Characterization of the Metabolome of Breast Tissues from Non-Hispanic Black and Non-Hispanic White Women Reveals Correlations between Microbial Dysbiosis and Enhanced Lipid Metabolism Pathways in Triple-Negative Breast Tumors.

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer that is non-responsive to hormonal therapies and disproportionately impact women of African ancestry. We previously showed that TN breast tumors have a distinct microbial signature that differs from less aggressive breast tumor subtypes and normal breast tissues. However, it is unknown whether these differences in breast tumor microbiota may be driven by alterations in microbial metabolites, leading to potentially protective or pathogenic consequences. The goal of this global metabolomic profiling study was to investigate alterations in microbial metabolism pathways in normal and breast tumor tissues, including TNBC, of non-Hispanic black (NHB) and non-Hispanic white (NHW) women. In this study, we profiled the microbiome (16S rRNA) from breast tumor tissues and analyzed 984 metabolites from a total of 51 NHB and NHW women. Breast tumor tissues were collected from 15 patients with TNBC, 12 patients with less aggressive luminal A-type (Luminal) breast cancer, and 24 healthy controls for comparison using UHPLC-tandem mass spectrometry. Principal component analysis and hierarchical clustering of the global metabolomic profiling data revealed separation between metabolic signatures of normal and breast tumor tissues. Random forest analysis revealed a unique biochemical signature associated with elevated lipid metabolites and lower levels of microbial-derived metabolites important in controlling inflammation and immune responses in breast tumor tissues. Significant relationships between the breast microbiome and the metabolome, particularly lipid metabolism, were observed in TNBC tissues. Further investigations to determine whether alterations in sphingolipid, phospholipid, ceramide, amino acid, and energy metabolism pathways modulate Fusobacterium and Tenericutes abundance and composition to alter host metabolism in TNBC are necessary to help us understand the risk and underlying mechanisms and to identify potential microbial-based targets.

Modulation of Estrogen α and Progesterone Receptors in Triple Negative Breast Cancer Cell Lines: The Effects of Vorinostat and Indole-3-Carbinol In Vitro.

BACKGROUND/AIM: Triple negative cancer (TNBC) is a subtype of breast cancer that is highly aggressive, with poor prognosis and responds differently to treatments. This study investigated the role of vorinostat and indole-3-carbinol (I3C) on regulating critical receptors that are not normally expressed in TNBC. MATERIALS AND METHODS: Using real-time PCR, immunostaining, and western blots, the re-expression of estrogen receptor α (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2) receptors was examined in four different TNBC cell types. RESULTS: ERα was re-expressed in three subtypes using vorinostat and I3C. Re-expression of the PR by vorinostat was also detected. Neither vorinostat nor I3C resulted in re-expression of the HER2 receptor. A significant decrease in growth and sensitivity to tamoxifen was also noted. CONCLUSION: The results of this study show that vorinostat and I3C modulate the re-expression of critical receptors in certain subtypes of TNBC through several pathways and these effects can be influenced by the molecular profiles of TNBCs.

Effect of titanium dioxide nanoparticles on DNA methylation in multiple human cell lines.

Nanoscale titanium dioxide (TiO2) is manufactured in wide scale, with a range of applications in consumer products. Significant toxicity of TiO2 nanoparticles has, however, been recognized, suggesting considerable risk to human health. To evaluate fully their toxicity, assessment of the epigenetic action of these nanoparticles is critical. However, only few studies are available examining capability of nanoparticles to alter epigenetic integrity. In the present study, the effect of TiO2 nanoparticles exposure on DNA methylation, a major epigenetic mechanism, was investigated in in vitro cellular model systems. A panel of cells relevant to portals of human exposure (Caco-2 (colorectal), HepG2 (liver), NL20 (lung), and A-431 (skin)) was exposed to TiO2 nanoparticles to assess effects on global methylation, gene-specific methylation, and expression levels of DNA methyltransferases, MBD2, and UHRF1. Global methylation was determined by enzyme-linked immunosorbent assay-based immunochemical analysis. Degree of promoter methylation across a defined panel of genes was evaluated using EpiTect Methyl II Signature PCR System Array technology. Expression of DNMT1, DNMT3a, DNMT3b, MBD2, and URHF1 was quantified by qRT-PCR. Decrease in global DNA methylation in cell lines Caco-2, HepG2, and A-431 exposed to TiO2 nanoparticles was shown. Across four cell lines, eight genes (CDKN1A, DNAJC15, GADD45A, GDF15, INSIG1, SCARA3, TP53, and BNIP3) were identified in which promotors were methylated after exposure. Altered expression of these genes is associated with disease etiology. The results also revealed aberrant expression of epigenetic regulatory genes involved in DNA methylation (DNMT1, DNMT3a, DNMT3b, MBD2, and UHRF1) in TiO2 exposed cells, which was cell type dependent. Findings from this study clearly demonstrate the impact of TiO2 nanoparticles exposure on DNA methylation in multiple cell types, supporting potential involvement of this epigenetic mechanism in the toxicity of TiO2 nanoparticles. Hence for complete assessment of potential risk from nanoparticle exposure, epigenetic studies are critical.

