siRNA-cyclin D1对乳腺癌MCF-7细胞增殖的抑制作用

研究小干扰RNA(small interfering RNA,siRNA)对乳腺癌MCF-7细胞株cyclin D1表达的抑制及对细胞增殖的影响。化学合成针对cyclin D1基因的siRNA,转染MCF-7细胞株;分别应用荧光定量PCR和免疫印迹测定cyclin D1 mRNA和蛋白的表达,CCK-8测定细胞的增殖活性,流式细胞仪检测细胞周期,软琼脂培养检测细胞克隆形成能力。在实验中,10、50、100 nmol/L siRNA-cyclin D1分别使MCF-7细胞cyclin D1 mRNA表达降低了57．85%、63．22%和68．02%,蛋白表达降低了51．13%、62．09%、77．68%。转染siRNA-cyclin D1后,细胞增殖受到抑制,细胞周期阻滞于G1期,软琼脂克隆形成率降低。结果提示siRNA可以有效抑制MCF-7细胞株中cyclin D1的表达,使细胞周期阻滞于G1期,从而抑制细胞增殖。     

Rho Isoform-specific Interaction with IQGAP1 Promotes Breast Cancer Cell Proliferation and Migration

We performed a proteomics screen for Rho isoform-specific binding proteins to clarify the tumor-promoting effects of RhoA and C that contrast with the tumor-suppressive effects of RhoB. We found that the IQ-motif-containing GTPase-activating protein IQGAP1 interacts directly with GTP-bound, prenylated RhoA and RhoC, but not with RhoB. Co-immunoprecipitation of IQGAP1 with endogenous RhoA/C was enhanced when RhoA/C were activated by epidermal growth factor (EGF) or transfection of a constitutively active guanine nucleotide exchange factor (GEF). Overexpression of IQGAP1 increased GTP-loading of RhoA/C, while siRNA-mediated depletion of IQGAP1 prevented endogenous RhoA/C activation by growth factors. IQGAP1 knockdown also reduced the amount of GTP bound to GTPase-deficient RhoA/C mutants, suggesting that IQGAP enhances Rho activation by GEF(s) or stabilizes Rho-GTP. IQGAP1 depletion in MDA-MB-231 breast cancer cells blocked EGF- and RhoA-induced stimulation of DNA synthesis. Infecting cells with adenovirus encoding constitutively active RhoA^L63 and measuring absolute amounts of RhoA-GTP in infected cells demonstrated that the lack of RhoA^L63-induced DNA synthesis in IQGAP1-depleted cells was not due to reduced GTP-bound RhoA. These data suggested that IQGAP1 functions downstream of RhoA. Overexpression of IQGAP1 in MDA-MB-231 cells increased DNA synthesis irrespective of siRNA-mediated RhoA knockdown. Breast cancer cell motility was increased by expressing a constitutively-active RhoC^V14 mutant or overexpressing IQGAP1. EGF- or RhoC-induced migration required IQGAP1, but IQGAP1-stimulated migration independently of RhoC, placing IQGAP1 downstream of RhoC. We conclude that IQGAP1 acts both upstream of RhoA/C, regulating their activation state, and downstream of RhoA/C, mediating their effects on breast cancer cell proliferation and migration, respectively.     

siRNA对乳腺癌细胞Cyclin E表达和生长抑制作用

研究siRNA对乳腺癌MCF-7细胞株cyclin E表达的抑制及对细胞生长的影响。化学合成针对cyclin E基因的小干扰RNA（siRNA）,转染MCF-7细胞株;分别应用荧光定量PCR和免疫印迹测定cyclin E mRNA和蛋白质的表达,CCK-8测定细胞的增殖活性,流式细胞仪检测细胞周期,软琼脂培养检测细胞克隆形成能力。10、50、100nmol／L siRNA-cyclin E分别使MCF-7细胞cyclin E基因表达降低了24．7％、62．5％和71．0％,蛋白质表达降低了40．8％、66．5％和71．3％。转染siRNA-cyclin E后,G1期细胞增多,S期减少,增殖受到抑制,软琼脂克隆形成率降低。结果提示,在MCF-7细胞株中,导入针对cyclin E的siRNA,可有效抑制cyclin E的表达,进而使细胞增殖减缓,逆转其恶性表型。     

