细胞自噬与肿瘤耐药

细胞自噬是一种细胞自我降解的过程,在适应代谢应激、保持基因组完整性及维持内环境稳定方面发挥重要作用. 在肿瘤治疗中,凋亡耐受是产生肿瘤耐药的重要机制. 细胞自噬可防止抗肿瘤药诱导的凋亡,促进肿瘤耐药. 然而,自噬性细胞死亡可能是凋亡耐受肿瘤细胞的一种死亡方式. 因此,细胞自噬对肿瘤细胞的耐药性有双重影响. 本文综述了细胞自噬的分子机制、细胞自噬与凋亡的关系、细胞自噬与肿瘤耐药以及治疗的主要研究进展.     

雷公藤甲素诱导HeLa细胞p53依赖的自噬和凋亡

自噬与凋亡被认为是细胞程序性死亡的两种重要途径,二者的交互联系对阐明药物的抗肿瘤机理有重要价值.众多的研究表明,雷公藤甲素对多种肿瘤细胞都具有显著的抑制作用.细胞凋亡与自噬可被相同的因素所诱导,p53蛋白可以同时对二者起调控作用,在自噬与凋亡的交互作用(crosstalk)中扮演着重要角色.本文以He La细胞为模型,研究雷公藤甲素诱导He La细胞发生自噬和凋亡的机制,并通过抑制p53依赖的转录,研究雷公藤甲素诱导He La细胞p53依赖的自噬和凋亡交互联系.     

小檗碱对乳腺癌MDA-MB-231细胞增殖抑制作用及其分子机制研究

本研究的目的是为了探索小檗碱对乳腺癌MDA-MB-231细胞增殖的影响以及阐明小檗碱促乳腺癌细胞凋亡的分子机制。在实验过程中,我们通过MTT检测小檗碱对乳腺癌MDA-MB-231细胞增殖的抑制作用,采用Annexin-V/PI染色定量考察小檗碱对肿瘤细胞凋亡的影响,运用Western Blot实验检测肿瘤相关通路蛋白表达来进行研究。实验表明小檗碱对乳腺癌MDA-MB-231细胞增殖具有抑制作用,使细胞中自噬因子Beclin 1表达增加,诱导细胞自噬泡的形成,导致肿瘤细胞发生凋亡。综上说明小檗碱是通过抑制AKT-mTOR通路,诱导MDA-MB-231细胞的自噬以及凋亡,从而发挥抗肿瘤作用。     

抗疟疾药物可以抗癌

在抗癌治疗当中,诱发癌细胞凋亡当然是最重要的策略之一,然而有些时候人们加入细胞凋亡的诱导剂却得不到理想的效果。例如在多种肿瘤组织中,Akt作为细胞凋亡的抑制蛋白往往都呈现过度激活的状态;而当研究者想通过抑制Akt的活性抗癌时,却往往发现肿瘤细胞的凋亡并不明显增加。最近的研究显示,Akt不仅是凋亡途径的抑制因子,也是自噬途径的抑制因子。Degtyarev等人发现,利用RNAi敲低Akt的表达时,细胞生长缓慢,细胞周期延长,也同时出现了自噬现象的一些典型改变,     

雷帕霉素对二种鳞翅目昆虫细胞自噬和凋亡的影响

以2种鳞翅目昆虫细胞为材料,采用雷帕霉素进行处理,初步研究自噬作用与昆虫细胞凋亡的关系。结果表明:雷帕霉素能够提高家蚕细胞系BMN-e细胞的自噬水平,并能诱导BMN-e细胞发生凋亡;自噬抑制剂3-甲基腺嘌呤能抑制雷帕霉素诱导的BMN-e细胞凋亡。相反,雷帕霉素虽能诱导斜纹夜蛾细胞系SL-HP细胞的自噬水平提高,但不能诱导斜纹夜蛾Spodoptera litura(Fabricius)细胞发生凋亡;雷帕霉素的预处理能抑制放线菌素D诱导的斜纹夜蛾细胞系SL-HP细胞发生凋亡;自噬抑制剂3-甲基腺嘌呤对放线菌素D诱导的细胞凋亡没有影响。因此家蚕Bombyx mori细胞自噬水平的提高与细胞凋亡具有正相关性,而斜纹夜蛾细胞自噬水平的提高与细胞凋亡不相关,相反还对细胞凋亡的诱导具有一定的抑制作用。     

