Demethylating Agent Research Articles

Abstract 5728: DNA demethylating agents and interferons as modulators of Wnt/β-catenin signaling in colorectal cancer

Abstract p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 12.0px 'Times New Roman'} Colorectal cancer (CRC) is the second leading cause of death from cancer in men and the third in women in Canada. There is accumulating evidence suggesting that CRC is organized in a hierarchical manner, at the apex of which are the Cancer-Initiating Cells (CIC). Furthermore, results from xenograft models and human clinical trials indicate a selective enrichment of CICs in tumours that are resistant to therapy, suggesting that targeting CICs may represent a new paradigm in cancer treatment. We have previously reported the treatment with low dose of the DNA demethylating agent, 5-Aza-2'-deoxycytidine (5-AZA-CdR), can target CICs through the activation of the RIG1-MDA5 viral sensing sensing pathway, leading to an anti-viral response in cancer cells. Although it is now known that 5-AZA-CdR induces a state of “viral mimicry” in these cancer cells, the mechanism by which 5-AZA-CdR can specifically target CICs is not well understood. In this study, we propose a novel intersection between RIG1-MDA5-IRF7 and Wnt/β-catenin pathways. We found that treatment of CIC enriched cancer cells with both 5-AZA-CdR and type I and III interferons reduced canonical Wnt/β-catenin signalling. Furthermore, treatment with type I and III interferons showed a reduced CIC frequency in colorectal cancer cells in vitro. Mechanistically, we found that IRF7 and β-catenin interact in the nucleus, giving rise to a novel, non-canonical pathway that can modulate WNT signalling. These findings may explain a novel pathway by which 5-AZA-CdR can specifically target CIC enriched populations in colorectal cancer, and highlights the importance of innate immune pathways in epigenetic therapy. Citation Format: Ilias Ettayebi, Parinaz Mehdipour, Rajat Singhania, Ankur Chakravarthy, Tiago Medina, Shu Yi Shen, Charles Ishak, David Roulois, Daniel De Carvalho. DNA demethylating agents and interferons as modulators of Wnt/β-catenin signaling in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5728.

Regulation of microRNA-129-2 expression by cytosine-phosphoric acid-guanine motif island methylation and its effect on the proliferation and colony formation in renal cell carcinoma

Objective To investigate the impact of methylation of the cytosine-phosphoric acid-guanine motif (CpG) island upstream of microRNA (miRNA, miR)-129-2 on regulation of expression and its effect on the proliferation and colony formation in renal cell carcinama. Methods The methylation specific polymerase chain reaction (MSP) and the bisulfite sequencing PCR (BSP) were implemented to detect CpG island methylation of miR-129-2 in the upstream region. Taqman real-time fluorescence quantitative polymerase chain reaction (Taqman-RT-PCR) was applied for identifying of miR-129-2 expression. The proliferation and colony formation of renal cell lines were detected by Thiazole blue (MTT) and colony formation assay. Results MSP results revealed that miR-129-2 was hypermethylated in tumor tissues, while BSP showed a decrease in the degree of methylation of miR-129-2 in renal cell lines (786-O, ACHN, Caki-1 and 796-P) treated with demethylating agents [(66.16±6.20)% vs. (85.63±6.27)%, (66.20±6.55)% vs. (86.74±4.42)%, (59.00±4.58)% vs. (76.85±9.40)%, (63.13±6.07)% vs. (80.32±8.39)%; t=3.824, 4.498, 2.956, 2.874; P=0.019, 0.011, 0.042, 0.045]. Compared with control group each cell line, demethylating agent elevated miR-129-2 expression (0.107±0.001 vs. 0.059±0.005, 0.084±0.007 vs. 0.027±0.005, 0.173±0.006 vs. 0.151±0.050, 0.114±0.006 vs. 0.079±0.004; t=9.793, 11.897, 3.093, 7.566; P=0.001, 0.000, 0.036, 0.002). Compared with control group above, the proliferation (0.97±0.16 vs. 1.42±0.13, 1.34±0.09 vs. 1.76±0.06, 0.92±0.04 vs. 1.16±0.07; t=3.804, 6.329, 5.220; P=0.019, 0.003, 0.006) and colony formation [(69±3) cells vs. (102±6) cells, (57±5) cells vs. (78±6) cells, (91±5) cells vs. (112±6) cells; t=8.498, 4.571, 5.157; P=0.001, 0.010, 0.007] significantly were inhibited in 786-O, ACHN and 769-P cell lines that were observed after treated with demethylating agent. Conclusion The expression of the miR-129-2 is regulated by the DNA methylation and closely related to the proliferation and colony formation ability. Key words: MicroRNA-129-2; Methylation; Renal cell carcinama; Proliferation; Colony formation

Global DNA methylation synergistically regulates the nuclear and mitochondrial genomes in glioblastoma cells.

