摘要
目的:鉴定中药来源通过靶向抑制拟素化(Neddylation)-Cullin-Ring泛素连接酶(CRL)通路发挥抗肺癌效应的活性成分。方法:利用酯蟾毒配基(RBG)处理肺癌细胞A549和H1299。通过三磷酸腺苷(ATP)发光检测试剂盒(ATPlite)进行细胞增殖实验确定RBG对肺癌细胞的半数抑制浓度(IC50),细胞计数试剂盒(CCK-8)进行细胞增殖实验检测其抑制肺癌细胞增殖能力;膜联蛋白V-异硫氰酸荧光素/碘化丙啶(AnnexinⅤ-FITC/PI)染色、流式细胞术检测其诱导肺癌细胞凋亡情况;蛋白质免疫印迹(Western blot)法检测其引起肺癌细胞相关死亡标志物表达情况。结果:ATPlite实验确定RBG在A549和H1299的IC50分别为0.136 6μmol/L和0.221 4μmol/L;CCK-8检测结果显示RBG显著抑制肺癌细胞生长;AnnexinⅤ-FITC/PI染色结果显示,RBG诱导肺癌细胞发生显著凋亡;Western blot结果显示,RBG显著抑制Neddylation关键催化酶的表达和Cullins蛋白家族的Neddylation修饰,并引起CRL经典底物佛波醇-12-肉豆蔻酸酯-13-乙酸酯诱导蛋白1(Noxa)、凋亡标志物蛋白剪切的多聚ADP核糖聚合酶(CleavedPARP)和DNA损伤应答相关蛋白磷酸化H2AX(p-H2AX)等的显著积聚。结论:RBG通过下调Neddylation关键催化酶表达抑制CRL泛素连接酶活性,导致DNA损伤应答和细胞凋亡,从而杀伤肺癌细胞。
Objective:To identify the active ingredients of traditional Chinese medicine that target the anti-lung cancer activity of Neddylation-Cullin-Ring E3 ligase(CRL).Methods:After resibufogenin(RBG)treatment of lung cancer cells A549 and H1299,adenosine triphosphate(ATP)luminescent cell viability assay kit(ATPlite)was performed to determine the half inhibitory rate(IC50)of RBG,and cell counting kit-8(CCK-8)was used to detect the effect of RBG on the growth of lung cancer cells;the effect of RBG on the morphology of lung cancer cells was observed,and apoptosis was detected by flow cytometry and annexin V-fluorescein isothiocyanate/propidium iodide dual staining(Annexin V-FITC/PI)assay;total proteins of A549 and H1299 cells were collected for Western blot analysis to explore the molecular mechanism of RBG inhibiting lung cancer cell growth.Results:The IC50 of RBG in A549 and H1299 was 0.1366μmol/L and 0.2214μmol/L,respectively,determined by ATPlite;CCK-8 assay showed that RBG significantly inhibited the growth of lung cancer cells;Annexin V-FITC/PI staining showed that RBG induced significant apoptosis of lung cancer cells;Western blot results showed that RBG significantly inhibited the expression of the key Neddylation catalytic enzyme and Neddylation of the Cullins protein family.It also caused significant accumulation of CRL classic substrate phorbol-12-myristate-13-acetate-induced protein 1(Noxa),Cleaved-Poly(ADPDOI ribose)polymerase(Cleaved-PARP),and phosphorylation of H2A histone family member X(p-H2AX),a protein associated with DNA damage response.Conclusion:RBG inhibits the activity of CRL ubiquitin ligase by unhibiting the expression of Neddylation key catalytic enzyme,leading to DNA damage response and apoptosis,thereby killing lung cancer cells.
作者
常改莉
胡莫媛
周赟杨
张颖
李蒙
李立辉
CHANG Gaili;HU Moyuan;ZHOU Yunyang;ZHANG Ying;LI Meng;LI Lihui(Institute of Traditional Chinese Medicine Oncology,Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 200032,China;Department of Traditional Chinese Medicine OncologyⅥ,Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 200032,China)
出处
《上海中医药大学学报》
2025年第4期35-44,共10页
Academic Journal of Shanghai University of Traditional Chinese Medicine
基金
上海市“科技创新行动计划”自然科学基金原创探索项目(21ZR1482200)。
作者简介
常改莉,女,在读硕士生,主要从事抗肿瘤靶点发现与中西医防治研究;通信作者:李立辉,研究员,硕士生导师,E-mail:dm-li@163.com。