ASPP2过表达可增强DRAM介导的自噬对HepG2细胞的促调亡作用

p53凋亡刺激蛋白2（apoptosis stimulating protein 2 of p53, ASPP2）能特异性地与p53蛋白结合并增强其促凋亡功能,进而发挥抗肿瘤作用.最近文献提示,自噬对肿瘤发生、发展及肿瘤细胞对抗肿瘤药物的反应都具有重要作用.在本研究中,甲基磺酸(MMS)处理HepG2细胞24 h后,用calcein AM/PI和M30染色检测细胞凋亡,可引起早期（M30免疫组化阳性）和晚期细胞凋亡（PI染色阳性）. 给HepG2细胞转染GFP-LC3质粒后,发现MMS处理24 h可引起自噬的发生. ASPP2腺病毒（rAd-ASPP2）感染HepG2细胞引起ASPP2过表达后,再用MMS处理24 h,能引起更明显的早期、晚期细胞凋亡和自噬. 荧光定量PCR检测发现,rAd-ASPP2诱导了更高的BCL-2相关X蛋白基因（BAX）和p53蛋白的目的基因p53诱导的自噬调节蛋白（p53-induced modulator of autophagy,DRAM）的表达. 但仅用rAd-ASPP2处理HepG2细胞不能引起自噬和凋亡.利用2条DRAM特异性的siRNA下调DRAM的表达,发现rAd-ASPP2引起的自噬被完全抑制, 早期和晚期凋亡均部分被抑制,同时BAX 的mRNA水平也明显下降. 以上结果说明,ASPP2可通过上调BAX和DRAM基因的转录而促进MMS引起的HepG2细胞凋亡; 另外,DRAM介导的自噬是ASPP2促进MMS引起的肿瘤细胞凋亡的机制之一. 该研究可为肝癌的基因治疗提供新的思路.     

自噬、自噬性细胞死亡与肿瘤的研究进展

自噬是绝大部分真核细胞中一种依赖于溶酶体的极度保守的生理过程,是细胞内物质再循环的有效机制。适度的自噬是细胞抵御不良环境的生存方式,但当发生过度的自噬时,将引起细胞的死亡,这种死亡方式称为自噬性细胞死亡。自噬性细胞死亡不同于凋亡,表现为细胞中出现大量包裹着细胞质和细胞器的自噬体。越来越多的研究提示,自噬与自噬性细胞死亡与某些恶性肿瘤的发生、发展关系密切。在各种不利环境下,自噬可作为一种应急机制维持肿瘤细胞的生存,而自噬性细胞死亡诱发后又可清除肿瘤细胞。现就自噬与肿瘤的研究进展作一综述。     

氯喹抑制自噬增强宫颈癌细胞SiHa对CPT处理的敏感性

自噬诱导是肿瘤细胞对化疗药物抵抗性的原因之一,该研究探讨溶酶体抑制剂氯喹对喜树碱(camptothecin,CPT)诱导的宫颈癌细胞Si Ha死亡的增敏效果。CPT和/或氯喹处理宫颈癌Si Ha细胞,MTT法检测细胞增殖,DAPI和TUNEL染色观察细胞凋亡,Western blot和免疫荧光检测自噬及凋亡相关蛋白。结果发现,CPT处理后,Si Ha细胞MAP1LC3B荧光点和LC3II(microtubuleassociated protein light chain 3II)蛋白水平增加,p62荧光点和蛋白质水平则减少;而采用氯喹特异抑制自噬后,可明显提高CPT诱导的细胞凋亡、caspase-9的激活和PARP(poly ADP-ribose polymerase)的切割,而全长caspase-2水平显著下降。以上结果提示,氯喹可通过抑制细胞自噬而增强宫颈癌细胞株Si Ha对CPT诱导细胞凋亡的敏感性。     

肿瘤治疗过程中凋亡与自噬的关系

凋亡和自噬是参与维持机体正常的生理平衡和内环境稳定重要机制,与正常生长发育以及肿瘤等多种疾病发展过程都有着密切的联系。对于肿瘤的治疗,传统的方法是诱导肿瘤细胞凋亡,然而,肿瘤细胞中凋亡抗性的出现成为肿瘤治疗的主要障碍。近来,通过诱导其它细胞死亡方式致肿瘤细胞死亡已经成为有潜力的新的抗肿瘤机制。自噬作为另外一种细胞程序性死亡方式与凋亡一样有着复杂的分子机制和调控机制,它们之间存在密切的联系,并且存在许多相同的调节蛋白。本文就凋亡和自噬在形态特征、分子机制、检测方法以及在肿瘤治疗过程两者之间的关系做一综述。     

