Asparanin A induces G2/M cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells

We recently established that asparanin A, a steroidal saponin extracted from Asparagus officinalis L., is an active cytotoxic component. The molecular mechanisms by which asparanin A exerts its cytotoxic activity are currently unknown. In this study, we show that asparanin A induces G₂/M phase arrest and apoptosis in human hepatocellular carcinoma HepG2 cells. Following treatment of HepG2 cells with asparanin A, cell cycle-related proteins such as cyclin A, Cdk1 and Cdk4 were down-regulated, while p21^WAF1/Cip1 and p-Cdk1 (Thr14/Tyr15) were up-regulated. Additionally, we observed poly (ADP-ribose) polymerase (PARP) cleavage and activation of caspase-3, caspase-8 and caspase-9. The expression ratio of Bax/Bcl-2 was increased in the treated cells, where Bax was also up-regulated. We also found that the expression of p53, a modulator of p21^WAF1/Cip1 and Bax, was not affected in asparanin A-treated cells. Collectively, our findings demonstrate that asparanin A induces cell cycle arrest and triggers apoptosis via a p53-independent manner in HepG2 cells. These data indicate that asparanin A shows promise as a preventive and/or therapeutic agent against human hepatoma.     

LYG-202 inhibits the proliferation of human colorectal carcinoma HCT-116 cells through induction of G1/S cell cycle arrest and apoptosis via p53 and p21(WAF1/Cip1) expression

We recently established that LYG-202, a new flavonoid with a piperazine substitution, exerts an anti-tumor effect in vivo and in vitro. In the present study, we demonstrate that LYG-202 induces G1/S phase arrest and apoptosis in human colorectal carcinoma HCT-116 cells. Data showed that the blockade of the cell cycle was associated with increased p21(WAF1/Cip1) and Rb levels and reduced expression of cyclin D1, cyclin E, and CDK4. Moreover, PARP cleavage, activation of caspase-3, caspase-8, and caspase-9, and an increased ratio of Bax/Bcl-2 were detected in LYG-202-induced apoptosis. Additionally, activation of p53 resulted in the up-regulation of its downstream targets PUMA and p21(WAF1/Cip1), as well as the down-regulation of its negative regulator MDM2, suggesting that the p53 pathway may play a crucial role in LYG-202-induced cell cycle arrest and apoptosis. Furthermore, siRNA knockdown of p53 attenuated the G1 cell cycle arrest and apoptosis induced by LYG-202, as the effects of LYG-202 on up-regulation of p21(WAF1/Cip1) and down-regulation of Bcl-2 and pro-caspase-3 were partly inhibited in p53 siRNA transfected cells compared with control siRNA transfected cells. Collectively, these data indicate that LYG-202 exerts its anti-tumor potency by activating the p53-p21 pathway for G1/S cell cycle arrest and apoptosis in colorectal cancer cells.     

Grape proanthocyanidins induce apoptosis by loss of mitochondrial membrane potential of human non-small cell lung cancer cells in vitro and in vivo

Lung cancer remains the leading cause of cancer-related deaths worldwide, and non-small cell lung cancer (NSCLC) represents approximately 80% of total lung cancer cases. The use of non-toxic dietary phytochemicals can be considered as a chemotherapeutic strategy for the management of the NSCLC. Here, we report that grape seed proanthocyanidins (GSPs) induce apoptosis of NSCLC cells, A549 and H1299, in vitro which is mediated through increased expression of pro-apoptotic protein Bax, decreased expression of anti-apoptotic proteins Bcl2 and Bcl-xl, disruption of mitochondrial membrane potential, and activation of caspases 9, 3 and poly (ADP-ribose) polymerase (PARP). Pre-treatment of A549 and H1299 cells with the caspase-3 inhibitor (z-DEVD-fmk) significantly blocked the GSPs-induced apoptosis of these cells confirmed that GSPs-induced apoptosis is mediated through activation of caspases-3. Treatments of A549 and H1299 cells with GSPs resulted in an increase in G1 arrest. G0/G1 phase of the cell cycle is known to be controlled by cyclin dependent kinases (Cdk), cyclin-dependent kinase inhibitors (Cdki) and cyclins. Our western blot analyses showed that GSPs-induced G1 cell cycle arrest was mediated through the increased expression of Cdki proteins (Cip1/p21 and Kip1/p27), and a simultaneous decrease in the levels of Cdk2, Cdk4, Cdk6 and cyclins. Further, administration of 50, 100 or 200 mg GSPs/kg body weight of mice by oral gavage (5 d/week) markedly inhibited the growth of s.c. A549 and H1299 lung tumor xenografts in athymic nude mice, which was associated with the induction of apoptotic cell death, increased expression of Bax, reduced expression of anti-apoptotic proteins and activation of caspase-3 in tumor xenograft cells. Based on the data obtained in animal study, human equivalent dose of GSPs was calculated, which seems affordable and attainable. Together, these results suggest that GSPs may represent a potential therapeutic agent for the non-small cell lung cancer.     

