Expression pattern analysis of the MAGE family genes in breast cancer patients and hypomethylation activation in the MCF-7 cells.

Melanoma antigen gene (MAGE) families are cancer-testis genes that normally show expression in the testes. However, their expressions have been linked with various types of human cancers, including BC. Therefore, the primary purposes of the present research were to assess the expression of MAGE-A, -B, and -C genes in Saudi female patients with BC and determine their regulation via the epigenetic mechanism. Ten BC samples were analyzed for the expression levels of nine MAGE-A genes, six MAGE-B genes, and three MAGE-C genes using the RT-PCR technique. All 18 evaluated genes except for MAGE-A1, -A3, -A4, and -B5 showed weak band expressions in some BC specimens. MAGE-A6 and -B2 were expressed in 40 % of the BC tissue samples, and MAGE-A9, -A10, and -B6 were expressed in 30 %. The lowest expression levels were found for MAGE-A11, -B1, -B3, -B4, -C1, and -C2 in 10 % of the BC specimens and for MAGE-A9,--B2, and --C3 in 20 % of the samples. The most frequently expressed gene was MAGE-A8 (found in 70 % of the BC samples), which suggests that it may serve as - a marker for screening of BC. In vitro treatment, the 5-aza-2'-deoxycytidine agent led to a significant rise in mRNA expressions for all tested genes related to the MAGE-A family, except for MAGE-A10. By contrast, among the genes in the MAGE-B and -C families, only MAGE-B1 and -C2 exhibited detectable mRNA expression levels after treatment.

Unravelling the impact of RNA methylation genetic and epigenetic machinery in the treatment of cardiomyopathy.

Cardiomyopathy (CM) represents a heterogeneous group of diseases primarily affecting cardiac structure and function, with genetic and epigenetic dysregulation playing a pivotal role in its pathogenesis. Emerging evidence from the burgeoning field of epitranscriptomics has brought to light the significant impact of various RNA modifications, notably N6-methyladenosine (m6A), 5-methylcytosine (m5C), N7-methylguanosine (m7G), N1-methyladenosine (m1A), 2'-O-methylation (Nm), and 6,2'-O-dimethyladenosine (m6Am), on cardiomyocyte function and the broader processes of cardiac and vascular remodelling. These modifications have been shown to influence key pathological mechanisms including mitochondrial dysfunction, oxidative stress, cardiomyocyte apoptosis, inflammation, immune response, and myocardial fibrosis. Importantly, aberrations in the RNA methylation machinery have been observed in human CM cases and animal models, highlighting the critical role of RNA methylating enzymes and their potential as therapeutic targets or biomarkers for CM. This review underscores the necessity for a deeper understanding of RNA methylation processes in the context of CM, to illuminate novel therapeutic avenues and diagnostic tools, thereby addressing a significant gap in the current management strategies for this complex disease.

Mono-quinoxaline-induced DNA structural alteration leads to ZBP1/RIP3/MLKL-driven necroptosis in cancer cells.

Evading the cellular apoptosis mechanism by modulating multiple pathways poses a sturdy barrier to effective chemotherapy. Cancer cell adeptly resists the apoptosis signaling pathway by regulating anti and pro-apoptotic proteins to escape cell death. Nevertheless, bypassing the apoptotic pathway through necroptosis, an alternative programmed cell death process, maybe a potential therapeutic modality for apoptosis-resistant cells. However, synthetic mono-quinoxaline-based intercalator-induced cellular necroptosis as an anti-cancer perspective remains under-explored. To address this concern, we undertook the design and synthesis of quinoxaline-based small molecules (3a-3l). Our approach involved enhancing the π-surface of the mandatory benzyl moiety to augment its ability to induce DNA structural alteration via intercalation, thereby promoting cytotoxicity across various cancer cell lines (HCT116, HT-29, and HeLa). Notably, the potent compound 3a demonstrated the capacity to induce DNA damage in cancer cells, leading to the induction of ZBP1-mediated necroptosis in the RIP3-expressed cell line (HT-29), where Z-VAD effectively blocked apoptosis-mediated cell death. Interestingly, we observed that 3a induced RIP3-driven necroptosis in combination with DNA hypomethylating agents, even in the RIP3-silenced cell lines (HeLa and HCT116). Overall, our synthesized compound 3a emerged as a promising candidate against various cancers, particularly in apoptosis-compromised cells, through the induction of necroptosis.

