Stancy Joseph

Abstract 4436: Metformin upregulates hENT1 expression and enhances gemcitabine efficacy in pancreatic cancer cells

Pancreatic cancer is the 4th leading cause of cancer death and is normally diagnosed at a late stage due to the lack of symptoms and early detection. Pancreatic cancer patients have one of the worst prognoses among all cancer types with a 5 year survival rate of less than 5%. Despite significant improvement in understanding molecular and epigenetic changes of this disease, the prognosis and management remained unchanged. The golden standard of advanced pancreatic cancer treatment is gemcitabine or gemcitabine with more targeted therapy. Patients treated with gemcitabine can, however, eventually develop resistance to this drug. Previously published data from our laboratory demonstrated enhanced efficacy of gemcitabine with the dietary agent, indole-3-carbinol (I3C) though up-regulation of the human equilibrative nucleoside transporter (hENT1). hENT1 is the major drug transporter for gemcitabine. One of the drugs currently being investigated for treatment of pancreatic cancer is metformin. Metformin is one of the most widely prescribed drugs for the treatment of type 2 diabetes mellitus. Metformin has exhibited both chemopreventive and chemotherapeutic activities in preclinical human pancreatic cancer cells and animal models. Currently this drug is in clinical trial for pancreatic cancer. Metformin has been found to be transported by a member of the equilibrative nucleoside transporter (ENT) family named ENT4.The current study examined the potential efficacy and regulation of hENT1 in pancreatic cancer cells when treated with gemcitabine and/or metformin. Several pancreatic cell lines (Mia PaCa-2, AsPC-1, Su86.86 and PANC-1) were examined for modulation of hENT1 expression when treated with gemcitabine or metformin or in combination for 24h and 72h. The results varied for each of the cell lines. After 24 h treatment, hENT1 expression analysis of Su86.86 and PANC-1 gemcitabine resistant cell lines showed no change in hENT1 expression levels. After 72h of treatment with gemcitabine and/or metformin hENT1 expression was up-regulated (p Citation Format: Stancy J. Joseph, Taylor Osborne, Beverly Word, Beverly Lyn-Cook. Metformin upregulates hENT1 expression and enhances gemcitabine efficacy in pancreatic cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4436. doi:10.1158/1538-7445.AM2015-4436

Abstract 4428: Metformin effects on ABCB1 expression and proliferation in pancreatic cancer cell lines with different ABCB1 genotypes/haplotypes

Pancreatic cancer carries a poor prognosis and survival rate. Developing effective agents or repurposing agents with low toxicity, such as metformin, an insulin-lowering drug, has recently been investigated for pancreatic cancer. Metformin has long been associated with decreased cancer risk, particularly in diabetic patients. Although its major action is inhibiting hepatic glucose production, it also improves insulin sensitivity in peripheral tissues. This study demonstrates that metformin modulates the ATP-binding cassette gene, ABCB1, expression in pancreatic cancer cell lines with different ABCB1 genotypes. Metformin (100μM or 200μM) alone (p = 0.0212 or p = 0.0161) or in combination with gemcitabine (15nM) (p = 0.0248 or p = 0.0174) significantly decreased ABCB1 expression in Mia pancreatic cells after 24 hr and/or 72 hr treatments. This correlated to increased cell death. Metformin in combination with indole-3-carbinol also significantly (p = 0.0317) decreased ABCB1 expression. However, a sex or genotype difference was noted when two female pancreatic cell lines, SU86.86 and AsPC1, were used. Metformin did not down-regulate ABCB1 expression and was chemoresistance in these two cell lines. AsPC1 and SU86.86 both carry the 2677GG-3435CC ABCB1 haplotype compared to MiaPaca-2 which carries the 2677TT-3134TT ABCB1 haplotype. The 2677TT and 3435TT genotypes/haplotypes are known to be associated with lower risk of developing pancreatic cancer but these data demonstrate that these genotypes are also more sensitive to metformin treatment compared to the 2677GG or 3435CC genotypes. Further research is needed to ascertain whether sex or genotype is contributing to different efficacy of metformin in pancreatic cancer cells with different genotypes. Citation Format: Beverly D. Lyn-Cook, Taylor Osborne, Stancy Joseph, Beverly Word, Li Pang, George Hammons. Metformin effects on ABCB1 expression and proliferation in pancreatic cancer cell lines with different ABCB1 genotypes/haplotypes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4428. doi:10.1158/1538-7445.AM2015-4428