昆虫细胞程序性死亡的研究进展

在昆虫发育和抵抗病原微生物的入侵过程中,细胞凋亡与自噬性死亡现象十分常见。昆虫细胞凋亡的研究已经取得了许多的成果,但是有关细胞自噬程序性死亡的研究还正在深入。昆虫细胞凋亡的信号通路至少有3条：一条类似于线虫细胞的凋亡信号通路,另一条类似于哺乳动物细胞的凋亡信号通路, 还有一条不依赖于胱天蛋白酶的凋亡信号通路。在昆虫的多种组织细胞中,细胞凋亡与自噬程序性死亡在信号通路上存在互串(cross talking),可以相互促进、抑制或替代。了解昆虫细胞程序性死亡对防治害虫具有一定的意义。     

Gastrointestinal stromal tumors (GIST): Facing cell death between autophagy and apoptosis

Autophagy and apoptosis are 2 fundamental biological mechanisms that may cooperate or be antagonistic, although both are involved in deciding the fate of cells in physiological or pathological conditions. These 2 mechanisms coexist simultaneously in cells and share common upstream signals and stimuli. Autophagy and apoptosis play pivotal roles in cancer development. Autophagy plays a key function in maintaining tumor cell survival by providing energy during unfavorable metabolic conditions through its recycling mechanism, and supporting the high energy requirement for metabolism and growth. This review focuses on gastrointestinal stromal tumors and cell death through autophagy and apoptosis, taking into account the involvement of both of these processes in tumor development and growth and as mechanisms of drug resistance. We also focus on the crosstalk between autophagy and apoptosis as an emerging field with major implications for the development of novel therapeutic options.     

MicroRNA-30a sensitizes tumor cells to cis-platinum via suppressing beclin 1-mediated autophagy

Autophagy is activated in cancer cells during chemotherapy and often contributes to tumor chemotherapy resistance. In this study, we characterized the role of microRNA-30a (miR-30a) in the coordination of cancer cell apoptosis and autophagy, which determines the sensitivity of cancer cells to chemotherapy. First, the autophagy activity in cancer cells increased after cis-dichloro-diamine platinum (cis-DDP) or Taxol treatment, as indicated by the enhanced expression of beclin 1, a key regulator of autophagy, and increased number of LC3-positive autophagosomes. Second, miRNA screening using a TaqMan probe-based quantitative RT-PCR assay identified that miR-30a, a miRNA that targets beclin 1, was significantly reduced in tumor cells by cis-DDP treatment. Forced expression of miR-30a significantly reduced beclin 1 and the autophagy activity of tumor cells induced by cis-DDP. Third, the blockade of tumor cell autophagy activity by miR-30a expression or 3-methyladenine significantly increased tumor cell apoptosis induced by cis-DDP treatment. Finally, an in vivo tumor implantation mouse model clearly showed that elevation of miR-30a in implanted tumor cells by administration of the recombinant lentivirus expressing miR-30a strongly enhanced cis-DDP-induced apoptosis of tumor cells. In conclusion, our results demonstrate for the first time that miR-30a can sensitize tumor cells to cis-DDP via reducing beclin 1-mediated autophagy and that increasing miR-30a level in tumor cells represents a novel approach to enhance the efficacy of chemotherapy during cancer treatment.     

Autophagy and EMT in cancer and metastasis: Who controls whom?

Metastasis consists of hallmark events, including Epithelial-Mesenchymal Transition (EMT), angiogenesis, initiation of inflammatory tumor microenvironment, and malfunctions in apoptosis. Autophagy is known to play a pivotal role in the metastatic process. Autophagy has pulled researchers towards it in recent times because of its dual role in the maintenance of cancer cells. Evidence states that cells undergoing EMT need autophagy in order to survive during migration and dissemination. Additionally, it orchestrates EMT markers in certain cancers. On the other side of the coin, autophagy plays an oncosuppressive role in impeding early metastasis. This review aims to project the interrelationship between autophagy and EMT. Targeting EMT via autophagy as a useful strategy is discussed in this review. Furthermore, for the first time, we have covered the possible reciprocating roles of EMT and autophagy and its consequences in cancer metastasis.     

