Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P08758 (annexin V)
 9,383 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Plasma membrane binding of annexin V was used to detect and quantitate apoptotic cells induced by cytotoxic drug treatment in epithelial cell cultures. Chinese hamster ovary (CHO) cells were incubated for 2 h with the ID90 concentration of Cisplatin (20 microM), and 24, 48, 72, and 96 h later the unfixed cells were stained with fluorescein isothiocyanate (FITC)-conjugated annexin V. The fluorescence signal was quantitated by flow cytometry (FCM). During the early phase of the apoptotic response, the annexin V-binding frequency histograms showed two separate cell populations, a dimly and a brightly fluorescent one. At t = 96 h after drug incubation, when the process of apoptosis was completed, only the brightly fluorescent population was present. A dose-effect relationship could be established between the Cisplatin concentration used in the 2 h incubation and the binding of annexin V on the cell membrane, as estimated by FITC fluorescence. The dimly and brightly fluorescent populations were sorted on the basis of annexin V binding, and assayed for 1) DNA breaks by in situ nick translation assay and DNA content by DNA-propidium iodine fluorescence in a bivariate analysis, 2) membrane integrity by dye exclusion, and 3) morphological characteristics of apoptosis. The dimly fluorescent cell population appeared to represent apoptotic cells in the early phase of the death process, as demonstrated by intact cell membranes, normal DNA content, few DNA breaks, and chromatin condensation. The brightly fluorescent cells predominantly had sub-G1 DNA content, nuclear fragmentation, leaky cell membranes, and probably represent late apoptotic cells. These results demonstrate that cytotoxic drug-induced apoptosis can be quantitated by annexin V binding and that by using this assay early and late apoptotic cells can be identified.
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PMID:Quantification of apoptotic cells with fluorescein isothiocyanate-labeled annexin V in chinese hamster ovary cell cultures treated with cisplatin. 872 61

Expression of the apoptosis-associated proteins Bcl-2 and Bax was quantitated by flow cytometry (FCM) in chemosensitive testicular germ-cell tumor NT2 cells, and the results were compared with those obtained by Western blotting. NT2 cells were incubated with cisplatin (3.1 microM for 2 h at 37 degrees C), and 24, 48, and 72 h later were analyzed for induction of apoptosis, and for modulation of the expression of cell death suppressing protein Bcl-2, as well as cell death promoting protein Bax. Apoptosis was quantitated by binding of annexin V conjugated with fluorescein isothiocyanate (FITC) to the cell membrane. Cisplatin-treatment induced apoptosis in NT2 cells. The apoptotic cell population increased in time, and at t = 72 h after drug incubation, about 90% of cells that were present in the cell culture were apoptotic. Subsequently, we determined the expression of the Bcl-2 and Bax proteins by FCM and Western blotting before and after drug treatment. NT2 cells had low constitutive expression levels of Bcl-2 and elevated constitutive expression levels of Bax protein, as determined by both methods. At t = 24 h and 48 h after drug treatment, no changes were observed in the expression of the Bcl-2 protein, as quantitated by FCM and Western blotting. Also, the expression of the Bax protein had not changed, based on Western blotting. However, FCM revealed that in a specific subpopulation of drug-treated NT2 cells, Bax expression was increased. On the basis of forward and perpendicular light-scatter this subpopulation, which consisted of large, early apoptotic, swollen cells with increased internal complexity, was sorted, and showed abundant Bax protein by FCM and Western blotting. Our results demonstrate that the chemosensitivity of NT2 cells is probably due to a low intrinsic threshold for drug-induced apoptosis that is accompanied by overexpression of the death-promoting Bax protein during the early stages of the apoptotic process. We conclude that FCM is superior to Western blotting for the detection of heterogeneous expression of Bax in a given cell population.
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PMID:Bax upregulation is an early event in cisplatin-induced apoptosis in human testicular germ-cell tumor cell line NT2, as quantitated by flow cytometry. 904 Nov 17

Cisplatin is a potent chemotherapeutic agent that is used to treat many human malignancies. Unfortunately, in addition to side effects such as ototoxicity, anaphylaxis, and bone marrow suppression, a significant percentage of patients receiving cisplatin develop severe nephrotoxicity. Mitochondrial dysfunction that is mediated via the generation of reactive oxygen species has been implicated in the pathogenesis of cisplatin-induced renal injury. To address the mechanism, it was hypothesized that overexpression of antioxidant enzymes, such as mitochondria-localized manganese superoxide dismutase (MnSOD) or mitochondria-targeted catalase (mito-Cat), would be cytoprotective in cisplatin-induced cell injury. To this end, human MnSOD or a mito-Cat vector were stably transfected into human embryonic kidney 293 cells. Cells that overexpressed MnSOD exhibited significantly less cell rounding and detachment compared with both mito-Cat and vector controls after exposure to 20 microM cisplatin. Cell injury as assessed by DNA fragmentation and annexin V binding assays was significantly decreased in the cells that overexpressed MnSOD compared with vector alone and mito-Cat. In addition, elevated levels of MnSOD were strongly associated with increased clonogenic potential after cisplatin challenge. Thus, overexpression of MnSOD, and not catalase, protects against cisplatin-induced renal epithelial cell injury. These results demonstrate the importance of reactive oxygen species in the mechanism that underlies cisplatin-induced renal injury and specifically implicate the superoxide radical, and not hydrogen peroxide, as the mediator.
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PMID:Manganese superoxide dismutase attenuates Cisplatin-induced renal injury: importance of superoxide. 1172 37

