scholarly journals Methylation Status of the miR-141-3p Promoter Regulates miR-141-3p Expression, Inflammasome Formation, and the Invasiveness of HTR-8/SVneo Cells

2021 ◽  
pp. 1-13
Author(s):  
Dongcai Wu ◽  
Li Shi ◽  
Fangrong Chen ◽  
Qing Lin ◽  
Jiao Kong
Keyword(s):  
Cell Growth ◽  
Cell Viability ◽  
Western Blotting ◽  
Promoter Methylation ◽  
Dna Methyltransferase ◽  
Methylation Status ◽  
Dna Methyltransferase Inhibitor ◽  
Nlrp3 Expression ◽  
Spontaneous Delivery

MicroRNA-141 (miR-141-3p) is upregulated in preeclampsia. This study investigated the effect of methylation of the miR-141-3p promoter on cell viability, invasion capability, and inflammasomes in vitro. The expression of miR-141-3p and methylation status of the miR-141-3p promoter were examined by RT-qPCR and pyrosequencing in villus tissues of women with spontaneous delivery (VTsd), villus tissues of women with preeclampsia (VTpe), and also in HTR-8/SVneo cells treated with a miR-141-3p inhibitor and 20 μmol/L 5-aza-2′-deoxycytidine (5-Aza), a DNA methyltransferase inhibitor. Cell viability and invasion were evaluated by CCK-8 and transwell assays. In addition, the levels of CXCL12, CXCR4, CXCR2, MMPs, NLRP3, and ASC expression were assessed by western blotting, and IL-1β and IL-18 concentrations were assayed by ELISA. miR-141-3p expression was upregulated, and the levels of miR-141-3p promoter methylation and CXCL12, CXCR4, and CXCR2 expression were decreased in VTpe relative to VTsd. In HTR-8/SVneo cells, hypomethylation caused by 5-Aza treatment increased miR-141-3p expression, while DNA methyltransferase 3 (DNMT3) transfection decreased miR-141-3p expression. miRNA-141-3p induced NLRP3, IL-1β, and IL-18 production, decreased CXCR4, MMP, and MMP2 production, and suppressed cell growth and invasion. Furthermore, we observed that NLRP3 plays an important mediatory role in the effects of miR-141-3p described above. Decreased methylation of the miR-141-3p promoter increases miR-141-3p expression, which in turn increases NLRP3 expression, resulting in higher IL-1β and IL-18 levels and lower levels of MMP2/9 and CXCR4. We conclude that modification of the miR-141-3p promoter might be a curial mediator in preeclampsia.

4-Acetylantrocamol LT3 Inhibits Glioblastoma Cell Growth and Downregulates DNA Repair Enzyme O6-Methylguanine-DNA Methyltransferase

2021 ◽  
pp. 1-17
Author(s):  
Shih-Yu Lee ◽  
I-Chuan Yen ◽  
Jang-Chun Lin ◽  
Min-Chieh Chung ◽  
Wei-Hsiu Liu
Keyword(s):  
Dna Repair ◽  
Cell Viability ◽  
Colony Formation ◽  
Dna Methyltransferase ◽  
Growth Factor Receptor ◽  
Repair Enzyme ◽  
U251 Cell ◽  
Methylguanine Dna Methyltransferase

Glioblastoma multiforme (GBM) is a deadly malignant brain tumor that is resistant to most clinical treatments. Novel therapeutic agents that are effective against GBM are required. Antrodia cinnamomea has shown antiproliferative effects in GBM cells. However, the exact mechanisms and bioactive components remain unclear. Thus, the present study aimed to investigate the effect and mechanism of 4-acetylantrocamol LT3 (4AALT3), a new ubiquinone from Antrodia cinnamomeamycelium, in vitro. U87 and U251 cell lines were treated with the indicated concentration of 4AALT3. Cell viability, cell colony-forming ability, migration, and the expression of proteins in well-known signaling pathways involved in the malignant properties of glioblastoma were then analyzed by CCK-8, colony formation, wound healing, and western blotting assays, respectively. We found that 4AALT3 significantly decreased cell viability, colony formation, and cell migration in both in vitro models. The epidermal growth factor receptor (EGFR), phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR), Hippo/yes-associated protein (YAP), and cAMP-response element binding protein (CREB) pathways were suppressed by 4AALT3. Moreover, 4AALT3 decreased the level of DNA repair enzyme O6-methylguanine-DNA methyltransferase and showed a synergistic effect with temozolomide. Our findings provide the basis for exploring the beneficial effect of 4AALT3 on GBM in vivo.

