Caffeine and Chlorogenic Acid Combination Attenuate Early-Stage Chemically Induced Colon Carcinogenesis in Mice: Involvement of oncomiR miR-21a-5p

Abstract

Colorectal cancer (CRC) is one of most common cancers worldwide, with high rates of mortality. Epidemiological findings demonstrate that coffee consumption reduces the risk of developing CRC by ~13%. In general, in vivo and in vitro findings demonstrate the antiproliferative, antioxidant and proapoptotic effects of brewed coffee or major bioavailable coffee compounds. Thus, it was assessed whether caffeine (CAF) and/or chlorogenic acid (CGA) attenuates the early-stage of chemically induced mouse colon carcinogenesis. Male Swiss mice were submitted to a 1,2-dimethylhydrazine/deoxycholic acid (DMH/DCA)-induced colon carcinogenesis model. These animals received CAF (50 mg/kg), CGA (25 mg/kg) or CAF+CGA (50 + 25 mg/kg) intragastrically for five times/week for ten weeks. CAF+CGA had the most pronounced effects on decreasing epithelial cell proliferation (Ki-67) and increasing apoptosis (cleaved caspase-3) in colonic crypts. This treatment also decreased the levels of proinflammatory cytokines IL-6, IL-17 and TNF-α, and downregulated the oncomiR miR-21a-5p in the colon. Accordingly, the analysis of miR-21a-5p targets demonstrated the genes involved in the negative regulation of proliferation and inflammation, and the positive regulation of apoptosis. Ultimately, CAF+CGA attenuated preneoplastic aberrant crypt foci (ACF) development. Our findings suggest that a combination of coffee compounds reduces early-stage colon carcinogenesis by the modulation of miR-21a-5p expression, highlighting the importance of coffee intake to prevent CRC.

Keywords: caffeine; chemically induced colon carcinogenesis; chlorogenic acid; miRNA expression; mice.

Figure 1

Representative methylene blue-stained colonic epithelium, showing (A) normal colon epithelium (scale bar: 100 µm, untreated group), (B) aberrant crypt foci with three crypts (scale bar: 50 µm, dotted line) and (C) aberrant crypt foci with >20 aberrant crypts (scale bar: 100 µm, dotted line). Effects of coffee compounds on the development of (D) AC or (E) ACF during DMH/DCA-induced colon carcinogenesis. n = 10 animals/group. Data are expressed as mean ± S.D. Untreated: 2×EDTA vehicle (i.p.)/balanced diet. DMH/DCA = 1,2-dimethylhydrazine (2 × 40 mg/kg b.wt., i.p.)/deoxycholic acid supplemented diet (0.02% w/w, 10 weeks). CAF = caffeine (50 mg/kg b.wt. intragastrical), CGA = chlorogenic acid (25 mg/kg b.wt. intragastrical) and CAF+CGA = caffeine + chlorogenic acid (50 and 25 mg/kg b.wt. intragastrical) for 10 weeks. * Statistical differences compared to the untreated group using ANOVA and Tukey’s test (p < 0.05).

Figure 2

Effects of coffee compounds on colonic proliferation (PI%) and apoptosis indexes (AI%) during DMH/DCA-induced colon carcinogenesis. n = 10 animals/group. Data are expressed as mean ± S.D. Representative photomicrographs of Ki-67-positive colonocytes (scale bar: 50 µm) and cleaved caspase-3-positive apoptotic bodies (scale bar: 25 µm, arrowheads) are also displayed. Untreated: 2 × EDTA vehicle (i.p.)/balanced diet. DMH/DCA = 2 × 1,2-dimethylhydrazine (40 mg/kg b.wt., i.p.)/deoxycholic acid supplemented diet (0.02% w/w, 10 weeks). CAF = caffeine (50 mg/kg b.wt. intragastrical), CGA = chlorogenic acid (25 mg/kg b.wt. intragastrical) and CAF+CGA = caffeine + chlorogenic acid (50 and 25 mg/kg b.wt. intragastrical) for 10 weeks. Asterisks correspond to statistical differences compared to the untreated (*) or DMH/DCA (**) counterparts using ANOVA and Tukey test (p < 0.05).

Figure 3

(A) Representative photomicrographs of HE-stained sections of colonic crypts (scale bar: 100 µm). * Detail on the inflammatory infiltrate present in the lamina propria of DMH/DCA group (scale bar: 20 µm). (B) Effects of coffee compounds on colonic levels of interleukins 6 (IL-6), 17 (IL-17) and the tumor necrosis factor alpha (TNF-α) during DMH/DCA-induced colon carcinogenesis. n = 7 animals/group. Data are expressed as mean ± S.D. Untreated: 2×EDTA vehicle (i.p.)/balanced diet. DMH/DCA = 2 × 1,2-dimethylhydrazine (40 mg/kg b.wt., i.p.)/deoxycholic acid supplemented diet (0.02% w/w, 10 weeks). CAF = caffeine (50 mg/kg b.wt. intragastrical), CGA = chlorogenic acid (25 mg/kg b.wt. intragastrical) and CAF+CGA = caffeine + chlorogenic acid (50 and 25 mg/kg b.wt. intragastrical) for 10 weeks. Asterisks correspond to statistical differences compared to the untreated (*) or DMH/DCA (**) counterparts using ANOVA and Tukey’s test (p < 0.05).

Figure 4

(A) The Venn diagram depicting the differentially expressed miRNAs (red: upregulated; blue: downregulated) in DMH/DCA (vs. untreated group) and CAF+CGA (vs. DMH/DCA). (B) STRING confidence network analysis of miR-21a-5p validated targets. Nodes in the correlated proteins are shown (with 3D structure inside). Edges correspond to the confidence of functional correlation (caption). DMH/DCA and CAF+CGA shared the differential expression of oncomiR miR-21-5p, which was upregulated in the DMH/DCA group and downregulated in CAF+CGA intervention. DMH/DCA = 2 × 1,2-dimethylhydrazine (40 mg/kg b.wt., i.p.)/deoxycholic acid supplemented diet (0.02% w/w, 10 weeks). CAF = caffeine (50 mg/kg b.wt. intragastrical), CGA = chlorogenic acid (25 mg/kg b.wt. intragastrical) and CAF+CGA = caffeine + chlorogenic acid (50 and 25 mg/kg b.wt. intragastrical) for 10 weeks.

Figure 5

Experimental design.