Clobenpropit enhances anti-tumor effect of gemcitabine in pancreatic cancer.

AIM: To evaluate the anti-tumor effect of clobenpropit, which is a specific H3 antagonist and H4 agonist, in combination with gemcitabine in a pancreatic cancer cell line. METHODS: Three kinds of human pancreatic cancer cell lines (Panc-1, MiaPaCa-2, and AsPC-1) were used in this study. Expression of H3 and H4 receptors in pancreatic cancer cells was identified with Western blotting. Effects of clobenpropit on cell proliferation, migration and apoptosis were evaluated. Alteration of epithelial and mesenchymal markers after administration of clobenpropit was analyzed. An in vivo study with a Panc-1 xenograft mouse model was also performed. RESULTS: H4 receptors were present as 2 subunits in human pancreatic cancer cells, while there was no expression of H3 receptor. Clobenpropit inhibited cell migration and increased apoptosis of pancreatic cancer cells in combination with gemcitabine. Clobenpropit up-regulated E-cadherin, but down-regulated vimentin and matrix metalloproteinase 9 in real-time polymerase chain reaction. Also, clobenpropit inhibited tumor growth (gemcitabine 294 ± 46 mg vs combination 154 ± 54 mg, P = 0.02) and enhanced apoptosis in combination with gemcitabine (control 2.5%, gemcitabine 25.8%, clobenpropit 9.7% and combination 40.9%, P = 0.001) by up-regulation of E-cadherin and down-regulation of Zeb1 in Panc-1 xenograft mouse. CONCLUSION: Clobenpropit enhanced the anti-tumor effect of gemcitabine in pancreatic cancer cells through inhibition of the epithelial-mesenchymal transition process.

The role of quantitative NPTX2 hypermethylation as a novel serum diagnostic marker in pancreatic cancer.

OBJECTIVES: The majority of pancreatic cancers are found to be unresectable, and the only chance for cure lies on early detection and complete resection. Several genes have been discovered to be aberrantly methylated in primary pancreatic cancer tissue, and this cancer DNA can be detected in the plasma. The aims of this study were to develop a novel diagnostic marker based on epigenetic characteristics of pancreatic cancer. METHODS: We enrolled 104 patients with pancreatic cancer, 60 with chronic pancreatitis, and 5 with benign biliary stone diseases. The blood samples were collected before surgery or any kinds of treatment modalities. DNA was extracted from the plasma of each patient, and NPTX2 (neuronal pentraxin II) CpG island hypermethylation was examined quantitatively by real-time polymerase chain reaction. RESULTS: NPTX2 hypermethylation levels were significantly higher compared with chronic pancreatitis (P = 0.016). The sensitivity and specificity were 80% and 76%, respectively (cutoff = 0.015). NPTX2 gene hypermethylation level was significantly elevated in correlation with higher American Joint Committee on Cancer stages. CONCLUSIONS: The aberrantly methylated NPTX2 gene may help to distinguish between chronic pancreatitis and pancreatic cancer with conventional diagnostic tools and could become a valuable diagnostic marker.

Effects and mechanisms of the combination of suberoylanilide hydroxamic acid and bortezomib on the anticancer property of gemcitabine in pancreatic cancer.

OBJECTIVES: Earlier studies that dealt with the combination therapy of gemcitabine and histone deacetylation inhibitors for pancreatic cancer revealed unsatisfactory results. The activation of nuclear factor κB (NF-κB) was referred as one of the attributable causes, and we attempted to overcome this resistance by the addition of a proteasome inhibitor. METHODS: The influences of suberoylanilide hydroxamic acid (vorinostat, SAHA), a histone deacetylase inhibitor, and bortezomib, a novel selective antagonist of 26S proteasome, with or without gemcitabine on cell growth and apoptosis and the expressions of related proteins were observed in pancreatic cancer cell lines (MiaPaCa-2 and ASPC-1). The xenograft model of pancreatic cancer was used to notice effects in vivo. RESULTS: Vorinostat and bortezomib had independent inhibitory effects and potentiated the antitumor property of gemcitabine in vitro. In the xenograft model, more augmented effects were achieved when bortezomib was combined with gemcitabine than gemcitabine alone. The down-regulation of pAkt and suppression of NF-κB activity was induced by the triple combination. CONCLUSIONS: The triple combination of vorinostat, bortezomib, and gemcitabine resulted in the strongest antitumor effects both in vitro and in vivo and pAkt and NF-κB seems to be involved in this process.

Determining the global DNA methylation status of rat according to the identifier repetitive elements.

