DNA methylation subtypes guiding prognostic assessment and linking to responses the DNA methyltransferase inhibitor SGI-110 in urothelial carcinoma.

BACKGROUND: At present, the extent and clinical relevance of epigenetic differences between upper tract urothelial carcinoma (UTUC) and urothelial carcinoma of the bladder (UCB) remain largely unknown. Here, we conducted a study to describe the global DNA methylation landscape of UTUC and UCB and to address the prognostic value of DNA methylation subtype and responses to the DNA methyltransferase inhibitor SGI-110 in urothelial carcinoma (UC). METHODS: Using whole-genome bisulfite sequencing (n = 49 samples), we analyzed epigenomic features and profiles of UTUC (n = 36) and UCB (n = 9). Next, we characterized potential links between DNA methylation, gene expression (n = 9 samples), and clinical outcomes. Then, we integrated an independent UTUC cohort (Fujii et al., n = 86) and UCB cohort (TCGA, n = 411) to validate the prognostic significance. Furthermore, we performed an integrative analysis of genome-wide DNA methylation and gene expression in two UC cell lines following transient DNA methyltransferase inhibitor SGI-110 treatment to identify potential epigenetic driver events that contribute to drug efficacy. RESULTS: We showed that UTUC and UCB have very similar DNA methylation profiles. Unsupervised DNA methylation classification identified two epi-clusters, Methy-High and Methy-Low, associated with distinct muscle-invasive statuses and patient outcomes. Methy-High samples were hypermethylated, immune-infiltrated, and enriched for exhausted T cells, with poor clinical outcome. SGI-110 inhibited the migration and invasion of Methy-High UC cell lines (UMUC-3 and T24) by upregulating multiple antitumor immune pathways. CONCLUSIONS: DNA methylation subtypes pave the way for predicting patient prognosis in UC. Our results provide mechanistic rationale for evaluating SGI-110 in treating UC patients in the clinic.

Dihydroartemisinin suppresses renal fibrosis in mice by inhibiting DNA-methyltransferase 1 and increasing Klotho.

Renal fibrosis is an unavoidable end result of all forms of progressive chronic kidney diseases (CKD). Discovery of efficacious drugs against renal fibrosis is in crucial need. In a preliminary study we found that a derivative of artemisinin, dihydroartemisinin (DHA), exerted strong renoprotection, and reversed renal fibrosis in adenine-induced CKD mouse model. In this study we investigated the anti-fibrotic mechanisms of DHA, particularly its specific target in renal cells. Renal fibrosis was induced in mice by unilateral ureteral obstruction (UUO) or oral administration of adenine (80 mg · kg-1), the mice received DHA (30 mg · kg-1 · d-1, i.g.) for 14 or 21 days, respectively. We showed that DHA administration markedly attenuated the inflammation and fibrotic responses in the kidneys and significantly improved the renal function in both the renal fibrosis mouse models. In adenine-treated mice, DHA was more effective than 5-azacytidine against renal fibrosis. The anti-fibrotic effects of DHA were also observed in TGF-ß1-treated HK-2 cells. In order to determine the target protein of DHA, we conducted pull-down technology coupled with shotgun proteomics using a small-molecule probe based on the structure of DHA (biotin-DHA). As a results, DNA methyltransferase 1 (DNMT1) was identified as the anti-fibrotic target of DHA in 3 different types of renal cell lines (HK-2, HEK293 and 3T3). We demonstrated that DHA directly bound to Asn 1529 and Thr 1528 of DNMT1 with a Kd value of 8.18 µM. In primary mouse renal tubular cells, we showed that DHA (10 µM) promoted DNMT1 degradation via the ubiquitin-proteasome pathway. DHA-reduced DNMT1 expression effectively reversed Klotho promoter hypermethylation, which led to the reversal of Klotho protein loss in the kidney of UUO mice. This subsequently resulted in inhibition of the Wnt/ß-catenin and TGF-ß/Smad signaling pathways and consequently conferred renoprotection in the animals. Knockdown of Klotho abolished the renoprotective effect of DHA in UUO mice. Our study reveals a novel pharmacological activity for DHA, i.e., renoprotection. DHA exhibits this effect by targeting DNMT1 to reverse Klotho repression. This study provides an evidence for the possible clinical application of DHA in the treatment of renal fibrosis.

