Caspase-2 triggers Bax-Bak-dependent and -independent cell death in colon cancer cells treated with resveratrol

Polyphenol phytoalexin (resveratrol), found in grapes and red wine is a strong chemopreventive agent with promising safety records with human consumption and unique forms of cell death induction in a variety of tumor cells. However, the mechanism of resveratrol-induced apoptosis upstream of mitochondria is still not defined. The results from this study suggest that caspase-2 activation occurs upstream of mitochondria in resveratrol-treated cells. The upstream activation of caspase-2 is not dependent on its antioxidant property or NF-kappaB inhibition. The activated caspase-2 triggers mitochondrial apoptotic events by inducing conformational changes in Bax/Bak with subsequent release of cytochrome c, apoptosis-inducing factor, and endonuclease G. Caspase-8 activation seems to be independent of these events and does not appear to be mediated by classical death receptor processing or downstream caspases. Both caspase-2 and caspase-8 contribute toward the mitochondrial translocation of Bid, since neither caspase-8 inhibition nor caspase-2 inhibition could prevent translocation of Bid DsRed into mitochondria. Caspase-2 inhibitors or antisense silencing of caspase-2 prevented cell death induced by resveratrol and partially prevented processing of downstream caspases, including caspase-9, caspase-3, and caspase-8. Studies using mouse embryonic fibroblasts deficient for both Bax and Bak indicate the contribution of both Bax and Bak in mediating cell death induced by resveratrol and the existence of Bax/Bak-independent cell death possibly through caspase-8- or caspase-2-mediated mitochondria-independent downstream caspase processing.     

Chemoprotective action of resveratrol and genistein from apoptosis induced in human peripheral blood lymphocytes

Extensive research is being carried out to analyse the importance of plant products such as resveratrol and genistein, which are known to exert a variety of pharmacological effects. This study aims at evaluating the protective role of these compounds against the apoptosis induced in normal cells by cytotoxic anticancer agents such as cisplatin and mytomycin C during therapy. Despite the broad antineoplastic action of cisplatin and mitomycin C, their genotoxicity in normal cell might lead to the induction of secondary malignancies. Therefore, the problem of identifying plant compounds, which might exert protective action in normal cells, gains lot of significance. We have analyzed the chemoprotective effect of plant compounds on peripheral blood human lymphocytes when exposed to cisplatin and mitomycin C by pre-treating and post-treating them with resveratrol and genistein at 100 microM concentration Biochemical alterations occurring in many cells during apoptosis include loss of plasma membrane phospholipid asymmetry, DNA fragmentation, and activation of caspase-3, et cetera, and have been assessed. Fluorescence microscopy, flow cytometric techniques have clearly demonstrated that resveratrol and genistein are efficient in protecting lymphocytes undergoing DNA damage when exposed to cisplatin and mitomycin C and exerted their activity by reducing the caspase 3 expression. An interesting observation is that, these compounds offered their protective effect by reducing their apoptotic potential on membrane and nucleic acids against cytotoxic agents, cisplatin, and mitomycin C. These results suggest that resveratrol and genistein might be useful for risk assessments in advance of clinical trials and could be considered as a strong candidate in pharmacogenomics or nutriprotective arena.     

Antiproliferative and pro-apoptotic activity of novel phenolic derivatives of resveratrol

Gloriosaols A-C, isolated from Yucca gloriosa (Agavaceae), are novel phenolic compounds structurally related to resveratrol. In the present study, we show that gloriosaols possess antiproliferative and pro-apoptotic activity on tumor cells of different histogenetic origin and that their cell growth inhibition potential is higher than that of resveratrol. Despite the close similarities in their structure, gloriosaols A-C exhibited different antiproliferative potency, as the EC(50) ascending order is: gloriosaol C, gloriosaol A, gloriosaol B. Further mechanisms of gloriosaol C cytotoxicity were elucidated in detail in U937 cells, the most sensitive of the cell lines tested. The effect of gloriosaol C on cell growth turned out to be strongly dependent upon the concentration. Gloriosaol C doses lower than the EC(50) value (8 mu-icroM) blocked the cell cycle in G(0)/G(1), with a concurrent decrease in the number of cells in the G(2)/M phases of the cell cycle. At higher doses, this arrest overlaps with the occurrence of apoptosis and necrosis. In the 10-25 microM range of doses, gloriosaol C caused cell death mainly by apoptosis, as measured by hypodiploidia induction, phosphatidyl serine externalization and disruption of mitochondrial transmembrane potential. A switch in the mode of death from apoptosis to necrosis occurred at doses of gloriosaol C higher than 30 microM. Gloriosaol C was found to induce production of reactive species dose-dependently, but also to counteract their elevation in stressed cells. Thus, the different fate of cells, that is cell cycle arrest or cell death, in response to different doses of gloriosaol C might be related to the extent of induced oxidative stress.     

