Resistant Starch-Encapsulated Probiotics Attenuate Colorectal Cancer Cachexia and 5-Fluorouracil-Induced Microbial Dysbiosis

Abstract

5-Fluorouracil (5-FU) is commonly used as the primary chemotherapy for colorectal cancer (CRC). However, it can lead to unwanted chemoresistance. Resistant starch (RS), which functions similarly to fermentable dietary fiber, has the potential to reduce the risk of CRC. The effects of RS on improving CRC-associated cachectic symptoms and 5-FU chemotherapy-induced microbial dysbiosis remain unknown. Female BALB/cByJNarl mice were randomly divided into four groups: one tumor group (with CT26 colonic carcinoma but no treatment) and three CT26 colonic carcinoma-bearing groups that were administered 20 mg/kg 5-FU (T+5-FU group), a probiotic cocktail (4 × 10⁸ CFUs) plus chemotherapy (T+5-FU+Pro), or resistant-starch-encapsulated probiotics plus chemotherapy (T+5-FU+RS-Pro). T+5-FU and T+5-FU+RS-Pro administration significantly suppressed tumor growth and activated apoptotic cell death in CT26-bearing mice. 5-FU-induced increases in inflammatory cytokines and NF-κB signaling were mitigated by the Pro or RS-Pro supplementation. A gut microbial composition comparison indicated that the abundance of intestinal bacteria in the T and T+5-FU groups decreased significantly, while the groups receiving Pro or RS-Pro maintained a greater abundance and healthy gut microbiota composition, suggesting that RS can reduce the microbial dysbiosis that occurs during 5-FU chemotherapy. The use of RS-Pro before chemotherapy should be considered for the regulation of chemotherapy-associated cachectic symptoms, inflammation, and chemotherapy-induced microbial dysbiosis.

Keywords: chemotherapy-enhanced inflammation; colon carcinoma-associated cachectic symptoms; microbiome.

Figure 1

The treatment schedule for 5-FU chemotherapy, probiotics, and RS-encapsulated probiotics in CT26 tumor-bearing mice and analysis timelines.

Figure 2

The impact of 5-FU chemotherapy, probiotics, and RS-encapsulated probiotics on tumor growth was assessed. (A) The tumor volume growth curve after various treatments. The blue arrow indicates CT26 cells were inoculated. (B) Tumor weights were measured at the study endpoint.

Figure 3

Quantification of mRNA levels of (A) Bax, (B) Bcl-2, (C) Bax/Bcl-2 ratio, (D) caspase 8, and (E) caspase 3 in the tumor tissues of mice treated with tumor inoculation, 5-FU chemotherapy, probiotics, and RS-encapsulated probiotics. The data are presented as means ± standard error of the mean (SEM). Statistical significance is denoted as follows: * p < 0.05, ** p < 0.01, and *** p < 0.001 when compared to the indicated group.

Figure 4

Effects of 5-FU chemotherapy, probiotics, and RS-encapsulated probiotics on the expression of inflammatory cytokines and NF-κB signaling in tumor tissue from CT26-inoculated BALB/cByJNarl mice. The protein levels of (A) IL-10, (B) TNF-α, and (C) IL-6 in the tumor tissue of mice were detected by means of ELISA. The data are presented as means ± SEM. Statistical significance is denoted as follows: * p < 0.05 and ** p < 0.01 when compared to the indicated group. (D) Semiquantitative analysis of colonic tissue protein levels of IKK-β, p-IKK-β, IκB-α, NF-κB, and GAPDH. GAPDH served as an internal control for equal loading.

Figure 5

Cachectic symptoms after treatment with 5-FU chemotherapy, probiotics, and RS-encapsulated probiotics in CT26-inoculated BALB/cByJNarl mice. (A) The body weights of tumor-bearing mice were monitored throughout the treatment course. Data were presented as mean ± SEM. (B) Weight of spleen tissues. (C) Weight of GM muscle tissues.

Figure 6

Changes in mean white blood cells (WBCs) (A), %neutrophils (B), %lymphocytes (C), and %monocytes (D) before (−7th) and the 8th and 22nd day after CT26 carcinoma were inoculated in BALB/cByJNarl mice. The data are presented as means ± SEM. Statistical significance is denoted as follows: * p < 0.05, ** p < 0.01, and *** p < 0.001 when compared to the indicated group.

Figure 7

Effects of 5-FU chemotherapy, probiotics, and RS-encapsulated probiotics on the expression of inflammatory cytokines in colon tissue from CT26-inoculated BALB/cByJNarl mice. The protein levels of (A) IL-10, (B) TNF-α, and (C) IL-6 in the colon tissue of mice were detected by means of ELISA. The data are presented as means ± SEM. Statistical significance is denoted as follows: * p < 0.05, ** p < 0.01 when compared to the indicated group.

Figure 8

Effects of 5-FU chemotherapy, probiotics, and RS-encapsulated probiotics on the structural and functional composition of the gut microbiota. (A) Changes in the relative phylum-level abundances of gut microbiota components in CT26-inoculated BALB/cByJNarl mice. (B) The ratio of Firmicutes to Bacteroidetes (F/B) boxplot showing the gut microbiota in CT26-inoculated BALB/cByJNarl mice. Boxes contain 50% of all values, and whiskers represent the 25th and 75th percentiles. * p < 0.05 indicates a significant difference from the compared group. (C–F) Box plots showing the species richness estimator-Chao1 (C) and Observed features (D), and the species evenness estimator-Shannon (E) and Simpson (F) of alpha diversity.

Figure 9

Heatmap depicting the relative abundance of the most abundant phyla (A), classes (B), and genera (C) (>0.1%) of the gut microbiota from different treatments. The color intensity of each sample is normalized to represent its relative ratio in CT26-inoculated BALB/cByJNarl mice. The colors from blue to red indicate the relative values of microbiota (−2 to 4). (D) The relative abundances of Enterorhabdus, RF39, Erysipelotrichaceae, Candidatus Saccharimonas, Oscillospiraceae uncultured, and Mucispirillum. The data are expressed as the means ± SEM. * p < 0.05, and ** p < 0.01 are significantly different from the compared group.