A novel anticancer agent, decursin, induces G1 arrest and apoptosis in human prostate carcinoma cells

We isolated a coumarin compound decursin (C(19)H(20)O(5); molecular weight 328) from Korean angelica (Angelica gigas) root and characterized it by spectroscopy. Here, for the first time, we observed that decursin (25-100 micromol/L) treatment for 24 to 96 hours strongly inhibits growth and induces death in human prostate carcinoma DU145, PC-3, and LNCaP cells. Furthermore, we observed that decursinol [where (CH(3))(2)-C=CH-COO- side chain of decursin is substituted with -OH] has much lower effects compared with decursin, suggesting a possible structure-activity relationship. Decursin-induced growth inhibition was associated with a strong G(1) arrest (P < 0.001) in DU145 and LNCaP cells, and G(1), S as well as G(2)-M arrests depending upon doses and treatment times in PC-3 cells. Comparatively, decursin was nontoxic to human prostate epithelial PWR-1E cells and showed only moderate growth inhibition and G(1) arrest. Consistent with G(1) arrest in DU145 cells, decursin strongly increased protein levels of Cip1/p21 but showed a moderate increase in Kip1/p27 with a decrease in cyclin-dependent kinases (CDK); CDK2, CDK4, CDK6, and cyclin D1, and inhibited CDK2, CDK4, CDK6, cyclin D1, and cyclin E kinase activity, and increased binding of CDK inhibitor (CDKI) with CDK. Decursin-caused cell death was associated with an increase in apoptosis (P < 0.05-0.001) and cleaved caspase-9, caspase-3, and poly(ADP-ribose) polymerase; however, pretreatment with all-caspases inhibitor (z-VAD-fmk) only partially reversed decursin-induced apoptosis, suggesting the involvement of both caspase-dependent and caspase-independent pathways. These findings suggest the novel anticancer efficacy of decursin mediated via induction of cell cycle arrest and apoptosis selectively in human prostate carcinoma cells.     

Molecular mechanisms for apigenin-induced cell-cycle arrest and apoptosis of hormone refractory human prostate carcinoma DU145 cells

Development of effective agents for treatment of hormone-refractory prostate cancer has become a national medical priority. We have reported recently that apigenin (4',5,7-trihydroxyflavone), found in many common fruits and vegetables, has shown remarkable effects in inhibiting cell growth and inducing apoptosis in many human prostate carcinoma cells. Here we demonstrate the molecular mechanism of inhibitory action of apigenin on androgen-refractory human prostate carcinoma DU145 cells that have mutations in the tumor suppressor gene p53 and pRb. Treatment of cells with apigenin resulted in a dose- and time-dependent inhibition of growth, colony formation, and G1 phase arrest of the cell cycle. This effect was associated with a marked decrease in the protein expression of cyclin D1, D2, and E and their activating partner, cyclin-dependent kinase (cdk)2, 4, and 6, with concomitant upregulation of WAF1/p21, KIP1/p27, INK4a/p16, and INK4c/p18. The induction of WAF1/p21 and its growth inhibitory effects by apigenin appears to be independent of p53 and pRb status of these cells. Apigenin treatment also resulted in alteration in Bax/Bcl2 ratio in favor of apoptosis, which was associated with the release of cytochrome c and induction of apoptotic protease-activating factor-1 (Apaf-1). This effect was found to result in a significant increase in cleaved fragments of caspase-9, -3, and poly(ADP-ribose) polymerase (PARP). Further, apigenin treatment resulted in downmodulation of the constitutive expression of nuclear factor-kappaB (NF-kappaB)/p65 and NF-kappaB/p50 in the nuclear fraction that correlated with an increase in the expression of IkappaB-alpha (IkappaBalpha) in the cytosol. Taken together, we concluded that molecular mechanisms during apigenin-mediated growth inhibition and induction of apoptosis in DU145 cells was due to (1) modulation in cell-cycle machinery, (2) disruption of mitochondrial function, and (3) NF-kappaB inhibition.     

Cell cycle regulatory protein expression in fresh acute myeloid leukemia cells and after drug exposure.

