Voelcker Academy

Research Symposium 2011

 

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Kevin Lee

Evaluating the protein expression of 11βhydroxysteroid dehydrogenase 2 in TPA-inducible JB6 Cells

Kevin Lee

Mentor(s): Dr. Thomas Slaga, Ph.D

Glucocorticoids (GCs) are involved in the regulation of nearly every physiological process and have been used for decades in many therapeutic capacities due to their anti-inflammatory and anti-proliferative properties such as arthritis, asthma, dermatitis, and cancer (Odermatt et. al, 2010; Ahmad et. al, 2011) Moreover, studies have shown GCs to be an efficacious treatment for skin inflammation, hyperplasia, and mouse skin tumor formation (Budunova et. al, 2003). Although GC receptor (GR) density is an important regulator of GC activity, the amount of active GC is also very important as well. GCs are activated (converted from cortisone to cortisol) by 11βhydroxysteroid dehydrogenase 1 (11βHSD1) and inactivated (converted from cortisol to cortisone) by 11βHSD2 (Krozowski et. al, 1999). Recently, many studies have shown a trend whereby 11βHSD2 is highly expressed in tumors and malignant cells when compared to their normal counterparts (Krozowski et. al, 1999; Sai et. al, 2011; Zhang et. al, 2009). Therefore, the role of these enzymes for chemotherapeutic purposes is currently being investigated in many different types of cancer. The purpose of this study was to first determine if the same trend of 11βHSD2 expression is present in the JB6 skin cancer in vitro model. The JB6 mouse epidermal model is a well-established inducible model that is very useful because it allows us to observe pre- and post-transformed cells. This model consists of the P- (Tetradecanoylphorbol 13-acetate (TPA) resistant), JB6 P+ (TPA inducible), and RT101 (transformed) cell lines. Future studies for this project will include evaluating different combinations of the phytonutrients: Ursolic Acid (UA), Resveratrol (Res), and 18β Glycyrrhetinic acid (GA) for their ability to potentially prevent or diminish transformation. For this purpose, we determined maximum tolerable doses of the phytonutrients. Resveratrol, found in red grapes, has been shown to have anti- proliferative effects (Jang, 1997). UA, found in apples, has exhibited anti-tumorigenic properties (Huang, 1994). GA is known to be an inhibitor of 11βHSD2 (Monder et. al, 1989). Based on the literature, we hypothesized that we would observe an increased expression of 11-βHSD2 in the TPA-induced JB6 P+ and RT101 cell lines. To test this hypothesis we determined maximum tolerable doses of various phytochemicals, namely GA, in order to carry out in vitro treatments for western blotting. We first evaluated the protein expression of 11βHSD2 within TPA- or vehicle-treated P+ cells to determine if there was a difference. We then evaluated 11βHSD2 expression with and without varying GA treatments in the presence of TPA to determine if GA could inhibit 11βHSD2 protein expression. Additionally, we measured protein expressions of sulfiredoxin (Srx) and osteopontin (OPN) as biomarkers for TPA-induced transformation in these cell lines, as they are widely used in the literature for this purpose. Our results show increased 11βHSD2, Srx, and OPN protein expression levels in already transformed RT101 cells and the TPA transformed P+ cells compared to the vehicle treated P+ cells. Contrary to our predictions, the protein expression of 11βHSD2 was increased by treatment with GA. Although these results are very preliminary, this may suggest that GA is not a suitable specific inhibitor for 11βHSD2. However, our results do show for the first time that 11βHSD2 protein expression is increased by TPA treatment, and when compared to normal cells, 11βHSD2 is expressed at higher levels in transformed JB6 cells. Future studies will involve evaluating the above mentioned phytochemicals for inhibition and prevention of transformation in this model, as well as RT-PCR of 11βHSD2 in order to determine if the same expression trend is observed at the gene level.

Collaborators: Kevin Lee, Anna Mancha, Jacob Junco, Dr. Huiyun Liang, Dr. Thomas Slaga