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1.  Trials and Tribulations of Interrogating Biomarkers to Define Efficacy of Cancer Risk Reductive Interventions 
The challenges of clinical screening of cancer risk reductive interventions (“chemopreventive”) have slowed progress in deployment of therapeutics to reverse or delay the carcinogenesis process. The preoperative or window of opportunity design clinical trial design enrolls subjects rapidly, has short study periods and quantifies tissue biomarkers that reflect both anticarcinogenesis mechanism of the risk reductive intervention and key molecular events of the carcinogenesis process for a specific epithelial target. High subject screened to on study ratios reduce the efficiency and increase cost of this research strategy. Small sized tissue samples obtained by minimally invasive endoscopic technologies limits the number of biomarkers that can be detected and quantified, forcing investigators into choosing either a broad based but superficial muti-mechanism exploration of signaling intermediates or a more focused analysis of multiple molecular events in a linear signaling specific pathway. More efficient strategies of the future might involve isolation and expansion of pluripotent cells from at risk epithelium or intraepithelial neoplastic lesions. Such a strategy would allow interrogation of key carcinogenesis associated pathways and mechanisms in representative primary single cell cultures amenable to genomic, proteomics, or transfection based technologies.
doi:10.1158/1940-6207.CAPR-12-0499
PMCID: PMC4139927  PMID: 23333813
2.  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 0.25-72 hours 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 6 subjects at each dose level. Serum samples were assayed for free curcumin, for its glucuronide and for it sulfate conjugate. The data were fit to a one-compartment absorption and elimination model.
Results
Using an HPLC 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 PK model, AUC for the 10g and 12 g doses was estimated (mean±standard error) to be 35.33±3.78 and 26.57±2.97 μg/mL × hr, respectively, while 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 hr and 6.77±0.83 hr. The ratio of glucuronide: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.
doi:10.1158/1055-9965.EPI-07-2693
PMCID: PMC4138955  PMID: 18559556
Chemoprevention; Pharmacokinetics; Curcumin; Metabolism; Clinical trial
3.  Discovery of sialyl Lewis A and Lewis X modified protein cancer biomarkers using high density antibody arrays 
Journal of proteomics  2013;96:291-299.
We report on a high-dimensional method to globally profile glycoproteins that are modified with sialyl Lewis A or Lewis X glycans. Specifically, glycoproteins in serum or plasma are fractionated on a high-density antibody microarray (i.e., each are localized to their specific antibody spot) and are specifically detected via fluorescently labeled anti-sialyl Lewis A or anti- Lewis X antibodies with quantification in a microarray scanner. Non-glycosylated proteins or glycoproteins with other glycan motifs do not interfere with this assay. The whole process is very rapid and applicable for high-throughput screening without the need for purification of glycoproteins from the samples. Using these methods, sialyl Lewis A or Lewis X moieties were found to be expressed on many previously unreported secreted or membrane associated proteins. Furthermore, the combination of sialyl Lewis A or Lewis X content with protein level increased the ability of certain glycoproteins to distinguish 30 patients with stage III and IV colon cancer from 60 control samples. Thus, this highly sensitive method is capable of discovering novel specific glycan modifications on proteins, many of which will likely be useful for disease detection and monitoring.
doi:10.1016/j.jprot.2013.10.030
PMCID: PMC3946870  PMID: 24185138
glycoproteins; glycans; sialyl Lewis A; Lewis X; cancer biomarker; antibody array
4.  Phase IIA Clinical Trial of Curcumin for the Prevention of Colorectal Neoplasia 
Curcumin is derived from the spice tumeric and has anti-inflammatory 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 acitivity in many sites, the reported poor bioavailability of 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 non-randomized, open-label clinical trial in 44 eligible smokers with 8 or more ACF on screening colonoscopy. We assessed pre- and post-treatment 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. 41 Subjects completed the study. Neither dose of curcumin reduced PGE2 or 5-HETE within ACF or normal mucosa or Ki-67 in normal mucosa. A significant 40% reduction in ACF number occurred with the 4 g dose (P < 0.005); while ACF were not reduced in the 2 g group. This reduction was associated with a significant change in plasma curcumin/conjugate levels pre- and post-treatmeeng (5-fold increase; P = 0.009) in the 4 g group. Curcumin was well tolerated at both 2 g and 4g. Our data suggest that curcumin can decrease ACF number, and this is potentially mediated by curcumin conjugates delivered systemically.
doi:10.1158/1940-6207.CAPR-10-0098
PMCID: PMC4136551  PMID: 21372035
ACF; Tobacco; Curcumin; Colon cancer
5.  Phase II trial of encapsulated ginger as a treatment for chemotherapy-induced nausea and vomiting 
Goals of work
Ginger has been used to treat numerous types of nausea and vomiting. Ginger has also been studied for its efficacy for acute chemotherapy-induced nausea and vomiting (CINV). However, its efficacy for delayed CINV in a diverse oncology population is unknown.
