Reversing Effects Of MK-2206 On MCF-7/ADR Multidrug Resistance

BackgroundBreast cancer is a common malignant tumor that threatens the physical and mental health of women. Currently, the incidence of breast cancer is highest among malignant tumors in women. The mortality lies in the sixth place. Chemotherapy is one of the most important steps in the comprehensive treatment of breast cancer. However, chemotherapy can induce multidrug resistance in the breast cancer cells, which results in therapy failure. Studies have shown that activation of phosphatidylinositol-3 kinase (PI3K) and protein kinase B (PKB) (AKT) signaling pathways is involved in the mechanism of multidrug resistance in breast cancer. Therefore, PI3K-AKT has been considered an important therapeutic target for breast cancer. A number of small molecule inhibitors of the PI3K-AKT pathway have entered the clinical study stage, with excellent prospects in the targeted treatment of breast cancer. MK-2206 is a new synthetic specific molecular AKT inhibitor with high selectivity and non-ATP competition, which has displayed effective tumor suppression in a variety of tumors and is being assessed in a Phase Ⅱ clinical study.PurposeThis study aimed to investigate the cytotoxicity and reversing effects of MK-2206 on MCF-7/ADR breast cancer cells at the cellular level, to verify whether antagonizing the expression of p-PRAS40-Thr246 in breast cancer cells can partially reverse the resistance to Doxorubicin. The findings described herein provide a basis for the clinical therapy of breast cancer.Methods1. Cell culture:MCF-7 and MCF-7/ADR cells were cultured in RPMI 1640 medium containing 10%fetal bovine serum,100 U/mL penicillin and 0.1 mg/mL streptomycin, at 37 ℃ in a humid environment containing 5% CO2. To maintain MCF-7/ADR cell resistance, doxorubicin at a final concentration of 1 μg/ml was added into the culture medium. Two weeks prior to the experiments, MCF-7/ADR cells were cultured under drug-free conditions, and logarithmic growth phase cells were assessed.2. CCK-8 measurement:To test drug resistance fold of MCF-7/ADR^ cytotoxicity of MK-2206 and Doxorubicin and MK-2206 reversing effects on MCF-7/ADR resistance.3. Flow cytometry measurements:MCF-7/ADR apoptosis and intracellular doxorubicin after treated with Doxorubicin and MK-2206 at a non-toxic dose.4. Western Blot:Assessment of p-(Thr246) PRAS40、p-(Thr308) AKT and P-gp protein expression levels.Results1. Both Doxorubicin and MK-2206 inhibited MCF-7 and MCF-7/ADR cell proliferation in a dose-dependent manner. Combination of Doxorubicin and MK-2206 (non-toxic dose) reduced the IC50 values for MCF-7 and MCF-7/ADR to a different degree; however, but the IC50 values for MCF-7/ADR were more significantly reduced compared with those obtained for MCF-7 (P< 0.01).2. As shown by Annexin V-FITC double staining, the apoptosis rate increased to different degrees after 24h treatment with Doxorubicin and MK-2206 at 0,5 and 10 nmol/L in MCF-7/ADR (P< 0.01). There were no significant differences between the experimental and control groups on intracellular doxorubicin. (P> 0.05).3. Western Blot data showed that the protein expression levels of p-(Thr246) PRAS40> p-(Thr308) and P-gp in MCF-7 are significantly lower than MCF-7/ADR. After treated with MK-2206 at a non-toxic dose for 24h, the protein expression levels of p-(Thr246) PRAS40 and p-(Thr308) AKT decreased in a dose-dependent manner in MCF-7/ADR cells, with a statistically significant difference compared with the control group (P< 0.01). There were no significant differences between the experimental and control groups on the protein expression level of P-gp.(P> 0.05).ConclusionMK-2206 partially reverses MCF-7/ADR cell resistance by antagonizing the expression of p-(Thr246) PRAS40 and inhibiting the PI3K/AKT signalling pathway.