Fusion of NY-ESO-1 epitope with heat shock protein 70 enhances its induced immune responses and antitumor activity against glioma in vitro.

Background: Glioma is the most common tumor originating in the brain and is difficult to cure. New York esophageal squamous cell carcinoma 1 (NY-ESO-1) is a promising cancer testis antigen (CTA) for tumor immunotherapy, and heat shock proteins (HSPs) can promote the antigen presentation of chaperoned peptides. This study investigates the therapeutic potential of HSP70 and NY-ESO-1 epitope fusion protein for glioma. Methods: Recombinant HSP70 protein was purified and fused to NY-ESO-1 epitope to generate HSP70/NY-ESO-1 p86-94. NY-ESO-1 expression was induced in U251 glioma cells via 5-Aza-2'-deoxycytidine (5-Aza-CdR) treatment. Dendritic cells (DCs) loaded with HSP70/NY-ESO-1 p86-94 or NY-ESO-1 protein stimulated NY-ESO-1-specific cytotoxic T lymphocytes (CTLs). The killing effect of NY-ESO-1 specific CTLs on U251 cells was detected by lactate dehydrogenase (LDH). Results: 5-Aza-CdR successfully induced NY-ESO-1 expression in U251 cells. NY-ESO-1-stimulated CTLs lysed more significantly with NY-ESO-1-positive U251 cells than with NY-ESO-1-negative cells. The immune response stimulated by a DC-based vaccine of HSP70/NY-ESO-1 p86-94 fusion protein was significantly enhanced compared with that induced by NY-ESO-1 alone. Conclusions: These findings indicate that the HSP70/NY-ESO-1 p86-94 may significantly enhance CTLs-mediated cytotoxicity and targeting ability against NY-ESO-1-expressing tumors in vitro. 5-Aza-CdR treatment with HSP70 binding to tumor antigen is a new strategy for immunotherapy of the tumors with poor CTA expression.

FTHL17, PRM2, CABYR, CPXCR1, ADAM29, and CABS1 are highly expressed in colon cancer patients and are regulated in vitro by epigenetic alterations.

Background: Colon cancer is a serious public health issue and a major cause of cancer-related mortality worldwide, including Saudi Arabia. Knowledge of genes associated with colon cancer development and progression is essential for identifying new cancer-specific biomarkers to improve the diagnosis of colon cancer. Methods: The expression levels of FTHL17, PRM2, CABYR, CPXCR1, ADAM29, and CABS1 in 15 adjacent colon cancer and normal colon tissue samples from male patients were investigated using reverse transcription polymerase chain reaction (RT-PCR) and quantitative RT-PCR (qRT-PCR) assays. qRT-PCR analysis was also used to determine whether reducing DNA methyltransferase (via 5-aza-2'-deoxycytidine treatment) or histone deacetylation (via trichostatin treatment) increased the expression levels of the tested genes. Results: The analysis of the 15 colon cancer and adjacent normal colon tissue samples revealed that all six genes were expressed in both groups, but their expression levels were significantly higher in the colon cancer group. Furthermore, the mRNA expression levels of the FTHL17, PRM2, CABYR, CPXCR1, and ADAM29 genes were considerably upregulated after treatment of HCT116 and Caco-2 cells with 5-aza-2'-deoxycytidine and trichostatin. However, the CABS1 gene was activated only with trichostatin treatment. Conclusions: The findings of this study suggest that FTHL17, PRM2, CABYR, CPXCR1, ADAM29, and CABS1 are suitable candidate biomarkers of colon cancer and their expressions are regulated by hypomethylation and hyperacetylation.

Hypermethylated ITGA8 Facilitate Bladder Cancer Cell Proliferation and Metastasis.

