Econazole nitrate reversed the resistance of breast cancer cells to Adriamycin through inhibiting the PI3K/AKT signaling pathway

Abstract

Activation of the phosphoinositide 3 kinase (PI3K)/AKT pathway is frequently implicated in resistance to anticancer therapies. PI3K inhibitors can restore sensitivity to standard breast cancer therapies, including endocrine therapy, HER2-targeted agents, and chemotherapy. Our previous research showed that econazole, a novel PI3Ka inhibitor, inhibits the PI3K/AKT pathway and induces apoptosis in lung cancer cells. In this study, econazole showed significant cytotoxic activity against Adriamycin-resistant breast cancer cells in vitro and in vivo. Additionally, econazole significantly sensitized MDA-MB-231 and MCF-7 cells to Adriamycin via inhibiting the PI3K/AKT pathway. Overexpression of constitutively active AKT1 abolished the function of econazole. The combination of econazole and Adriamycin exerted synergistic inhibitory effects in breast cancer cells in vitro and in vivo. Taken together, the PI3K inhibitor econazole could effectively overcome Adriamycin resistance and showed synergistic effects with chemotherapy on breast cancer.

Keywords: AKT; Adriamycin; Econazole; PI3K; breast cancer; drug resistance.

Figure 1

The cytotoxic effects of econazole and Adriamycin in two ADR and sensitive breast cancer cell lines. A. Adriamycin (1, 3, 10, and 30 μM) efficiently decreased the viability of sensitive breast cancer cells (MDA-MB-231 and MCF-7) at 24, 48, and 72 hours, as measured by MTT assays. MDA-MB-231/ADR and MCF-7/ADR cells showed resistance to low concentrations (<10 μM) of Adriamycin. B. Econazole (1, 3, 10, and 30 μM) efficiently decreased the viability of ADR breast cancer cells (MDA-MB-231/ADR and MCF-7/ADR) at 24, 48, and 72 hours, as measured by MTT assays. Interestingly, MDA-MB-231 and MCF-7 cells were not sensitive to low concentrations (<10 μM) of econazole. C. The combination of econazole (0.3-10 μM) and Adriamycin (0.3-10 μM) synergistically decreased the viability of ADR breast cancer cells, as measured by MTT assays at 72 hours. Statistical analysis was performed by one-way ANOVA (Analysis Of Variance), and the difference was considered significant if P<0.05, which is marked with an asterisk (*) in the figures; if P<0.01, the results are marked with two asterisks (**).

Figure 2

Econazole and Adriamycin induced apoptosis in ADR and sensitive breast cancer cell lines. (A) Econazole (3 μM), Adriamycin (3 μM), and the combination of econazole (3 μM) with Adriamycin (3 μM) treatment (24 hours) induced apoptosis in MDA-MB-231/ADR and MDA-MB-231 breast cancer cells. Apoptosis was measured by Annexin V/PI staining and flow cytometry (P<0.05). (B) The quantitative results of (A). (C) Econazole (3 μM), Adriamycin (3 μM), and the combination of econazole (3 μM) with Adriamycin (3 μM) treatment (24 hours) induced apoptosis in MCF-7/ADR and MCF-7 breast cancer cells. Apoptosis was measured by Annexin V/PI staining and flow cytometry (P<0.05). (D) The quantitative results of (C).

Figure 3

Econazole inhibited the PI3K/AKT pathway in ADR and sensitive breast cancer cell lines. A. The expression levels of pAKT (S473 and T308), P-gp, and Bcl-2 were decreased by econazole in MDA-MB-231/ADR and MDA-MB-231 cells in a dose-dependent manner. The cells were treated with increasing concentrations of econazole (0, 1, 3, 10, and 30 μM) for 24 hours. Econazole had no effect on PI3K p110α or t-AKT. b-actin was used as a loading control. B. The expression levels of pAKT (S473 and T308), P-gp, and Bcl-2 were decreased by econazole in MCF-7/ADR and MCF-7 cells in a dose-dependent manner. The cells were treated with increasing concentrations of econazole (0, 1, 3, 10, and 30 μM) for 24 hours. Econazole had no effect on PI3K p110α or t-AKT. b-actin was used as a loading control. C. Econazole and BYL719 significantly inhibited PI3Kα kinase activity in a dose dependent manner (IC₅₀: 79.29±6.97 nM and 8.68±2.13).

Figure 4

The overexpression of AKT1ca gene decreased econazole-induced apoptosis in MDR breast cancer cell lines. (A) AKT1ca overexpression decreased econazole-induced decreases of P-gp and Bcl-2 protein levels in two ADR breast cancer cell lines (MDA-MB-231/ADR and MCF-7/ADR), as measured by Western blotting. pGSK3β (S9) was used as a positive control because it is phosphorylated by AKT. β-actin was used as a loading control. (B) AKT1ca overexpression significantly decreased the econazole-induced loss of cell viability in MDA-MB-231/ADR, as measured by MTT assays at 72 hours (P<0.05). (C) AKT1ca overexpression significantly decreased the econazole-induced loss of cell viability in MCF-7/ADR, as measured by MTT assays at 72 hours (P<0.05). (D) AKT1ca overexpression significantly decreased econazole (3 μM, 24 hours)-induced apoptosis in both MDA-MB-231/ADR and MCF-7/ADR cells. Apoptosis was measured by Annexin V/PI staining and flow cytometry. (E) Quantitative results of (D) (P<0.05).

Figure 5

Econazole significantly suppressed MDA-MB-231/ADR tumor growth in nude mice and sensitized cancer cells to Adriamycin in vivo. A. Econazole (40 mg/kg) significantly suppressed MDA-MB-231/ADR tumor growth in nude mice, as measured by tumor weights. The combination of econazole (40 mg/kg) and Adriamycin (40 mg/kg) further decreased tumor weights. Adriamycin (40 mg/kg) treatment for 21 days had no effect on tumor weights. B. Econazole alone or in combination with Adriamycin reduced MDA-MB-231/ADR tumor masses in nude mice. C. Econazole alone or in combination with Adriamycin had little effect on mouse body weights at different time points. D. Econazole alone or in combination with Adriamycin significantly suppressed MDA-MB-231/ADR tumor growth in nude mice, as measured by tumor volume over the course of 21 days (P<0.05).