Structure-activity relationship study of novel anticancer aspirin-based compounds

We performed a structure-activity relationship (SAR) study of a novel aspirin (ASA) derivative, which shows strong anticancer activity in vitro and in vivo. A series of ASA-based benzyl esters (ABEs) were synthesized and their inhibitory activity against human colon (HT-29 and SW480) and pancreatic (BxPC-3 and MIA PaCa-2) cancer cell lines was evaluated. The ABEs that we studied largely comprise organic benzyl esters bearing an ASA or acyloxy group (X) at the meta or para position of the benzyl ring and one of four different leaving groups. The nature of the salicyloyl/acyloxy function, the leaving group, and the additional substituents affecting the electron density of the benzyl ring, all were influential determinants of the inhibitory activity on cancer cell growth for each ABE. Positional isomerism also played a significant role in this effect. The mechanism of action of these compounds appears consistent with the notion that they generate either a quinone methide or an m-oxybenzyl zwitterion (or an m-hydroxybenzyl cation), which then reacts with a nucleophile, mediating their biological effect. Our SAR study provides an insight into the biological properties of this novel class of compounds and underscores their potential as anticancer agents.

Epigenome-Wide Association (DNA Methylation) Study of Sex Differencesin Normal Human Kidney

Studies have identified epigenetic sex differences in several human tissues and have implicated epigenetic factors in the regulation of tissue-specific expression. Studies have also shown that women and men respond differently to various drugs, thereby influencing the pharmacokinetics, pharmacodynamics, adverse reactions, efficacy, and safety of a drug. Using Illumina Human Methylation450 BeadChip kit, we investigated the influence of sex on DNA methylation patterns in normal human kidneys (16 females and 15 males). We then related the methylome to mRNA expression levels in kidney structure/function and Drug Metabolizing Enzyme and Transporter (DMET) genes (32 females and 59 males). Our findings indicate that 429 methylated sites on autosomal chromosomes had significant sex-specific differences in the normal human kidney. Methylated sites in/near regions associated with DMET genes or with genes involved in renal structure/function and disease were identified for subsequent analysis. Validation of 2 DMETs genes (POR and ABCA3) and 2 renal structure/function/disease genes (LAMA5 and PLAT) exhibited significant sex-specific differences in mRNA expression. Our results highlight sitespecific sexual dimorphisms (epigenetic-based) in normal human kidney. Importantly, we provide a reference methylome for normal human kidney, which may be utilized to improve our understanding of renal disease and assessing the overall safety and effectiveness of a drug in the kidney.

Epigenome-wide association study of peripheral blood mononuclear cells in systemic lupus erythematosus: Identifying DNA methylation signatures associated with interferon-related genes based on ethnicity and SLEDAI

Systemic lupus erythematosus (SLE or lupus) is a heterogeneous autoimmune disease characterized by the involvement of multiple organs and the production of antinuclear antibodies. DNA methylation plays an important role in the pathogenesis of lupus. We have performed an epigenome-wide DNA methylation study in lupus and healthy control (non-lupus) subjects to identify epigenetic patterns in lupus characterized ethnicity and SLE disease activity index (SLEDAI). A total of fifty-seven lupus patients (39 African American (AA) and 18 European American (EA)) and 33 healthy controls (17 AA and 16 EA) were studied. Differential DNA methylation between lupus patients and controls was assessed for approximately 485,000 CpG sites across the genome. We identified 41 differentially methylated sites (associated with 30 genes) between lupus and control s subjects, 85% of which were hypomethylated. Significant hypomethylation of differentially methylated sites was associated with several interferon-related genes, including MX1, IFI44L, PARP9, DT3XL, IFIT1, IFI44, RSAD2, PLSCR1, and IRF7. Several of these associated genes were also hypomethylated in comparisons between AA lupus and AA non-lupus subjects and between lupus patients with SLEDAI>6 and non-lupus subjects. Our analysis of gene expression data through RT-PCR confirmed these findings. Thus, the results indicate epigenetics susceptibility in lupus, which may be associated with SLEDAI score and ethnicity. In addition, our findings support the importance of the Type 1 interferon pathway in lupus pathogenesis.