Replication of mitochondrial DNA is strictly regulated during differentiation and development allowing each cell type to acquire its required mtDNA copy number to meet its specific needs for energy. Undifferentiated cells establish the mtDNA set point, which provides low numbers of mtDNA copy but sufficient template for replication once cells commit to specific lineages. However, cancer cells, such as those from the human glioblastoma multiforme cell line, HSR-GBM1, cannot complete differentiation as they fail to enforce the mtDNA set point and are trapped in a ‘pseudo-differentiated’ state. Global DNA methylation is likely to be a major contributing factor, as DNA demethylation treatments promote differentiation of HSR-GBM1 cells. To determine the relationship between DNA methylation and mtDNA copy number in cancer cells, we applied whole genome MeDIP-Seq and RNA-Seq to HSR-GBM1 cells and following their treatment with the DNA demethylation agents 5-azacytidine and vitamin C. We identified key methylated regions modulated by the DNA demethylation agents that also induced synchronous changes to mtDNA copy number and nuclear gene expression. Our findings highlight the control exerted by DNA methylation on the expression of key genes, the regulation of mtDNA copy number and establishment of the mtDNA set point, which collectively contribute to tumorigenesis.

Attenuation of TGFBR2 expression and tumour progression in prostate cancer involve diverse hypoxia-regulated pathways

BackgroundDysregulation of transforming growth factor β (TGF-β) signaling and hypoxic microenvironment have respectively been reported to be involved in disease progression in malignancies of prostate. Emerging evidence indicates that downregulation of TGFBR2, a pivotal regulator of TGF-β signaling, may contribute to carcinogenesis and progression of prostate cancer (PCa). However, the biological function and regulatory mechanism of TGFBR2 in PCa remain poorly understood. In this study, we propose to investigate the crosstalk of hypoxia and TGF-β signaling and provide insight into the molecular mechanism underlying the regulatory pathways in PCa.MethodsProstate cancer cell lines were cultured in hypoxia or normoxia to evaluate the effect of hypoxia on TGFBR2 expression. Methylation specific polymerase chain reaction (MSP) and demethylation agents was used to evaluate the methylation regulation of TGFBR2 promoter. Besides, silencing of EZH2 via specific siRNAs or chemical inhibitor was used to validate the regulatory effect of EZH2 on TGFBR2. Moreover, we conducted PCR, western blot, and luciferase assays which studied the relationship of miR-93 and TGFBR2 in PCa cell lines and specimens. We also detected the impacts of hypoxia on EZH2 and miR-93, and further examined the tumorigenic functions of miR-93 on proliferation and epithelial-mesenchymal transition via a series of experiments.ResultsTGFBR2 expression was attenuated under hypoxia. Hypoxia-induced EZH2 promoted H3K27me3 which caused TGFBR2 promoter hypermethylation and contributed to its epigenetic silencing in PCa. Besides, miR-93 was significantly upregulated in PCa tissues and cell lines, and negatively correlated with the expression of TGFBR2. Ectopic expression of miR-93 promoted cell proliferation, migration and invasion in PCa, and its expression could also be induced by hypoxia. In addition, TGFBR2 was identified as a bona fide target of miR-93.ConclusionsOur findings elucidate diverse hypoxia-regulated pathways including EZH2-mediated hypermethylation and miR-93-induced silencing contribute to attenuation of TGFBR2 expression and promote cancer progression in prostate cancer.

Correction: Chidamide and decitabine can synergistically induce apoptosis of Hodgkin lymphoma cells by up-regulating the expression of PU.1 and KLF4.

[This corrects the article DOI: 10.18632/oncotarget.20659.].

