neurons and glial cells in comparison to surrounding muscle tissue. Furthermore, in vivo liposome administration by intramuscular or intravenous injection confirmed that the particles were delivered to myelinated peripheral nerves. Within the CNS, we detected the liposomes in choroid epithelium, but not in myelinated white matter regions or in brain parenchyma. The described nanoparticles represent a novel neurophilic delivery vehicle for targeting small therapeutic compounds, biological molecules, or imaging reagents into peripheral neurons and Schwann cells, and provide a major advancement toward developing effective therapies for peripheral

Targeting regulatory T cells in cancer.

LENUS (Irish Health Repository)

Byrne, William L

2012-01-31

Infiltration of tumors by regulatory T cells confers growth and metastatic advantages by inhibiting antitumor immunity and by production of receptor activator of NF-kappaB (RANK) ligand, which may directly stimulate metastatic propagation of RANK-expressing cancer cells. Modulation of regulatory T cells can enhance the efficacy of cancer immunotherapy. Strategies include depletion, interference with function, inhibition of tumoral migration, and exploitation of T-cell plasticity. Problems with these strategies include a lack of specificity, resulting in depletion of antitumor effector T cells or global interruption of regulatory T cells, which may predispose to autoimmune diseases. Emerging technologies, such as RNA interference and tetramer-based targeting, may have the potential to improve selectivity and efficacy.

The mechanism of gene targeting in human somatic cells.

Directory of Open Access Journals (Sweden)

Yinan Kan

2014-04-01

Full Text Available Gene targeting in human somatic cells is of importance because it can be used to either delineate the loss-of-function phenotype of a gene or correct a mutated gene back to wild-type. Both of these outcomes require a form of DNA double-strand break (DSB repair known as homologous recombination (HR. The mechanism of HR leading to gene targeting, however, is not well understood in human cells. Here, we demonstrate that a two-end, ends-out HR intermediate is valid for human gene targeting. Furthermore, the resolution step of this intermediate occurs via the classic DSB repair model of HR while synthesis-dependent strand annealing and Holliday Junction dissolution are, at best, minor pathways. Moreover, and in contrast to other systems, the positions of Holliday Junction resolution are evenly distributed along the homology arms of the targeting vector. Most unexpectedly, we demonstrate that when a meganuclease is used to introduce a chromosomal DSB to augment gene targeting, the mechanism of gene targeting is inverted to an ends-in process. Finally, we demonstrate that the anti-recombination activity of mismatch repair is a significant impediment to gene targeting. These observations significantly advance our understanding of HR and gene targeting in human cells.

Nipah virus infection and glycoprotein targeting in endothelial cells

Directory of Open Access Journals (Sweden)

Maisner Andrea

2010-11-01

Full Text Available Abstract Background The highly pathogenic Nipah virus (NiV causes fatal respiratory and brain infections in animals and humans. The major hallmark of the infection is a systemic endothelial infection, predominantly in the CNS. Infection of brain endothelial cells allows the virus to overcome the blood-brain-barrier (BBB and to subsequently infect the brain parenchyma. However, the mechanisms of NiV replication in endothelial cells are poorly elucidated. We have shown recently that the bipolar or basolateral expression of the NiV surface glycoproteins F and G in polarized epithelial cell layers is involved in lateral virus spread via cell-to-cell fusion and that correct sorting depends on tyrosine-dependent targeting signals in the cytoplasmic tails of the glycoproteins. Since endothelial cells share many characteristics with epithelial cells in terms of polarization and protein sorting, we wanted to elucidate the role of the NiV glycoprotein targeting signals in endothelial cells. Results As observed in vivo, NiV infection of endothelial cells induced syncytia formation. The further finding that infection increased the transendothelial permeability supports the idea of spread of infection via cell-to-cell fusion and endothelial cell damage as a mechanism to overcome the BBB. We then revealed that both glycoproteins are expressed at lateral cell junctions (bipolar, not only in NiV-infected primary endothelial cells but also upon stable expression in immortalized endothelial cells. Interestingly, mutation of tyrosines 525 and 542/543 in the cytoplasmic tail of the F protein led to an apical redistribution of the protein in endothelial cells whereas tyrosine mutations in the G protein had no effect at all. This fully contrasts the previous results in epithelial cells where tyrosine 525 in the F, and tyrosines 28/29 in the G protein were required for correct targeting. Conclusion We conclude that the NiV glycoprotein distribution is responsible for

Prodrug strategy for cancer cell-specific targeting: A recent overview.

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Zhang, Xian; Li, Xiang; You, Qidong; Zhang, Xiaojin

2017-10-20

The increasing development of targeted cancer therapy provides extensive possibilities in clinical trials, and numerous strategies have been explored. The prodrug is one of the most promising strategies in targeted cancer therapy to improve the selectivity and efficacy of cytotoxic compounds. Compared with normal tissues, cancer cells are characterized by unique aberrant markers, thus inactive prodrugs targeting these markers are excellent therapeutics to release active drugs, killing cancer cells without damaging normal tissues. In this review, we explore an integrated view of potential prodrugs applied in targeted cancer therapy based on aberrant cancer specific markers and some examples are provided for inspiring new ideas of prodrug strategy for cancer cell-specific targeting. Copyright © 2017. Published by Elsevier Masson SAS.

DNA fragmentation: manifestation of target cell destruction mediated by cytotoxic T-cell lines, lymphotoxin-secreting helper T-cell clones, and cell-free lymphotoxin-containing supernatant

International Nuclear Information System (INIS)

Schmid, D.S.; Tite, J.P.; Ruddle, N.H.

1986-01-01

A Lyt-2 + , trinitrophenyl-specific, lymphotoxin-secreting, cytotoxic T-cell line, PCl 55, mediates the digestion of target cell DNA into discretely sized fragments. This phenomenon manifests itself within 30 min after effector cell encounter as measured by the release of 3 H counts from target cells prelabeled with [ 3 H]deoxythymidine and occurs even at very low effector to target cell ratios (0.25:1). A Lyt-1 + , ovalbumin-specific, lymphotoxin-secreting T-helper cell clone, 5.9.24, is also able to mediate fragmentation of target cell DNA over a time course essentially indistinguishable from the cytotoxic T lymphocyte-mediated hit. Cell-free lymphotoxin-containing supernatants also cause release of DNA from targets, although they require a longer time course, on the order of 24 hr. In contrast, lysis of cells by antibody plus complement or Triton X-100 does not result in DNA release even after extended periods of incubation (24 hr). All three treatments that result in the release of DNA from cells cause fragmentation of that DNA into discretely sized pieces that are multiples of 200 base pairs. The results thus suggest that cytotoxic T cells, lymphotoxin-secreting helper clones with cytolytic activity, and lymphotoxin all effect target cell destruction by means of a similar mechanism and that observed differences in time course and the absence of target cell specificity in killing mediated by lymphotoxin may simply reflect differences in the mode of toxin delivery

Targeting vaccines to dendritic cells

DEFF Research Database (Denmark)

Foged, Camilla; Sundblad, Anne; Hovgaard, Lars

2002-01-01

delivery systems (DDS) with adjuvant effect that target DC directly and induce optimal immune responses. This paper will review the current knowledge of DC physiology as well as the progress in the field of novel vaccination strategies that directly or indirectly aim at targeting DC....... to be far superior to that of B-cells and macrophages. DC are localized at strategic places in the body at sites used by pathogens to enter the organism, and are thereby in an optimal position to capture antigens. In general, vaccination strategies try to mimic the invasiveness of the pathogens. DC...

Pros and Cons of Antigen-Presenting Cell Targeted Tumor Vaccines

Directory of Open Access Journals (Sweden)

Cleo Goyvaerts

2015-01-01

Full Text Available In therapeutic antitumor vaccination, dendritic cells play the leading role since they decide if, how, when, and where a potent antitumor immune response will take place. Since the disentanglement of the complexity and merit of different antigen-presenting cell subtypes, antitumor immunotherapeutic research started to investigate the potential benefit of targeting these subtypes in situ. This review will discuss which antigen-presenting cell subtypes are at play and how they have been targeted and finally question the true meaning of targeting antitumor-based vaccines.

Oncolytic viral therapy: targeting cancer stem cells

Directory of Open Access Journals (Sweden)

Smith TT

2014-02-01

Full Text Available Tyrel T Smith,1 Justin C Roth,1 Gregory K Friedman,1 G Yancey Gillespie2 1Department of Pediatrics, The University of Alabama at Birmingham, Birmingham, AL, USA; 2Department of Neurosurgery, The University of Alabama at Birmingham, Birmingham, AL, USA Abstract: Cancer stem cells (CSCs are defined as rare populations of tumor-initiating cancer cells that are capable of both self-renewal and differentiation. Extensive research is currently underway to develop therapeutics that target CSCs for cancer therapy, due to their critical role in tumorigenesis, as well as their resistance to chemotherapy and radiotherapy. To this end, oncolytic viruses targeting unique CSC markers, signaling pathways, or the pro-tumor CSC niche offer promising potential as CSCs-destroying agents/therapeutics. We provide a summary of existing knowledge on the biology of CSCs, including their markers and their niche thought to comprise the tumor microenvironment, and then we provide a critical analysis of the potential for targeting CSCs with oncolytic viruses, including herpes simplex virus-1, adenovirus, measles virus, reovirus, and vaccinia virus. Specifically, we review current literature regarding first-generation oncolytic viruses with their innate ability to replicate in CSCs, as well as second-generation viruses engineered to enhance the oncolytic effect and CSC-targeting through transgene expression. Keywords: oncolytic virotherapy, cancer stem cell niche

Purification-Free, Target-Selective Immobilization of a Protein from Cell Lysates.

Science.gov (United States)

Cha, Jaehyun; Kwon, Inchan

2018-02-27

Protein immobilization has been widely used for laboratory experiments and industrial processes. Preparation of a recombinant protein for immobilization usually requires laborious and expensive purification steps. Here, a novel purification-free, target-selective immobilization technique of a protein from cell lysates is reported. Purification steps are skipped by immobilizing a target protein containing a clickable non-natural amino acid (p-azidophenylalanine) in cell lysates onto alkyne-functionalized solid supports via bioorthogonal azide-alkyne cycloaddition. In order to achieve a target protein-selective immobilization, p-azidophenylalanine was introduced into an exogenous target protein, but not into endogenous non-target proteins using host cells with amber codon-free genomic DNAs. Immobilization of superfolder fluorescent protein (sfGFP) from cell lysates is as efficient as that of the purified sfGFP. Using two fluorescent proteins (sfGFP and mCherry), the authors also demonstrated that the target proteins are immobilized with a minimal immobilization of non-target proteins (target-selective immobilization). © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Studies on ADCC (antibody-dependent cell-mediated cytotoxicity) using sheep red blood cells as target cells, 2

International Nuclear Information System (INIS)

Ichikawa, Yukinobu; Takaya, Masatoshi; Arimori, Shigeru

1979-01-01

A non-specific cytotoxic mediator from effector cells (human peripheral blood leukocytes) was investigated in the ADCC (antibody-dependent cell-mediated cytotoxicity) system using antibody-coated sheep red blood cells (SRBC) as target cells. 51 Cr-labelled homologous (sheep) or heterologous (human) red blood cells were used as adjacent cells. Either crude lymphocyte fraction, phagocyte depleted fraction or granulocyte rich fraction separated from human peripheral leukocytes showed moderate cytotoxic effect on homologous adjacent cells, however no cytotoxic activity on heterologous adjacent cells was demonstrated in any leukocyte fraction. This suggests that the cytotoxic effects on homologous adjacent cells were resulted from the translocation of antibody molecules to adjacent cells from antibody-coated target cells. We concluded that the cytotoxic mechanism in this ADCC system was not mediated by non-specific soluble factors released from either human peripheral lymphocytes, monocytes or granulocytes. (author)

Cyclosporin A inhibits the propagation of influenza virus by interfering with a late event in the virus life cycle.

Science.gov (United States)

Hamamoto, Itsuki; Harazaki, Kazuhiro; Inase, Naohiko; Takaku, Hiroshi; Tashiro, Masato; Yamamoto, Norio

2013-01-01

Influenza is a global public health problem that causes a serious respiratory disease. Influenza virus frequently undergoes amino acid substitutions, which result in the emergence of drug-resistant viruses. To control influenza viruses that are resistant to currently available drugs, it is essential to develop new antiviral drugs with a novel molecular target. Here, we report that cyclosporin A (CsA) inhibits the propagation of influenza virus in A549 cells by interfering with a late event in the virus life cycle. CsA did not affect adsorption, internalization, viral RNA replication, or synthesis of viral proteins in A549 cells, but inhibited the step(s) after viral protein synthesis, such as assembly or budding. In addition, siRNA-mediated knockdown of the expression of the major CsA targets, namely cyclophilin A (CypA), cyclophilin B (CypB), and P-glycoprotein (Pgp), did not inhibit influenza virus propagation. These results suggest that CsA inhibits virus propagation by mechanism(s) independent of the inhibition of the function of CypA, CypB, and Pgp. CsA may target an unknown molecule that works as a positive regulator in the propagation of influenza virus. Our findings would contribute to the development of a novel anti-influenza virus therapy and clarification of the regulatory mechanism of influenza virus multiplication.

The polarized double cell target of the SMC

International Nuclear Information System (INIS)

Adams, D.; Adeva, B.; Arik, E.; Arvidson, A.; Badelek, B.; Ballintijn, M.K.; Bardin, G.; Baum, G.; Berglund, P.; Betev, L.; Bird, I.G.; Birsa, R.; Bjoerkholm, P.; Bonner, B.E.; Botton, N. de; Boutemeur, M.; Bradamante, F.; Bravar, A.; Bressan, A.; Bueltmann, S.; Burtin, E.; Cavata, C.; Crabb, D.; Cranshaw, J.; Cuhadar, T.; Torre, S. Dalla; Dantzig, R. van; Derro, B.; Deshpande, A.; Dhawan, S.; Dulya, C.; Dyring, A.; Eichblatt, S.; Faivre, J.C.; Fasching, D.; Feinstein, F.; Fernandez, C.; Forthmann, S.; Frois, B.; Gallas, A.; Garzon, J.A.; Gaussiran, T.; Gilly, H.; Giorgi, M.; Goeler, E. von; Goertz, S.; Gracia, G.; Groot, N. de; Perdekamp, M. Grosse; Guelmez, E.; Haft, K.; Harrach, D. von; Hasegawa, T.; Hautle, P.; Hayashi, N.; Heusch, C.A.; Horikawa, N.; Hughes, V.W.; Igo, G.; Ishimoto, S.; Iwata, T.; Kabuss, E.M.; Kageya, T.; Karev, A.; Kessler, H.J.; Ketel, T.J.; Kiryluk, J.; Kishi, A.; Kisselev, Yu.; Klostermann, L.; Kraemer, D.; Krivokhijine, V.; Kroeger, W.; Kurek, K.; Kyynaeraeinen, J.; Lamanna, M.; Landgraf, U.; Layda, T.; Le Goff, J.M.; Lehar, F.; Lesquen, A. de; Lichtenstadt, J.; Lindqvist, T.; Litmaath, M.; Lowe, M.; Magnon, A.; Mallot, G.K.; Marie, F.; Martin, A.; Martino, J.; Matsuda, T.; Mayes, B.; McCarthy, J.S.; Medved, K.; Meyer, W.; Middelkoop, G. van; Miller, D.; Miyachi, Y.; Mori, K.; Moromisato, J.; Nassalski, J.; Naumann, L.; Neganov, B.; Niinikoski, T.O.; Oberski, J.E.J.; Ogawa, A.; Ozben, C.; Parks, D.P.; Pereira, H.; Penzo, A.; Perrot-Kunne, F.; Peshekhonov, D.; Piegaia, R.; Pinsky, L.; Platchkov, S.; Plo, M.; Pose, D.; Postma, H.; Pretz, J.; Pussieux, T.; Pyrlik, J.; Raedel, G.; Reyhancan, I.; Reicherz, G.; Rieubland, J.M.; Rijllart, A.; Roberts, J.B.; Rock, S.; Rodriguez, M.; Rondio, E.; Rosado, A.; Roscherr, B.; Sabo, I.; Saborido, J.; Sandacz, A.; Savin, I.; Schiavon, P.; Schiller, A.; Schueler, K.P.; Segel, R.; Seitz, R.; Semertzidis, Y.; Sever, F.; Shanahan, P.; Sichtermann, E.P.; Simeoni, F.; Smirnov, G.I.; Staude, A.; Steinmetz, A.; Stiegler, U.; Stuhrmann, H.; Szleper, M.; Teichert, K.M.; Tessarotto, F.; Thers, D.; Tlaczala, W.; Trentalange, S.; Tripet, A.; Unel, G.; Velasco, M.; Vogt, J.; Voss, R.; Weinstein, R.; Whitten, C.; Windmolders, R.; Willumeit, R.; Wislicki, W.; Witzmann, A.; Zanetti, A.M.; Zaremba, K.; Zhao, J.

1999-01-01

The polarized target of the Spin Muon Collaboration at CERN was used for deep inelastic muon scattering experiments during 1993-1996 with a polarized muon beam to investigate the spin structure of the nucleon. Most of the experiments were carried out with longitudinal target polarization and 190 GeV muons, and some were done with transverse polarization and 100 GeV muons. Protons as well as deuterons were polarized by dynamic nuclear polarization (DNP) in three kinds of solid materials -- butanol, ammonia, and deuterated butanol -- with maximum degrees of polarization of 94%, 91% and 60%, respectively. Considerable attention was paid to the accuracies of the NMR polarization measurements and their analyses, the accuracies achieved were between 2.0% and 3.2%. The SMC target system with two cells of opposite polarizations, each cell 65 cm long and 5 cm in diameter, constitutes the largest polarized target system ever built and facilitates accurate spin asymmetry measurements. The design considerations, construction and performance of the target are reviewed

Multidisciplinary Analysis of Cyclophilin A Function in Human Breast Cancer

Science.gov (United States)

2011-03-01

following washing and medium change; at concen- trations of z30 Ag/mL, these effects were not reversible and cell death ensued (data not shown). To assess...ml) inhibiting proliferation and higher doses (30 mg CsA/ml and above) inducing cell death [50]. CsA treatment also decreased breast cancer cell...Genes Dev. 11, 167–178 7 Horseman , N.D. et al. (1997) Defective mammopoiesis, but normal hematopoiesis, in mice with a targeted disruption of the

T-REX on-demand redox targeting in live cells.

Science.gov (United States)

Parvez, Saba; Long, Marcus J C; Lin, Hong-Yu; Zhao, Yi; Haegele, Joseph A; Pham, Vanha N; Lee, Dustin K; Aye, Yimon

2016-12-01

This protocol describes targetable reactive electrophiles and oxidants (T-REX)-a live-cell-based tool designed to (i) interrogate the consequences of specific and time-resolved redox events, and (ii) screen for bona fide redox-sensor targets. A small-molecule toolset comprising photocaged precursors to specific reactive redox signals is constructed such that these inert precursors specifically and irreversibly tag any HaloTag-fused protein of interest (POI) in mammalian and Escherichia coli cells. Syntheses of the alkyne-functionalized endogenous reactive signal 4-hydroxynonenal (HNE(alkyne)) and the HaloTag-targetable photocaged precursor to HNE(alkyne) (also known as Ht-PreHNE or HtPHA) are described. Low-energy light prompts photo-uncaging (t 1/2 <1-2 min) and target-specific modification. The targeted modification of the POI enables precisely timed and spatially controlled redox events with no off-target modification. Two independent pathways are described, along with a simple setup to functionally validate known targets or discover novel sensors. T-REX sidesteps mixed responses caused by uncontrolled whole-cell swamping with reactive signals. Modification and downstream response can be analyzed by in-gel fluorescence, proteomics, qRT-PCR, immunofluorescence, fluorescence resonance energy transfer (FRET)-based and dual-luciferase reporters, or flow cytometry assays. T-REX targeting takes 4 h from initial probe treatment. Analysis of targeted redox responses takes an additional 4-24 h, depending on the nature of the pathway and the type of readouts used.

Risky business: target choice in adoptive cell therapy.

Science.gov (United States)

Morgan, Richard A

2013-11-14

In this issue of Blood, Casucci et al present an elegant study that describes a potential new target for adoptive cell transfer (ACT), in this case CD44 splice variant 6 (CD44v6), and detail why it may be a good target for ACT and how to manage expected off-tumor/on-target toxicities.

Targeting senescence cells in pancreatic cancer | IDRC ...

International Development Research Centre (IDRC) Digital Library (Canada)

Targeting senescence cells in pancreatic cancer. Cellular senescence is a programmed response to oncogenic (tumour-causing) stress that aims to halt the expansion of cells with malignant potential. It does this by stopping the proliferation of pre-cancerous lesions and recruitment of the immune system for their elimination.

The cancer cell adhesion resistome: mechanisms, targeting and translational approaches.

Science.gov (United States)

Dickreuter, Ellen; Cordes, Nils

2017-06-27

Cell adhesion-mediated resistance limits the success of cancer therapies and is a great obstacle to overcome in the clinic. Since the 1990s, where it became clear that adhesion of tumor cells to the extracellular matrix is an important mediator of therapy resistance, a lot of work has been conducted to understand the fundamental underlying mechanisms and two paradigms were deduced: cell adhesion-mediated radioresistance (CAM-RR) and cell adhesion-mediated drug resistance (CAM-DR). Preclinical work has evidently demonstrated that targeting of integrins, adapter proteins and associated kinases comprising the cell adhesion resistome is a promising strategy to sensitize cancer cells to both radiotherapy and chemotherapy. Moreover, the cell adhesion resistome fundamentally contributes to adaptation mechanisms induced by radiochemotherapy as well as molecular drugs to secure a balanced homeostasis of cancer cells for survival and growth. Intriguingly, this phenomenon provides a basis for synthetic lethal targeted therapies simultaneously administered to standard radiochemotherapy. In this review, we summarize current knowledge about the cell adhesion resistome and highlight targeting strategies to override CAM-RR and CAM-DR.

The kinematics of cytotoxic lymphocytes influence their ability to kill target cells.

Directory of Open Access Journals (Sweden)

Purnima Bhat

Full Text Available Cytotoxic lymphocytes (CTL have been reported to show a range of motility patterns from rapid long-range tracking to complete arrest, but how and whether these kinematics affect their ability to kill target cells is not known. Many in vitro killing assays utilize cell lines and tumour-derived cells as targets, which may be of limited relevance to the kinetics of CTL-mediated killing of somatic cells. Here, live-cell microscopy is used to examine the interactions of CTL and primary murine skin cells presenting antigens. We developed a qualitative and quantitative killing assay using extended-duration fluorescence time-lapse microscopy coupled with large-volume objective software-based data analysis to obtain population data of cell-to-cell interactions, motility and apoptosis. In vivo and ex vivo activated antigen-specific cytotoxic lymphocytes were added to primary keratinocyte targets in culture with fluorometric detection of caspase-3 activation in targets as an objective determinant of apoptosis. We found that activated CTL achieved contact-dependent apoptosis of non-tumour targets after a period of prolonged attachment - on average 21 hours - which was determined by target cell type, amount of antigen, and activation status of CTL. Activation of CTL even without engagement of the T cell receptor was sufficient to mobilise cells significantly above baseline, while the addition of cognate antigen further enhanced their motility. Highly activated CTL showed markedly increased vector displacement, and velocity, and lead to increased antigen-specific target cell death. These data show that the inherent kinematics of CTL correlate directly with their ability to kill non-tumour cells presenting cognate antigen.

Glypican-3 Targeting of Liver Cancer Cells Using Multifunctional Nanoparticles

Directory of Open Access Journals (Sweden)

James O. Park

2011-01-01

Full Text Available Imaging is essential in accurately detecting, staging, and treating primary liver cancer (hepatocellular carcinoma [HCC], one of the most prevalent and lethal malignancies. We developed a novel multifunctional nanoparticle (NP specifically targeting glypican-3 (GPC3, a proteoglycan implicated in promotion of cell growth that is overexpressed in most HCCs. Quantitative real-time polymerase chain reaction was performed to confirm the differential GPC3 expression in two human HCC cells, Hep G2 (high and HLF (negligible. These cells were treated with biotin-conjugated GPC3 monoclonal antibody (αGPC3 and subsequently targeted using superparamagnetic iron oxide NPs conjugated to streptavidin and Alexa Fluor 647. Flow cytometry demonstrated that only GPC3-expressing Hep G2 cells were specifically targeted using this αGPC3-NP conjugate (fourfold mean fluorescence over nontargeted NP, and magnetic resonance imaging (MRI experiments showed similar findings (threefold R2 relaxivity. Confocal fluorescence microscopy localized the αGPC3 NPs only to the cell surface of GPC3-expressing Hep G2 cells. Further characterization of this construct demonstrated a negatively charged, monodisperse, 50 nm NP, ideally suited for tumor targeting. This GPC3-specific NP system, with dual-modality imaging capability, may enhance pretreatment MRI, enable refined intraoperative HCC visualization by near-infrared fluorescence, and be potentially used as a carrier for delivery of tumor-targeted therapies, improving patient outcomes.

Chemosensitization of cancer cells by siRNA using targeted nanogel delivery

International Nuclear Information System (INIS)

Dickerson, Erin B; Blackburn, William H; Smith, Michael H; Kapa, Laura B; Lyon, L Andrew; McDonald, John F

2010-01-01

Chemoresistance is a major obstacle in cancer treatment. Targeted therapies that enhance cancer cell sensitivity to chemotherapeutic agents have the potential to increase drug efficacy while reducing toxic effects on untargeted cells. Targeted cancer therapy by RNA interference (RNAi) is a relatively new approach that can be used to reversibly silence genes in vivo by selectively targeting genes such as the epidermal growth factor receptor (EGFR), which has been shown to increase the sensitivity of cancer cells to taxane chemotherapy. However, delivery represents the main hurdle for the broad development of RNAi therapeutics. We report here the use of core/shell hydrogel nanoparticles (nanogels) functionalized with peptides that specially target the EphA2 receptor to deliver small interfering RNAs (siRNAs) targeting EGFR. Expression of EGFR was determined by immunoblotting, and the effect of decreased EGFR expression on chemosensitization of ovarian cancer cells after siRNA delivery was investigated. Treatment of EphA2 positive Hey cells with siRNA-loaded, peptide-targeted nanogels decreased EGFR expression levels and significantly increased the sensitivity of this cell line to docetaxel (P < 0.05). Nanogel treatment of SK-OV-3 cells, which are negative for EphA2 expression, failed to reduce EGFR levels and did not increase docetaxel sensitivity (P > 0.05). This study suggests that targeted delivery of siRNAs by nanogels may be a promising strategy to increase the efficacy of chemotherapy drugs for the treatment of ovarian cancer. In addition, EphA2 is a viable target for therapeutic delivery, and the siRNAs are effectively protected by the nanogel carrier, overcoming the poor stability and uptake that has hindered clinical advancement of therapeutic siRNAs

Mutations in PPIB (cyclophilin B) delay type I procollagen chain association and result in perinatal lethal to moderate osteogenesis imperfecta phenotypes.

Science.gov (United States)

Pyott, Shawna M; Schwarze, Ulrike; Christiansen, Helena E; Pepin, Melanie G; Leistritz, Dru F; Dineen, Richard; Harris, Catharine; Burton, Barbara K; Angle, Brad; Kim, Katherine; Sussman, Michael D; Weis, Maryann; Eyre, David R; Russell, David W; McCarthy, Kevin J; Steiner, Robert D; Byers, Peter H

2011-04-15

Recessive mutations in the cartilage-associated protein (CRTAP), leucine proline-enriched proteoglycan 1 (LEPRE1) and peptidyl prolyl cis-trans isomerase B (PPIB) genes result in phenotypes that range from lethal in the perinatal period to severe deforming osteogenesis imperfecta (OI). These genes encode CRTAP (encoded by CRTAP), prolyl 3-hydroxylase 1 (P3H1; encoded by LEPRE1) and cyclophilin B (CYPB; encoded by PPIB), which reside in the rough endoplasmic reticulum (RER) and can form a complex involved in prolyl 3-hydroxylation in type I procollagen. CYPB, a prolyl cis-trans isomerase, has been thought to drive the prolyl-containing peptide bonds to the trans configuration needed for triple helix formation. Here, we describe mutations in PPIB identified in cells from three individuals with OI. Cultured dermal fibroblasts from the most severely affected infant make some overmodified type I procollagen molecules. Proα1(I) chains are slow to assemble into trimers, and abnormal procollagen molecules concentrate in the RER, and bind to protein disulfide isomerase (PDI) and prolyl 4-hydroxylase 1 (P4H1). These findings suggest that although CYPB plays a role in helix formation another effect is on folding of the C-terminal propeptide and trimer formation. The extent of procollagen accumulation and PDI/P4H1 binding differs among cells with mutations in PPIB, CRTAP and LEPRE1 with the greatest amount in PPIB-deficient cells and the least in LEPRE1-deficient cells. These findings suggest that prolyl cis-trans isomerase may be required to effectively fold the proline-rich regions of the C-terminal propeptide to allow proα chain association and suggest an order of action for CRTAP, P3H1 and CYPB in procollagen biosynthesis and pathogenesis of OI.