Abstract 256: Analysis of combined drug effects on hENT1 and hENT4 in pancreatic cancer cells

Pancreatic cancer is the 4th leading cause of cancer death and diagnosis usually occurs at late stages due to the lack of symptoms and early detection, making surgical intervention almost unfeasible due to low survival rates. Pancreatic cancer patients have one of the worst prognoses among all cancer types with a 5 year survival rate of less than 5%. Despite significant improvement in understanding molecular and epigenetic changes of this disease, the prognosis and management remained unchanged. Gemcitabine, a deoxycytidine nucleoside analog, is the golden standard of advanced pancreatic cancer treatment for patients with locally advanced or metastatic cancer of the pancreas. Patients treated with gemcitabine can, however, eventually develop resistance to this drug. Previously published data from our laboratory demonstrated enhanced efficacy of gemcitabine with the dietary agent, indole-3-carbinol (I3C) though up-regulation of the human equilibrative nucleoside transporter 1 (hENT1). hENT1 (SLC29A1) is the major drug transporter for gemcitabine. One of the drugs currently being investigated for treatment of pancreatic cancer is metformin. Metformin is most commonly used for the treatment of type 2 diabetes mellitus and has exhibited both chemopreventive and chemotherapeutic activities in preclinical human pancreatic cancer cells and animal models. Metformin has been found to be transported by another member of the equilibrative nucleoside transporter (ENT) family named hENT4 (SLC29A4). The current study examined the combined drug effects of gemcitabine, metformin and I3C on hENT1 and hENT4 in pancreatic cancer cells. Several pancreatic cell lines were examined for cell viability, drug synergy and modulation of hENT1 and hENT4 expression when treated with either gemcitabine, metformin, I3C or in combination for 24h and 72h. The results varied for each of the cell lines. After 24h and 72h treatment there was a significant decrease in cell viability when pancreatic cancer cells were treated with metformin or gemcitabine or 500μM I3C alone or in combination. The decrease in cell viability for these treatment conditions was also time dependent, where pancreatic cancer cells treated for 72h exhibited lower cell viability compared to 24h treatment. Pancreatic cancer cells treated with 250μM I3C alone or in combination did not decrease cell viability after 24h; however after 72h the cell viability varies based on the cell line. In most cases the drug activity exhibited antagonistic effect in combination therapy treatment. hENT1 and hENT4 expression levels varied between cell lines, where gemcitabine resistant cell lines exhibited lower hENT levels. Our initial findings showed that gemcitabine may also modulate hENT4 expression in gemcitabine sensitive cell lines and I3C modulations hENT4 expression levels. Citation Format: Stancy J. Joseph, Beverly Word, Beverly Lyn-Cook. Analysis of combined drug effects on hENT1 and hENT4 in pancreatic cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 256.

Abstract C26: Epigenetics and pancreatic cancer: The role of nutrigenomics in cancer health disparities

