METHODS: Cell viability was measured using a cell counting kit-8 (CCK-8) assay. Protein expression and phosphorylation levels were determined using western blotting. The interactions of mTOR and Raptor with RagC were determined by co-immunoprecipitation. Cells overexpressing RagC wild-type (RagCWT) and RagC Thr90Asn (RagCT90N) were generated by lentiviral infection. Raptor knockdown was performed by lentivirus-mediated shRNA transduction. The in vivo anti-tumour effect of cardamonin was assessed in a xenograft model.
RESULTS: Cardamonin disrupted mTOR complex interactions by decreasing Raptor protein levels. RagCT90N overexpression via lentiviral infection increased cell proliferation and mTORC1 activation. The viability and tumour growth rate of RagCT90N-mutant cells were more sensitive to cardamonin treatment than those of normal and RagCWT cells. Cardamonin also exhibited a stronger inhibitory effect on the phosphorylation of mTOR and p70 S6 kinase 1 in RagCT90N-mutant cells. Raptor knockdown abolishes the inhibitory effects of cardamonin on mTOR. An in vivo xenograft model demonstrated that the RagCT90N-mutant showed significantly higher sensitivity to cardamonin treatment.
CONCLUSIONS: Cardamonin exerts selective therapeutic effects on RagCT90N-mutant cells. Cardamonin can serve as a drug for individualised therapy for follicular lymphoma with RRAGC mutations.
方法:使用细胞计数试剂盒-8(CCK-8)测定法测量细胞活力。使用蛋白质印迹法测定蛋白质表达和磷酸化水平。通过免疫共沉淀确定mTOR和Raptor与RagC的相互作用。通过慢病毒感染产生过表达RagC野生型(RagCWT)和RagCThr90Asn(RagCT90N)的细胞。通过慢病毒介导的shRNA转导进行Raptor敲低。在异种移植模型中评估了豆蔻素的体内抗肿瘤作用。
结果:Cardamonin通过降低Raptor蛋白水平破坏mTOR复合物相互作用。经由慢病毒感染的RagCT90N过表达增加了细胞增殖和mTORC1活化。与正常细胞和RagCWT细胞相比,RagCT90N突变细胞的活力和肿瘤生长速率对豆蔻素治疗更敏感。在RagCT90N-突变细胞中,Cardamonin对mTOR和p70S6激酶1的磷酸化也表现出更强的抑制作用。Raptor敲除消除了豆蔻素对mTOR的抑制作用。体内异种移植模型证明RagCT90N-突变体显示出对豆蔻素治疗的显著更高的敏感性。
结论:Cardamonin对RagCT90N突变细胞具有选择性治疗作用。Cardamonin可作为RRAGC突变滤泡性淋巴瘤个体化治疗的药物。