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Temozolomide (TMZ) is an important first-line treatment for glioblastoma (GBM), but there are limitations to TMZ response in terms of durability and dependence on the promoter methylation status of the DNA repair gene O6-methylguanine DNA methyltransferase (MGMT). MGMT-promoter-hypermethylated (MGMT-M) GBMs are more sensitive to TMZ than MGMT-promoter-hypomethylated (MGMT-UM) GBMs. Moreover, TMZ resistance is inevitable even in TMZ-sensitive MGMT-M GBMs. Hence, epigenetic reprogramming strategies are desperately needed in order to enhance TMZ response in both MGMT-M and MGMT-UM GBMs. In this study, we present novel evidence that the epigenetic reactivation of Tumor Suppressor Candidate 3 (TUSC3) can reprogram sensitivity of GBM stem cells (GSCs) to TMZ irrespective of MGMT promoter methylation status. Interrogation of TCGA patient GBM datasets confirmed TUSC3 promoter regulation of TUSC3 expression and also revealed a strong positive correlation between TUSC3 expression and GBM patient survival. Using a combination of loss-of-function, gain-of-function and rescue studies, we demonstrate that TUSC3 reactivation is associated with enhanced TMZ response in both MGMT-M and MGMT-UM GSCs. Further, we provide novel evidence that the demethylating agent 5-Azacitidine (5-Aza) reactivates TUSC3 expression in MGMT-M GSCs, whereas the combination of 5-Aza and MGMT inhibitor Lomeguatrib is necessary for TUSC3 reactivation in MGMT-UM GSCs. Lastly, we propose a pharmacological epigenetic reactivation strategy involving TUSC3 that leads to significantly prolonged survival in MGMT-M and MGMT-UM orthotopic GSCs models. Collectively, our findings provide a framework and rationale to further explore TUSC3-mediated epigenetic reprogramming strategies that could enhance TMZ sensitivity and outcomes in GBM. Mechanistic and translational evidence gained from such studies could contribute towards optimal design of impactful trials for MGMT-UM GBMs that currently do not have good treatment options. [ABSTRACT FROM AUTHOR]

Abstract Background Colorectal cancer (CRC) represents a common malignancy in gastrointestinal tract. Iodine-125 (125I) seed implantation is an emerging treatment technology for unresectable tumors. This study investigated the mechanism of 125I seed in the function of CRC cells. Methods The CRC cells were irradiated with different doses of 125I seed (0.4, 0.6 and 0.8 mCi). miR-615 expression in CRC tissues and adjacent tissues was detected by RT-qPCR. miR-615 expression was intervened with miR-615 mimic or miR-615 inhibitor, and then the CRC cells were treated with 5-AZA (methylation inhibitor). The CRC cell growth, invasion and apoptosis were measured. The methylation level of miR-615 promoter region was detected. The xenograft tumor model irradiated by 125I seed was established in nude mice. The methylation of miR-615, Ki67 expression and CRC cell apoptosis were detected. Results 125I seed irradiation repressed the growth and facilitated apoptosis of CRC cells in a dose-dependent manner. Compared with adjacent tissues, miR-615 expression in CRC tissues was downregulated and miR-615 was poorly expressed in CRC cells. Overexpression of miR-615 suppressed the growth of CRC cells. 125I seed-irradiated CRC cells showed increased miR-615 expression, reduced growth rate and enhanced apoptosis. The methylation level of miR-615 promoter region in CRC cells was decreased after 125I seed treatment. In vivo experiments confirmed that 125I seed-irradiated xenograft tumors showed reduced methylation of the miR-615 promoter and increased miR-615 expression, as well as decreased Ki67 expression and enhanced apoptosis. The target genes of miR-615 and its regulatory downstream pathway were further predicted by bioinformatics analysis. Conclusions 125I seed repressed the growth and facilitated the apoptosis of CRC cells by suppressing the methylation of the miR-615 promoter and thus activating miR-615 expression. The possible mechanism was that miR-615-5p targeted MAPK13, thus affecting the MAPK pathway and the progression of CRC.