Characteristics of treatment-induced cell cycle arrest are important for in vitro and in vivo sensitivity of acute myeloid leukemia (AML) cells to cytotoxic drugs. We analyzed the expression of the major G1 cell cycle regulators (p21Cip1, p27Kip1, cyclins D, cyclin E and pRb) in 41 fresh AML cell samples. The level of p27 expression was the only factor correlated with the response to chemotherapy, a high level of p27 expression being predictive of complete remission. There was a close relation between expression of pRb, cyclin D2 and FAB subtype, illustrated by the absence of both proteins in most samples having a monocytic component (M4, M5). We also assessed the expressions of pRb, cyclin E, p21 and p27 and the activity of cdk2, the major regulator of S-phase entry, after exposure to cytosine-arabinoside (AraC) and daunorubicin (DNR), and found these proteins could characterize time- and dose-dependent cellular response to each drug. We observed hyperphosphorylated pRb, increased levels of cyclin E and a high cdk2 activity, but no p21 induction, in AML cells exposed to 10(-6) M AraC. After exposure to 10(-5) M AraC, corresponding to the serum concentration reached in high-dose AraC regimens (HDAraC), a strong p21 induction was observed, associated with similarly overexpressed cyclin E and even higher cdk2 activity than after 10(-6) M AraC, while apoptosis was significantly increased. These data suggest that cdk2 activity is likely to play a role in AraC-induced apoptosis in AML cells. This mechanism may account for high efficacy of HDAraC in cells showing little sensitivity to conventional AraC doses.     

Interaction of retinoblastoma protein and D cyclins during cell-growth inhibition by hexamethylenebisacetamide in TM2H mouse epithelial cells

To explore the regulation and function of D-type cyclins in breast cancer cells, the mouse mammary hyperplastic epithelial cell line TM2H was treated with 5 mM hexamethylenebisacetamide (HMBA), a polar differentiation factor. The resulting growth-inhibitory effect of HMBA was completely reversible and was analyzed in terms of percent cells in G₁; association of D-type cyclins with cyclin-dependent kinase (cdk) 4 and cdk6; G₁ kinase activity; association of retinoblastoma protein (pRb) and phosphorylated pRb with D-type cyclins; and association of p16^INK4a, p15^INK4b, and p27^Kip1 with cdk4 and cdk6. Synchronized TM2H cells were examined at 0, 3, 5, 9, 12, and 24 h after exposure to 5 mM HMBA. Inhibition of DNA synthesis, as measured by thymidine uptake, was first observed at 5 h (40%) and peaked at 24 h (80%). Flow cytometry at 9 h showed treated cells to be in G₁ arrest. Western blot analysis showed weakly detectable cyclin D1 but readily detectable cyclin D2 and D3 proteins at 0 h; thereafter, cyclin D2 and D3 protein levels remained higher while cyclin D1 levels declined significantly in treated versus untreated cells. By 5 h (early G₁), HMBA had markedly inhibited cdk4 and cdk6 kinase activity (67% and 75%, respectively) in treated versus untreated cells. By 9 and 12 h, pRb levels had increased 3.4-fold in treated versus untreated cells. At 5 h, cyclin D–associated pRb was totally hypophosphorylated in treated cells and hyperphosphorylated in untreated cells. The levels of pRb associated with cyclin D2 and D3 increased 2.89-fold and 4.6-fold, respectively, in treated versus untreated cells. At 5 h, treated cells showed a fivefold increase in cdk4-associated p27^Kip1 and, at 9 h, a fourfold increase in cdk6-associated p27^Kip1 over control levels. In confirmation of these data, HMBA was found to inhibit the growth of Rb-positive Du/145Rb cells but not their Rb-negative parental Du/145 cells. The data suggest that HMBA-induced growth inhibition is due to multifactorial mechanisms involving decreases in total cyclin D1 and inhibition of cdk4 and cdk6 kinase activities through elevation of levels of cdk4- and cdk6-associated p27^Kip1 and concomitant increases in hypophosphorylated pRb and stable cyclin D2/pRb and cyclin D3/pRb complexes that help maintain pRb in a functional state. Mol. Carcinog. 22:128–143, 1998. © 1998 Wiley-Liss, Inc.     

