Nonsteroidal anti-inflammatory drugs inhibit growth of human neuroendocrine tumor cells via G1 cell-cycle arrest

Therapeutic options to inhibit growth of human NETs of the GEP system are limited. Since NSAIDs might provide an antiproliferative treatment alternative with acceptable toxicity, we examined the effects of different NSAIDs on growth and survival in a representative set of human GEP NET cell lines. Growth and apoptosis were determined based on cell numbers, cell-cycle analyses, kinase assays, DNA fragmentation and PARP cleavage. Expression of COX and cell cycle-regulatory molecules was examined by immunoblotting and reporter gene assays. Depending on the drug and cell line investigated, NSAID treatment resulted in profound growth inhibition of GEP NET cells. Growth-inhibitory effects were achieved with either COX-2 selective (NS398) or unselective (indomethacin, sulindac) compounds. Cell-cycle analyses documented a G1 arrest in NSAID-treated GEP NET populations. In addition, 100 microM sulindac or indomethacin induced apoptosis. All 3 COX inhibitors prevented CDK-2 activation. In parallel to the NSAID-mediated reduction of CDK-2 activity, p21(cip-1) promoter activity and cellular p21(cip-1) levels increased and p21(cip-1) was sequestered into CDK-2 complexes. Thus, the G1 arrest likely resulted from p21(cip-1)-dependent inhibition of CDK-2 activity. At therapeutically relevant concentrations, sulindac significantly reduced GEP NET cell numbers, whereas IFN-alpha and octreotide remained ineffective. The extent of growth inhibition in GEP NETs was comparable to the antiproliferative effects of sulindac in established NSAID-sensitive cell models. NSAIDs acted as potent antiproliferative agents in GEP NET cells via G1 cell-cycle arrest and might therefore offer a therapeutic alternative to current treatment modalities.     

Anti-proliferative effect of apigenin and its apoptotic induction in human Hep G2 cells

Apigenin, a common dietary flavonoid abundantly present in fruits and vegetables, is believed to possess preventive and therapeutic potential against cancers. In this study, the anti-hepatoma property of apigenin was evaluated on three different human hapatoma cells, namely Hep G2, Hep 3B, and PLC/PRF/5 cells. Results showed that apigenin exhibited a significant growth inhibition against the three selected hepatoma cell lines but not the normal murine liver BNL CL.2 cells. Interestingly, it was shown to possess a similar potency as a commercial anti-hepatoma agent 5-flurouracil (5-FU: positive control) against Hep G2 cells, with IC50 value of 8.02+/-1.30 microg/ml. Therefore, we conducted our study further to investigate the cellular mechanism of apigenin effect on Hep G2 cell death. Using DNA ladder and flow cytometric analysis, apigenin was found to induce apoptosis in Hep G2 cells. It also increased the accumulation of p53 and further enhanced the level of p21/WAF1. Together, it was shown that the apoptosis induced by apigenin in Hep G2 cells was possibly mediated through the p53-dependent pathway and the induction of p21 expression, which was probably associated with the cell cycle arrest in G2/M phase. The present study concludes that the anti-hepatoma activity of apigenin is as effective as 5-FU and its apoptotic mechanism might be mediated through the p53-dependent pathway and the induction of p21 expression.     

蛋白酶体抑制剂MG132诱导HL-60细胞凋亡前G2/M期阻滞及机制

背景和目的:蛋白酶体(proteasome)抑制剂能够诱导多种肿瘤细胞凋亡,是一种潜在的有应用前景的抗肿瘤剂.本研究旨在探讨蛋白酶体抑制剂MGl32(Z-Leu-Leu-Leu-CHO)诱导白血病细胞HL-60凋亡和C2/M期阻滞的机制.方法:采用荧光显微镜观察、流式细胞术和免疫印迹研究测定MG132诱导HL-60细胞凋亡和周期阻滞及机制.结果:2μmol/L的MG132能够有效地诱导HL-60细胞凋亡,用药后24 h就显现有细胞凋亡;在MG132诱导HL-60细胞凋亡出现之前有一个明显的G2/M期阻滞,加MG132后12 h时G2/M期时相百分比为63.42±2.02;24 h时加MG132组细胞凋亡为16.67±1.48,与对照组G2/M期时相百分比为7.29±3.01及细胞凋亡为0相比,两者之间有显著性差别(P＜0.01);咖啡因CAF能够减少MG132诱导HL-60细胞出现的G2/M期阻滞,同时也减少凋亡细胞的比例;细胞周期检查点的负调控因子p21waf/cip1蛋白在加MG132处理后3 h有明显的表达,但并未能检测到p53和p27蛋白.结论:MG132诱导HL-60细胞凋亡之前有一个明显的G2/M期阻滞,p21蛋白表达明显上调提示:是p21waf/cip1而不是p53或其同源蛋白参与了其中的调控.     

