Study Of A Novel EGFR Tyrosine Kinase Inhibitor In The Treatment Of Prostate Cancer

Prostate cancer is one of the most common malignant tumors in Western countries.Currently,the treatment of advanced prostate cancer is limited,and can only delay the progress of the disease,but not cure it.The incidence of prostate cancer in China is on the rise.Most of the prostate cancer patients in China have metastasis at the initial diagnosis.When the disease progresses to metastatic castrated resistant prostate cancer,the prognosis of the patients is poor.Overexpression of EGFR plays an important role in the genesis and development of prostate cancer.EGFR signaling pathway is the key molecular mechanism in the genesis and development of prostate cancer.Overexpression of EGFR suggests poor prognosis in patients with prostate cancer.The positive expression rate of EGFR in prostate cancer was significantly higher than that in benign prostatic hyperplasia.The positive expression rate of EGFR was positively correlated with Gleason grade,clinical stage and lymph node metastasis.Clinical data showed that EGFR signaling pathway could activate androgen receptor and enhance receptor-mediated tumor growth.These results suggest that EGFR plays an important role in the occurrence,development and metastasis of prostate cancer.More and more attention has been paid to the research and development of drugs targeting EGFR.The research and development of new EGFR inhibitors is a hotspot in the research and development of new drugs.Nowadays,the application of computer-aided screening technology in the study of FDA approved drugs for clinical use,this new drug screening mechanism can not only enrich active compounds,but also reduce costs and save time,and discover new indications of these drugs.In this study,EGFR was selected as the target protein,and idock software simulated the protein-ligand docking molecule.Drugs that could successfully dock with EGFR molecule were screened from FDA approved drugs.The inhibition of these drugs on prostate cancer cells was preliminarily detected by MTT assay.In vitro cell experiments(flow cytometry and Western blotting)and in vivo animal experiments(nude mice transplanted tumors)further verify the inhibitory effect of the new EGFR inhibitor on prostate cancer and explore its anti-cancer mechanism.In this study,computer screening and biological experiments confirmed that the new EGFR inhibitors can effectively inhibit the EGFR signaling pathway,block the growth and proliferation of prostate cancer cells,and promote apoptosis.This new EGFR inhibitor may become a small molecule targeted drug for prostate cancer,realizing the new value of its old drugs,and laying a theoretical foundation for the research and development of targeted drugs for prostate cancer and the optimization of treatment schemes.Through the study of EGFR inhibitors,we can provide a new direction for the screening of anti-cancer drugs and the discovery of new drugs in China.It may be a new and effective treatment strategy for advanced prostate cancer.Objective:To identify the new EGFR inhibitor can inhibit the growth and proliferation of prostate cancer cells,inhibit the EGFR signaling pathway,and exhibite additive and synergistic effects with chemotherapy,and explore the underlying mechanisms.Methods:(1)The idock software simulated the molecular docking between the EGFR target protein and ligand,then screened the EGFR inhibitors.(2)MTT assay was used to identify the EGFR inhibitors that inhibits the prostate cancer cell proliferation.(3)Western blotting was performed to examine the expression of the critical EGFR signaling pathway proteins and other pathway protein of ERK after the treatment of EGFR inhibitor.(4)Flow cytometry was used to measure apoposis of prostate cancer cells induced by EGFR inhibitor.Western blotting was performed to examine the apoptosis related proteins expression of PARP and Caspase-3.(5)Prostate cancer xenografts models in nude mice were used to evaluate the effects of EGFR inhibitor in vivo.Results:(1)The database including 3167 FDA-approved drugs were docked to the ATP binding pocket of EGFR and ranked according to their average predicted binding affinity across structures of EGFR.The visual docking results are available athttp://istar.cse.cuhk.edu.hk/idock/iview/?4JPS-dbap+fda+npc.Six compounds were selected for subsequent investigation,including Dibromopropamidine Dihydrochloride,Lenperone Avanafil,Droperidol,Alfuzosin HCl and Domperidone,.Their idock scores were-9.68?-6.95 kcal/mol,and RF-scores were 6.74?7.90 pKd.(2)Six compounds were tested the anticancer effects by MTT assay,and two compounds decreased prostate cancer cell viability.Among them,dibromopropamidine dihydrochloride exhibited the strongest anticancer effect in a dose-dependent manner(ICso=3.28 ?M in PC-3 cell line,2.81 ?M in DU 145 cell line).Moreover,dibromopropamidine dihydrochloride(ICso=3.28 ?M in PC-3 cell line)was more potent than two known EGFR inhibitors(Gefitinib)(IC50=5.92 ?M)and(Lapatinib)(IC50=6.92?M)in terms of cytotoxicity for 72 hours.(3)The predicted conformation of dibromopropamidine dihydrochloride in complex with EGFR protein was shown that dibromopropamidine dihydrochloride binds to EGFR by forming a hydrogen bond with GLY796 and ASP855.(4)Western blotting assay showed that dibromopyridinium dihydrochloride inhibited EGFR target protein in prostate cancer cell lines(PC-3 and DU145),inhibited EGFR phosphorylation,and inhibited downstream proteins AKT,ERK and Bcl-2 expression.(5)Dibromopropamidine dihydrochloride significantly and dose-dependently increased the cell apoptosis percentage of prostate cancer cells by Annexin V staining.Dibromopropamidine dihydrochloride induced PARP and caspase-3 cleavage in a dose-dependent manner by western blotting.(6)In vivo experiments were performed to establish a nude mouse model of PC-3 prostate cancer cell xenografts,and intraperitoneal injection of dibromopyridinium dihydrochloride and gefitinib(40 mg/kg/d)for 21 days.Intraperitoneal injection of dibromopyridinium dihydrochloride(experimental group)significantly inhibited tumor growth compared with the control group.The inhibitory effect of the dibromopyridinium dihydrochloride group was better than that of the gefitinib group.There were no deaths in the three groups of nude mice,and there was no significant difference in body weight.Conclusion:Dibromopropamidine dihydrochloride,an antifungal agent,was identified as a promising EGFR inhibitor.Dibromopropamidine dihydrochloride exhibited the highest activity in decreasing cell viability in pathological types of prostate cancer cell lines.Furthermore,dibromopropamidine dihydrochloride significantly inhibited downstream target of the EGFR signaling pathway and promoted apoptosis.More importantly,dibromopropamidine dihydrochloride(40mg/kg)significantly suppresses PC-3 tumor growth in nude mice.Dibromopropamidine dihydrochloride may serve as a new EGFR inhibitor for treatment of prostate cancer and reevaluate the new anti-cancer value of an old medicine.