The chemotherapeutic cisplatin causes renal dysfunction and renal proximal tubular cell (RPTC) apoptosis. The goal of these studies was to examine the role of p53, caspase 3, 8, and 9, and mitochondria in the signaling of cisplatin-induced apoptosis. Cisplatin (50 microM) produced time-dependent apoptosis in RPTCs, causing cell shrinkage, a 50-fold increase in caspase 3 activity, a 4-fold increase in phosphatidylserine externalization, and 5- and 15-fold increases in chromatin condensation and DNA hypoploidy, respectively. Mitochondrial membrane potential and ATP levels did not change at any time during cisplatin exposure. Caspase 8 and 9 activities also did not increase during treatment. Cisplatin increased nuclear p53 expression 4 h after treatment, preceding both caspase 3 activation and chromatin condensation. Treatment with the p53 inhibitor alpha-2-(2-imino-4,5,6,7-tetrahydrobenzothiazol-3-yl)-1-p-tolylethanone (PFT) before cisplatin exposure inhibited p53 nuclear expression at 4, 8, and 12 h and inhibited phosphatidylserine externalization and caspase 3 activation at 12 h. Neither DEVD-fmk nor ZVAD-fmk inhibited cisplatin-induced p53 nuclear expression. Both DEVD-fmk and ZVAD-fmk completely inhibited caspase 3 activity but, like PFT, partially inhibited cisplatin-induced chromatin condensation, annexin V labeling, and DNA hypoploidy after 24 h. These data demonstrate that at least 50% of cisplatin-induced apoptosis in RPTC is mediated by p53 and that p53 activates caspase 3 independently of either caspase 9 or 8 or mitochondrial dysfunction. Furthermore, 50% of cisplatin-induced RPTC apoptosis is independent of p53 and caspases 3, 8, and 9.
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PMID:Cisplatin-induced renal cell apoptosis: caspase 3-dependent and -independent pathways. 1206 94

It has been demonstrated recently that rabbit renal proximal tubule cells (RPTC) express a novel Ca(2+)-independent phospholipase A(2) (iPLA(2)) whose activity localizes to the endoplasmic reticulum (ER-iPLA(2)) and is similar to group VIB PLA(2). In this study, the expression of group VIB PLA(2) was examined and the role of ER-iPLA(2) in cisplatin-induced apoptosis was determined. Cisplatin induced both time- and concentration-dependent RPTC apoptosis as determined by p53 nuclear localization, annexin V staining, caspase 3 activity, and chromatin condensation. Inhibition of ER-iPLA(2) with bromoenol lactone (5 microM) reduced cisplatin-induced annexin V binding 40%, chromatin condensation 55%, and caspase 3 activity 42%, but had no effect on p53 nuclear localization. Treatment of RPTC with the protein kinase C stimulator phorbol 12-myristate 13-acetate increased the activity of ER-iPLA(2) 2-fold and increased cisplatin-induced RPTC apoptosis. These studies demonstrate that group VIB PLA(2) is expressed in RPTC and suggest that RPTC ER-iPLA(2) is the rabbit homolog of group VIB PLA(2). These data also demonstrate that ER-iPLA(2) acts downstream of p53 and upstream of caspase 3 to mediate cisplatin-induced RPTC apoptosis. Finally, ER-iPLA(2) seems to be regulated by protein kinase C.
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PMID:Role of an endoplasmic reticulum Ca2+-independent phospholipase A2 in cisplatin-induced renal cell apoptosis. 1463 37

