自噬在索拉菲尼诱导肝癌细胞凋亡中的作用

目的：研究索拉菲尼（Sorafenib）诱导的自噬在肝癌细胞凋亡中的作用。方法：体外培养肝癌细胞Huh7和 LH86细胞株。分为对照组、Sorafenib 处理组、Bafilomycin 处理组、Sorafenib + Bafilomycin 处理组。将构建的质粒表达载体 pEGFP - N1- LC3分别转染 Huh7和 LH86细胞株,用索拉菲尼或者索拉菲尼加 Bafilo-mycin 同时处理细胞,通过荧光显微镜观察对照组和处理组细胞内细胞自噬小体形成情况。免疫印迹（West-ern blot）方法检测细胞自噬作用标志蛋白 LC3以及细胞凋亡蛋白 Caspase 3切割条带在各组中的表达, Heochst 33342染料对各组细胞核进行染色,以评估细胞自噬作用对索拉菲尼诱导细胞凋亡的影响。结果：在荧光显微镜下,可以观察到 Sorafenib 能够显著诱导两种肝癌细胞株发生自噬作用。抑制剂 Balfilomycin 能够显著抑制 Sorafenib 诱导的自噬作用。Balfilomycin 抑制自噬能够显著增强 Sorafenib 诱导的细胞凋亡。结论：Sorafenib 能够诱导肝癌细胞产生自噬作用。这种自噬作用在 Sorafenib 诱导的肝癌细胞凋亡中起到负调控作用。     

细胞自噬在肝癌发生发展中的作用

细胞自噬是细胞内高度保守的细胞自我消化和分解代谢过程,是维持细胞内稳态的重要机制之一。在肝癌发生发展的不同阶段,细胞自噬起着不同作用。在肝癌的起始阶段,经常存在自噬缺陷,这也可说明细胞自噬的正常运转可抑制肝癌的发生;但是在肝癌的发展过程中,随着肿瘤微环境的变化,肝癌细胞也会利用自噬来保护自己。故自噬所扮演的角色也在改变,起着保护肝癌细胞的作用。另外,自噬与肝癌细胞的转移及耐药也密切相关。     

细胞自噬在肿瘤中的作用

自噬为一种细胞的自我消耗过程,其具有潜在的抗肿瘤生物学活性,亦与肿瘤耐药有关.该文从自噬的调节机制入手,综述自噬参与肿瘤发生的机制,自噬在肿瘤治疗中的作用及应用,以期为自噬在临床上的应用提供依据.     

大黄素在体外诱导人肝癌细胞HepG2发生凋亡的初步研究

背景与目的:大黄素(3-甲基-1,6,8-三羟蒽醌)是大黄等多种中药的有效成分之一。研究表明大黄素对于乳腺癌细胞、肺癌细胞的生长具有抑制作用,但目前大黄素抗肿瘤的作用机理尚不明确。本研究拟讨论大黄素在体外对肝癌细胞HepG2的作用及其机理。方法:应用MTT、软琼脂克隆形成、DNAladder凝胶电泳及流式细胞术等方法,研究中药单体大黄素对肝癌细胞HepG2生长增殖的影响以及作用机理。结果:大黄素在较低浓度可抑制肿瘤细胞的生长,MTT实验测得的半数抑制浓度(IC50)为(36±2.6)μg/ml。在软琼脂实验中,大黄素可以浓度依赖的方式抑制细胞克隆的形成。经DNAladder及流式细胞仪检测发现,大黄素能诱导HepG2细胞凋亡,与阴性对照相比,随着药物浓度从10μg/ml增加到20μg/ml,AnnexinV染色细胞显著增多,由27.3%增至59.6%;当药物浓度增至40μg/ml时,培养液中几乎无活细胞,由碘化丙啶和AnnexinV双重标记的凋亡后期以继发性坏死细胞为主。结论:中药单体大黄素在体外能抑制肝癌细胞HepG2的生长增殖,并能诱导该细胞凋亡。     

倍半萜类化合物Hirsutanol激活ROS诱导肝癌细胞自噬性死亡

Background and Objective:Hirsutanol A is a novel sesquiterpene compound purified from fungus chondrostereum sp in Sarcophyton tortuosum.Its pharmacologic effect has not been reported yet.This study aimed to investigate cytotoxic effect of Hirsutanol A on hepatocellular carcinoma(HCC) cells and its mechanism.Methods:Hep3B cells were treated with different concentrations of Hirsutanol A.Cell proliferation was detected by MTT assay.The protein expression of LC3 was determined by Western blot.The generation of ...     

