Biologic sequelae of c-Jun NH(2)-terminal kinase (JNK) activation in multiple myeloma cell lines

Although c-Jun NH(2)-terminal kinase (JNK) is activated by treatment with therapeutic agents, the biologic sequelae of inhibiting constitutive activation of JNK has not yet been clarified. In this study, we examine the biologic effect of JNK inhibition in multiple myeloma (MM) cell lines. JNK-specific inhibitor SP600125 induces growth inhibition via induction of G1 or G2/M arrest in U266 and MM.1S multiple myeloma cell lines, respectively. Neither exogenous IL-6 nor insulin-like growth factor-1 (IGF-1) overcome SP600125-induced growth inhibition, and IL-6 enhances SP600125-induced G2/M phase in MM.1S cells. Induction of growth arrest is mediated by upregulation of p27(Kip1), without alteration of p53 and JNK protein expression. Importantly, SP600125 inhibits growth of MM cells adherent to bone marrow stromal cells (BMSCs). SP600125 induces NF-kappaB activation in a dose-dependent fashion, associated with phosphorylation of IkappaB kinase alpha (IKKalpha) and degradation of IkappaBalpha. In contrast, SP600125 does not affect phosphorylation of STAT3, Akt, and/or ERK. IKK-specific inhibitor PS-1145 inhibits SP600125-induced NF-kappaB activation and blocks the protective effect of SP600125 against apoptosis. Our data therefore demonstrate for the first time that inhibiting JNK activity induces growth arrest and activates NF-kappaB in MM cells.     

Bcl-2 overexpression attenuates SP600125-induced apoptosis in human leukemia U937 cells

SP600125 is a specific inhibitor of c-Jun N-terminal kinase (JNK) that is known to strongly induce apoptosis and block cell cycle progression in G₂/M phase. In this study, we demonstrated that treatment of U937 cells with SP600125 resulted in significant G₂/M cell cycle arrest that was due to decreased cyclin B1 and cdc25c protein levels. Moreover, SP600125 promoted LDH release and DNA fragmentation that was associated with caspase-3 activation and degradation of its substrates. In contrast, overexpression of the antiapoptotic protein Bcl-2 rendered leukemia cells resistant to SP600125-induced apoptosis, but more sensitive to G₂/M phase arrest and endoreduplication (>4N DNA). Overexpression of Bcl-2 significantly inhibited SP600125-induced caspase-3 activation and degradation of its substrates, and sustained expression levels of the IAP-2 proteins following SP600125 treatment. The inhibitory effect of Bcl-2 on apoptosis was attenuated by treatment with the small molecule Bcl-2 inhibitor, HA14-1. These data provide important mechanistic insights related to Bcl-2-mediated resistance to SP600125-induced apoptosis, and induction of G₂/M phase arrest and endoreduplication.     

Moscatilin induces apoptosis in human colorectal cancer cells: a crucial role of c-Jun NH2-terminal protein kinase activation caused by tubulin depolymerization and DNA damage

PURPOSE: To study the effect of moscatilin (purified from the stem of orchid Dendrobrium loddigesii) on the proliferation of human colorectal cancer HCT-116 cells in vitro and in vivo. EXPERIMENTAL DESIGN: The growth inhibition of moscatilin was screened on several human cancer cell lines. The effect of moscatilin on tubulin was detected in vitro. Following moscatilin treatment on HCT-116 cells, c-Jun NH(2)-terminal protein kinase (JNK) and caspase activation was studied by Western blot analysis, and DNA damage was done by Comet assay. Specific JNK inhibitor SP600125 was cotreated to reverse moscatilin-induced apoptosis. Tumor growth inhibition of moscatilin was done on HCT-116 xenograft models. RESULTS: Moscatilin induced a time-dependent arrest of the cell cycle at G(2)-M, with an increase of cells at sub-G(1). Moscatilin inhibited tubulin polymerization, suggesting that it might bind to tubulins. Moscatilin also induced the phosphorylation of JNK1/2. SP600125 significantly inhibited the activation of caspase-9 and caspase-3 and the subsequent moscatilin-induced apoptosis. The data suggest that JNK activation may contribute to moscatilin-mediated apoptosis signaling. A parallel experiment showed that SP600125 significantly inhibits Taxol- and vincristine-induced HCT-116 cell apoptosis. This suggests that the JNK activation may be a common mechanism for tubulin-binding agents. Moreover, moscatilin induces DNA damage, phosphorylation of H2AX and p53, and up-regulation of p21. Our HCT-116 xenograft models show the in vivo efficacy of moscatilin. CONCLUSIONS: In summary, our results suggest that moscatilin induces apoptosis of colorectal HCT-116 cells via tubulin depolymerization and DNA damage stress and that this leads to the activation of JNK and mitochondria-involved intrinsic apoptosis pathway.     

