Li Pang

Comprehensive Translational Assessment of Human Induced Pluripotent Stem Cell Derived Cardiomyocytes for Evaluating Drug-Induced Arrhythmias

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) hold promise for assessment of drug-induced arrhythmias and are being considered for use under the comprehensive in vitro proarrhythmia assay (CiPA). We studied the effects of 26 drugs and 3 drug combinations on 2 commercially available iPSC-CM types using high-throughput voltage-sensitive dye and microelectrode-array assays being studied for the CiPA initiative and compared the results with clinical QT prolongation and torsade de pointes (TdP) risk. Concentration-dependent analysis comparing iPSC-CMs to clinical trial results demonstrated good correlation between drug-induced rate-corrected action potential duration and field potential duration (APDc and FPDc) prolongation and clinical trial QTc prolongation. Of 20 drugs studied that exhibit clinical QTc prolongation, 17 caused APDc prolongation (16 in Cor.4U and 13 in iCell cardiomyocytes) and 16 caused FPDc prolongation (16 in Cor.4U and 10 in iCell cardiomyocytes). Of 14 drugs that cause TdP, arrhythmias occurred with 10 drugs. Lack of arrhythmic beating in iPSC-CMs for the four remaining drugs could be due to differences in relative levels of expression of individual ion channels. iPSC-CMs responded consistently to human ether-a-go-go potassium channel blocking drugs (APD prolongation and arrhythmias) and calcium channel blocking drugs (APD shortening and prevention of arrhythmias), with a more variable response to late sodium current blocking drugs. Current results confirm the potential of iPSC-CMs for proarrhythmia prediction under CiPA, where iPSC-CM results would serve as a check to ion channel and in silico modeling prediction of proarrhythmic risk. A multi-site validation study is warranted.

Evaluation of Batch Variations in Induced Pluripotent Stem Cell-Derived Human Cardiomyocytes from 2 Major Suppliers

Drug-induced proarrhythmia is a major safety issue in drug development. Developing sensitive in vitro assays that can predict drug-induced cardiotoxicity in humans has been a challenge of toxicology research for decades. Recently, induced pluripotent stem cell-derived human cardiomyocytes (iPSC-hCMs) have become a promising model because they largely replicate the electrophysiological behavior of human ventricular cardiomyocytes. Patient-specific iPSC-hCMs have been proposed for personalized cardiac drug selection and adverse drug response prediction; however, many procedures are involved in cardiomyocytes differentiation and purification process, which may result in large line-to-line and batch-to-batch variations. Here, we examined the purity, cardiac ion channel gene expression profile, and electrophysiological response of 3 batches of iPSC-hCMs from each of 2 major cell suppliers. We found that iPSC-hCMs from both vendors had similar purities. Most of the cardiac ion channel genes were expressed uniformly among different batches of iCells, while larger variations were found in Cor.4U cells, particularly in the expression of CACNA1C, KCND2, and KCNA5 genes, which could underlie the differences in baseline beating rate (BR) and field potential duration (FPD) measurements. Although, in general, the electrophysiological responses of different batches of cells to Na+, Ca2+, Ikr, and Iks channel blockers were similar, with Ikr blocker-induced proarrhythmia, the sensitivities were depended on baseline BR and FPD values: cells that beat slower had longer FPD and greater sensitivity to drug-induced proarrhythmia. Careful evaluation of the performance of iPSC-hCMs and methods of data analysis is warranted for shaping regulatory standards in qualifying iPSC-hCMs for drug safety testing.

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

Sex-related Differences in Drug-induced QT Prolongation and Torsades de Pointes: a New Model System with Human iPSC-CMs

