Chemopreventive effects of citrus hystrix leaf ethanolic extract on colitis-associated cancer in mice

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder that causes inflammation of the intestine, predominantly in the elderly. Patients with IBD are associated with high risk of colorectal cancer (CRC), namely, colitis associated cancer (CAC). Therefore, fluorouracil (5-FU) is the most prescribed medication for CRC to kill cancer cells and abate the cancer from proliferating. However, patients often develop side effects towards 5-FU. Thus, herbal medicines for CRC that have anti-inflammatory and antioxidant properties may offer safer alternative. This study aims to investigate the efficacy of Citrus hystrix leaf extract (CLE) in alleviating CAC. The CLE extract was subjected to UHPLC/MS/MS while MTT assay was employed to assess the in vitro cytotoxic effect of the CLE. The in vivo study utilized male Balb/c mice that were randomly assorted into six groups (n=12): normal control, tumour control Azoxymethane (AOM)/Dextran Sulfate Sodium (DSS), positive control (Limonin; 50 mg/kg body weight) and treated CLE at 100, 200 and 300 mg/kg body weight. Mice were intraperitoneally injected with AOM (10 mg/kg body weight). A week post-injection of AOM, mice received 2% DSS for 7 days via drinking water, followed by regular drinking water during the recovery period. Treatments were orally administered through drinking water for 17 weeks post-induction with AOM/DSS. The CAC development was monitored via macroscopic, histopathological observations, protein biomarkers, mRNA expression and immunophenotyping analysis. The UHPLC/MS/MS showed CLE contains taxifolin, hesperidin, diosmin, tangeritin, quercitrin, catechin, isovitexin, apiin, apigenin, isovitexin, rutin, luteolin, torachrysone, pelargonidin, kaempferol, xanthotoxol, and their glucosides. The in vitro study showed that CLE exerted cytotoxic effects on HT-29 cells growth at IC50 = 18.93 μg/mL after 24 hours incubation. CLE also did not cause any cytotoxicity towards normal 3T3 cells. Thus, CLE effect on colitis associated cancer was further investigated in an animal model to see how the body respond to CLE absorption in the body of mice. In the animal study, the CLE administration has altered disease activity index (DAI) score, colon shortening, spleen weight and histological damage (colon, kidney, and liver). The CLE treatment alleviated AOM/DSS-induced colitis-associated cancer (CAC) by significantly (p<0.05) suppressed the pro-inflammatory (TNF-α, iNOS and PGE2), cell proliferation and cell cycle (β-catenin and Cyclin-D1), oxidative stress (MDA) and colon cancer markers (CCA). These findings were supported with anti-tumour effects of CLE which significantly (p<0.05) increased anti-inflammatory (TGF-β1), antioxidant (Nrf2, Sod2, GSH), immune (IFN-γ, CD8a and CD11b) and tumour suppressor (p53) biomarkers. The results also indicated that CLE enhanced tissue healing or regeneration of healthy tissues (Stat3, Myc, CD4, Vegfa, Hif-1α, Caspase-3 and Bcl-2). In addition, the CLE significantly (p<0.05) increased T cells (CD4 and CD8) and B cells (CD19) and significantly reduced (p<0.05) natural killer cells (CD335) in spleen tissues. Altogether, this study can conclude that CLE has great potential in attenuating colitis-associated cancer (CAC) in colon cancer induced mice via multiple pathways including inflammation, oxidative stress, antioxidant activity and immunity. CLE also protected colon through the reduction in tumor incidence and histological damage.

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