Hedgehog pathway regulators influence cervical cancer cell proliferation, survival and migration

IQGAP1, a ubiquitously expressed scaffold protein, has been identified in a wide range of organisms. It participates in multiple aspects of cellular events by binding to and regulating numerous interacting proteins. In our present study, we identified a new IQGAP1 binding protein named Aurora-A which is an oncogenic protein and overexpressed in various types of human tumors. In vitro analysis with GST-Aurora-A fusion proteins showed a physical interaction between Aurora-A and IQGAP1. Moreover, the binding also occurred in HeLa cells as endogenous Aurora-A co-immunoprecipitated with IQGAP1 from the cell lysates. Overexpression of IQGAP1 resulted in an elevation of both expression and activity of Aurora-A kinase. Endogenous IQGAP1 knockdown by siRNA promoted Aurora-A degradation whereas IQGAP1 overexpression enhanced the stability of Aurora-A. Additionally, we documented that the IQGAP1-induced cell proliferation was suppressed by knocking down Aurora-A expression. Taken together, our results showed an unidentified relationship between Aurora-A and IQGAP1, and provided a new insight into the molecular mechanism by which IQGAP1 played a regulatory role in cancer.     

Downregulation of RPL6 by siRNA inhibits proliferation and cell cycle progression of human gastric cancer cell lines

Our previous study revealed that human ribosomal protein L6 (RPL6) was up-regulated in multidrug-resistant gastric cancer cells and over-expression of RPL6 could protect gastric cancer from drug-induced apoptosis. It was further demonstrated that up-regulation of RPL6 accelerated growth and enhanced in vitro colony forming ability of GES cells while down-regulation of RPL6 exhibited the opposite results. The present study was designed to investigate the potential role of RPL6 in therapy of gastric cancer for clinic. The expression of RPL6 and cyclin E in gastric cancer tissues and normal gastric mucosa was evaluated by immunohistochemisty. It was found that RPL6 and cyclin E were expressed at a higher level in gastric cancer tissues than that in normal gastric mucosa and the two were correlative in gastric cancer. Survival time of postoperative patients was analyzed by Kaplan- Meier analysis and it was found that patients with RPL6 positive expression showed shorter survival time than patients that with RPL6 negative expression. RPL6 was then genetically down-regulated in gastric cancer SGC7901 and AGS cell lines by siRNA. It was demonstrated that down-regulation of RPL6 reduced colony forming ability of gastric cancer cells in vitro and reduced cell growth in vivo. Moreover, down-regulation of RPL6 could suppress G1 to S phase transition in these cells. Further, we evidenced that RPL6 siRNA down-regulated cyclin E expression in SGC7901 and AGS cells. Taken together, these data suggested that RPL6 was over-expressed in human gastric tissues and caused poor prognosis. Down-regulation of RPL6 could suppress cell growth and cell cycle progression at least through down-regulating cyclin E and which might be used as a novel approach to gastric cancer therapy.     

