雷公藤红素靶向抑制STAT3抗结直肠癌的机制  

Celastrol exerts anti-colorectal cancer effect via STAT3 inhibition

作　　者：范若兰 陈海兰 赖斌 李志利 徐伟[1] 严国鸿 许少华[1] 樊志敏 FAN Ruo-lan;CHEN Hai-lan;LAI Bin;LI Zhi-li;XU Wei;YAN Guo-hong;XU Shao-hua;FAN Zhi-min(School of Pharmacy,Fujian University of Traditional Chinese Medicine,Fujian Key Laboratory for Research and Development of TCM Resources,Fuzhou 350122,China;Nanjing Traditional Chinese Medicine Hospital Affiliated to Nanjing University of Traditional Chinese Medicine,Nanjing 210022,China;the Affiliated People’s Hospital of Fujian University of Traditional Chinese Medicine,Fuzhou 350004,China)

机构地区：[1]福建中医药大学药学院,福建省中药资源研究与开发利用重点实验室,福建福州350122 [2]南京中医药大学附属南京中医院,江苏南京210022 [3]福建中医药大学附属人民医院,福建福州350004

出　　处：《中国药理学通报》2022年第11期1673-1680,共8页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No.82104321);福建省自然科学基金项目(No.2020J05062);福建省教育厅中青年教师教育科研项目(No.JAT190240);福建省大学生创新训练计划项目(No.201910393036)。

摘　　要：目的探究雷公藤红素(celastrol,CEL)通过靶向抑制STAT3发挥抗结肠癌作用。方法体外培养HCT-116细胞、HepG2细胞、A549细胞,经不同浓度的CEL处理,CCK-8检测体外抗增殖活性;选择HCT-116细胞,利用Western blot检测给药前后STAT3及其上下游蛋白(JAK2、Survivin、MCL-1)的表达;应用表面等离子共振(SPR)以及分子对接技术分析CEL与人源STAT3重组蛋白(rhSTAT3)的结合;采用流式细胞仪检测HCT-116细胞凋亡与细胞周期阻滞情况;建立4例患者来源的结直肠肿瘤类器官(colorectal cancer organoid,CCO)模型(CCO-1、CCO-2、CCO-3、CCO-4)和1例正常结直肠组织类器官模型(colorectal normal organoid,CNO),评价化合物对CCOs和CNO的抑制作用。结果CEL对3种肿瘤细胞的半数抑制浓度分别为(A549:IC_(50)=2.37±0.02μmol·L^(-1)、HCT-116:IC_(50)=1.40±0.21μmol·L^(-1)、HepG2:IC_(50)=2.52±0.02μmol·L^(-1));HCT-116细胞经IL-6诱导后各蛋白水平均有明显的升高,差异具有统计学意义(P<0.05),CEL给药6 h后,使磷酸化信号转导及转录激活因子(p-STAT3)、细胞凋亡相关蛋白(Survivin、MCL-1)的表达量下调(P<0.05),对STAT3总蛋白以及磷酸化酪氨酸激酶(p-JAK2)的表达量无明显影响(P>0.05);CEL能够阻滞HCT-116细胞周期并诱导细胞凋亡;SPR分析CEL与rhSTAT3蛋白的平衡解离常数K_(D)=6.038×10^(-5) mol·L^(-1),具有良好的结合力,分子对接预测其结合位点位于STAT3的SH2结构域;CEL抗结直肠癌活性在CCOs上得到进一步验证,且作用强于阳性药奥沙利铂(L-OHP)。结论CEL具有显著的抗肿瘤活性,其可能是通过直接靶向抑制STAT3,进而诱导细胞凋亡、阻滞细胞周期来发挥抗结直肠癌作用。Aim To investigate the anti-tumor effect of celastrol(CEL)on colorectal cancer and the possible targets/mechanisms.Methods The cytotoxic activities of CEL were evaluated against A549,HCT-116,HepG2 by CCK-8 method.Western blotting was used to detect the expression level of STAT3 and its upstream and downstream proteins(JAK2,Survivin,MCL-1)in HCT-116 cells before and after CEL treatment Flow cytometry was applied to assess CEL’s apoptosis and cell-cycle arrest effect in HCT-116 cells.SPR detection and molecular docking analysis were performed to further assess the binding ability between CEL and STAT3 protein.Lastly,human colorectal cancer organoid culture was constructed to verify the anti-tumor effect of CEL.Results CEL showed significant cytotoxicity to A549(IC_(50)=2.37±0.02μmol·L^(-1)),HCT-116(IC_(50)=1.40±0.21μmol·L^(-1))and HepG2(IC_(50)=2.52±0.02μmol·L^(-1)).Additionally,CEL could effectively decrease the level of p-STAT3 and the downstream gene expression of STAT3(Survivin and MCL-1)in a concentration-dependent manner;however,CEL did not affect the total level of STAT3 and upstream kinases JAK2.Moreover,CEL could induce apoptosis of HCT-116 cells concentration-dependently and arrest the cell cycle.According to the SPR analysis,CEL showed a strong binding affinity with the K_(D) value(the equilibrium dissociation constant)of 60.38μmol·L^(-1).Molecular docking analysis also suggested that CEL bound to the SH2 domain of STAT3.Lastly,CEL showed much better activity than the positive drug oxaliplatin(L-OHP)on all the colorectal cancer organoids.Conclusions CEL shows a significant anti-colorectal cancer effect,potentially caused by a direct target inhibiting STAT3,inducing apoptosis,and blocking the cell cycle.

关 键 词：雷公藤红素 结直肠癌 STAT3 分子对接 SPR 肿瘤类器官 

分 类 号：R282.71[医药卫生—中药学] R329.25[医药卫生—中医学] R329.28R735.35R979.1