Brian J. Doyle

Aberrant DNA methylation in hereditary nonpolyposis colorectal cancer without mismatch repair deficiency.

BACKGROUND & AIMS Approximately half of the families that fulfill Amsterdam criteria for Lynch syndrome or hereditary nonpolyposis colorectal cancer (HNPCC) do not have evidence of the germline mismatch repair gene mutations that define this syndrome and result in microsatellite instability (MSI). The carcinogenic pathways and the best diagnostic approaches to detect microsatellite stable (MSS) HNPCC tumors are unclear. We investigated the contribution of epigenetic alterations to the development of MSS HNPCC tumors. METHODS Colorectal cancers were divided into 4 groups: (1) microsatellite stable, Amsterdam-positive (MSS HNPCC) (N = 22); (2) Lynch syndrome cancers (identified mismatch repair mutations) (N = 21); (3) sporadic MSS (N = 92); and (4) sporadic MSI (N = 46). Methylation status was evaluated for CACNAG1, SOCS1, RUNX3, NEUROG1, MLH1, and long interspersed nucleotide element-1 (LINE-1). KRAS and BRAF mutation status was analyzed. RESULTS MSS HNPCC tumors displayed a significantly lower degree of LINE-1 methylation, a marker for global methylation, than any other group. Although most MSS HNPCC tumors had some degree of CpG island methylation, none presented a high index of methylation. MSS HNPCC tumors had KRAS mutations exclusively in codon 12, but none harbored V600E BRAF mutations. CONCLUSIONS Tumors from Amsterdam-positive patients without mismatch repair deficiency (MSS HNPCC) have certain molecular features, including global hypomethylation, that distinguish them from all other colorectal cancers. These characteristics could have an important impact on tumor behavior or treatment response. Studies are underway to further assess the cause and effects of these features.

Susceptibility Genetic Variants Associated With Colorectal Cancer Risk Correlate With Cancer Phenotype

BACKGROUND & AIMS Ten common low-penetrant genetic variants have been consistently associated with colorectal cancer (CRC) risk; little is known about the correlation between these variants and CRC phenotype. Characterization of such a correlation would improve CRC management and prevention programs. We assessed the association between these genetic variants and CRC phenotype in patients and modeled pairwise combinations to detect epistasis. METHODS The validation population corresponded to a prospective, multicenter, population-based cohort (EPICOLON I) of 1096 patients with newly diagnosed CRC. The replication set was an independent, prospective, multicenter Spanish cohort (EPICOLON II) of 895 patients with newly diagnosed CRC. For individual single nucleotide polymorphism (SNP) association analyses, a multivariate method using logistic regression was applied in EPICOLON I and subsequently prospectively validated in EPICOLON II. Interactions between SNPs were assessed using the likelihood ratio test. RESULTS Validated results confirmed that the C allele on 8q23.3 (rs16892766) was significantly associated with advanced-stage tumors (odds ratio [OR], 1.48; 95% confidence interval [CI], 1.15-1.90; P value = 4.9 x 10(-3)). The G allele on 8q24.21 (rs6983267) was more common in patients with a familial history of CRC (OR, 2.02; 95% CI, 1.35-3.03; P value = 3.9 x 10(-4)). The combination of rs6983267 on 8q24.21 and rs9929218 on 16q22.2 was associated with a history of colorectal adenoma (carriers of GG and AA, respectively; OR, 2.28; 95% CI, 1.32-3.93; P = 5.0 x 10(-4)). CONCLUSIONS CRC susceptibility variants at 8q23.3, 8q24.21, and 16q22.2 appear to be associated with cancer phenotype. These findings might be used to develop screening and surveillance strategies.

Effects of cranberry extracts and ursolic acid derivatives on P-fimbriated Escherichia coli, COX-2 activity, pro-inflammatory cytokine release and the NF-κβ transcriptional response in vitro

Cranberry, the fresh or dried ripe fruit of Vaccinium macrocarpon Ait. (Ericaceae), is currently used as adjunct therapy for the prevention and symptomatic treatment of urinary tract infections. Data from clinical trials suggest that extracts of cranberry or cranberry juice reduce the bacterial load of E. coli and also suppress the inflammatory symptoms induced by E. coli infections. A methanol extract prepared from 10 kg of dehydrated cranberries did not directly inhibit the growth of E coli strains ATCC 700336 or ATCC 25922 in concentrations up to 256 μg/mL in vitro. However, the methanol extract (CR-ME) inhibited the activity of cyclooxygenase-2, with an IC50 of 12.8 μg/mL. Moreover, CR-ME also inhibited the NF-κβ transcriptional activation in human T lymphocytes with an IC50 of 19.4 μg/mL, and significantly (p < 0.01) inhibited the release of interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor-α from E. coli lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells in vitro, at a concentration of 50 μg/mL. The extract had no effect on inducible nitric oxide synthase activity in the murine macrophage cell line RAW 264.7. The compounds responsible for this activity were identified using a novel LC-MS based assay as ursolic acid and ursolic acid derivatives. Taken together, these data suggest CR-ME and its constituent chemical compounds target specific pathways involved in E. coli-induced inflammation.

