Induction of p21CIP/WAF-1 and G2 arrest by ionizing irradiation impedes caspase-3-mediated apoptosis in human carcinoma cells

There is an ongoing controversy regarding the relevance of apoptosis induction by ionizing irradiation as compared with other end points including transient or permanent cell cycle arrest of damaged cells. Here, we show that such permanent cell cycle arrest and apoptosis represent two sides of the same coin. MCF-7 cells fail to express procaspase-3, which results in resistance to apoptosis induced by anticancer drugs. Conversely, restoration of procaspase-3 sensitizes MCF-7 cells to chemotherapeutics including epirubicine, etoposide and taxol. In contrast, irradiation does not trigger apoptotic cell death but results in prolonged arrest in the G2 phase of the cell division cycle regardless of procaspase-3 expression. This suggested that the propensity of MCF-7 cells to arrest at the G2 checkpoint results in resistance to apoptosis upon gamma-irradiation. This G2 arrest was associated with upregulation of p21CIP/WAF-1. Inhibition of DNA-damage-induced stress kinases and p21CIP/WAF-1 expression by caffeine abrogated G2 arrest and induced apoptosis of the irradiated cells in a caspase-3-dependent manner. Inhibition of cell cycle progression by adenoviral expression of the cyclin dependent kinase inhibitor p21CIP/WAF-1 prevented apoptosis upon caffeine treatment indicating that cell cycle progression, that is, G2-release, is required for induction of apoptosis. Likewise, cells homozygously deleted for p21CIP/WAF-1 (HCT116 p21-/-) display enhanced irradiation-induced apoptosis via a caspase-3-dependent mechanism. These data indicate that the disruption of G2 checkpoint control overcomes cell cycle arrest and resistance to gamma-irradiation-induced cell death. Thus, DNA damage may trigger a permanent G2 arrest as an initial inactivation step of tumor cells where the phenomenon of apoptosis is hidden unless cell cycle arrest is overcome. The efficient induction of apoptosis upon G2 release thereby depends on the propensity to activate the key executioner caspase-3. This finding is of crucial importance for the understanding of molecular steps underlying the efficacy of ionizing radiation to delete tumor cells.     

Loss of p21 disrupts p14 ARF-induced G1 cell cycle arrest but augments p14 ARF-induced apoptosis in human carcinoma cells

The human INK4a locus encodes two structurally unrelated tumor suppressor proteins, p16 INK4a and p14 ARF (p19 ARF in the mouse), which are frequently inactivated in human cancer. Both the proapoptotic and cell cycle-regulatory functions of p14 ARF were initially proposed to be strictly dependent on a functional p53/mdm-2 tumor suppressor pathway. However, a number of recent reports have implicated p53-independent mechanisms in the regulation of cell cycle arrest and apoptosis induction by p14 ARF. Here, we show that the G1 cell cycle arrest induced by p14 ARF entirely depends on both p53 and p21 in human HCT116 and DU145 carcinoma cells. In contrast, neither loss of p53 nor p21 impaired apoptosis induction by p14 ARF as evidenced by nuclear DNA fragmentation, phosphatidyl serine exposure, and caspase activation, which included caspase-3/7- and caspase-9-like activities. However, lack of functional p21 resulted in the accumulation of cells in G2/M phase of the cell cycle and markedly enhanced p14 ARF-induced apoptosis that was, nevertheless, efficiently inhibited by the cell permeable broad-spectrum caspase inhibitor zVAD-fmk (valyl-alanyl-aspartyl-(O)-methyl)-fluoromethylketone). Thus, loss of cell cycle restriction point control in the absence of p21 may interfere with p14 ARF-induced apoptosis. Finally, these data indicate that the signaling events required for G1 cell cycle arrest and apoptosis induction by p14 ARF dissociate upstream of p53.     