DNA methylation plays a vital role during the development of tumorigenesis. The purpose of this study is to identify candidate DNA methylation drivers during progression of bladder cancer (BLCA). The methylation spectrum in bladder cancer tissues was detected by CHARM analysis, and methylated ITGA8 was selected for further study due to its low expression. Methylation levels in BLCA tissues and cells were detected with methylated-specific PCR (MSP), while mRNA expression and methylation of ITGA8 were detected by qRT-PCR and MSP. After treatment with 5-Aza-dC (DNA methylation inhibitor), the proliferation, migration, and invasion abilities of BLCA cells were determined by MTT, wound healing, and transwell assays, respectively. Flow cytometric analysis was performed to evaluate any variance in the cell cycle. In addition, the effect of demethylated ITGA8 on BLCA tumor growth was verified with an in vivo xenograft tumor model. Based on the methylation profiling of BLCA, ITGA8 was identified to be hypermethylated. ITGA8 methylation levels in BLCA tissues and cells were upregulated, and 5-Aza-dC significantly suppressed ITGA8 methylation levels and increased ITGA8 mRNA expression. Furthermore, after treatment with 5-Aza-dC, the propagation, migration, and invasiveness of the cancer cells were inhibited, and more cancer cells were arrested at the G0/G1 phase. In vivo assays further demonstrated that 5-Aza-dC could impede BLCA tumor growth by repressing methylation levels of ITGA8 and increasing ITGA8 mRNA expression. Hypermethylated ITGA8 facilitated BLCA progression, and 5-Aza-dC treatment inhibited BLCA cell propagation and metastasis by decreasing methylation levels of ITGA8 and inducing cell cycle arrest.

5-Aza-2'-Deoxycytidine Alters the Methylation Profile of Bortezomib-Resistant U266 Multiple Myeloma Cells and Affects Their Proliferative Potential.

Multiple myeloma (MM) is a plasma cell malignancy that accounts for 1% of all cancers and is the second-most-common hematological neoplasm. Bortezomib (BTZ) is a proteasome inhibitor widely implemented in the treatment of MM alone or in combination with other agents. The development of resistance to chemotherapy is one of the greatest challenges of modern oncology. Therefore, it is crucial to discover and implement new adjuvant therapies that can bypass therapeutic resistance. In this paper, we investigated the in vitro effect of methylation inhibitor 5-Aza-2'-deoxycytidine on the proliferative potential of MM cells and the development of resistance to BTZ. We demonstrate that alterations in the DNA methylation profile are associated with BTZ resistance. Moreover, the addition of methylation inhibitor 5-Aza-2'-deoxycytidine to BTZ-resistant MM cells led to a reduction in the proliferation of the BTZ-resistant phenotype, resulting in the restoration of sensitivity to BTZ. However, further in vitro and ex vivo studies are required before adjuvant therapy can be incorporated into existing treatment regimens.

Combination of verteporfin-photodynamic therapy with 5-aza-2'-deoxycytidine enhances the anti-tumour immune response in triple negative breast cancer.

Introduction: Triple negative breast cancer (TNBC) is a subtype of breast cancer characterised by its high tumourigenic, invasive, and immunosuppressive nature. Photodynamic therapy (PDT) is a focal therapy that uses light to activate a photosensitizing agent and induce a cytotoxic effect. 5-aza-2'-deoxycytidine (5-ADC) is a clinically approved immunomodulatory chemotherapy agent. The mechanism of the combination therapy using PDT and 5-ADC in evoking an anti-tumour response is not fully understood. Methods: The present study examined whether a single dose of 5-ADC enhances the cytotoxic and anti-tumour immune effect of low dose PDT with verteporfin as the photosensitiser in a TNBC orthotopic syngeneic murine model, using the triple negative murine mammary tumour cell line 4T1. Histopathology analysis, digital pathology and immunohistochemistry of treated tumours and distant sites were assessed. Flow cytometry of splenic and breast tissue was used to identify T cell populations. Bioinformatics were used to identify tumour immune microenvironments related to TNBC patients. Results: Functional experiments showed that PDT was most effective when used in combination with 5-ADC to optimize its efficacy. 5-ADC/PDT combination therapy elicited a synergistic effect in vitro and was significantly more cytotoxic than monotherapies on 4T1 tumour cells. For tumour therapy, all types of treatments demonstrated histopathologically defined margins of necrosis, increased T cell expression in the spleen with absence of metastases or distant tissue destruction. Flow cytometry and digital pathology results showed significant increases in CD8 expressing cells with all treatments, whereas only the 5-ADC/PDT combination therapy showed increase in CD4 expression. Bioinformatics analysis of in silico publicly available TNBC data identified BCL3 and BCL2 as well as the following anti-tumour immune response biomarkers as significantly altered in TNBC compared to other breast cancer subtypes: GZMA, PRF1, CXCL1, CCL2, CCL4, and CCL5. Interestingly, molecular biomarker assays showed increase in anti-tumour response genes after treatment. The results showed concomitant increase in BCL3, with decrease in BCL2 expression in TNBC treatment. In addition, the treatments showed decrease in PRF1, CCL2, CCL4, and CCL5 genes with 5-ADC and 5-ADC/PDT treatment in both spleen and breast tissue, with the latter showing the most decrease. Discussion: To our knowledge, this is the first study that shows which of the innate and adaptive immune biomarkers are activated during PDT related treatment of the TNBC 4T1 mouse models. The results also indicate that some of the immune response biomarkers can be used to monitor the effectiveness of PDT treatment in TNBC murine model warranting further investigation in human subjects.