Materials and methods
We performed a randomized, double-blind, placebo-controlled trial in 162 patients with cancer who were receiving chemotherapy and had experienced CINV during at least one previous round of chemotherapy. All participants were receiving a 5-HT3 receptor antagonists and/or aprepitant. Participants were randomized to receive either 1.0 g ginger, 2.0 g ginger daily, or matching placebo for 3 days. The primary outcome was change in the prevalence of delayed CINV. Secondary outcomes included acute prevalence of CINV, acute and delayed severity of CINV, and assessment of blinding.
Main results
There were no differences between groups in the prevalence of delayed nausea or vomiting, prevalence of acute CINV, or severity of delayed vomiting or acute nausea and vomiting. Participants who took both ginger and aprepitant had more severe acute nausea than participants who took only aprepitant. Participants were able to accurately guess which treatment they had received. Ginger appeared well tolerated, with no difference in all adverse events (AEs) and significantly less fatigue and miscellaneous AEs in the ginger group.
Conclusions
Ginger provides no additional benefit for reduction of the prevalence or severity of acute or delayed CINV when given with 5-HT3 receptor antagonists and/or aprepitant.
doi:10.1007/s00520-008-0528-8
PMCID: PMC4131259  PMID: 19005687
Ginger; Apripetant; Chemotherapy-induced nausea and vomiting
6.  Human colonic crypts in culture: segregation of immunochemical markers in normal versus adenoma-derived 
In order to advance a culture model of human colonic neoplasia, we developed methods for the isolation and in vitro maintenance of intact colonic crypts from normal human colon tissue and adenomas. Crypts were maintained in three-dimensional Matrigel culture with a simple, serum-free, low Ca2+ (0.15 mM) medium. Intact colonic crypts from normal human mucosa were viably maintained for 3–5 days with preservation of the in situ crypt-like architecture, presenting a distinct base and apex. Abnormal structures from adenoma tissue could be maintained through multiple passages (up to months), with expanding buds/tubules. Immunohistochemical markers for intestinal stem cells (Lgr5), growth (Ki67), differentiation (E-cadherin, cytokeratin 20 (CK20) and mucin 2 (MUC2)) and epithelial turnover (Bax, cleaved Caspase-3), paralleled the changes in function. The epithelial cells in normal crypts followed the physiological sequence of progression from proliferation to differentiation to dissolution in a spatially and temporally appropriate manner. Lgr5 expression was seen in a few basal cells of freshly isolated crypts, but was not detected after 1–3 days in culture. After 24 h in culture, crypts from normal colonic tissue continued to show strong Ki67 and MUC2 expression at the crypt base, with a gradual decrease over time such that by days 3–4 Ki67 was not expressed. The differentiation marker CK20 increased over the same period, eventually becoming intense throughout the whole crypt. In adenoma-derived structures, expression of markers for all stages of progression persisted for the entire time in culture. Lgr5 showed expression in a few select cells after months in culture. Ki67 and MUC2 were largely associated with the proliferative budding regions while CK20 was localized to the parent structure. This ex vivo culture model of normal and adenomatous crypts provides a readily accessible tool to help understand the growth and differentiation process in human colonic epithelium.
doi:10.1038/labinvest.2013.145
PMCID: PMC4108175  PMID: 24365748
adenoma; apoptosis; chemoprevention; colon crypt culture; cytokeratin 20; Lgr5; mucin 2
7.  Relationships between Serum and Colon Concentrations of Carotenoids and Fatty Acids in a Randomized Dietary Intervention Trial 
Little is known about dietary effect on colonic nutrient concentrations associated with preventive foods. This study observed 120 persons at increased risk of colon cancer randomized to a Mediterranean versus a Healthy Eating diet for six months. The former targeted increases in whole grains, fruits, vegetables, monounsaturated and n3 fats. Healthy Eating diet was based on Healthy People 2010 recommendations. At baseline, dietary fat and carotenoid intakes were poorly associated (Spearman ρ < 0.4) with serum and colon concentrations. Strong associations were observed between serum and colon measurements of β-cryptoxanthin (ρ = 0.58, p-value < 0.001), α-carotene (ρ = 0.48, p-value < 0.001), and β-carotene (ρ = 0.45, p-value < 0.001). After six months, the Healthy Eating arm increased serum lutein, β- and α-carotene significantly (p-value < 0.05). In the Mediterranean arm the significant increases were in serum lutein, β-cryptoxanthin, β-carotene, monounsaturated and n3 fats. A significant group-by-time interaction (p-value = 0.03) was obtained for monounsaturated fats. Colonic increases in carotenoids and n3 fats were significant only in Healthy Eating arm, while group-by-time interaction were significant for β-carotene (p-value = 0.02), and α-carotene (p-value = 0.03). Changes in colon concentrations were not significantly associated with reported dietary changes. Changes in colon and serum concentrations were strongly associated for β-cryptoxanthin (ρ = 0.56, p-value < 0.001), and α-carotene (ρ = 0.40, p-value < 0.001). The associations between colonic and serum concentrations suggest the potential utility of using serum concentration as a target in dietary interventions aimed at reducing colon cancer risk.