Anticarcinogenic effect of FTY720 in human prostate carcinoma DU145 cells: modulation of mitogenic signaling, FAK, cell-cycle entry and apoptosis

Despite the high frequency of prostate cancer, therapeutic options for advanced disease are limited to chemotherapy, radiation or hormonal therapy and eventually fail in all patients. Therefore, alternative approaches need to be developed. We previously reported that FTY720, a metabolite from Isaria sinclarii, is a unique antitumor agent for an androgen-independent prostate cancer cell line and requires caspase-3 activation in apoptosis. In our study, we have evaluated the effect of FTY720 on a family of mitogen-activated protein kinases (MAPKs), focal adhesion kinase (FAK), mitochondrial transmembrane potential, caspase-9 and caspase-8 and analyzed the expression of some cell-cycle regulator proteins in DU145 cells in order to understand the various antitumor effects of FTY720. Apoptosis was quantified by phosphatidylserine exposure. Activation of MAPKs, cleavage of caspase-9 and caspase-8, status of cyclin-dependent kinases (CDKs) and Cip1/p21, a cyclin-dependent kinase inhibitor, were evaluated by Western blot analysis, in addition to FAK and phospho-FAK immunoprecipitation and cell-cycle analysis by FACScan. We found that in DU145 cells, 40 microM FTY720 caused activation of p38 MAPK and the upstream kinase MKK3/MKK6 but not SAPK/JNK. Mitochondrial transmembrane potential, FAK and ERK1/2 were reduced while caspase-9 and caspase-8 were cleaved. The p38-specific inhibitor had no effect on apoptosis induced by FTY720, whereas z-VAD.FMK, a broad-spectrum caspase inhibitor, did not inhibit the p38 MAPK activation. An amount of 20 microM FTY720 resulted in G(1) arrest and a decrease of CDK2 as well as CDK4, whereas it induced Cip1/p21. FTY720 may exert anticarcinogenic effects against prostate cancer cells possibly involving modulation of mitogenic signaling, cell-cycle regulators, induction of G(1) arrest and apoptotic death in DU145 cells.     

Involvement of p27Kip1 in G1 arrest by high dose 5 alpha-dihydrotestosterone in LNCaP human prostate cancer cells

The cell cycle is governed by cyclin dependent kinases (cdks), which are activated by binding of cyclins, inhibited by cdk inhibitors and regulated by phosphorylation and dephosphorylation. Exposure to high dose dihydrotestosterone (DHT) inhibits population growth of the human prostate carcinoma cell line, LNCaP. To determine the mechanism of growth arrest by high dose DHT, we assayed the changes in cell cycle profile and the cell cycle regulators that mediate these effects. Treatment of asynchronously growing LNCaP cells with 100 nM DHT caused a G1 arrest. The proportion of cells in S phase fell from 22 to 2%, while the G1 fraction rose from 74 to 92% by 24 h. Loss of phosphorylation of the retinoblastoma protein was noted and cdk4 and cyclin E/ cdk2 activities fell. Inhibition of these G1 cyclin dependent kinases was not due to loss of either cyclin or cdk proteins nor to increases in the cdk inhibitors p16INK4A and p21CiP1. p21Cip1 protein levels remained constant, and cyclin E-associated p21CiP1 fell, suggesting that p21CiP1 is not relevant to this form of cyclin E/cdk2 inhibition. Of note, total p27KiP1 levels and cyclin E-associated p27Kip1 increased as cells arrested and the amount of the CAK activated cdk2 bound to cyclin E decreased. p27KiP1 immunodepletion experiments demonstrated that the DHT-mediated increase in p27Kip1 was sufficient to fully saturate and inhibit target cyclin E/ cdk2. The inhibition of cyclin E/cdk2 by p27Kip1 contributes to G1 arrest of LNCaP following high dose DHT. p27KiP1 may be a key effector of androgen dependent growth modulation in prostate cancer cells.     

P21(Cip1) induced by Raf is associated with increased Cdk4 activity in hematopoietic cells

To investigate the functions of the different Raf genes in hematopoietic cell proliferation, the capacities of beta-estradiol-regulated Delta Raf:ER genes to induce cell cycle regulatory gene expression and cell cycle progression in FDC-P1 cells were examined. Raf activation increased the expression of Cdk2, Cdk4, cyclin A, cyclin D, cyclin E, p21(Cip1) and c-Myc and decreased the expression of p27(Kip1) which are associated with G(1) progression. However only the cell clones with moderate Raf activation, i.e. FD/Delta Raf-1:ER and FD/Delta A-Raf:ER, successfully underwent cell proliferation. The cell clones with the highest Delta Raf activity, FD/Delta B-Raf:ER, underwent apoptosis before cell proliferation. p21(Cip1) induced by Raf activation specifically bound with Cdk4/cyclin D complexes but not Cdk2/cyclin E complexes and this binding was associated with the increased Cdk4 activity. However, no binding of p27(Kip1) with either Cdk2/cyclin E or Cdk4/cyclin D was observed. Thus Raf mediated growth was associated with elevated p21(Cip1) expression, which may specifically bind with and activate Cdk4/cyclin D complexes and with decreased p27(Kip1) expression.     