硫氧还蛋白还原酶1(TrxR1)在胃癌中的表达及对胃癌细胞生长的影响*

目的: 探讨胃癌组织硫氧还蛋白还原酶1(TrxR1)表达与生存时间的关系及其对胃癌细胞生长的影响。方法: 用Real-time PCR法检测76例胃癌组织及癌旁TrxR1 mRNA表达,并分析其与胃癌患者临床病理特征及预后的关系;随机选取3例胃癌组织及癌旁组织,采用免疫组化法、Western blot法检测TrxR1蛋白表达。采用Western blot法和Real-time PCR法检测胃癌细胞系及人胃粘膜上皮细胞中TrxR1的表达。采用小RNA干扰序列(siRNA)处理AGS细胞,根据处理方法不同将AGS细胞分为3组:阴性对照组:转染NC-siRNA、TRXR1 siRNA干扰1组:转染TRXR1-siRNA1、TRXR1 siRNA干扰2组:转染TRXR1-siRNA2。使用Real-time PCR法检测各组AGS细胞中TrxR1 mRNA的表达,克隆形成试验和MTT法检测AGS细胞生长情况。结果: 胃癌组织中TrxR1 mRNA和蛋白表达量均显著性上调,TrxR1主要定位于细胞质中。TrxR1高表达与患者TNM分期及淋巴结转移有关,且TrxR1高表达组患者的中位生存时间短于低表达组(P＜0.05)。胃癌细胞中TrxR1表达量高于人胃粘膜上皮细胞系中的表达。TRXR1-siRNA1组AGS细胞和TRXR1-siRNA2组AGS细胞中TrxR1 mRNA和蛋白与NC-siRNA组相比均显著性降低(P＜0.05),且AGS细胞克隆形成与增殖能力均降低(P＜0.05)。结论: 胃癌组织中TrxR1高表达提示患者预后不良,沉默TrxR1能抑制胃癌细胞的增殖。     

小干扰RNA抑制LRP16基因表达限制了MCF-7乳腺癌细胞增殖

雌激素雌二醇上调人乳腺癌细胞MCF 7中LRP16基因表达 ,该基因过表达促进MCF 7细胞增殖 .为进一步探讨LRP16基因不同表达水平对MCF 7细胞增殖的影响以及对雌激素的反应性增殖能力 ,采用针对LRP16基因特异的小干扰RNA策略 ,通过逆转录病毒介导及抗性筛选构建了LRP16基因被稳定抑制的 2个MCF 7细胞系 ,针对绿色荧光蛋白的干扰序列作为阴性对照 .Northern印迹实验检测了LRP16基因在各个细胞株中mNRA的水平 ,与对照组细胞比较 ,针对LRP16基因不同位置的 2个小干扰RNA可分别将该基因抑制 90 %和 6 0 % .细胞增殖试验结果显示 ,MCF 7细胞中LRP16基因表达抑制率越高 ,细胞增殖速率减慢越显著 (P <0 0 5 ) ;软琼脂集落形成试验结果显示 ,抑制LRP16基因在MCF 7细胞中表达 ,限制了细胞锚定非依赖性生长 ;细胞周期分析结果表明 ,LRP16基因抑表达使MCF 7细胞G1 S周期转换受抑 ;Western印迹结果表明 ,LRP16基因表达抑制的细胞中细胞周期蛋白E及细胞周期蛋白D1蛋白水平显著下调 ,但未检测到P5 3及Rb蛋白表达水平的影响 .雌二醇刺激的增殖实验结果显示 ,抑制LRP16基因表达没有消除MCF 7细胞的反应性增殖特征 .上述结果表明 ,LRP16基因表达量与MCF 7细胞增殖能力密切相关 ,抑制其表达可有效限制MCF 7细胞的增殖能力 ,提     

Adrenomedullin promotes cell cycle transit and up-regulates cyclin D1 protein level in human glioblastoma cells through the activation of c-Jun/JNK/AP-1 signal transduction pathway

Adrenomedullin is a secreted peptide hormone with multiple functions. Although a number of reports have indicated that adrenomedullin may be involved in tumor progression, its mechanism of action remains obscure. In this study, we have analysed the signal transduction pathway activated by adrenomedullin in human glioma cells. Our results revealed that adrenomedullin induced the phosphorylation of both c-Jun and JNK in glioblastoma cells. Silencing JNK expression with siRNA reversed the phosphorylation of c-Jun induced by adrenomedullin, indicating that JNK is responsible of c-Jun activation. In addition, electrophoretic mobility-shift assays showed that the increase in phosphorylation of c-Jun was associated with increased AP-1 DNA binding activity. Supershift assays and co-immunoprecipitation demonstrated that c-Jun and JunD are part of the AP-1 complex, indicating that activated c-Jun is dimerized with JunD in response to adrenomedullin. Furthermore, adrenomedullin was shown to promote cell transit beyond cell cycle phases with a concomittant increase in cyclin D1 protein level, suggesting that adrenomedullin effect cell proliferation through up-regulation of cyclin D1. The inhibition of JNK activation or the suppression of c-Jun or JunD expression with siRNA impaired the effects of adrenomedullin on cell proliferation and on cyclin D1. Taken together, these data demonstrate that activation of cJun/JNK pathway is involved in the growth regulatory activity of adrenomedullin in glioblastoma cells.     

