PO.MCB03.01 · 分子与细胞生物学
评估 Pan-KRas 抑制剂 RMC-6236 对 KRas 突变型结直肠癌细胞的效应
Evaluating the effects of the Pan-KRas inhibitor RMC-6236 in KRas-mutant colorectal cancer cells
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：KRas 突变发生在约 40-45% 的结直肠癌（CRC）中，且具有高度异质性，存在不同的密码子变异。这种多样性使 KRas 成为 CRC 中一个具有挑战性的治疗靶点。RMC-6236 是一种新型非共价 pan-KRas 抑制剂，可选择性靶向多种 KRas 变异中活性的、GTP 结合形式的 KRas。本研究评估其对 KRas 突变型 CRC 细胞的活力、凋亡和细胞周期进展的效应。
方法：用递增浓度的 RMC-6236（0、10、25 和 50 nM）处理 SW480 细胞。使用台盼蓝排斥法和 XTT 法评估细胞活力。DNA 片段化和吉姆萨染色评估形态学变化和凋亡。流式细胞术用于评估细胞周期分布（PI 染色）和凋亡（Annexin V/PI）。所有实验均一式三份进行。使用 Western blot 评估凋亡标志物（procaspase-3 和剪切型 caspase-3）的表达水平。使用单因素方差分析（ANOVA）确定统计学显著性（p < 0.05）。
结果：台盼蓝和 XTT 实验表明，RMC-6236 处理的 SW480 细胞活力呈显著的剂量依赖性降低（p < 0.001）。DNA 片段化增加（p = 0.03），吉姆萨染色显示特征性凋亡形态。流式细胞术显示剂量依赖性的 G0/G1 细胞周期阻滞、sub-G0 累积增加以及 S 期和 G2/M 期细胞群减少。Annexin V/PI 分析证实凋亡细胞呈剂量依赖性增加。Western blot 显示剪切型 caspase-3 增加、procaspase-3 减少，与凋亡通路的激活一致。
结论：RMC-6236 在 KRas 突变型 CRC 细胞中诱导细胞毒性、凋亡和细胞周期紊乱，支持将其作为一种能够应对 KRas 突变异质性的治疗方法进一步评估。该抑制剂对下游 KRas 致癌通路（包括 PI3K 和 MAPK 信号传导）的影响也将在未来研究中进行探讨。
查看英文原文 English abstract
Background: KRas mutations occur in ~40-45% of colorectal cancers (CRC) and are highly heterogeneous, with distinct codon variants. This diversity makes KRas a challenging therapeutic target in CRC. RMC-6236 is a novel, non-covalent pan-KRas inhibitor that selectively targets active, GTP-bound KRas across multiple KRas variants. This study evaluates its effects on viability, apoptosis, and cell-cycle progression in KRas-mutant CRC cells.
Methods: SW480 cells were treated with increasing RMC-6236 concentrations (0, 10, 25 and 50 nM). Cell viability was assessed using Trypan Blue exclusion and XTT assays. DNA fragmentation and Giemsa staining evaluated morphological changes and apoptosis. Flow cytometry was used to assess cell-cycle distribution (PI staining) and apoptosis (Annexin V/PI). All experiments were performed in triplicate. Expression levels of apoptotic markers (procaspase-3 and cleaved caspase-3) were assessed using western blotting. Statistical significance was determined using one-way ANOVA (p < 0.05).
Results: Trypan Blue and XTT assays demonstrated a significant, dose-dependent reduction in cell viability in RMC-6236-treated SW480 cells (p < 0.001). DNA fragmentation increased (p = 0.03), and Giemsa staining showed characteristic apoptotic morphology. Flow cytometry demonstrated dose-dependent G0/G1 cell-cycle arrest, increased sub-G0 accumulation, and reduced S and G2/M phase populations. Annexin V/PI analysis confirmed a dose-dependent increase in apoptotic cells. Western blotting showed increased cleaved caspase-3 and decreased procaspase-3, consistent with activation of apoptotic pathways.
Conclusion: RMC-6236 induces cytotoxicity, apoptosis, and cell-cycle disruption in KRas-mutant CRC cells, supporting its further evaluation as a therapeutic approach capable of addressing KRas mutation heterogeneity. The impact of the inhibitor on downstream KRas oncogenic pathways, including PI3K and MAPK signaling will also be investigated in future studies.
利益披露 Disclosure
S. nadir, None..
S. B. Hamid, None..
A. Shafiq, None.