| Background:Multiple myeloma(MM)is a plasma cell malignancy mainly residing in the bone marrow.Recently,new chemotherapeutic drugs and regimens have substantially improved the prognosis of patients with MM,with an increase in median survival from 3-5 years to8-10 years.However,MM still remains largely incurable.Most patients eventually relapse due to chemotherapeutic drug resistance.Therefore,the investigation of the mechanisms underlying MM drug resistance may have important clinical significance.Proteasome inhibitors,such as bortezomib(Bor)and carfilzomib,are highly effective and widely used for the treatment of MM.The ubiquitination and degradation of p53 and IκB plays an important role in MM cell survival and tumor progression.Proteasome inhibitors promote the apoptosis of MM cells through inhibiting proteasome-mediated degradation of the ubiquitinated p53 and IκB.Although proteasome inhibitors have achieved clinical success,most patients eventually develop resistance to the therapy.And the underlying mechanisms need to be further investigated.Tetrahydrobiopterin(BH4)is a co-factor of nitric oxide synthase(NOS),tyrosine hydroxylase,tryptophan hydroxylase et al.,which are involved in the biosynthesis of nitric oxide and monoamine neurotransmitters and pain sensitivity.BH4 promotes tumor growth and modulates ubiquitination and proteosome activity through NOS-mediated S-nitrosation of target proteins.Loss of BH4 triggers the increase of ubiquitinated proteins and subsequent degradation in cells.However,the role of BH4 in MM drug resistance and progression remains unknown.In this study,we will explore the effect of BH4 on MM drug resistance and progression to provide new strategies for clinical treatment.Our study is mainly divided into the following five parts:Part 1 The role of BH4 in MM progression in vivo Objective: To investigate the effects of BH4 on MM cell proliferation and tumor growth in vivo.Methods:(1)MPC-11 and MOPC-315 MM cells(1 × 106 cell per mouse)were labeled with CFSE and injected into Balb/c mice through the tail veins.Mice were injected intraperitoneally(i.p.)with BH4(60μg/mouse)on day 0,day 2 and day 4 after tumor challenge.Mice treated with PBS served as controls.On day 5,cells were isolated from lung tissues and MM cell proliferation were analyzed by flowcytometry.(2)Balb/c mice were injected subcutaneously(s.c.)with MPC-11 or MOPC-315 cells(1 × 106 per mouse).From day 0,mice were injected i.p.with BH4 every two days.Mice treated with PBS served as controls.Tumor growth was compared between the different groups.Results:(1)BH4 treatment increased MM cell proliferation in MPC-11 and MOPC-315 MM mouse model as compared with PBS control;(2)BH4 promoted MPC-11 and MOPC-315 MM tumor growth as compared with PBS control in Balb/c mouse model.Conclusion: BH4 promotes MM progression in vivo.Part 2 Effects of BH4 on MM drug resistance Objective: To examined the role of BH4 in Bor-induced MM therapy.Methods:(1)MPC-11 and MOPC-315 cells were cultured in the presence of BH4,Bortezomib(Bor)or their combinations(BH4+Bor)for 24 hours.Cells treated with PBS served as controls.Cell apoptosis was analyzed by Flowcytometry;In addition,human MM cell lines(ARP-1 and CAG)were similarly processed and analyzed;(2)Balb/c mice were injected s.c.with MPC-11 or MOPC-315 cells(1 × 106 per mouse).From day 6,mice were injected i.p.with Bor or BH4+Bor every two days.Mice received PBS served as controls.Tumor growth was compared between the different groups.Results:(1)In MPC-11 and MOPC-315 cell lines,the addition of Bor efficiently induced MPC-11 MM cell apoptosis in the cell culture as compared with PBS control,while the addition of BH4 significantly inhibited Bor-induced MM cell apoptosis;and,BH4 treatment also significantly inhibited Bor-induced MM cell apoptosis in both ARP-1 and CAG MM cell models;(2)By using Balb/c mouse MPC-11 and MOPC-315 MM tumor model,Bor treatment potently inhibited MPC-11 MM tumor growth as compared with PBS control,while BH4 treatment largely abolished Bor-induced inhibition of MPC-11and MOPC-315 MM tumor growth.Conclusion: BH4 increases Bor resistance in MM therapy.Part 3 Mechanism of BH4 promoting MM cell survival Objective: To investigate the molecular mechanism underlying BH4-induced MM cell survival.Methods:(1)MPC-11 and MOPC-315 were cultured with or without(PBS)the