Study Of Mechanisms Of Jurkat And Raji Cell Death Induced By Resveratrol

Resveratrol is a naturally occurring phytoalexin produced by some higher plants in response to injury or fungal infection. Resveratrol has demonstrated inhibition of growth of several cancer cell lines and tumors, suggesting that it has an inhibitory effect on cancer promotion/progression. Incidence of leukema is 2-4 to 100 thousand population in our country, which is one of principal malignant tumor to result in death. The common and frequently therapy-resistant leukemic are among the most notorious. Accordingly, natural food constituents capable of inhibiting, delaying or reversing events associated with tumor initiation, promotion, and progression have attracted much attention. The natural plant polyphenol resveratrol present in some foods including grapes, wine, and peanuts, has been implicated in the inhibition, delay, and reversion of cellular events associated with many diseases and tumorigenesis. Recent work has suggested that the cancer chemoprotective effect of the compound is primarily linked to its ability to induce cell division cycle arrest and apoptosis.The purpose of this work was to investigate resveratrol's effects on leukemic cell line for determining its therapeutic value in preventing or treating this disease. Thus, we examined the apoptosis and autophagy pathway involved in resveratrol-induced cell death in human T-cell lymphoblastic leukemia Jurkat cell line and B-cell Burkitt lymphoma Raji cell line.Our research showed that:Resveratrol inhibited the growth of Jurkat cells in a dose-and time-dependent manner. At 12h, typical features of apoptosis were observed under light and electron microscope in all treatment groups, and these features are more prominent with the time. After stained by Hoechest 33258, the majority of the cellular nuclei in the median lethal dosage (0.16mmol/L) of resveratrol treated Jurkat cells at 24h presented even weak blu fluorescence, the minority presented light blue. Cell shrinkage, chromatin condensation, and marginalization were observed by Wrigh-Gimsa staining and transmission electron microscope. DNA "ladder" was displayed in 0.16mmol/L treatment group at 24h,36h and 48h, while the control groups was negative through agarrose electrophoresis. At 48h, sub-G1 peak was detected in all groups by flow cytometry. The rate of apoptotic cells to total cells was 5.49%,6.78%,12.01% in 0.015, 0.03,0.06mmol/L treatment groups, respectively, and that in the control groups was 2.05%. Resveratrol arrested the cells in the S phase of the cell cycle in a dose-dependent manner. After exposure to 0.06mmol/L resveratrol,64.6% of the cells were arrested in the S phase.Resveratrol also inhibited the growth of Raji cells in a dose-and time-dependent manner. But the majority of resveratrol-treated Raji cells did not display typical morphology of apoptosis. Raji cells that exposed to 0.22mmol/L resveratrol, typical features of apoptosis were not observed, such as cytoplasmic condensation, nuclear fragmentation, or chromatin marginalization. DNA "ladder" was absent in 0.22mmol/L treatment group for 12h,24h,36h and 48h through agarose electrophoresis; all of these morphological characteristics support the hypothesis that nonapoptotic death mechanisms can mediate the response to resveratrol. Electron microscopic characterization, which has been the gold standard for most precisely determining the mode of cell death, was next used to distinguish these possibilities.After 48h of treatment, resveratrol resulted in the appearance of autophagic vesicle in Raji cells. Autophagy vesicle contained extensively degraded organelles. This suggested that the Raji cells were undergoing autophagic cell death. Autophagic vesicle contained extensively degraded organelles. MDC, a fluorescent compound that can be selectively taken up by autophagosomes, was used to obtain independent evidence supporting the conclusion that resveratrol triggered autophagy. MDC was applied to Raji cells after resveratrol treatment, and when these labeled cells were imaged by using fluorescence microscopy, treated cells demonstrated an intense, punctate fluorescence pattern. In contrast, control cells and Jurkat cells treated with resveratrol had minimal fluorescence.Immunoblotting analysis showed that resveratrol also aroused the release of cytochrome c from mitochondrion, while the amount decreased distinctly compared with Jurkat cells and can't induce the activation of caspase-3, This suggested that resveratrol induced autophagic cell death of Raji cell through caspase-independent path.Cathepsin D (CatD) is a lysosomal aspartic proteinase and plays an important role in the degradation of protein and the apoptotic processe induced by oxidative stress, cytokines, and aging. Therefore, we investigated the role of Cath D in Jurkat cell apoptosis and Raji cell autophagy. Cells were incubated with or without resveratrol (Raji: 0.22mmol/L; Jurkat:0.16mmol/L) for 12h,24h,36h and 48h, and then harvested and processed by western blot analysis. In jurkat cells, we detected Cath D as a cleaved product (32kDa) but the change was not distinct. In Raji cells, expression levels of Cath D (32kDa) decreased with time. Maybe Cath D decreased, so in the tumour cells produced resistance to the anticancer chemical drugs, the impairment to mitochondrion is alight and cell death is induced by autophagy. Conclusion:①Resveratrol inhibit the proliferation, cause S-phage arrest and induce the apoptosis cells death in Jurkat cells.②Reaveratrol inhibit the proliferation and induces autophagy cells death in Raji cells.③Resveratrol can not only induce apoptosis cell death through caspase-dependent pathway, but also induce autophagic cell death through caspase-independent pathway, which suggest the mechanism of reveratrol inducing different tumour cell death has distinction.④Resveratrol may induce autophagy cell death through Cath D.⑤Reaveratrol may be used to treat leukemia through a new mechanism through inducing autophagy cell death, which provides new targets for the therapy for leukemia.The role of apoptosis in restraining abnormal cell proliferation has been extensively studied, leading to the emergence of apoptosis-based therapies of cancer. However, even upon inhibition of apoptosis, cell death may still proceed due to the activation of nonapoptotic pathways. In some cases, inhibition of apoptosis might even enhance the activation of the alternative cell death mechanism. Both apoptosis and autophagic cell death induction by resveratrol underlines the potential utility of its induction as a new cancer treatment modality. Molecules that were capable of inducing cell death of both autophagic and apoptosis would have the value of a'golden bullet'. The mechanism of resveratrol treating different leukemia has distinction, which involved in inducing the different pathways of apoptosis and autophagy. This study provided an important model for further investagation apoptosis and autophgy cell death. In this report, we have identified a new activity for resveratrol to treat leukemia, the ability to induce autophagy, that to offer a pattern as a potential therapeutic tumor for resveratrol. Our findingd further support existence of different pathways through which different cells can execute death; provide new targets in controlling cell death pathway in various diseases through the difference analysis between apoptosis cell death and autophgic cell death; provide new hypothesis--the study of autophagy for investigating the mechanism that anticancer chemical drugs induced apoptosis has no effect. But the role of autophagic cell death in the anticancer effect of the drugs remains to be further explored.