Natural Products As Antibacterial Agents

Abstract For thousands of years medicinal plants have played a significant role in the treatment of a wide range of medical conditions, including infectious diseases. Some naturally occurring chemical compounds serve as models for a large percentage clinically proven drugs, and many are now being re-assessed as antimicrobial agents. The primary reason for this renaissance is the fact that infectious disease remains a significant cause of morbidity and mortality worldwide, accounting for approximately 50% of all deaths in tropical countries and as much as 20% of deaths in the Americas. Despite the significant progress made in microbiology and the control of microorganisms, sporadic incidents of epidemics due to drug resistant microorganisms and previously unknown disease-causing microbes pose an enormous threat to global public health. These negative health trends call for a global initiative for the development of new strategies for the prevention and treatment of infectious disease, including natural products. Literally thousands of plant species have been tested against hundreds of bacterial strains in vitro, and many medicinal plants are active against a wide range of gram-positive and gram-negative bacteria. However, very few of these medicinal plant extracts have been tested in animal or human studies to determine safety and efficacy. This review focuses on the medicinal plants and phytochemical for which there is significant published in vitro, in vivo and clinical data available. The examples provided in this review such as St. Johns wort, tree tea oil, and green tea demonstrate that even commonly used plant extracts may offer prospective new treatment of bacterial infections, including multi-drug resistant bacteria. One interesting example is St. Johns wort (Hypericum perforatum) and its active constituent hyperforin, both of which have significant activity against MRSA in microgram concentrations. This antibacterial discovery was based on the ethnomedical use of St. Johns wort to treat skin infections and wounds. Review of the published data indicates that medicinal plants offer significant potential for the development of novel antibacterial therapies and adjunct treatments (i.e. MDR pump inhibitors). However, new investigations should employ modern methodology, including using nationally recognized protocols and standards for microbial testing, the generation of minimum inhibitory concentrations, as well as standardization of the quality of plant materials used for testing.

Analysis of the Oxidative Damage Repair Genes NUDT1, OGG1, and MUTYH in Patients from Mismatch Repair Proficient HNPCC Families (MSS-HNPCC)

Purpose: Several studies have described molecular differences between microsatellite stable hereditary nonpolyposis colorectal cancer (MSS-HNPCC) and microsatellite unstable Lynch syndrome tumors (MSI-HNPCC). These differences highlight the possibility that other instability forms could explain cancer susceptibility in this group of families. The base excision repair (BER) pathway is the major DNA repair pathway for oxidative DNA damage. A defect in this pathway can result in DNA transversion mutations and a subsequent increased cancer risk. Mutations in MUTYH have been associated with increased colorectal cancer (CRC) risk while no association has been described for OGG1 or NUDT1. Experimental Design: We performed mutational screening of the three genes involved in defense against oxidative DNA damage in a set of 42 MSS-HNPCC families. Results: Eight rare variants and 5 frequent variants were found in MSS-HNPCC patients. All variants were previously described by other authors except variant c.285C>T in OGG1. Segregation studies were done and in silico programs were used to estimate the level of amino acid conservation, protein damage prediction, and possible splicing alterations. Variants OGG1 c.137G>A; MUTYH c.1187G>A were detected in Amsterdam I families and cosegregate with cancer. Analysis of OGG1 c.137G>A transcripts showed an inactivation of the splicing donor of exon 1. Conclusions: Two rare variants (OGG1 c.137G>A; MUTYH c.1187G>A) and one common polymorphism (NUDT1 c.426C>T) were associated with CRC risk. We show that the BER pathway can play a significant role in a number of MSS-HNPCC colorectal cancers. More studies could be of interest in order to gain further understanding of yet unexplained CRC susceptibility cases. Clin Cancer Res; 17(7); 1701–12. ©2011 AACR.

