The kinase Mirk/Dyrk1B mediates cell survival in pancreatic ductal adenocarcinoma

Ductal adenocarcinoma of the pancreas is almost uniformly lethal as this cancer is invariably detected at an advanced stage and is resistant to treatment. The serine/threonine kinase Mirk/Dyrk1B has been shown to be antiapoptotic in rhabdomyosarcomas. We have now investigated whether Mirk might mediate survival in another cancer in which Mirk is widely expressed, pancreatic ductal adenocarcinoma. Mirk was an active kinase in each pancreatic cancer cell line where it was detected. Mirk knockdown by RNA interference (RNAi) reduced the clonogenicity of Panc1 pancreatic cancer cells 4-fold and decreased tumor cell number, showing that Mirk mediates survival in these cells. Mirk knockdown by synthetic duplex RNAis in Panc1, AsPc1, and SU86.86 pancreatic cancer cells induced apoptosis and enhanced the apoptosis induced by gemcitibine. Mirk knockdown did not increase the abundance or activation of Akt. However, four of five pancreatic carcinoma cell lines exhibited either elevated Mirk activity or elevated Akt activity, suggesting that pancreatic cancer cells primarily rely on Mirk or Akt for survival signaling. Mirk protein was detected by immunohistochemistry in 25 of 28 cases (89%) of pancreatic ductal adenocarcinoma, with elevated expression in 11 cases (39%). Increased expression of Mirk was seen in pancreatic carcinomas compared with primary cultures of normal ductal epithelium by serial analysis of gene expression and by immunohistochemistry. Thus, Mirk is a survival factor for pancreatic ductal adenocarcinoma. Because knockout of Mirk does not cause embryonic lethality, Mirk is not essential for normal cell growth and may represent a novel therapeutic target.     

BRCA1-mediated G2/M cell cycle arrest requires ERK1/2 kinase activation

Germline mutations in the BRCA1 gene are associated with an increased susceptibility to the development of breast and ovarian cancers. Evidence suggests that BRCA1 protein plays a key role in mediating DNA damage-induced checkpoint responses. Several studies have shown that ectopic expression of BRCA1 in human cells can trigger cellular responses similar to those induced by DNA damage, including G2/M cell cycle arrest and apoptosis. While the effects of ectopic BRCA1 expression on the G2/M transition and apoptosis have been extensively studied, the factors that dictate the balance between these two responses remain poorly understood. We have recently shown that ectopic expression of BRCA1 in MCF-7 human breast cancer cells resulted in activation of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) and G2/M cell cycle arrest. Furthermore, inhibition of BRCA1-induced ERK1/2 activation using mitogen-activated protein kinase kinase 1 and 2 (MEK1/2)-specific inhibitors resulted in increased apoptosis, suggesting a potential role of ERK1/2 kinases in BRCA1-mediated G2/M checkpoint response. In this study, we assessed the role of ERK1/2 kinases in the regulation of BRCA1-mediated G2/M cell cycle arrest. Results indicate that BRCA1-induced G2/M cell cycle arrest and ERK1/2 activation correlate with changes in the level and/or activity of several key regulators of the G2/M checkpoint, including activation of Chk1 and Wee1 kinases, induction of 14-3-3, and down-regulation of Cdc25C. Furthermore, inhibition of ERK1/2 kinases using MEK1/2-specific inhibitors results in a marked attenuation of the BRCA1-induced G2/M arrest. Biochemical studies established that ERK1/2 inhibition abolished the effects of BRCA1 on components of the G2/M checkpoint, including regulation of Cdc25C expression and activation of Wee1 and Chk1 kinases. These results implicate a critical role of ERK1/2 signaling in the regulation of BRCA1 function on controlling the G2/M checkpoint responses.     

