The influence of isothiocyanates on colorectal cancer: an experimental study

Colorectal carcinoma (CRC) has poor prognosis, and current treatment strategies including therapy regimens based on 5-fluorouracil (5-FU) combined with irinotecan (CPT-11) or bevacizumab (BVZ), do not yield the desired outcomes. Therefore, exploring alternative drug combinations is encouraged, including the use of food-derived bioactive compounds, which antitumor potential remains unexplored. Hence, we evaluated whether allyl (AITC) and phenethyl (PEITC) isothiocyanates enhance the antitumor effects of current therapy regimens for CRC. Heterotypic spheroids (3D) formed by HCT-116 CRC cells and CCD-18Co colonic fibroblasts (in ultra-low adherence plates, 2:1 ratio), or HCT-116 monolayers (2D) were treated with AITC, PEITC, BVZ, CPT-11, or 5-FU to determine the half-maximal effective concentration (EC₅₀) by MTT assay (24, 48 h). Then, 1/5 or 1/10 EC₅₀ of AITC (1513 and 757 µM) and PEITC (8.8 and 4.4 µM) were combined with 5-FU+CPT-11 or 5-FU+BVZ doublets at 1/5 (1+30 µM or 1+15 µM) and 1/10 (0.5+15 µM or 0.5+7.5 µM) of the EC₅₀ - similarly to plausible serum and tissue levels - cell viability was also evaluated, and the Combinatory Index (CI, 1= additive, <1 synergism, >1 antagonism) was calculated. Data were analyzed by one-way ANOVA and post hoc Tukey test (p<0.05). AITC promoted the BVZ+5-FU-induced reduction in spheroid viability, whilst it did not alter CPT-11+5-FU effects. Indeed, the CI indicated antagonism between the PEITC and the therapies (PEITC+5-FU+BVZ: 7.64; PEITC+5-FU+CPT-11: 1.06), while AITC had synergistic effects with 5-FU+BVZ doublet (AITC+5-FU+BVZ: 0.97; AITC+5-FU+CPT-11: 1.77). In the HCT-116/CCD-18Co transwell model (2D), 1/5 EC₅₀ AITC (188 µM) was combined with 1/5 EC₅₀ 5-FU+BVZ (11+7 µM) to assess motility, migration and colony formation after 36 hours. The combination reduced CRC cell motility but did not alter invasion or colony formation. In the cell-line derived xenograft (CDX) model, HCT-116 (1x10⁶) and CCD-18Co (1x10⁵) were inoculated into male BALB/c nude mice, which were then treated with AITC (1, 5, and 10 mg/kg) and 5-FU+BVZ (40+10 mg/kg) doublet for two weeks, the tumor volume was measured every two days to monitor tumor growth (Ethics committee #1486/2026). After euthanasia, serum biochemical parameters (ALT, AST, and creatinine) were assessed, and histological analyses were performed to quantify apoptosis and Ki-67 (i.e., proliferation) in tumor tissues.  RNA sequencing of tumor samples was also performed. The AITC+5-FU+BVZ combination neither modified further therapy-induced reduction in tumor growth, nor altered apoptosis and proliferation. In accordance, AITC+5-FU+BVZ combination had minimal transcriptomic impact in xenograft tumors. Overall, AITC enhanced the antitumor effects of 5-FU+BVZ in vitro, but this effect was not reproduced in vivo. While previous findings reported antitumor effects of AITC as a single agent in CRC models, its ability to enhance the efficacy of therapy remains to be clarified.

This study was supported by the São Paulo Research Foundation (#22/06082-8; #2023/05411-0; #25/15456-7).