Induction of G2/M phase arrest and apoptosis by a novel enediyne derivative, THDA, in chronic myeloid leukemia (K562) cells

We studied the effect of 2-(6-(2-thieanisyl)-3(Z)-hexen-1,5-diynyl)aniline(THDA), a newly developed anti-cancer agent, on cell proliferation, cell cycle progression, and induction of apoptosis in K562 cells. THDA was found to inhibit the growth of K562 cells in a time-and dose-dependent manner. Cell cycle analysis showed G2/M phase arrest and apoptosis in K562 cells following 24 h exposure to THDA. During the G2/M arrest, cyclin-dependent kinase inhibitors (CDKIs), p21 and p27 were increased in a time-dependent manner. Analysis of the cell cycle regulatory proteins demonstrated that THDA did not change the steady-state levels of cyclin B1, cyclin D3 and Cdc25C, but decreased the protein levels of Cdk1, Cdk2 and cyclin A. THDA also caused a marked increase in apoptosis, which was associated with activation of caspase-3 and proteolytic cleavage of poly (ADP-ribose) polymerase. These molecular alterations provide an insight into THDA-caused growth inhibition, G2/M arrest and apoptotic death of K562 cells.     

Helicobacter pylori γ-glutamyltranspeptidase induces cell cycle arrest at the G1-S phase transition

In our previous study, we showed that Helicobacter pylori γ-glutamyltranspeptidase (GGT) is associated with H. pylori-induced apoptosis through a mitochondrial pathway. To better understand the role of GGT in apoptosis, we examined the effect of GGT on cell cycle regulation in AGS cells. To determine the effect of recombinant GGT (rGGT) on cell cycle distribution and apoptosis, rGGT-treated and untreated AGS cells were analyzed in parallel by flow cytometry using propidium iodide (PI). We found that rGGT inhibited the growth of AGS cells in a time-dependent manner, and that the pre-exposure of cells to a caspase-3 inhibitor (z-DEVD-fmk) effectively blocked GGT-induced apoptosis. Cell cycle analysis showed G1 phase arrest and apoptosis in AGS cells following rGGT treatment. The rGGT-mediated G1 phase arrest was found to be associated with down-regulation of cyclin E, cyclin A, Cdk 4, and Cdk 6, and the up-regulation of the cyclindependent kinase (Cdk) inhibitors p27 and p21. Our results suggest that H. pylori GGT induces cell cycle arrest at the G1-S phase transition.     

Hyperoxia induces S-phase cell-cycle arrest and p21(Cip1/Waf1)-independent Cdk2 inhibition in human carcinoma T47D-H3 cells