Molecular mechanisms of G0/G1 cell-cycle arrest and apoptosis induced by terfenadine in human cancer cells

Terfenadine (TF), a highly potent histamine H1 receptor antagonist, has been shown to exert no significant central nervous system side effects in clinically effective doses. In this study, we demonstrated that TF induced significant growth inhibition of human cancer cells, including Hep G2, HT 29, and COLO 205 cells, through induction of G(0)/G(1) phase cell-cycle arrest. The minimal dose of TF induced significant G(0)/G(1) arrest in these cells was 1-3 microM. The protein levels of p53, p21/Cip1, and p27/Kip1 were significantly elevated, whereas the kinase activities of cyclin-dependent kinase 2 (CDK2) and CDK4 were inhibited simultaneously in the TF-treated cells. On the other hand, significant apoptosis, but not G(0)/G(1) arrest, was induced in the HL 60 (p53-null) or Hep 3B (with deleted p53) cells when treated with TF (3-5 microM). To clarify the roles of p21/Cip1 and p27/Kip1 protein expression, which was involved in G(0)/G(1) arrest and apoptosis induced by TF in human cancer cells, antisense oligodeoxynucleotides (ODNs) specific to p21/Cip1 and p27/Kip1 were used, and the expression of the p21/Cip1 and p27/Kip1 were monitored by immunoblotting analysis. Our data demonstrated that the percentage of the apoptotic cells detected by annexin V/PI analysis in the TF-treated group was clearly attenuated by pretreatment with p27/Kip1-specific ODNs. These results indicated that p27/Kip1 (but not p21/Cip1) protein indeed played a critical role in the TF-induced apoptosis. We also demonstrated that the TF-induced G(0)/G(1) cell-cycle arrest effect was not reversed by TF removal, and this growth inhibition lasted for at least 7 d. Importantly, the occurrence of apoptosis and cell growth arrest was not observed in the TF-treated normal human fibroblast, even at a dose as high as 25 microM. Our study showed the molecular mechanisms for TF-induced cell growth inhibition and the occurrence of apoptosis in human cancer cells.     

Inhibition of human vascular endothelial cells proliferation by terbinafine

We have demonstrated previously that terbinafine (TB), an oral antifungal agent used in the treatment of superficial mycosis, suppresses proliferation of various cultured human cancer cells in vitro and in vivo by inhibiting DNA synthesis and activating apoptosis. In our study, we further demonstrated that TB at a range of concentrations (0-120 microM) dose-dependently decreased cell number in cultured human umbilical vascular endothelial cells (HUVEC). Terbinafine was not cytotoxic at a concentration of 120 microM, indicating that it may have an inhibitory effect on the cell proliferation in HUVEC. The TB-induced inhibition of cell growth rate is reversible. [(3)H]thymidine incorporation revealed that TB reduced the [(3)H]thymidine incorporation into HUVEC during the S-phase of the cell-cycle. Western blot analysis demonstrated that the protein levels of cyclin A, but not cyclins B, D1, D3, E, CDK2 and CDK4, decreased after TB treatment. The TB-induced cell-cycle arrest in HUVEC occurred when the cyclin-dependent kinase 2 (CDK2) activity was inhibited just as the protein level of p21 was increased and cyclin A was decreased. Pretreatment of HUVEC with a p21 specific antisense oligonucleotide reversed the TB-induced inhibition of [(3)H]thymidine incorporation. Taken together, these results suggest an involvement of the p21-associated signaling pathway in the TB-induced antiproliferation in HUVEC. Capillary-like tube formation and chick embryo chorioallantoic membrane (CAM) assays further demonstrated the anti-angiogenic effect of TB. These findings demonstrate for the first time that TB can inhibit the angiogenesis.     

