Heat shock protein 27 mediates bortezomib-resistance of multiple myeloma cells / 肿瘤

ABSTRACT

Objective: To investigate the expression of heat shock protein 27 (HSP27) in multiple myeloma (MM) cells, and to explore its relationship with bortezomib (BTZ) resistance. Methods: The expression levels of HSP27 mRNA and protein in myeloma cells (CD38+/CD138+ plasma cells) of 22 patients with MM (12 newly treated patients and 10 relapsed patients after BTZ treatment) were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The BTZresistant U266/BTZ cell line was established, and CCK-8 assay was used to detect the effects of different concentrations of BTZ, doxorubicin and etoposide on the proliferation of U266/BTZ cells and the parent U266 cells. The expression levels of HSP27 mRNA and protein in U266/BTZ cells and U266 cells were also examined by real-time fluorescent quantitative PCR and Western blotting, respectively. The specific small interfering RNA (siRNA) was used to silence HSP 27 gene expression in U266/BTZ cells, and the impact of silencing HSP 27 gene expression on BTZ-induced apoptosis of U266/BTZ cells was assessed by FCM. Additionally, the U266 cells were treated with BTZ alone or in combination with p38 mitogen-activated protein kinase (MAPK) signaling pathway inhibitor SB203580, then the expression levels of HSP27, phospho-HSP27 (p-HSP27), p38 MAPK, and p-p38 MAPK were measured by Western blotting. Finally, the recombinant lentivirus pCDH-CMV-MCS-EF1-copGFP-T2A-Puro/ HSP27 was constructed and infected into U266 cells to induce the overexpression of HSP 27 gene, then the activity of 20S proteasome in U266 cells with HSP 27 gene overexpression after treatment with different concentrations of BTZ was determined by fluorescence substrate method. Results: The expression levels of HSP27 mRNA and protein in myeloma cells from MM-relapsed patients were significantly higher than those in newly treated patients (both P < 0.01). BTZ-resistant myeloma cell line was successfully established, the half-maximal inhibitory concentration (IC50) of BTZ, doxorubicin and etoposide in BTZ-resistant U266/BTZ cells was significantly increased as compared with its parent U266 cells (all P < 0.01). The expressions of HSP27 mRNA and protein in U266/BTZ cells were significantly up-regulated (both P < 0.01). Silencing HSP 27 gene expression significantly increased BTZ-induced apoptotic rate of U266/BTZ cells (P < 0.01). Treatment with BTZ alone considerably up-regulated the expressions of HSP27, p-HSP27 and p-p38 MAPK in U266 cells, while this effect could be dramatically counteracted by pre-treatment with SB203580 (all P < 0.01). Furthermore, the overexpression of HSP 27 gene significantly reduced the inhibitory effects of BTZ at different concentrations on the activity of 20S proteasome in U266 cells (P < 0.01). Conclusion: The expression of HSP27 is closely related to BTZ-resistance of MM cells, which may be related to the BTZ exposure activating the p38 MAPK signaling pathway and up-regulating HSP27 expression, consequently decreasing the inhibitory effect of BTZ on proteasome activity through a feedback mechanism.