Altered expression of G1/S regulatory genes occurs early and frequently in lung carcinogenesis in transforming growth factor-beta1 heterozygous mice

We developed the AJBL6 transforming growth factor-beta 1 (TGF-beta1) heterozygous (HT) mouse by mating A/J mice with C57BL/6 TGF-beta1 HT mice that shows increased carcinogen-induced lung lesions with decreased latency to examine progressive events in lung tumorigenesis. Mouse cDNA macroarrays were used to identify cell cycle genes that are differentially regulated in ethyl carbamate-induced lung adenocarcinomas compared with normal lung tissue in AJBL6 TGF-beta1 HT mice using probes that were generated from tissues isolated using laser capture microdissection. While expression of the genes for cyclin D1, CDK4, and E2F1 increased in lung adenocarcinomas relative to normal lung, expression of p15(Ink4b), p16(Ink4a), p21(Cip1), p27(Kip1), p57(Kip2), and pRb genes decreased in comparison. Competitive RT-PCR showed that the levels of cyclin D1 and CDK4 mRNAs were 2- and 3-fold higher, respectively, in lung adenocarcinomas than in normal lung, while the mRNAs for p15(Ink4b), p16(Ink4a), p21(Cip1), p27(Kip1), and pRb were 3- to 4-fold lower in adenocarcinomas than in normal lung, thus validating the macroarray findings. Competitive RT-PCR of microdissected lesions also showed that the levels of cyclin D1 and CDK4 mRNAs increased significantly, while the mRNAs for p15(Ink4b) and p27(Kip1) decreased significantly as lung tumorigenesis progressed. Immunohistochemical staining for cyclin D1 and CDK4 showed staining in >80% of nuclei in adenocarcinomas compared with fewer than 20% of nuclei staining positively in normal lung. In contrast, while >60% of normal lung cells showed immunostaining for p15(Ink4b), p16(Ink4a), p21(Cip1), p27(Kip1), and pRb, staining for these proteins decreased in hyperplasias, adenomas, and adenocarcinomas. These data show that multiple components of the cyclin D1/CDK4/p16(Ink4a)/pRb signaling pathway are frequently altered early in lung lesions of AJBL6 TGF-beta1 HT mice that are induced by ethyl carbamate as a function of progressive lung carcinogenesis, suggesting that components of this pathway may be potential targets for gene therapy.     

PI3K/AKT信号通路阻断药联合小檗碱对前列腺癌DU145细胞生长的抑制作用

目的 探讨采用磷脂酰肌醇3-激酶(PI3K)抑制药——LY294002抑制磷脂酰肌醇-3-激酶/丝苏氨酸蛋白激酶(PI3K/AKT)信号通路联合小檗碱对前列腺癌DU145细胞生长的抑制作用.方法 采用MTT法检测细胞增殖情况;采用流式细胞仪检测细胞凋亡率和细胞周期分布情况;采用Western blot检测细胞中PI3K、p-AKT、cyclin D1、bcl-2、BAX蛋白表达情况.结果 随着小檗碱作用浓度的增加,DU145细胞的增殖率逐渐降低;不同浓度小檗碱与LY294002联合处理对DU145细胞增殖的抑制作用均较相同浓度小檗碱单独处理增强(P﹤0.05).与空白对照组相比,小檗碱组DU145细胞的凋亡率增高(P﹤0.05),G0/G1期细胞所占比例增高(P﹤0.05),PI3K、p-AKT、cyclin D1、bcl-2蛋白表达水平均下调(P﹤0.05),BAX蛋白表达水平上调(P﹤0.05).与小檗碱组和空白对照组相比,小檗碱+LY294002组DU145细胞的凋亡率增高(P﹤0.05),G0/G1期细胞所占比例增高(P﹤0.05),PI3K、p-AKT、cyclin D1、bcl-2蛋白表达水平均下调(P﹤0.05),BAX蛋白表达水平上调(P﹤0.05).结论 小檗碱能够抑制DU145细胞增殖,并促进细胞凋亡和细胞周期阻滞,采用LY294002阻断PI3K/AKT信号通路能够明显增强小檗碱对前列腺癌DU145细胞生长的抑制作用.     

