Radiotherapy is widely used in the management of advanced colorectal cancer(CRC).However,the clinical efficacy is limited by the safe irradiated dose.Sensitizing tumor cells to radiotherapy via interrupting DNA repair...Radiotherapy is widely used in the management of advanced colorectal cancer(CRC).However,the clinical efficacy is limited by the safe irradiated dose.Sensitizing tumor cells to radiotherapy via interrupting DNA repair is a promising approach to conquering the limitation.The BRCA1-BARD1 complex has been demonstrated to play a critical role in homologous recombination(HR)DSB repair,and its functions may be affected by HERC2 or BAP1.Accumulated evidence illustrates that the ubiquitination-deubiquitination balance is involved in these processes;however,the precise mechanism for the cross-talk among these proteins in HR repair following radiation hasn’t been defined.Through activity-based profiling,we identified PT33 as an active entity for HR repair suppression.Subsequently,we revealed that BAP1 serves as a novel molecular target of PT33 via a CRISPR-based deubiquitinase screen.Mechanistically,pharmacological covalent inhibition of BAP1 with PT33 recruits HERC2 to compete with BARD1 for BRCA1 interaction,interrupting HR repair.Consequently,PT33 treatment can substantially enhance the sensitivity of CRC cells to radiotherapy in vitro and in vivo.Overall,these findings provide a mechanistic basis for PT33-induced HR suppression and may guide an effective strategy to improve therapeutic gain.展开更多
目的:探讨二甲双胍是否能够逆转卵巢癌细胞对顺铂的耐药性,以及其可能的作用机制。方法:CCK-8法检测二甲双胍作用后卵巢癌细胞顺铂耐药株C13K和CP70细胞对顺铂的耐药性变化,以及转染针对切除修复交叉互补基因1(excision repair cross-co...目的:探讨二甲双胍是否能够逆转卵巢癌细胞对顺铂的耐药性,以及其可能的作用机制。方法:CCK-8法检测二甲双胍作用后卵巢癌细胞顺铂耐药株C13K和CP70细胞对顺铂的耐药性变化,以及转染针对切除修复交叉互补基因1(excision repair cross-complemention 1,ERCC1)的特异性小干扰RNA(small interfering RNA,si RNA)后C13K和CP70细胞耐药性的变化。实时荧光定量PCR法检测不同浓度二甲双胍作用后,C13K和CP70细胞中ERCC1基因表达的变化。蛋白质印迹法检测二甲双胍作用后C13K和CP70细胞中腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)信号通路的激活和p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase,p38MAPK)信号通路的抑制情况,以及联合或不联合AMPK信号通路阻断剂Compound C作用后ERCC1蛋白的表达情况。同时,蛋白质印迹法检测p38MAPK信号通路抑制剂SB203580作用后耐药细胞中ERCC1蛋白表达的变化,以及二甲双胍作用联合p38MAPK si RNA转染前后耐药细胞中ERCC1蛋白的表达变化。结果:二甲双胍能够逆转C13K和CP70细胞对顺铂的耐药性(P<0.05)。二甲双胍作用能够降低C13K和CP70细胞中ERCC1 m RNA和蛋白的表达水平(P<0.05),并激活AMPK信号通路(P<0.05);而联合应用AMPK信号通路阻断剂Compound C可以阻断二甲双胍的这种作用(即ERCC1蛋白表达水平升高)(P<0.01)。p38MAPK信号通路抑制剂SB203580作用后,C13K和CP70细胞中ERCC1蛋白表达水平降低(P<0.05);而二甲双胍作用可降低p38MAPK的磷酸化水平(P<0.01),抑制p38MAPK信号通路。二甲双胍作用转染了p38MAPK si RNA的C13K和CP70细胞后,ERCC1蛋白的表达水平无明显变化(P>0.05)。结论:二甲双胍可能逆转卵巢癌顺铂耐药株对顺铂的耐药性,并且这一作用可能是通过抑制p38MAPK信号通路和降低细胞中ERCC1的表达而实现的。展开更多