Relative non-steroidal anti-inflammatory drug (NSAID) antiproliferative activity is mediated through p21-induced G1 arrest and E2F inhibition

This study was performed to compare the relative antineoplastic activity of 10 different non-steroidal anti-inflammatory drugs (NSAIDs) in clinical use, and to investigate the underlying mechanisms of this activity in a squamous cell carcinoma of the head and neck model (SCCHN). A standard 5-day MTT assay was used to calculate IC(50) values in UM-SCC-1 cells for 10 NSAIDs, including celecoxib, rofecoxib, sulindac sulfide, sulindac sulfone, indomethacin, ketoprofen, flurbiprofen, naproxen, piroxicam, and aspirin. Celecoxib, a COX-2 specific inhibitor, was by far the most potent NSAID, with an IC(50) of 39.9 +/- 1.1 microM, followed by sulindac sulfide (116.5 +/- 2.34 microM). Celecoxib and sulindac sulfide also induced more activation of caspase-3 than any other NSAID. Cell cycle analysis showed that celecoxib and sulindac sulfide both induced a 3-fold increase in G(1) phase distribution, and this correlated with strong induction of p21(waf1/cip1), inhibition of cyclin D1, and hypophosphorylation of Rb. Celecoxib and sulindac sulfide treatment induced strong downstream inhibition of E2F transactivating activity as determined by a luciferase reporter assay. These data demonstrate the wide range of activity of various NSAID agents, and reveal a mechanism of action through cell cycle inhibition and induction of apoptosis.     

siRNA-mediated silencing of Notch-1 enhances docetaxel induced mitotic arrest and apoptosis in prostate cancer cells

Purpose: Notch is an important signaling pathway that regulates cell fate, stem cell maintenance and theinitiation of differentiation in many tissues. It has been reported that activation of Notch-1 contributes totumorigenesis. However, whether Notch signaling might have a role in chemoresistance of prostate cancer isunclear. This study aimed to investigate the effects of Notch-1 silencing on the sensitivity of prostate cancercells to docetaxel treatment. Methods: siRNA against Notch-1 was transfected into PC-3 prostate cancer cells.Proliferation, apoptosis and cell cycle distribution were examined in the presence or absence of docetaxel byMTT and flow cytometry. Expression of p21waf1/cip1 and Akt as well as activation of Akt in PC-3 cells were detectedby Western blot and Real-time PCR. Results: Silencing of Notch-1 promoted docetaxel induced cell growthinhibition, apoptosis and cell cycle arrest in PC-3 cells. In addition, these effects were associated with increasedp21waf1/cip1 expression and decreased Akt expression and activation in PC-3 cells. Conclusion: Notch-1 promoteschemoresistance of prostate cancer and could be a potential therapeutic target.     

槲皮素通过调控p21/cip1和p27/kip1蛋白的稳定性抑制口腔鳞状细胞癌细胞增殖

目的:探究槲皮素通过调控p21/cip1和p27/kip1蛋白的稳定性对口腔鳞状细胞癌细胞增殖的影响.方法:CCK-8试剂盒检测口腔鳞状细胞癌细胞(SCC-15)增殖情况;流式细胞术检测SCC-15细胞周期分布;Western blot检测CyclinD1/CDK复合体、p21/cip1、p27/kip1、p-GSK3...     

P21-dependent g(1)arrest with downregulation of cyclin D1 and upregulation of cyclin E by the histone deacetylase inhibitor FR901228

Depsipeptide, FR901228, a novel cyclic peptide inhibitor of histone deacetylase with a unique cytotoxicity profile is currently in phase I clinical trials. Here we demonstrate that, in addition to G2/M arrest, FR901228 causes G1 arrest with Rb hypophosphorylation. In vitro kinase assays demonstrated no direct inhibition of CDK activity, however, an inhibition was observed in CDKs extracted from cells exposed to FR901228. Cyclin D1 protein disappeared between 6 and 12 hours after treatment with FR901228, whereas cyclin E was upregulated. While it did not induce wt p53, FR901228 did induce p21(WAF1/CIP1)in a p53-independent manner. Cell clones lacking p21 were not arrested in G1 phase, but continued DNA synthesis and were arrested in G2/M phase following FR901228 treatment. Finally, FR901228 blunted ERK-2/MAPK activation by EGF whereas early signal transduction events remained intact since overall cellular tyrosine phosphorylation after EGF stimulation was unaffected. Thus, FR901228, while not directly inhibiting kinase activity, causes cyclin D1 downregulation and a p53-independent p21 induction, leading to inhibition of CDK and dephosphorylation of Rb resulting in growth arrest in the early G1 phase. In contrast to the G1 arrest, the G2/M arrest is p21-independent, but is associated with significant cytotoxicity.     