Resveratrol inhibits oral squamous cell carcinoma through induction of apoptosis and G2/M phase cell cycle arrest

The present study was performed to investigate the effect of resveratrol (trans-3,4′,5-trihydroxystilbene) present as a natural phytoalexin in grapes, peanuts, and red wine on oral squamous cancer cell lines, SCC-VII, SCC-25, and YD-38. MTS assay and flow cytometry, respectively, were used for the analysis of inhibition of cell proliferation and apoptosis. Western blot analysis was performed to examine the effect of resveratrol on the expression of proteins associated with cell cycle regulation. The results revealed a concentration- and time-dependent inhibition of proliferation in all the three tested cell lines on treatment with resveratrol. The IC₅₀ of resveratrol for SCC-VII, SCC-25, and YD-38 cell lines was found to be 0.5, 0.7, and 1.0 μg/ml, respectively, after 48-h treatment. Examination of the cell cycle analysis showed that resveratrol treatment induced cell cycle arrest in the G2/M phase and enhanced the expression of phospho-cdc2 (Tyr 15), cyclin A2, and cyclin B1 in the oral squamous cell carcinoma (OSCC) cells. It also caused a marked increase in the percentage of apoptotic cells as revealed by the fluorescence-activated cell sorting analysis. Thus, resveratrol exhibits inhibitory effect on the proliferation of OSCC oral cancer cells through the induction of apoptosis and G2/M phase cell cycle arrest.     

Cooperative effect of p21Cip1/WAF-1 and 14-3-3sigma on cell cycle arrest and apoptosis induction by p14ARF

P14(ARF) (p19(ARF) in the mouse) plays a central role in the regulation of cellular proliferation. Although the capacity of p14(ARF) to induce a cell cycle arrest in G1 phase depends on a functional p53/p21-signaling axis, the G2 arrest triggered by p14(ARF) is p53/p21-independent. Using isogeneic HCT116 cells either wild-type or homozygously deleted for p21, 14-3-3sigma or both, we further investigated the cooperative effect of p21 and 14-3-3sigma on cell cycle regulation and apoptosis induction by p14(ARF). In contrast to DNA damage, which induces mitotic catastrophe in 14-3-3sigma-deficient cells, we show here that the expression of p14(ARF) triggers apoptotic cell death, as evidenced by nuclear DNA fragmentation and induction of pan-caspase activities, irrespective of the presence or absence of 14-3-3sigma. The activation of the intrinsic mitochondrial apoptosis pathway by p14(ARF) was confirmed by cytochrome c release from mitochondria and induction of caspase-9- (LEHDase) and caspase-3/7-like (DEVDase) activities. Moreover, 14-3-3sigma/p21 double-deficient cells were exceedingly sensitive to apoptosis induction by p14(ARF) as compared to wild-type cells or cells lacking either gene alone. Notably, p14(ARF)-induced apoptosis was preceded by an arrest in the G2 phase of cell cycle, which coincided with downregulation of cdc2 (cdk1) protein expression and lack of its nuclear localization. This indicates that p14(ARF) impairs mitotic entry by targeting the distal DNA damage-signaling pathway and induces apoptotic cell death, rather than mitotic catastrophe, out of a transient G2 arrest. Furthermore, our data delineate that the disruption of G2/M cell cycle checkpoint control critically determines the sensitivity of the cell toward p14(ARF)-induced mitochondrial apoptosis.     

Up-regulation of p21WAF1/Cip1 by saRNA induces G1-phase arrest and apoptosis in T24 human bladder cancer cells

Very recent studies have reported that chemically synthesized small duplex RNAs complementary to the promoters of target genes can activate gene expression in different cancer cell lines. Such dsRNA have been referred to as saRNA for small activating RNA. The present study was conducted to evaluate the potential of p21(WAF1/Cip1) (p21) induction by small activating RNA targeting the p21 promoter in the treatment of bladder cancer. Using T24 human bladder cancer cells, we found that p21 saRNA caused dose- and time-dependent inhibition of cell proliferation and viability which was associated with induced G1-phase cell cycle arrest and apoptosis. The decreased anti-apoptotic protein Bcl-xL and activation of caspase-3 and PARP also supported the efficacy of the treatment. These data suggest that up-regulation of p21 by saRNA may be an effective way for treating human bladder and other types of cancers.     