Strange attractors: DAMPs and autophagy link tumor cell death and immunity

Resistance to ‘apoptotic'' cell death is one of the major hallmarks of cancer, contributing to tumor development and therapeutic resistance. Damage-associated molecular patterns (DAMPs) are molecules released or exposed by dead, dying, injured, or stressed non-apoptotic cells, with multiple roles in inflammation and immunity. Release of DAMPs not only contributes to tumor growth and progression but also mediates skewing of antitumor immunity during so-called immunogenic tumor cell death (ICD). Autophagy is a lysosome-mediated homeostatic degradation process in which cells digest their own effete organelles and macromolecules to meet bioenergetic needs and enable protein synthesis. For tumor cells, autophagy is a double-edged sword. Autophagy, in balance with apoptosis, can function as a tumor suppressor; autophagy deficiency, associated with alterations in apoptosis, initiates tumorigenesis in many settings. In contrast, autophagy-related stress tolerance generally promotes cell survival, which enables tumor growth and promotes therapeutic resistance. Most anticancer therapies promote DAMP release and enhance autophagy. Autophagy not only regulates DAMP release and degradation, but also is triggered and regulated by DAMPs. This interplay between autophagy and DAMPs, serving as ‘strange attractors'' in the dynamic system that emerges in cancer, regulates the effectiveness of antitumor treatment. This interplay also shapes the immune response to dying cells upon ICD, culling the least fit tumor cells and promoting survival of others. Thus, DAMPs and autophagy are suitable emergent targets for cancer therapy, considering their more nuanced role in tumor progression.     

Cleavage of Atg3 protein by caspase-8 regulates autophagy during receptor-activated cell death

Autophagy is an evolutionarily conserved mechanism contributing to cell survival under stress conditions including nutrient and growth factor deprivation. Connections and cross-talk between cell death mechanisms and autophagy is under investigation. Here, we describe Atg3, an essential regulatory component of autophagosome biogenesis, as a new substrate of caspase-8 during receptor-mediated cell death. Both, tumor necrosis factor α and tumor necrosis factor-related apoptosis inducing ligand induced cell death was accompanied by Atg3 cleavage and this event was inhibited by a pan-caspase inhibitor (zVAD) or a caspase-8-specific inhibitor (zIETD). Indeed, caspase-8 overexpression led to Atg3 degradation and this event depended on caspase-8 enzymatic activity. Mutation of the caspase-8 cleavage site on Atg3 abolished its cleavage both in vitro and in vivo, demonstrating that Atg3 was a direct target of caspase-8. Autophagy was inactive during apoptosis and blockage of caspases or overexpression of a non-cleavable Atg3 protein reestablished autophagic activity upon death receptor stimulation. In this system, autophagy was important for cell survival since inhibition of autophagy increased cell death. Therefore, Atg3 provides a novel link between apoptosis and autophagy during receptor-activated cell death.     

Mechanisms of autophagy and apoptosis: Recent developments in breast cancer cells

Autophagy,the pathway whereby cell components are degraded by lysosomes,is involved in the cell response to environmental stresses,such as nutrient deprivation,hypoxia or exposition to chemotherapeutic agents.Under these conditions,which are reminiscent of certain phases of tumor development,autophagy either promotes cell survival or induces cell death. This strengthens the possibility that autophagy could be an important target in cancer therapy,as has been proposed.Here,we describe the regulation of survival and death by autophagy and apoptosis,especially in cultured breast cancer cells.In particular,we discuss whether autophagy represents an apoptosis-independent process and/or if they share common pathways. We believe that understanding in detail the molecular mechanisms that underlie the relationships between autophagy and apoptosis in breast cancer cells could improve the available treatments for this disease.     