Over-expression of insulin-like growth factor binding protein-related protein-1(IGFBP-rP1/mac25) in the M12 prostate cancer cell line alters tumor growth by a delay in G1 and cyclin A associated apoptosis.

In the present study, we examined the effects of over-expression of the potential tumor suppressor gene IGFBP-rP1/mac25 on cell-cycle kinetics in prostate cancer cells. The majority of the high expressing IGFBP-rP1/mac25 cell population was located in the G1 and sub-G0/G1 peaks; synchronizing cells in G2/M with nocodazole demonstrated the high expressing IGFBP-rP1/mac25 clones were delayed in the G1 phase of the cell cycle. Unscheduled expression of cyclin A in the sub-G0/G1 peak occurred in the IGFBP-rP1/mac25 clones. Immunoblots showed decreased cyclin D1 and p21 and increased cyclin E, p16, and p27 in the high expressing IGFBP-rP1/mac25 clones compared to the control cells. Cyclin D1/cdk-4,6 and cyclin E/cdk-2 kinase activities decreased but cyclin A/cdk-2 kinase activity increased for the high expressing IGFBP-rP1/mac25 clones compared to control cells. A pRb immunoprecipitation demonstrated more binding of E2F-1 to pRb in the high expressing IGFBP-rP1/mac25 clones than in control cells. Finally, cell senescence, as assessed by senescence-associated beta-galactosidase, demonstrated significantly more staining in the IGFBP-rP1/mac25 cells than control cells. These results suggest that IGFBP-rP1/mac25 alters the cell cycle kinetics of the M12 prostate cell line by delaying the cells in the G1 phase of the cell cycle. In addition, the appearance of cyclin A in the sub-G0/G1 phase of the cell cycle and the increased kinase activity of cyclin A/cdk-2 in the IGFBP-rP1/mac25 clones suggests that cyclin A is associated with the apoptotic cells.     

Anticarcinogenic effect of a polyphenolic fraction isolated from grape seeds in human prostate carcinoma DU145 cells: modulation of mitogenic signaling and cell-cycle regulators and induction of G1 arrest and apoptosis

There is an increasing interest in identifying potent cancer preventive and therapeutic agents against prostate cancer (PCA). In a recent study, we showed that a polyphenolic fraction isolated from grape seeds (hereafter referred to as GSP) that is substantially rich in antioxidant procyanidins exerts exceptionally high preventive effects against tumorigenesis in a murine skin model. In the present study, we investigated the anticarcinogenic effect of GSP against PCA by employing DU145 human prostate carcinoma cells. GSP treatment (10-100 microg/mL doses for 2-6 d) of cells resulted in a highly significant (P < 0.01-0.001) inhibition of cell growth in both dose- and time-dependent manner. Compared with the vehicle, 2 d of GSP treatment resulted in 27, 39, and 76% growth inhibition at 50, 75, and 100 microg/mL doses, respectively, whereas 28-97% and 12-98% inhibition was evident at 10-100 microg/mL doses of GSP after 4 and 6 d of treatment, respectively. These doses of GSP also resulted in dose- and time-dependent cell death (6-50%, P <0.1-0. 001) that was later characterized as apoptotic death. In molecular mechanistic studies, treatment of DU145 cells with GSP at 25-75 microg/mL doses for 24, 48, and 72 h resulted in 77-88%, 65-93%, and 38-98% reduction, respectively (P < 0.001), in phospho-extracellular signal-regulated protein kinase (ERK) 1 and 78%, 19-76%, and 63-71% reduction (P < 0.1-0.001) in phospho-ERK2 levels, respectively. In other studies, similar doses of GSP showed up to 1.9-fold increases in Cip1/p21 and a significant (P < 0.001) decrease in cyclin-dependent kinase (CDK) 4 (up to 90% decrease), CDK2 (up to 50% decrease), and cyclin E (up to 60% decrease). GSP treatment of DU145 cells also resulted in a significant (P < 0.001) G1 arrest in cell-cycle progression in a dose-dependent manner. The growth-inhibitory and cell-death effects of GSP were also observed in another human PCA line, LNCaP. Together, these results suggest that GSP may exert strong anticarcinogenic effect against PCA and that this effect possibly involves modulation of mitogenic signaling and cell-cycle regulators and induction of G1 arrest, cell-growth inhibition, and apoptotic death. Mol. Carcinog. 28:129-138, 2000.     

