Abstract A004: Experimental and Computational Validation of Alcea rosea Compoundss in Blocking Colorectal Cancer Progression

Background: RAS mutations are major oncogenic drivers in colorectal cancer, but their therapeutic targeting remains challenging due to the complex, tissue-specific signaling networks they create. To explore alternative strategies for KRAS-mutant colorectal cancer, bioactive metabolites from Alcea rosea were evaluated for their effects on RAS-linked pathways. Building on earlier findings showing anti-inflammatory and pro-apoptotic activity, these compounds were tested in HCT116 (KRAS-mutant) and HTC9 colorectal cancer cells, with additional molecular docking and molecular dynamics (MD) simulations to predict interactions with key RAS-associated proteins. Methods: Alcea rosea compounds were assessed using MTT viability, colony formation, wound healing, spheroid formation, DAPI–PI staining, AO/EtBr staining, ROS measurement, and rhodamine-123 mitochondrial membrane potential assays in HCT116 and HTC9 cells. Western blotting examined cleaved PARP, Caspase-3, phospho-ERK, phospho-MEK, and stemness-associated markers. Molecular docking was performed against MEK, ERK, PI3K, and RAF to evaluate binding affinities. MD simulations (100 ns) assessed complex stability, conformational behavior, RMSD, RMSF, and hydrogen-bonding patterns. Results: Alcea rosea metabolites inhibited proliferation, colony formation, migration, and spheroid growth in both cell lines. Apoptosis was confirmed by DAPI-PI and AO/EtBr staining and increased cleavage of PARP and Caspase-3. Treatment elevated ROS and disrupted mitochondrial membrane potential, consistent with oxidative stress mediated apoptosis. Western blotting showed reduced MEK (2.7-fold) and ERK (1.8-fold) phosphorylation, indicating suppression of KRAS-driven signaling. Stemness markers were downregulated, supporting inhibition of self-renewal pathways. Docking studies revealed strong predicted binding of the metabolites to MEK, ERK, and PI3K, suggesting interference with RAS–MAPK and PI3K signaling. MD simulations demonstrated stable ligand protein interactions with low RMSD fluctuations and persistent hydrogen bond occupancy. Conclusions: Alcea rosea metabolites exert multifaceted anticancer activity in KRAS mutant colorectal cancer by inducing apoptosis, elevating ROS, impairing mitochondrial function, reducing stemness, and suppressing RAS dependent signaling. Docking and MD analyses further support their potential to target KRAS associated pathways. These findings justify further in-vivo validation toward possible translational development. Agreement on Use of Generative AI: Generative artificial intelligence was used solely for language refinement and restructuring of author-written text; no AI tools were used in the design, execution, analysis, or interpretation of the underlying study. Citation Format: Ruhban Ansar. Parry. Experimental and Computational Validation of Alcea rosea Compoundss in Blocking Colorectal Cancer Progression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RAS Oncogenesis and Therapeutics; 2026 Mar 5-8; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(5_Suppl_1):Abstract nr A004.