MicroRNA-944 Affects Cell Growth by Targeting EPHA7 in Non-Small Cell Lung Cancer

Directory of Open Access Journals (Sweden)

Minxia Liu

2016-09-01

Full Text Available MicroRNAs (miRNAs have critical roles in lung tumorigenesis and development. To determine aberrantly expressed miRNAs involved in non-small cell lung cancer (NSCLC and investigate pathophysiological functions and mechanisms, we firstly carried out small RNA deep sequencing in NSCLC cell lines (EPLC-32M1, A549 and 801D and a human immortalized cell line 16HBE, we then studied miRNA function by cell proliferation and apoptosis. cDNA microarray, luciferase reporter assay and miRNA transfection were used to investigate interaction between the miRNA and target gene. miR-944 was significantly down-regulated in NSCLC and had many putative targets. Moreover, the forced expression of miR-944 significantly inhibited the proliferation of NSCLC cells in vitro. By integrating mRNA expression data and miR-944-target prediction, we disclosed that EPHA7 was a potential target of miR-944, which was further verified by luciferase reporter assay and microRNA transfection. Our data indicated that miR-944 targets EPHA7 in NSCLC and regulates NSCLC cell proliferation, which may offer a new mechanism underlying the development and progression of NSCLC.

MicroRNA-944 Affects Cell Growth by Targeting EPHA7 in Non-Small Cell Lung Cancer.

Science.gov (United States)

Liu, Minxia; Zhou, Kecheng; Cao, Yi

2016-09-26

MicroRNAs (miRNAs) have critical roles in lung tumorigenesis and development. To determine aberrantly expressed miRNAs involved in non-small cell lung cancer (NSCLC) and investigate pathophysiological functions and mechanisms, we firstly carried out small RNA deep sequencing in NSCLC cell lines (EPLC-32M1, A549 and 801D) and a human immortalized cell line 16HBE, we then studied miRNA function by cell proliferation and apoptosis. cDNA microarray, luciferase reporter assay and miRNA transfection were used to investigate interaction between the miRNA and target gene. miR-944 was significantly down-regulated in NSCLC and had many putative targets. Moreover, the forced expression of miR-944 significantly inhibited the proliferation of NSCLC cells in vitro. By integrating mRNA expression data and miR-944-target prediction, we disclosed that EPHA7 was a potential target of miR-944, which was further verified by luciferase reporter assay and microRNA transfection. Our data indicated that miR-944 targets EPHA7 in NSCLC and regulates NSCLC cell proliferation, which may offer a new mechanism underlying the development and progression of NSCLC.

Cell targeting peptides as smart ligands for targeting of therapeutic or diagnostic agents: a systematic review.

Science.gov (United States)

Mousavizadeh, Ali; Jabbari, Ali; Akrami, Mohammad; Bardania, Hassan

2017-10-01

Cell targeting peptides (CTP) are small peptides which have high affinity and specificity to a cell or tissue targets. They are typically identified by using phage display and chemical synthetic peptide library methods. CTPs have attracted considerable attention as a new class of ligands to delivery specifically therapeutic and diagnostic agents, because of the fact they have several advantages including easy synthesis, smaller physical sizes, lower immunogenicity and cytotoxicity and their simple and better conjugation to nano-carriers and therapeutic or diagnostic agents compared to conventional antibodies. In this systematic review, we will focus on the basic concepts concerning the use of cell-targeting peptides (CTPs), following the approaches of selecting them from peptide libraries. We discuss several developed strategies for cell-specific delivery of different cargos by CTPs, which are designed for drug delivery and diagnostic applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Aptamer-Mediated Polymeric Vehicles for Enhanced Cell-Targeted Drug Delivery.

Science.gov (United States)

Tan, Kei X; Danquah, Michael K; Sidhu, Amandeep; Yon, Lau Sie; Ongkudon, Clarence M

2018-02-08

The search for smart delivery systems for enhanced pre-clinical and clinical pharmaceutical delivery and cell targeting continues to be a major biomedical research endeavor owing to differences in the physicochemical characteristics and physiological effects of drug molecules, and this affects the delivery mechanisms to elicit maximum therapeutic effects. Targeted drug delivery is a smart evolution essential to address major challenges associated with conventional drug delivery systems. These challenges mostly result in poor pharmacokinetics due to the inability of the active pharmaceutical ingredients to specifically act on malignant cells thus, causing poor therapeutic index and toxicity to surrounding normal cells. Aptamers are oligonucleotides with engineered affinities to bind specifically to their cognate targets. Aptamers have gained significant interests as effective targeting elements for enhanced therapeutic delivery as they can be generated to specifically bind to wide range of targets including proteins, peptides, ions, cells and tissues. Notwithstanding, effective delivery of aptamers as therapeutic vehicles is challenged by cell membrane electrostatic repulsion, endonuclease degradation, low pH cleavage, and binding conformation stability. The application of molecularly engineered biodegradable and biocompatible polymeric particles with tunable features such as surface area and chemistry, particulate size distribution and toxicity creates opportunities to develop smart aptamer-mediated delivery systems for controlled drug release. This article discusses opportunities for particulate aptamer-drug formulations to advance current drug delivery modalities by navigating active ingredients through cellular and biomolecular traffic to target sites for sustained and controlled release at effective therapeutic dosages while minimizing systemic cytotoxic effects. A proposal for a novel drug-polymer-aptamer-polymer (DPAP) design of aptamer-drug formulation with

Therapeutic targeting strategies using endogenous cells and proteins.

Science.gov (United States)

Parayath, Neha N; Amiji, Mansoor M

2017-07-28

Targeted drug delivery has become extremely important in enhancing efficacy and reducing the toxicity of therapeutics in the treatment of various disease conditions. Current approaches include passive targeting, which relies on naturally occurring differences between healthy and diseased tissues, and active targeting, which utilizes various ligands that can recognize targets expressed preferentially at the diseased site. Clinical translation of these mechanisms faces many challenges including the immunogenic and toxic effects of these non-natural systems. Thus, use of endogenous targeting systems is increasingly gaining momentum. This review is focused on strategies for employing endogenous moieties, which could serve as safe and efficient carriers for targeted drug delivery. The first part of the review involves cells and cellular components as endogenous carriers for therapeutics in multiple disease states, while the second part discusses the use of endogenous plasma components as endogenous carriers. Further understanding of the biological tropism with cells and proteins and the newer generation of delivery strategies that exploits these endogenous approaches promises to provide better solutions for site-specific delivery and could further facilitate clinical translations. Copyright © 2017 Elsevier B.V. All rights reserved.

Enhancing oral vaccine potency by targeting intestinal M cells.

Directory of Open Access Journals (Sweden)

Ali Azizi

2010-11-01

Full Text Available The immune system in the gastrointestinal tract plays a crucial role in the control of infection, as it constitutes the first line of defense against mucosal pathogens. The attractive features of oral immunization have led to the exploration of a variety of oral delivery systems. However, none of these oral delivery systems have been applied to existing commercial vaccines. To overcome this, a new generation of oral vaccine delivery systems that target antigens to gut-associated lymphoid tissue is required. One promising approach is to exploit the potential of microfold (M cells by mimicking the entry of pathogens into these cells. Targeting specific receptors on the apical surface of M cells might enhance the entry of antigens, initiating the immune response and consequently leading to protection against mucosal pathogens. In this article, we briefly review the challenges associated with current oral vaccine delivery systems and discuss strategies that might potentially target mouse and human intestinal M cells.

Cell cycle and anti-estrogen effects synergize to regulate cell proliferation and ER target gene expression.

Directory of Open Access Journals (Sweden)

Mathieu Dalvai

Full Text Available Antiestrogens are designed to antagonize hormone induced proliferation and ERalpha target gene expression in mammary tumor cells. Commonly used drugs such as OH-Tamoxifen and ICI 182780 (Fulvestrant block cell cycle progression in G0/G1. Inversely, the effect of cell cycle stage on ER regulated gene expression has not been tested directly. We show that in ERalpha-positive breast cancer cells (MCF-7 the estrogen receptor gene and downstream target genes are cell cycle regulated with expression levels varying as much as three-fold between phases of the cell cycle. Steroid free culture conditions commonly used to assess the effect of hormones or antiestrogens on gene expression also block MCF-7 cells in G1-phase when several ERalpha target genes are overexpressed. Thus, cell cycle effects have to be taken into account when analyzing the impact of hormonal treatments on gene transcription. We found that antiestrogens repress transcription of several ERalpha target genes specifically in S phase. This observation corroborates the more rapid and strong impact of antiestrogen treatments on cell proliferation in thymidine, hydroxyurea or aphidicolin arrested cells and correlates with an increase of apoptosis compared to similar treatments in lovastatin or nocodazol treated cells. Hence, cell cycle effects synergize with the action of antiestrogens. An interesting therapeutic perspective could be to enhance the action of anti-estrogens by associating hormone-therapy with specific cell cycle drugs.

ATR-dependent bystander effects in non-targeted cells

International Nuclear Information System (INIS)

Burdak-Rothkamm, S.

2007-01-01

Complete text of publication follows. Radiation induced non-targeted bystander effects have been reported for a range of endpoints including the induction of γH2AX foci which serve as a marker for DNA double strand breaks. We have recently reported the induction of γH2AX foci in non-targeted bystander cells up to 48 hours after irradiation and the involvement of reactive oxygen species (ROS) and TGF-beta 1 in the induction of γH2AX foci (Oncogene (2007) 26:993-1002). Here, we wanted to determine the role of the PI3-like kinases ATM, ATR and DNA-PK in DNA damage signalling in bystander cells. Conditioned medium from T98G cells irradiated with 2 Gy of X-rays was transferred onto non-irradiated cells that were subsequently analysed for the induction of γH2AX, ATR and 53BP1 foci as well as clonogenic survival. Irradiated T98G glioma cells generated signals that induced γH2AX and 53BP1 foci in cells treated with the conditioned medium from irradiated cells. These foci co-localised with ATR foci. Inhibition of ATM and DNA-PK could not suppress the induction of bystander γH2AX foci whereas the mutation of ATR in Seckel cells abrogated bystander foci induction. A restriction of bystander foci to the S-phase of the cell cycle both in T98G cells and in ATR- proficient fibroblasts was observed. These results identify ATR as a central player within the bystander signalling cascade leading to γH2AX and 53BP1 foci formation, and suggest a mechanism of DNA damage induction in non-targeted cells. Further investigations have shown decreased clonogenic cell survival in bystander T98G and ATR wild-type fibroblasts. ATR mutated Seckel cells and also ATM-/- fibroblasts were resistant to this effect suggesting a role for both ATR and ATM in the bystander signalling cascade with regard to cell survival. Taken together, these observations support a hypothesis of DNA damage-induced accumulation of stalled replication forks in bystander cells which are subsequently processed by

A new prospect in cancer therapy: targeting cancer stem cells to eradicate cancer.

Science.gov (United States)

Chen, Li-Sha; Wang, An-Xin; Dong, Bing; Pu, Ke-Feng; Yuan, Li-Hua; Zhu, Yi-Min

2012-12-01

According to the cancer stem cell theory, cancers can be initiated by cancer stem cells. This makes cancer stem cells prime targets for therapeutic intervention. Eradicating cancer stem cells by efficient targeting agents may have the potential to cure cancer. In this review, we summarize recent breakthroughs that have improved our understanding of cancer stem cells, and we discuss the therapeutic strategy of targeting cancer stem cells, a promising future direction for cancer stem cell research.

The polarized double cell target of the SMC

CERN Document Server

Adams, D; Adeva, B; Arik, E; Arvidson, A; Badelek, B; Ballintijn, M K; Bardin, G; Baum, G; Berglund, P; Betev, L; Bird, I G; Birsa, R; Björkholm, P; Bonner, B E; De Botton, N R; Boutemeur, M; Bradamante, Franco; Bravar, A; Bressan, A; Bültmann, S; Burtin, E; Cavata, C; Crabb, D; Cranshaw, J; Çuhadar-Dönszelmann, T; Dalla Torre, S; Van Dantzig, R; Derro, B R; Deshpande, A A; Dhawan, S K; Dulya, C M; Dyring, A; Eichblatt, S; Faivre, Jean-Claude; Fasching, D; Feinstein, F; Fernández, C; Forthmann, S; Frois, Bernard; Gallas, A; Garzón, J A; Gaussiran, T; Gilly, H; Giorgi, M A; von Goeler, E; Görtz, S; Gracia, G; De Groot, N; Grosse-Perdekamp, M; Gülmez, E; Haft, K; Von Harrach, D; Hasegawa, T; Hautle, P; Hayashi, N; Heusch, C A; Horikawa, N; Hughes, V W; Igo, G; Ishimoto, S; Iwata, T; Kabuss, E M; Kageya, T; Karev, A G; Kessler, H J; Ketel, T; Kiryluk, J; Kishi, A; Kiselev, Yu F; Klostermann, L; Krämer, Dietrich; Krivokhizhin, V G; Kröger, W; Kurek, K; Kyynäräinen, J; Lamanna, M; Landgraf, U; Layda, T; Le Goff, J M; Lehár, F; de Lesquen, A; Lichtenstadt, J; Lindqvist, T; Litmaath, M; Loewe, M; Magnon, A; Mallot, G K; Marie, F; Martin, A; Martino, J; Matsuda, T; Mayes, B W; McCarthy, J S; Medved, K S; Meyer, W T; Van Middelkoop, G; Miller, D; Miyachi, Y; Mori, K; Moromisato, J H; Nassalski, J P; Naumann, Lutz; Neganov, B S; Niinikoski, T O; Oberski, J; Ogawa, A; Ozben, C; Parks, D P; Pereira, H; Penzo, Aldo L; Perrot-Kunne, F; Peshekhonov, V D; Piegaia, R; Pinsky, L; Platchkov, S K; Pló, M; Pose, D; Postma, H; Pretz, J; Pussieux, T; Pyrlik, J; Rädel, G; Reyhancan, I; Reicherz, G; Rijllart, A; Roberts, J B; Rock, S E; Rodríguez, M; Rondio, Ewa; Rosado, A; Roscherr, B; Sabo, I; Saborido, J; Sandacz, A; Savin, I A; Schiavon, R P; Schiller, A; Schüler, K P; Segel, R E; Seitz, R; Semertzidis, Y K; Sever, F; Shanahan, P; Sichtermann, E P; Simeoni, F; Smirnov, G I; Staude, A; Steinmetz, A; Stiegler, U; Stuhrmann, H B; Szleper, M; Teichert, K M; Tessarotto, F; Thers, D; Tlaczala, W; Trentalange, S; Tripet, A; Ünel, G; Velasco, M; Vogt, J; Voss, Rüdiger; Weinstein, R; Whitten, C; Windmolders, R; Willumeit, R; Wislicki, W; Witzmann, A; Zanetti, A M; Zaremba, K; Zhao, J

1999-01-01

The polarized target of the Spin Muon Collaboration at CERN was used for deep inelastic muon scattering experiments during 1993 to 1996 with a polarized muon beam to investigate the spin structure of the nucleon. Most of the experiments were carried out with longitudinal target polarization and 190 GeV muons, and some were done with transverse polarization and 100 GeV muons. Protons as well as deuterons were polarized by dynamic nuclear polarization (DNP) in three kinds of solid materials $-$ butanol, ammonia, and deuterated butanol, with maximum degrees of polarization of 94, 91, and 60 \\%, respectively. Considerable attention was paid to the accuracies of the NMR polarization measurements and their analyses. The achieved accuracies were between 2.0 and 3.2 \\%. The SMC target system with two cells of opposite polarizations, each cell 65 cm long and 5 cm in diameter, constitutes the largest polarized target system ever built and facilitates accurate spin asymmetry measurements. The design considerations, the ...

Endothelial Cell-Targeted Adenoviral Vector for Suppressing Breast Malignancies

National Research Council Canada - National Science Library

Huang, Shuang

2004-01-01

.... Our proposal is designed to develop an endothelial cell-targeted adenoviral vector and to use the targeted vector to express high levels of anticancer therapeutic genes in the sites of angiogenenic...

Luteolin suppresses cancer cell proliferation by targeting vaccinia-related kinase 1.

Directory of Open Access Journals (Sweden)

Ye Seul Kim

Full Text Available Uncontrolled proliferation, a major feature of cancer cells, is often triggered by the malfunction of cell cycle regulators such as protein kinases. Recently, cell cycle-related protein kinases have become attractive targets for anti-cancer therapy, because they play fundamental roles in cellular proliferation. However, the protein kinase-targeted drugs that have been developed so far do not show impressive clinical results and also display severe side effects; therefore, there is undoubtedly a need to investigate new drugs targeting other protein kinases that are critical in cell cycle progression. Vaccinia-related kinase 1 (VRK1 is a mitotic kinase that functions in cell cycle regulation by phosphorylating cell cycle-related substrates such as barrier-to-autointegration factor (BAF, histone H3, and the cAMP response element (CRE-binding protein (CREB. In our study, we identified luteolin as the inhibitor of VRK1 by screening a small-molecule natural compound library. Here, we evaluated the efficacy of luteolin as a VRK1-targeted inhibitor for developing an effective anti-cancer strategy. We confirmed that luteolin significantly reduces VRK1-mediated phosphorylation of the cell cycle-related substrates BAF and histone H3, and directly interacts with the catalytic domain of VRK1. In addition, luteolin regulates cell cycle progression by modulating VRK1 activity, leading to the suppression of cancer cell proliferation and the induction of apoptosis. Therefore, our study suggests that luteolin-induced VRK1 inhibition may contribute to establish a novel cell cycle-targeted strategy for anti-cancer therapy.

ErbB-targeted CAR T-cell immunotherapy of cancer.

Science.gov (United States)

Whilding, Lynsey M; Maher, John

2015-01-01

Chimeric antigen receptor (CAR) based immunotherapy has been under development for the last 25 years and is now a promising new treatment modality in the field of cancer immunotherapy. The approach involves genetically engineering T cells to target malignant cells through expression of a bespoke fusion receptor that couples an HLA-independent antigen recognition domain to one or more intracellular T-cell activating modules. Multiple clinical trials are now underway in several centers to investigate CAR T-cell immunotherapy of diverse hematologic and solid tumor types. The most successful results have been achieved in the treatment of patients with B-cell malignancies, in whom several complete and durable responses have been achieved. This review focuses on the preclinical and clinical development of CAR T-cell immunotherapy of solid cancers, targeted against members of the ErbB family.

GEM-loaded magnetic albumin nanospheres modified with cetuximab for simultaneous targeting, magnetic resonance imaging, and double-targeted thermochemotherapy of pancreatic cancer cells.

Science.gov (United States)

Wang, Ling; An, Yanli; Yuan, Chenyan; Zhang, Hao; Liang, Chen; Ding, Fengan; Gao, Qi; Zhang, Dongsheng

2015-01-01

Targeted delivery is a promising strategy to improve the diagnostic imaging and therapeutic effect of cancers. In this paper, novel cetuximab (C225)-conjugated, gemcitabine (GEM)-containing magnetic albumin nanospheres (C225-GEM/MANs) were fabricated and applied as a theranostic nanocarrier to conduct simultaneous targeting, magnetic resonance imaging (MRI), and double-targeted thermochemotherapy against pancreatic cancer cells. Fe3O4 nanoparticles (NPs) and GEM co-loaded albumin nanospheres (GEM/MANs) were prepared, and then C225 was further conjugated to synthesize C225-GEM/MANs. Their morphology, mean particle size, GEM encapsulation ratio, specific cell-binding ability, and thermal dynamic profiles were characterized. The effects of discriminating different EGFR-expressing pancreatic cancer cells (AsPC-1 and MIA PaCa-2) and monitoring cellular targeting effects were assessed by targeted MRI. Lastly, the antitumor efficiency of double/C225/magnetic-targeted and nontargeted thermochemotherapy was compared with chemotherapy alone using 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and flow cytometry (FCM) assay. When treated with targeted nanospheres, AsPC-1 cells showed a significantly less intense MRI T2 signal than MIA PaCa-2 cells, while both cells had similar signal strength when incubated with nontargeted nanospheres. T2 signal intensity was significantly lower when magnetic and C225 targeting were combined, rather than used alone. The inhibitory and apoptotic rates of each thermochemotherapy group were significantly higher than those of the chemotherapy-alone groups. Additionally, both MTT and FCM analysis verified that double-targeted thermochemotherapy had the highest targeted killing efficiency among all groups. The C225-GEM/MANs can distinguish various EGFR-expressing live pancreatic cancer cells, monitor diverse cellular targeting effects using targeted MRI imaging, and efficiently mediate double-targeted thermochemotherapy

Phenotypic high-throughput screening elucidates target pathway in breast cancer stem cell-like cells.

Science.gov (United States)

Carmody, Leigh C; Germain, Andrew R; VerPlank, Lynn; Nag, Partha P; Muñoz, Benito; Perez, Jose R; Palmer, Michelle A J

2012-10-01

Cancer stem cells (CSCs) are resistant to standard cancer treatments and are likely responsible for cancer recurrence, but few therapies target this subpopulation. Due to the difficulty in propagating CSCs outside of the tumor environment, previous work identified CSC-like cells by inducing human breast epithelial cells into an epithelial-to-mesenchymal transdifferentiated state (HMLE_sh_ECad). A phenotypic screen was conducted against HMLE_sh_ECad with 300 718 compounds from the Molecular Libraries Small Molecule Repository to identify selective inhibitors of CSC growth. The screen yielded 2244 hits that were evaluated for toxicity and selectivity toward an isogenic control cell line. An acyl hydrazone scaffold emerged as a potent and selective scaffold targeting HMLE_sh_ECad. Fifty-three analogues were acquired and tested; compounds ranged in potency from 790 nM to inactive against HMLE_sh_ECad. Of the analogues, ML239 was best-in-class with an IC(50)= 1.18 µM against HMLE_sh_ECad, demonstrated a >23-fold selectivity over the control line, and was toxic to another CSC-like line, HMLE_shTwist, and a breast carcinoma cell line, MDA-MB-231. Gene expression studies conducted with ML239-treated cells showed altered gene expression in the NF-κB pathway in the HMLE_sh_ECad line but not in the isogenic control line. Future studies will be directed toward the identification of ML239 target(s).

Detecting drug-target binding in cells using fluorescence-activated cell sorting coupled with mass spectrometry analysis

Science.gov (United States)

Wilson, Kris; Webster, Scott P.; Iredale, John P.; Zheng, Xiaozhong; Homer, Natalie Z.; Pham, Nhan T.; Auer, Manfred; Mole, Damian J.

2018-01-01

The assessment of drug-target engagement for determining the efficacy of a compound inside cells remains challenging, particularly for difficult target proteins. Existing techniques are more suited to soluble protein targets. Difficult target proteins include those with challenging in vitro solubility, stability or purification properties that preclude target isolation. Here, we report a novel technique that measures intracellular compound-target complex formation, as well as cellular permeability, specificity and cytotoxicity-the toxicity-affinity-permeability-selectivity (TAPS) technique. The TAPS assay is exemplified here using human kynurenine 3-monooxygenase (KMO), a challenging intracellular membrane protein target of significant current interest. TAPS confirmed target binding of known KMO inhibitors inside cells. We conclude that the TAPS assay can be used to facilitate intracellular hit validation on most, if not all intracellular drug targets.

Targeted cancer cell death induced by biofunctionalized magnetic nanowires

KAUST Repository

Contreras, Maria F.; Ravasi, Timothy; Kosel, Jü rgen

2014-01-01

Magnetic micro and nanomaterials are increasingly interesting for biomedical applications since they possess many advantageous properties: they can become biocompatible, they can be functionalized to target specific cells and they can be remotely manipulated by magnetic fields. The goal of this study is to use antibody-functionalized nickel nanowires (Ab-NWs) as an alternative method in cancer therapy overcoming the limitations of current treatments that lack specificity and are highly cytotoxic. Ab-NWs have been incubated with cancer cells and a 12% drop on cell viability was observed for a treatment of only 10 minutes and an alternating magnetic field of low intensity and low frequency. It is believed that the Ab-NWs vibrate transmitting a mechanical force to the targeted cells inducing cell death. © 2014 IEEE.

Targeted cancer cell death induced by biofunctionalized magnetic nanowires

KAUST Repository

Contreras, Maria F.

2014-02-01

Magnetic micro and nanomaterials are increasingly interesting for biomedical applications since they possess many advantageous properties: they can become biocompatible, they can be functionalized to target specific cells and they can be remotely manipulated by magnetic fields. The goal of this study is to use antibody-functionalized nickel nanowires (Ab-NWs) as an alternative method in cancer therapy overcoming the limitations of current treatments that lack specificity and are highly cytotoxic. Ab-NWs have been incubated with cancer cells and a 12% drop on cell viability was observed for a treatment of only 10 minutes and an alternating magnetic field of low intensity and low frequency. It is believed that the Ab-NWs vibrate transmitting a mechanical force to the targeted cells inducing cell death. © 2014 IEEE.

Profiling the Targets of Protective CD8+ T Cell Responses to Infection

Directory of Open Access Journals (Sweden)

Joseph T. Bruder

2017-12-01

Full Text Available T cells are critical effectors of host immunity that target intracellular pathogens, such as the causative agents of HIV, tuberculosis, and malaria. The development of vaccines that induce effective cell-mediated immunity against such pathogens has proved challenging; for tuberculosis and malaria, many of the antigens targeted by protective T cells are not known. Here, we report a novel approach for screening large numbers of antigens as potential targets of T cells. Malaria provides an excellent model to test this antigen discovery platform because T cells are critical mediators of protection following immunization with live sporozoite vaccines and the specific antigen targets are unknown. We generated an adenovirus array by cloning 312 highly expressed pre-erythrocytic Plasmodium yoelii antigens into adenovirus vectors using high-throughput methodologies. The array was screened to identify antigen-specific CD8+ T cells induced by a live sporozoite vaccine regimen known to provide high levels of sterile protection mediated by CD8+ T cells. We identified 69 antigens that were targeted by CD8+ T cells induced by this vaccine regimen. The antigen that recalled the highest frequency of CD8+ T cells, PY02605, induced protective responses in mice, demonstrating proof of principle for this approach in identifying antigens for vaccine development.

A novel murine T-cell receptor targeting NY-ESO-1.

Science.gov (United States)

Rosati, Shannon F; Parkhurst, Maria R; Hong, Young; Zheng, Zhili; Feldman, Steven A; Rao, Mahadev; Abate-Daga, Daniel; Beard, Rachel E; Xu, Hui; Black, Mary A; Robbins, Paul F; Schrump, David A; Rosenberg, Steven A; Morgan, Richard A

2014-04-01

Cancer testis antigens, such as NY-ESO-1, are expressed in a variety of prevalent tumors and represent potential targets for T-cell receptor (TCR) gene therapy. DNA encoding a murine anti-NY-ESO-1 TCR gene (mTCR) was isolated from immunized HLA-A*0201 transgenic mice and inserted into a γ-retroviral vector. Two mTCR vectors were produced and used to transduce human PBL. Transduced cells were cocultured with tumor target cell lines and T2 cells pulsed with the NY-ESO-1 peptide, and assayed for cytokine release and cell lysis activity. The most active TCR construct was selected for production of a master cell bank for clinical use. mTCR-transduced PBL maintained TCR expression in short-term and long-term culture, ranging from 50% to 90% efficiency 7-11 days after stimulation and 46%-82% 10-20 days after restimulation. High levels of interferon-γ secretion were observed (1000-12000 pg/mL), in tumor coculture assays and recognition of peptide-pulsed cells was observed at 0.1 ng/mL, suggesting that the new mTCR had high avidity for antigen recognition. mTCR-transduced T cells also specifically lysed human tumor targets. In all assays, the mTCR was equivalent or better than the comparable human TCR. As the functional activity of TCR-transduced cells may be affected by the formation of mixed dimers, mTCRs, which are less likely to form mixed dimers with endogenous hTCRs, may be more effective in vivo. This new mTCR targeted to NY-ESO-1 represents a novel potential therapeutic option for adoptive cell-transfer therapy for a variety of malignancies.

Neuraminidase treatment of respiratory syncytial virus-infected cells or virions, but not target cells, enhances cell-cell fusion and infection

International Nuclear Information System (INIS)

Barretto, Naina; Hallak, Louay K.; Peeples, Mark E.