doi:10.1158/1940-6207.CAPR-13-0019
PMCID: PMC4021591  PMID: 23592741
Mediterranean diet; Healthy People diet; Carotenoids; Fatty Acids; Spearman Correlation
8.  Rectal Mucosal Quantitative Galactose Oxidase-Schiff Reaction as an Early Detection Biomarker for Colorectal Cancer: Comparison to Fecal Occult Stool Blood Test 
The galactose oxidase-Schiff (GOS) reaction detects D-galactose-β-[1,3]-N-acetyl-D-galactosamine. This is a T-antigen expressed in mucus from malignant cells and colonic mucosa adjacent to cancer but not in normal mucosa. Previous studies using a qualitative GOS assay proved to be of limited value for the detection of colorectal neoplasia. We used a newly developed quantitative GOS assay to determine its potential as an early detection biomarker for colorectal cancer. We completed a multi-center, prospective, cross-sectional cohort validation study consisting of 70 normal controls, 23 high-risk normal patients (polyp history or family history of colorectal cancer (CRC) with currently normal colonoscopy), 137 patients with adenomatous polyps, and 69 with colorectal cancers. Prior to colonoscopy, two samples of stool were collected via a rectal exam: one for FOBT, and one for GOS. The area under the ROC curve (AUC) for detecting colonic adenomas and cancer for normal colons, computed with logistic regression was 0.69 for GOS, 0.62 for FOBT, and 0.73 for GOS combined with FOBT. Adding GOS to FOBT did not significantly change the ROC of FOBT alone. GOS does not appear to be a suitable marker of colorectal neoplasia.
doi:10.3233/CBM-2011-0206
PMCID: PMC4016824  PMID: 21896998
Colorectal Neoplasms/diagnosis; Colorectal Neoplasms/prevention & control; Carcinoma/diagnosis; Galactose Oxidase; Predictive Value of Tests
9.  Effects of Ginger Supplementation on Cell Cycle Biomarkers in the Normal-Appearing Colonic Mucosa of Patients at Increased Risk for Colorectal Cancer: Results from a Pilot, Randomized, Controlled Trial 
To estimate the effects of ginger on apoptosis, proliferation, and differentiation in the normal-appearing colonic mucosa, we randomized 20 people at increased risk for colorectal cancer to 2.0 g of ginger or placebo daily for 28 days in a pilot trial. Overall expression and distributions of Bax, Bcl-2, p21, hTERT and MIB-1 (Ki-67) in colorectal crypts in rectal mucosa biopsies were measured using automated immunohistochemistry and quantitative image analysis. Relative to placebo, Bax expression in the ginger group decreased 15.6% (p = 0.78) in the whole crypts, 6.6% (p = 0.95) in the upper 40% (differentiation zone) of crypts, and 21.7% (p = 0.67) in the lower 60% (proliferative zone) of crypts; however, there was a 19% increase (p = 0.14) in Bax expression in the upper 40% relative to the whole crypt. While p21 and Bcl-2 expression remained relatively unchanged, hTERT expression in the whole crypts decreased by 41.2% (p = 0.05); the estimated treatment effect on hTERT expression was larger in the upper 40% of crypts (−47.9%; p = 0.04). In the ginger group, MIB-1 expression decreased in the whole crypts, upper 40% of crypts, and lower 60% of crypts by 16.9% (p = 0.39), 46.8% (p = 0.39), and 15.3% (p = 0.41), respectively. These pilot study results suggest that ginger may reduce proliferation in the normal-appearing colorectal epithelium and increase apoptosis and differentiation relative to proliferation—especially in the differentiation zone of the crypts, and support a larger study to further investigate these results.
doi:10.1158/1940-6207.CAPR-12-0327
PMCID: PMC3618532  PMID: 23303903
Differentiation (p21waf1/cip1); Apoptosis (Bax and Bcl-2); Proliferation (MIB-1/Ki-67 and hTERT); Colorectal Cancer; Ginger
10.  Flaxseed-Derived Enterolactone Is Inversely Associated with Tumor Cell Proliferation in Men with Localized Prostate Cancer 
Journal of Medicinal Food  2013;16(4):357-360.
Abstract
Enterolactone and enterodiol, mammalian lignans derived from dietary sources such as flaxseed, sesame seeds, kale, broccoli, and apricots, may impede tumor proliferation by inhibiting activation of nuclear factor kappa B (NFκB) and vascular endothelial growth factor (VEGF). We examined the associations between urinary enterolactone and enterodiol with prostatic tumor expression of NFκB, VEGF, and Ki67 among 147 patients with prostate cancer who participated in a presurgical trial of flaxseed supplementation (30 g/day) for ∼30 days. Urinary enterolignans and tissue biomarkers were determined by high-performance liquid chromatography and immunohistochemistry, respectively. After supplementation, we observed significant correlations between intakes of plant lignan and urinary concentrations of total enterolignans (ρ=0.677, P<.0001), enterolactone (ρ=0.676, P<.0001), and enterodiol (ρ=0.628, P<.0001). Importantly, we observed that total urinary enterolignans and enterolactone were significantly and inversely correlated with Ki67 in the tumor tissue (ρ=−0.217, P=.011, and ρ=−0.230, P=.007, respectively), and a near-significant inverse association was observed for enterodiol (ρ=−0.159, P=.064). An inverse association was observed between enterolactone and VEGF (ρ=−0.143, P=.141), although this did not reach statistical significance. We did not observe an association between enterolignans and NFκB. In conclusion, flaxseed-derived enterolignans may hinder cancer cell proliferation via VEGF-associated pathways.
