Effect Of The Hsp70 Inhibitor 2-phenylethynesulfonamide On Replication And Carcinogenicity Of Epstein-Barr Virus And The Underlying Molecular Mechanism

Background and Objective Epstein-Barr virus(EBV),also known as human herpesvirus 4(HHV-4),is a member of the herpesvirus gamma subfamily.EBV can infect and establish long-term latency in human.EBV also cause various malignancies and other non-malignant virus diseases,such as Nasopharyngeal carcinoma(NPC),gastric cancer,Burkitt’s lymphoma,Hodgkin’s lymphoma,infectious mononucleosis syndrome,lymphoproliferative diseases.Acyclovir and ganciclovir are the main anti-EBV drugs currently for treating EBV-associated diseases.Acyclovir and ganciclovir mainly act on the viral DNA polymerase,but they also disturb replication of DNAs in normal cells,which results in many toxic side effects.Treatment with acyclovir and ganciclovir is easy to cause drug-resistant mutations of human cells,so the clinical effect is relatively unsatisfactory.Therefore,it is of great clinical significance to find new drugs that have good treatment results,but less side effects.2-phenylethynesulfonamide(PES),a small molecular inhibitor of heat shock protein 70(Hsp70),can interact with.Hsp70 and disrupt bindings of Hsp70 to its co-chaperone and substrate proteins.Studies in recent years have shown that PES can selectively kill tumor cells and may be a promising anticancer drug.This research will investigate the effects of PES on EBV-positive cells and explore the underlying molecular mechanisms.Method Cell Counting Kit-8(CCK-8)activity assay kit was used to detect the effects of PES on the cell viability in EBV-positive cells(HONE1/Akata,HKl/Akata,and B95-8)after time-and dose-independent treatments.The effect of changes in Hsp70 expression on the viability of EBV-positive cells was examined using the CCK-8 activity assay kit.The effect of PES on the long-term proliferation of EBV-positive cells(HONE1/Akata and HK1/Akata)was examined by cell plate colony formation assay.The effect of PES on the migration ability of EBV-positive cells was examined by cell scratch assay.Flow cytometry was used to detect whether PES can induce cell cycle arrest or apoptosis in EBV-positive cells.The expression levels of the target proteins in cells treated with PES or other various conditions,were analyzed by Western blotting assay.The effects of PES on EBV mRNA levels and production of EBV genomic DNA in EBV-positive cells were detected by quantitative polymerase chain reaction(Q-PCR).The effect of PES on the fluorescence intensity of GFP in the lytic HONE1/Akata cells was examined by flow cytometry.The interaction between Hsp70 and EBNA1,and the effect of PES on this interaction were examined by co-immunoprecipitation(IP)and immunofluorescence(IF).BALB/c nude mice with HONE1/Akata tumor were treated with PES or control(0.006%DMSO).The effects of PES on body weight,tumor weight,tumor volume and the expression level of EB virus nuclear antigen 1(EBNA1)in tumor tissues were investigated.Results PES effectively inhibited the cell viability of EBV-positive HONE1/Akata,HK1/Akata and B95-8 cells,but had no significant effect on the proliferation inhibition of EBV-negative normal renal proximal tubular epithelial cells HK2.Over-expression of Hsp70 reduced the inhibitory effect of PES on cell proliferation.However,silencing of Hsp70 increased the inhibitory effect of PES on cell proliferation.PES inhibited the migration abilities of EBV-positive HONEl/Akata and HK1/Akata cells.PES resulted in G2/M cell-cycle arrest and induced apoptosis of EBV-positive HONE1/Akata,HKl/Akata and B95-8 cells by inhibition of autophagosome lysosomal pathway.PES significantly decreased EBNA1 expression without reducing EBNA1 transcript level in EBV-positive cells.The inhibitory effect of PES on EBNA1 was not achieved by acting on EBNA1 Gly-Ala repeats.PES did not induce proteasomal degradation of EBNA1 and did not impair half-life of EBNA1.PES reduced production of EBV genomic DNA in lytic HONE1/Akata and B95-8 cells.Overexpression of Hsp70 enhanced EBNA1 expression and intracellular viral genomic DNA production,but PES inhibited this effect in a dose-dependent manner.Silencing of Hsp70 inhibited the expression of EBNA1 and the production of viral genomic DNA in cells,while PES enhanced this effect in a dose-dependent manner.Hsp70 interacted with EBNA1,but PES can interfere with this interaction.PES significantly inhibited the growth of HONE1/Akata tumors and the replication of EB viral genomic DNA in BALB/c nude mice.Conclusions and significances My research results show that PES reduces expression of EBNA1 in EBV-positive cells,inhibits proliferation and migration of EBV-positive cells,induces cell cycle arrest,and promotes cell apoptosis by inhibiting autophagosome lysosomal pathway.Hsp70 can increase expression of EBV-encoded protein EBNA1,promote replication of viral genomic DNA and increase production of viral genomic DNA.By inhibiting function of Hsp70,PES can inhibit expression of EBNA1 and replication of viral genomic DNA.PES can inhibit the interaction between Hsp70 and EBNA1.The researches on anti-viral and anti-tumor effects of PES in EBV-positive cells provide theoretical basis and practical application values for use of PES for to treat EBV-related malignant tumors.