Lori Horton

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.

Selenomethionine and α-Tocopherol Do Not Inhibit Prostate Carcinogenesis in the Testosterone plus Estradiol-Treated NBL Rat Model

Previous studies with selenium and/or vitamin E in prostate carcinogenesis animal models have been negative, but these models may not involve oxidative stress mechanisms. In this study, we examined the potential of selenomethionine and α-tocopherol to modulate prostate cancer development in the testosterone plus estradiol–treated NBL rat, a model that does involve sex hormone–induced oxidative stress mechanisms and prostatic inflammation. One week following the implantation with hormone-filled Silastic implants, rats were fed diets containing l-selenomethionine (1.5 or 3.0 mg/kg), DL-α-tocopherol acetate (2,000 or 4,000 mg/kg), or a natural ingredient control diet (NIH-07). The development of prostate carcinomas was not affected by dietary treatment with either agent. Food intake, body weight, and mortality were also not affected. The high dose of selenomethionine reduced the severity of epithelial dysplasia in the lateral prostate that was not associated with inflammation, and α-tocopherol reduced in a dose-related fashion the incidence of marked inflammation and marked epithelial dysplasia in the lateral prostate, regardless of whether these lesions were associated with inflammation. α-Tocopherol significantly increased the incidence of adenocarcinomas of the mammary glands at both dietary concentrations. Collectively, our findings suggest that selenomethionine and α-tocopherol supplementation does not prevent prostate cancer in rats fed diets with nutritionally adequate levels of selenium and vitamin E. Importantly, the results of the current animal studies and those reported previously were fully predictive of the outcome of the Selenium and Vitamin E Cancer Prevention Trial. Cancer Prev Res; 3(3); 371–80

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.

MULTIPLE PATHWAYS OF PROSTATE CARCINOGENESIS ANALYZED BY USING CULTURED CELLS ISOLATED FROM RATS TREATED WITH N-METHYL-N-NITROSOUREA AND TESTOSTERONE

Treatment of rats with N‐methyl‐N‐nitrosourea (MNU) and testosterone results in a high incidence of metastasizing dorsolateral prostate tumors. In previous studies, a high frequency (≥70%) of a G35 → A transition mutation at the second position of codon 12 of the Ki‐ras oncogene was found in these tumors. This was confirmed in the study reported here, and the frequency of this mutation appeared similar in tumors induced in four different rat strains, regardless of differences in sensitivity among these strains to the induction of prostate cancers by MNU and testosterone: Wistar Furth (62% incidence of grossly visible prostate tumors) > Lobund Wistar (55%) > Fisher 344 (40%) > Copenhagen (37%). A method was developed to isolate and separately culture epithelial and stromal cells from these rat prostate carcinomas. Of 20 primary cell cultures established from histologically confirmed rat prostate carcinomas, 19 (95%) displayed one or more of the following characteristics: the Ki‐ras mutation (17 of 20; 85%), anchorage‐independent growth in soft agar at early passage (12 of 20; 60%), or tumorigenicity at later passage (eight of eight; 100%). One epithelial cell culture and all five stromal cell cultures established from prostate tumors had none of these characteristics. Epithelial cultures that had the Ki‐ras mutation and grew in soft agar constitute the predominant genotype/phenotype (55%), cultures with the mutation that did not grow in soft agar were less frequent (30%), 10% of the cultures had neither characteristic, and only one grew in soft agar but did not have the mutation. These findings suggest that there are at least two and perhaps more different molecular pathways of prostate carcinogenesis in rats treated with MNU plus testosterone. Furthermore, these data suggest that these pathways and the mechanisms determining strain differences in sensitivity to prostate cancer induction are unrelated. Mol. Carcinog. 25:179–186, 1999.

Acute high-level exposure to WTC particles alters expression of genes associated with oxidative stress and immune function in the lung.