Use of 5-azacytidine in a proof-of-concept study to evaluate the impact of pre-natal and post-natal exposures, as well as within generation persistent DNA methylation changes in Daphnia

Short-term exposures at critical stages of development can lead to delayed adverse effects long after the initial stressor has been removed, a concept referred to as developmental origin of adult disease. This indicates that organisms’ phenotypes may epigenetically reflect their past exposure history as well as reflecting chemicals currently present in their environment. This concept has significant implications for environmental monitoring. However, there is as yet little or no implementation of epigenetics in environmental risk assessment. In a proof-of-principle study we exposed Daphnia magna to 5-azacytidine, a known DNA de-methylating agent. Exposures covered combinations of prenatal and postnatal exposures as well as different exposure durations and recovery stages. Growth, the transcription of genes and levels of metabolites involved in regulating DNA methylation, and methylation levels of several genes were measured. Our data shows that prenatal exposures caused significant changes in the methylome of target genes, indicating that prenatal stages of Daphnia are also susceptible to same level of change as post-natal stages of Daphnia. While the combination of pre- and postnatal exposures caused the most extreme reduction in DNA methylation compared to the control group. Furthermore, some of the changes in the methylation patterns were persistent even after the initial stressor was removed. Our results suggest that epigenetic biomarkers have the potential to be used as indicators of past chemical exposure history of organisms and provide strong support for implementing changes to the current regimes for chemical risk assessment to mimic realistic environmental scenarios.

Abstract A171: Epigenetic modification of α-N-acetylgalactosaminidase (NAGA) enhances cisplatin resistance in ovarian cancer

Abstract Although cisplatin is one of the most effective antitumor drugs for ovarian cancer, the emergence of chemoresistance to cisplatin in over 80% of initially responsive patients is a major barrier to successful therapy. The precise mechanisms underlying the development of cisplatin resistance are not fully understood, but alteration of DNA methylation associated with aberrant gene silencing may play a role. To identify epigenetically regulated genes directly associated with ovarian cancer cisplatin resistance, we compared the expression and methylation profiles of cisplatin-sensitive and -resistant human ovarian cancer cell lines. We identified α-N-acetylgalactosaminidase (NAGA) as one of the key candidate genes for cisplatin drug response. Interestingly, in cisplatin-resistant cell lines, NAGA was significantly downregulated and hypermethylated at a promoter CpG site at position +251 relative to the transcriptional start site. Low NAGA expression in cisplatin-resistant cell lines was restored by treatment with a DNA demethylation agent, indicating transcriptional silencing by hyper-DNA methylation. Furthermore, overexpression of NAGA in cisplatin-resistant lines induced cytotoxicity in response to cisplatin, whereas depletion of NAGA expression increased cisplatin chemoresistance, suggesting an essential role of NAGA in sensitizing ovarian cells to cisplatin. These findings indicate that NAGA acts as a cisplatin sensitizer and its gene silencing by hypermethylation confers resistance to cisplatin in ovarian cancer. Therefore, we suggest NAGA may be a promising potential therapeutic target for improvement of sensitivity to cisplatin in ovarian cancer. Citation Format: Woong Ju, Hye Youn Sung, Jung-Hyuck Ahn. Epigenetic modification of α-N-acetylgalactosaminidase (NAGA) enhances cisplatin resistance in ovarian cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A171.

Epigenetic modification of α-N-acetylgalactosaminidase enhances cisplatin resistance in ovarian cancer.

Although cisplatin is one of the most effective antitumor drugs for ovarian cancer, the emergence of chemoresistance to cisplatin in over 80% of initially responsive patients is a major barrier to successful therapy. The precise mechanisms underlying the development of cisplatin resistance are not fully understood, but alteration of DNA methylation associated with aberrant gene silencing may play a role. To identify epigenetically regulated genes directly associated with ovarian cancer cisplatin resistance, we compared the expression and methylation profiles of cisplatin-sensitive and -resistant human ovarian cancer cell lines. We identified α-Nacetylgalactosaminidase (NAGA) as one of the key candidate genes for cisplatin drug response. Interestingly, in cisplatin-resistant cell lines, NAGA was significantly downregulated and hypermethylated at a promoter CpG site at position +251 relative to the transcriptional start site. Low NAGA expression in cisplatin-resistant cell lines was restored by treatment with a DNA demethylation agent, indicating transcriptional silencing by hyper-DNA methylation. Furthermore, overexpression of NAGA in cisplatin-resistant lines induced cytotoxicity in response to cisplatin, whereas depletion of NAGA expression increased cisplatin chemoresistance, suggesting an essential role of NAGA in sensitizing ovarian cells to cisplatin. These findings indicate that NAGA acts as a cisplatin sensitizer and its gene silencing by hypermethylation confers resistance to cisplatin in ovarian cancer. Therefore, we suggest NAGA may be a promising potential therapeutic target for improvement of sensitivity to cisplatin in ovarian cancer.

