结合肽P201与5-氟尿嘧啶联合用药对肝癌HepG2细胞的协同杀伤作用及抗耐药分子机制
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摘要
目的:采用多种方法探究FoxM1/mdr1信号调控的结合肽P201与5-氟尿嘧啶(5FU)联合用药对肝癌细胞HepG2的协同杀伤作用及抗耐药分子机制。方法:CCK-8法测定联合用药对HepG2细胞的抑制杀伤作用;吖啶橙/溴化乙锭(AO-EB)荧光双染、AnnexinⅤ-FITC/PI流式细胞术检测细胞凋亡;细胞划痕和Transwell细胞迁移实验检测HepG2细胞迁移能力;最后通过qRT-PCR和Western印迹检测HepG2细胞中FoxM1、mdr1和ABCG2等耐药相关基因在mRNA水平和蛋白水平的表达量。结果:联合用药[P201(45.0μg/mL)+5FU(100.0μg/mL)]作用48 h抑制率达83.8%,作用24 h的抑制率为77.8%,显著高于单独用药(P<0.001);流式细胞术检测联合用药细胞凋亡率达43.4%,而单独用药分别为19.4%、25.1%;联合用药在mRNA水平可显著下调HepG2细胞中的FoxM1、mdr1耐药基因,与蛋白水平结果一致;联合用药可显著抑制HepG2细胞的迁移。结论:联合用药对HepG2细胞有强的抑制杀伤作用,在促进细胞凋亡的同时可显著抑制HepG2细胞迁移,且通过下调FoxM1、mdr1和ABCG2等耐药基因和蛋白的表达,增加HepG2细胞对5FU的敏感性。提示P201可提升肿瘤细胞对化疗药物的敏感性,减少抗癌药物的副作用。
Objective: A series of experiments were conducted to study the synergistic killing effects and mechanisms of anti-drug resistance of P201+5-fluorouracil(5 FU) combined treatment for liver cancer HepG2 cells regulated by Foxm1/mdr1 signal pathway. Methods: CCK-8 assay was used to explore the inhibitory and killing effect of combined drugs on HepG2 cells. AO-EB staining and AnnexinⅤ-FITC/PI FACS were explored the apoptosis of HepG2 treated with P201+5 FU. Then migration ability was detected by scratch and transwell experiments. Finally,the mRNA and protein levels of FoxM1, mdr1 and ABCG2 in HepG2 were detected by qRT-PCR and Western blot. Results: After 48 hours, the inhibition rate of the combined treatment[P201(45.0 μg/mL)+5 FU(100.0 μg/mL)] was about 83.0%, which was significantly higher than those of P201 or 5 FU alone(P<0.001). In addition,the apoptosis rate of the combination was 43.4%, while the apoptosis rate of 5 FU and P201 alone was 19.4% and25.1%, respectively. The mRNA expression levels of FoxM1 and mdr1 were remarkably down-regulating in HepG2 after the combined treatment. And it could inhibit the migration of HepG2 significantly. Conclusion: The combination therapy has strong inhibitory effect on HepG2 and can significantly inhibit the migration of HepG2 cells while promoting apoptosis. In addition, the sensitivity of HepG2 cells to 5 FU was increased by down-regulating the expression of FoxM1, mdr1 and ABCG2. It is suggested that P201 can reverse the sensitivity of tumor cells to5 FU and reduce the side effects of anticancer drugs.
Objective: A series of experiments were conducted to study the synergistic killing effects and mechanisms of anti-drug resistance of P201+5-fluorouracil(5 FU) combined treatment for liver cancer HepG2 cells regulated by Foxm1/mdr1 signal pathway. Methods: CCK-8 assay was used to explore the inhibitory and killing effect of combined drugs on HepG2 cells. AO-EB staining and AnnexinⅤ-FITC/PI FACS were explored the apoptosis of HepG2 treated with P201+5 FU. Then migration ability was detected by scratch and transwell experiments. Finally,the mRNA and protein levels of FoxM1, mdr1 and ABCG2 in HepG2 were detected by qRT-PCR and Western blot. Results: After 48 hours, the inhibition rate of the combined treatment[P201(45.0 μg/mL)+5 FU(100.0 μg/mL)] was about 83.0%, which was significantly higher than those of P201 or 5 FU alone(P<0.001). In addition,the apoptosis rate of the combination was 43.4%, while the apoptosis rate of 5 FU and P201 alone was 19.4% and25.1%, respectively. The mRNA expression levels of FoxM1 and mdr1 were remarkably down-regulating in HepG2 after the combined treatment. And it could inhibit the migration of HepG2 significantly. Conclusion: The combination therapy has strong inhibitory effect on HepG2 and can significantly inhibit the migration of HepG2 cells while promoting apoptosis. In addition, the sensitivity of HepG2 cells to 5 FU was increased by down-regulating the expression of FoxM1, mdr1 and ABCG2. It is suggested that P201 can reverse the sensitivity of tumor cells to5 FU and reduce the side effects of anticancer drugs.
