Celecoxib acts in a cyclooxygenase-2-independent manner and in synergy with emodin to suppress rat cholangiocarcinoma growth in vitro through a mechanism involving enhanced Akt inactivation and increased activation of caspases-9 and -3

Emodin, a tyrosine kinase inhibitor, effectively blocked tyrosine phosphorylation of p185(neu) overexpressed in cultured rat C611B cholangiocarcinoma (ChC) cells and in neu-transformed WB-F344 rat-liver epithelial stem-like cells (WBneu cells). Celecoxib, a cyclooxygenase-2 inhibitor, markedly decreased prostaglandin (PG) levels overproduced by these respective neoplastically transformed liver cell types but was without effect in inhibiting PG production by untransformed WB-F344 cells that do not express detectable cyclooxygenase-2 protein. Notably, in combination, emodin (30 micro M) and celecoxib (35 micro M) acted synergistically to significantly suppress anchorage-dependent and -independent growth of C611B ChC cells and of WBneu cells over treatments with either agent alone. This prominent suppression of cell growth correlated with significant increases in the activation of caspases-9 and -3 and induction of apoptosis in the combination-treated cells, which was associated with an enhanced suppression of Akt activation. Here it is important to note that the concentration of celecoxib needed to suppress growth and induce apoptosis in the C611B and WBneu cells was markedly higher than that needed to effectively inhibit PG production by these malignant cell types. Thus, our data indicate that celecoxib is acting independently of its ability to inhibit cyclooxygenase-2 activity in suppressing growth of C611B and WBneu cells in vitro. Furthermore, our findings strongly suggest that increased inhibition of the antiapoptotic kinase Akt activation produced by the emodin/celecoxib combination treatment plays a key role in the mechanism by which this drug combination acts to enhance cell growth suppression and apoptosis in cultured C611B ChC cells and WBneu cells.     

Motexafin gadolinium modulates levels of phosphorylated Akt and synergizes with inhibitors of Akt phosphorylation

Motexafin gadolinium (MGd, Xcytrin) is a tumor-selective expanded porphyrin that targets oxidative stress-related proteins. MGd treatment of the follicular lymphoma-derived cell line HF-1 resulted in growth suppression and apoptosis whereas MGd treatment of the Burkitt's lymphoma-derived cell line Ramos resulted in growth suppression but not apoptosis. Because phosphorylation status of Akt/protein kinase B is regulated by oxidative stress, we monitored total and phosphorylated Akt (pAkt) in MGd-treated HF-1 and Ramos cells. Levels of pAkt increased within 30 minutes after MGd treatment of HF-1 but after 4 hours began to show a progressive decline to below baseline levels before cells underwent apoptosis. In MGd-treated Ramos cells, pAkt increased approximately 2-fold within 4 hours and remained persistently elevated. Because pAkt activates survival pathways, we determined if MGd-induced cell death could be enhanced by inhibiting phosphorylation of Akt. The addition of specific inhibitors of Akt phosphorylation (Akt inhibitor 1 or SH-5) reduced pAkt levels in MGd-treated HF-1 and Ramos cells and synergistically enhanced MGd-induced cell death. MGd was also evaluated in combination with celecoxib, an inhibitor of Akt phosphorylation, or docetaxel, a microtubule inhibitor that can decrease Akt phosphorylation. The combination of MGd/celecoxib or MGd/docetaxel resulted in decreased Akt phosphorylation and in synergistic cytotoxicity compared with either agent alone. These data point to a potential protective role for pAkt in MGd-induced apoptosis and suggest that MGd activity may be enhanced by combining it with agents that inhibit Akt phosphorylation.     

The alkylphospholipid perifosine induces apoptosis of human lung cancer cells requiring inhibition of Akt and activation of the extrinsic apoptotic pathway

