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

TL;DR

This study tests dacarbazine, temozolomide, and BRAF siRNA in melanoma cells to see if combining them improves treatment effectiveness.

Contribution

The study introduces a novel combination of BRAF(V600E) siRNA with dacarbazine and temozolomide for melanoma treatment.

Findings

Combining BRAF siRNA with chemotherapy drugs reduced cell viability significantly.

Apoptosis increased in all treated groups compared to the control.

The siRNA+DTIC+TMZ group showed the most effective enhancement in CASP3 expression.

Abstract

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.

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).

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.

These findings suggest that the siRNA-transfected treatment groups have the potential to provide therapeutic effects comparable to those of chemotherapy.

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