Cyclin G2 is degraded through the ubiquitin-proteasome pathway and mediates the antiproliferative effect of activin receptor-like kinase 7

We have previously reported that Nodal, a member of the TGF-β superfamily, acts through activin receptor-like kinase 7 (ALK7) to inhibit ovarian cancer cell proliferation. To determine the mechanism underlying their effects, a cell cycle gene array was performed and cyclin G2 mRNA was found to be strongly up-regulated by Nodal and ALK7. To study the function and regulation of cyclin G2 in ovarian cancer cells, expression constructs were generated. We found that cyclin G2 protein level decreased rapidly after transfection, and this decrease was prevented by 26S proteasome inhibitors. Immunoprecipitation and pull-down studies showed that ubiquitin, Skp1, and Skp2 formed complexes with cyclin G2. Knockdown of Skp2 by siRNA increased, whereas overexpression of Skp2 decreased cyclin G2 levels. Nodal and ALK7 decreased the expression of Skp1 and Skp2 and increased cyclin G2 levels. Overexpression of cyclin G2 inhibited cell proliferation whereas cyclin G2-siRNA reduced the antiproliferative effect of Nodal and ALK7. Taken together, these findings provide strong evidence that cyclin G2 is degraded by the ubiquitin–proteasome pathway and that Skp2 plays a role in regulating cyclin G2 levels. Furthermore, our results also demonstrate that the antiproliferative effect of Nodal/ALK7 on ovarian cancer cells is in part mediated by cyclin G2.     

沉默PRR14基因抑制结肠癌HCT116细胞增殖

已有报道显示,富脯氨酸蛋白 14（proline-rich protein 14,PRR14）促进肿瘤的发生发展,但具体作用机制仍不清楚。本文以结肠癌细胞为模型,探索其对细胞增殖和细胞周期进程的影响。qPCR和Western 印迹检测发现,PRR14在4个结肠癌细胞系中呈现高水平表达。合成特异靶向PRR14基因的siRNA,转染结肠癌HCT116细胞。检测发现,PRR14基因表达下调约70%。CCK8结果显示,沉默PRR14后各时间点细胞增殖能力均显著降低,克隆形成实验细胞克隆数减少约40%;流式细胞仪结果显示,沉默PRR14后,G1期细胞比例升高约10%,S期细胞比例降低约14%;BrdU标记免疫荧光检测结果显示,BrdU阳性细胞比例减少约50%,表明细胞DNA合成速率显著降低。机制分析表明：促G1/S期转换基因周期蛋白依赖性激酶2（cyclin dependent kinase 2, CDK2）mRNA水平降低约85%,对应的蛋白质水平也明显降低,G1/S期转换抑制因子周期蛋白依赖性激酶抑制因子1A（cyclin dependent kinase inhibitor 1A,CDKN1A/P21）和周期蛋白依赖性激酶抑制因子1B（cyclin dependent kinase inhibitor 1B,CDKN1B/P27）mRNA水平分别升高约1.8倍和5倍,对应的蛋白质水平也明显升高。沉默PRR14表达,G1/S期相关基因表达紊乱,导致细胞G1期阻滞并抑制细胞增殖。结肠癌细胞中PRR14高表达可促进癌细胞恶性增殖。     