Antiglioma action of xanthones from Gentiana kochiana: Mechanistic and structure-activity requirements

The present study identifies xanthones gentiakochianin and gentiacaulein as the active principles responsible for the in vitro antiglioma action of ether and methanolic extracts of the plant Gentiana kochiana. Gentiakochianin and gentiacaulein induced cell cycle arrest in G(2)/M and G(0)/G(1) phases, respectively, in both C6 rat glioma and U251 human glioma cell lines. The more efficient antiproliferative action of gentiakochianin was associated with its ability to induce microtubule stabilization in a cell-free assay. Both the xanthones reduced mitochondrial membrane potential and increased the production of reactive oxygen species in glioma cells, but only the effects of gentiakochianin were pronounced enough to cause caspase activation and subsequent apoptotic cell death. The assessment of structure-activity relationship in a series of structurally related xanthones from G. kochiana and Gentianella austriaca revealed dihydroxylation at positions 7, 8 of the xanthonic nucleus as the key structural feature responsible for the ability of gentiakochianin to induce microtubule-associated G(2)/M cell block and apoptotic cell death in glioma cells.     

Antiproliferative activities of resveratrol and related compounds in human hepatocyte derived HepG2 cells are associated with biochemical cell disturbance revealed by fluorescence analyses

Resveratrol is a well known polyphenol largely produced in grapevine. It is a strong antioxidant and a free radical scavenger. It exhibits several beneficial effects for health including cancer. Resveratrol antioxidant activity is essential in the prevention of chemical-induced cancer by inhibiting initiation step of carcinogenesis process but it is also considered to inhibit cancer promotion and progression steps. While the effects of resveratrol on cancer cells are widely described, the data available on the antiproliferative potential of resveratrol derivatives remain weak. Nevertheless, resveratrol analogs could exhibit stronger potentials than the parent molecule. So, we compared the cellular effects of trans-resveratrol, trans-epsilon-viniferin and their respective acetate derivatives, as well as a polyphenol mixture extracted from grapevine shoots, called vineatrol. We studied their abilities to interfere with cell proliferation, their uptake and their effects on parameters of cellular state in human hepatoma cells (HepG2). Cell growth experiments show that resveratrol triacetate presents a slightly better antiproliferative potential than resveratrol. The dimer epsilon-viniferin,as well as its pentaacetate analog, is less powerful than resveratrol, although a similar uptake kinetics in cells. Interestingly, among the tested polyphenols, vineatrol is the most potent solution, indicating a possible synergistic effect of both resveratrol and epsilon-viniferin. We took advantage of the fluorescence properties of these compounds to evidence cellular uptake by using flow cytometry. In addition, by competition assay, we demonstrate that resveratrol triacetate enters in hepatic HepG2 cells by the same way as resveratrol. By autofluorescence in situ measurement we observed that resveratrol and related compounds induce deep changes in cells activity. These changes occur mainly by increasing NADPH cell content and the number of green fluorescent cytoplasmic granular structures which may be related to an induction of detoxifying enzyme mechanisms.     

白藜芦醇以Caspase依赖和非依赖两种方式导致Jurkat细胞死亡

应用形态学观察、流式细胞仪检测、Western印迹和DNA凝胶电泳等方法研究白藜芦醇对Jurkat细胞的作用。发现白藜芦醇处理组中细胞有皱缩、出泡、染色质边集等现象,但染色质浓缩呈散在团块状且不致密。细胞质结构疏松,线粒体肿胀,脊消失。少见凋亡小体。在白藜芦醇处理组,Western印迹可检测到弱的17kDacaspase-3条带,DNA凝胶电泳可以检测到梯状DNA和弥散条带;流式细胞仪在白藜芦醇处理组检测到大量PI单阳性细胞和少量膜联蛋白V单阳性细胞。Z-VAD-FMK干预后可以发现细胞死亡率降低,同时该组梯状DNA消失,但是大分子量弥散DNA条带依然可以检测到。结果表明白藜芦醇可以通过caspase依赖和非依赖途径导致Jurkat细胞死亡。此分子机制的明确将为白藜芦醇应用于临床白血病的治疗打下理论基础。     