Repurposing the Damage Repair Protein Methyl Guanine Methyl Transferase as a Ligand Inducible Fusion Degron.

We successfully repurpose the DNA repair protein methylguanine methyltransferase (MGMT) as an inducible degron for protein fusions. MGMT is a suicide protein that removes alkyl groups from the O6 position of guanine (O6G) and is thereafter quickly degraded by the ubiquitin proteasome pathway (UPP). Starting with MGMT pseudosubstrates (benzylguanine and lomeguatrib), we first demonstrate that these lead to potent MGMT depletion while affecting little else in the proteome. We then show that fusion proteins of MGMT undergo rapid UPP-dependent degradation in response to pseudosubstrates. Mechanistic studies confirm the involvement of the UPP, while revealing that at least two E3 ligase classes can degrade MGMT depending on cell-line and expression type (native or ectopic). We also demonstrate the technique's versatility with two clinically relevant examples: degradation of KRASG12C and a chimeric antigen receptor.

5-aza-2'-deoxycytidine potentiates anti-tumor immunity in colorectal peritoneal metastasis by modulating ABC A9-mediated cholesterol accumulation in macrophages.

Background: 5-aza-2'-deoxycytidine (5Aza), a DNA methyltransferase (DNMT) inhibitor, could activate tumor adaptive immunity to inhibit tumor progression. However, the molecular mechanisms by which 5Aza regulates tumor immune microenvironment are still not fully understood. Methods: The role of 5Aza in immune microenvironment of peritoneal carcinomatosis (PC) of colorectal cancer (CRC) was investigated. The effects of 5Aza on macrophage activation were studied by flow cytometry, real-time PCR, Western blotting assays, and Drug Affinity Responsive Target Stability (DARTS). The effects of 5Aza on tumor immunity were validated in stromal macrophages and T cells from CRC patients. Results: 5Aza could stimulate the activation of macrophages toward an M1-like phenotype and subsequent activation of T cells in premetastatic fat tissues, and ultimately suppress CRC-PC in immune-competent mouse models. Mechanistically, 5Aza stimulated primary mouse macrophages toward to a M1-like phenotype characterized by the increase of p65 phosphorylation and IL-6 expression. Furthermore, we screened and identified ATP-binding cassette transporter A9 (ABC A9) as a binding target of 5Aza. 5Aza induced cholesterol accumulation, p65 phosphorylation and IL-6 expression in an ABC A9-dependent manner. Pharmacological inhibition of NF-κB, or genetic depletion of IL-6 abolished the antitumor effect of 5Aza in mice. In addition, the antitumor effect of 5Aza was synergistically potentiated by conventional chemotherapeutic drugs 5-Fu or OXP. Finally, we validated the reprogramming role of 5Aza in antitumor immunity in stromal macrophages and T cells from CRC patients. Conclusions: Taken together, our findings showed for the first time that 5Aza suppressed CRC-PC by regulating macrophage-dependent T cell activation in premetastatic microenvironment, meanwhile uncovered a DNA methylation-independent mechanism of 5Aza in regulating ABC A9-associated cholesterol metabolism and macrophage activation.

Acridine-O6-benzylguanine hybrids: Synthesis, DNA binding, MGMT inhibition and antiproliferative activity.

