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Abstract：
BACKGROUND: mTORC1 (mechanistic target of rapamycin complex 1) is associated with lymphoma progression. Oncogenic RRAGC (Rag guanosine triphosphatase C) mutations identified in patients with follicular lymphoma facilitate the interaction between Raptor (regulatory protein associated with mTOR) and Rag GTPase. It promotes the activation of mTORC1 and accelerates lymphomagenesis. Cardamonin inhibits mTORC1 by decreasing the protein level of Raptor. In the present study, we investigated the inhibitory effect and possible mechanism of action of cardamonin in RRAGC-mutant lymphoma. This could provide a precise targeted therapy for lymphoma with RRAGC mutations.
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.
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.
摘要：
背景：mTORC1（雷帕霉素复合物1的机制靶标）与淋巴瘤进展相关。在滤泡性淋巴瘤患者中发现的致癌RRAGC（Rag鸟苷三磷酸酶C）突变促进Raptor（与mTOR相关的调节蛋白）和RagGTP酶之间的相互作用。它促进mTORC1的激活并加速淋巴生成。Cardamonin通过降低Raptor的蛋白质水平来抑制mTORC1。在本研究中,我们研究了豆蔻素对RRAGC突变淋巴瘤的抑制作用和可能的作用机制。这可以为具有RRAGC突变的淋巴瘤提供精确的靶向治疗。
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。
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。
