Tumour necrosis factor-α (TNF-α) enhances dietary carcinogen-induced DNA damage in colorectal cancer epithelial cells through activation of JNK signaling pathway

Abstract

Colorectal cancer (CRC) is the third most common cancer worldwide and the second leading cause of cancer death. Benzo[a]pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazol [4,5-b] pyridine (PhIP) present in cooked meat are pro-carcinogens and considered to be potential risk factors for CRC. Their carcinogenic and mutagenic effects require metabolic activation primarily by cytochrome P450 1 family enzymes (CYPs); the expression of these enzymes can be modulated by aryl hydrocarbon receptor (AhR) activation and the tumour microenvironment, involving mediators of inflammation. In this study, we hypothesized that tumour necrosis factor-α (TNF-α), a key mediator of inflammation, modulates BaP- and PhIP-induced DNA damage in colon cancer epithelial cells. Importantly, we observed that TNF-α alone (0.1-100 pg/ml) induced DNA damage (micronuclei formation) in HCT-116 cells and co-treatment of TNF-α with BaP or PhIP showed higher levels of DNA damage compared to the individual single treatments. TNF-α alone or in combination with BaP or PhIP did not affect the expression levels of CYP1A1 and CYP1B1 (target genes of AhR signaling pathways). The DNA damage induced by TNF-α was elevated in p53 null HTC-116 cells compared to wild type cells, suggesting that TNF-α-induced DNA damage is suppressed by functional p53. In contrast, p53 status failed to affect BaP and PhIP induced micronucleus frequency. Furthermore, JNK and NF-κB signaling pathway were activated by TNF-α treatment but only inhibition of JNK significantly reduced TNF-α-induced DNA damage. Collectively, these findings suggest that TNF-α induced DNA damage involves JNK signaling pathway rather than AhR and NF-κB pathways in colon cancer epithelial cells.

Keywords: CYP1A1/1B1; Cytotoxicity; DNA damage; Genotoxicity; JNK pathway; TNF-α.

Fig. 1

Cytotoxicity of HCT116 cells. (A) Cells treated with 10 μM BaP, TNF-α at doses of 0.1, 10, 100 pg/mL, or a combination of both for 48 h. (B) Cells treated with 100 μM PhIP, TNF-α at doses of 0.1, 10, 100 pg/mL, or a combination of both for 48 h. Cytotoxicity is expressed as % of viable cells relative to the negative control. Significance was calculated using one-way ANOVA with a Dunnett test. The data are presented as SEM from three independent cultures.

Fig. 2

Genotoxicity of HCT116 cells. DNA damage measured by micronucleus frequency in (A) cells co-treated with TNF-α and BaP or (B) cells co-treated with TNF-α and PhIP for 48 h. The treatment concentrations of BaP and PhIP were 10 μM and 100 μM, respectively. Statistically significant differences are shown as following: carcinogen treatment vs. co-treatment (p < 0.05, ++p < 0.01, +++p < 0.001, ++++ p < 0.0001), TNF-α vs. co-treatment (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001), negative control Vs. carcinogens or TNF-α treatment (#p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001). Significance was calculated using one-way ANOVA with a Dunnett test. The data are presented as SEM from three independent cultures.

Fig. 3

CYP1A1/1B1 gene expression in HCT116. (A) Cells were treated with TNF-α dose range of (0.01-100 pg/mL) over 72 h. (B) cells co- treated with BaP and PhIP. Gene expression were measured by RT-qPCR. Data are shown as fold change. Significance was calculated using one-way ANOVA with a Dunnett test (GraphPad Prism 8). The data are presented as SEM from three independent cultures.

Fig. 4

Genotoxicity in HCT116 cells. DNA damage measured by Micronucleus frequency in HCT116 p53-/- and HCT116 p53+/+ cells induced by (A) HCT116 p53-/- treated with TNF-α dose range of (0.01-100 pg/mL) and (B) HCT116 p53-/- treated with BaP dose range of (0.1, 1,10μM) and PhIP dose range of (1-100μM), (C) HCT116 p53+/+ treated with BaP and PhIP. Statistically Significant differences are shown for comparisons between treated samples Vs. vehicle (*p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001). Significance was calculated using one-way ANOVA with a Dunnett test (GraphPad Prism 8). The data are presented as SEM from three independent cultures.

Fig. 5

Effect of TNF-α on NF-kB activation in colon cancer cells HCT116. Cells were treated with TNF-α dose range of (0.01-100 pg/mL) for 24 h. IkB and phosphorylated IkB (p-IkB) protein expression were determined by immunoblotting and quantified using Image J. Beta-actin was a loading control. Significance was assessed using one-way ANOVA (GraphPad Prism 8, (*p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001) compared to control. Data are presented as the ratio of p-IkB/total IkB with SEM from three independent cultures(n=3).

Fig. 6

Effect of TNF-α on JNK activation in colon cancer cells HCT116. Levels of phosphorylated JNK are increased upon TNF-α treatment. Cells were treated with TNF-α dose range of (0.01-100 pg/mL) for 24 h. JNK and phosphorylated JNK (p-JNK) protein expression were determined by immunoblotting and quantified using Image J. Beta-actin was a loading control. Significance was assessed using one-way ANOVA (GraphPad Prism 8, (*p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001) compared to control. Data are presented as the ratio of p-IkB/total IkB with SEM from three independent cultures(n=3).

Fig. 7

JNK and NF-kB involvement in TNF-α induced DNA damage. (A) genotoxicity of HCT116, cells were treated with 0.13μM of Bortezomib (BZ) with TNF-α dose range of (0.01-100 pg/mL) for 24 h. (B) genotoxicity of HCT116, cells were treated with 30μM of JNK inhibitor (SP600125) with TNF-α dose range of (0.01-100 pg/mL) for 24 h. Statistically Significant differences are shown for comparisons between inhibitor treated cells Vs. untreated cells. Significance was assessed using one-way ANOVA (GraphPad Prism 8, (**** p < 0.0001) and (++++ p < 0.0001) compared to control. Data are presented as a mean and error bars represent the SEM for independent cultures(n=3).