O6-Methylguanine-DNA-methyltransferase (MGMT) is a key DNA repair enzyme involved in chemoresistance to DNA-alkylating anti-cancer drugs such as Temozolomide (TMZ) through direct repair of drug-induced O6-methylguanine residues in DNA. MGMT substrate analogues, such as O6-benzylguanine (BG), efficiently inactivate MGMT in vitro and in cells; however, these drugs failed to reach the clinic due to adverse side effects. Here, we designed hybrid drugs combining a BG residue covalently linked to a DNA-interacting moiety (6-chloro-2-methoxy-9-aminoacridine). Specifically, two series of hybrids, encompassing three compounds each, were obtained by varying the position of the attachment point of BG (N9 of guanine vs. the benzyl group) and the length and nature of the linker. UV/vis absorption and fluorescence data indicate that all six hybrids adopt an intramolecularly stacked conformation in aqueous solutions in a wide range of temperatures. All hybrids interact with double-stranded DNA, as clearly evidenced by spectrophotometric titrations, without intercalation of the acridine ring and do not induce thermal stabilization of the duplex. All hybrids, as well as the reference DNA intercalator (6-chloro-2-methoxy-9-aminoacridine 8), irreversibly inhibit MGMT in vitro with variable efficiency, comparable to that of BG. In a multidrug-resistant glioblastoma cell line T98G, benzyl-linked hybrids 7a-c and the N9-linked hybrid 19b are moderately cytotoxic (GI50 ≥ 15 µM after 96 h), while N9-linked hybrids 19a and 19c are strongly cytotoxic (GI50 = 1-2 µM), similarly to acridine 8 (GI50 = 0.6 µM). Among all compounds, hybrids 19a and 19c, similarly to BG, display synergic cytotoxic effect upon co-treatment with subtoxic doses of TMZ, with combination index (CI) values as low as 0.2-0.3. In agreement with in vitro results, compound 19a inactivates cellular MGMT but, unlike BG, does not induce significant levels of DNA damage, either alone or in combination with TMZ, as indicated by the results of γH2AX immunostaining experiments. Instead, and unlike BG, compound 19a alone induces significant apoptosis of T98G cells, which is not further increased in a combination with TMZ. These results indicate that molecular mechanisms underlying the cytotoxicity of 19a and its combination with TMZ are distinct from that of BG. The strongly synergic properties of this combination represent an interesting therapeutic opportunity in treating TMZ-resistant cancers.

High-Throughput-Methyl-Reading (HTMR) assay: a solution based on nucleotide methyl-binding proteins enables large-scale screening for DNA/RNA methyltransferases and demethylases.

Epigenetic therapy has significant potential for cancer treatment. However, few small potent molecules have been identified against DNA or RNA modification regulatory proteins. Current approaches for activity detection of DNA/RNA methyltransferases and demethylases are time-consuming and labor-intensive, making it difficult to subject them to high-throughput screening. Here, we developed a fluorescence polarization-based 'High-Throughput Methyl Reading' (HTMR) assay to implement large-scale compound screening for DNA/RNA methyltransferases and demethylases-DNMTs, TETs, ALKBH5 and METTL3/METTL14. This assay is simple to perform in a mix-and-read manner by adding the methyl-binding proteins MBD1 or YTHDF1. The proteins can be used to distinguish FAM-labelled substrates or product oligonucleotides with different methylation statuses catalyzed by enzymes. Therefore, the extent of the enzymatic reactions can be coupled with the variation of FP binding signals. Furthermore, this assay can be effectively used to conduct a cofactor competition study. Based on the assay, we identified two natural products as candidate compounds for DNMT1 and ALKBH5. In summary, this study outlines a powerful homogeneous approach for high-throughput screening and evaluating enzymatic activity for DNA/RNA methyltransferases and demethylases that is cheap, easy, quick, and highly sensitive.

Development and biological evaluation of AzoBGNU: A novel hypoxia-activated DNA crosslinking prodrug with AGT-inhibitory activity.

