背景:在线粒体质量控制(MQC)的机制中,线粒体衍生的囊泡(MDV)的产生是避免线粒体完全失败的过程,决定线粒体受损蛋白的溶酶体降解。在这种情况下,RAB7,一种晚期内吞小GTP酶,控制MDV向晚期内体的递送,以进行随后的溶酶体降解。我们先前证明,RAB7在响应顺铂(CDDP)调节细胞外囊泡(EV)分泌对药物的耐药性方面具有关键作用。
方法:采用Westernblot和免疫荧光分析方法分析CDDP化疗敏感和化疗耐药的卵巢癌细胞株内体和溶酶体的结构和功能。通过超速离心或免疫分离从化学敏感性和化学抗性细胞中纯化EV,以分析其线粒体DNA和蛋白质含量。用氰化物间氯苯腙(CCCP)和RAB7调制处理,分别,了解线粒体和晚期内体/溶酶体改变对MDV分泌的作用。使用来自化学抗性细胞的条件培养基,测定MDV对CDDP处理后的存活力的影响。海马测定和免疫荧光分析用于研究MDV的生化作用以及MDV的摄取和细胞内定位,分别。
结果:我们观察到CDDP耐药细胞的特征是MDV分泌增加,晚期内吞交通受损,RAB7下调,电动汽车中RAB7的增加,与化学敏感细胞相比,以及TFEB-mTOR途径的下调,监督溶酶体和线粒体的生物发生和周转。我们确定MDV可以分泌而不是递送到溶酶体,并且能够将CDDP递送到细胞外。我们显示了由EV中RAB7的挤出最终引起的化学抗性细胞的MDV分泌增加,导致其细胞内含量急剧下降,作为调节RAB7水平的新机制。我们证明了从化学抗性细胞中纯化的MDV在RAB7调节的过程中诱导化学抗性,and,从受体细胞摄取后,MDV定位于线粒体并减慢线粒体活性。
结论:化疗耐药细胞中功能失调的MQC决定了MDV溶酶体降解及其随后分泌的阻滞,这表明MQC不能消除受损的线粒体,其成分被分泌成为化学抗性的效应子和潜在标志物。
Among the mechanisms of mitochondrial quality control (MQC), generation of mitochondria-derived vesicles (MDVs) is a process to avoid complete failure of mitochondria determining lysosomal degradation of mitochondrial damaged proteins. In this context,
RAB7, a late endocytic small GTPase, controls delivery of MDVs to late endosomes for subsequent lysosomal degradation. We previously demonstrated that
RAB7 has a pivotal role in response to cisplatin (CDDP) regulating resistance to the drug by extracellular vesicle (EVs) secretion.
Western blot and immunofluorescence analysis were used to analyze structure and function of endosomes and lysosomes in CDDP chemosensitive and chemoresistant ovarian cancer cell lines. EVs were purified from chemosensitive and chemoresistant cells by ultracentrifugation or immunoisolation to analyze their mitochondrial DNA and protein content. Treatment with cyanide m-chlorophenylhydrazone (CCCP) and
RAB7 modulation were used, respectively, to understand the role of mitochondrial and late endosomal/lysosomal alterations on MDV secretion. Using conditioned media from chemoresistant cells the effect of MDVs on the viability after CDDP treatment was determined. Seahorse assays and immunofluorescence analysis were used to study the biochemical role of MDVs and the uptake and intracellular localization of MDVs, respectively.
We observed that CDDP-chemoresistant cells are characterized by increased MDV secretion, impairment of late endocytic traffic, RAB7 downregulation, an increase of
RAB7 in EVs, compared to chemosensitive cells, and downregulation of the TFEB-mTOR pathway overseeing lysosomal and mitochondrial biogenesis and turnover. We established that MDVs can be secreted rather than delivered to lysosomes and are able to deliver CDDP outside the cells. We showed increased secretion of MDVs by chemoresistant cells ultimately caused by the extrusion of RAB7 in EVs, resulting in a dramatic drop in its intracellular content, as a novel mechanism to regulate RAB7 levels. We demonstrated that MDVs purified from chemoresistant cells induce chemoresistance in
RAB7-modulated process, and, after uptake from recipient cells, MDVs localize to mitochondria and slow down mitochondrial activity.
Dysfunctional MQC in chemoresistant cells determines a block in lysosomal degradation of MDVs and their consequent secretion, suggesting that MQC is not able to eliminate damaged mitochondria whose components are secreted becoming effectors and potential markers of chemoresistance.