Jason Kinyon
Abstract
There has been evidence that shows inhibiting Cyclo-oxygense 2 (COX-2) enzymes may decrease angiogenic factors such as Vascular Endothelial Growth Factor (VEGF), which may prove beneficial in halting angiogenesis and metastasis, stopping cancer progression. It has been shown that there is a significant positive correlation between COX-2 and VEGF mRNA expression in breast cancer specimens. 17-Estradiol (E2) has shown to increase VEGF production by binding to estrogen receptor positive breast cancer cell lines such as MCF-7. The COX-2 inhibitor celecoxib can be added to E2 treated MCF-7 cells to determine its effect on VEGF production using the Enzyme Linked Immunoabsorbent Assay (ELISA).
Introduction
Cancer is the second leading cause of death in the United States. Over one million people are diagnosed with cancer each year. Approximately one out of every two American men and one out of every three American women will be diagnosed with cancer at some point during their lifetime. Recent advances in cancer research have concentrated on angiogenesis and its process of generating new blood vessels into tumors which eventually leads to tumor growth and metastasis. One protein that is a main player in blood vessel recruitment is VEGF which is expressed by metastatic tumors. VEGF expression has recently been found to be upregulated in MCF-7 breast cancer cell line in the presence E2. E2 is able to upregulate transcriptional activation of VEGF by binding to estrogen receptors and and inducing its signal pathway.3 VEGF has also been recently shown to be reduced by COX-2 enzyme inhibition in other cancer cell lines. Cyclo-oxegenases (COX) are the rate limiting enzymes in the pathway of converting arachidonic acid to prostaglandins which are responsible for inflammation and have been linked to cancer. In breast cancer, a product of the COX-2 pathway prostaglandin E2 (PGE-2) has been shown to increase VEGF. COX-2 mRNA has shown to be overexpressed in both human breast cancer and adjacent non-cancerous tissue.2 However, there has been no study known to show if COX-2 inhibition affects VEGF production in the estrogen dependant breast cancer cell line MCF-7. If in fact COX-2 inhibition does play a role in limiting E2 stimulated VEGF production, it would be beneficial to breast cancer patients diagnosed with estrogen receptor positive tumors. My hypothesis is that COX-2 inhibition plays a role in reducing VEGF production and metastasis in the estrogen receptor positive cancer line MCF-7.
Methods
Cell Culture- MCF-7 (breast adenocarcinoma) cells were cultured in Minimum Essential Media (Hyclone, Logan, UT) supplemented with 10% Bovine Calf Serum and maintained at 37oC in 5% CO2. Cells were passaged when 90% confluent with 0.1% Trypsin.
Chemicals- E2 was purchased through Sigma (St. Louis, MO) and was dissolved in 100% ethanol. Two 1:1000 dilutions were made to make a final stock solution of 2 µM. Celecoxib was a gift from Pharmacia/Pfizer (St. Louis, MO) and was dissolved in DMSO. Stock solutions of 100 mM, 60 mM, and 20mM were used. Combined Solvents (ETOH and DMSO) never exceeded .1% v/v in the treatment media.
Alamar Blue (AB)- Alamar Blue (Biosource, Camarillo, CA) was used to measure cell proliferation during treatment of celecoxib. 96 well plates were seeded at 2,500 MCF-7 cells/well and left to attach to the plate. On the second day after seeding, cells were treated with 1nm E2, and 1nm E2 along with 50µM, 30µM, and 10 µM of Celecoxib. Metabolic rate was measured using 10% AB and monitored for 24 hours using a plate reader at two different wavelengths of 570nm and 610nm. The metabolic rate was compared to the media control using the equation (Test 570-(610*R))/(Control 570-(610*R)) where R is a ratio of readings of media with no cells over media with 10% AB.
VEGF Competitive Enzyme Linked Immunoabsorbent Assay- The amount of VEGF from supernatant media from treated and control MCF-7 cells was measured by a Competitive ELISA kit (Chemicon, Carlsbad, CA).
Results
Alamar Blue- Alamar Blue (AB) was used to determine if cells would remain in exponential phase and to determine if the celecoxib and E2 treatments were cytotoxic to the MCF-7 cells. Alamar Blue data indicated each of the individual treatments of 1nm E2 along with 50 µM, 30 µM, or 10 µM were in exponential and above 90% of the metabolic rate compared to the control (Figure 1). This indicates that the concentrations that were used were not cytoxic to the cells.
VEGF Competitive Enzyme Linked Immunoabsorbent Assay- The supernatants of five experiments were analyzed using a VEGF competitive ELISA kit. The positive control of cell treated solely with E2 did not significantly increase the VEGF produced as previously predicted. Test concentrations were not significantly different as seen in figure 3.
Discussion
COX-2 inhibition has become an area of interest in cancer research for the last five years. Studies in tumors treated with COX-2 inhibitors have generally shown their anti-angiogenesis properties. MCF-7 cells generally express minimal COX-2 proteins. One of the goals of this experiment was to see if E2 stimulated MCF-7 cells produced VEGF via a COX-2 related pathway. As shown by the ELISA, the MCF-7 cells used for this experiment were not sensitive to the 1nm E2 treatment that the cells were exposed to and thus the cells did not increase VEGF levels. According to Hamlers et al., different MCF-7 cell lines mitogenic response to E2 may vary and may even be absent. I suggest that this may have been the case for the MCF-7 used in this experiment. A more highly E2 responsive MCF-7 cell line would be necessary to see if in fact celecoxib has an effect on E2 induced VEGF production.
References
- Iniguez MA et al. Cyclooxygenase-2: a therapeutic target in angiogenesis. Trends Mol Med. 2003 Feb; 9(2): 73-78.
- Kirkpatrick K et al. The mRNA expression of cyclo-oxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) in human breast cancer. Curr Med Res Opin. 2002;18(4):237-41.
- Buteau-Lozano H et al. Transcriptional regulation of vascular endothelial growth factor by estradiol and tamoxifen in breast cancer cells: a complex interplay between estrogen receptors alpha and beta. Cancer Res. 2002 Sep 1;62(17):4977-84.
- Hamlers IH et al. 17beta-Estradiol responsiveness of MCF-7 laboratory strains is dependent on an autocrine signal activating the IGF type I receptor.
Cancer Cell Int. 2003 Jul 11;3(1):10.