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.     

异构体ΔASPP2可拮抗ASPP2对p53（细胞）凋亡功能的增强作用

p53 凋亡刺激蛋白2(apoptosis stimulating protein 2 of p53, ASPP2)能够与p53 蛋白结合特异性地增强其促细胞凋亡功能,进而发挥肿瘤抑制作用．我们发现的1个比ASPP2少300多个N端氨基酸的异构体ΔASPP2.目前,ΔASPP2对p53起何种作用尚不清楚．在本研究中,我们构建了rAd-ASPP2、rAd-ΔASPP2腺病毒,利用rAd-p53、rAd-ASPP2、rAd-ΔASPP2 感染p53缺失的细胞系H1299,在MMS的作用下研究ASPP2 和 ΔASPP2 对p53介导的细胞凋亡的影响．结果发现,p53自身过表达能明显促进肿瘤细胞的凋亡;ASPP2可显著增强p53介导的MMS引起的H1299细胞凋亡的作用;然而,ΔASPP2对p53介导的细胞凋亡没有明显影响但却显著抑制rAd-ASPP2 增强的rAd-p53的促细胞凋亡作用．p53-ASPP2 复合体可能改变p53 蛋白的构象,促进p53 和增强子Bax的结合活性．p53 转录调控基因的表达研究显示,ΔASPP2的存在可显著抑制ASPP2增强p53 介导的bax基因转录活性, 提示ΔASPP2可能与ASPP2结合后来抑制p53的凋亡基因转录活性．     

ASPP2增强结肠癌HCT116细胞对奥沙利铂的化疗敏感性

为研究ASPP2对奥沙利铂诱导的结肠癌细胞系HCT116 p53+/+(野生型)凋亡及周期的影响.利用ASPP2（rAd-ASPP2）及p53腺病毒（rAd-p53）感染HCT116 p53+/+细胞,经奥沙利铂50 μmol/L诱导细胞凋亡及周期改变.Western印迹检测ASPP2及p53的表达水平;MTT法检测ASPP2腺病毒对奥沙利铂诱导的HCT116细胞活性的影响;Calcein/PI吸收试验检测细胞凋亡情况;流式细胞术分析细胞周期分布. 结果显示,ASPP2、p53共同过表达,或者ASPP2单独过表达均能增强奥沙利铂诱导的HCT116 p53+/+细胞增殖抑制,以及S期抑制并伴有细胞凋亡水平的升高;而无奥沙利铂诱导时,ASPP2对HCT116 p53+/+细胞的活性、细胞周期及细胞凋亡水平的影响无统计学意义. 上述结果表明,ASPP2能够增强奥沙利铂诱导HCT116 p53+/+细胞的增殖抑制、细胞周期抑制和细胞凋亡.     

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.     

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。     

抑制自噬促进冬凌草甲素诱导的多发性骨髓瘤细胞凋亡涉及胞内ROS产生

目的本实验主要研究冬凌草甲素诱导多发性骨髓瘤发生自噬、凋亡,两者之间的关系以及所涉及的相关机制。方法利用MTT比色法检测冬凌草甲素对多发性骨髓瘤RPMI8226细胞的增殖活性影响;透视电镜观察细胞内凋亡和自噬的形态学改变;TUNEL检测细胞凋亡;分别利用以下技术检测处理后的细胞内的自噬变化:使用QDs605nm-Anti-LC3荧光探针以及免疫荧光技术定位细胞胞内LC3Ⅰ和LC3Ⅱ蛋白,利用western blot免疫印记技术检测Beclin 1蛋白表达水平;利用DCFH-DA探针以及流式细胞术检测细胞胞内ROS水平。结果冬凌草甲素能明显抑制RPMI8226细胞增殖,其抑制作用呈时间、剂量依赖性;冬凌草甲素能同时诱发细胞凋亡、自噬和胞内ROS产生;NAC完全抑制胞内ROS产生后冬凌草甲素诱导的细胞凋亡消失;3-MA抑制自噬后,冬凌草甲素诱导的胞内ROS产生进一步增多,凋亡增多。结论冬凌草甲素能明显抑制RPMI8226细胞增殖;冬凌草甲素同时诱发细胞凋亡和自噬;胞内ROS产生介导冬凌草甲素诱导的凋亡;凋亡为细胞死亡的主要途径,而自噬通过下调胞内ROS产生抑制凋亡。     