Adriamycin induced G2/M cell cycle arrest in transitional cell cancer cells with wt p53 and p21(WAF1/CIP1) genes

Adriamycin (ADM), widely used for systemic and local treatment of bladder tumors, triggers apoptosis in bladder cancer cells. Here we investigated the effect of ADM on cell cycle progression and expression of cell cycle regulating proteins in bladder cancer cell lines with various p53 and p21(WAF1/CIP1) status. Flowcytometric analysis was used to estimate the cell cycle distribution of T24, HT-1376, RT4, and SCaBER bladder cancer cell lines. Cell cycle regulating proteins were analyzed by Immunoblot. Treatment of RT4 cells, bearing wild type p53 and p21(WAF1/CIP1), with ADM induced expression of both proteins and cell cycle arrest, not in G1, as was anticipated, but in the G2 phase. Simultaneously, Retinoblastoma (Rb) protein expression was decreased. Expression of PCNA, which is a target gene of E2F, was not changed. The results suggest that even if the tumor cells bear wild type (wt) p53 and wt p21(WAF1/CIP1) and both proteins accumulate due to genotoxic stimuli, the cell cycle arrest might happen not in the G1 but in the G2 phase.     

Bromelain inhibits nuclear factor kappa-B translocation, driving human epidermoid carcinoma A431 and melanoma A375 cells through G(2)/M arrest to apoptosis

Bromelain, obtained from pineapple, is already in use clinically as adjunct in chemotherapy. Our objective was to test its ability to act as a sole anti-cancer agent. Therefore, we describe its anti-proliferative, anti-inflammatory and subsequent anti-cancer effects in vitro, against human epidermoid carcinoma-A431 and melanoma-A375 cells. Bromelain exhibited reduction in proliferation of both these cell-lines and suppressed their potential for anchorage-independent growth. Further, suppression of inflammatory signaling by bromelain was evident by inhibition of Akt regulated-nuclear factor-kappaB activation via suppression of inhibitory-kappaBα phosphorylation and concomitant reduction in cyclooxygenase-2. Since, the inflammatory cascade is well-known to be closely allied to cancer; we studied the effect of bromelain on events/molecules central to it. Bromelain caused depletion of intracellular glutathione and generation of reactive oxygen-species followed by mitochondrial membrane depolarization. This led to bromelain-induced cell-cycle arrest at G(2)/M phase which was mediated by modulation of cyclin B1, phospho-cdc25C, Plk1, phospho-cdc2, and myt1. This was subsequently followed by induction of apoptosis, indicated by membrane-blebbing, modulation of Bax-Bcl-2 ratio, Apaf-1, caspase-9, and caspase-3; chromatin-condensation, increase in caspase-activity and DNA-fragmentation. Bromelain afforded substantial anti-cancer potential in these settings; hence we suggest it as a potential prospect for anti-cancer agent besides only an additive in chemotherapy.     

Down-regulation of miRNA-106b inhibits growth of melanoma cells by promoting G1-phase cell cycle arrest and reactivation of p21/WAF1/Cip1 protein

MiR-106b is overexpressed in various types of cancers and is associated with the regulation of the carcinogenic processes. Using RT-PCR, we have identified overexpression of miRNA-106b in various melanoma cell lines (A375, Hs294t, SK-Mel28, SK-Mel 119, Mel 1241, Mel 1011 and Mel 928) as compared to its expression in normal human epidermal melanocytes (NHEM). The overexpression of miR-106b in melanoma cells (A375, Hs294t) was associated with greater cell proliferation capacity than NHEM. Treatment of A375 and Hs294t cells with anti-miR-106b resulted in inhibition of cell proliferation as well as G1-phase arrest. We determined the effects of grape seed proanthocyanidins (GSPs) on the expression of miRNA-106b and its underlying molecular targets. Treatment of A375 and Hs294t cells with GSPs resulted in suppression of the levels of miRNA-106b, cytotoxicity, G1-phase arrest and reactivation of p21/WAF1/Cip1. Dietary GSPs significantly inhibited growth of A375 melanoma cell tumor xenografts in nude mice, which was associated with reduction in the levels of miRNA-106b, tumor cell proliferation and increases in the levels of p21/WAF1/Cip1 protein. These studies suggest that miRNA-106b plays a crucial role in melanoma growth and that GSPs act as an inhibitor of miR-106b thereby blocking melanoma growth in vitro and in vivo models.     