A large portion of the genome represents repetitive elements. Identifier (ID) elements, the major elements of short interspersed repetitive elements, are widespread with about 150 000 copies in the rat genome. Each ID element contains six CpG dinucleotides, which might account for the global methylation status of rat. We validated the CpG methylation of the ID elements by various methods. The methylation of one CpG site (CpG-3) of the ID element was investigated by performing pyrosequencing. The methylation percentage of the CpG-3 site was 53.6% (SD = 2.2) on average from six rat tissues with blood, but 24.6% (SD = 1.0) in rat pheochromocytoma, PC-12, cell line. This CpG-3 methylation was further verified by whole genome amplification (WGA), 5-azacytidine treatment, and proportional mixing of rat WGA genomic DNA (gDNA) with liver gDNA. Methylation-sensitive restriction enzyme PCR method showed that three other CpG sites (CpG-1, CpG-4, and CpG-5) within the ID element were also methylated (about 60%) in rat gDNA, but not in WGA gDNA. The ID elements may be good candidates for routine analysis of the global DNA methylation changes of rat for pharmaceutical treatment and their use can make basic epigenetic research possible with high accuracy.

Quantification of CpG methylation at the 5'-region of XIST by pyrosequencing from human serum.

Aberrant methylation of X (inactive)-specific transcript (XIST) is common in serum derived from human prostate and testicular germ cell tumors. The direct quantification of XIST methylation is urgently required for clinical application because human serum contains both normal and cancer-originated XIST DNA. We directly quantitated the methylation percentage of three CpG sites (+947, +956, +971) from the 5'-region of XIST by pyrosequencing. The average methylation percentages at three CpG sites were 88% (+/-5.8) at CpG1, 98% (+/-3.4) at CpG2, and 92% (+/-5.6) at CpG3 from normal male (N = 19). From prostate cancer-derived sera, the average methylation percentage of XIST was 65% (+/-8.3) at CpG1, 82% (+/-10.9) at CpG2, and 74% (+/-4.4) at CpG3, which is lower than the normal XY serum DNA, but greater than normal XX serums. The methylation status of XIST also correlated with its gene expression in B-lymphoblastoid and prostate cancer cell lines. This method is sensitive for quantifying the small percentage change in the methylation status of XIST, and may be used for early diagnosis and monitoring of cancer in men using serum.

Estimating the total mouse DNA methylation according to the B1 repetitive elements.

Measuring the degree of methylation of the B1 element in mouse may represent the global DNA methylation status because about 30,000 copies of the B1 element are randomly dispersed in the total mouse genome. Six CpG dinucleotides are located within each 163 bp size of B1 element, and each CpG dinucleotide was partially methylated. We quantitated the DNA methylation of the B1 repetitive elements by performing PCR for the methylation specific PCR (MSP) and also by the pyrosequencing. Each CpG dinucleotide was methylated at an average of 9% in the mouse genome by the pyrosequencing and MSP. Especially, we checked whether CpG methylation of the B1 element could respond to a treatment of the DNA methylation inhibitor, 5-azacytidine (5-AzaC). Consequently, the calibration graph resulting from measuring the relative CpG methylation percentage of the B1 element is linearly decreased with the increasing amount of 5-AzaC (up to 50 ng/ml concentration) in the NIH3T3 cells with a standard deviation of only 1.73% between three independent assays. Our methods can be applied to the routine analysis of the global DNA methylation changes in mouse in vivo and in vitro in pharmaceuticals and basic epigenetic research with efforts being less labor-intensive.

The analysis of X-chromosome inactivation-related gene expression from single mouse embryo with sex-determination.

Chromatin remodeling by histone and DNA modification is important for the initiation of X-chromosome inactivation (XCI). In this study, a thorough transcriptional analysis of five XCI-related genes was performed by single cell reverse-transcribed PCR. An analysis of the XCI-related gene (Xist, Tsix, SUV39H1, SET7, and DNMT1) expression was performed to investigate the initiation process of XCI from early mouse single embryo (1-cell, 2-cell, 4-cell, 8-cell, and blastocyst). Detection of the expression of Xist and Tsix from single 2-cell embryo was feasible, although the expression of those genes was very low in single 1-cell embryo. Transcription of those genes may be activated from single 2-cell embryo. After determining the sex of single embryo by Y-chromosome-specific Zfy expression, we found that Tsix could be detected from both male and female single embryos, but it was only possible to detect Xist from female single embryo. XCI chromatin-remodeling genes, such as histone H3 methylation enzymes (SUV39H1 and SET7) and DNA methylation enzyme (DNMT1), were expressed during all early phases of embryogenesis. The expression of those genes in single embryo was not dependent on sex. Our study illustrated that the expression of these chromatin-remodeling genes, SUV39H1, DNMT1, and SET7, may be originated from germ cells, which were not dependent on zygotic activation of Xist from female single embryo.