Metformin alleviates adriamycin resistance of osteosarcoma by declining YY1 to inhibit MDR1 transcriptional activity

Abstract

Chemotherapy resistance hinders the successful treatment of osteosarcoma (OS) to some extent. Previous studies have confirmed that metformin (Met) enhances apoptosis induced by chemotherapeutic drugs, but the underlying mechanism remains unclear. To establish adriamycin (ADM)-resistant MG-63 (MG-63/ADM) cells, the dosage of ADM was progressively increased. The results of qRT-PCR and Western blotting demonstrated that the expression level of Yin Yang 1 (YY1) and multi-drug resistance-1 (MDR1) in MG-63/ADM cells were remarkably increased compared with those in MG-63 cells. Met dramatically enhanced ADM cytotoxicity and accelerated apoptosis of MG-63/ADM cells. Moreover, Met suppressed the expressions of YY1 and MDR1 in MG-63/ADM cells. YY1 promoted its transcriptional expression by directly binding to the MDR1 promoter. Furthermore, the effects of Met on ADM sensitivity in MG-63/ADM cells was reversed due to overexpression of YY1 or MDR1. Collectively, these findings suggested that Met inhibited YY1/MDR1 pathway to reverse ADM resistance in OS, providing a new insight into the mechanism of Met in ADM resistance of OS.

Keywords: Adriamycin resistant; Metformin; Multi-drug resistance-1; Osteosarcoma; Yin Yang 1.

Fig. 1

Relative expressions of YY1 and MDR1 in ADM resistant OS cells. (A) MG-63 and MG-63/ADM cells were treated with different concentrations of ADM ranging from 2 µg/mL to 64 µg/mL, and cell viability was assessed by MTT assay. (B) IC50 value was calculated in MG-63 and MG-63/ADM cells. (C) The mRNA expression levels of YY1 (C) and MDR1 (D) in MG-63 and MG-63/ADM cells were detected via qRT-PCR. (E) YY1 and P-gp protein expressions in MG-63 and MG-63/ADM cells were detected by Western blotting. (F) Quantitative analysis of Western blot and GAPDH as internal reference. All the results were shown as mean ± SD (n = 3), which were three separate experiments performed in triplicate. **P < 0.01, ***P < 0.001

Fig. 2

Met increased sensitivity of MG-63/ADM cells to ADM and accelerated cell apoptosis. (A) MG-63/ADM cells were treated with different concentrations of Met ranging from 1mM to 10 mM and cell viability was assessed by MTT assay. (B) IC50 value was calculated MG-63/ADM cells with Met treatment. (C) ADM induced apoptosis of MG-63/ADM cells after Met induction was determined by flow cytometry. (D) The apoptosis rate was calculated. All the results were shown as mean ± SD (n = 3), which were three separate experiments performed in triplicate. *P < 0.05, ***P < 0.001

Fig. 3

Met suppressed the expression of YY1 and MDR1 in MG-63/ADM cells. The mRNA expression levels of YY1 (A) and MDR1 (B) after Met treatment were detected via qRT-PCR. (C) The protein expression levels of YY1, P-gp, Bcl-2 and Bax following Met exposure were detected by Western blotting. (D) Quantitative analysis of Western blot and GAPDH as internal reference. All the results were shown as mean ± SD (n = 3), which were three separate experiments performed in triplicate. *P < 0.05, ***P < 0.001

Fig. 4

YY1 promoted MDR1 expression by directly binding to its promoter (A) The expression of YY1 after transfection with YY1 overexpression vector was measured via qRT-PCR. (B) The protein expression of YY1 after transfection with YY1 overexpression vector was detected by Western blotting. (C) The mRNA levels of MDR1 were measured via qRT-PCR. (D) P-gp protein expressions were detected by western blotting. (E) Dual-luciferase reporter detection was carried out to measure the transcriptional activity of MDR1 with YY1 overexpression. (F) The targeting relationship between YY1 and MDR1 was determined by ChIP-qPCR assay. (G) Agarose gel electrophoresis of ChIP-qPCR results were assayed. All the results were shown as mean ± SD (n = 3), which were three separate experiments performed in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

Overexpression of YY1 or MDR1 reversed the effect of Met on ADM sensitivity of MG-63/ADM cells. (A) The cytotoxic effects of Met treatment on ADM-induced apoptosis of MG-63/ADM cells after overexpression of YY1 or MDR1 were determined by flow cytometry. (B) The apoptosis rate was calculated. (C) Western blotting was performed to detect the protein levels P-gp, Bcl-2 and Bax in MG-63/ADM cells with YY1 or MDR1 overexpression. (D) Quantitative analysis of Western blot and GAPDH as internal reference. All the results were shown as mean ± SD (n = 3), which were three separate experiments performed in triplicate. **P < 0.01, ***P < 0.001

Fig. 6

A schema of Met in OS ADM resistance. Met alleviated ADM resistance of OS by decreasing YY1 to directly inhibit transcription and expression of MDR1.