Abstract First responders (FR) present at Ground Zero in the first 72 h after the World Trade Center (WTC) collapsed have progressively exhibited significant respiratory injuries. The few toxicology studies performed to date evaluated effects from just fine (< 2.5 µm) WTC dusts; none examined health effects/toxicities from atmospheres bearing larger particle sizes, despite the fact the majority (> 96%) of dusts were > 10 µm and most FR likely entrained dusts by mouth breathing. Using a system that generated/delivered supercoarse (10–53 µm) WTC dusts to F344 rats (in a manner that mimicked FR exposures), this study sought to examine potential toxicities in the lungs. In this exploratory study, rats were exposed for 2 h to 100 mg WTC dust/m3 (while under isoflurane [ISO] anesthesia) or an air/ISO mixture; this dose conservatively modeled likely exposures by mouth-breathing FR facing ≈750–1000 mg WTC dust/m3. Lungs were harvested 2 h post-exposure and total RNA extracted for subsequent global gene expression analysis. Among the >  1000 genes affected by WTC dust (under ISO) or ISO alone, 166 were unique to the dust exposure. In many instances, genes maximally-induced by the WTC dust exposure (relative to in naïve rats) were unchanged/inhibited by ISO only; similarly, several genes maximally inhibited in WTC dust rats were largely induced/unchanged in rats that received ISO only. These outcomes reflect likely contrasting effects of ISO and the WTC dust on lung gene expression. Overall, the data show that lungs of rats exposed to WTC dust (under ISO) – after accounting for any impact from ISO alone – displayed increased expression of genes related to lung inflammation, oxidative stress, and cell cycle control, while several involved in anti-oxidant function were inhibited. These changes suggested acute inflammogenic effects and oxidative stress in the lungs of WTC dust-exposed rats. This study, thus, concludes that a single very high exposure to WTC dusts could potentially have adversely affected the respiratory system – in terms of early inflammatory and oxidative stress processes. As these changes were not compared with other types of dusts, the uniqueness of these WTC-mediated effects remains to be confirmed. It also still remains to be determined if these effects might have any relevance to chronic lung pathologies that became evident among FR who encountered the highest dust levels on September 11, 2001 and the 2 days thereafter. Ongoing studies using longer-range post-exposure analyses (up to 1-year or more) will help to determine if effects seen here on genes were acute, reversible, or persistent, and associated with corresponding histopathologic/biochemical changes in situ.

The Source and Secretion of Immunoactive Relaxin in Rat Milk

The milk of many mammalian species contains hormones and growth factors in addition to nutrients and immuocompetent substances. These factors can be absorbed into the circulation of suckling neonates to exert important effects on metabolism and promote tissue and organ growth. Frequently, there is uncertainty as to whether such substances are gene products of the mammary glands themselves or are produced elsewhere and concentrated from the systemic circulation. The 6 kD polypeptide, relaxin, appears in milk of several mammalian species, including that of the rat, but proof of its source of secretion (corpus luteum vs. mammary gland) is so far lacking. The specific monoclonal anti-rat relaxin antibody MCA1 has previously been utilized successfully to investigate many of relaxin’s actions in the rat, including those affecting the development of the mammary apparatus. In this report, MCA1 was utilized to aid in the identification of the source of relaxin in rat milk. Treatment of lactating rats with MCA1 completely neutralized the luteal relaxin circulating in serum but did not decrease the concentration of immunoactive relaxin secreted in milk. Moreover, the antibody did not appear to reach the mammary epithelium. The evidence thus supports the view that in the rat, the relaxin secreted in milk is primarily a product of the mammary glands and not concentrated from the systemic circulation.

A novel system to generate WTC dust particles for inhalation exposures

First responders (FRs) present at Ground Zero within the critical first 72 h after the World Trade Center (WTC) collapse have progressively exhibited significant respiratory injury. The majority (>96%) of WTC dusts were >10 μm and no studies have examined potential health effects of this size fraction. This study sought to develop a system to generate and deliver supercoarse (10–53 μm) WTC particles to a rat model in a manner that mimicked FR exposure scenarios. A modified Fishing Line generator was integrated onto an intratracheal inhalation (ITIH) system that allowed for a bypassing of the nasal passages so as to mimic FR exposures. Dust concentrations were measured gravimetrically; particle size distribution was measured via elutriation. Results indicate that the system could produce dusts with 23 μm mass median aerodynamic diameter (MMAD) at levels up to ≥1200 mg/m3. To validate system utility, F344 rats were exposed for 2 h to ≈100 mg WTC dust/m3. Exposed rats had significantly increased lung weight and levels of select tracer metals 1 h after exposure. Using this system, it is now possible to conduct relevant inhalation exposures to determine adverse WTC dusts impacts on the respiratory system. Furthermore, this novel integrated Fishing Line–ITIH system could potentially be used in the analyses of a wide spectrum of other dusts/pollutants of sizes previously untested or delivered to the lungs in ways that did not reflect realistic exposure scenarios.

Impact of acute exposure to WTC dust on ciliated and goblet cells in lungs of rats

Abstract Clinical studies and the World Trade Center (WTC) Health Registry have revealed increases in the incidence of chronic (non-cancer) lung disorders among first responders (FR) who were at Ground Zero during the initial 72 h after the collapse. Our previous analyses of rats exposed to building-derived WTC dusts using exposure scenarios/levels that mimicked FR mouth-breathing showed that a single WTC dust exposure led to changes in expression of genes whose products could be involved in the lung ailments, but few other significant pathologies. We concluded that rather than acting as direct inducers of many of the FR health effects, it was more likely inhaled WTC dusts instead may have impacted on toxicities induced by other rescue-related co-pollutants present in Ground Zero air. To allow for such effects to occur, we hypothesized that the alkaline WTC dusts induced damage to the normal ability of the lungs to clear inhaled particles. To validate this, rats were exposed on two consecutive days (2 h/d, by intratracheal inhalation) to WTC dust (collected 12–13 September 2001) and examined over a 1-yr period thereafter for changes in the presence of ciliated cells in the airways and hyperplastic goblet cells in the lungs. WTC dust levels in the lungs were assessed in parallel to verify that any changes in levels of these cells corresponded with decreases in host ability to clear the particles themselves. Image analyses of the rat lungs revealed a significant decrease in ciliated cells and increase in hyperplastic goblet cells due to the single series of WTC dust exposures. The study also showed there was only a nominal non-significant decrease (6–11%) in WTC dust burden over a 1-yr period after the final exposure. These results provide support for our current hypothesis that exposure to WTC dusts caused changes in airway morphology/cell composition; such changes could, in turn, have led to potential alterations in the clearance/toxicities of other pollutants inhaled at Ground Zero in the critical initial 72-h period.