Differential inhibition of human cytochrome P450 enzymes by epsilon-viniferin,the dimer of resveratrol: comparison with resveratrol and polyphenols from alcoholized beverages

epsilon-Viniferin, a dimer of resveratrol, was isolated in wine at concentration between 0.5 and 5 microM. As resveratrol and polyphenols from red wine were reported to inhibit cytochrome P450 (CYP) activities, this led us to investigate the inhibitory effects of epsilon-viniferin on human CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2E1, CYP3A4 and CYP4A activities. These effects were compared to those of resveratrol and non volatiles compounds from red wine or various Cognac(R) beverages (enriched with oak-polyphenols). Assays were carried out on human liver microsomes and heterologously expressed CYPs. Ethoxyresorufin, coumarin, benzoxyresorufin, chlorzoxazone, testosterone and lauric acid were used as selective substrates for CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2E1, CYP3A4 and CYP4A, respectively. epsilon-viniferin displayed a more potent inhibitory effect than resveratrol for all the CYP activities tested (Ki 0.5 to 20 microM vs. 10 to 100 microM, respectively). This effect was not due to an inhibition of the NADPH reductase. A particularly potent inhibitory effect was shown for CYP1A1, CYP1B1 and CYP2B6 which are involved in bioactivation of numerous carcinogens. epsilon-viniferin was not a mechanism-based inhibitor of human CYPs. It displayed, like resveratrol, mixed-type inhibitions for all the CYP tested, except for CYP2E1 (non-competitive). Comparison of the inhibitory effects exerted on CYP activities by epsilon-viniferin, resveratrol and non volatile components from red wine or various Cognac beverages showed that neither resveratrol, nor epsilon-viniferin is the main CYP inhibitor present in red wine solids.     

Protective effect of resveratrol on beta-amyloid-induced oxidative PC12 cell death

Beta-amyloid peptide is considered to be responsible for the formation of senile plaques that accumulate in the brains of patients with Alzheimer's disease. There has been compelling evidence supporting the idea that beta-amyloid-induced cytotoxicity is mediated through the generation of reactive oxygen intermediates (ROIs). Considerable attention has been focused on identifying phytochemicals that are able to scavenge excess ROIs, thereby protecting against oxidative stress and cell death. Resveratrol (3,5,4'-trihydroxy-trans-stilbene), a phytoalexin found in the skin of grapes, has strong antioxidative properties that have been associated with the protective effects of red wine consumption against coronary heart disease ("the French paradox"). In this study, we have investigated the effects of resveratrol on beta-amyloid-induced oxidative cell death in cultured rat pheochromocytoma (PC12) cells. PC12 cells treated with beta-amyloid exhibited increased accumulation of intracellular ROI and underwent apoptotic death as determined by characteristic morphological alterations and positive in situ terminal end-labeling (TUNEL staining). Beta-amyloid treatment also led to the decreased mitochondrial membrane potential, the cleavage of poly(ADP-ribose)polymerase, an increase in the Bax/Bcl-X(L) ratio, and activation of c-Jun N-terminal kinase. Resveratrol attenuated beta-amyloid-induced cytotoxicity, apoptotic features, and intracellular ROI accumulation. Beta-amyloid transiently induced activation of NF-kappaB in PC12 cells, which was suppressed by resveratrol pretreatment.     

Stilbene oligomers in roots of Sophora davidii

Three stilbene oligomers, davidiols A-C were isolated from the roots of Sophora davidii in addition to the seven known phenols, leachianone A, sophoraflavanones G, H and I, miyabenol C, alpha-viniferin and epsilon-viniferin. Their structures and relative configurations were established by means of 2D-NMR spectroscopy including COLOC and PSNOESY.     

