Faculty Bibliography

BACKGROUND: Development and progression of multiple myeloma is dependent on the bone marrow (BM) microenvironment, and within the BM, a number of factors are secreted, including the Wnt ligands. Bone marrow stromal cells (BMSC) secrete Wnt ligands that activate Wnt signaling in multiple myeloma. The canonical Wnt pathway which is mediated through the transcriptional effector beta-catenin (beta-cat) is commonly de-regulated in many cancers. Cells with active beta-cat-regulated transcription (CRT) are protected against apoptosis; conversely, inhibition of CRT may prevent cell proliferation. MATERIALS AND METHODS: In this study, we tested the efficacy of recently described inhibitors of CRT (iCRTs; oxazole and thiazole) for their selective antagonistic effect on Wnt-beta-cat response in MM cells MM.1, U266, BMSC and primary BMMC obtained from patient samples (n=16). RESULTS: We demonstrated that iCRTs we used, block Wnt/beta-cat reporter activity, down regulate beta-cat expression and inhibit cell proliferation in a dose-dependent manner with an optimal dose closer to 15 muM. Our data further indicate that iCRTs do not influence the expression of the upstream components of the Wnt pathway DKK1 at the optimal dose, suggesting that iCRTs may specifically target beta-cat in MM cells. Additionally, iCRT-treatment of MM cells, co-cultured with BMSC, showed an inhibitory effect on VEGF and cell migration. CONCLUSION: This study provides the first in vitro data evaluation of newly-described iCRTs as potential Wnt-beta-cat/VEGF pathway antagonists in multiple myeloma.

Cervical cancer constitutes the second most common cancer in women. It is evident from earlier studies that epidermal growth factor (EGF) is a mitogen, in that it mimics the function of estrogen by mediating cross-talk with other oncoproteins. Although epidermal growth factor receptor (EGFR) is highly expressed in breast and ovarian tumor tissues, its regulation by the exogenous source of its ligand EGF in human papillomavirus (HPV)-associated cervical cancer remains unclear. In this study, we addressed the question of whether EGF is required for the proliferation of HPV-positive cervical cancer cells and what mechanisms are involved. To determine this, we conducted a series of studies using HPV-positive human cervical cancer cells, CaSki and HeLa, and stimulated the cells with EGF. Our findings suggest that 6 h of stimulation with 10 ng/ml of EGF is sufficient to induce cell cycle progression associated with a significant increase in DNA synthesis, EGFR, COX-2 and cyclin D1 levels. Consistently, cellular localization and Western blot analysis for p-EGFR (Try-1045) protein showed an increase after EGF stimulation. Using siRNA gene knockdown assays we have shown that cyclin D1 siRNA has a significant negative effect on EGFR and inhibit cell growth independent of COX-2 levels. In summary, our findings reveal that an exogenous EGF stimulation may enhance HPV-related cervical cancer cell proliferation by activating EGFR and cyclin D1 that is independent of COX-2 levels, suggesting that the inhibitors of EGFR and cyclin D1 may be effective against cervical cancer cell proliferation

BACKGROUND: Although significant accumulation of prostaglandin E(2) (PGE(2)) in the human prostate cancer tissues has been reported, there is lack of substantial evidence regarding the key role of PGE(2)-induced E-prostanoid-4 receptor (EP4) on Snail, a master regulator of epithelial mesenchymal transition (EMT). In this study, we investigated a novel connection between PGE(2)-induced EP4 and Snail (encodes DNA binding zinc finger protein that acts as transcriptional repressor) signaling in prostate cancer. MATERIALS AND METHODS: To investigate the key role of serum PGE(2), EP4, p-Akt and Snail in prostate cancer progression, we used prostate-specific phosphatase and tensin homolog (PTEN)-knockout (PTEN-KO) mice of different age groups from 4 to 28 weeks. To determine the EP4-specific interaction with Snail in prostate cancer, we used cell-based assays, including siRNA knockdown, and treatment with EP4 antagonist. RESULTS: An interaction between EP4 with Snail was evident in prostate-specific PTEN-KO mice that showed an elevated level of PGE(2) in the serum and of EP4, p-Akt and Snail in the tissues. Prostate cancer cells transfected with EP4-siRNA and treatments with EP4 antagonist suggest a link between EP4, and Snail activation, potentially via p-Akt. Cells treated with EP4 antagonist exhibited a significant decrease in Snail, mesenchymal markers and cell migration, and cell cycle arrest with a gain in E-cadherin levels. CONCLUSION: Our findings provide key evidence that support there being a role of PGE(2)/EP4/p-Akt in Snail signaling and conferring cell survival advantage. Cancer progression via EMT can be reversed by an EP4 antagonist in this model of prostate cancer.

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

BACKGROUND: 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. METHODS: To determine whether activated macrophage (infiltrating monocytes), iNOS and NF-kappaB are primary mediators of inflammation, besides COX-2, in prostate carcinogenesis, we examined tissue sections of rat prostate tumor induced by N-methyl-N-nitrosourea (MNU) plus testosterone in a follow-up study. We performed H&E and immunohsitochemical staining of the prostate tissue to detect specific markers of inflammation. RESULTS: We report an increase in infiltrating monocyte, iNOS, NF-kappaBp65, VEGF and TNF-alpha at the early and advanced stages of tumor growth in MNU plus testosterone treated rats. Monocyte infiltration was often found in the stromal and perivascular regions of the DL prostate. We conclude for the first time that prostate cancer induced by MNU plus testosterone partly involves mediators of inflammation which could trigger the process of carcinogenesis and cause loss of apoptosis. Selective COX-2 inhibitor celecoxib at a dose of 500 mg/kg/bw administered for 52 weeks reduced infiltrating monocytes, inhibited iNOS, NF-kappaB p65 expression, induced apoptosis and tumor growth inhibition. CONCLUSION: Carcinogen plus testosterone induced prostate carcinogenesis showing activation of macrophage, iNOS and NF-kappaBp65 could be prevented by celecoxib or related anti-inflammatory agents

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-alpha 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-alpha indicated anti-inflammatory effects. A strong inhibition of total and phosphorylated form of EGFR (Tyr(992) and Tyr(845)) and Akt (Ser(473)) 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

BACKGROUND: Licofelone, a potent antiinflammatory agent has been reported to interfere with the cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) signaling pathways with few side-effects. However, the underlying mechanism of licofelone against human cancer is not understood. MATERIALS AND METHODS: Human and mouse prostate cancer cells were exposed to licofelone in a time- and dose-dependent manner. Cell growth/cell viability, apoptosis, and expression of COX-2 and 5-LOX at the gene and protein levels were investigated. RESULTS: For the first time, it was demonstrated that licofelone inhibited prostate cancer cell growth and significantly down-regulated COX-2 and 5-LOX expression. A weak inhibitory effect on COX-1 protein was also observed. CONCLUSION: Licofelone inhibited COX-2 and 5-LOX simultaneously and prevented overall cancer cell growth by enhancing apoptosis in both androgen-dependent and androgen-independent prostate cancer cells. Validating the dual role of licofelone in animal models of prostate cancer is critical for promoting its use as a potential chemopreventive or therapeutic agent

Chemoprevention has the potential to be a major component of colon, breast, prostate and lung cancer control. Epidemiological, experimental, and clinical studies provide evidence that antioxidants, anti-inflammatory agents, n-3 polyunsaturated fatty acids and several other phytochemicals possess unique modes of action against cancer growth. However, the mode of action of several of these agents at the gene transcription level is not completely understood. Completion of the human genome sequence and the advent of DNA microarrays using cDNAs enhanced the detection and identification of hundreds of differentially expressed genes in response to anticancer drugs or chemopreventive agents. In this review, we are presenting an extensive analysis of the key findings from studies using potential chemopreventive agents on global gene expression patterns, which lead to the identification of cancer drug targets. The summary of the study reports discussed in this review explains the extent of gene alterations mediated by more than 20 compounds including antioxidants, fatty acids, NSAIDs, phytochemicals, retinoids, selenium, vitamins, aromatase inhibitor, lovastatin, oltipraz, salvicine, and zinc. The findings from these studies further reveal the utility of DNA microarray in characterizing and quantifying the differentially expressed genes that are possibly reprogrammed by the above agents against colon, breast, prostate, lung, liver, pancreatic and other cancer types. Phenolic antioxidant resveratrol found in berries and grapes inhibits the formation of prostate tumors by acting on the regulatory genes such as p53 while activating a cascade of genes involved in cell cycle and apoptosis including p300, Apaf-1, cdk inhibitor p21, p57 (KIP2), p53 induced Pig 7, Pig 8, Pig 10, cyclin D, DNA fragmentation factor 45. The group of genes significantly altered by selenium includes cyclin D1, cdk5, cdk4, cdk2, cdc25A and GADD 153. Vitamine D shows impact on p21(Waf1/Cip1) p27 cyclin B and cyclin A1. Genomic expression profile with vitamin D indicated differential expression of gene targets such as c-JUN, JUNB, JUND, FREAC-1/FoxF1, ZNF-44/KOX7, plectin, filamin, and keratin-13, involved in antiproliferative, differentiation pathways. The agent UBEIL has a remarkable effect on cyclin D1. Curcumin mediated NrF2 pathway significantly altered p21(Waf1/Cip1) levels. Aromatase inhibitors affected the expression of cyclin D1. Interestingly, few dietary compounds listed in this review also have effect on APC, cdk inhibitors p21(Waf1/Cip1) and p27. Tea polyphenol EGCG has a significant effect on TGF-beta expression, while several other earlier studies have shown its effect on cell cycle regulatory proteins. This review article reveals potential chemoprevention drug targets, which are mainly centered on cell cycle regulatory pathway genes in cancer

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 muM 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. (c) 2006 Wiley-Liss, Inc