2003-01-01

Respiratory syncytial virus (RSV) infection of HeLa cells induces fusion, but transient expression of the three viral glycoproteins induces fusion poorly, if at all. We found that neuraminidase treatment of RSV-infected cells to remove sialic acid (SA) increases fusion dramatically and that the same treatment of transiently transfected cells expressing the three viral glycoproteins, or even cells expressing the fusion (F) protein alone, results in easily detectable fusion. Neuraminidase treatment of the effector cells, expressing the viral glycoproteins, enhanced fusion while treatment of the target cells did not. Likewise, infectivity was increased by treating virions with neuraminidase, but not by treating target cells. Reduction of charge repulsion by removal of the negatively charged SA is unlikely to explain this effect, since removal of negative charges from either membrane would reduce charge repulsion. Infection with neuraminidase-treated virus remained heparan-sulfate-dependent, indicating that a novel attachment mechanism is not revealed by SA removal. Interestingly, neuraminidase enhancement of RSV infectivity was less pronounced in a virus expressing both the G and the F glycoproteins, compared to virus expressing only the F glycoprotein, possibly suggesting that the G protein sterically hinders access of the neuraminidase to its fusion-enhancing target

Evaluation of Cytochalasin B-Induced Membrane Vesicles Fusion Specificity with Target Cells

Directory of Open Access Journals (Sweden)

Marina Gomzikova

2018-01-01

Full Text Available Extracellular vesicles (EV represent a promising vector system for biomolecules and drug delivery due to their natural origin and participation in intercellular communication. As the quantity of EVs is limited, it was proposed to induce the release of membrane vesicles from the surface of human cells by treatment with cytochalasin B. Cytochalasin B-induced membrane vesicles (CIMVs were successfully tested as a vector for delivery of dye, nanoparticles, and a chemotherapeutic. However, it remained unclear whether CIMVs possess fusion specificity with target cells and thus might be used for more targeted delivery of therapeutics. To answer this question, CIMVs were obtained from human prostate cancer PC3 cells. The diameter of obtained CIMVs was 962,13 ± 140,6 nm. We found that there is no statistically significant preference in PC3 CIMVs fusion with target cells of the same type. According to our observations, the greatest impact on CIMVs entry into target cells is by the heterophilic interaction of CIMV membrane receptors with the surface proteins of target cells.

MiR-223 suppresses cell proliferation by targeting IGF-1R.

Directory of Open Access Journals (Sweden)

Cheng You Jia

Full Text Available To study the roles of microRNA-223 (miR-223 in regulation of cell growth, we established a miR-223 over-expression model in HeLa cells infected with miR-223 by Lentivirus pLL3.7 system. We observed in this model that miR-223 significantly suppressed the proliferation, growth rate, colony formation of HeLa cells in vitro, and in vivo tumorigenicity or tumor formation in nude mice. To investigate the mechanisms involved, we scanned and examined the potential and putative target molecules of miR-223 by informatics, quantitative PCR and Western blot, and found that insulin-like growth factor-1 receptor (IGF-1R was the functional target of miR-223 inhibition of cell proliferation. Targeting IGF-1R by miR-223 was not only seen in HeLa cells, but also in leukemia and hepatoma cells. The downstream pathway, Akt/mTOR/p70S6K, to which the signal was mediated by IGF-1R, was inhibited as well. The relative luciferase activity of the reporter containing wild-type 3'UTR(3'untranslated region of IGF-1R was significantly suppressed, but the mutant not. Silence of IGF-1R expression by vector-based short hairpin RNA resulted in the similar inhibition with miR-223. Contrarily, rescued IGF-1R expression in the cells that over-expressed miR-223, reversed the inhibition caused by miR-223 via introducing IGF-1R cDNA that didn't contain the 3'UTR. Meanwhile, we also noted that miR-223 targeted Rasa1, but the downstream molecules mediated by Rasa1 was neither targeted nor regulated. Therefore we believed that IGF-1R was the functional target for miR-223 suppression of cell proliferation and its downstream PI3K/Akt/mTOR/p70S6K pathway suppressed by miR-223 was by targeting IGF-1R.

Mitochondria-targeted vitamin E analogs inhibit breast cancer cell energy metabolism and promote cell death

International Nuclear Information System (INIS)

Cheng, Gang; Zielonka, Jacek; McAllister, Donna M; Mackinnon, A Craig Jr; Joseph, Joy; Dwinell, Michael B; Kalyanaraman, Balaraman

2013-01-01

Recent research has revealed that targeting mitochondrial bioenergetic metabolism is a promising chemotherapeutic strategy. Key to successful implementation of this chemotherapeutic strategy is the use of new and improved mitochondria-targeted cationic agents that selectively inhibit energy metabolism in breast cancer cells, while exerting little or no long-term cytotoxic effect in normal cells. In this study, we investigated the cytotoxicity and alterations in bioenergetic metabolism induced by mitochondria-targeted vitamin E analog (Mito-chromanol, Mito-ChM) and its acetylated ester analog (Mito-ChMAc). Assays of cell death, colony formation, mitochondrial bioenergetic function, intracellular ATP levels, intracellular and tissue concentrations of tested compounds, and in vivo tumor growth were performed. Both Mito-ChM and Mito-ChMAc selectively depleted intracellular ATP and caused prolonged inhibition of ATP-linked oxygen consumption rate in breast cancer cells, but not in non-cancerous cells. These effects were significantly augmented by inhibition of glycolysis. Mito-ChM and Mito-ChMAc exhibited anti-proliferative effects and cytotoxicity in several breast cancer cells with different genetic background. Furthermore, Mito-ChM selectively accumulated in tumor tissue and inhibited tumor growth in a xenograft model of human breast cancer. We conclude that mitochondria-targeted small molecular weight chromanols exhibit selective anti-proliferative effects and cytotoxicity in multiple breast cancer cells, and that esterification of the hydroxyl group in mito-chromanols is not a critical requirement for its anti-proliferative and cytotoxic effect

Designing and modeling a centrifugal microfluidic device to separate target blood cells

Science.gov (United States)

Shamloo, Amir; Selahi, AmirAli; Madadelahi, Masoud

2016-03-01

The objective of this study is to design a novel and efficient portable lab-on-a-CD (LOCD) microfluidic device for separation of specific cells (target cells) using magnetic beads. In this study the results are shown for neutrophils as target cells. However, other kinds of target cells can be separated in a similar approach. The designed microfluidics can be utilized as a point of care system for neutrophil detection. This microfluidic system employs centrifugal and magnetic forces for separation. After model validation by the experimental data in the literature (that may be used as a design tool for developing centrifugo-magnetophoretic devices), two models are presented for separation of target cells using magnetic beads. The first model consists of one container in the inlet section and two containers in the outlets. Initially, the inlet container is filled with diluted blood sample which is a mixture of red blood cells (RBCs) plus neutrophils which are attached to Magnetic beads. It is shown that by using centrifugal and magnetic forces, this model can separate all neutrophils with recovery factor of ~100%. In the second model, due to excess of magnetic beads in usual experimental analysis (to ensure that all target cells are attached to them) the geometry is improved by adding a third outlet for these free magnetic beads. It is shown that at angular velocity of 45 rad s-1, recovery factor of 100% is achievable for RBCs, free magnetic beads and neutrophils as target cells.

Systematic identification of combinatorial drivers and targets in cancer cell lines.

Directory of Open Access Journals (Sweden)

Adel Tabchy

Full Text Available There is an urgent need to elicit and validate highly efficacious targets for combinatorial intervention from large scale ongoing molecular characterization efforts of tumors. We established an in silico bioinformatic platform in concert with a high throughput screening platform evaluating 37 novel targeted agents in 669 extensively characterized cancer cell lines reflecting the genomic and tissue-type diversity of human cancers, to systematically identify combinatorial biomarkers of response and co-actionable targets in cancer. Genomic biomarkers discovered in a 141 cell line training set were validated in an independent 359 cell line test set. We identified co-occurring and mutually exclusive genomic events that represent potential drivers and combinatorial targets in cancer. We demonstrate multiple cooperating genomic events that predict sensitivity to drug intervention independent of tumor lineage. The coupling of scalable in silico and biologic high throughput cancer cell line platforms for the identification of co-events in cancer delivers rational combinatorial targets for synthetic lethal approaches with a high potential to pre-empt the emergence of resistance.

Systematic identification of combinatorial drivers and targets in cancer cell lines.

Science.gov (United States)

Tabchy, Adel; Eltonsy, Nevine; Housman, David E; Mills, Gordon B

2013-01-01

There is an urgent need to elicit and validate highly efficacious targets for combinatorial intervention from large scale ongoing molecular characterization efforts of tumors. We established an in silico bioinformatic platform in concert with a high throughput screening platform evaluating 37 novel targeted agents in 669 extensively characterized cancer cell lines reflecting the genomic and tissue-type diversity of human cancers, to systematically identify combinatorial biomarkers of response and co-actionable targets in cancer. Genomic biomarkers discovered in a 141 cell line training set were validated in an independent 359 cell line test set. We identified co-occurring and mutually exclusive genomic events that represent potential drivers and combinatorial targets in cancer. We demonstrate multiple cooperating genomic events that predict sensitivity to drug intervention independent of tumor lineage. The coupling of scalable in silico and biologic high throughput cancer cell line platforms for the identification of co-events in cancer delivers rational combinatorial targets for synthetic lethal approaches with a high potential to pre-empt the emergence of resistance.

Microchimeric cells in systemic lupus erythematosus: targets or innocent bystanders?

Science.gov (United States)

Stevens, A M

2006-01-01

During pregnancy maternal and fetal cells commute back and forth leading to fetal microchimerism in the mother and maternal microchimerism in the child that can persist for years after the birth. Chimeric fetal and maternal cells can be hematopoietic or can differentiate into somatic cells in multiple organs, potentially acting as targets for 'autoimmunity' and so have been implicated in the pathogenesis of autoimmune diseases that resemble graft-versus-host disease after stem cell transplantation. Fetal cells have been found in women with systemic lupus erythematosus, both in the blood and a target organ, the kidney, suggesting that they may be involved in pathogenesis. Future studies will address how the host immune system normally tolerates maternal and fetal cells or how the balance may change during autoimmunity.

Myeloid derived suppressor cells as therapeutic target in hematological malignancies

Directory of Open Access Journals (Sweden)

Kim eDe Veirman

2014-12-01

Full Text Available Myeloid derived suppressor cells (MDSC are a heterogeneous population of immature myeloid cells that accumulate during pathological conditions such as cancer and are associated with a poor clinical outcome. MDSC expansion hampers the host anti-tumor immune response by inhibition of T cell proliferation, cytokine secretion and recruitment of regulatory T cells. In addition, MDSC exert non-immunological functions including the promotion of angiogenesis, tumor invasion and metastasis. Recent years, MDSC are considered as a potential target in solid tumors and hematological malignancies to enhance the effects of currently used immune modulating agents. This review focuses on the characteristics, distribution, functions, cell-cell interactions and targeting of MDSC in hematological malignancies including multiple myeloma, lymphoma and leukemia.

Vesicle-associated membrane protein 7 (VAMP-7) is essential for target cell killing in a natural killer cell line

International Nuclear Information System (INIS)

Marcet-Palacios, Marcelo; Odemuyiwa, Solomon O.; Coughlin, Jason J.; Garofoli, Daniella; Ewen, Catherine; Davidson, Courtney E.; Ghaffari, Mazyar; Kane, Kevin P.; Lacy, Paige; Logan, Michael R.; Befus, A. Dean; Bleackley, R. Chris; Moqbel, Redwan

2008-01-01

Natural killer cells recognize and induce apoptosis in foreign, transformed or virus-infected cells through the release of perforin and granzymes from secretory lysosomes. Clinically, NK-cell mediated killing is a major limitation to successful allo- and xenotransplantation. The molecular mechanisms that regulate the fusion of granzyme B-containing secretory lysosomes to the plasma membrane in activated NK cells, prior to target cell killing, are not fully understood. Using the NK cell line YT-Indy as a model, we have investigated the expression of SNAP REceptors (SNAREs), both target (t-) and vesicular (v-) SNAREs, and their function in granzyme B-mediated target cell killing. Our data showed that YT-Indy cells express VAMP-7 and SNAP-23, but not VAMP-2. VAMP-7 was associated with granzyme B-containing lysosomal granules. Using VAMP-7 small interfering RNA (siRNA), we successfully knocked down the expression of VAMP-7 protein in YT-Indy to less than 10% of untreated cells in 24 h. VAMP7-deficient YT-Indy cells activated via co-culture with Jurkat cells released <1 ng/mL of granzyme B, compared to 1.5-2.5 μg/mL from controls. Using Jurkat cells as targets, we showed a 7-fold reduction in NK cell-mediated killing by VAMP-7 deficient YT-Indy cells. Our results show that VAMP-7 is a crucial component of granzyme B release and target cell killing in the NK cell line YT-Indy. Thus, targeting VAMP-7 expression specifically with siRNA, following transplantation, may be a viable strategy for preventing NK cell-mediated transplant rejection, in vivo

Targeting of porous hybrid silica nanoparticles to cancer cells

NARCIS (Netherlands)

Rosenholm, J.M.; Meinander, A.; Peuhu, E.; Niemi, R.; Eriksson, J.E.; Sahlgren, C.; Lindén, M.

2009-01-01

Mesoporous silica nanoparticles functionalized by surface hyperbranching polymerization of polyethylene imine), PEI, were further modified by introducing both fluorescent and targeting moieties, with the aim of specifically targeting cancer cells. Owing to the high abundance of folate receptors in

Liver cell-targeted delivery of therapeutic molecules.

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Kang, Jeong-Hun; Toita, Riki; Murata, Masaharu

2016-01-01

The liver is the largest internal organ in mammals and is involved in metabolism, detoxification, synthesis of proteins and lipids, secretion of cytokines and growth factors and immune/inflammatory responses. Hepatitis, alcoholic or non-alcoholic liver disease, hepatocellular carcinoma, hepatic veno-occlusive disease, and liver fibrosis and cirrhosis are the most common liver diseases. Safe and efficient delivery of therapeutic molecules (drugs, genes or proteins) into the liver is very important to increase the clinical efficacy of these molecules and to reduce their side effects in other organs. Several liver cell-targeted delivery systems have been developed and tested in vivo or ex vivo/in vitro. In this review, we discuss the literature concerning liver cell-targeted delivery systems, with a particular emphasis on the results of in vivo studies.

Human B cells induce dendritic cell maturation and favour Th2 polarization by inducing OX-40 ligand

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Maddur, Mohan S.; Sharma, Meenu; Hegde, Pushpa; Stephen-Victor, Emmanuel; Pulendran, Bali; Kaveri, Srini V.; Bayry, Jagadeesh

2015-01-01

Dendritic cells (DCs) play a critical role in immune homeostasis by regulating the functions of various immune cells, including T and B cells. Notably, DCs also undergo education on reciprocal signalling by these immune cells and environmental factors. Various reports demonstrated that B cells have profound regulatory functions, although only few reports have explored the regulation of human DCs by B cells. Here we demonstrate that activated but not resting B cells induce maturation of DCs with distinct features to polarize Th2 cells that secrete interleukin (IL)-5, IL-4 and IL-13. B-cell-induced maturation of DCs is contact dependent and implicates signalling of B-cell activation molecules CD69, B-cell-activating factor receptor, and transmembrane activator and calcium-modulating cyclophilin ligand interactor. Mechanistically, differentiation of Th2 cells by B-cell-matured DCs is dependent on OX-40 ligand. Collectively, our results suggest that B cells have the ability to control their own effector functions by enhancing the ability of human DCs to mediate Th2 differentiation. PMID:24910129

Enhancing Natural Killer Cell Mediated Targeting and Responses to Myeloid Leukemias

Science.gov (United States)

2017-10-01

AWARD NUMBER: W81XWH-16-1-0380 TITLE: Enhancing Natural Killer Cell Mediated Targeting and Responses to Myeloid Leukemias PRINCIPAL...2017 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Enhancing Natural Killer Cell Mediated Targeting and Responses to Myeloid Leukemias 5b. GRANT NUMBER...leukemias still have poor prognosis, particularly in the elderly, and require hematopoietic cell transplants to fully kill the tumor, which is both

Stem Cell-Based Cell Carrier for Targeted Oncolytic Virotherapy: Translational Opportunity and Open Questions

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Janice Kim

2015-11-01

Full Text Available Oncolytic virotherapy for cancer is an innovative therapeutic option where the ability of a virus to promote cell lysis is harnessed and reprogrammed to selectively destroy cancer cells. Such treatment modalities exhibited antitumor activity in preclinical and clinical settings and appear to be well tolerated when tested in clinical trials. However, the clinical success of oncolytic virotherapy has been significantly hampered due to the inability to target systematic metastasis. This is partly due to the inability of the therapeutic virus to survive in the patient circulation, in order to target tumors at distant sites. An early study from various laboratories demonstrated that cells infected with oncolytic virus can protect the therapeutic payload form the host immune system as well as function as factories for virus production and enhance the therapeutic efficacy of oncolytic virus. While a variety of cell lineages possessed potential as cell carriers, copious investigation has established stem cells as a very attractive cell carrier system in oncolytic virotherapy. The ideal cell carrier desire to be susceptible to viral infection as well as support viral infection, maintain immunosuppressive properties to shield the loaded viruses from the host immune system, and most importantly possess an intrinsic tumor homing ability to deliver loaded viruses directly to the site of the metastasis—all qualities stem cells exhibit. In this review, we summarize the recent work in the development of stem cell-based carrier for oncolytic virotherapy, discuss the advantages and disadvantages of a variety of cell carriers, especially focusing on why stem cells have emerged as the leading candidate, and finally propose a future direction for stem cell-based targeted oncolytic virotherapy that involves its establishment as a viable treatment option for cancer patients in the clinical setting.

Concise Review: Cell Surface N-Linked Glycoproteins as Potential Stem Cell Markers and Drug Targets.

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Boheler, Kenneth R; Gundry, Rebekah L

2017-01-01

Stem cells and their derivatives hold great promise to advance regenerative medicine. Critical to the progression of this field is the identification and utilization of antibody-accessible cell-surface proteins for immunophenotyping and cell sorting-techniques essential for assessment and isolation of defined cell populations with known functional and therapeutic properties. Beyond their utility for cell identification and selection, cell-surface proteins are also major targets for pharmacological intervention. Although comprehensive cell-surface protein maps are highly valuable, they have been difficult to define until recently. In this review, we discuss the application of a contemporary targeted chemoproteomic-based technique for defining the cell-surface proteomes of stem and progenitor cells. In applying this approach to pluripotent stem cells (PSCs), these studies have improved the biological understanding of these cells, led to the enhanced use and development of antibodies suitable for immunophenotyping and sorting, and contributed to the repurposing of existing drugs without the need for high-throughput screening. The utility of this latter approach was first demonstrated with human PSCs (hPSCs) through the identification of small molecules that are selectively toxic to hPSCs and have the potential for eliminating confounding and tumorigenic cells in hPSC-derived progeny destined for research and transplantation. Overall, the cutting-edge technologies reviewed here will accelerate the development of novel cell-surface protein targets for immunophenotyping, new reagents to improve the isolation of therapeutically qualified cells, and pharmacological studies to advance the treatment of intractable diseases amenable to cell-replacement therapies. Stem Cells Translational Medicine 2017;6:131-138. © 2016 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Intracellular CXCR4+ cell targeting with T22-empowered protein-only nanoparticles

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Unzueta, Ugutz; Céspedes, María Virtudes; Ferrer-Miralles, Neus; Casanova, Isolda; Cedano, Juan; Corchero, José Luis; Domingo-Espín, Joan; Villaverde, Antonio; Mangues, Ramón; Vázquez, Esther

2012-01-01

Background Cell-targeting peptides or proteins are appealing tools in nanomedicine and innovative medicines because they increase the local drug concentration and reduce potential side effects. CXC chemokine receptor 4 (CXCR4) is a cell surface marker associated with several severe human pathologies, including colorectal cancer, for which intracellular targeting agents are currently missing. Results Four different peptides that bind CXCR4 were tested for their ability to internalize a green fluorescent protein-based reporter nanoparticle into CXCR4+ cells. Among them, only the 18 mer peptide T22, an engineered segment derivative of polyphemusin II from the horseshoe crab, efficiently penetrated target cells via a rapid, receptor-specific endosomal route. This resulted in accumulation of the reporter nanoparticle in a fully fluorescent and stable form in the perinuclear region of the target cells, without toxicity either in cell culture or in an in vivo model of metastatic colorectal cancer. Conclusion Given the urgent demand for targeting agents in the research, diagnosis, and treatment of CXCR4-linked diseases, including colorectal cancer and human immunodeficiency virus infection, T22 appears to be a promising tag for the intracellular delivery of protein drugs, nanoparticles, and imaging agents. PMID:22923991

Targeting cancer stem cells: emerging role of Nanog transcription factor

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Wang ML

2013-09-01

Full Text Available Mong-Lien Wang,1 Shih-Hwa Chiou,2,3 Cheng-Wen Wu1,4–61Institute of Biochemistry and Molecular Biology, 2Institute of Pharmacology, National Yang Ming University, Taipei, Taiwan; 3Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan; 4Institute of Microbiology and Immunology, 5Institute of Clinical Medicine, National Yang Ming University, Taipei, Taiwan; 6Institute of Biomedical Science, Academia Sinica, Taipei, TaiwanAbstract: The involvement of stemness factors in cancer initiation and progression has drawn much attention recently, especially after the finding that introducing four stemness factors in somatic cells is able to reprogram the cells back to an embryonic stem cell-like state. Following accumulating data revealing abnormal elevated expression levels of key stemness factors, like Nanog, Oct4, and Sox2, in several types of cancer stem cells; the importance and therapeutic potential of targeting these stemness regulators in cancers has turned to research focus. Nanog determines cell fate in both embryonic and cancer stem cells; activating Nanog at an inappropriate time would result in cancer stem cells rather than normal pluripotent stem cells or differentiated somatic cells. Upregulated Nanog is correlated with poor survival outcome of patients with various types of cancer. The discoveries of downstream regulatory pathways directly or indirectly mediated by Nanog indicate that Nanog regulates several aspects of cancer development such as tumor cell proliferation, self-renewal, motility, epithelial-mesenchymal transition, immune evasion, and drug-resistance, which are all defined features for cancer stem cells. The current review paper illustrates the central role of Nanog in the regulatory networks of cancer malignant development and stemness acquirement, as well as in the communication between cancer cells and the surrounding stroma. Though a more defined model is needed to test the

Designing and modeling a centrifugal microfluidic device to separate target blood cells

International Nuclear Information System (INIS)

Shamloo, Amir; Selahi, AmirAli; Madadelahi, Masoud

2016-01-01

The objective of this study is to design a novel and efficient portable lab-on-a-CD (LOCD) microfluidic device for separation of specific cells (target cells) using magnetic beads. In this study the results are shown for neutrophils as target cells. However, other kinds of target cells can be separated in a similar approach. The designed microfluidics can be utilized as a point of care system for neutrophil detection. This microfluidic system employs centrifugal and magnetic forces for separation. After model validation by the experimental data in the literature (that may be used as a design tool for developing centrifugo-magnetophoretic devices), two models are presented for separation of target cells using magnetic beads. The first model consists of one container in the inlet section and two containers in the outlets. Initially, the inlet container is filled with diluted blood sample which is a mixture of red blood cells (RBCs) plus neutrophils which are attached to Magnetic beads. It is shown that by using centrifugal and magnetic forces, this model can separate all neutrophils with recovery factor of ∼100%. In the second model, due to excess of magnetic beads in usual experimental analysis (to ensure that all target cells are attached to them) the geometry is improved by adding a third outlet for these free magnetic beads. It is shown that at angular velocity of 45 rad s −1 , recovery factor of 100% is achievable for RBCs, free magnetic beads and neutrophils as target cells. (paper)

Identification of human embryonic progenitor cell targeting peptides using phage display.

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Paola A Bignone

Full Text Available Human pluripotent stem (hPS cells are capable of differentiation into derivatives of all three primary embryonic germ layers and can self-renew indefinitely. They therefore offer a potentially scalable source of replacement cells to treat a variety of degenerative diseases. The ability to reprogram adult cells to induced pluripotent stem (iPS cells has now enabled the possibility of patient-specific hPS cells as a source of cells for disease modeling, drug discovery, and potentially, cell replacement therapies. While reprogramming technology has dramatically increased the availability of normal and diseased hPS cell lines for basic research, a major bottleneck is the critical unmet need for more efficient methods of deriving well-defined cell populations from hPS cells. Phage display is a powerful method for selecting affinity ligands that could be used for identifying and potentially purifying a variety of cell types derived from hPS cells. However, identification of specific progenitor cell-binding peptides using phage display may be hindered by the large cellular heterogeneity present in differentiating hPS cell populations. We therefore tested the hypothesis that peptides selected for their ability to bind a clonal cell line derived from hPS cells would bind early progenitor cell types emerging from differentiating hPS cells. The human embryonic stem (hES cell-derived embryonic progenitor cell line, W10, was used and cell-targeting peptides were identified. Competition studies demonstrated specificity of peptide binding to the target cell surface. Efficient peptide targeted cell labeling was accomplished using multivalent peptide-quantum dot complexes as detected by fluorescence microscopy and flow cytometry. The cell-binding peptides were selective for differentiated hPS cells, had little or no binding on pluripotent cells, but preferential binding to certain embryonic progenitor cell lines and early endodermal hPS cell derivatives. Taken

Identification and Regulation of c-Myb Target Genes in MCF-7 Cells

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O'Rourke John P

2011-01-01

Full Text Available Abstract Background The c-Myb transcription factor regulates differentiation and proliferation in hematopoietic cells, stem cells and epithelial cells. Although oncogenic versions of c-Myb were first associated with leukemias, over expression or rearrangement of the c-myb gene is common in several types of solid tumors, including breast cancers. Expression of the c-myb gene in human breast cancer cells is dependent on estrogen stimulation, but little is known about the activities of the c-Myb protein or what genes it regulates in estrogen-stimulated cells. Methods We used chromatin immunoprecipitation coupled with whole genome promoter tiling microarrays to identify endogenous c-Myb target genes in human MCF-7 breast cancer cells and characterized the activity of c-Myb at a panel of target genes during different stages of estrogen deprivation and stimulation. Results By using different antibodies and different growth conditions, the c-Myb protein was found associated with over 10,000 promoters in MCF-7 cells, including many genes that encode cell cycle regulators or transcription factors and more than 60 genes that encode microRNAs. Several previously identified c-Myb target genes were identified, including CCNB1, MYC and CXCR4 and novel targets such as JUN, KLF4, NANOG and SND1. By studying a panel of these targets to validate the results, we found that estradiol stimulation triggered the association of c-Myb with promoters and that association correlated with increased target gene expression. We studied one target gene, CXCR4, in detail, showing that c-Myb associated with the CXCR4 gene promoter and activated a CXCR4 reporter gene in transfection assays. Conclusions Our results show that c-Myb associates with a surprisingly large number of promoters in human cells. The results also suggest that estradiol stimulation leads to large-scale, genome-wide changes in c-Myb activity and subsequent changes in gene expression in human breast cancer

Identification and Regulation of c-Myb Target Genes in MCF-7 Cells

International Nuclear Information System (INIS)

Quintana, Anita M; Liu, Fan; O'Rourke, John P; Ness, Scott A

2011-01-01

The c-Myb transcription factor regulates differentiation and proliferation in hematopoietic cells, stem cells and epithelial cells. Although oncogenic versions of c-Myb were first associated with leukemias, over expression or rearrangement of the c-myb gene is common in several types of solid tumors, including breast cancers. Expression of the c-myb gene in human breast cancer cells is dependent on estrogen stimulation, but little is known about the activities of the c-Myb protein or what genes it regulates in estrogen-stimulated cells. We used chromatin immunoprecipitation coupled with whole genome promoter tiling microarrays to identify endogenous c-Myb target genes in human MCF-7 breast cancer cells and characterized the activity of c-Myb at a panel of target genes during different stages of estrogen deprivation and stimulation. By using different antibodies and different growth conditions, the c-Myb protein was found associated with over 10,000 promoters in MCF-7 cells, including many genes that encode cell cycle regulators or transcription factors and more than 60 genes that encode microRNAs. Several previously identified c-Myb target genes were identified, including CCNB1, MYC and CXCR4 and novel targets such as JUN, KLF4, NANOG and SND1. By studying a panel of these targets to validate the results, we found that estradiol stimulation triggered the association of c-Myb with promoters and that association correlated with increased target gene expression. We studied one target gene, CXCR4, in detail, showing that c-Myb associated with the CXCR4 gene promoter and activated a CXCR4 reporter gene in transfection assays. Our results show that c-Myb associates with a surprisingly large number of promoters in human cells. The results also suggest that estradiol stimulation leads to large-scale, genome-wide changes in c-Myb activity and subsequent changes in gene expression in human breast cancer cells

Neuroblastoma cell lines contain pluripotent tumor initiating cells that are susceptible to a targeted oncolytic virus.

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Yonatan Y Mahller

Full Text Available Although disease remission can frequently be achieved for patients with neuroblastoma, relapse is common. The cancer stem cell theory suggests that rare tumorigenic cells, resistant to conventional therapy, are responsible for relapse. If true for neuroblastoma, improved cure rates may only be achieved via identification and therapeutic targeting of the neuroblastoma tumor initiating cell. Based on cues from normal stem cells, evidence for tumor populating progenitor cells has been found in a variety of cancers.Four of eight human neuroblastoma cell lines formed tumorspheres in neural stem cell media, and all contained some cells that expressed neurogenic stem cell markers including CD133, ABCG2, and nestin. Three lines tested could be induced into multi-lineage differentiation. LA-N-5 spheres were further studied and showed a verapamil-sensitive side population, relative resistance to doxorubicin, and CD133+ cells showed increased sphere formation and tumorigenicity. Oncolytic viruses, engineered to be clinically safe by genetic mutation, are emerging as next generation anticancer therapeutics. Because oncolytic viruses circumvent typical drug-resistance mechanisms, they may represent an effective therapy for chemotherapy-resistant tumor initiating cells. A Nestin-targeted oncolytic herpes simplex virus efficiently replicated within and killed neuroblastoma tumor initiating cells preventing their ability to form tumors in athymic nude mice.These results suggest that human neuroblastoma contains tumor initiating cells that may be effectively targeted by an oncolytic virus.

