甘氨鹅脱氧胆酸钠通过MAPK/ERK1/2介导Bcl-2在Ser70位点的磷酸化引起人肝细胞癌的抗药

B细胞淋巴瘤-2（Bcl-2）是一种重要的抗凋亡蛋白质,在多种人类肿瘤中普遍过表达。甘氨鹅脱氧胆酸钠（GCDA）与消化道肿瘤的发生发展密切相关,并能介导肝癌细胞对化疗药物的抵抗。本文旨在探讨在GCDA介导的人肝细胞癌（HCC）耐药性中Bcl-2的作用及其机制。本研究以肝癌细胞系为研究对象,Western印迹结果显示,Bcl-2在多种肝癌细胞系中均有表达。设计靶向Bcl-2的siRNA沉默HCC细胞系内源性Bcl-2的表达,发现Bcl-2沉默之后促进了化疗药物5-FU介导的HCC细胞凋亡。机制上,GCDA可介导Bcl-2在Ser70位点的磷酸化,而Ser70位点的磷酸化能够被PD98059（MAPK/ERK1/2抑制剂）所抑制。构建huBcl2-WT和huBcl2-S70A真核表达载体,脂质体转染HCC细胞系。用Annexin V-FITC/PI流式细胞术检测凋亡细胞。结果显示,huBcl2-WT过表达能抑制5 FU介导的凋亡,S70位点失活突变成A后,Bcl-2的过表达不能抑制5-FU介导的凋亡。本研究提示,GCDA通过MAPK/ERK1/2通路介导的Bcl-2 Ser70位点的磷酸化,在肝癌细胞的存活和抗药中发挥重要作用。抑制Bcl-2能够促进化疗药物5-FU介导的HCC细胞凋亡,该结果为治疗GCDA介导的耐药性肝癌提供新的思路。     

姜黄素通过MAPK信号通路诱导人肝癌SMMC-7721细胞凋亡

本文阐述了姜黄素(Curcumin)对体外培养的人肝癌SMMC-7721细胞增殖和凋亡的影响,并探讨了其诱导凋亡的信号转导机制。采用MTT法和细胞计数法检测不同浓度姜黄素对人肝癌细胞株SMMC-7721增殖的影响,利用流式细胞术检测姜黄素对人肝癌SMMC-7721细胞凋亡的影响,通过RT-PCR及Western blot检测姜黄素对人肝癌SMMC-7721细胞中凋亡相关蛋白Caspase-3、Survivin、Bcl-2和Bax表达的影响,最后通过检测MAPK的磷酸化水平分析姜黄素诱导SMMC-7721细胞凋亡的信号转导机制,通过MAPK抑制剂实验进一步证实诱导凋亡的分子机制。研究结果显示,姜黄素呈时间和剂量依赖性抑制人肝癌SMMC-7721细胞的增殖,其中40μmol/L姜黄素可明显诱导SMMC-7721细胞的凋亡,并呈时间依赖性上调促凋亡蛋白Caspase-3和Bax的表达、下调抗凋亡蛋白Survivin和Bcl-2的表达,姜黄素对凋亡相关蛋白表达的调节及诱导凋亡可以通过激活JNK、抑制ERK和p38 MAPK信号通路实现。表明姜黄素可诱导人肝癌SMMC-7721细胞凋亡,其机制与姜黄素激活JNK、抑制ERK和p38 MAPK信号通路从而上调Caspase-3和Bax的表达,下调Survivin和Bcl-2的表达有关。     

ERK1/2在食管鳞状细胞癌中促进细胞增殖、抑制凋亡及其调控机制的研究

探讨ERK1/2在食管鳞状细胞癌(ESCC)中对肿瘤细胞增殖、凋亡的调控及其机制。平板克隆、细胞凋亡和细胞周期实验结果发现ERK1/2 MAPK通路抑制可减弱Eca109细胞克隆形成和增殖,促进细胞凋亡,减慢细胞周期;进一步发现ERK1/2 MAPK通路抑制可以反转由mi R-21过表达诱导的Eca109细胞增殖、凋亡和周期的变化;q RT-PCR和Western-blot免疫印迹结果发现ERK1/2 MAPK信号通路抑制可以下调内源性mi R-21表达和反转外源性mi R-21诱导的ERK1/2 MAPK信号通路活化。实验结果提示ERK1/2 MAPK通路抑制可能通过下调Eca109细胞中mi R-21表达阻碍Eca109细胞增殖、促进细胞凋亡和减慢细胞周期,最终导致ESCC细胞生长抑制。     