Differential protective effects of quercetin, resveratrol, rutin and epigallocatechin gallate against mitochondrial dysfunction induced by indomethacin in Caco-2 cells

The beneficial effects of dietary polyphenols on health are due not only to their antioxidant properties but also to their antibacterial, anti-inflammatory and/or anti-tumoral activities. It has recently been proposed that protection of mitochondrial function (which is altered in several diseases such as Alzheimer, Parkinson, obesity and diabetes) by these compounds, may be important in explaining the beneficial effects of polyphenols on health. The aim of this study was to evaluate the protective effects of dietary polyphenols quercetin, rutin, resveratrol and epigallocatechin gallate against the alterations of mitochondrial function induced by indomethacin (INDO) in intestinal epithelial Caco-2 cells, and to address the mechanism involved in such damaging effect by INDO, which generates oxidative stress. INDO concentration dependently decreases cellular ATP levels and mitochondrial membrane potential in Caco-2 cells after 20min of incubation. INDO also inhibits the activity of mitochondrial complex I and causes accumulation of NADH; leading to overproduction of mitochondrial O(2)()(-), since it is prevented by pyruvate. Quercetin (0.01mg/ml), resveratrol (0.1mg/ml) and rutin (1mg/ml) protected Caco-2 cells against INDO-induced mitochondrial dysfunction, while no protection was observed with epigallocatechin gallate. Quercetin was the most efficient in protecting against mitochondrial dysfunction; this could be due to its ability to enter cells and accumulate in mitochondria. Additionally its structural similarity with rotenone could favor its binding to the ubiquinone site of complex I, protecting it from inhibitors such as INDO or rotenone. These findings suggest a possible new protective role for dietary polyphenols for mitochondria, complementary of their antioxidant property. This new role might expand the preventive and/or therapeutic use of PPs in conditions involving mitochondrial dysfunction and associated with increased oxidative stress at the cellular or tissue levels.     

Apoptosis of human malignant lymphoid NAMALWA cells induced by resveratrol and quercetin

The effects of naturally-occuring polyphenols, resveratrol and quercetin, on cell viability and apoptosis were studied in Namalwa B-cell lymphoma line. Apoptotic cells were identified using DNA flow cytometric analysis and 1H NMR spectroscopy. The effects of the agents on the cell cycle kinetics and activation of caspase-3 were examined. Both resveratrol and quercetin induced apoptosis in Namalwa cells as demonstrated by the increased number of hypodiploid cells, elevated level of mobile lipid domains and caspase-3 activation. Treatment with 40 microM of resveratrol for 48 h resulted in time-dependent cell-cycle arrest at G0/G1. In contrast, upon quercetin treatment Namalwa cells accumulated in G2/M. Obtained results suggest that resveratrol and quercetin induced caspase-dependent apoptosis in human malignant lymphoid cells in vitro. These findings provide a rationale for further studies of in vivo effects of those polyphenols.     

Resveratrol induces p53-independent, X-linked inhibitor of apoptosis protein (XIAP)-mediated Bax protein oligomerization on mitochondria to initiate cytochrome c release and caspase activation

Resveratrol, a naturally occurring phytoalexin, is known to induce apoptosis in multiple cancer cell types, but the underlying molecular mechanisms remain unclear. Here, we show that resveratrol induced p53-independent, X-linked inhibitor of apoptosis protein (XIAP)-mediated translocation of Bax to mitochondria where it underwent oligomerization to initiate apoptosis. Resveratrol treatment promoted interaction between Bax and XIAP in the cytosol and on mitochondria, suggesting that XIAP plays a critical role in the activation and translocation of Bax to mitochondria. This process did not involve p53 but required accumulation of Bim and t-Bid on mitochondria. Bax primarily underwent homo-oligomerization on mitochondria and played a major role in release of cytochrome c to the cytosol. Bak, another key protein that regulates the mitochondrial membrane permeabilization, did not interact with p53 but continued to associate with Bcl-xL. Thus, the proapoptotic function of Bak remained suppressed during resveratrol-induced apoptosis. Caspase-9 silencing inhibited resveratrol-induced caspase activation, whereas caspase-8 knockdown did not affect caspase activity, suggesting that resveratrol induces caspase-9-dependent apoptosis. Together, our findings characterize the molecular mechanisms of resveratrol-induced caspase activation and subsequent apoptosis in cancer cells.     

