白鲜碱靶向调控PI3K/KEAP1信号抑制卵巢癌细胞增殖并诱导自噬及凋亡  

Dictamnine inhibits proliferation and induces autophage and apoptosis by targeting PI3K/KEAP1 signaling in ovarian cancer cells

作　　者：吕思慧 梁金兰 邓明晶 刘萤照 王琪[1,2,3] LÜSihui;LIANG Jinlan;DENG Mingjing;LIU Yingzhao;WANG Qi(Department of Experimental Research,Guangxi Medical University Cancer Hospital;Key Laboratory of Early Prevention and Treatment for Regional High Frequency Tumor(Guangxi Medical University),Ministry of Education;Guangxi Key Laboratory of Early Prevention and Treatment for Regional High Frequency Tumor;Institute of Life Sciences,Guangxi Medical University,Nanning 530021,China)

机构地区：[1]广西医科大学附属肿瘤医院实验研究部,南宁530021 [2]区域性高发肿瘤早期防治研究教育部重点实验室(广西医科大学) [3]广西区域性高发肿瘤早期防治研究重点实验室 [4]广西医科大学生命科学研究院

出　　处：《中国癌症防治杂志》2023年第3期285-291,共7页CHINESE JOURNAL OF ONCOLOGY PREVENTION AND TREATMENT

基　　金：国家自然科学基金项目(82260484);广西创新驱动发展专项(桂科AA18242040);区域性高发肿瘤早期防治研究教育部重点实验室自主课题(GKE-ZZ202016;GKE-ZZ202122;GKE-ZZ202236)。

摘　　要：目的探讨白鲜皮活性单体白鲜碱(Dictamnine)对卵巢癌细胞增殖、凋亡和自噬的影响及其作用机制。方法人卵巢癌细胞系(A2780、PA-1)及人正常卵巢上皮细胞株(IOSE80)分别用不同浓度白鲜碱(0、32、65、125、250μmol/L)处理,CCK-8实验检测各细胞IC50值。利用网络药理学筛选出白鲜碱治疗卵巢癌的核心靶点,分子对接评估结合能力。选取A2780细胞用白鲜碱(0、50、75μmol/L)处理后,利用CCK-8实验、HE染色、Annexin V-FITC/PI双染法、Western blot法分析白鲜碱对A2780细胞增殖、凋亡和自噬的影响。裸鼠皮下移植瘤模型观察白鲜碱对卵巢癌移植瘤生长的影响,HE染色和透射电子显微镜观察移植瘤组织形态及超微结构,免疫组化染色(IHC)检测移植瘤组织中Cleaved PARP、Ki67的表达水平。结果CCK-8实验结果显示,白鲜碱作用于A2780细胞、PA-1细胞的IC50显著低于IOSE80细胞,其中对A2780细胞的抑制作用最强;白鲜碱(50μmol/L、75μmol/L)处理组的A2780细胞增殖能力较未处理细胞明显下降(均P<0.05)。网络药理学筛选出10个白鲜碱抗卵巢癌的核心靶点,结合能力较强的靶点为PIK3CD、KEAP1、AHR。流式细胞术结果显示白鲜碱处理48 h、72 h后A2780细胞凋亡率均明显上升(均P<0.01)。Western blot实验显示,与未处理组相比,白鲜碱处理组的A2780细胞中凋亡相关蛋白Cleaved PARP、PARP表达水平明显升高;自噬相关蛋白SQSTM1/p62、KEAP1表达水平下降,LC3BⅡ/Ⅰ比例升高;PI3K信号通路蛋白PI3K表达水平下降(均P<0.05)。白鲜碱处理后,裸鼠移植瘤体积明显减小(P=0.0053);镜下可见瘤组织有明显凋亡细胞;IHC染色结果显示Ki67表达水平降低,Cleaved PARP表达水平上升(均P<0.001)。HE染色结果显示白鲜碱处理后A2780细胞呈短纺锤形或卵圆形,且体积变小;移植瘤组织中存在凋亡/坏死细胞。结论白鲜碱可能靶向调控PI3K/KEAP1信号转导抑制卵巢癌细胞增殖并诱导自噬�Objective To investigate the effect of Dictamnine on proliferation,apoptosis and autophagy of ovarian cancer cells and its mechanism.Methods Human ovarian cancer cell lines(A2780,PA⁃1)and human normal ovarian epithelial cell lines(IOSE80)were treated with Dictamnine of different concentrations(0,32,65,125,250μmol/L),and the IC50 values of each cell were detected by CCK⁃8 assay.The core targets of Dictamnine for ovarian cancer were screened out by network pharmacology,and molecular docking was used to assess the binding ability.A2780 cells were selected and treated with Dictamnine(0,50,75μmol/L).The effects of Dictamnine on cell proliferation,apoptosis and autophagy of A2780 cells were analyzed by CCK⁃8 assay,HE staining,Annexin V⁃FITC/PI double staining and Western blot.The effect of Dictamnine on the growth of ovarian cancer grafts in nude mice was observed.The tissue morphology and ultrastructure of the grafts were observed by HE staining and transmission electron microscopy(TEM),and the expression levels of Cleaved PARP and Ki67 in the grafts were detected by immunohistochemical staining(IHC).Results The results of CCK⁃8 assay showed that the IC50 of Dictamnine on A2780 cells and PA⁃1 cells were significantly lower than those of IOSE80 cells,with the strongest inhibitory effect on A2780 cells.The proliferation ability of A2780 cells in Dictamnine(50μmol/L,75μmol/L)treatment groups was significantly decreased compared with untreated cells(all P<0.05).The ten core targets of Dictamnine against ovarian cancer were screened by network pharmacology,and the targets with strong binding ability were PIK3CD,KEAP1 and AHR.Flow cytometry results showed a significant increase in apoptosis rate in A2780 cells after treatment with Dictamnine for 48 h and 72 h(all P<0.01).Western blot analysis showed that compared with the untreated group,the expression levels of A2780 cells apoptosis⁃related proteins Cleaved PARP and PARP were significantly increased,the expression levels of autophagy⁃related proteins SQ

关 键 词：卵巢癌 白鲜碱 增殖 凋亡 自噬 网络药理学 

分 类 号：R737.3[医药卫生—肿瘤]