Distinct microbial communities that differ by race, stage, or breast-tumor subtype in breast tissues of non-Hispanic Black and non-Hispanic White women.

Growing evidence highlights an association between an imbalance in the composition and abundance of bacteria in the breast tissue (referred as microbial dysbiosis) and breast cancer in women. However, studies on the breast tissue microbiome have not been conducted in non-Hispanic Black (NHB) women. We investigated normal and breast cancer tissue microbiota from NHB and non-Hispanic White (NHW) women to identify distinct microbial signatures by race, stage, or tumor subtype. Using 16S rRNA gene sequencing, we observed that phylum Proteobacteria was most abundant in normal (n = 8), normal adjacent to tumor (normal pairs, n = 11), and breast tumors from NHB and NHW women (n = 64), with fewer Firmicutes, Bacteroidetes, and Actinobacteria. Breast tissues from NHB women had a higher abundance of genus Ralstonia compared to NHW tumors, which could explain a portion of the breast cancer racial disparities. Analysis of tumor subtype revealed enrichment of family Streptococcaceae in TNBC. A higher abundance of genus Bosea (phylum Proteobacteria) increased with stage. This is the first study to identify racial differences in the breast tissue microbiota between NHB and NHW women. Further studies on the breast cancer microbiome are necessary to help us understand risk, underlying mechanisms, and identify potential microbial targets.

LXR/RXR pathway signaling associated with triple-negative breast cancer in African American women.

BACKGROUND: Triple-negative breast cancer (TNBC) is more prevalent in African and African American (AA) women compared to European American (EA) women. African and AA women diagnosed with TNBC experience high frequencies of metastases and less favorable outcomes. Emerging evidence indicates that this disparity may in fact be the result of the uniquely aggressive biology of African and AA disease. PURPOSE: To understand the reasons for TNBC in AA aggressive biology, we designed the present study to examine the proteomic profiles of TNBC and luminal A (LA) breast cancer within and across patients' racial demographic groups in order to identify proteins or molecular pathways altered in TNBC that offer some explanation for its aggressiveness and potential targets for treatment. MATERIALS AND METHODS: Proteomic profiles of TNBC, LA tumors, and their adjacent normal tissues from AA and EA women were obtained using 2-dimensional gel electrophoresis and bioinformatics, and differentially expressed proteins were validated by Western blot and immunohistochemistry. Our data showed that a number of proteins have significantly altered in expression in LA tumors compared to TNBC, both within and across patients' racial demographic groups. The differentially overexpressed proteins in TNBC (compared to LA) of AA samples were distinct from those in TNBC (compared to LA) of EA women samples. Among the signaling pathways altered in AA TNBC compared to EA TNBC are innate immune signaling, calpain protease, and pyrimidine de novo synthesis pathways. Furthermore, liver LXR/RXR signaling pathway was altered between LA and TNBC in AA women and may be due to the deficiency of the CYP7B1 enzyme responsible for cholesterol degradation. CONCLUSION: These findings suggest that TNBC in AA women enriched in signaling pathways that are different from TNBC in EA women. Our study draws a link between LXR/RXR expression, cholesterol, obesity, and the TNBC in AA women.

In vitro analysis of factors influencing CYP1A2 expression as potential determinants of interindividual variation.

