Indu Sinha

Dietary methionine restriction inhibits prostatic intraepithelial neoplasia in TRAMP mice

Prostate cancer (PCa) is a major aging‐related disease for which little progress has been made in developing preventive strategies. Over the past several years, methionine restriction (MR), the feeding of a diet low in methionine (Met), has been identified as an intervention which significantly extends lifespan and reduces the onset of chronic diseases, including cancer, in laboratory animals. We, therefore, hypothesized that MR may be an effective strategy for inhibiting PCa.

Methylseleninic acid downregulates hypoxia-inducible factor-1α in invasive prostate cancer.

Alternative strategies are needed to control growth of advanced and hormone refractory prostate cancer. In this regard, we investigated the efficacy of methylseleninic acid (MSeA), a penultimate precursor to the highly reactive selenium metabolite, methylselenol, to inhibit growth of invasive and hormone refractory rat (PAIII) and human (PC‐3 and PC‐3M) prostate cancer cells. Our results demonstrate that MSeA inhibits PAIII cell growth in vitro as well as reduces weights of tumors generated by PAIII cells treated ex vivo. A significant reduction in the number of metastatic lung foci by MSeA treatment was also noted in Lobund‐Wistar rats. The PAIII cells along with PC‐3, DU145 and PC‐3M cells undergo apoptosis after MSeA treatments in both normoxia and hypoxia. Treatment of metastatic rat and human prostate cancer cell lines with MSeA decreased hypoxia‐inducible factor‐1α (HIF‐1α) levels in a dose‐dependent manner. Additionally, HIF‐1α transcription activity both in normoxic and hypoxic conditions is reduced after MSeA treatment of prostate cancer cells. Furthermore, VEGF and GLUT1, downstream targets of HIF‐1α, were also reduced in prostate cancer cells after MSeA treatment. Our study illustrates the efficacy of MSeA in controlling growth of hormone refractory prostate cancer by downregulating HIF‐1α, which is possibly occurring through stabilization or increase in prolyl hydroxylase activity.

Synthesis and antitumor properties of selenocoxib‐1 against rat prostate adenocarcinoma cells

Hormone refractory prostate cancer poses a huge problem and standard of care chemotherapy has not been very successful. We used a novel strategy to combine properties of 2 well‐studied class of compounds (selenium and COX‐2 inhibitor) and examined the resulting effectiveness against prostate cancer. Bearing in mind that sulfonamide moiety and pyrazole ring is important for the proapoptotic activity of Celecoxib, we synthesized a selenium derivative, Selenocoxib‐1, by modifying Celecoxib at position 3 of the pyrazole ring. The PAIII cells derived from a metastatic prostate tumor that arose spontaneously in a Lobund‐Wistar (LW) rat were used to examine the efficacy of Selenocoxib‐1 in vitro. In addition, human metastatic prostate cancer cells, PC‐3M, were tested for antitumor effect of Selenocoxib‐1 in vitro. The IC50 in PAIII and PC‐3M cells for Selenocoxib‐1 was about 5 μM, while for Celecoxib it was more than 20 μM. Selenocoxib‐1 induced apoptosis in a dose‐dependent manner in the PAIII cells. COX‐2 expression in PAIII cells was downregulated by Celecoxib and Selenocoxib‐1 at 20 and 5 μM, respectively; the COX‐2 activity was, however, not affected by Selenocoxib‐1. Following treatment with Selenocoxib‐1, PAIII cells resulted in dose‐dependent decrease in HIF‐1α, p‐AKT and Bcl‐2 levels. A reduction in weights was observed in subcutaneous tumors produced by PAIII cells pretreated with Selenocoxib‐1 as compared to Celecoxib in LW rats. Further, following 1 week Selenocoxib‐1 treatment of PAIII tumors resulted in significant reduction of tumor weights. This study demonstrates that Selenocoxib‐1 is more effective against prostate cancer than Celecoxib.

