cytochrome-c-t and phenethyl-isothiocyanate

Phenethyl isothiocyanate (PEITC), a potential cancer chemopreventive constituent of cruciferous vegetables, including watercress, has been reported to inhibit cancer cell growth by arresting the cell cycle and inducing apoptosis in various human cancer cell models. However, the role of PEITC in the inhibition of human chronic myeloid leukemia (CML) K562 cell growth and its underlying mechanisms have yet to be elucidated. In the present study, PEITC was found to induce cell death through the induction of reactive oxygen species (ROS) stress and oxidative damage. Heme oxygenase‑1 (HO‑1), which participates in the development of numerous tumors and the sensitivity of these tumors to chemotherapeutic drugs, plays a protective role by modulating oxidative injury. Therefore, the present study assessed the inhibitory effect of PEITC on K562 cells and whether HO‑1 facilitated cell apoptosis and ROS generation. PEITC was found to suppress cell growth and cause apoptosis by promoting Fas and Fas ligand expression, increasing ROS generation and by the successive release of cytochrome c as well as the activation of caspase‑9 and caspase‑3. PEITC was also combined with the HO‑1 inhibitor zinc protoporphyrin IX and the inducer hemin to assess whether HO‑1 determines cell survival and ROS generation. The results of the present study suggest that PEITC may be a potential anti‑tumor compound for CML therapy, and that HO‑1 has a critical function in PEITC‑induced apoptosis and ROS generation.

Topics: Anticarcinogenic Agents; Apoptosis; Caspase 3; Caspase 9; Caspases; Cell Proliferation; Cytochromes c; Heme Oxygenase-1; Humans; Isothiocyanates; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Reactive Oxygen Species; RNA, Messenger

Heat shock proteins (HSPs) are chaperones for several client proteins involved in transcriptional regulation, signal transduction, and cell cycle control. HSPs (27, 70, and 90) are abundantly expressed in a wide range of cancers and are transcriptionally regulated by heat shock factor (HSF1). Most of the synthetic HSP inhibitors exhibit toxicity, therefore, searching for inhibitors with limited or no toxicity will be of help. The objective of the present study was to determine the effect of natural isothiocyanate (phenethyl isothiocyanate; PEITC) on different HSPs (27, 70, and 90) and HSF1 in 2 breast cancer cell lines, namely breast adenocarcinoma MCF-7 (with wild type p53) and highly metastatic breast cancer cell MDA-MB-231 (with mutated p53). PEITC significantly inhibited the expression of HSPs (particularly HSP 90) and HSF1. Molecular consequences due to HSP inhibition were downregulation of cell-cycle regulatory proteins like Cyclin B1, CDK1, Cdc25C, PLK-1, and upregulation of p21 irrespective of p53 status. These modulations were accompanied by cell-cycle arrest at G2/M phase and apoptosis by activation of caspases 3 and 9. PEITC therefore may be regarded as a potent HSP inhibitor and an antitumor agent in the treatment of breast cancer.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes c; DNA Fragmentation; Heat-Shock Proteins; Humans; Isothiocyanates; MCF-7 Cells; NF-kappa B; Proto-Oncogene Proteins c-bcl-2; Tumor Suppressor Protein p53

Phenethyl isothiocyanate (PEITC), one of many compounds found in cruciferous vegetables, has been reported as a potential anticancer agent. In earlier studies, PEITC was shown to inhibit cell growth and induction of apoptosis in many cancer cell lines. However, no report has shown whether PEITC can induce apoptosis in human prostate cancer cells. Herein, we aimed to determine whether PEITC has anticancer activity in DU 145 human prostate cancer cells. As a result, we found that PEITC induced a dose-dependent decrease in cell viability through induction of cell apoptosis and cell cycle arrest in the G(2)/M phase of DU 145 cells. PEITC induced morphological changes and DNA damage in DU 145 cells. The induction of G(2)/M phase arrest was mediated by the increase of p53 and WEE1 and it reduced the level of CDC25C protein. The induction of apoptosis was mediated by the activation of caspase-8-, caspase-9- and caspase-3-depedent pathways. Results also showed that PEITC caused mitochondrial dysfunction, increasing the release of cytochrome c and Endo G from mitochondria, and led cell apoptosis through a mitochondria-dependent signaling pathway. This study showed that PEITC might exhibit anticancer activity and become a potent agent for human prostate cancer cells in the future.