Cystatin A suppresses tumor cell growth through inhibiting epithelial to mesenchymal transition in human lung cancer.

Cystatin A (CSTA), belonging to type 1 cystatin super-family, is expressed primarily in epithelial and lymphoid tissues for protecting cells from proteolysis of cytoplasmic and cytoskeletal proteins by cathepsins B, H and L. CSTA acts as a tumor suppressor in esophageal cancer, however, its role in lung cancer has not yet been elucidated. Here we found that CSTA was down-regulated in all lung cancer cell lines compared to normal lung epithelial cells. CSTA was restored in most lung cancer cell lines after treatment with demethylation agent 5-aza-2-deoxycytidine and deacetylation agent Trichostatin. Bisulfite sequencing revealed that CSTA was partially methylated in the promoter and exon 1. In primary lung tumors, squamous cell carcinoma (SCC) significantly expressed more CSTA compared to adenocarcinoma (p<0.00001), and higher expression of CSTA was significantly associated with lower tumor grade (p<0.01). CSTA stable transfection reduced the activity of cathepsin B and inhibited the ability of colony formation, migration and invasion, and enhanced gemcitabine-induced apoptosis. CSTA overexpression resulted in reduced activity of ERK, p-38, and AKT. Additionally, CSTA overexpression led to a mesenchymal to epithelial transition (MET) and prevented the TGF-β1-induced epithelial to mesenchymal transition (EMT) through inhibiting the ERK/MAPK pathway. In conclusion, our date indicate 1) epigenetic regulation is associated with CSTA gene silencing; 2) CSTA exerts tumor suppressive function through inhibiting MAPK and AKT pathways; 3) Overexpression of CSTA leads to MET and prevents TGF-β1-induced EMT by modulating the MAPK pathway; 4) CSTA may be a potential biomarker for lung SCC and tumor differentiation.

The 3p14.2 tumour suppressor ADAMTS9 is inactivated by promoter CpG methylation and inhibits tumour cell growth in breast cancer.

Chromosome region 3p12‐14 is an important tumour suppressor gene (TSG) locus for multiple cancers. ADAMTS9, a member of the metalloprotease large family, has been identified as a candidate 3p14.2 TSG inactivated by aberrant promoter CpG methylation in several carcinomas, but little known about its expression and function in breast cancer. In this report, ADAMTS9 expression and methylation was analysed in breast cancer cell lines and tissue samples. ADAMTS9 RNA was significantly down‐regulated in breast cancer cell lines (6/8). After treating the cells with demethylation agent Aza and TSA,ADAMTS9 expression was dramatically increased. Bisulphite genomic sequencing and methylation‐specific PCR detected promoter methylation, which was associated with decreased ADAMTS9 expression. Hypermethylation was also detected in 130/219 (59.4%) of primary tumours but only in 4.5% (2/44) of paired surgical margin tissues. Ectopic expression of ADAMTS9 in tumor cells induced significant growth suppression, cell cycle arrest at the G0/G1 phase, enhanced apoptosis and reduced cell migration and invasion. Conditioned culture medium from ADAMTS9‐transfected BT549 cells markedly disrupted tube formation ability of human umbilical vein endothelial cell (HUVEC) in Matrigel. Furthermore, ADAMTS9 inhibited AKT signaling and its downstream targets (MDM2, p53, p21, p27, E‐cadherin, VIM, SNAIL, VEGFA, NFκB‐p65 and MMP2). In addition, we demonstrated, for the first time, that ADAMTS9 inhibits AKT signaling, through suppressing its upstream activators EGFR and TGFβ1/TβR(I/II) in breast cancer cells. Our results suggest that ADAMTS9 is a TSG epigenetically inactivated in breast cancer, which functions through blocking EGFR‐ and TGFβ1/TβR(I/II)‐activated AKT signaling.