LPS-induced apoptosis is dependent upon mitochondrial dysfunction

Bacterial infection induces apoptotic cell death in human monoblastic U937 cells that have been pretreated with interferon gamma (U937IFN). Apoptosis occurs in a manner that is independent of bacterial virulence proteins. In the present study, we show that lipopolysaccharide (LPS), a membrane constituent of gram-negative bacteria, also induces apoptosis in U937IFN cells. LPS treatment led to the appearance of characteristic markers of apoptosis such as nuclear fragmentation and activation of caspases. While the caspase inhibitor Z-VAD-fmk prevented LPS-induced apoptosis as judged by its inhibition of nuclear fragmentation, it failed to inhibit cytochrome c release and loss of mitochondrial membrane potential. Transfection of peptides containing the BH4 (Bcl-2 homology 4) domain derived from the anti-apoptotic protein Bcl-XL blocked LPS-induced nuclear fragmentation and the limited digestion of PARP. These results suggest that LPS does not require caspase activation to induce mitochondrial dysfunction and that mitochondria play a crucial role in the regulation of LPS-mediated apoptosis in U937IFN cells.     

Requirement of Apaf-1 for mitochondrial events and the cleavage or activation of all procaspases during genotoxic stress-induced apoptosis

Sequential activation of caspases is critical for the execution of apoptosis. Recent evidence suggests caspase 2 is a significant upstream caspase capable of initiating mitochondrial events, such as the release of cytochrome c. In particular, in vitro studies using recombinant proteins have shown that cleaved caspase 2 can induce mitochondrial outer membrane permeabilization directly or by cleaving the BH3-only protein BID (BH3 interacting domain death agonist). However, whether interchain cleavage or activation of procaspase 2 occurs prior to Apaf-1-mediated procaspase 9 activation under more natural conditions remains unresolved. In the present study, we show that Apaf-1-deficient Jurkat T-lymphocytes and mouse embryonic fibroblasts were highly resistant to DNA-damage-induced apoptosis and failed to cleave or activate any apoptotic procaspase, including caspase 2. Significantly, drug-induced cytochrome c release and loss of mitochondrial membrane potential were inhibited in cells lacking Apaf-1. By comparison, procaspase proteolysis and apoptosis were only delayed slightly in Apaf-1-deficient Jurkat cells upon treatment with anti-Fas antibody. Our data support a model in which Apaf-1 is necessary for the cleavage or activation of all procaspases and the promotion of mitochondrial apoptotic events induced by genotoxic drugs.     

DPI induces mitochondrial superoxide-mediated apoptosis

The iodonium compounds diphenyleneiodonium (DPI) and diphenyliodonium (IDP) are well-known phagocyte NAD(P)H oxidase inhibitors. However, it has been shown that at high concentrations they can inhibit the mitochondrial respiratory chain as well. Since inhibition of the mitochondrial respiratory chain has been shown to induce superoxide production and apoptosis, we investigated the effect of iodonium compounds on mitochondria-derived superoxide and apoptosis. Mitochondrial superoxide production was measured on both cultured cells and isolated rat-heart submitochondrial particles. Mitochondria function was examined by monitoring mitochondrial membrane potential. Apoptotic pathways were studied by measuring cytochrome c release and caspase 3 activation. Apoptosis was characterized by detecting DNA fragmentation on agarose gel and measuring propidium iodide- (PI-) stained subdiploid cells using flow cytometry. Our results showed that DPI could induce mitochondrial superoxide production. The same concentration of DPI induced apoptosis by decreasing mitochondrial membrane potential and releasing cytochrome c. Addition of antioxidants or overexpression of MnSOD significantly reduced DPI-induced mitochondrial damage, cytochrome c release, caspase activation, and apoptosis. These observations suggest that DPI can induce apoptosis via induction of mitochondrial superoxide. DPI-induced mitochondrial superoxide production may prove to be a useful model to study the signaling pathways of mitochondrial superoxide.     

The IAP-antagonist ARTS initiates caspase activation upstream of cytochrome C and SMAC/Diablo

ARTS (Sept4_i2) is a pro-apoptotic tumor suppressor protein that functions as an antagonist of X-linked IAP (XIAP) to promote apoptosis. It is generally thought that mitochondrial outer membrane permeabilization (MOMP) occurs before activation of caspases and is required for it. Here, we show that ARTS initiates caspase activation upstream of MOMP. In living cells, ARTS is localized to the mitochondrial outer membrane. In response to apoptotic signals, ARTS translocates rapidly to the cytosol in a caspase-independent manner, where it binds XIAP and promotes caspase activation. This translocation precedes the release of cytochrome C and SMAC/Diablo, and ARTS function is required for the normal timing of MOMP. We also show that ARTS-induced caspase activation leads to cleavage of the pro-apoptotic Bcl-2 family protein Bid, known to promote MOMP. We propose that translocation of ARTS initiates a first wave of caspase activation that can promote MOMP. This leads to the subsequent release of additional mitochondrial factors, including cytochrome C and SMAC/Diablo, which then amplifies the caspase cascade and causes apoptosis.     

