Cellular and Molecular Neurobiology

, Volume 37, Issue 3, pp 555–562 | Cite as

RLIP76 Depletion Enhances Autophagic Flux in U251 Cells

  • Chenran Zhang
  • Zheng Cai
  • Qiang Liang
  • Qi Wang
  • Yicheng Lu
  • Liuhua HuEmail author
  • Guohan HuEmail author
Original Research


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.


RLIP76 PI3K–Akt Glioma Autophagy Apoptosis 



This work was generously supported by the National Natural Sciences Fund Project of China (NSFC Nos. 81500601), Shanghai Municipal Natural Science Foundation (14ZR1413800) and Shanghai Municipal Health Bureau Project (201440383 and 20154Y0036).

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they do not have any financial or non-financial competing interests.

Supplementary material

10571_2016_410_MOESM1_ESM.pdf (901 kb)
Supplementary material 1 (PDF 900 kb)
10571_2016_410_MOESM2_ESM.pdf (91 kb)
Supplementary material 2 (PDF 90.8 kb)


  1. Bhutia SK et al (2011) Autophagy switches to apoptosis in prostate cancer cells infected with melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24). Autophagy 7:1076–1077CrossRefPubMedPubMedCentralGoogle Scholar
  2. Bordin DL et al (2013) DNA alkylation damage and autophagy induction. Mutat Res 753:91–99CrossRefPubMedGoogle Scholar
  3. Carmo A, Carvalheiro H, Crespo I, Nunes I, Lopes MC (2011) Effect of temozolomide on the U-118 glioma cell line. Oncol Lett 2:1165–1170PubMedPubMedCentralGoogle Scholar
  4. Chen Y et al (2015) MP8 facilitates cellular proliferation and temozolomide resistance in human glioma cells. Neuro Oncol 17:407–418PubMedGoogle Scholar
  5. Dubrovska A et al (2009) The role of PTEN/Akt/PI3K signaling in the maintenance and viability of prostate cancer stem-like cell populations. Proc Natl Acad Sci USA 106:268–273CrossRefPubMedGoogle Scholar
  6. Eyler CE, Foo WC, LaFiura KM, McLendon RE, Hjelmeland AB, Rich JN (2008) Brain cancer stem cells display preferential sensitivity to Akt inhibition. Stem Cells 26:3027–3036CrossRefPubMedPubMedCentralGoogle Scholar
  7. Gewirtz DA, Hilliker ML, Wilson EN (2009) Promotion of autophagy as a mechanism for radiation sensitization of breast tumor cells. Radiother Oncol 92:323–328CrossRefPubMedGoogle Scholar
  8. Hambardzumyan D, Becher OJ, Rosenblum MK, Pandolfi PP, Manova-Todorova K, Holland EC (2008) PI3K pathway regulates survival of cancer stem cells residing in the perivascular niche following radiation in medulloblastoma in vivo. Genes Dev 22:436–448CrossRefPubMedPubMedCentralGoogle Scholar
  9. Hariharan N, Zhai P, Sadoshima J (2011) Oxidative stress stimulates autophagic flux during ischemia/reperfusion. Antioxid Redox Signal 14:2179–2190CrossRefPubMedPubMedCentralGoogle Scholar
  10. Imai Y, Yamagishi H, Ono Y, Ueda Y (2012) Versatile inhibitory effects of the flavonoid-derived PI3K/Akt inhibitor, LY294002, on ATP-binding cassette transporters that characterize stem cells. Clin Transl Med 1:24CrossRefPubMedPubMedCentralGoogle Scholar
  11. Ito H et al (2006) Autophagic cell death of malignant glioma cells induced by a conditionally replicating adenovirus. J Natl Cancer Inst 98:625–636CrossRefPubMedGoogle Scholar
  12. Kimura S, Noda T, Yoshimori T (2007) Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy 3:452–460CrossRefPubMedGoogle Scholar
  13. Klionsky DJ et al (2008) Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 4:151–175CrossRefPubMedGoogle Scholar
  14. Knizhnik AV et al (2013) Survival and death strategies in glioma cells: autophagy, senescence and apoptosis triggered by a single type of temozolomide-induced DNA damage. PLoS One 8:e55665. doi: 10.1371/journal.pone.0055665 CrossRefPubMedPubMedCentralGoogle Scholar