Numerous drugs have the potential to prolong the QT interval and may cause accidental cardiac arrest (torsades de pointes [TdP]). Women are at a higher risk than men for experiencing drug-induced TdP. Due to the lack of appropriate tools, few studies have investigated whether genetic differences between men and women have any effects on drug-induced proarrhythmia. Sex hormones are believed to play a predominant role in the induction of TdP. Recently, progress in induced pluripotent stem cell (iPSC) technologies has made it possible to utilize human iPSC-derived cardiomyocytes (hiPSC-CMs) to investigate the influence of both genetics and sex hormones on cardiac ion channel gene expression and cardiomyocyte function. In this study, we investigated genetic and hormonal effects on sex differences of drug-induced QT prolongation and TdP with hiPSC-CMs from healthy male and female donors. We found that despite batch variations in beating rates and field potential durations (FPD), female-derived hiPSC-CMs showed steeper slopes of FPD to interspike interval ratios and were more sensitive to IKr blocker-induced FPD prolongation. 17β-estradiol increased FPD and 5α-dihydrotestosterone shortened FPD, but the addition of sex hormones had limited effect on the responses of hiPSC-CMs to IKr blockades. The differential expression of KCNE1 gene and reduced repolarization reserve in female-derived hiPSC-CMs compared with male-derived hiPSC-CMs may partially explain why females are more susceptible to proarrhythmias. Human iPSC-CMs can be a useful new model to study mechanisms of sex differences in cardiomyocyte repolarization processes and aid in the prediction of drug-induced proarrhythmias in both men and women.

Abstract 759: ATP-binding cassette (ABC) genes genotype and expression: A potential association with pancreatic cancer development and chemoresistance

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Genetic polymorphisms in ATP-Binding Cassette (ABC) transporter genes are associated with different responses to chemotherapy in various cancers including pancreatic cancer. In this study, four single nucleotide polymorphisms (SNPs) in the ABCB1, ABCC1 and ABCG2 genes were investigated in normal and pancreatic cancer specimens. The potential association with expression of the efflux pumps was also analyzed. The TT genotypes of G2677T and C3435T in ABCB1 gene were associated with lower risk of developing pancreatic cancer in the investigated populations (p=0.013, OR=0.35 and p=0.015, OR=0.29, respectively). To our knowledge, this is the first report of the common polymorphisms in ABCB1 gene affecting the genetic risk of developing pancreatic cancer. Moreover, compared to ABCB1 G2677T and C3435T wild-type homozygotes and heterozygotes, the expression of ABCB1 in mutant-type homozygotes (2677TT and 3435TT) was lower in normal pancreatic tissue (64.3% and 68.9%, respectively, p<0.05), and the same trend was also found in pancreatic cancer specimens. Interestingly, the pancreatic cancer cells MIA1, BXPC3 and CFPAC1 are ABCB1 2677TT-3435TT homozygotes. Cell viability assay revealed that these three cell lines were much more sensitive to gemcitabine than PANC1, SU86.86, PL-45, and ASPC1, which are either ABCB1 G2677T-C3435T wild type homozygotes or heterozygotes. These results are consistent with the clinical observation that for patients receiving post-operative gemcitabine chemotherapy, the overall survival is tended to be longer in the ABCB1 2677TT and 3435TT carriers. Conclusion: Polymorphisms in ABCB1 G2677T and G3435T were associated with different susceptibility to pancreatic cancer and may predict responses to chemotherapy. Citation Format: Li Pang, Beverly Word, Joshua Xu, George Hammons, Shiew-Mei Huang, Beverly Lyn-Cook. ATP-binding cassette (ABC) genes genotype and expression: A potential association with pancreatic cancer development and chemoresistance. [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 759. doi:10.1158/1538-7445.AM2014-759

Abstract 5376: Expression of histone deacetylases (HDACs) 1, 2, 3 and 7 in pancreatic cancer cell lines: The role of indole-3-carbinol.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Pancreatic cancer has the poorest prognosis of most cancers, mainly due to its resistant phenotype. Early or late biomarkers are greatly needed in this cancer. Scientists have shown that epigenetic mechanisms may be involved in the etiology and progression of pancreatic cancer. Epigenetic changes involve modification of the DNA through hyper- and hypo-methylation, histone modification and modulation in expression of microRNAs. Histone deacetylases modify tails on the histones, which can result in repressive chromatin formation and inactivation of specific genes. This study, using real-time PCR, examined the level of HDACs 1, 2, 3 and 7 in four pancreatic cancer cell lines (BXPC, CFPAC,ASPC and MIA). The highest level of expression of HDAC 1 and 3 was found in CFPAC and MIA cells compared to BXPC and ASPC cells. Indole-3 carbinol, in previously published studies from our laboratory, have shown several anti-cancer activities, including the re-expression of p16 in MIA cells (Lyn-Cook et al, 2010 Anticancer Res., 30:4907-4914). The current study demonstrated that indole-3-carbinol at concentrations of 20 μM and 100 μM significantly decreased expression of HDAC 3 by 2-fold and 3-fold in MIA cells, respectively. Results further showed that indole-3 carbinol decreased expression of HDAC 1 in CFPAC cells and HDAC 7 in BXPC cells by 2-fold. No change in expression was shown in any of the cell lines on HDAC 2. This study showed that indole-3-carbinol may exert histone deacetylase inhibitory effects. Further studies are needed to confirm these results. Specific HDACs as potential targets for pancreatic cancer are growing among the scientific community. HDACs are known to be regulators of growth, differentiation and cell death and dysfunction of transcriptional repression mediated by HDACs may lead to the development of cancer. Citation Format: Beverly D. Lyn-Cook, Li Pang, Lascelles E. Lyn-Cook, Beverly Word, George Hammons. Expression of histone deacetylases (HDACs) 1, 2, 3 and 7 in pancreatic cancer cell lines: The role of indole-3-carbinol. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5376. doi:10.1158/1538-7445.AM2013-5376