From a Better Understanding of the Mechanisms of Action of Histone Deacetylases Inhibitors to the Progress of the Treatment of Malignant Lymphomas and Plasma Cell Myeloma.

Notable progress has been made in chemo- and immunotherapy of B-cell lymphomas, but less in the treatment of T-cell lymphomas. Histone deacetylases inhibitors are a potentially useful therapeutic mean, as an epigenetic dysregulation is present in lymphomas, and especially in T-cell types. We aimed to study the progress made in this area. A mini-review was achieved using the articles published in PubMed in the last two years and the new patents made in this field. Histone deacetylases inhibitors are involved in the derepression of tumor suppressor genes through a histone deacetylase-mediated transcriptional process. Their inhibition is followed by cell cycle arrest, cell differentiation, apoptosis, sometimes autophagy, and a reversal of the transformed phenotype. They can also remove the resistance to chemo- or immunotherapy through different pathways. Some of them, as romidepsin, may decrease the protein level of multi-drug resistance associated protein 1, followed by a decrease in cellular drug export activity for DNA alkylating agents. Some compounds are approved for relapsed/refractory T-cell lymphomas or multiple myeloma treatment. The recent patents and the clinical trials with a histone deacetylases inhibitor administred in a synergistic drug combination with a demethylating, immunomodulatory, or anticancer agent as well as the discovery of more selective histone deacetylases inhibitors, with fewer side effects, could be a way to increase the treatment efficacy. New and more effective histone deacetylases inhibitors given alone or in drug combination are a solution for an improved response to the treatment of patients with relapsed/refractory lymphoproliferative disorders.

Methylatedclaudin-11associated with metastasis and poor survival of colorectal cancer

As one of crucial epigenetic modification, DNA methylation plays an important role during the carcinogenesis of colorectal cancer (CRC). In the current study, we used a human genome methylation array to detect the aberrant methylation genes in CRC. We further identified the hypermethylation of claudin-11 (CLDN11) and proved inverse correlation between CLDN11 methylation and its expression in CRC. In vitro experiments showed debased migration ability of colonic cancer cells in accompany with the converted methylation of CLDN11 after colonic cancer cells treated with demethylation agent, 5-aza-2’-deoxycytidine. Besides, our results also represented that hypermethylation of CLDN11 was associated with increased metastatic potential of CRC and with low progression free survival (PFS) of CRC. In conclusion, our findings supported that the hypermethylated CLDN11 is associated with metastasis of CRC and prognosis of poor survival of CRC.

Epigenetic mediated zinc finger protein 671 downregulation promotes cell proliferation and tumorigenicity in nasopharyngeal carcinoma by inhibiting cell cycle arrest

BackgroundEpigenetic abnormalities play important roles in nasopharyngeal cancer (NPC), however, the epigenetic changes associated with abnormal cell proliferation remain unclear.MethodsWe detected epigenetic change of ZNF671 in NPC tissues and cell lines by bisulfite pyrosequencing. We evaluated zinc finger protein 671 (ZNF671) expression in NPC cell lines and clinical tissues using real-time PCR and western blotting. Then, we established NPC cell lines that stably overexpressed ZNF671 and knocked down ZNF671 expression to explore its function in NPC in vitro and in vivo. Additionally, we investigated the potential mechanism of ZNF671 by identifying the mitotic spindle and G2/M checkpoint pathways pathway downstream genes using gene set enrichment analysis, flow cytometry and western blotting.ResultsZNF671 was hypermethylated in NPC tissues and cell lines. The mRNA and protein expression of ZNF671 was down-regulated in NPC tissues and cell lines and the mRNA expression could be upregulated after the demethylation agent 5-aza-2′-deoxycytidine treatment. Overexpression of ZNF671 suppressed NPC cell proliferation and colony formation in vitro; silencing ZNF671 using a siRNA had the opposite effects. Additionally, overexpression of ZNF671 reduced the tumorigenicity of NPC cells in xenograft model in vivo. The mechanism study determined that overexpressing ZNF671 induced S phase arrest in NPC cells by upregulating p21 and downregulating cyclin D1 and c-myc.ConclusionsEpigenetic mediated zinc finger protein 671 downregulation promotes cell proliferation and enhances tumorigenicity by inhibiting cell cycle arrest in NPC, which may represent a novel potential therapeutic target.