We reported that 50% of cisplatin-induced apoptosis in primary cultures of rabbit renal proximal tubule cells (RPTC) proceeded via caspase-independent mechanisms. This study determined whether caspase-independent apoptosis, using multiple and diverse endpoints, could be produced by toxicants other than cisplatin and in cell models other than RPTC. Cisplatin, staurosporine, vincristine, and A23187 induced RPTC apoptosis after 24 h as indicated by 2- to 2.5-fold increases in annexin V and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining, and 2- to 10-fold increases in cell shrinkage. All toxicants induced 8- to 50-fold increases in caspase-3 activities, which were completely inhibited by the pan caspase inhibitor ZVAD-fmk. However, ZVAD-fmk only decreased cisplatin- and staurosporine-induced annexin V staining and cell shrinkage 30 to 50%, staurosporine-induced TUNEL staining 30%, and did not affect vincristine- or A23187-induced RPTC apoptosis. All toxicants tested induced apoptotic RPTC nuclear morphology. However, similar to its effect on annexin V and TUNEL staining, ZVAD-fmk only partially inhibited toxicant-induced apoptotic nuclear morphology. Cisplatin and staurosporine also induced annexin V staining in the human epithelial cancer cell lines Caki-1 (kidney carcinoma), A549 (lung carcinoma), A172 (glioblastoma), and murine lymphocytic leukemia L1210 cells. Pretreatment with ZVAD-fmk inhibited cisplatin-induced annexin V staining in Caki-1, A172, and A549 cells but had no affect in L1210 cells. Pretreatment with ZVAD-fmk did not decrease staurosporine-induced annexin V staining in Caki-1, A549, L1210, and A172 cells. These results suggest that a significant fraction of apoptosis induced by diverse toxicants in renal epithelial cells and in four different cancer cell lines is caspase-independent.
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PMID:Identification of caspase-independent apoptosis in epithelial and cancer cells. 1502 82

Cisplatin treatment induces extensive death of the proximal tubules in mice. We also demonstrated that treatment of immortalized mouse proximal tubule cells (TKPTS) with 25 microM cisplatin induces apoptotic death in vitro. Here, we demonstrate that members of the MAPKs such as ERK, JNK, and p38 are all activated after cisplatin treatment both in vivo and in vitro. Because MAPKs mediate cell survival and death, we studied their role in cisplatin-induced cell death in vitro. Apoptosis was confirmed by cell morphology, fluorescence-activated cell-sorting analysis, annexin V/propidium iodide binding, and caspase-3 activation in TKPTS cells. Inhibition of ERK, but not JNK or p38, abolished caspase-3 activation and apoptotic death, suggesting a prodeath role of ERK in cisplatin-induced injury. We also determined that cisplatin-induced ERK as well as caspase-3 activation are epidermal growth factor receptor (EGFR) and c-src dependent because inhibition of these genes inhibited ERK and caspase-3 activation and attenuated apoptotic death. These results suggest that caspase-3 mediates cisplatin-induced cell death in TKPTS cells via an EGFR/src/ERK-dependent pathway. We also suggest that the prodeath effect of ERK is injury type dependent because during oxidant injury, ERK supports survival rather than death in the same cells. We propose that injury-specific outcome diverges downstream from ERK in cisplatin- or H(2)O(2)-mediated cell survival and death.
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PMID:Cisplatin-induced cell death is EGFR/src/ERK signaling dependent in mouse proximal tubule cells. 1514 69

Cisplatin has a significant role in the treatment of selected human tumors including advanced melanoma, but new platinum compounds are still in focus of search for better properties. Modern drug design is often based on studies detecting the abilities of tested drug to induce apoptosis and disturb cell cycle. Aim of the study was to establish the influence of a platinum complex Pt-rib-1 on cell cycle and apoptosis occurrence in mouse melanoma B16 and ClS91 cells. Pt-rib-1 is a ribavirin derivative. previously characterized and described as cytotoxic to B16 and ClS91 mouse melanoma cells in vitro. The new platinum complex (Pt-rib-1); cis- dichloro (dimethylsulphoxide) (1- beta- D-ribofuranosyl- 1,2,4-triazolo -3- carboxyamide) platinum (II) was supplied. Cisdiaminodichloroplatinum (II), (cisplatin) was used in control groups. To detect apoptotic and necrotic cells, Annexin V- conjugated with fluorescein isothiocyanate (Annexin V-FITC, Immunotech) and propidium iodide (IP, Immunotech) were used. Apoptosis detection were done using fluorescence microscopy and flow cytometry. The total DNA content within the cell indicated phase of the cell cycle. DNA content was measured using flow cytometry. Values given represent the mean from three determinations. Results were presented as mean +/- standard deviation (SD). Statistical analysis was done using t-Student test. There were 70.4% of apoptotic cells in the ClS91 culture after 24 h incubation with Pt-rib-1 at a concentration of 2.04 x 10(-3) M (4 x IC50). In B16 group, 83.2 per cent of apoptotic cells was found after 24h incubation with Pt-rib-1 at high concentration (2.30 x 10(-3) M). A 24-h experiment shows a threshold at a concentration higher than 3 x IC50 responsible for apoptosis induction in B16 and ClS91 cells. After 48 h incubation with Pt-rib-1 the per cent of apoptotic cells increased gradually with rising concentrations of Pt-rib-1 up to a final concentration of 2.04 x 10(-3) M and 0.92 x 10(-3) M in ClS91 and B16 groups, respectively. Cell accumulation was observed in S phase after 48 h incubation with Pt-rib-1. The per cent of cells in S phase increased from 31 to 51.1% and 38.8 to 50.0% in ClS91 and B16 culture, respectively. There were no B16 and ClS91 cells in G2/M phase after incubation with higher concentrations of Pt-rib-1 (from 0.2 to 2.0 x 10(-5) M/dm3). Pt-rib-1 partially exhibits action of cisplatin. which has no specific influence on cell cycle and ribavirin. probably responsible for DNA synthesis delay.
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PMID:Influence of a novel platinum compound--cis-dichloro (dimethylsulphoxide) (1-beta-D-rybofuranosyl-1,2,4-triazolo-3-arboxyamide) platinum (II)--"Pt-rib-1"--on cell cycle and apoptosis in ClS91 and B16 mouse melanoma in vitro. 1525 56