Remarkable inhibition of mTOR signaling by the combination of rapamycin and 1,4-phenylenebis(methylene)selenocyanate in human prostate cancer cells

Preclinical studies and clinical analyses have implicated the mammalian target of rapamycin (mTOR) pathway in the progression of prostate cancer, suggesting mTOR as a potential target for new therapies. mTOR, a serine/threonine kinase, belongs to two distinct signaling complexes: mTORC1 and mTORC2. We previously showed that the synthetic organoselenium compound, p‐XSC, effectively inhibits viability and critical signaling molecules (e.g., androgen receptor, Akt) in androgen responsive (AR) and androgen independent (AI) human prostate cancer cells. On the basis of its inhibition of Akt, we hypothesized that p‐XSC modulates mTORC2, an upstream regulator of the kinase. We further hypothesized that combining p‐XSC with rapamycin, an mTORC1 inhibitor, would be an effective combinatory strategy for the inhibition of prostate cancer. The effects of p‐XSC and rapamycin, alone or in combination, on viability and mTOR signaling were examined in AR LNCaP prostate cancer cells and AI C4‐2 and DU145 cells. Phosphorylation of downstream targets of mTORC1 and mTORC2 was analyzed by immunoblotting. The interaction of mTORC1‐ and mTORC2‐specific proteins with mTOR was probed through immunoprecipitation and immunoblotting. p‐XSC inhibited phosphorylation of mTORC2 downstream targets, Akt and PCKα, and decreased the levels of rictor, an mTORC2‐specific protein, coimmunoprecipitated with mTOR in C4‐2 cells. The combination of p‐XSC and rapamycin more effectively inhibited viability and mTOR signaling in C4‐2, LNCaP and DU145 cells than either agent individually.

Gene expression profiles in HPV-immortalized human cervical cells treated with the nicotine-derived carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

Human papillomavirus (HPV) infection is an established etiological factor for cervical cancer. Epidemiological studies suggest that smoking in combination with HPV infection plays a significant role in the etiology of this disease. We have previously shown that the tobacco carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is present in human cervical mucus. Here, we hypothesized that treatment of HPV-16-immortalized human ectocervical cells (Ecto1/E6E7) with NNK would alter the expression of genes involved in cellular transformation. Ecto1/E6E7 cells were treated with water (vehicle control) alone or with 1 microM, 10 microM, and 100 microM of NNK in water for 12 weeks. The colony-forming efficiency increased following NNK treatment; the maximum effect was observed after 12 weeks with 100 microM NNK. Microarray analysis revealed that, independent of the dose of NNK, expression of 30 genes was significantly altered; 22 of these genes showed a dose-response pattern. Genes identified are categorized as immune response (LTB4R), RNA surveillance pathway (SMG1), metabolism (ALDH7A1), genes frequently expressed in later stages of neoplastic development (MT1F), DNA binding (HIST3H3 and CHD1L), and protein biosynthesis (UBA52). Selected genes were confirmed by qRT-PCR. Western blot analysis indicates that phosphorylation of histone 3 at serine 10, a marker of cellular transformation, was up-regulated in cells treated with NNK. This is the first study showing that NNK can alter gene expression that may, in part, account for transformation of HPV-immortalized human cervical cells. The results support previous epidemiological observations that, in addition to HPV, tobacco smoking also plays an important role in the development of cervical cancer.

Selenium-responsive proteins in the sera of selenium-enriched yeast-supplemented healthy African American and Caucasian men.