Individual differences in drug metabolism contribute to interindividual variation that characterizes responses to drugs and risk in exposure to foreign chemicals. Large individual differences are found in expression levels of CYP1A2, a major drug-metabolizing enzyme. Underlying causes for this variation are not well understood. Several factors, including tobacco smoking, consumption of cruciferous vegetables, and sex, have been associated with modulating CYP1A2 expression. To understand factors regulating expression of CYP1A2 in establishing a causal relationship, this study examined effects of cigarette smoke condensate (CSC), indole-3-carbinol (I3C), and 17ß-estradiol (estradiol) on CYP1A2 expression in in vitro systems using human liver and lung cells. Treatment with CSC (2-25 µg/mL) significantly increased levels of CYP1A2 in six cell lines examined, in a concentration- and time-dependent manner. Fold changes in expression levels relative to controls varied among cell lines. CYP1A2 enzymatic activity also increased with CSC exposure. Treatment of H1299 and HepB3 cells with dietary agent I3C (50 and 100 µmol/L) increased CYP1A2 expression. In human cell lines H1299 and H1395, treatment with estradiol (10 and 100 nmol/L) significantly reduced expression of CYP1A2. Using ChIP assays, effects of CSC on histone modifications were analyzed. Increases in H3K4me3 and H4K16ac were observed at several segments in the CYP1A2 gene, whereas H3K27me3 decreased, following CSC treatment. These results suggest that CYP1A2 expression is affected epigenetically by CSC. Additional studies will be needed to further establish regulatory mechanisms underlying effects of various environmental, dietary, and endogenous factors on CYP1A2 expression in better predicting individual variation.

Prolactin and Dehydroepiandrosterone Levels in Women with Systemic Lupus Erythematosus: The Role of the Extrapituitary Prolactin Promoter Polymorphism at -1149G/T.

Systemic lupus erythematosus (SLE) has shown an association with high levels of prolactin, low levels of dehydroepiandrosterone (DHEA), and induction of inflammatory cytokines in the serum of patients with the disease. This preliminary study examined the relevance of a -1149G/T functional single-nucleotide polymorphism (SNP) (rs1341239) in the promoter of the extrapituitary prolactin gene in a cohort of African American and European American women with lupus. Examination of this SNP revealed that the -1149TT genotype was correlated with higher levels of prolactin in serum and prolactin gene expression (p = 0.0001) in peripheral blood mononuclear cells (PBMCs). Lower levels of DHEA in serum were demonstrated in lupus patients (p = 0.001); those with the -1149TT genotype had the lowest levels of DHEA. Furthermore, a small subset of women who were on DHEA therapy and had a TT genotype showed a significant decrease in prolactin gene expression and lower disease activity scores (SLEDAI). Lupus patients, particularly African Americans, had significantly higher levels of IL-6 (p = 0.0001) and TNF-α (p = 0.042). This study suggests that the -1149TT genotype may be a risk factor for lupus and may predict who could possibly benefit from DHEA therapy; therefore, these results should be validated in a larger cohort with all ethnic groups.

Cytotoxicity of chronic exposure to 4 cigarette smoke condensates in 2 cell lines.

Tobacco use is the leading preventable cause of death. The cytotoxicity of cigarette smoke condensate (CSC), the particulate fraction of cigarette smoke without the vapor phase, has mostly been tested in short-term in vitro studies lasting from a few hours to a few days. Here, we assessed the toxicity of CSCs from 2 reference cigarettes, 3R4F and CM6, using a primary human small airway epithelial (PSAE) cell line by quantifying adenosine 5'-triphosphate (ATP), 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), total glutathione (reduced glutathione [GSH] + oxidized glutathione [GSSG]), and lactate dehydrogenase (LDH) release over the course of 28 days. The CSCs, 0.3 to 10 µg/mL, promoted cell proliferation at 120 hours of exposure, but demonstrated cytotoxicity at days 14 and 28. Interestingly, CSCs, 0.3 to 3 µg/mL, showed a cell death effect at day 14 but induced cell proliferation at day 28. Consistently, transformation associated with morphological changes began by day 14 and the transformed cells grew dramatically at day 28. The LDH assay appeared to be sensitive for assessing early cell damage, whereas the ATP, MTS, and GSH assays were more suitable for determining later stage CSCs-induced cytotoxicity. The ATP assay showed greater sensitivity than the MTS and GSH assays. We also assessed the toxicity of CSCs in an human Telomerase Reverse Transcriptase (hTERT)-immortalized Barrett esophagus cell line (CP-C). The CP-C cells demonstrated dose- and time-dependent cytotoxicity over the course of 28 days but displayed higher resistance to CSCs than PSAE cells. This study demonstrates that CSCs cause cytotoxicity and induce transformation related to cell resistance and cell invasion properties.