Inhibition of JNK reduces G2/M transit independent of p53, leading to endoreduplication, decreased proliferation, and apoptosis in breast cancer cells

c-Jun N-terminal kinase (JNK) is activated by diverse cell stimuli, including stress, growth factors, and cytokines. Traditionally, activation of JNK by stress treatment is thought to induce cell death. However, our recent data indicate that JNK's ability to sensitize cells to apoptosis may be, in part, cell cycle dependent. Here, we show that the majority of both paclitaxel- and UV-induced apoptosis can be inhibited by the pharmacological JNK inhibitor, SP600125, in MCF-7 cells. However, inhibition of JNK does little to reverse doxorubicin-induced apoptosis in MCF-7 cells or doxorubicin- and UV-mediated death in MDA MB-231 cells. SP treatment causes G2/M arrest of three breast cancer cell lines and results in the endoreduplication (cellular DNA content >4N) of MCF-7 and MDA MB-231 cells. These effects on cell cycle and apoptosis are not significantly altered by the inhibition of p53, indicating that JNK is functioning independently of p53. Lastly, inhibition of JNK using both SP and antisense oligonucleotides targeted to JNK1 and JNK2 reduced proliferation of all three breast cancer cell lines. Taken together, these results suggest that the activation of JNK is important for the induction of apoptosis following stresses that function at different cell cycle phases, and that basal JNK activity is necessary to promote proliferation and maintain diploidy in breast cancer cells.     

Induction of apoptosis and cell cycle arrest by a specific c-Jun NH2-terminal kinase (JNK) inhibitor, SP-600125, in gastrointestinal cancers

The c-Jun NH(2)-terminal kinase (JNK) is activated in several tumor cell lines. The aim of this study was to determine the effects of SP-600125, a specific JNK inhibitor, on the viability, apoptosis, cell cycle distribution of gastrointestinal cancer cells, and the potential anti-tumor mechanisms. Three gastric cancer cell lines, AGS, BCG-823 and MKN-45, and three colorectal cancer cell lines, SW1116, COLO205 and HT-29, were used. Cells were treated with SP-600125, and cell viability, apoptosis and cell cycle distribution, caspase-3 activity, expression of JNK and apoptosis related proteins were detected. SP-600125 inhibited cell proliferation by 10-80% for the different cell lines, and increased apoptosis by 1.5-4.5 folds for COLO205, BCG-823, MKN-45, AGS cells. Caspase-8 and caspase-3 were involved in the induction of apoptosis. SP-600125 caused G2/M cell cycle arrest and elevation of cyclin B1 and p27(kip). The differential response in cells to SP-600125 was associated with the basal level of phosphorylated JNK2. It is concluded that SP-600125 inhibits proliferation, induces apoptosis and causes cell cycle arrest in gastrointestinal cancer cells, indicating that JNK inhibitors have an anti-tumor effect and are potential therapeutic agents for cancers.     

c-Jun NH2-terminal kinase-mediated up-regulation of death receptor 5 contributes to induction of apoptosis by the novel synthetic triterpenoid methyl-2-cyano-3,12-dioxooleana-1, 9-dien-28-oate in human lung cancer cells

Death receptor (DR) 4 or 5, on binding to its ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), triggers apoptosis via activating the caspase-8-mediated caspase cascade. Certain anticancer drugs up-regulate the expression of these receptors and thereby induce apoptosis or enhance TRAIL-induced apoptosis. In this study, we explored the ability of methyl-2-cyano-3,12-dioxooleana-1,9-dien-28-oate (CDDO-Me) to activate the extrinsic DR-mediated apoptotic pathway in human lung cancer cells. We found that CDDO-Me not only activated caspase-8 but also induced expression of DRs, particularly DR5, in a p53-independent mechanism. Correspondingly, CDDO-Me augmented TRAIL-induced apoptosis in these cells regardless of p53 status as evidenced by enhanced DNA fragmentation and activation of caspase cascades, suggesting that CDDO-Me-induced DRs are functionally active. Moreover, silencing of DR5 expression using small interfering RNA suppressed apoptosis induced by CDDO-Me alone or by combination of CDDO-Me and TRAIL, indicating that DR5 up-regulation is required for induction of apoptosis by CDDO-Me and for enhancement of TRAIL-induced apoptosis by CDDO-Me. CDDO-Me rapidly activated c-Jun NH(2)-terminal kinase (JNK) before DR up-regulation and caspase-8 activation. Moreover, application of the JNK-specific inhibitor SP600125 blocked CDDO-Me-induced increases in JNK activation, DR up-regulation, caspase-8 activation, and DNA fragmentation. These results show that activation of JNK pathway results in CDDO-Me-induced DR up-regulation, caspase-8 activation, and apoptosis. Collectively, we conclude that CDDO-Me induces apoptosis via the JNK-mediated DR up-regulation in human lung cancer cells.     

