Mechanisms of apoptosis in colon cancer cells induced by IDO1 inhibitor and irradiation: A metabolomic study

Background: Indoleamine 2,3-dioxygenase 1 (IDO1) catalyzes the conversion of tryptophan to kynurenine, promoting tumor progression via immune suppression and activation of the Wnt/β-catenin pathway. As the activation of kynurenine pathway also produces key metabolites such as nicotinamide adenine dinucleotide (NAD ⁺ ), its inhibition may influence tumor cell metabolism and contribute to anti-tumor effects. This study investigated the mechanisms underlying the combined effects of IDO1 inhibition and radiation (Rad) on colon cancer cells, with a focus on metabolic alterations. Methods: Colon26 murine colon cancer cells were treated with an IDO1 inhibitor, 1-methyltryptophan (1-MT), Rad (10 Gy/10 fractions), or both. Apoptosis was evaluated in cultured cells and subcutaneous tumors. Apoptosis-related proteins in cultured Colon26 cells treated with 1-MT and/or Rad were evaluated by Western blot analysis. Metabolome analysis assessed intracellular changes in metabolites including NAD ⁺ of kynurenine pathways caused by these treatments. The additional effect of β-nicotinamide mononucleotide (NMN) was evaluated. Results: Combination treatment with 1-MT and Rad increased apoptosis compared to Rad alone in vitro (7.0% vs. 5.5%, p = 0.0495) and in vivo (0.19% vs. 0.13%, p = 0.025). Western blotting showed elevations of cleaved caspase 3, 8, and 9 by dual treatments. NAD ⁺ levels decreased by additional 1-MT in radiated Colon26 cells (452 vs. 1027 nmol/g, p = 0.0495). Supplementation with NMN (500 μM) restored NAD ⁺ levels and suppressed apoptosis in cells treated with 1-MT and Rad. Conclusions: IDO1 inhibition enhanced Rad-induced apoptosis in Colon26 cell through NAD ⁺ depletion. Targeting NAD ⁺ synthesis may represent a therapeutic approach in colorectal cancer.