Colorectal cancer (CRC), mostly categorized as a low immunogenic microsatellite-stable phenotype bearing complex immunosuppressive tumor microenvironment (TME), is highly resistant to immunotherapy. Seeking safe and efficient alternatives aimed at modulating tumor immunosuppressive TME to improve outcome of CRC is highly anticipated yet remains challenging.

Adjuvant chemotherapy based on 5-fluorouracil (5-FU), such as FOLFOX, is suggested as a treatment for gastrointestinal cancer. Yet, intestinal damage continues to be a prevalent side effect for which there are no practical prevention measures. We investigated whether Babao Dan (BBD), a Traditional Chinese Medicine, protects against intestinal damage induced by 5-FU by controlling immune response and gut microbiota. 5-FU was injected intraperitoneally to establish the mice model, then 250 mg/kg BBD was gavaged for five days straight. 5-FU led to marked weight loss, diarrhea, fecal blood, and histopathologic intestinal damage. Administration of BBD reduced these symptoms, inhibited proinflammatory cytokine (IL-6, IL-1β, IFN-γ, TNF-α) secretion, and upregulated the ratio of CD3(+) T cells and the CD4(+)/CD8(+) ratio. According to 16S rRNA sequencing, BBD dramatically repaired the disruption of the gut microbiota caused in a time-dependent way, and increased the Firmicutes/Bacteroidetes (F/B) ratio. Transcriptomic results showed that the mechanism is mainly concentrated on the NF-κB pathway, and we found that BBD reduced the concentration of LPS in the fecal suspension and serum, and inhibited TLR4/MyD88/NF-κB pathway activation. Furthermore, at the genus level on the fifth day, BBD upregulated the abundance of unidentified_Corynebacteriaceae, Aerococcus, Blautia, Jeotgalicoccus, Odoribacter, Roseburia, Rikenella, Intestinimonas, unidentified_Lachnospiraceae, Enterorhabdus, Ruminiclostridium, and downregulated the abundance of Bacteroides, Parabacteroides, Parasutterella, Erysipelatoclostridium, which were highly correlated with intestinal injury or the TLR4/MyD88/NF-κB pathway. In conclusion, we established a network involving 5-FU, BBD, the immune response, gut microbiota, and key pathways to explain the pharmacology of oral BBD in preventing 5-FU-induced intestinal injury.

Combination chemotherapy with chemosensitizers can exert synergistic therapeutic effects, reduce toxicity, and delay the induction of drug resistance. In the present study, the antitumor effects were investigated, and the possible underlying mechanisms of kaempferol combined with 5‑fluorouracil (5‑FU) in colorectal cancer cells were explored. HCT‑8 or HCT‑116 cells were treated with various concentrations of kaempferol and/or 5‑FU for the indicated time‑points. An MTT assay was used to determine cell viability, whereas the synergistic effects were assessed by calculating the combination indices of kaempferol and 5‑FU. Annexin V analysis and Hoechst staining were used to determine cell apoptosis. q‑PCR and western blotting were performed to determine the expression levels of Bax, Bcl‑2, thymidylate synthase (TS), PTEN, PI3K, AKT, and p‑AKT. The combination of kaempferol and 5‑FU was determined to be more effective in inhibiting cell viability than either of the agents alone. The inhibition of tumors in response to kaempferol and 5‑FU was associated with the reduction in proliferation ability and stimulation of apoptosis. The protein results indicated that kaempferol and 5‑FU could significantly upregulate the expression levels of Bax and downregulate the expression levels of Bcl‑2 and TS. Furthermore, the combination treatment greatly inhibited the activation of the PI3K/Akt pathway, suggesting the involvement of this pathway in the synergistic effects. The present study demonstrated that kaempferol has a synergistic effect with 5‑FU by inhibiting cell proliferation and inducing apoptosis in colorectal cancer cells via suppression of TS or attenuation of p‑Akt activation. The combination of kaempferol and 5‑FU may be used as an effective therapeutic strategy for colorectal cancer.

