Dominick Nargi

Liposome encapsulation of curcumin and resveratrol in combination reduces prostate cancer incidence in PTEN knockout mice.

Increasing interest in the use of phytochemicals to reduce prostate cancer led us to investigate 2 potential agents, curcumin and resveratrol as preventive agents. However, there is concern about the bioavailability of these agents pertinent to the poor absorption and thereby limiting its clinical use. With the view to improve their bioavailability, we used the liposome encapsulated curcumin, and resveratrol individually and in combination in male B6C3F1/J mice. Further, we examined the chemopreventive effect of liposome encapsulated curcumin and resveratrol in combination in prostate‐specific PTEN knockout mice. In vitro assays using PTEN‐CaP8 cancer cells were performed to investigate the combined effects curcumin with resveratrol on (i) cell growth, apoptosis and cell cycle (ii) impact on activated p‐Akt, cyclin D1, m‐TOR and androgen receptor (AR) proteins involved in tumor progression. HPLC analysis of serum and prostate tissues showed a significant increase in curcumin level when liposome encapsulated curcumin coadministered with liposomal resveratrol (p < 0.001). Combination of liposomal forms of curcumin and resveratrol significantly decreased prostatic adenocarcinoma in vivo (p < 0.001). In vitro studies revealed that curcumin plus resveratrol effectively inhibit cell growth and induced apoptosis. Molecular targets activated due to the loss of phosphatase and tensin homolog (PTEN) including p‐Akt, cyclin D1, mammalian target of rapamycin and AR were downregulated by these agents in combination. Findings from this study for the first time provide evidence on phytochemicals in combination to enhance chemopreventive efficacy in prostate cancer. These findings clearly suggest that phytochemicals in combination may reduce prostate cancer incidence due to the loss of the tumor suppressor gene PTEN.

RNA interference–mediated cyclooxygenase-2 inhibition prevents prostate cancer cell growth and induces differentiation: modulation of neuronal protein synaptophysin, cyclin D1, and androgen receptor

Cyclooxygenase-2 (COX-2) plays an important role in tumor development and progression. Inconsistent reports on the expression of COX-2 in early versus advanced prostate cancer raised the question on whether COX-2 inhibition affects prostate carcinogenesis. Evidence from recent studies indicates that prostate carcinogenesis depends on the altered expression of several factors including androgen receptor signaling, proinflammatory, and cell cycle regulatory genes. Very often, the outcome of androgen ablation treatment is not effective and, eventually, the cancer becomes androgen independent followed by activation of several survival genes and transcription factors. Most importantly, the extent of the influence of COX-2 on the regulation of the androgen receptor, cyclin D1, and other factors involved in cancer growth is not known. Using RNA interference–mediated COX-2 inhibition in metastatic prostate cancer cells, this study has shown that the silencing of COX-2 at the mRNA level can induce cell growth arrest and down-regulate androgen receptor and cyclin D1. We have further shown for the first time that COX-2 knockdown prostate cancer cells depict morphologic changes associated with enhanced expression of differentiation markers, particularly the neuronal protein synaptophysin along with activation of p21(Waf1/Cip1) and p27(Kip1). In summary, our findings determined the role of COX-2 in prostate carcinogenesis and its control on COX-2-independent targets. Second, abrogation of COX-2 and activation of synaptophysin provide evidence for the control of COX-2 on the expression of a neuronal protein. Finally, our findings provide evidence of COX-2-independent targets promoting cell growth arrest and differentiation in cells lacking COX-2 expression at the mRNA level. [Mol Cancer Ther 2006;5(5):1117–25]

Docosahexaenoic acid in combination with celecoxib modulates HSP70 and p53 proteins in prostate cancer cells

The role of cyclooxygenase‐2 (COX‐2) and the mechanism by which it influences the development and behavior of prostate cancer is unclear. Selective COX‐2 inhibitors may be effective against prostate cancer via COX‐2‐independent mechanisms. But administration of high doses of COX‐2 inhibitors over longer period of time may not be devoid of side effects. There is increasing interest in using COX‐2 inhibitors in combination with other chemopreventive agents to overcome the issue of toxicity. However, the molecular mechanisms underlying their combined actions are not well understood. Therefore, the present study was designed to determine the effects of low doses of docosahexaenoic acid (DHA) in combination with celecoxib on the molecular targets at the proteins level in rat prostate cancer cells. Two‐dimensional gel electrophoresis, in combination with mass spectrometry analysis, was used for protein identification. Western blot analysis confirmed the proteins identified. Paraffin‐embedded tissue sections from the rat prostate tumor were used to detect base level expression of heat shock protein 70 (HSP70) and p53. The rate of cancer cell growth was inhibited more effectively (p < 0.01) by DHA in combination with celecoxib at lower doses (2.5 μM each). A total number of twelve proteins were differentially expressed by the combined action of DHA and celecoxib at low doses. It was interesting to note that these agents activated both HSP70 and p53 proteins. Activation of HSP70 by the combined actions of DHA and celecoxib in the presence of wild‐type p53 reveals a unique COX‐2 independent mode of action against prostate cancer.

