Original Article | Published:

Sirolimus induces apoptosis and reverses multidrug resistance in human osteosarcoma cells in vitro via increasing microRNA-34b expression

Acta Pharmacologica Sinica volume 37, pages 519529 (2016) | Download Citation

Subjects

Abstract

Aim:

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.

Conclusion:

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.

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Acknowledgements

This work was supported by grants from National Natural Science Foundation of China (No 81172548 and 81372873).

Author information

Author notes

    • Yan Zhou
    •  & Rui-hua Zhao

    These authors contributed equally to this work.

Affiliations

  1. Department of Oncology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China

    • Yan Zhou
    • , Kun-peng Li
    • , Kun Han
    • , Zhi-hua Gan
    • , Shu-chen Lin
    • , Hai-yan Hu
    •  & Da-liu Min
  2. Department of Oncology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China

    • Rui-hua Zhao
  3. Department of Hematology, Zhujiang Hospital Affiliated with Southern Medical University, Guangzhou 510282, China

    • Zhi-gang Lu
    •  & Yuan Liu
  4. Department of Biophysics, Oregon State University, ALS-2139 Corvallis, OR 97330, USA

    • Kuo-Fu Tseng

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Correspondence to Hai-yan Hu or Da-liu Min.

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DOI

https://doi.org/10.1038/aps.2015.153

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