Metformin selectively targets 4T1 tumorspheres and enhances the antitumor effects of doxorubicin by downregulating the AKT and STAT3 signaling pathways

Abstract

Recent studies have reported that metformin (Met), the first-line medication for the treatment of type 2 diabetes, exhibited anticancer and chemoprotective effects in diverse cancer cells. In this study, we investigated the effects of Met on the drug-resistance of 4T1 murine breast cancer tumorspheres (TS) and the mechanism responsible for its drug-resistance. 4T1 TS exhibited accumulations of cells at the G₀/G₁ phase compared with cells in monolayer culture, which suggested the majority of cells in TS were quiescent. Furthermore, it was identified that activations of the signal transducer and activator of transcription 3 (STAT3) and protein kinase B (AKT) signaling pathways in 4T1 TS conferred drug-resistance to doxorubicin (Dox) and lapatinib (Lapa). However, Met selectively targeted TS rather than cells in monolayer culture and increased the cytotoxic effect of Dox on TS by inhibiting activations of the STAT3 and AKT signaling pathways. These observations suggested that inhibitions of STAT3 and AKT underlie the selective cytotoxic effects of Met on TS. In addition, Met exhibited synergistic antitumor effects with Dox on 4T1 tumor-bearing BALB/c mice. Our findings suggest that combinations of Met and cytotoxic anticancer drugs may offer an advantage for treating drug-resistant breast cancer.

Keywords: 4T1 breast cancer cells; drug-resistance; metformin; quiescence; tumorspheres.

Figure 1.

Majority of cells in 4T1 TS are quiescent. (A) Images of TS generated from 4T1 cells. TS were cultured in non-adherent culture plates for 4 days. Scale bar, 100 µm. (B) TS exhibited slower cell proliferation rates than cells grown in monolayers (2D). Cell absorbances measured using WST-8 reagent after incubation for 4 days. (C) Cell cycle analysis of cells grown as 2D cultures or as TS showed accumulations of cells in the G0/G1 phase in TS. (D) mRNA expression of cyclin D1 was diminished in cells grown in TS. TS, tumorspheres.

Figure 2.

Resistances of 4T1 TS to Dox and Lapa are due to upregulation of the STAT3 and AKT signaling pathways. Chemotherapeutic responses to (A) Dox or (B) Lapa in 2D or TS cultured cells. After treatment with (A) Dox or (B) Lapa for 3 days, cell viabilities were assessed using WST-8 reagent. Results are the means of three independent experiments performed in triplicate. Error bars indicate the standard deviations of the mean. (C) Comparison of p-STAT3 and p-AKT expression levels in 2D or TS cultured cells as determined by western blot analysis. **P<0.01 vs. 2D. (D) Dox and Lapa had no inhibitory effects of p-STAT3 and p-AKT expression levels in TS cultured cells. Lapa, lapatinib; Dox, doxorubicin; STAT3, signal transducer and activator of transcription 3; AKT, protein kinase B; TS, tumorspheres; p, phosphorylated.

Figure 3.

Metformin selectively targets TS rather than 2D cultured cells. (A) Chemotherapeutic responses of 2D and TS cultured cells to metformin. After treatment with metformin for 3 days, cell viabilities were assessed using WST-8 reagent. Results represent three independent experiments performed in triplicate. Error bars indicate the standard deviations of the means. (B) Whole cell lysates of TS treated with metformin were analyzed by western blotting for p-STAT3 and p-AKT. *P<0.05, **P<0.01 vs. control (0 mM metformin). TS, tumorspheres; p, phosphorylated; STAT3, signal transducer and activator of transcription 3; AKT, protein kinase.

Figure 4.

Met increases the cytotoxicity of Dox to TS by inhibiting the STAT3 and AKT signaling pathways. Synergistic cytotoxic effects of Met on (A) Lapa-treated TS cultured cells and (B) Dox-treated TS cultured cells. 4T1 cells were treated with various concentrations of Lapa or Dox in the presence of 1 mM Met for 3 days and viabilities were assessed using WST-8 reagent. Results represent three independent experiments performed in triplicate. Error bars represent the standard deviations of the means. (C) Inhibitory effect of Met on the expressions of p-STAT3 and p-AKT in Dox-treated TS. TS were treated with 0.5 µM Dox and/or 1 mM Met for 3 days and then western blotted. **P<0.01 vs. Con; ^##P<0.01 vs. Dox group. STAT3, signal transducer and activator of transcription 3; AKT, protein kinase; TS, tumorspheres; Lapa, lapatinib; Dox, doxorubicin; Met, metformin; DM, doxorubicin+metformin; p, phosphorylated; Con, control.

Figure 5.

Met enhances the ability of Dox to suppress tumor growth. (A) Experimental scheme for testing the antitumor effects of Dox and Met in vivo. (B) No loss of body weight occurred in mice treated with Dox or Met alone or in combination. (C) Synergistic antitumor effects of Met and Dox in 4T1 tumor-bearing BALB/c mice. *P<0.05 vs. control at day 10. Dox, doxorubicin; Met, metformin; i.p. intraperitoneal injection.