scholarly journals Prostaglandin E2 Reverses the Effects of DNA Methyltransferase Inhibitor and TGFB1 on the Conversion of Naive T Cells to iTregs

2019 ◽  
Vol 47 (3) ◽  
pp. 244-253
Author(s):  
Mehmet Sahin ◽  
Emel Sahin
Keyword(s):  
T Cells ◽  
Prostaglandin E2 ◽  
Dna Methyltransferase ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Naive T Cells ◽  
Naïve T Cells ◽  
Dna Methyltransferase Inhibitor

Naturally occurring regulatory T cells (nTregs) are produced under thymic (tTregs) or peripherally induced (pTregs) conditions in vivo. On the other hand, Tregs generated from naive T cells in vitro under some circumstances, such as treatment with transforming growth factor-β (TGFB), are called induced Tregs (iTregs). Tregs are especially characterized by FOXP3 expression, which is mainly controlled by DNA methylation. nTregs play important roles in the suppression of immune response and self-tolerance. The prostaglandin E2 (PGE2) pathway was reported to contribute to regulatory functions of tumor-infiltrating nTregs. In this study, we examined whether PGE2 contributes to the formation of iTregs treated with TGFB1 and 5-aza-2′-deoxycytidine (5-aza-dC), which is a DNA methyltransferase inhibitor. We found that the protein and gene expression levels of FOXP3 and IL-10 were increased in 5-aza-dC and TGFB1-treated T cells in vitro. However, the addition of PGE2 to these cells reversed these increments significantly. In CFSE-based cell suppression assays, we demonstrated that PGE2 decreased the suppressive functions of 5-aza-dC and TGFB1-treated T cells.

scholarly journals VEGF/Flk1 Mechanism is Involved in Roxarsone Promotion of Rat Endothelial Cell Growth and B16F10 Xenograft Tumor Angiogenesis

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Shihao Chen ◽  
Jinge Xu ◽  
Qianhan Wei ◽  
Zeting Zhao ◽  
Xin Chen ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cell Viability ◽  
Vascular Endothelial Cells ◽  
Growth Promotion ◽  
Xenograft Model ◽  
Feed Additive ◽  
Vascular Endothelial ◽  
Mouse Xenograft Model ◽  
Significant Difference

AbstractThe potential angiogenic effect of roxarsone, a feed additive widely used to promote animal growth worldwide, was demonstrated recently. We explored the mechanism of vascular endothelial growth factor (VEGF) and its receptor (VEGFR) in roxarsone promotion of rat vascular endothelial cells (ECs) and B16F10 mouse xenografts. ECs were treated with 0.1–50 μM roxarsone or with roxarsone plus 10 ng/mL VEGF, VEGFR1 (Flt1), or VEGFR2 (Flk1) antibodies for 12–48 h to examine their role in cell growth promotion. Small interfering RNA (siRNA) targeting Vegf, Flt1, and Flk1 were transfected in the ECs, and we measured the expression level, cell proliferation, migration, and tube formation ability. The siRNA targeting Vegf or Flk1 were injected intratumorally in the B16F10 xenografts of mice that received 25 mg/kg roxarsone orally. Cell viability and VEGF expression following roxarsone treatment were significantly higher than that of the control (P < 0.05), peaking following treatment with 1.0 μM roxarsone. Compared to roxarsone alone, the VEGF antibody decreased cell promotion by roxarsone (P < 0.05), and the Flk1 antibody greatly reduced cell viability compared to the Flt1 antibody (P < 0.01). Roxarsone and Flk1 antibody co-treatment increased supernatant VEGF significantly, while cellular VEGF was obviously decreased (P < 0.01), whereas there was no significant difference following Flt1 antibody blockade. The siRNA against Vegf or Flk1 significantly attenuated the roxarsone promotion effects on EC proliferation, migration, and tube-like formation (P < 0.01), whereas the siRNA against Flt1 effected no obvious differences. Furthermore, the RNA interference significantly weakened the roxarsone-induced increase in xenograft weight and volume, and VEGF and Flk1 expression. Roxarsone promotion of rat EC growth, migration, and tube-like formation in vitro and of B16F10 mouse xenograft model tumor growth and angiogenesis involves a VEGF/Flk1 mechanism.