Mitochondrial DNA haplotypes induce differential patterns of DNA methylation that result in differential chromosomal gene expression patterns

Mitochondrial DNA copy number is strictly regulated during development as naive cells differentiate into mature cells to ensure that specific cell types have sufficient copies of mitochondrial DNA to perform their specialised functions. Mitochondrial DNA haplotypes are defined as specific regions of mitochondrial DNA that cluster with other mitochondrial sequences to show the phylogenetic origins of maternal lineages. Mitochondrial DNA haplotypes are associated with a range of phenotypes and disease. To understand how mitochondrial DNA haplotypes induce these characteristics, we used four embryonic stem cell lines that have the same set of chromosomes but possess different mitochondrial DNA haplotypes. We show that mitochondrial DNA haplotypes influence changes in chromosomal gene expression and affinity for nuclear-encoded mitochondrial DNA replication factors to modulate mitochondrial DNA copy number, two events that act synchronously during differentiation. Global DNA methylation analysis showed that each haplotype induces distinct DNA methylation patterns, which, when modulated by DNA demethylation agents, resulted in skewed gene expression patterns that highlight the effectiveness of the new DNA methylation patterns established by each haplotype. The haplotypes differentially regulate α-ketoglutarate, a metabolite from the TCA cycle that modulates the TET family of proteins, which catalyse the transition from 5-methylcytosine, indicative of DNA methylation, to 5-hydroxymethylcytosine, indicative of DNA demethylation. Our outcomes show that mitochondrial DNA haplotypes differentially modulate chromosomal gene expression patterns of naive and differentiating cells by establishing mitochondrial DNA haplotype-specific DNA methylation patterns.

Chidamide and decitabine can synergistically induce apoptosis of Hodgkin lymphoma cells by up-regulating the expression of PU.1 and KLF4.

Epigenetic abnormalities play important roles in the pathogenesis of Hodgkin lymphoma (HL). Highly expressed class I histone acetyltransferase (HDAC) and hyper-methylation of the promoter region of tumor suppressor genes have been demonstrated in Hodgkin lymphoma. In this paper, we investigated the synergistic effects of combination treatment of chidamide, a selective HDAC inhibitor, and decitabine, a demethylation agent, for HL cell lines and explored a new strategy for treating HL. The apoptosis rates, cell cycle, mitochondrial transmembrane potentials, epigenetic changes and gene expression of HL cell lines treated with chidamide as a single agent and in combination with decitabine were tested. We found that chidamide inhibited the proliferation of HL cells through increased apoptosis. Interestingly, after combined with decitabine, the inhibition rate and apoptotic death in HL cells were significantly increased. Further studies demonstrated that when combined with decitabine the expression of acetylated histone H3 and H3K9 induced by chidamide in HL cells increased, and also the expression of tumor suppressor genes PU.1 and KLF4, which exert inhibition through apoptosis pathway. Therefore, we could come to a conclusion that chidamide and decitabine can synergistically induce apoptosis of Hodgkin lymphoma cells by up-regulating the expression of PU.1 and KLF4. These results provide a new sight in combining two different epigenetic regulatory agents for treating HL.

Modulation of Mitochondrial DNA Copy Number to Induce Hepatocytic Differentiation of Human Amniotic Epithelial Cells.