Cisplatin (CDDP) is among the most widely used and most effective chemotherapeutic agent for many types of human cancer. Because killing cancer cells by chemotherapy is principally executed by apoptosis, a defective apoptotic program might acquire drug resistance. Flow cytometric Annexin V assay demonstrated that HEp-2 cells (human laryngeal cancer) were persistently resistant to CDDP as compared to HeLa cells (human uterine cervical cancer), despite the same histological type and wild-type p53 status. CDDP treatment caused steady induction of p53 protein in both cancer cell types, although it was more dramatic in CDDP-resistant HEp-2 cells, which was correlated well with p53 Ser15 phosphorylation, but not with the expression level of HPV type 18 E6 oncoprotein in these cells. Importantly, CDDP differently activated caspase cascades between HEp-2 and HeLa cells. CDDP activated the caspase-8 pathway through TNFR superfamily receptors such as Fas, but not caspase-9 in HeLa cells. On the other hand, the caspase-9 pathway was significantly activated in HEp-2 cells, although the activation of caspase-8 by CDDP was deficient. This different response to CDDP in caspase-8 activation was not related with the expression level of either Fas or FasL in these cells. We concluded from these results that loss of the caspase-8 activation pathway in HEp-2 cells was a possible mechanism for its resistance to CDDP-induced apoptosis. The caspase-8 pathway might play an important role in CDDP-induced apoptosis in HPV-positive human squamous cell carcinomas.
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PMID:Loss of caspase-8 activation pathway is a possible mechanism for CDDP resistance in human laryngeal squamous cell carcinoma, HEp-2 cells. 1528 75

We demonstrate that cells derived from primary cultures of rabbit proximal tubules (RPTC), human embryonic kidney (HEK293) and human kidney carcinomas (Caki-1) express microsomal Ca(2+)-independent phospholipase A(2) (iPLA(2)gamma) and cytosolic Ca(2+)-independent phospholipase A(2) (iPLA(2)beta). Inhibition of iPLA(2) activity in these cells using the iPLA(2) inhibitor bromoenol lactone (BEL) (0-5.0microM) for 24h did not induce cell death as determined by annexin V and propidium iodide (PI) staining. However, BEL treatment prior to cisplatin (50muM) or vincristine (2microM) exposure reduced apoptosis 30-50% in all cells tested (RPTC, HEK293 and Caki-1 cells). To identify the phospholipids altered during cell death electrospray ionization-mass spectrometry and lipidomic analysis of HEK293 and Caki-1 cells was performed. Cisplatin treatment reduced 14:0-16:0 and 16:0-16:0 phosphatidylcholine (PtdCho) 50% and 35%, respectively, in both cell lines, 16:0-18:2 PtdCho in Caki-1 cells and increased 16:1-22:6 plasmenylcholine (PlsCho). BEL treatment prior to cisplatin exposure further decreased 14:0-16:0 PtdCho, 16:0-16:1 PlsCho and 16:0-18:1 PlsCho in HEK293 cells, and inhibited cisplatin-induced increases in 16:1-22:6 PlsCho in Caki-1 cells. Treatment of cells with BEL prior to cisplatin exposure also increased the levels of several arachidonic containing phospholipids including 16:0-20:4, 18:1-20:4, and 18:0-20:4 PtdCho, compared to cisplatin only treated cells. These data demonstrate that inhibition of iPLA(2) protects against chemotherapeutic-induced cell death in multiple human renal cell models, identifies specific phospholipids whose levels are altered during cell death, and demonstrates that alterations in these phospholipids correlate to the protection against cell death in the presence of iPLA(2) inhibitors.
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PMID:The effect of inhibition of Ca2+-independent phospholipase A2 on chemotherapeutic-induced death and phospholipid profiles in renal cells. 1622 24


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