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Autophagy inhibition enhances RAD001-induced cytotoxicity in human bladder cancer cells.

ABSTRACT: Mammalian target of rapamycin (mTOR), involved in PI3K/AKT/mTOR pathway, is known to play a central role in regulating the growth of cancer cells. The PI3K/AKT/mTOR pathway enhances tumor survival and proliferation through suppressing autophagy, which sustains energy homeostasis by collecting and recycling cellular components under stress conditions. Conversely, inhibitors of the mTOR pathway such as RAD001 induce autophagy, leading to promotion of tumor survival and limited antitumor efficacy. We thus hypothesized that the use of autophagy inhibitor in combination with mTOR inhibition improves the cytotoxicity of mTOR inhibitors in bladder cancer.The cytotoxicity of RT4, 5637, HT1376, and T24 human bladder cancer cells treated with RAD001 alone or combined with autophagy inhibitors (3-methyladenine (3-MA), bafilomycin A1 (Baf A1), chloroquine, or hydroxychloroquine) was assessed using the WST-8 cell viability kit. The autophagy status in cells was analyzed by the detection of microtubule-associated light chain 3 form II (LC3-II), using immunofluorescent staining and Western blot. Acidic vesicular organelle (AVO) formation in treated cells was determined by acridine orange vital staining. Inhibition of mTOR pathway by RAD001 was monitored by using a homemade quantitative polymerase chain reaction gene array, while phospho-mTOR was detected using Western blot. Induced apoptosis was determined by measurement of caspase 3/7 activity and DNA fragmentation in cells after treatment.Advanced bladder cancer cells (5637, HT1376, and T24) were more resistant to RAD001 than RT4. Autophagy flux detected by the expression of LC3-II showed RAD001-induced autophagy. AVO formation was detected in cells treated with RAD001 and was inhibited by the addition of 3-MA or Baf A1. Cotreatment of RAD001 with autophagy inhibitors further reduced cell viability and induced apoptosis in bladder cancer cells.Our results indicate that simultaneous inhibition of the mTOR and autophagy pathway significantly enhances apoptosis, and it is suggested to be a new therapeutic paradigm for the treatment of bladder cancer.

SUBMITTER: Lin JF

PROVIDER: S-EPMC4841413 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Autophagy inhibition enhances RAD001-induced cytotoxicity in human bladder cancer cells.

Lin Ji-Fan JF Lin Yi-Chia YC Yang Shan-Che SC Tsai Te-Fu TF Chen Hung-En HE Chou Kuang-Yu KY Hwang Thomas I-Sheng TI

Drug design, development and therapy 20160418

<h4>Background</h4>Mammalian target of rapamycin (mTOR), involved in PI3K/AKT/mTOR pathway, is known to play a central role in regulating the growth of cancer cells. The PI3K/AKT/mTOR pathway enhances tumor survival and proliferation through suppressing autophagy, which sustains energy homeostasis by collecting and recycling cellular components under stress conditions. Conversely, inhibitors of the mTOR pathway such as RAD001 induce autophagy, leading to promotion of tumor survival and limited a ...[more]

PMID: 27143856

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Project description:IntroductionmiR-99a-5p, known to play an important role in mammalian target of rapamycin (mTOR) regulation, is downregulated in human bladder cancer. The study aimed to investigate the anticancer activity of miR-99a-5p and the possible mechanism associated with mTOR in bladder cancer cells.Materials and methodsVectors expressing miR-99a-5p were transfected into human urinary bladder urothelial carcinoma (5637 and T24) cells. The level of miR-99a-5p was monitored by microRNA (miRNA) quantitative polymerase chain reaction (QPCR). Luciferase reporter assays were performed to verify the direct binding of miR-99a-5p to mTOR transcripts. The mTOR transcripts and protein levels were measured by QPCR and Western blot, respectively. Cell viability of miR-99a-5p-transfected cells was detected by tetrazolium salt (WST-1). Inhibition of mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) signaling was detected by the phosphorylation of mTOR and AKT using Western blot. The ability of miR-99a-5p to enhance RAD001-induced apoptosis was determined as the expression of cleaved caspase 3 and levels of DNA fragmentation.ResultsTransfection of miR-99a-5p-expressing vector elevated the expression level of miR-99a-5p up to sixfold compared to vector-only controls. The results from luciferase assay verified that miR-99a-5p directly binds to the predicted sequence in the 3' untranslated region (3'-UTR) of mTOR. The levels of mTOR RNA and protein were decreased in miR-99a-5p-transfected cells. Dual inhibition of mTORC1 and mTORC2 by miR-99a-5p was confirmed by the decreased phosphorylation of mTOR (at Ser2448 and Ser2481), phospho-rpS6 and phospho-4EBP1. The phosphorylation of AKT was significantly inhibited in miR-99a-5p-transfected cells upon RAD001 treatment. Enforced expression of miR-99a-5p potentiated RAD001-induced apoptosis in these cells.ConclusionThis is the first study showing that miR-99a-5p markedly inhibits the growth of bladder cancer cells via dual inhibition of mTORC1 and mTORC2. Our data demonstrated that forced expression of miR-99a-5p inhibits the feedback of AKT survival pathway and enhances the induction of apoptosis in RAD001-treated bladder cancer cells.

Dataset Information

Autophagy inhibition enhances RAD001-induced cytotoxicity in human bladder cancer cells.

Publications

Autophagy inhibition enhances RAD001-induced cytotoxicity in human bladder cancer cells.

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