Abstract B57: Gnetin C, a novel resveratrol dimer, targets pancreatic cancer metabolism

Background: Pancreatic ductal adenocarcinoma (PDAC) is aggressively invasive and treatment-resistant malignancy, and is the fourth leading cause of cancer deaths in the United States. The pancreatic cancer cells alter specific metabolic pathways to meet their tremendous energy and biomass demands which contribute to the progression and dissemination of this disease. More importantly, the oncogenic signaling enables cancer cells to reprogram the cellular metabolism that afford growth and proliferative advantages over normal cells and, thus, may contribute to pancreatic cancer pathophysiology. There is an increasing interest to investigate the oncogenic signaling that controls the metabolic reprogramming of cancer cells. Objectives: Mammalian Target of Rapamycin (mTOR), a central downstream target of Akt, is a highly conserved protein kinase, and is frequent activated in PDAs. Earlier studies reported that mTOR, a master regulator of cell growth and proliferation downstream of oncogenic signaling pathways, controls specific aspects of cellular metabolism through the induction of metabolic gene expression. In our earlier microarray studies, we observed differential expression of genes associated with glucose and lipid metabolic pathways, with concomitant increase in mTOR expression. However, the mechanism and critical link between mTOR and the pancreatic cancer metabolism has not been fully understood. Methodology: In the present study, we evaluated the effectiveness of Melinjo (Indonesian name; Gnetum gnemon L.) seed extract (MSE) and gnetin C (GC), a resveratrol dimer found abundantly in MSE in human (Aspc-1 and PANC-1) and mouse (Pan-02) pancreatic cancer cells. MSE has been reported to exert antioxidant, lipase and amylase inhibition, anti-metabolic syndrome effects and anticancer activities. A recent clinical study reported that MSE decreased serum uric acid and increased HDL cholesterol levels in humans, suggesting that MSE may improve lipid metabolism. To test the anti-tumor activities, pancreatic cancer cells were treated with various concentrations of MSE (0 - 400μg/ml) and GC (0 – 100μM) for 48 h. MTS cell proliferation and apoptotic assays were performed to determine the cell growth inhibition and induction of apoptosis. Trans-resveratrol (RES) that is abundant in grapes and wine was used to compare the effects of GC. Real-time PCR was performed to evaluate the effect of a MSE or GC on mTOR and its downstream targets, as well selected glucose and lipid metabolic pathway genes. Western blot was performed to confirm their expression at the protein level. Results and conclusion: Human (ASPC-1 and PANC-1), and mouse (Pan-02) pancreatic cancer cells treated with MSE, GC and RES showed a dose-dependent decrease in cancer cell proliferation. The IC50 values of MSE, GC and RES related to their anti-proliferative effects revealed that GC, the major metabolite of MSE has superior anti-proliferative properties than resveratrol. Further, flow cytometry analysis showed a pre-G1 peak of apoptosis (>10%) with GC. We observed that GC significantly downregulated mTOR complex, namely (mTORC1 and mTOC2). mTORC1 is a sensor of systemic and local levels of nutrients that regulates cancer cell proliferation and survival. mTORC2 is associated with the ribosome and insulin-stimulated oncogenic PI3K signaling. We also found that GC downregulated p-ribosomal protein S6 (p-RPS6), a downstream target of mTOR that plays important role in both neoplastic and inflammatory processes. In addition, GC also downregulated glycogen synthase kinase 3 alpha and beta (GSK3α and β) that is linked to glycogen metabolism and 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1), which is associated with lipids and lipoproteins metabolism (cholesterol biosynthesis). Further analysis on key genes of the lipid metabolism is in progress. Given the preliminary evidence that GC has superior anti-proliferative activities and its ability to inhibit mTOR and metabolic/lipid pathway genes can be expected to yield more potent therapies against this deadly disease. The future goal will be to test the potentials of GC on the antitumor efficacy and the effect on metabolic pathway targets in preclinical animal models of pancreatic cancer. Citation Format: Narayanan K. Narayanan, Kazuhiro Kunimasa, Di Tian, Lori Horton, Igor Dolgaev, Adriana Heguy, George Miller, Amit Tiwari, Bhagavathi A. Narayanan. Gnetin C, a novel resveratrol dimer, targets pancreatic cancer metabolism. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B57.