David R. Nielsen and Dr. Merril Christensen, Food and Science and Nutrition
Introduction
Prostate cancer is the second leading cause of cancer-related deaths in American men, with current estimates projecting 179,300 new cases and 37,000 deaths this year [1]. The development of prostate cancer may be divided into three stages: initiation, promotion, and progression. During the initiation and early promotion stages, the cancer is clinically undetectable. This period of latency may last for decades and displays little geographic or ethnic variation among males. In contrast, there is an approximate 30-fold difference between countries with the lowest rates of prostate cancer mortality and countries with the highest rates [2]. Once the cancer has advanced to the progression stage, or metastasis, there is little variation among mortality incidences. This suggests that the stage at which prostate cancer is most affected is the promotion stage. In 1983, Clark and Brown began a double-blind, placebo-controlled, randomized trial to study of the effects of selenium on epithelial melanoma, and discovered that selenium, although it had little effect on melanoma, reduced the incidence of prostate cancer by 63%. The blinded phase of the trial was terminated prematurely in January 1996 so that those taking the placebo supplements could begin taking selenium supplements to aid in prostate cancer prevention [3,4,5]. Previous research indicates that human prostate tumor cells are highly responsive to steroid hormones, particularly androgens and estrogens [6]. Because of the high responsiveness of prostate tumor cells to steroid hormones and because selenium has been shown to significantly decrease the incidence of prostate cancer, a study of the relationship between selenium supplementation and steroid hormone-mediated gene expression is outlined below.
Methods and Materials
In this study, human prostate cancer epithelial cells, LNCaP (American Type Culture Collection) were
treated with adequate and high amounts of selenium in the form of sodium selenite, Na2SeO3 (Sigma). To
determine appropriate adequate and high levels of selenium supplementation, a titration with LNCaP cells
was performed using concentrations of 4-600 ng Se /ml. Cell viability was determined using the Trypan
blue assay, and enzymatic assays were used to detect the level of GPX1 and GST activity. As GPX1
activity was maximized at 4 ng Se/ml and cell viability was significantly reduced with more than 100 ng
Se/ml, treatment amounts of 4 ng Se/ml and 100 ng Se/ml were chosen for adequate and high
concentrations, respectively. Cells were cultured in RPMI 1640 (Gibco) medium with 10% fetal bovine
serum (Hyclone), or FBS , 2 mM glutamine, and á-tocopherol . Cells were grown for 48 hours in medium
until they reached nearly 60% confluency. The cells were then treated with the appropriate concentrations of selenium for a period of 24 hours. After the treatment they were harvested and the RNA was isolated using TRIzol reagent (Gibco).
The following five genes were studied:
· AR (androgen receptor)
· ER (estrogen receptor)
· 5-á-reductase
· SHGB (sex hormone binding globulin)
· PSA (prostate specific antigen)
The expression for these genes were analyzed using a relative multiplex RT-PCR method developed in our laboratory [7]. This method facilitates the verification of original differences in gene expression. The gene of interest is PCR coamplified with a housekeeping gene—in this case 18S rRNA. The PCR product is run on a 2% agarose electrophoresis gel, stained with ethidium bromide, and exposed under UV fluorescence.
The fluorescence is then normalized and plotted as a function of cycle number. Differences in gene expression are determined by the number of cycles necessary to reach a given threshold intensity.
Results
Gene expression of three genes, 5-á-reductase, SHBG, and PSA were shown to be down-regulated in high selenium supplementation. There was no significant difference in the expression of AR. ERá and the six isoforms of ERâ were studied individually with little expression of each in LNCaP cells. There has been some disagreement in the literature as to whether or not LNCaP cells express ER. At this point it seems that those papers which note the expression of ER in LNCaP cells were amplifying sequences common to all isoforms rather than sequences unique to each. The data from this experiment suggest that a nearly nondetectable amount of each isoform is expressed in the LNCaP cells. These results have yet to be confirmed.
Discussion
5-á-reductase converts testosterone to its active form. This is consistent with my hypothesis that selenium has a chemopreventive effect on prostate cancer cells by reducing the expression of this gene, thereby reducing proliferation of prostate tumor cells which are highly responsive to steroid hormones. SHGB was also down-regulated, suggesting that the mechanism by which prostate cancer is retarded does not involve the binding of SHBG to steroid hormones. PSA levels in LNCaP cells were reduced slightly, although in vivo, increased PSA levels are often used as a tool in diagnosing prostate cancer. The expression of the various ER isoforms has yet to be confirmed. Our laboratory anticipates confirming the expression of these and other genes in LNCaP cells using the Roche LightCycler, which allows for a more accurate method of determining gene expression.
References
- Prostate Cancer-Overview. www3.cancer.org/cancer info/documents/overviews/prosover.asp?ct+36. 1999.
- Clinton SK, Giovannucci E. Diet, nutrition, and prostate cancer. Annual Reviews, Nutrition. 1998; 18:413-40.
- Clark LC, Combs GF, et al. Decreased incidence of prostate cancer with selenium supplementation: results of a double-blind cancer prevention trial. British Journal of Urology. 1998; 81:730-34.
- Clark LC, Combs GF, et al. Effects of Selenium Supplementation for Cancer Prevention in Patients With Carcinoma of the Skin. Journal of the American Medical Association. 1996; 276:1957-1963.
- Clark LC, Combs GF, et al. Reduction of Cancer Risk With and Oral Supplement of Selenium. Biomedical and Environmental Sciences. 1997; 10:227-234.
- Navone MN, et al. Model systems of prostate cancer: Uses and limitations. Cancer and Metastasis Review. 1999; 17:361-371.
- Spencer WE, Christensen MJ. A Multiplex Relative RT-PCR Method for Verification for Verification of Differential Gene Expression. Biotechniques. 1999; 27:in press.