Abstract
Ribonucleotide reductase (RR) is the rate-limiting enzyme that controls the deoxynucleotide triphosphate synthesis and it is an important target of cancer treatment, since it is expressed in tumor cells in proportion to their proliferation rate, their invasiveness and poor prognosis. Didox, a derivative of hydroxyurea (HU), is one of the most potent pharmaceutical inhibitors of this enzyme, with low in vivo side effects. It inhibits the activity of the subunit RRM2 and deoxyribonucleotides (dNTPs) synthesis, and it seems to show iron-chelating activity. In the present work, we mainly investigated the iron-chelating properties of didox using the HA22T/VGH cell line, as a model of hepatocellular carcinoma (HCC). We confirmed that didox induced cell death and that this effect was suppressed by iron supplementation. Interestingly, cell treatments with didox caused changes of cellular iron content, TfR1 and ferritin levels comparable to those caused by the iron chelators, deferoxamine (DFO) and deferiprone (DFP). Chemical studies showed that didox has an affinity binding to Fe3+ comparable to that of DFO and DFP, although with slower kinetic. Structural modeling indicated that didox is a bidentated iron chelator with two theoretical possible positions for the binding and among them that with the two hydroxyls of the catechol group acting as ligands is the more likely one. The iron chelating property of didox may contribute to its antitumor activity not only blocking the formation of the tyrosil radical on Tyr122 (such as HU) on RRM2 (essential for its activity) but also sequestering the iron needed by this enzyme and to the cell proliferation.
Highlights
Ribonucleotide reductase (RR) is one of the fifty genes reported to be overexpressed in highly malignant tumors with poor prognosis [1,2,3,4,5]
Ribonucleotide reductase (RR) is the rate-limiting enzyme that controls the deoxynucleotide triphosphate synthesis and it is an important target of cancer treatment, since it is expressed in tumor cells in proportion to their proliferation rate, their invasiveness and poor prognosis
The iron chelating property of didox may contribute to its antitumor activity blocking the formation of the tyrosil radical on Tyr122 on RRM2 and sequestering the iron needed by this enzyme and to the cell proliferation
Summary
Ribonucleotide reductase (RR) is one of the fifty genes reported to be overexpressed in highly malignant tumors with poor prognosis [1,2,3,4,5]. This compound, targeting RRM2 subunits, was found to trigger cell apoptosis with a different extenTthdisepcoemndpinogunodn,tthaergceltlintygpResR[M132–s1u5b] ubyniitnsc, rweaassinfoguthnedlteovetrlsigogfetrhceepllroa-paoppotpotsoitsicwpirtohteaindiBffaexreantd erxetleenatsedeopf ecnydtoinchgronmtehCe cfreollmtytpheesm[1i3to–c1h5o] nbdyriinac[r1e6a]s.inDgidtohxe rlevelasleodf tghoeopdreoffi-acpaocpytoatgicaipnrsot tmeiunltBipaxle amndyerloelmeaasceelolsf [c1y5t]o, pchrorostmatee tCumfroorm[16t]h,ebrmeaistot cahnocnedrrcieall[s1[61]7.]Daniddoaxcurteevaenadlecdhrgoonoidc meyffeicloaicdyleaugkaeinmsita mHuLlt-i6p0leanmdyKel5o6m2 acecllellisne[s15[]1,4p].roMstoautesetummodorel[s1h6a],vbersehaostwcnanthcaetr dciedllosx[s1i7g]naifincdanatcluytecaaunsdedcghrroownitch minyheilboitdionleoufkbemreiaast HcaLn-c6e0r [a1n7d] aKnd56h2umcealln leinueksem[1i4a][.18M].oFuuserthmeromdeolrse, hinavpehassheoIw/InI cltihnaictaldtirdioaxls, sdigidniofxicsahnotlwyecdaumseindimgraolwtotxhicinithyibinitcioancoefrbpraetaiestnctsan[1c9e,r20[1].7] and human leukemia [18]. SDuildtsox Suppresses the Viability of Hepatocellular Carcinoma HA22T/VGH Cell Line. The hepatocellular carcinoma (HCC) HA22T/VGH cell line was chosen as a model to study 2d.1i.dDoxidaonxtSituupmproerssaecsttihveitVy.iaIbtilhitays oaf dHeetpeacttoacbellleullearveClaorcfiRnoRmMa2HaAn2d2Th/iVghGHlevCeellsl oLfinientracellular iron and iron-Trheleatheedppartootceeilnlus,lainr lcinaercwiniothmiats (hHepCaCti)c HorAig2i2nT(/nVoGt sHhocwelnl).liTnheewcealslscwhoesreeninacus baatmedodweilthtodisffteurdeynt dciodnocxenantrtaittuiomnsorofacdtiidvoitxy.(1I,t 1h0a,s2a5,d5e0t,e1c0ta0b, 2le00leavnedl o5f0R0 RμMM2) faonrd24h–i4g8halnevde7ls ohfainndtrathceelnlutlhaeririrvoinabainlidty iraonna-lryezleadtebdy parnoMteTinTs,asinsayli.nDeidwoixthreidtsucheedpcaetlilcvoiarbigiliinty (innodtosshe-oawnnd).tiTmhee-dceepllesndweenrteminacnunbear t(eFdigwurieth2) dwififtehreinnctrceoanscinengtpraottieonncsyoaftdtihdeoxco(n1,c1en0,tr2a5t,io50n, o1f001,0200, 020a0nadn5d0050μ0Mμ)Mfoarn2d4–a4f8tearn4d87a2nhd a7n2dht(hFeinguthreei2r).
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