沉默PRR14基因抑制结肠癌HCT116细胞增殖

已有报道显示,富脯氨酸蛋白 14（proline-rich protein 14,PRR14）促进肿瘤的发生发展,但具体作用机制仍不清楚。本文以结肠癌细胞为模型,探索其对细胞增殖和细胞周期进程的影响。qPCR和Western 印迹检测发现,PRR14在4个结肠癌细胞系中呈现高水平表达。合成特异靶向PRR14基因的siRNA,转染结肠癌HCT116细胞。检测发现,PRR14基因表达下调约70%。CCK8结果显示,沉默PRR14后各时间点细胞增殖能力均显著降低,克隆形成实验细胞克隆数减少约40%;流式细胞仪结果显示,沉默PRR14后,G1期细胞比例升高约10%,S期细胞比例降低约14%;BrdU标记免疫荧光检测结果显示,BrdU阳性细胞比例减少约50%,表明细胞DNA合成速率显著降低。机制分析表明：促G1/S期转换基因周期蛋白依赖性激酶2（cyclin dependent kinase 2, CDK2）mRNA水平降低约85%,对应的蛋白质水平也明显降低,G1/S期转换抑制因子周期蛋白依赖性激酶抑制因子1A（cyclin dependent kinase inhibitor 1A,CDKN1A/P21）和周期蛋白依赖性激酶抑制因子1B（cyclin dependent kinase inhibitor 1B,CDKN1B/P27）mRNA水平分别升高约1.8倍和5倍,对应的蛋白质水平也明显升高。沉默PRR14表达,G1/S期相关基因表达紊乱,导致细胞G1期阻滞并抑制细胞增殖。结肠癌细胞中PRR14高表达可促进癌细胞恶性增殖。     

RNA干扰Ｃyclin D1基因表达对瘢痕疙瘩成纤维细胞增殖和G1期调控的影响

为研究siRNA干扰瘢痕疙瘩成纤维细胞cyclin D1基因表达,对瘢痕疙瘩成纤维细胞的增殖、细胞周期和G1期调控的影响,构建了靶向cyclin D1的siRNA表达质粒.利用LipofecmmineTM2000转染体外培养的瘢痕疙瘩成纤维细胞,应用荧光定量PCR、RT-PCR检测cyclin D1 mRNA的干扰效果,应用MTT法、流式细胞仪检测细胞增殖和细胞周期的变化,应用免疫组织化学染色检测成纤维细胞中cyclin D1、CDK4、P16、pRb蛋白表达的影响.主要结果如F:a.靶向cyclin D1的特异性siRNA序列可以高效地抑制成纤维细胞cyclin D1基因表达,对照组与实验组在mRNA水平其表达抑制率分别为63.68%和92.83%(P<0.01);b.可以显著抑制瘢痕疙瘩成纤维细胞的增殖,改变细胞周期分布,G0/G1期细胞比例显著高于各对照组(P<0.05),细胞分裂被阻滞;c.免疫组化染色发现,转染72 h后,过表达的cyclin D1、CDK4和pRb蛋白,在瘢痕疙瘩成纤维细胞中均出现了不同程度的表达下调,而低表达的P16则呈上调表现.由上述结果可见,构建的靶向cyclin D1的RNAi表达质粒,可有效地抑制瘢痕疙瘩成纤维细胞cyclin D1基因表达,通过改变Gl期相关周期蛋白的水平,影响G1/S期的进程,显著地抑制成纤维细胞的增殖.     

支架蛋白IQGAP1参与气道上皮细胞损伤修复过程

气道上皮损伤修复过程包括细胞延伸、迁移和增殖.IQGAP1 (IQ domain GTPase-activating protein 1)是一个在许多细胞生命活动中非常有意义的蛋白,但其在肺上皮细胞中的作用尚未阐述清楚.本文采用目前广泛应用的刮伤气道上皮细胞的体外模型来研究IQGAP1的功能.结果显示,IQGAP1在小鼠、大鼠、猪和人气道上皮细胞中有丰富表达.它与微管骨架共定位,可被微管解聚剂nocodazole破坏.刮伤6～9h后,IQGAP1 mRNA及蛋白表达上调.过表达外源性IQGAP1导致β-catenin核转位,从而活化Tcf/Lef信号.此外,刮伤还影响IQGAP1与β-catenin、结肠腺瘤病(adenomatous polyposis coli, APC)蛋白及细胞质连接蛋白-170 (cytoplasmic linker protein-170, CLIP-170)之间的相互作用.通过小干扰RNA (small interference RNA, siRNA)沉默IQGAP1表达则明显延迟损伤愈合.结果提示,IQGAP1信号参与气道上皮细胞损伤修复过程.     

