HGF/c-Met信号通路在克唑替尼诱导不同肺癌细胞株凋亡中的作用

 目的: 观察克唑替尼(crizotinib)诱导不同肺癌细胞株凋亡中HGF/c-Met信号通路的变化并探讨其调控机制。方法: 采用噻唑蓝(MTT)法检测克唑替尼对H1993(c-Met扩增的肺腺癌细胞)、H2228(含有EML4-ALK融合基因的肺癌细胞)和A549细胞的活力抑制情况;采用流式细胞术检测3种细胞在克唑替尼作用后24 h、48 h和72 h的凋亡率;采用Western blot检测细胞在克唑替尼作用前后HGF/c-Met信号通路中MET蛋白及其磷酸化形式p-MET的水平,同时观察其下游通路关键蛋白AKT、ERK、p-AKT和p-ERK的变化情况。结果: MTT结果表明克唑替尼作用72 h后,H1993、H2228和A549细胞株的细胞活力抑制率均呈剂量依赖性升高。流式细胞术检测发现随着克唑替尼作用时间的延长,细胞凋亡率呈时间依赖性增加(P<0.05)。Western blot检测结果提示在H1993细胞株和H2228细胞株中,p-MET、p-AKT和p-ERK随着时间的延长蛋白水平呈现下降趋势。而在A549细胞株中p-AKT、p-ERK和p-MET在药物作用72 h后的变化趋势不明显。结论: 初步证实HGF/c-Met信号通路与克唑替尼诱导肺癌细胞株H1993和H2228凋亡相关。     

mTOR信号通路与细胞凋亡

哺乳类动物雷帕霉素靶蛋白(mTOR)主要通过上游信号转导通路磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(PKB/Akt)/mTOR信号通路及下游信号通路mTOR/ eIF4E结合蛋白1(4EBP1)、mTOR/p70S6激酶(p70S6K)在细胞生长、增值与分化和在血管再生、蛋白合成与降解中发挥作用.细胞凋亡是细胞的一种程序性死亡,在机体发育、组织代谢中有着重要作用,而细胞凋亡的异常调节与许多疾病的发生和发展紧密相连.近年研究发现,mTOR信号通路在细胞凋亡过程中扮演了重要角色,并已被作为新的药物治疗靶点.     

旁路信号通路激活介导的EML4-ALK融合基因阳性肺癌细胞株H3122对alectinib继发耐药的研究

目的:本研究旨在探讨肝细胞生长因子(HGF)、表皮生长因子(EGF)及转化生长因子α(TGF-α)是否以旁路激活的方式诱导EML4-ALK融合基因阳性肺癌细胞株H3122对alectinib的耐药,并进一步探讨旁路信号激活在alectinib耐药中的作用。方法:用不同浓度的alectinib、克唑替尼(crizotinib)、17-DMAG或(和)HGF(50μg/L)、EGF(100μg/L)、TGF-α(100μg/L)处理EML4-ALK阳性肺癌细胞株H3122,采用CCK-8法检测细胞活力,流式细胞术检测细胞凋亡,应用Western blot技术检测细胞中ALK、c-Met、EGFR及相应磷酸化蛋白的表达,观察其下游通路关键蛋白AKT、ERK、p-AKT和p-ERK水平。结果:Alectinib作用72 h后,H3122细胞株的活力随着alectinib药物浓度的增加而逐渐下降,呈剂量依赖性。HGF、EGF和TGF-α诱导后,alectinib抑制H3122细胞的生长曲线往右移,HGF、EGF和TGF-α处理能够降低alectinib对肺癌细胞活力的抑制作用。0.05μmol/L alectinib作用H3122细胞株48 h后的凋亡率为(20.12±1.36)%,而alectinib联合HGF、EGF和TGF-α后的凋亡率分别为(7.85±1.03)%、(5.60±0.79)%和(4.58±1.00)%,显著低于alectinib单药处理(P0.05)。Alectinib单药成功抑制p-ALK及其下游信号通路,HGF明显增加细胞中p-Met及其下游p-AKT、p-ERK的蛋白水平,EGF和TGF-α明显增加细胞中p-EGFR及其下游p-AKT、p-ERK的表达,alectinib抑制p-ALK,但不能抑制HGF、EGF和TGF-α诱导的pAKT和p-ERK的蛋白表达。此外,联合应用crizotinib和17-DMAG可以抑制因HGF和EGFR配体而导致的H3122耐药细胞的活力。结论:HGF、EGF和TGF-α可通过旁路激活的方式诱导EML4-ALK阳性肺癌细胞H3122对alectinib耐药,其机制可能与HGF激活c-Met磷酸化、EGF和TGF-α激活EGFR磷酸化有关。     