Epigenetic PPARγ preservation attenuates temporomandibular joint osteoarthritis.

OBJECTIVE: Previous studies have demonstrated that PPARγ deficiency is associated with osteoarthritis in the knee joint. However, whether epigenetic PPARγ dysregulation has any effect on temporomandibular joint osteoarthritis (TMJOA) is unknown. This study aims to determine the role and mechanism of epigenetic PPARγ dysregulation in TMJOA. METHODS: Partial TMJ discectomy was performed to induce TMJOA in rat. Primary condylar chondrocytes were isolated, and TNF-α-induced inflammatory condition was created in vitro. The expressions of PPARγ and DNA methyltransferase were investigated in vivo and in vitro. The association of PPARγ and DNA methylation was further studied by treating chondrocytes with DNA demethylation agent 5-Aza-2'-deoxycytidine (5Aza) and transfecting with siRNA of DNA methyltransferase (DNMT)1 and DNMT3a, and the methylation level of PPARγ promoter was evaluated by Bisulfite-sequencing PCR. The chondroprotective effects of 5Aza were explored in vitro and in vivo. RESULTS: PPARγ suppression and upregulated DNMT1/DNMT3a expression exist in TMJOA cartilage in vivo and primary condylar chondrocytes under TNF-α-induced inflammatory conditions in vitro. DNMT1 and DNMT3a elevation contributes to PPARγ-promoter hypermethylation in TMJ chondrocytes under TNF-α-induced inflammation conditions. DNA demethylation intervention by 5Aza protects chondrocytes from inflammation response in vitro. Mechanistically, 5Aza reversed the hypermethylation of the PPARγ promoter and subsequently resulted in PPARγ restoration and decreased expression of cartilage-catabolic factors in chondrocytes. Rat TMJOA model revealed that 5Aza, by reversing PPARγ suppression, effectively attenuated cartilage degeneration and stabilized cartilage homeostasis by balancing anabolic factor and catabolic factor expression. CONCLUSION: Epigenetic PPARγ suppression may play a causal role in TMJOA pathogenesis, which can be alleviated by DNA demethylation with 5Aza treatment. This study provides new insights into the pathogenic mechanism and therapeutic strategy of TMJOA.

Combination of a MIP3α-antigen fusion therapeutic DNA vaccine with treatments of IFNα and 5-Aza-2'Deoxycytidine enhances activated effector CD8+ T cells expressing CD11c in the B16F10 melanoma model.

Previous studies in the B16F10 mouse melanoma model have demonstrated that combining a DNA vaccine comprised of regions of gp100 and tyrosinase-related protein 2 fused to Macrophage-inflammatory protein 3-alpha (MIP3α) with recombinant Interferon alpha (IFN) and 5-Aza-2'-Deoxycytidine (5Aza) treatments resulted in significantly greater anti-tumor activity and immunogenicity in the tumor microenvironment (TME). This brief report details that the combination of vaccine with treatments IFN and 5Aza results in both the upregulation of genes expressing CD11c-interacting proteins and an increase in the TME of a distinct CD11c+ CD8+ T cell population. This cell population correlates with tumor size, is primarily comprised of effector or effector memory T cells, and has a more robust response to ex vivo stimulation as compared to CD11c- CD8+ T cells as measured by surface activation markers 4-1BB (CD137) and KLRG1 (Killer cell lectin-like receptor G1) and intracellular IFNγ production. In conclusion, this combination therapy results in greater presence of highly active effector CD8+ T-cells expressing CD11c in the TME that correlate with and are likely primary contributors to treatment efficacy.

Molecular predictors for decitabine efficacy in meningiomas - a pilot study.