Triazole analog 1-(1-benzyl-5-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)-2-(4-bromophenylamino)-1-(4-chlorophenyl)ethanol induces reactive oxygen species and autophagy-dependent apoptosis in both in vitro and in vivo breast cancer models

Autophagy is considered as an important cell death mechanism that closely interacts with other common cell death programs like apoptosis. Critical role of autophagy in cell death makes it a promising, yet challenging therapeutic target for cancer. We identified a series of 1,2,3-triazole analogs having significant breast cancer inhibition property. Therefore, we attempted to study whether autophagy and apoptosis were involved in the process of cancer cell inhibition. The lead molecule, 1-(1-benzyl-5-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)-2-(4-bromophenylamino)-1-(4-chlorophenyl)ethanol (T-12) induced significant cell cycle arrest, mitochondrial membrane depolarization, apoptosis and autophagy in MCF-7 and MDA-MB-231 cells. T-12 increased reactive oxygen species and its inhibition by N-acetyl-l-cysteine protected breast cancer cells from autophagy and apoptosis. Autophagy inhibitor, 3-methyladenine abolished T-12 induced apoptosis, mitochondrial membrane depolarization and reactive oxygen species generation. This suggested that T-12 induced autophagy facilitated cell death rather than cell survival. Pan-caspase inhibition did not abrogate T-12 induced autophagy, suggesting that autophagy precedes apoptosis. In addition, T-12 inhibited cell survival pathway signaling proteins, Akt, mTOR and Erk1/2. T-12 also induced significant regression of tumor with oral dose of as low as 10 mg/kg bodyweight in rat mammary tumor model without any apparent toxicity. In presence of reactive oxygen species inhibitor (N-acetyl-l-cysteine) and autophagy inhibitor (chloroquine), T-12 induced tumor regression was significantly decreased. In conclusion, T-12 is a potent inducer of autophagy-dependent apoptosis in breast cancer cells both in vitro and in vivo and can serve as an important lead in development of new anti-tumor therapy.     

A novel autophagy/mitophagy inhibitor liensinine sensitizes breast cancer cells to chemotherapy through DNM1L-mediated mitochondrial fission

Autophagy inhibition has been widely accepted as a promising therapeutic strategy in cancer, while the lack of effective and specific autophagy inhibitors hinders its application. Here we found that liensinine, a major isoquinoline alkaloid, inhibits late-stage autophagy/mitophagy through blocking autophagosome-lysosome fusion. This effect is likely achieved via inhibiting the recruitment of RAB7A to lysosomes but not to autophagosomes. We further investigated the effects of autophagy inhibition by liensinine on the therapeutic efficacy of chemotherapeutic drugs and found that cotreatment of liensinine markedly decreased the viability and increased apoptosis in breast cancer cells treated with various chemotherapeutic agents. Mechanistically, we found that inhibition of autophagy/mitophagy by liensinine enhanced doxorubicin-mediated apoptosis by triggering mitochondrial fission, which resulted from dephosphorylation and mitochondrial translocation of DNM1L. However, blocking autophagosome/mitophagosome formation by pharmacological or genetic approaches markedly attenuated mitochondrial fission and apoptosis in cells with combinatatorial treatment. Moreover, liensinine was synergized with doxorubicin to inhibit tumor growth in MDA-MB-231 xenograft in vivo. Our findings suggest that liensinine could potentially be further developed as a novel autophagy/mitophagy inhibitor, and a combination of liensinine with classical chemotherapeutic drugs could represent a novel therapeutic strategy for treatment of breast cancer.     

Autophagy Plays a Critical Role in ChLym-1-Induced Cytotoxicity of Non-Hodgkin’s Lymphoma Cells

Autophagy is a critical mechanism in both cancer therapy resistance and tumor suppression. Monoclonal antibodies have been documented to kill tumor cells via apoptosis, antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In this study, we report for the first time that chLym-1, a chimeric anti-human HLA-DR monoclonal antibody, induces autophagy in Raji Non-Hodgkin’s Lymphoma (NHL) cells. Interestingly, inhibition of autophagy by pharmacological inhibitors (3-methyladenine and NH₄Cl) or genetic approaches (siRNA targeting Atg5) suppresses chLym-1-induced growth inhibition, apoptosis, ADCC and CDC in Raji cells, while induction of autophagy could accelerate cytotoxic effects of chLym-1 on Raji cells. Furthermore, chLym-1-induced autophagy can mediate apoptosis through Caspase 9 activation, demonstrating the tumor-suppressing role of autophagy in antilymphoma effects of chLym-1. Moreover, chLym-1 can activate several upstream signaling pathways of autophagy including Akt/mTOR and extracellular signal-regulated kinase 1/2 (Erk1/2). These results elucidate the critical role of autophagy in cytotoxicity of chLym-1 antibody and suggest a potential therapeutic strategy of NHL therapy by monoclonal antibody chLym-1 in combination with autophagy inducer.