The Akt inhibitor, perifosine, is an alkylphospholipid exhibiting antitumor properties and is currently in phase II clinical trials for various types of cancer. The mechanisms by which perifosine exerts its antitumor effects, including the induction of apoptosis, are not well understood. The current study focused on the effects of perifosine on the induction of apoptosis and its underlying mechanisms in human non-small cell lung cancer (NSCLC) cells. Perifosine, at clinically achievable concentration ranges of 10 to 15 micromol/L, effectively inhibited the growth and induced apoptosis of NSCLC cells. Perifosine inhibited Akt phosphorylation and reduced the levels of total Akt. Importantly, enforced activation of Akt attenuated perifosine-induced apoptosis. These results indicate that Akt inhibition is necessary for perifosine-induced apoptosis. Despite the activation of both caspase-8 and caspase-9, perifosine strikingly induced the expression of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor, death receptor 5, and down-regulated cellular FLICE-inhibitory protein (c-FLIP), an endogenous inhibitor of the extrinsic apoptotic pathway, with limited modulatory effects on the expression of other genes including Bcl-2, Bcl-X(L), PUMA, and survivin. Silencing of either caspase-8 or death receptor 5 attenuated perifosine-induced apoptosis. Consistently, further down-regulation of c-FLIP expression with c-FLIP small interfering RNA sensitized cells to perifosine-induced apoptosis, whereas enforced overexpression of ectopic c-FLIP conferred resistance to perifosine. Collectively, these data indicate that activation of the extrinsic apoptotic pathway plays a critical role in perifosine-induced apoptosis. Moreover, perifosine cooperates with TRAIL to enhance the induction of apoptosis in human NSCLC cells, thus warranting future in vivo and clinical evaluation of perifosine in combination with TRAIL in the treatment of NSCLC.     

Cytotoxic synergy between the multikinase inhibitor sorafenib and the proteasome inhibitor bortezomib in vitro: induction of apoptosis through Akt and c-Jun NH2-terminal kinase pathways

This study was undertaken to characterize preclinical cytotoxic interactions for human malignancies between the multikinase inhibitor sorafenib (BAY 43-9006) and proteasome inhibitors bortezomib or MG132. Multiple tumor cell lines of varying histiotypes, including A549 (lung adenocarcinoma), 786-O (renal cell carcinoma), HeLa (cervical carcinoma), MDA-MB-231 (breast), K562 (chronic myelogenous leukemia), Jurkat (acute T-cell leukemia), MEC-2 (B-chronic lymphocytic leukemia), and U251 and D37 (glioma), as well as cells derived from primary human glioma tumors that are likely a more clinically relevant model were treated with sorafenib or bortezomib alone or in combination. Sorafenib and bortezomib synergistically induced a marked increase in mitochondrial injury and apoptosis, reflected by cytochrome c release, caspase-3 cleavage, and poly(ADP-ribose) polymerase degradation in a broad range of solid tumor and leukemia cell lines. These findings were accompanied by several biochemical changes, including decreased phosphorylation of vascular endothelial growth factor receptor-2, platelet-derived growth factor receptor-beta, and Akt and increased phosphorylation of stress-related c-Jun NH2-terminal kinase (JNK). Inhibition of Akt was required for synergism, as a constitutively active Akt protected cells against apoptosis induced by the combination. Alternatively, the JNK inhibitor SP600125 could also protect cells from apoptosis induced by the combination, indicating that both inhibition of Akt and activation of JNK were required for the synergism. These findings show that sorafenib interacts synergistically with bortezomib to induce apoptosis in a broad spectrum of neoplastic cell lines and show an important role for the Akt and JNK pathways in mediating synergism. Further clinical development of this combination seems warranted.     

Inhibition of the phosphatidylinositol 3-kinase/Akt pathway sensitizes MDA-MB468 human breast cancer cells to cerulenin-induced apoptosis

Fatty acid synthase is overexpressed in cancer especially in tumors with a poor prognosis. The specific fatty acid synthase inhibitor cerulenin can induce apoptosis in cancer cells. Likewise, phosphatidylinositol 3-kinase (PI3K)/Akt kinase activities are elevated in primary tumors and cancer cell lines. Here, we tested whether inhibition of PI3K/Akt pathway would sensitize cancer cells to cerulenin-induced apoptosis. We show that LY294002, an inhibitor of PI3K, sensitized MDA-MB468 breast cancer cells to cerulenin-induced apoptosis. In MDA-MB468 cells, cerulenin- and LY294002-mediated apoptosis was associated with caspase-3 activation and the release of cytochrome c from mitochondria to cytosol. In addition, we observed additional species of Bak in mitochondria, suggesting a possible Bak activation. Treatment of cells with cerulenin and LY294002 down-regulated the protein levels of X chromosome-linked inhibitor of apoptosis (XIAP), cellular inhibitor of apoptosis 1 (cIAP-1), and Akt, whereas the levels of mitogen-activated protein/extracellular signal-regulated kinase kinase and other antiapoptotic Bcl-2 family proteins (Bcl-2 and Bcl-xl) did not change. Interestingly, the nonspecific caspase inhibitor, z-VAD-FMK, inhibited the down-regulation of Akt, XIAP, and cIAP-1 in cerulenin- and LY294002-treated cells. In conclusion, these studies show that inhibition of PI3K can sensitize cerulenin-induced apoptosis in MBA-MB468 breast cancer cells via activation of caspases, down-regulation of antiapoptotic proteins, such as XIAP, cIAP-1 and Akt, and possibly, activation of Bak in mitochondria.     