Estrogenic and Progestagenic effects of extracts of Justicia pectoralis Jacq., an herbal medicine from Costa Rica used for the treatment of Menopause and PMS

OBJECTIVES To investigate the biological activities of Justicia pectoralis Jacq. (Acanthaceae), an herbal medicine used in Costa Rica (CR) for the management of menopausal symptoms and dysmenorrhea. STUDY DESIGN The aerial parts of J. pectoralis were collected, dried and extracted in methanol. To establish possible mechanisms of action of JP for the treatment of menopausal symptoms, the estrogenic and progesterone agonists, and antiinflammatory activities were investigated. MAIN OUTCOME MEASURES The methanol extract (JP-M) was tested in ER and PR binding assays, a COX-2 enzyme inhibition assay, the ERbeta-CALUX assay in U2-OS cells, as well as reporter and endogenous gene assays in MCF-7 K1 cells. RESULTS The JP-M extract inhibited COX-2 catalytic activity (IC(50) 4.8 microg/mL); bound to both ERalpha and ERbeta (IC(50) 50 microg/mL and 23.1 microg/mL, respectively); induced estrogen-dependent transcription in the ERbeta-CALUX; and bound to the progesterone receptor (IC(50) 22.8 microg/mL). The extract also modulated the expression of endogenous estrogen responsive genes pS2, PR, and PTGES in MCF-7 cells at a concentration of 20 microg/mL. Activation of a 2 ERE-construct in transiently transfected MCF-7 cells by the extract was inhibited by the estrogen receptor antagonist ICI 182,780, indicating that the effects were mediated through the estrogen receptor. Finally, the extract weakly enhanced the proliferation of MCF-7 cells, however this was not statistically significant as compared with DMSO controls. CONCLUSIONS Extracts of J. pectoralis have estrogenic, progestagenic and anti-inflammatory effects, and thus have a plausible mechanism of action, explaining its traditional use for menopause and PMS.

Quartz crystal microbalance study of bovine serum albumin adsorption onto self-assembled monolayer-functionalized gold with subsequent ligand binding

Adsorption characteristics of the model protein bovine serum albumin (BSA) onto gold surfaces were examined using a 5 MHz quartz crystal microbalance. Protein immobilization was executed in the presence and absence of a homogenous self-assembled monolayer (SAM) of NHS-terminated alkanethiols. BSA concentrations in the range of 3.2 × 10(-6) to 1.0 × 10(-3)mol/L were found to saturate both SAM-functionalized and non-functionalized surfaces with similar densities of 450 ± 26 ng/cm(2). The lack of functionalization dependence is attributed to the large protein size relative to the density of available binding sites in either surface condition. The BSA ligand 8-anilino-1-naphthalenesulfonic acid (ANS) was subsequently introduced to the immobilized BSA to determine any effects of the protein immobilization conditions on ligand binding. The rate of ANS binding to BSA was found to increase with increasing BSA concentration used in the immobilization step. This suggests that protein concentration affects morphology and ligand binding affinity without significantly altering adsorption quantity.

Black cohosh (Actaea racemosa) for the mitigation of menopausal symptoms: recent developments in clinical safety and efficacy

The purpose of this article is to assess recent data supporting the safety and efficacy of black cohosh products for the mitigation of menopause-related symptoms. Searches of the published literature in Napralert, Cochrane Library and PubMed databases were performed from 2003 to 2006. Information from drug regulatory agencies from five different countries was obtained to evaluate safety. While there are a few contradictory studies, the majority of the clinical trials indicate that extracts of black cohosh (Actaea racemosa L.) improve menopause-related symptoms. However, to date, at least 50 cases of possible hepatotoxicity have been reported. Although previous safety reviews suggest that black cohosh is well tolerated, the increasing numbers of these case reports indicates that further preclinical toxicological evaluations of black cohosh are urgently needed. At this time, it appears prudent to advise menopausal women with underlying liver disease, autoimmune diseases or those taking medications that may impact liver function not to use products containing black cohosh.

Menopause in Latin America: Symptoms, attitudes, treatments and future directions in Costa Rica

Similar to their US counterparts, Costa Rican women enter menopause at ∼50 years of age, have similar symptoms, including hot flashes and night sweats, as well as an overall negative attitude toward the menopausal transition. One study of rural women in Monteverde reported that women knew little about the menopausal transition, as the subject was not discussed. Similar to other Latin American women, the use of hormone therapy by Costa Rican women is low and instead they use alternative therapies, including massage, dietary changes and herbal medicines. A wide variety of herbal therapies are used, and some of these herbs have estrogenic activities in vitro. However, clinical data on the safety and efficacy of any of these treatments is lacking. Recently, a disturbing increase in the incidence of human papilloma virus infections in menopausal women has been reported, due in part to more sexual freedom after menopause. Fortunately, the strain of HPV infecting these women is not associated with cervical cancer. Overall, there is a significant lack of scientific and medical research on menopausal women in Costa Rica. Considering the aging population, the high use of herbal medicines by menopausal women and the lack of clinical studies on these treatments, future research should focus on gaining a better understanding of menopause in this population. Furthermore, new educational programs for these women and the health professionals who serve them are necessary, as well as investigations of the safety and efficacy of the herbal supplements women use to manage their menopausal symptoms.

Menopause, A Universal Female Experience: Lessons from Mexico and Central America

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