Smad4分子在ERK/MAPK信号转导通路中的作用

为了探讨在人永生化支气管上皮细胞BEP2D细胞中,Smad4分子对 ERK/MAPK通路的作用,我们用RNA干扰的方法分别设计了两对Smad4 siRNA,并使BEP2D细胞中Smad4靶向沉默,用Western印迹分析了细胞内ERK激酶和MEK激酶磷酸化水平的变化.结果发现,当Smad4表达沉默后,ERK激酶磷酸化水平未变,MEK激酶磷酸化水平有所降低;再加TGF-β1诱导后ERK激酶和MEK激酶磷酸化水平均显著降低至基础水平以下.结果表明在BEP2D细胞中,Smad4的缺失抑制TGF-β1对ERK/MAPK通路的活化,故提出TGF β活化ERK/MAPK通路需要Smad4存在的假设.     

褪黑素联合顺铂对A549细胞凋亡相关基因的影响

观察褪黑素以及褪黑素联合顺铂是否可以抑制A549细胞增殖并促进细胞凋亡,探讨ERK1/2MAPK信号通路在这一过程中的作用,为肺腺癌的研究提供实验依据。用不同浓度的褪黑素、不同浓度的顺铂、褪黑素联合顺铂刺激体外培养的肺腺癌细胞系A549细胞,MTT法检测各组细胞的活性,运用Real-time RT-PCR检测凋亡相关基因Bcl-2、Bax、p53的表达水平,Western-blot检测ERK1/2MAPK的活性变化。结果:1)单纯使用褪黑素和顺铂均可抑制A549细胞增殖,褪黑素联合顺铂时,抑制作用更加显著,一定浓度的褪黑素可降低顺铂的使用浓度;2)未经处理的A549细胞Bcl-2/Bax很高,p53基因表达较低,经褪黑素以及褪黑素联合顺铂刺激后A549细胞中Bcl-2/Bax明显下降,p53基因表达明显升高,并有浓度依赖;3)经褪黑素和顺铂刺激后A549细胞中磷酸化ERK1/2MAPK水平明显降低。褪黑素联合顺铂可抑制A549细胞增殖并且诱导凋亡相关基因表达,褪黑素和顺铂对A549细胞的抑制作用可能与ERK1/2MAPK信号通路有关。     

SIRT6基因沉默对人肝癌细胞凋亡的影响及其机制研究

该文旨在探讨慢病毒介导的沉默信息调节因子6(silent information regulator 6,SIRT6)基因沉默对人肝癌细胞凋亡的影响及其机制。逆转录PCR(RT-PCR)和Western blot分别检测人肝癌细胞系(SK-Hep-1、Huh-7、PLC/PRF/5、Hep G2)和永生化肝细胞系(MIHA)中SIRT6基因的表达水平;利用慢病毒介导的sh RNA干扰技术靶向沉默SIRT6的表达,并通过RT-PCR和Western blot验证其沉默效率;流式细胞术检测SIRT6基因沉默对人肝癌细胞凋亡的影响,进一步应用RT-PCR和Western blot检测SIRT6基因沉默对凋亡抑制蛋白基因(inhibitor of apoptosis proteins,IAPs)家族m RNA和蛋白质水平的影响;最后,应用流式细胞术分析X连锁凋亡抑制蛋白基因(X-linked inhibitor of apoptosis protein gene,XIAP)在SIRT6基因沉默诱导的肝癌细胞凋亡中的作用。结果显示,SIRT6基因在人肝癌细胞系中表达上调;慢病毒介导的sh RNA能抑制人肝癌细胞中SIRT6基因的表达;沉默SIRT6基因的表达能诱导人肝癌细胞凋亡,并降低XIAP的m RNA和蛋白质水平;过表达XIAP能逆转SIRT6基因沉默所诱导的人肝癌细胞凋亡。该研究结果提示,SIRT6基因沉默可能通过调节XIAP的表达从而诱导人肝癌细胞凋亡。     

