Inhibition of receptor signaling and bioenergetic modulation in antimelanoma treatment

Introduction

Accumulation of alterations in tumor cells leads to appearance of metastatic cancer cells. They become invasive after releasing from the primary tumor, because they acquire ability to penetrate the bloodstream or the lymph system, to grow and thrive in their new location and to induce angiogenesis. We have previously described new derived hypermetastatic human melanoma cell line EGFP A375/Rel251, which is highly metastatic and tumorigenic.

Materials/methods

Hypermetastatic malignant melanoma cells EGFP A375/ Rel251 were cultivated in standard DMEM with 5% fetal bovine serum along with parental EGFP-A375 cells. Cells were treated with kinase or glycolytic inhibitors and evaluated by luminometric assay (LUMIstar OPTIMA) and live-cell kinetic imaging (IncuCyte ZOOMTM Kinetic Imaging System). The expression of growth factors and their cognate receptors was analyzed by quantitative real-time PCR (BioRad CFX96 PCR Detection System). Biochemical analysis for the function of intracellular pathways was performed by standard western blotting.

Results and conclusions

Melanoma cells were substantially influenced by different signaling pathway inhibitors such as MEK inhibitor PD98059 or c-MET/HGF inhibitor SU11274, and we observed changes in cell proliferation and morphology. We have also observed changes induced by glycolytic inhibitors 3-BrPA (3-bromopyruvate) or DCA (dichloroacetate). According to our findings it appears that interfering with the c-Met/HGF signaling pathway plays a role in our aggressive metastatic cell line. We will track the changes at the level of gene expression, cell proliferation, morphology and responses to inhibition. We will try to show the mechanism which contributes to the invasive and aggressive behavior of metastatic malignant melanoma cell line EGFP/A375/ Rel251. Better understanding of the receptor signaling in metastatic melanoma cells can unravel more suitable therapeutic combinations for advanced disease.

This work was supported by Cancer Research Foundation RFL 2009, RFL 2012 and the Slovak Research and Development Agency under the contract No. APVV-0230-11, VEGA 2/0087/15.