stilbenes and Mesothelioma

Intracellular calcium (Ca(2+)) is known to play an important role in cancer development and growth. Resveratrol (Res) is a stilbene polyphenol occurring in several plant species and known for various possible beneficial effects, including its ability to inhibit proliferation and to induce apoptosis in cancer cells. This study was designed to determine whether Res affects Ca(2+) signaling in cancer cells.. We used the REN human mesothelioma cell line, as an in vitro cancer cell model, and the non-malignant human mesothelial MeT5A cell line, as normal cell model. Cytosolic Ca(2+) concentration was measured by the fluorescent indicator Fura-2. Immunofluorescence, Western blot, and siRNA technique were employed to assess the involvement of T-type Ca(2+) channels. Cell viability was determined by the calcein assay.. REN cells transiently exposed to 1-10μM Res showed increasing peaks of Ca(2+) that were absent in Ca(2+)-free medium and were reduced by non-selective (Ni(2+)), and highly selective (NNC 55-0396) T-type Ca(2+) channels antagonist, and by siRNA knockout of Cav3.2T-type Ca(2+) channel gene. Dose-dependent curve of Res-induced Ca(2+) peaks showed a rightward shift in normal MeT-5A mesothelial cells (EC50=4.9μM) with respect to REN cells (EC50=2.7μM). Moreover, incubation with 3 and 10μM Res for 7days resulted in cell growth inhibition for REN, but not for MeT-5A cells.. Res induces Ca(2+) influx, possibly mediated through T-type Ca(2+) channels, with significant selectivity towards mesothelioma cells, suggesting a possible use as an adjuvant to chemotherapy drugs for mesothelioma clinical treatment.

Topics: Calcium; Calcium Channels, T-Type; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Humans; Intracellular Fluid; Mesothelioma; Resveratrol; Stilbenes

Malignant mesothelioma (MM) is characterized by poor responsiveness to current chemotherapeutic drugs, usually owing to high resistance to apoptosis. Here, we investigated chemosensitizing effects of phytochemical resveratrol, in combination with cisplatin, on MM cells. The combination treatment of cisplatin and resveratrol (CDDP/RSV) synergistically induced apoptosis, as evidenced by typical cell morphological changes, the appearance of sub-G0/G1 peak, an increase in the Annexin V(+) cells and the cleavage of caspase-3 and PARP. CDDP/RSV increased ROS production and depolarization of mitochondrial membrane potential with an increase in the Bax/Bcl-2 ratio. These changes were attenuated by pretreatment with N-acetylcysteine, suggesting that CDDP/RSV induced apoptosis through oxidative mitochondrial damage. Compared with MSTO-211H cells, CDDP/RSV was less efficient in killing H-2452 cells. H-2452 cells exhibited an enhanced autophagy to CDDP/RSV, as observed by an increase in viable cells exhibiting intense LysoTracker Red staining and up-regulation of Beclin-1 and LC3A. Inhibition of autophagy by bafilomycin A1 rendered cells more sensitive to CDDP/RSV-induced cytotoxicity and this was associated with induction of apoptosis. These data indicate that the increased resistance of H-2452 cells to CDDP/RSV is closely related to the activation of self-defensive autophagy, and provide the rationale for targeting the autophagy regulation in the treatment of MM.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Autophagy; Caspase 3; Cisplatin; Humans; Lung Neoplasms; Mesothelioma; Mesothelioma, Malignant; Oxidative Stress; Resveratrol; Stilbenes; Tumor Cells, Cultured

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cisplatin; Drug Synergism; Humans; Lung Neoplasms; Mesothelioma; Mesothelioma, Malignant; Models, Biological; Resveratrol; Stilbenes

We previously demonstrated that resveratrol and clofarabine elicited a marked cytotoxicity on malignant mesothelioma (MM) MSTO-211H cells but not on the corresponding normal mesothelial MeT-5A cells. Little is known of the possible molecules that could be used to predict preferential chemosensitivity on MSTO-211H cells. Resveratrol and clofarabine induced down-regulation of Mcl-1 protein level in MSTO-211H cells. Treatment of cells with cycloheximide in the presence of proteasome inhibitor MG132 suggested that Mcl-1 protein levels were regulated at the post-translational step. The siRNA-based knockdown of Mcl-1 in MSTO-211H cells triggered more growth-inhibiting and apoptosis-inducing effects with the resultant cleavages of procaspase-3 and its substrate PARP, increased caspase-3/7 activity, and increased percentage of apoptotic propensities. However, the majority of the observed changes were not shown in MeT-5A cells. Collectively, these studies indicate that the preferential activation of caspase cascade in malignant cells might have important applications as a therapeutic target for MM.

