The Effect Of NVP-BKM120 Combined With NVP-RAD001 On Triple-negative Breast Cancer Stem Cells

Objective1. To evaluate the effects of pan-class IA PI3 K inhibitor NVP-BKM120 on triple-negative breast cancer total cells and stem cells in vitro, including growth,proliferation and other biological characteristics.2. To explore and analyze the effects of NVP-BKM120 combined with m TOR inhibitor NVP-RAD001 on triple-negative breast cancer total cells and stem cells in vitro.3. To investigate the combining effects of NVP-BKM120 and NVP-RAD001 in CSCs derived xenografts to provide further evidence of clinical combination therapy.Methods1. In vitro, triple-negative breast cancer(TNBC) stem cells(SCs) were cultured and enriched in serum-free stem cell culture medium. ESA+CD44+CD24-/low cells were defined as cancer stem cells(CSCs) and sorted using flow cytometry. The inhibitory effects of NVP-BKM120 and NVP-RAD001 were evaluated by MTT assay and the combination index(CI) were calculated. The cytocidal effects were investigated through mammosphere formation assay and drug-resistance colony formation assay. Western blotting analysis was used to verify the expression of key protein in PI3K/AKT/m TOR cell signaling.2. In vivo, triple-negative breast cancer stem cells were enriched in serum-free stem cell culture medium. The BALB/c mice were injected with stem cells in the left inguinal mammary fat pads. All the nude mice were divided into four groups by random assignment and different treatments were administered subsequently.Volume of tumor xenografts and bodyweight of the mice were measured to evaluate the effect of the compounds.Results1. NVP-BKM120 or NVP-RAD001 alone could inhibit the growth, proliferation,drug-colony formation and mammosphere formation of TNBC cells in a dose-depend manner. Compared with total cells, stem cells(SCs) do exhibit resistance to either compound alone. Combined NVP-BKM120 with NVP-RAD001 exerted dramatic anti-tumor effect both in total cells and stem cells. Also, combination therapy almost block the signal transduction in PI3K/Akt/m TOR pathway completely. Validated by the CI calculation, the two compounds displayed synergistic effect.2. NVP-BKM120 plus NVP-RAD001 was more effective on reducing tumor volume in CSCs derived xenografts while not significantly decreasing body weight of the nude mice.Conclusions1. NVP-BKM120 combined with NVP-RAD001 could inhibit the growth,proliferation both in the total cells and stem cells, reduce drug-resistance colony formation and mammosphere formation, block the signal transduction in PI3K/Akt/m TOR pathway synergistically.2. Combined NVP-BKM120 with NVP-RAD001 could reduce tumor volume in CSCs derived xenografts, meanwhile not decrease body weight of the nude mice.