Quinoxaline 1,4-dioxides induce G2/M cell cycle arrest and apoptosis in human colon cancer cells

We have recently shown that quinoxaline 1,4-dioxide (QdNO) derivatives, namely 2-benzoyl-3-phenyl-6,7-dichloroquinoxaline 1,4-dioxide (DCQ), 2-benzoyl-3-phenyl-quinoxaline 1,4-dioxide (BPQ) and 2-acetyl-3-methyl-quinoxaline 1,4-dioxide (AMQ), suppress the growth of T-84 human colon cancer cells. Here we show that the growth-suppressive effects of QdNOs are due to their ability to induce cell cycle arrest and/or apoptosis. While AMQ blocked more than 60% of cells at the G₂/M phase without inducing apoptosis, DCQ caused a significant increase in apoptotic cells with no noticeable effects on the cycling of cells. Treatment with BPQ resulted in G₂/M cell cycle arrest and induction of apoptosis. With regard to the effects of QdNOs on molecules that regulate apoptosis and the G₂ to M transition, both BPQ and AMQ inhibited the expression of cyclin B, while DCQ significantly decreased the levels of Bcl-2 and increased Bax expression. Next, we investigated whether transforming growth factor-beta1 (TGF-1) and/or extracellular signal-regulated kinase (ERK) mediate the antiproliferative and apoptotic effects of QdNOs in colon cancer cells. Interestingly, the above QdNOs increased differentially total TGF1 mRNA expression and decreased TGF mRNA and ERK phosphorylation. None of these QdNOs induced changes in TGF-2 mRNA expression. The addition of a specific inhibitor of MEK greatly enhanced apoptosis in cells treated with DCQ, suggesting that the inhibition of ERK phosphorylation may explain, to an extent, the apoptogenic effects of this compound. Taken together, these findings provide insights into possible molecular mechanisms of growth inhibition by QdNOs that could aid in their evaluation for anticancer therapy.     

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.     

Griseofulvin potentiates antitumorigenesis effects of nocodazole through induction of apoptosis and G2/M cell cycle arrest in human colorectal cancer cells

In this study, we demonstrate that apoptosis and G2/M cell cycle arrest were easily induced by treatment with the oral-antifungal agent, griseofulvin (GF). The mechanisms of GF-induced G2/M arrest were characterized as (a) induction of abnormal mitotic spindle formation, (b) elevation of cyclin B1/cdc2 kinase activity and (c) down-regulation of myt-1 protein expression. On the other hand, caspase 3 activation, Bcl-2 hyperphosphorylation and inhibition of the normal function of Bcl-2 associated with Bax were demonstrated to be the mechanisms of GF-induced apoptosis. DNA fragmentation and flow cytometry analyses demonstrated that combined treatment of GF with the cancer chemotherapeutic agent, nocodazole (ND), strongly potentiates the apoptotic effect and arrest of the G2/M cell cycle in 5 types of human cancer cells, but not in normal human keratinocytes (#76 KhGH). The combined treatment of GF and ND triggered the polymerization of purified tubulin in HT 29 but not in #76 KhGH cells. To further confirm these observations, the therapeutic efficacy was further examined in vivo by treating athymic mice bearing COLO 205 tumor xenografts, with GF (50 mg/kg), ND (5 mg/kg) or GF + ND. Combined treatment of GF and ND significantly enhanced the effect of ND, and led to cessation of tumor growth. These results suggest that chemotherapeutic agents (such as ND) administered in the presence of GF might provide a novel therapy for colorectal cancer.     

Genistein inactivates bcl-2, delays the G2/M phase of the cell cycle, and induces apoptosis of human breast adenocarcinoma MCF-7 cells

The aim of this study was to identify the molecular mechanism of action of the isoflavone, genistein. Genistein at 0.15 mM caused MCF-7 apoptotic cell death, which was accompanied by cell cycle delay in the G2/M phase. Twenty-four hours post-treatment, 47.3% of the MCF-7 cells accumulated at G2/M, compared with 19.9% in the untreated controls. At 0.15 mM, genistein caused an increase in the steady-state levels of the wild-type tumour suppressor p53, which was attributed to stabilising the tumour suppressor protein, since p53 mRNA levels did not increase. Prior to the upregulation of p53, which became evident within 6 h of genistein treatment, there was increased bcl-2 phosphorylation at 30 min post-treatment. Although early changes (30-120 min) in the phosphotyrosine peptide patterns were not detected, after 24h, genistein inhibited phosphorylation of several peptides. These results suggest that genistein's dual roles of protein tyrosine kinase inhibitor and topoisomerase II inhibitor are essential for the initiation of apoptosis.     