引文
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[15]Sun L,Chen W,Qu L,et al.Icaritin reverses multidrug resistance of HepG2/ADR human hepatoma cells via downregulation of MDR1 and P-glycoprotein expression[J].Mol Med Rep,2013,8(6):1883-1887.
[16]Liu W,Ding R,Zhang Y,et al.Transcriptome profiling analysis of differentially expressed mRNAs and lncRNAs in HepG2 cells treated with peptide 9R-P201[J].Biotechnol Lett,2017,39:1639-1647.
[2]孙慧伟,谢辉,李瑞生,等.新型肝脏肿瘤介入治疗后复发与转移预测方法的建立[J].生物技术通讯,2018,29(3):404-408.
[3]Holohan C,Van Schaeybroeck S,Longley D B,et al.Cancer drug resistance:an evolving paradigm[J].Nat Rev Cancer,2013,13(10):714-726.
[4]Higuchi E,Oridate N,Furuta Y,et al.Differentially expressed genes associated with CIS-diamminedichloroplatinum(Ⅱ)resistance in head and neck cancer using differential display and CDNA microarray[J].Head Neck,2003,25(3):187-193.
[5]Xie T,Geng J,Wang Y,et al.FOXM1 evokes 5-fluorouracil resistance in colorectal cancer depending on ABCC10[J].Oncotarget,2016,8(5):8574-8589.
[6]Roh Y G,Mun M H,Jeong M S,et al.Drug resistance of bladder cancer cells through activation of ABCG2 by FOXM1[J].BMB Rep,2018,51(2):98-103.
[7]Liao G B,Li X Z,Zeng S,et al.Regulation of the master regulator FOXM1 in cancer[J].Cell Commun Signal,2018,16(1):57.
[8]Arceci A,Bonacci T,Wang X,et al.FOXM1 deubiquitination by USP21 regulates cell cycle progression and paclitaxel sensitivity in Basal-like breast cancer[J].Cell Rep,2019,26(11):3076-3086.
[9]Lam E W,Brosens J J,Gomes A R,et al.Forkhead box proteins:tuning forks for transcriptional harmony[J].Nat Rev Cancer,2013,13(7):482.429
[10]Cui J,Huang J,Guo T,et al.Expression and selection of human Foxm1c binding peptides and their inhibitions on MCF7 cancer cells[J].Int J Peptide Res Ther,2014,20(4):447-456.
[11]Bi Z,Liu W,Ding R,et al.A novel peptide,9R-P201,strongly inhibits the viability,proliferation and migration of liver cancer HepG2 cells and induces apoptosis by down-regulation of FoxM1 expression[J].Eur J Pharmacol,2016,796:175-189.
[12]Yuan R,Hou Y,Sun W,et al.Natural products to prevent drug resistance in cancer chemotherapy:a review[J].Ann N Y Acad Sci,2017,1401(1):19-27.
[13]Varghese V,Magnani L,Harada-Shoji N,et al.FOXM1modulates 5-FU resistance in colorectal cancer through regulating TYMS expression[J].Sci Rep,2019,9(1):1505.
[14]Okada K,Fujiwara Y,Takahashi T,et al.Overexpression of forkhead box M1 transcription factor(FOXM1)is a potential prognostic marker and enhances chemoresistance for docetaxel in gastric cancer[J].Ann Surg Oncol,2013,20(3):1035-1043.
[15]Sun L,Chen W,Qu L,et al.Icaritin reverses multidrug resistance of HepG2/ADR human hepatoma cells via downregulation of MDR1 and P-glycoprotein expression[J].Mol Med Rep,2013,8(6):1883-1887.
[16]Liu W,Ding R,Zhang Y,et al.Transcriptome profiling analysis of differentially expressed mRNAs and lncRNAs in HepG2 cells treated with peptide 9R-P201[J].Biotechnol Lett,2017,39:1639-1647.