Snow, Dallin The Derivatives of Resveratrol and Prostate Cancer Faculty Mentor: Merrill Christensen, Nutrition Dietetics and Food Science Introduction Resveratrol has been shown to be a very effective anticarcinogenic compound, in addition to its anti-viral, anti-aging, anti-inflammatory, and life prolonging effects. Resveratrol binds the estrogen receptor beta which in turn down regulates the androgen receptor, which is active in prostate cancer. Past studies of the effects of resveratrol on human prostate cancer cells have shown that it increases the rate of apoptosis and decreases proliferation in a dose and time dependent manner (Benitez, 2007). Also, a 2004 Figure 1. Molecular study exploring the absorption and metabolism of resveratrol in humans structure of resveratrol. indicated that it is metabolized at a rapid rate by humans. It is mostly excreted in the urine and only trace amounts remain in the plasma (Walle, 2004). Because it is metabolized so quickly in the body, it is not around long enough to have a strong effect in cancer prevention. Dr. Merritt Andrus of the department of Chemistry and Biochemistry has synthesized derivatives of the compound resveratrol that are less Figure 2. Molecular soluble in water, which slows their metabolism and excretion. If the structure of resveratrol derivative is, as we suspect, less water soluble and has the same cancer derivative. preventive effects as the parent compound of resveratrol, then it will increase its ability to act in the human body. Testing the effects of the combination of two different compounds is becoming more common in cancer research. In his research, Dr. Merrill Christensen has studied many of the effects and mechanisms pertaining to the mineral selenium (Se). He has previously shown that Se down- regulates the androgen receptor. Combining Se and a resveratrol derivative could have greater effects against prostate cancer. Materials and Methods LNCaP cells, an androgen sensitive prostate cancer cell line with a mutant androgen receptor, were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). Cells were grown in RPMI-1640 Medium supplemented with 10% heat inactivated fetal bovine serum, 1% Penicillin-Streptomycin, 0.5 mL of a 0.03M solution of alpha tocopherol, and 0.5 mL of a 50 µM solution Na2SeO3. LNCaP cells were incubated at 37° C in 5% CO2. Cells were seeded into 96 well plates at a density of 2.0 x 104 cells/well in quadruplicate. Cells were allowed to adhere for 72 hours and were treated with increasing concentrations of resveratrol, resveratrol derivative, selinite, and combinations of all for an additional 72 hours. Cell viability was determined by colorimeteric MTS using Cell Titer 96(R) Aqueous Solution Assay from Promega (Madison, WI) following the manufacturer's instructions—20 µL MTS for every 100 µL of cultured media. In this assay, viable cells convert MTS tetrazolium into a formazan-colorproduct which is read at an OD of 492 nm 4 hours after addition of MTS. Results When comparing the effect of resveratrol to its derivative on LNCaP cells, resveratrol decreased cell number by approximately 15% at a concentration of 25µM, while its derivative decreased cell number by approximately 75% at the same concentration. When adding selenium in the form of Na2SeO3 to the resveratrol treatments, no significant decrease in cell number was seen when using the standard resveratrol form. However, the combination of selenium and the resveratrol derivative showed a decrease in cell number by nearly 40% at 25 µM. Discussion Even though positive results were seen regarding the resveratrol derivative, problems have arisen that have compromised the results and have slowed the work. The main problem that has compromised the results is that when adding the resveratrol treatment to the cells, it is added in different concentrations. As the concentration increases, less media is added to the cells. We suspect that less media has a greater effect than our treatment, thus confounding the results. The alternatives being used to combat this problem are using PBS to make all conditions equal with all treatments and dissolving the treatments in the cell media. As we have tried different methods, results have been inconsistent. When we obtain consistent and reliable results, we will move forward with the project. The future for this project will be to examine at what step inhibition takes place. This can be done by measuring activation of the estrogen and the androgen receptors, levels of mRNA for estrogen- and androgen-regulated genes, and protein levels. Acknowledgements Thanks to Dr. Christensen who has mentored me and instilled in me a strong interest in cancer research. Also, thanks to Susan Reese who helped with much of the preliminary procedures, and thanks to Jason Morris who will be continuing this project in my absence. References Benitez, Dixan A., et al. "Mechanisms Involved in Resveratrol-Induced Apoptosis and Cell Cycle Arrest in Prostate Cancer-Derived Cell Lines." Journal of andrology 28.2 (2007): 282-93. Walle, Thomas, et al. "High Absorption but very Low Bioavailability of Oral Resveratrol in Humans." Drug Metabolism and Disposition: The Biological Fate of Chemicals 32.12 (2004): 1377-82.
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