Chloroethylnitrosoureas (CENUs) are an important family of chemotherapies in clinical treatment of cancers, which exert antitumor activity by inducing the formation of DNA interstrand crosslinks (dG-dC ICLs). However, the drug resistance mediated by O6-alkylguanine-DNA alkyltransferase (AGT) and absence of tumor-targeting ability largely decrease the antitumor efficacy of CENUs. In this study, we synthesized an azobenzene-based hypoxia-activated combi-nitrosourea prodrug, AzoBGNU, and evaluated its hypoxic selectivity and antitumor activity. The prodrug was composed of a CENU pharmacophore and an O6-benzylguanine (O6-BG) analog moiety masked by a N,N-dimethyl-4-(phenyldiazenyl)aniline segment as a hypoxia-activated trigger, which was designed to be selectively reduced via azo bond break in hypoxic tumor microenvironment, accompanied with releasing of an O6-BG analog to inhibit AGT and a chloroethylating agent to induce dG-dC ICLs. AzoBGNU exhibited significantly increased cytotoxicity and apoptosis-inducing ability toward DU145 cells under hypoxia compared with normoxia, indicating the hypoxia-responsiveness as expected. Predominant higher cytotoxicity was observed in the cells treated by AzoBGNU than those by traditional CENU chemotherapy ACNU and its combination with O6-BG. The levels of dG-dC ICLs in DU145 cells induced by AzoBGNU was remarkably enhanced under hypoxia, which was approximately 6-fold higher than those in the AzoBGNU-treated groups under normoxia and those in the ACNU-treated groups. The results demonstrated that azobenzene-based combi-nitrosourea prodrug possessed desirable tumor-hypoxia targeting ability and eliminated chemoresistance compared with the conventional CENUs.

Inhibition of DNA methylation in newborns reprograms ischemia-sensitive biomarkers resulting in development of a heart ischemia-sensitive phenotype late in life.

Adverse environmental stress exposure at critical perinatal stages can alter cardiovascular development, which could persist into adulthood and develop a cardiovascular dysfunctional phenotype late in life. However, the underlying molecular mechanisms remain largely unknown. The present study provided a direct evidence that DNA methylation is a key epigenetic mechanism contributing to the developmental origins of adult cardiovascular disease. We hypothesized that DNA hypomethylation at neonatal stage alters gene expression patterns in the heart, leading to development of a cardiac ischemia-sensitive phenotype late in life. To test this hypothesis, a DNA methylation inhibitor 5-Aza-2-deoxycytidine (5-Aza) was administered in newborn rats from postnatal day 1-3. Cardiac function and related key genes were measured in 2-week- and 2-month-old animals, respectively. 5-Aza treatment induced an age- and sex-dependent inhibition of global and gene-specific DNA methylation levels in left ventricles, resulting in a long-lasting growth restriction but an asymmetry increase in the heart-to-body weight ratio. In addition, treatment with 5-Aza enhanced ischemia and reperfusion-induced cardiac dysfunction and injury in adults as compared with the saline controls, which was associated with up-regulations of miRNA-181a and angiotensin II receptor type 1 & 2 gene expressions, but down-regulations of PKCε, Atg5, and GSK3ß gene expressions in left ventricles. In conclusion, our results provide compelling evidence that neonatal DNA methylation deficiency is a key mechanism contributing to differentially reprogram cardiac gene expression patterns, leading to development of a heart ischemia-sensitive phenotype late in life.

Targeting cancer epigenetic pathways with small-molecule compounds: Therapeutic efficacy and combination therapies.

Epigenetics mainly refers to covalent modifications to DNA or histones without affecting genomes, which ultimately lead to phenotypic changes in cells or organisms. Given the abundance of regulatory targets in epigenetic pathways and their pivotal roles in tumorigenesis and drug resistance, the development of epigenetic drugs holds a great promise for the current cancer therapy. However, lack of potent, selective, and clinically tractable small-molecule compounds makes the strategy to target cancer epigenetic pathways still challenging. Therefore, this review focuses on epigenetic pathways, small molecule inhibitors targeting DNA methyltransferase (DNMT) and small molecule inhibitors targeting histone modification (the main regulatory targets are histone acetyltransferases (HAT), histone deacetylases (HDACs) and histone methyltransferases (HMTS)), as well as the combination strategies of the existing epigenetic therapeutic drugs and more new therapies to improve the efficacy, which will shed light on a new clue on discovery of more small-molecule drugs targeting cancer epigenetic pathways as promising strategies in the future.

Epigenetics and therapeutic targets in gastrointestinal malignancies.

Gastrointestinal (GI) malignancies account for substantial mortality and morbidity worldwide. They are generally promoted by dysregulated signal transduction and epigenetic pathways, which are controlled by specific enzymes. Recent studies demonstrated that histone deacetylases (HDACs) together with DNA methyltransferases (DNMTs) have crucial roles in the signal transduction/epigenetic pathways in GI regulation. In this review, we discuss various enzyme targets and their functional mechanisms responsible for the regulatory processes of GI malignancies. We also discuss the epigenetic therapeutic targets that are mainly facilitated by DNMT and HDAC inhibitors, which have functional consequences and clinical outcomes for GI malignancies.

