Indole alkaloid corynan isolated from <i>Petasites hybridus</i> as a potential antitumor agent.

Abstract

e15158 Background: The aim of this study was to evaluate the antitumor activity of the alkaloid corynan, which we obtained, in silico and in vitro on H1299 and HT29 tumor cell lines. Methods: Corynan was extracted from the plant material Petasites hybridus using preparative chromatography. Its structure was confirmed by Nuclear Magnetic Resonance (NMR) as (2S,3R,12bS)-2,3-diethyl-12b-methyl-1,2,3,4,6,7,7a,12,12a,12b-decahydroindolo[2,3-a]quinolizine. Molecular docking of corynan with five receptors involved in carcinogenesis — EGFR, PDGFR, MET, MRP2, and NOX4—was performed using AutoDock Vina 4.0. PDB structures were imported into Discovery Studio Visualizer 4.0. AlphaFoldDB structures were already cleaned. These structures were optimized prior to docking using Chimera 1.6.2. The resulting .pdb files were validated using the PROCHECK server and aligned with the original structure. These files were used to prepare the grid in AutoDockTools-1.5.7. H1299 (non-small cell lung cancer) and HT29 (colorectal cancer) cells were cultured under standard conditions in MEM medium. Upon reaching 75-80% confluence, the medium was replaced with one containing corynan (128 µM concentration), and the cells were cultured for 72 hours. Cell viability was determined using a NanoEnTek JuliFl (Korea) counter in the presence of 0.4% trypan blue. The assessment of expression levels after treatment was performed by RT-PCR using a gene panel: CASP9, CASP7, CASP3, TP53, MDM2, BAX, BCL2, PTEN, ATM, MTCH1, CYCS, DIABLO, VDAC1. Results: Corynan demonstrated the highest affinity for the MET protein (-8.6 kcal/mol), followed by PDGFR (-7.2 kcal/mol), EGFR and MRP2 (-6.4 kcal/mol and -6.3 kcal/mol), and NOX4 (-5.9 kcal/mol). Corynan at a concentration of 128 µM and 72-hour exposure caused death in 60% of HT29 cells and 36% of H1299 cells. Under the same conditions, corinane caused a 22-fold increase in CASP9 expression in H1299 cells, and in HT29 cells, a 9.5-fold increase in CASP9 expression and a 12.5-fold increase in CASP7 expression. In HT29 cells, it caused an 82-fold increase in BAX expression and a 64-fold increase in MTCH1 expression. No statistically significant changes were observed in the expression of the other investigated genes. Conclusions: Molecular docking of corynan showed that it could be an effective inhibitor of the MET protein—a key factor in invasion and metastasis. The inhibition of viability in HT29 and H1299 cell lines in vitro and the changes in the expression of caspase genes, BAX , and MTCH1 suggest that corynan possesses antitumor activity and provides a basis for its further study.