Tetrathiomolybdate Inhibits The Reaction Of Cisplatin With Human Copper Chaperone Atox1

Cisplatin is a widely used anticancer drug in clinic.It is well known that cisplatin induces cell apoptosis by binding to DNA in the nucleus;however,only a very small portion of cellular platinum is able to form DNA crosslinks in the nucleus.Increasing evidence indicates that also some protein targets are involved in the mechanism of cisplatin action.The human copper chaperone Atox1 was found to play a role in cisplatin intracellular transport.The CXXC copper-binding motif of Atoxl can also bind Pt(?).In-cell NMR spectroscopy indicated that cisplatin reacts quantitatively with Atox1,while in vitro assays showed that cisplatin binding can induce unfolding and aggregation of Atoxl.Thus,it has been proposed that Atox1 can be a candidate for cisplatin resistance by competing with DNA platination.Ammonium tetrathiomolybdate([(NH4)2MoS4],TM)is a copper chelator used in clinic for the treatment of Wilson's disease.TM also showed potency in the inhibition of tumor growth by its anti-angiogenic effects.Mechanistic investigations indicated that TM modulates copper levels by binding to copper proteins,such as serum albumin,ceruloplasmin and metallothioneins.A number of researches showed that TM can enhance the therapeutic effect and circumvent the drug resistance of cisplatin;In addition,TM can enhance the therapeutic effect of cisplatin by selectively increasing DNA platination in cancerous but not in normal tissues.However,the origin of this effect is not clear.It has been proposed that the synergistic effect of TM and cisplatin could be due to the copper chelation ability of TM.Recently,a X-ray crystal structure showed that TM binds to a yeast Cu chaperone Atx 1,forming a stable[TM·Cu·(Cu-Atx1)3]complex.Interestingly,Atx1 is the analogue of human Cu chaperone Atox1,and Atoxl is found to be associated with the drug resistance of cisplatin.Based on these finding,we hypothesized that TM could interfere with the reaction between Cu-Atox1 and cisplatin,and this reaction could reduce the drug resistance of cisplatin.In this work,we have investigated the influence of TM on the platination of Atox1.Results confirmed that TM can induce protein trimer of Atox 1 in solution,and inhibit the platination of Cu-Atox1.Consequently,TM prevents protein unfolding and aggregation induced by cisplatin.The reaction was also performed on apo-Atox1 and Ag-Atox1.Interestingly,TM only inhibits the platination of Cu-Atox1,while it does not prevent the reaction of Ag-Atox1 with cisplatin under the same experimental condition.This result indicates that the formation of the Mo-Cu-centered trimeric protein,which impedes the attack of cisplatin to cysteine residues in Atox1,is a key factor in the overcoming of the Atox1-dependent cisplatin resistance.These results help to understand the synergistic effect of TM and cisplatin in cancer chemotherapy.In chapter 1,the mechanism of platinum drugs are reviewed.The relationship between the resistance of platinum based drugs and the copper transporter system have been discussed,including the structures and functions of copper transport protein CTR1,human copper chaperone Atox1 and P type ATPases.The anticancer functions of TM are also reviewed.In chapter 2,the influence of TM on the platination of Cu-Atox1 in vitro was investigated.Chromatographic analyses and ESI-MS demonstrate that TM inhibits the reaction of cisplatin with Cu-Atox1.NMR spectra indicate that TM binds to Cu-Atoxl and this reaction could prevent the reaction of cisplatin with Cu-Atoxl.Circular dichroism and gel electrophoresis analyses confirmed that protein unfolding and aggregation occurred on the reaction of cisplatin with Cu-Atoxl,but not with TM-Cu-Atox1.Unlike Cu-Atoxl,apo-Atox1 can still react with cisplatin in the presence of TM.Notably,although Ag(?)binds to Atox1 in a way similar to Cu-Atox1,TM does not prevent the reaction of Ag-Atoxl with cisplatin since TM cannot induce a protein trimer of Ag-Atox1.These results indicate that the formation of Mo-centered tetra-copper cluster in the TM-Cu-Atox1 complex plays a key role in inhibition of the platination of Atox1.