Inhibitory Effect And Anti-drug Resistance Mechanisms Of Combined Teatment With 9R-P201/DOX To Hepatocellular Cells

Cancer is one of the major diseases endangering human life and health,especially for the Asian population,the prevention and treatment of HCC is particularly important.As a first-line clinical drug for liver cancer,DOX has been unable to meet the treatment needs of HCC patients due to its high drug resistance,dose-dependent efficacy and strong toxicity.Therefore,it has become a new development direction for the research of liver cancer's treatment to seek new molecular targeted drugs and design reasonable drug combination.FoxM1 is a transcriptional activator,highly expressed in various tumor tissues and widely involved in the occurrence and development of tumors.It is known as the " Achilles' heel of cancer" and one of the important targets with great potential for discoverying new drugs.P201 is a molecular targeted polypeptide targeted FoxM-DBD,screened from phage random dodecapeptide library in the early stage of our laboratory.The experiment found that the 9R-P201 had a strong inhibitory effect on liver cancer cells.In this study,HepG2-C3 A was taken as the main research object,and to reduce DOX dose and drug resistance were taken as the main purpose.Firstly,in vitro cell proliferation inhibition experiment,it was found that 9R-P201 and DOX could inhibit the proliferation of HepG2-C3 A cells.DOX showed a dose-dependent and time-dependent relationship with the proliferation inhibition rate of HepG2-C3 A cells.The optimal combination of 9R-P201 and DOX was as follows: 9R-P201(60.0 ?g/mL)was added for 12 hours,followed by DOX(0.2 ?g/mL)for 24 hours,a total of 36 hours.Secondly though AO/EB double dyed,FCM apoptosis assay,qRT-PCR,Western blotting,it it was confirmed that compared with the separate treatment group of DOX,9R-P201 and DOX combined treatment group can obviously promote the HepG2-C3 A cell apoptosis,and through the lower FoxM1 to downgrade the expression of MDR1 and ABCG2 drug-resistant related proteins,reducing the dose and drug resistance of DOX.To further investigate the regulation of FoxM1,we constructed stable cell lines overexpressing FoxM1-c by lentivirus transfection.The sequence information obtained by the sequencing was compared with the NCBI database,and the results showed that there were only three homonym mutation sites in the PCR amplification sequence,and the others were consistent.The titer of the concentrated virus collected in the lentivirus packaging experiment was 5.6×108 TU/mL by the multiple dilution method.After transfection,HepG2-C3 A cells were screened by puromycin,and green fluorescence was observed under fluorescence microscope.HepG2-C3 A cells that overexpressed FoxM1-c were successfully obtained.Finally,in order to obtain a small molecular targeting peptide with higher inhibitory effect than P201,15 peptide sequences were selected from the phage random dodecapeptide library by four rounds of biological screening with recombinant FoxM1-DBDp as the target.Five peptide sequences were found to have higher P/N values by reverse sieving,and DS3.0 molecular dynamics docking simulation showed that P15 sequence and FoxM1-DBD had the lowest binding free energy,and had further research value.In conclusion,,9R-P201 and DOX combined treatment group can obviously promote the HepG2-C3 A cell apoptosis,and through the lower FoxM1 to downgrade the expression of MDR1 and ABCG2 drug-resistant related proteins,reducing the dose and drug resistance of DOX,indicating that 9R-P201 combined with DOX has great potential in the field of optimal treatment of liver cancer.At the same time,HepG2-C3 A cells that overexpressed FoxM1-c were successfully obtained,laying a foundation for further research.15 peptide sequences were screened from the phage random dodecapeptide library by targeting FoxM1-DBDp,and providing a foundation of finding more effective molecular targeted peptides.