MGMT-inhibitor in combination with TGF-BRI inhibitor or CDK 4/6 inhibitor increases temozolomide sensitivity in temozolomide-resistant glioblastoma cells

Background Glioblastoma (GB) remains an incurable and deadly brain malignancy that often proves resistant to upfront treatment with temozolomide. Nevertheless, temozolomide remains the most commonly prescribed FDA-approved chemotherapy for GB. The DNA repair protein methylguanine-DNA methyl transferase (MGMT) confers resistance to temozolomide. Unsurprisingly temozolomide-resistant tumors tend to possess elevated MGMT protein levels or lack inhibitory MGMT promotor methylation. In this study, cultured human temozolomide resistance GB (43RG) cells were introduced to the MGMT inhibitor O6-benzylguanine combined with temozolomide and either LY2835219 (CDK 4/6 inhibitor) or LY2157299 (TGF-βRI inhibitor) seeking to overcome GB treatment resistance. Methods Treatment effects were assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, western blot, cell viability, and cell cycle progression. Results Our in vitro study demonstrated that sequential treatment of O6-Benzylguanine with either LY2385219 or LY2157299-enhanced temozolomide enhanced sensitivity in MGMT+ 43RG cells. Importantly, normal human neurons and astrocytes remained impervious to the drug therapies under these conditions. Furthermore, LY2835219 has additional anti-proliferative effects on cell cycling, including induction of an RB-associated G (1) arrest via suppression of cyclin D-CDK4/6-Rb pathway. LY2157299 enhances anti-tumor effect by disrupting TGF-β–dependent HIF-1α signaling and by activating both Smad and PI3K-AKT pathways towards transcription of S/G2 checkpoints. Conclusion This study establishes the groundwork for the development of a combinatorial pharmacologic approach by using either LY2385219 or LY2157299 inhibitor plus O6-Benzylguanine to augment temozolomide response in temozolomide-resistant GB cells (AU)

5-Aza-2'-deoxycytidine induces a greater inflammatory change, at the molecular levels, in normoxic than hypoxic tumor microenvironment.

Hypoxia is associated with tumor aggressiveness and poor prognosis, including breast cancer. Low oxygen levels induces global genomic hypomethylation and hypermethylation of specific loci in tumor cells. DNA methylation is a reversible epigenetic modification, usually associated with gene silencing, contributing to carcinogenesis and tumor progression. Since the effects of DNA methyltransferase inhibitor are context-dependent and as there is little data comparing their molecular effects in normoxic and hypoxic microenvironments in breast cancer, this study aimed to understand the gene expression profiles and molecular effects in response to treatment with DNA methyltransferase inhibitor in normoxia and hypoxia, using the breast cancer model. For this, a cDNA microarray was used to analyze the changes in the transcriptome upon treatment with DNA methyltransferase inhibitor (5-Aza-2'-deoxycytidine: 5-Aza-2'-dC), in normoxia and hypoxia. Furthermore, immunocytochemistry was performed to investigate the effect of 5-Aza-2'-dC on NF-κB/p65 inflammation regulator subcellular localization and expression, in normoxia and hypoxia conditions. We observed that proinflammatory pathways were upregulated by treatment with 5-Aza-2'-dC, in both conditions. However, treatment with 5-Aza-2'-dC in normoxia showed a greater amount of overexpressed proinflammatory pathways than 5-Aza-2'-dC in hypoxia. In this sense, we observed that the NF-κB expression increased only upon 5-Aza-2'-dC in normoxia. Moreover, nuclear staining for NF-κB and NF-κB target genes upregulation, IL1A and IL1B, were also observed after 5-Aza-2'-dC in normoxia. Our results suggest that 5-Aza-2'-dC induces a greater inflammatory change, at the molecular levels, in normoxic than hypoxic tumor microenvironment. These data may support further studies and expand the understanding of the DNA methyltransferase inhibitor effects in different tumor contexts.