CHOP双重调控衣霉素诱导的DU-145细胞凋亡及自噬的研究

目的研究内质网应激分子CHOP调控细胞凋亡与自噬的作用和机制。 方法利用衣霉素诱导DU-145细胞产生内质网应激,Western Blot法检测内质网应激相关分子Grp78、Grp94、p-eIF2α和CHOP及自噬蛋白LC3Ⅱ、Atg5和Beclin1的表达;用流式细胞术检测细胞凋亡水平;沉默CHOP基因,用Western Blot法检测凋亡蛋白PARP、Caspase3的表达,流式细胞术检测细胞凋亡;并利用免疫荧光检测自噬标志性蛋白LC3B的表达。 结果衣霉素诱导DU-145细胞内质网应激能诱导一定程度的细胞凋亡,衣霉素处理8、12、24?h的细胞凋亡率分别为3.27﹪±1.02﹪,8.97﹪±0.71﹪和11.67﹪±1.41﹪,处理12?h及24?h的细胞凋亡率与对照组相比差异具有统计学意义（P < 0.01）。同时也能通过抑制PI3K/AKt/mTOR信号通路激活DU-145细胞自噬。CHOP基因沉默抑制细胞凋亡,shCtrl组细胞凋亡率为32.17﹪±3.93﹪,shCHOP-1组细胞凋亡率为23.53﹪±3.41﹪,两组相比差异具有统计学意义（P < 0.05）。且CHOP基因沉默能促进细胞自噬分子LC3B的表达。 结论衣霉素诱导DU-145细胞内质网应激状态下,CHOP在细胞凋亡与自噬之间发挥双重调控作用。     

干扰PKM2对人白血病细胞增殖和凋亡的影响及潜在机制

目的:探讨糖酵解酶丙酮酸激酶M2型(PKM2)对人白血病细胞体外增殖和凋亡的影响及潜在机制。方法:将靶向PKM2的慢病毒载体转染人K562细胞株(sh PKM2组),同时设立空载体转染组为对照(Vector组)。采用qRT-PCR和Western blot技术分别检测Vector组和sh PKM2组PKM2 mRNA和蛋白的表达以及自噬标志物的变化; CCK-8实验检测细胞体外增殖能力;流式细胞术检测细胞周期和凋亡情况; Western blot检测凋亡相关蛋白Bax、Bcl-2表达水平。结果:在稳定干扰PKM2后,PKM2的mRNA(t=11. 58,P=0. 000 3)和蛋白水平(t=11. 88,P=0. 000 3)均明显降低。与Vector组比较,shPKM2组细胞体外增殖能力显著降低(F=118. 87,P 0. 000 1)。同时,干扰PKM2可使K562细胞周期阻滞在G1期,细胞凋亡率显著增加(t=37. 23,P 0. 000 1);使促凋亡蛋白Bax表达增加(t=15. 36,P=0. 000 1)、抗凋亡蛋白Bcl-2表达降低(t=9. 965,P=0. 000 6)。此外,干扰PKM2可减弱K562细胞自噬水平,表现为LC3Ⅱ降低(t_(LC3Ⅱ)=10. 32,P_(LC3Ⅱ)=0. 000 5),而p62水平增加(t_(p62)=14. 59,P_(p62)=0. 000 1)。还发现自噬诱导剂能逆转shPKM2引起的白血病细胞体外增殖能力减弱(F=96. 32,P 0. 000 1)。结论:以上结果表明干扰PKM2可抑制人白血病K562细胞的体外增殖并促进其凋亡,其机制可能与PKM2介导的自噬活性降低有关,提示PKM2可能作为白血病诊疗的一个潜在靶点。     

磷酸腺苷激活的蛋白激酶(AMPK)参与了曲格列酮诱导的HeLa细胞自噬和凋亡

【目的】明确磷酸腺苷激活的蛋白激酶(AMPK)在细胞自噬和凋亡中的作用。【方法】利用电镜、荧光显微镜、蛋白免疫杂交、siRNA干扰、流式细胞计数、MTS细胞活性检测等对曲格列酮(troglitazone,TZ)处理的HeLa细胞自噬和凋亡情况进行了检测。【结果】不同检测方法均表明TZ增加了HeLa细胞的自噬,这种自噬的发生伴随着AMPK的磷酸化的降低;抑制AMPK增加基础细胞自噬,而阻断了TZ引起的自噬标记物LC3-II的增加,同时也减少了TZ引起的凋亡分子PARP的切割;用自噬抑制剂3-MA和干扰细胞自噬基因,不仅PARP的切割明显地受到抑制,而且也部分阻断了TZ引起的细胞活性丧失。【结论】AMPK直接参与了TZ引起的HeLa细胞自噬过程,这种自噬发生促进了其诱导的细胞凋亡。     