调控肿瘤细胞自噬相关分子的研究进展

自噬是真核细胞中的重要生理过程,有助于细胞成分的正常更新。机体处于饥饿状态时,自噬不仅可降解蛋白质产生氨基酸、ATP等,为细胞存活提供条件,还能清除受损的细胞器,维持细胞正常机能。自噬功能的异常与肿瘤的形成发展密切相关,其程度过高或过低均会抑制肿瘤。现已发现自噬受多种分子调控,且作用结果各异。因此,对这些自噬相关分子作用机制的深入研究将成为进一步认识自噬的出发点和治疗肿瘤的突破口。     

细胞自噬与肝脏疾病关系的研究进展

细胞自噬是细胞利用溶酶体自身消化的代谢过程。近年来,研究发现细胞自噬在肝纤维化、肝炎、肝细胞癌的发生、发展中具有重要作用,本文就细胞自噬与肝脏疾病相关性的研究进展作一综述。     

自噬在胃肠道肿瘤的研究

自噬是由溶酶体介导的细胞降解程序,参与维持细胞稳定.在胃肠道肿瘤早期阶段,自噬清除毒性损伤以对抗肿瘤进展;而肿瘤进展期则呈现细胞杀伤与细胞保护的两面性.在自噬与细胞凋亡之间亦存在拮抗与协同的双重作用.合理调控自噬有可能成为胃肠道肿瘤预防或治疗的一种新手段.     

塞来昔布诱导胃癌SGC-7901细胞凋亡和自噬

目的:观察塞来昔布对人胃癌细胞株SGC-7901凋亡和自噬的影响,并探讨其凋亡的机制。方法:不同浓度塞来昔布处理SGC-7901细胞后,MTT法检测SGC-7901细胞的增殖,TUNEL法检测SGC-7901细胞的凋亡,透射电镜观察SGC-7901细胞超微结构的改变,流式细胞术检测SGC-7901细胞的凋亡率,实时定量荧光PCR法检测SGC-7901细胞中caspase-8和caspase-9 mRNA的表达。结果:塞来昔布时间(24、48、72 h)和剂量(50、75、100、125μmol/L)依赖性抑制SGC-7901细胞的增殖,125μmol/L塞来昔布作用SGC-7901 72 h细胞的增殖抑制率高达(85.6±4.51)%。塞来昔布可诱导SGC-7901细胞凋亡,透射电镜下观察到典型的凋亡小体和自噬体,细胞凋亡率从(2.2±1.32)%上升到(35.7±5.73)%(P<0.01)。塞来昔布作用后,SGC-7901细胞中caspase-8和caspase-9 mRNA表达明显增加,呈时间和剂量依赖性(P<0.05)。结论:塞来昔布通过激活依赖caspase-8的死亡受体途径和依赖caspase-9的线粒体途径诱导胃癌SGC-7901细胞凋亡,同时诱发自噬性细胞死亡。     

Inhibition of autophagy enhances cisplatin cytotoxicity through endoplasmic reticulum stress in human cervical cancer cells

The function of autophagy in cisplatin-treated cancer cells is not fully understood. Cisplatin treatment induced degradation of ubiquitinated proteins by autophagy, which reduced apoptosis induced by endoplasmic reticulum (ER) stress and downregulated the mitochondrial pathway of apoptosis. Inhibition of autophagy using 3-methyladenine (3-MA) or chloroquine (CQ) increased the levels of intracellular misfolded proteins, which enhanced cellular apoptosis. We found that tunicamycin, an ER stress inducer, augmented cisplatin cytotoxicity by upregulating ER stress-mediated apoptosis. Our data indicates that autophagy plays an important role in preventing cisplatin-induced apoptosis in HeLa cells, thus inhibition of autophagy may improve cisplatin chemotherapy.     

Synergistic inhibition of autophagy and neddylation pathways as a novel therapeutic approach for targeting liver cancer

Liver cancer is the second-most frequent cause of cancer death in the world and is highly treatment resistant. We reported previously that inhibition of neddylation pathway with specific NAE inhibitor MLN4924, suppressed the malignant phenotypes of liver cancer. However, during the process, MLN4924 induces pro-survival autophagy as a mechanism of drug resistance. Here, we report that blockage of autophagy with clinically-available autophagy inhibitors (e.g. chloroquine) significantly enhanced the efficacy of MLN4924 on liver cancer cells by triggering apoptosis. Mechanistically, chloroquine enhanced MLN4924-induced up-regulation of pro-apoptotic proteins (e.g. NOXA) and down-regulation of anti-apoptotic proteins. Importantly, the down-regulation of NOXA expression via siRNA silencing substantially attenuated apoptosis of liver cancer cells. Further mechanistic studies revealed that blockage of autophagy augmented MLN4924-induced DNA damage and reactive oxygen species (ROS) generation. The elimination of DNA damage or blockage of ROS production significantly reduced the expression of NOXA, and thereby attenuated apoptosis and reduced growth inhibition of liver cancer cells. Moreover, blockage of autophagy enhanced the efficacy of MLN4924 in an orthotopic model of human liver cancer, with induction of NOXA and apoptosis in tumor tissues. These findings provide important preclinical evidence for clinical investigation of synergistic inhibition of neddylation and autophagy in liver cancer.     