EGFR突变的非小细胞肺癌患者EML4-ALK融合基因的检测及其临床特征分析

目的: 检测EML4-ALK融合基因在表皮生长因子受体(epidermal growth factor receptor, EGFR)突变的非小细胞肺癌(non-small-cell lung cancer, NSCLC)人群中的突变率,并分析其与临床特征的关系。方法: 入选的102例NSCLC患者均为中国人,且至少满足以下1个入选条件:女性、不吸烟/少吸烟和肺腺癌。将102例患者的组织标本采用多重逆转录聚合酶链反应 (multiplex RT-PCR)的方法检测其EML4-ALK融合基因的突变率;对EML4-ALK阳性患者的组织标本采用DNA扩增后直接测序的方法来检测其EGFR(18~21号外显子)及Kirsten鼠肉瘤基因(Kirsten rat sarcoma,KRAS)(1、2号外显子)的突变情况。结果: 102例非小细胞肺癌患者的组织标本,有8例(7.8%)存在EML4-ALK融合基因突变,其中7例为突变体1(variant 1,V1),1例为突变体2(variant 2,V2);这8例EML4-ALK阳性患者组织标本的EGFR(18~21号外显子)及KRAS(1、2号外显子)均为野生型。8例阳性患者中,5例患者的年龄小于总体患者的平均年龄(59±10)岁,占62.5%(5/8);女性患者6例,占75%(6/8);不吸烟患者7例,占87.5%(7/8);腺癌患者5例,占62.5%(5/8)。结论: EML4-ALK融合基因突变代表了NSCLC的一个新的分子亚型,EML4-ALK突变与EGFR及KRAS突变是不共存的。     

克唑替尼在ALK阳性中晚期非小细胞肺癌中的疗效观察

目的：探讨克唑替尼治疗晚期间变淋巴瘤激酶(anaplastic lymphoma kinase,ALK)融合基因阳性中晚期非小细胞肺癌(non-small cell lung cancer,NSCLC)的近期疗效及毒副反应。方法：回顾性分析43例ALK阳性的中晚期NSCLC患者,服用克唑替尼治疗,服用至病情进展或出现不可耐受的毒副反应,随访12个月,观察疗效。结果：克唑替尼治疗ALK阳性NSCLC的疾病控制率(disease control rate,DCR)为93%(3/43),客观缓解率(objective response rate,ORR)为62%(26/43),中位无进展生存时间(progression free survival,PFS)为7.0个月(95% CI,6.0~8.0月),不良反应主要为消化道症状,其次是谷丙转氨酶升高,视觉障碍,大部分为1~2级。结论：克唑替尼作为NSCLC患者的多靶点靶向治疗,具有良好的疗效及安全性,不良反应轻微。     

卡博替尼、克唑替尼和奥西替尼 对小鼠髓源抑制性细胞亚型和凋亡的影响

目的探讨卡博替尼、克唑替尼和奥斯替尼(AZD9291)对小鼠骨髓或脾脏源性抑制细胞(MDSCs)亚群比例、凋亡及增殖的影响。方法免疫磁珠法分离BALB/c小鼠(雄性)骨髓G-MDSCs和M-MDSCs,分别加入卡博替尼(0.01、0.1、0.3和0.9μmol/L)、克唑替尼(0.2、2、20和200μg/mL)或AZD9291(0.01、0.1、1和10μmol/L)培养24 h,采用流式细胞术(FCM)检测3种靶向药物对MDSCs亚型的影响,CCK-8法检测MDSCs增殖;流式细胞分选仪分选小鼠骨髓Gr-1~+CD11b~+细胞,检测Gr-1~+CD11b~+细胞凋亡。结果克唑替尼处理组骨髓粒细胞型MDSCs(G-MDSCs)、单核细胞型MDSCs(M-MDSCs)比例均下降(P0.05);卡博替尼处理组脾脏G-MDSCs比例下降(P0.05);卡博替尼、克唑替尼处理骨髓MDSCs后早期凋亡比例增加(P0.05),AZD9291处理的MDSCs凋亡比例无明显变化。结论卡博替尼和克唑替尼可能通过诱导MDSCs凋亡降低MDSCs比例。     

