siRNA - Mediated LRP/LR knock-down reduces cellular viability of malignant melanoma cells through the activation of apoptotic caspases

Highlights: • Late stage malignant A375SM melanoma cells exhibit higher cell surface and total LRP/LR levels in comparison to the early stage malignant A375 melanoma cells. • siRNA technology lead to successful down-regulation of LRP expression in early and late stage malignant melanoma cells. • siRNA- mediated knock-down of LRP expression results in significantly reduced cellular viability of A375 and A375SM cells. • Knock-down of LRP levels results in nuclear morphological changes indicating apoptotic induction in A375 and A375SM cells. • Increased levels of apoptotic induction post siRNA-mediated LRP knock-down in A375 and A375SM melanoma cells. • Significant increase in caspase-3 activity in both early and late stage malignant melanoma cells post siRNA transfection. • Apoptosis occurrs via the extrinsic pathway in A375 cells and via the intrinsic pathway inA375SM cells. • High correlation between total LRP levels post siRNA transfection and induction of apoptosis in both melanoma cell lines. • Apoptosis occurred via decreased laminin-1/LRP/LR interaction and activation of survival pathways as well as activated caspase-3. • siRNAs targeting LRP expression may act ter as a alternative therapeutic tool for treatment of A375 and A375SM cells. The 37 kDa/67 kDa laminin receptor (LRP/LR) is over-expressed in tumor cells and has been implicated in several tumourigenic processes such as metastasis and telomerase activation, however, more importantly the focus of the present study is on the maintenance of cellular viability and the evasion of apoptosis. The aim of the study was to investigate the role of LRP/LR on the cellular viability of early (A375) and late stage (A375SM) malignant melanoma cells. Flow cytometry and western blot analysis revealed that A375SM cells contain more cell-surface and total LRP/LR levels in comparison to the A375 cells, respectively. In order to determine the effect of LRP/LR on cell viability and apoptosis, LRP was down-regulated via siRNA technology. MTT assays revealed that LRP knock-down led to significant reductions in the viability of A375 and A375SM cells. Confocal microscopy indicated nuclear morphological changes suggestive of apoptotic induction in both cell lines and Annexin-V FITC/PI assays confirmed this observation. Additionally, caspase-3 activity assays revealed that apoptosis was induced in both cell lines after siRNA-mediated down-regulation of LRP. Caspase-8 and −9 activity assays suggested that post LRP knock-down; A375 cells undergo apoptosis solely via the extrinsic pathway, while A375SM cells undergo apoptosis via the intrinsic pathway. Implications: siRNAs mediated LRP knock-down might represent a powerful alternative therapeutic strategy for the treatment of malignant melanoma through the induction of apoptosis.