Abstract
Prostate cancer (PCa) is a malignant tumor for which there are no effective treatment strategies. In this study, we developed a targeted strategy for prostate-specific membrane-antigen (PSMA)-positive PCa in vitro based on 2-(3-((S)-5-amino-1-carboxypentyl)ureido) pentanedioic acid (ACUPA) modified polyethylene glycol (PEG)-Cholesterol micelles containing wogonin (WOG), which was named ACUPA-M-WOG. ACUPA-M-WOG was conventionally prepared using a self-assembling method, which produced stable particle size and ζ potential. Moreover, ACUPA-M-WOG showed good drug encapsulating capacity and drug release profiles. Fluorescence activated cell sorting (FACS) results suggested that ACUPA modified PEG-Cholesterol micelles could effectively enhance the drug uptake on PSMA(+) PCa cells, and the cytotoxicity of ACUPA-M-WOG was stronger than other controls according to in vitro cellular proliferation and apoptosis assays, separately through methyl thiazolyl tetrazolium (MTT) and Annexin V/Propidium Iodide (PI) staining. Finally, the molecular mechanisms of ACUPA-M-WOG’s effects on human PSMA(+) PCa were investigated, and were mainly the intrinsic or extrinsic apoptosis signaling pathways. The Western blot results suggested that ACUPA-M-WOG could enhance the WOG-induced apoptosis, which was mainly via the intrinsic signaling pathway rather than the extrinsic signaling pathway. In conclusion, ACUPA-M-WOG was successfully developed for WOG-selective delivery to PSMA(+) PCa cells and had stronger inhibition than free drugs, which might make it an effective strategy for PSMA(+) PCa.
Highlights
Introduction1. InAtrsoda umctaijoonr cause of cancer-related death in male patients in Europe and USA, prostate cancer (P(saPPunCCCrdagaa,reA))erslyisiuass,ratrigaaavmedegrgaliyryarje,oetaeirraotfatfcndbea,bcuiuaaturisntdvrieddoeeonenaffn,nofcrdaoafnrlornoodrccbgeaoebarlntoni-hzrtddeehprledaopaptgPtreaieiCdevtnniaaedttnd[sei1otea]sanpt.nhrHatdihnivonedapwrtmuaieppobavunylleiebc(rtAl,phimcaDeaferttmTiadee)prinecaydatarsielp(ciAtsaenhyrlDieEsostTusyedt)rmsaootanespfrd.meeAaIsanrDt.hdnTtedth,hIsePUnetCraSeatnaaAhprdec,elayauprentrdsaiocttrasrtlpathgyarneeotssrecofateoacpfdgraePmnueuCcrtoeetiaocsrf, pcarsotcreadtiuorne-sreasnisdtarnetlaptirvoesltyateeffceacntciveer f(oCrRlPoCca),lizweidthPhCiagh[1]p.otHenocwyeovferm, aefttaesrtaasips earniodd doefvAelDopTm, PeCnta
The multiple peaks around δ 2.0 ppm (d) are assigned to the methylene of the glutamate group in the ACUPA fragment, and the adjacent two single peaks at δ 1.40 and 1.38 ppm (e) are assigned to the tert-butyl ester group in the ACUPA fragment. These results further show that the conjugate has reacted well and target molecule was successfully synthesized.In general, the preparation of ACUPA-M-WOG is utilized a self-assembly method, and the mean size of wogonin loaded micelles is about 24 ̆ 5 nm (Figure 3A), with an optimal drug/polymer (w/w) ratio of 1:19, and optimal drug loading (DL) and entrapped efficiency (EE) of 4.8% ̆ 0.2% and 95.8% ̆ 2.6%, respectively
