Investigating therapeutic efficacy of dacarbazine and temozolomide, alone and in combination with BRAF(V600E) siRNA in A375 human melanoma cell line

Abstract

Objective(s): Melanoma is one of the most aggressive and deadly skin cancers. Despite advances, effective melanoma treatment is challenging, often requiring a shift from individual therapies to combination approaches. This study explores whether combining dacarbazine (DTIC) and temozolomide (TMZ) with the siRNA approach holds promise for melanoma treatment. Materials and Methods: To determine the IC50 values of DTIC and TMZ, the A375 cell line was treated with different drug concentrations for 24-72 hr. The best exposure time of BRAF siRNA transfection was performed. Subsequently, cell viability (using the MTT assay), apoptosis (by flow cytometry), and gene expression levels of B-Raf proto-oncogene, serine/threonine kinase (BRAF), caspase 3 (CASP3), and phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3) genes (by quantitative real-time PCR) were assessed in the treated groups (i.e., control, negative controls, DTIC alone, TMZalone, DTIC+ TMZ, BRAF(V600E)siRNA alone, siRNA+ DTIC, siRNA+ TMZ, and siRNA+ DTIC+TMZ groups). Results: Cell viability significantly decreased in the chemotherapy-only and siRNA+drug groups, although no difference was observed between them. The apoptosis percentage in all treated groups indicated a significant difference compared to the control group. The expression of the BRAF gene notably decreased in the BRAF (V600E) siRNA +drug groups compared to the chemotherapy groups. Despite overexpression of CASP3 in the chemotherapy-treated groups, the most effective enhancement was noted in the siRNA+DTIC+TMZ group (P<0.0001). The mean expression of the PIK3R3 gene in siRNA+chemotherapy groups revealed a notable reduction. Conclusion: These findings suggest that the siRNA-transfected treatment groups have the potential to provide therapeutic effects comparable to those of chemotherapy.