Sprouty-related, EVH1 domain-containing protein 1 (SPRED1) has been identified as a novel tumor suppressor gene in acute myeloid leukemia (AML). Previous studies showed that SPRED1 methylation levels were significantly increased in AML patients, making it an interesting candidate for further investigations. To confirm the association of SPRED1 methylation, clinical parameters, and known molecular prognosticators and to identify the impact of methylation level on treatment outcome, we conducted this study in a larger cohort of 75 AML patients. Significantly increased methylation levels of SPRED1 were detected at four of ten CpG units by quantitative high-resolution mass spectrometry-based approach (MassARRAY) in AML patients. Whereas overall survival (OS) and relapse-free survival (RFS) showed no statistical difference between hypermethylation and hypomethylation subgroups, the relationship between methylation level and treatment response was indicated in paired samples from pre- and post-induction. To determine the possible mechanism of SPRED1 methylation in AML, we performed in vitro experiments using THP-1 cells, as the latter showed the highest methylation level (determined by utilizing bisulfite modification) among the three AML cell lines we tested. When treated with 5-AZA and lentivirus transfection, upregulated SPRED1 expression, decreased cell proliferation, increased cell differentiation and apoptosis, and inactivated phosphorylated extracellular signal-regulated kinase (p-ERK) were detected in THP-1 cells. These results show that demethylation of SPRED1 can inhibit the proliferation of AML cells and promote their differentiation and apoptosis, possibly by the ERK pathway. The hypermethylation of SPRED1 is a potential therapeutic target for AML. [ABSTRACT FROM AUTHOR]

Background: Colorectal cancer (CRC) represents a common malignancy in gastrointestinal tract. Iodine-125 (125I) seed implantation is an emerging treatment technology for unresectable tumors. This study investigated the mechanism of 125I seed in the function of CRC cells.Methods: The CRC cells were irradiated with different doses of 125I seed (0.4, 0.6 and 0.8 mCi). miR-615 expression in CRC tissues and adjacent tissues was detected by RT-qPCR. miR-615 expression was intervened with miR-615 mimic or miR-615 inhibitor, and then the CRC cells were treated with 5-AZA (methylation inhibitor). The CRC cell growth, invasion and apoptosis were measured. The methylation level of miR-615 promoter region was detected. The xenograft tumor model irradiated by 125I seed was established in nude mice. The methylation of miR-615, Ki67 expression and CRC cell apoptosis were detected.Results: 125I seed irradiation repressed the growth and facilitated apoptosis of CRC cells in a dose-dependent manner. Compared with adjacent tissues, miR-615 expression in CRC tissues was downregulated and miR-615 was poorly expressed in CRC cells. Overexpression of miR-615 suppressed the growth of CRC cells. 125I seed-irradiated CRC cells showed increased miR-615 expression, reduced growth rate and enhanced apoptosis. The methylation level of miR-615 promoter region in CRC cells was decreased after 125I seed treatment. In vivo experiments confirmed that 125I seed-irradiated xenograft tumors showed reduced methylation of the miR-615 promoter and increased miR-615 expression, as well as decreased Ki67 expression and enhanced apoptosis. The target genes of miR-615 and its regulatory downstream pathway were further predicted by bioinformatics analysis.Conclusions: 125I seed repressed the growth and facilitated the apoptosis of CRC cells by suppressing the methylation of the miR-615 promoter and thus activating miR-615 expression. The possible mechanism was that miR-615-5p targeted MAPK13, thus affecting the MAPK pathway and the progression of CRC. [ABSTRACT FROM AUTHOR]