A link between benzyl isothiocyanate-induced cell cycle arrest and apoptosis: involvement of mitogen-activated protein kinases in the Bcl-2 phosphorylation

In the present study, we clarified the molecular mechanism underlying the relationship between benzyl isothiocyanate (BITC)-induced cell cycle arrest and apoptosis and the involvement of mitogen-activated protein kinases (MAPKs). The exposure of Jurkat human T-cell leukemia cells to BITC resulted in the inhibition of the G(2)-M progression that coincided with the apoptosis induction. The experiment using the phase-specific synchronized cells demonstrated that the G(2)-M phase-arrested cells are more sensitive to undergoing apoptotic stimulation by BITC than the cells in other phases. We also confirmed that BITC activated c-Jun N-terminal kinase (JNK) and p38 MAPK, but not extracellular signal-regulated kinase, at the concentration required for apoptosis induction. An experiment using a JNK-specific inhibitor SP600125 or a p38 MAPK inhibitor SB202190 indicated that BITC-induced apoptosis might be regulated by the activation of these two kinases. Conversely, BITC is likely to confine the Jurkat cells in the G(2)-M phase mainly through the p38 MAPK pathway because only the p38 MAPK inhibitor significantly attenuated the accumulation of inactive phosphorylated Cdc2 protein and the G(2)-M-arrested cell numbers. We reported here for the first time that the antiapoptotic Bcl-2 protein was phosphorylated by the BITC treatment without significant alteration of the Bcl-2 total protein amount. This was abrogated by a JNK specific inhibitor SP600125 at the concentration required for specific inhibition of the c-Jun phosphorylation. Moreover, the spontaneous phosphorylation of antiapoptotic Bcl-2 in the G(2)-M synchronized cells was enhanced synergistically by the BITC treatment. Involvement of the MAPK activation in the Bcl-2 phosphorylation and apoptosis induction also was observed in HL-60 and HeLa cells. Thus, we identified the phosphorylated Bcl-2 as a key molecule linking the p38 MAPK-dependent cell cycle arrest with the JNK activation by BITC.     

Mechanisms of 2-methoxyestradiol-induced apoptosis and G2/M cell-cycle arrest of nasopharyngeal carcinoma cells

2-Methoxyestradiol (2ME2) is an endogenous metabolite of 17beta-estradiol (E(2)). This study aims to examine the anti-tumour activities of 2ME2 on the poorly differentiated HONE-1 NPC cell line. At the concentration of 1muM, 2ME2 was found to induce a short-term reversible G2/M cell-cycle arrest. Further 10-fold increase to 10muM, 2ME2 induced both irreversible G2/M phase cell-cycle arrest and apoptosis. Induction of apoptosis and G2/M cell-cycle arrest was due to oxidative stress as both apoptosis and the proportion of cells arresting at G2/M phase could be reduced by the superoxide dismutase (SOD) mimetic, TEMPO. Induction of apoptosis was accompanied with proteolytic cleavage of caspase-9 and -3, but not caspase-8. Kinetics studies revealed that 2ME2 induced a time-dependent inhibition of extracellular signal-regulated protein kinase (ERK) and an activation of c-jun N-terminal kinases (JNKs). The chemical inhibitor of JNKs, SP600125, was found to reduce 2ME2-induced apoptosis of the HONE-1 cells. Confocal microscopy revealed that the induction of G2/M cell-cycle arrest was associated with the presence of immunoreactivity of p-cdc2 (Tyr15) in the nucleus. The G2/M cell-cycle arrest is also correlated with an increased level of inactive p-cdc25C (Ser216) in 2ME2-treated HONE-1 cells. Results from this study indicate that production of superoxide anions might be involved in 2ME2-induced apoptosis and G2/M cell-cycle arrest of the HONE-1 cells.     