Mitochondrial deoxyribonucleic acid (mtDNA) copy number is tightly regulated during pluripotency and differentiation. There is increased demand of cellular adenosine triphosphate (ATP) during differentiation for energy-intensive cell types such as hepatocytes and neurons to meet the cell's functional requirements. During hepatocyte differentiation, mtDNA copy number should be synchronously increased to generate sufficient ATP through oxidative phosphorylation. Unlike bone marrow mesenchymal cells, mtDNA copy number failed to increase by 28 days of differentiation of human amniotic epithelial cells (hAEC) into hepatocyte-like cells (HLC) despite their expression of some end-stage hepatic markers. This was due to higher levels of DNA methylation at exon 2 of POLGA, the mtDNA-specific replication factor. Treatment with a DNA demethylation agent, 5-azacytidine, resulted in increased mtDNA copy number, reduced DNA methylation at exon 2 of POLGA, and reduced hepatic gene expression. Depletion of mtDNA followed by subsequent differentiation did not increase mtDNA copy number, but reduced DNA methylation at exon 2 of POLGA and increased expression of hepatic and pluripotency genes. We encapsulated hAEC in barium alginate microcapsules and subsequently differentiated them into HLC. Encapsulation resulted in no net increase of mtDNA copy number but a significant reduction in DNA methylation of POLGA. RNAseq analysis showed that differentiated HLC express hepatocyte-specific genes but also increased expression of inflammatory interferon genes. Differentiation in encapsulated cells showed suppression of inflammatory genes as well as increased expression of genes associated with hepatocyte function pathways and networks. This study demonstrates that an increase in classical hepatic gene expression can be achieved in HLC through encapsulation, although they fail to effectively regulate mtDNA copy number.

Effects of acute stress and placebo intervention on subjective and objective nausea measures

Dysregulated expression of cystatin B (CSTB) has been implicated in various cancers. The aims of this study were to analyze the CSTB expression and investigate the epigenetic regulation of CSTB in lung and colon cancer cell lines, and also evaluate the clinical outcome of CSTB in primary lung and colorectal tumors.CSTB expression in lung and colon cancer cell lines was analyzed by real-time RT-PCR and western blotting. Epigenetic regulation of CSTB was examined by demethylation, deacetylation tests and bisulfite sequencing (BS). In primary lung and colorectal tumors, the protein expression of CSTB was evaluated by immunohistochemistry on tissue microarray.CSTB was downregulated in lung cancer cell lines on mRNA and protein levels compared to human bronchial epithelial cells (HBEC). In colon cancer cell lines, CSTB was weakly expressed in Caco2, CX2 and HCT-16 and highly expressed in HT-29, WiDr, SW480 and HRT-18 on mRNA level compared to normal colonic fibroblast cells CCD33Co. After treatment with demethylation agent 5-aza-2′-deoxycytidine, increased CSTB mRNA expression was found in 7 out of 11 lung cancer cell lines including H226, H157, H2170, H1299, COLO677, A549 and H1975, while no obvious alteration was found in colon cancer cell lines. No DNA methylation could be found in the selected CpG islands in two types of cancer cell lines by bisulfite sequencing. In primary tumors, CSTB expression was significantly and inversely correlated with lung tumor stage (pN) and tumor grade (p = 0.022 and 0.047, respectively). Kaplan-Meier survival curve revealed a tendency that lung tumors with high CSTB expression had a more favourable prognosis (p = 0.062). In colorectal tumors, CSTB was not linked to any clinicopathological parameters including age, size of tumor, lymph node metastasis and tumor grading.CSTB might be a potential prognostic marker for patients with primary lung cancer.

Expression and epigenetic regulation of cystatin B in lung cancer and colorectal cancer

AimsDysregulated expression of cystatin B (CSTB) has been implicated in various cancers. The aims of this study were to analyze the CSTB expression and investigate the epigenetic regulation of CSTB in lung and colon cancer cell lines, and also evaluate the clinical outcome of CSTB in primary lung and colorectal tumors. MethodsCSTB expression in lung and colon cancer cell lines was analyzed by real-time RT-PCR and western blotting. Epigenetic regulation of CSTB was examined by demethylation, deacetylation tests and bisulfite sequencing (BS). In primary lung and colorectal tumors, the protein expression of CSTB was evaluated by immunohistochemistry on tissue microarray. ResultsCSTB was downregulated in lung cancer cell lines on mRNA and protein levels compared to human bronchial epithelial cells (HBEC). In colon cancer cell lines, CSTB was weakly expressed in Caco2, CX2 and HCT-16 and highly expressed in HT-29, WiDr, SW480 and HRT-18 on mRNA level compared to normal colonic fibroblast cells CCD33Co. After treatment with demethylation agent 5-aza-2′-deoxycytidine, increased CSTB mRNA expression was found in 7 out of 11 lung cancer cell lines including H226, H157, H2170, H1299, COLO677, A549 and H1975, while no obvious alteration was found in colon cancer cell lines. No DNA methylation could be found in the selected CpG islands in two types of cancer cell lines by bisulfite sequencing. In primary tumors, CSTB expression was significantly and inversely correlated with lung tumor stage (pN) and tumor grade (p=0.022 and 0.047, respectively). Kaplan-Meier survival curve revealed a tendency that lung tumors with high CSTB expression had a more favourable prognosis (p=0.062). In colorectal tumors, CSTB was not linked to any clinicopathological parameters including age, size of tumor, lymph node metastasis and tumor grading. ConclusionsCSTB might be a potential prognostic marker for patients with primary lung cancer.

