Recombinant Carboxypeptidase G2on Toxicity And Anti-Cancer Activity Of Methotrexate

Methotrexate (MTX) is clinically used for the treatment of pediatric leukemia,osteosarcoma, choriocarcinoma, breast carcinoma and other cancer. It also showscurative effects for autoimmune diseases, such as psoriasis and rheumatoid arthritis.However, serious adverse reactions, including myelosuppression, orointestinalmucositis and renal dysfunction, limit its clinical application. MTX is primarilyexcreted via the kidneys as the prototype drug and nephrotoxicity is one of the mostserious adverse reactions and one of the most common cause of death resulted byMTX. Once MTX results in the damage of kidney, the elimination of MTX would beimpaired more and more and nephrotoxicity would become more and more lethal.CPDG2is a bacterial enzyme that rapidly cleaves MTX to two inactive metabolites:amino acid glutamate and2,4-diamino-N10-methylpteroic acid (DAMPA). DAMPAis primarily excreted via the liver. Conversion of MTX to DAMPA relieves stress onthe kidneys for elimination of MTX and generates a relatively nontoxic compound,reducing HDMTX risks in clinical.The objectives of this study were to observe MTX’s antitumor effects withfollow-up CPG2treatment both in vivo and in vitro, to explore reasonable dose andschedule of CPG2administration, and to make sure whether CPG2could reduce ofMTX’s toxicity but not reduce MTX’s antitumor activity, with an eventual aim tomake a suggestion for the combinational use of CPG2and MTX in clinical.In vitro, MTT method was used to evaluate the effect of CPG2on MTX’stoxicity to MCF-7and JEG-3cancer cell lines. In vivo, mice models bearing S180sarcoma homograft and nude mice models bearing human breast cancer MCF-7xenografts were used to evaluate the effects of CPG2on HDMTX’s anti-tumoractivity and toxicity. Anti-tumor effect observed in this study included maximuminhibition rate, tumor weight inhibition rate, tumors volume inhibition rate and tumordistribution, etc; MTX toxicity index mainly included animals survival, body weight, serum creatinine, serum urea and pathological test.In vitro, MTX showed a strong inhibitory effect on MCF-7and JEG-3cells on aconcentration-dependent manner following treatment with concentrations of0.15625,0.3125,0.625,1.25,2.5,5and10μg/ml for2hours. Next, CPG2at concertration of0.01,0.1,1,25,50and100U/ml were used to catalyze MTX and terminate itsanticancer effects. As expected, CPG2could not impair the existed anticancer effectsof MTX, but could stop its further toxic effects on cancer cells.In the toxicity test using KM mice with S180sarcoma homografts, four micefrom500mg/kg MTX group died during the experiment. After MTX treatment, allmice lost their weights and showed the lowest weights on the seventh day. Thereafter,body weight recovered gradually. As to MTX+24hCPG2group, only one mouse diedand other mice showed their lowest weight on the fiveth day after MTX treatment.From Day6, their body weight recovered gradually. For MTX+12hCPG2group, nomouse died during the experiment.In the toxicity test using nude mice with human breast cancer MCF-7xenografts,three mice from800mg/kg MTX group died during the experiment. After MTXtreatment, all nude mice lost their weights and recovered slowly from the seventhday.For MTX+12hCPG2group, no animals died and body weight of animalsrecovered completely on the seventh day. As to MTX+16hCPG2group, one mousedied and body weight of animals recovered completely on the eighth day.For KM mice with S180sarcoma homografts, the inhibition rate came up to58.5%when treated with500mg/kg MTX. If400U/kg or100U/kg CPG2wasadministered following the treatment with500mg/kg MTX for16hours, theinhibition rate of MTX was44.0%and59.0%, respectively. Four of8mice died whenadministrated with MTX alone. However, all mice survived with follow-upadministration of CPG2. The highter dose of CPG2was used, the smaller loss ofweight and faster recovery of MTX-treated mice were observed. Repeatedexperiments showed the similar results.For nude mice with human breast cancer MCF-7xenografts, the inhibition rate came up to32.4%when treated with800mg/kg MTX. If200U/kg,100U/kg or50U/kg CPG2was administered following the treatment with800mg/kg MTX for12hours, the inhibition rate of MTX was37.1%,37.6%and59.0%, respectively. Four of8mice died when administrated with MTX alone. However, only one mouse diedwith follow-up administration of200U/kg or50U/kg CPG2. Taken together, thetoxicity of MTX was largely reduced by follow-up administration of CPG2.No obvious nephrotoxicity effects were observed from serum creatinine and ureatest, when the MTX administrated concentration was500-800mg/kg. Renalpathological test neither showed obvious damage on kidney. Collectively, theincidence of renal injury caused by MTX was too small to be observed in the periodof our experiments.Above all, in vitro, CPG2could not impair the existed anticancer effects of MTX,but could stop its further toxic effects on cancer cells. In vivo, the toxicity of MTX(500-800mg/kg) could be reduced by12-16h later fellow-up administration of50-100U/kg CPG2, without reduction of MTX’s anticancer activity. All these resultswould make a suggestive indication for the combinational use of CPG2and MTX inclinical...