The Effect And Mechanism Of Rh-EPO On The Growth Of Human MDA-MA-231Breast Cancer Cells In Nude Mice

Objective: to observe the effect of rh-EPO on the growth of breast cancerMDA-MA-231cells in Vitro and in Vivo of nude mice and to investigate the regulatoryrole and mechanism on tumor growth, angiogenesis and apoptosis of human breastcancer.Method:1. The proliferation abilities of MDA-MB-231cells induced by rh-EPO in differentconcentration and the appropriate dose of p38MAPK inhibitors (SB203580), ERKinhibitors (U0126), JNK inhibitors (SP600125), NF-κ B inhibitors (PDTC)weredetected by MTT assay.2. The inflammatory mediator TNF-α, PGE-2in MDA-MB-231cell lines wereobserved after treated with rh-EPO in terms of an effective dose by Enzyme LinkedImmunosorbent Assay (ELISA).3. MDA-MB-231cells were injected respectively into the oxter of the nude mice toestablish the xenograft． After2weeks, nude mice randomly divided into four groups,respectively, rh-EPO group, negative control group, EPO antagonist group, EPO+EPO antagonist group, and each group has six mice. Administered every three dayswith a total of five times. Then nude mouse were executed in broken neck to measurethe size and weight of tumor.Histopathological changes was tested byhematoxylin-eosin staining．The mRNA and protein expression of EPO，EPO-R，TNF-α，IL-10，COX-2，BCL-2were measured by RT-PCR and western blot．Themicrovessel density(MVD)and VEGF were detected by immunohistochemisty．Theapoptosis of cells were investigated by terminal deoxynucleotidyl transferase mediateddutp-Biotin nick end-labeling assay (TUNEL).Results:1. Rh-EPO could promote the proliferation of MDA-MB-231cells.This function wasdecreased when the cells were treatmented by NF-kappa B inhibitors (PDTC) and p38MAPK inhibitors (SB203580), and no influence after treatmenting by JNK inhibitors (SP600125) and MAPK kinase inhibitors (U0126).2. The ELISA results revealed that rh-EPO could increase the expression of TNF-α,PGE-2of MDA-MB-231cells in a dose and time-dependent manner.3. Compared to the other three groups, the tumor in rh-EPO group grew fastly，both thetumor volume and weight increased obviously(P<0.05).4. RT-PCR and western blot indicated that the mRNA and protein expression of EPO inthe rh-EPO group were evidently higher than those in the negative controlgroups(P<0.01)．As compared with the negative control group, the mRNA and proteinexpression of EPO in the rh-EPO group, EPO antagonist group and EPO+EPOantagonists of EPOR, TNF-α, IL-10were evidently higher than those in the negativecontrol groups(P<0.05)．was significantly higher (P <0.05).5. Immunohistochemical results show that the MVD and VEGF in the rh-EPO groupwere significantly higher than those in the other three groups(P<0.05).6It was found that apoptosis index of rh-EPO group was lower compared with theother three groups （P<0.05）.Conclusion:1. rh-EPO can promote the proliferation of MDA-MB-231cells and the formation ofmicrovessel while suppress apoptosis of breast cancer，which may be played throughthe NF-κB, MAPK pathway.2.The inflammatory mediators as COX-2、 PGE-2andTNFα may participate in theeffection of rh-EPO promoting the proliferation of MDA-MB-231breast cancer cells.