addition of BH4 for 24 hours.Cells were analyzed by RNA-seq;(2)MPC-11 and MOPC-315 were cultured in the presence or absence(PBS)of BH4,Bor or their combinations(BH4+Bor)for 24 hours.q PCR assessed the m RNA levels of Usp7 and Usp46;(3)MPC-11 and MOPC-315 cells were cultured in the presence or absence(PBS)of Bor,P22077(USP inhibitor,USPi),BH4+Bor,Bor+USPi or their combinations(BH4+Bor+USPi).Cell apoptosis were analyzed by Flowcytometry;(4)MPC-11 were treated by Usp7(si-Usp7),Usp46(si-Usp46)or control si RNA(si-CT)for 24 hours.q PCR assessed m RNA levels of Usp7 and Usp46;(5)MPC-11 treated with si-Usp7 or si-CT were cultured in the presence of BH4+Bor combinations for 24 hours.Cell apoptosis were analyzed by Flowcytometry;(6)MPC-11 treated with si-Usp46 or control si-CT were cultured in the presence of BH4+Bor combinations for 24 hours.Cell apoptosis were analyzed by Flowcytometry.Results:(1)RNA-seq analysis were performed in MPC-11 and MOPC-315 MM cells with or without BH4 treatment.32 differentially expressed genes were identified,including Usp7 and Usp46.(2)RNA-seq analysis revealed that the addition of BH4 increased the expression of Usp7 and Usp46 in MPC-11 and MOPC-315 MM cells,and the addition of BH4 also increased the expression of Usp7 and Usp46 in Bor-treated MPC-11 MM cells;(3)The addition of USPi increased MPC-11 and MOPC-315 MM cell apoptosis as compared with PBS control,and the addition of USPi further increased the apoptosis of Bor-treated MM cells.Furthermore,the addition of USPi partially abolished BH4-induced MM cell survival in Bor-treated MM cells.Furthermore,the addition of USPi also partially abolished the survival of cells treated with BH4 plus Bor compared to Bor alone;(4)By using si RNAs to specifically silence Usp7 or Usp46 in MPC-11 cells,knockdown of either Usp7 or Usp46 reduced BH4-mediated Bor resistance in MPC-11 cells.Conclusion: BH4 increases MM cell survival via USP7 and USP46.Part 4 Signaling pathways of BH4 mediated MM cell survival Objective: To investigate Usp7 and Usp46 signaling of BH4 promoting MM cell survival.Methods:(1)MPC-11 were cultured with or without the additions of BH4,Bor or their combinations(BH4+Bor)for 2 hours.Western-blots examined the protein levels of p-Ik Bα,Ik Bα and p53.The nuclear protein levels of P50 and P65 were examined by western-blots after 5 hours’ culture;(2)MPC-11 were cultured with USPi,Bor,BH4+Bor or BH4+Bor+USPi for 2 hours.Western-blots examined the protein levels of p Ik Bα,Ik Bα and p53.The nuclear protein levels of P50 and P65 were examined by western-blots after 5 hours’ culture.Results:(1)BH4 treatment exhibited minor effects on the protein level of p53 in MM cells as compared to PBS control.Bor treatment increased the protein level of p53 in MM cells as compared to PBS control,and the addition of BH4 almost completely abolished the increase of p53 in Bor-treated MM cells.Similarly,BH4 treatment exhibited minor effects on the protein levels of p-IκBα and IκBα,but slightly increased the nuclear translocation of P50 and P65 in MM cells as compared to PBS control.And,Bor treatment increased the protein levels of p-IκBα and IκBα,and decreased the nuclear translocation of P50 and P65 in MM cells as compared with PBS control.However,BH4 treatment markedly decreased the protein levels of p-Ik Bα and Ik B-α and increased the nuclear translocation of P50 and P65 in Bor-treated MM cells;(2)USPi partially abolished BH4-induced decrease of p53,p-IκBαand IκBα in Bor-treated MM cells.Furthermore,USPi partially abolished BH4-induced increase of P50 and P65 nuclear translocation in Bor treated MM cells.Conclusion: BH4 increases p53 degradation and NF-κB activation via USP7 and USP46.Part 5 The role of USPs in MM therapy Objective: To examined the efficacy of USP inhibition in Bor-mediated MM treatment.Methods: Balb/c mice were injected s.c.with MPC-11 or MOPC-315 cells(1 × 106 per mouse).From day 6,mice were injected i.p.with Bor,USP7 i or their combinations(Bor+USP7i)every two days.Tumor growth was compared between the different groups.Results: While USPi treatment exhibited minor effects on MM tumor growth as compared to PBS control,the addition of USPi increased Bor-mediated inhibition of MPC-11 and MOPC-315 MM tumor growth.Conclusion: The inhibition of USPs increased Bor antitumor effects in MM. |
PDF Full Text Request