基金supported by the National Natural Science Foundation of China(NSFC)(No.82272743 to Xin Yue(82172812)of NSFC to Ran-yi Liu+4 种基金81871996 to Ran-yi Liu82003218 to Xuecen Wang82072029 to Zhenwei Peng and 81973174 to Xianzhang Bu)Guangdong Basic and Applied Basic Research Foundation(No.2021A1515012496 to Xin Yue and 2022A1515012221 to Xianzhang Bu)Basic Scientific Research Operation of Sun Yat-sen University(No.19ykpy192 to Xin Yue)。
文摘Radiotherapy is widely used in the management of advanced colorectal cancer(CRC).However,the clinical efficacy is limited by the safe irradiated dose.Sensitizing tumor cells to radiotherapy via interrupting DNA repair is a promising approach to conquering the limitation.The BRCA1-BARD1 complex has been demonstrated to play a critical role in homologous recombination(HR)DSB repair,and its functions may be affected by HERC2 or BAP1.Accumulated evidence illustrates that the ubiquitination-deubiquitination balance is involved in these processes;however,the precise mechanism for the cross-talk among these proteins in HR repair following radiation hasn’t been defined.Through activity-based profiling,we identified PT33 as an active entity for HR repair suppression.Subsequently,we revealed that BAP1 serves as a novel molecular target of PT33 via a CRISPR-based deubiquitinase screen.Mechanistically,pharmacological covalent inhibition of BAP1 with PT33 recruits HERC2 to compete with BARD1 for BRCA1 interaction,interrupting HR repair.Consequently,PT33 treatment can substantially enhance the sensitivity of CRC cells to radiotherapy in vitro and in vivo.Overall,these findings provide a mechanistic basis for PT33-induced HR suppression and may guide an effective strategy to improve therapeutic gain.
文摘目的:探讨二甲双胍是否能够逆转卵巢癌细胞对顺铂的耐药性,以及其可能的作用机制。方法:CCK-8法检测二甲双胍作用后卵巢癌细胞顺铂耐药株C13K和CP70细胞对顺铂的耐药性变化,以及转染针对切除修复交叉互补基因1(excision repair cross-complemention 1,ERCC1)的特异性小干扰RNA(small interfering RNA,si RNA)后C13K和CP70细胞耐药性的变化。实时荧光定量PCR法检测不同浓度二甲双胍作用后,C13K和CP70细胞中ERCC1基因表达的变化。蛋白质印迹法检测二甲双胍作用后C13K和CP70细胞中腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)信号通路的激活和p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase,p38MAPK)信号通路的抑制情况,以及联合或不联合AMPK信号通路阻断剂Compound C作用后ERCC1蛋白的表达情况。同时,蛋白质印迹法检测p38MAPK信号通路抑制剂SB203580作用后耐药细胞中ERCC1蛋白表达的变化,以及二甲双胍作用联合p38MAPK si RNA转染前后耐药细胞中ERCC1蛋白的表达变化。结果:二甲双胍能够逆转C13K和CP70细胞对顺铂的耐药性(P<0.05)。二甲双胍作用能够降低C13K和CP70细胞中ERCC1 m RNA和蛋白的表达水平(P<0.05),并激活AMPK信号通路(P<0.05);而联合应用AMPK信号通路阻断剂Compound C可以阻断二甲双胍的这种作用(即ERCC1蛋白表达水平升高)(P<0.01)。p38MAPK信号通路抑制剂SB203580作用后,C13K和CP70细胞中ERCC1蛋白表达水平降低(P<0.05);而二甲双胍作用可降低p38MAPK的磷酸化水平(P<0.01),抑制p38MAPK信号通路。二甲双胍作用转染了p38MAPK si RNA的C13K和CP70细胞后,ERCC1蛋白的表达水平无明显变化(P>0.05)。结论:二甲双胍可能逆转卵巢癌顺铂耐药株对顺铂的耐药性,并且这一作用可能是通过抑制p38MAPK信号通路和降低细胞中ERCC1的表达而实现的。
