Chemopreventive effects of Ficus dubia Latex extract on initiation of colorectal carcinogenesis

Abstract

The effects of Ficus dubia latex extract (FDLE) on rat colonic carcinogenesis and molecular mechanism were investigated in this study. Firstly, DMH was used to induce aberrant crypt foci (ACF) formation in rat colon in the initiation stage. Rats were fed orally by 100 and 500 mg/kg bw of FDLE continuously from the first week before DMH injection (40 mg/kg bodyweight, once a week for 2 weeks) until the end of the experiment. The results indicated that FDLE administration at dose of 500 mg/kg bw significantly reduced the total number of ACF (37.3% inhibition) when compared to the DMH-treated alone. Rats treated with FDLE showed the inhibition of liver phase I enzyme, correlated to lowering the phase II enzyme, including glutathione-S-transferase; GST and UDP glucuronyl transferase; UDPGT. These inhibitions caused the reduction of O 6 - methylguanine in rat liver and metylazozymethanol (MAM)-glucuronide conjugate secreted to colonic lumen. Moreover, FDLE exhibited competitive inhibition of bacterial β-glucuronidase in rat colon leading to the reduction of reactivated-MAM and colonic DNA adduct formation. These results affected the initiative formation of ACF in rat colon.Secondly, DMH was also used to induce rat colorectal carcinogenesis in the post- initiation stage. Rats were initially injected with DMH once a week for 2 weeks to induce ACF formation, after 2 weeks of the second DMH injection, FDLE administration at doses of 100 and 500 mg/kg bw were started and continued for 4 weeks until the end of the experiment during the ACF progression. The results revealed that FDLE administration at doses of 100 and 500 mg/kg bw also significantly reduced the total number of ACF (32,5% and 48.0% inhibition, respectively) and significantly decreased the average crypt multiplicity (AC/F) (16.7% and 18.4% inhibition, respectively) when compared to the rats that were only treated with DMH. These results were caused from modulating the aberrant colonic mucosal cell proliferation (PCNA expression) and recovering apoptosis of aberrant colonic mucosal cells (caspase-3 activation). For this reason, FDLE administration reduced the mRNA levels of pro-inflammatory cytokines, including IL-1β, IL-6, TNF-α and inflammation-related enzymes including iNOS and COX-2 in rat colonic mucosa when compared to the rats that were only treated with DMH. Therefore, the retardation of ACF growth was related to suppression of the inflammatory process in colonic mucosal cells. In order to explore whether FDLE inhibit colorectal cancer by reducing inflammation and to evaluate the different mechanisms of FDLE on colorectal cancer in normal and inflammatory conditions, human colorectal cancer cell lines (HCT-116 and HT-29) were used for experiments in vitro. HCT116 and HT-29 were treated with a mixture of TNF-α, IFN- γ and LPS (10 ng/ml each) for 2 hours before FDLE intervention. After that, the cells were cultured in various concentrations of FDLE in DMEM. Cell proliferation was measured by the MTT and colony formation assay while cell cycle arresting and apoptosis were measured by flow cytometry. The results showed that FDLE. exhibited anti-proliferative activity in both normal growth and inflamed HCT-116 and HT-29 cells, and this anti-proliferative activity was more effective in the inflammatory condition. In normal conditions. FDLE only induced cell cycle arrest by down-regulating NF-kB, cychn D1, CDK4 but up-regulating p21 in both HCT-116 and HT-29 cells. In inflammatory conditions, the cytokines induced hyperproliferation by activating NF-kB signaling pathway. However. FDLE administration not only induced cell cycle arrest by down-regulating NF-kB, cyclin D1 and CDK4, and down-regulating p21 in both HCT- 116 and HT-29 cells, but also induced apoptosis by down-regulating NF-kB and Bcl-xl and up-regulating Bid, Bak, cleaved caspase-7 and caspase-3 in HCT-116 cells Finally, the effect of FDLE on inflammation was further determined in lipopolysaccharide (LPS)-activated RAW 264.7 macrophage cells. The results showed that treatment of FDLE after LPS induction significantly reduced the secreted cells. inflammatory cytokines including TNF-α, IL-1β and IL-6 in culture supematant. Moreover, the inhibitions of FDLE on inflammatory response in colorectal cancer cell lines induced by the mixture of cytokines were also investigated. They also demonstrated that treatment of FDLE after cytokines induction significantly reduced the mRNA level of TNF-α, IL-1, and IL-6 in HCT-116 cells, and TNF-α and IL-1 in HT-29 cells. The results indicated that FDLE also inhibited the responsibility of colorectal cancer cell lines to inflammation which might be related to the growth promotion of colorectal cancer cells In conclusion, Ficus dubia latex exhibited the preventive effect in DMH-induced rat colorectal carcinogenesis in early stage by alteration of carcinogen metabolism in liver and colon in the initiation stage. Moreover, Ficus dubia latex suppressed ACF progression by decreasing inflammation. leading to a decrease of cell proliferation and increase of apoptosis in colon mucous. Furthermore, in vitro experiments demonstrated that the extract of Ficus dubia latex exhibited anti-proliferative activity in both normal growth and inflamed colorectal cancer cell lines. This anti-proliferative activity was more effective in the inflammatory condition due to its more effective regulation of NF-kB inactivation and some proteins related to cell cycle progression and apoptosis induction. Therefore, the phytochemicals in latex of Ficus dubia could potentially be used for prevention and treatment of colorectal cancer, especially in inflammation-induced hyperproliferation progression. The applications of Ficus dubia latex might be in the form of instant drink or functional tea because the extract of Ficus dubia latex could be solubilized in water and might be well absorbed in human gut.