Triptolide-induced cell cycle arrest and apoptosis in human renal cell carcinoma cells

Renal cell carcinoma (RCC) is the most frequent type of renal-originated malignancy. Although nephrectomy is successfully used to save the lives of patients with localized RCC, treatment of advanced and other refractory RCCs is poor and still inadequate. Here, we show that triptolide, a small molecule and a well-known anti-inflammatory and anti-immunity agent used in the clinic, is capable of inducing cell apoptosis via the mitochondrial pathway in the 786-0 RCC cell line. This induction occurred in concert with reduced expression of genes related to the stabilization of mitochondria such as Bcl-2 and Bcl-XL. Cell cycle analysis showed that exposure to triptolide decreased the proportion of cells in the G0/G1 and G2/M phases, and increased the proportion of cells in the S phase. Cell accumulation in the S phase can be attributed to reduced expression of cell cycle checkpoint regulators such as cyclin A, cyclin B, CDK1, CDK2 and retinoblastoma proteins (Rb). These results raise the possibility that triptolide-induced apoptosis is mediated by cell cycle arrest. Similarly, in another human RCC cell line, OS-RC-2, triptolide-induced apoptosis and cell accumulation in S phase were also observed. Therefore, triptolide emerges as a stimulator of apoptosis by influencing coordinate regulation of proliferation and apoptosis, and may be applicable to the treatment of human renal cell carcinoma.     

5'-nitro-indirubinoxime induces G2/M cell cycle arrest and apoptosis in human KB oral carcinoma cells

Our previous study demonstrated that the novel indirubin derivative, 5'-nitro-indirubinoxime (5'-NIO), effectively arrested the tumor growth through the inhibition of cell proliferation and the induction of apoptosis. However, the precise molecular mechanisms underlying 5'-NIO-induced antitumor activity remain unclear. Here, we report that 5'-NIO inhibits the proliferation of human KB oral carcinoma cells via the cell cycle arrest in G2/M phase. 5'-NIO reduced the activity of Cdc2/cyclin B complex through the inhibition of the PLK1 expression. Partially, 5'-NIO also arrested cell cycle in G1/S phase via the reduction of CDK4 and cyclin D1/D3 levels by p16 and induction of the level of p21waf1. Using flow cytometry analysis, we showed that 5'-NIO-induced cell cycle arrest is followed by apoptosis. We determined further that 5'-NIO-induced apoptosis is accomplished by the mitochondria-dependent activation of the caspase cascade. Overall, these observations suggest the potential value of 5'-NIO as a candidate for a therapeutic modality for the treatment of oral cancer.     

Induction of cell cycle arrest and p21(CIP1/WAF1) expression in human lung cancer cells by isoliquiritigenin

Isoliquiritigenin is a natural flavonoid isolated from licorice, shallot and bean sprouts. The effect of isoliquiritigenin on cell proliferation and cell cycle progression was examined in the A549 human lung cancer cell line. Isoliquiritigenin significantly inhibited the proliferation of lung cancer cells in a dose- and time-dependent manner. Flow cytometric analysis demonstrated that isoliquiritigenin restrained the cell cycle progression at G2/M phase. Further examinations using cDNA arrays and real-time quantitative RT-PCR revealed that isoliquiritigenin enhanced the expression of p21(CIP1/WAF1), a universal inhibitor of cyclin-dependent kinases. These results suggest that isoliquiritigenin will be a promising agent for use in chemopreventive or therapeutics against lung cancer.     

p53-independent G1 cell cycle arrest of human colon carcinoma cells HT-29 by sulforaphane is associated with induction of p21CIP1 and inhibition of expression of cyclin D1

