Characterization of adiponectin receptor expression and function in TRAMP prostate tumors and the TRAMP-C2 cell line Free

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Introduction: Obesity is associated with increased risk for more aggressive prostate cancer (Pca) as defined by an increase in the risk of Pca death and an increased chance of progression after surgery. Obesity may mediate its effects on Pca in part due to factors secreted from adipose tissue. One factor potentially involved in the interaction between Pca and obesity is adiponectin, also known as adipocyte complement-related protein of 30 kDa (Acrp30). Lower serum Acrp30 levels have been reported for Pca patients compared to patients with benign prostatic hyperplasia or healthy controls. In addition, lower expression levels of Acrp30 receptors are found in prostate tumors as compared to healthy prostate tissue. Here we assessed how Acrp30 impacted cell growth in vitro in the TRAMP-C2 cell line which is derived from a TRAMP prostate tumor and determined Acrp30 receptor expression in the TRAMP model.
 >Procedures: TRAMP-C2 cells (ATCC) were used in growth assays (CC8 kit Dojindo Laboratories). Whole cell extracts were obtained using Phosphosafe extraction reagent from Novagen for determination of adiponectin receptors (AdipoR1 and R2) and signaling proteins by western blot. Antibodies were from Santa Cruz Biotechnology except antibodies to AdipoR1 (Abcam Inc), AdipoR2 (Phoenix Pharmaceuticals, Inc.) and anti-rabbit secondary (Cell Signaling Inc.).TRAMP mice were euthanized at 50 weeks and urogenital tracts plus abnormal growths/tumors removed. Sections were stained with the rabbit ABC staining system for AdipoR1 and R2.
 >Results: There was a dose-related reduction in proliferation of the TRAMP-C2 cells after 48 hours in response to the addition of Acrp30. The difference in proliferation was statistically significant at physiological Acrp30 concentrations of 10 and 20 ug/ml (Student’s t-test p<0.03 and 0.02 respectively) compared to untreated cells. Western blots indicated that AdipoR1 and AdipoR2 are both expressed by TRAMP-C2 cells. We also identified increases or decreases in phosphorylation of several growth associated signaling proteins with western blots. Acrp30 increased levels for both ERK1 and ERK2. The phosphorylation of Stat3 was decreased by the addition of fetal calf serum but this decrease was blocked by Acrp30.
 >We also found that tumors from TRAMP mice expressed the two receptors for Acrp30, AdipoR1 and AdipoR2. Using immunohistochemical analysis we found expression of AdipoR1 in prostate tumor tissue from TRAMP mice was mostly in epithelial cells on the apical membrane. AdipoR2 was present in the same areas as AdipoR1 but the staining was lower. Western blot analysis of frozen tissue from the same mice also indicated expression of AdipoR1 and AdipoR2 in prostate tumor tissue.
 >Conclusions: Here,we are the first to report the presence of AdipoR1 and AdipoR2 in prostate tumor tissues from TRAMP mice and in the TRAMP-C2 cell line which is derived from the prostate tumor of a TRAMP mouse. The receptors appear to be functional since proliferation of TRAMP-C2 cells was inhibited by addition of Acrp30. This decrease in cell growth may be attributable to increased signaling through ERK 1/2 since Acrp30 increased the phosphorylation of ERK 1/2. We are currently investigating the levels of Acrp30 in vivo with ongoing mouse studies in relationship to body weight and Pca development. Support from DOD PC 050284 and The Hormel Foundation.