Cigarette smoke condensate and individual constituents modulate DNA methyltransferase expression in human liver cells.

OBJECTIVES: Previous studies found higher expression levels of DNA methyltransferase 1 in liver samples from smokers compared to those from non-smokers. In contrast, expression levels of DNA methyltransferase 3a and DNA methyltransferase 3b were similar in smokers and non-smokers. This study extends these studies to establish a causal linkage to cigarette smoke exposure by examining whether DNA methyltransferase expression is modulated by cigarette smoke condensate. METHODS: These experiments were conducted in an in vitro system using HepG2 human liver cells. The dose range of cigarette smoke condensate was 0.1-120 µg/mL. The duration of exposure was up to 72 h. RESULTS: In a 24-h exposure, DNA methyltransferase 1 expression was found to increase significantly in a dose-dependent manner (greater than threefold at 100 µg/mL cigarette smoke condensate). Expression levels of DNA methyltransferase 3a and DNA methyltransferase 3b were, however, not affected under these conditions. The effect of two cigarette constituents, nicotine and cotinine, on DNA methyltransferase 1 expression was also examined. Nicotine exposure significantly increased DNA methyltransferase 1 expression in a dose-dependent manner (greater than twofold at 50 µM). However, under these conditions, cotinine did not increase DNA methyltransferase 1 expression. CONCLUSION: These results clearly provide additional support of the modulating effect of cigarette smoke on DNA methyltransferase 1 expression. Given the potential of alterations in DNA methyltransferase expression to affect cellular function, this pathway may play a critical role in cigarette smoke-induced toxicity.

Effect of cigarette smoke condensate on gene promoter methylation in human lung cells.

BACKGROUND: In lung cancer, an association between tobacco smoking and promoter DNA hypermethylation has been demonstrated for several genes. However, underlying mechanisms for promoter hypermethylation in tobacco-induced cancer are yet to be fully established. METHODS: Promoter methylation was evaluated in control and cigarette smoke condensate (CSC) exposed human lung cells using the Methyl-Profiler DNA Methylation PCR System. PSAE cells were exposed to 0.3 or 1.0 µg/ml CSC for 72 hours and longer term for 14 and 30 days. NL-20 cells were exposed for 30 days to 10 or 100 µg/ml CSC. RESULTS: Promoters of several genes, including hsa-let-7a-3, CHD1, CXCL12, PAX5, RASSF2, and TCF21, were highly methylated (>90%); hsa-let-7a-3 was affected in both cell lines and under all exposure conditions. Level of methylation tended to increase with CSC concentration and exposure duration (statistical differences were not determined). Percentage methylation of TCF21, which was >98% at exposures of 10 or 100 µg/ml CSC, was found to be reduced to 28% and 42%, respectively, in the presence of the dietary agent genistein. CONCLUSIONS: Using array techniques, several tumor suppressor genes in human lung cells were identified that undergo promoter hypermethylation, providing further evidence of their potential involvement in tobacco smoke-induced lung carcinogenesis and their use as potential biomarkers of harm in tobacco smoke exposure. Results from the study also demonstrated the potential of a dietary agent to exert chemopreventive activity in human tissue against tobacco smoke related diseases through modulation of DNA methylation. Additional studies are needed to confirm these findings.

ATP-Binding Cassette Genes Genotype and Expression: A Potential Association with Pancreatic Cancer Development and Chemoresistance?

Genetic polymorphisms in ABC (ATP-binding cassette) transporter genes are associated with differential responses to chemotherapy in various cancers including pancreatic cancer. In this study, four SNPs in the ABCB1, ABCC1, and ABCG2 genes were investigated in normal and pancreatic cancerous specimens. The expression of the three transporters was also analyzed. The TT genotypes of G2677T and C3435T in ABCB1 gene were associated with lower risk of developing pancreatic cancer (P = 0.013, OR = 0.35 and P = 0.015, OR = 0.29, resp.). To our knowledge, this is the first report of the common polymorphisms in the ABCB1 gene affecting the genetic risk of developing pancreatic cancer. Moreover, the expression of ABCB1 in 2677TT and 3435TT carriers was lower compared to the wild-type homozygotes and heterozygotes. A cell viability assay, using standard pancreatic cancer cell lines, revealed that the ABCB1 2677TT-3455TT haplotype was more sensitive than the other haplotypes to gemcitabine. Conclusion. Polymorphisms in ABCB1 G2677T and G3435T were associated with differential susceptibility to pancreatic cancer and may predict responses to chemotherapy.