盐霉素增强吉非替尼诱导人肺腺癌细胞株A549凋亡的作用

 目的：探讨盐霉素联合吉非替尼诱导人肺腺癌细胞株A549凋亡的协同作用。方法：采用MTT的方法检测盐霉素对A549细胞生长的抑制作用;流式细胞术检测盐霉素对A549细胞凋亡和线粒体膜电位的影响;比色法检测caspase-3、-8和-9活性;Western bloting 分析细胞色素C、Bcl-2、p-EGFR、p-Akt和p-ERK蛋白水平。结果：盐霉素与吉非替尼单用均出现不同程度的细胞增殖抑制作用和诱导细胞凋亡作用;而盐霉素与吉非替尼联合作用,能更显著地抑制细胞增殖,且凋亡细胞显著增加（P<0.05）。盐霉素单独作用A549细胞,线粒体膜电位显著下降,细胞内活性氧和Ca2+在短期内显著升高,胞浆细胞色素C含量以及caspase-3、-8和-9活性均显著增加,与对照组比较差异均有统计学意义;吉非替尼单用则主要表现为对p-EGFR、p-Akt和p-ERK蛋白表达的抑制作用,而对胞浆细胞色素C含量以及caspase-3、-8和-9活性影响较少。Western blotting检测发现,联合用药组的Bcl- 2、p-EGFR、p-Akt和p-ERK蛋白表达量明显减少,但是对EGFR、Akt和ERK总蛋白水平无显著影响。结论：盐霉素与吉非替尼联用具有较好的协同作用,可能通过Bcl-2途径及线粒体凋亡途径诱导人肺腺癌A549细胞凋亡,提高A549细胞对吉非替尼的敏感性。     

克唑替尼对ALK阳性非小细胞肺癌脑转移的疗效分析

目的：非小细胞肺癌(non-small cell lung cancer,NSCLC)脑转移是影响NSCLC患者生存期及生存质量的重要因素,本研究将探讨克唑替尼在间变性淋巴瘤激酶(anaplastic lymphoma kinase, ALK)阳性NSCLC脑转移患者中的疗效。方法：筛选20例ALK阳性NSCLC脑转移患者,给予克唑替尼治疗,观察临床疗效。结果：克唑替尼治疗前基线有脑转移患者,颅内疗效部分缓解(partial response,PR)12例,疾病稳定(stable disease,SD)7例,疾病进展(progressive disease,PD)1例。客观缓解率(objective response rate,ORR)为60%,疾病控制率(disease control rate,DCR)为95%。中位无进展生存期(progression free survival,PFS)为6个月(95%CI：3.92~8.08)。结论：克唑替尼治疗ALK阳性NSCLC脑转移患者有效。     

哺乳动物雷帕霉素靶蛋白信号通路在帕金森病中的作用

哺乳动物雷帕霉素靶蛋白（mTOR）是一种保守的丝氨酸/ 苏氨酸蛋白激酶,调控细胞生长、增殖、运 动、存活和自噬等生物学活动,作为细胞代谢和生存的关键调节剂,现已成为帕金森病（PD）的新型治疗靶点。 越来越多的研究表明,在PD模型中恢复受干扰的mTOR信号转导可以预防神经元的死亡。然而,在PD中通过 mTOR介导产生作用的分子机制尚未完全清楚。现将mTOR信号通路与PD关系的最新进展进行综述,并进一步 讨论mTOR信号通路在PD中的潜在作用。     

EML4-ALK fusion gene in Korean non-small cell lung cancer

A fusion gene between echinoderm microtubule-associated protein-like 4 (EML4) and the anaplastic lymphoma kinase (ALK) has been identified in non-small cell lung cancers (NSCLCs). Although a few studies have evaluated EML4-ALK fusion genes in Korean NSCLCs, the prevalence of different EML4-ALK fusion variants has yet to be clearly assessed. Herein, we have examined the profiles of EML4-ALK fusion gene variants in Korean patients of NSCLCs. EML4-ALK fusion genes have been detected in 10 (6.0%) of 167 patients of NSCLCs and in 9 (7.4%) of 121 patients of adenocarcinoma. Of the 10 patients with fusion genes identified, 8 (80%) were E13;A20 (variant 1) and 2 (20%) were E6;A20, with an additional 33-bp sequence derived from intron 6 of EML4 (variant 3b). These results indicate that the profiles of EML4-ALK fusion gene variants in Korean patients of NSCLC may differ from those in other ethnic populations. Herein, we describe for the first time the profiles of EML4-ALK fusion variants of Korean patients with NSCLCs.     

Impact of mTOR signaling pathway on CD8+ T cell immunity through Eomesodermin in response to invasive candidiasis

BackgroundWe investigated the effect of the mammalian target of rapamycin (mTOR) pathway on CD8+ T cell immunity through Eomesodermin (Eomes) in intensive care unit (ICU) patients with invasive candidiasis (IC) and in a mouse model.MethodsWe evaluated quantitative changes in parameters of the mTOR/phosphorylated ribosomal S6 kinase (pS6K) pathway and immune system at the onset of infection in ICU patients. The study was registered on 28 February 2017 at chictr.org.cn (ChiCTR-ROC-17010750). We also used a mouse model of Candida infection and constructed T-cell-specific mTOR and T-cell-specific tuberous sclerosis complex (TSC) 1 conditional knockout mice to elucidate the molecular mechanisms.ResultsWe enrolled 88 patients, including 8 with IC. The IC group had lower CD8+ T cell counts, higher serum levels of mTOR, pS6K, Eomes and interleukin (IL)-6. The mouse model with IC showed results consistent in the clinical study. The CD8+ T cell immune response to IC seemed to be weakened in TSC1 knockout mice compared with wild-type IC mice, demonstrating that mTOR activation resulted in the impaired CD8+ T cell immunity in IC.ConclusionsIn IC, the mTOR activation may play a vital role in impaired CD8+ T cell immunity through enhancing expression of Eomes.The study was registered on 28 February 2017 at chictr.org.cn (identifier ChiCTR-ROC-17010750).     