Targeted delivery of celastrol to mesangial cells is effective against mesangioproliferative glomerulonephritis.

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Guo, Ling; Luo, Shi; Du, Zhengwu; Zhou, Meiling; Li, Peiwen; Fu, Yao; Sun, Xun; Huang, Yuan; Zhang, Zhirong

2017-10-12

Mesangial cells-mediated glomerulonephritis is a frequent cause of end-stage renal disease. Here, we show that celastrol is effective in treating both reversible and irreversible mesangioproliferative glomerulonephritis in rat models, but find that its off-target distributions cause severe systemic toxicity. We thus target celastrol to mesangial cells using albumin nanoparticles. Celastrol-albumin nanoparticles crosses fenestrated endothelium and accumulates in mesangial cells, alleviating proteinuria, inflammation, glomerular hypercellularity, and excessive extracellular matrix deposition in rat anti-Thy1.1 nephritis models. Celastrol-albumin nanoparticles presents lower drug accumulation than free celastrol in off-target organs and tissues, thereby minimizing celastrol-related systemic toxicity. Celastrol-albumin nanoparticles thus represents a promising treatment option for mesangioproliferative glomerulonephritis and similar glomerular diseases.Mesangial cell-mediated glomerulonephritis is a frequent cause of kidney disease. Here the authors show that celastrol loaded in albumin nanoparticles efficiently targets mesangial cells, and is effective in rat models.

Identification and validation nucleolin as a target of curcumol in nasopharyngeal carcinoma cells.

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Wang, Juan; Wu, Jiacai; Li, Xumei; Liu, Haowei; Qin, Jianli; Bai, Zhun; Chi, Bixia; Chen, Xu

2018-06-30

Identification of the specific protein target(s) of a drug is a critical step in unraveling its mechanisms of action (MOA) in many natural products. Curcumol, isolated from well known Chinese medicinal plant Curcuma zedoary, has been shown to possess multiple biological activities. It can inhibit nasopharyngeal carcinoma (NPC) proliferation and induce apoptosis, but its target protein(s) in NPC cells remains unclear. In this study, we employed a mass spectrometry-based chemical proteomics approach reveal the possible protein targets of curcumol in NPC cells. Cellular thermal shift assay (CETSA), molecular docking and cell-based assay was used to validate the binding interactions. Chemical proteomics capturing uncovered that NCL is a target of curcumol in NPC cells, Molecular docking showed that curcumol bound to NCL with an -7.8 kcal/mol binding free energy. Cell function analysis found that curcumol's treatment leads to a degradation of NCL in NPC cells, and it showed slight effects on NP69 cells. In conclusion, our results providing evidences that NCL is a target protein of curcumol. We revealed that the anti-cancer effects of curcumol in NPC cells are mediated, at least in part, by NCL inhibition. Many natural products showed high bioactivity, while their mechanisms of action (MOA) are very poor or completely missed. Understanding the MOA of natural drugs can thoroughly exploit their therapeutic potential and minimize their adverse side effects. Identification of the specific protein target(s) of a drug is a critical step in unraveling its MOA. Compound-centric chemical proteomics is a classic chemical proteomics approach which integrates chemical synthesis with cell biology and mass spectrometry (MS) to identify protein targets of natural products determine the drug mechanism of action, describe its toxicity, and figure out the possible cause of off-target. It is an affinity-based chemical proteomics method to identify small molecule-protein interactions

Targeting nanoparticles to dendritic cells for immunotherapy.

NARCIS (Netherlands)

Cruz, L.J.; Tacken, P.J.; Rueda, F.; Domingo, J.C.; Albericio, F.; Figdor, C.G.

2012-01-01

Dendritic cells (DCs) are key players in the initiation of adaptive immune responses and are currently exploited in immunotherapy for treatment of cancer and infectious diseases. Development of targeted nanodelivery systems carrying vaccine components, including antigens and adjuvants, to DCs in

Radioprotection of targeted and bystander cells by methylproamine

Energy Technology Data Exchange (ETDEWEB)

Burdak-Rothkamm, Susanne [Queen' s University Belfast, Centre for Cancer Research and Cell Biology, Belfast (United Kingdom); Oxford University Hospitals, Cellular Pathology, Oxford (United Kingdom); Smith, Andrea; Lobachevsky, Pavel; Martin, Roger [Peter MacCallum Cancer Centre, Molecular Radiation Biology Laboratory, Melbourne (Australia); University of Melbourne, The Sir Peter MacCallum Department of Oncology, Melbourne (Australia); Prise, Kevin M. [Queen' s University Belfast, Centre for Cancer Research and Cell Biology, Belfast (United Kingdom)

2014-09-23

Radioprotective agents are of interest for application in radiotherapy for cancer and in public health medicine in the context of accidental radiation exposure. Methylproamine is the lead compound of a class of radioprotectors which act as DNA binding anti-oxidants, enabling the repair of transient radiation-induced oxidative DNA lesions. This study tested methylproamine for the radioprotection of both directly targeted and bystander cells. T98G glioma cells were treated with 15 μM methylproamine and exposed to {sup 137}Cs γ-ray/X-ray irradiation and He{sup 2+} microbeam irradiation. Radioprotection of directly targeted cells and bystander cells was measured by clonogenic survival or γH2AX assay. Radioprotection of directly targeted T98G cells by methylproamine was observed for {sup 137}Cs γ-rays and X-rays but not for He{sup 2+} charged particle irradiation. The effect of methylproamine on the bystander cell population was tested for both X-ray irradiation and He{sup 2+} ion microbeam irradiation. The X-ray bystander experiments were carried out by medium transfer from irradiated to non-irradiated cultures and three experimental designs were tested. Radioprotection was only observed when recipient cells were pretreated with the drug prior to exposure to the conditioned medium. In microbeam bystander experiments targeted and nontargeted cells were co-cultured with continuous methylproamine treatment during irradiation and postradiation incubation; radioprotection of bystander cells was observed. Methylproamine protected targeted cells from DNA damage caused by γ-ray or X-ray radiation but not He{sup 2+} ion radiation. Protection of bystander cells was independent of the type of radiation which the donor population received. (orig.) [German] Radioprotektive Agenzien sind sowohl in der Strahlentherapie von Krebserkrankungen als auch im Strahlenschutz im Zusammenhang mit akzidenteller Exposition von Bedeutung. Methylproamine ist die Leitsubstanz einer Klasse von

Cyclophilins facilitate dissociation of the human papillomavirus type 16 capsid protein L1 from the L2/DNA complex following virus entry.

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Bienkowska-Haba, Malgorzata; Williams, Carlyn; Kim, Seong Man; Garcea, Robert L; Sapp, Martin

2012-09-01

Human papillomaviruses (HPV) are composed of the major and minor capsid proteins, L1 and L2, that encapsidate a chromatinized, circular double-stranded DNA genome. At the outset of infection, the interaction of HPV type 16 (HPV16) (pseudo)virions with heparan sulfate proteoglycans triggers a conformational change in L2 that is facilitated by the host cell chaperone cyclophilin B (CyPB). This conformational change results in exposure of the L2 N terminus, which is required for infectious internalization. Following internalization, L2 facilitates egress of the viral genome from acidified endosomes, and the L2/DNA complex accumulates at PML nuclear bodies. We recently described a mutant virus that bypasses the requirement for cell surface CyPB but remains sensitive to cyclosporine for infection, indicating an additional role for CyP following endocytic uptake of virions. We now report that the L1 protein dissociates from the L2/DNA complex following infectious internalization. Inhibition and small interfering RNA (siRNA)-mediated knockdown of CyPs blocked dissociation of L1 from the L2/DNA complex. In vitro, purified CyPs facilitated the dissociation of L1 pentamers from recombinant HPV11 L1/L2 complexes in a pH-dependent manner. Furthermore, CyPs released L1 capsomeres from partially disassembled HPV16 pseudovirions at slightly acidic pH. Taken together, these data suggest that CyPs mediate the dissociation of HPV L1 and L2 capsid proteins following acidification of endocytic vesicles.

Death receptor pathways mediate targeted and non-targeted effects of ionizing radiations in breast cancer cells

International Nuclear Information System (INIS)

Luce, A.; Courtin, A.; Levalois, C.; Altmeyer-Morel, S.; Chevillard, S.; Lebeau, J.; Romeo, P.H.

2009-01-01

Delayed cell death by mitotic catastrophe is a frequent mode of solid tumor cell death after γ-irradiation, a widely used treatment of cancer. Whereas the mechanisms that underlie the early γ-irradiation-induced cell death are well documented, those that drive the delayed cell death are largely unknown. Here we show that the Fas, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and tumor necrosis factor (TNF)-α death receptor pathways mediate the delayed cell death observed after γ-irradiation of breast cancer cells. Early after irradiation, we observe the increased expression of Fas, TRAIL-R and TNF-R that first sensitizes cells to apoptosis. Later, the increased expression of FasL, TRAIL and TNF-α permit the apoptosis engagement linked to mitotic catastrophe. Treatments with TNF-α, TRAIL or anti-Fas antibody, early after radiation exposure, induce apoptosis, whereas the neutralization of the three death receptors pathways impairs the delayed cell death. We also show for the first time that irradiated breast cancer cells excrete soluble forms of the three ligands that can induce the death of sensitive bystander cells. Overall, these results define the molecular basis of the delayed cell death of irradiated cancer cells and identify the death receptors pathways as crucial actors in apoptosis induced by targeted as well as non-targeted effects of ionizing radiation. (authors)

Targeting NK cells for anti-cancer immunotherapy: clinical and pre-clinical approaches

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Sebastian eCarotta

2016-04-01

Full Text Available The recent success of checkpoint blockade has highlighted the potential of immunotherapy approaches for cancer treatment. While the majority of approved immunotherapy drugs target T cell subsets, it is appreciated that other components of the immune system have important roles in tumor immune-surveillance as well and thus represent promising additional targets for immunotherapy. Natural killer cells are the body’s first line of defense against infected or transformed cells as they kill target cells in an antigen-independent manner. Although several studies have clearly demonstrated the active role of NK cells in cancer-immune surveillance, only few clinically approved therapies currently exist that harness their potential. Our increased understanding of NK cell biology over the past few years has renewed the interest in NK cell based anti-cancer therapies, which has lead to a steady increase of NK cell based clinical and pre-clinical trials. Here, the role of NK cells in cancer immunesurveillance is summarized and several novel approaches to enhance NK cell cytotoxicity against cancer are discussed.

Therapeutic targeting of the p53 pathway in cancer stem cells

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Prabhu, Varun V.; Allen, Joshua E.; Hong, Bo; Zhang, Shengliang; Cheng, Hairong; El-Deiry, Wafik S.

2013-01-01

Introduction Cancer stem cells are a high profile drug target for cancer therapeutics due to their indispensable role in cancer progression, maintenance, and therapeutic resistance. Restoring wild-type p53 function is an attractive new therapeutic approach for the treatment of cancer due to the well-described powerful tumor suppressor function of p53. As emerging evidence intimately links p53 and stem cell biology, this approach also provides an opportunity to target cancer stem cells. Areas covered Therapeutic approaches to restore the function of wild-type p53, cancer and normal stem cell biology in relation to p53, and the downstream effects of p53 on cancer stem cells. Expert opinion The restoration of wild-type p53 function by targeting p53 directly, its interacting proteins, or its family members holds promise as a new class of cancer therapies. This review examines the impact that such therapies may have on normal and cancer stem cells based on the current evidence linking p53 signaling with these populations. PMID:22998602

Engineering of Systematic Elimination of a Targeted Chromosome in Human Cells.

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Sato, Hiroshi; Kato, Hiroki; Yamaza, Haruyoshi; Masuda, Keiji; Nguyen, Huong Thi Nguyen; Pham, Thanh Thi Mai; Han, Xu; Hirofuji, Yuta; Nonaka, Kazuaki

2017-01-01

Embryonic trisomy leads to abortion or congenital genetic disorders in humans. The most common autosomal chromosome abnormalities are trisomy of chromosomes 13, 18, and 21. Although alteration of gene dosage is thought to contribute to disorders caused by extra copies of chromosomes, genes associated with specific disease phenotypes remain unclear. To generate a normal cell from a trisomic cell as a means of etiological analysis or candidate therapy for trisomy syndromes, we developed a system to eliminate a targeted chromosome from human cells. Chromosome 21 was targeted by integration of a DNA cassette in HeLa cells that harbored three copies of chromosome 21. The DNA cassette included two inverted loxP sites and a herpes simplex virus thymidine kinase (HSV-tk) gene. This system causes missegregation of chromosome 21 after expression of Cre recombinase and subsequently enables the selection of cells lacking the chromosome by culturing in a medium that includes ganciclovir (GCV). Cells harboring only two copies of chromosome 21 were efficiently induced by transfection of a Cre expression vector, indicating that this approach is useful for eliminating a targeted chromosome.

A drug development perspective on targeting tumor-associated myeloid cells.

Science.gov (United States)

Majety, Meher; Runza, Valeria; Lehmann, Christian; Hoves, Sabine; Ries, Carola H

2018-02-01

Despite decades of research, cancer remains a devastating disease and new treatment options are needed. Today cancer is acknowledged as a multifactorial disease not only comprising of aberrant tumor cells but also the associated stroma including tumor vasculature, fibrotic plaques, and immune cells that interact in a complex heterotypic interplay. Myeloid cells represent one of the most abundant immune cell population within the tumor stroma and are equipped with a broad functional repertoire that promotes tumor growth by suppressing cytotoxic T cell activity, stimulating neoangiogenesis and tissue remodeling. Therefore, myeloid cells have become an attractive target for pharmacological intervention. In this review, we summarize the pharmacological approaches to therapeutically target tumor-associated myeloid cells with a focus on advanced programs that are clinically evaluated. In addition, for each therapeutic strategy, the preclinical rationale as well as advantages and challenges from a drug development perspective are discussed. © 2017 Federation of European Biochemical Societies.

Glycan Markers as Potential Immunological Targets in Circulating Tumor Cells.

Science.gov (United States)

Wang, Denong; Wu, Lisa; Liu, Xiaohe

2017-01-01

We present here an experimental approach for exploring a new class of tumor biomarkers that are overexpressed by circulating tumor cells (CTCs) and are likely targetable in immunotherapy against tumor metastasis. Using carbohydrate microarrays, anti-tumor monoclonal antibodies (mAbs) were scanned against a large panel of carbohydrate antigens to identify potential tumor glycan markers. Subsequently, flow cytometry and fiber-optic array scanning technology (FAST) were applied to determine whether the identified targets are tumor-specific cell-surface markers and are, therefore, likely suitable for targeted immunotherapy. Finally, the tumor glycan-specific antibodies identified were validated using cancer patients' blood samples for their performance in CTC-detection and immunotyping analysis. In this article, identifying breast CTC-specific glycan markers and targeting mAbs serve as examples to illustrate this tumor biomarker discovery strategy.

Targeting Myeloid-Derived Suppressor Cells to Bypass Tumor-Induced Immunosuppression

Directory of Open Access Journals (Sweden)

Viktor Fleming

2018-03-01

Full Text Available The immune system has many sophisticated mechanisms to balance an extensive immune response. Distinct immunosuppressive cells could protect from excessive tissue damage and autoimmune disorders. Tumor cells take an advantage of those immunosuppressive mechanisms and establish a strongly immunosuppressive tumor microenvironment (TME, which inhibits antitumor immune responses, supporting the disease progression. Myeloid-derived suppressor cells (MDSC play a crucial role in this immunosuppressive TME. Those cells represent a heterogeneous population of immature myeloid cells with a strong immunosuppressive potential. They inhibit an antitumor reactivity of T cells and NK cells. Furthermore, they promote angiogenesis, establish pre-metastatic niches, and recruit other immunosuppressive cells such as regulatory T cells. Accumulating evidences demonstrated that the enrichment and activation of MDSC correlated with tumor progression, recurrence, and negative clinical outcome. In the last few years, various preclinical studies and clinical trials targeting MDSC showed promising results. In this review, we discuss different therapeutic approaches on MDSC targeting to overcome immunosuppressive TME and enhance the efficiency of current tumor immunotherapies.

GEM-loaded magnetic albumin nanospheres modified with cetuximab for simultaneous targeting, magnetic resonance imaging, and double-targeted thermochemotherapy of pancreatic cancer cells

Directory of Open Access Journals (Sweden)

Wang L

2015-03-01

Full Text Available Ling Wang,1 Yanli An,2 Chenyan Yuan,3 Hao Zhang,2 Chen Liang,2 Fengan Ding,2 Qi Gao,1 Dongsheng Zhang4 1Department of Ultrasonography, Zhong Da Hospital, Medical School, Southeast University, Nanjing, People’s Republic of China; 2Medical School, Southeast University, Nanjing, People’s Republic of China; 3Department of Clinical Laboratory, Zhong Da Hospital, Medical School, Southeast University, Nanjing, People’s Republic of China; 4Jiangsu Key Laboratory for Biomaterials and Devices, Medical School, Southeast University, Nanjing, People’s Republic of China Background: Targeted delivery is a promising strategy to improve the diagnostic imaging and therapeutic effect of cancers. In this paper, novel cetuximab (C225-conjugated, gemcitabine (GEM-containing magnetic albumin nanospheres (C225-GEM/MANs were fabricated and applied as a theranostic nanocarrier to conduct simultaneous targeting, magnetic resonance imaging (MRI, and double-targeted thermochemotherapy against pancreatic cancer cells. Methods: Fe3O4 nanoparticles (NPs and GEM co-loaded albumin nanospheres (GEM/MANs were prepared, and then C225 was further conjugated to synthesize C225-GEM/MANs. Their morphology, mean particle size, GEM encapsulation ratio, specific cell-binding ability, and thermal dynamic profiles were characterized. The effects of discriminating different EGFR-expressing pancreatic cancer cells (AsPC-1 and MIA PaCa-2 and monitoring cellular targeting effects were assessed by targeted MRI. Lastly, the antitumor efficiency of double/C225/magnetic-targeted and nontargeted thermochemotherapy was compared with chemotherapy alone using 3-(4, 5-dimethyl-2-thiazolyl-2,5-diphenyl-2H-tetrazolium bromide (MTT and flow cytometry (FCM assay. Results: When treated with targeted nanospheres, AsPC-1 cells showed a significantly less intense MRI T2 signal than MIA PaCa-2 cells, while both cells had similar signal strength when incubated with nontargeted nanospheres. T2 signal

PEGylated anticancer-carbon nanotubes complex targeting mitochondria of lung cancer cells

Science.gov (United States)

Kim, Sang-Woo; Lee, Yeon Kyung; Lee, Jong Yeon; Hong, Jeong Hee; Khang, Dongwoo

2017-11-01

Although activating apoptosis in cancer cells by targeting the mitochondria is an effective strategy for cancer therapy, insufficient targeting of the mitochondria in cancer cells restricts the availability in clinical treatment. Here, we report on a polyethylene glycol-coated carbon nanotube (CNT)-ABT737 nanodrug that improves the mitochondrial targeting of lung cancer cells. The polyethylene glycol-coated CNT-ABT737 nanodrug internalized into the early endosomes via macropinocytosis and clathrin-mediated endocytosis in advance of early endosomal escape and delivered into the mitochondria. Cytosol release of the nanodrug led to apoptosis of lung cancer cells by abruption of the mitochondrial membrane potential, inducing Bcl-2-mediated apoptosis and generating intracellular reactive oxygen species. As such, this study provides an effective strategy for increasing the anti-lung cancer efficacy by increasing mitochondria accumulation rate of cytosol released anticancer nanodrugs.

Targeted DNA vaccines for enhanced induction of idiotype-specific B and T cells

International Nuclear Information System (INIS)

Fredriksen, Agnete B.; Sandlie, Inger; Bogen, Bjarne

2012-01-01

Background: Idiotypes (Id) are antigenic determinants localized in variable (V) regions of Ig. Id-specific T and B cells (antibodies) play a role in immunotherapy of Id + tumors. However, vaccine strategies that enhance Id-specific responses are needed. Methods: Id + single-chain fragment variable (scFv) from multiple myelomas and B cell lymphomas were prepared in a fusion format that bivalently target surface molecules on antigen-presenting cells (APC). APC-specific targeting units were either scFv from APC-specific mAb (anti-MHC II, anti-CD40) or chemokines (MIP-1α, RANTES). Homodimeric Id-vaccines were injected intramuscularly or intradermally as plasmids in mice, combined with electroporation. Results: (i) Transfected cells secreted plasmid-encoded Id + fusion proteins to extracellular fluid followed by binding of vaccine molecules to APC. (ii) Targeted vaccine molecules increased Id-specific B and T cell responses. (iii) Bivalency and xenogeneic sequences both contributed to enhanced responses. (iv) Targeted Id DNA vaccines induced tumor resistance against challenges with Id + tumors. (v) Human MIP-1α targeting units enhanced Id-specific responses in mice, due to a cross reaction with murine chemokine receptors. Thus, targeted vaccines designed for humans can be quality tested in mice. (vi) Human Id + scFv from four multiple myeloma patients were inserted into the vaccine format and were successfully tested in mice. (vii) Human MIP-1α vaccine proteins enhanced human T cell responses in vitro. (viii) A hypothetical model for how the APC-targeted vaccine molecules enhance Id-specific T and B cells is presented. Conclusion: Targeted DNA Id-vaccines show promising results in preclinical studies, paving the way for testing in patients.

Targeted DNA vaccines for enhanced induction of idiotype-specific B and T cells

Energy Technology Data Exchange (ETDEWEB)

Fredriksen, Agnete B.; Sandlie, Inger; Bogen, Bjarne, E-mail: bjarne.bogen@medisin.uio.no [Centre for Immune Regulation, Institute of Immunology, University of Oslo and Oslo University Hospital, Oslo (Norway)

2012-10-30

Background: Idiotypes (Id) are antigenic determinants localized in variable (V) regions of Ig. Id-specific T and B cells (antibodies) play a role in immunotherapy of Id{sup +} tumors. However, vaccine strategies that enhance Id-specific responses are needed. Methods: Id{sup +} single-chain fragment variable (scFv) from multiple myelomas and B cell lymphomas were prepared in a fusion format that bivalently target surface molecules on antigen-presenting cells (APC). APC-specific targeting units were either scFv from APC-specific mAb (anti-MHC II, anti-CD40) or chemokines (MIP-1α, RANTES). Homodimeric Id-vaccines were injected intramuscularly or intradermally as plasmids in mice, combined with electroporation. Results: (i) Transfected cells secreted plasmid-encoded Id{sup +} fusion proteins to extracellular fluid followed by binding of vaccine molecules to APC. (ii) Targeted vaccine molecules increased Id-specific B and T cell responses. (iii) Bivalency and xenogeneic sequences both contributed to enhanced responses. (iv) Targeted Id DNA vaccines induced tumor resistance against challenges with Id{sup +} tumors. (v) Human MIP-1α targeting units enhanced Id-specific responses in mice, due to a cross reaction with murine chemokine receptors. Thus, targeted vaccines designed for humans can be quality tested in mice. (vi) Human Id{sup +} scFv from four multiple myeloma patients were inserted into the vaccine format and were successfully tested in mice. (vii) Human MIP-1α vaccine proteins enhanced human T cell responses in vitro. (viii) A hypothetical model for how the APC-targeted vaccine molecules enhance Id-specific T and B cells is presented. Conclusion: Targeted DNA Id-vaccines show promising results in preclinical studies, paving the way for testing in patients.

Single-cell analysis of targeted transcriptome predicts drug sensitivity of single cells within human myeloma tumors.

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Mitra, A K; Mukherjee, U K; Harding, T; Jang, J S; Stessman, H; Li, Y; Abyzov, A; Jen, J; Kumar, S; Rajkumar, V; Van Ness, B

2016-05-01

Multiple myeloma (MM) is characterized by significant genetic diversity at subclonal levels that have a defining role in the heterogeneity of tumor progression, clinical aggressiveness and drug sensitivity. Although genome profiling studies have demonstrated heterogeneity in subclonal architecture that may ultimately lead to relapse, a gene expression-based prediction program that can identify, distinguish and quantify drug response in sub-populations within a bulk population of myeloma cells is lacking. In this study, we performed targeted transcriptome analysis on 528 pre-treatment single cells from 11 myeloma cell lines and 418 single cells from 8 drug-naïve MM patients, followed by intensive bioinformatics and statistical analysis for prediction of proteasome inhibitor sensitivity in individual cells. Using our previously reported drug response gene expression profile signature at the single-cell level, we developed an R Statistical analysis package available at https://github.com/bvnlabSCATTome, SCATTome (single-cell analysis of targeted transcriptome), that restructures the data obtained from Fluidigm single-cell quantitative real-time-PCR analysis run, filters missing data, performs scaling of filtered data, builds classification models and predicts drug response of individual cells based on targeted transcriptome using an assortment of machine learning methods. Application of SCATT should contribute to clinically relevant analysis of intratumor heterogeneity, and better inform drug choices based on subclonal cellular responses.

Glycoengineering of Human Cell Lines Using Zinc Finger Nuclease Gene Targeting

DEFF Research Database (Denmark)

Steentoft, Catharina; Bennett, Eric Paul; Clausen, Henrik

2013-01-01

Lectin affinity chromatography is a powerful technique for isolation of glycoproteins carrying a specific glycan structure of interest. However, the enormous diversity of glycans present on the cell surface, as well as on individual proteins, makes it difficult to isolate an entire glycoproteome...... with one or even a series of lectins. Here we present a technique to generate cell lines with homogenous truncated O-glycans using zinc finger nuclease gene targeting. Because of their simplified O-glycoproteome, the cells have been named SimpleCells. Glycoproteins from SimpleCells can be isolated...... in a single purification step by lectin chromatography performed on a long lectin column. This protocol describes Zinc finger nuclease gene targeting of human cells to simplify the glycoproteome, as well as lectin chromatography and isolation of glycopeptides from total cell lysates of SimpleCells....

Development of novel strategy for the synthesis of organometallic compounds usable as protein ligands: application to the human cyclophilin hCyp-18; Developpement de ligands de proteines par assemblage combinatoire autour d'un coeur de rhenium{sup V}: application a la cyclophiline hCyp-18

Energy Technology Data Exchange (ETDEWEB)

Clavaud, C

2006-02-15

This thesis describes a new strategy for the development of bioactive organometallic compounds, basing on the combinatorial assembly of sub-chemical libraries (A and B) independent but complementary and able to coordinate a metallic heart M to form A-M-B complex potential ligands of biomolecules. The coordination of metals, well adapted to the production of molecular variety is usually used in medicinal chemistry, in diagnostic and therapeutic nuclear medicine. Among the useful elements, the rhenium and the technetium are metals of choice for the development of the assembly strategy because of their chemical and radiochemical properties and of the structure analogy of their complexes. This strategy was validated in vitro. The protein chosen for this purpose was the cyclophilin hCyp-18. (N.C.)

Splenocytes cultured in low concentrations of IL-2 generate NK cell specificities toward syngenic and allogenic targets

DEFF Research Database (Denmark)

Nissen, Mogens Holst; Jeppesen, M; Claesson, M H

2000-01-01

Splenocytes cultured in the presence of 30-60 units/ml IL-2 for 5 days develop natural killer activity toward syngeneic and allogeneic tumor cell targets. The IL-2 activated splenocytes, themselves, are partially resistant, whereas concanavalin A-activated T blast cells are completely resistant...... to killing. Surprisingly, major histocompatibility complex (MHC)-I-negative target cells are also resistant to natural killer (NK)-cell-mediated killing. Cells resistant to killing were unable to block NK-cell-mediated killing of sensitive targets as judged from cold target cell inhibition experiments......, and one type of target cells sensitive to killing did generally not cross-block killing of other killing-sensitive target cell types. Alloantigen exposure of splenocytes, i.e., one-way mixed lymphocyte cultures, partially prevents the development of NK-cell activity. Our data suggest that target...

Orchestration of transplantation tolerance by regulatory dendritic cell therapy or in-situ targeting of dendritic cells.