Expression of cyclins A and D and p21(waf1/cip1) proteins in renal cell cancer and their relation to clinicopathological variables and patient survival.

We have studied 118 renal cell carcinomas to analyse the expressions of cyclins A and D1 and p21(waf1/cip1), and their relationship to clinical and histopathological parameters as well as to clinical outcome. Cyclins A and D1 and cyclin-dependent kinase inhibitor p21 (waf1/cip1) were not expressed in normal renal tissue. Staining signals of cyclin D1 and p21(waf1/cip1) were always nuclear but cyclin A was also expressed in the cytoplasm of the tumour cells. The mean (range) fractions of cyclin A, cyclin D1 and p21(waf1/cip1)-positive tumour cells were 2.2% (range 0-20%), 23.3% (range 0-90%) and 6.8% (range 0-70%) respectively. The expression of cyclin A was related to venous invasion, high nuclear grade, high mitotic rate, high Ki-67 and high PCNA expressions (P < or = 0.006 for all). The expression of cyclin D1 was linked with age over 65 years, low nuclear grade and high p53 expression (P < or = 0.05 for all). An inverse correlation was present between p21(waf1/cip1) and cyclin D1 (P = 0.011). Cyclin A predicted survival in the entire study group (P = 0.0014), in T1-4/N0-2/M0 (P = 0.0007) and in T1-2/N0/M0 tumours (P = 0.0007). Cyclin A was also a powerful predictor of disease-free survival in T1-4/N0/M0 (P = 0.0027) tumours (P = 0.0007). Cyclin D1 and p21(waf1/cip1) were not significantly related to survival or disease-free survival in any of the groups. In the entire material the independent prognostic factors were the presence of distant metastases (relative risk (RR) 5.16, P < 0.001), T category (RR 2.68, P < 0.001), Ki-67 expression (RR 1.02, P = 0.026) and cyclin A expression (RR 1.12, P = 0.001). The independent predictors in T1-4/N0/M0 tumours were T-category (RR 2.67, P = 0.001) and cyclin A (RR 1.21, P < 0.001), and in T1-2/N0/M0 tumours the only significant predictor was cyclin A (RR 1.19, P = 0.0002). In renal cell carcinoma, cyclin A is a powerful and independent prognostic factor in all clinical stages of the disease, whereas cyclin D1 and p21(waf1/cip1) have no prognostic value.     

Cyclin E、CDK2和p21WAF1在食管上皮癌变过程中的表达及意义

目的探讨食管上皮癌变过程中细胞周期调控因子cyclin E、CDK2和p21WAF1的表达状况及其意义.方法应用免疫组化SP法和原位杂交方法分别检测48例食管癌组织、31例非典型增生组织和17例正常食管粘膜中cyclin E、CDK2和p21WAF1蛋白及mRNA表达.应用半定量RT-PCR和Western blot检测22例新鲜食管癌及相应癌旁组织的mRNA和蛋白表达.结果从食管正常粘膜、非典型增生组织到癌组织,cyclin E和CDK2蛋白和mRNA阳性表达率逐渐上升,差异具有统计学意义(P＜0.01或P＜0.05).食管癌组织中cyclin E、CDK2和p21WAF1蛋白及mRNA高表达,与癌旁组织或切缘正常食管粘膜有显著性差异(P＜0.01).cyclin E、CDK2和p21WAF1基因表达显著正相关(P＜0.01或P＜0.05).结论食管上皮癌变过程中,细胞周期相关基因cyclin E和CDK2表达逐渐增强.cyclin E基因表达异常是食管癌变过程中的早期事件.p21WAF1基因在食管癌中高表达,可能与细胞周期调控的反馈机制有关.     

Suppression of ABCG2 inhibits cancer cell proliferation

The ATP‐binding cassette efflux transporter, ABCG2, is widely expressed in a variety of normal tissues, stem cells, as well as cancer cells. Existing data suggest that ABCG2 plays an important role in the maintenance of the stem cell phenotype and multidrug resistance of cancer cells. However, the potential role of ABCG2 in other cellular processes remains speculative and poorly understood. Here, we demonstrated that ABCG2 is involved in the proliferation of cancer cells. We used RNA interference approach to efficiently and specifically down‐regulate ABCG2 protein levels in MCF‐7/MX and A549 cells. We showed that knockdown of ABCG2 significantly inhibited the proliferation of these cells. Suppression of ABCG2 reduced the percentage of cells in the S phase of the cell cycle and enhanced G0/G1 accumulation. The G0/G1 growth arrest was associated with down‐regulation of cyclin D3 and up‐regulation of p21. Furthermore, blocking of ABCG2 function by chemical inhibitor fumitremorgin C also inhibited cell proliferation via the prolonged G0/G1 interval. Taken together, these findings suggest that ABCG2 correlates with cell cycle progression, highlighting a novel function of ABCG2 in cancer cell proliferation.     

