The Mechanisms Of Mitochondria On Resveratrol Inducing HepG2 Cell Apoptosis

Backgroud and objectiveResveratrol, a natural polyphenolic compound found in various plants, has been reported to posses a potential function including anti-inflammatory, anti-oxygen, suppression of platelet aggregation, protection of hear-blood vessel and anti-tumor. Recent research has shown that resveratrol posses a cancer chemopreventive potential through its ability to trigger apoptosis in tumor cells and against various major stages of carcinogenesis including inhibition of iNOS expression, inhibition of cytochrome P450 1A1 transcription, phosphorylation of PKC, and interruption of TNF inducing NF-kappaB activation with dose and time-dependently. There are also report that both ERK1/2 and p38 MAPK mediate resveratrol-induced activation of p53 and apoptosis through phosphorylation of p53 at serine-15. But recently, the molecular mechanisms of these actions is still not well defined, especially few studies have evaluated the chemopreventive effects of resveratrol against liver cancer.Mitochondria, the cells powerhouses, are essential for maintaining cell life, and they also play a major role in producing various kinds of energy. Except that, recent research has shown that mitochondria have possessed other significant functions and the key role of mitochondria in cell apoptosis have become the issue. Many events happened in apoptotic process have been tightly related to mitochondria, including caspases activation, cytochrome c (Cyt. c) release, changes of electron transfer chain, the depletion of mitochondrial membrane potential (mitochondrialΔΨ_m), alteration of redox state, participation of Bcl-2 family and so on. Finally, different signal conductions converged on mitochondria, and triggered or inhibited these events. Many studies have been found that in early apoptosis mitochondria could be kept integration, while in death, mitochondra could be swelled. This is the important distinction between apoptosis and death. The alteration of mitochondrial structure and function could accelerate the appearance of apoptotic nuclear and it is the key step in this process. More and more evidence indicated that mitochondria play the key role in cell apoptotic death. It is also well recognized that mitochondfia is the performer of apoptosis.In the current study, we investigated resveratrol-induced apoptosis in human hepatoblastoma-derived HepG2 cell line and the potential mechanisms involving mitochondfia of resveratrol-induced HepG2 cell apoptosis. In addition, we also identified the differentially expressed proteins of mitochondria between resveratrol-treated HepG2 cells and untreated. Our purpose was to investigate the potential mechanisms involving mitochondria in resveratrol-induceded HepG2 cell apoptosis and further provide the more extensive, and comprehensive experimental and theoretical foundation in theraping hepatom liver cancer.MethodsIn the current study, the growth and proliferation of HepG2 cell were evaluated by MTT assay and the effect of resveratrol on cell cycle was measured by flow cytometry. Resveratrol-induced apoptosis in HepG2 was investigated by Annexin V-FITC/PI stained and flow cytometry. Morphological changes of HepG2 were observed by inverted microscope and electronmicroscope. MitochondrialΔΨ_m in whole cells was measured using two fluorimetric probes in separate experiments, rhodamine123 (Rh123) and tetramethylrhodamine ethyl ester (TMRE). HepG2 cells stained with Rh123 were analyzed by flow cytometry and cells stained with TMRE were analyzed by confocal microscope. Another two fluorimetric probes, calcein and TMRE, were employed to detect the MPTP opening in intact cells. Cyt. c release was also detected by flow cytometry. In order to further investigate the mechanism of MPTP opening, the effect of resveratrol on intracellular calcium was measured by confocal microscopy. In addition, we established a model of permeabilized HepG2 cell to observe the role of calcium in resveratrol-induced MPTP opening. In particular, the relationship among MPTP opening, Cyt. c release and calcium-induced calcium release from mitochondria in resveratrol-induced apoptosis was analyzed. Our study investigated resveratrol-induced apoptosis in human hepatoblastoma-derived HepG2 cell line and the potential mechanisms involving mitochondria. Furthermore, changes in the mitochondrial proteome of resveratrol-treated cells compared to untreated cells were analyzed by two-dimensional gel electrophoresis and mass spectrometry.Results1. The effect of resveratrol on proliferation and apoptotic death in HepG2 cells(1) Inhibited Growth of HepG2 cells by resveratrolThe results of MTT showed that the proliferation activity of resveratrol-treated cells was weak than that of untreated cells (P＜0.01). It indicated that resveratrol could obviously inhibit the proliferatrion of HepG2 cells with dose and time-dependent (F=8.113, P＜0.01).