title: Sirolimus induces apoptosis and reverses multidrug resistance in human osteosarcoma cells in vitro via increasing microRNA-34b expression.
abstract: OBJECTIVE
  Multi-drug resistance poses a critical bottleneck in chemotherapy. Given the up-regulation of mTOR pathway in many chemoresistant cancers, we examined whether sirolimus (rapamycin), a first generation mTOR inhibitor, might induce human osteosarcoma (OS) cell apoptosis and increase the sensitivity of OS cells to anticancer drugs in vitro.
  
  
  METHODS
  Human OS cell line MG63/ADM was treated with sirolimus alone or in combination with doxorubicin (ADM), gemcitabine (GEM) or methotrexate (MTX). Cell proliferation and apoptosis were detected using CCK-8 assay and flow cytometry, respectively. MiRNAs in the cells were analyzed with miRNA microarray. The targets of miR-34b were determined based on TargetScan analysis and luciferase reporter assays. The expression of relevant mRNA and proteins was measured using qRT-PCR and Western blotting. MiR-34, PAK1 and ABCB1 levels in 40 tissue samples of OS patients were analyzed using qRT-PCR and in situ hybridization assays.
  
  
  RESULTS
  Sirolimus (1-100 nmol/L) dose-dependently suppressed the cell proliferation (IC50=23.97 nmol/L) and induced apoptosis. Sirolimus (10 nmol/L) significantly sensitized the cells to anticancer drugs, leading to decreased IC50 values of ADM, GEM and MTX (from 25.48, 621.41 and 21.72 μmol/L to 4.93, 73.92 and 6.77 μmol/L, respectively). Treatment of with sirolimus increased miR-34b levels by a factor of 7.5 in the cells. Upregulation of miR-34b also induced apoptosis and increased the sensitivity of the cells to the anticancer drugs, whereas transfection with miR-34b-AMO, an inhibitor of miR-34b, reversed the anti-proliferation effect of sirolimus. Two key regulators of cell cycle, apoptosis and multiple drug resistance, PAK1 and ABCB1, were demonstrated to be the direct targets of miR-34b. In 40 tissue samples of OS patients, significantly higher miR-34 ISH score and lower PAK5 and ABCB1 scores were detected in the chemo-sensitive group.
  
  
  CONCLUSIONS
  Sirolimus increases the sensitivity of human OS cells to anticancer drugs in vitro by up-regulating miR-34b interacting with PAK1 and ABCB1. A low miR-34 level is an indicator of poor prognosis in OS patients.
authors.affiliation: Department of Oncology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Department of Oncology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China.
authors.forename: Rui-hua
authors.initials: RH
authors.lastname: Zhao
authors.affiliation: Department of Biophysics, Oregon State University, ALS-2139 Corvallis, OR 97330, USA.
authors.forename: Kuo-fu
authors.initials: KF
authors.lastname: Tseng
authors.affiliation: Department of Oncology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China.
authors.forename: Kun-peng
authors.initials: KP
authors.lastname: Li
authors.affiliation: Department of Hematology, Zhujiang Hospital Affiliated with Southern Medical University, Guangzhou 510282, China.
authors.forename: Zhi-gang
authors.initials: ZG
authors.lastname: Lu
authors.affiliation: Department of Hematology, Zhujiang Hospital Affiliated with Southern Medical University, Guangzhou 510282, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Oncology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China.
authors.forename: Kun
authors.initials: K
authors.lastname: Han
authors.affiliation: Department of Oncology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China.
authors.forename: Zhi-hua
authors.initials: ZH
authors.lastname: Gan
authors.affiliation: Department of Oncology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China.
authors.forename: Shu-chen
authors.initials: SC
authors.lastname: Lin
authors.affiliation: Department of Oncology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China.
authors.forename: Hai-yan
authors.initials: HY
authors.lastname: Hu
authors.affiliation: Department of Oncology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China.
authors.forename: Da-liu
authors.initials: DL
authors.lastname: Min
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001859:Bone Neoplasms / Q000188:drug therapy*; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000494:pharmacology; D004317:Doxorubicin / Q000494:pharmacology; D018432:Drug Resistance, Multiple / Q000187:drug effects; D019008:Drug Resistance, Neoplasm / Q000187:drug effects; D004357:Drug Synergism; D057809:HEK293 Cells; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D012516:Osteosarcoma / Q000188:drug therapy*; D020123:Sirolimus / Q000494:pharmacology*; D000093542:Gemcitabine
chemical_list: D000970:Antineoplastic Agents; C522425:MIRN34 microRNA, human; D035683:MicroRNAs; D003841:Deoxycytidine; D004317:Doxorubicin; D020123:Sirolimus; D008727:Methotrexate; D000093542:Gemcitabine