PURPOSE: Effective chemotherapeutical agents for the treatment of meningiomas are still lacking. Previous in-vitro analyses revealed efficacy of decitabine (DCT), a DNA methyltransferase (DNMT) inhibitor established in the treatment of leukemia, in a yet undefined subgroup of meningiomas. METHODS: Effects of DCT on proliferation and viability was analyzed in primary meningioma cells by immunofluorescence and MTT assays, and cases were classified as drug responders and non-responders. Molecular preconditions for efficacy were analyzed using immunofluorescence for Ki67, DNMT1, and five oncogenes (TRIM58, FAM84B, ELOVL2, MAL2, LMO3) previously found to be differentially methylated after DCT exposition, as well as by genome-wide DNA methylation analyses. RESULTS: Efficacy of DCT (10µM) was found in eight (62%) of 13 meningioma cell lines 48 h after drug exposition (p < .05). DCT significantly reduced DNMT1 expression in all but two cell lines, and median ΔDNMT1 reduction 48 h after drug exposition was lower in DCT-resistant (-11.1%) than in DCT-sensitive (-50.5%, p = .030) cells. Rates of cell lines responsive to DCT exposition distinctly decreased to 25% after 72 h. No significant correlation of the patients´ age, sex, histological subtype, location of the paternal tumor, expression of Ki67, DNMT1 or the analyzed oncogenes with treatment response was found (p > .05, each). DCT efficacy was further independent of the methylation class and global DNA methylation of the paternal tumor. CONCLUSION: Early effects of DCT in meningiomas are strongly related with DNMT1 expression, while clinical, histological, and molecular predictors for efficacy are sparse. Kinetics of drug efficacy might indicate necessity of repeated exposition and encourage further analyses.

The Methylation Analysis of the Glucose-Dependent Insulinotropic Polypeptide Receptor (GIPR) Locus in GH-Secreting Pituitary Adenomas.

The glucose-dependent insulinotropic polypeptide receptor (GIPR) is aberrantly expressed in about one-third of GH-secreting pituitary adenomas (GH-PAs) and has been associated with a paradoxical increase of GH after a glucose load. The reason for such an overexpression has not yet been clarified. In this work, we aimed to evaluate whether locus-specific changes in DNA methylation patterns could contribute to this phenomenon. By cloning bisulfite-sequencing PCR, we compared the methylation pattern of the GIPR locus in GIPR-positive (GIPR+) and GIPR-negative (GIPR-) GH-PAs. Then, to assess the correlation between Gipr expression and locus methylation, we induced global DNA methylation changes by treating the lactosomatotroph GH3 cells with 5-aza-2'-deoxycytidine. Differences in methylation levels were observed between GIPR+ and GIPR- GH-PAs, both within the promoter (31.9% vs. 68.2%, p < 0.05) and at two gene body regions (GB_1 20.7% vs. 9.1%; GB_2 51.2% vs. 65.8%, p < 0.05). GH3 cells treated with 5-aza-2'-deoxycytidine showed a ~75% reduction in Gipr steady-state level, possibly associated with the observed decrease in CpGs methylation. These results indicate that epigenetic regulation affects GIPR expression in GH-PAs, even though this possibly represents only a part of a much more complex regulatory mechanism.

Higher Expression Levels of SSX1 and SSX2 in Patients with Colon Cancer: Regulated In Vitro by the Inhibition of Methylation and Histone Deacetylation.

Background and Objectives: Colon cancer (CC) has a high mortality rate and is often diagnosed at an advanced stage in Saudi Arabia. Thus, the identification and characterization of potential new cancer-specific biomarkers are imperative for improving the diagnosis of CC by detecting it at an early stage. Cancer-testis (CT) genes have been identified as potential biomarkers for the early diagnosis of various cancers. Among the CT genes are those belonging to the SSX family. In order to assess the usefulness of SSX family genes as cancer biomarkers for the detection of early-stage CC, the goal of this research was to validate the expressions of these genes in patients with CC and in matched patients with normal colons (NCs). Materials and Methods: RT-PCR assays were used to analyze the SSX1, SSX2, and SSX3 family gene expression levels in 30 neighboring NC and CC tissue samples from male Saudi patients. Epigenetic alterations were also tested in vitro using qRT-PCR analysis to determine whether reduced DNA methyltransferase or histone deacetylation could stimulate SSX gene expression via 5-aza-2'-deoxycytidine and trichostatin treatments, respectively. Results: The RT-PCR results showed SSX1 and SSX2 gene expression in 10% and 20% of the CC tissue specimens, respectively, but not in any of the NC tissue specimens. However, no SSX3 expression was detected in any of the examined CC or NC tissue samples. In addition, the qRT-PCR results showed significantly higher SSX1 and SSX2 expression levels in the CC tissue samples than in the NC tissue samples. The 5-aza-2'-deoxycytidine and trichostatin treatments significantly induced the mRNA expression levels of the SSX1, SSX2, and SSX3 genes in the CC cells in vitro. Conclusions: These findings suggest that SSX1 and SSX2 are potentially suitable candidate biomarkers for CC. Their expressions can be regulated via hypomethylating and histone deacetylase treatments, subsequently providing a potential therapeutic target for CC.