The transmission electron microscopy (TEM) image of ACUPA-M-WOG is demonstrated in Figure 3C, which implies that the ACUPA-M-WOG is nearly spherical in shape and its diameter is about 30 nm
Summary
1. InAtrsoda umctaijoonr cause of cancer-related death in male patients in Europe and USA, prostate cancer (P(saPPunCCCrdagaa,reA))erslyisiuass,ratrigaaavmedegrgaliyryarje,oetaeirraotfatfcndbea,bcuiuaaturisntdvrieddoeeonenaffn,nofcrdaoafnrlornoodrccbgeaoebarlntoni-hzrtddeehprledaopaptgPtreaieiCdevtnniaaedttnd[sei1otea]sanpt.nhrHatdihnivonedapwrtmuaieppobavunylleiebc(rtAl,phimcaDeaferttmTiadee)prinecaydatarsielp(ciAtsaenhyrlDieEsostTusyedt)rmsaootanespfrd.meeAaIsanrDt.hdnTtedth,hIsePUnetCraSeatnaaAhprdec,elayauprentrdsaiocttrasrtlpathgyarneeotssrecofateoacpfdgraePmnueuCcrtoeetiaocsrf, pcarsotcreadtiuorne-sreasnisdtarnetlaptirvoesltyateeffceacntciveer f(oCrRlPoCca),lizweidthPhCiagh[1]p.otHenocwyeovferm, aefttaesrtaasips earniodd doefvAelDopTm, PeCnta. CNaonwtraadnasyfso,rmchteomcoatshteraratipoyn-risesitshteanmt porsot stcaotme mcaonncerus(CedRPaCp)p,rwoaitchh htioghcpomotbenatcyCoRfPmCe, tadsetsapsiisteanitds wApddaflofswsaAupbdpPhoeopepnnhiaenahEeelo2wnoouvetslcdovcaesaG7ghfggacenpalarper-8iodovi-oorymdlbdimttlect0iatnuiaaoefnlofiesies,alaotixcieragpitssfnloH(tccnaihrecaaii,hmRetuebsede[lvcsseeCd(e-u4OplpiuitteFexabanlug–ohTmtoaistnieiSatin6ctnrigrl,c-icrlry)tcsiayano]1oisatleu.tbea.fiirpsi1agulnnttridpneloc6athslRsegNcgillifiaounxteieastsbhe-acr1nptyeaofltdcrpphacienaAartolrasoiweitasnl-aerdtceoecspeyab)uniccmtttaoaafogceeaoiulHhtlicandtminatylungsportelatyic.teeaayaihyvttdmaseit,voidIlhtyxoii.eccantibvcebfeaseantnhlcoeheiniIi,clrnosogpehtttnfueaoeaaeauiyxrcfmeaenlplfotidatxhyrlmdccsucoslnyppaewgeflapiacurioairlepoea[mecetnnraiko8guruactyoenlmsclch]lgeoloaxnvtsue.ridpsdiepeofiatetoeidrclnpharofurllnrraamfatycxsoeekeuetsereihieptofecnvdreneescpfieltliaylaidltutoieeisrcossipmoittxslovvicnmaicsuluysy[getaloeac(ti7nbsisdtRaornecyrfu]laiitefiikii,iioosOilnnfneensmdfrptedefaiotstasaoStuc.ocuyc,isghSftbna)-ttettieomdf.itceiioeooinr,nvoavominutienn-fttSaeoerrciamfctlslttKsgcmxeayoeeacryil,aalipeameonratnOlmuciincmatsaacssdvenlstaVlenrucksstiuniaehouinxeeervdli3atccmomeainrlliiii,baoneamcssfptotbLimeapiciitu.maantroettitutlNastoMlsieohcsinsatcumurmodllnpeeaCngoiaroohnldnsrlrboaagyneacefraaaitiantlhtnaeeiPgchppcoscinnlo,iaassotapaaeelntuieiAdalsvuanhnsccrucrslcspp,ealltaeeeoc2tutiheairrposwicceto7dvad,ndrcaia,thrp8efeiusircsoxvtpiot0uctvupeptacyeehgoape,inrvtetgovplecseorlioHsadelectieciipahnlscnaesersidpiCnorlfaaipnnftr,t[tioinoedyof7Tlehltrtccdofaraoh]ea-yrr-aot(uSat,1cxlareoseeFoieftch1nmoespfunp[iixp.huce6olo3gsngogcrioatoytp]nncu[.aoyareho.in,d4odieatlbnnlrtSl–scaroiIrcbre[yalctdfinKee63nieonohirlofiaer1[n]e]ipdOHgym2.s.adccstAdhsah.d]utRVert.uiibPIM)bieroecvlcetnlA3caaaomEhihcasxce,ccoiltpeulGetecacitaLietoorecshahntelhCssseyNiyxddtslletittsoeeoldlRbyllyisaniCdaytufvaafglercPhti,essa[[nsugeiaeeeeia28iCatwsrsddPdnhnngyaas]]tt,,,.., FFiigguurree 11.. ((AA)) The chemical structure of wogonin; (BB) Schematic representation of sseellff--assembled. 