Increased expression of Toll-like receptors (TLRs) 7 and 9 and other cytokines in systemic lupus erythematosus (SLE) patients: ethnic differences and potential new targets for therapeutic drugs.

Increased expression of pro-inflammatory cytokines such as interferon, tumor necrosis factors (TNFs) and specific interleukins (ILs) has been found in a number of autoimmune diseases, including systemic lupus erythematous (SLE). These cytokines are induced by toll-like receptors (TLRs). Toll-like receptors are activated in response to accumulation of apoptotic bodies. These receptors play critical roles in innate immune systems. Increased levels of interferon-alpha (INF-α) have also been found in many SLE patients and often correlate with disease severity. The objectives of this study were to examine the expression of selected TLRs and cytokines that have been identified in animal models and some limited human studies in a group of African Americans (AA) and European Americans (EA) women with lupus in comparison to age-matched non-lupus women. Blood samples were consecutively obtained by informed consent from 286 patients, 153 lupus and 136 non-lupus, seen in the rheumatology clinics at East Carolina University. Cytokines were analyzed from blood serum using enzyme linked immunoassay (ELISA) for IL-6 and INF-α. Total RNA was isolated, using a Paxgene kit, from peripheral blood mononuclear cells of African American and European American women blood samples. Quantitative real-time PCR using the CFX real-time system was conducted on all samples to determine TLRs 7 and 9, as well as INF-α expression. Toll-like receptor 7 (p<0.01) and 9 (p=0.001) expression levels were significantly increased in lupus patients compared to age-matched controls. African American women with lupus had a 2-fold increase in TLR-9 expression level when compared to their healthy controls or European American lupus patients. However, there was no ethnic difference in expression of TLR-7 in lupus patients. INF-α expression was significantly higher in lupus patients (p<0.0001) and also showed ethnic difference in expression. Serum levels revealed significant increases in expression of IL-6, IFN-γ and TNF-α in lupus patients compared to non-lupus patients. African American women with lupus had significantly higher serum levels of IL-6 and TNF-α. African American women with lupus demonstrated increased levels of specific pro-inflammatory cytokines and Toll-like receptors when compared to EA women. Increased expression in these lupus patients provides an opportunity for targeting with antagonist as a new therapy for systemic lupus erythematous.

Restoration of the methylation status of hypermethylated gene promoters by microRNA-29b in human breast cancer: A novel epigenetic therapeutic approach.

It is well established that transcriptional silencing of critical tumor-suppressor genes by DNA methylation is a fundamental component in the initiation of breast cancer. However, the involvement of microRNAs (miRNAs) in restoring abnormal DNA methylation patterns in breast cancer is not well understood. Therefore, we investigated whether miRNA-29b, due to its complimentarity to the 3'- untranslated region of DNA methyltransferase 3A (DNMT3A) and DNMT3B, could restore normal DNA methylation patterns in human breast cancers and breast cancer cell lines. We demonstrated that transfection of pre-miRNA-29b into less aggressive MCF-7 cells, but not MDA-MB-231 mesenchymal cells, inhibited cell proliferation, decreased DNMT3A and DNMT3B messenger RNA (mRNA), and decreased promoter methylation status of ADAM23 , CCNA1, CCND2, CDH1, CDKN1C, CDKN2A, HIC1, RASSF1, SLIT2, TNFRSF10D, and TP73 tumor-suppressor genes. Using methylation polymerase chain reaction (PCR) arrays and real-time PCR, we also demonstrated that the methylation status of several critical tumor-suppressor genes increased as stage of breast disease increased, while miRNA-29b mRNA levels were significantly decreased in breast cancers versus normal breast. This increase in methylation status was accompanied by an increase in DNMT1 and DNMT3B mRNA in advanced stage of human breast cancers and in MCF-7, MDA-MB-361, HCC70, Hs-578T, and MDA-MB-231 breast cancer cells as compared to normal breast specimens and MCF-10-2A, a non-tumorigenic breast cell line, respectively. Our findings highlight the potential for a new epigenetic approach in improving breast cancer therapy by targeting DNMT3A and DNMT3B through miRNA-29b in non-invasive epithelial breast cancer cells.

