Effect of VAL-083, a novel N7 alkylating agent, on growth of temolozomide-resistant primary adult glioblastoma multiforme (GBM) cells providing a new potential treatment option for GBM.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e13123-e13123
Author(s):  
Sandra E. Dunn ◽  
Kaiji Hu ◽  
Abbas Fotovati ◽  
James Chen ◽  
Joanna Triscott ◽  
...  
Keyword(s):  
Dna Methyltransferase ◽  
Alkylating Agent ◽  
Standard Of Care ◽  
Clinical Activity ◽  
Front Line ◽  
New Agents ◽  
Methylguanine Dna Methyltransferase ◽  
The Usa ◽  
Intrinsic Drug Resistance

e13123 Background: Glioblastoma (GBM) remains one of the most difficult tumors to treat first because many new agents fail to cross the blood brain barrier (BBB), and second due to intrinsic drug resistance. Temozolomide (TMZ) is a front-line therapy for the treatment of GBM; however, it is often ineffective due to drug inactivation by O6-methylguanine-DNA methyltransferase (MGMT). Cancer stem cells (CSC) are a subpopulation of the tumor that resist therapy and give rise to relapse. Here we describe VAL-083, a novel alkylating agent that creates N7 methylation on DNA, and readily crosses the BBB. VAL-083 is currently undergoing human clinical trials in the USA in refractory GBM patients. We previously described how VAL-083 can overcome resistance associated with MGMT in cell lines, and targets brain tumor CSCs. This demonstrates that VAL-083 has the potential to surpass the standard-of-care. Methods: To provide further preclinical support for VAL-083 we examined the effect of VAL-083 on primary adult GBM cells in culture, isolated fresh following surgical resection, in cases where clinical activity of TMZ was known to be limited and high expression of MGMT had been observed. Results: VAL-083 (5uM) inhibited cell growth in these primary adult GBMs that did not show significant sensitivity to TMZ in vitro. VAL-083 also inhibited the growth of CSCs by 100% in neurosphere growth assays. Conclusions: In summary, VAL-083 demonstrates in vitro efficacy against primary adult GBM cells where TMZ has limited activity thereby further supporting the potential of VAL-083 to surpas the standard-of-care.

scholarly journals Temozolomide antagonizes oncolytic immunovirotherapy in glioblastoma

2020 ◽  
Vol 8 (1) ◽  
pp. e000345 ◽  
Author(s):  
Dipongkor Saha ◽  
Samuel D Rabkin ◽  
Robert L Martuza
Keyword(s):  
Dna Methyltransferase ◽  
Standard Of Care ◽  
Interleukin 12 ◽  
Current Standard ◽  
Antitumor Effects ◽  
Methylguanine Dna Methyltransferase ◽  
Beneficial Effects ◽  
Simplex Virus

BackgroundTemozolomide (TMZ) chemotherapy is a current standard of care for glioblastoma (GBM), however it has only extended overall survival by a few months. Because it also modulates the immune system, both beneficially and negatively, understanding how TMZ interacts with immunotherapeutics is important. Oncolytic herpes simplex virus (oHSV) is a new class of cancer therapeutic with both cytotoxic and immunostimulatory activities. Here, we examine the combination of TMZ and an oHSV encoding murine interleukin 12, G47Δ-mIL12, in a mouse immunocompetent GBM model generated from non-immunogenic 005 GBM stem-like cells (GSCs.MethodsWe first investigated the cytotoxic effects of TMZ and/or G47Δ-IL12 treatments in vitro, and then the antitumor effects of combination therapy in vivo in orthotopically implanted 005 GSC-derived brain tumors. To improve TMZ sensitivity, O6-methylguanine DNA methyltransferase (MGMT) was inhibited. The effects of TMZ on immune cells were evaluated by flow cytometery and immunohistochemistry.ResultsThe combination of TMZ+G47Δ-IL12 kills 005 GSCs in vitro better than single treatments. However, TMZ does not improve the survival of orthotopic tumor-bearing mice treated with G47Δ-IL12, but rather can abrogate the beneficial effects of G47Δ-IL12 when the two are given concurrently. TMZ negatively affects intratumor T cells and macrophages and splenocytes. Addition of MGMT inhibitor O6-benzylguanine (O6-BG), an inactivating pseudosubstrate of MGMT, to TMZ improved survival, but the combination with G47Δ-IL12 did not overcome the antagonistic effects of TMZ treatment on oHSV therapy.ConclusionsThese results illustrate that chemotherapy can adversely affect oHSV immunovirotherapy. As TMZ is the standard of care for GBM, the timing of these combined therapies should be taken into consideration when planning oHSV clinical trials with chemotherapy for GBM.

CBIO-02. IN VITRO TUMOR TREATING FIELDS (TTFields) REDUCE PROLIFERATION AND ALTER MLH1 EXPRESSION IN TEMOZOLOMIDE RESISTANT (TMZR) PATIENT-DERIVED GLIOBLASTOMA (GBM) CELLS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi27-vi27
Author(s):  
Sharon Michelhaugh ◽  
Katherine Degen ◽  
Blake Walker ◽  
Sandeep Mittal
Keyword(s):  
Dna Methyltransferase ◽  
Clonogenic Assay ◽  
Control Cell ◽  
Acquired Resistance ◽  
Dna Mismatch ◽  
Methylguanine Dna Methyltransferase ◽  
Tumor Treating Fields ◽  
Cell Counts ◽  
Repair Protein