The Ubiquitin-Proteasome Pathway and Epigenetic Modifications in Cancer.

BACKGROUND: The ubiquitin-proteasome pathway is involved in almost all cellular processes (cell cycle, gene transcription and translation, cell survival and apoptosis, cell metabolism and protein quality control) mainly through the specific degradation of the majority of intracellular proteins (>80%) or partial processing of transcription factors (e.g., NF-κB). A growing amount of evidence now indicates that epigenetic changes are also regulated by the ubiquitin-proteasome pathway. Recent studies indicate that epigenetic regulations are equally crucial for almost all biological processes as well as for pathological conditions such as tumorigenesis, as compared to non-epigenetic control mechanisms (i.e., genetic alterations or classical signal transduction pathways). OBJECTIVE: Here, we reviewed the recent work highlighting the interaction of the ubiquitin-proteasome pathway components (e.g., ubiquitin, E1, E2 and E3 enzymes and 26S proteasome) with epigenetic regulators (histone deacetylases, histone acetyltransferases and DNA methyltransferases). RESULTS: Alterations in the regulation of the ubiquitin-proteasome pathway have been discovered in many pathological conditions. For example, a 2- to 32-fold increase in proteasomal activity and/or subunits has been noted in primary breast cancer cells. Although proteasome inhibitors have been successfully applied in the treatment of hematological malignancies (e.g., multiple myeloma), the clinical efficacy of the proteasomal inhibition is limited in solid cancers. Interestingly, recent studies show that the ubiquitin-proteasome and epigenetic pathways intersect in a number of ways through the regulation of epigenetic marks (i.e., acetylation, methylation and ubiquitylation). CONCLUSION: It is therefore believed that novel treatment strategies involving new generation ubiquitinproteasome pathway inhibitors combined with DNA methyltransferase, histone deacetylase or histone acetyltransferase inhibitors may produce more effective results with fewer adverse effects in cancer treatment as compared to standard chemotherapeutics in hematological as well as solid cancers.

MGMT-inhibitor in combination with TGF-ßRI inhibitor or CDK 4/6 inhibitor increases temozolomide sensitivity in temozolomide-resistant glioblastoma cells.

BACKGROUND: Glioblastoma (GB) remains an incurable and deadly brain malignancy that often proves resistant to upfront treatment with temozolomide. Nevertheless, temozolomide remains the most commonly prescribed FDA-approved chemotherapy for GB. The DNA repair protein methylguanine-DNA methyl transferase (MGMT) confers resistance to temozolomide. Unsurprisingly temozolomide-resistant tumors tend to possess elevated MGMT protein levels or lack inhibitory MGMT promotor methylation. In this study, cultured human temozolomide resistance GB (43RG) cells were introduced to the MGMT inhibitor O6-benzylguanine combined with temozolomide and either LY2835219 (CDK 4/6 inhibitor) or LY2157299 (TGF-ßRI inhibitor) seeking to overcome GB treatment resistance. METHODS: Treatment effects were assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, western blot, cell viability, and cell cycle progression. RESULTS: Our in vitro study demonstrated that sequential treatment of O6-Benzylguanine with either LY2385219 or LY2157299-enhanced temozolomide enhanced sensitivity in MGMT+ 43RG cells. Importantly, normal human neurons and astrocytes remained impervious to the drug therapies under these conditions. Furthermore, LY2835219 has additional anti-proliferative effects on cell cycling, including induction of an RB-associated G (1) arrest via suppression of cyclin D-CDK4/6-Rb pathway. LY2157299 enhances anti-tumor effect by disrupting TGF-ß-dependent HIF-1α signaling and by activating both Smad and PI3K-AKT pathways towards transcription of S/G2 checkpoints. CONCLUSION: This study establishes the groundwork for the development of a combinatorial pharmacologic approach by using either LY2385219 or LY2157299 inhibitor plus O6-Benzylguanine to augment temozolomide response in temozolomide-resistant GB cells.