  15. Kumar D et al (2015) Andrographolide analogue induces apoptosis and autophagy mediated cell death in U937 cells by inhibition of PI3K/Akt/mTOR pathway. PLoS One 10:e0139657. doi: 10.1371/journal.pone.0139657 CrossRefPubMedPubMedCentralGoogle Scholar
  16. Leake K, Singhal J, Nagaprashantha LD, Awasthi S, Singhal SS (2012) RLIP76 regulates PI3K/Akt signaling and chemo-radiotherapy resistance in pancreatic cancer. PLoS One 7:e34582. doi: 10.1371/journal.pone.0034582 CrossRefPubMedPubMedCentralGoogle Scholar
  17. Messaoudi K, Clavreul A, Lagarce F (2015) Toward an effective strategy in glioblastoma treatment. Part I: resistance mechanisms and strategies to overcome resistance of glioblastoma to temozolomide. Drug Discov Today 20:899–905CrossRefPubMedGoogle Scholar
  18. Mizushima N (2007) Autophagy: process and function. Genes Dev 21:2861–2873CrossRefPubMedGoogle Scholar
  19. Natsumeda M et al (2015) Targeting notch signaling and autophagy increases cytotoxicity in glioblastoma neurospheres. Brain Pathol. doi: 10.1111/bpa.12343 Google Scholar
  20. Pratt J, Annabi B (2014) Induction of autophagy biomarker BNIP3 requires a JAK2/STAT3 and MT1-MMP signaling interplay in concanavalin-A-activated U87 glioblastoma cells. Cell Signal 26:917–924CrossRefPubMedGoogle Scholar
  21. Tsai JP et al (2015) Licochalcone A induces autophagy through PI3K/Akt/mTOR inactivation and autophagy suppression enhances licochalcone A-induced apoptosis of human cervical cancer cells. Oncotarget 6:28851–28866PubMedPubMedCentralGoogle Scholar
  22. Wang Q et al (2013a) Knockdown of RLIP76 expression by RNA interference inhibits invasion, induces cell cycle arrest, and increases chemosensitivity to the anticancer drug temozolomide in glioma cells. J Neuro Oncol 112:73–82CrossRefGoogle Scholar
  23. Wang Q et al (2013b) RLIP76 is overexpressed in human glioblastomas and is required for proliferation, tumorigenesis and suppression of apoptosis. Carcinogenesis 34:916–926CrossRefPubMedGoogle Scholar
  24. Wang X et al (2014) Histone deacetylase 4 selectively contributes to podocyte injury in diabetic nephropathy. Kidney Int 86:712–725CrossRefPubMedGoogle Scholar
  25. Wang KF, Yang H, Jiang WQ, Li S, Cai YC (2015) Puquitinib mesylate (XC-302) induces autophagy via inhibiting the PI3K/AKT/mTOR signaling pathway in nasopharyngeal cancer cells. Int J Mol Med 36:1556–1562PubMedPubMedCentralGoogle Scholar
  26. Yan Y, Xu Z, Dai S, Qian L, Sun L, Gong Z (2016) Targeting autophagy to sensitive glioma to temozolomide treatment. J Exp Clin Cancer Res 35:23CrossRefPubMedPubMedCentralGoogle Scholar
  27. Yang J, Song Q, Cai Y, Wang P, Wang M, Zhang D (2015) RLIP76-dependent suppression of PI3K/Akt/Bcl-2 pathway by miR-101 induces apoptosis in prostate cancer. Biochem Biophys Res Commun 463:900–906CrossRefPubMedGoogle Scholar
  28. Yousefi S et al (2006) Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis. Nat Cell Biol 8:1124–1132CrossRefPubMedGoogle Scholar
  29. Zhang C et al (2015) RIZ1: a potential tumor suppressor in glioma. BMC Cancer 15:990CrossRefPubMedPubMedCentralGoogle Scholar
  30. Zhou J et al (2007) Activation of the PTEN/mTOR/STAT3 pathway in breast cancer stem-like cells is required for viability and maintenance. Proc Natl Acad Sci USA 104:16158–16163CrossRefPubMedPubMedCentralGoogle Scholar
  31. Zou Y, Wang Q, Li B, Xie B, Wang W (2014) Temozolomide induces autophagy via ATM-AMPK-ULK1 pathways in glioma. Mol Med Rep 10:411–416PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Neurosurgery, Changzheng HospitalSecond Military Medical UniversityShanghaiChina
  2. 2.Department of Pediatric Neurosurgery, Xinhua Hospital, School of MedicineShanghai Jiao Tong UniversityShanghaiChina
  3. 3.Department of NeurosurgeryPLA No. 322 HospitalShanxiChina
  4. 4.Department of Cardiology, Ren Ji Hospital, School of MedicineShanghai Jiao Tong UniversityShanghaiChina

Personalised recommendations