Inhibition of 5-lipoxygenase by MK886 augments the antitumor activity of celecoxib in human colon cancer cells

Cyclooxygenase (COX)-2 and 5-lipoxygenase (5-LOX) are key enzymes involved in arachidonic acid metabolism. Their products, prostaglandins and leukotrienes, are involved in colorectal tumor development. We aimed at evaluating whether combined blocking of the COX-2 and 5-LOX pathways might have additive antitumor effects in colorectal cancer. The expression/activity of COX-2 and 5-LOX were assessed in 24 human colorectal cancer specimens. The effects of the COX-2 inhibitor celecoxib and the 5-LOX inhibitor MK886 on prostaglandin E(2) and cysteinyl leukotriene production, tumor cell proliferation, cell apoptosis, and Bcl-2/Bax expression were evaluated in the Caco-2 and HT29 colon cancer cells. We also investigated the effect of the enzymatic inhibition on mitochondrial membrane depolarization, one of the most important mechanisms involved in ceramide-induced apoptosis. Up-regulation of the COX-2 and 5-LOX pathways was found in the tumor tissue in comparison with normal colon mucosa. Inhibition of either COX-2 or 5-LOX alone resulted in activation of the other pathway in colon cancer cells. Combined treatment with 10 micromol/L celecoxib and MK886 could prevent this activation and had additive effects on inhibiting tumor cell proliferation, inducing cell apoptosis, decreasing Bcl-2 expression, increasing Bax expression, and determining mitochondrial depolarization in comparison with treatment with either inhibitor alone. The administration of the ceramide synthase inhibitor fumonisin B1 could prevent some of these antineoplastic effects. In conclusion, our study showed that inhibition of 5-LOX by MK886 could augment the antitumor activity of celecoxib in human colorectal cancer.     

EFFECTS OF p53 GENE THERAPY COMBINED WITH CYCLOOXYGENASE-2 INHIBITOR ON CYCLOOXYGENASE-2 GENE EXPRESSION AND GROWTH INHIBITION OF HUMAN LUNG CANCER CELLS

1 INTRODUCTIONCOXis the key enzyme involved in the synthesis of prostanoids,a collective termfor the PGs andthromboxanes.Of the two major isoforms of the COXenzyme,COX-1 is ubiquitous and constitutively ex-pressedin virtually all normal tissues.In contras…     

Inhibition of both focal adhesion kinase and insulin-like growth factor-I receptor kinase suppresses glioma proliferation in vitro and in vivo

Multiple genetic aberrations in human gliomas contribute to their highly infiltrative and rapid growth characteristics. Focal adhesion kinase (FAK) regulates tumor migration and invasion. Insulin-like growth factor-I receptor (IGF-IR), whose expression correlates with tumor grade, is involved in proliferation and survival. We hypothesized that inhibiting the phosphorylation of FAK and IGF-IR by NVP-TAE226 (hereafter called TAE226), a novel dual tyrosine kinase inhibitor of FAK and IGF-IR, would suppress the growth and invasion of glioma cells. In culture, TAE226 inhibited extracellular matrix-induced autophosphorylation of FAK (Tyr(397)). TAE226 also inhibited IGF-I-induced phosphorylation of IGF-IR and activity of its downstream target genes such as MAPK and Akt. TAE226 retarded tumor cell growth as assessed by a cell viability assay and attenuated G(2)-M cell cycle progression associated with a decrease in cyclin B1 and phosphorylated cdc2 (Tyr(15)) protein expression. TAE226 treatment inhibited tumor cell invasion by at least 50% compared with the control in an in vitro Matrigel invasion assay. Interestingly, TAE226 treatment of tumor cells containing wild-type p53 mainly exhibited G(2)-M arrest, whereas tumor cells bearing mutant p53 underwent apoptosis. Induction of apoptosis by TAE226 was substantiated by detection of caspase-3/7 activation and poly(ADP-ribose) polymerase cleavage and by an Annexin V apoptosis assay. More importantly, TAE226 treatment significantly increased the survival rate of animals in an intracranial glioma xenograft model. Collectively, these data show that blocking the signaling pathways of FAK and IGF-IR with TAE226 has the potential to be an efficacious treatment for human gliomas.     