mTOR信号通路与组蛋白乙酰化在胃癌细胞中的相互作用

目的研究哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路与组蛋白乙酰化在人胃癌细胞生存活力、细胞周期、相关基因及蛋白表达方面的相互作用。方法分别单独或联合脱乙酰化酶抑制剂曲古抑菌素(trichostatin A,TSA)、mTOR抑制剂雷帕霉素和PI3K抑制剂LY294002干预人胃癌MKN45和SGC7901细胞,以噻唑蓝法检测细胞生存活力,流式细胞术检测细胞周期,实时定量PCR检测p21WAF1基因表达情况,蛋白免疫印迹检测Akt、p70S6K及4E-BP1蛋白表达情况。结果联合用药比单独用药对胃癌细胞生长抑制作用更明显(P<0.01);不管单独或联合用药,均可使MKN45细胞阻滞在G2期(P<0.05),使SGC7901细胞阻滞在G1或G2期(P<0.05);联合用药比单独用药更能提高抑癌基因p21WAF1的表达;联合用药使Akt、p70S6K及4E-BP1磷酸化表达较单独用药显著下降(P<0.01),联合用药后组蛋白H4/H3乙酰化也较单独用药表达升高(P<0.01)。结论mTOR信号通路与组蛋白乙酰化在胃癌的发生发展中存在相互关系,mTOR抑制剂与脱乙酰化酶抑制剂联合应用具有协同作用,可望成为肿瘤基因防治的新靶点。     

Role of TM4SF1 in regulating breast cancer cell migration and apoptosis through PI3K/AKT/mTOR pathway

Purpose: The purpose of this study was to investigate the effect of transmembrane-4-l-six-family-1 (TM4SF1) on breast cancer cell line MDA-MB-231 invasion and apoptosis and its mechanism through PI3K/AKT/mTOR pathway. Methods: siRNA-TM4SF1 and pcDNA-TM4SF1 plasma were constructed and then transfected into MDA-MB-231 cells respectively. Real time (RT)-PCR was used to measure the mRNA expression of TM4SF1 in each group. Also, matrigel method and Annexin V-FITC were used to detect the effect of TM4SF1 expression on MDA-MB-231 cell migration and apoptosis respectively. Besides, western blotting analyze was used to assay the effects of TM4SF1 expression on PI3K/AKT/mTOR pathway associated proteins expressions. Results: The results showed that after being transfected with siRNA-TM4SF1, TM4SF1 expression was significantly declined, while it was significantly increased after cells were transfected with pcDNA-TM4SF1 (P<0.05). Compared with the controls, TM4SF1 overexpression significantly contributed MDA-MB-231 cell migration but decreased apoptotic cells (P<0.05), which were opposite to the results when TM4SF1 was sliced in cells. Moreover, TM4SF1 slicing significantly decreased the expressions of phosphorylated (p)-AKT, p-mTOR, and p-P70 (P<0.05). Conclusion: Our study suggested that TM4SF1 may be a therapeutic target for breast cancer treatment and may loan insight into the mechanisms behind the development and metastasis of advanced breast cancer.     

Hydrogen peroxide induces autophagic cell death in C6 glioma cells via BNIP3-mediated suppression of the mTOR pathway

Oxidative stress by exposure to H₂O₂ induces various types of cell death depending on cell type and conditions. We report herein on a study of the mechanisms underlying H₂O₂-induced cell death in C6 glioma cells. The findings show that H₂O₂ triggers a caspase-independent autophagic cell death in these cells. The findings also show that H₂O₂ induces the dephosphorylation of the mammalian target of rapamycin (mTOR) at Ser 2481 and the p70 ribosomal protein S6 kinase (p70S6K) at Thr389 in a Bcl-2/E1B 19 kDa interacting protein 3 (BNIP3)-dependent manner. BNIP3 has the capacity to inhibit mTOR activity and mTOR inhibition plays a role in autophagic induction. This suggests that BNIP3 may mediate H₂O₂-induced autophagic cell death through the suppression of mTOR. The findings show that the down-regulation of BNIP3 by BNIP3 siRNA prevents C6 cells from undergoing H₂O₂-induced autophagic cell death. Collectively, these results suggest that H₂O₂ induces autophagic cell death in C6 cells via the BNIP3-mediated suppression of the mTOR pathway.