Up-Regulation of PSMA Expression In Vitro as Potential Application in Prostate Cancer Therapy.

Possibilities to improve the therapeutic efficacy of Lu-177-PSMA-617 radionuclide therapy by modulation of target expression are being investigated. Knowledge on regulatory factors that promote prostate cancer (PCa) progression may contribute to targeting prostate cancer more effectively. We aimed at the stimulation of PCa cell lines using the substances 5-aza-2'-deoxycitidine (5-aza-dC) and valproic acid (VPA) to achieve increased prostate-specific membrane antigen (PSMA) expression. PC3, PC3-PSMA, and LNCaP cells were incubated with varying concentrations of 5-aza-dC and VPA to investigate the cell-bound activity of Lu-177-PSMA-617. Stimulation effects on both the genetically modified cell line PC3-PSMA and the endogenously PSMA-expressing LNCaP cells were demonstrated by increased cellular uptake of the radioligand. For PC3-PSMA cells, the fraction of cell-bound radioactivity was enhanced by about 20-fold compared to that of the unstimulated cells. Our study reveals an increased radioligand uptake mediated by stimulation for both PC3-PSMA and LNCaP cell lines. In perspective of an enhanced PSMA expression, the present study might contribute to advanced radionuclide therapy approaches that improve the therapeutic efficacy, as well as combined treatment options.

Dataset on DNA methylation and gene expression changes induced by 5-aza-2'-deoxycytidine in Syrian golden hamster fetal cell cultures.

The Syrian hamster (SH) is an animal model used in virology, toxicology, and carcinogenesis, where a better understanding of epigenetic mechanisms is required. Finding genetic loci regulated by DNA methylation may assist in the development of DNA methylation-based in vitro assays for the identification of carcinogens. This dataset informs on the regulation of gene expression by DNA methylation. Primary cultures of SH male fetal cells (sex determined by differences in kdm5 loci on the X and Y chromosome) were exposed for 7 days to the carcinogen benzo[a]pyrene (20 µM) from which a morphologically transformed colony was collected and reseeded. The colony bypassed senescence and sustained growth. After 210 days of culture, the cells were collected and divided in 16 aliquots to create 4 experimental groups to test the effects of the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5adC). The experiment was initiated 24 h after cell seeding in 10 cm plates. The groups are naïve cells (N), cells exposed for 48 h to either 0.05% DMSO as vehicle (V), or to 5adC at 1 µM and 5 µM. DNA and RNA libraries were sequenced on an Illumina NextSeq 500. Gene expression was analysed by RNAseq and differentially methylated DNA regions (DMRs: clusters of 200 base pairs (bp), read depth >20, q< 0.05, methylation difference >|25%|) were identified by reduce representation bisulfite sequencing (RRBS). Global genome DNA methylation was similar between the N (mean±SD, 47.3%±0.02) and V groups (47.3%±0.01). Although 5adC reduced methylation, the reduction was larger in the 1 µM (39.2%±0.002) than in the 5 µM group (44.3%±0.01). 5adC induced a total of 612 and 190 DMRs by 1 µM and 5 µM, among which 79 and 23 were in the promoter regions (±3,000 bp from the transcription start site), respectively. 5adC induced a total of 1,170 and 1,797 differentially expressed genes (DEGs) by 1 µM and 5 µM, respectively. The 5 µM treatment induced statistically significant toxicity (% cell viability: group N 97%±8, V 98.8%±1.3, 1 µM 97.3%±0.5, 5 µM 93.8%±1.5), which perhaps reduced cell division and daughter cell numbers with inherited changes in methylation, but increased number of DEGs due to both toxicity and methylation changes. As usually observed in the literature, a small portion of DEGs (4% and 4% at 1 µM and 5 µM, respectively) are associated with DMRs in their promoters. These promoter DMRs by themselves are sufficient among other epigenetic marks to induce DEGs. The dataset provides the genomic coordinates of the DMRs and an opportunity to further examine their roles in distal putative promoters or enhancers (yet to be described in the SH) in contributing to gene expression changes, senescence bypass and sustained proliferation as essential carcinogenic events (see companion paper [1]). Finally, this experiment confirms the possibility in future experiments to use 5adC as a positive control for effects on DNA methylation in cells derived from SH.

