(E)-N-(2-(3, 5-Dimethoxystyryl) phenyl) furan-2-carboxamide (BK3C231) induces cytoprotection in CCD18-Co human colon fibroblast cells through Nrf2/ARE pathway activation

Abstract

Cytoprotection involving the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway is an important preventive strategy for normal cells against carcinogenesis. In our previous study, the chemopreventive potential of (E)-N-(2-(3, 5-Dimethoxystyryl) phenyl) furan-2-carboxamide (BK3C231) has been elucidated through its cytoprotective effects against DNA and mitochondrial damages in the human colon fibroblast CCD-18Co cell model. Therefore this study aimed to investigate the molecular mechanisms underlying BK3C231-induced cytoprotection and the involvement of the Nrf2/ARE pathway. The cells were pretreated with BK3C231 before exposure to carcinogen 4-nitroquinoline N-oxide (4NQO). BK3C231 increased the protein expression and activity of cytoprotective enzymes namely NAD(P)H:quinone oxidoreductase 1 (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 a potential chemopreventive agent.

Figure 1

Effect of BK3C231 pretreatment on the activity of cytoprotective enzymes namely NQO1 (a), GST (b), and HO-1 (c). The enzymatic activity of cytoprotective enzymes was observed at basal level in untreated control cells (CON) and cells treated only with BK3C231 (BK3C231 24 h). However, in cells treated only with 4NQO (4NQO 1 h), there was a decrease in the activity of cytoprotective enzymes as compared to that of untreated control cells. Pretreatment of cells with BK3C231 before 4NQO exposure increased the activity of cytoprotective enzymes, significantly at 24 h as compared to that of cells treated with 4NQO only. Treated whole cell lysates were subjected to spectrophotometric and microplate reader data analysis as mentioned in “Results” to determine the activity of NQO1, GST and HO-1. Each data point was expressed as mean ± SEM from at least three independent experimental replicates. * p < 0.05 against 4NQO 1 h.

Figure 2

Effect of BK3C231 pretreatment on the protein expression of cytoprotective enzymes namely NQO1 (a), GST (b), and HO-1 (c). Basal-level constitutive expressions of NQO1, GST and HO-1 were observed in untreated control cells and cells treated only with BK3C231. In cells treated only with 4NQO, the expression level of cytoprotective enzymes decreased as compared to that of untreated control. Pretreatment of cells with BK3C231 before 4NQO exposure significantly increased the protein expression of cytoprotective enzymes at 12 h and 24 h for NQO1; 6 h, 12 h and 24 h for GST; and 24 h for HO-1 as compared to that of cells treated with 4NQO only. Treated whole cell lysates were subjected to immunoblotting analysis to determine the protein expression level of NQO1, GST, and HO-1. Each data point was expressed as mean ± SEM from at least three independent experimental replicates. *p < 0.05 against 4NQO 1 h. The blots displayed in the figure are cropped. Full-length blots are presented in Supplementary Figure S2.

Figure 3

Effect of BK3C231 pretreatment on Nrf2 (a) and Keap1 (b) protein expression levels and dissociation level of Nrf2 from Keap1 (c). (a, b) Constitutive expressions of Nrf2 and Keap1 at the basal level were observed in untreated control cells. 4NQO reduced Nrf2 expression and increased Keap1 expression over untreated control. Cells treated only with BK3C231 showed a significant reduction of Keap1 expression over 4NQO-treated cells whereas Nrf2 expression remained at basal level. BK3C231 pretreatment for 6 h, 12 h, and 24 h significantly increased Nrf2 expression over 4NQO-treated cells. Keap1 expression was significantly reduced in cells pretreated with BK3C231 at 4 h and 24 as compared to that of 4NQO-treated cells. (c) The Keap1/Nrf2 ratio was increased in 4NQO-treated cells over untreated control. BK3C231 pretreatment from as early as 4 h till 24 h significantly reduced Keap1/Nrf2 ratio over 4NQO-treated cells and untreated control, thereby inducing dissociation of Nrf2 and Keap1 leading to Nrf2 activation. Treated whole cell lysates were subjected to immunoblotting analysis to determine the expression level of Nrf2 and Keap1. Treated whole cell lysates were subjected to co-immunoprecipitation as mentioned in “Results” and immunoblotting analysis to determine the Keap1 to Nrf2 ratio. Each data point was expressed as mean ± SEM from at least three independent experimental replicates. *p < 0.05 against 4NQO 1 h. The blots displayed in the figure are cropped. Full-length blots are presented in Supplementary Fig. S3.

Figure 4

Effect of BK3C231 pretreatment on ARE promoter activity. The relative luciferase activity which indicates ARE promoter activity was observed at basal level in untreated control cells (CON). Treatment of cells with 4NQO only (4NQO 1 h) decreased ARE promoter activity over untreated control. However, in cells treated only with BK3C231 (BK3C231 24 h), relative luciferase activity significantly increased over 4NQO-treated cells (4NQO 1 h). BK3C231 treatment alone also enhanced relative luciferase activity over the basal level in the untreated control (CON). BK3C231 pretreatment significantly increased relative luciferase activity from 4 h till 24 h over 4NQO-treated cells (4NQO 1 h). Cells were transfected with ARE reporter construct and pretreated with BK3C231 before 4NQO exposure as mentioned in “Results”. Subsequently, the dual-luciferase assay was performed to determine the firefly and Renilla luciferase activities. Cells transfected with Cignal negative control and positive control reporter constructs were used as the negative control (NC) and positive control (PC) for this assay. Each data point was expressed as mean ± SEM from at least three independent experimental replicates. *p < 0.05 against 4NQO 1 h.

Figure 5

Schematic illustration representing the cytoprotective effects of BK3C231 through Nrf2 activation which leads to ARE-mediated upregulation of cytoprotecive enzymes. Upon exposure of cells to 4NQO, BK3C231 pretreatment increased protein expression and activity of the cytoprotective enzymes namely NQO1, GST and HO-1, as well as maintaining GCLC at basal level. BK3C231 pretreatment prior to 4NQO exposure induced Nrf2 activation by increasing Nrf2 protein level and decreasing Keap1 level, as well as promoting disruption of Keap1-Nrf2 complex thus leading to an increase in ARE promoter activity upon Nrf2 activation and upregulation of cytoprotective enzymes. Interestingly, reduction in Keap1 level which lead to an increase in the dissociation level of Nrf2 from Keap1 as well as ARE promoter activity were observed in cells treated only with BK3C231, hereby suggesting that BK3C231 is able to sensitize the cells for its endogenous cytoprotective responses and elevate the cytoprotective capacity of cells upon exposure to stress inducers or toxicants such as 4NQO.