Shaiju K. Vareed

Phase IIA Clinical Trial of Curcumin for the Prevention of Colorectal Neoplasia

Curcumin is derived from the spice tumeric and has antiinflammatory and antineoplastic effects in vitro and in animal models, including preventing aberrant crypt foci (ACF) and adenomas in murine models of colorectal carcinogenesis. Inhibiting the production of the procarcinogenic eicosanoids prostaglandin E2 (PGE2) and 5-hydroxyeicosatetraenoic acid (5-HETE) can suppress carcinogenesis in rodents. Curcumin reduces mucosal concentrations of PGE2 (via inhibition of cyclooxygenases 1 and 2) and 5-HETE (via inhibition of 5-lipoxygenase) in rats. Although preclinical data support curcumin activity in many sites, the poor bioavailability reported for this agent supports its use in the colorectum. We assessed the effects of oral curcumin (2 g or 4 g per day for 30 days) on PGE2 within ACF (primary endpoint), 5-HETE, ACF number, and proliferation in a nonrandomized, open-label clinical trial in 44 eligible smokers with eight or more ACF on screening colonoscopy. We assessed pre- and posttreatment concentrations of PGE2 and 5-HETE by liquid chromatography tandem mass spectroscopy in ACF and normal-tissue biopsies; ACF number via rectal endoscopy; proliferation by Ki-67 immunohistochemistry; and curcumin concentrations by high-performance liquid chromatography in serum and rectal mucosal samples. Forty-one subjects completed the study. Neither dose of curcumin reduced PGE2 or 5-HETE within ACF or normal mucosa or reduced Ki-67 in normal mucosa. A significant 40% reduction in ACF number occurred with the 4-g dose (P < 0.005), whereas ACF were not reduced in the 2-g group. The ACF reduction in the 4-g group was associated with a significant, five-fold increase in posttreatment plasma curcumin/conjugate levels (versus pretreatment; P = 0.009). Curcumin was well tolerated at both 2 g and 4 g. Our data suggest that curcumin can decrease ACF number, and this is potentially mediated by curcumin conjugates delivered systemically. Cancer Prev Res; 4(3); 354–64. ©2011 AACR.

Insulin Secretion by Bioactive Anthocyanins and Anthocyanidins Present in Fruits

Anthocyanins are responsible for a variety of bright colors including red, blue, and purple in fruits, vegetables, and flowers and are consumed as dietary polyphenols. Anthocyanin-containing fruits are implicated in a decrease in coronary heart disease and are used in antidiabetic preparations. In the present study, we have determined the ability of anthocyanins, cyanidin-3-glucoside (1), delphinidin-3-glucoside (2), cyanidin-3-galactoside (3), and pelargonidin-3-galactoside (4), and anthocyanidins, cyanidin (5), delphinidin (6), pelargonidin (7), malvidin (8), and petunidin (9), to stimulate insulin secretion from rodent pancreatic beta-cells (INS-1 832/13) in vitro. The compounds were tested in the presence of 4 and 10 mM glucose concentrations. Our results indicated that 1 and 2 were the most effective insulin secretagogues among the anthocyanins and anthocyanidins tested at 4 and 10 mM glucose concentrations. Pelargonidin-3-galactoside is one of the major anthocyanins, and its aglycone, pelargonidin, caused a 1.4-fold increase in insulin secretion at 4 mM glucose concentration. The rest of the anthocyanins and anthocyanidins tested in our assay had only marginal effects on insulin at 4 and 10 mM glucose concentrations.