Exisulind in Combination with Celecoxib Modulates Epidermal Growth Factor Receptor, Cyclooxygenase-2, and Cyclin D1 against Prostate Carcinogenesis: In vivo Evidence

Purpose: Nonsteroidal anti-inflammatory drugs mediate anticancer effects by modulating cyclooxygenase-2 (COX-2)-dependent and/or COX-2–independent mechanism(s); however, the toxicity issue is a concern with single agents at higher doses. In this study, we determined the combined effect of celecoxib, a COX-2 inhibitor, along with exisulind (sulindac sulfone/Aptosyn) at low doses in prostate cancer. Experimental Design: We used a sequential regimen of N-methyl-N-nitrosourea + testosterone to induce prostate cancer in Wistar-Unilever rats. Following carcinogen treatment, celecoxib and exisulind individually and their combination at low doses were given in NIH-07 diet for 52 weeks. We determined the incidence of prostatic intraepithelial neoplasia, adenocarcinomas, rate of tumor cell proliferation, and apoptosis. Immunohistochemical and Western blot analysis were done to determine COX-2, epidermal growth factor receptor (EGFR), Akt, androgen receptor, and cyclin D1 expression. Serum prostaglandin E2 and tumor necrosis factor-α levels were determined using enzyme immunoassay/ELISA assays. Results: The rats that received celecoxib in combination with exisulind at low doses showed a significant decrease in prostatic intraepithelial neoplasia and adenocarcinomas as well as an enhanced rate of apoptosis. An overall decrease in COX-2, EGFR, Akt, androgen receptor, and cyclin D1 expression was found associated with tumor growth inhibition. Reduced serum levels of COX-2 protein, prostaglandin E2, and tumor necrosis factor-α indicated anti-inflammatory effects. A strong inhibition of total and phosphorylated form of EGFR (Tyr992 and Tyr845) and Akt (Ser473) was significant in rats given with these agents in combination. Conclusions: In this study, we show for the first time that the combination of celecoxib with exisulind at low doses could prevent prostate carcinogenesis by altering key molecular events.

Inflammatory processes of prostate tissue microenvironment drive rat prostate carcinogenesis: Preventive effects of celecoxib

Prostate tissue microenvironment is susceptible to several risk factors including carcinogens, dietary factors, hormones, cytokines and growth factors that could induce chronic inflammation. Because of the difference in the serum levels and the intrinsic ability of monocytes/macrophages to cause harm, the transcriptional responses triggered by inflammatory stimuli must be controlled. Unfortunately, an in‐depth association between prostate cancer and potential mediators of inflammation has not been completely investigated.

Abstract B97: Combination chemoprevention and underlying mechanisms of finasteride with NSAIDs using in vitro and in vivo models (Pten-/-) for prostate cancer

B97 Background The Prostate Cancer Prevention Trial (PCPT) in a total of 1,626 men demonstrated that finasteride, a 5α reductase inhibitor reduces prostate cancer risk by 24.8%, and however, it was associated with a risk of small increase in high-Gleason grade tumors. This suggests that although finasteride is a potential chemopreventive agent, prostate cancer risk factors are not limited its target. To enhance the efficacy of finasteride, earlier studies have successfully used the combination with flutamide for the treatment of advanced disease. Based on our successful attempts in testing the chemopreventive effects of NSAIDs in combination against prostate cancer, in this study, we tested the efficacy of finasteride with anti-inflammatory agents at low doses in preclinical models to synergistically decrease prostate cancer incidence. Methods To accomplish our goals, we examined the effects of finasteride individually and in combination with celecoxib or aspirin using in vitro and in vivo models. We used murine prostate cancer cells (CaP8) deleted for Pten-/- and human benign prostate hyperplastic epithelial cells (BPH1). The cells were pretreated for 24 h with testosterone before incubating with different doses of finasteride (15μM) or aspirin (40μM) or celecoxib (40μM) individually and in combination (half of each of the single dose) for 48 h. Effect on cell growth, cell cycle, apoptosis, molecular targets AR, AKT, Cyclin D1, COX-2 and serum PGE2 levels were analyzed with MTT, flow cytometry, Western blot and ELISA assays. To determine in vivo efficacy, four week old PTEN/KO mice (an immune-competent, orthotopic model) were administered (n=5/group) orally with finasteride (15mg/kg bw) or celecoxib (200 mg/kg bw) or aspirin (200 mg/kg bw) individually or in combinations with half of the single dose for a period of six weeks. Histopathological analysis of the DL prostate tissues was performed to determine the effect on the incidence of mPIN or adenocarcinoma stages. Specific immunohistochemical staining was performed to detect the molecular targets. Results Findings from MTT assays showed inhibition of CaP8 cell (Pten-/-) growth by 56% (P Conclusion Finasteride in combination with anti-inflammatory agents, aspirin or celecoxib at low doses, synergistically prevents prostate cancer in preclinical models. Citation Information: Cancer Prev Res 2008;1(7 Suppl):B97.