Study of the Effect of Antioxidant on Oxidative Stress in Molecular and Cellular Models

Abstract

This study was designed to study various classes of synthetic organic compounds for their antioxidant activities in molecular and cellular models of oxidative stress. Etiology of numerous types of cancers has been linked with oxidative stress, especially, metastatic melanoma is known as a reactive oxygen species (ROS) driven tumor. In this study, five metastatic melanoma cell lines (A375, WM266-4, SK-Mel-28, BLM, and MV3), and two non-metastatic melanoma cell lines (WM-115 and Mel-HO) were used to detect ROS association with neoplastic transformation relative to normal cell lines wound healing fibroblast (CPD95) and normal human melanocytes (NHEM). However, corelation of ROS production with metastatic propensity of malignant melanoma cell lines could be found. Out of over 1,000 compounds evaluated during this study, 250 synthetic organic compounds showed free radical scavenging activity in the DPPH radical scavenging assay. From them 34 synthetic organic compounds (17 free radical scavengers with low to high free radical scavenging activity, along with 17 non-radical scavenger homologues) were selected for further biomolecular studies. These compounds were evaluated for their cytotoxicity, using the MTT assay. Eight different synthetic organic compounds of four different classes, pyrimidine derivatives 1 and 4, biscoumarin derivatives 10 and 11, thiazole derivatives 6 and 8, and aryl Schiff’s bases 13 and 15, were identified as potential anti-melanoma agents due to their cytotoxicity against both metastatic (A375, and SK-Mel-28), and non-metastatic cell lines (WM-115) cell lines. The identified potential anti-melanoma agents were further evaluated against normal cells lines wound healing fibroblast (CPD95) and normal human melanocytes (NHEM), and all of them were found to be non-cytotoxic against (NHEM). However, one of the bis-coumarin derivatives 11, one thiazole derivative 8, and both of the pyrimidine, derivatives 1 and 4 were found non-cytotoxic to the fibroblast cells. These compounds were further tested for their anti-migration properties. Both derivatives of the class thiazole, aryl Schiff’s bases, and bis-coumarins were found to behave as antimigration agents, wherase none of them affected intracellular ROS production of A375 cells. DNA synthesis inhibition activity of these compounds was also evaluated using the BrdU incorporation assay; all compounds were found to inhibit DNA synthesis in the metastatic melanoma (A375 cell line) cancer cells. A non-radical scavenger, bis-coumarin derivative 11 with anti-migration and anti-proliferation activities, was tested on 43 different kinases phosphorylation sites, and 2 related proteins (heat shock and tumor supressor) at two different time ponits using proteome profiler human phospho-kinase array assay. Compound 11 was found to target 40 different phosphorylation sites of 25 different kinases. The mammalian target of rapamycin (mTOR) pathway kinases (mTOR and PRS40), as well as tumor suppressor proteins (Tp53 and Chk-2 kinase) that are involved in melanoma cell growth and tumor suppression, respectively, were found to be the significant targets of compound 11. Compound 11 was also found to inhibit melanoma cell survival, and growth at gene level via inhibiting phosphorylation of STATs family of transcription factors through the JAK- STAT pathway. Compound 11 was also found to inhibit the melanoma cell survival and proliferation by inhibiting phosphorylation of ERK1/2, MSK1/2, and CREB via EGF and PDGF receptor-dependent, and intracellular redox-independent signaling pathways. Members of Src family of kinases implicated in melanoma migration/metastasis were found to be additional targets of this compound. Compound 11 was effective in inhibiting metastatic melanoma proliferation, and cell migration. For the first time it was identified as a novel anti-melanoma agent of the bis-coumarin class that could suppress the melanoma cell growth along with its metastatic propensity without affecting normal cells. Based on these findings, the lead compound 3,3'((3,5Dichlorophenyl) methylene) bis (4-hydroxy-2-H-chromen-2-one) (11) was identified as a potential anti-melanoma and anti-metastatic drug candidate.