Caffeic acid phenethyl ester induced cell cycle arrest and growth inhibition in androgen-independent prostate cancer cells via regulation of Skp2, p53, p21Cip1 and p27Kip1

Prostate cancer (PCa) patients receiving the androgen ablation therapy ultimately develop recurrent castration-resistant prostate cancer (CRPC) within 1–3 years. Treatment with caffeic acid phenethyl ester (CAPE) suppressed cell survival and proliferation via induction of G1 or G2/M cell cycle arrest in LNCaP 104-R1, DU-145, 22Rv1, and C4–2 CRPC cells. CAPE treatment also inhibited soft agar colony formation and retarded nude mice xenograft growth of LNCaP 104-R1 cells. We identified that CAPE treatment significantly reduced protein abundance of Skp2, Cdk2, Cdk4, Cdk7, Rb, phospho-Rb S807/811, cyclin A, cyclin D1, cyclin H, E2F1, c-Myc, SGK, phospho-p70S6kinase T421/S424, phospho-mTOR Ser2481, phospho-GSK3α Ser21, but induced p21^Cip1, p27^Kip1, ATF4, cyclin E, p53, TRIB3, phospho-p53 (Ser6, Ser33, Ser46, Ser392), phospho-p38 MAPK Thr180/Tyr182, Chk1, Chk2, phospho-ATM S1981, phospho-ATR S428, and phospho-p90RSK Ser380. CAPE treatment decreased Skp2 and Akt1 protein expression in LNCaP 104-R1 tumors as compared to control group. Overexpression of Skp2, or siRNA knockdown of p21^Cip1, p27^Kip1, or p53 blocked suppressive effect of CAPE treatment. Co-treatment of CAPE with PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or Bcl-2 inhibitor ABT737 showed synergistic suppressive effects. Our finding suggested that CAPE treatment induced cell cycle arrest and growth inhibition in CRPC cells via regulation of Skp2, p53, p21^Cip1, and p27^Kip1.     

Polyamine-depletion induces p27Kip1 and enhances dexamethasone-induced G1 arrest and apoptosis in human T lymphoblastic leukemia cells

Glucocorticoid-induced apoptosis is preceded by G1 arrest and supposed to be up-regulated by polyamine-depletion, which also induces G1, arrest. In CEM leukemia cells, dexamethasone showed an antileukemic effect by inducing G1 arrest and apoptosis. DFMO, which depleted cellular polyamines by inhibiting ornithine decarboxylase, induced G1 arrest but without apoptosis, though it enhanced dexamethasone-induced G1 arrest and apoptosis. The G1 arrest was associated with hypophosphorylation of pRb. Dexamethasone inhibited the increase of mutated p53 expression but had little effect on p2Wafl/Cip1 expression. The p27Kip1, level was increased by dexamethasone or and DFMO in line with the kinetics of G1 arrest. Therefore, the up-regulation of dexamethasone-induced apoptosis by polyamine-depletion may be associated with additive down-regulation of G1 progression via the p27Kip1-pRb pathtway.     

Pharmacologic inhibition of RAF-->MEK-->ERK signaling elicits pancreatic cancer cell cycle arrest through induced expression of p27Kip1

Expression of mutationally activated RAS is a feature common to the vast majority of human pancreatic adenocarcinomas. RAS elicits its effects through numerous signaling pathways including the RAF-->mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase [MEK]-->ERK MAP kinase pathway. To assess the role of this pathway in regulating cell proliferation, we tested the effects of pharmacologic inhibition of MEK on human pancreatic cancer cell lines. In eight cell lines tested, MEK inhibition led to a cessation of cell proliferation accompanied by G0-G1 cell cycle arrest. Concomitant with cell cycle arrest, we observed induced expression of p27Kip1, inhibition of cyclin/cyclin-dependent kinase 2 (cdk2) activity, accumulation of hypophosphorylated pRb, and inhibition of E2F activity. Using both antisense and RNA interference techniques, we assessed the role of p27Kip1 in the observed effects of MEK inhibition on pancreatic cancer cell proliferation. Inhibition of p27Kip1 expression in Mia PaCa-2 cells restored the activity of cyclin/cdk2, phosphorylation of pRb, and E2F activity and partially relieved the effects of U0126 on pancreatic cancer cell cycle arrest. Consistent with the effects of p27Kip1 on cyclin/cdk2 activity, inhibition of CDK2 expression by RNA interference also led to G0-G1 cell cycle arrest. These data suggest that the expression of p27Kip1 is downstream of the RAF-->MEK-->ERK pathway and that the regulated expression of this protein plays an important role in promoting the proliferation of pancreatic cancer cells. Moreover, these data suggest that pharmacologic inhibition of the RAF-->MEK-->ERK signaling pathway alone might tend to have a cytostatic, as opposed to a cytotoxic, effect on pancreatic cancer cells.     

