Triple-negative breast cancer (TNBC) is an aggressive subtype that lacks progesterone (PR), estrogen (ER) and human epidermal growth factor receptor 2 (HER2). Gemcitabine (GEM) is a drug commonly used for chemotherapy, but GEM resistance often develops, leading to more severe outcomes and relapse. This highlights the need for new targeted therapies, especially in GEM-resistant TNBC cases. The PI3K/AKT/mTOR pathway and anti-apoptotic Bcl-2 family proteins play crucial roles in cancer cell survival. Targeting both pathways might be an effective therapeutic approach. In this study, we explored the combination of NVP-BEZ235, a dual PI3K/mTOR inhibitor, with ABT-737, a BH3-mimetic Bcl-2 inhibitor, as a strategy to sensitize triple-negative breast cancer (TNBC) cells to gemcitabine (GEM) and overcome resistance.

MDA-MB-231 cells were treated with increasing doses of GEM for over 8 months to generate GEM-resistant cells MDA-MB-231GEMR. The resistance factor was calculated, and qPCR assessed the expression of resistant related gene. Parental MDA-MB-231 and MDA-MB-231GEMR cell lines were used to find the cytotoxicity and the IC50 values by Resazurin assay. Cell migration and growth ability were analyzed with Scratch and Colony Formation Assays, respectively. Cell cycle analysis was conducted by flow cytometry, and gene expression of mTOR pathway and apoptotic related genes were analyzed by qPCR.

MDA-MB-231GEMR cells were 5.06 times more resistant to GEM than parental MDA-MB-231 cells according to fold resistant calculation. qPCR showed significantly reduced expression of hENT1 (0.5 ± 0.1) in MDA-MB-231GEMR cells. The IC50 values for ABT-737 and NVP-BEZ235 were 9.923µM and 0.345µM, respectively. Treatment with the combination of NVP-BEZ235 and ABT-737 significantly inhibited cell growth and migration in MDA-MB-231GEMR cells, while cell cycle analysis revealed a notable SubG0 arrest, with a significant increase compared to the negative controls in resistant cells. Gene expression analysis revealed increased levels of Caspase-3 and decreased level of Bcl-2, and Mcl-1 expression compared to control, indicating the activation of apoptotic mechanisms. Consistent with expectations, the expression of PI3K and mTOR was reduced following treatment. Additionally, the increased expression of hENT1 suggests a potential reversal of resistance.

Our results suggest that the combination of ABT-737 and NVP-BEZ235 is a promising therapeutic strategy for overcoming Gemcitabine resistance in TNBC. This combinational approach enhances apoptosis and inhibits cell proliferation and migration by targeting both the PI3K/mTOR pathway and Bcl-2 family proteins. Further studies are needed to assess the potential of this strategy for GEM-resistant TNBC.