Erythropoietin accelerates tumor growth through increase of erythropoietin receptor (EpoR) as well as by the stimulation of angiogenesis in DLD-1 and Ht-29 xenografts

Abstract

Anemia is a relatively common symptom coexisting with colorectal carcinoma. Besides having a positive impact on hematological parameters, erythropoietin (Epo) has the serious adverse effect of promoting the neoplastic process. The role of Epo in colon cancer has not been clearly shown. The aim of this study was to assess the effects of Epo therapy on colorectal carcinoma cells both in in vitro and in animal models. Human colon adenocarcinoma cells DLD-1 and Ht-29 were cultured in medium with Epo beta in normoxia. Cell proliferation was measured with an automated cell counter. Expression of erythropoietin receptor (EpoR) mRNA, Akt mRNA, and their proteins were assessed by RT-PCR and confocal microscopy, respectively. Nude mice were inoculated with adenocarcinoma cells and treated with a therapeutic dose of Epo. Expression of EpoR, VEGF, Flt-1 and CD31 was evaluated in xenograft tumors. We identified that Epo through EpoR activates Akt, which promotes colon cancer cell growth and proliferation. Epo, and high levels of phosphorylated EpoR, directly accelerates tumor growth through its proliferative and proangiogenic effects. This study demonstrated that Epo had enhanced carcinogenesis through increase of EpoR and Flt-1 expression, and thereby contributed to tumor development. These results suggest that both EpoR-positive and EpoR-negative cancer cells could be regulated by exogenous Epo. However, an increased response to erythropoietin was observed in the EpoR-positive cells. Thus, erythropoietin increases the risk of tumor progression in colon cancer and should not be used to treat anemia in this type of cancer.

Simultaneous use of erythropoietin and LFM‐A13 as a new therapeutic approach for colorectal cancer

“…Previously, we proved that Epo has varied effects on cells dependent on the presence of the Epo receptor (Tankiewicz‐Kwedlo et al, ). Here, we wanted to examine whether the various effects would be maintained after incubation with Epo, LFM‐A13 and both of these compounds together.…”

“…Quantitative‐real‐time‐PCR was performed using a standard method described previously (Tankiewicz‐Kwedlo et al ., ). Primers were designed using PRIMER‐ blast software (http://www.ncbi.nlm.nih.gov/tools/primer-blast).…”

“…DLD‐1 (ATCC, Cat# CCL‐221, RRID:CVCL_0248) and HT‐29 (ATCC, Cat# HTB‐38, RRID:CVCL_0320) , cell lines of human colorectal adenocarcinoma, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The characteristics of these cell lines were as presented previously (Tankiewicz‐Kwedlo et al, ).…”

“…Immunofluorescence was performed as described previously (Tankiewicz‐Kwedlo et al, ). The cells were incubated with mouse monoclonal D‐5 antibody against EpoR (Santa Cruz Biotechnology, Cat# sc‐365662, RRID:AB_10841725), rabbit polyclonal antibody against phospho EpoR (Santa Cruz Biotechnology, Cat# sc‐20236‐R, RRID:AB_2098548), rabbit monoclonal H‐136 antibody against Akt1/2/3 (Santa Cruz Biotechnology, Cat# sc‐8312, RRID:AB_671714) and rabbit polyclonal Ser 473 antibody against phospho Akt1/2/3 (Santa Cruz Biotechnology, Cat# sc‐7985 also sc‐7985‐R, RRID:AB_667741) and then incubated with fluorescent (FITC) secondary goat polyclonal antibody against mouse (Sigma‐Aldrich, Cat# F0257, RRID:AB_259378) or fluorescent (FITC) secondary goat polyclonal antibody against rabbit (Sigma‐Aldrich, Cat# F9887, RRID:AB_259816) for 60 min in the dark.…”

“…In light of our previous research and other evidence, erythropoietin (Epo) is regarded as a promoter of the neoplastic process (Yasuda et al, ; Hardee et al, ; Tankiewicz‐Kwedlo et al, ; Tankiewicz‐Kwedlo et al, ). Epo, when used to treat anaemia promotes the survival, proliferation and differentiation of the progenitors of erythropoiesis, as well as exerting a pro‐angiogenic and an anti‐apoptotic effect.…”