doi:10.1089/jmf.2012.0159
PMCID: PMC3624628  PMID: 23566060
diet; flaxseed; lignans; phytoestrogens; prostatic neoplasia
11.  A Mediterranean Dietary Intervention in Persons at High Risk of Colon Cancer: Recruitment and Retention to an Intensive Study Requiring Biopsies 
Contemporary clinical trials  2012;33(5):881-888.
This study recruited persons at increased risk of colon cancer to an intensive dietary intervention study that required biopsies of the colon by flexible sigmoidoscopy at baseline and after six months of intervention. A total of 1314 individuals contacted the study, and only 16 individuals indicated that the sigmoidoscopy procedure was an obstacle to study participation. A total of 270 individuals completed a screening visit and signed a screening consent form. Inquiries about the study tended to be fewer in the winter and late summer. Failure to return food records was the most common reason for exclusion. Dietary recall at enrollment indicated that subjects were consuming significantly more vegetables, lower sodium and a lower glycemic load on the day before starting the study versus during the eligibility phase which might have an impact on biomarker measures. This makes it important to capture dietary changes in the period between determination of eligibility and enrollment. Subjects (n=120) were randomized to follow a Healthy Eating or a Mediterranean Diet, each of which required substantial dietary record-keeping. The study completion rate was 78%, and subjects reported high satisfaction with study participation. Of the 93 individuals who completed the study, only one refused the flexible sigmoidoscopy at the final visit. These findings suggest that flexible sigmoidoscopy does not appear to be a barrier for recruitment of high-risk individuals to an intensive dietary intervention trial, but that completing food records can be.
doi:10.1016/j.cct.2012.05.006
PMCID: PMC3408796  PMID: 22640923
colon biopsy; recruitment; dietary intervention; cancer prevention
12.  Effect of ginger root on cyclooxygenase-1 and 15-hydroxyprostaglandin dehydrogenase expression in colonic mucosa of humans at normal and increased risk of colorectal cancer 
Objectives
Elevated tissue levels of prostaglandin E2 (PGE2), produced by cyclooxygenase (COX) are an early event in colorectal cancer (CRC). Data suggest the efficacy of non-steroidal anti-inflammatory (NSAIDs) drugs, which inhibit COX activity, as cancer preventives; however, side effects of NSAIDs indicate unacceptable limitations. Ginger has been reported to have anti-inflammatory activities with significant CRC preventive potential. We investigated if consumption of 2.0 g ginger daily regulated the level of two key enzymes, which control PGE2 production, COX-1 and NAD+- dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH).
Methods
Thirty participants at normal and twenty participants at increased risk of CRC were randomized and given 2.0 g/day ginger or placebo for 28 days. Flexible sigmoidoscopy was used to obtain colon biopsies at baseline and end of the study. Tissue level of COX-1 and 15-PGDH were assessed using Western Blotting.
Results
After ginger consumption participants at increased risk of CRC, had significantly reduced colonic COX-1 protein level (23.8%± 41) compared to the placebo group (18.9%± 52; p=0.03). Protein levels of 15-PGDH in the colon were unchanged. In normal risk for CRC participants, neither protein levels of COX-1 nor 15-PGDH in the colon were altered by ginger consumption.
Conclusion
Ginger significantly lowered COX-1 protein expression in increased risk participants, but not in normal risk participants at normal for CRC. Ginger did not alter 15-PGDH protein expression in either increased or normal risk participants. Further investigation, in larger studies with a longer ginger intervention is needed to examine the ability of ginger to impact tissue level of prostaglandin.