Oral RKS262 reduces tumor burden in a neuroblastoma xenograft animal model and mediates cytotoxicity through SAPK/JNK and ROS activation in vitro

Patients diagnosed with high-risk neuroblastoma (NB), an extracranial solid tumor in children, have metastases and low survival (30%) despite aggressive multi-modal therapy. Therefore new therapies are urgently needed. We show significant in vitro and in vivo antitumor efficacy of RKS262 in NB. RKS262 showed superior cytotoxicity (IC(50) = 6-25 μM) against six representative NB cell lines compared to its parent analog Nifurtimox (currently in phase 2). Pre-formulated RKS262 (150 mg/kg/daily) pellets administered orally, suppressed tumor growth (60%, p = 0.021) in NB xenograft mice within 28 days. RKS262-treated SMSKCNR cells showed TUNEL-positive DNA nicks and activation of ROS, MAPKs (SAPK/JNK), caspase-3, and p53, along with suppression of the IGF-1R/PI3K/PKC pathway and the Bcl2 family of proteins. RKS262 caused G(2)/M-phase arrest and suppressed cdc-2, cyclin B1, p21, and cyclin D1/D4 expression. N-acetyl-cysteine (NAC; 10 mM) pre-treatment rescued cell viability of RKS262 (23 μM)-treated SMSKCNR cells, and pre-treatment with ascorbic acid (100 μM) and a MAPK inhibitor SB203580 (20 μM) reversed SAPK/JNK, caspase-3 activation, PARP-1 cleavage, and suppression of IGF-1R, PI3K, and PKC phosphorylation. Further, treatment with exogenous BDNF (50 nM) did not suppress SAPK/JNK or ROS activation due to RKS262. Rather, BDNF (50 nM), EGF (100 nM) and IGF-1 (100 nM) co-treatment with RKS262 induced a remarkable S-phase arrest rather than a G(2)/M phase arrest when RKS262 was used alone. In summary, RKS262 shows oral efficacy in NB xenograft animals, and induces apoptosis in vitro in SMSKCNR cells via cell cycle arrest, MAPK and ROS activation, and suppression of IGF-1R/PI3K/PKC and Bcl2 family proteins in a growth factor (BDNF/EGF/IGF-1)-independent fashion.     

TPA-induced p21 expression augments G2/M arrest through a p53-independent mechanism in human breast cancer cells

The tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), has a differential role on the regulation of the cell cycle in a variety of tumor cells. The mechanism between TPA and the cell cycle in breast cancer is not fully understood. Therefore, we investigated the regulatory mechanism of TPA on control of the cell cycle of breast cancer cells. Our results showed that TPA increased the level of p21 expression in MCF-7 cells with wild-type p53 and MDA-MB-231 cells with mutant p53 in a dose-dependent manner. In contrast, TPA decreased the expression of p53 in MCF-7 cells, but did not affect MDA-MB-231 cells. We next examined the regulatory mechanism of TPA on p21 and p53 expression. Our results showed that the TPA-induced up-regulation of p21 and down-regulation of p53 was reversed by UO126 (a MEK1/2 inhibitor), but not by SP600125 (a JNK inhibitor) or SB203580 (a p38 inhibitor), although TPA increased the phosphorylation of ERK and JNK in MCF-7 cells. In addition, the TPA-induced arrest of the G2/M phase was also recovered by UO126 treatment. To confirm the expression of p21 through the MEK/ERK pathway, cells were transfected with constitutively active (CA)-MEK adenovirus. Our results showed that the expression of p21 was significantly increased by CA-MEK overexpression. Taken together, we suggest that TPA reciprocally regulates the level of p21 and p53 expression via a MEK/ERK-dependent pathway. The up-regulation of p21 in response to TPA is mediated through a p53-independent mechanism in breast cancer cells.     