(2) The effect of resveratrol on HepG2 cell cycleThe results of flow cytometry showed that resveratrol (25, 50, 100μmol/L) caused an increase of cells in the S phase and a corresponding decrease of cells in the G₂/M phase compared to control. It indicated that resveratrol showed both cell cycle arrested in S-phase as well as stimulatory effects on DNA synthesis in HepG2 cells. (3) Resveratrol-induces HepG2 cells apoptosisThe results showed that there is an increase in the number of apoptotic cells after resveratrol treatment as suggested by exhibiting cell shrinkage, chromatin condensation, nuclear fragmentation and shedding. Annexin-V binding and flow cytometry also showed that resveratrol-treated in HepG2 cells had a significant concentration-dependent increase in Annexin-V binding populations.2. The effect of resveratrol on mitochondrialΔΨ_m,MPTP,Cyt. cå’Œ[Ca²⁺]_c(1) Resveratrol caused mitochondrialΔΨ_m depolarizationAfter treatment of cells with 25, 50, 100, 150 and 200μmol/L resveratrol, the mitochondrialΔΨ_m decreased by 16.2%, 19.5%, 25.5%, 29.8%, and 34.2%, which indicated that resveratrol could cause cell dysfunction through decreasing mitochondrialΔΨ_m.(2) Resveratrol induced MPTP openingAfter 10h exposure to resveratrol, approximately one-third of the mitochondria began to lost TMRE fluorescence and was simultaneously filled with calcein fluorescence, indicating mitochondrial depolarization and inner membrane permeabilization, corresponding to onset of the MPTP. After12h, virtually all mitochondria were depolarized and permeable to calcein.(3) Resveratrol induced Cyt. c releaseCyt. c release was found to occur after resveratrol treated HepG2 cells for 12h. These findings implied that the mitochondrial depolarization and onset of MPTP induced by resveratrol preceded Cyt. c release, and then trigger cell apoptotic death. CsA could obviously reduce this resveratrol-induced apoptosis, but not completely inhibit it. These demonstrated that resveratrol could directly target the MPTP and induce MPTP opening in a CsA-inhibitable manner.(4) Resveratrol promoted [Ca²⁺]_c elevation Our data demonstrated that resveratrol (100μmol/L) caused a sustained elevation of intracellular [Ca²⁺] when compared to control. In addition, BAPTA/AM completely suppressed the mitochondrial depolarization and cell apoptotic death induced by resveratrol. In contrast, EDTA alone revealed a weaker effect comparing to BAPTA/AM, suggesting that the elevation of intracellular free Ca²⁺ concentration was responsible for the collapse of mitochondrialΔΨ_m and the increased intracellular free Ca²⁺ concentration in HepG2 cells was largely contributed by the redistribution of intracellular Ca²⁺. Ca²⁺ may be necessary in resveratrol induced mitochondrial membrane depolarization and MPTP opening.3. Resveratrol promoted Ca²⁺-mediated MPTP opening(1) Resveratrol promoted Ca²⁺-mediated activation of MPTPThe addition ofresveratrol and Ca²⁺ to the permeabilized cells yielded increases of [Ca²⁺]_c which were transient and the decrease of mitochondrialΔΨ_m which means the activation of MPTP. There occurred no decrease of mitochondrialΔΨ_m when cells were stimulated with 100μmol/L resveratrol only, or treated with 100μmol/L resveratrol plus 10μmol/L Ca²⁺ after pretreated with 0.5mmol/L EGTA to chelate the intracellular Ca²⁺. However, if precultured with 1.0mmol/L Ca²⁺ after 0.5mmol/L EGTA, the resveratrol-induced mitochondrialΔΨ_m decrease occurred. Further experiments demonstrated that 100μmol/L resveratrol plus 1μmol/L Ca²⁺ induced no changes in mitochondrialΔΨ_m, while 10～20μmol/L Ca²⁺ plus 100μmol/L resveratrol could potentiate the fall of mitochondrialΔΨ_m.