沉默信息调节因子3促进肝癌细胞凋亡及其机制研究

该文旨在探讨沉默信息调节因子3(sirtuin 3,SIRT3)对肝癌细胞凋亡的影响,并研究SIRT3调节肝癌细胞凋亡的分子机制。运用流式细胞术检测SIRT3过表达对肝癌细胞系(SMMC-7721和SK-Hep-1)凋亡的影响;通过si RNA靶向沉默SIRT3并检测SIRT3沉默对肝癌细胞凋亡的影响;实时荧光定量PCR(Real-time PCR)分析SIRT3对Bcl-2家族成员m RNA水平的影响,筛选受SIRT3调节的Bcl-2家族成员;Western blot进一步检测SIRT3对目标Bcl-2家族成员蛋白水平的影响;流式细胞术分析目标Bcl-2家族成员在SIRT3诱导肝癌细胞凋亡中的作用。结果显示,SIRT3过表达促进肝癌细胞凋亡并引起Bax mRNA和蛋白水平升高;SIRT3沉默抑制肝癌细胞凋亡,同时也抑制Bax蛋白水平表达,Bax沉默显著减少了SIRT3过表达细胞中的凋亡数目。该研究结果提示,SIRT3通过凋亡调节基因Bax诱导肝癌细胞凋亡。     

Mcl-1在胆盐（GCDA）诱导的肝癌细胞耐药中的作用

目的：探讨抗凋亡蛋白Mcl-1在GCDA诱导的肝癌细胞耐药中的作用及其机制。方法：培养3种肝癌细胞系,用免疫荧光法和Western blot技术检测Mcl-1的表达;GCDA±CYC处理HepG2细胞,采用Western blot技术检测Mcl-1的半衰期变化;用抗癌药物Irinotecan与GCDA对HepG2细胞进行处理,采用MTT法和Western blot技术分别检测细胞增殖抑制率和Mcl-1的表达变化;用RNA干扰技术下调Mcl-1,检测化疗药物对HepG2细胞的敏感性。结果：Mcl-1在肝癌细胞中广泛表达;GCDA能延长Mcl-1的半衰期至6h以上,并明显减弱化疗药物对抗凋亡蛋白Mcl-1的抑制作用,降低癌细胞的药物敏感性;RNA干扰下调Mcl-1能增加癌细胞的药物敏感性。结论：胆盐（GCDA）能诱导HepG2细胞产生耐药性,其作用机制可能是通过延长Mcl-1半衰期增加其蛋白稳定性和抗凋亡作用来促使肝癌细胞抗药的。     

RNAi沉默Snail增加结肠癌对5氟尿嘧啶敏感性研究

目的:研究Snail的抑制是否能增加耐药结肠癌细胞对5-FU的敏感性,评估其可能的信号转导通路。方法:使用5-氟尿嘧啶耐药HCT116细胞(HCT116/5-FU),评估细胞形态及分子的变化。通过靶向人Snail基因小干扰RNA(si RNA)抑制Snail的表达。Annexin V/PI染色用于评估5-FU诱导的细胞凋亡。Western blot检测caspase以及可能的丝裂原活化蛋白激酶(MAPK)和线粒体途径。结果:HCT116细胞对5-Fu耐药性的获得诱导了与EMT一致的形态学变化。RNA干扰沉默Snail逆转HCT116/5-FU细胞EMT并增加了5-FU耐药HCT116细胞对5-FU的敏感性。可能的机制涉及JNK与线粒体途径的激活。结论:EMT样表型的改变与HCT116细胞对5-FU耐药相关;si RNA介导的Snail下调可能是一个潜在的克服5-FU化疗耐药的治疗方法。     

花生四烯酸细胞色素P450表氧化酶对凋亡内皮细胞Bcl-2表达、Caspase-3及MAPK活性的影响

花生四烯酸经细胞色素P450表氧化酶代谢产生的内皮来源超极化因子(EDHF)[表氧化二十烷烯酸(EETs)]对内皮细胞具有保护作用。研究了转染细胞色素P450表氧化酶基因CYPBM3·F87V、CYP2C11OR及CYP2J2产生内源性EETs,通过检测内皮细胞中Bcl-2表达、caspase-3的活性及MAPK磷酸化水平探讨内源性EDHF的内皮细胞保护效应及其抗TNF-α诱导内皮细胞凋亡的作用机制。原代培养的牛主动脉血管内皮细胞转染CYP450表氧化酶基因24h后,加入TNF-α作用一定时间诱导内皮细胞凋亡,用Western印迹方法检测Bcl-2的表达,MAPK磷酸化水平,同时测定caspase-3的活性。结果显示转染表氧化酶基因能抑制TNF-α诱导的时间依赖性Bcl-2下调,抑制Caspase-3的激活。TNF-α使细胞内磷酸化MAPK水平呈时间依赖性减低,转染表氧化酶基因后细胞内的磷酸化MAPK水平较对照组升高。因此,转染表氧化酶基因CYPBM3·F87V、CYP2C11OR以及CYP2J2使内皮细胞产生内源性EETs(EDHF)通过激活MAPK(ERK1/2)途径,抑制抗凋亡基因Bcl-2的降解,抑制caspase-3的激活,从而抑制TNF-α诱导的内皮细胞凋亡,因而具有内皮保护效应。     