doi:10.1097/CEJ.0b013e32835c829b
PMCID: PMC3720765  PMID: 23222413
ginger root extract; cyclooxygenase; 15-hydroxyprostaglandin dehydrogenase; colon cancer risk; cancer risk reduction
13.  EFFECT OF CYLOOXYGENASE GENOTYPE AND DIETARY FISH OIL ON COLONIC EICOSANOIDS IN MICE 
Dietary ω3 fatty acids can modulate substrate availability for cyclooxygenases and lipoxygenases, thus modulating downstream eicosanoid formation. This could be an alternative approach to using NSAIDs and other COX inhibitors for limiting PGE2 synthesis in colon cancer prevention. The aims of this study were to evaluate to what extent cyclooxygenase- and lipoxygenase-derived products could be modulated by dietary fish oil in normal colonic mucosa, and to evaluate the role of COX-1 and COX-2 in formation of these products. Mice (wild-type, COX-1 null, or COX-2 null) were fed a diet supplying a broad mixture of fatty acids present in European/American diets, supplemented with either olive oil (oleate control diet) or menhaden (fish) oil ad libitum for 9–11 wk. Colonic eicosanoid levels were measured by LC-MS/MS, and proliferation was assessed by Ki67 immunohistochemistry. Dietary alteration of colonic arachidonic acid: eicosapentaenoic ratios resulted in large shifts in formation of cyclooxygenase and lipoxygenase metabolites. COX-1 knockout virtually abolished PGE2 formation but interestingly 12-HETE and 15-HETE formation was increased. The large changes in eicosanoid profiles were accompanied by relatively small changes in colonic crypt proliferation, but such changes in eicosanoid formation might have greater biological impact upon carcinogen challenge. These results indicate that in normal colon, inhibition of COX-2 would have little effect on reducing PGE2 levels.
doi:10.1016/j.jnutbio.2011.05.003
PMCID: PMC3246564  PMID: 21937210
colon cancer; fish oil; cyclooxygenase; prostaglandin E2; hydroxyeicosatetraenoic acids; EPA
14.  Characterization of vitamin D receptor (VDR) in lung adenocarcinoma 
Purpose
The anti-proliferative effects of 1α,25-dihydroxyvitamin D3 (1,25-D3, calcitriol, the active form of vitamin D) are mediated by the nuclear vitamin D receptor (VDR). In the present study, we characterized VDR expression in lung adenocarcinoma (AC).
Experimental Design
We examined VDR mRNA expression using a quantitative real-time PCR (qRT-PCR) in 100 patients who underwent surgery for lung AC. In a subset of these patients (n = 89), we examined VDR protein expression using immunohistochemistry. We also examined the association of VDR protein expression with circulating serum levels of 25-hydroxyvitamin D3 (25-D3) and 1,25-D3. The antiproliferative effects and cell cycle arrest of 1,25-D3 were examined using lung cancer cell lines with high (SKLU-1) as well as low (A549) expression of VDR mRNA.
Results
Higher VDR expression correlates with longer survival after adjusting for age, sex, disease stage and tumor grade (HR 0.73, 95% CI 0.58–0.91). In addition, there was a positive correlation (r = 0.38) between serum 1,25-D3 and tumor VDR protein expression. A greater anti-proliferative effect of 1,25-D3 was observed in high compared to low VDR-expressing cell lines; these effects corresponded to G1 cell cycle arrest; this was associated with a decline in cyclin D1, S-phase kinase protein 2 (Skp2), retinoblastoma (Rb) and minichromosome maintenance 2 (MCM2) proteins involved in S-phase entry.
Conclusions
Increased VDR expression in lung AC is associated with improved survival. This may relate to a lower proliferative status and G1 arrest in high VDR-expressing tumors.
doi:10.1016/j.lungcan.2012.04.010
PMCID: PMC3396768  PMID: 22564539
VDR; Vitamin D; 1,25-D3; Lung Adenocarcinoma; Survival
15.  Increased plasma levels of the APC-interacting protein MAPRE1, LRG1 and IGFBP2 preceding a diagnosis of colorectal cancer in women 
Longitudinal blood collections from cohort studies provide the means to search for proteins associated with disease prior to clinical diagnosis. We investigated plasma samples from the Women’s Health Initiative (WHI) cohort to determine quantitative differences in plasma proteins between subjects subsequently diagnosed with colorectal cancer (CRC) and matched controls that remained cancer free during the period of follow-up. Proteomic analysis of WHI samples collected prior to diagnosis of CRC resulted in the identification of six proteins with significantly (p <0.05) elevated concentrations in cases compared to controls. Proteomic analysis of two colorectal cancer cell lines showed 5 of the 6 proteins were produced by cancer cells. MAPRE1, IGFBP2, LRG1 and CEA were individually assayed by enzyme linked immunosorbent assay (ELISA) in 58 pairs of newly diagnosed CRC samples and controls and yielded significant elevations (p <0.05) among cases relative to controls. A combination of these four markers resulted in an ROC with an AUC=0.841 and 57% sensitivity at 95% specificity. This combination rule was tested in an independent set of WHI samples collected within 7 months prior to diagnosis from cases and matched controls resulting in 41% sensitivity at 95% specificity. A panel consisting of CEA, MAPRE1, IGFBP2 and LRG1 has predictive value in pre-diagnostic colorectal cancer plasmas.