Suppression of autophagy enhances preferential toxicity of paclitaxel to folliculin-deficient renal cancer cells

Background

Paclitaxel, a widely used chemotherapeutic drug, can induce apoptosis in variety of cancer cells. A previous study has shown preferential toxicity of paclitaxel to FLCN-deficient kidney cancer cell line, UOK257. In this report, we investigate the cellular and molecular mechanism of paclitaxel-induced autophagy and apoptosis in renal cancer cells with and without FLCN expression.

Methods

Two pairs of cell lines were used: FLCN siRNA-silenced ACHN cell line (ACHN-5968) and scrambled ACHN cell line (ACHN-sc); FLCN-null UOK257 cell line and UOK257-2 cell line restored with ectopic expression of FLCN. Autophagy was examined by western blot, GFP-LC3, transmission electron microscopy, and MDC assay. Cell viability and apoptosis were detected using MTT assay, DAPI stain and TUNEL assay. After inhibition of autophagy with 3-Methyladenine (3-MA) or Beclin 1 siRNA, cell viability and apoptosis were measured by MTT assay and TUNEL assay.

Results

After paclitaxel treatment, a dose-dependent decrease in cell viability and increase in apoptosis were observed in FLCN-deficient UOK257 and ACHN-5968 cells compared to their FLCN-expressing counterparts, suggesting that renal cancer cells without FLCN were more sensitive to paclitaxel. Enhanced autophagy was found to be associated with paclitaxel treatment in FLCN-deficient RCC cells. The MAPK pathway was also identified as a key pathway for the activation of autophagy in these kidney cancer cells. Inhibition of phosphorylated ERK with ERK inhibitor U0126 showed a significant decrease in autophagy. Furthermore, after inhibition of autophagy with 3-Methyladenine (3-MA) or Beclin 1 siRNA, apoptosis induced by paclitaxel was significantly increased in FLCN-deficient UOK257 and ACHN-5968 cells.

Conclusions

Preferential toxicity of paclitaxel to FLCN-deficient kidney cancer cells is associated with enhanced autophagy. Suppression of autophagy further enhances paclitaxel-induced apoptosis in FLCN-deficient renal cancer cells. Our results suggest that paclitaxel combined with an autophagy inhibitor might be a potentially more effective chemotherapeutic approach for FLCN-deficient renal cancer.

     

Inhibiting autophagy increases epirubicin's cytotoxicity in breast cancer cells

Chemotherapy, radiotherapy, and endocrinotherapy are documented to induce autophagy among breast cancer cells, but the role of autophagy in this disease has been attributed as cytoprotective as well as tumor‐suppressing. Thus we studied MDA‐MB‐231 and SK‐BR‐3 breast cancer cell lines treated with epirubicin (EPI) to assess autophagy and apoptosis. We found out that EPI induced apoptosis and autophagy in both cell lines. The lysosomal inhibitor bafilomycin A1 inhibited cellular autophagy and enhanced EPI‐triggered apoptosis, perhaps due to inhibition of autolysosome formation, which then inhibited autophagic effects of engulfing and clearing damaged mitochondria. This inhibition increased mitochondrial cytochrome C release which augmented epirubicin‐induced caspase‐dependent apoptosis and cytotoxicity. In addition, the lysosomal neutralizing agent ammonia chloride (AC), and Atg7 knockdown by siRNA, could inhibit epirubicin‐triggered autophagy, enhance cytotoxicity, and increase caspase‐9‐ and caspase‐3‐dependent apoptosis. Thus, autophagy plays a prosurvival role in EPI‐treated MDA‐MB‐231 and SK‐BR‐3 cells, and autophagy inhibition can potentially reverse this effect and increase the cytotoxicity of EPI.     