Apigenin induces apoptosis in human leukemia cells and exhibits anti-leukemic activity in vivo

In this study, we investigated the functional role of Akt and c-jun-NH(2)-kinase (JNK) signaling cascades in apigenin-induced apoptosis in U937 human leukemia cells and anti-leukemic activity of apigenin in vivo. Apigenin induced apoptosis by inactivation of Akt with a concomitant activation of JNK, Mcl-1 and Bcl-2 downregulation, cytochrome c release from mitochondria, and activation of caspases. Constitutively active myristolated Akt prevented apigenin-induced JNK, caspase activation, and apoptosis. Conversely, LY294002 and a dominant-negative construct of Akt potentiated apigenin-induced apoptosis in leukemia cells. Interruption of the JNK pathway showed marked reduction in apigenin-induced caspase activation and apoptosis in leukemia cells. Furthermore, in vivo administration of apigenin resulted in attenuation of tumor growth in U937 xenografts accompanied by inactivation of Akt and activation of JNK. Attenuation of tumor growth in U937 xenografts by apigenin raises the possibility that apigenin may have clinical implications and can be further tested for incorporating in leukemia treatment regimens.     

Enhancing antitumor effects in pancreatic cancer cells by combined use of COX-2 and 5-LOX inhibitors

Cyclooxygenase (COX)-2 and lipoxygenase (LOX)-5 are involved in carcinogenesis of pancreatic cancer. COX-2 inhibitor celecoxib displays inhibitory effects in pancreatic cancer cell growth. Recently, it has been reported that COX-2 inhibitor may not be able to suppress pancreatic tumor growth in vivo and its application is further limited by untoward side effects. The present study provides evidence that combined use of celecoxib and 5-LOX inhibitor MK886 markedly suppresses pancreatic tumor cell growth in vitro. Compared to the single inhibitor treatment, dual treatment with celecoxib and MK886 exerted additive antitumor effects in pancreatic tumor cells. We found that MK886 reversed celecoxib-induced increases in 5-LOX gene expression and Erk1/2 activation in pancreatic tumor cells. Moreover, Dual treatment of pancreatic tumor cells with celecoxib and MK886 inhibited the levels of LBT4 receptor BLT1 and vascular endothelial growth factor. Our results imply that combined use of celecoxib and MK886 might be an effective way to treat clinical patients with pancreatic cancer.     

Rapamycin and UCN-01 synergistically induce apoptosis in human leukemia cells through a process that is regulated by the Raf-1/MEK/ERK, Akt, and JNK signal transduction pathways

Interactions between the protein kinase C and Chk1 inhibitor UCN-01 and rapamycin in human leukemia cells have been investigated in relation to apoptosis induction. Treatment of U937 monocytic leukemia cells with rapamycin (10 nmol/L) in conjunction with a minimally toxic concentration of UCN-01 (100 nmol/L) for 36 hours resulted in marked potentiation of mitochondrial injury (i.e., loss of mitochondrial membrane potential and cytosolic release of cytochrome c, AIF, and Smac/DIABLO), caspase activation, and apoptosis. The release of cytochrome c, AIF, and Smac/DIABLO were inhibited by BOC-D-fmk, indicating that their release was caspase dependent. These events were associated with marked down-regulation of Raf-1, MEK, and ERK phosphorylation, diminished Akt activation, and enhanced phosphorylation of c-Jun NH2-terminal kinase (JNK). Coadministration of UCN-01 and rapamycin reduced the expression levels of the antiapoptotic members of the Bcl-2 family Mcl-1 and Bcl-xL and diminished the expression of cyclin D1 and p34(cdc2). Furthermore, enforced expression of a constitutively active MEK1 or, to a lesser extent, myristoylated Akt construct partially but significantly attenuated UCN-01/rapamycin-mediated lethality in both U937 and Jurkat cell systems. Finally, inhibition of the stress-related JNK by SP600125 or by the expression of a dominant-negative mutant of c-Jun significantly attenuated apoptosis induced by rapamycin/UCN-01. Together, these findings indicate that the mammalian target of rapamycin inhibitor potentiates UCN-01 cytotoxicity in a variety of human leukemia cell types and suggest that inhibition of both Raf-1/MEK/ERK and Akt cytoprotective signaling pathways as well as JNK activation contribute to this phenomenon.     