Cigarette smoke condensate induces differential expression and promoter methylation profiles of critical genes involved in lung cancer in NL-20 lung cells in vitro: short-term and chronic exposure.

Establishing early diagnostic markers of harm is critical for effective prevention programs and regulation of tobacco products. This study examined effects of cigarette smoke condensate (CSC) on expression and promoter methylation profile of critical genes (DAPK, ECAD, MGMT, and RASSF1A) involved in lung cancer development in different human lung cell lines. NL-20 cells were treated with 0.1-100 µg/ml of CSC for 24 to 72 hrs for short-term exposures. DAPK expression or methylation status was not significantly affected. However, CSC treatment resulted in changes in expression and promoter methylation profile of ECAD, MGMT, and RASSF1A. For chronic studies, cells were exposed to 1 or 10 µg/ml CSC up to 28 days. Cells showed morphological changes associated with transformation and changes in invasion capacities and global methylation status. This study provides critical data suggesting that epigenetic changes could serve as an early biomarker of harm due to exposure to cigarette smoke.

Characterization of UDP-glucuronosyltransferase (UGT1A1) Promoter Polymorphisms and Gene Expression on Ethnicity, Stage of Disease, and Menopausal Status in Breast Cancer.

Estrogen metabolism, catalyzed by UGTs, is a major drug-metabolic pathway that results in inactivation of estrogens and their metabolites. Alterations in UGTs involved in estrogen metabolism, has been suggested to play a role in breast cancer risk. The purpose of this study was to: 1) compare the mRNA expression levels of UGTs involved in estrogen metabolism in human breast tissues from women; 2) compare UGT1A1 mRNA expression to tumor stage, ethnicity, and menopausal status in a group of human breast tumors and normal breast tissues, and 3) investigate the association between variations in the number of TA repeats in the promoter region of UGT1A1 to gene expression. Quantification of UGT mRNA in breast tissues revealed that UGT1A4, UGT1A10, and UGT2B7 mRNA levels were decreased in breast cancers as compared to normal breast tissues. UGT1A1 mRNA levels were also significantly decreased in breast cancers as compared to normal breast tissues (Tumor: 0.5 ± 0.2; Normal: 4.1 ± 1.3, p = 0.0006). UGT1A1 mRNA down-regulation was strongly correlated with postmenopausal status in breast cancer versus controls (p = 0.04). In all the UGT1A1 genotypes observed in our study, the mean mRNA levels was significantly decreased among breast cancer cases as compared to controls for UGT1A1*1/*1 (p = 0.004), UGT1A1*28/*28 (p = 0.03) and UGT1A1*28/*37 (p = 0.06). Our findings demonstrate that further investigations are necessary to determine the role of UGT1A1 in breast carcinogenesis.

Enhanced efficacy of gemcitabine by indole-3-carbinol in pancreatic cell lines: the role of human equilibrative nucleoside transporter 1.

Pancreatic cancer patients treated with gemcitabine (2',2'-difluorodeoxycytidine) can eventually develop resistance. Recently, published data from our laboratory demonstrated enhanced efficacy of gemcitabine with the dietary agent, indole-3-carbinol (I3C). The current study examined the possible mechanism for this I3C-enhanced efficacy. Several pancreatic cell lines (BxPC-3, Mia Paca-2, PL-45, AsPC-1 and PANC-1) were examined for modulation of human equilibrative nucleoside transporter 1 (hENT1) expression, the major transporter for gemcitabine, by I3C alone and combined with gemcitabine. I3C significantly (p<0.01) up-regulated hENT1 expression in several cell lines. Gemcitabine alone showed no effect on hENT1 expression. However, combining gemcitabine with I3C further increased hENT1 expression. Cell viability assays revealed no effect of I3C on normal cells, hTERT-HPNE. hENT1-specific inhibitor, nitrobenzylthioinosine, significantly abrogated I3C-induced gemcitabine cytotoxicity, further demonstrating its specificity. This study demonstrates that up-regulation of hENT1 expression may be a novel mechanism involved in the additive effect of I3C and gemcitabine.