doi:10.1158/1940-6207.CAPR-11-0412
PMCID: PMC3419141  PMID: 22277732
colorectal cancer; risk markers; Pre-Diagnostic samples
16.  Microsatellite Instability and DNA Mismatch Repair Protein Deficiency in Lynch Syndrome Colorectal Polyps 
Colorectal cancers associated with Lynch syndrome (LS) are characterized by deficient DNA mismatch repair (MMR) function. Our aim was to evaluate the prevalence of microsatellite instability (MSI) and loss of MMR protein expression in LS-associated polyps. Sixty two colorectal polyps – 37 adenomas (APs), 23 hyperplastic polyps (HPs), and 2 sessile serrated polyps (SSPs) – from 34 subjects with germline MMR gene mutations were tested for MSI using a single pentaplex PCR for five mononucleotide repeat microsatellite markers, and also for expression of MLH1, MSH2, MSH6, and PMS2 proteins by immunohistochemistry (IHC). High-level MSI (MSI-H) was seen in 15/37 (41%) APs, 1/23 (4%) HPs, and 1/2 (50%) SSPs. Loss of MMR protein expression was seen in 18/36 (50%) APs, 0/21 HPs, and 0/2 SSPs. APs ≥8 mm were significantly more likely to demonstrate MSI-H (OR = 9.98, 95% CI: 1.52-65.65, p = 0.02) and deficient MMR protein expression (OR = 3.17, 95% CI: 1.20-8.37, p = 0.02) compared with those <8 mm. All (6/6) APs ≥10 mm demonstrated both MSI-H and loss of MMR protein expression by IHC. Our finding that the prevalence of MMR deficiency increases with the size of APs suggests that loss of MMR function is a late event in LS-associated colorectal neoplasia. Although testing large APs may be of value in the diagnostic evaluation of patients with suspected LS, the absence of an MMR deficient phenotype in an adenoma cannot be considered strong evidence against LS, as it is with colorectal carcinomas.
doi:10.1158/1940-6207.CAPR-11-0519
PMCID: PMC3461594  PMID: 22262812
Lynch Syndrome; adenomas; microsatellite instability
17.  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 cyclooxygenase (COX) indicate that up-regulation of inflammatory eicosanoids produced by COX, and in particular prostaglandin E2 (PGE2), are early events in the development of colorectal cancer (CRC). Ginger has demonstrated down regulation of COX in vitro and decreased incidence/ multiplicity of adenomas in rats. This study was conducted to determine if 2.0 g/day of ginger could decrease the levels of PGE2, 13-hydroxy-octadecadienoic acids (13-HODE), and 5-, 12-, & 15-hydroxyeicosatetraenoic acid (5-, 12-, & 15-HETE), in the colon mucosa of healthy volunteers. To investigate this aim we randomized 30 subjects to 2.0 g/day 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 (AE) (p=0.55). Based on these results, it appears that Ginger has the potential to decrease eicosanoid levels, perhaps by inhibiting their synthesis from arachidonic acid. Ginger also appeared to be tolerable and safe. Further investigation in people at high risk for CRC seems warranted.
doi:10.1158/1940-6207.CAPR-11-0224
PMCID: PMC3208778  PMID: 21990307
Cancer Risk Reductive; Eicosanoids; Colorectal Cancer; Inflammation; and Ginger
18.  EFFECT OF FISH OIL ON LEVELS OF R- AND S-ENANTIOMERS OF 5-, 12- AND 15-HYDROXYEICOSATETRAENOIC ACIDS IN MOUSE COLONIC MUCOSA 
Nutrition and cancer  2011;64(1):163-172.
The balance of putative pro- and anti-inflammatory lipoxygenase (LOX)-derived S-hydroxyeicosatetraenoic acids (S-HETEs) in colon mucosa is a potential target for modulating colon cancer risk and progression. The biological effects of S-HETEs and R-HETEs (produced by distinct pathways) may differ, but levels of these compounds in colon are unknown. The objective of this study was to develop chiral methods to characterize HETE enantiomers in colonic mucosa and evaluate the effects of fish oil on HETE formation. C57BL/6 mice (COX-1 null, COX-2 null, wild-type) were fed a diet supplemented with either olive oil or menhaden oil for 11 weeks, and R/S-HETEs in colonic mucosa were quantified by chiral LC-MS/MS. The R-enantiomer comprised 60-72% of 5-HETE, 18-58% of 15-HETE and 1-16% of 12-HETE in colonic mucosa, suggesting that non-LOX sources contribute to HETE profiles. Fish oil reduced levels of both R- and S-HETEs, and increased the preponderance of the R-enantiomers (particularly 12- and 15-HETEs). There was apparent shunting of arachidonic acid to12/15-LOX in the COX-1 null animals. This is the first report of the enantiomeric composition of HETEs in the colon in vivo.
doi:10.1080/01635581.2012.630168
PMCID: PMC3410550  PMID: 22149144
experimental; lipoxygenase; hydroxyeicosatetraenoic acid; enantiomer; colon
20.  Regulatory Approval of Cancer Risk-reducing (Chemopreventive) Drugs: Moving What We Have Learned into the Clinic 
This paper endeavors to clarify the current requirements and status of regulatory approval for chemoprevention (risk reduction) drugs and discusses possible improvements to the regulatory pathway for chemoprevention. Covering a wide range of topics in as much depth as space allows, this report is written in a style to facilitate the understanding of non-scientists and to serve as a framework for informing the directions of experts engaged more deeply with this issue. Key topics we cover here are as follows: a history of definitive cancer chemoprevention trials and their influence on the evolution of regulatory assessments; a brief review of the long-standing success of pharmacologic risk reduction of cardiovascular diseases and its relevance to approval for cancer risk reduction drugs; the use and limitations of biomarkers for developing and the approval of cancer risk reduction drugs; the identification of individuals at a high(er) risk for cancer and who are appropriate candidates for risk reduction drugs; business models that should incentivize pharmaceutical-industry investment in cancer risk reduction; a summary of scientific and institutional barriers to development of cancer risk reduction drugs; and a summary of major recommendations that should help facilitate the pathway to regulatory approval for pharmacologic cancer risk reduction drugs.