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Morelli, Adrian E; Thomson, Angus W

2014-08-01

Extensive research in murine transplant models over the past two decades has convincingly demonstrated the ability of regulatory dendritic cells (DCregs) to promote long-term allograft survival. We review important considerations regarding the source of therapeutic DCregs (donor or recipient) and their mode of action, in-situ targeting of DCregs, and optimal therapeutic regimens to promote DCreg function. Recent studies have defined protocols and mechanisms whereby ex-vivo-generated DCregs of donor or recipient origin subvert allogeneic T-cell responses and promote long-term organ transplant survival. Particular interest has focused on how donor antigen is acquired, processed and presented by autologous dendritic cells, on the stability of DCregs, and on in-situ targeting of dendritic cells to promote their tolerogenic function. New evidence of the therapeutic efficacy of DCregs in a clinically relevant nonhuman primate organ transplant model and production of clinical grade DCregs support early evaluation of DCreg therapy in human graft recipients. We discuss strategies currently used to promote dendritic cell tolerogenicity, including DCreg therapy and in-situ targeting of dendritic cells, with a view to improved understanding of underlying mechanisms and identification of the most promising strategies for therapeutic application.

Therapeutic Approaches to Target Cancer Stem Cells

International Nuclear Information System (INIS)

Diaz, Arlhee; Leon, Kalet

2011-01-01

The clinical relevance of cancer stem cells (CSC) remains a major challenge for current cancer therapies, but preliminary findings indicate that specific targeting may be possible. Recent studies have shown that these tumor subpopulations promote tumor angiogenesis through the increased production of VEGF, whereas the VEGF neutralizing antibody bevacizumab specifically inhibits CSC growth. Moreover, nimotuzumab, a monoclonal antibody against the epidermal growth factor receptor (EGFR) with a potent antiangiogenic activity, has been shown by our group to reduce the frequency of CSC-like subpopulations in mouse models of brain tumors when combined with ionizing radiation. These studies and subsequent reports from other groups support the relevance of approaches based on molecular-targeted therapies to selectively attack CSC. This review discusses the relevance of targeting both the EGFR and angiogenic pathways as valid approaches to this aim. We discuss the relevance of identifying better molecular markers to develop drug screening strategies that selectively target CSC

Killing cancer cells by targeted drug-carrying phage nanomedicines

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Yacoby Iftach

2008-04-01

Full Text Available Abstract Background Systemic administration of chemotherapeutic agents, in addition to its anti-tumor benefits, results in indiscriminate drug distribution and severe toxicity. This shortcoming may be overcome by targeted drug-carrying platforms that ferry the drug to the tumor site while limiting exposure to non-target tissues and organs. Results We present a new form of targeted anti-cancer therapy in the form of targeted drug-carrying phage nanoparticles. Our approach is based on genetically-modified and chemically manipulated filamentous bacteriophages. The genetic manipulation endows the phages with the ability to display a host-specificity-conferring ligand. The phages are loaded with a large payload of a cytotoxic drug by chemical conjugation. In the presented examples we used anti ErbB2 and anti ERGR antibodies as targeting moieties, the drug hygromycin conjugated to the phages by a covalent amide bond, or the drug doxorubicin conjugated to genetically-engineered cathepsin-B sites on the phage coat. We show that targeting of phage nanomedicines via specific antibodies to receptors on cancer cell membranes results in endocytosis, intracellular degradation, and drug release, resulting in growth inhibition of the target cells in vitro with a potentiation factor of >1000 over the corresponding free drugs. Conclusion The results of the proof-of concept study presented here reveal important features regarding the potential of filamentous phages to serve as drug-delivery platform, on the affect of drug solubility or hydrophobicity on the target specificity of the platform and on the effect of drug release mechanism on the potency of the platform. These results define targeted drug-carrying filamentous phage nanoparticles as a unique type of antibody-drug conjugates.

Killing cancer cells by targeted drug-carrying phage nanomedicines

Science.gov (United States)

Bar, Hagit; Yacoby, Iftach; Benhar, Itai

2008-01-01

Background Systemic administration of chemotherapeutic agents, in addition to its anti-tumor benefits, results in indiscriminate drug distribution and severe toxicity. This shortcoming may be overcome by targeted drug-carrying platforms that ferry the drug to the tumor site while limiting exposure to non-target tissues and organs. Results We present a new form of targeted anti-cancer therapy in the form of targeted drug-carrying phage nanoparticles. Our approach is based on genetically-modified and chemically manipulated filamentous bacteriophages. The genetic manipulation endows the phages with the ability to display a host-specificity-conferring ligand. The phages are loaded with a large payload of a cytotoxic drug by chemical conjugation. In the presented examples we used anti ErbB2 and anti ERGR antibodies as targeting moieties, the drug hygromycin conjugated to the phages by a covalent amide bond, or the drug doxorubicin conjugated to genetically-engineered cathepsin-B sites on the phage coat. We show that targeting of phage nanomedicines via specific antibodies to receptors on cancer cell membranes results in endocytosis, intracellular degradation, and drug release, resulting in growth inhibition of the target cells in vitro with a potentiation factor of >1000 over the corresponding free drugs. Conclusion The results of the proof-of concept study presented here reveal important features regarding the potential of filamentous phages to serve as drug-delivery platform, on the affect of drug solubility or hydrophobicity on the target specificity of the platform and on the effect of drug release mechanism on the potency of the platform. These results define targeted drug-carrying filamentous phage nanoparticles as a unique type of antibody-drug conjugates. PMID:18387177

Tumor initiating cells and chemoresistance: which is the best strategy to target colon cancer stem cells?

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Paldino, Emanuela; Tesori, Valentina; Casalbore, Patrizia; Gasbarrini, Antonio; Puglisi, Maria Ausiliatrice

2014-01-01

There is an emerging body of evidence that chemoresistance and minimal residual disease result from selective resistance of a cell subpopulation from the original tumor that is molecularly and phenotypically distinct. These cells are called "cancer stem cells" (CSCs). In this review, we analyze the potential targeting strategies for eradicating CSCs specifically in order to develop more effective therapeutic strategies for metastatic colon cancer. These include induction of terminal epithelial differentiation of CSCs or targeting some genes expressed only in CSCs and involved in self-renewal and chemoresistance. Ideal targets could be cell regulators that simultaneously control the stemness and the resistance of CSCs. Another important aspect of cancer biology, which can also be harnessed to create novel broad-spectrum anticancer agents, is the Warburg effect, also known as aerobic glycolysis. Actually, little is yet known with regard to the metabolism of CSCs population, leaving an exciting unstudied avenue in the dawn of the emerging field of metabolomics.

CD147-mediated chemotaxis of CD4+CD161+ T cells may contribute to local inflammation in rheumatoid arthritis.

Science.gov (United States)

Lv, Minghua; Miao, Jinlin; Zhao, Peng; Luo, Xing; Han, Qing; Wu, Zhenbiao; Zhang, Kui; Zhu, Ping

2018-01-01

CD161 is used as a surrogate marker for Th17 cells, which are implicated in the pathogenesis of rheumatoid arthritis (RA). In this study, we evaluated the percentage, clinical significance, and CD98 and CD147 expression of CD4 + CD161 + T cells. The potential role of CD147 and CD98 in cyclophilin A-induced chemotaxis of CD4 + CD161 + T cells was analyzed. Thirty-seven RA patients, 15 paired synovial fluid (SF) of RA, and 22 healthy controls were recruited. The cell populations and surface expression of CD98 and CD147 were analyzed by flow cytometry. Spearman's rank correlation coefficient and multiple linear regression were applied to calculate the correlations. Chemotaxis assay was used to investigate CD4 + CD161 + T cell migration. We found that the percentage of CD4 + CD161 + T cells and their expression of CD147 and CD98 in SF were higher than in the peripheral blood of RA patients. Percentage of SF CD4 + CD161 + T cells was positively correlated with 28-Joint Disease Activity Score (DAS28). CD147 monoclonal antibody (HAb18) attenuated the chemotactic ability of CD4 + CD161 + T cells. An increased CD4 + CD161 + T cell percentage and expression of CD147 and CD98 were shown in RA SF. Percentage of SF CD4 + CD161 + T cells can be used as a predictive marker of disease activity in RA. CD147 block significantly decreased the chemotactic index of CD4 + CD161 + cells induced by cyclophilin A (CypA). These results imply that the accumulation of CD4 + CD161 + T cells in SF and their high expression of CD147 may be associated with CypA-mediated chemotaxis and contribute to local inflammation in RA.

Engineering of Systematic Elimination of a Targeted Chromosome in Human Cells

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Hiroshi Sato

2017-01-01

Full Text Available Embryonic trisomy leads to abortion or congenital genetic disorders in humans. The most common autosomal chromosome abnormalities are trisomy of chromosomes 13, 18, and 21. Although alteration of gene dosage is thought to contribute to disorders caused by extra copies of chromosomes, genes associated with specific disease phenotypes remain unclear. To generate a normal cell from a trisomic cell as a means of etiological analysis or candidate therapy for trisomy syndromes, we developed a system to eliminate a targeted chromosome from human cells. Chromosome 21 was targeted by integration of a DNA cassette in HeLa cells that harbored three copies of chromosome 21. The DNA cassette included two inverted loxP sites and a herpes simplex virus thymidine kinase (HSV-tk gene. This system causes missegregation of chromosome 21 after expression of Cre recombinase and subsequently enables the selection of cells lacking the chromosome by culturing in a medium that includes ganciclovir (GCV. Cells harboring only two copies of chromosome 21 were efficiently induced by transfection of a Cre expression vector, indicating that this approach is useful for eliminating a targeted chromosome.

Towards The Generation of Functionalized Magnetic Nanowires to Target Leukemic Cells

KAUST Repository

Alsharif, Nouf

2016-01-01

. In addition the NWs can be coated and functionalized to target cells of interest and, upon exposure to an alternating magnetic field, have been shown to induce cell death on several types of adherent cells, including several cancer cell types. For suspension

Targeting cancer cells using 3-bromopyruvate for selective cancer treatment

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Hussam H Baghdadi

2017-01-01

Full Text Available Cancer treatment deserves more research efforts despite intensive conventional treatment modalities for many types of malignancies. Metastasis and resistance to chemotherapy and radiotherapy receive a lot of global research efforts. The current advances in cancer biology may improve targeting the critical metabolic differences that distinguish cancer cells from normal cells. Cancer cells are highly glycolytic for energy production, exhibit the Warburg effect, establish aggressive acidic microenvironment, maintain cancer stem cells, exhibit resistance to chemotherapy, have low antioxidant systems but different ΔΨm (delta psi, mitochondrial transmembrane potential, express P-glycoprotein for multidrug resistance, upregulate glucose transporters and monocarboxylate transporters and are under high steady-state reactive oxygen species conditions. Normal cells differ in all these aspects. Lactate produced through the Warburg effect helps cancer metastasis. Targeting glycolysis reactions for energy production in cancer cells seems promising in decreasing the proliferation and metastasis of cancer cells. 3-bromopyruvate makes use of cancer biology in treating cancer cells, cancer stem cells and preventing metastasis in human cancer as discussed in this review. Updated advances are analyzed here, which include research analysis of background, experience, readings in the field of cancer biology, oncology and biochemistry.

Slp-76 is a critical determinant of NK cell-mediated recognition of missing-self targets

Science.gov (United States)

Lampe, Kristin; Endale, Mehari; Cashman, Siobhan; Fang, Hao; Mattner, Jochen; Hildeman, David; Hoebe, Kasper

2015-01-01

Absence of MHC class I expression is an important mechanism by which NK cells recognize a variety of target cells, yet the pathways underlying “missing-self” recognition, including the involvement of activating receptors, remain poorly understood. Using ENU mutagenesis in mice, we identified a germline mutant, designated Ace, with a marked defect in NK cell-mediated recognition and elimination of “missing-self” targets. The causative mutation was linked to chromosome 11 and identified as a missense mutation [Thr428Ile] in the SH2 domain of Slp-76—a critical adapter molecule downstream of ITAM-containing surface receptors. The Slp-76 Ace mutation behaved as a hypomorphic allele—while no major defects were observed in conventional T cell development/function, a marked defect in NK cell-mediated elimination of β2-Microglobulin (β2M)-deficient target cells was observed. Further studies revealed Slp-76 to control NK cell receptor expression and maturation, however, activation of Slp-76ace/ace NK cells through ITAM-containing NK cell receptors or allogeneic/tumor target cells appeared largely unaffected. Imagestream analysis of the NK-β2M−/− target cell synapse, revealed a specific defect in actin recruitment to the conjugate synapse in Slp-76ace/ace NK cells. Overall these studies establish Slp-76 as a critical determinant of NK cell development and NK cell-mediated elimination of missing-self target cells. PMID:25929249

Slp-76 is a critical determinant of NK-cell mediated recognition of missing-self targets.

Science.gov (United States)

Lampe, Kristin; Endale, Mehari; Cashman, Siobhan; Fang, Hao; Mattner, Jochen; Hildeman, David; Hoebe, Kasper

2015-07-01

Absence of MHC class I expression is an important mechanism by which NK cells recognize a variety of target cells, yet the pathways underlying "missing-self" recognition, including the involvement of activating receptors, remain poorly understood. Using ethyl-N-nitrosourea mutagenesis in mice, we identified a germline mutant, designated Ace, with a marked defect in NK cell mediated recognition and elimination of "missing-self" targets. The causative mutation was linked to chromosome 11 and identified as a missense mutation (Thr428Ile) in the SH2 domain of Slp-76-a critical adapter molecule downstream of ITAM-containing surface receptors. The Slp-76 Ace mutation behaved as a hypomorphic allele-while no major defects were observed in conventional T-cell development/function, a marked defect in NK cell mediated elimination of β2-microglobulin (β2M) deficient target cells was observed. Further studies revealed Slp-76 to control NK-cell receptor expression and maturation; however, activation of Slp-76(ace/ace) NK cells through ITAM-containing NK-cell receptors or allogeneic/tumor target cells appeared largely unaffected. Imagestream analysis of the NK-β2M(-/-) target cell synapse revealed a specific defect in actin recruitment to the conjugate synapse in Slp-76(ace/ace) NK cells. Overall these studies establish Slp-76 as a critical determinant of NK-cell development and NK cell mediated elimination of missing-self target cells in mice. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Targeting of follicle stimulating hormone peptide-conjugated dendrimers to ovarian cancer cells

Science.gov (United States)

Modi, Dimple A.; Sunoqrot, Suhair; Bugno, Jason; Lantvit, Daniel D.; Hong, Seungpyo; Burdette, Joanna E.

2014-02-01

Ovarian cancer is the most lethal gynecological malignancy. Current treatment modalities include a combination of surgery and chemotherapy, which often lead to loss of fertility in premenopausal women and a myriad of systemic side effects. To address these issues, we have designed poly(amidoamine) (PAMAM) dendrimers to selectively target the follicle stimulating hormone receptor (FSHR), which is overexpressed by tumorigenic ovarian cancer cells but not by immature primordial follicles and other non-tumorigenic cells. Fluorescein-labeled generation 5 (G5) PAMAM dendrimers were conjugated with the binding peptide domain of FSH (FSH33) that has a high affinity to FSHR. The targeted dendrimers exhibited high receptor selectivity to FSHR-expressing OVCAR-3 cells, resulting in significant uptake and downregulation of an anti-apoptotic protein survivin, while showing minimal interactions with SKOV-3 cells that do not express FSHR. The selectivity of the FSH33-targeted dendrimers was further validated in 3D organ cultures of normal mouse ovaries. Immunostaining of the conjugates revealed their selective binding and uptake by ovarian surface epithelium (OSE) cells that express FSHR, while sparing the immature primordial follicles. In addition, an in vivo study monitoring tissue accumulation following a single intraperitoneal (i.p.) injection of the conjugates showed significantly higher accumulation of FSH33-targeted dendrimers in the ovary and oviduct compared to the non-targeted conjugates. These proof-of-concept findings highlight the potential of these FSH33-targeted dendrimers to serve as a delivery platform for anti-ovarian cancer drugs, while reducing their systemic side effects by preventing nonspecific uptake by the primordial follicles.Ovarian cancer is the most lethal gynecological malignancy. Current treatment modalities include a combination of surgery and chemotherapy, which often lead to loss of fertility in premenopausal women and a myriad of systemic side

Receptor-Targeted Nipah Virus Glycoproteins Improve Cell-Type Selective Gene Delivery and Reveal a Preference for Membrane-Proximal Cell Attachment.

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Ruben R Bender

2016-06-01

Full Text Available Receptor-targeted lentiviral vectors (LVs can be an effective tool for selective transfer of genes into distinct cell types of choice. Moreover, they can be used to determine the molecular properties that cell surface proteins must fulfill to act as receptors for viral glycoproteins. Here we show that LVs pseudotyped with receptor-targeted Nipah virus (NiV glycoproteins effectively enter into cells when they use cell surface proteins as receptors that bring them closely enough to the cell membrane (less than 100 Å distance. Then, they were flexible in receptor usage as demonstrated by successful targeting of EpCAM, CD20, and CD8, and as selective as LVs pseudotyped with receptor-targeted measles virus (MV glycoproteins, the current standard for cell-type specific gene delivery. Remarkably, NiV-LVs could be produced at up to two orders of magnitude higher titers compared to their MV-based counterparts and were at least 10,000-fold less effectively neutralized than MV glycoprotein pseudotyped LVs by pooled human intravenous immunoglobulin. An important finding for NiV-LVs targeted to Her2/neu was an about 100-fold higher gene transfer activity when particles were targeted to membrane-proximal regions as compared to particles binding to a more membrane-distal epitope. Likewise, the low gene transfer activity mediated by NiV-LV particles bound to the membrane distal domains of CD117 or the glutamate receptor subunit 4 (GluA4 was substantially enhanced by reducing receptor size to below 100 Å. Overall, the data suggest that the NiV glycoproteins are optimally suited for cell-type specific gene delivery with LVs and, in addition, for the first time define which parts of a cell surface protein should be targeted to achieve optimal gene transfer rates with receptor-targeted LVs.

Quantitative ultrasound characterization of tumor cell death: ultrasound-stimulated microbubbles for radiation enhancement.

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Hyunjung Christina Kim

Full Text Available The aim of this study was to assess the efficacy of quantitative ultrasound imaging in characterizing cancer cell death caused by enhanced radiation treatments. This investigation focused on developing this ultrasound modality as an imaging-based non-invasive method that can be used to monitor therapeutic ultrasound and radiation effects. High-frequency (25 MHz ultrasound was used to image tumor responses caused by ultrasound-stimulated microbubbles in combination with radiation. Human prostate xenografts grown in severe combined immunodeficiency (SCID mice were treated using 8, 80, or 1000 µL/kg of microbubbles stimulated with ultrasound at 250, 570, or 750 kPa, and exposed to 0, 2, or 8 Gy of radiation. Tumors were imaged prior to treatment and 24 hours after treatment. Spectral analysis of images acquired from treated tumors revealed overall increases in ultrasound backscatter intensity and the spectral intercept parameter. The increase in backscatter intensity compared to the control ranged from 1.9±1.6 dB for the clinical imaging dose of microbubbles (8 µL/kg, 250 kPa, 2 Gy to 7.0±4.1 dB for the most extreme treatment condition (1000 µL/kg, 750 kPa, 8 Gy. In parallel, in situ end-labelling (ISEL staining, ceramide, and cyclophilin A staining demonstrated increases in cell death due to DNA fragmentation, ceramide-mediated apoptosis, and release of cyclophilin A as a result of cell membrane permeabilization, respectively. Quantitative ultrasound results indicated changes that paralleled increases in cell death observed from histology analyses supporting its use for non-invasive monitoring of cancer treatment outcomes.

Targeting poly (ADP-ribose polymerase partially contributes to bufalin-induced cell death in multiple myeloma cells.

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He Huang

Full Text Available Despite recent pharmaceutical advancements in therapeutic drugs, multiple myeloma (MM remains an incurable disease. Recently, ploy(ADP-ribose polymerase 1 (PARP1 has been shown as a potentially promising target for MM therapy. A previous report suggested bufalin, a component of traditional Chinese medicine ("Chan Su", might target PARP1. However, this hypothesis has not been verified. We here showed that bufalin could inhibit PARP1 activity in vitro and reduce DNA-damage-induced poly(ADP-ribosylation in MM cells. Molecular docking analysis revealed that the active site of bufalin interaction is within the catalytic domain of PAPR1. Thus, PARP1 is a putative target of bufalin. Furthermore, we showed, for the first time that the proliferation of MM cell lines (NCI-H929, U266, RPMI8226 and MM.1S and primary CD138(+ MM cells could be inhibited by bufalin, mainly via apoptosis and G2-M phase cell cycle arrest. MM cell apoptosis was confirmed by apoptotic cell morphology, Annexin-V positive cells, and the caspase3 activation. We further evaluated the role of PARP1 in bufalin-induced apoptosis, discovering that PARP1 overexpression partially suppressed bufalin-induced cell death. Moreover, bufalin can act as chemosensitizer to enhance the cell growth-inhibitory effects of topotecan, camptothecin, etoposide and vorinostat in MM cells. Collectively, our data suggest that bufalin is a novel PARP1 inhibitor and a potentially promising therapeutic agent against MM alone or in combination with other drugs.

Mechanoresponsive stem cells to target cancer metastases through biophysical cues.

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Liu, Linan; Zhang, Shirley X; Liao, Wenbin; Farhoodi, Henry P; Wong, Chi W; Chen, Claire C; Ségaliny, Aude I; Chacko, Jenu V; Nguyen, Lily P; Lu, Mengrou; Polovin, George; Pone, Egest J; Downing, Timothy L; Lawson, Devon A; Digman, Michelle A; Zhao, Weian

2017-07-26

Despite decades of effort, little progress has been made to improve the treatment of cancer metastases. To leverage the central role of the mechanoenvironment in cancer metastasis, we present a mechanoresponsive cell system (MRCS) to selectively identify and treat cancer metastases by targeting the specific biophysical cues in the tumor niche in vivo. Our MRCS uses mechanosensitive promoter-driven mesenchymal stem cell (MSC)-based vectors, which selectively home to and target cancer metastases in response to specific mechanical cues to deliver therapeutics to effectively kill cancer cells, as demonstrated in a metastatic breast cancer mouse model. Our data suggest a strong correlation between collagen cross-linking and increased tissue stiffness at the metastatic sites, where our MRCS is specifically activated by the specific cancer-associated mechano-cues. MRCS has markedly reduced deleterious effects compared to MSCs constitutively expressing therapeutics. MRCS indicates that biophysical cues, specifically matrix stiffness, are appealing targets for cancer treatment due to their long persistence in the body (measured in years), making them refractory to the development of resistance to treatment. Our MRCS can serve as a platform for future diagnostics and therapies targeting aberrant tissue stiffness in conditions such as cancer and fibrotic diseases, and it should help to elucidate mechanobiology and reveal what cells "feel" in the microenvironment in vivo. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Crispr-mediated Gene Targeting of Human Induced Pluripotent Stem Cells.

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Byrne, Susan M; Church, George M

2015-01-01

CRISPR/Cas9 nuclease systems can create double-stranded DNA breaks at specific sequences to efficiently and precisely disrupt, excise, mutate, insert, or replace genes. However, human embryonic stem or induced pluripotent stem cells (iPSCs) are more difficult to transfect and less resilient to DNA damage than immortalized tumor cell lines. Here, we describe an optimized protocol for genome engineering of human iPSCs using a simple transient transfection of plasmids and/or single-stranded oligonucleotides. With this protocol, we achieve transfection efficiencies greater than 60%, with gene disruption efficiencies from 1-25% and gene insertion/replacement efficiencies from 0.5-10% without any further selection or enrichment steps. We also describe how to design and assess optimal sgRNA target sites and donor targeting vectors; cloning individual iPSC by single cell FACS sorting, and genotyping successfully edited cells.

Melanocytes from patients affected by Ullrich congenital muscular dystrophy and Bethlem myopathy have dysfunctional mitochondria that can be rescued with cyclophilin inhibitors

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Alessandra eZulian

2014-11-01

Full Text Available Ullrich congenital muscular dystrophy and Bethlem myopathy are caused by mutations in collagen VI genes, which encode an extracellular matrix protein; yet mitochondria play a major role in disease pathogenesis through a short circuit caused by inappropriate opening of the permeability transition pore, a high conductance channel which causes a shortage in ATP production. We find that melanocytes do not produce collagen VI yet they bind it at the cell surface, suggesting that this protein may play a trophic role and that its absence may cause lesions similar to those seen in skeletal muscle. We show that mitochondria in melanocytes of Ullrich congenital muscular dystrophy and Bethlem myooathy patients display increased size, reduced matrix density and disrupted cristae, findings that suggest a functional impairment. In keeping with this hypothesis, mitochondria (i underwent anomalous depolarization after inhibition of the F-ATP synthase with oligomycin, and (ii displayed decreased respiratory reserve capacity. The non-immunosuppressive cyclophilin inhibitor NIM811 prevented mitochondrial depolarization in response to oligomycin in melanocytes from both Ullrich congenital muscular dystrophy and Bethlem myopathy patients, and partially restored the respiratory reserve of melanocytes from one Bethlem myopathy patient. These results match our recent findings on melanocytes from patients affected by Duchenne muscular dystrophy (Pellegrini et al., 2013 Melanocytes--a novel tool to study mitochondrial dysfunction in Duchenne muscular dystrophy. J Cell Physiol 228, 1323-1331, and suggest that skin biopsies may represent a minimally invasive tool to investigate mitochondrial dysfunction and to evaluate drug efficacy in collagen VI-related myopathies and possibly in other muscle wasting conditions like aging sarcopenia.

Targeted destruction of murine macrophage cells with bioconjugated gold nanorods

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Pissuwan, Dakrong; Valenzuela, Stella M.; Killingsworth, Murray C.; Xu, Xiaoda; Cortie, Michael B.

2007-12-01

Gold nanorods manifest a readily tunable longitudinal plasmon resonance with light and consequently have potential for use in photothermal therapeutics. Recent work by others has shown how gold nanoshells and rods can be used to target cancer cells, which can then be destroyed using relatively high power laser radiation (˜1×105 to 1×1010 W/m2). Here we extend this concept to demonstrate how gold nanorods can be modified to bind to target macrophage cells, and show that high intensity laser radiation is not necessary, with even 5×102 W/m2 being sufficient, provided that a total fluence of ˜30 J/cm2 is delivered. We used the murine cell line RAW 264.7 and the monoclonal antibody CD11b, raised against murine macrophages, as our model system and a 5 mW solid state diode laser as our energy source. Exposure of the cells labeled with gold nanorods to a laser fluence of 30 J/cm2 resulted in 81% cell death compared to only 0.9% in the control, non-labeled cells.

Myeloid Conditioning with c-kit-Targeted CAR-T Cells Enables Donor Stem Cell Engraftment.

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Arai, Yasuyuki; Choi, Uimook; Corsino, Cristina I; Koontz, Sherry M; Tajima, Masaki; Sweeney, Colin L; Black, Mary A; Feldman, Steven A; Dinauer, Mary C; Malech, Harry L

2018-05-02

We report a novel approach to bone marrow (BM) conditioning using c-kit-targeted chimeric antigen receptor T (c-kit CAR-T) cells in mice. Previous reports using anti-c-kit or anti-CD45 antibody linked to a toxin such as saporin have been promising. We developed a distinctly different approach using c-kit CAR-T cells. Initial studies demonstrated in vitro killing of hematopoietic stem cells by c-kit CAR-T cells but poor expansion in vivo and poor migration of CAR-T cells into BM. Pre-treatment of recipient mice with low-dose cyclophosphamide (125 mg/kg) together with CXCR4 transduction in the CAR-T cells enhanced trafficking to and expansion in BM (c-kit + population (9.0%-0.1%). Because congenic Thy1.1 CAR-T cells were used in the Thy1.2-recipient mice, anti-Thy1.1 antibody could be used to deplete CAR-T cells in vivo before donor BM transplant. This achieved 20%-40% multilineage engraftment. We applied this conditioning to achieve an average of 28% correction of chronic granulomatous disease mice by wild-type BM transplant. Our findings provide a proof of concept that c-kit CAR-T cells can achieve effective BM conditioning without chemo-/radiotherapy. Our work also demonstrates that co-expression of a trafficking receptor can enhance targeting of CAR-T cells to a designated tissue. Published by Elsevier Inc.

Targeting Gallium to Cancer Cells through the Folate Receptor

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Nerissa Viola-Villegas

2008-01-01

Full Text Available The development of gallium(III compounds as anti-cancer agents for both treatment and diagnosis is a rapidly developing field of research. Problems remain in exploring the full potential of gallium(III as a safe and successful therapeutic agent or as an imaging agent. One of the major issues is that gallium(III compounds have little tropism for cancer cells. We have combined the targeting properties of folic acid (FA with long chain liquid polymer poly(ethylene glycol (PEG 'spacers’. This FA-PEG unit has been coupled to the gallium coordination complex of 1,4,7,10-tetraazacyclo-dodecane-N, N′, N′, N′′-tetraacetic acid (DOTA through amide linkages for delivery into target cells overexpressing the folate receptor (FR. In vitro cytotoxicity assays were conducted against a multi-drug resistant ovarian cell line (A2780/AD that overexpresses the FR and contrasted against a FR free Chinese hamster ovary (CHO cell line. Results are rationalized taking into account stability studies conducted in RPMI 1640 media and HEPES buffer at pH 7.4.

