4α,5α-epoxy-10α,14H-1-epi-inuviscolide induces mitochondrial-mediated apoptosis and suppresses SW480 colorectal cancer cell growth in vitro and in vivo

Abstract 4 α ,5 α -epoxy-10 α ,14H-1-epi-inuviscolide (INV), a guaianolide sesquiterpene lactone, has received limited pharmacological investigation beyond its reported antifungal and antibacterial activities; its anticancer effects remain entirely uncharacterized. Cytotoxicity, cellular and nuclear morphology, and clonogenic potential of INV were evaluated. Network pharmacology and Venn analysis cross-referencing INV’s targets with SW480 transcriptomic data (GEO: GSE345612) identified five core apoptotic targets ( BAX , BCL2 , CASP3 , CASP9 , and CYCS ). Binding of INV to the predicted pockets of BAX and BCL2 was evaluated using molecular docking. Apoptosis, cell-cycle distribution, and key associated regulatory proteins were assessed by flow cytometry and Western blotting. In vivo antitumor efficacy at 40 and 80 mg/kg (MPK) was evaluated in a SW480 tumour-bearing mouse model. Preliminary safety was assessed using a single acute oral toxicity study (OECD 423; 300 and 2000 MPK). INV exhibited antiproliferative activity across a panel of CRC cell lines, with the best activity against SW480 cells (IC 50 : 4.65 µ M). INV upregulated BAX, cleaved caspase 3, and cleaved PARP, and downregulated BCL2 and cell-cycle regulatory proteins, cyclin D1 and CDK4. Elevated reactive oxygen species (ROS) disrupted mitochondrial membrane potential and triggered cytochrome C release, implicating the intrinsic mitochondrial apoptotic pathway. Orally administered INV demonstrated significant tumour growth inhibition of 78.26% and 96.71% at 40 and 80 MPK, respectively. Acute toxicity evaluation at 300 and 2000 MPK revealed no clinical signs or mortality, indicating an LD 50 > 5000 MPK. Collectively, these findings provide new evidence of INV efficacy in SW480 cells, supporting further investigation of INV as a potential anticancer lead.