doi:10.1158/1940-6207.CAPR-09-0014
PMCID: PMC3059243  PMID: 21372031
21.  CYP24A1 Is an Independent Prognostic Marker of Survival in Patients with Lung Adenocarcinoma 
Purpose
The active form of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25-D3) exerts antiproliferative effects in cancers, including lung adenocarcinoma (AC). CYP24A1 is overexpressed in many cancers and catabolizes 1,25-D3. The purpose of our study was to assess CYP24A1 as a prognostic marker and to study its relevance to antiproliferative activity of 1,25-D3 in lung AC cells.
Experimental Design
Tumors and corresponding normal specimens from 86 patients with lung AC (stages I–III) were available. AffymetrixR array data and subsequent confirmation by quantitative real time-PCR were used to determine CYP24A1 mRNA expression. A subsequent validation set of 101 lung AC was used to confirm CYP24A1 mRNA expression and its associations with clinical variables. The antiproliferative effects of 1,25-D3 were examined using lung cancer cell lines with high as well as low expression of CYP24A1 mRNA.
Results
CYP24A1 mRNA was elevated 8–50 fold in lung AC (compared to normal nonneoplastic lung) and significantly higher in poorly-differentiated cancers. At 5 years of follow-up, the probability of survival was 42% (high CYP24A1, n = 29) versus 81% (low CYP24A1, n = 57) (P = 0.007). The validation set of 101 tumors showed that CYP24A1 was independently prognostic of survival (multivariate Cox model adjusted for age, gender and stage, P = 0.001). A549 cells (high CYP24A1) were more resistant to antiproliferative effects of 1,25-D3 compared with SKLU-1 cells (low CYP24A1).
Conclusions
CYP24A1 overexpression is associated with poorer survival in lung AC. This may relate to abrogation of antiproliferative effects of 1,25-D3 in high CYP24A1 expressing lung AC.
doi:10.1158/1078-0432.CCR-10-1789
PMCID: PMC3058389  PMID: 21169243
22.  Enhanced Discrimination of Malignant from Benign Pancreatic Disease by Measuring the CA 19-9 Antigen on Specific Protein Carriers 
PLoS ONE  2011;6(12):e29180.
The CA 19-9 assay detects a carbohydrate antigen on multiple protein carriers, some of which may be preferential carriers of the antigen in cancer. We tested the hypothesis that the measurement of the CA 19-9 antigen on individual proteins could improve performance over the standard CA 19-9 assay. We used antibody arrays to measure the levels of the CA 19-9 antigen on multiple proteins in serum or plasma samples from patients with pancreatic adenocarcinoma or pancreatitis. Sample sets from three different institutions were examined, comprising 531 individual samples. The measurement of the CA 19-9 antigen on any individual protein did not improve upon the performance of the standard CA 19-9 assay (82% sensitivity at 75% specificity for early-stage cancer), owing to diversity among patients in their CA 19-9 protein carriers. However, a subset of cancer patients with no elevation in the standard CA 19-9 assay showed elevations of the CA 19-9 antigen specifically on the proteins MUC5AC or MUC16 in all sample sets. By combining measurements of the standard CA 19-9 assay with detection of CA 19-9 on MUC5AC and MUC16, the sensitivity of cancer detection was improved relative to CA 19-9 alone in each sample set, achieving 67–80% sensitivity at 98% specificity. This finding demonstrates the value of measuring glycans on specific proteins for improving biomarker performance. Diagnostic tests with improved sensitivity for detecting pancreatic cancer could have important applications for improving the treatment and management of patients suffering from this disease.
doi:10.1371/journal.pone.0029180
PMCID: PMC3248411  PMID: 22220206
23.  Targeting Breast Stem Cells with the Cancer Preventive Compounds Curcumin and Piperine 
Background
The cancer stem cell hypothesis asserts that malignancies arise in tissue stem and/or progenitor cells through the dysregulation or acquisition of self-renewal. In order to determine whether the dietary polyphenols, curcumin and piperine, are able to modulate the self-renewal of normal and malignant breast stem cells, we examined the effects of these compounds on mammosphere formation, on expression of the breast stem cell marker aldehyde dehydrogenase (ALDH), and on Wnt signaling.
Design
Mammosphere formation assays were performed after curcumin, piperine and control treatment in unsorted normal breast epithelial cells and normal stem and early progenitor cells, selected by ALDH positivity. Wnt signaling was examined using a Topflash assay.
