目的探讨小豆蔻明(Cardamonin,CAR)抑制Raptor(Regulatory associated protein of mTOR)蛋白表达而调控HepG2细胞增殖和凋亡的作用。方法生物信息学分析TCGA(The Cancer Genome Atlas)数据库中肝癌患者RPTOR基因表达的差异及其与患者总...目的探讨小豆蔻明(Cardamonin,CAR)抑制Raptor(Regulatory associated protein of mTOR)蛋白表达而调控HepG2细胞增殖和凋亡的作用。方法生物信息学分析TCGA(The Cancer Genome Atlas)数据库中肝癌患者RPTOR基因表达的差异及其与患者总体生存率、无进展生存期等预后指标关系;CCK-8检测细胞活力;EdU核染色检测细胞增殖;流式细胞术检测细胞凋亡;Western Blot检测凋亡蛋白cleaved caspase 3、mTORC1信号通路关键蛋白mTOR、p-mTOR、S6K1、p-S6K1及mTORC1特异性结合蛋白Raptor的表达。结果RPTOR基因在肝癌组织中的表达水平高于正常组织(P<0.05),且高表达患者的总体生存率和无进展生存期显著低于低表达的患者(P<0.05);CAR抑制HepG2细胞增殖,诱导HepG2细胞凋亡,增加凋亡相关蛋白cleaved caspase3的表达;CAR降低肝癌HepG2细胞Raptor蛋白的表达,抑制mTOR、S6K1蛋白的磷酸化,但对其总蛋白的表达无明显影响。结论RPTOR基因在肝癌组织中高表达,与总体生存率和无进展生存期呈负相关;CAR可抑制肝癌HepG2细胞的增殖,促进其凋亡,机制与CAR降低Raptor的表达抑制mTORC1通路活性有关。展开更多
Background:Acetaminophen(APAP)overdose can cause liver injury and liver failure,which is one of the most common causes of drug-induced liver injury in the United States.Pharmacological activation of autophagy by inhib...Background:Acetaminophen(APAP)overdose can cause liver injury and liver failure,which is one of the most common causes of drug-induced liver injury in the United States.Pharmacological activation of autophagy by inhibiting mechanistic target of rapamycin(mTOR)protects against APAP-induced liver injury likely via autophagic removal of APAP-adducts and damaged mitochondria.In the present study,we aimed to investigate the role of genetic ablation of mTOR pathways in mouse liver in APAP-induced liver injury and liver repair/regeneration.Methods:Albumin-Cre(Alb-Cre)mice,mTOR^(f/f) and Raptor^(f/f) mice(C57BL/6J background)were crossbred to produce liver-specific mTOR knockout(L-mTOR KO,Alb Cret/-,mTOR^(f/f))and liver-specific Raptor KO(L-Raptor,Alb Cret/-,Raptor^(f/f))mice.Alb-Cre littermates were used as wild-type(WT)mice.These mice were treated with APAP for various time points for up to 48 h.Liver injury,cell proliferation,autophagy and mTOR activation were determined.Results:We found that genetic deletion of neither Raptor,an important adaptor protein in mTOR complex 1,nor mTOR,in the mouse liver significantly protected against APAP-induced liver injury despite increased hepatic autophagic flux.Genetic deletion of Raptor or mTOR in mouse livers did not affect APAP metabolism and APAP-induced c-Jun N-terminal kinase(JNK)activation,but slightly improved mouse survival likely due to increased hepatocyte proliferation.Conclusions:Our results indicate that genetic ablation of mTOR in mouse livers does not protect against APAP-induced liver injury but may slightly improve liver regeneration and mouse survival after APAP overdose.展开更多
文摘目的探讨小豆蔻明(Cardamonin,CAR)抑制Raptor(Regulatory associated protein of mTOR)蛋白表达而调控HepG2细胞增殖和凋亡的作用。方法生物信息学分析TCGA(The Cancer Genome Atlas)数据库中肝癌患者RPTOR基因表达的差异及其与患者总体生存率、无进展生存期等预后指标关系;CCK-8检测细胞活力;EdU核染色检测细胞增殖;流式细胞术检测细胞凋亡;Western Blot检测凋亡蛋白cleaved caspase 3、mTORC1信号通路关键蛋白mTOR、p-mTOR、S6K1、p-S6K1及mTORC1特异性结合蛋白Raptor的表达。结果RPTOR基因在肝癌组织中的表达水平高于正常组织(P<0.05),且高表达患者的总体生存率和无进展生存期显著低于低表达的患者(P<0.05);CAR抑制HepG2细胞增殖,诱导HepG2细胞凋亡,增加凋亡相关蛋白cleaved caspase3的表达;CAR降低肝癌HepG2细胞Raptor蛋白的表达,抑制mTOR、S6K1蛋白的磷酸化,但对其总蛋白的表达无明显影响。结论RPTOR基因在肝癌组织中高表达,与总体生存率和无进展生存期呈负相关;CAR可抑制肝癌HepG2细胞的增殖,促进其凋亡,机制与CAR降低Raptor的表达抑制mTORC1通路活性有关。
基金This work is partially funded by the USA National Institutes of Health R01 DK 102142(W.-X.Ding)We thank Margitta Lebofsky for technical assistance in the measurement of hepatic GSH.
文摘Background:Acetaminophen(APAP)overdose can cause liver injury and liver failure,which is one of the most common causes of drug-induced liver injury in the United States.Pharmacological activation of autophagy by inhibiting mechanistic target of rapamycin(mTOR)protects against APAP-induced liver injury likely via autophagic removal of APAP-adducts and damaged mitochondria.In the present study,we aimed to investigate the role of genetic ablation of mTOR pathways in mouse liver in APAP-induced liver injury and liver repair/regeneration.Methods:Albumin-Cre(Alb-Cre)mice,mTOR^(f/f) and Raptor^(f/f) mice(C57BL/6J background)were crossbred to produce liver-specific mTOR knockout(L-mTOR KO,Alb Cret/-,mTOR^(f/f))and liver-specific Raptor KO(L-Raptor,Alb Cret/-,Raptor^(f/f))mice.Alb-Cre littermates were used as wild-type(WT)mice.These mice were treated with APAP for various time points for up to 48 h.Liver injury,cell proliferation,autophagy and mTOR activation were determined.Results:We found that genetic deletion of neither Raptor,an important adaptor protein in mTOR complex 1,nor mTOR,in the mouse liver significantly protected against APAP-induced liver injury despite increased hepatic autophagic flux.Genetic deletion of Raptor or mTOR in mouse livers did not affect APAP metabolism and APAP-induced c-Jun N-terminal kinase(JNK)activation,but slightly improved mouse survival likely due to increased hepatocyte proliferation.Conclusions:Our results indicate that genetic ablation of mTOR in mouse livers does not protect against APAP-induced liver injury but may slightly improve liver regeneration and mouse survival after APAP overdose.