Topics: Anticarcinogenic Agents; Apoptosis; Blotting, Western; Caspases; Cell Division; Cell Proliferation; Comet Assay; Cytochromes c; DNA Damage; Flow Cytometry; Fluorescent Antibody Technique; G2 Phase; Humans; Isothiocyanates; Male; Membrane Potential, Mitochondrial; Mitochondria; Prostatic Neoplasms; Reactive Oxygen Species; Signal Transduction; Tumor Cells, Cultured

Prostate cancer is the leading cause of cancer-related death in men, incidences of which are increasing gradually in India. Protein kinase C (PKC), an enzyme, gets overexpressed in prostate cancer and results in a resistance to chemotherapy. Telomerase, a reverse transcriptase, is highly activated in prostate cancer cells. Both of these enzymes can be considered as potential molecular markers for prostate cancer. The present study investigates the effects of natural isothiocyanate phenethyl isothiocyanate (PEITC) in modulating the activities of PKC and telomerase in the androgen-independent human prostate adenocarcinoma (PC-3) cell line. We observed that PEITC downregulated the antiapoptotic isoforms (PKC alpha and epsilon) efficiently and zeta moderately. Basal level of PKC delta, a proapoptotic form, was very poor and its modulation was not significant. PEITC also inhibited the activity of telomerase. Studies were conducted to measure the degree of apoptotic cell death induced either by PEITC alone or in combination with adriamycin or etoposide. Apoptosis was evident from the release of mitochondrial cytochrome c, apoptotic index, and by the induction of caspases 3 and 8. PEITC exhibited remarkable efficacy in sensitizing PC-3 cells to undergo cell death by adriamycin and etoposide, which might prove to be of considerable value in synergistic therapy of cancer.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biomarkers, Tumor; Caspases; Cell Line, Tumor; Cytochromes c; Down-Regulation; Doxorubicin; Drug Resistance, Neoplasm; Etoposide; Humans; Isothiocyanates; Male; Prostatic Neoplasms; Protein Kinase C; Telomerase

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia, and resistance to fludarabine-based therapies is a major challenge in CLL treatment. Because CLL cells are known to have elevated levels of reactive oxygen species (ROS), we aimed to test a novel ROS-mediated strategy to eliminate fludarabine-resistant CLL cells based on this redox alteration. Using primary CLL cells and normal lymphocytes from patients (n = 58) and healthy subjects (n = 12), we showed that both fludarabine-resistant and -sensitive CLL cells were highly sensitive to beta-phenylethyl isothiocyanate (PEITC) with mean IC(50) values of 5.4 microM and 5.1 microM, respectively. Normal lymphocytes were significantly less sensitive to PEITC (IC(50) = 27 microM, P < .001). CLL cells exhibited intrinsically higher ROS level and lower cellular glutathione, which were shown to be the critical determinants of CLL sensitivity to PEITC. Exposure of CLL cells to PEITC induced severe glutathione depletion, ROS accumulation, and oxidation of mitochondrial cardiolipin leading to massive cell death. Such ROS stress also caused deglutathionylation of MCL1, followed by a rapid degradation of this cell survival molecule. Our study demonstrated that the natural compound PEITC is effective in eliminating fludarabine-resistant CLL cells through a redox-mediated mechanism with low toxicity to normal lymphocytes, and warrants further clinical evaluation.

Topics: Antineoplastic Agents; Caspase 3; Cell Death; Cytochromes c; Drug Resistance, Neoplasm; Glutathione; Glutathione Peroxidase; Humans; In Vitro Techniques; Isothiocyanates; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphocytes; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; Oxidation-Reduction; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Vidarabine

Although isothiocyanates have been shown to inhibit carcinogen-induced tumorigenesis, no studies have been made to determine their therapeutic potential for the treatment of breast cancer. In the present study, we evaluated the apoptotic activities of phenethyl isothiocyanate (PEITC) in human breast cancer MCF-7 cells. Exposure to PEITC potently reduced cell viability. In addition, DNA fragments and TUNEL positive nuclei were detected in PEITC-treated cells. Furthermore, PEITC induced apoptosis via activation of caspases 7 and 9 and the cleavage of PARP, and these effects were reversed by treatment with the caspase inhibitor, Z-VAD-fmk. PEITC also caused a decrease in the levels of Bcl-2 with a concomitant increase in Bax levels, which resulted in the release of cytochrome c. XIAP suppression and Smac translocation also contributed to the PEITC-induced apoptosis. However, PEITC did not increase the expressions of p53 and p21. Taken together, the results of this study demonstrate that PEITC significantly induces apoptosis via a mitochondrial pathway. Specifically, PEITC induced a change in the Bax/Bcl-2 ratios, XIAP levels and Smac translocation that was conjunction with the release of cytochrome c and following caspase activation. Therefore, PEITC has the potential for use as a therapeutic agent for the treatment of breast cancer.

