The Impacts Of Nimotuzumab On Pancreatic Cancer Cell Growth In Vitro And In Vivo And Its Potential Mechanisms

Objection:The morbidity and mortality ofpancreatic cancer are both high in China.It is necessary to develop new drugs or optimize exsisting regimes to improvethe outcome ofpancreatic cancer patients.In present study,the impacts of epidermal growth factor receptor(EGFR)expression and Kirsten rat sarcoma viral oncogene(KRAS)status on the efficacy of nimotuzumab inhibiting pancreatic cancer cells growth both in vitro and in vivo were evaluated,and its potential molecular mechanisms were also investigated.Methods:1)EGFR expression and KRAS mutation status of pancreatic cancer cell lines were detected by Western Blot(WB),immunocytochemistry staining(ICC),as well asgene sequencing test,respectively.2)CCK-8assay andflowcytometrywere used to assesscellproliferation,cell cycleand apoptosis of pancreatic cancer cellstreated by nimotuzumab in vitro.The concentration of epidermal growth factor(EGF)and interleukin-6(IL-6)in supernatant of pancreatic cancer cells treated by nimotuzumab were detected by enzyme-linked immunosorbent assay(ELISA),and the expression of EGFR、phospholated EGFR(pEGFR),pErk,pAKTwere detectedby Western Blot.3)Pancreatic cancer cells were subcutaneously inoculated into nude mice to establish tumor xenografts,mice were treated by nimotuzumab intraperitoneally every 3 days,and the control group was treated by saline.The volumes of xenogragfts were measured weekly,and growth curves of the tumors were calculated.Peripheral blood of mice was collected at the end of treatment.EGF and IL-6levels in peripheral blood were detectedby ELISA.Polymerase chain reaction(PCR)was used to evaluate the level of IL-6 in xenografts.The expression of EGFR,pEGFR,pErk,pAKT and IL-6 were determined by Western Blot.Results:1)Pancreatic cancer cell lineBxPc3 withKRAS wild type and EGFRhigh expression,Panc-1 with KRAS mutant type and EGFR low expression,Patu-8988withKRAS mutant type and EGFR high expression were selected.2)Incell proliferation assay,therewere no difference after nimotuzumab treatment comparing with PBS treatment(P>0.05).Cell cycle progression and cell apoptosis in nimotuzumab group and PBS group were also similar(P>0.05).3)For BxPc3 and Patu-8988 cells,the volumes of subcutaneous xenografts innimotuzumab groups were significantsmaller than those in control group(nimouzumab group versus control group,tumor volume at the forth week:BxPc3group:51.05±18.59mm~3vs108.35±30.43mm~3,P<0.05;Patu-8988:103.86±31.64mm~3 vs326.67±142.35mm~3,P<0.05),while there was no difference between nimotuzumab group and control group inPanc-1 group(tumor volumes at the forth week:1992.01±1406.50mm~3 vs1988.82±1359.26mm~3,P>0.05).4)ELISA results showed that compared with those in control group,the levels of EGFand IL-6 in cell supernatant afternimotuzumab treatmentwerenot changed significantly in all three cell lines(allP>0.05).In peripheral blood of mice,EGF levels in nimotuzumab treatment group were significantly decreased compared with those in control group(nimotuzumab group vs control:BxPc3:52.18±3.20pg/ml vs.185.54±5.32pg/ml,P<0.05;Panc-1:49.31±6.16pg/ml vs.80.21±2.30pg/ml,P<0.05;Patu-8988:220.21±7.13pg/ml vs239.59±11.02pg/ml,P<0.05).5)Western blot showed that in vitro,the expression of pEGFR in BxPc3 cell was upregulated compared with control while there were no significant differences in Panc-1 and Patu-8988 cell lines.The expression of EGFR,pErk,pAKT did not change in three cell lines.In vivo,nimotuzumabinhibitedexpression of EGFRand IL-6 inBxPc3 and Patu-8988 xenografts while had no impact on Panc-1xenografts.The expression of pEGFR,pErk,pAKT in BxPc3 and Panc-1 xenografts were upregulated while downregulated in Patu-8988 xenografts after nimotuzumabtreatment.Conclusion:The efficacy of nimotuzumab inhibitingpancreaticcancer cells growth in vivowas correlated with EGFR expression,while was not influenced by KRAS status.The downregulation of IL-6 could participate in the inhibition of pancreatic cancer cells by nimotuzumab in vivo.