benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and pifithrin

Malignant gliomas are the leading cause of morbidity and mortality in brain and central nervous system tumors. Recently, casticin has drawn wide attention to its critical role in tumor progression. However, the effect of casticin on glioma remains undefined.. Following treatment with casticin, cell viability, apoptosis, and cell cycle arrest were examined in U251 glioma cells. Additionally, the involved molecular mechanism was assessed by western blotting and flow cytometry.. Casticin triggered an obvious dose-dependent decrease in U251, U87 and U373 glioma cell viability, and the growth inhibitory effect of casticin was correlated with cell cycle arrest and cell apoptosis. Further mechanistic analysis indicated that casticin induced G2/M phase arrest by attenuating the polymerization of tubulin. Furthermore, striking apoptosis was also confirmed, accompanied by the up-regulation of caspase-3, p53 and proapoptotic protein Bax. These effects were absent when the caspase inhibitor z-VAD-fmk or p53 inhibitor PFTα were applied, suggesting that casticin could trigger cell apoptosis in a caspase-3 and p53-dependent manner.. These findings provide a prominent insight into how casticin abrogates the pathogenesis of glioma, and support its potential clinical prospect for further development of anti-brain cancer therapy.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; bcl-2-Associated X Protein; Benzothiazoles; Caspase 3; Cell Cycle Checkpoints; Cell Line, Tumor; Flavonoids; G2 Phase Cell Cycle Checkpoints; Glioma; Humans; M Phase Cell Cycle Checkpoints; Toluene; Tubulin; Tumor Suppressor Protein p53; Up-Regulation

We demonstrate the role of p53-mediated caspase-2 activation in the mitochondrial release of apoptosis-inducing factor (AIF) in cisplatin-treated renal tubular epithelial cells. Gene silencing of AIF with its small interfering RNA (siRNA) suppressed cisplatin-induced AIF expression and provided a marked protection against cell death. Subcellular fractionation and immunofluorescence studies revealed cisplatin-induced translocation of AIF from the mitochondria to the nuclei. Pancaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone or p53 inhibitor pifithrin-alpha markedly prevented mitochondrial release of AIF, suggesting that caspases and p53 are involved in this release. Caspase-2 and -3 that were predominantly activated in response to cisplatin provided a unique model to study the role of these caspases in AIF release. Cisplatin-treated caspase-3 (+/+) and caspase-3 (-/-) cells exhibited similar AIF translocation to the nuclei, suggesting that caspase-3 does not affect AIF translocation, and thus, caspase-2 may be involved in the translocation. Caspase-2 inhibitor benzyloxycarbonyl-Val-Asp-Val-Ala-Asp-fluoromethylketone or down-regulation of caspase-2 by its siRNA significantly prevented translocation of AIF. Caspase-2 activation was a critical response from p53, which was markedly induced and phosphorylated in cisplatin-treated cells. Overexpression of p53 not only resulted in caspase-2 activation but also mitochondrial release of AIF. The p53 inhibitor pifithrin-alpha or p53 siRNA prevented both cisplatin-induced caspase-2 activation and mitochondrial release of AIF. Caspase-2 activation was dependent on the p53-responsive gene, PIDD, a death domain-containing protein that was induced by cisplatin in a p53-dependent manner. These results suggest that caspase-2 activation mediated by p53 is an important pathway involved in the mitochondrial release of AIF in response to cisplatin injury.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Agents; Apoptosis Inducing Factor; Benzothiazoles; Carrier Proteins; Caspase 2; Caspase 3; Caspase Inhibitors; Caspases; Cell Nucleus; Cisplatin; Cysteine Proteinase Inhibitors; Death Domain Receptor Signaling Adaptor Proteins; Enzyme Activation; Epithelial Cells; Flavoproteins; Kidney Tubules; Membrane Proteins; Mice; Mice, Knockout; Mitochondria; RNA, Small Interfering; Thiazoles; Toluene; Tumor Suppressor Protein p53

Oxidative stress has been implicated in the pathogenesis of stroke, traumatic brain injuries, and neurodegenerative diseases affecting both neuronal and glial cells in the central nervous system (CNS). The tumor suppressor protein p53 plays a pivotal function in neuronal apoptosis triggered by oxidative stress. We investigated the role of p53 and related molecular mechanisms that support oxidative stress-induced apoptosis in glia. For this purpose, we exposed C6 glioma cells and primary cultures of rat cortical astrocytes to an H(2)O(2)-induced oxidative stress protocol followed by a recovery period. We evaluated the effects of pifithrin-alpha (PF-alpha), which has been reported to protect neurons from ischemic insult by specifically inhibiting p53 DNA-binding activity. Strikingly, PF-alpha was unable to prevent oxidative stress-induced astrocyte apoptosis. We demonstrate that p53 is able to mediate an apoptotic response by direct signaling at mitochondria, despite its transcriptional activity. The z-VAD-fmk-sensitive apoptotic response requires a caspase-dependent MDM-2 degradation, leading to p53 mitochondrial targeting accompanied by cytochrome c release and nucleosomal fragmentation.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Astrocytes; Benzothiazoles; Blotting, Northern; Cell Survival; Cells, Cultured; Cytochromes c; Embryo, Mammalian; Fluorescent Antibody Technique; Genes, bcl-2; Hydrogen Peroxide; Microscopy, Confocal; Mitochondria; Models, Biological; Neuroprotective Agents; Nuclear Proteins; Nucleosomes; Oxidants; Oxidative Stress; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Rats; Recovery of Function; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thiazoles; Time Factors; Toluene; Tumor Suppressor Protein p53

