Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer characterized by the lack of expression of HER2, estrogen and progesterone receptor (1). Due to limited targeted therapies and frequent development of chemoresistance, effective treatments remain a significant clinical challenge (2). Gemcitabine, a cytotoxic antimetabolite commonly used in chemotherapy, often develops resistance over time, decreasing the therapeutic efficacy (3). PI3K/AKT/mTOR pathway plays an important role in cell survival, growth, proliferation and motility. NVP-BEZ235, a dual PI3K and mTOR kinase inhibitor, decreases the proliferation of chemotherapy-resistant cancer cells (4). Phase II clinical trials of NVP-BEZ235 show anti-tumor efficacy in solid tumors however, side effects were also observed. Thus, employing targeted nanoparticle-based drug delivery systems may help overcome this obstacle. In this study, we aimed to overcome gemcitabine resistance in TNBC cell line (MDA-MB-231GEM) by delivering NVP-BEZ235 drug conjugated to targeted gold nanoparticle (t/BEZ@NP) developed in our laboratory.

MDA-MB-231GEM was developed through incremental exposure to gemcitabine over a year. The resistance factor and the doubling time were analyzed to confirm the newly developed drug resistant cell line. Cytotoxic effect of t/BEZ@NP were evaluated via resazurin assay, while colony formation and wound healing assays were used to asses the growth and migration inhibition, respectively. Cell cycle and apoptotic assay were used to analyze the cell cycle arrest and apoptotic cell death induction.

MDA-MB-231GEM cell line was successfully established with a fold resistance value of 116,04. t/BEZ@NP significantly reduced cell viability in MDA-MB-231GEM cells, with an IC50 of 8.3 nM, compared to free NVP-BEZ235 (IC50 = 10403 nM) at 48h. Colony formation assays demonstrated a reduction in both colony number and size following t/BEZ@NP treatment. Wound healing assays showed impaired migration, with the lowest closure rate observed in the t/BEZ@NP group. Flow cytometry analysis indicated an increase in the SubG0 phase and a reduction in S phase progression, highlighting the antiproliferative and pro-apoptotic effects of t/BEZ@NP.

The combination of dual PI3K/mTOR inhibition with nanoparticle-based targeted therapy effectively overcame gemcitabine resistance in TNBC. t/BEZ@NP significantly enhanced cytotoxicity, suppressed proliferation, and impaired migration. This approach may offer a new avenue for improving treatment outcomes in patients with aggressive, drug-resistant breast cancer subtypes. This project is funded by TUBITAK (122S737).