Azacitidine, as a DNMT Inhibitor Decreases hTERT Gene Expression and Telomerase Activity More Effective Compared with HDAC Inhibitor in Human Head and Neck Squamous Cell Carcinoma Cell Lines.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is one of the most fatal malignancies worldwide and despite using various therapeutic strategies for the treatment of HNSCC, the surveillance rate is low. Telomerase has been remarked as the primary target in cancer therapy. Considering the key regulatory role of epigenetic mechanisms in controlling genome expression, the present study aimed to investigate the effects of two epigenetic modulators, a DNA methylation inhibitor and a histone deacetylase inhibitor on cell migration, proliferation, hTERT gene expression, and telomerase activity in HNSCC cell lines. METHODS: Human HNSCC cell lines were treated with Azacitidine and Trichostatin A to investigate their effects on telomerase gene expression and activity. Cell viability, migration, hTERT gene expression, and telomerase activity were studied using MTT colorimetric assay, scratch wound assay, qRT-PCR, and TRAP assay, respectively. RESULTS: Azacitidine at concentrations of ≤1µM and Trichostatin A at 0.1 to 0.3nM concentrations significantly decreased FaDu and Cal-27 cells migration. The results showed that Azacitidine significantly decreased hTERT gene expression and telomerase activity in FaDu and Cal-27 cell lines. However, there were no significant changes in hTERT gene expression at different concentrations of Trichostatin A in both cell lines. Trichostatin A treatment affected telomerase activity at the high dose of 0.3 nM Trichostatin A. CONCLUSION: The findings revealed that unlike histone deacetylase inhibitor, Azacitidine as an inhibitor of DNA methylation decreases telomerase expression in HNSCC cells. This might suggest the potential role of DNA methyltransferase inhibitors in telomerase-based therapeutic approaches in squamous cell carcinoma.

Combination of levetiracetam and IFN-α increased temozolomide efficacy in MGMT-positive glioma.

PURPOSE: Glioma, especially glioblastoma (GBM), is the most aggressive malignant brain tumor and its standard therapy is often ineffective because of temozolomide (TMZ) resistance. Reversal of the TMZ resistance might improve the prognosis of glioma patients. We previously found that interferon-α (IFN-α) and anti-epileptic drug levetiracetam (LEV) could sensitize glioma to TMZ, respectively. In this study, we further investigated the efficiency of combining of LEV and IFN-α for improving the efficacy of TMZ. METHODS: We evaluated whether LEV and IFN-α could increase TMZ efficacy using colony formation assay and cell viability assay with MGMT-positive and MGMT-negative glioma cell lines in vitro. Subcutaneous xenografts and orthotopic xenografts mice models were used in vivo to observe the tumor growth and mice survival upon treatments with TMZ, TMZ + IFN-α, TMZ + LEV, or TMZ + LEV + IFN-α. The expression levels of MGMT, markers of pro-apoptotic and anti-apoptotic in tumor samples were analyzed by Western blotting. RESULTS: The combinational use of IFN-α, LEV, and TMZ showed the best anti-tumor activity in MGMT-positive cell lines (U138, GSC-1, U118, and T98 G). TMZ + LEV + IFN-α further obviously increased TMZ + LEV or TMZ + IFN-α efficiency in MGMT-positive cell lines, while not in negative cell lines (SKMG-4, U87, U373, and U251) in vitro, which were also observed in subcutaneous mice models (U138, GSC-1 compared to SKMG-4, U87) and orthotopic models (GSC-1) in vivo. Strikingly, the combination of LEV and IFN-α together with TMZ significantly prolonged the survival of mice with orthotopic GSC-1 glioma. Furthermore, we confirmed that the combination of LEV and IFN-α enhanced the inhibition of MGMT and the activation of apoptosis in U138 tumor on the basis of TMZ treatment. CONCLUSIONS: The combination use of LEV and IFN-α could be an optimal method to overcome TMZ resistance through obvious MGMT inhibition in MGMT-positive glioma.

Demetylation of the sex-determining region Y gene promoter and incidence of disorder of sex development in cloned dog males.