丝裂原活化蛋白激酶信号转导系统对Vp-16诱导分化作用的影响

目的:探讨丝裂原活化蛋白激酶(mitogen-acti-vated protein kinases,MAPKs)信号转导系统对依托泊苷(Vp-16)诱导K562细胞分化作用的影响。方法:采用四甲基偶氮唑盐(MTT)法测定细胞增殖活性;流式细胞仪解析细胞周期;硝基四氮唑蓝(NBT)还原实验检测细胞向单核/巨噬系统分化。结果:0·1~0·8μg/mL的Vp-16抑制K562细胞增殖,引起细胞G2/M期阻滞,诱导细胞向单核/巨噬系统分化;细胞外信号调节激酶(extracellular signal-regulated kinases,ERK)抑制剂PD98059降低Vp-16的诱导分化作用,P<0·05;p38丝裂原活化蛋白激酶(p38mitogen-activated protein kina-ses,p38MAPK)抑制剂SB203580增强Vp-16的作用,P<0·05;而C-JUN氨基末端激酶(c-jun N-terminal ki-nases,JNK)抑制剂SP600125对Vp-16的诱导分化作用无明显影响,P>0·05。结论:在Vp-16诱导K562细胞向单核/巨噬系统分化过程中,ERK正向,p38MAPK负向调节Vp-16的诱导分化作用。     

Overcoming the radioresistance of prostate cancer cells with a novel Bcl-2 inhibitor

Bcl-2 overexpression is an important mechanism underlying the aggressive behavior of prostate cancer cells and their resistance to radio- or chemotherapy. HA14-1, a recently discovered organic Bcl-2 inhibitor, potently induces apoptosis in various human cancer cells. Sequential exposure of radioresistant LNCaP (wild-type (wt) p53), LNCaP/Bcl-2 (wt p53) and PC3 (mutant p53) prostate cancer cells to a minimally cytotoxic concentration of 10 microM HA14-1 for 1 h followed by 1-6 Gy gamma radiation, resulted in a highly synergistic (combination index <1.0) induction of cell death as determined by an apoptosis assay at 72 h, and a clonogenicity assay at 12 days, after the initial treatment. The reverse treatment sequence did not cause a synergistic induction of cell death. When compared to individual treatments, cell death induced by the combined treatment was associated with dramatically increased reactive oxygen species (ROS) generation, c-Jun N-terminal kinase (JNK) activation, Bcl-2 phosphorylation, cytochrome c release, caspase-3 activation and DNA fragmentation. Exposure to either 200 microg/ml of the antioxidant alpha-tocopherol or 10 microM JNK inhibitor SP600125 before the combined treatment resulted in decreased activation of JNK and caspase-3 as well as decreased DNA fragmentation. However, treatment with the pancaspase inhibitor carbobenzoxyl-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone before the combined treatment inhibited apoptosis without affecting JNK activation, and this inhibitory effect was enhanced in the presence of alpha-tocopherol or SP600125. Taken together, our results indicate that HA14-1 potently sensitizes radioresistant LNCaP and PC3 cells to gamma radiation, regardless of the status of p53. ROS and JNK are important early signals that trigger both caspase-dependent and -independent cell death pathways and contribute to the apoptotic synergy induced by the combined treatments.     

Jaceosidin induces p53-dependent G2/M phase arrest in U87 glioblastoma cells

Flavonoid compounds have been shown to trigger cell cycle arrest at G0/G1, S and G2/M checkpoints, allowing cells to repair DNA damage before entry into mitosis. Jaceosidin, a flavonoid compound, has been reported to induce apoptosis in various cancer cell lines. In our previous study, we established that jaceosidin induces apoptosis in U87 glioblastoma cells through G2/M phase arrest. However the molecular mechanisms oremained unclear. In the present study, mRNA and protein expression levels of major cell cycle regulatory genes were analyzed by semi-quantitative RT-PCR and Western blot studies respectively. The results demonstrated that jaceosidin-induced G2/M phase arrest in U87 cells is associated with DNA fragmentation, up-regulation of p53 and p21 and subsequent down-regulation of cyclin B1 and CDK1 expression at mRNA as well as at protein level. These findings provide insights into jaceosidin-induced G2/M phase arrest in U87 glioblastoma cells.     

The importance of drug scheduling and recovery phases in determining drug activity. Improving etoposide efficacy in BCR-ABL-positive CML cells