scholarly journals TrxR2 overexpression alleviates inflammation-mediated neuronal death via reducing the oxidative stress and activating the Akt–Parkin pathway

2019 ◽  
Vol 8 (5) ◽  
pp. 641-653 ◽  
Author(s):  
Jinbao Gao ◽  
Yunjun Li ◽  
Wende Li ◽  
Haijiang Wang
Keyword(s):  
Oxidative Stress ◽  
Cell Death ◽  
Cell Viability ◽  
Western Blotting ◽  
Neuronal Death ◽  
Protective Effects ◽  
Mitochondrial Apoptosis ◽  
Inflammation Response ◽  
N2a Cells

Abstract Neuronal death caused by inflammatory cytokine-mediated neuroinflammation is being extensively explored. Thioredoxin reductase (TrxR) 2 is a novel mediator of inflammation response. In the current study, we focus on the mechanisms of TrxR2 overexpression in inflammation-mediated neuronal death. LPS was used to induce neuroinflammation in N2a cells in vitro. Adenovirus-loaded TrxR2 was transfected into N2a cells to up-regulate TrxR2 expression. Then, cell viability was determined via MTT assay and TUNEL assay. Apoptosis was measured via western blotting and ELISA. Oxidative stress was detected via ELISA and flow cytometry. A pathway inhibitor was used to verify the role of the Akt–Parkin pathway in the LPS-mediated N2a cell death in the presence of TrxR2 overexpression. With the help of immunofluorescence assay and western blotting, we found that TrxR2 expression was significantly reduced in response to LPS treatment, and this effect was associated with N2a cell death via apoptosis. At the molecular level, TrxR2 overexpression elevated the activity of the Akt–Parkin pathway, as evidenced by the increased expression of p-Akt and Parkin. Interestingly, inhibition of the Akt–Parkin pathway abolished the regulatory effect of TrxR2 on LPS-treated N2a cells, as evidenced by the decreased cell viability and increased apoptotic ratio. Besides, TrxR2 overexpression also reduced oxidative stress, inflammation factor transcription and mitochondrial apoptosis. However, inhibition of Akt–Parkin axis abrogated the protective effects of TrxR2 on redox balance, mitochondrial performance and cell survival. LPS-mediated neuronal death was linked to a drop in TrxR2 overexpression and the inactivation of the Akt–Parkin pathway. Overexpression of TrxR2 sustained mitochondrial function, inhibited oxidative stress, repressed inflammation response, and blocked mitochondrial apoptosis, finally sending a pro-survival signal for the N2a cells in the setting of LPS-mediated inflammation environment.

scholarly journals Methylation in p14ARF is Frequently Observed in Colorectal Cancer with Low-level Microsatellite Instability

2009 ◽  
Vol 37 (4) ◽  
pp. 1038-1045 ◽  
Author(s):  
K Kominami ◽  
T Nagasaka ◽  
HM Cullings ◽  
N Hoshizima ◽  
H Sasamoto ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Microsatellite Instability ◽  
Promoter Methylation ◽  
Dna Methyltransferase ◽  
Methylation Status ◽  
Gene Promoters ◽  
Braf Mutations ◽  
Low Level ◽  
Methylguanine Dna Methyltransferase ◽  
High Level

Colorectal cancer (CRC) can be classified as high-level microsatellite instability (MSI-H), low-level MSI (MSI-L) and microsatellite stable (MSS) depending on levels of MSI. MSI-H CRC relies on a distinct molecular pathway due to the mismatch repair (MMR) deficiency and shows methylation in multiple gene promoters. The genetic pathway leading to MSI-L is unknown, although higher levels of promoter methylation are observed in this group compared with MSS CRCs. This study explored how promoter methylation affects MSI phenotype, by analysing the methylation status of eight CRC-related promoters, MSI phenotype and KRAS/BRAF mutations in a series of 234 CRCs. Promoter methylation of p14ARF was significantly related to MSI-L CRC with KRAS mutation. The MSI-H phenotype was related to methylation of MLH1 as expected, while the MSS phenotype was related to methylation of p16INK4a and O6-methylguanine-DNA methyltransferase, although this was not statistically significant. Thus, promoter methylation of p14ARF could be a significant alteration leading to CRC with MSI-L.