Targeting Gallium to Cancer Cells through the Folate Receptor

Directory of Open Access Journals (Sweden)

Nerissa Viola-Villegas

2008-01-01

Full Text Available The development of gallium(III compounds as anti-cancer agents for both treatment and diagnosis is a rapidly developing field of research. Problems remain in exploring the full potential of gallium(III as a safe and successful therapeutic agent or as an imaging agent. One of the major issues is that gallium(III compounds have little tropism for cancer cells. We have combined the targeting properties of folic acid (FA with long chain liquid polymer poly(ethylene glycol (PEG ‘spacers’. This FA-PEG unit has been coupled to the gallium coordination complex of 1,4,7,10-tetraazacyclo-dodecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA through amide linkages for delivery into target cells overexpressing the folate receptor (FR. In vitro cytotoxicity assays were conducted against a multi-drug resistant ovarian cell line (A2780/AD that overexpresses the FR and contrasted against a FR free Chinese hamster ovary (CHO cell line. Results are rationalized taking into account stability studies conducted in RPMI 1640 media and HEPES buffer at pH 7.4.

Evaluating Cytotoxicity of Hyaluronate Targeted Solid Lipid Nanoparticles of Etoposide on SK-OV-3 Cells

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Parviz Mohammadi Ghalaei

2014-01-01

Full Text Available The epithelial ovarian carcinoma is one of the most fatal gynecological cancers. Etoposide is used in treating platinum-resistant ovarian cancer. Sodium hyaluronate is a substance that binds to the CD44 receptors overexpressed in SK-OV-3 cells of epithelial ovarian carcinoma. The aim of the present work was to study the cytotoxicity effect of hyaluronate targeted solid lipid nanoparticles (SLNs of etoposide on SK-OV-3 cells. The cytotoxicity of the targeted and nontargeted SLNs of etoposide was compared to free drug on the SK-OV-3 cells by MTT assay method. The cellular uptake of the targeted and nontargeted nanoparticles containing sodium fluorescein was also studied. The difference of cell vitality between nontargeted nanoparticles and also targeted nanoparticles with free drug was significant. Targeted nanoparticles also caused more toxicity than nontargeted nanoparticles (P<0.05. After 4 hours of incubating, the fluorescence was remarkably higher in the cells treated by targeted SLNs rather than nontargeted ones, and there was no observable fluorescence in cells incubated with pure sodium fluorescein. Hyaluronate targeted SLNs containing etoposide increased the cytotoxicity of etoposide on SK-OV-3 cells which may be a worthwhile potential method for reducing the prescribed dose and systemic side effects of this drug in epithelial ovarian carcinoma.

Gene targeting and cloning in pigs using fetal liver derived cells.

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Waghmare, Sanjeev K; Estrada, Jose; Reyes, Luz; Li, Ping; Ivary, Bess; Sidner, Richard A; Burlak, Chris; Tector, A Joseph

2011-12-01

Since there are no pig embryonic stem cells, pig genetic engineering is done in fetal fibroblasts that remain totipotent for only 3 to 5 wk. Nuclear donor cells that remain totipotent for longer periods of time would facilitate complicated genetic engineering in pigs. The goal of this study was to test the feasibility of using fetal liver-derived cells (FLDC) to perform gene targeting, and create a genetic knockout pig. FLDC were isolated and processed using a human liver stem cell protocol. Single copy α-1,3-galactosyl transferase knockout (GTKO) FLDCs were created using electroporation and neomycin resistant colonies were screened using PCR. Homozygous GTKO cells were created through loss of heterozygosity mutations in single GTKO FLDCs. Double GTKO FLDCs were used in somatic cell nuclear transfer (SCNT) to create GTKO pigs. FLDCs grew for more than 80 population doublings, maintaining normal karyotype. Gene targeting and loss of heterozygosity mutations produced homozygous GTKO FLDCs. FLDCs used in SCNT gave rise to homozygous GTKO pigs. FDLCs can be used in gene targeting and SCNT to produce genetically modified pigs. The increased life span in culture compared to fetal fibroblasts may facilitate genetic engineering in the pig. Copyright © 2011 Elsevier Inc. All rights reserved.

Chimeric Antigen Receptor (CAR) T Cells: Lessons Learned from Targeting of CD19 in B-Cell Malignancies.

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Hay, Kevin A; Turtle, Cameron J

2017-03-01

Adoptive immunotherapy with chimeric antigen receptor-modified (CAR)-T cells is a rapidly growing therapeutic approach to treating patients with refractory cancer, with over 100 clinical trials in various malignancies in progress. The enthusiasm for CAR-T cells has been driven by the clinical success of CD19-targeted CAR-T cell therapy in B-cell acute lymphoblastic leukemia, and the promising data in B-cell non-Hodgkin's lymphoma and chronic lymphocytic leukemia. Despite the success of targeting CD19 with CAR-T cells in early clinical studies, many challenges remain to improve outcomes, reduce toxicity, and determine the appropriate settings for CAR-T cell immunotherapy. Reviewing the lessons learned thus far in CD19 CAR-T cell trials and how some of these challenges may be overcome will help guide the development of CAR-T cell therapy for malignancies of B-cell origin, as well as for other hematopoietic and non-hematopoietic cancers.

Near-IR laser-triggered target cell collection using a carbon nanotube-based cell-cultured substrate.

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Sada, Takao; Fujigaya, Tsuyohiko; Niidome, Yasuro; Nakazawa, Kohji; Nakashima, Naotoshi

2011-06-28

Unique near-IR optical properties of single-walled carbon nanotube (SWNTs) are of interest in many biological applications. Here we describe the selective cell detachment and collection from an SWNT-coated cell-culture dish triggered by near-IR pulse laser irradiation. First, HeLa cells were cultured on an SWNT-coated dish prepared by a spraying of an aqueous SWNT dispersion on a glass dish. The SWNT-coated dish was found to show a good cell adhesion behavior as well as a cellular proliferation rate similar to a conventional glass dish. We discovered, by near-IR pulse laser irradiation (at the laser power over 25 mW) to the cell under optical microscopic observation, a quick single-cell detachment from the SWNT-coated surface. Shockwave generation from the irradiated SWNTs is expected to play an important role for the cell detachment. Moreover, we have succeeded in catapulting the target single cell from the cultured medium when the depth of the medium was below 150 μm and the laser power was stronger than 40 mW. The captured cell maintained its original shape. The retention of the genetic information of the cell was confirmed by the polymerase chain reaction (PCR) technique. A target single-cell collection from a culture medium under optical microscopic observation is significant in wide fields of single-cell studies in biological areas.

Integrin Targeting and Toxicological Assessment of Peptide-Conjugated Liposome Delivery Systems to Activated Endothelial Cells

DEFF Research Database (Denmark)

Kermanizadeh, Ali; Villadsen, Klaus; Østrem, Ragnhild Garborg

2017-01-01

constructed with the aim of targeting integrins (i.e. vitronectin and/or fibronectin receptors) on activated endothelial cells. The peptide-conjugated liposomes induced only cytotoxicity at the highest concentration in non-activated or activated endothelial cells, as well as in co-culture of endothelial cells...... and macrophages. There was unaltered secretion of cytokines following exposure of peptide-conjugated liposomes to endothelial cells, indicating that the materials were not inflammogenic. Liposomes with a peptide targeting the fibronectin receptor (integrin α5β1) were more effective in targeting of activated....... Therefore, this study demonstrates the feasibility of constructing a peptide-conjugated cationic liposome, which displays targeting to activated endothelial cells at concentrations that are not cytotoxic or inflammogenic to the cells....

The cell's nucleolus: an emerging target for chemotherapeutic intervention.

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Pickard, Amanda J; Bierbach, Ulrich

2013-09-01

The transient nucleolus plays a central role in the up-regulated synthesis of ribosomal RNA (rRNA) to sustain ribosome biogenesis, a hallmark of aberrant cell growth. This function, in conjunction with its unique pathohistological features in malignant cells and its ability to mediate apoptosis, renders this sub-nuclear structure a potential target for chemotherapeutic agents. In this Minireview, structurally and functionally diverse small molecules are discussed that have been reported to either interact with the nucleolus directly or perturb its function indirectly by acting on its dynamic components. These molecules include all major classes of nucleic-acid-targeted agents, antimetabolites, kinase inhibitors, anti-inflammatory drugs, natural product antibiotics, oligopeptides, as well as nanoparticles. Together, these molecules are invaluable probes of structure and function of the nucleolus. They also provide a unique opportunity to develop novel strategies for more selective and therefore better-tolerated chemotherapeutic intervention. In this regard, inhibition of RNA polymerase-I-mediated rRNA synthesis appears to be a promising mechanism for killing cancer cells. The recent development of molecules targeted at G-quadruplex-forming rRNA gene sequences, which are currently undergoing clinical trials, seems to attest to the success of this approach. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Targeting Gas6/TAM in cancer cells and tumor microenvironment.

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Wu, Guiling; Ma, Zhiqiang; Cheng, Yicheng; Hu, Wei; Deng, Chao; Jiang, Shuai; Li, Tian; Chen, Fulin; Yang, Yang

2018-01-31

Growth arrest-specific 6, also known as Gas6, is a human gene encoding the Gas6 protein, which was originally found to be upregulated in growth-arrested fibroblasts. Gas6 is a member of the vitamin K-dependent family of proteins expressed in many human tissues and regulates several biological processes in cells, including proliferation, survival and migration, by binding to its receptors Tyro3, Axl and Mer (TAM). In recent years, the roles of Gas6/TAM signalling in cancer cells and the tumour microenvironment have been studied, and some progress has made in targeted therapy, providing new potential directions for future investigations of cancer treatment. In this review, we introduce the Gas6 and TAM receptors and describe their involvement in different cancers and discuss the roles of Gas6 in cancer cells, the tumour microenvironment and metastasis. Finally, we introduce recent studies on Gas6/TAM targeting in cancer therapy, which will assist in the experimental design of future analyses and increase the potential use of Gas6 as a therapeutic target for cancer.

Chimeric Antigen Receptor (CAR) T cells: Lessons Learned from Targeting of CD19 in B cell malignancies

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Hay, Kevin A; Turtle, Cameron J

2017-01-01

Adoptive immunotherapy with chimeric antigen receptor-modified T (CAR-T) cells is a rapidly growing therapeutic approach to treating patients with refractory cancer, with over 100 clinical trials in various malignancies in progress. The enthusiasm for CAR-T cells has been driven by the clinical success of CD19-targeted CAR-T therapy in B-cell acute lymphoblastic leukemia, and the promising data in B-cell non-Hodgkin’s lymphoma and chronic lymphocytic leukemia. Despite the success of targeting CD19 with CAR-T cells in early clinical studies, many challenges remain to improve outcomes, reduce toxicity, and determine the appropriate settings for CAR-T cell immunotherapy. Reviewing the lessons learned thus far in CD19 CAR-T cell trials and how some of these challenges may be overcome will help guide the development of CAR-T cell therapy for malignancies of B-cell origin, as well as for other hematopoietic and non-hematopoietic cancers. PMID:28110394

MicroRNA-145 targets YES and STAT1 in colon cancer cells

DEFF Research Database (Denmark)

Gregersen, Lea H; Jacobsen, Anders B; Frankel, Lisa

2010-01-01

miRNA overexpression. Gene Ontology analysis showed an overrepresentation of genes involved in cell death, cellular growth and proliferation, cell cycle, gene expression and cancer. A number of the identified miRNA targets have previously been implicated in cancer, including YES, FSCN1, ADAM17, BIRC2......, VANGL1 as well as the transcription factor STAT1. Both YES and STAT1 were verified as direct miR-145 targets. CONCLUSIONS/SIGNIFICANCE: The study identifies and validates new cancer-relevant direct targets of miR-145 in colon cancer cells and hereby adds important mechanistic understanding of the tumor......BACKGROUND: MicroRNAs (miRNAs) have emerged as important gene regulators and are recognized as key players in tumorigenesis. miR-145 is reported to be down-regulated in several cancers, but knowledge of its targets in colon cancer remains limited. METHODOLOGY/PRINCIPAL FINDINGS: To investigate...

Membrane Targeting of P-type ATPases in Plant Cells

International Nuclear Information System (INIS)

Harper, Jeffrey F.

2004-01-01

How membrane proteins are targeted to specific subcellular locations is a very complex and poorly understood area of research. Our long-term goal is to use P-type ATPases (ion pumps), in a model plant system Arabidopsis, as a paradigm to understand how members of a family of closely related membrane proteins can be targeted to different subcellular locations. The research is divided into two specific aims. The first aim is focused on determining the targeting destination of all 10 ACA-type calcium pumps (Arabidopsis Calcium ATPase) in Arabidopsis. ACAs represent a plant specific-subfamily of plasma membrane-type calcium pumps. In contrast to animals, the plant homologs have been found in multiple membrane systems, including the ER (ACA2), tonoplast (ACA4) and plasma membrane (ACA8). Their high degree of similarity provides a unique opportunity to use a comparative approach to delineate the membrane specific targeting information for each pump. One hypothesis to be tested is that an endomembrane located ACA can be re-directed to the plasma membrane by including targeting information from a plasma membrane isoform, ACA8. Our approach is to engineer domain swaps between pumps and monitor the targeting of chimeric proteins in plant cells using a Green Fluorescence Protein (GFP) as a tag. The second aim is to test the hypothesis that heterologous transporters can be engineered into plants and targeted to the plasma membrane by fusing them to a plasma membrane proton pump. As a test case we are evaluating the targeting properties of fusions made between a yeast sodium/proton exchanger (Sod2) and a proton pump (AHA2). This fusion may potentially lead to a new strategy for engineering salt resistant plants. Together these aims are designed to provide fundamental insights into the biogenesis and function of plant cell membrane systems

Targeting myeloid cells to the brain using non-myeloablative conditioning.

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Chotima Böttcher

Full Text Available Bone marrow-derived cells (BMDCs are able to colonize the central nervous system (CNS at sites of damage. This ability makes BMDCs an ideal cellular vehicle for transferring therapeutic genes/molecules to the CNS. However, conditioning is required for bone marrow-derived myeloid cells to engraft in the brain, which so far has been achieved by total body irradiation (TBI and by chemotherapy (e.g. busulfan treatment. Unfortunately, both regimens massively disturb the host's hematopoietic compartment. Here, we established a conditioning protocol to target myeloid cells to sites of brain damage in mice using non-myeloablative focal head irradiation (HI. This treatment was associated with comparatively low inflammatory responses in the CNS despite cranial radiation doses which are identical to TBI, as revealed by gene expression analysis of cytokines/chemokines such as CCL2, CXCL10, TNF-α and CCL5. HI prior to bone marrow transplantation resulted in much lower levels of blood chimerism defined as the percentage of donor-derived cells in peripheral blood ( 95% or busulfan treatment (> 50%. Nevertheless, HI effectively recruited myeloid cells to the area of motoneuron degeneration in the brainstem within 7 days after facial nerve axotomy. In contrast, no donor-derived cells were detected in the lesioned facial nucleus of busulfan-treated animals up to 2 weeks after transplantation. Our findings suggest that myeloid cells can be targeted to sites of brain damage even in the presence of very low levels of peripheral blood chimerism. We established a novel non-myeloablative conditioning protocol with minimal disturbance of the host's hematopoietic system for targeting BMDCs specifically to areas of pathology in the brain.

Characterization of aldehyde dehydrogenase 1 high ovarian cancer cells: Towards targeted stem cell therapy.

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Sharrow, Allison C; Perkins, Brandy; Collector, Michael I; Yu, Wayne; Simons, Brian W; Jones, Richard J

2016-08-01

The cancer stem cell (CSC) paradigm hypothesizes that successful clinical eradication of CSCs may lead to durable remission for patients with ovarian cancer. Despite mounting evidence in support of ovarian CSCs, their phenotype and clinical relevance remain unclear. We and others have found high aldehyde dehydrogenase 1 (ALDH(high)) expression in a variety of normal and malignant stem cells, and sought to better characterize ALDH(high) cells in ovarian cancer. We compared ALDH(high) to ALDH(low) cells in two ovarian cancer models representing distinct subtypes: FNAR-C1 cells, derived from a spontaneous rat endometrioid carcinoma, and the human SKOV3 cell line (described as both serous and clear cell subtypes). We assessed these populations for stem cell features then analyzed expression by microarray and qPCR. ALDH(high) cells displayed CSC properties, including: smaller size, quiescence, regenerating the phenotypic diversity of the cell lines in vitro, lack of contact inhibition, nonadherent growth, multi-drug resistance, and in vivo tumorigenicity. Microarray and qPCR analysis of the expression of markers reported by others to enrich for ovarian CSCs revealed that ALDH(high) cells of both models showed downregulation of CD24, but inconsistent expression of CD44, KIT and CD133. However, the following druggable targets were consistently expressed in the ALDH(high) cells from both models: mTOR signaling, her-2/neu, CD47 and FGF18/FGFR3. Based on functional characterization, ALDH(high) ovarian cancer cells represent an ovarian CSC population. Differential gene expression identified druggable targets that have the potential for therapeutic efficacy against ovarian CSCs from multiple subtypes. Copyright © 2016 Elsevier Inc. All rights reserved.

Development of novel strategy for the synthesis of organometallic compounds usable as protein ligands: application to the human cyclophilin hCyp-18

International Nuclear Information System (INIS)

Clavaud, C.

2006-02-01

This thesis describes a new strategy for the development of bioactive organometallic compounds, basing on the combinatorial assembly of sub-chemical libraries (A and B) independent but complementary and able to coordinate a metallic heart M to form A-M-B complex potential ligands of biomolecules. The coordination of metals, well adapted to the production of molecular variety is usually used in medicinal chemistry, in diagnostic and therapeutic nuclear medicine. Among the useful elements, the rhenium and the technetium are metals of choice for the development of the assembly strategy because of their chemical and radiochemical properties and of the structure analogy of their complexes. This strategy was validated in vitro. The protein chosen for this purpose was the cyclophilin hCyp-18. (N.C.)

A Phenotypic Cell-Binding Screen Identifies a Novel Compound Targeting Triple-Negative Breast Cancer.

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Chen, Luxi; Long, Chao; Youn, Jonghae; Lee, Jiyong

2018-06-11

We describe a "phenotypic cell-binding screen" by which therapeutic candidate targeting cancer cells of a particular phenotype can be isolated without knowledge of drug targets. Chemical library beads are incubated with cancer cells of the phenotype of interest in the presence of cancer cells lacking the phenotype of interest, and then the beads bound to only cancer cells of the phenotype of interest are selected as hits. We have applied this screening strategy in discovering a novel compound (LC129-8) targeting triple-negative breast cancer (TNBC). LC129-8 displayed highly specific binding to TNBC in cancer cell lines and patient-derived tumor tissues. LC129-8 exerted anti-TNBC activity by inducing apoptosis, inhibiting proliferation, reversing epithelial-mesenchymal transition, downregulating cancer stem cell activity and blocking in vivo tumor growth.

Protocells and their use for targeted delivery of multicomponent cargos to cancer cells

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Brinker, C Jeffrey; Ashley, Carlee Erin; Jiang, Xingmao; Liu, Juewen; Peabody, David S; Wharton, Walker Richard; Carnes, Eric; Chackerian, Bryce; Willman, Cheryl L

2015-03-31

Various embodiments provide materials and methods for synthesizing protocells for use in targeted delivery of cargo components to cancer cells. In one embodiment, the lipid bilayer can be fused to the porous particle core to form a protocell. The lipid bilayer can be modified with targeting ligands or other ligands to achieve targeted delivery of cargo components that are loaded within the protocell to a target cell, e.g., a type of cancer. Shielding materials can be conjugated to the surface of the lipid bilayer to reduce undesired non-specific binding.

Targeting tissue factor on tumour cells and angiogenic vascular endothelial cells by factor VII-targeted verteporfin photodynamic therapy for breast cancer in vitro and in vivo in mice

International Nuclear Information System (INIS)

Hu, Zhiwei; Rao, Benqiang; Chen, Shimin; Duanmu, Jinzhong

2010-01-01

The objective of this study was to develop a ligand-targeted photodynamic therapy (tPDT) by conjugating factor VII (fVII) protein with photosensitiser verteporfin in order to overcome the poor selectivity and enhance the effect of non-targeted PDT (ntPDT) for cancer. fVII is a natural ligand for receptor tissue factor (TF) with high affinity and specificity. The reason for targeting receptor TF for the development of tPDT is that TF is a common but specific target on angiogenic tumour vascular endothelial cells (VEC) and many types of tumour cells, including solid tumours and leukaemia. Murine factor VII protein (mfVII) containing a mutation (Lys341Ala) was covalently conjugated via a cross linker EDC with Veterporfin (VP) that was extracted from liposomal Visudyne, and then free VP was separated by Sephadex G50 spin columns. fVII-tPDT using mfVII-VP conjugate, compared to ntPDT, was tested in vitro for the killing of breast cancer cells and VEGF-stimulated VEC and in vivo for inhibiting the tumour growth of breast tumours in a mouse xenograft model. We showed that: (i) fVII protein could be conjugated with VP without affecting its binding activity; (ii) fVII-tPDT could selectively kill TF-expressing breast cancer cells and VEGF-stimulated angiogenic HUVECs but had no side effects on non-TF expressing unstimulated HUVEC, CHO-K1 and 293 cells; (iii) fVII targeting enhanced the effect of VP PDT by three to four fold; (iii) fVII-tPDT induced significantly stronger levels of apoptosis and necrosis than ntPDT; and (iv) fVII-tPDT had a significantly stronger effect on inhibiting breast tumour growth in mice than ntPDT. We conclude that the fVII-targeted VP PDT that we report here is a novel and effective therapeutic with improved selectivity for the treatment of breast cancer. Since TF is expressed on many types of cancer cells including leukaemic cells and selectively on angiogenic tumour VECs, fVII-tPDT could have broad therapeutic applications for other solid cancers

Analysis of the role of homology arms in gene-targeting vectors in human cells.

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Ayako Ishii

Full Text Available Random integration of targeting vectors into the genome is the primary obstacle in human somatic cell gene targeting. Non-homologous end-joining (NHEJ, a major pathway for repairing DNA double-strand breaks, is thought to be responsible for most random integration events; however, absence of DNA ligase IV (LIG4, the critical NHEJ ligase, does not significantly reduce random integration frequency of targeting vector in human cells, indicating robust integration events occurring via a LIG4-independent mechanism. To gain insights into the mechanism and robustness of LIG4-independent random integration, we employed various types of targeting vectors to examine their integration frequencies in LIG4-proficient and deficient human cell lines. We find that the integration frequency of targeting vector correlates well with the length of homology arms and with the amount of repetitive DNA sequences, especially SINEs, present in the arms. This correlation was prominent in LIG4-deficient cells, but was also seen in LIG4-proficient cells, thus providing evidence that LIG4-independent random integration occurs frequently even when NHEJ is functionally normal. Our results collectively suggest that random integration frequency of conventional targeting vectors is substantially influenced by homology arms, which typically harbor repetitive DNA sequences that serve to facilitate LIG4-independent random integration in human cells, regardless of the presence or absence of functional NHEJ.

PET imaging of T cells: Target identification and feasibility assessment.

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Auberson, Yves P; Briard, Emmanuelle; Rudolph, Bettina; Kaupmann, Klemen; Smith, Paul; Oberhauser, Berndt

2018-06-01

Imaging T cells using positron emission tomography (PET) would be highly useful for diagnosis and monitoring in immunology and oncology patients. There are however no obvious targets that can be used to develop imaging agents for this purpose. We evaluated several potential target proteins with selective expression in T cells, and for which lead molecules were available: PKC , Lck, ZAP70 and Itk. Ultimately, we focused on Itk (interleukin-2-inducible T cell kinase) and identified a tool molecule with properties suitable for in vivo imaging of T cells, (5aR)-5,5-difluoro-5a-methyl-N-(1-((S)-3-(methylsulfonyl)-phenyl)(tetrahydro-2H-pyran-4-yl)methyl)-1H-pyrazol-4-yl)-1,4,4a,5,5a,6-hexahydro-cyclopropa[f]-indazole-3-carboxamide (23). While not having the optimal profile for clinical use, this molecule indicates that it might be possible to develop Itk-selective PET ligands for imaging the distribution of T cells in patients. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tumor Initiating Cells and Chemoresistance: Which Is the Best Strategy to Target Colon Cancer Stem Cells?

Directory of Open Access Journals (Sweden)

Emanuela Paldino

2014-01-01

Full Text Available There is an emerging body of evidence that chemoresistance and minimal residual disease result from selective resistance of a cell subpopulation from the original tumor that is molecularly and phenotypically distinct. These cells are called “cancer stem cells” (CSCs. In this review, we analyze the potential targeting strategies for eradicating CSCs specifically in order to develop more effective therapeutic strategies for metastatic colon cancer. These include induction of terminal epithelial differentiation of CSCs or targeting some genes expressed only in CSCs and involved in self-renewal and chemoresistance. Ideal targets could be cell regulators that simultaneously control the stemness and the resistance of CSCs. Another important aspect of cancer biology, which can also be harnessed to create novel broad-spectrum anticancer agents, is the Warburg effect, also known as aerobic glycolysis. Actually, little is yet known with regard to the metabolism of CSCs population, leaving an exciting unstudied avenue in the dawn of the emerging field of metabolomics.

Nitric oxide mediated bystander responses induced by microbeam targeted cells

International Nuclear Information System (INIS)

Shao, C.; Prise, K.M.; Folkard, M.; Michael, B.D.

2003-01-01

Considerable evidence has recently been accumulated in support of the existence of a 'bystander effect', which cells having received no irradiation show biological consequences from their vicinal irradiated cells. The application of microbeams is providing new insights into the radiation-induced bystander effect. The present study found that when a fraction of radioresistant human glioblastoma cells were individually targeted with a precise number of helium ions generated from the Gray Cancer Institute Charged Particle Microbeam, micronucleus (MN) induction significantly exceeded the expected value that was calculated from the number of MN observed when all of the cells were targeted assuming no bystander effect occurring. Even when only a single cell within a population was hit by one helium ion, the MN induction in the population could be increased by 16%. These results provide direct evidence of radiation-induced bystander effect. Moreover, MN was effectively induced in the unirradiated primary human fibroblasts and glioblastoma cells either co-cultured with irradiated cells or treated with the medium harvested from irradiated cells, indicating a signal molecule was produced from the irradiated cells. However, when c-PTIO, a nitric oxide (NO)-specific scavenger, was present in the medium during and after irradiation until MN analysis, the production of MN in all of the above cases was reduced to low levels. Consequently, NO plays an important role in the radiation-induced bystander effect

Immunologic targeting of FOXP3 in inflammatory breast cancer cells.

Directory of Open Access Journals (Sweden)

Smita Nair

Full Text Available The forkhead transcription factor FOXP3 is necessary for induction of regulatory T lymphocytes (Tregs and their immunosuppressive function. We have previously demonstrated that targeting Tregs by vaccination of mice with murine FOXP3 mRNA-transfected dendritic cells (DCs elicits FOXP3-specific T cell responses and enhances tumor immunity. It is clear that FOXP3 expression is not restricted to T-cell lineage and herein, using RT-PCR, flow cytometry, and western immunoblot we demonstrate for the first time that FOXP3 is expressed in inflammatory breast cancer (IBC cells, SUM149 (triple negative, ErbB1-activated and SUM190 (ErbB2-overexpressing. Importantly, FOXP3-specific T cells generated in vitro using human FOXP3 RNA-transfected DCs as stimulators efficiently lyse SUM149 cells. Interestingly, an isogenic model (rSUM149 derived from SUM149 with an enhanced anti-apoptotic phenotype was resistant to FOXP3-specific T cell mediated lysis. The MHC class I cellular processing mechanism was intact in both cell lines at the protein and transcription levels suggesting that the resistance to cytolysis by rSUM149 cells was not related to MHC class I expression or to the MHC class I antigen processing machinery in these cells. Our data suggest that FOXP3 may be an effective tumor target in IBC cells however increased anti-apoptotic signaling can lead to immune evasion.

Targeted destruction of murine macrophage cells with bioconjugated gold nanorods

Energy Technology Data Exchange (ETDEWEB)

Pissuwan, Dakrong [University of Technology Sydney, Institute for Nanoscale Technology (Australia); Valenzuela, Stella M. [University of Technology Sydney, Department of Medical and Molecular Biosciences (Australia)], E-mail: stella.valenzuela@uts.edu.au; Killingsworth, Murray C. [Sydney South West Pathology Service (Australia)], E-mail: murray.killingsworth@swsahs.nsw.gov.au; Xu, Xiaoda; Cortie, Michael B. [University of Technology Sydney, Institute for Nanoscale Technology (Australia)], E-mail: michael.cortie@uts.edu.au

2007-12-15

Gold nanorods manifest a readily tunable longitudinal plasmon resonance with light and consequently have potential for use in photothermal therapeutics. Recent work by others has shown how gold nanoshells and rods can be used to target cancer cells, which can then be destroyed using relatively high power laser radiation ({approx}1x10{sup 5} to 1x10{sup 10} W/m{sup 2}). Here we extend this concept to demonstrate how gold nanorods can be modified to bind to target macrophage cells, and show that high intensity laser radiation is not necessary, with even 5x10{sup 2} W/m{sup 2} being sufficient, provided that a total fluence of {approx}30 J/cm{sup 2} is delivered. We used the murine cell line RAW 264.7 and the monoclonal antibody CD11b, raised against murine macrophages, as our model system and a 5 mW solid state diode laser as our energy source. Exposure of the cells labeled with gold nanorods to a laser fluence of 30 J/cm{sup 2} resulted in 81% cell death compared to only 0.9% in the control, non-labeled cells.

