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Erythropoietin and Breast Cancer Progression: An in vitro Study Joy Obayemi**, Alyssa Calabro*, Craig Queenan*, David Becker*, and Donna Leonardi** * Bergen County Academies, Nano-Structural Imaging Lab, 200 Hackensack Avenue, Hackensack, NJ 07601 ** Bergen County Academies, Biotechnology Lab, 200 Hackensack Avenue, Hackensack, NJ 07601 Introduction Erythropoietin (EPO) is a glycoprotein hormone that is the main regulator of erythropoiesis, the production of red blood cells (RBCs) in most mammals. Advances in biotechnology have made it possible to create recombinant erythropoietin than can induce the normal erythropoietic response in patients suffering from anemia [1]. In 2007, the US Food and Drug Administration issued a Black Box Warning on all Erythropoiesis-Stimulating Agents (ESAs) due to the growing concern that recombinant EPO, when used as treatment for chemotherapy-induced anemia, was actually increasing the risk of tumor progression in patients with cancer of the breast, lung (non-small cell ), head, neck, lymph, and cervix [2]. Interestingly, reports in the literature indicate that members of the statin family of drugs typically used for the regulation of cholesterol, have shown some potential as antitumorigenic agents [3]. Statins primary mechanism of action is to effectively inhibit the mevalonate pathway, required for the production of cholesterol. Members of this same pathway are necessary to produce functional receptors for ESAs through geranylgeranylation. It has been suggested that statins may be able to inhibit the production of functional EPO receptors, with the potential to render ESAs safe with regard to tumor progression [4]. In this study, two breast cancer cell lines were used: triple-negative MDA-MB-231 and estrogen receptor positive MCF-7. Assays were carried out in both normoxia and hypoxia (3%) in order to model the physiological conditions at the center of a dense tumor mass. To maximize bioavailability of the statin (Lovastatin), gold nanoparticle-cyclodextrin conjugates as well as liposomes as carrier molecules for drug delivery were evaluated. Cyclodextrin, an oligosaccharide with a cylinder-shaped cavity that has already shown potential as a drug carrier molecule [5],was impregnated with Lovastatin and conjugated to gold nanoparticles. Gold nanoparticles have recently become the focus of the pharmacological community because of their size, stability, and biocompatibility, characteristics that make them ideal molecules for drug-delivery [6]. Liposomes, which were also evaluated, have the ability to carry more drug. Results & Discussion Figure 1: Concentrations of EPO secreted from breast cancer cell lines under normoxia and hypoxia. Bars are means ± STD DEV (n=5). * = p < 0.05 Figure 2: Viability of MCF-7 cells treated with EPO, Cisplatin, and a combination of the two drugs. Bars are means ± STD DEV (n=5). * = p < 0.05 Figure 3: The effect of Lovastatin treatment on EPO receptors in breast cancer cell lines. OD450 measured. Bars are means ± STD DEV (n=5). * = p < 0.05 vs. control Figure 4: Lovastatin treatment on control HaCat cells under normoxia and hypoxia. Bars are means ± STD DEV (n=5). * = p < 0.05 vs. control Figure 5: Lovastatin treatment on breast cancer cell lines under normoxia. Bars are means ± STD DEV (n=5). * = p < 0.05 vs. control Figure 6: Lovastatin treatment on breast cancer cell lines under hypoxia. Bars are means ± STD DEV (n=5). * = p < 0.05 vs. control Materials & Methods Cell Culture MDA-MB-231 and MCF-7 (ATCC) breast cancer cells were cultured in DMEM/F12 medium supplemented with GlutaMAXTM-1 (Invitrogen), 10% fetal bovine serum, and penicillin/streptomycin, and incubated at 37°C and 5% CO2. For experiments at normoxia cells were kept in 21% O2 environment; hypoxia experiments used 3% O2. All cell viability testing was performed using the CellTiter 96® AQueous MTS cell proliferation assay (Promega). Erythropoietin Assay An EPO ELISA (R&D Systems) was conducted on both cell lines in both normoxic and hypoxic conditions. After 24 h, the EPO levels in the cells’ media were measured at 450nm. Effect of Erythropoietin on Cisplatin Efficacy Both cell lines were seeded in 96 well plates at 20,000cells/well and incubated for 24 h. 1U/mL EPO (R&D Systems) was added to the sample wells and the plates were incubated for another 24 h. 20µg/ml of cisplatin LD50 was added to the sample wells . After another 24 h of incubation, an MTS assay was performed. Erythropoietin Receptor (EPOR) Assay Cells were treated with 50μM Lovastatin (Sigma Aldrich) in both normoxia and hypoxia (3%). After 24h incubation the cells were lysed and the supernatants collected for ELISA and measured at 450nm (R&D Systems) . Effects of Lovastatin on Breast Cancer Cells and Non-Cancerous Cells Lovastatin was diluted in ethanol (1, 5, 10, 20, 50, 100µM). MDA-MB-231, MCF-7, and HaCat human keratinocytes (control), were aliquoted into 96-well plates at a concentration of 20,000cells/well, incubated for 24h in both normoxia and hypoxia, and then treated with the Lovastatin dilutions. After another 24h incubation a cell proliferation assay was performed (as described above). Gold Nanoparticle-Cyclodextrin Conjugates Cyclodextrin molecules , with and without Lovastatin (maximum concentration of 2x108μM), were conjugated to 40nm spherical gold nanoparticles by NanoPartz, Inc. Lovastatin and control conjugates were aliquoted into 96-well plates containing both cell lines (20,000cells/well) and incubated for 24h in both normoxia and hypoxia. Cell viability was tested as described above. Liposome-Encapsulated Lovastatin Lovastatin (10mg/mL) was added to an aliquot of Pre-Liposome Formulation 4 (Sigma Aldrich). 0.2 mL of water was added, followed by one minute of vortexing. 