Therapeutic effects of dihydroartemisinin in multiple stages of colitis-associated colorectal cancer

Abstract

Colitis-associated colorectal cancer (CAC) develops from chronic intestinal inflammation. Dihydroartemisinin (DHA) is an antimalarial drug exhibiting anti-inflammatory and anti-tumor effects. Nonetheless, the therapeutic effects of DHA on CAC remain unestablished. Methods: Mice were challenged with azoxymethane (AOM) and dextran sulfate sodium (DSS) to establish CAC models. DHA was administered via oral gavage in different stages of CAC models. Colon and tumor tissues were obtained from the AOM/DSS models to investigate inflammatory responses and tumor development. Inflammatory cytokines in the murine models were detected through qRT-PCR and ELISA. Toll-like receptor 4 (TLR4) signaling-related proteins were detected by western blot. Macrophage infiltration was measured using immunostaining analysis, and apoptosis in the colon cancer cells was detected by flow cytometry and western blot. Results: DHA inhibited inflammatory responses in the early stage of the AOM/DSS model and subsequent tumor formation. In the early stage, DHA reversed macrophage infiltration in colon mucosa and decreased the expression of pro-inflammatory cytokines. DHA inhibited the activation of macrophage by suppressing the TLR4 signal pathway. In the late stage of CAC, DHA inhibited tumor growth by enhancing cell cycle arrest and apoptosis in tumor cells. Administration of DHA during the whole period of the AOM/DSS model generated an addictive effect based on the inhibition of inflammation and tumor growth, thereby improving the therapeutic effect of DHA on CAC. Conclusion: Our study indicated that DHA could be a potent agent in managing the initiation and development of CAC without obvious side effects, warranting further clinical translation of DHA for CAC treatment.

Keywords: Dihydroartemisinin; anti-inflammation; anti-tumor; colitis-associated colorectal cancer; macrophage.

Figure 1

DHA alleviates DSS induced colitis. (A) The survival rate of DSS-induced lethal colitis treated with PBS or DHA. A log-rank test was used to verify the Kapla-Meier curves. N = 8 in each group, p < 0.001. (B) Flowchart for establishing the early stage (the stage of chronic colitis) of CAC models. (C) Weight changes were monitored during the process of AOM/DSS murine model. (D) Disease activity index was measured for each treatment group. (E) The macroscopic appearance of the colon. (F) The colon length was measured. (G) Representative H&E staining for colon mucosa from each treatment group. N = 5 in control and DHA groups. N = 10 in AOM+DSS and AOM+DSS+DHA groups. Data are presented as mean ± SD. ** p < 0.01, **** p < 0.0001. ns: non-significantly.

Figure 2

DHA suppresses inflammatory responses in the early CAC stages. Inflammatory cytokine expression levels in LPMC (A) and serum (B) from different treatment groups on day 22 of the CAC murine model. Colonic cytokines levels were determined by qRT-PCR while serum levels were determined by ELISA. (C) The expression levels of indicated proteins from LPMC. (D) IHC analysis of immune cell markers expressed on the colonic mucosa in the early CAC stage. (E) Quantitative analysis of positive cells was performed by Image-Pro Plus 5.0. (F) IF analysis of iNOS expressed on the colonic mucosa. (G) iNOS positive cells were counted. N = 3 in each group. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns: non-significantly.

Figure 3

DHA inhibits the activation of macrophages in vitro. (A) qRT-PCR analysis for mRNA levels of inflammatory cytokines in BMDM and RAW264.7 treated with LPS and DHA. The analysis of TLR4-related protein expression in BMDM (B) and RAW264.7 (C). (D) The analysis for cell migration in THP-1 and RAW264.7. (E) The number of migrating cells was measured. Data are presented as mean ± SD. * p < 0.05, *** p < 0.001, **** p < 0.0001. All experiments were repeated three times.

Figure 4

DHA inhibits the initiation and development of CAC. (A) The flowchart for establishing a CAC model. DHA was administered in the early stage, late stage, and the whole stage of CAC. Colon tumors were analyzed on day 101. (B) Macroscopic appearance of colon images. The yellow arrows indicate colon tumors. (C) The tumor number, (D) sum of tumor diameters, tumor size, and (E) the percentage of different tumor sizes were measured. (F) Representative H&E staining for colon epithelium from each treatment group. N = 10 in each group. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01. **** p < 0.0001. ns: non-significantly.

Figure 5

DHA exhibits anti-tumor effects on tumor cells from the CAC model. (A) IHC analysis of c-PARP in tumor tissues from CAC models treated with DHA in different stages. (B) Quantitative analysis of positive cells was performed by Image-Pro Plus 5.0. (C) Tunel analysis in tumor cells from CAC model. (D) Tunel positive cells were counted. (E) Apoptosis and cell cycle-related proteins in tumor tissues were detected by western blot. N = 3 in each group. Data are presented as mean ± SD. * p < 0.05, *** p < 0.001. **** p < 0.0001.

Figure 6

DHA exhibits anti-tumor effects in colon cancer cells. (A) Viabilities of HCT116 and RKO cells treated with various concentrations of DHA at different times. (B) Expression levels of apoptosis-associated proteins in HCT116 and RKO cell lines treated with DHA. (C) Flow cytometric analysis of apoptosis in HCT116 and RKO cell lines treated with DHA and Z-VAD-FMK. (D) The percentage of apoptotic cells was measured. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001. All experiments were repeated three times.

Figure 7

A schematic diagram illustrating the therapeutic effects of DHA in multiple stages of CAC. In CAC murine model, macrophages are activated and infiltrated in the colonic mucosa. Thereafter, the colonic epithelium will gradually transform from adenoma to adenocarcinoma due to inflammatory response. DHA inhibits CAC development both in the early and late stage.