Abstract B15: Expression of ABC transporters in human pancreatic cancer cell lines and correlation with gemcitabine sensitivity.

Pancreatic cancer is one of the deadliest malignancies with very poor prognosis. Because of no symptoms or nonspecific and varied symptoms, pancreatic cancer is often not diagnosed until it is advanced. Chemotherapy is the main treatment, but due to chemoresistance, the efficacy of chemotherapy is limited. Even for Gemcitabine, the golden standard for advance pancreatic cancer treatment, the tumor response rate is only about 15-20% and the 1-year survival rate is less than 25%. Although intensively investigated, the understanding of drug insensitivity is still fragmentary. Of the many different, unrelated mechanisms, we are particularly interested in abnormal expression of ATP-binding cassette (ABC) transporters, as the multidrug efflux pumps play an important role in the uptake and distribution of therapeutic drugs. We examined multiple ABC transporters gene expression in 8 human pancreatic carcinoma cell lines and found that Mia Paca-2 has the lowest expressions of ABCB1, ABCC1 and ABCG2, PANC1 has highest expressions of ABCC1 and ABCG2, and CFPAC1 has the highest expression of ABCB1. AsPC-1, PL-45 and HPAF-II also show significant amounts of ABCB1, ABCC1 and ABCG2 expression. We have previously noted that Mia Paca-2 is significantly more sensitive to gemcitabine than PANC1, PL-45 and ASPC-1. The expressions of the ABC transporters may correlate with the differential sensitivity of the cells to gemcitabine. The potential correlations of ABC transporters9 expression with the chemosensitivity to other chemotherapy drugs are currently being investigated. Citation Format: Li Pang, Beverly Word, Honggang Wang, Beverly Lyn-Cook. Expression of ABC transporters in human pancreatic cancer cell lines and correlation with gemcitabine sensitivity. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B15.

Abstract A44: Individual variation in hENT expression in normal and malignant pancreatic tissues: The role of dietary agents in enhancing efficacy in gemcitabine treatment.

Equilibrative nucleoside transporters (ENTs) are responsible for the uptake of a large number of nucleosides and nucleoside analogs. hENT1 is the most abundant and is widely distributed in human cells. It is the major transporter by which gemcitabine enters cells and decreased levels is an established resistance mechanism in vitro. However, high levels represent an increase in positive predictive factors for patients’ response to gemcitabine. hENT expression is, therefore, a prognostic marker for patients with resected cancer but is it also a biomarker for pancreatic cancer? This study demonstrated that hENT expression in pancreatic tissue was highly variable among individuals and that moderate to high levels of hENT was expressed in malignant pancreatic cancers but very little to any in normal pancreatic tissues. Recent published studies in our laboratory have shown that the non-toxic dietary agent, indole-3-carbinol (IC3), can modulate hENT expression in pancreatic cancer cell lines, increasing expression of this transporter and further enhancing the efficacy of gemicitabine. Moderate to high level of expression of hENT in tumor tissues and not normal pancreatic tissues suggest that it could also be a biomarker, however larger number of samples are needed for validation. Modulation of hENT expression by I3C could prove to be an important agent for combination therapy in patients expressing low levels of hENT. Citation Format: Beverly Lyn-Cook, Honggang Wang, Li Pang, Beverly Word. Individual variation in hENT expression in normal and malignant pancreatic tissues: The role of dietary agents in enhancing efficacy in gemcitabine treatment. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A44.