MRTX1133联合SMAD3抑制剂作用于KRAS G12D突变胰腺癌细胞的机制研究OA
Effects of MRTX1133 combined with SMAD3 inhibitor on pancreatic carcinoma cells with KRAS G12D mutation and their mechanism
目的:本实验将KRAS(Kirsten rat sarcoma)G12D突变蛋白小分子抑制剂MRTX1133和转化生长因子β(transforming growth factor-β,TGF-β)/SMAD3通路抑制剂SIS3联合作用于KRAS G12D突变胰腺癌细胞系,并探索两药联合的作用机制.方法:使用分子对接软件计算MRTX1133与KRAS G12D突变蛋白的结合能;并使用CCK-8实验测定药物的IC50;后续将两种细胞系按实验需要分组,测定细胞活力分析联合效应;通过EdU实验和流式细胞术测定subG1期细胞比例和细胞周期以验证两药联合对细胞增殖的影响;Transwell实验验证两药联合对肿瘤细胞迁移和侵袭的影响;Western blot实验检测Jagged1(JAG1)和NOTCH4的蛋白表达水平以探索联合机制;最后通过Western blot实验检测醛脱氢酶1家族成员A1(aldehyde dehydrogenase 1 family member A1,ALDH1A1)蛋白表达水平及流式细胞术测定CD133+细胞比例来验证两药对肿瘤细胞干性的影响.结果:MRTX1133对PANC-1和SW1990细胞系的IC50分别为0.894 µmol/L和1.115 µmol/L;KRAS G12D突变胰腺癌细胞系中TGF-β蛋白的表达水平相对于正常胰腺导管细胞显著升高.两药联用后,通过细胞活力计算出联合指数和HAS(highest single-agent)指数,均表明两药具有联合效应;两药联用对肿瘤细胞增殖有明显抑制作用,且明显减少细胞周期中S期细胞比例;肿瘤细胞迁移侵袭能力亦有明显降低;两药联用可抑制NOTCH通路配体JAG1的生成,从而抑制NOTCH4通路;两药联用明显降低CD133+细胞比例及ALDH1A1的蛋白表达水平,表明两药通过抑制肿瘤细胞干性而达到联合作用.结论:MRTX1133联合SIS3通过抑制JAG1生成,干扰NOTCH4通路,从而减弱肿瘤细胞干性,在KRAS G12D突变胰腺癌细胞系中发挥协同抑制作用.
AIM:To investigate the combining effect of a specific Kirsten rat sarcoma(KRAS)G12D inhibi-tor MRTX1133 in combination with an SMAD3 inhibitor SIS3 against pancreatic carcinoma cells with KRAS G12D muta-tion,and to explore the underlying combining mechanism.METHODS:The potential affinity of MRTX1133 with KRAS G12D mutant protein was analyzed using molecular docking software.Cell viability was measured using Cell Counting Kit-8,and then the dose-effect curve and IC50 value were plotted.The combination index of MRTX1133 with SIS3 was calcu-lated using CompuSyn.The EdU incorporation assay was used to assess cell proliferation.Cell cycle profile and CD133-positive cell population was analyzed by flow cytometry.The invasion and migration of pancreatic carcinoma cells were de-tected using Transwell assays.Western blot was used to evaluated Jagged1(JAG1),NOTCH4 and aldehyde dehydroge-nase 1 family member A1(ALDH1A1)expression.RESULTS:The IC50 values of MRTX1133 were 0.894 µmol/L and 1.115 µmol/L in PANC-1 and SW1990 pancreatic carcinoma cells,respectively.Combined treatment with MRTX1133 and SIS3 showed the synergic effect.The combination of MRTX1133 with SIS3 reduced cell proliferation,delayed the pro-gression of cell cycle,and decreases the invasion and migration of pancreatic carcinoma cells.The CD133-positive cell population and ALDH1A1 expression were decreased in the combination group,suggesting that the combination of MRTX1133 with SIS3 can inhibit pancreatic carcinoma cell stemness.The mechanism study found that MRTX1133 in combination of SIS3 can inhibit JAG1 of NOTCH ligand expression.CONCLUSION:The combination of MRTX1133 with SIS3 affects NOTCH4 pathway and inhibits tumor stemness,thus showing the synergic effect against pancreatic carci-noma with KRAS G12D mutation.
张榜梅;蔡梅;王宏;章俊
贵州医科大学病理学教研室,贵州 贵阳 561113贵州医科大学病理学教研室,贵州 贵阳 561113贵州医科大学病理学教研室,贵州 贵阳 561113贵州医科大学病理学教研室,贵州 贵阳 561113||贵州医科大学附属医院病理科,贵州 贵阳 550004
医药卫生
胰腺癌MRTX1133KRAS G12D突变SMAD3蛋白
pancreatic carcinomaMRTX1133KRAS G12D mutationSMAD3 protein
《中国病理生理杂志》 2026 (3)
417-427,11
国家自然科学基金资助项目(No.82060565)病理形态及分子实验室项目(No.2025FYYJ-001)
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