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

Ethnic differences in DNA methyltransferases expression in patients with systemic lupus erythematosus

Systemic lupus erythematous (SLE) is a systemic autoimmune inflammatory disease with both genetic and epigenetic etiologies. Evidence suggests that deregulation of specific genes through epigenetic mechanisms may be a contributing factor to SLE pathology. There is increasing evidence that DNA methyltransferase activity may be involved. This study demonstrated modulation in expression of DNA methyltransferases (DNMTs) according to ethnicity in patients diagnosed with SLE. Furthermore, differential expression in one of the DNMTs was found in a subset of lupus patients on dehydroepiandrosterone (DHEA) therapy. Real-time PCR analyses of DNMT1, DNMT3A and DNMT3B in peripheral blood mononuclear cells from a cohort of African American and European American lupus and non-lupus women were conducted. Also, global DNA methylation was assessed using the MethylFlash.sup.TM methylated quantification colorimetric assay. These findings suggest that epigenetic changes may play a critical role in the manifestations of the disease observed among ethnic groups, particularly African American women who often have a higher incidence of lupus. DHEA therapy effects on DNMT3A expression in AA women warrant further investigation in a larger population

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

Abstract 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.</jats:p

Abstract B19: The role of ABCB1 genotypes and targeting cancer stem cells in pancreatic cancer: Effects of metformin and dietary agents

Abstract 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. Cancer stem cells have been identified in pancreatic tumors and have been shown to contribute to its progression and resistance to standard treatments. Pancreatic cancer stem cells are subject to regulation by key embryonic stem cell transcription factors that are known to be aberrantly expressed in pancreatic cancer, such as SOX2, OCT4 and NANOG. Overexpression of OCT4, SOX2 and NANOG together or separately leads to tumor transformation and tumor metastasis. These stem cell factors are known to promote self-renewal by interacting with other transcription factors. Using a human embryonic stem cell RT2 Profiler gene array, numerous stem cell genes were expressed in pancreatic cancer cell lines (MIAPaca2, Panc1). Both cell lines expressed high levels of SOX2, NANOG, CD44, GATA2, POU5F1 (OCT4) and other genes. Panc1 expressed high levels of SOX 17, while MIAPaCa2 expressed high levels of SOX 3, 15 and 17. Treatment of cells with the dietary agent, indole-3-carbinol alone at 100 or 200 µM inhibited or decreased the expression of SOX2, NANOG, CD44, SOX15 and STAT3. However, treatment with metformin alone increased expression of SOX15 and STAT3 in Panc1 cells but not in MIAPaCa2. Combination of metformin and indole-3-carbinol inhibited the expression of SOX2, NANOG, STAT3, and CD44. Expression levels of drug transporters in pancreatic tissue can also affect potential treatment of pancreatic cancer. High level of expression of ABCB1 causes multidrug-resistance. Metformin modulated the ATP-binding cassette gene, ABCB1, expression in pancreatic cancer cell lines with different ABCB1 genotypes. Metformin (100 µM or 200 µM) 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 MIAPaCa2 pancreatic cells after 24 hr 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. These data demonstrated 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. This study have shown that the dietary agent, indole-3-carbinol alone and in combination with metformin down-regulate critical stem cell genes involved in maintenance of pluripotency and self-renewal of cancer stem cells. Furthermore, downregulation of ABCB1 expression varied with treatment with specificity to certain genotypes. Citation Format: Beverly D. Lyn-Cook, Beverly Word, George Hammons. The role of ABCB1 genotypes and targeting cancer stem cells in pancreatic cancer: Effects of metformin and dietary agents [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr B19.</jats:p

Abstract B41: Metformin and indole-3-carbinol (I3C) modulate regulation of genes involved in dedifferentiation in chemo-resistant pancreatic cancer cells