谷氧还蛋白1调节高糖诱导的心肌细胞自噬

谷氧还蛋白1（Grx1）在体内具有广泛的抗氧化、抗凋亡作用,与氧化应激损伤导致的糖尿病和心肌病等多种疾病的发病机制密切相关. 研究表明,糖尿病心血管病与自噬调节异常密切相关,但糖尿病心血管病变时自噬水平如何调节才能够保护受损的心肌还尚未定论.为研究自噬在高糖诱导心肌细胞凋亡中的作用及其与Grx1的关系,以明确Grx1对高糖诱导的心肌细胞凋亡的抑制作用及相关机制,本研究以高糖诱导大鼠心肌细胞H9c2建立高糖损伤模型,采用氧化还原蛋白免疫印迹法检测蛋白质的氧化水平.免疫印迹检测活性caspase 3蛋白和自噬蛋白Beclin1和LC3以及抗凋亡蛋白Bcl 2的表达水平.研究发现,高糖可诱导蛋白质的氧化水平增加,而Grx1可拮抗高糖诱导的H9c2细胞中蛋白质的氧化.并且含血清的高糖（25和50 mmol/L）作用H9c2心肌细胞后,自噬蛋白Beclin 1表达水平在6～48 h显著上调.同时发现,活性caspase 3水平也呈时间依赖性表达上调,caspase 3和自噬蛋白表达水平的同趋势增加,说明升高的自噬水平与心肌细胞凋亡的调节有关.Grx1保护组的自噬蛋白及活性caspase 3表达水平均显著下调,Grx1抑制剂镉组可拮抗Grx1调节的自噬蛋白和凋亡蛋白水平,说明Grx 1通过抑制自噬及caspase 3水平抑制高糖诱导的心肌细胞凋亡.以上研究结果提示,通过提高Grx1/GSH抗氧化系统功能,调节氧化还原稳态,可以有效减少高糖诱导的心肌损伤,保护糖尿病心脏功能.     

Phosphorylated AKT inhibits the apoptosis induced by DRAM-mediated mitophagy in hepatocellular carcinoma by preventing the translocation of DRAM to mitochondria

Increasing autophagy is beneficial for curing hepatocellular carcinoma (HCC). Damage-regulated autophagy modulator (DRAM) was recently reported to induce apoptosis by mediating autophagy. However, the effects of DRAM-mediated autophagy on apoptosis in HCC cells remain unclear. In this study, normal hepatocytes (7702) and HCC cell lines (HepG2, Hep3B and Huh7) were starved for 48 h. Starvation induced apoptosis and autophagy in all cell lines. We determined that starvation also induced DRAM expression and DRAM-mediated autophagy in both normal hepatocytes and HCC cells. However, DRAM-mediated autophagy was involved in apoptosis in normal hepatocytes but not in HCC cells, suggesting that DRAM-mediated autophagy fails to induce apoptosis in hepatoma in response to starvation. Immunoblot and immunofluorescence assays demonstrated that DRAM translocated to mitochondria and induced mitophagy, which led to apoptosis in 7702 cells. In HCC cells, starvation also activated the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, which blocks the translocation of DRAM to mitochondria through the binding of p-AKT to DRAM in the cytoplasm. Inactivation of the PI3K/AKT pathway rescued DRAM translocation to mitochondria; subsequently, mitochondrial DRAM induced apoptosis in HCC cells by mediating mitophagy. Our findings open new avenues for the investigation of the mechanisms of DRAM-mediated autophagy and suggest that promoting DRAM-mediated autophagy together with PI3K/AKT inhibition might be more effective for autophagy-based therapy in hepatoma.     

DRAM, a p53-induced modulator of autophagy, is critical for apoptosis

Inactivation of cell death is a major step in tumor development, and p53, a tumor suppressor frequently mutated in cancer, is a critical mediator of cell death. While a role for p53 in apoptosis is well established, direct links to other pathways controlling cell death are unknown. Here we describe DRAM (damage-regulated autophagy modulator), a p53 target gene encoding a lysosomal protein that induces macroautophagy, as an effector of p53-mediated death. We show that p53 induces autophagy in a DRAM-dependent manner and, while overexpression of DRAM alone causes minimal cell death, DRAM is essential for p53-mediated apoptosis. Moreover, analysis of DRAM in primary tumors revealed frequent decreased expression often accompanied by retention of wild-type p53. Collectively therefore, these studies not only report a stress-induced regulator of autophagy but also highlight the relationship of DRAM and autophagy to p53 function and damage-induced programmed cell death.     