Molecular mechanisms of resveratrol (3,4,5-trihydroxy-trans-stilbene) and its interaction with TNF-related apoptosis inducing ligand (TRAIL) in androgen-insensitive prostate cancer cells

Although resveratrol, an active ingredient derived from grapes and red wine, possesses chemopreventive properties against several cancers, the molecular mechanisms by which it inhibits cell growth and induces apoptosis have not been clearly understood. Here, we examined the molecular mechanisms of resveratrol and its interactive effects with TRAIL on apoptosis in prostate cancer PC-3 and DU-145 cells. Resveratrol inhibited cell viability and colony formation, and induced apoptosis in prostate cancer cells. Resveratrol downregulated the expression of Bcl-2, Bcl-X_L and survivin and upregulated the expression of Bax, Bak, PUMA, Noxa, and Bim, and death receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5). Treatment of prostate cancer cells with resveratrol resulted in generation of reactive oxygen species (ROS), translocation of Bax to mitochondria and subsequent drop in mitochondrial membrane potential, release of mitochondrial proteins (cytochrome c, Smac/DIABLO, and AIF) to cytosol, activation of effector caspase-3 and caspase-9, and induction of apoptosis. Resveratrol-induced ROS production, caspase-3 activity and apoptosis were inhibited by N-acetylcysteine. Bax was a major proapoptotic gene mediating the effects of resveratrol as Bax siRNA inhibited resveratrol-induced apoptosis. Resveratrol enhanced the apoptosis-inducing potential of TRAIL, and these effects were inhibited by either dominant negative FADD or caspase-8 siRNA. The combination of resveratrol and TRAIL enhanced the mitochondrial dysfunctions during apoptosis. These properties of resveratrol strongly suggest that it could be used either alone or in combination with TRAIL for the prevention and/or treatment of prostate cancer.     

Rotenone-induced G2/M cell cycle arrest and apoptosis in a human B lymphoma cell line PW.

Concentrations of rotenone (ROT) that block electron flow through mitochondrial complex I (100 nM) did not significantly alter either cell viability or the growth of PW cells. However, 10- to 50-fold higher concentrations (1-5 microM) were found to induce a dose-dependent cell cycle arrest predominantly at the G2/M stage of the cycle and apoptosis. Apoptosis was dependent on the cell cycle arrest, since apoptosis but not the G2/M arrest was prevented with the broad spectrum caspase inhibitor zVADfmk. Biochemical features of apoptosis included mitochondrial cytochrome c release, reactive oxygen species generation, and the activation of procaspase 3. Thus, ROT inhibition of mitochondrial electron transport may be insufficient to induce apoptosis in PW cells. Instead, apoptosis in these cells occurs as a consequence of disruption of the cell cycle and is only indirectly dependent upon mitochondrial electron transport.     

Methylglyoxal induces apoptosis in Jurkat leukemia T cells by activating c-Jun N-terminal kinase

Methylglyoxal (MG) is a physiological metabolite, but it is known to be toxic, inducing stress in cells and causing apoptosis. This study examines molecular mechanisms in the MG-induced signal transduction leading to apoptosis, focusing particularly on the role of JNK activation. We first confirmed that MG caused apoptosis in Jurkat cells and that it was cell type dependent because it failed to induce apoptosis in MOLT-4, HeLa, or COS-7 cells. A caspase inhibitor, Z-DEVD-fmk, completely blocked MG-induced poly(ADP-ribose)polymerase (PARP) cleavage and apoptosis, showing the critical role of caspase activation. Inhibition of JNK activity by a JNK inhibitor, curcumin, remarkably reduced MG-induced caspase-3 activation, PARP cleavage, and apoptosis. Stable expression of the dominant negative mutant of JNK also protected cells against apoptosis notably, although not completely. Correspondingly, loss of the mitochondrial membrane potential induced by MG was decreased by the dominant negative JNK. These results confirmed a crucial role of JNK working upstream of caspases, as well as an involvement of JNK in affecting the mitochondrial membrane potential.