Targeted destruction of murine macrophage cells with bioconjugated gold nanorods

International Nuclear Information System (INIS)

Pissuwan, Dakrong; Valenzuela, Stella M.; Killingsworth, Murray C.; Xu, Xiaoda; Cortie, Michael B.

2007-01-01

Gold nanorods manifest a readily tunable longitudinal plasmon resonance with light and consequently have potential for use in photothermal therapeutics. Recent work by others has shown how gold nanoshells and rods can be used to target cancer cells, which can then be destroyed using relatively high power laser radiation (∼1x10 5 to 1x10 10 W/m 2 ). Here we extend this concept to demonstrate how gold nanorods can be modified to bind to target macrophage cells, and show that high intensity laser radiation is not necessary, with even 5x10 2 W/m 2 being sufficient, provided that a total fluence of ∼30 J/cm 2 is delivered. We used the murine cell line RAW 264.7 and the monoclonal antibody CD11b, raised against murine macrophages, as our model system and a 5 mW solid state diode laser as our energy source. Exposure of the cells labeled with gold nanorods to a laser fluence of 30 J/cm 2 resulted in 81% cell death compared to only 0.9% in the control, non-labeled cells

Multifunctional Polymer Nanoparticles for Dual Drug Release and Cancer Cell Targeting

Directory of Open Access Journals (Sweden)

Yu-Han Wen

2017-06-01

Full Text Available Multifunctional polymer nanoparticles have been developed for cancer treatment because they could be easily designed to target cancer cells and to enhance therapeutic efficacy according to cancer hallmarks. In this study, we synthesized a pH-sensitive polymer, poly(methacrylic acid-co-histidine/doxorubicin/biotin (HBD in which doxorubicin (DOX was conjugated by a hydrazone bond to encapsulate an immunotherapy drug, imiquimod (IMQ, to form dual cancer-targeting and dual drug-loaded nanoparticles. At low pH, polymeric nanoparticles could disrupt and simultaneously release DOX and IMQ. Our experimental results show that the nanoparticles exhibited pH-dependent drug release behavior and had an ability to target cancer cells via biotin and protonated histidine.

Designing nanoconjugates to effectively target pancreatic cancer cells in vitro and in vivo.

Directory of Open Access Journals (Sweden)

Jameel Ahmad Khan

Full Text Available Pancreatic cancer is the fourth leading cause of cancer related deaths in America. Monoclonal antibodies are a viable treatment option for inhibiting cancer growth. Tumor specific drug delivery could be achieved utilizing these monoclonal antibodies as targeting agents. This type of designer therapeutic is evolving and with the use of gold nanoparticles it is a promising approach to selectively deliver chemotherapeutics to malignant cells. Gold nanoparticles (GNPs are showing extreme promise in current medicinal research. GNPs have been shown to non-invasively kill tumor cells by hyperthermia using radiofrequency. They have also been implemented as early detection agents due to their unique X-ray contrast properties; success was revealed with clear delineation of blood capillaries in a preclinical model by CT (computer tomography. The fundamental parameters for intelligent design of nanoconjugates are on the forefront. The goal of this study is to define the necessary design parameters to successfully target pancreatic cancer cells.The nanoconjugates described in this study were characterized with various physico-chemical techniques. We demonstrate that the number of cetuximab molecules (targeting agent on a GNP, the hydrodynamic size of the nanoconjugates, available reactive surface area and the ability of the nanoconjugates to sequester EGFR (epidermal growth factor receptor, all play critical roles in effectively targeting tumor cells in vitro and in vivo in an orthotopic model of pancreatic cancer.Our results suggest the specific targeting of tumor cells depends on a number of crucial components 1 targeting agent to nanoparticle ratio 2 availability of reactive surface area on the nanoparticle 3 ability of the nanoconjugate to bind the target and 4 hydrodynamic diameter of the nanoconjugate. We believe this study will help define the design parameters for formulating better strategies for specifically targeting tumors with nanoparticle

Simultaneous targeting of prostate stem cell antigen and prostate-specific membrane antigen improves the killing of prostate cancer cells using a novel modular T cell-retargeting system.

Science.gov (United States)

Arndt, Claudia; Feldmann, Anja; Koristka, Stefanie; Cartellieri, Marc; Dimmel, Maria; Ehninger, Armin; Ehninger, Gerhard; Bachmann, Michael

2014-09-01

Recently, we described a novel modular platform technology in which T cell-recruitment and tumor-targeting domains of conventional bispecific antibodies are split to independent components, a universal effector module (EM) and replaceable monospecific/monovalent target modules (TMs) that form highly efficient T cell-retargeting complexes. Theoretically, our unique strategy should allow us to simultaneously retarget T cells to different tumor antigens by combining the EM with two or more different monovalent/monospecific TMs or even with bivalent/bispecific TMs, thereby overcoming limitations of a monospecific treatment such as the selection of target-negative tumor escape variants. In order to advance our recently introduced prostate stem cell antigen (PSCA)-specific modular system for a dual-targeting of prostate cancer cells, two additional TMs were constructed: a monovalent/monospecific TM directed against the prostate-specific membrane antigen (PSMA) and a bivalent/bispecific TM (bsTM) with specificity for PSMA and PSCA. The functionality of the novel dual-targeting strategies was analyzed by performing T cell activation and chromium release assays. Similar to the PSCA-specific modular system, the novel PSMA-specific modular system mediates an efficient target-dependent and -specific tumor cell lysis at low E:T ratios and picomolar Ab concentrations. Moreover, by combination of the EM with either the bispecific TM directed to PSMA and PSCA or both monospecifc TMs directed to either PSCA or PSMA, dual-specific targeting complexes were formed which allowed us to kill potential escape variants expressing only one or the other target antigen. Overall, the novel modular system represents a promising tool for multiple tumor targeting. © 2014 Wiley Periodicals, Inc.

Nanoscale mapping and organization analysis of target proteins on cancer cells from B-cell lymphoma patients

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Li, Mi [State Key Laboratory of Robotics, Shenyang Institute of Automation, Chinese Academy of Sciences, Shenyang 110016 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Xiao, Xiubin [Department of Lymphoma, Affiliated Hospital of Military Medical Academy of Sciences, Beijing 100071 (China); Liu, Lianqing, E-mail: lqliu@sia.cn [State Key Laboratory of Robotics, Shenyang Institute of Automation, Chinese Academy of Sciences, Shenyang 110016 (China); Xi, Ning, E-mail: xin@egr.msu.edu [State Key Laboratory of Robotics, Shenyang Institute of Automation, Chinese Academy of Sciences, Shenyang 110016 (China); Department of Mechanical and Biomedical Engineering, City University of Hong Kong, Hong Kong (China); Wang, Yuechao; Dong, Zaili [State Key Laboratory of Robotics, Shenyang Institute of Automation, Chinese Academy of Sciences, Shenyang 110016 (China); Zhang, Weijing, E-mail: zhangwj3072@163.com [Department of Lymphoma, Affiliated Hospital of Military Medical Academy of Sciences, Beijing 100071 (China)

2013-11-01

CD20, a membrane protein highly expressed on most B-cell lymphomas, is an effective target demonstrated in clinical practice for treating B-cell non-Hodgkin's lymphoma (NHL). Rituximab is a monoclonal antibody against CD20. In this work, we applied atomic force microscopy (AFM) to map the nanoscale distribution of CD20 molecules on the surface of cancer cells from clinical B-cell NHL patients under the assistance of ROR1 fluorescence recognition (ROR1 is a specific cell surface marker exclusively expressed on cancer cells). First, the ROR1 fluorescence labeling experiments showed that ROR1 was expressed on cancer cells from B-cell lymphoma patients, but not on normal cells from healthy volunteers. Next, under the guidance of ROR1 fluorescence, the rituximab-conjugated AFM tips were moved to cancer cells to image the cellular morphologies and detect the CD20-rituximab interactions on the cell surfaces. The distribution maps of CD20 on cancer cells were constructed by obtaining arrays of (16×16) force curves in local areas (500×500 nm{sup 2}) on the cell surfaces. The experimental results provide a new approach to directly investigate the nanoscale distribution of target protein on single clinical cancer cells. - Highlights: • Cancer cells were recognized from healthy cells by ROR1 fluorescence labeling. • The nanoscale distribution of CD20 on cancer cells was characterized. • The distribution of CD20 was non-uniform on the surface of cancer cells.

Targeting of phage particles towards endothelial cells by antibodies selected through a multi-parameter selection strategy.

Science.gov (United States)

Mandrup, Ole A; Lykkemark, Simon; Kristensen, Peter

2017-02-10

One of the hallmarks of cancer is sustained angiogenesis. Here, normal endothelial cells are activated, and their formation of new blood vessels leads to continued tumour growth. An improved patient condition is often observed when angiogenesis is prevented or normalized through targeting of these genomically stable endothelial cells. However, intracellular targets constitute a challenge in therapy, as the agents modulating these targets have to be delivered and internalized specifically to the endothelial cells. Selection of antibodies binding specifically to certain cell types is well established. It is nonetheless a challenge to ensure that the binding of antibodies to the target cell will mediate internalization. Previously selection of such antibodies has been performed targeting cancer cell lines; most often using either monovalent display or polyvalent display. In this article, we describe selections that isolate internalizing antibodies by sequential combining monovalent and polyvalent display using two types of helper phages, one which increases display valence and one which reduces background. One of the selected antibodies was found to mediate internalization into human endothelial cells, although our results confirms that the single stranded nature of the DNA packaged into phage particles may limit applications aimed at targeting nucleic acids in mammalian cells.

Target-specific delivery of doxorubicin to human glioblastoma cell ...

Indian Academy of Sciences (India)

Abdullah Tahir Bayraç

2018-01-29

Jan 29, 2018 ... was previously selected for specific recognition of glioblastoma and represented many advantageous ... antigens, receptors or any 3-D structure on the target cells ..... both PSMA (?) and PSMA (-) prostate cancers.

Imaging and Targeting of Hypoxic Tumor Cells with Use of HIF-1-2

International Nuclear Information System (INIS)

Kizaka-Kondoh, Shinae; Harada, Hiroshi; Tanaka, Shotaro; Hiraoka, Masahiro

2006-01-01

This paper describes imaging (visualization) of transplanted tumor cells under hypoxia in vivo and molecular targeting to kill those cells by inducing their apoptosis. HIF (hypoxia inducible factor) concerned with angiogenesis is induced specifically in hypoxic tumor cells and its activity can be visualized by transfection of reporter vector construct of fluorescent protein GFP or luciferase. Authors established the transfected tumor cells with the plasmid p5HRE-luciferase and when transplanted in the nude mouse, those cells emitted light dependently to their hypoxic conditions, which could be visualized by in vivo imaging system (IVIS) with CCD camera. Authors prepared the oxygen-dependent degradation-procaspase 3-fusion protein (TOP3) to target the hypoxic tumor cells for enhancing their apoptotic signaling, whose apoptosis was actually observed by the IVIS. Reportedly, radiation transiently activates HIF-1 and combination treatment of radiation and TOP3 resulted in the enhanced death of tumor cells. Interestingly, the suppression of tumor growth lasted longer than expected, probably due to inhibition of angiogenesis. Authors called this anti-tumor strategy as the micro-environmental targeting. (T.I.)

Intracellular CXCR4+ cell targeting with T22-empowered protein-only nanoparticles

Directory of Open Access Journals (Sweden)

Unzueta U

2012-08-01

Full Text Available Ugutz Unzueta,1–3 María Virtudes Céspedes,3,4 Neus Ferrer-Miralles,1–3 Isolda Casanova,3,4 Juan Cedano,5 José Luis Corchero,1–3 Joan Domingo-Espín,1–3 Antonio Villaverde,1–3 Ramón Mangues,3,4 Esther Vázquez1–31Institut de Biotecnologia i de Biomedicina, 2Departamento de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, 3CIBER en Bioingeniería, Biomateriales y Nanomedicina, Bellaterra, Barcelona, 4Oncogenesis and Antitumor Drug Group, Biomedical Research Institute Sant Pau, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; 5Laboratory of Immunology, Regional Norte, Universidad de la Republica, Salto, UruguayBackground: Cell-targeting peptides or proteins are appealing tools in nanomedicine and innovative medicines because they increase the local drug concentration and reduce potential side effects. CXC chemokine receptor 4 (CXCR4 is a cell surface marker associated with several severe human pathologies, including colorectal cancer, for which intracellular targeting agents are currently missing.Results: Four different peptides that bind CXCR4 were tested for their ability to internalize a green fluorescent protein-based reporter nanoparticle into CXCR4+ cells. Among them, only the 18 mer peptide T22, an engineered segment derivative of polyphemusin II from the horseshoe crab, efficiently penetrated target cells via a rapid, receptor-specific endosomal route. This resulted in accumulation of the reporter nanoparticle in a fully fluorescent and stable form in the perinuclear region of the target cells, without toxicity either in cell culture or in an in vivo model of metastatic colorectal cancer.Conclusion: Given the urgent demand for targeting agents in the research, diagnosis, and treatment of CXCR4-linked diseases, including colorectal cancer and human immunodeficiency virus infection, T22 appears to be a promising tag for the intracellular delivery of protein drugs, nanoparticles

Odin (ANKS1A is a Src family kinase target in colorectal cancer cells

Directory of Open Access Journals (Sweden)

Feller Stephan M

2008-10-01

Full Text Available Abstract Background Src family kinases (SFK are implicated in the development of some colorectal cancers (CRC. One SFK member, Lck, is not detectable in normal colonic epithelium, but becomes aberrantly expressed in a subset of CRCs. Although SFK have been extensively studied in fibroblasts and different types of immune cells, their physical and functional targets in many epithelial cancers remain poorly characterised. Results 64 CRC cell lines were tested for expression of Lck. SW620 CRC cells, which express high levels of Lck and also contain high basal levels of tyrosine phosphorylated (pY proteins, were then analysed to identify novel SFK targets. Since SH2 domains of SFK are known to often bind substrates after phosphorylation by the kinase domain, the LckSH2 was compared with 14 other SH2s for suitability as affinity chromatography reagent. Mass spectrometric analyses of LckSH2-purified pY proteins subsequently identified several proteins readily known as SFK kinase substrates, including cortactin, Tom1L1 (SRCASM, GIT1, vimentin and AFAP1L2 (XB130. Additional proteins previously reported as substrates of other tyrosine kinase were also detected, including the EGF and PDGF receptor target Odin. Odin was further analysed and found to contain substantially less pY upon inhibition of SFK activity in SW620 cells, indicating that it is a formerly unknown SFK target in CRC cells. Conclusion Rapid identification of known and novel SFK targets in CRC cells is feasible with SH2 domain affinity chromatography. The elucidation of new SFK targets like Odin in epithelial cancer cells is expected to lead to novel insight into cancer cell signalling mechanisms and may also serve to indicate new biomarkers for monitoring tumor cell responses to drug treatments.

MicroRNA-429 induces tumorigenesis of human non-small cell lung cancer cells and targets multiple tumor suppressor genes

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Lang, Yaoguo; Xu, Shidong; Ma, Jianqun; Wu, Jun [Department of Thoracic Surgery, Harbin Medical University Cancer Hospital, 150 Haping Road, Harbin, Heilongjiang 150081 (China); Jin, Shi; Cao, Shoubo [Department of Medical Oncology, Harbin Medical University Cancer Hospital, 150 Haping Road, Harbin, Heilongjiang 150081 (China); Yu, Yan, E-mail: yuyan@hrbmu.edu.cn [Department of Medical Oncology, Harbin Medical University Cancer Hospital, 150 Haping Road, Harbin, Heilongjiang 150081 (China)

2014-07-18

Highlights: • MiR-429 expression is upregulated in non-small cell lung cancer (NSCLC). • MiR-429 inhibits PTEN, RASSF8 and TIMP2 expression. • MiR-429 promotes metastasis and proliferation. • We report important regulatory mechanisms involved in NSCLC progression. • MiR-429 is a potential therapeutic target and diagnostic marker. - Abstract: Lung cancer is the major cause of cancer death globally. MicroRNAs are evolutionally conserved small noncoding RNAs that are critical for the regulation of gene expression. Aberrant expression of microRNA (miRNA) has been implicated in cancer initiation and progression. In this study, we demonstrated that the expression of miR-429 are often upregulated in non-small cell lung cancer (NSCLC) compared with normal lung tissues, and its expression level is also increased in NSCLC cell lines compared with normal lung cells. Overexpression of miR-429 in A549 NSCLC cells significantly promoted cell proliferation, migration and invasion, whereas inhibition of miR-429 inhibits these effects. Furthermore, we demonstrated that miR-429 down-regulates PTEN, RASSF8 and TIMP2 expression by directly targeting the 3′-untranslated region of these target genes. Taken together, our results suggest that miR-429 plays an important role in promoting the proliferation and metastasis of NSCLC cells and is a potential target for NSCLC therapy.

Robotic Automation of In Vivo Two-Photon Targeted Whole-Cell Patch-Clamp Electrophysiology.

Science.gov (United States)

Annecchino, Luca A; Morris, Alexander R; Copeland, Caroline S; Agabi, Oshiorenoya E; Chadderton, Paul; Schultz, Simon R

2017-08-30

Whole-cell patch-clamp electrophysiological recording is a powerful technique for studying cellular function. While in vivo patch-clamp recording has recently benefited from automation, it is normally performed "blind," meaning that throughput for sampling some genetically or morphologically defined cell types is unacceptably low. One solution to this problem is to use two-photon microscopy to target fluorescently labeled neurons. Combining this with robotic automation is difficult, however, as micropipette penetration induces tissue deformation, moving target cells from their initial location. Here we describe a platform for automated two-photon targeted patch-clamp recording, which solves this problem by making use of a closed loop visual servo algorithm. Our system keeps the target cell in focus while iteratively adjusting the pipette approach trajectory to compensate for tissue motion. We demonstrate platform validation with patch-clamp recordings from a variety of cells in the mouse neocortex and cerebellum. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Intracellular Delivery of Nanobodies for Imaging of Target Proteins in Live Cells.

Science.gov (United States)

Röder, Ruth; Helma, Jonas; Preiß, Tobias; Rädler, Joachim O; Leonhardt, Heinrich; Wagner, Ernst

2017-01-01

Cytosolic delivery of nanobodies for molecular target binding and fluorescent labeling in living cells. Fluorescently labeled nanobodies were formulated with sixteen different sequence-defined oligoaminoamides. The delivery of formulated anti-GFP nanobodies into different target protein-containing HeLa cell lines was investigated by flow cytometry and fluorescence microscopy. Nanoparticle formation was analyzed by fluorescence correlation spectroscopy. The initial oligomer screen identified two cationizable four-arm structured oligomers (734, 735) which mediate intracellular nanobody delivery in a receptor-independent (734) or folate receptor facilitated (735) process. The presence of disulfide-forming cysteines in the oligomers was found critical for the formation of stable protein nanoparticles of around 20 nm diameter. Delivery of labeled GFP nanobodies or lamin nanobodies to their cellular targets was demonstrated by fluorescence microscopy including time lapse studies. Two sequence-defined oligoaminoamides with or without folate for receptor targeting were identified as effective carriers for intracellular nanobody delivery, as exemplified by GFP or lamin binding in living cells. Due to the conserved nanobody core structure, the methods should be applicable for a broad range of nanobodies directed to different intracellular targets.

Targeting melanoma stem cells with the Vitamin E derivative δ-tocotrienol.

Science.gov (United States)

Marzagalli, Monica; Moretti, Roberta Manuela; Messi, Elio; Marelli, Marina Montagnani; Fontana, Fabrizio; Anastasia, Alessia; Bani, Maria Rosa; Beretta, Giangiacomo; Limonta, Patrizia

2018-01-12

The prognosis of metastatic melanoma is very poor, due to the development of drug resistance. Cancer stem cells (CSCs) may play a crucial role in this mechanism, contributing to disease relapse. We first characterized CSCs in melanoma cell lines. We observed that A375 (but not BLM) cells are able to form melanospheres and show CSCs traits: expression of the pluripotency markers SOX2 and KLF4, higher invasiveness and tumor formation capability in vivo with respect to parental adherent cells. We also showed that a subpopulation of autofluorescent cells expressing the ABCG2 stem cell marker is present in the A375 spheroid culture. Based on these data, we investigated whether δ-TT might target melanoma CSCs. We demonstrated that melanoma cells escaping the antitumor activity of δ-TT are completely devoid of the ability to form melanospheres. In contrast, cells that escaped vemurafenib treatment show a higher ability to form melanospheres than control cells. δ-TT also induced disaggregation of A375 melanospheres and reduced the spheroidogenic ability of sphere-derived cells, reducing the expression of the ABCG2 marker. These data demonstrate that δ-TT exerts its antitumor activity by targeting the CSC subpopulation of A375 melanoma cells and might represent a novel chemopreventive/therapeutic strategy against melanoma.

Low-Dose Irradiation Enhances Gene Targeting in Human Pluripotent Stem Cells.

Science.gov (United States)

Hatada, Seigo; Subramanian, Aparna; Mandefro, Berhan; Ren, Songyang; Kim, Ho Won; Tang, Jie; Funari, Vincent; Baloh, Robert H; Sareen, Dhruv; Arumugaswami, Vaithilingaraja; Svendsen, Clive N

2015-09-01

Human pluripotent stem cells (hPSCs) are now being used for both disease modeling and cell therapy; however, efficient homologous recombination (HR) is often crucial to develop isogenic control or reporter lines. We showed that limited low-dose irradiation (LDI) using either γ-ray or x-ray exposure (0.4 Gy) significantly enhanced HR frequency, possibly through induction of DNA repair/recombination machinery including ataxia-telangiectasia mutated, histone H2A.X and RAD51 proteins. LDI could also increase HR efficiency by more than 30-fold when combined with the targeting tools zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats. Whole-exome sequencing confirmed that the LDI administered to hPSCs did not induce gross genomic alterations or affect cellular viability. Irradiated and targeted lines were karyotypically normal and made all differentiated lineages that continued to express green fluorescent protein targeted at the AAVS1 locus. This simple method allows higher throughput of new, targeted hPSC lines that are crucial to expand the use of disease modeling and to develop novel avenues of cell therapy. The simple and relevant technique described in this report uses a low level of radiation to increase desired gene modifications in human pluripotent stem cells by an order of magnitude. This higher efficiency permits greater throughput with reduced time and cost. The low level of radiation also greatly increased the recombination frequency when combined with developed engineered nucleases. Critically, the radiation did not lead to increases in DNA mutations or to reductions in overall cellular viability. This novel technique enables not only the rapid production of disease models using human stem cells but also the possibility of treating genetically based diseases by correcting patient-derived cells. ©AlphaMed Press.

Mitochondrial targets of photodynamic therapy and their contribution to cell death

Science.gov (United States)

Oleinick, Nancy L.; Usuda, Jitsuo; Xue, Liang-yan; Azizuddin, Kashif; Chiu, Song-mao; Lam, Minh C.; Morris, Rachel L.; Nieminen, Anna-Liisa

2002-06-01

In response to photodynamic therapy (PDT), many cells in culture or within experimental tumors are eliminated by apoptosis. PDT with photosensitizers that localize in or target mitochondria, such as the phthalocyanine Pc 4, causes prompt release of cytochrome c into the cytoplasm and activation of caspases-9 and -3, among other caspases, that are responsible for initiating cell degradation. Some cells appear resistant to apoptosis after PDT; however, if they have sustained sufficient damage, they will die by a necrotic process or through a different apoptotic pathway. In the case of PDT, the distinction between apoptosis and necrosis may be less important than the mechanism that triggers both processes, since critical lethal damage appears to occur during treatment and does not require the major steps in apoptosis to be expressed. We earlier showed, for example, that human breast cancer MCF-7 cells that lack caspase-3 are resistant to the induction of apoptosis by PDT, but are just as sensitive to the loss of clonogenicity as MCF-7 cells stably expressing transfected procaspase-3. Many photosensitizers that target mitochondria specifically attack the anti-apoptotic protein Bcl-2, generating a variety of crosslinked and cleaved photoproducts. Recent evidence suggests that the closely related protein Bcl-xL is also a target of Pc 4-PDT. Transient transfection of an expression vector encoding deletion mutants of Bcl-2 have identified the critical sensitive site in the protein that is required for photodamage. This region contains two alpha helices that form a secondary membrane anchorage site and are thought to be responsible for pore formation by Bcl-2. As specific protein targets are identified, we are becoming better able to model the critical events in PDT-induced cell death.

MiRNA-125a-5p inhibits glioblastoma cell proliferation and promotes cell differentiation by targeting TAZ

Energy Technology Data Exchange (ETDEWEB)

Yuan, Jian; Xiao, Gelei [Department of Neurosurgery, Xiangya Hospital, Central South University, Changsha, Hunan 410008 (China); The Institute of Skull Base Surgery & Neuro-oncology at Hunan, Changsha, Hunan 410008 (China); Peng, Gang [Department of Neurosurgery, Xiangya Hospital, Central South University, Changsha, Hunan 410008 (China); Liu, Dingyang [Department of Neurosurgery, Xiangya Hospital, Central South University, Changsha, Hunan 410008 (China); The Institute of Skull Base Surgery & Neuro-oncology at Hunan, Changsha, Hunan 410008 (China); Wang, Zeyou [Cancer Research Institute, Central South University, Changsha, Hunan 410008 (China); Liao, Yiwei; Liu, Qing [Department of Neurosurgery, Xiangya Hospital, Central South University, Changsha, Hunan 410008 (China); The Institute of Skull Base Surgery & Neuro-oncology at Hunan, Changsha, Hunan 410008 (China); Wu, Minghua [The Institute of Skull Base Surgery & Neuro-oncology at Hunan, Changsha, Hunan 410008 (China); Cancer Research Institute, Central South University, Changsha, Hunan 410008 (China); Yuan, Xianrui, E-mail: xry69@163.com [Department of Neurosurgery, Xiangya Hospital, Central South University, Changsha, Hunan 410008 (China); The Institute of Skull Base Surgery & Neuro-oncology at Hunan, Changsha, Hunan 410008 (China)

2015-02-06

Highlights: • Expression of miR-125a-5p is inversely correlated with that of TAZ in glioma cells. • MiR-125a-5p represses TAZ expression in glioma cells. • MiR-125a-5p directly targets the 3′ UTR of TAZ mRNA and promotes its degradation. • MiR-125a-5p represses CTGF and survivin via TAZ, and inhibits glioma cell growth. • MiR-125a-5p inhibits the stem cell features of HFU-251 MG cells. - Abstract: Glioblastoma (GBM) is the most lethal brain tumor due to the resistance to conventional therapies, such as radiotherapy and chemotherapy. TAZ, an important mediator of the Hippo pathway, was found to be up-regulated in diverse cancers, including in GBM, and plays important roles in tumor initiation and progression. However, little is known about the regulation of TAZ expression in tumors. In this study, we found that miR-125a-5p is an important regulator of TAZ in glioma cells by directly targeting the TAZ 3′ UTR. MiR-125a-5p levels are inversely correlated with that of TAZ in normal astrocytes and a panel of glioma cell lines. MiR-125a-5p represses the expression of TAZ target genes, including CTGF and survivin, and inhibits cell proliferation and induces the differentiation of GBM cells; whereas over-expression of TAZ rescues the effects of miR-125a-5p. This study revealed a mechanism for TAZ deregulation in glioma cells, and also demonstrated a tumor suppressor role of miR-125a-5p in glioblastoma cells.

MiRNA-125a-5p inhibits glioblastoma cell proliferation and promotes cell differentiation by targeting TAZ

International Nuclear Information System (INIS)

Yuan, Jian; Xiao, Gelei; Peng, Gang; Liu, Dingyang; Wang, Zeyou; Liao, Yiwei; Liu, Qing; Wu, Minghua; Yuan, Xianrui

2015-01-01

Highlights: • Expression of miR-125a-5p is inversely correlated with that of TAZ in glioma cells. • MiR-125a-5p represses TAZ expression in glioma cells. • MiR-125a-5p directly targets the 3′ UTR of TAZ mRNA and promotes its degradation. • MiR-125a-5p represses CTGF and survivin via TAZ, and inhibits glioma cell growth. • MiR-125a-5p inhibits the stem cell features of HFU-251 MG cells. - Abstract: Glioblastoma (GBM) is the most lethal brain tumor due to the resistance to conventional therapies, such as radiotherapy and chemotherapy. TAZ, an important mediator of the Hippo pathway, was found to be up-regulated in diverse cancers, including in GBM, and plays important roles in tumor initiation and progression. However, little is known about the regulation of TAZ expression in tumors. In this study, we found that miR-125a-5p is an important regulator of TAZ in glioma cells by directly targeting the TAZ 3′ UTR. MiR-125a-5p levels are inversely correlated with that of TAZ in normal astrocytes and a panel of glioma cell lines. MiR-125a-5p represses the expression of TAZ target genes, including CTGF and survivin, and inhibits cell proliferation and induces the differentiation of GBM cells; whereas over-expression of TAZ rescues the effects of miR-125a-5p. This study revealed a mechanism for TAZ deregulation in glioma cells, and also demonstrated a tumor suppressor role of miR-125a-5p in glioblastoma cells

Novel targets for sensitizing breast cancer cells to TRAIL-induced apoptosis with siRNA delivery.