Abstract Pancreatic ductal adenocarcinoma remains one of the deadliest cancers and is projected to become the second leading cause of cancer death by 2020. More alarming, is the projection of death in minority populations for this cancer. Although several risk factors have been associated with the etiology of this cancer, such as smoking, diet, obesity and recently nicotine, it continues to steadily increase. Numerous studies have shown the importance of cancer stem cells in cancer resistance and self-renewal. Dedifferentiation of cancer cells requires key factors to be present in the tumor environment. These factors are regulated by key transcription factors that are known to control cancer stem cells and dedifferentiation. Using a human embryonic stem cell RT2 Profiler gene array, numerous stem cell genes expressed in pancreatic cancer cell lines (MIAPaca2, Panc1) can be identified. However, this study focused on those involved in dedifferentiation of cancer cells. Although both cell lines, Panc-1 and MIAPaCa2 (Mia), expressed high levels of SOX 2, NANOG, CD44, GATA 2, POU5F1 (OCT4), and other genes, differences were noted in the expression of GATA6. GATA6 is a zinc finger transcription factor that is known to play a role in regulating cellular differentiation and organogenesis in gut, lung and heart development and has been found to be overexpressed in pancreatic, gastric and esophageal cancers. A pathogenic gene signature has emerged for genes regulated by GATA6 that could provide potential drug targets. Mia cells, which are less resistance to the standard drug used for pancreatic cancer, gemcitabine, expressed higher levels of GATA6 compared to Panc1, a more resistance phenotype. Treatment of cells with I3C alone at 100 or 200 µM inhibited the expression of GATA6 in Mia cells, compared to Panc 1 cells. Treatment with metformin alone and in combination with I3C showed similar results. Modulation of other gene expressions was noted with treatment of I3C and metformin, such as DPPA2, THY1, CCNA2 and DPPA2. This study has shown that the dietary agent, I3C, alone and in combination with metformin down-regulate critical stem cell genes involved in dedifferentiation of cancer cells that should affect the maintenance of their pluripotency and self-renewal. Citation Format: Beverly Lyn-Cook, George Cooper, Beverly Word, George Hammons. Metformin and indole-3-carbinol (I3C) modulate regulation of genes involved in dedifferentiation in chemo-resistant pancreatic cancer cells. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B41.</jats:p

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

Abstract In vivo, in vitro and epidemiological studies have shown that an individual's 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.</jats:p

Abstract 2556: Indole-3-carbinol (I3C) and gemcitabine as potential combinatorial therapy in pancreatic cancer

Abstract Pancreatic cancer has a poor prognosis, mainly due to lack of effective therapies. The use of non-toxic dietary agents in combination with standard treatment is showing promise in several highly deadly cancers. This study demonstrated the ability of dietary agent, indole-3-carbinol (I3C), to lower the LD50 of gemcitabine (Gemzar) and significantly decrease the growth of both male (MiaPaca2) and female (SU86.86) pancreatic cancer cells. In the male cell line (MiaPaca2) I3C enhanced the anti-proliferative effect at lower doses, decreasing gemcitabine IC50 from 30nM alone to 3nM in combination, a ten-fold decrease in the amount of gemcitabine needed to kill a significant number of cancer cells. Sex difference in response to gemcitabine was also demonstrated. Pancreatic cancer cells from the female cell line (SU86.86) were highly resistant to gemcitabine alone and growth was not affected even at doses as high as 500 μM compared to another powerful anti-cancer drug, doxorubicin (IC50=2.47 µM). However, I3C in combination with gemcitabine exerted powerful anti-cancer effects. The IC50 with 200 µM I3C was 0.388 µM and 1.13 µM with 100 µM I3C, which were lower than that of doxorubicin. Gemcitabine is the current FDA approved single agent for first-line treatment for patients with stage II, III and metastatic adenocarcinoma of the pancreas. This study demonstrated the potential of I3C, a possible non-toxic hypomethylating agent, combined with the anti-cancer agent, gemcitabine, to be a powerful strategy for treating pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2556. doi:10.1158/1538-7445.AM2011-2556</jats:p