Background: Studies have shown that supplementation of adult men with selenium-enriched yeast (SY) was protective against prostate cancer (PCa) and also reduced oxidative stress and levels of prostate-specific antigen. Here, we determined the effect of SY supplementation on global serum protein expression in healthy men to provide new insights into the mechanism of selenium chemoprevention; such proteins may also serve as biomarkers of disease progression. Methods: Serum samples from 36 adult men were obtained from our previous SY clinical trial, 9 months after supplementation with either SY (247 μg/d; n = 17) or placebo (nonenriched yeast; n = 19). Results: Proteomic profiling using two-dimensional difference in gel electrophoresis followed by liquid chromatography-tandem mass spectrometry revealed a total of 1,496 candidate proteins, of which, 11 were differentially expressed in the SY group as compared with placebo. Eight proteins were upregulated [clusterin isoform 1 (CLU), transthyretin, α-1B-glycoprotein, transferrin, complement component 4B proprotein, isocitrate dehydrogenase, haptoglobin, and keratin 1] and three proteins were downregulated [α-1 antitrypsin (AAT), angiotensin precursor, and albumin precursor] by SY. All of the identified proteins were redox-sensitive or involved in the regulation of redox status. Because both AAT and CLU have been previously linked to PCa development, their identities were confirmed by two-dimensional Western blot analysis. Conclusions: We identified AAT and CLU as potential candidate proteins involved in the mechanism of PCa prevention by SY. Collectively, proteins identified in this study might serve as potential new biomarkers for monitoring and comparing responses to selenium-based chemopreventive agents. Impact: Proteomic analysis of serum might be useful for the early detection and monitoring efficacy of chemopreventive agents. Cancer Epidemiol Biomarkers Prev; 19(9); 2332–40. ©2010 AACR.

Methylseleninic acid elevates REDD1 and inhibits prostate cancer cell growth despite AKT activation and mTOR dysregulation in hypoxia

Methylseleninic acid (MSeA) is a monomethylated selenium metabolite theoretically derived from subsequent β‐lyase or transamination reactions of dietary Se‐methylselenocysteine that has potent antitumor activity by inhibiting cell proliferation of several cancers. Our previous studies showed that MSeA promotes apoptosis in invasive prostate cancer cells in part by downregulating hypoxia‐inducible factor HIF‐1α. We have now extended these studies to evaluate the impact of MSeA on REDD1 (an mTOR inhibitor) in inducing cell death of invasive prostate cancer cells in hypoxia. In both PTEN+ and PTEN− prostate cancer cells we show that MSeA elevates REDD1 and phosphorylation of AKT along with p70S6K in hypoxia. Furthermore, REDD1 induction by MSeA is independent of AKT and the mTOR inhibition in prostate cancer cells causes partial resistance to MSeA‐induced growth reduction in hypoxia. Our data suggest that MSeA induces REDD1 and inhibits prostate cancer cell growth in hypoxia despite activation of AKT and dysregulation of mTOR.

4-(Methylnitrosamino)-I-(3-Pyridyl)-1-Butanone Enhances the Expression of Apolipoprotein A-I and Clara Cell 17-kDa Protein in the Lung Proteomes of Rats Fed a Corn Oil Diet but not a Fish Oil Diet

The nicotine-derived nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the most potent lung carcinogens in rodents. Several epidemiologic studies indicated that the development of lung cancer in smokers is influenced by the type and amount of dietary polyunsaturated fatty acids. A high corn oil diet has been shown to increase lung tumor volume and to decrease tumor latency in rats treated with NNK. In this study, we investigated the effects of dietary polyunsaturated fatty acids in the form of corn oil or fish oil on lung proteomes in F344 rats treated with or without NNK. The fish oil diet contained 17% fish oil and 3% corn oil, and the corn oil diet contained 20% corn oil. Rats were sacrificed after 3 months, and lungs were excised. Whole lung tissue proteins were separated by two-dimensional liquid chromatography, and differentially expressed proteins were identified by trypsin digestion and tandem mass spectrometry. Apolipoprotein A-I and Clara cell 17-kDa protein were overexpressed in the lungs of rats fed corn oil diet, compared with fish oil diet. NNK further enhanced their expression in rats fed corn oil diet; this effect was not observed in animals fed fish oil diet. The results suggest that the elevated levels of apolipoprotein A-I and Clara cell 17-kDa protein may be involved in the development of NNK-induced lung cancer in rats fed a high corn oil diet. Therefore, we propose that both proteins may serve as potential biomarkers in future molecular epidemiologic and clinical chemoprevention intervention studies. (Cancer Epidemiol Biomarkers Prev 2007;16(2):228–35)

Oral supplementation with liposomal glutathione elevates body stores of glutathione and markers of immune function