Artepillin C in Brazilian propolis induces G(0)/G(1) arrest via stimulation of Cip1/p21 expression in human colon cancer cells

Potential chemopreventive agents exist in foods. Artepillin C in Brazilian propolis was investigated for its effects on colon carcinogenesis. We had found that artepillin C was a bioavailable antioxidant, which could be incorporated into intestinal Caco-2 and hepatic HepG2 cells without any conjugation and inhibited the oxidation of intracellular DNA. Artepillin C was then added to human colon cancer WiDr cells. It dose-dependently inhibited cell growth, inducing G(0)/G(1) arrest. The events involved a decrease in the kinase activity of a complex of cyclin D/cyclin-dependent kinase 4 and in the levels of retinoblastoma protein phosphorylated at Ser 780 and 807/811. The inhibitors of the complex, Cip1/p21 and Kip1/p27, increased at the protein level. On the other hand, Northern blotting showed that artepillin C did not affect the expression of Kip1/p27 mRNA. According to the experiments using isogenic human colorectal carcinoma cell lines, artepillin C failed to induce G(0)/G(1) arrest in the Cip1/p21-deleted HCT116 cells, but not in the wild-type HCT116 cells. Artepillin C appears to prevent colon cancer through the induction of cell-cycle arrest by stimulating the expression of Cip1/p21 and to be a useful chemopreventing factor in colon carcinogenesis.     

组织芯片/免疫组化检测CDC2/CyclinB1在胶质瘤中的联合表达及意义

目的:探讨CDC2/CyclinB1在胶质瘤组织芯片中的表达及意义。方法:构建布有各级别人脑胶质瘤、人脑胶质瘤体外细胞系球体及其移植瘤组织、脑肿瘤干细胞、神经干细胞和相应的对照标本共118例的组织芯片,进行CDC2、CyclinB1的EnVision法免疫组化染色,分析其表达率、表达强度及联合表达情况。结果:CDC2、CyclinB1表达阳性率在71例人脑胶质瘤组织中分别为54.9%和52.1%。11例正常成人脑组织中分别为18.2%和9.0%,两者比较P<0.05。CDC2和CyclinB1的联合表达率为89.7%,两者表达呈正相关(r=0.90,P<0.01)。CDC2、CyclinB1的表达在不同级别人脑胶质瘤中也有差异(P<0.05),与病理级别分别呈正相关(r=0.982,P=0.018;r=0.959,P=0.041)。在体外培养的胶质瘤细胞球体、裸鼠皮下移植瘤、人胚胎脑和裸小鼠骨髓中均高表达,而脑肿瘤干细胞和神经干细胞球体未见表达。结论:CDC2和CyclinB1,随着脑胶质瘤恶性度增高而表达量增加;在神经干细胞、脑肿瘤干细胞低表达,而在SHG44和GBM细胞球体中高表达,表明其在肿瘤细胞分化抑制中起作用。这对进一步研究胶质瘤分子病因有重要价值。     

PAK4促进人乳腺肿瘤细胞G1/S期转化机制探讨

目的研究p21蛋白激活激酶4(p21protein activated kinase 4,PAK4)对人乳腺肿瘤细胞生长的影响。方法人乳腺肿瘤细胞MCF-7转染不同剂量PAK4真核表达载体或慢病毒敲除PAK4表达,收集细胞提取蛋白进行蛋白质印迹法检测PAK4和Cyclin D1表达。流式细胞仪检测PAK4过表达和敲除后对细胞生长周期影响。结果过表达PAK4明显上调人乳腺肿瘤细胞MCF-7中的Cyclin D1蛋白表达,引起G1期细胞减少(从80%下降到55%),导致G1/S期转化细胞增多。慢病毒敲除人乳腺肿瘤细胞MCF-7中的PAK4表达降低了的Cyclin D1蛋白表达,使G1期的细胞增加12%,而S期的细胞下调7%。结论 PAK4调节Cyclin D1表达,促进人乳腺肿瘤细胞MCF-7G1/S期转化,揭示PAK4可能是重要的人乳腺肿瘤治疗靶点。