A novel dihydroisoxazole derivative DHI9 suppresses colorectal cancer cell survival by inducing oxidative stress, DNA damage, and mitochondrial apoptosis while targeting Akt and Wnt/β-catenin signalling.

Colorectal cancer is one of the most common malignancies worldwide and remains a major cause of cancer-related mortality. Therefore, the identification of novel compounds with selective anticancer activity represents an important research priority. Dihydroisoxazole derivatives represent a promising class of heterocyclic compounds with potential antitumour properties. This study evaluated the anticancer effects of the dihydroisoxazole derivative DHI9 in human colorectal cancer cell lines HCT116 and Caco-2 and investigated the molecular mechanisms involved. The cytotoxic effect of DHI9 was determined using the MTT assay, revealing IC50 values of 2.5 µM in HCT116 cells and 10.0 µM in Caco-2 cells after 72 h treatment. In non-tumour CCD-18Co cells, the IC50 value was 127.3 µM, indicating promising selective cytotoxicity toward the tested colorectal cancer cells. All subsequent biological and mechanistic experiments were performed exclusively in HCT116 and Caco-2 cancer cells. In these cancer cells, DHI9 inhibited cell proliferation, induced G2/M cell cycle arrest after 24 h, and caused a time-dependent increase in the sub-G0/G1 cell population. Furthermore, DHI9 also increased p21 expression, ROS production, and phospho γH2A.X levels, indicating oxidative stress and activation of the DNA damage response. Apoptosis induction was confirmed by phosphatidylserine externalization, loss of mitochondrial membrane potential, cytochrome c release, and increased caspase-9 and caspase-3/7 activity. In addition, fluorescence microscopy showed changes in nuclear morphology and actin cytoskeleton organization. Western blot analysis demonstrated that DHI9 modulated selected proteins involved in Wnt/β-catenin and Akt signalling pathways, apoptosis, and cell cycle regulation, including PCNA, Dvl2, p-Akt, p-GSK-3β, total GSK-3β, β-catenin, non-phosphorylated β-catenin, PARP, p-Rb, p-Wee1, p-Cyclin B1, and p-Cdc2. Moreover, DHI9 also reduced the migration of both colorectal cancer cell lines and enhanced the cytotoxic activity of 5-fluorouracil, with predominantly synergistic interactions according to the Highest Single Agent model.