塞来昔布对人白血病细胞株HL-60细胞增殖及凋亡的影响及其作用机制

本研究旨在探讨塞来昔布（celecoxib）对人急性髓系白血病细胞株HL-60细胞增殖、凋亡的影响及其可能的作用机制。不同浓度塞来昔布作用于HL-60细胞24h后,CCK-8法测定细胞增殖活性,流式细胞技术分析细胞凋亡及细胞周期分布的变化,定量RT—PCR的方法检测细胞周期蛋白DJ、EI及COX-2mRNA的表达。结果表明,不同浓度塞来昔布作用于HL-60细胞24h后,细胞增殖明显受抑,且呈-定的浓度依赖性（r=0．955）,24h的IC50值为63．037μmol／L。塞来昔布可诱导HL-60细胞的凋亡,也呈剂量依赖性（r=0．988）。塞来昔布可使HL-60细胞明显阻滞于G0／G1期,可下调cyclinD1、cyclinE1mRNA的表达。塞来昔布可使细胞COX-2mRNA表达水平降低。结论：塞来昔布呈浓度依赖性抑制HL-60细胞增殖,并可能通过下调cyclinD1、cyclinE1的表达引起细胞G0／G1,期阻滞,下调COX-2的表达诱导细胞凋亡。     

Proteasome-mediated degradation of cell division cycle 25C and cyclin-dependent kinase 1 in phenethyl isothiocyanate-induced G2-M-phase cell cycle arrest in PC-3 human prostate cancer cells

Phenethyl isothiocyanate (PEITC), a constituent of many cruciferous vegetables, offers significant protection against cancer in animals induced by a variety of carcinogens. The present study demonstrates that PEITC suppresses proliferation of PC-3 cells in a dose-dependent manner by causing G(2)-M-phase cell cycle arrest and apoptosis. Interestingly, phenyl isothiocyanate (PITC), which is a structural analogue of PEITC but lacks the -CH(2) spacers that link the aromatic ring to the -N=C=S group, neither inhibited PC-3 cell viability nor caused cell cycle arrest or apoptosis. These results indicated that even a subtle change in isothiocyanate (ITC) structure could have a significant impact on its biological activity. The PEITC-induced cell cycle arrest was associated with a >80% reduction in the protein levels of cyclin-dependent kinase 1 (Cdk1) and cell division cycle 25C (Cdc25C; 24 h after treatment with 10 micro M PEITC), which led to an accumulation of Tyr(15) phosphorylated (inactive) Cdk1. On the other hand, PITC treatment neither reduced protein levels of Cdk1 or Cdc25C nor affected Cdk1 phosphorylation. The PEITC-induced decline in Cdk1 and Cdc25C protein levels and cell cycle arrest were significantly blocked on pretreatment of PC-3 cells with proteasome inhibitor lactacystin. A 24 h exposure of PC-3 cells to 10 micro M PEITC, but not PITC, resulted in about 56% and 44% decrease in the levels of antiapoptotic proteins Bcl-2 and Bcl-X(L), respectively. However, ectopic expression of Bcl-2 failed to alter sensitivity of PC-3 cells to growth inhibition or apoptosis induction by PEITC. Treatment of cells with PEITC, but not PITC, also resulted in cleavage of procaspase-3, procaspase-9, and procaspase-8. Moreover, the PEITC-induced apoptosis was significantly attenuated in the presence of general caspase inhibitor and specific inhibitors of caspase-8 and caspase-9. In conclusion, our data indicate that PEITC-induced cell cycle arrest in PC-3 cells is likely due to proteasome-mediated degradation of Cdc25C and Cdk1, and ectopic expression of Bcl-2 fails to confer resistance to PEITC-induced apoptosis. Furthermore, the results of the present study point toward involvement of both caspase-8- and caspase-9-mediated pathways in apoptosis induction by PEITC.     

Mifepristone pretreatment overcomes resistance of prostate cancer cells to tumor necrosis factor alpha-related apoptosis-inducing ligand (TRAIL)

Examination of the effects of TRAIL (tumor necrosis factor alpha-related apoptosis-inducing ligand) showed higher apoptotic response in LNCaP C4-2, whereas LNCaP were resistant. However, treatment of LNCaP with Mifepristone, an antiprogestin, before TRAIL induced significant apoptosis, similar to the levels observed in LNCaP C4-2. Experiments to determine the reasons for altered response of the cell lines showed no significant differences in death/decoy receptors and caspase-8 activity. However, treatment induced increased truncation of Bid and activation of caspases -9, -7, and -3 in LNCaP C4-2. Time course experiments showed that caspase-8 was activated before the involvement of mitochondrial pathway, and caspase-9 was responsible for activation of caspases -7 and -3. Use of specific caspase inhibitors demonstrated the presence of a short-loop feedback activation of Bid. Published reports suggested that increased phosphorylation of Akt was responsible for resistance of LNCaP to TRAIL. However, no significant differences were noticed in the levels of phosphorylated Akt in TRAIL-resistant LNCaP and TRAIL-sensitive LNCaP C4-2. On the basis of our results, it is suggested that the differences in response of the two cell lines to TRAIL is at the mitochondrial level.     

