Abstract:
One of the most important factors limiting cancer treatment is the lack of specificity of chemotherapeutic agents to cancerous tissue. Therefore, new studies are conducted every day to identify new molecules specific to cancer cells. In recent studies, the high expression of the CAIX enzyme in various cancer cells and its limited expression in normal tissues have made it a selective target for cancer treatment. CAIX enables tumor cells to adapt to hypoxic conditions and promote metastasis by regulating intracellular and extracellular pH. Recent studies have shown that pharmacological interventions targeting the catalytic activity of CAIX using CAIX-specific small molecule inhibitors disrupt pH regulation in cancer cells, resulting in reduced primary tumor growth and metastasis. Therefore, CAIX inhibitors are proposed as a potential new class of anti-tumor agents. This study aimed to investigate the anticancer activity of 2-bromo-n-(4-sulfamoyl phenyl) propanamide (MMH-1), which has CAIX inhibitory properties and has previously shown anticancer efficacy in breast cancer, in combination with commonly used chemotherapeutic drugs (epirubicin, doxorubicin, and paclitaxel) in an in vitro setting using 4T1 cells. The cytotoxic effects of MMH-1, paclitaxel, epirubicin, and doxorubicin were examined by applying them alone and in combination at different doses to 4T1 cells using the MTT method. The apoptotic effect of the combination with the lowest IC50 dose, determined to be epirubicin and MMH1, was evaluated using the annexin-V and acridine orange methods. Additionally, the effects on cell cycle, mitochondrial membrane potential, and intracellular reactive oxygen species (ROS) were analyzed using flow cytometry. The study results revealed the IC50 values as follows: MMH-1 (5.859 μM/mL), Epirubicin (1.852 μM/mL), Doxorubicin (2.388 μM/mL), and Paclitaxel (3.298 μM/mL), MMH 1+Epirubicin (1.395 μM/mL), MMH-1+Doxorubicin (1.398 μM/mL), and MMH 1+Paclitaxel (1.763 μM/mL). The combination treatment of MMH-1+Epirubicin exhibited the strongest cytotoxic effect, disrupting the membrane potential and inducing an increase in intracellular ROS, leading to apoptosis in 4T1 cells. Furthermore, the combination treatment arrested 4T1 cells in the G0/G1 phase, halting the cell cycle. Based on the obtained data, it was determined that the MMH 1+Epirubicin combination treatment could be effective in breast cancer therapy, and it is believed to have the potential to be a new chemotherapeutic drug for cancer treatment. Further studies in an in vivo setting are required to validate these findings.