Topics: Adenine Nucleotides; Apoptosis; Arabinonucleosides; Caspase 3; Cell Line, Tumor; Clofarabine; Down-Regulation; Enzyme Activation; Humans; Mesothelioma; Myeloid Cell Leukemia Sequence 1 Protein; Resveratrol; Stilbenes

Resveratrol is a naturally occurring polyphenolic phytoalexin with chemopreventive properties. We previously reported a synergistic anti-proliferative effect of resveratrol and clofarabine against malignant mesothelioma (MM) cells. Here, we further investigated molecular mechanisms involved in the synergistic interaction of these compounds in MM MSTO-211H cells. Resveratrol, in combination with clofarabine, time-dependently induced a strong cytotoxic effect with the nuclear accumulation of phospho-p53 (p-p53) in MSTO-211H cells, but not in normal mesothelial MeT-5A cells. Combination treatment up-regulated the levels of p-p53, cleaved caspase-3, and cleaved PARP proteins. Gene silencing with p53-targeting siRNA attenuated the sensitivity of cells to the combined treatment of two compounds. Analyses of p53 DNA binding assay, p53 reporter gene assay, and RTP-CR toward p53-regulated genes, including Bax, PUMA, Noxa and p21, demonstrated that induced p-p53 is transcriptionally active. These results were further confirmed by the siRNA-mediated knockdown of p53 gene. Combination treatment significantly caused the accumulation of cells at G1 phase with the increases in the sub-G0/G1 peak, DNA ladder, nuclear fragmentation, and caspase-3/7 activity. Taken together, these results demonstrate that resveratrol and clofarabine synergistically elicit apoptotic signal via a p53-dependent pathway, and provide a scientific rationale for clinical evaluation of resveratrol as a promising chemopotentiator in MM.

Topics: Adenine Nucleotides; Anticarcinogenic Agents; Antineoplastic Agents; Arabinonucleosides; Base Sequence; Cell Line, Tumor; Clofarabine; DNA Primers; G1 Phase; Genes, p53; Humans; Mesothelioma; Oncogene Proteins; Polymerase Chain Reaction; Resveratrol; Stilbenes

Mcl-1 and Bcl-xL, key anti-apoptotic proteins of the Bcl-2 family, have attracted attention as important molecules in the cell survival and drug resistance. In this study, we investigated whether inhibition of Bcl-xL influences cell growth and apoptosis against simultaneous treatment of resveratrol and clofarabine in the human malignant mesothelioma H-2452 cells. Resveratrol and clofarabine decreased Mcl-1 protein levels but had little effect on Bcl-xL levels. In the presence of two compounds, any detectable change in the Mcl-1 mRNA levels was not observed in RT-PCR analysis, whereas pretreatment with the proteasome inhibitor MG132 led to its accumulation to levels far above basal levels. The knockdown of Bcl-xL inhibited cell proliferation with cell accumulation at G2/M phase and the appearance of sub-G0/G1 peak in DNA flow cytometric assay. The suppression of cell growth was accompanied by an increase in the caspase-3/7 activity with the resultant cleavages of procaspase-3 and its substrate poly (ADP-ribose) polymerase, and increased percentage of apoptotic propensities in annexin V binding assay. Collectively, our data represent that the efficacy of resveratrol and clofarabine for apoptosis induction was substantially enhanced by Bcl-xL-lowering strategy in which the simultaneous targeting of Mcl-1 and Bcl-xL could be a more effective strategy for treating malignant mesothelioma.

Topics: Adenine Nucleotides; Antimetabolites, Antineoplastic; Apoptosis; Arabinonucleosides; bcl-X Protein; Caspase 3; Caspase 7; Cell Line, Tumor; Cell Proliferation; Clofarabine; G2 Phase Cell Cycle Checkpoints; Gene Knockdown Techniques; Humans; Leupeptins; Lung Neoplasms; M Phase Cell Cycle Checkpoints; Mesothelioma; Mesothelioma, Malignant; Myeloid Cell Leukemia Sequence 1 Protein; Resveratrol; RNA Interference; RNA, Messenger; RNA, Small Interfering; Stilbenes