Title: Promoter Methylation of Selected Genes and Response to Azacitidine (AZ) Therapy in Patients with Non-BCR-ABL Myeloproliferative Disorders (MPDs)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4625-4625
Author(s):  
Nicholas Achille ◽  
Laura Michaelis ◽  
Scott E. Smith ◽  
Eliza Germano ◽  
Nancy J. Zeleznik-Le ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Promoter Methylation ◽  
Research Funding ◽  
Dna Methyltransferase ◽  
Methylation Status ◽  
Cpg Islands ◽  
Myeloproliferative Disorders ◽  
Response To Treatment ◽  
Promoter Regions ◽  
Days Of Therapy

Abstract Abstract 4625 Background: Gene silencing via methylation of CpG islands in the promoter regions of many genes but specifically of APAF1, p15INK4B, p16INK4A, RARB, and CDH1 appears to play a role in pathogenesis of myeloid malignancies. Azacitidine (AZ) causes demethylation by inhibiting DNA methyltransferase and has already been shown to be an effective therapy for myelodysplastic syndromes. The demethylation induced by AZ is detectable in about 48 hours and increases significantly after 5 days of therapy. After that, the effect tends to plateau. Methods: We initiated a Phase 2 study of patients with non-BCR-ABL MPDs to determine clinical response to AZ therapy and correlate it with promoter DNA methylation and gene re-expression. The protocol was approved by the institutional IRB. Patients received AZ 75mg/m2 s/c for days 1–7 and repeated every 28 days for a minimum of 4 cycles. Responders were allowed to continue treatment until disease progression. Pretreatment and D 7 peripheral blood samples were analyzed for promoter methylation status and expression of the 5 genes mentioned above. Bisulfite conversion of DNA was followed by quantitative PCR using primers specific for methylated or for unmethylated promoter regions. For gene re-expression analysis, quantitative RT-PCR was performed with RNA isolated from the same patient samples and the same time points as the DNA methylation analyses. Results: Seven patients were enrolled before the study closed due to lack of accrual. The diagnoses were: Myelofibrosis (MF) 4, essential thrombocythemia 1, unclassified MPD with dysplasia 2. One patient with MF and one with unclassified MPD responded, the latter with normalization of marrow karyotype. Both responses were accompanied by significant decrease in APAF1 promoter methylation and surprisingly, an increase in promoter methylation of RARB. In three of the non-responders, APAF1 methylation increased. In patients with decreased Apaf1 methylation, a statistically significant increase in mRNA expression was observed. Conclusions: Within its limitations, this small trial shows that the methylation status of selected genes, particularly of APAF1 and RARB (inversely) is associated with response to treatment with azacitidine in patients with MPDs. In non-responders, Apaf1 methylation appears to increase. A larger study will be necessary to confirm these preliminary observations. Disclosures: Smith: Seattle Genetics, Inc.: Research Funding; Cephalon: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Spectrum: Consultancy; GSK: Speakers Bureau. Nand:Celgene Corporation: Research Funding.

scholarly journals Phase I/IIa Study of Cilengitide and Temozolomide With Concomitant Radiotherapy Followed by Cilengitide and Temozolomide Maintenance Therapy in Patients With Newly Diagnosed Glioblastoma

2010 ◽  
Vol 28 (16) ◽  
pp. 2712-2718 ◽  
Author(s):  
Roger Stupp ◽  
Monika E. Hegi ◽  
Bart Neyns ◽  
Roland Goldbrunner ◽  
Uwe Schlegel ◽  
...  
Keyword(s):  
Phase I ◽  
Promoter Methylation ◽  
Dna Methyltransferase ◽  
Progression Free Survival ◽  
Recurrent Glioblastoma ◽  
Mgmt Promoter Methylation ◽  
Newly Diagnosed ◽  
Mgmt Promoter ◽  
Newly Diagnosed Glioblastoma