Survivin inhibition is critical for Bcl-2 inhibitor-induced apoptosis in hepatocellular carcinoma cells

Our study aims to study the therapeutic effects of a novel Bcl-2 inhibitor, ABT-263, on hepatocellular carcinoma (HCC) and to provide primary preclinical data for future clinical trial with ABT-263. In this study we showed that Bcl-xL and survivin were up-regulated in HCC cell lines and human liver cancer tissues. Clinic used ABT-263 single treatment had no apoptotic effects on HCC cells whereas higher doses of ABT-263 did. Interestingly, the combination treatment of ABT-263 with survivin inhibitor YM-155 could result in significant apoptosis in HCC cells. Survivin inhibition through gene silencing significantly enhanced ABT-263 to induce apoptosis in HCC cells. We found that low dose of ABT-263 single treatment resulted in ERK activation and survivin up-regulation, which might be involved in the resistance of HCC cells to ABT-263 since blockade of ERK activation sensitized ABT-263-induced apoptosis. Importantly, ABT-263 and YM-155 combination treatment had no apoptotic effects on normal human hepatocytes. Taken together, these data suggest the combination treatment of Bcl-2 inhibitor and survivin inhibition may have a great potential for liver cancer therapy.     

MEK/ERK-dependent uPAR expression is required for motility via phosphorylation of P70S6K in human hepatocarcinoma cells

Motility and invasiveness events require specific intracellular signaling cascade activations. In cancer liver cells, one of these mechanisms could involve the MAPK MEK/ERK cascade activation which has been shown over expressed and activated in hepatocellular carcinoma. To study whether the MEK/ERK cascade is involved in the motility of HCC, we examined the effect of MEK inhibitor and ERK2 silencing using monolayer wound-healing assays and fluoroblock invasion systems. Evidence was provided that the MAPK cascade is a key transduction pathway which controls HCC cells motility and invasiveness. We could disconnect proliferation to motility using mitomycin C and we established that RNAi-mediated inhibition of ERK2 led to strongly reduced cell motility. To improve our understanding, we analysed the regulation and the role of urokinase receptor (uPAR) in this process. We provided evidence that uPAR was under a MEK/ERK dependent mechanism and blocking uPAR activity using specific antagonist or inhibiting its expression by RNA interference which resulted in complete inhibition of motility. Moreover, we found in MAPK inhibited cultures and in uPAR silencing cells that p70S6K phosphorylation on residue Thr-389 was significantly reduced, whereas Ser-421/Thr-424 phosphorylation did not change. We highlighted that the FRAP/mTOR pathway did not affect motility and Thr-389 phosphorylation. Furthermore, we demonstrated that p70S6K inhibition by RNA interference completely inhibited hepatocarcinoma cell motility. Therefore, targeting uPAR and/or MEK/ERK/S6K by RNA interference could be a major therapeutic strategy for the future treatment of invasive hepatocarcinoma cells.     

激活mGlu5通过MAPK信号通路调节肝癌细胞HepG2生长

代谢型谷氨酸受体5（mGlu5）与神经元存活及脑肿瘤发生关系密切.近年发现,mGlu5在肝组织中有表达,并且在肝的病理过程中发挥重要的调节作用.而 mGlu5 是否在肝癌中起作用,目前尚未见报道.本研究选用代谢型谷氨酸受体特异性激动剂二羟基苯甘氨酸(dihydroxyphenylglycine,DHPG)处理肝癌细胞HepG2,从而探讨激活mGlu5对肝癌细胞生长的影响及其机制.结果显示,激活mGlu5能够促进HepG2细胞生长,并激活ERK/JNK通路,抑制p38通路,进而激活转录因子CREB/Elk1和NF-κB.本文揭示了MAPK通路可能参与mGlu5对肝癌细胞生长的调控,为临床提供以mGlu5作为药物靶位点的肝癌治疗新思路.     