In vivo, in vitro and epidemiological studies have shown that an individual9s diet may contribute to their susceptibility to develop cancer. Pancreatic cancer remains a very complex and challenging disease that is increasing in African Americans. This cancer carries one of the worst prognosis of any major malignancy, mainly due to its lack of early detection and lack of effective therapeutic agents. Although improvements in imaging technology has aided in diagnosis and identification of patients with the disease, these new technologies have not greatly improved the mortality rate of pancreatic cancer. Evidence has shown that epigenetic mechanisms play an important role early in this cancer and furthermore, epigenetic modifications can be altered by external or internal environmental factors, such as components found in diets, and have the potential to also be reversed. We and others have shown that dietary agents found in cruciferous vegetables, such as indole-3-carbinol, have tremendous anti-cancer effects and can modulate key signals in pancreatic cancer through various mechanisms, such as inactivation of STAT3, reactivation of p16, the ability to up-regulate critical drug transporters such as hENT, to increase drug efficacy and recently modulate Wnt signaling pathways. All of these targets are potentially significant in developing chemopreventive strategies for reducing cancer health disparities among higher risk populations. Results will be shown where indole-3-carbinol has demonstrated effects on all of these targets in pancreatic cancer. Educational strategies are needed to increase knowledge of the importance of proper diet to vulnerable populations. Citation Format: Beverly Lyn-Cook, Beverly Word, Stancy Joseph, Goorge Hammons. Epigenetics and pancreatic cancer: The role of nutrigenomics in cancer health disparities. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr C26.

Abstract 770: Gender differences in the expression of drug metabolizing enzymes in human liver tissues

Differences in pharmacokinetics and drug metabolism present a major challenge in treatment of diseases, such as cancer, as they contribute to variations that characterizes how well drugs respond. Variation in the expression of major drug metabolizing enzymes (DMEs) is associated with significant differences in the bioavailability, distribution, metabolism and clearance of drugs and other xenobiotics. Previous mice/rats studies have shown genetic variation, or polymorphism, in these DMEs is one of the most important causes of variable drug response. It is essential not just in terms of how they affects drug efficacy but in how drugs affect the enzymes. A particular drug may inhibit the metabolic activity of certain DMEs which may lead to deadly levels of drug concentration and an increase in the severity of adverse side effects. On the contrary, when enzymatic activity is induced, drug concentration may decrease, severely limiting its performance. Given the important role of DMEs in regulating the pharmacological and biological activity of drugs it is essential to understand the regulatory features that lead to individual differences in the DMEs expression. Several factors contribute to individual differences in DME expression and drug metabolism. One of which is the differences contributed due the sex/gender of the subjects. In order to understand how sex differences in human may differ in terms of expression in DME, we compared gene expression in male and female human liver tissues, the liver being an important drug metabolizing tissue. Microarray analysis and real time PCR were utilized to identify significant changes in DME expression in hepatic tissues. Microarray analysis identified 34 sex-differential metabolizing enzymes in normal human livers at the RNA level. Of these, we focused on the expression of five metabolizing enzymes: CYP2B6, CYP1A1, CYP11A1, UGT2B15, GST-alpha. These five genes play potential roles in how anti-cancer drugs are metabolized and eliminated from the liver. mRNA expression for these five metabolizing enzymes were significantly higher in normal male human livers compared to female. At the protein levels, significant differences were only shown in CYP2B6 (p Citation Format: Stancy J. Joseph, Tamara Nicolson, Honggang Wang, Beverly R. Word, George Hammons, Beverly Lyn-Cook. Gender differences in the expression of drug metabolizing enzymes in human liver tissues. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 770. doi:10.1158/1538-7445.AM2014-770

Abstract 745: Structure-activity relationship study of the novel aspirin-based anticancer compounds

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC We performed a structure-activity relationship (SAR) study of novel derivatives of aspirin (ASA) which show strong anticancer activity in vitro and in vivo. We synthesized a series of ASA-based benzyl esters (ABEs) and evaluated their inhibitory activity against human colon (HT-29 and SW480) and pancreatic cancer cell lines (BxPC-3 and MIA PaCa-2). The ABEs that we studied largely comprise organic benzyl esters bearing an ASA or acyloxy group (X) at the meta or para position of the benzyl ring and one of four different leaving groups. The nature of the salicyloyl/acyloxy function, the leaving group, and the additional substituents affecting the electron density of the benzyl ring, all were influential determinants of the cancer cell growth inhibitory activity of each ABE. Positional isomerism also played a significant role in this effect. Our SAR study provides an insight into the biological properties of this novel class of compounds and underscores their potential as anticancer agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 745.