Pharmacokinetics of Curcumin Conjugate Metabolites in Healthy Human Subjects

Background: Curcumin is a polyphenol, found in the spice turmeric, that has promising anticancer properties, but previous studies suggest that absorption of curcumin may be limited. Methods: This study examined the pharmacokinetics of a curcumin preparation in healthy human volunteers 0.25 to 72 h after a single oral dose. Curcumin was administered at doses of 10 g (n = 6) and 12 g (n = 6). Subjects were randomly allocated to dose level for a total of six subjects at each dose level. Serum samples were assayed for free curcumin, for its glucuronide, and for its sulfate conjugate. The data were fit to a one-compartment absorption and elimination model. Results: Using a high-performance liquid chromatography assay with a limit of detection of 50 ng/mL, only one subject had detectable free curcumin at any of the 14 time points assayed, but curcumin glucuronides and sulfates were detected in all subjects. Based on the pharmacokinetic model, the area under the curve for the 10 and 12 g doses was estimated (mean ± SE) to be 35.33 ± 3.78 and 26.57 ± 2.97 μg/mL × h, respectively, whereas Cmax was 2.30 ± 0.26 and 1.73 ± 0.19 μg/mL. The Tmax and t1/2 were estimated to be 3.29 ± 0.43 and 6.77 ± 0.83 h. The ratio of glucuronide to sulfate was 1.92:1. The curcumin conjugates were present as either glucuronide or sulfate, not mixed conjugates. Conclusion: Curcumin is absorbed after oral dosing in humans and can be detected as glucuronide and sulfate conjugates in plasma. (Cancer Epidemiol Biomarkers Prev 2008;17(6):1411–7)

Metabolomic Profiling Reveals a Role for Androgen in Activating Amino Acid Metabolism and Methylation in Prostate Cancer Cells

Prostate cancer is the second leading cause of cancer related death in American men. Development and progression of clinically localized prostate cancer is highly dependent on androgen signaling. Metastatic tumors are initially responsive to anti-androgen therapy, however become resistant to this regimen upon progression. Genomic and proteomic studies have implicated a role for androgen in regulating metabolic processes in prostate cancer. However, there have been no metabolomic profiling studies conducted thus far that have examined androgen-regulated biochemical processes in prostate cancer. Here, we have used unbiased metabolomic profiling coupled with enrichment-based bioprocess mapping to obtain insights into the biochemical alterations mediated by androgen in prostate cancer cell lines. Our findings indicate that androgen exposure results in elevation of amino acid metabolism and alteration of methylation potential in prostate cancer cells. Further, metabolic phenotyping studies confirm higher flux through pathways associated with amino acid metabolism in prostate cancer cells treated with androgen. These findings provide insight into the potential biochemical processes regulated by androgen signaling in prostate cancer. Clinically, if validated, these pathways could be exploited to develop therapeutic strategies that supplement current androgen ablative treatments while the observed androgen-regulated metabolic signatures could be employed as biomarkers that presage the development of castrate-resistant prostate cancer.

Metabolomic profiling identifies biochemical pathways associated with castration-resistant prostate cancer.

Despite recent developments in treatment strategies, castration-resistant prostate cancer (CRPC) is still the second leading cause of cancer-associated mortality among American men, the biological underpinnings of which are not well understood. To this end, we measured levels of 150 metabolites and examined the rate of utilization of 184 metabolites in metastatic androgen-dependent prostate cancer (AD) and CRPC cell lines using a combination of targeted mass spectrometry and metabolic phenotyping. Metabolic data were used to derive biochemical pathways that were enriched in CRPC, using Oncomine concept maps (OCM). The enriched pathways were then examined in-silico for their association with treatment failure (i.e., prostate specific antigen (PSA) recurrence or biochemical recurrence) using published clinically annotated gene expression data sets. Our results indicate that a total of 19 metabolites were altered in CRPC compared to AD cell lines. These altered metabolites mapped to a highly interconnected network of biochemical pathways that describe UDP glucuronosyltransferase (UGT) activity. We observed an association with time to treatment failure in an analysis employing genes restricted to this pathway in three independent gene expression data sets. In summary, our studies highlight the value of employing metabolomic strategies in cell lines to derive potentially clinically useful predictive tools.