Transcriptional activity of DNMT3B in pancreatic cancer cells: effects of -149 (C→T) promoter polymorphism.

Polymorphic C-to-T change in the promoter region of DNA-methyltransferase-3B (DNMT3B) gene is associated with risk of several cancers. The aim of this study was to investigate the effect of DNMT3B promoter genetic variant on its transcriptional activity and to compare activity in several pancreatic cell lines. DNMT3B promoter constructs carrying either -149C allele or -149T allele were transiently transfected into pancreatic cancer cells. In promoter assaying, carriage of -149T allele showed only a slight activity (1.1-fold) in Mia cells (p=0.462). In contrast, significant increase (3.8-fold) in activity of -149T allele was shown in SU86.86 pancreatic cancer cells (p=0.0001). These preliminary findings suggest that genetic variance may influence DNMT3B expression in pancreatic cancer. Further studies are needed.

Food contaminant acrylamide increases expression of Cox-2 and nitric oxide synthase in breast epithelial cells.

Acrylamide has been discovered in foods cooked at high temperature. A potentially harmful effect of this dietary component has been suggested by data indicating its association with increased breast cancer. This study investigated the potential effects of acrylamide in nontumorigenic breast cells by assessing expression levels of inducible nitric oxide synthase (iNOS) and cycloogenase-2 (Cox-2) and NOS activity, which are known to be early molecular changes in disease formation. Treatment of cells with acrylamide increased levels of iNOS (both expression and activity) and Cox-2. Its potent metabolite, glycidamide, also induced both iNOS and Cox-2, with induction of iNOS occurring at a lower concentration. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), another food-borne carcinogen, was found to induce Cox-2 expression. Combining acrylamide with PhIP did not result in a further increase. These studies suggest that further research is needed to determine the role of carcinogens formed from cooking foods in inducing early molecular changes associated with breast cancer.

Gender differences in gemcitabine (Gemzar) efficacy in cancer cells: effect of indole-3-carbinol.

Pancreatic cancer has a poor prognosis, mainly due to lack of effective therapies. This study demonstrated the ability of dietary agent, indole-3-carbinol (I3C), to lower the LD(50) of gemcitabine (Gemzar) in decreasing growth of both male (MiaPaca2) and female (SU86.86) pancreatic cancer cells. Female pancreatic cancer cells were more resistant to gemcitabine alone. Additionally, RT-PCR analysis of MiaPaca2 cells treated with 1, 10 or 100 µM of I3C showed that I3C reactivated the tumor suppressor gene p16INK4a in pancreatic cancer cells. Methylated-specific PCR analysis indicated that I3C demethylated the promoter region of p16 INK4a, which was methylated in the untreated cancer cells. p16INK4a inactivation through promoter hypermethylation is considered an early event in pancreatic carcinogenesis. A positive control using 5-azacytidine also reactivated p16INK4a. This study demonstrated the potential of I3C, a possible non-toxic hypomethylating agent, combined with the anticancer agent, gemcitabine, to be a powerful strategy for treating pancreatic cancer.

Acrylamide decreased dopamine levels and increased 3-nitrotyrosine (3-NT) levels in PC 12 cells.

Acrylamide is a chemical known to produce neurotoxicity in animals, as well as in humans. The mechanism of acrylamide-induced neurotoxicity is not fully known. However, recent studies have revealed that acrylamide affects the dopaminergic system. Therefore, the aim of this study was to investigate the effect of acrylamide on dopamine (DA) and the metabolites, 3,4-dihydroxy phenylacetic acid (DOPAC) and homovanillicacid (HVA), levels in Pheochromocytoma (PC 12) cells. In addition, the generation of peroxynitrite (ONOO(-)), measured by 3-nitrotyrosine (3-NT), was investigated as a possible mechanism in acrylamide-induced neurotoxicity. HPLC-coupled to electrochemical detection (ECD) was used to determine DA, DOPAC, HVA and 3-NT levels. Acrylamide (0.01-5mM) exposure produced a dose- and time (1-42h)-dependent decrease in DA levels. The decrease (P<0.05) in DA levels was noted at 24h after exposure to acrylamide. The study also revealed that 3-NT levels in PC 12 increased as a result of treatment with acrylamide. Thus, these data suggest that acrylamide-induced decrease in DA levels in PC 12 cells may be associated with peroxynitrite formation, measured as 3-NT levels.