Date of Award

11-18-2025

Degree Type

Thesis

Degree Name

MPhil in Biological and Biomedical Sciences

First Advisor

Dr. Arooj Shafi

Second Advisor

Dr. Kulsoom Ghias

Third Advisor

Dr. Azhar Hussain

Department

Biological and Biomedical Sciences

Abstract

Background: In 40–45% of colorectal cancer (CRC) cases K-Ras mutations occur and are associated with poor response to standard therapies. RMC-6236 is a newly developed, non-covalent inhibitor that specifically binds to the active, GTP-bound state of K-Ras, demonstrating efficacy in lung and pancreatic cancers. This study evaluates its effect on cell viability, apoptosis, and cell cycle progression via the K-Ras pathway and its associated down-stream targets such as Ral pathway in the K-RAS-mutant CRC cell line SW480.
Methods SW480 cells were treated with an increasing concentration of RMC-6236 in a time dependent manner. Cell viability was measured using Trypan Blue and XTT assays. DNA fragmentation and Giemsa staining assessed genotoxicity and morphology. Flow cytometry (PI, Annexin V/PI) evaluated cell cycle and apoptosis. RalA/B expression and apoptotic markers (procaspase, cleaved caspase and PRP) were analyzed via Western blotting. Cell migration was assessed using a wound healing assay, conducted under FBS-free conditions to minimize cell proliferation. A one-way ANOVA was performed to assess whether there were significant differences between the groups.
Results: XTT and Trypan Blue exclusion assays demonstrated a clear reduction in cell viability proportional to the dose (p < 0.01). DNA fragmentation was markedly elevated in treated groups compared with controls (p = 0.03), while Giemsa staining revealed progressive, dose-related morphological changes consistent with apoptosis. Flow cytometric cell cycle analysis indicated an v accumulation of cells in the sub-G0 fraction, accompanied by a reduction in G0/G1, S, and G2/M phase populations at higher concentrations of RMC-6236. Annexin V/PI staining further confirmed increased apoptotic cell death in response to treatment. Western blot analysis revealed enhanced levels of cleaved caspase-3, together with reduced levels of procaspase-3 a, supporting the induction of caspase-dependent apoptosis. Western blot analysis showed no significant alterations in RalA or RalB expression. The wound healing experiment showed that drug treatment notably hindered cell migration, evidenced by a greater unclosed wound area relative to the untreated control.
Conclusion: RMC-6236 induces dose-dependent cytotoxicity and apoptosis in K-Ras-mutant colorectal cancer cells, accompanied by pronounced cell cycle arrest and impaired cell migration. These findings support further investigation of RMC-6236 as a potential targeted therapy for colorectal cancer. These results highlight the need to investigate the impact of RMC-6236 on downstream K-Ras signaling pathways to better define its therapeutic potential in colorectal cancer.

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1

Last Page

52

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