The novel BH-3 mimetic apogossypolone induces Beclin-1- and ROS-mediated autophagy in human hepatocellular cells

Apogossypolone (ApoG2), a novel derivative of gossypol, exhibits superior antitumor activity in Bcl-2 transgenic mice, and induces autophagy in several cancer cells. However, the detailed mechanisms are not well known. In the present study, we showed that ApoG2 induced autophagy through Beclin-1- and reactive oxygen species (ROS)-dependent manners in human hepatocellular carcinoma (HCC) cells. Incubating the HCC cell with ApoG2 abrogated the interaction of Beclin-1 and Bcl-2/xL, stimulated ROS generation, increased phosphorylation of ERK and JNK, and HMGB1 translocation from the nucleus to cytoplasm while suppressing mTOR. Moreover, inhibition of the ROS-mediated autophagy by antioxidant N-acetyl-cysteine (NAC) potentiates ApoG2-induced apoptosis and cell killing. Our results show that ApoG2 induced protective autophagy in HCC cells, partly due to ROS generation, suggesting that antioxidant may serve as a potential chemosensitizer to enhance cancer cell death through blocking ApoG2-stimulated autophagy. Our novel insights may facilitate the rational design of clinical trials for Bcl-2-targeted cancer therapy.     

Bile salt exposure increases proliferation through p38 and ERK MAPK pathways in a non-neoplastic Barrett's cell line

Bile reflux has been implicated in the neoplastic progression of Barrett's esophagus (BE). Bile salts increase proliferation in a Barrett's-associated adenocarcinoma cell line (SEG-1 cells) by activating ERK and p38 MAPK pathways. However, it is not clear that these findings in cancer cells are applicable to non-neoplastic cells of benign BE. We examined the effect of bile salts on three human cell lines: normal esophageal squamous (NES) cells, non-neoplastic Barrett's cells (BAR cells), and SEG-1 cells. We hypothesized that bile salt exposure activates proproliferative and antiapoptotic pathways to promote increased growth in BE. NES, BAR, and SEG-1 cells were exposed to glycochenodeoxycholic acid (GCDA) at a neutral pH for 5 min. Proliferation was measured by Coulter counter cell counts and a 5-bromo-2'-deoxyuridine (BrdU) incorporation assay. GCDA-induced MAPK activation was examined by Western blot analysis for phosphorylated ERK and p38. Apoptosis was measured by TdT-mediated dUTP nick-end labeling and annexin V staining after GCDA and UV-B exposure. Statistical significance was determined by ANOVA. NES cells exposed to 5 min of GCDA did not increase cell number. In BAR cells, GCDA exposure increased cell number by 31%, increased phosphorylated p38 and ERK levels by two- to three-fold, increased BrdU incorporation by 30%, and decreased UV-induced apoptosis by 15-20%. In conclusion, in a non-neoplastic Barrett's cell line, GCDA exposure induces proliferation by activation of both ERK and p38 MAPK pathways. These findings suggest a potential mechanism whereby bile reflux may facilitate the neoplastic progression of BE.     

CDC20 regulates the cell proliferation and radiosensitivity of P53 mutant HCC cells through the Bcl-2/Bax pathway

Purpose: The incidence of hepatocellular carcinoma (HCC) is extremely high, and China accounts for approximately 50% of global liver cancer cases. Previous studies reported that CDC20 is involved in the occurrence and progression of a variety of malignant tumors. So, whether CDC20 will affect the development of HCC, we have conducted in-depth research on this.Methods: We selected Hep3B and HepG2 for cell culture, and performed siRNA transfection, lentiviral infection, western blot, MTS determination, cell cycle determination, apoptosis test, immunodeficiency test, clone survival test and subcutaneous parthenogenesis in nude mice.Results: Knockdown of CDC20 greatly enhanced the radiation efficacy on the growth retardation in HepG2, and protein level of CDC20 was decreased for the activation of P53 by radiation. Downregulation of CDC20 combined with radiation can inhibit proliferation, aggravate DNA damage, increase G2/M arrest, and promote apoptosis of HCC cells to a greater extent, and the relative survival fraction of HCC cells was gradually reduced with radiation dose increased in P53 mutated Hep3B cells. After knocking down CDC20 in HCC, Bcl-2 was down-regulated and Bax expression increased. Down-regulation of CDC20 can inhibit further invasion by promoting the radiosensitivity of HCC.Conclusion: In this study, we found that that CDC20 was highly expressed in HCC and participated in radio resistance of HCC cells with P53 mutation Bcl-2/Bax via signaling pathway. This study is the first to present evidence that CDC20 may play a role in improving the efficacy of radiotherapy in HCC.