Inflammation is a frequent finding in benign prostatic hyperplasia (BPH) pathology. To dissect the role of inflammatory chemokines and cytokines in BPH pathology, in the present study, total RNA was isolated from exponentially growing BPH-1 cells (a non-tumorigenic cell line), normal prostate epithelial cells (PrEC) and Paraffin-embedded tissue specimen from six patients diagnosed with BPH that were obtained from transurethral resections of the prostate. Real-time RT PCR was performed using the SuperArray inflammatory cytokine array. BPH-1 cells showed elevated levels of monocyte chemotactic protein-1 (MCP-1 or CCL2) followed by IL-8RB and IL-10RA compared to PrEC cells. Similar results were obtained in BPH tissue samples. MCP-1 promoter analysis indicated the presence of several NF\#954;B sites and hence, we used Curcumin (Cur) and Thymoquinone (Thy) to potently inhibit the expression of MCP-1 since Cur is known to inhibit NF\#954;B and Thy is a potent repressor of ARE activity. Thy potently inhibited the expression of MCP-1 followed by Cur alone. When Thy was combined with Cur, the fold change decreased further than Cur alone but not with Thy alone. Cur was the potent inhibitor of IL8RB and exerted its inhibitory effect when combined with Thy, since Thy was found to induce IL-8RB. On the contrary, IL-10RA was significantly induced in response to Cur or Thy or Cur plus Thy. The effect of Cur or Thy or Cur plus Thy on growth of exponentially growing BPH-1 cells was assessed using Real-Time Cell Electronic Sensing system. Increasing reduction in cell growth was observed with increasing doses of Cur or Thy. Further, complete inhibition of growth was observed when Cur was combined with Thy. The effect of these agents on intrinsic NF\#954;B activity were analyzed by using 3x \#954;B-CAT reporter construct. BPH-1 cells showed elevated constitutive NF\#954;B activity. Thy alone suppressed the basal NF\#954;B activity (p<0.001) by 1.7 folds. When combined with Cur, the NF\#954;B activity was further reduced by 4.8 folds (p<0.00001) compared to untreated cells and by 3-folds compared to Thy treatment. Effects of these agents at the protein level were assessed 24 h after treatment in BPH-1 cells by Western blot analysis. High levels of MCP-1 protein expression were detected in the untreated cells. Treatment with Cur significantly inhibited the basal protein expression levels and the inhibition was more effective with Thy treatment. Further, combination treatment completely abrogated the levels of MCP-1 protein expression suggesting that these agents are potent inhibitors of MCP-1 expression and this effect is exerted by inhibiting NF\#954;B function as the promoter of MCP-1 harbors several NF\#954;B sites. Results demonstrate that natural agents disrupting cytokine mediated inflammation such as inhibiting the production of MCP-1 represent an adequate molecular targeted strategy to contain the development and progression of BPH.
Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 2987.
100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO