A pilot, randomized, double-blind, placebo-controlled trial to assess the safety and efficacy of a novel Boswellia serrata extract in the management of osteoarthritis of the knee.

A double-blind, placebo-controlled human trial was conducted to evaluate the safety and efficacy of a standardized oral supplementation of Boswellin®, a novel extract of Boswellia serrata extract (BSE) containing 3-acetyl-11-keto-ß-boswellic acid (AKBBA) with ß-boswellic acid (BBA). A total of 48 patients with osteoarthritis (OA) of the knee were randomized and allocated to the BSE and placebo groups for intervention. Patients were administered BSE or placebo for a period of 120 days. The trial results revealed that BSE treatment significantly improved the physical function of the patients by reducing pain and stiffness compared with placebo. Radiographic assessments showed improved knee joint gap and reduced osteophytes (spur) confirming the efficacy of BSE treatment. BSE also significantly reduced the serum levels of high-sensitive C-reactive protein, a potential inflammatory marker associated with OA of the knee. No serious adverse events were reported. This is the first study with BSE conducted for a period of 120 days, longer than any other previous clinical trial on patients with OA of the knee. The findings provide evidence that biologically active constituents of BSE, namely, AKBBA and BBA, act synergistically to exert anti-inflammatory/anti-arthritic activity showing improvement in physical and functional ability and reducing the pain and stiffness.

[Retracted] Epidermal growth factor-stimulated human cervical cancer cell growth is associated with EGFR and cyclin D1 activation, independent of COX-2 expression levels.

Following the publication of this article, which was concerned with the expression of phosphorylated epidermal growth factor receptor (pEGFR) and cyclin D1 activation independently of the expression levels of cyclo-oxygenase-2, an interested reader drew to our attention apparent anomalies associated with the western blot data shown in Fig. 2C. Following an internal investigation at the New York University School of Medicine, we were requested to produce the original film, or the scan of the image of the film, for verification. Unfortunately, we were unable to provide the original film or scanned image to disprove the allegation, since the original pEGFR image could not be found. Therefore, the Investigation Committee recommended that this article be retracted, and we are withdrawing the article in line with the request. All the authors agree to the retraction of this paper. We sincerely regret any inconvenience this has caused. [the original article was published in the International Journal of Oncology 40: 13-20, 2012; DOI: 10.3892/ijo.2011.1211].

Antitumor activity of melinjo (Gnetum gnemon L.) seed extract in human and murine tumor models in vitro and in a colon-26 tumor-bearing mouse model in vivo.

Melinjo (Gnetum gnemon L.) seed extract (MSE) and its active ingredient gnetin C (GC), a resveratrol dimer, have been shown to possess a broad spectrum of pharmacological activities. In this study, we investigated the antitumor activity of MSE and GC using human and murine tumor cell culture models in vitro. The antitumor activity of GC was compared with trans-resveratrol (tRV), a stilbenoid polyphenol. Our results show that MSE and GC at clinically achievable concentrations significantly inhibited the proliferation of pancreatic, prostate, breast, and colon cancer cell types (P < 0.05), without affecting normal cells. Interestingly, GC exerts enhanced antitumor activity than that of tRV (P < 0.05). MSE and GC significantly induced apoptosis in all the cancer cells, indicating MSE and GC inhibit tumor cell growth by inducing apoptosis (P < 0.001). Our findings provide evidence that MSE might induce apoptosis in cancer cells via caspase-3/7-dependent and -independent mechanisms. However, GC might trigger both early and late stage apoptosis in cancer cells, at least in part by activating caspase 3/7-dependent mechanisms. Furthermore, the antitumor efficacy of MSE observed in vitro was also validated in a widely used colon-26 tumor-bearing mouse model. Oral administration of MSE at 50 and 100 mg/kg per day significantly inhibited tumor growth, intratumoral angiogenesis, and liver metastases in BALB/c mice bearing colon-26 tumors (P < 0.05). In conclusion, our findings provide evidence that MSE and GC have potent antitumor activity. Most importantly, we provide the first evidence that MSE inhibits tumor growth, intratumoral angiogenesis, and liver metastasis in a colon-26 tumor-bearing mice.

Proline-rich AKT substrate of 40-kDa (PRAS40) in the pathophysiology of cancer.

Dysregulation of PI3K-AKT-mTOR pathway has been reported in various pathologies, such as cancer and insulin resistance. The proline-rich AKT substrate of 40-kDa (PRAS40), also known as AKT substrate 1 (AKT1S1), lies at the crossroads of these cascades and inhibits the activity of the mTOR complex 1 (mTORC1) kinase. This review discusses the role of PRAS40 and possible feedback mechanisms, and alterations in AKT/PRAS40/mTOR signaling that have been implicated in the pathogenesis of tumor progression. Additionally, we probed new datasets extracted from Oncomine, a cancer microarray database containing datasets derived from patient samples, to further understand the role of PRAS40 (AKT1S1). These data strongly supports the hypothesis that PRAS40 may serve as a potential therapeutic target for various cancers.

IND-2, a pyrimido[1″,2″:1,5]pyrazolo[3,4-b]quinoline derivative, circumvents multi-drug resistance and causes apoptosis in colon cancer cells.

Naturally occurring condensed quinolines have anticancer properties. In efforts to find active analogues, we designed and synthesized eight polycyclic heterocycles with a pyrimido[1″,2″:1,5]pyrazolo[3,4-b]quinoline framework (IND series). The compounds were evaluated for activity against colon (HCT-116 and S1-MI-80), prostate (PC3 and DU-145), breast (MCF-7 and MDAMB-231), ovarian (ov2008 and A2780), and hepatocellular (HepG2) cancer cells and against non-cancerous Madin Darby canine kidney (MDCK), mouse embryonic fibroblast (NIH/3T3), and human embryonic kidney cells (HEK293). IND-2, a 4-chloro-2-methyl pyrimido[1″,2″:1,5]pyrazolo[3,4-b]quinoline, exhibited more than ten-fold selectivity and potent cytotoxic activity against colon cancer cells relative to the other cancer and non-cancer cells. With five additional colon cancer cell lines (HT-29, HCT-15, LS-180, LS-174, and LoVo), IND-2 had similar cytotoxicity and selectivity, and sub-micromolar concentrations caused changes in the morphology of HCT-116 and HCT-15 cells. IND-2 did not activate the transactivating function of the pregnane X receptor (PXR), indicating that it does not induce PXR-regulated ABCB1 or ABCG2 transporters. Indeed, IND-2 was not a substrate of ABCB1 or ABCG2, and it induced cytotoxicity in HEK293 cells overexpressing ABCB1 or ABCG2 to the same extent as in normal HEK293 cells. IND-2 was cytotoxic to resistant colon carcinoma S1-MI-80 cells, approximately three- and five-fold more than SN-38 and topotecan, respectively. In HCT-116 colon cancer cells, IND-2 produced concentration-dependent changes in mitochondrial membrane potential, leading to apoptosis, and sub-micromolar concentrations caused chromosomal DNA fragmentation. These findings suggest that, by increasing apoptosis, IND-2 has potential therapeutic efficacy for colorectal cancer.

Characterization of multiple myeloma clonal cell expansion and stromal Wnt/ß-catenin signaling in hyaluronic acid-based 3D hydrogel.

BACKGROUND: Emerging interest on three-dimensional (3D) cell culture models to replace two-dimensional cultures of cancer cells and their xenografts in immunocompromised animal hosts prompted us to investigate the use of new biodegradable gels to recapitulate the physiological conditions of the microenvironment of multiple myeloma (MM) cells. MATERIALS AND METHODS: In the present study, for the first time, we used a new 3D model of hyaluronic acid (HA)-based hydrogels with difference in their matrix composition and stiffness. RESULTS: We demonstrated that hyaluronic acid (HA)-based hydrogels perfectly accommodate MM cells; confirmed by cell survival, migration, colony forming units and expression of cell adhesion proteins of the Wnt signaling pathways over a period of time. CONCLUSION: This study provides the first 3D microenvironment data that HA-based hydrogels could provide with a suitable 3D substratum for MM cells to comprehensively analyze phenotypic changes and the influence of bone marrow stromal stem cells on Wnt/ß catenin signaling in response to targeted drug treatments.

Antagonistic effect of small-molecule inhibitors of Wnt/ß-catenin in multiple myeloma.

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 ß-catenin (ß-cat) is commonly de-regulated in many cancers. Cells with active ß-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-ß-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/ß-cat reporter activity, down regulate ß-cat expression and inhibit cell proliferation in a dose-dependent manner with an optimal dose closer to 15 µM. 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 ß-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-ß-cat/VEGF pathway antagonists in multiple myeloma.

Epidermal growth factor-stimulated human cervical cancer cell growth is associated with EGFR and cyclin D1 activation, independent of COX-2 expression levels.

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.

Modulation of PGE2-induced EP4 expression on snail signaling and the impact on epithelial-mesenchymal transition: significance of EP4 antagonism.

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.

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.

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

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.

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-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.

Anticancer effects of licofelone (ML-3000) in prostate cancer cells.

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.

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.

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 microM 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.

Adenocarcina of the mouse prostate growth inhibition by celecoxib: downregulation of transcription factors involved in COX-2 inhibition.

BACKGROUND: Epidemiological studies have shown a decreased risk of prostate cancer among men who regularly take aspirin or other non-steroidal anti-inflammatory drugs (NSAIDs). In this study, we examined a dose-dependent effect of a cyclooxygenase-2 (COX-2) inhibitor, celecoxib against transgenic adenocarcinoma of the mouse prostate. METHODS: Efficacy of four different doses in parts per million of celecoxib, such as 200 ppm, 400 ppm, 600 ppm, and 1,000 ppm representing very low, moderate, and high doses, respectively were tested against adenocarcinoma of the mouse prostate using a transgenic adenocarcinoma of the mouse prostate (TRAMP) model assay. RESULTS: Dietary supplement of celecoxib at doses of 400 ppm, 600 ppm, and 1,000 ppm are most effective against mPIN (mouse prostatic intraepithelial neoplasia) and adenocarcinoma of the prostate. Tumor growth inhibition by celecoxib was associated with increased rate of apoptosis. At 1,000 ppm, a complete inhibition of the PIN lesions was extended to limit the growth of adenocarcinoma (from 85% to 15%) and metastasis of the mouse prostate. The chemopreventive effect was significant (P<0.01) at 400 ppm, 600 ppm, and 1,000 ppm doses compared to that at the lowest dose of 200 ppm and control. A dose-dependent effect on tumor growth inhibition was associated with reduced expression of NF-kappaBp65 and COX-2. CONCLUSIONS: Dietary supplementation of celecoxib at different doses provides evidence for the suppression of prostate adenocarcinoma tumor growth in a dose-dependent manner. Suppression of adenocarcinoma by celecoxib further limits the growth of metastatic prostate cancer.

A combination of docosahexaenoic acid and celecoxib prevents prostate cancer cell growth in vitro and is associated with modulation of nuclear factor-kappaB, and steroid hormone receptors.

Epidemiological studies have provided evidence that high intake of saturated fat and/or animal fat increases the risk of prostate cancer, but on the other hand, diets rich in omega-3 polyunsaturated fatty acids (n-3 PUFAs), present in fish oils were found to reduce the risk. There are indications of an increased expression of immunoreactive PPARgamma in prostatic intraepithelial neoplasia (PIN) and prostate cancer, suggesting that PPARgamma ligands may exert their own potent anti-proliferative effect against prostate cancer. The experimental evidence for the role of cyclooxygenase-2 (COX-2) in prostate carcinogenesis is well established through several investigations. It clearly suggests the need for development of strategies to inhibit COX-2 mediated prostate carcinogenesis. However, administration of high doses of COX-2 inhibitors, such as celecoxib, over longer periods may not be devoid of side effects. We assessed the efficacy of DHA and celecoxib individually and in combination at low doses in three prostate cancer cell lines (LNCaP, DU145 and PC-3) measuring cell growth inhibition and apoptosis, and on the levels of expression of COX-2, nuclear factor-kappaB (NF-kappaBp65), and nuclear receptors, such as PPARgamma and retinoid X receptors (RXR), all of which presumably participate in prostate carcinogenesis. A 48-h incubation of prostate cancer cells with 5 microM each of DHA or celecoxib induced cell growth inhibition and apoptosis, and altered the expression of the above molecular parameters. Interestingly, the modulation of these cellular and molecular parameters was more pronounced in cells treated with low doses of DHA and celecoxib (2.5 microM each) in combination than in cells treated with the higher doses of individual agents. In conclusion, the present study demonstrates for the first time that a combination of lower doses of the n-3 PUFA, and DHA with the selective COX-2 inhibitor celecoxib effectively modulates the above cellular and molecular parameters that are relevant to prostate cancer. This raises the intriguing prospect that the use of low doses of a COX-2 inhibitor in combination with an n-3 PUFA could be a highly promising strategy for prostate cancer chemoprevention while minimizing undesired side effects.

Resveratrol-induced cell growth inhibition and apoptosis is associated with modulation of phosphoglycerate mutase B in human prostate cancer cells: two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry evaluation.

Several studies provide evidence for the anti-carcinogenic activity of resveratrol, a phytoalexin present in grapes and berries, but the precise mechanisms involved in the modulation of prostate carcinogenesis by resveratrol remain to be elucidated. The inhibitory effects induced by resveratrol in human prostate cancer cells impact diverse cellular mechanisms associated with tumor initiation, promotion, and progression. In our earlier studies with prostate cancer cells using cDNA microarray analysis, we indicated the importance of p53-mediated molecular targets of resveratrol. The present study based on two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D-SDS-PAGE) followed by mass spectrometry analysis of human prostate cells that have been treated with resveratrol clearly identifies the role of phosphoglycerate mutase B. For the first time, we report on phosphoglycerate mutase B in the resveratrol-treated prostate cancer cells LNCaP, DU145, and PC-3 at the transcription level. Our observations raise the possibility of its effect on metabolic enzymes in cancer cells without affecting the normal cells.

Regression of mouse prostatic intraepithelial neoplasia by nonsteroidal anti-inflammatory drugs in the transgenic adenocarcinoma mouse prostate model.