Canine cloning is occasionally accompanied by abnormal sexual development. Some male donor cells produce cloned pups with female external genitalia and complete male gonadal dysgenesis, which is classified as an XY disorder of sex development (XY DSD). In this study, we examine the potential of 5-aza-2'-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, to reduce the phenotypic abnormality XY DSD in somatic cell nuclear transfer (SCNT)- derived pups. We used a 9-year-old normal male German Shepherd dog as a cell donor. Donor cells were treated with 10 nM 5-aza-dC for 4 days before being used for SCNT. At the same stage of cell development, significantly lower levels of DNA methylation of the sex-determining region Y (SRY) promoter was observed in the treated donor cells compared to that in the untreated cells (95.2% versus 53.3% on day 4 for the control and treated groups, respectively). No significant differences were observed in the control or treatment groups concerning fusion rate, pregnancy rate (30 days or entire period), the number of pups, or the incidence of XY DSD. However, more XY DSD dogs were observed in the control group (31.25%) than in the treatment group (14.29%). Hypermethylation of the SRY promoter was observed in the XY DSD cloned pups in both the treatment (84.8%) and control groups (91.1 ± 1.4%) compared to the methylation level in the phenotypically normal male pups of the treatment (23.2 ± 20.9%) and control groups (39.1 ± 20.1%). These results suggest that 5-aza-dC treatment of donor cells can reduce the methylation level of the SRY promoter in donor cells, and thus, 5-aza-dC is advantageous for reducing the incidence of XY DSD in canine cloning.

A Methyltransferase Inhibitor (Decitabine) Alleviates Intergenerational Effects of Paternal Neonatal Exposure to Anesthesia With Sevoflurane.

BACKGROUND: Neonatal exposure to sevoflurane induces neurobehavioral and neuroendocrine abnormalities in exposed male rats (generation F0) and neurobehavioral, but not neuroendocrine, abnormalities in their male, but not female, offspring (generation F1). These effects of sevoflurane are accompanied by a hypermethylated neuron-specific K-2Cl (Kcc2) Cl exporter gene in the F0 spermatozoa and the F1 male hypothalamus, while the gene's expression is reduced in the F0 and F1 hypothalamus. We investigated whether inhibition of deoxyribonucleic acid methyltransferases (DNMTs) before paternal sevoflurane exposure could alleviate the anesthetic's F0 and F1 effects. METHODS: Sprague-Dawley male rats were anesthetized with 2.1% sevoflurane for 5 hours on postnatal day (P) 5 and mated with control females on P90 to generate offspring. The nonselective DNMT inhibitor decitabine (0.5 mg/kg, intraperitoneally) was administered 30 minutes before sevoflurane exposure. The F0 and F1 male rats were evaluated in in vivo and in vitro tests in adulthood. RESULTS: Paternal exposure to sevoflurane induced impaired prepulse inhibition of the acoustic startle response and exacerbated corticosterone responses to stress in F0 males and impaired prepulse inhibition of the startle responses in F1 males. These effects were accompanied in both generations by reduced and increased expressions of hypothalamic Kcc2 and Dnmt3a/b, respectively. Decitabine deterred the effects of paternal exposure to sevoflurane in F0 and F1 males. CONCLUSIONS: These results suggest that similar decitabine-sensitive mechanisms regulating expression of multiple genes are involved in the mediation of neurobehavioral abnormalities in sires neonatally exposed to sevoflurane and in their future unexposed male offspring.

Augmenting engineered T-cell strategies in solid cancers through epigenetic priming.

T-cell receptor (TCR)- and chimeric antigen receptor (CAR)-based adoptive cell transfer (ACT) has shown promising results in hematological malignancies, but remains immature in solid cancers. The challenges associated with identification of tumor-specific targets, the heterogenic antigen expression, limited T-cell trafficking to tumor sites and the hostile tumor microenvironment (TME), are all factors contributing to the limited efficacy of ACT therapies against solid tumors. Epigenetic priming of tumor cells and the microenvironment may be a way of overcoming these obstacles and improving the clinical efficacy of adoptive T-cell therapies in the future. Here, we review the current literature and suggest combining epigenetic modulators and ACT strategies as a way of augmenting the efficacy of TCR- and CAR-engineered T cells against solid tumors.