The issue of p53 requirement for the caspase-mediated apoptosis induced by selenium in a cancer chemoprevention or chemotherapy context has not been critically addressed. We and others have shown that selenite induces apoptotic DNA laddering in the p53-mutant DU145 prostate cancer cells and the p53-null HL60 leukemia cells without the cleavage of poly(ADP-ribose) polymerase (PARP; i.e., caspase-independent apoptosis), whereas selenium compounds leading to the formation of methylselenol induce caspase-mediated apoptosis in these cells. Because selenite induces DNA single strand breaks, and because certain types of DNA damage activate p53, we investigated whether the human LNCaP prostate cancer cells, which contain a wild-type p53, execute selenite-induced apoptosis through caspase pathways. The results showed that exposure of LNCaP cells for 24 hours to lower micromolar concentrations of selenite led to DNA laddering, and to the cleavage of PARP and several pro-caspases. In contrast to this apoptosis sensitivity, LNCaP cells were rather resistant to similar concentrations of the methylselenol precursor methylseleninic acid. Selenite treatment led to a significant increase in p53 phosphorylation on Ser-15 (Ser15P). Time course experiments showed that p53 Ser15P occurred several hours before caspase activation and PARP cleavage. The general caspase inhibitor zVADfmk completely blocked PARP cleavage, and significantly decreased DNA laddering, but did not affect p53 Ser15P. An inhibitor for caspase-8 was equally as protective as that for caspase-9 against the selenite-induced apoptosis. Attenuating p53 by a chemical inhibitor pifithrin-alpha decreased the selenite-induced p53 Ser15P and led to concordant reductions of PARP cleavage and apoptosis. In summary, selenite-induced p53 Ser15P appeared to be important for activating the caspase-mediated apoptosis involving both the caspase-8 and the caspase-9 pathways in the LNCaP cells.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Benzothiazoles; Caspase Inhibitors; Caspases; Cell Line, Tumor; Enzyme Activation; Humans; Male; Phosphorylation; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Serine; Sodium Selenite; Thiazoles; Toluene; Tumor Suppressor Protein p53

Tubular damage by cisplatin leads to acute renal failure, which limits its use in cancer therapy. In tubular cells, a primary target for cisplatin is presumably the genomic DNA. However, the pathway relaying the signals of DNA damage to tubular cell death is unclear. In response to DNA damage, the tumor suppressor gene p53 is induced and is implicated in subsequent DNA repair and cell death by apoptosis. The current study was designed to examine the role of p53 in cisplatin-induced apoptosis in cultured rat kidney proximal tubular cells. Cisplatin at 20 microM induced apoptosis in approximately 70% of cells, which was partially suppressed by carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone (VAD), a general caspase inhibitor. Of interest, cisplatin-induced apoptosis was also suppressed by pifithrin-alpha, a pharmacological inhibitor of p53. Cisplatin-induced caspase activation was completely inhibited by VAD, but only partially by pifithrin-alpha. Early during cisplatin treatment, p53 was phosphorylated and upregulated. The p53 activation was blocked by pifithrin-alpha, but not by VAD. Bcl-2 expression abolished cisplatin-induced apoptosis without blocking p53 phosphorylation or induction. The results suggest that p53 activation might be an early signal for apoptosis during cisplatin treatment. To further determine the role of p53, tubular cells were stably transfected with a dominant-negative mutant of p53 with diminished transcriptional activity. Expression of the mutant attenuated cisplatin-induced apoptosis and caspase activation. In conclusion, the results support an important role for p53 in cisplatin-induced apoptosis in renal tubular cells. p53 May regulate apoptosis through the transcription of apoptotic genes.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Benzothiazoles; Caspase Inhibitors; Caspases; Cell Line; Cisplatin; Enzyme Inhibitors; Genes, p53; Kidney Tubules, Proximal; Mutation; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Rats; Thiazoles; Toluene; Transcription, Genetic; Transfection; Tumor Suppressor Protein p53

The molecular basis for the sensitivity of tumor cells to chemopreventive natural food compounds and commonly used chemotherapeutic agents is not well understood, not least because studies are frequently confounded by the diversity among cell lines or rely on experimental protein overexpression. Here we investigated the effects of n-butyrate, a cancer-preventive short-chain fatty acid produced by anaerobic bacteria in the gastrointestinal tract, on the human wild-type p53 and p21 expressing HCT116 colon carcinoma cell line and on HCT116 cells with either p53 or p21 alleles inactivated by homologous recombination. The effects of n-butyrate were then compared with those elicited by cytotoxic drugs and the natural chemopreventive phytoalexin of wine and grapes, resveratrol. We document that physiological concentrations of n-butyrate stimulate p21 expression and induce apoptosis independently of p53, and that the absence of p21 increases apoptosis drastically. The apoptosis is mediated through the mitochondria and is accompanied by mitochondrial proliferation and membrane potential changes. Adriamycin, etoposide, cisplatinum, colcemid and resveratrol induce distinct cellular responses; however, absence of p21 favors apoptosis-induction by adriamycin, etoposide and colcemid. Thus, control of p21 expression may support chemoprevention and certain tumor therapies.

Topics: Adenocarcinoma; Alleles; Amino Acid Chloromethyl Ketones; Anticarcinogenic Agents; Antineoplastic Agents; Apoptosis; Benzothiazoles; Butyrates; Cisplatin; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cysteine Proteinase Inhibitors; Demecolcine; Doxorubicin; Drug Resistance, Neoplasm; Etoposide; Fluorouracil; Gene Expression Regulation, Neoplastic; Genes, p53; Humans; Intracellular Membranes; Membrane Potentials; Mitochondria; Neoplasm Proteins; Recombination, Genetic; Resveratrol; Stilbenes; Thiazoles; Toluene; Tumor Cells, Cultured; Tumor Suppressor Protein p53