The safety, efficacy, and treatment outcomes of a combination of low-dose decitabine treatment in patients with recurrent ovarian cancer

ABSTRACTPurpose: DNA demethylating agents have shown clinical effectiveness in hematological and solid tumors. This trial tested the safety, efficacy, and treatment outcomes of decitabine-based chemotherapy or combined with immunotherapy in recurrent ovarian cancer patients.Patients and methods: Fifty-five patients with recurrent ovarian cancer were enrolled and 52 were assessable for clinical response and survival. Patients either received 5-d decitabine treatment, followed by reduced-dose of paclitaxel/carboplatin administration (DTC cohort), or the aforementioned regimen combined with cytokine-induced killer cells therapy (DTC+CIK cohort). The primary end point was clinical response rate and progression-free survival (PFS). Secondary evaluation included safety assessment and overall survival (OS).Results: Disease control rate (DCR) and objective response rate (ORR) were 73.91% and 23.91% in disease measurable patients by RECIST criteria, totally 76.92% and 30.77%, including disease non-measurable patients, which were higher in platinum-resistant/refractory patients. Clinical benefits could be associated with the number of DAC treatment cycles and the inclusion of CIK immunotherapy. In DTC+CIK cohort, DCR and ORR reached 100% and 58.30%, respectively. Notably, DTC+CIK treatment in platinum-resistant/refractory patients had an ORR of 87.50%. Consistently, PFS was longer in platinum-resistant/refractory patients comparing with that of platinum-sensitive patients. PFS and OS were 8 and 19 mo in platinum-resistant/refractory patients with DTC+CIK therapy. The most common toxicities were nausea, anorexia, fatigue, neutropenia, and anemia; many of which were grade 1–2.Conclusion: Low-dose DAC/paclitaxel/carboplatin regimen demonstrates disease benefit, especially in patients with platinum-resistant/refractory ovarian cancer, and might show remarkable clinical response when combined with adoptive immunotherapy in platinum-resistant/refractory ovarian cancer patients.

Restoring PU.1 induces apoptosis and modulates viral transactivation via interferon-stimulated genes in primary effusion lymphoma.

Primary effusion lymphoma (PEL), which is an aggressive subgroup of B-cell lymphoma associated with Kaposi sarcoma-associated herpes virus/human herpes virus-8, is refractory to the standard treatment, and exhibits a poor survival. Although PU.1 is downregulated in PEL, the potential role of its reduction remains to be elucidated. In this investigation, we analyzed the DNA methylation of PU.1 cis-regulatory elements in PEL and the effect of restoring PU.1 on PEL cells. The mRNA level of PU.1 was downregulated in PEL cells. The methylated promoter and enhancer regions of the PU.1 gene were detected in PEL cells. Suppression of cell growth and apoptosis were caused by the restoration of PU.1 in PEL cells. A microarray analysis revealed that interferon-stimulated genes (ISGs) including pro-apoptotic ISGs were strongly increased in BCBL-1 cells after the induction of PU.1. Reporter assays showed that PU.1 transactivated pro-apoptotic ISG promoters, such as the XAF1, OAS1 and TRAIL promoters. Mutations at the PU.1 binding sequences suppressed its transactivation. We confirmed the binding of PU.1 to the XAF1, OAS1 and TRAIL promoters in a chromatin immunoprecipitation assay. PU.1 suppressed ORF57 activation by inducing IRF7. The reinduction of PU.1 reduced formation of ascites and lymphoma cell infiltration of distant organs in PEL xenograft model mice. Collectively, PU.1 has a role in tumor suppression in PEL and its down-regulation is associated with PEL development. Restoring PU.1 with demethylation agents may be a novel therapeutic approach for PEL.