High nuclear expression of DNMT1 in correlation with inactivation of TET1 portray worst prognosis among the cervical carcinoma patients: clinical implications.

In this study, we aimed to understand the interplay of the epigenetic modifier genes DNMT1 and TET1 along with HPV infection in the cervical epithelium and how it changes during tumorigenesis. For this purpose, initially the bioinformatical analysis (methylation and expression profile) of DNMT1 and TET1 was analyzed in the TCGA dataset. Next genetic (deletion) and epigenetic profiling (promoter methylation) of DNMT1 and TET1 were done in our sample pool and also validated in CACX cell lines as well. The results were further correlated with different clinicopathological parameters. Our data revealed that HPV infection in basal/parabasal layers of cervical epithelium actually disrupts the epigenetic homeostasis of DNMT1 and TET1 proteins which ultimately leads to the high expression of DNMT1 along with further reduction in TET1 protein during the development of carcinoma. Further, in-depth look into the results revealed that comparatively low methylation frequency of DNMT1 coupled with high promoter methylation and deletion frequency [22-46%] of TET1 were the plausible reasons of their antagonistic expression profile during the progression of the disease. Interestingly, the prevalence of DNMT1 [9.1%] and TET1 promoter methylation [22.7%] found in both the plasma DNA of the respective CACX patients implicated its diagnostic importance in this study. Lastly, molecular alteration of TET1 alone or in combination with DNMT1 showed the worst overall survival among the patients. Hence, it may be concluded that an inverse molecular profile of DNMT1 and TET1 genes seen in the proliferative basal-parabasal layers of the cervical epithelium was aggravated during the development of CACX along with genetic and epigenetic changes due to HPV infection.

DNA hypomethylation promotes learning and memory recovery in a rat model of cerebral ischemia/reperfusion injury.

Cerebral ischemia/reperfusion injury impairs learning and memory in patients. Studies have shown that synaptic function is involved in the formation and development of memory, and that DNA methylation plays a key role in the regulation of learning and memory. To investigate the role of DNA hypomethylation in cerebral ischemia/reperfusion injury, in this study, we established a rat model of cerebral ischemia/reperfusion injury by occlusion of the middle cerebral artery and then treated the rats with intraperitoneal 5-aza-2'-deoxycytidine, an inhibitor of DNA methylation. Our results showed that 5-aza-2'-deoxycytidine markedly improved the neurological function, and cognitive, social and spatial memory abilities, and dose-dependently increased the synaptic density and the expression of SYP and SHANK2 proteins in the hippocampus in a dose-dependent manner in rats with cerebral ischemia/reperfusion injury. The effects of 5-aza-2'-deoxycytidine were closely related to its reduction of genomic DNA methylation and DNA methylation at specific sites of the Syp and Shank2 genes in rats with cerebral ischemia/reperfusion injury. These findings suggest that inhibition of DNA methylation by 5-aza-2'-deoxycytidine promotes the recovery of learning and memory impairment in a rat model of cerebral ischemia/reperfusion injury. These results provide theoretical evidence for stroke treatment using epigenetic methods.

Comparison the effects of 5-Aza-2'-deoxycytidine and zebularine on the in vitro development, blastocyst quality, methylation pattern and conception rate on handmade cloned buffalo embryos.