Little is known about cell-cycle checkpoint activation by oxidative stress in mammalian cells. The effects of hyperoxia on cell-cycle progression were investigated in asynchronous human T47D-H3 cells, which contain mutated p53 and fail to arrest at G1/S in response to DNA damage. Hyperoxic exposure (95% O(2), 40-64 h) induced an S-phase arrest associated with acute inhibition of Cdk2 activity and DNA synthesis. In contrast, exit from G2/M was not inhibited in these cells. After 40 h of hyperoxia, these effects were partially reversible during recovery under normoxic conditions. The inhibition of Cdk2 activity was not due to degradation of Cdk2, cyclin E or A, nor impairment of Cdk2 complex formation with cyclin A or E and p21(Cip1). The loss of Cdk2 activity occurred in the absence of induction and recruitment of cdk inhibitor p21(Cip1) or p27(Kip1) in cyclin A/Cdk2 or cyclin E/Cdk2 complexes. In contrast, Cdk2 inhibition was associated with increased Cdk2-Tyr15 phosphorylation, increased E2F-1 recruitment, and decreased PCNA contents in Cdk2 complexes. The latter results indicate a p21(Cip1)/p27(Kip1)-independent mechanism of S-phase checkpoint activation in the hyperoxic T47D cell model investigated.     

Receptor for advanced glycation end products inhibits proliferation in osteoblast through suppression of Wnt, PI3K and ERK signaling

We previously reported the potential of a novel small molecule 3-amino-6-(3-methoxyphenyl)thieno[2.3-b]pyridine-2-carboxamide (SKLB70326) as an anticancer agent. In the present study, we investigated the anticancer effects and possible mechanisms of SKLB70326 in vitro. We found that SKLB70326 treatment significantly inhibited human hepatic carcinoma cell proliferation in vitro, and the HepG2 cell line was the most sensitive to its treatment. The inhibition of cell proliferation correlated with G(0)/G(1) phase arrest, which was followed by apoptotic cell death. The SKLB70326-mediated cell-cycle arrest was associated with the downregulation of cyclin-dependent kinase (CDK) 2, CDK4 and CDK6 but not cyclin D1 or cyclin E. The phosphorylation of the retinoblastoma protein (Rb) was also observed. SKLB70326 treatment induced apoptotic cell death via the activation of PARP, caspase-3, caspase-9 and Bax as well as the downregulation of Bcl-2. The expression levels of p53 and p21 were also induced by SKLB70326 treatment. Moreover, SKLB70326 treatment was well tolerated. In conclusion, SKLB70326, a novel cell-cycle inhibitor, notably inhibits HepG2 cell proliferation through the induction of G(0)/G(1) phase arrest and subsequent apoptosis. Its potential as a candidate anticancer agent warrants further investigation.     

Noscapine induces mitochondria-mediated apoptosis in human colon cancer cells in vivo and in vitro

Noscapine, a phthalide isoquinoline alkaloid derived from opium, has been widely used as a cough suppressant for decades. Noscapine has recently been shown to potentiate the anti-cancer effects of several therapies by inducing apoptosis in various malignant cells without any detectable toxicity in cells or tissues. However, the mechanism by which noscapine induces apoptosis in colon cancer cells remains unclear. The signaling pathways by which noscapine induces apoptosis were investigated in colon cancer cell lines treated with various noscapine concentrations for 72 h, and a dose-dependent inhibition of cell viability was observed. Noscapine effectively inhibited the proliferation of LoVo cells in vitro (IC(50)=75 μM). This cytotoxicity was reflected by cell cycle arrest at G(2)/M and subsequent apoptosis, as indicated by increased chromatin condensation and fragmentation, the upregulation of Bax and cytochrome c (Cyt-c), the downregulation of survivin and Bcl-2, and the activation of caspase-3 and caspase-9. Moreover, in a xenograft tumor model in mice, noscapine injection clearly inhibited tumor growth via the induction of apoptosis, which was demonstrated using a TUNEL assay. These results suggest that noscapine induces apoptosis in colon cancer cells via mitochondrial pathways. Noscapine may be a safe and effective chemotherapeutic agent for the treatment of human colon cancer.     

