Synergistic Killing Effect And Anti-drug Resistance Mechanism Of Peptide P201 Combined Treated With 5-Fu On HepG2 Liver Cancer Cells

Cancer is a fatal malignant tumor.Therefore,it is urgent to find treatment with low-toxic side effects,the current treatment for cancer is chemotherapy and radiotherapy with serious side effects and drug resistance which limits the clinical use of chemotherapy.There are many mechanisms for drug resistance,including inhibition of apoptosis,increased drug excretion,and increased proliferation,thereby reducing the effectiveness of the drug.A broader mechanism of tumor cell drug spillover can be attributed to the dysregulation of the mdr1 gene,resulting in abnormally high expression of this protein.FoxM1 is a promising prognostic marker because of highly expressed in a variety of cancers and is involved in the malignant proliferation,invasion and drug resistance of tumor cells.In our previous work,we had successfully obtained the binding peptide P201 with high affinity for FoxM1c-DBD through phage randomized dodecapeptide library screening.We had confirmed that the modified peptide 9R-P201 can inhibit the proliferation,migration and invasion of HepG2 cells and induce cell apoptosis.In this study,the modified peptide 9R-P201 combined treated with 5-Fu was used to investigate the synergistic killing effect and anti-drug resistance mechanism on HepG2 and HepG2/R(drug resistance to 5-Fu)liver cancer cells.At first,HepG2 and HepG2/R cells were used to explore the inhibitory effect of the combined drug(P201+5-Fu).The result of morphological observation and CCK-8 showed that the combined drug had significantly stronger killing effect on both cells than those of 5-Fu and P201 alone.Moreover,at a concentration of P201 of 45.0 ?g/mL,the inhibition of P201 on HepG2/R cells(67%)was significantly stronger than that on the parental HepG2(43%).By plate cell cloning,cell scratching,and transwell chamber experiments,we found that combined therapy can effectively inhibit the proliferation and migration ability of these two cells.At the same time,the results of AO/EB fluorescence double staining and Annexin V-FITC flow cytometry assay indicated that the combined drug can promote the apoptosis of HepG2 cells and HepG2/R cell.The above results were confirmed by qRT-PCR and Western blot experiments: the expression levels of FoxM1,mdr1,and bax were significantly down-regulated in the two cells.The expression levels of FoxM1 and mdr1 in HepG2 cells were significantly lower than those in drug-resistant cells,and the difference was extremely significant(P<0.001).The foxo3 a was significantly up-regulated in both cells.The above results were also confirmed at the protein level.Moreover,ABCG2 was down-regulated at the protein level with statistically significant(P<0.05).Finally,the differential expression gene was analyzed by transcriptome sequencing.There were 2686 differentially expressed genes between parental HepG2 cells and HepG2/R cells.There were 1596 differentially expressed genes in the P201 and 2998 differentially expressed genes in the combination compared with the control,only 784 differentially expressed genes in the 5-Fu treatment.The differentially expressed genes in each treatment is mostly involved in cellular processes,various metabolic activities,cell proliferation,apoptosis,and tumor-related signaling pathways.In summary,9R-P201 can reverse the expression of mdr1 in 5-Fu-resistant HepG2/R cells by inhibit FoxM1,so that the sensitivity of 5-Fu is improved and the concentration of 5-Fu in cells could be increased,thereby enhancing killing effect.The inhibition of combined treatment of cells is significantly stronger than those of the single drugs.