Manilkara zapota (L.) P. Royen Leaf Mitigates Colitis-Associated Colon Cancer through Anti-inflammatory Modulation in BALB/C Mice

Abstract

Colitis-associated colon cancer (CAC) arises from prolonged inflammation of the inner colon lining. An alternative approach to treating or preventing CAC involves the use of natural products such as Manilkara zapota (L.) P. Royen or M. zapota, which has been studied for its medicinal and pharmacological properties. Previous research has demonstrated the anticancer effects of M. zapota leaf aqueous extract (MZLAE) on colon cancer cells. However, no animal study has investigated the effects of MZLAE on CAC. Therefore, this study aimed to assess the potential anti-inflammatory effects of MZLAE on CAC in mice. In the present study, CAC was induced using azoxymethane (AOM) and dextran sodium sulphate (DSS). The mice were randomly assigned into five groups: (a) normal, (b) AOM/DSS, (c) AOM/DSS + 50 mg/kg MZLAE, (d) AOM/DSS + 100 mg/kg MZLAE, and (e) AOM/DSS + 200 mg/kg MZLAE. Various parameters including disease activity index (DAI), colon length and weight, reactive oxygen species (ROS), superoxide, superoxide dismutase (SOD), histopathological assessment, and proinflammatory cytokines expression were analysed. The results indicated that MZLAE improved DAI scores, colon length, colon histological dysplasia and inflammation scores, and SOD level, while also reducing ROS production and expression of proinflammatory cytokines (tumour necrosis factor-alpha (TNF- α) and interleukin 6 (IL-6)). In conclusion, this study suggests that MZLAE may serve as a promising source of antioxidants and anti-inflammatory agents for alleviating CAC.

Figure 1

Schematic overview of the experimental timeline.

Figure 2

DAI score of CAC mice (n = 6). ^aValues with different letters on the same line (group) indicate significant differences between weeks at p <  0.05. ^∗∗ indicates a significant difference compared to the AOM/DSS group (p < 0.05). ^  ^ indicates a significant difference compared to the normal group (p < 0.05). Week 1: AOM administration; week 2: first DSS cycle; week 5: second DSS cycle; week 8: third DSS cycle; week 10: end of the study. AOM: azoxymethane; DSS: dextran sodium sulphate; MZLAE: Manilkara zapota leaf aqueous extract.

Figure 3

Histological evaluation of mouse colonic dysplasia using H&E staining ( n = 3): (a) normal group, (b) AOM/DSS group (arrow shows distorted crypts in high-grade dysplasia), (c) AOM/DSS + 50 mg/kg MZLAE group (arrow shows low-grade dysplasia), (d) AOM/DSS + 100 mg/kg MZLAE, and (e) AOM/DSS + 200 mg/kg MZLAE group (100 µm = magnification or 20X). AOM: azoxymethane; DSS: dextran sodium sulphate; MZLAE: Manilkara zapota leaf aqueous extract.

Figure 4

Histological evaluation of colonic inflammation in CAC mice (n = 3): (a) normal, (b) AOM/DSS (long arrow shows infiltration of inflammatory cells and short arrow shows destruction of the mucosal cell architecture), (c) AOM/DSS + 50 mg/kg MZLAE (arrow shows inflammation), (d) AOM/DSS + 100 mg/kg MZLAE, and (e) AOM/DSS + 200 mg/kg MZLAE (100 µm = magnification or 20X). AOM: azoxymethane; DSS: dextran sodium sulphate; MZLAE: Manilkara zapota leaf aqueous extract.

Figure 5

Effect of MZLAE on ROS production (a), superoxide production (b), and SOD level of CAC mice (c) (n = 3). ^aValues with different letters indicate significant differences at p < 0.05. AOM: azoxymethane; DSS: dextran sodium sulphate; MZLAE: Manilkara zapota leaf aqueous extract; SOD: superoxide dismutase.

Figure 6

Effect of MZLAE on the expression of (a) TNF-α and (b) IL-6 by Western blot (n = 3). ^aValues with different letters indicate significant differences at p < 0.05. AOM: azoxymethane; DSS: dextran sodium sulphate; MZLAE: Manilkara zapota leaf aqueous extract.

Figure 7

Summation diagram that illustrates the possible mechanism of MZLAE in CAC. AOM: azoxymethane; DSS: dextran sodium sulphate; TNF-α: tumour necrosis factor-alpha; IL-6: interleukin 6; DNA: deoxyribonucleic acid; ROS: reactive oxygen species; NF-κβ: nuclear factor kappa beta; STAT3: signal transducer and activator of transcription 3.