NCX-4016, a nitro-derivative of aspirin,inhibits EGFR and STAT3 signaling and mdulates Bcl-2 proteins in cisplatin-resistant human ovarian cancer cells and xenografts

We have previously reported the inhibitory effect of NCX-4016, a nitro derivative of aspirin, on the proliferation of cisplatin-resistant human ovarian cancer cells, in vitro (Bratasz et al., Proc Natl Acad Sci USA 2006; 103:3914-9). In this report we present the results of our study on the mechanistic aspects of drug action including the molecular and signaling pathways involved in an in vitro cell line, as well as in a murine tumor xenograft. We report, for the first time, that NCX-4016 significantly inhibited the growth of cisplatin-resistant human ovarian cancer xenografts in mice. We observed that the inhibitory effect of NCX-4016 on cell proliferation was associated with G1 phase cell-cycle arrest with increased activity of p53, p21 and p27 proteins. NCX-4016 modulated the Bcl-2 family of proteins, and induced apoptosis by activating Bax and cytochrome c release in a time-dependent manner. In addition, NCX-4016 selectively down-regulated the phosphorylated forms of EGFR (Tyr845, Tyr992), pAkt (Ser473, Thr305), and STAT3 (Tyr705, Ser727), in vitro and in vivo. Taken together, the results clearly suggested that NCX-4016 causes significant induction of cell-cycle arrest and apoptosis in cisplatin-resistant human ovarian cancer cells via down-regulation of EGFR/PI3K/STAT3 signaling and modulation of Bcl-2 family proteins. Thus, NCX-4016 appears to be a potential therapeutic agent for treating recurrent human ovarian carcinoma.     

Cdk5 activator-binding protein C53 regulates apoptosis induced by genotoxic stress via modulating the G2/M DNA damage checkpoint

In response to DNA damage, the cellular decision of life versus death involves an intricate network of multiple factors that play critical roles in regulation of DNA repair, cell cycle, and cell death. DNA damage checkpoint proteins are crucial for maintaining DNA integrity and normal cellular functions, but they may also reduce the effectiveness of cancer treatment. Here we report the involvement of Cdk5 activator p35-binding protein C53 in regulation of apoptosis induced by genotoxic stress through modulating Cdk1-cyclin B1 function. C53 was originally identified as a Cdk5 activator p35-binding protein and a caspase substrate. Importantly, our results demonstrated that C53 deficiency conferred partial resistance to genotoxic agents such as etoposide and x-ray irradiation, whereas ectopic expression of C53 rendered cells susceptible to multiple genotoxins that usually trigger G(2)/M arrest. Furthermore, we found that Cdk1 activity was required for etoposide-induced apoptosis of HeLa cells. Overexpression of C53 promoted Cdk1 activity and nuclear accumulation of cyclin B1, whereas C53 deficiency led to more cytoplasmic retention of cyclin B1, suggesting that C53 acts as a pivotal player in modulating the G(2)/M DNA damage checkpoint. Finally, C53 and cyclin B1 co-localize and associate in vivo, indicating a direct role of C53 in regulating the Cdk1-cyclin B1 complex. Taken together, our results strongly indicate that in response to genotoxic stress, C53 serves as an important regulatory component of the G(2)/M DNA damage checkpoint. By overriding the G(2)/M checkpoint-mediated inhibition of Cdk1-cyclin B1 function, ectopic expression of C53 may represent a novel approach for chemo- and radio-sensitization of cancer cells.     

New alternative phosphorylation sites on the cyclin dependent kinase 1/cyclin a complex in p53-deficient human cells treated with etoposide: possible association with etoposide-induced apoptosis

Cell cycle arrest is a major cellular response to DNA damage preceding the decision to repair or die. Many malignant cells have non-functional p53 rendering them more “aggressive” in nature. Arrest in p53-negative cells occurs at the G2M cell cycle checkpoint. Failure of DNA damaged cells to arrest at G2 results in entry into mitosis and potential death through aberrant mitosis and/or apoptosis. The pivotal kinase regulating the G2M checkpoint is Cdk1/cyclin B whose activity is controlled by phosphorylation. The p53-negative myeloid leukemia cell lines K562 and HL-60 were used to determine Cdk1 phosphorylation status during etoposide treatment. Cdk1 tyrosine 15 phosphorylation was associated with G2M arrest, but not with cell death. Cdk1 tyrosine 15 phosphorylation also led to suppression of nuclear cyclin B-associated Cdk1 kinase activity. However cell death, associated with broader tyrosine phosphorylation of Cdk1 was not attributed to tyrosine 15 alone. This broader phosphoryl isoform of Cdk1 was associated with cyclin A and not cyclin B. Alternative phosphorylations sites were predicted as tyrosines 4, 99 and 237 by computer analysis. No similar pattern was found on Cdk2. These findings suggest novel Cdk1 phosphorylation sites, which appear to be associated with p53-independent cell death following etoposide treatment.     