Purpose Invasion and migration are key processes of glioblastoma and are tightly linked to tumor recurrence. Integrin inhibition using cilengitide has shown synergy with chemotherapy and radiotherapy in vitro and promising activity in recurrent glioblastoma. This multicenter, phase I/IIa study investigated the efficacy and safety of cilengitide in combination with standard chemoradiotherapy in newly diagnosed glioblastoma. Patients and Methods Patients (age ≥ 18 to ≤ 70 years) were treated with cilengitide (500 mg) administered twice weekly intravenously in addition to standard radiotherapy with concomitant and adjuvant temozolomide. Treatment was continued until disease progression or for up to 35 weeks. The primary end point was progression-free survival (PFS) at 6 months. Results Fifty-two patients (median age, 57 years; 62% male) were included. Six- and 12-month PFS rates were 69% (95% CI, 54% to 80%) and 33% (95% CI, 21% to 46%). Median PFS was 8 months (95% CI, 6.0 to 10.7 months). Twelve- and 24-month overall survival (OS) rates were 68% (95% CI, 53% to 79%) and 35% (95% CI, 22% to 48%). Median OS was 16.1 months (95% CI, 13.1 to 23.2 months). PFS and OS were longer in patients with tumors with O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation (13.4 and 23.2 months) versus those without MGMT promoter methylation (3.4 and 13.1 months). The combination of cilengitide with temozolomide and radiotherapy was well tolerated, with no additional toxicity. No pharmacokinetic interactions between temozolomide and cilengitide were identified. Conclusion Compared with historical controls, the addition of concomitant and adjuvant cilengitide to standard chemoradiotherapy demonstrated promising activity in patients with glioblastoma with MGMT promoter methylation.

scholarly journals Recycling drug screen repurposes hydroxyurea as a sensitizer of glioblastomas to temozolomide targeting de novo DNA synthesis, irrespective of molecular subtype

2017 ◽  
Vol 20 (5) ◽  
pp. 642-654 ◽  
Author(s):  
Jian Teng ◽  
Seyedali Hejazi ◽  
Lotte Hiddingh ◽  
Litia Carvalho ◽  
Mark C de Gooijer ◽  
...  
Keyword(s):  
Promoter Methylation ◽  
Dna Methyltransferase ◽  
Methylation Status ◽  
Primary Cultures ◽  
Mgmt Promoter Methylation ◽  
In Vivo Models ◽  
Promoter Methylation Status ◽  
Mgmt Promoter ◽  
Mgmt Promoter Methylation Status

Abstract Background Glioblastoma (GBM) is the most common and most aggressive primary malignant brain tumor. Standard-of-care treatment involves maximal surgical resection of the tumor followed by radiation and chemotherapy (temozolomide [TMZ]). The 5-year survival rate of patients with GBM is &lt;10%, a colossal failure that has been partially attributed to intrinsic and/or acquired resistance to TMZ through O6-methylguanine DNA methyltransferase (MGMT) promoter methylation status in the tumor. Methods A drug screening aimed at evaluating the potential recycling and repurposing of known drugs was conducted in TMZ-resistant GBM cell lines and primary cultures of newly diagnosed GBM with different MGMT promoter methylation status, phenotypic/genotypic background and subtype, and validated with sphere formation, cell migration assays, and quantitative invasive orthotopic in vivo models. Results We identified hydroxyurea (HU) to synergize with TMZ in GBM cells in culture and in vivo, irrespective of MGMT promoter methylation status, subtype, and/or stemness. HU acts specifically on the S-phase of the cell cycle by inhibiting the M2 unit of enzyme ribonucleotide reductase. Knockdown of this enzyme using RNA interference and other known chemical inhibitors exerted a similar effect to HU in combination with TMZ both in culture and in vivo. Conclusions We demonstrate preclinical efficacy of repurposing hydroxyurea in combination with TMZ for adjuvant GBM therapy. This combination benefit is of direct clinical interest given the extensive use of TMZ and the associated problems with TMZ-related resistance and treatment failure.

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