0.8 mL of water was added and the solution was agitated for 30 minutes resulting in the formation of large multilamellar liposomes or vesicles (MLV). Cells were treated with both Lovastatin (20mM) and liposomes containing the same Lovastatin concentrations. Cell viability was tested as described above. Transmission Electron Microscopy Cells treated with gold nanoparticle carriers were fixed in 4% glutaraldehyde/2% formaldehyde in 0.2M sodium cacodylate buffer, pH 7.4, scraped from the wells with Teflon® and gently spun down to form a pellet. The pellet was post-fixed in 2% osmium tetroxide, dehydrated in a graded series of acetone, infiltrated and cured in epoxy resin. 100nm sections were collected onto 200 mesh copper grids and post-stained with 4% uranyl acetate and 0.5% lead citrate. A 2 µl aliquot of liposomal carriers in suspension was placed on a 200 mesh formvar/carbon coated copper grid and negatively stained with 2% uranyl acetate prior to imaging. The first phase of this study was to identify the relationship between cancer cells, EPO, and chemotherapeutic agents (Cisplatin). In normoxia and hypoxia, it was determined that: exogenous EPO significantly increased cell viability in both normoxia and hypoxia (data not shown) and there was a significant increase in endogenous EPO secretion in hypoxic cells (Figure 1). The effect of this increased EPO concentration on chemotherapeutics was then tested. This demonstrated that cancer cells treated with a combination of Cisplatin and EPO demonstrated a significant increase in viability over those treated with Cisplatin alone (Figure 2; results for MDA-MB-231 cells showed similar results, data not shown). This indicates that EPO treatments do significantly decrease the efficacy of chemotherapeutic drugs. The next phase of this study examined the anti-tumorigenic effect of Lovastatin on these cells. Lovastatin was first tested on HaCat keratinocytes (which do not have EPORs) to show the drug could be used safely and as a targeted chemotherapeutic agent against cancer cells bearing EPOR’s. The results demonstrated that in normoxia and hypoxia, there was no significant cell death until the highest concentration (100µM) of Lovastatin was administered (Figure 4). When the same concentrations of Lovastatin were tested on both of the cancer cell lines in hypoxia and normoxia, results showed significant cell death in both cancer lines by the 50µM concentration treatment (Figures 5 and 6). This data shows that cancer cells are susceptible to Lovastatin at lower concentrations than non-cancer cells. The effect of the 50µM concentration on EPOR was then tested. There was a significant reduction in the relative amount of receptors found in treated cells versus control cells (Figure 3). This data suggests there may be a connection between the reduction in the number of EPORs and the viability of cancer cells treated with Lovastatin. The final phase of the study was to determine if the efficacy of Lovastatin could be increased by drug carriers. Gold nanoparticle-cyclodextrin conjugates were the first tested. TEM micrographs of MDA-MB-231 cells after treatment showed that the cells internalized the nanoparticles, compartmentalizing them within vesicles (Figure 7). Although the nanoparticle conjugates were taken in by the cells, viability tests showed greater cell death in cells treated with the nanoparticle conjugates without the Lovastatin than the carriers with Lovastatin (data not shown). The second carrier tested was liposomes (20µM). TEM micrographs of liposomes after combination with Lovastatin were used to understand the size and structure of the carriers (Figure 8A). The liposomes appeared globular in shape with an apparent bilayer membrane, and ranged in size from 100nm-400nm in diameter. Viability tests were then performed, which showed a significant decrease in cell viability of cells treated with the liposome-Lovastatin complex as compared to Lovastatin alone (Figure 8B). Overall, this study shows that EPO does decrease the efficacy of cisplatin treatment by increasing the proliferation of the breast cancer cells tested, and that Lovastatin has the potential to reverse this effect. Furthermore, Lovastatin could itself be used as a targeted anti-tumorigenic agent as it is more cytotoxic to cancer cells than non-cancer cells. This effect could be related to Lovastatin’s ability to inhibit the post-translational modifications needed to make functional EPORs. Finally, it was determined that the effective dose of Lovastatin needed to kill cancer cells could be reduced through the use of liposomal carriers. References [1] H. Corwin, et al., The New England Journal of Medicine. 357(10) (2007) 965-975. [2] R. Steinbrook, et al., The New England Journal of Medicine. 356 (2007) 24482451. [3] K. Hindler, et al., The Oncologist. 11 (2006) 306-315. [4] S. Hamadmad, et al., The Journal of Pharmacology and Experimental Therapeutics. 316(1) (2005) 403-409. [5] R. Patel, et al., Dhaka University Journal of Pharmaceutic Science. 6(1) (2007) 2536. [6] C. Park, et al., Journal of Materials Chemistry. 19 (2009) 2310-2315. Figure 7: TEM micrographs of gold nanoparticle-cyclodextrin conjugates internalized in MDA-MB231 cells. Scale bars = 5µm Figure 8: A) TEM micrograph of liposomal carriers containing Lovastatin. B) Viability of breast cancer cell lines after treatment with Lovastatin and the Lovastatin encapsulated liposomal carriers. Bars are means ± STD DEV (n=5). * = p < 0.05 vs. control Presented at Microscopy & Microanalysis 2011 August 7-11, Nashville, TN Poster Number: 211 Paper Number: 81490 Acknowledgements • Dr. Howard Lerner, Superintendent, Bergen County Technical Schools & Special Services • Edmund Hayward, Technology Director, Bergen County Technical Schools • Russell Davis, Principal, Bergen County Academies