Phase II Study of the Effects of Ginger Root Extract on Eicosanoids in Colon Mucosa in People at Normal Risk for Colorectal Cancer

Inhibitors of COX indicate that upregulation of inflammatory eicosanoids produced by COX, and in particular prostaglandin E2 (PGE2), are early events in the development of colorectal cancer (CRC). Ginger has shown downregulation of COX in vitro and decreased incidence/multiplicity of adenomas in rats. This study was conducted to determine if 2.0 g/d of ginger could decrease the levels of PGE2, 13-hydroxy-octadecadienoic acids, and 5-, 12-, and 15-hydroxyeicosatetraenoic acid (5-, 12-, and 15-HETE), in the colon mucosa of healthy volunteers. To investigate this aim, we randomized 30 subjects to 2.0 g/d ginger or placebo for 28 days. Flexible sigmoidoscopy at baseline and day 28 was used to obtain colon biopsies. A liquid chromatography mass spectrometry method was used to determine eicosanoid levels in the biopsies, and levels were expressed per protein or per free arachidonic acid. There were no significant differences in mean percent change between baseline and day 28 for any of the eicosanoids, when normalized to protein. There was a significant decrease in mean percent change in PGE2 (P = 0.05) and 5-HETE (P = 0.04), and a trend toward significant decreases in 12-HETE (P = 0.09) and 15-HETE (P = 0.06) normalized to free arachidonic acid. There was no difference between the groups in terms of total adverse events P = 0.55). On the basis of these results, it seems that ginger has the potential to decrease eicosanoid levels, perhaps by inhibiting their synthesis from arachidonic acid. Ginger also seemed to be tolerable and safe. Further investigation in people at high risk for CRC seems warranted. Cancer Prev Res; 4(11); 1929–37. ©2011 AACR.

Identification of metabolites in Withania sominfera fruits by liquid chromatography and high-resolution mass spectrometry

RATIONALE Withania somnifera is a rich source of biologically active secondary metabolites. Our earlier investigations on the fruits of this plant have yielded novel compounds, withanamides, with potent antioxidant activity and protective effect on β-amyloid-induced cytotoxicity in vitro. However, several minor compounds present in the fruits have not been characterized previously which may contribute to the observed biological activities. METHODS Liquid chromatography (LC) coupled with high-resolution mass spectrometry (HRMS) with data-dependent and targeted MS/MS experiments were conducted to elucidate the structure of observed metabolites. RESULTS A total of 62 metabolites identified included 32 withanamides, 22 withanolides, 3 steroidal saponins, 2 lignanamides, feruloyl tyramine, methoxy feruloyl tyramine and a diglucoside of hydroxyl palmitic acid. The structures of these compounds were proposed based on accurate masses of the molecular and fragment ions. Several of these new compounds identified from the profile were derived from withanamides with variations in aliphatic and/or glycosyl moieties. In addition, six new withanolides, a new hydroxy fatty acid diglucoside and several known compounds in the extract were identified. CONCLUSIONS The current study revealed the presence of several new and known compounds in Withania somnifera fruits. High-resolution MS and MS/MS experiments provide an extremely effective approach to derive the structures of plant secondary metabolites including isomeric compounds.

Metabolites of Purine Nucleoside Phosphorylase (NP) in Serum Have the Potential to Delineate Pancreatic Adenocarcinoma

Pancreatic Adenocarcinoma (PDAC), the fourth highest cause of cancer related deaths in the United States, has the most aggressive presentation resulting in a very short median survival time for the affected patients. Early detection of PDAC is confounded by lack of specific markers that has motivated the use of high throughput molecular approaches to delineate potential biomarkers. To pursue identification of a distinct marker, this study profiled the secretory proteome in 16 PDAC, 2 carcinoma in situ (CIS) and 7 benign patients using label-free mass spectrometry coupled to 1D-SDS-PAGE and Strong Cation-Exchange Chromatography (SCX). A total of 431 proteins were detected of which 56 were found to be significantly elevated in PDAC. Included in this differential set were Parkinson disease autosomal recessive, early onset 7 (PARK 7) and Alpha Synuclein (aSyn), both of which are known to be pathognomonic to Parkinsons disease as well as metabolic enzymes like Purine Nucleoside Phosphorylase (NP) which has been exploited as therapeutic target in cancers. Tissue Microarray analysis confirmed higher expression of aSyn and NP in ductal epithelia of pancreatic tumors compared to benign ducts. Furthermore, extent of both aSyn and NP staining positively correlated with tumor stage and perineural invasion while their intensity of staining correlated with the existence of metastatic lesions in the PDAC tissues. From the biomarker perspective, NP protein levels were higher in PDAC sera and furthermore serum levels of its downstream metabolites guanosine and adenosine were able to distinguish PDAC from benign in an unsupervised hierarchical classification model. Overall, this study for the first time describes elevated levels of aSyn in PDAC as well as highlights the potential of evaluating NP protein expression and levels of its downstream metabolites to develop a multiplex panel for non-invasive detection of PDAC.