5-Fluorouracil (5-FU)-based chemotherapy often causes several drawbacks including weight loss, diarrhea, myelosuppression, and the intestinal mucositis. This study aimed to evaluate the protective effect of Qingjie Fuzheng Granule (QFG) on 5-FU-induced intestinal mucositis in CT-26 tumor-bearing xenograft mice and investigated the possible molecular mechanism. Tumor xenograft models of CT-26 cells were generated in BALB/c nude mice, the mice were randomly divided into 4 groups including control, QFG, 5-FU and 5-FU combined QFG groups. The body weight, volume of tumor and diarrhea score of each group were recorded daily. On the fifth day, the blood of mice was collected, the mice were subsequently euthanized and their thymus, spleen, intestine and tumor were removed for the following analysis. QFG alleviated severe diarrhea and reversed the decrease in the number of white blood cell including granulocyte and lymphocyte induced by 5-FU. QFG could also significantly improve 5-FU-induced several intestinal mucosal damages, and characterized by integrity villus and crypts, the reduction of necrotic cells. QFG decreased the serum levels of TNF-α, IL-1β, and IL-6 and increased the levels of IL-10. Furthermore, QFG inhibited the cellular apoptosis in the jejunum tissue caused by 5-FU via the increasing Bcl-2 expression and decreasing Bax expression. In addition, QFG promoted the cell proliferation via elevating the expression of Cyclin D1 and CDK4 and reducing p21 expression. Meanwhile, QFG could not further impact on the cell apoptosis and proliferation of tumors caused by 5-FU. QFG attenuated the intestinal mucositis and diarrhea induced by 5-FU via preventive effect on inflammation and its improvement of the intestinal barrier function, inhibiting cell apoptosis and promoting cell proliferation, and without affecting the 5-FU treatment efficiency. The results suggest that QFG may act as a potential agent against chemotherapy-induced intestinal mucositis.

Chemotherapy-induced intestinal injury limits the clinical use of drugs. Intestinal injury involves multiple signaling pathways and gut microbiota disruption.

To investigate the effect of Qingjie Fuzheng Granule (QFG) on lymphangiogenesis and lymphatic metastasis in colorectal cancer. The effects of QFG on the expression and secretion of vascular endothelial growth factor-C (VEGF-C) in HCT-116 cells were investigated both in vitro and in vivo. HCT-116 cells were treated with different concentrations (0.2, 0.5, and 1.0 mg/mL) of QFG. The VEGF-C expression level was determined using RT-qPCR and western blotting, and the VEGF-C concentration in supernatant was measured by ELISA. Tumor xenograft models of HCT-116 cells were generated using BALB/c nude mice, and the mice were randomly divided into a control group (gavaged with normal saline) and QFG group (gavaged with 2 g/kg QFG). The effect of QFG on tumor growth was evaluated by comparing the volume and weight of tumors between two groups. Immunohistochemistry (IHC) and RT-qPCR were performed to detect the expression levels of VEGF-C, vascular endothelial growth factor receptor 3 (VEGFR-3), and LYVE-1 (lymphatic vessel endothelial hyaluronan receptor 1). ELISA was performed to measure the concentration of serum VEGF-C. TMT proteomics technology and Reactome pathway analysis were used to explore the mechanism of QFG inhibiting lymphangiogenesis in tumor. The VEGF-C (5 ng/mL)-stimulated human lymphatic endothelial cell (HLEC) model was conducted to evaluate the effect of QFG on lymphangiogenesis in vitro. The model cells were treated with different concentrations (0.2, 0.5, and 1.0 mg/mL) of QFG. Cell viability was then determined using an MTT assay. The cell migration, invasion, and tube-formation ability were analyzed using transwell migration, matrigel invasion and tube formation assays, respectively. The underlying mechanism was uncovered, the levels of VEGFR-3, matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9), p-PI3K/PI3K, p-AKT/AKT and p-mTOR/ mTOR were detected using western blotting. QFG significantly reduced VEGF-C expression and secretion in HCT-116 cells. QFG evidently suppressed in vivo tumor growth and the expression of VEGF-C, VEGFR-3, and LYVE-1. The serum VEGF-C level was also reduced by QFG. Moreover, TMT proteomics technology and Reactome pathway analysis identified 95 differentially expressed protein and multiple enriched pathway about matrix metalloproteinase and extracellular matrix, which is direct associate with lymphangiogenesis. In vitro experiment, QFG inhibited the viability, migration, invasion and tube formation of HLECs. Additionally, QFG reduced the VEGFR-3, MMP-2, MMP-9 expression levels, and the p-PI3K/PI3K, p-AKT/AKT, p-mTOR/ mTOR ratios. QFG can exert its effect on both tumor cells and HLECs, exhibiting ani- lymphangiogenesis in colorectal cancer via the VEGF-C/VEGFR-3 dependent PI3K/AKT pathway pathway.