Dietary phytochemicals as adjuvants have been suggested to play important roles in enhancing chemotherapeutic potential owing to multitargeted chemopreventive properties and lack of substantial toxicity. Here, we investigated the efficacy of the combined treatment of various phytochemicals with the anticancer drug clofarabine in malignant mesothelioma MSTO-211H cells and normal mesothelial MeT-5A cells. The combined treatment of resveratrol and clofarabine produced a synergistic antiproliferative effect in MSTO-211H cells, but not in MeT-5A cells. In MSTO-211H cells, the nuclear accumulation of Sp1 and the levels of p-Akt, Sp1, c-Met, cyclin D1, and p21 were effectively decreased by the combined treatment of them. In combination with clofarabine, the ability of resveratrol to reduce the contents of Sp1 and its target gene products was also evident in a time- and dose-dependent experiment. The inhibition of phosphoinositide 3-kinase using Ly294002 augmented a decrease in the p21 level induced by their combination, but it showed no significant effects on expression of Sp1 and cyclin D1. Taken together, the data provide evidence that the synergistic antiproliferative effect of resveratrol and clofarabine is linked to the inhibition of Akt and Sp1 activities, and suggest that this combination may have therapeutic value in treatment of malignant mesothelioma.

Topics: Adenine Nucleotides; Antineoplastic Combined Chemotherapy Protocols; Arabinonucleosides; Cell Line, Tumor; Cell Proliferation; Chromones; Clofarabine; Cyclin D1; Enzyme Inhibitors; Epithelial Cells; Humans; Mesothelioma; Morpholines; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-met; Resveratrol; Sp1 Transcription Factor; Stilbenes

Resveratrol (Res), from the skin of red grapes, induces apoptosis in some malignant cells, but there are no reports on the apoptotic effect of Res on human malignant pleural mesothelioma. We found that Res interacts with specificity protein 1 (Sp1). The IC50 for Res was 17 µM in MSTO-211H cells. Cell viability was decreased and apoptotic cell death was increased by Res (0-60 µM). Res increased the Sub-G1 population in MSTO-211H cells and significantly suppressed Sp1 protein levels, but not Sp1 mRNA levels. Res modulated the expression of Sp1 regulatory proteins including p21, p27, cyclin D1, Mcl-1 and survivin in mesothelioma cells. After treatment with Res, apoptosis signaling cascades were activated by the activation of Bid, Bim, caspase-3 and PARP, upregulation of Bax and downregulation of Bcl-xL. Res (20 mg/kg daily for 4 weeks) effectively suppressed tumor growth in vivo in BALB/c athymic (nu+/nu+) mice injected with MSTO-211H cells, an effect that was mediated by inhibition of Sp1 expression and induction of apoptotic cell death. Our results strongly suggest that Sp1 is a novel molecular target of Res in human malignant pleural mesothelioma.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Bcl-2-Like Protein 11; bcl-X Protein; BH3 Interacting Domain Death Agonist Protein; Caspase 3; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Humans; Inhibitor of Apoptosis Proteins; Membrane Proteins; Mesothelioma; Mice; Mice, Inbred BALB C; Mice, Nude; Myeloid Cell Leukemia Sequence 1 Protein; Pleural Neoplasms; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Resveratrol; RNA, Messenger; Sp1 Transcription Factor; Stilbenes; Survivin; Transplantation, Heterologous

We previously reported that MSTO-211H cells have a higher capacity to regulate Nrf2 activation in response to changes in the cellular redox environment. To further characterize its biological significance, the response of Nrf2, a transcription factor that regulates ARE-containing genes, on the synergistic cytotoxic effect of clofarabine and resveratrol was investigated in mesothelioma cells. The combination treatment showed a marked growth-inhibitory effect, which was accompanied by suppression of Nrf2 activation and decreased expression of heme oxygenase-1 (HO-1). While transient overexpression of Nrf2 conferred protection against the cytotoxicity caused by their combination, knockdown of Nrf2 expression using siRNA enhanced their cytotoxic effect. Pretreatment with Ly294002, a PI3K inhibitor, augmented the decrease in HO-1 level by their combination, whereas no obvious changes were observed in Nrf2 levels. Altogether, these results suggest that the synergistic cytotoxic effect of clofarabine and resveratrol was mediated, at least in part, through suppression of Nrf2 signaling.

Topics: Adenine Nucleotides; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Arabinonucleosides; Blotting, Western; Cell Proliferation; Clofarabine; Down-Regulation; Drug Synergism; Heme Oxygenase-1; Humans; Mesothelioma; NF-E2-Related Factor 2; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Resveratrol; RNA, Small Interfering; Signal Transduction; Stilbenes; Tumor Cells, Cultured