Breviscapine reverses doxorubicin resistance in breast cancer and its related mechanisms

Weijiang Fu, Jie Song, Haiying Li, Weijiang Fu, Jie Song, Haiying Li

Abstract

Background: Based on the effect of breviscapine (BRE) on reversing drug resistance of human breast cancer cell line MCF-7/doxorubicin (Dox), the mechanism was preliminarily explored.

Methods: The methyl thiazolyl tetrazolium (MTT) method was introduced to detect inhibitory effect of Dox alone or in combination with BRE on MCF-7 (M) and MCF-7/Dox (MD) cells, and the inhibitory concentration (IC50 ) was obtained. Cell apoptosis and Dox concentration was assessed by flow cytometry. The drug resistance multiple and reversal fold were calculated. Western blot was performed to evaluate the expression of Bcl-2, Bax, EGFR, p-EGFR, P-gp, caspase-3, and cleaved-caspase-3 protein in cells. The efflux of Rho-123 was measured by flow cytometry and fluorescence microscopy.

Results: The IC50 of Dox on MD and M cells was 16.67 and 0.71 μg/mL, respectively, with a drug resistance ratio of 23.48 times. The IC50 of Dox combined with BRE on MD cells was 5.62 μg/mL, with a reversal ratio of 2.97 times. BRE greatly enhanced Dox-induced apoptosis of MD cells. Bax and cleaved-caspase-3 (proapoptotic protein) expression were obviously increased, while Bcl-2 (antiapoptotic protein) expression was significantly decreased after BRE treatment. BRE inhibited EGFR activation and P-gp expression. BRE increased the intracellular accumulation of Dox in MD cells by P-gp.

Conclusion: BRE could increase the MD sensitivity to Dox via increasing Bax and cleaved-caspase-3 expression and inhibiting Bcl-2 expression, thereby promoting cell apoptosis. BRE reversed Dox resistance of MD cells by increasing Dox intracellular accumulation through inhibiting P-gp expression via EGFR.

Keywords: P-gp; apoptosis; breast cancer; breviscapine; doxorubicin.

Conflict of interest statement

The authors declare that they have no competing interests.

© 2023 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.

Figures

FIGURE 1
FIGURE 1
Breviscapine (BRE) inhibited the proliferation of MCF‐7 (M) and MCF‐7/Dox (MD) cells and reversed the drug resistance of MD cells. (a) Effect of treatment with different concentrations of BRE on the viability of M and MD cells. (b) Effect of BRE combined with Dox on the viability of M and MD cells.
FIGURE 2
FIGURE 2
Breviscapine (BRE) enhanced cell apoptosis induced by Dox in MCF‐7/Dox (MD) cells. (a, b) The effect of Dox and BRE on apoptosis of MCF‐7 (M) and MD cells was detected by flow cytometry. (c) Western blot was used to detect the effect of BRE on apoptosis‐related proteins in M and MD cells.
FIGURE 3
FIGURE 3
Effect of breviscapine (BRE) treatment on the amount of intracellular Dox in MCF‐7 (M) and MCF‐7/Dox (MD) cells. (a, b) The intracellular Dox accumulation of M and MD cells was detected by flow cytometry. (c, d) The intracellular Rho‐123 accumulation of M and MD cells was detected by fluorescence microscopy and flow cytometry. *p < 0.05, compared with control.
FIGURE 4
FIGURE 4
Epidermal growth factor receptor (EGFR), phospho‐epidermal growth factor receptor (p‐EGFR) and P‐glycoprotein (P‐gp) protein expression levels in MCF‐7 (M) and MCF‐7/Dox (MD) cells after cotreatment with breviscapine (BRE) and doxycycline (Dox).
FIGURE 5
FIGURE 5
Breviscapine (BRE) reversed multiple drug resistance (MDR) by increasing intracellular drug accumulation through P‐gp. (a) The transfection efficiency of PCDNA3.1‐P‐gp in MD cells was detected by Western blot. (b) Effect of P‐gp overexpression on the viability of MD cells treated by doxycycline (Dox) and BRE. (c, d) P‐gp overexpression could reverse the effects of BRE treatment on Dox accumulation in MD cells. **p < 0.01, ***p < 0.001, compared with Dox or Dox + BRE group.

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