In this study we treated the handmade cloned (HMC) buffalo embryos with the DNA methylation inhibitors; 5-aza-2'-deoxycytidine (AzadC) or Zebularine individually after post-fusion and during in vitro culture till eighth day. The blastocysts production rate significantly improved (p < .01) after treating embryos independently with 5 nM AzadC and 5 nM zebularine compared with 2 and 10 nM AzadC or zebularine groups, respectively. The highest cleavage rates were obtained for 5 nM treatment of AzadC and zebularine compared with other treatments and untreated control group. Quality of blastocysts were evaluated using total cell number (TCN) and the ratio of number of inner cell mass (ICM) cells/total cell number (ICM/TCN). Zebularine treatments (2/5/10 nM) significantly improved both TCN and ICM/TCN ratio compared with AzadC treatments (2/5/10 nM); however, control group TCN and ICM/TCN ratio was found lower. The methylation percentage of pDS4.1 and B. bubalis satellite DNA were comparatively more attenuated with 5 nM zebularine than 5 nM AzadC treatment. The increased in vitro development rates of the treated embryos were correlated with the decreased level of DNA methylation and the improved blastocyst quality. Following transfer of 5 nM zebularine treated embryos to 6 recipients, 4 were found to be pregnant, though the pregnancies were not carried to full term.

Epigenetic therapy reprograms M2-type tumor-associated macrophages into an M1-like phenotype by upregulating miR-7083-5p.

Reprogramming M2-type, pro-tumoral tumor-associated macrophages (TAMs) into M1-type, anti-tumoral macrophages is a key strategy in cancer therapy. In this study, we exploited epigenetic therapy using the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) and the histone deacetylation inhibitor trichostatin A (TSA), to reprogram M2-type macrophages into an M1-like phenotype. Treatment of M2-type macrophages with the combination of 5-aza-dC and TSA decreased the levels of M2 macrophage cytokines while increasing those of M1 macrophage cytokines, as compared to the use of either therapy alone. Conditioned medium of M2 macrophages treated with the combination of 5-aza-dC and TSA sensitized the tumor cells to paclitaxel. Moreover, treatment with the combination inhibited tumor growth and improved anti-tumor immunity in the tumor microenvironment. Depletion of macrophages reduced the anti-tumor growth activity of the combination therapy. Profiling of miRNAs revealed that the expression of miR-7083-5p was remarkably upregulated in M2 macrophages, following treatment with 5-aza-dC and TSA. Transfection of miR-7083-5p reprogrammed the M2-type macrophages towards an M1-like phenotype, and adoptive transfer of M2 macrophages pre-treated with miR-7083-5p into mice inhibited tumor growth. miR-7083-5p inhibited the expression of colony-stimulating factor 2 receptor alpha and CD43 as candidate targets. These results show that epigenetic therapy upon treatment with the combination of 5-aza-dC and TSA skews M2-type TAMs towards the M1-like phenotype by upregulating miR-7083-5p, which contributes to the inhibition of tumor growth.

SLC22A3 methylation-mediated gene silencing predicts adverse prognosis in acute myeloid leukemia.

BACKGROUND: We screened out several hypermethylated solute carrier (SLC) family genes in acute myeloid leukemia by reduced representation bisulfite sequencing. SLC22A3 encodes an organic cation transport protein, which is critical for drug transportation and cellular detoxification. SLC22A3 is significantly downregulated and associated with tumor progression and worse prognosis in a variety of solid tumors. However, there are no data available regarding the role of SLC22 in AML. This study aimed to explore the regulatory mechanism of DNA methylation on SLC22A3 expression, as well as its clinical significance in AML prognosis. RESULTS: SLC22A3 was identified as the sole prognosis-associated gene among SLCs based on TCGA and Beat AML databases. Bone marrow mononuclear cells (BMMNCs) from AML, MDS patients, and healthy donors were enrolled in this study. SLC22A3 methylation was significantly increased in AML compared with controls and MDS patients; meanwhile, the expression level of SLC22A3 was decreased. SLC22A3 hypermethylation presented an obvious association with some specific clinical characteristics and affected the survival time of AML patients as an independent risk indicator. SLC22A3 expression changed regularly as the disease complete remissions and relapses. Demethylation drug 5-aza-2'-deoxycytidine (DAC) activated transcription and increased mRNA expression of SLC22A3 in leukemia cell lines and AML fresh BMMNCs. Knockdown of SLC22A3 in leukemia cells enhanced cell proliferation and suppressed cell apoptosis. Data from public programs were used for auxiliary screening of probable molecular mechanisms of SLC22A3 in the antileukemia effect. CONCLUSIONS: Our results showed that increased methylation and decreased expression of SLC22A3 may be indicators of poor prognosis in AML. Methylation-silenced SLC22A3 expression may have potential guiding significance on antileukemia effect of DAC.