Isothiocyanate sulforaphane (SFN) is a potent cancer chemopreventive agent. We investigated the mechanisms underlying the anti-proliferative effects of SFN in the human colon carcinoma cell line, HT-29. We demonstrate that SFN inhibits the growth of HT-29 cells in a dose- and time-dependent manner. Treatment of serum-stimulated HT-29 cells with SFN suppressed the re-initiation of cell cycle by inducing a G₁ phase cell cycle arrest. At high doses (>25 μM), SFN dramatically induces the expression of p21^CIP1 while significantly inhibits the expression of the G₁ phase cell cycle regulatory genes such as cyclin D1, cyclin A, and c-myc. This regulation can be detected at both the mRNA and protein levels as early as 4 h post-treatment of SFN at 50 μM. Additionally, SFN activates MAPKs pathways, including ERK, JNK and p38. Exposure of HT-29 cells with both SFN and an antioxidant, either NAC or GSH, completely blocked the SFN-mediated activation of these MAPK signaling cascades, regulation of cyclin D1and p21^CIP1 gene expression, and G₁phase cell cycle arrest. This finding suggests that SFN-induced oxidative stress plays a role in these observed effects. Furthermore, the activation of the ERK and p38 pathways by SFN is involved in the upregulation of p21^CIP1 and cyclin D1, whereas the activation of the JNK pathway plays a contradictory role and may be partially involved in the downregulation of cyclin D1. Because cyclin D1 and p21^CIP1 play opposing roles in G₁ phase cell cycle progression regulation, blocking the activation of each MAPK pathway with specific MAPK inhibitors, is unable to rescue the SFN-induced G₁ phase cell cycle arrest in HT-29 cells.     

Tetrandrine-induced cell cycle arrest and apoptosis in A549 human lung carcinoma cells

Tetrandrine, isolated from the root of Stephania tetrandra, has been used in Chinese medicine for the treatment of silicosis and arthritis, and it also has anti-tumor/growth activities. However, the signaling pathways of tetrandrine-induced growth arrest and apoptosis in cancer cells remain unclear. We investigated the molecular mechanisms of tetrandrine-induced apoptosis and growth arrest in human lung carcinoma cells. Upon treatment with tetrandrine, a time-dependent inhibition of cell growth was observed and cells developed many of the hallmark features of apoptosis. Flow cytometry analysis confirmed that tetrandrine increased populations of both apoptotic sub-G1 and G1 phase. Tetrandrine-induced growth inhibition was associated with induction of Cdk inhibitor p21, inhibition of cyclin D1 and activation of caspase-3. Tetrandrine also affected the expression patterns of cytoskeletons including distribution of F-actin and expression level of microtubule. These results suggest that tetrandrine merits further investigation as a cell cycle blocker as well as a cancer chemopreventive agent.     

Rapid induction of p21WAF1 but delayed down-regulation of Cdc25A in the TGF-beta-induced cell cycle arrest of gastric carcinoma cells.

Transforming growth factor-beta (TGF-beta) is a multifunctional polypeptide that inhibits cellular proliferation in most epithelial cells. cdk4 and several cyclin-dependent kinase (cdk) inhibitors (p15INK4B, p21WAF1/Cip1 and p27Kip1) have been implicated in the TGF-beta-induced cell cycle arrest. More recently, down-regulation of Cdc25A, a cdk activator, was additionally suggested as a mechanism underlying growth inhibition by TGF-beta. The existence of diverse cellular mediators of TGF-beta, however, raises the question of whether their involvement might occur in a redundant manner or coordinately in a certain cell type. Using two TGF-beta-sensitive gastric carcinoma cell lines (SNU-16 and -620), we addressed the contributory roles of several cdk inhibitors, and of cdk4 and Cdc25A, in TGF-beta-induced cell cycle arrest by comparing their temporal expression pattern in response to TGF-beta. Among the cdk inhibitors examined, p21 mRNA was most rapidly (in less than 1 h) and prominently induced by TGF-beta. In contrast, p15 mRNA was more slowly induced than p21 in SNU-620 cells, and not expressed in SNU-16 cells harbouring homozygous deletion of p15. Western blotting results confirmed the rapid increase of p21, while opposite patterns of p27 expression were observed in the two cell lines. The down-regulation of Cdc25A mRNA occurred, but was more delayed than that of p15 or p21. Until G1 arrest was established, changes in the protein levels of both Cdc25A and cdk4 were marginal. Co-immunoprecipitation with anti-cdk4 antibody showed that induced p21 associates with cdk4 and that its kinase activity is reduced by TGF-beta, which kinetically correlates closely with G1 arrest following TGF-beta treatment of both cell lines. These results suggest that in certain human epithelial cells, p21 may play an early role in TGF-beta-induced cell cycle arrest, and its cooperation with other cdk inhibitors is different depending on cell type. Delayed down-regulation of Cdc25A and cdk4 may contribute to cell adaptation to the quiescent state in the two gastric carcinoma cell lines studied.     

