目的:在成功制备小鼠抗人IL-13Rα2高亲和力单克隆抗体(mAb)的基础上,通过分子克隆方法获得该抗体可变区基因序列。方法:从1株小鼠抗人IL-13Rα2 mAb杂交瘤细胞LX147-7中提取总RNA,以此为模板反转录获得cDNA,用针对小鼠mAb重链和轻链可...目的:在成功制备小鼠抗人IL-13Rα2高亲和力单克隆抗体(mAb)的基础上,通过分子克隆方法获得该抗体可变区基因序列。方法:从1株小鼠抗人IL-13Rα2 mAb杂交瘤细胞LX147-7中提取总RNA,以此为模板反转录获得cDNA,用针对小鼠mAb重链和轻链可变区基因序列的特异性引物分别进行PCR反应。将PCR产物连入克隆载体,经筛选阳性克隆,PCR和酶切鉴定正确后送测序,测序结果进行生物信息学分析。结果:成功克隆了抗人IL-13Rα2 mAb LX147-7重链和轻链的可变区基因。结论:获得了抗人IL-13Rα2 mAb LX147-7重链和轻链的可变区基因序列,为构建相关基因工程抗体打下了良好基础。展开更多
Objective:Androgen deprivation therapy(ADT)is still the principal treatment option for prostate cancer(PCa).In addition to reactivation of androgen receptor signaling,the resistance of PCa to apoptosis during ADT also...Objective:Androgen deprivation therapy(ADT)is still the principal treatment option for prostate cancer(PCa).In addition to reactivation of androgen receptor signaling,the resistance of PCa to apoptosis during ADT also contributes to castration resistant PCa(CRPC).A previous study reported that gene transfer of IL-13Rα2 into PCa cells sensitized the cells to the IL-13R-targeted cytotoxin IL13Rα1,leading to apoptosis.Compared with IL-13Rα2,IL13Rα1 is more constitutively expressed in PCa cells,but its function in PCa remains to be established.Methods:We determined the role and expression of IL13Rα1 in PCa cancer cells using western blotting,flow cytometry,and cell proliferation assays.Co-immunoprecipitation and mass spectrometry were used to identify the proteins that interacted with IL13Rα1,to elucidate its function.Results:In this study,we showed that IL13Rα1 was selectively suppressed in androgen-deprived PCa cells and that its suppression tended to be associated with poor prognoses of PCa patients.IL13Rα1 overexpression promoted apoptosis and inhibited tumor growth under androgen-deprived or castrated conditions(P<0.01).Mechanistically,IL13Rα1 recruited and facilitated ubiquitin protein ligase E3C-mediated ubiquitination and degradation of hexokinase 2(HK2),resulting in glycolytic inhibition and eventually leading to PCa cell apoptosis.Furthermore,our data revealed that mutated ataxia-telangiectasia kinase phosphorylated and facilitated the selective ubiquitin proteasome-mediated degradation of HK2.Notably,IL13Rα1-overexpressing PCa cells were more susceptible to apoptosis and exhibited reduced tumor growth after exposure to the HK2 inhibitor,2-deoxy-D-glucose(P<0.01).Conclusions:Our data identified a tumor suppressor role for IL13Rα1 in preventing the resistance of PCa cells to apoptosis during androgen deprivation by inhibiting glycolysis.IL13Rα1-mediated signaling involving HK2 may therefore provide a novel treatment target and strategy for CRPC.展开更多
基金Supported by the grant from the National Natural Science Foundation of China ( No .30571631 30872209)the grant from key provincial Natural Science Foundation of Anhui(KJ2009A80)
文摘目的:在成功制备小鼠抗人IL-13Rα2高亲和力单克隆抗体(mAb)的基础上,通过分子克隆方法获得该抗体可变区基因序列。方法:从1株小鼠抗人IL-13Rα2 mAb杂交瘤细胞LX147-7中提取总RNA,以此为模板反转录获得cDNA,用针对小鼠mAb重链和轻链可变区基因序列的特异性引物分别进行PCR反应。将PCR产物连入克隆载体,经筛选阳性克隆,PCR和酶切鉴定正确后送测序,测序结果进行生物信息学分析。结果:成功克隆了抗人IL-13Rα2 mAb LX147-7重链和轻链的可变区基因。结论:获得了抗人IL-13Rα2 mAb LX147-7重链和轻链的可变区基因序列,为构建相关基因工程抗体打下了良好基础。
基金supported by the National Natural Science Foundation of China(Grant Nos.81772760 and 82072850)the Natural Science Foundation of Shandong Province(Grant Nos.ZR2020YQ55 and ZR2020QH327),the Shandong Taishan Scholarship(Grant No.tsqn20161076)+1 种基金the Innovation Project of Shandong Academy of Medical Sciences(2020)the program for Outstanding PhD candidate of Shandong University(2020)and Academic promotion programme of Shandong First Medical University(LJ001).
文摘Objective:Androgen deprivation therapy(ADT)is still the principal treatment option for prostate cancer(PCa).In addition to reactivation of androgen receptor signaling,the resistance of PCa to apoptosis during ADT also contributes to castration resistant PCa(CRPC).A previous study reported that gene transfer of IL-13Rα2 into PCa cells sensitized the cells to the IL-13R-targeted cytotoxin IL13Rα1,leading to apoptosis.Compared with IL-13Rα2,IL13Rα1 is more constitutively expressed in PCa cells,but its function in PCa remains to be established.Methods:We determined the role and expression of IL13Rα1 in PCa cancer cells using western blotting,flow cytometry,and cell proliferation assays.Co-immunoprecipitation and mass spectrometry were used to identify the proteins that interacted with IL13Rα1,to elucidate its function.Results:In this study,we showed that IL13Rα1 was selectively suppressed in androgen-deprived PCa cells and that its suppression tended to be associated with poor prognoses of PCa patients.IL13Rα1 overexpression promoted apoptosis and inhibited tumor growth under androgen-deprived or castrated conditions(P<0.01).Mechanistically,IL13Rα1 recruited and facilitated ubiquitin protein ligase E3C-mediated ubiquitination and degradation of hexokinase 2(HK2),resulting in glycolytic inhibition and eventually leading to PCa cell apoptosis.Furthermore,our data revealed that mutated ataxia-telangiectasia kinase phosphorylated and facilitated the selective ubiquitin proteasome-mediated degradation of HK2.Notably,IL13Rα1-overexpressing PCa cells were more susceptible to apoptosis and exhibited reduced tumor growth after exposure to the HK2 inhibitor,2-deoxy-D-glucose(P<0.01).Conclusions:Our data identified a tumor suppressor role for IL13Rα1 in preventing the resistance of PCa cells to apoptosis during androgen deprivation by inhibiting glycolysis.IL13Rα1-mediated signaling involving HK2 may therefore provide a novel treatment target and strategy for CRPC.