K562 leukaemic cells are known to be less sensitive to etoposide than other cell lines, despite having similar topo II mRNA levels and cleavable complex formation. We have investigated the effect of etoposide schedule on cell cycle distribution, apoptosis and p21(waf1) and cdk1(p34) status in two bcr-abl-positive chronic myeloid leukaemia (CML) cell lines (K562 and KU812) and two small cell lung cancer (SCLC) cell lines (H69 and GLC4). During a continuous 5-day exposure, the SCLC cell lines showed a time and concentration-dependent loss of cell viability, with an initial block in the G2/M phase of the cell cycle followed by apoptosis. In contrast, the two CML cell lines showed no significant apoptosis or loss of viability after a similar block in G2/M. However, when K562 or KU812 cells were placed in drug-free medium following a 3-day drug exposure there was marked, concentration-dependent apoptosis (% apoptosis after release at 1 microM etoposide in K562, 10% at 24 h, 30% at 48 h). Our data also show that p21(waf1) does not increase after etoposide treatment in either H69 or GLC4 (both with mutated-p53). Although K562 and KU812 cells are null-p53, the arrest in G2/M during drug exposure was associated with increased p21(waf1) and a decrease in cdk1 (both P<0.001 compared with controls). Upon release of these cells from drug-medium, p21(waf1) gradually returned to control levels, which was associated with an easing of the block at G2/M and an induction of apoptosis. This study highlights the importance of cell cycle regulatory proteins in drug sensitivity and resistance, and suggests that in cells such as K562 and KU812, a pulsed schedule may be more active than a single prolonged exposure.     

Knockdown of a novel lincRNA AATBC suppresses proliferation and induces apoptosis in bladder cancer

Long intergenic noncoding RNAs (lincRNAs) play important roles in regulating various biological processes in cancer, including proliferation and apoptosis. However, the roles of lincRNAs in bladder cancer remain elusive. In this study, we identified a novel lincRNA, which we termed AATBC. We found that AATBC was overexpressed in bladder cancer patient tissues and positively correlated with tumor grade and pT stage. We also found that inhibition of AATBC resulted in cell proliferation arrest through G1 cell cycle mediated by cyclin D1, CDK4, p18 and phosphorylated Rb. In addition, inhibition of AATBC induced cell apoptosis through the intrinsic apoptosis signaling pathway, as evidenced by the activation of caspase-9 and caspase-3. The investigation for the signaling pathway revealed that the apoptosis following AATBC knockdown was mediated by activation of phosphorylated JNK and suppression of NRF2. Furthermore, JNK inhibitor SP600125 could attenuate the apoptotic effect achieved by AATBC knockdown, confirming the involvement of JNK signaling in the induced apoptosis. Moreover, mouse xenograft model revealed that knockdown of AATBC led to suppress tumorigenesis in vivo. Taken together, our study indicated that AATBC might play a critical role in pro-proliferation and anti-apoptosis in bladder cancer by regulating cell cycle, intrinsic apoptosis signaling, JNK signaling and NRF2. AATBC could be a potential therapeutic target and molecular biomarker for bladder cancer.     

SP600125对人宫颈癌HeLa细胞增殖和侵袭的影响

目的 探讨SP600125对人宫颈癌HeLa细胞的增殖周期、凋亡以及侵袭的影响.方法 采用CCK-8法检测不同时间点不同浓度的SP600125作用后HeLa细胞的增殖状态.确定20μmol/L的SP600125用于后续实验.利用平板克隆形成实验检测细胞增殖能力,DAPI染色观察细胞核形态,流式细胞仪检测细胞周期和凋亡,...     

Caspase-mediated Cdk2 activation is a critical step to execute transforming growth factor-beta1-induced apoptosis in human gastric cancer cells

Although TGF-beta1, a growth inhibitor, is known to also induce apoptosis, the molecular mechanism of this apoptosis is largely undefined. Here, we identify the mechanism of TGF-beta1-induced apoptosis in SNU-16 human gastric cancer cells. Cell cycle and TUNEL analysis showed that, upon TGF-beta1 treatment, cells were initially arrested at the G1 phase and then driven into apoptosis. Of note, caspase-3 was activated in accordance with TGF-beta1-induced G1 arrest. Activated caspase-3 is targeted to cleave p21(cip1), p27(kip1), and Rb, which play important roles in TGF-beta-induced G1 arrest, into inactive fragments. Subsequently, Cdk2 was aberrantly activated due to the cleavage of p21 and p27. We found that the inhibition of Cdk2 activity efficiently blocks TGF-beta1-induced apoptosis, whereas it did not prevent caspase-3 activation or the subsequent cleavage of target proteins. In contrast, the suppression of caspase-3 activity inhibited the cleavage of target proteins, the activation of Cdk2, and the induction of apoptosis. Taken together, our results suggest that activation of caspase-3 by TGF-beta1 may initiate the conversion from G1 cell cycle arrest to apoptosis via the cleavage of p21, p27 and Rb, which in turn causes Cdk2 activation and, most significantly, Cdk2 activation as a downstream effector of caspase is a critical step for the execution of TGF-beta1-induced apoptosis.