CIL-102 interacts with microtubule polymerization and causes mitotic arrest following apoptosis in the human prostate cancer PC-3 cell line

There have been no therapeutic agents that provide a survival advantage in hormone-refractory prostate cancer. Recently, the Food and Drug Administration approved docetaxel combined with prednisone for the treatment of patients with advanced metastatic prostate cancer, and it does show a survival benefit. Hence, anti-microtubule drugs might be of benefit in chemotherapy of hormone-refractory prostate cancer. We used metastatic hormone-refractory prostate cancer PC-3 cells to investigate potential molecular mechanisms for CIL-102, a semisynthetic alkaloid derivative. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide and sulforhodamine B assays indicated that CIL-102 inhibits cell growth dose-dependently. Immunofluorescence microscopy and in vitro tubulin assembly assays indicated that CIL-102 binds to tubulin and disrupts microtubule organization. Flow cytometry showed that CIL-102 causes cells to accumulate in G(2)/M phase and sub-G(0)/G(1) phase. CIL-102-induced apoptosis was also characterized by immunofluorescence microscopy. Western blotting and kinase assays showed that CIL-102 exposure induced up-regulation of cyclin B1 and p34(cdc2) kinase activity and olomoucine, a p34(cdc2) inhibitor, profoundly reduced the number of cells accumulated in mitotic phase. Moreover, Bcl-2 phosphorylation, Cdc25C phosphorylation, and survivin expression were increased. CIL-102-induced apoptosis was associated with activation of caspase-3, but a noncaspase pathway may also be involved, since benzyloxycarbonyl-VAD-fluoromethyl ketone, a pancaspase inhibitor, only partially inhibited the apoptosis, and apoptosis-inducing factor was translocated from mitochondria to cytosol. We conclude that CIL-102 induces mitotic arrest and apoptosis by binding to tubulin and inhibiting tubulin polymerization. CIL-102 causes mitotic arrest, at least partly, by modulating cyclin-dependent kinases and then apoptosis executed by caspase and noncaspase pathways.     

Sulindac-derived reactive oxygen species induce apoptosis of human multiple myeloma cells via p38 mitogen activated protein kinase-induced mitochondrial dysfunction

Non-steroidal anti-inflammatory drugs are well known to induce apoptosis of cancer cells independent of their ability to inhibit cyclooxygenase-2, but the molecular mechanism for this effect has not yet been fully elucidated. The purpose of this study was to elucidate the potential signaling components underlying sulindac-induced apoptosis in human multiple myeloma (MM) cells. We found that sulindac induces apoptosis by promoting ROS generation, accompanied by opening of mitochondrial permeability transition pores, release of cytochrome c and apoptosis inducing factor from mitochondria, followed by caspase activation. Bcl-2 cleavage and down-regulation of the inhibitor of apoptosis proteins (IAPs) family including cIAP-1/2, XIAP, and survivin, occurred downstream of ROS production during sulindac-induced apoptosis. Forced expression of survivin and Bcl-2 blocked sulindac-induced apoptosis. Most importantly, sulindac-derived ROS activated p38 mitogen-activated protein kinase and p53. SB203580, a p38 mitogen-activated protein kinase inhibitor, and RNA inhibition of p53 inhibited the sulindac-induced apoptosis. Furthermore, p53, Bax, and Bak accumulated in mitochondria during sulindac-induced apoptosis. All of these events were significantly suppressed by SB203580. Our results demonstrate a novel mechanism of sulindac-induced apoptosis in human MM cells, namely, accumulation of p53, Bax, and Bak in mitochondria mediated by p38 MAPK activation downstream of ROS production.     