IQGAP1基因干扰对人食管癌细胞同质粘附能力的影响

目的：研究IQGAP1基因干扰对人食管癌细胞同质粘附能力的影响。方法：体外培养人食管癌KYSE150和 EC9706细胞,利用Western blot方法检测两株细胞IQGAP1蛋白的表达,利用缓慢聚集和细胞分离实验比较两株细胞同质粘附能力的差异;进一步在KYSE150和EC9706细胞中构建IQGAP1基因干扰的稳定细胞系,观察IQGAP1基因干扰后细胞同质粘附能力的改变。结果：KYSE150细胞IQGAP1蛋白表达量低于EC9706细胞,而同质粘附能力高于EC9706细胞;IQGAP1基因干扰后,其蛋白表达量明显降低,而细胞同质粘附能力明显增强。结论：IQGAP1 基因干扰能够显著增强食管癌细胞的同质粘附能力,从而降低肿瘤细胞的恶性表型。     

Role of PI3K and AKT specific isoforms in ovarian cancer cell migration, invasion and proliferation through the p70S6K1 pathway

Ovarian cancer is the leading cause of death from gynecological malignancy for women. The amplification of the PI3K catalytic subunit (p110) and the lost function of PTEN are frequently detected in ovarian cancer cells. PI3K plays an important role in tumorigenesis. To specifically inhibit PI3K activity in ovarian cancer cells, we constructed small interfering RNA (siRNA) against p110. The expression of p110 siRNA significantly decreased cell migration, invasion, and proliferation compared to the siSCR control cells. The expression of p110 siRNA induced CDK inhibitor p27^KIP1 levels, and decreased levels of cyclin D1, CDK4, and phosphorylated retinoblastoma protein. PI3K transmits the mytogenic signal through AKT. AKT has three isoforms in the cells: AKT1, AKT2 and AKT3. We found that inhibition of AKT1 is sufficient to affect cell migration, invasion, and proliferation. Expression of AKT1 siRNA had a similar effect as p110 siRNA in the cells. We showed the roles of specific PI3K and AKT isoforms in the cells, which are important to understanding the mechanism of PI3K/AKT signaling in ovarian cancer cells. Both p110 and AKT1 siRNA-expressing cells decreased the activation of p70S6K1. Inhibition of p70S6K1 activity by its siRNA also decreased cell migration, invasion, and proliferation associated with the induction of p27^KIP1 levels, and with the inhibition of cell cycle-associated proteins including cyclin D1, CDK2, and phosphorylated retinoblastoma protein. This study demonstrates the important role of the PI3K/AKT/mTOR/p70S6K1 pathway in cell proliferation, migration, and invasion in ovarian cancer cells by using siRNA-mediated gene silencing as a reverse genetic method.     

Epstein-Barr Virus-Encoded BARF1 Promotes Proliferation of Gastric Carcinoma Cells through Regulation of NF-κB

