Erythropoietin receptors (EPORs) are present not only in erythrocyte precursors but also in non-hematopoietic cells including cancer cells. In this study, we determined the effect of fetal bovine serum (FBS) in culture medium on the EPOR expression and viability of the estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 breast cancer cells. Using flow cytometry, we showed that the inclusion of 10% FBS in the medium increased the EPOR expressions and viabilities of MDA-MB-231 and MCF-7 cells. The MDA-MB-231 showed greater EPOR expression than MCF-7 cells, suggesting that the presence of ERs on cells is associated with poor expression of EPOR. Culture medium containing 10% FBS also caused increased number of breast cancer cells entering the synthesis phase of the cell cycle. The study also showed that rHuEPO treatment did not affect viability of breast cancer cells. In conclusion, it was shown that the inclusion of FBS in culture medium increased expression of EPOR in breast cancer cells and rHuEPO treatment had no effect on the proliferation of these cancer cells.
Nanomedicine is an emerging area in the medical field, particularly in the treatment of cancers. Nanostructured lipid carrier (NLC) was shown to be a good nanoparticulated carrier for the delivery of tamoxifen (TAM). In this study, the tamoxifen-loaded erythropoietin-coated nanostructured lipid carriers (EPO-TAMNLC) were developed to enhance the anti-cancer properties and targetability of TAM, using EPO as the homing ligand for EPO receptors (EpoRs) on breast cancer tissue cells. Tamoxifen-loaded NLC (TAMNLC) was used for comparison. The LA7 cells and LA7 cell-induced rat mammary gland tumor were used as models in the study. Immunocytochemistry staining showed that LA7 cells express estrogen receptors (ERs) and EpoRs. EPO-TAMNLC and TAMNLC significantly (p<0.05) inhibited proliferation of LA7 in dose- and time-dependent manner. EPO-TAMNLC induced apoptosis and G0/G1 cell cycle arrest of LA7 cells. Both drug delivery systems showed anti-mammary gland tumor properties. At an intravenous dose of 5 mg kg-1 body weight, EPO-TAMNLC and TAMNLC were not toxic to rats, suggesting that both are safe therapeutic compounds. In conclusion, EPO-TAMNLC is not only a unique drug delivery system because of the dual drug-loading feature, but also potentially highly specific in the targeting of breast cancer tissues positive for ERs and EpoRs. The incorporation of TAM into NLC with and without EPO coat had significantly (p<0.05) improved specificity and safety of the drug carriers in the treatment of mammary gland tumors.
Tamoxifen (TAM) has been used in the treatment of breast cancers and is supplemented with erythropoietin (EPO) to alleviate the cancer-related anemia. The purported deleterious effects caused by the use of EPO with chemotherapeutic agents in the treatment of cancer-related anemia vary across studies and remain controversial. The use of nanoparticles as a drug delivery system has the potential to improve the specificity of anticancer drugs. In this study, we simultaneously incorporated two pharmacological active ingredients in one nanocarrier to develop EPO-conjugated TAM-loaded lipid nanoparticles (EPO-TAMNLC), a targeted delivery system, to enhance the cytotoxic activity while reducing the side effects of the ingredients. The effect of temperature in modulating the thermodynamic parameters associated with the binding of EPO and TAMNLC was assessed using isothermal titration calorimetry, while the unfolding of EPO structure was determined using fluorescence-quenching approach. The association efficiency of EPO and TAMNLC was 55.43%. Unlike binding of albumin to TAMNLC, the binding of EPO to TAMNLC occurred through endothermic and entropy-driven reaction. The EPO-TAMNLC formulation was stable because of the hydrophobic interaction and the high free energy, suggesting the spontaneity of the interactions between EPO and TAMNLC. The EPO-TAMNLC enhanced the in vitro cytotoxicity of TAM to MCF-7 cells. The EPO surface-functionalized TAMNLC could sequentially deliver EPO and TAM as well as improving site-specific delivery of these therapeutic compounds.