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Thapa, Bindu; Bahadur Kc, Remant; Uludağ, Hasan

2018-02-01

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in variety of cancer cells without affecting most normal cells, which makes it a promising agent for cancer therapy. However, TRAIL therapy is clinically not effective due to resistance induction. To identify novel regulators of TRAIL that can aid in therapy, protein targets whose silencing sensitized breast cancer cells against TRAIL were screened with an siRNA library against 446 human apoptosis-related proteins in MDA-231 cells. Using a cationic lipopolymer (PEI-αLA) for delivery of library members, 16 siRNAs were identified that sensitized the TRAIL-induced death in MDA-231 cells. The siRNAs targeting BCL2L12 and SOD1 were further evaluated based on the novelty and their ability to sensitize TRAIL induced cell death. Silencing both targets sensitized TRAIL-mediated cell death in MDA-231 cells as well as TRAIL resistant breast cancer cells, MCF-7. Combination of TRAIL and siRNA silencing BCL2L12 had no effect in normal human umbilical vein cells and human bone marrow stromal cell. The silencing of BCL2L12 and SOD1 enhanced TRAIL-mediated apoptosis in MDA-231 cells via synergistically activating capsase-3 activity. Hence, here we report siRNAs targeting BCL2L12 and SOD1 as a novel regulator of TRAIL-induced cell death in breast cancer cells, providing a new approach for enhancing TRAIL therapy for breast cancer. The combination of siRNA targeting BCL2L12 and TRAIL can be a highly effective synergistic pair in breast cancer cells with minimal effect on the non-transformed cells. © 2017 UICC.

Dendritic cell based PSMA immunotherapy for prostate cancer using a CD40-targeted adenovirus vector.

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Briana Jill Williams

Full Text Available Human prostate tumor vaccine and gene therapy trials using ex vivo methods to prime dendritic cells (DCs with prostate specific membrane antigen (PSMA have been somewhat successful, but to date the lengthy ex vivo manipulation of DCs has limited the widespread clinical utility of this approach. Our goal was to improve upon cancer vaccination with tumor antigens by delivering PSMA via a CD40-targeted adenovirus vector directly to DCs as an efficient means for activation and antigen presentation to T-cells. To test this approach, we developed a mouse model of prostate cancer by generating clonal derivatives of the mouse RM-1 prostate cancer cell line expressing human PSMA (RM-1-PSMA cells. To maximize antigen presentation in target cells, both MHC class I and TAP protein expression was induced in RM-1 cells by transduction with an Ad vector expressing interferon-gamma (Ad5-IFNγ. Administering DCs infected ex vivo with CD40-targeted Ad5-huPSMA, as well as direct intraperitoneal injection of the vector, resulted in high levels of tumor-specific CTL responses against RM-1-PSMA cells pretreated with Ad5-IFNγ as target cells. CD40 targeting significantly improved the therapeutic antitumor efficacy of Ad5-huPSMA encoding PSMA when combined with Ad5-IFNγ in the RM-1-PSMA model. These results suggest that a CD-targeted adenovirus delivering PSMA may be effective clinically for prostate cancer immunotherapy.

PSMA-targeted bispecific Fab conjugates that engage T cells.

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Patterson, James T; Isaacson, Jason; Kerwin, Lisa; Atassi, Ghazi; Duggal, Rohit; Bresson, Damien; Zhu, Tong; Zhou, Heyue; Fu, Yanwen; Kaufmann, Gunnar F

2017-12-15

Bioconjugate formats provide alternative strategies for antigen targeting with bispecific antibodies. Here, PSMA-targeted Fab conjugates were generated using different bispecific formats. Interchain disulfide bridging of an αCD3 Fab enabled installation of either the PSMA-targeting small molecule DUPA (SynFab) or the attachment of an αPSMA Fab (BisFab) by covalent linkage. Optimization of the reducing conditions was critical for selective interchain disulfide reduction and good bioconjugate yield. Activity of αPSMA/CD3 Fab conjugates was tested by in vitro cytotoxicity assays using prostate cancer cell lines. Both bispecific formats demonstrated excellent potency and antigen selectivity. Copyright © 2017. Published by Elsevier Ltd.

Pretargeting vs. direct targeting of human betalox5 islet cells subcutaneously implanted in mice using an anti-human islet cell antibody

International Nuclear Information System (INIS)

Liu Guozheng; Dou Shuping; Akalin, Ali; Rusckowski, Mary; Streeter, Philip R.; Shultz, Leonard D.; Greiner, Dale L.

2012-01-01

Introduction: We previously demonstrated MORF/cMORF pretargeting of human islets and betalox 5 cells (a human beta cell line) transplanted subcutaneously in mice with the anti-human islet antibody, HPi1. We now compare pretargeting with direct targeting in the beta cell transplant model to evaluate the degree to which target/non-target (T/NT) ratios may be improved by pretargeting. Methods: Specific binding of an anti-human islet antibody HPi1 to the beta cells transplanted subcutaneously in mice was examined against a negative control antibody. We then compared pretargeting by MORF-HPi1 plus 111 In-labeled cMORF to direct targeting by 111 In-labeled HPi1. Results: HPi1 binding to betalox5 human cells in the transplant was shown by immunofluorescence. Normal organ 111 In backgrounds by pretargeting were always lower, although target accumulations were similar. More importantly, the transplant to pancreas and liver ratios was, respectively, 26 and 10 by pretargeting as compared to 9 and 0.6 by direct targeting. Conclusions: Pretargeting greatly improves the T/NT ratios, and based on the estimated endocrine to exocrine ratio within a pancreas, pretargeting may be approaching the sensitivity required for successful imaging of human islets within this organ.

CD19-Targeted CAR T Cells as Novel Cancer Immunotherapy for Relapsed or Refractory B-Cell Acute Lymphoblastic Leukemia

OpenAIRE

Davila, Marco L.; Brentjens, Renier J.

2016-01-01

Immunotherapy has demonstrated significant potential for the treatment of patients with chemotherapy-resistant hematologic malignancies and solid tumors. One type of immunotherapy involves the adoptive transfer of T cells that have been genetically modified with a chimeric antigen receptor (CAR) to target a tumor. These hybrid proteins are composed of the antigen-binding domains of an antibody fused to T-cell receptor signaling machinery. CAR T cells that target CD19 recently have made the ju...

Cyclophilin A secreted from fibroblast-like synoviocytes is involved in the induction of CD147 expression in macrophages of mice with collagen-induced arthritis

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Nishioku Tsuyoshi

2012-11-01

Full Text Available Abstract Background Cyclophilin A (CypA, a member of the immunophilin family, is a ubiquitously distributed intracellular protein. Recent studies have shown that CypA is secreted by cells in response to inflammatory stimuli. Elevated levels of extracellular CypA and its receptor, CD147 have been detected in the synovium of patients with RA. However, the precise process of interaction between CypA and CD147 in the development of RA remains unclear. This study aimed to investigate CypA secretion from fibroblast-like synoviocytes (FLS isolated from mice with collagen-induced arthritis (CIA and CypA-induced CD147 expression in mouse macrophages. Findings CIA was induced by immunization with type II collagen in mice. The expression and localization of CypA and CD147 was investigated by immunoblotting and immunostaining. Both CypA and CD147 were highly expressed in the joints of CIA mice. CD147 was expressed in the infiltrated macrophages in the synovium of CIA mice. In vitro, spontaneous CypA secretion from FLS was detected and this secretion was increased by stimulation with lipopolysaccharide. CypA markedly increased CD147 levels in macrophages. Conclusions These findings suggest that an interaction in the synovial joints between extracellular CypA and CD147 expressed by macrophages may be involved in the mechanisms underlying the development of arthritis.

Engineered Proteins Program Mammalian Cells to Target Inflammatory Disease Sites.

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Qudrat, Anam; Mosabbir, Abdullah Al; Truong, Kevin

2017-06-22

Disease sites in atherosclerosis and cancer feature cell masses (e.g., plaques/tumors), a low pH extracellular microenvironment, and various pro-inflammatory cytokines such as tumor necrosis factor α (TNFα). The ability to engineer a cell to seek TNFα sources allows for targeted therapeutic delivery. To accomplish this, here we introduced a system of proteins: an engineered TNFα chimeric receptor (named TNFR1chi), a previously engineered Ca 2+ -activated RhoA (named CaRQ), vesicular stomatitis virus glycoprotein G (VSVG), and thymidine kinase. Upon binding TNFα, TNFR1chi generates a Ca 2+ signal that in turn activates CaRQ-mediated non-apoptotic blebs that allow migration toward the TNFα source. Next, the addition of VSVG, upon low pH induction, causes membrane fusion of the engineered and TNFα source cells. Finally, after ganciclovir treatment cells undergo death via the thymidine kinase suicide mechanism. Hence, we assembled a system of proteins that forms the basis of engineering a cell to target inflammatory disease sites characterized by TNFα secretion and a low-pH microenvironment. Copyright © 2017 Elsevier Ltd. All rights reserved.

MicroRNA-424 suppresses estradiol-induced cell proliferation via targeting GPER in endometrial cancer cells.

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Zhang, H; Wang, X; Chen, Z; Wang, W

2015-11-30

Endometrial carcinoma (EC) is the most common gynecologic malignancy with increasing morbidity in recent years. MicroRNAs (miRNAs), a type of non-coding RNA, have been proven to be critical in the process of tumorigenesis. miR-424 has been reported to play a protective role in various type of cancer including endometrial carcinoma. It has been reported that high levels of estrogen increase morbidity of EC by promoting cell growth ability. The current research was designed to delineate the mechanism of miR-424 in regulating E2 (17β-estradiol)-induced cell proliferation in endometrial cancer. In this study, we confirmed that cell proliferation is increased significantly in E2-treated endometrial cancer cell lines. Moreover, miR-424 overexpression dramatically decreased E2-induced cell proliferation, indicating a pivotal role in endometrial cancer cell growth. In addition, the results suggest that miR-424 up-regulation inactivated the PI3K/AKT signaling, which was mediated by G-protein-coupled estrogen receptor-1 (GPER) in endometrial cancer. Furthermore, the luciferase report confirmed the targeting reaction between miR-424 and GPER. After transfection with the GPER overexpression vector into E2-induced endometrial cancer cells, we found that GPER significantly attenuated the inhibition effect of miR-424 in E2-induced cell growth in EC. Taken together, our study suggests that increased miR-424 suppresses E2-induced cell growth, and providing a potential therapeutic target for estrogen-associated endometrial carcinoma.

How immunoglobulin G antibodies kill target cells: revisiting an old paradigm.

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Biburger, Markus; Lux, Anja; Nimmerjahn, Falk

2014-01-01

The capacity of immunoglobulin G (IgG) antibodies to eliminate virtually any target cell has resulted in the widespread introduction of cytotoxic antibodies into the clinic in settings of cancer therapy, autoimmunity, and transplantation, for example. More recently, it has become apparent that also the protection from viral infection via IgG antibodies may require cytotoxic effector functions, suggesting that antibody-dependent cellular cytotoxicity (ADCC) directed against malignant or virally infected cells is one of the most essential effector mechanisms triggered by IgG antibodies to protect the host. A detailed understanding of the underlying molecular and cellular pathways is critical, therefore, to make full use of this antibody effector function. Several studies over the last years have provided novel insights into the effector pathways and innate immune effector cells responsible for ADCC reactions. One of the most notable outcomes of many of these reports is that cells of the mononuclear phagocytic system rather than natural killer cells are critical for removal of IgG opsonized target cells in vivo. © 2014 Elsevier Inc. All rights reserved.

Fuel cell and hydrogen R and D targets and funding : a comparative analysis

International Nuclear Information System (INIS)

Adamson, K.A.; Jollie, D.; Baker, A.

2005-01-01

Substantial research and development is needed if fuel cells and hydrogen are to become a mass market reality. Setting research and development targets are central to the long term development of the market. An overview of fuel cell research in the United States, the European Union, and parts of Asia was presented. Research and development targets were analyzed, as well as funding levels for fuel cells and hydrogen. The time frames of targets were considered, as well as the levels of ambition and overall program goals of various countries. Funding barriers and challenges were also considered. It was noted that some governments, such as Japan and Korea, have set a number of very ambitious, highly focused long term targets with substantial funding. The European Union has taken a more integrated approach, wrapping fundamental research and development into large integrated projects which run in combination with a number of other market aspects, such as public acceptance and roadmapping. The United States has a number of long term programmes and targets, but levels of funding are set annually with the passing of each year's Fiscal Budget. The overall goal of the paper was to provide a clearer picture of regional fuel cell research in order to discover areas for potential international collaboration

Targeting neuroblastoma stem cells with retinoic acid and proteasome inhibitor.

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Barbara Hämmerle

Full Text Available Neuroblastma cell lines contain a side-population of cells which express stemness markers. These stem-like cells may represent the potential underlying mechanism for resistance to conventional therapy and recurrence of neuroblastoma in patients.To develop novel strategies for targeting the side-population of neurobastomas, we analyzed the effects of 13-cis-retinoic acid (RA combined with the proteasome inhibitor MG132. The short-term action of the treatment was compared with effects after a 5-day recovery period during which both chemicals were withdrawn. RA induced growth arrest and differentiation of SH-SY5Y and SK-N-BE(2 neuroblastoma cell lines. Inhibition of the proteasome caused apoptosis in both cell lines, thus, revealing the critical role of this pathway in the regulated degradation of proteins involved in neuroblastoma proliferation and survival. The combination of RA with MG132 induced apoptosis in a dose-dependent manner, in addition to promoting G2/M arrest in treated cultures. Interestingly, expression of stem cell markers such as Nestin, Sox2, and Oct4 were reduced after the recovery period of combined treatment as compared with untreated cells or treated cells with either compound alone. Consistent with this, neurosphere formation was significantly impaired by the combined treatment of RA and MG132.Given that stem-like cells are associated with resistant to conventional therapy and are thought to be responsible for relapse, our results suggest that dual therapy of RA and proteasome inhibitor might be beneficial for targeting the side-population of cells associated residual disease in high-risk neuroblastoma.

Targeting Cellular Calcium Homeostasis to Prevent Cytokine-Mediated Beta Cell Death.

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Clark, Amy L; Kanekura, Kohsuke; Lavagnino, Zeno; Spears, Larry D; Abreu, Damien; Mahadevan, Jana; Yagi, Takuya; Semenkovich, Clay F; Piston, David W; Urano, Fumihiko

2017-07-17

Pro-inflammatory cytokines are important mediators of islet inflammation, leading to beta cell death in type 1 diabetes. Although alterations in both endoplasmic reticulum (ER) and cytosolic free calcium levels are known to play a role in cytokine-mediated beta cell death, there are currently no treatments targeting cellular calcium homeostasis to combat type 1 diabetes. Here we show that modulation of cellular calcium homeostasis can mitigate cytokine- and ER stress-mediated beta cell death. The calcium modulating compounds, dantrolene and sitagliptin, both prevent cytokine and ER stress-induced activation of the pro-apoptotic calcium-dependent enzyme, calpain, and partly suppress beta cell death in INS1E cells and human primary islets. These agents are also able to restore cytokine-mediated suppression of functional ER calcium release. In addition, sitagliptin preserves function of the ER calcium pump, sarco-endoplasmic reticulum Ca 2+ -ATPase (SERCA), and decreases levels of the pro-apoptotic protein thioredoxin-interacting protein (TXNIP). Supporting the role of TXNIP in cytokine-mediated cell death, knock down of TXNIP in INS1-E cells prevents cytokine-mediated beta cell death. Our findings demonstrate that modulation of dynamic cellular calcium homeostasis and TXNIP suppression present viable pharmacologic targets to prevent cytokine-mediated beta cell loss in diabetes.

Chlorin e6 Conjugated Interleukin-6 Receptor Aptamers Selectively Kill Target Cells Upon Irradiation

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Sven Kruspe

2014-01-01

Full Text Available Photodynamic therapy (PDT uses the therapeutic properties of light in combination with certain chemicals, called photosensitizers, to successfully treat brain, breast, prostate, and skin cancers. To improve PDT, current research focuses on the development of photosensitizers to specifically target cancer cells. In the past few years, aptamers have been developed to directly deliver cargo molecules into target cells. We conjugated the photosensitizer chlorin e6 (ce6 with a human interleukin-6 receptor (IL-6R binding RNA aptamer, AIR-3A yielding AIR-3A-ce6 for application in high efficient PDT. AIR-3A-ce6 was rapidly and specifically internalized by IL-6R presenting (IL-6R+ cells. Upon light irradiation, targeted cells were selectively killed, while free ce6 did not show any toxic effect. Cells lacking the IL-6R were also not affected by AIR-3A-ce6. With this approach, we improved the target specificity of ce6-mediated PDT. In the future, other tumor-specific aptamers might be used to selectively localize photosensitizers into cells of interest and improve the efficacy and specificity of PDT in cancer and other diseases.

Lipoproteins tethered dendrimeric nanoconstructs for effective targeting to cancer cells

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Jain, Anupriya; Jain, Keerti; Mehra, Neelesh Kumar; Jain, N. K.

2013-10-01

In the present investigation, poly (propylene imine) dendrimers up to fifth generation (PPI G5.0) were synthesized using ethylene diamine and acrylonitrile. Lipoproteins (high-density lipoprotein; HDL and low-density lipoprotein; LDL) were isolated from human plasma by discontinuous density gradient ultracentrifugation, characterized and tethered to G5.0 PPI dendrimers to construct LDL- and HDL-conjugated dendrimeric nanoconstructs for tumor-specific delivery of docetaxel. Developed formulations showed sustained release characteristics in in vitro drug release and in vivo pharmacokinetic studies. The cancer targeting potential of lipoprotein coupled dendrimers was investigated by ex vivo cytotoxicity and cell uptake studies using human hepatocellular carcinoma cell lines (HepG2 cells) and biodistribution studies in albino rats of Sprague-Dawley strain. Lipoprotein anchored dendrimeric nanoconstructs showed significant uptake by cancer cells as well as higher biodistribution of docetaxel to liver and spleen. It is concluded that these precisely synthesized engineered dendrimeric nanoconstructs could serve as promising drug carrier for fighting with the fatal disease, i.e., cancer, attributed to their defined targeting and therapeutic potential.

Lipoproteins tethered dendrimeric nanoconstructs for effective targeting to cancer cells

Energy Technology Data Exchange (ETDEWEB)

Jain, Anupriya; Jain, Keerti, E-mail: keertijain02@gmail.com; Mehra, Neelesh Kumar, E-mail: neelesh81mph@gmail.com; Jain, N. K., E-mail: dr.jnarendr@gmail.com [Dr. H. S. Gour University, Pharmaceutics Research Laboratory, Department of Pharmaceutical Sciences (India)

2013-10-15

In the present investigation, poly (propylene imine) dendrimers up to fifth generation (PPI G5.0) were synthesized using ethylene diamine and acrylonitrile. Lipoproteins (high-density lipoprotein; HDL and low-density lipoprotein; LDL) were isolated from human plasma by discontinuous density gradient ultracentrifugation, characterized and tethered to G5.0 PPI dendrimers to construct LDL- and HDL-conjugated dendrimeric nanoconstructs for tumor-specific delivery of docetaxel. Developed formulations showed sustained release characteristics in in vitro drug release and in vivo pharmacokinetic studies. The cancer targeting potential of lipoprotein coupled dendrimers was investigated by ex vivo cytotoxicity and cell uptake studies using human hepatocellular carcinoma cell lines (HepG2 cells) and biodistribution studies in albino rats of Sprague-Dawley strain. Lipoprotein anchored dendrimeric nanoconstructs showed significant uptake by cancer cells as well as higher biodistribution of docetaxel to liver and spleen. It is concluded that these precisely synthesized engineered dendrimeric nanoconstructs could serve as promising drug carrier for fighting with the fatal disease, i.e., cancer, attributed to their defined targeting and therapeutic potential.

Targeting Aberrant Glutathione Metabolism to Eradicate Human Acute Myelogenous Leukemia Cells*

Science.gov (United States)

Pei, Shanshan; Minhajuddin, Mohammad; Callahan, Kevin P.; Balys, Marlene; Ashton, John M.; Neering, Sarah J.; Lagadinou, Eleni D.; Corbett, Cheryl; Ye, Haobin; Liesveld, Jane L.; O'Dwyer, Kristen M.; Li, Zheng; Shi, Lei; Greninger, Patricia; Settleman, Jeffrey; Benes, Cyril; Hagen, Fred K.; Munger, Joshua; Crooks, Peter A.; Becker, Michael W.; Jordan, Craig T.

2013-01-01

The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular, primitive leukemia cells, often termed leukemia stem cells, are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34+) leukemic versus normal specimens. Our data indicate that CD34+ AML cells have elevated expression of multiple glutathione pathway regulatory proteins, presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation, CD34+ AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34+ cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise, we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34+ AML cells. Importantly, these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34+ cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism, which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1), as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism, an intrinsic property of primary human AML cells. PMID:24089526

Engineering Multi-Walled Carbon Nanotube Therapeutic Bionanofluids to Selectively Target Papillary Thyroid Cancer Cells.

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Idit Dotan

Full Text Available The incidence of papillary thyroid carcinoma (PTC has risen steadily over the past few decades as well as the recurrence rates. It has been proposed that targeted ablative physical therapy could be a therapeutic modality in thyroid cancer. Targeted bio-affinity functionalized multi-walled carbon nanotubes (BioNanofluid act locally, to efficiently convert external light energy to heat thereby specifically killing cancer cells. This may represent a promising new cancer therapeutic modality, advancing beyond conventional laser ablation and other nanoparticle approaches.Thyroid Stimulating Hormone Receptor (TSHR was selected as a target for PTC cells, due to its wide expression. Either TSHR antibodies or Thyrogen or purified TSH (Thyrotropin were chemically conjugated to our functionalized Bionanofluid. A diode laser system (532 nm was used to illuminate a PTC cell line for set exposure times. Cell death was assessed using Trypan Blue staining.TSHR-targeted BioNanofluids were capable of selectively ablating BCPAP, a TSHR-positive PTC cell line, while not TSHR-null NSC-34 cells. We determined that a 2:1 BCPAP cell:α-TSHR-BioNanofluid conjugate ratio and a 30 second laser exposure killed approximately 60% of the BCPAP cells, while 65% and >70% of cells were ablated using Thyrotropin- and Thyrogen-BioNanofluid conjugates, respectively. Furthermore, minimal non-targeted killing was observed using selective controls.A BioNanofluid platform offering a potential therapeutic path for papillary thyroid cancer has been investigated, with our in vitro results suggesting the development of a potent and rapid method of selective cancer cell killing. Therefore, BioNanofluid treatment emphasizes the need for new technology to treat patients with local recurrence and metastatic disease who are currently undergoing either re-operative neck explorations, repeated administration of radioactive iodine and as a last resort external beam radiation or chemotherapy, with

Cyclophilin D deficiency rescues Aβ-impaired PKA/CREB signaling and alleviates synaptic degeneration.

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Du, Heng; Guo, Lan; Wu, Xiaoping; Sosunov, Alexander A; McKhann, Guy M; Chen, John Xi; Yan, Shirley ShiDu

2014-12-01

The coexistence of neuronal mitochondrial pathology and synaptic dysfunction is an early pathological feature of Alzheimer's disease (AD). Cyclophilin D (CypD), an integral part of mitochondrial permeability transition pore (mPTP), is involved in amyloid beta (Aβ)-instigated mitochondrial dysfunction. Blockade of CypD prevents Aβ-induced mitochondrial malfunction and the consequent cognitive impairments. Here, we showed the elimination of reactive oxygen species (ROS) by antioxidants probucol or superoxide dismutase (SOD)/catalase blocks Aβ-mediated inactivation of protein kinase A (PKA)/cAMP regulatory-element-binding (CREB) signal transduction pathway and loss of synapse, suggesting the detrimental effects of oxidative stress on neuronal PKA/CREB activity. Notably, neurons lacking CypD significantly attenuate Aβ-induced ROS. Consequently, CypD-deficient neurons are resistant to Aβ-disrupted PKA/CREB signaling by increased PKA activity, phosphorylation of PKA catalytic subunit (PKA C), and CREB. In parallel, lack of CypD protects neurons from Aβ-induced loss of synapses and synaptic dysfunction. Furthermore, compared to the mAPP mice, CypD-deficient mAPP mice reveal less inactivation of PKA-CREB activity and increased synaptic density, attenuate abnormalities in dendritic spine maturation, and improve spontaneous synaptic activity. These findings provide new insights into a mechanism in the crosstalk between the CypD-dependent mitochondrial oxidative stress and signaling cascade, leading to synaptic injury, functioning through the PKA/CREB signal transduction pathway. Copyright © 2013 Elsevier B.V. All rights reserved.

Organelle targeting: third level of drug targeting

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Sakhrani NM

2013-07-01

Full Text Available Niraj M Sakhrani, Harish PadhDepartment of Cell and Molecular Biology, BV Patel Pharmaceutical Education and Research Development (PERD Centre, Gujarat, IndiaAbstract: Drug discovery and drug delivery are two main aspects for treatment of a variety of disorders. However, the real bottleneck associated with systemic drug administration is the lack of target-specific affinity toward a pathological site, resulting in systemic toxicity and innumerable other side effects as well as higher dosage requirement for efficacy. An attractive strategy to increase the therapeutic index of a drug is to specifically deliver the therapeutic molecule in its active form, not only into target tissue, nor even to target cells, but more importantly, into the targeted organelle, ie, to its intracellular therapeutic active site. This would ensure improved efficacy and minimize toxicity. Cancer chemotherapy today faces the major challenge of delivering chemotherapeutic drugs exclusively to tumor cells, while sparing normal proliferating cells. Nanoparticles play a crucial role by acting as a vehicle for delivery of drugs to target sites inside tumor cells. In this review, we spotlight active and passive targeting, followed by discussion of the importance of targeting to specific cell organelles and the potential role of cell-penetrating peptides. Finally, the discussion will address the strategies for drug/DNA targeting to lysosomes, mitochondria, nuclei and Golgi/endoplasmic reticulum.Keywords: intracellular drug delivery, cancer chemotherapy, therapeutic index, cell penetrating peptides

Magnetic targeting as a strategy to enhance therapeutic effects of mesenchymal stromal cells.

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Silva, Luisa H A; Cruz, Fernanda F; Morales, Marcelo M; Weiss, Daniel J; Rocco, Patricia R M

2017-03-09

Mesenchymal stromal cells (MSCs) have been extensively investigated in the field of regenerative medicine. It is known that the success of MSC-based therapies depends primarily on effective cell delivery to the target site where they will secrete vesicles and soluble factors with immunomodulatory and potentially reparative properties. However, some lesions are located in sites that are difficult to access, such as the heart, spinal cord, and joints. Additionally, low MSC retention at target sites makes cell therapy short-lasting and, therefore, less effective. In this context, the magnetic targeting technique has emerged as a new strategy to aid delivery, increase retention, and enhance the effects of MSCs. This approach uses magnetic nanoparticles to magnetize MSCs and static magnetic fields to guide them in vivo, thus promoting more focused, effective, and lasting retention of MSCs at the target site. In the present review, we discuss the magnetic targeting technique, its principles, and the materials most commonly used; we also discuss its potential for MSC enhancement, and safety concerns that should be addressed before it can be applied in clinical practice.

Emerging Therapeutic Strategies for Targeting Chronic Myeloid Leukemia Stem Cells

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Ahmad Hamad

2013-01-01

Full Text Available Chronic myeloid leukemia (CML is a clonal myeloproliferative disorder. Current targeted therapies designed to inhibit the tyrosine kinase activity of the BCR-ABL oncoprotein have made a significant breakthrough in the treatment of CML patients. However, CML remains a chronic disease that a patient must manage for life. Although tyrosine kinase inhibitors (TKI therapy has completely transformed the prognosis of CML, it has made the therapeutic management more complex. The interruption of TKI treatment results in early disease progression because it does not eliminate quiescent CML stem cells which remain a potential reservoir for disease relapse. This highlights the need to develop new therapeutic strategies for CML to achieve a permanent cure, and to allow TKI interruption. This review summarizes recent research done on alternative targeted therapies with a particular focus on some important signaling pathways (such as Alox5, Hedgehog, Wnt/b-catenin, autophagy, and PML that have the potential to target CML stem cells and potentially provide cure for CML.