In Epstein-Barr virus (EBV)-infected gastric carcinoma, EBV-encoded BARF1 has been hypothesized to function as an oncogene. To evaluate cellular changes induced by BARF1, we isolated the full-length BARF1 gene from gastric carcinoma cells that were naturally infected with EBV and transfected BARF1 into EBV-negative gastric carcinoma cells. BARF1 protein was primarily secreted into culture supernatant and only marginally detectable within cells. Compared with gastric carcinoma cells containing empty vector, BARF1-expressing gastric carcinoma cells exhibited increased cell proliferation (P < 0.05). There were no significant differences in apoptosis, invasion, or migration between BARF1-expressing gastric carcinoma cells and empty vector-transfected cells. BARF1-expressing gastric carcinoma cells demonstrated increased nuclear expression of nuclear factor kappa B (NF-κB) RelA protein and increased NF-κB-dependent cyclin D1. The expression of p21^WAF1 was diminished by BARF1 transfection and increased by NF-κB inhibition. Proliferation of naturally EBV-infected gastric carcinoma cells was suppressed by BARF1 small interfering RNA (siRNA) (P < 0.05). Immunohistochemical analysis of 120 human gastric carcinoma tissues demonstrated increased expression of cyclin D1 and reduced expression of p21^WAF1 in EBV-positive samples versus EBV-negative gastric carcinomas (P < 0.05). In conclusion, the secreted BARF1 may stimulate proliferation of EBV-infected gastric carcinoma cells via upregulation of NF-κB/cyclin D1 and reduction of the cell cycle inhibitor p21^WAF1, thereby facilitating EBV-induced cancer progression.     

HMG盒转录因子1(HBP1)参与H2O2诱导的细胞衰老过程

为探讨HMG盒转录因子1 (HBP1)在过氧化氢（H2O2）诱导的细胞衰老中所起的作用,通过慢病毒感染得到稳定表达HBP1的MDA-MB-231细胞,以H2O2处理细胞．采用Western免疫印迹杂交试验和实时PCR检测HBP1、p16和细胞周期蛋白D1（cyclinD1）表达水平的变化．用荧光免疫试验检测H2O2对HBP1表达的影响,以及HBP1在H2O2的诱导下对于p16和细胞周期蛋白D1启动子的影响．用细胞增殖试验检测H2O2对于细胞增殖的影响． 用基因敲减实验和衰老相关β半乳糖苷酶(SA-β-Gal)染色检测在H2O2诱导的细胞衰老中HBP1所起的作用．Western和免疫荧光实验结果显示,细胞经H2O2处理后,HBP1表达增高的同时促进了p16的表达,降低了细胞周期蛋白D1的表达．细胞增殖实验结果显示,H2O2显著抑制了细胞的增殖．基因敲减实验和SA-β-Gal染色实验说明,H2O2可诱导HBP1表达正常的MDA-MB-231细胞衰老,而HBP1的敲减则抑制了H2O2诱导的细胞衰老过程．本研究结果提示,在H2O2诱导的衰老中,HBP1的表达显著增加,并通过促进衰老相关基因p16的表达和抑制生长因子cyclinD1的表达来阻碍细胞增殖,促进细胞衰老．HBP1在H2O2诱导的细胞衰老过程中起着重要作用,H2O2诱导的细胞衰老必须在HBP1存在的情况下才能发生．     

Antiproliferative mechanism of a cannabinoid agonist by cell cycle arrest in human gastric cancer cells

For gastric cancers, the antineoplastic activity of cannabinoids has been investigated in only a few reports and knowledge regarding the mechanisms involved is limited. We have reported previously that treatment of gastric cancer cells with a cannabinoid agonist significantly decreased cell proliferation and induced apoptosis. Here, we evaluated the effects of cannabinoids on various cellular mediators involved in cell cycle arrest in gastric cancer cells. AGS and MKN-1 cell lines were used as human gastric cancer cells and WIN 55,212-2 as a cannabinoid agonist. Cell cycles were analyzed by flow cytometry and western blotting. Treatment with WIN 55,212-2 arrested the cell cycle in the G0/G1 phase. WIN 55,212-2 also upregulated phospho-ERK1/2, induced Kip1/p27 and Cip1/WAF1/p21 expression, decreased cyclin D1 and cyclin E expression, decreased Cdk 2, Cdk 4, and Cdk 6 expression levels, and decreased phospho-Rb and E2F-1 expression. ERK inhibitor decreased the proportion of G0/G1 phase which was induced by WIN 55,212-2. Inhibition of pAKT led to cell cycle arrest in gastric cancer cells. Cell cycle arrest preceded apoptotic response. Thus, this cannabinoid agonist can reduce gastric cancer cell proliferation via G1 phase cell cycle arrest, which is mediated via activation of the MAPK pathway and inhibition of pAKT.