Effects of oestradiol and tamoxifen on VEGF, soluble VEGFR-1, and VEGFR-2 in breast cancer and endothelial cells (Cynthia Chuang)
Effects of oestradiol and tamoxifen on VEGF, soluble VEGFR-1, and VEGFR-2 in breast cancer and endothelial cells
S. Garvin, U.W. Nilsson, and C. Dabrosin
Division of Gynecologic Oncology, University Hospital, SE-581 85 Linko¨ping, Sweden
British Journal of Cancer (2005) 93, 10005-1010. Published online 18 October 2005.
Summary
This paper sought to discover the relation between cancer and endothelial cells and the effect of oestradiol and tamoxifen on both types of cells. Vascular endothelial growth factor (VEGF) is a key mediator of tumor angiogenesis, which includes neovascularisation in human breast cancer, and acts via two tyrosine kinase receptors VEGFR-1 and VEGFR-2. Oestradiol has been proven to increase levels of VEGF while tamoxifen inhibits the secretion of VEGF in breast cancer in vivo. Ratio of sVEGFR-1 to VEGF is a strong indicator of disease-free and overall survival in breast cancer patients.
In order to investigate the effects of oestradiol and tamoxifen on sVEGFR-1 and VEGFR-2 in human cell lines in vitro and a mouse model of breast cancer in vivo. Human umbilical vein endolethial cells were isolated and grown in Dulbecco's modified Eagle's medium. Cell were used from passages 2-3. Mice were implanted with pellets that either either continuously released oestradiol or released a placebo. MCF-7 cells were cultured in Dulbecco's modified Eagle's medium, trypsinised, seeded into Petri dishes, and incubated. The cells were then treated with or without oestradiol, or a combination of oestradiol and tamoxifen. The medium was changed every day, and on day 7 secreted VEGF and sVEGFR-1 was quantified using Bio-Rad DC Protein Assay. Tumor growth was determined through volume calculation.
Figure 1: Tumor sections from nude mice with MCF-7 stained with anti-von Willebrand's factor. (A) oestradiol group (B) oestradiol+tamoxifen-treated groups. Arrows indicate examples of positively stained vessels.
Results indicated that oestradiol decreased secreted sVEGFR-1, increased secreted VEGF, and decreased the ratio of sVEGFR-1/VEGF in MCF-7 human breast cancer cells. Addition of tamoxifen significantly countered the effects of oestradiol. Additionally, tamoxifen and oestradiol exert dual effects on the angiogenic environment in breast cancer by regulating cancer cell-secreted angiogenic ligands (e.g. VEGF and sVEGFR-1) and by affecting VEGFR-2 expression of endothelial cells.
Significance
Breast cancer is one of the most common forms of cancer in women and one of the leading causes of cancer death. This study demonstrates the advances being made to better understand the linkage between breast cancer cells' individual molecular components, oestradiol, and tamoxifen. It is an example of the use of tissue engineering as it attempts to better understand the principles of breast cancer cell growth. Application of results can be used to develop therapeutic strategies aimed at replacement and repair of tumor cells. In this case, combination of tamoxifen and oestradiol delivered to breast tumor cells is a viable solution that should be further explored to eliminate or reduce breast cancer.
My lab group is interested in conducting an experiment based on the understanding of oestradiol's relationship with MCF-7 breast cancer cells and VEGF secretion to determine gene expression and optimal VEGF secretion rate. This paper is a valuable resource as it gives (1) a protocol and conditions to grow MCF-7 cells and (2) amount of oestradiol that can be added to physiologically mimic local production and accumulation of oestradiol in human breast tumors in vivo. Additionally, this paper's reference section will serve as additional resources for exploration.
7 comments:
"Ratio of sVEGFR-1 to VEGF is a strong indicator of disease-free and overall survival in breast cancer patients."
What is "sVEGFR-1"? Does a higher or lower ratio correspond to survival? Are MCF-7 cells affected by VEGFR-2, or only endothelial cells? Does one VEGF activate sVEGFR-1 and VEGFR-2, or it must select one to activate? What is the mechanism of oestradiol--does it block the active site of sVEGFR-1, therefore inhibiting sVEGFR-1 and allowing more free VEGF?
Hi Cynthia,
Interesting paper -- my group also looked at VEGF secretion for our final project. I have a question about the "pellets" that were implanted in the mice. I haven't heard about this before... is it just like a long-release pill, or a drug-eluting capsul of some sort? Also, was there a reason they chose day 7 to do the protein assay? I would have liked to see a range of days, to be able to track any changed in secretion. Also, it would have been cool to couple this with a nucleic acid assay of some sort. Thanks for the paper.
Jason
Sorry, *changes.
This may be self-evident but can you explain what sVEGFR is (is this the secreted ligand for VEGFR?).
Also are tamoxifen and oestradiol produced naturally by breast cells and if so what is the natural secretion profile compared to cancer profile?
How did your experiment turn out?
Alisa -
VEGF exerts its angiogenic effects via the tyrosine kinase receptors VEGFR-1 and VEGFR-2. sVEGFR-1 is the soluble form of VEGFR-1 that has been found to bind to all VEGF isoforms and to inhibit endolethlial cell proliferation induced by VEGF. sVEGFR-1 is believed to be a negative regulator of VEGF in breast cancer. VEGFR-2 expression has been identified in epithelial cells in breast cancer.
Generally, the majority of primary breast cancers are oestrogen dependent. What's currently not understand is how exactly oestrogen and antioestrogen (tamoxifen) affect the VEGF receptors and the angiogenic factors of breast cancer. This study attempted to investigate the effects of oestraidol and tamoxifen on sVEGFR-1 and VEGFR-2.
Dan -
The goal of our experiment was to find the ideal range of estradiol concentration that would affect MCF-7 cells' VEGF secretion rate. There were numerous design and experimental errors that resulted in inconclusive data - we could not reasonably conclude a trend due to large standard error and small data pool that could not be used for viable statistical analysis. What we learned from our experiment was that it was a good first step in identifying errors in experiment that needed to be re-designed and conducted again in the future.
It was great using this paper for our experiment on measuring the rate of VEGF secretion from MCF-7 breast cancer cells. It was unfortunate for us that we could not completely mimic their protocol due to the lack of resources and limited budget.
Is there anything in this paper that can be useful for a 3-D model experiment in measuring VEGF in serum?
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