Download Erythropoietin and Breast Cancer Progression: An in vitro Study

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