Cyclin D1, p53, and p21Waf1/Cip1 expression is predictive of poor clinical outcome in serous epithelial ovarian cancer.

PURPOSE: Dysregulation of cell cycle control, in particular G(1)-S-phase transition, is implicated in the pathogenesis of most human cancers, including epithelial ovarian cancer (EOC). However, the prognostic significance of aberrant cell cycle gene expression in EOC remains unclear. EXPERIMENTAL DESIGN: The expression of selected genes from the pRb pathway that regulates G(1)-S-phase progression, including cyclin D1, p16(Ink4a), cyclin E, p27(Kip1), p21(Waf1/Cip1), and p53, was examined in a consecutive series of 134 serous EOC using immunohistochemistry and the results correlated to disease outcome. RESULTS: Molecular markers predictive of reduced overall survival in univariate analysis were overexpression of cyclin D1 (P = 0.03) and p53 (P = 0.03) and reduced expression of p27(Kip1) (P = 0.05) and p21(Waf1/Cip1) (P = 0.02), with the latter three also being prognostic for a shorter progression-free interval. In addition, patients displaying overexpression of p53 with concurrent loss of p21(Waf1/Cip1) had a significantly shorter overall (P = 0.0008) and progression-free survival (P = 0.0001). On multivariate analysis, overexpression of cyclin D1 and combined loss of p21(Waf1/Cip1) in the presence of p53 overexpression were independent predictors of overall survival. Similarly, the combination of p21(Waf1/Cip1) loss and p53 overexpression was independently predictive of a shorter progression-free interval. Overexpression of p53 and cyclin E and reduced expression of p27(Kip1) and p21(Waf1/Cip1) were significantly associated with increasing tumor grade. CONCLUSIONS: This study confirms that dysregulation of cell cycle genes is common in EOC, and that aberrant expression of critical cell cycle regulatory proteins can predict patient outcome in serous EOC.     

德士康对前列腺癌激素非依赖型细胞株细胞周期的抑制作用

目的探讨中草药混合制剂德士康(暂定名)对雄激素非依赖型前列腺癌细胞株PC 3、DU145细胞周期的抑制作用和机制.方法MTT法检测用药后细胞增殖的改变,流式细胞仪(FCM)测定细胞周期的改变,Western blot方法分析细胞周期相关蛋白表达的改变.结果德士康可抑制PC-3、DU145细胞的生长,PC-3、DU145分别主要受阻于G1期和G2/M期.用药组p16Ink4a蛋白表达增高,Rb磷酸化蛋白表达降低,伴有非磷酸化Rb蛋白的相应增多;而p21waf1、p27Kip1和细胞周期蛋白cyclin D1的表达均无明显改变.结论德士康可能是通过影响p16Ink4a蛋白的表达,改变细胞周期蛋白依赖蛋白激酶的活性,最后作用于Rb,抑制其磷酸化而发挥作用,从而抑制PC-3、DU145细胞的生长,具有应用于临床治疗的前景.     

AKAP12 induces apoptotic cell death in human fibrosarcoma cells by regulating CDKI-cyclin D1 and caspase-3 activity

AKAP12 (A-Kinase anchoring protein 12) is a protein kinase C substrate and a potential tumor suppressor. AKAP12 is down-regulated by several oncogenes and strongly suppressed in various cancers including prostate, ovarian and breast cancers. AKAP12 acts as a regulator of mitogenesis by anchoring key signal proteins such as PKA, PKC, and cyclins. In this study, AKAP12 was found to suppress tumor cell viability by inducing apoptosis via caspase-3 in HT1080 cells. This AKAP12-induced apoptosis was associated with a decreased expression of Bcl-2 and increased expression of Bax. Moreover, AKAP12-transfectant strongly induced the expression of Cip1/p21 and Kip1/p27, but resulted in a decrease in cyclin D1 involved in G(1) progression. Accordingly, these results suggest that AKAP12 may play an important role in tumor growth suppression by inducing apoptosis with the regulation of multiple molecules in the cell cycle progression.