PURPOSE: Epidemiologic studies have revealed a decreased risk of colon cancer among people who have regularly taken cyclooxygenase (COX)-2 inhibitors such as aspirin or other nonsteroidal anti-inflammatory drugs (NSAIDs). Whereas the selective COX-2 inhibitor celecoxib and exisulind, a metabolic product of sulindac, have gained increasing attention as efficacious chemopreventive agents against colon and prostate cancer, not much is known about the underlying molecular targets and mechanisms. Moreover, the side effects of NSAIDs are a major obstacle for large-scale application to the prevention of cancer in humans; for example, in the United States in 1998, there were 16,550 deaths from NSAID-induced gastrointestinal complications. The toxicity associated with these compounds is raising concerns, and more needs to be known about their mode of action and molecular targets. EXPERIMENTAL DESIGN: We used the transgenic mouse prostate (TRAMP) model, which exhibits similarities with human prostate cancer, including epithelial origin, progression from the PIN stage to adenocarcinoma, and metastasis by a transgene that is hormonally regulated by androgens. In addition to histologically analyzing the PIN lesions of the dorsolateral prostate from TRAMP mice, we delineated the molecular targets and mechanisms of celecoxib and exisulind against mouse PIN lesions. We performed Western blot analysis of the total protein lysate from the tissues of mouse PIN lesions to measure the level of expression of androgen receptor, vascular endothelial growth factor, nuclear factor-kappaB p65, BclII, AKT (total and phosphorylated Ser473), p53, cyclin-dependent kinase inhibitor p21WAF1/CIP1, p27, BAX, and caspase-3 to demonstrate the COX-2-independent mechanism involved in the inhibition of PIN lesions of the dorsolateral prostate by both celecoxib and exisulind. RESULTS: We found for the first time that (a) both celecoxib and exisulind as dietary supplements induce strong inhibitory effects against prostate cancer at doses of 800 and 500 ppm, respectively, after 16 weeks; (b) the histologic analysis of the dorsolateral prostate after 2 weeks of treatment indicated a reduction of PIN lesions from 75% to 19% with celecoxib and to 16% with exisulind; (c) more importantly, those few PINs and adenocarcinomas in the groups treated with celecoxib or exisulind showed more apoptotic cells, lower levels of proliferating cell nuclear antigen, and a lower number of mitotic cells. To understand the molecular mechanisms involved in the inhibition of PIN lesions, first, we examined the expression of molecular targets involved in angiogenesis and inflammatory processes. It was clearly evident from Western blot analysis of the total protein lysate derived from the dorsolateral prostate tissues with PIN lesions that expression of androgen receptor, vascular endothelial growth factor, nuclear factor-kappaB p65, and BclII is down-regulated more effectively by celecoxib. Down-regulation of AKT protein (total and phosphorylated at Ser473) signaling by celecoxib clearly indicates an inhibition of the survival gene and the pathological process that could otherwise lead to adenocarcinoma. CONCLUSIONS: Overall, the findings from this study clearly show the effectiveness of celecoxib and exisulind in reducing the PIN lesions by modulating a cascade of molecular targets involved in COX-2-dependent and -independent mechanisms. Whereas these agents are already in clinical trial or in use as chemopreventive agents, findings from this study demonstrate the difference in their mode of action, thus helping us to understand the side effects.

Effects of a combination of docosahexaenoic acid and 1,4-phenylene bis(methylene) selenocyanate on cyclooxygenase 2, inducible nitric oxide synthase and beta-catenin pathways in colon cancer cells.

Epidemiological and preclinical studies suggest that diets that are rich in n-3 polyunsaturated fatty acids (PUFAs) and selenium (Se) reduce the risk of colon cancer. Studies conducted in our laboratory have indicated that synthetic organoselenium 1,4-phenylene bis(methylene) selenocyanate (p-XSC) is less toxic and more effective than inorganic Se and selenomethionine, the major Se compound in natural selenium yeast. Through cDNA microarray analysis, we have demonstrated earlier that the n-3 PUFA docosahexaenoic acid (DHA), modulated more than one signaling pathway by altering several genes involved in colon cancer growth. There is increasing interest in the use of combinations of low doses of chemopreventive agents that differ in their specific modes of action as this approach can minimize toxicity and increase efficacy in model assays. In the present study we assessed the efficacy of DHA and p-XSC individually and in combination at low doses in CaCo-2 colon cancer cells, using cell growth inhibition and apoptosis as measures of chemopreventive efficacy. On the basis of western blot and RT-PCR analysis, we also determined the effects of DHA and p-XSC on the levels of expression of cyclooxygenase-2, inducible nitric oxide synthase, cyclin D1, beta-catenin and nuclear factor kappaB, all of which presumably participate in colon carcinogenesis. A 48 h incubation of CaCo-2 cells with 5 microM each DHA or p-XSC induced cell growth inhibition and apoptosis and altered the expression of the above molecular parameters. Interestingly, the modulation of these cellular and molecular parameters was more pronounced in cells treated with low doses of DHA and p-XSC (2.5 microM each) in combination than in cells treated with these agents individually at higher concentrations (5.0 microM each). These findings are viewed as highly significant since they will provide the basis for the development of combinations of low dose regimens of DHA and p-XSC in preclinical models against colon carcinogenesis and, ultimately, in human clinical trials.