Ginsenoside F2 induces apoptosis accompanied by protective autophagy in breast cancer stem cells

Ginsenoside F2 (F2) was assessed for its antiproliferative activity against breast cancer stem cells (CSCs). F2 induced apoptosis in breast CSCs by activating the intrinsic apoptotic pathway and mitochondrial dysfunction. Concomitantly, F2 induced the formation of acidic vesicular organelles, recruitment of GFP-LC3-II to autophagosomes, and elevation of Atg-7 levels, suggesting that F2 initiates an autophagic progression in breast CSCs. Treatment with an inhibitor of autophagy enhanced F2-induced cell death. Our findings provide new insights into the anti-cancer activity of F2 and may contribute to the rational use and pharmacological study of F2.     

内质网应激在肝癌中的作用

内质网应激(Endoplasmic Reticulum Stress,ERS)是一种极其重要的信号反应通路。内质网应激时,首先启动生存途径即未折叠蛋白反应(Unfolded protein response,UPR),而长时间的内质网应激将会启动细胞凋亡途径。目前越来越多的研究表明,肝癌与内质网应激密切相关,并且肝癌的肿瘤微环境可使内质网产生应激已得到证实。根据相关文献报道的内质网双重作用,内质网应激既可以诱导转化肝癌,又可以抑制肝癌增殖、转移等,故内质网应激为肝癌新的治疗方法提供可能。本文就以内质网应激与肝癌关系的研究进展进行综述。     

肝癌干细胞与肝癌的研究进展

肿瘤起源于干细胞的假说已在人类许多实体瘤中得到证实,近来亦发现肝癌中存在肝癌干细胞.肿瘤干细胞被认为是肿瘤产生的根源,对肿瘤的发生、发展、转移、复发及耐药具有关键作用.因此,如何分离鉴定肝癌干细胞对于改善预防方法、促进早期检测以及研发新的治疗方法都是一个非常紧迫的课题.本文就肝癌干细胞的来源、表面标志、分选方法、应用前景及存在的问题作一综述.     

大黄素对肺癌细胞系A973增殖的影响

目的了解大黄素对肺癌细胞系A973细胞生长、增殖的影响.方法采用台盼蓝染色排斥法进行细胞计数,MTT法测定大黄素对肺癌细胞系A973细胞增殖的抑制率.结果大黄素IC50=1775.54μg/mL.结论大黄素的抗肿瘤作用较弱.     

circRNA MYO1C对肺癌细胞自噬和肿瘤干细胞分化的影响

目的 探讨环状RNA(circRNA)MYO1C对非小细胞肺癌(NSCLC)细胞自噬和肿瘤干细胞(Cancer stem cells,CSCs)分化的影响。方法 通过RT-qPCR检测正常肺上皮细胞和NSCLC A549细胞中circRNA MYO1C水平。将A549分为对照组、NC组和siMYO1C组,通过转染si-circRNA MYO1C质粒干扰circRNA MYO1C。用CCK-8法和Transwell实验检测细胞生长活力和迁移能力。通过Western blot检测CSCs分化标志蛋白(OCT4、SOX2和NANOG)以及自噬标志蛋白(P62和IC3II/LC3I)的表达水平。结果 相对正常肺上皮细胞,circRNA MYO1C在A549细胞中表达明显上调(0.99±0.06 vs. 4.87±0.88,P=0.002)。与NC组比较,siMYO1C组细胞中circRNA MYO1C水平、细胞活力、迁移、OCT4、SOX2、NANOG和IC3II/LC3I蛋白水平显著降低,而P62蛋白水平显著升高(P＜0.001)。结论 下调circRNA MYO1C能够抑制NSLCLC细胞的CSCs分化和自噬相关蛋白表达,并抑制肺癌细胞迁移。     

大黄素诱导胆管癌QBC939细胞凋亡的实验研究

 目的 探讨大黄素在体外诱导人胆管癌QBC939细胞凋亡的作用及机制。方法 采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)法检测大黄素对细胞增殖的抑制作用;在荧光显微镜下观察凋亡细胞的形态学变化;运用流式细胞技术检测细胞凋亡率和细胞内活性氧水平;通过比色法测定细胞内caspase-9及caspase-3的相对活性。结果 大黄素以时间-剂量的方式抑制胆管癌QBC939细胞的增殖;在荧光显微镜下,凋亡的细胞呈现亮色,或核呈分叶、碎片状,细胞皱缩;QBC939细胞在30 µmol/L和50 µmol/L浓度的大黄素作用下,24 h的凋亡率分别为38.9%±9.07%及67.09%±4.08%（P<0.05）,30 min后细胞内活性氧的水平分别为对照组的1.65±0.08及2.28±0.04倍（P<0.05）;大黄素可激活caspase-9及caspase-3,导致其活性升高（P<0.05）。结论 大黄素能通过诱导凋亡来抑制胆管癌QBC939细胞的增殖,其机制与细胞的活性氧水平升高及caspase-9及caspase-3活化有关。