Apigenin inhibits pancreatic cancer cell proliferation through G2/M cell cycle arrest

Background  

Many chemotherapeutic agents have been used to treat pancreatic cancer without success. Apigenin, a naturally occurring flavonoid, has been shown to inhibit growth in some cancer cell lines but has not been studied in pancreatic cancer. We hypothesized that apigenin would inhibit pancreatic cancer cell growth in vitro.     

Expression of AIE-75 PDZ-domain protein induces G2/M cell cycle arrest in human colorectal adenocarcinoma SW480 cells

AIE-75 has been known as a 75-kDa autoantigen detected in the serum of autoimmune enteropathy (AIE) and as a colon cancer-related antigen, and now designated as a gene causative of Usher syndrome type 1C hereditary syndromic hearing loss. It binds to a novel putative tumor suppressor MCC2 that is homologous to MCC (mutated in colon cancer) through a PSD-95/Dlg/ZO-1 (PDZ) domain. To clarify the functional role in colon cancer cells, we transfected AIE-75 gene into SW480 colon cancer cells which do not express AIE-75. Expression of AIE-75 suppressed growth of SW480 cells in vitro in correlation with the expression levels. It was due mainly to G2/M phase cell cycle arrest associated with mitotic slippage, resulting in emergence of hyperploid giant-nucleated or multi-nucleated cells. Screening of proteins that bound to PDZ domains of AIE-75 by a yeast two hybrid system showed that three serine/threonine phosphatase catalytic subunits (PP2AC-alpha, PP2AC-beta, and PPP6C) could bind to AIE-75. Since PP2AC is known to regulate G2/M checkpoint, we suggest that AIE-75 interacts with PP2AC and prevent cells to transit mitotic phase.     

下调PAR基因表达对前列腺癌PC3细胞周期及Cdc25C蛋白表达的影响

背景与目的:前列腺雄激素调节基因(prostate androgen regulated gene,PAR)在雄激素非依赖性前列腺癌中特异性高表达,可能与细胞恶性转化相关。本研究用RNA干扰技术下调PAR基因表达,并探讨其对前列腺癌PC3细胞细胞周期及调节因子Cdc25C表达的影响。方法:针对PAR基因的siRNA转染PC3细胞,以RT-PCR验证其对PAR基因表达抑制的有效性,用流式细胞术检测细胞周期,Western blot检测Cdc25C表达水平。结果:siRNA转染可抑制PC3细胞PAR基因表达,同时G2/M期细胞及凋亡细胞比例分别由(23.79±3.16)%及(1.49±0.13)%增加至(29.95±3.25)%和(20.61±2.73)%,Cdc25C蛋白表达水平明显下降。结论:PAR基因表达下调可诱导细胞G2/M期阻滞和凋亡,抑制细胞周期调节因子Cdc25C的表达。     

G2/M cell cycle arrest and apoptosis induced by COH-203 in human promyelocytic leukemia HL-60 cells

The combretastatin A-4/oltipraz hybrid (COH), 5-(3-amino-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-3H−1,2-dithiole-3-one (COH-203) is one of the COH compounds synthesized by our previous study, which has been reported to affect a number of cancer cell lines, such as SGC-7901, KB, HT-1080, HepG2, SMMC-7721 and BEL-7402. The sensitivity of human acute leukemia cell lines to COH-203, and the mechanism underlying its anti-proliferative effects remain unknown, which was investigated in the present study. In the present study, it was demonstrated that COH-203 had notable time- and dose-dependent antiproliferative effects on the human acute promyelocytic leukemia HL-60 cell line. Furthermore, COH-203 treatment resulted in cell cycle arrest at G₂/M phase in a dose-dependent manner, and subsequently induced apoptosis. Western blot analysis revealed that upregulation of cyclin B was associated with G₂/M arrest. In addition, treatment with COH-203 resulted in downregulated expression of Bcl-2. This result revealed that COH-203-induced apoptosis in HL-60 cells may occur via the mitochondrial pathway in a caspase-dependent manner.     