Inhibition of cell growth and induction of G1-phase cell cycle arrest in hepatoma cells by steroid extract from Meretrix meretrix

In this study, we report that the steroid extract 5alpha, 8alpha-epidioxycholest-6-ene-3beta-ol (MME) from Meretrix meretrix has the ability to inhibit growth of hepatoma cells and to induce G1-phase cell cycle arrest in two human hepatoma cell lines, HepG2 and Hep3B. HepG2 cells were more sensitive than Hep3B to MME. The extract markedly up-regulated the expression of p53 and p21WAF1/CIP1 in HepG2, suggesting that MME-induced G1 phase cell cycle arrest in HepG2 might be p53-dependent. Therefore, the up-regulation of p27KIP1and p16INK4A in both cell lines indicates that a p53-independent pathway might be involved in the mechanism of MME inducing cell cycle arrest. In conclusion, MME induces G1 phase cell cycle arrest via both p53-dependent and p53-independent pathways.     

Activation of ATM/Chk1 by curcumin causes cell cycle arrest and apoptosis in human pancreatic cancer cells

Curcumin has been shown to inhibit the growth of various types of cancer cells; however, at concentrations much above the clinically achievable levels in humans. The concentration of curcumin achieved in the plasma after oral administration in humans was estimated to be around 1.8 μM. Here, we report that treatment of BxPC-3 human pancreatic cancer cells with a low and single exposure of 2.5 μM curcumin for 24 h causes significant arrest of cells in the G2/M phase and induces significant apoptosis. Immunoblot studies revealed increased phosphorylation of H2A.X at Ser-139 and Chk1 at Ser-280 and a decrease in DNA polymerase-β level in curcumin-treated cells. Phosphorylation of H2A.X and Chk1 proteins are an indicator of DNA damage whereas DNA polymerase-β plays a role in the repair of DNA strand breaks. Normal immortalised human pancreatic ductal epithelial (HPDE-6) cells remained unaffected by curcumin treatment. In addition, we also observed a significant increase in the phosphorylation of Chk1 at Ser-345, Cdc25C at Ser-216 and a subtle increase in ATM phosphorylation at Ser-1981. Concomitant decrease in the expressions of cyclin B1 and Cdk1 were seen in curcumin-treated cells. Further, curcumin treatment caused significant cleavage of caspase-3 and PARP in BxPC-3 but not in HPDE-6 cells. Silencing ATM/Chk1 expression by transfecting BxPC-3 cells with ATM or Chk1-specific SiRNA blocked the phosphorylation of ATM, Chk1 and Cdc25C and protected the cells from curcumin-mediated G2/M arrest and apoptosis. This study reflects the critical role of ATM/Chk1 in curcumin-mediated G2/M cell cycle arrest and apoptosis in pancreatic cancer cells.     

Hypothermia causes a reversible, p53-mediated cell cycle arrest in cultured fibroblasts.

Normal human fibroblasts grown in cell culture undergo a reversible growth arrest when incubated at 28 degrees C. During incubation at 28 degrees C, levels of p53 and p21 rise in these cells and cell cycle analysis shows that they have undergone a cell cycle arrest. To examine the importance of p53 in mediating this arrest, mouse embryo fibroblasts that are either wild-type or that are defective in p53 were also subjected to hypothermia. Only those cells with wild-type p53 undergo a cell cycle arrest, indicating that p53 has a role in mediating this response. Because many tumor cells have defective p53, this suggests that hypothermia may increase the selective toxicity of chemotherapeutic agents for tumor cells.