Reduced expression of DRAM2/TMEM77 in tumor cells interferes with cell death

Although the role of autophagy in tumorigenesis remains controversial, recent reports support the notion that inhibition of autophagy promotes tumor formation. Damage-regulated autophagy regulator (DRAM) has been identified as an effector molecule that is critical for p53-mediated apoptosis, and we investigated whether there might be other DRAM-like molecules linking autophagy and apoptosis. In this study, we cloned a novel DRAM-homologous protein, DRAM2, and showed that the expression of DRAM2 is down-regulated in ovarian tumors. DRAM2 is mainly localized in the lysosome, and co-localizes with DRAM. While expression of DRAM or DRAM2 individually did not induce cell death, co-expression of DRAM2 with DRAM significantly induced cell death, while the silencing of endogenous DRAM2 attenuated cell death, suggesting that DRAM2 is involved in cell death. Thus, we propose that reduced expression of DRAM2 may contribute to enhanced cell survival in tumor cells.     

The expression of damage-regulated autophagy modulator 2 (DRAM2) contributes to autophagy induction

Autophagy is a membrane trafficking process involved in intracellular degradation and recycling in eukaryotic cells. DRAM2 (damage-regulated autophagy modulator 2) is a homologue of DRAM that regulates p53-mediated cell death. As its name implies, DRAM expression induces autophagy in a p53-dependent manner; however, the role of DRAM2 in autophagy is not clear. In this study, we report that DRAM2 expression contributes to autophagy induction. Overexpression of DRAM2 induces cytoplasmic GFP-LC3 punctuates, and increases the level of endogenous LC3-II. Moreover, the silencing of endogenous DRAM2 interferes with starvation-induced autophagy. Thus, we propose that DRAM2 as well as DRAM are involved in autophagy.     

Deficiency of apoptosis‐stimulating protein two of p53 ameliorates acute kidney injury induced by ischemia reperfusion in mice through upregulation of autophagy

Acute kidney injury (AKI) has become a common disorder with a high risk of morbidity and mortality, which remains major medical problem without reliable and effective therapeutic intervention. Apoptosis‐stimulating protein two of p53 (ASPP2) is a proapoptotic member that belongs to p53 binding protein family, which plays a key role in regulating apoptosis and cell growth. However, the role of ASPP2 in AKI has not been reported. To explore the role of ASPP2 in the progression of AKI, we prepared an AKI mouse model induced by ischaemia reperfusion (I/R) in wild‐type (ASPP2^+/+) mice and ASPP2 haploinsufficient (ASPP2^+/?) mice. The expression profile of ASPP2 were examined in wild‐type mice. The renal injury, inflammation response, cellular apoptosis and autophagic pathway was assessed in ASPP2^+/+ and ASPP2^+/? mice. The renal injury, inflammation response and cellular apoptosis was analysed in ASPP2^+/+ and ASPP2^+/? mice treated with 3‐methyladenine or vehicle. The expression profile of ASPP2 showed an increase at the early stage while a decrease at the late stage during renal injury. Compared with ASPP2^+/+ mice, ASPP2 deficiency protected mice against renal injury induced by I/R, which mainly exhibited in slighter histologic changes, lower levels of blood urea nitrogen and serum creatinine, and less apoptosis as well as inflammatory response. Furthermore, ASPP2 deficiency enhanced autophagic activity reflecting in the light chain 3‐II conversion and p62 degradation, while the inhibition of autophagy reversed the protective effect of ASPP2 deficiency on AKI. These data suggest that downregulation of ASPP2 can ameliorate AKI induced by I/R through activating autophagy, which may provide a novel therapeutic strage for AKI.     

新的抑癌基因ASPP对p53作用的研究进展

p53是一个肿瘤抑制蛋白,它是通过调节相关基因表达,诱导细胞凋亡。p53诱导细胞凋亡的机制多年来一直不太清楚,而最近发现的ASPP(apoptosis stimulating protein of p53)蛋白家族对p53诱导细胞凋亡的机制研究有了新的进展。本文就此作一综述。