(2) Resveratrol promoted calcium-induced calcium release from mitochondria -mediated MPTP opening100μmol/L resveratrol plus 10μmol/L Ca²⁺ triggered mitochondrial Ca²⁺ overloaded rapidly then declined over the starting baseline gradually. Ascend and descent of mitochondrial Ca²⁺ in the permeabilized cells were the proof of the free Ca²⁺ influx into mitochondria first and then effiux .from mitochondria, indicating there occurred calcium-induced calcium release from mitochondria during resveratrol-induced MPTP opening.The further results showed 100μmol/L resveratrol plus 1μmol/L Ca²⁺ can not induce changes of mitochondrial Ca²⁺ andΔΨ_m in the permeabilized cells. Similarly, 10μmol/L or 20μmol/L Ca²⁺ plus 100μmol/L resveratrol, which can induce ascend and descent of mitochondrial Ca²⁺ in permeabilized cells, induced mitochondrialΔΨ_m decrease as well. In attempt to demonstrate this result, we study the relationship between calcium-induced calcium release from mitochondria and MPTP induced by resveratrol. Resveratrol-induced calcium-induced calcium release from mitochondria was inhibited or weakened in the presence of 1μmol/L RR, an inhibitor of mitochondrial Ca²⁺ uniporter, and mitochondrialΔΨ_m decrease was inhibited too. Moreover, with 1μmol/L trifluoperazine, an inhibitor of Ca²⁺/Na⁺ exchanger that is one of the mitochondrial Ca²⁺ extrusion pathways, resveratrol-induced calcium-induced calcium release from mitochondria and the mitochondrialΔΨ_m decline were unchangeable. These results indicated that part or full inhibition of calcium-induced calcium release from mitochondria could result in part or full inhibition of MPTP. In addition, we found that the mitochondrial depolarization, Cyt. c release and cell apoptosis induced by resveratrol were inhibited by RR in intact cells.(3) Resveratrol induced MPTP opening with dose and time-dependentPermeabilized cells were pretreated with resveratrol at the concentration of 100, 120, and 150μmol/L for 4min prior to the addition of 10μmol/L Ca²⁺ which induce mitochondrialΔΨ_m decrease, respectively, and the decline accelerated along with the resveratrol concentrations, indicating that the effect of resveratrol on MPTP is dose-dependent. On the other hand, pretreating permeabilized cells with resveratrol for 0, 4, 8, 12min, 10μmol/L Ca²⁺ induce decrease of mitochondrialΔΨ_m at different level, indicating the effects of resveratrol on MPTP is time-dependent. These results proved that resveratrol could facilitate MPTP opening with dose and time-dependent manner in the presence of Ca²⁺.4. Differentially expressed proteins by proteomics analysis(1) Fractionation of mitochondria from HepG2 cellsOur research established a better approach to extract mitochondria. We validate this method through the choice of homogenizer, component of buffer solution, the methods of differential centrifugation and the degree of mixing other cellular organ.(2) Protein identification by 2D gel and mass spectrometryTo facilitate to analyze protein spots by mass spectrometry, 2D gels were stained by coomassie brilliant blue. Image analysis revealed that 860±60 protein spots can be detected in 2D gels. Four significant difference proteins were identified by mass spectrometry. In contrast to the control group, three down-regulated and one up-regulated protein were expressed in resveratrol-treated HepG2 cells. The four proteins were A1 (Kinesin protein), A2 (Mitochondrial ribosomal protein L7/L12), A3 (CENP-E), A4 (Peptidase (mitochondrial processing) beta).Conclusion1. Resveratrol could obviously inhibit the proliferatrion of HepG2 cells with dose and time-dependent. Resveratrol showed both cell cycle arrest as well as stimulatory effects on DNA synthesis in HepG2 cells. In addition, resveratrol could induce cell apotposis with dose-dependent.2. Resveratrol triggered cell apoptotic death through depolarizing mitochondrialΔΨ_m, promoting the CsA-dependent MPTP opening and inducing Cyt. c release. Moreover, resveratrol caused a sustained elevation of intracellular [Ca²⁺] when compared to control and the increased intracellular free Ca²⁺ concentration in HepG2 cells was largely contributed by the redistribution of intracellular Ca²⁺.3. Ca²⁺ and calcium-induced calcium release from mitochondria was necessary for resveratrol-induced mitochondrialΔΨ_m depolarized and MPTP opening. Resveratrol promoted Ca²⁺ and calcium-induced calcium release from mitochondria mediated MPTP opening. Resveratrol also could facilitate MPTP opening with dose and time-dependent manner in the presence of Ca²⁺.4. Four significant difference proteins were identified by mass spectrometry. In contrast to the control group, three down-regulated and one up-regulated protein were expressed in resveratrol-treated HepG2 cells. The down-regulated proteins were A1 (Kinesin protein), A3 (CENP-E), A4 (Peptidase (mitochondrial processing) beta). The up-regulated protein was A2 (Mitochondrial ribosomal protein L7/L12).