Methyl protodioscin induces G2/M cell cycle arrest and apoptosis in HepG2 liver cancer cells

Methyl protodioscin (NSC-698790) is one of the main bioactive components in the traditional Chinese medicine Dioscorea collettii var. hypoglauca (Dioscoreaceae). In this study, we investigated the anti-proliferative effect of methyl protodioscin on the HepG2 cells and the mechanism of the induced cytotoxicity. Treatment of methyl protodioscin resulted in G2/M arrest and apoptosis in HepG2 cells. These effects were attributed to down-regulation of Cyclin B1 and the signaling pathways leading to up-regulation of Bax and down-regulation of BCL2, suggesting that methyl protodioscin may be a novel anti-mitotic agent.     

Delta9-tetrahydrocannabinol inhibits cell cycle progression in human breast cancer cells through Cdc2 regulation

It has been proposed that cannabinoids are involved in the control of cell fate. Thus, these compounds can modulate proliferation, differentiation, and survival in different manners depending on the cell type and its physiopathologic context. However, little is known about the effect of cannabinoids on the cell cycle, the main process controlling cell fate. Here, we show that Delta(9)-tetrahydrocannabinol (THC), through activation of CB(2) cannabinoid receptors, reduces human breast cancer cell proliferation by blocking the progression of the cell cycle and by inducing apoptosis. In particular, THC arrests cells in G(2)-M via down-regulation of Cdc2, as suggested by the decreased sensitivity to THC acquired by Cdc2-overexpressing cells. Of interest, the proliferation pattern of normal human mammary epithelial cells was much less affected by THC. We also analyzed by real-time quantitative PCR the expression of CB(1) and CB(2) cannabinoid receptors in a series of human breast tumor and nontumor samples. We found a correlation between CB(2) expression and histologic grade of the tumors. There was also an association between CB(2) expression and other markers of prognostic and predictive value, such as estrogen receptor, progesterone receptor, and ERBB2/HER-2 oncogene. Importantly, no significant CB(2) expression was detected in nontumor breast tissue. Taken together, these data might set the bases for a cannabinoid therapy for the management of breast cancer.     

Induction of G2/M phase cell cycle arrest by carnosol and carnosic acid is associated with alteration of cyclin A and cyclin B1 levels

Carnosol and carnosic acid, two antioxidant polyphenols present in Rosmarinus officinalis (rosemary), were investigated for their antiproliferative properties toward Caco-2 cells. Twenty hours of treatment with both carnosol and carnosic acid inhibited 3H-thymidine incorporation in a dose-dependent manner, with a 50% inhibitory concentration of 23 microM and significantly increased the doubling time of Caco-2 cells from 29.5 to 140 and 120 h, respectively. These effects were associated with accumulation of treated cells in the G2/M phase of the cell cycle. Carnosol was found to exert its major cell cycle effect after prometaphase, and caused an increase in cyclin B1 protein levels whereas carnosic acid arrested cells prior to prometaphase, and caused a reduction in cyclin A levels. These structurally related phytochemicals, therefore, appear to arrest cells at different phases of the cell cycle possibly through influencing the levels of different cyclin proteins.     

Paclitaxel induced apoptosis in breast cancer cells requires cell cycle transit but not Cdc2 activity

Purpose Paclitaxel (PTX) is a widely used chemotherapy agent and may cause cell death by apoptosis subsequent to microtubule (MT) disruption. In this paper, we have investigated whether cell cycle transit and or Cdc2 (Cdk1) activity is required for the apoptosis induced by PTX.Methods Cell cycle was analyzed by flow cytometry, Cdc2 was assayed bio chemically. Cdc2 activity was decreased by siRNA and dominant negative (dn) Cdc2 expression. Cells were arrested by chemical or biological inhibitors in a G₁or S phase. Apoptosis was measured by DNA fragmentation and examination of nuclei by microscopy. JNK and AKT activations were assessed as well.Results Cell cycle inhibition was highly effective in decreasing PTX induced apoptosis. MT morphology was not altered by these inhibitors. PTX induced JNK activity or AKT mediated BAD phosphorylation was unaffected by cell cycle inhibitors. Abrogation of Cdc 2 activity was without effect on PTX induced apoptosis.Conclusions While cell cycle transit is required for PTX induced apoptosis; Cdc2 activity is not required.     