22--((33--((((SS))--55--aammiinnoo--11--ccaarrbbooxxyyppeennttyyll))uurreeiiddoo))ppeennttaanneeddiiooiicc aacciidd ((AACCUUPPAA)) mmooddiififieedd ppoollyyeetthhyylleennee ggllyyccooll ((PPEEGG))--CChhoolleesstteerrooll mmiicceelllleess ccoonnttaaiinniinngg wwooggoonniinn ((WWOOGG)) ((AACCUUPPAA--MM--WWOOGG)). PPrroossttaattee--ssppeecciifificc mmeemmbbrraannee--aannttiiggeenn((PPSSMMAA))iiss aa mmeemmbbeerr ooff ttyyppee IIII ttrraannssmmeemmbbrraannee ggllyyccoossyyllaatteedd pprrootteeiinn wwiitthh aa mmoolleeccuullaarr wweeiigghhtt ooff aabboouutt 110000 kkDDaa. IIttiissmmaaiinnllyyccoommppoosseeddoofftthhrreeeeffuunnccttiioonnaallddoommaaiinnss:: aa CC--tteerrmmiinnaallddoommaainin, ,a aprportoeatesaesdeodmoaminaianndanadn aapnicaaplidcaolmdaoinm[a9i–n13[9].–T1h3]e. ExTphreesesxiopnrelsesvieolnoflePvSeMl oAf PisSoMbvAioius solbyvuipou-rselgyuulapt-erdeginulparteodstaintepcraonscteartececlalsnctherancenllosrmthaalnpnroosrtmatael ipssruosetoatreeinsdsuotehoerliaelncdeolltsh.eTlihael cgeelnlso.mTihces greesneoamrcihcessreasnedarpcahtehsoalongdicpaaltahnoalolygsiicsahl aavnealpyrsoisvhedavtehaptrPovSMedAthiastoPnSeMofAthiseomnoesotfinthceremasoesdt ienxcprreeassseiodnexpproretesisniosninprPoCteai,nasnidn PthCeaP, aSnMdAtheexpPrSeMssAionexlpevreeslssiohnowlesveal pshosoiwtivseacpoorsreitliavteiocnorwreitlhatitohne wpoitthenthcye pooftetuncmyoorf tpurmogorrepssroogrremsseotarsmtaestiasst[a1s4i–s1[91]4.–1In9].rIencernect eynetayresa, rsse, vseevraelrasltsrtartaetgeigeiseshhaavvee bbeeeenn ddeevveellooppeedd ttoo aacchhiieevvee ttaarrggeettiinngg ccaappaacciittyy ffoorr PPCCaa iinn PPSSMMAA ttaarrggeetteedd pprrooddrruuggss oorr nnaannoommeeddiicciinneess,, ssuucchh aass ppeeppttiiddeess,, RRNNAA aappttaammeerrss aanndd mmoonnoocclloonnaall aannttiibbooddiieess ((mmAAbb)). TThhee pphhaaggee ddiissppllaayy tteecchhnnoollooggyy iiss uusseedd ttoo ssccrreeeennaannddidideenntitfiyfybbininddininggpeppetpidtiedsefsofroPrSPMSAM,Aa,nadntdhethreesureltssusltusgsguegstgethsat tthKaYtLKAYYLPADYSPVDHSIWVHaInWd WanQdPDWTQAPHDHTWAHATHLWcaAnTbLindcatno PbSiMndA twoithPShMigAh afwfinitihty hanigdhinahfifbinititiyts galnudtaminahteibciatrbitosxygpleuptatimdaastee acacrtibvoixtyyp[2e0p–t2id2a].seSimacitliavriltyy, L[u20p–o2l2d].etSailm. AAftecranbinredlienagsetoDPUSMPAA,iDntUoPcAytcoapnlabsemimamndedtihaetenlyreetnudroncytotostehde into clathrin-coated pits, and PSMA can release DUPA into cytoplasm and return to the cell
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