Results
Both curcumin and piperine inhibited mammosphere formation, serial passaging and percent of ALDH+ cells, by 50% at 5 μM and completely at 10 μM concentration in normal and malignant breast cells. There was no effect on cellular differentiation. Wnt signaling was inhibited by both curcumin and piperine by 50% at 5 μM and completely at 10μM.
Conclusion
Curcumin and piperine separately, and in combination, inhibit breast stem cell self renewal but do not cause toxicity to differentiated cells. These compounds could be potential cancer preventive agents. Mammosphere formation assays may be a quantifiable biomarker to assess cancer preventive agent efficacy and Wnt signaling assessment a mechanistic biomarker for use in human clinical trials.
doi:10.1007/s10549-009-0612-x
PMCID: PMC3039120  PMID: 19898931
24.  Repeat Dose Study of the Cancer Chemopreventive Agent Resveratrol in Healthy Volunteers: Safety, Pharmacokinetics and Effect on the Insulin-like Growth Factor Axis 
Cancer research  2010;70(22):9003-9011.
Resveratrol, a naturally occurring polyphenol, has cancer chemopreventive properties in preclinical models. It has been shown to downregulate levels of insulin-like growth factor-1 (IGF-1) in rodents. The purpose of the study was to assess its safety, pharmacokinetics and effects on circulating levels of IGF-1 and insulin-like growth factor binding protein-3 (IGFBP-3) after repeated dosing. Forty healthy volunteers ingested resveratrol at 0.5, 1.0, 2.5 or 5.0g daily for 29 days. Levels of resveratrol and its metabolites were measured by HPLC-UV in plasma obtained before and up to 24h after a dose between days 21 and 28. IGF-1 and IGFBP-3 were measured by enzyme-linked immunosorbent assay in plasma taken pre-dosing and on day 29. Resveratrol was safe, but the 2.5 and 5g doses caused mild to moderate gastrointestinal symptoms. Resveratrol-3-O-sulfate, resveratrol-4′-O-glucuronide and resveratrol-3-O-glucuronide were major plasma metabolites. Maximal plasma levels and areas under the concentration versus time curve (AUC) for the metabolites dramatically exceeded those for resveratrol, in the case of the AUC by up to 20.3-fold. Ingestion of resveratrol caused a decrease in circulating IGF-1 and IGFBP-3 (P<0.04 for both), respectively, compared to pre-dosing values, in all volunteers. At the 2.5g dose level the decrease was most marked. The results suggest that repeated administration of high doses of resveratrol generates micromolar concentrations of parent and much higher levels of glucuronide and sulfate conjugates in the plasma. The observed decrease in circulating IGF-1 and IGFBP-3 may contribute to cancer chemopreventive activity.
doi:10.1158/0008-5472.CAN-10-2364
PMCID: PMC2982884  PMID: 20935227
Resveratrol; chemoprevention; pharmacokinetics; pharmacodynamics
25.  Repeat Dose Study of the Cancer Chemopreventive Agent Resveratrol in Healthy Volunteers: Safety, Pharmacokinetics and Effect on the Insulin-like Growth Factor Axis 
Cancer research  2010;70(22):9003-9011.
Resveratrol, a naturally occurring polyphenol, has cancer chemopreventive properties in preclinical models. It has been shown to downregulate levels of insulin-like growth factor-1 (IGF-1) in rodents. The purpose of the study was to assess its safety, pharmacokinetics and effects on circulating levels of IGF-1 and insulin-like growth factor binding protein-3 (IGFBP-3) after repeated dosing. Forty healthy volunteers ingested resveratrol at 0.5, 1.0, 2.5 or 5.0g daily for 29 days. Levels of resveratrol and its metabolites were measured by HPLC-UV in plasma obtained before and up to 24h after a dose between days 21 and 28. IGF-1 and IGFBP-3 were measured by enzyme-linked immunosorbent assay in plasma taken pre-dosing and on day 29. Resveratrol was safe, but the 2.5 and 5g doses caused mild to moderate gastrointestinal symptoms. Resveratrol-3-O-sulfate, resveratrol-4′-O-glucuronide and resveratrol-3-O-glucuronide were major plasma metabolites. Maximal plasma levels and areas under the concentration versus time curve (AUC) for the metabolites dramatically exceeded those for resveratrol, in the case of the AUC by up to 20.3-fold. Ingestion of resveratrol caused a decrease in circulating IGF-1 and IGFBP-3 (P<0.04 for both), respectively, compared to pre-dosing values, in all volunteers. At the 2.5g dose level the decrease was most marked. The results suggest that repeated administration of high doses of resveratrol generates micromolar concentrations of parent and much higher levels of glucuronide and sulfate conjugates in the plasma. The observed decrease in circulating IGF-1 and IGFBP-3 may contribute to cancer chemopreventive activity.
doi:10.1158/0008-5472.CAN-10-2364
PMCID: PMC2982884  PMID: 20935227
Resveratrol; chemoprevention; pharmacokinetics; pharmacodynamics

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