title: RLIP76 Depletion Enhances Autophagic Flux in U251 Cells.
abstract: Our previous study showed that RalA-binding protein 1 (RLIP76) is overexpressed in gliomas and is associated with higher tumour grade and decreased patient survival. Furthermore, RLIP76 downregulation increases chemosensitivity of glioma cells to temozolomide by inducing apoptosis. However, other mechanisms underlying RLIP76-associated chemoresistance are unknown. In this study, we investigated the effect of RLIP76 depletion on autophagy. RLIP76 was knocked down in U251 glioma cells using shRNA and autophagy-related proteins, and PI3K/Akt signalling components were evaluated. RLIP76 depletion significantly increased cell autophagy as demonstrated by a significant increase in LC3 II, autophagy protein 5 (ATG-5), and Beclin1, and a decrease in p62 expression levels. Furthermore, RLIP76 knockdown increased autophagic flux in U251 cells as autolysosome numbers increased relative to autophagosome numbers. Autophagy induced by RLIP76 knockdown resulted in increased apoptosis that was independent of temozolomide treatment. Moreover, RLIP76 knockdown decreased PI3K and Akt activation. RLIP76 depletion also resulted in decreased levels of the anti-apoptotic protein Bcl2. LY294002, a PI3K/Akt pathway inhibitor, led to increased autophagy and apoptosis in U251 RLIP76-depleted cells. Therefore, RLIP76 knockdown increased autophagic flux and apoptosis in U251 glioma cells, possibly through inhibition of the PI3K/Akt pathway. Thus, this study provides a novel mechanism for the role of RLIP76 in glioma pathogenesis and chemoresistance.
authors.affiliation: Department of Neurosurgery, Changzheng Hospital, Second Military Medical University, Shanghai, 200003, China.|Department of Pediatric Neurosurgery, Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200092, China.
authors.forename: Chenran
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Department of Neurosurgery, Changzheng Hospital, Second Military Medical University, Shanghai, 200003, China.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Cai
authors.affiliation: Department of Neurosurgery, Changzheng Hospital, Second Military Medical University, Shanghai, 200003, China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Liang
authors.affiliation: Department of Neurosurgery, Changzheng Hospital, Second Military Medical University, Shanghai, 200003, China.|Department of Neurosurgery, PLA No. 322 Hospital, 2 Yunzhong Road, Shanxi, 03700, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Department of Neurosurgery, Changzheng Hospital, Second Military Medical University, Shanghai, 200003, China.
authors.forename: Yicheng
authors.initials: Y
authors.lastname: Lu
authors.affiliation: Department of Cardiology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, China. iam565@126.com.
authors.forename: Liuhua
authors.initials: L
authors.lastname: Hu
authors.affiliation: Department of Neurosurgery, Changzheng Hospital, Second Military Medical University, Shanghai, 200003, China. huguohan@163.com.
authors.forename: Guohan
authors.initials: G
authors.lastname: Hu
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D017209:Apoptosis / Q000187:drug effects; D000071182:Autophagosomes / Q000187:drug effects / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects; D001932:Brain Neoplasms / Q000473:pathology; D045744:Cell Line, Tumor; D003606:Dacarbazine / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D005910:Glioma / Q000378:metabolism* / Q000473:pathology*; D006801:Humans; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D008561:Membrane Fusion / Q000187:drug effects; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D000077204:Temozolomide
chemical_list: D018528:ATP-Binding Cassette Transporters; D020690:GTPase-Activating Proteins; C095637:RALBP1 protein, human; D034741:RNA, Small Interfering; D003606:Dacarbazine; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D000077204:Temozolomide
keywords: Apoptosis; Autophagy; Glioma; PI3K–Akt; RLIP76