Previously, we described the link between C-X-C motif chemokine 12 (Cxcl12) gene induction and DNA hypomethylation in the absence of poly(ADP-ribose) polymerase 1 (PARP-1). We have now firmly established that demethylation is the primary cause of gene induction on the basis of Cxcl12 gene upregulation upon treatment with the demethylating agent 5-azacytidine (5-aza). Since the demethylation state of Cxcl12 is favored by PARP-1 absence, we investigated the presence of ten-eleven translocation (TET) proteins on the Cxcl12 promoter in order to corroborate the relationship between the demethylation process and increased gene expression that occurs in the absence of PARP-1. Analysis was performed on the promoter region within CpG islands of Cxcl12 from control mouse embryonic fibroblasts (NIH3T3) and PARP-1 knock-out mouse embryonic fibroblasts (PARP1-/-). The lack of PARP-1 increased the abundance of TET2 on the Cxcl12 promoter, suggesting that TET-mediated demethylation provoked by the absence of PARP-1 could account for the observed increased expression of this chemokine. Deciphering the regulation of DNA (de)methylation factors that control Cxcl12 expression may provide an additional therapeutic approach in pharmacological interventions where gene switching on or off based on targeted stimulation or inhibition is necessary. [ABSTRACT FROM AUTHOR]

Background: Dietary polyphenols, such as those found in green tea and red wine, are linked to antitumor activity. They are known to influence many signaling pathways epigenetically within the human body. In this regard, CPUK02 (15-Oxosteviol benzyl ester) is a new ent-kaurenoid derivative of stevioside and exhibits strong anti-cancer activity in vitro and in vivo. Nowadays, the role of epigenetics in cancer has been the subject of intensive study and DNA methylation targeting represents a relevant strategy for cancer treatment. There are no reports regarding the effects of CPUK02 on epigenetic alterations in colorectal cancer cell line. This study was an attempt to compare CPUK02 with 5-AZA as DNMT inhibitor agent and evaluate whether it can induce its anti-cancer effects via altering the level of DNMT3b mRNA, MGMT and SFRP2 methylation pattern in HCT 116 cell line. Methods: To evaluate DNMT3b expression, DNMT3B mRNA levels in HCT116 CRC cell line were quantified by real-time reverse-transcriptase Polymerase Chain Reaction (PCR) assay after 24 hr of incubation time with CPUK02 and 5-AZA. In addition, the methylation patterns of 2 CpG islands in this cell line were examined by methylationspecific PCR methods. Results: CPUK02 surprisingly, decreased the DNMT3b mRNA level. The average expression levels of DNMT3b in HCT116 treated with CPUK02 and 5-AZA relative to the GAPDH expression level in control were 0.16 and 0.5%, respectively. Furthermore, CPUK02 could decrease the methylated allele of MGMT and SFRP2 genes in HCT 116 after 24 hr. Conclusion: In this study, positive correlation was found between mRNA expression of DNMT3b and gene promoter hypermethylation after treatment with CPUK02 and 5-AZA. Our data confirmed that CPUK02 like 5-AZA exhibits demethylating properties. [ABSTRACT FROM AUTHOR]

Ovarian cancer, as well as other cancers, is primarily caused by methylation at cytosines in CpG islands, but the current marker for ovarian cancer is low in sensitivity and failed in early-stage detection. Fourier transform infrared (FT-IR) spectroscopy is powerful in analysis of functional groups within molecules, and infrared microscopy illustrates the location of specific groups within single cells. In this study, we applied HPLC and FT-IR microspectrometry to study normal epithelial ovarian cell line immortalized ovarian surface epithelium (IOSE), two epithelial ovarian cell lines (A2780 and CP70) with distinct properties, and the effect of a cancer drug 5-aza-2'-deoxycytidine (5-aza) without labeling. Our results reveal that inhibition of methylation on cytosine with 5-aza initiates the protein expression. Furthermore, paraffin-adsorption kinetic study allows us to distinguish hypermethylated and hypomethyated cells, and this assay can be a potential diagnosis method for cancer screening. [ABSTRACT FROM AUTHOR]