Expression of survivin, a novel inhibitor of apoptosis and cell cycle regulatory protein, in pancreatic adenocarcinoma

Survivin is unique for its expression in human malignancies but not in normal adult cells. It has been implicated in sensitisation to chemotherapy and as a prognostic marker in several common cancers. Immunohistochemistry for Survivin, P53 and BCL-2 expression as well as cell proliferative index (Ki-67) and apoptosis index (TUNEL) was conducted on 52 pancreatic and 12 ampullary adenocarcinomas. Survivin was detected in the cytoplasm of carcinoma cells in 46 (88%) of pancreatic tumours. P53 and BCL-2 were detected in 54% and 12% of pancreatic tumours, respectively. Proliferative index was 26.2+/-10.5% and apoptosis index was 1.38+/-0.69%. Prevalence of Survivin expression was significantly higher in P53-positive than in P53-negative cases (P=0.05) but was not associated with BCL-2 expression. Incrementally higher weighted scores of Survivin expression were associated with increased proliferative index (P=0.001). Furthermore, there was linear correlation between increased proliferative index and higher apoptosis index (P<0.001). Surprisingly, higher scores of Survivin expression were associated with increased apoptosis index (P=0.007). Survival characteristics were not influenced by Survivin, P53 or BCL-2 expression, apoptosis index or proliferative index. Ampullary carcinoma showed Survivin expression in 83% of cases. However, unlike pancreatic carcinoma, there was no correlation between Survivin and P53 expression or proliferative index. In conclusion, Survivin is expressed in the majority of pancreatic adenocarcinomas and correlates with both cellular proliferation and apoptosis. Molecular manipulation of Survivin expression may enhance chemotherapy and radiation therapy for pancreatic cancer.     

Genistein inhibits radiation-induced activation of NF-κB in prostate cancer cells promoting apoptosis and G2/M cell cycle arrest

Background  

New cancer therapeutic strategies must be investigated that enhance prostate cancer treatment while minimizing associated toxicities. We have previously shown that genistein, the major isoflavone found in soy, enhanced prostate cancer radiotherapy in vitro and in vivo. In this study, we investigated the cellular and molecular interaction between genistein and radiation using PC-3 human prostate cancer cells.     

Calcium/calmodulin-dependent kinase inhibitor induces growth inhibition, cell cycle arrest, and apoptosis in human choriocarcinoma cells

KN-93, a membrane-permeant calcium/calmodulin- dependent kinase-selective inhibitor, induces apoptosis in some lines of human tumor cells. We investigated the effect of KN-93 in the choriocarcinoma cell line, BeWo. BeWo cells were treated with various concentrations of KN-93, and changes in cell growth, the cell cycle, apoptosis, and related parameters were examined. A WST-1 assay showed that BeWo cells were sensitive to the growth inhibitory effect of KN-93. Cell cycle analysis indicated that exposure to KN-93 decreased the proportion of cells in the S phase and increased the proportion in the G0/G1 phases of the cell cycle. Induction of apoptosis was confirmed by Annexin V staining of externalized phosphatidylserine, by the loss of mitochondrial transmembrane potential, and by antibodies directed against histones from fragmented DNA. This induction occurred in conjunction with the altered expression of genes related to cell growth, malignant phenotype, and apoptosis. These results suggest that KN-93 may serve as a therapeutic agent for the treatment of choriocarcinoma.     

Induction of apoptosis and G2/M cell cycle arrest by DCC.

The Deleted in Colorectal Cancer gene (DCC) encodes a cell surface receptor that belongs to the Ig superfamily. Inactivation of the DCC gene has been implicated in human tumor progression. However, little is known about the biological function of the DCC protein. In the present study, we demonstrated that expression of DCC activated caspase-3 and programmed cell death, or induced G2/M cell cycle arrest in tumor cells. In some cell lines, apoptosis was evident within 24 h of DCC expression. Timing of the appearance of apoptotic cells coincided with that of the cleavage of poly (ADP-ribose) polymerase, a substrate of caspase-3. Expression of the apoptosis inhibitory gene Bcl-2 was not able to abrogate the DCC-induced apoptosis. In the G2/M cycle arrest cells, cdk1 activity was inhibited. Our results suggest that the DCC protein may transduce signals resulting in activation of caspases or inhibition of Cdk1. These data provide a possible mechanism by which DCC suppresses tumorigenesis.