摘要
目的探讨靶向分子CD24单克隆抗体3E10调节肝癌HuH-7细胞对化疗药顺铂敏感度的作用及其分子机制。方法 Western blot和流式细胞术检测抗CD24抗体3E10的特异性;Annexin V-FITC/PI双染色检测细胞凋亡;Real-time PCR和Western blot检测耐药基因和肿瘤干细胞特性基因的表达水平;Western blot检测JAK/STAT3和PI3K/AKT信号通路的活性水平。结果 3E10可有效识别CD24蛋白及HuH-7细胞。3E10显著增强HuH-7细胞对顺铂的敏感度(P<0.05),抑制率由(10.3±3.0)%提高至(34.4±10.8)%。3E10显著降低HuH-7细胞耐药基因ABCB1、ABCB5、ABCC1和肿瘤干性基因NANOG、CD24的表达水平及干性基因β-catenin的磷酸化水平(P<0.05)。3E10显著降低STAT3和AKT的磷酸化水平(P<0.05)。结论靶向CD24分子3E10通过降低HuH-7细胞的耐药性、肿瘤干性和JAK/STAT3、PI3K/AKT信号通路活性来增强HuH-7细胞对化疗药顺铂的敏感度。
Objective To investigate the function and molecular mechanism of 3E10 molecule targeting CD24 in regulating the chemosensitivity of liver cancer HuH-7 cells to cisplatin. Methods Western blot and flow cytometry were used to verify 3E10 recognition specificity for CD24. Annexin V-FITC/PI double staining was used to detect cell apoptosis. The expression level of drug resistance gene and tumor stem cell gene were analyzed by real-time PCR and Western blot, and the activity of JAK/STAT3 and PI3K/AKT signaling pathways were measured by Western blot. Results 3E10 could effectively identify CD24 protein and HuH-7 cells. 3E10 significantly enhanced the sensitivity of HuH-7 cells to cisplatin(P<0.05), and the inhibition rate was increased from(10.3±3.0)% to(34.4±10.8)%. 3E10 significantly decreased the expression level of drug resistance gene ABCB1, ABCB5, ABCC1 and tumor stem gene NANOG, CD24, as well as the phosphorylation level of β-catenin in HuH-7 cells(P<0.05). 3E10 significantly down-regulated the phosphorylation of STAT3 and AKT(P<0.05). Conclusion 3E10 molecule targeting CD24 could enhance the sensitivity of HuH-7 cells to cisplatin through reducing the drug resistance, stemness and JAK/STAT3, PI3K/AKT signaling pathway activity of HuH-7 cells.
引文
[1]Stewart BW,Wild CP.World Cancer Report 2014[M].WHO:International Agency for Research on Cancer,2014:403-13.
[2]Le Grazie M,Biagini MR,Tarocchi M,et al.Chemotherapy for hepatocellular carcinoma:The present and the future[J].World J Hepatol,2017,9(21):907-20.
[3]Chiba T,Iwama A,Yokosuka O.Cancer stem cells in hepatocellular carcinoma:Therapeutic implications based on stem cell biology[J].Hepatol Res,2016,46(1):50-7.
[4]Castelli G,PelosiE,Testa U.Liver Cancer:Molecular Characterization,Clonal Evolution and Cancer Stem Cells[J].Cancers(Basel),2017,9(9),pii:E127.
[5]Muinao T,Deka Boruah HP,Pal M.Diagnostic and prognostic biomarkers in ovarian cancer and the potential roles of cancer stem cells-An updated review[J].Exp Cell Res,2018,362(1):1-10.
[6]Liu G,Liu GX,Fang Y,et al.Clinicopathological and prognostic value of CD24 expression in breast cancer:a metaanalysis[J].Int J Biol Markers,2017,32(2):e182-9.
[7]Tan Y,Zhao M,Xiang B,et al.CD24:from a hematopoietic differ entiation antigen to a genetic risk factor for multiple autoimmune diseases[J].Clin Rev Allergy Immunol,2016,50(1):70-83.
[8]van Kessel KE,Zuiverloon TC,Alberts AR,et al.Targeted therapies in bladder cancer:an overview of in vivo research[J].Nat Rev Urol,2015,12(12):681-94.
[9]Shapira S,Pleban S,Kazanov D,et al.Terpinen-4-ol:A Novel and Promising Therapeutic Agent for Human Gastrointestinal Cancers[J].PLo S One,2016,11(6):e0156540.
[10]Jain A,Jahagirdar D,Nilendu P,et al.Molecular approaches to potentiate cisplatin responsiveness in carcinoma therapeutics[J].Expert Rev Anticancer Ther,2017,17(9):815-25.
[11]Begicevic RR,Falasca M.ABC Transporters in Cancer Stem Cells:Beyond Chemoresistance[J].Int J Mol Sci,2017,18(11):E2362.
[12]Huynh J,Etemadi N,Hollande F,et al.The JAK/STAT3 axis:A comprehensive drug target for solid malignancies[J].Semin Cancer Biol,2017,45:13-22.
[13]Lv D,Guo L,Zhang T,et al.PRAS40 signaling in tumor[J].Oncotarget,2017,8(40):69076-85.
