PI3K/Akt inhibition and down-regulation of BCRP re-sensitize MCF7 breast cancer cell line to mitoxantrone chemotherapy

Abstract

Objectives: Multidrug resistance (MDR) of cancer cells is a major obstacle to successful chemotherapy. Overexpression of breast cancer resistance protein (BCRP) is one of the major causes of MDR. In addition, it has been shown that PI3K/Akt signaling pathway involves in drug resistance. Therefore, we evaluated the effects of novel approaches including siRNA directed against BCRP and targeted therapy against PI3K/Akt signaling pathway using LY294002 (LY) to re-sensitize breast cancer MCF7 cell line to mitoxantrone (MTX) chemotherapy.

Materials and methods: Anticancer effects of MTX, siRNA, and LY alone and in combination were evaluated in MCF7 cells using MTT cytotoxicity assay and flow cytometry analysis of cell cycle distribution and apoptosis induction.

Results: MTT and apoptosis assays showed that both MTX and LY inhibited cell proliferation and induced apoptosis in MCF7 cells. Results indicated that inhibition of BCRP by siRNA or PI3K/Akt signaling pathway by LY significantly increased sensitivity of MCF7 cells to antiproliferation and apoptosis induction of MTX. Furthermore, MTX showed G2/M arrest, whereas LY induced G0/G1 arrest in cell cycle distribution of MCF7 cells. Combination of siRNA or LY with MTX chemotherapy significantly increased accumulation of MCF7 cells in the G2/M phase of cell cycle.

Conclusion: Combination of MTX chemotherapy with BCRP siRNA and PI3K/Akt inhibition can overcome MDR in breast cancer cells. This study furthermore suggests that novel therapeutic approaches are needed to enhance anticancer effects of available drugs in breast cancer.

Keywords: BCRP; Breast cancer; Combination therapy; Mitoxantrone; PI3K/Akt; siRNA.

Figure 1

Chemical structure of LY294002

Figure 2

Cytotoxic effects of the MTX and LY on MCF7 cells The MCF7 cells were treated with different concentrations of MTX (A) and LY (B). After 48 hr and 72 hr of exposure, the cell proliferation was determined using MTT assay. Each experiment was repeated at least three times in quadruplicates for each concentration and the results were expressed as mean±SD. *denotes P<0.001 for significant difference between treatments in comparison to control RPMI

Figure 3

Effect of siRNA against BCRP on the sensitivity of MCF cells to mitoxantrone The MCF7 cells were exposed to Chitosan + Alginate (Polymer), siRNA, siRNA-polymer (si+P) complex as well as combination of siRNA-polymer (si+P) complex with MTX 250 nM for 6 hr as described in the methods. Then MCF7 cells were incubated with RPMI or MTX for 48 hr and then cytotoxicity was determined by MTT assay. Each experiment was repeated at least three times in quadruplicates for each treatment and the results are expressed as mean±SD. *denotes P<0.001 for significant difference between treatments in comparison to control RPMI

Figure 4

Effects of LY on mitoxantrone cytotoxicity in MCF7 cells The MCF7 cells were co-treated with specified concentrations of LY + MTX. After 48 hr and 72 hr of exposure, the cell proliferation was determined using MTT assay. Each experiment was repeated at least three times in quadruplicates for each treatment and the results are expressed as mean±SD. *denotes P<0.001 for significant difference between treatments in comparison to control RPMI

Figure 5

Apoptosis induction in MCF7 cells The MCF7 cells were treated with mitoxantrone, LY, and siRNA-polymer (si+P) complex alone and in combination for 48 hr to evaluate induction of apoptosis using Annexin V-FITC/PI double-staining by flow cytometry. The percentage of early and late apoptotic cells were presented for each treatment group. Data are expressed as means ± SD of three independent experiments. *denotes P<0.001 and # P<0.01 for significant difference between treatments in comparison to control RPMI

Figure 6

Cell cycle alterations in MCF7 cells The MCF7 cells were treated with mitoxantrone, LY, and siRNA-polymer (si+P) complex alone and in combination for 48 hr and stained with DAPI and analyzed by flow cytometry to determine cell cycle distribution pattern. Data are presented as the mean±SD and each experiment was repeated at least three times in triplicate formats. *denotes P<0.001, # P<0.01 and & P<0.05 for significant difference between treatments in comparison to control RPMI