Antitumor Effect And Mechanisms Research Of The PI3K Inhibitor BKM120 On Human Breast Cancer Multidrug Resistance Cells

Objective:1. The multidrug resistance(MDR) phenotype always accompanies activation of the phosphatidylinositol 3-kinase(PI3K)/AKT pathway, which renders a survival signal to cytotoxic anti-cancer drugs and enhances cancer stem cell(CSC) characteristics.This study was to evaluate the effect of apoptosis promoting and CSC eliminating when NVP-BKM120(BKM120), a selective inhibitor of PI3 K, was treated individually in MDR breast cancer cell lines.2. The activity of BKM120 synergistically with doxorubicin on MDR breast cancer cells was evaluated both in vitro and in vivo. Methods:1. After treatment of BKM120, cytotoxic activity was measured by MTT assay and drug resistant clones formation assay, Annexin V/PI staining, tests of caspase-3/7 and caspase 9 activities and apoptosis related genes expression were used to evaluate cell apoptosis, p-Akt, total Akt, NF-κB p65 in nuclei, total NF-κB p65 subunit were observed by western blot analysis and immunofluorescence staining. We also investigated the effect on CSC population and characteristics through mammosphere formation assay, soft agar colony formation assay, CD44+/CD24- and ALDH+detecting by flow cytometry. In vivo studies, the BALB/c mice were injected with MDR cells and the different treatments were administered.After necropsy, the expression of p-Akt, total Akt, NF-κB p65 in nuclei, total NF-κB p65 subunit was analyzed by western-blot.2. The combination effect of BKM120 and doxorubicin on MDR breast cancer cells was evaluated by MTT assay in vitro. For drugcombination experiments, a combination index(CI) number wascalculated using the CalcuSyn software based on theChou and Talalay method. In vivo assay, tumor cells wereinjectedinto the mammary fat pad of nude mice, and mice were equally distributed according to tumor burden into four groups to receive BKM120, doxorubicin, BKM120 combined with doxorubicin or vehicle control.Drugs were injected every there days and tumor volume were monitored till the mice were sacrificed. Result:1. The IC50 values for BKM120 in drug sensitive and resistant cells were not significantly different. The differences of IC50 values only ranged from 1.2 to 6.8 folds, and BKM120 reduced cell viability in a dose dependent manner in both of chemosensitive and chemoresistant breast cancer cells. Moreover, BKM120 significantly decreased the capacity of MDR cells to survive after one week treatment. BKM120 exhibited potent effects against MDR breast cancer cells, comparing with most conventional chemotherapeutic agents.2. The apoptosis assay was performed to evaluate whether BKM120 treatment promotes cell apoptosis by using Annexin V/Propidium stain assay, caspase-3/7 activity test and caspase-9 activity test. After BKM120 treatment, the percentage of viable MCF-7/A02 cells decreased remarkably. BKM120 activity induced the blockage of PI3 K signaling, decreased Akt phosphorylation and NF-κB expression which leading to concomitant caspase 3/7 and caspase 9 activation, as well as changes of several apoptosis related genes expression.3. Comparing with MCF-7 and Cal51, both MCF-7/A02 and CalDOX cell lines were composed of much higher population of cells exhibiting ALDH activity, respectively, and the ALDH activity in MDR cell lines were inhibited remarkably after exposure to BKM120 with doses lower than IC50.Moreover, BKM120 eliminated MCF-7/A02 and CalDOX cells’ abilities to form tumor-spheres, as well as the abilities to produce colonies.4. In vivo study, single-agent BKM120 significantly reduced tumor progression in both xenogragft models relative to vehicle control. BKM120 effectively blocks the aberrant activity of the PI3K/Akt/NF-κB signaling pathway in vivo, which consequently inducing tumor regression in chemoresistant breast cancer xenograft models.5. When doxorubicin was combined with BKM120, strong synergistic anti-proliferative effect on chemo-resistant breast cancer cells was noted both in vitro and in vivo. Conclusion:1. Our findings suggested that BKM120 effectively inhibits the PI3K/Akt signaling pathway, which resulted in apoptosis induction and CSC abolishing through PI3K/Akt/ NF-κB axis.2. Furthermore, simultaneous combination of doxorubicin and BKM120 induce synergistic cytotoxicity and partially overcome MDR phenotype in chemo-resistant breast cancer. This offers a strong rationale to explore therapeutic strategy of BKM120 in chemotherapy non-responsive breast cancer patients alone or in combination therapies.