Cantharidin analogue NCTD-01 inhibits AOM/DSS-induced orthotopic colorectal carcinogenesis in mice by suppressing inflammation-driven tumorigenesis.

Although cantharidin (CTD) exhibits significant clinical efficacy against various cancers, its clinical utility is severely hampered by profound systemic toxicity. Thus, structural modification of CTD to obtain analogues with potent anticancer activity and lower toxicity is essential for advancing CTD-based therapeutics. Building on our previous synthesis of a series of C-5 alkynyl-substituted CTD analogues with potent anti-inflammatory properties, this study investigates the anticancer potential of a new analogue NCTD-01 demonstrated a favorable safety profile in mice, with the maximum tolerated dose (MTD) for oral administration exceeding 1852 mg/kg. In addition to exhibiting potent in vitro and in vivo anti-inflammatory activities comparable to its analogues, NCTD-01 displayed cytotoxicity against colon cancer cell lines and exhibited therapeutic efficacy in an azoxymethane/dextran sulfate sodium-induced orthotopic mouse model of colorectal cancer. Molecular analyses revealed that NCTD-01 reduced Ki-67 expression, suppressed the mRNA levels of pro-inflammatory and immunosuppressive mediators (IL-1β, COX-2, IL-6, IL-10, and TGF-β) in colonic tumor tissues, and decreased serum levels of TNF-α and IL-1β. Furthermore, NCTD-01 treatment led to reduced expression of Foxp3, CD25, PD-1, and PD-L1, alongside enhanced CD8 and GITR expression in colorectal tissues, suggesting an effective modulation of tumor-associated immune responses. This study suggests that NCTD-01, a new C-5 alkynyl-substituted CTD analogue, may help alleviate the inflammation-to-cancer transition through tumor-associated immune modulation while maintaining a favorable safety profile, pointing to its potential as a candidate for anticancer drug discovery.