Cellular, biochemical, and genetic analysis of mechanism of small molecule IAP inhibitors

XIAP is member of the IAP family of anti-apoptotic proteins and is known for its ability to bind and suppress caspase family cell death proteases. A phenylurea series of chemical inhibitors of XIAP was recently generated by our laboratories (Schimmer, A. D., Welsh, K., Pinilla, C., Bonneau, M., Wang, Z., Pedersen, I. M., Scott, F. L., Glinsky, G. V., Scudiero, D. A., Sausville, E., Salvesen, G., Nefzi, A., Ostresh, J. M., Houghten, R. A., and Reed, J. C. (2004) Cancer Cell 5, 25-35). We examined the mechanisms of action of these chemical compounds using biochemical, molecular biological, and genetic methods. Active phenylurea-based compounds dissociated effector protease caspase-3 but not initiator protease caspase-9 from XIAP in vitro and restored caspase-3 but not caspase-9 enzymatic activity. When applied to tumor cell lines in culture, active phenylurea-based compounds induced apoptosis in a rapid, concentration-dependent manner, associated with activation of cellular caspases. Apoptosis induced by active phenylurea-based compounds was blocked by chemical inhibitors of caspases, with inhibitors of downstream effector caspases displaying more effective suppression than inhibitors of upstream initiator caspases. Phenylurea-based XIAP antagonists induced apoptosis (defined by annexin V staining) prior to mitochondrial membrane depolarization, in contrast to cytotoxic anticancer drugs. Consistent with these findings, apoptosis induced by phenylurea-based compounds was not altered by genetic alterations in the expression of Bcl-2 family proteins that control mitochondria-dependent cell death pathways, including over-expression of anti-apoptotic proteins Bcl-2 or Bcl-X(L) and genetic ablation of pro-apoptotic proteins Bax and Bak. Conversely, conditional over-expression of an active fragment of XIAP or genetic ablation of XIAP expression altered the apoptosis dose-response of the compounds. Altogether, these findings indicate that phenylurea-based XIAP antagonists block interaction of downstream effector caspases with XIAP, thus inducing apoptosis of tumor cell lines through a caspase-dependent, Bcl-2/Bax-independent mechanism.     

Phosphorylation of Bcl-2 in G2/M phase-arrested cells following photodynamic therapy with hypericin involves a CDK1-mediated signal and delays the onset of apoptosis

The role of Bcl-2 in photodynamic therapy (PDT) is controversial, and some photosensitizers have been shown to induce Bcl-2 degradation with loss of its protective function. Hypericin is a naturally occurring photosensitizer with promising properties for the PDT of cancer. Here we show that, in HeLa cells, photoactivated hypericin does not cause Bcl-2 degradation but induces Bcl-2 phosphorylation in a dose- and time-dependent manner. Bcl-2 phosphorylation is induced by sublethal PDT doses; increasing the photodynamic stress promptly leads to apoptosis, during which Bcl-2 is neither phosphorylated nor degraded. Bcl-2 phosphorylation involves mitochondrial Bcl-2 and correlates with the kinetics of a G(2)/M cell cycle arrest, preceding apoptosis. The co-localization of hypericin with alpha-tubulin and the aberrant mitotic spindles observed following sublethal PDT doses suggest that photodamage to the microtubule network provokes the G(2)/M phase arrest. PDT-induced Bcl-2 phosphorylation is not altered by either the overexpression or inhibition of p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun NH(2)-terminal protein kinase 1 (JNK1) nor by inhibiting the extracellular signal-regulated kinases (ERKs) or protein kinase C. By contrast, Bcl-2 phosphorylation is selectively suppressed by the cyclin-dependent protein kinase (CDK)-inhibitor roscovitine, completely blocked by the protein synthesis inhibitor cycloheximide and enhanced by the overexpression of CDK1, suggesting a role for this pathway. However, in an in vitro kinase assay, active CDK1/cyclin B1 complex failed to phosphorylate immunoprecipitated Bcl-2, suggesting that this protein kinase may not directly modify Bcl-2. Mutation of serine-70 to alanine in Bcl-2 abolishes PDT-induced phosphorylation and restores the caspase-3 activation to the same levels of the vector-transfected cells, indicating that Bcl-2 phosphorylation may be a signal to delay apoptosis in G(2)/M phase-arrested cells.