Ficus dubia latex extract prevent DMH-induced rat early colorectal carcinogenesis through the regulation of xenobiotic metabolism, inflammation, cell proliferation and apoptosis

Abstract

Ficus dubia latex is recognized as a remedy in Asian traditional medicine with various therapeutic effects. The present study aimed to determine the preventive action of Ficus dubia latex extract (FDLE) on 1,2-dimethylhydrazine (DMH)-induced rat colorectal carcinogenesis and its mechanisms. The experiment included an initiation model in which rats were orally administered with FDLE daily for 1 week before DMH injection until the end of the experiment, while only after DMH injection until the end in the post-initiation model. The results firstly indicated that FDLE treatment could reduce the level of methylazoxymethanol (MAM) in rat colonic lumen by inhibition of the activities of both phase I xenobiotic metabolizing enzymes in the liver and β-glucuronidase in the colon, leading to reduced DNA methylation in colonic mucosal cells, related to the number of ACF in the initiation stage. Besides, FDLE modulated the inflammation which could suppress the growth and induce apoptosis of aberrant colonic mucosal cells, leading to retardation of ACF multiplicity. Therefore, FDLE showed the ability to suppress the DMH-induced rat ACF formation and inflammation promoted growth of ACF. In conclusion, FDLE had the potential to prevent carcinogens-induced rat colorectal carcinogenesis in the initiation stage.

Figure 1

HPLC chromatograms of (a) FDLE, (b) the standard of chlorogenic acid and apigenin and (c) FDLE with standard.

Figure 2

The experimental protocol and treatment schedule. (a) Initiation stage and (b) post-initiation stage of DMH-induced ACF formation.

Figure 3

Effect of FDLE on the metabolism of dimethylhydrazine and DNA adduct formation in rat liver and colon (a) scheme of inhibitory effect of Ficus dubia latex extract on DMH-induced carcinogenesis. (b) The level of O⁶-MeG DNA adducts in liver tissue and colonic mucosa. (c) The activity of phase I and II xenobiotic metabolizing enzymes in rat liver tissue, cytochrome P4502E1 (CYP2E1) catalyzed PNP-hydroxylation, (d) UDP-glucuronosyltransferase (UDPGT) and (e) Glutathione-S-transferase (GST), phase II detoxifying enzymes. (f) The activity of bacterial β-glucuronidase in rat feces. (g) Lineweaver–Burk plot of E. coli β-glucuronidase in the presence of FDLE with data representing three independent experiments with similar results. Rat experiments data are presented as the mean ± SD of five rats per group. *p < 0.05, **p < 0.01.

Figure 4

Anti-inflammation effects of FDLE (a) Relative mRNA level of pro-inflammatory cytokines and enzymes including IL-1β, IL-6, TNF-α, iNOS and COX-2 in rat colonic mucosa. Data are presented as the mean ± SD of 4 rats per group. (b) The secretion of pro-inflammatory cytokines including TNF-α, IL-1β and IL-6 in LPS induce RAW 264.7 cells treated with FDLE. Relative mRNA levels of pro-inflammatory cytokines, including TNF-α, IL-1β and IL-6 in cytokines (TNF-α, IFN-γ and LPS each 10 ng/mL) induced colorectal cancer cell lines at 48 h. (c) HT-29 cells. (d) HCT-116 cells. Data are presented as the mean ± SD of three independent experiments which are similar results. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 5

Effect of FDLE on cell proliferation and apoptosis in rat colonic mucosa and human colorectal cancer cells. (a) Immunoblot analysis of PCNA, cleaved caspase-3 and β-actin proteins. (b) Relative expression of PCNA to β-actin proteins. (c) Relative expression of cleaved caspase-3 to β-actin protein. Data are presented as the mean ± SD of four rats per group. Relative cell proliferation (%zerotime) of colorectal cancer cell lines with cytokines (TNF-α, IFN-γ and LPS each 10 ng/mL) or together with FDLE for 24 and 48 h. (d) HT-29 cells. (e) HCT-116 cells. Data are presented as the mean ± SD of three independent experiments with similar results, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 6

The chemopreventive mechanism of FDLE in DMH-induced rat colorectal carcinogenesis in the early stage.