Background/Objectives:Glutathione (GSH) is the most abundant endogenous antioxidant and a critical regulator of oxidative stress. Maintenance of optimal tissues for GSH levels may be an important strategy for the prevention of oxidative stress-related diseases. We investigated if oral administration of liposomal GSH is effective at enhancing GSH levels in vivo.Subjects/Methods:A 1-month pilot clinical study of oral liposomal GSH administration at two doses (500 and 1000 mg of GSH per day) was conducted in healthy adults. GSH levels in whole blood, erythrocytes, plasma and peripheral blood mononuclear cells (PBMCs) were assessed in 12 subjects at the baseline and after 1, 2 and 4 weeks of GSH administration.Results:GSH levels were elevated after 1 week with maximum increases of 40% in whole blood, 25% in erythrocytes, 28% in plasma and 100% in PBMCs occurring after 2 weeks (P<0.05). GSH increases were accompanied by reductions in oxidative stress biomarkers, including decreases of 35% in plasma 8-isoprostane and 20% in oxidized:reduced GSH ratios (P<0.05). Enhancements in immune function markers were observed with liposomal GSH administration including Natural killer (NK) cell cytotoxicity, which was elevated by up to 400% by 2 weeks (P<0.05), and lymphocyte proliferation, which was elevated by up to 60% after 2 weeks (P<0.05). Overall, there were no differences observed between dose groups, but statistical power was limited due to the small sample size in this study.Conclusions:Collectively, these preliminary findings support the effectiveness of daily liposomal GSH administration at elevating stores of GSH and impacting the immune function and levels of oxidative stress.

Abstract 574: 1,4-phenylenebis(methylene)selenocyanate (p-XSC) inhibits mammalian target of rapamycin complex 2 (mTORC2) signaling in prostate cancer cells

The lack of treatment for worried-well patients with high-grade prostatic intraepithelial neoplasia combined with issues of recurrence and hormone resistance in prostate cancer survivors remains a major public health obstacle. The long latency of prostate cancer development provides an opportunity to intervene with mechanistically-based agents at various stages of disease progression. Preclinical studies and immunohistochemical analyses of human prostate adenocarcinoma tissues have implicated the mammalian target of rapamycin (mTOR) signaling pathway in the progression of prostate cancer and its transition to androgen independence, suggesting mTOR as a potential target for new therapies. Preclinical studies in human prostate cancer cells also show that there is cross-talk between mTOR and androgen receptor signaling, a process critical for prostate cancer growth and survival. We have previously shown that the synthetic organoselenium compound p-XSC effectively inhibits cell viability and down-regulates androgen receptor expression and transcriptional activity in androgen responsive (AR) and androgen independent (AI) human prostate cancer cells; the naturally occurring selenomethionine was either ineffective or weakly active at non-physiological levels. Therefore, we focused our study on the effects of p-XSC and we hypothesized that it inhibits prostate cancer cell growth by interfering with mTOR signaling and that inhibition of this pathway may be linked to down-regulation of androgen receptor signaling in these cells. Here we show that p-XSC inhibits phosphorylation of mTOR in both AR LNCaP and AI LNCaP C4-2 human prostate cancer cells. This inhibition is attenuated by stimulation with androgens in AR LNCaP but not AI C4-2 cells, emphasizing a cross-talk between the two pathways. p-XSC also decreases mTOR binding to mTOR complex 2 (mTORC2) protein Rictor in AR LNCaP and AI C4-2 cells and inhibits the phosphorylation of mTORC2 downstream target Akt. Since p-XSC appears to be inhibiting mTORC2 as well as androgen signaling, we further hypothesized that the combination of p-XSC with rapamycin, which primarily targets mTORC1, may be a superior approach to inhibit prostate cancer cell growth than either agent alone. Our data clearly support this hypothesis. Validation of mTOR as a target of selenium in prostate cancer will provide evidence for its potential role against advanced and hormone refractory prostate disease either individually or in combination with therapies that target other signaling pathways. Support: NIH CA111842, DOD W81XWH-08-1-0297 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 574.