Targeting P-glycoprotein function, p53 and energy metabolism: Combination of metformin and 2-deoxyglucose reverses the multidrug resistance of MCF-7/Dox cells to doxorubicin

Abstract

Multidrug resistance(MDR) is a major obstacle to efficiency of breast cancer chemotherapy. We investigated whether combination of metformin and 2-deoxyglucose reverses MDR of MCF-7/Dox cells and tried to elucidate the possible mechanisms. The combination of metformin and 2-deoxyglucose selectively enhanced cytotoxicity of doxorubicin against MCF-7/Dox cells. Combination of the two drugs resumed p53 function via inhibiting overexpression of murine doubleminute 2(MDM2) and murine doubleminute 4(MDM4) leading to G2/M arrest and apoptosis in MCF-7/Dox cells. Combination of the two drugs had no effect on P-glycoprotein mRNA expression and P-glycoprotein ATPase activity but increased doxorubicin accumulation in MCF-7/Dox cells. The increased doxorubicin accumulation maybe associate with metabolic stress. Combination of metformin and 2-deoxyglucose initiated a strong metabolic stress in MCF-7/Dox cells via inhibiting glucose uptake, lactate, fatty acid, ATP production and protein kinase B(AKT)/ mammalian target of rapamycin(mTOR) pathway. Taken together, combination of metformin and 2-deoxyglucose reverses MDR of MCF-7/Dox cells by recovering p53 function and increasing doxorubicin accumulation. Furthermore, doxorubicin selectively increases MCF-7/Dox apoptosis via aggravating metabolic stress induced by metformin plus 2-deoxyglucose. The mutually reinforcing effect made the combination of metformin and 2DG had a better effect on reversing MDR.

Keywords: P-glycoprotein; energy metabolism; multidrug resistance; p53.

Figure 1. Metformin and 2-deoxyglucose(2DG) combination enhanced the cytotoxicity of doxorubicin in MCF-7/Dox cells

A, B. MCF-7 and MCF-7/Dox cells were treated with metformin(0.3, 0.5, 1 mM) and 2DG(0.3, 0.5, 1 mM) for 24h, and the cell viability was determined by MTT assay. C, D. Cells were pretreated with indicated drugs followed by incubation with various concentrations of doxorubicin for an additional 24h. Concentrations of doxorubicin were 0, 0.10, 0.30, 0.50, 1.00, 3.00, 5.00 μM for MCF-7 cells and 0, 1, 5, 10, 20, 30, 50 μM for MCF-7/Dox cells. Columns, data are expressed as mean ± SD. *P < 0.05 Significantly different from control group. M0.5: 0.5 mM metformin, D0.5: 0.5 mM 2DG, MD0.5: 0.5 mM metformin plus 0.5 mM 2DG.

Figure 2. Metformin and 2-deoxyglucose(2DG) combination increased p53 expression to enhance the cytotoxicity of doxorubicin

A. Cell cycle was determined by flow cytometric after treating with indicated drugs for 24h. B. Metformin 2DG combination and AB143228 increased p53 expression via inhibiting the overexpression of MDM2 and MDM4, as well as its effectors Cyclin B1, Cyclin D1, BAX, Bcl-2, caspase9. C. Metformin 2DG combination and AB143228 increased caspase3 activity. D. Cells were pretreated with AB143228(1μM) followed by incubation with various concentrations of doxorubicin(1, 5, 10, 20, 30, 50 μM) for an additional 24h. Columns, data are expressed as mean ± SD. *P < 0.05 Significantly different from control group. M0.5: 0.5 mM metformin, D0.5: 0.5 mM 2DG, MD0.5: 0.5 mM metformin plus 0.5 mM 2DG.

Figure 3. Metformin and 2-deoxyglucose(2DG) combination increased doxorubicin accumulation in MCF-7/Dox cells

A. mRNA expression of MDR1 was tested by quantitative real-time PCR analysis(the control group is set at 1). Verapamil(10μM) was positive group in B, C and D. (B) P-gp ATP enzyme activity was tested by Pgp-Glo™ Assay Systems. Metformin(0.5mM) plus 2DG(0.5mM) had no effect on P-gp mRNA expression and P-gp ATP enzyme activity. (C) Doxorubicin accumulation was determined by flow cytometric. (D) DOX fluorescence intensity analysis of C. Columns, data are expressed as mean ± SD. *P < 0.05 Significantly different from control group. M0.5: 0.5 mM metformin, D0.5: 0.5 mM 2DG, MD0.5: 0.5 mM metformin plus 0.5 mM 2DG, M1D0.5: 1mM metformin plus 0.5 mM 2DG.

Figure 4. The inhibitory effect of metformin plus 2-deoxyglucose(2DG) on energy metabolism

Cells were treated with indicated drugs for 24h. A, B, C, D. Combination of metformin and 2DG inhibited glucose uptake, lactate, fatty acid and ATP production in both cell lines. E. Metformin plus 2DG increased p-AKT expression and down-regulated mTOR and p- mTOR, as well as FASN and ACC1 expression. Columns, data are expressed as mean ± SD. *P < 0.05 Significantly different from control group. M0.5: 0.5 mM metformin, D0.5: 0.5 mM 2DG, MD0.5: 0.5 mM metformin plus 0.5 mM 2DG.

Figure 5. Interaction effects between P-gp substrate and combination of metformin and 2-deoxyglucose(2DG) on energy metabolism

A, B, C. Glucose uptake, lactate and ATP production was determined after the addition of indicated drugs for 24h. D. Caspase3 activity was determined by flow cytometry. Columns, data are expressed as mean ± SD. *P < 0.05 Significantly different from control group. VER: 10 μM verapamil, DIG: 10 μM digoxin, DOX: 10 μM doxorubici, M0.5: 0.5 mM metformin, D0.5: 0.5 mM 2DG, MD0.5: 0.5 mM metformin plus 0.5 mM 2DG.