Blood–Brain Barrier Permeability of Bioactive Withanamides Present in Withania somnifera Fruit Extract

The neuroprotective effect of Withania somnifera L. Dunal fruit extract, in rodent models, is known. Withanamides, the primary active constituents in W. somnifera fruit extract exhibited neuroprotective effects against β‐amyloid‐induced cytotoxicity in neuronal cell culture studies. Therefore, we investigated the blood–brain barrier permeability of withanamides in W. somnifera fruit extract in mice using HPLC coupled with high resolution quadrupole time of flight mass spectrometer (Q‐TOF/MS) detection. Mice were administered with 250 mg/kg of W. somnifera extract by intraperitoneal injection, and the blood and brain samples analyzed by Q‐TOF/MS detection. Four major withanamides were detected in brain and blood of mice administered with W. somnifera extract. The results suggested that the withanamides crossed the blood–brain barrier. These results may help to develop W. somnifera fruit extract as a preventive or therapeutic botanical drug for stress‐induced neurological disorders. Copyright

Abstract B92: Metabolomic landscape of African American prostate cancer: Insights into the biologic basis of the racial disparity

African American (AA) men have an approximately 60% higher incidence of prostate cancer (PCa) and have about two times greater risk of dying of the disease than their European American (EA) counterparts. Despite this staggeringly unequal burden of PCa incidence and outcome between AA and EA men, there is limited insight into the molecular mechanisms associated with this racial disparity. Using state-of-the-art mass spectrometry platform, we uncovered the very first metabolic and lipidomics landscape in PCa/benign adjacent tissue pairs and paired plasma and urine containing epidemiologic meta-data from ancestry-typed AA and EA men. A total of 190 polar and mid-polar metabolites and 495 lipids were measured in tissues and 183 metabolites and lipids were examined in the plasma and urine samples. AA PCa tissue and plasma signatures had unique alterations in metabolites and key enzymes associated with the methionine-homocysteine pathway, including adenosine and inosine levels, compared to corresponding tissues and plasma from EA case controls and EA PCa tissues. Intriguingly, methionine levels in AA PCa were also portrayed by distinct dietary practices in these patients. In addition, AA PCa tissues demonstrated unique patterns of bioenergetic metabolites and accumulated lipids reflecting impaired mitochondrial activity and TCA cycle. Further characterization of these first-in-the-field findings demonstrating reprogrammed metabolism in AA PCa relative to EA tumors could reveal insights into the biologic basis of PCa disparities and novel areas for therapeutic intervention. Citation Format: Stacy M. Lloyd*, Jie H. Gohlke*, Sumanta Basu*, Vasanta Putluri,Shaiju K Vareed, Rebeca San Martin, Thekkelnaycke Rajendiran, Tiffany A. Dorsey, Bandana Prasad, Rajni Sonavane, Uttam Rasaily, James Henderson, Balasubramanyam Karanam, Harene Venghatakrishnan, Salil Bhowmik, Alexander Zaslavsky,Nilanjan Guha, Rick Kittles, Stefan Ambs, Michael Ittmann,David Rowley, Ganesh Palapattu, Nagireddy Putluri,George Michailidis, Arun Sreekumar. Metabolomic landscape of African American prostate cancer: Insights into the biologic basis of the racial disparity [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr B92.