Caspase-mediated apoptosis and caspase-independent cell death induced by irofulven in prostate cancer cells

Irofulven (hydroxymethylacylfulvene) is a novel antitumor drug, which acts by alkylating cellular macromolecular targets. The drug is a potent inducer of apoptosis in various types of tumor cells, whereas it is nonapoptotic in normal cells. This study defined molecular responses to irofulven involving mitochondrial dysfunction and leading to death of prostate tumor LNCaP-Pro5 cells. Irofulven caused early (2-5 hours) translocation of the proapoptotic Bax from cytosol to mitochondria followed by the dissipation of mitochondrial membrane potential and cytochrome c release at 4 to 12 hours. These effects preceded caspase activation and during the first 6 hours were not affected by caspase inhibitors. Processing of caspase-9 initiated the caspase cascade at approximately 6 hours and progressed over time. The activation of the caspase cascade provided a positive feedback loop that enhanced Bcl-2-independent translocation and cytochrome c release. General and specific caspase inhibitors abrogated irofulven-induced apoptotic DNA fragmentation with the following order of potency: pan-caspase > or = caspase-9 > caspase-8/6 > caspase-2 > caspase-3/7 > caspase-1/4. Abrogation of caspase-mediated DNA fragmentation failed to salvage irofulven-treated cells from growth inhibition and loss of viability, demonstrating a substantial contribution of a caspase-independent cell death. Monobromobimane, an inhibitor of alternative caspase-independent apoptotic pathway that is mediated by mitochondrial permeability transition, antagonized both apoptosis, measured as phosphatidylserine externalization, and cytotoxicity of irofulven. Collectively, the results indicate that irofulven-induced signaling is integrated at the level of mitochondrial dysfunction. The induction of both caspase-dependent and caspase-independent death pathways is consistent with pleiotropic effects of irofulven, which include targeting of cellular DNA and proteins.     

Silymarin induces apoptosis primarily through a p53-dependent pathway involving Bcl-2/Bax, cytochrome c release, and caspase activation

Silymarin, a plant flavonoid, has been shown to inhibit skin carcinogenesis in mice. However, the mechanism responsible for the anti-skin carcinogenic effects of silymarin is not clearly understood. Here, we report that treatment of JB6 C141 cells (preneoplastic epidermal keratinocytes) and p53+/+ fibroblasts with silymarin and silibinin (a major constituent of silymarin) resulted in a dose-dependent inhibition of cell viability and induction of apoptosis in an identical manner. Silymarin-induced apoptosis was determined by fluorescence staining (8-64% apoptosis) and flow cytometry (12-76% apoptosis). The silymarin-induced apoptosis was primarily p53 dependent because apoptosis occurred to a much greater extent in the cells expressing wild-type p53 (p53+/+, 9-61%) than in p53-deficient cells (p53-/-, 6-20%). The induction of apoptosis in JB6 C141 cells was associated with increased expression of the tumor suppressor protein, p53, and its phosphorylation at Ser15. The constitutive expression of antiapoptotic proteins Bcl-2 and Bcl-xl were decreased after silymarin treatment, whereas the expression of the proapoptotic protein Bax was increased. There was a shift in Bax/Bcl-2 ratio in favor of apoptotic signal in silymarin-treated cells, which resulted in increased levels of cytochrome c release, apoptotic protease-activating factor-1, and cleaved caspase-3 and poly(ADP-ribose) polymerase in JB6 C141 cells. The shift in Bax/Bcl-2 ratio was more prominent in p53+/+ fibroblasts than in p53-/- cells. Silymarin-induced apoptosis was blocked by the caspase inhibitor (Z-VAD-FMK) in JB6 C141 cells which suggested the role of caspase activation in the induction of apoptosis. These observations show that silymarin-induced apoptosis is primarily p53 dependent and mediated through the activation of caspase-3.