Topics: Anticarcinogenic Agents; Apoptosis; Blotting, Western; Caspases; Cell Line, Tumor; Cell Survival; Cytochromes c; DNA Fragmentation; Down-Regulation; Enzyme Activation; Gene Expression; Genes, bcl-2; Genes, p53; Humans; In Situ Nick-End Labeling; Isothiocyanates; Mitochondria; Poly(ADP-ribose) Polymerases; Signal Transduction; X-Linked Inhibitor of Apoptosis Protein

The initiating events that lead to the induction of apoptosis mediated by the chemopreventative agent beta-phenyethyl isothiocyanate (PEITC) have yet to be elucidated. In the present investigation, we examined the effects of PEITC on mitochondrial function and apoptotic signaling in hepatoma HepG2 cells and isolated rat hepatocyte mitochondria. PEITC induced a conformational change in Bax leading to its translocation to mitochondria in HepG2 cells. Bax accumulation was associated with a rapid loss of mitochondrial membrane potential (Deltapsim), impaired respiratory chain enzymatic activity, release of mitochondrial cytochrome c and the activation of caspase-dependent cell death. Caspase inhibition did not prevent Bax translocation, the release of cytochrome c or the loss of Deltapsim, but blocked caspase-mediated DNA fragmentation and cell death. To determine whether PEITC dependent Bax translocation caused loss of Deltapsim by the activation of the mitochondrial permeability transition (MPT), we examined the effects of PEITC in isolated rat hepatocyte mitochondria. Interestingly, PEITC did not induce MPT in isolated rat mitochondria. Accordingly, using pharmacological inhibitors of MPT namely cyclosporine A, trifluoperazine and Bongkrekic acid we were unable to block PEITC mediated apoptosis in HepG2 cells, this suggesting that mitochondrial permeablisation is a likely consequence of Bax dependent pore formation. Taken together, our data suggest that mitochondria are a key target in PEITC induced apoptosis in HepG2 cells via the pore forming ability of pro-apoptotic Bax.

Topics: Animals; Anticarcinogenic Agents; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Caspases; Cell Line, Tumor; Cell Membrane Permeability; Cytochromes c; DNA Fragmentation; Humans; Intracellular Membranes; Isothiocyanates; Membrane Potentials; Mitochondria, Liver; Proto-Oncogene Proteins c-bcl-2; Rats

Phenylethyl isothiocyanate (PEITC) is a well recognized potential chemopreventive compound against human cancers. In this study, the molecular mechanism of PEITC-induced apoptosis was examined with two antioxidants (N-acetyl-cysteine and vitamin E) and a caspase-3 inhibitor (z-DEVD-fmk). Results demonstrated that PEITC significantly induced human hepatoma PLC/PRF/5 (CD95-negative) cells undergoing apoptosis. Treatment with 0 approximately 10 microM PEITC-triggered cell apoptosis as revealed by the externalization of annexin V-targeted phosphatidylserine and the subsequent appearance of sub-G1 population. Results also displayed that PEITC-induced apoptosis involves the up-regulation of p53 and Bax protein, down-regulation of the XIAP, Bcl-2, Bcl-(XL) and Mcl-1 proteins, cleavage of Bid, and the release of cytochrome c and Smac/Diablo, which were accompanied by the activation of caspases -9, -3 and -8. PEITC-induced the generation of reactive oxygen species and the decrease of mitochondrial membrane potential (Deltapsim) in a time-dependent pattern. N-acetyl-cysteine and vitamin E at 100 microM, and z-DEVD-fmk at 50 microM markedly blocked PEITC-induced apoptosis, which was demonstrated by a decline in the reactive oxygen species generation and the release of the cytochrome c and Smac/Diablo from mitochondria to the cytosol. N-acetyl-cysteine, vitamin E and z-DEVD-fmk also prevented the PEITC in inducing the loss of Deltapsim. They also affected the activity of XIAP and Bax proteins. Taken together, these studies suggest that PEITC is an apoptotic inducer that acts on the mitochondria and the feedback amplification loop of caspase-8/Bid pathways in PLC/PRF/5 cells.

Topics: Acetylcysteine; Antioxidants; Apoptosis; Apoptosis Regulatory Proteins; Apoptotic Protease-Activating Factor 1; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Blotting, Western; Carcinoma, Hepatocellular; Carrier Proteins; Caspase 3; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cell Proliferation; Cysteine Proteinase Inhibitors; Cytochromes c; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Activation; fas Receptor; Flow Cytometry; Humans; Intracellular Membranes; Intracellular Signaling Peptides and Proteins; Isothiocyanates; Liver Neoplasms; Membrane Potentials; Mitochondria; Mitochondrial Proteins; Oligopeptides; Proteins; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Time Factors; Tumor Suppressor Protein p53; Up-Regulation; Vitamin E; X-Linked Inhibitor of Apoptosis Protein