Molecular mechanisms of BK3C231-induced cytoprotection in normal human colon fibroblast ccd-18co cells

Abstract

Cytoprotection via preservation of DNA and mitochondrial function involving the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signalling pathway is an important preventive strategy for normal cells against carcinogenesis. Stilbenes are a group of chemicals characterized with the presence of 1, 2-diphenylethylene. Previously, synthesized (E)-N-(2-(3, 5-dimethoxystyryl) phenyl) furan-2-carboxamide (BK3C231) has been demonstrated to possess potential chemopreventive activity specifically inducing NAD(P)H:quinone oxidoreductase 1 (NQO1) protein expression and activity. This study aimed to investigate the molecular mechanisms underlying BK3C231-induced cytoprotection against 4-nitroquinoline Noxide (4NQO)-induced DNA and mitochondrial damages through the involvement of the Nrf2/ARE pathway in normal human colon fibroblast CCD-18Co cells. The cells were pretreated with BK3C231 at 6.25 μM till 50 μM for 2 hours prior to exposure to the carcinogen 4NQO for subsequent 22 hours. BK3C231 was able to inhibit 4NQOinduced cytotoxicity. Cells treated with 4NQO alone caused high level of DNA and mitochondrial damages. However, pretreatment with BK3C231 protected against these damages by reducing DNA strand breaks and micronucleus formation as well as decreasing losses of mitochondrial membrane potential (ΔΨm) and cardiolipin in a concentration-dependent manner. Interestingly, our study has demonstrated that nitrosative stress instead of oxidative stress was involved in 4NQO-induced DNA and mitochondrial damages. Inhibition of 4NQO-induced nitrosative stress by BK3C231 was observed through a decrease in nitric oxide (NO) level and an increase in glutathione (GSH) level. We further elucidated the role of BK3C231 in the upstream cytoprotective signalling pathway which is the Nrf2/ARE pathway. BK3C231 increased the protein expression and activity of cytoprotective enzymes namely NQO1, glutathione S-transferase (GST) and heme oxygenase-1 (HO-1), as well as restoring the expression of glutamate-cysteine ligase catalytic subunit (GCLC) back to the basal level. Furthermore, dissociation of Nrf2 from its inhibitory protein, Keap1, and ARE promoter activity were upregulated in cells pretreated with BK3C231. Taken together, our findings suggest that BK3C231 exerts cytoprotection by activating the Nrf2 signaling pathway which leads to ARE-mediated upregulation of cytoprotective proteins. This study provides new mechanistic insights into BK3C231 chemopreventive activities and highlights the importance of stilbene derivatives upon development as potential chemopreventive agents