Abstract BACKGROUND TTFields therapy is approved as a monotherapy for the treatment of recurrent GBM, tumors that often have acquired resistance to TMZ. To examine TTFields efficacy in the context of TMZR in vitro, TTFields were applied to TMZR GBM cells to assess proliferation, clonogenicity, and changes in expression of O6-methylguanine-DNA methyltransferase (MGMT) that confers TMZ resistance and DNA Mismatch Repair Protein Mlh1 (MLH1) that may confer TMZ sensitivity. METHODS 15-037 GBM cells were isolated from a newly-diagnosed patient tumor (IDH-WT, unmethylated MGMT promoter. Cells were grown in DMEM/F12 media with 10% FBS and gentamicin. U-251 MG cells and 15-037 cells were incubated with 100 µM TMZ for three 5-day cycles to establish TMZR. TMZR cells were plated on coverslips (1×104cells/coverslip) and incubated overnight prior to TTFields application. TTFields were applied at 200 kHz (field intensity of ~1.6 V/cm) for 14 days. Then coverslips (n=3/group) were harvested, counted, and replated for clonogenic assay at 1000 cells/35mm2 well. Clonogenic assay plates were stained with crystal violet, imaged, and colonies counted. Additional coverslips (n=3/group) were harvested and MGMT and MLH1 expression were assessed by qRT-PCR with the delta-delta Ct method using GAPDH as housekeeping control. Cell counts and colony counts were compared with two-tailed t-test with significance at p< 0.05. RESULTS In U-251 MG and 15-037 TMZR cells, TTFields application significantly reduced both cell counts and clonogenic assay colony counts compared to untreated controls (U-251 MG: 45.4% and 26.5%; 15-037: 96.2% and 60.9%, respectively, p< 0.05). In both TMZR cell lines, MGMT expression was unchanged by TTFields, but MLH1 expression increased 2.2 fold in U-251 MG cells and 7.1 fold in 15-037 cells after TTFields application. CONCLUSIONS Not only are TMZR GBM cells sensitive to TTFields in vitro, but TTFields increased expression of MLH1 which may be able to reduce resistance to TMZ.

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 MGMT genomic rearrangements contribute to chemotherapy resistance in gliomas

2020 ◽  
Author(s):  
Barbara Oldrini ◽  
Nuria Vaquero-Siguero ◽  
Quanhua Mu ◽  
Paula Kroon ◽  
Ying Zhang ◽  
...  
Keyword(s):  
Dna Methyltransferase ◽  
Chemotherapy Resistance ◽  
Glioma Cells ◽  
Promoter Hypermethylation ◽  
Dna Adduct ◽  
Genomic Rearrangements ◽  
Low Grade ◽  
Methylguanine Dna Methyltransferase

AbstractTemozolomide (TMZ) is an oral alkylating agent used for the treatment of glioblastoma and is now becoming a chemotherapeutic option in patients diagnosed with high-risk low-grade gliomas. The O-6-methylguanine-DNA methyltransferase (MGMT) is responsible for the direct repair of the main TMZ-induced toxic DNA adduct, the O6-Methylguanine lesion. MGMT promoter hypermethylation is currently the only known biomarker for TMZ response in glioblastoma patients. Here we show that a subset of recurrent gliomas carries MGMT genomic rearrangements that lead to MGMT overexpression, independently from changes in its promoter methylation. By leveraging the CRISPR/Cas9 technology we generated some of these MGMT rearrangements in glioma cells and demonstrated that the MGMT genomic rearrangements contribute to TMZ resistance both in vitro and in vivo. Lastly, we showed that such fusions can be detected in tumor-derived exosomes and could potentially represent an early detection marker of tumor recurrence in a subset of patients treated with TMZ.

scholarly journals MGMT-activated DUB3 stabilizes MCL1 and drives chemoresistance in ovarian cancer

2019 ◽  
Vol 116 (8) ◽  
pp. 2961-2966 ◽  
Author(s):  
Xiaowei Wu ◽  
Qingyu Luo ◽  
Pengfei Zhao ◽  
Wan Chang ◽  
Yating Wang ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cells ◽  
Dna Methyltransferase ◽  
Essential Role ◽  
Histone Deacetylase Inhibitors ◽  
Combined Treatment ◽  
Ovarian Cancer Cells ◽  
Methylguanine Dna Methyltransferase

Chemoresistance is a severe outcome among patients with ovarian cancer that leads to a poor prognosis. MCL1 is an antiapoptotic member of the BCL-2 family that has been found to play an essential role in advancing chemoresistance and could be a promising target for the treatment of ovarian cancer. Here, we found that deubiquitinating enzyme 3 (DUB3) interacts with and deubiquitinates MCL1 in the cytoplasm of ovarian cancer cells, which protects MCL1 from degradation. Furthermore, we identified that O6-methylguanine-DNA methyltransferase (MGMT) is a key activator of DUB3 transcription, and that the MGMT inhibitor PaTrin-2 effectively suppresses ovarian cancer cells with elevated MGMT-DUB3-MCL1 expression both in vitro and in vivo. Most interestingly, we found that histone deacetylase inhibitors (HDACis) could significantly activate MGMT/DUB3 expression; the combined administration of HDACis and PaTrin-2 led to the ideal therapeutic effect. Altogether, our results revealed the essential role of the MGMT-DUB3-MCL1 axis in the chemoresistance of ovarian cancer and identified that a combined treatment with HDACis and PaTrin-2 is an effective method for overcoming chemoresistance in ovarian cancer.

scholarly journals Riluzole enhances the antitumor effects of temozolomide via suppression of MGMT expression in glioblastoma

2020 ◽  
pp. 1-10 ◽  
Author(s):  
Tetsuya Yamada ◽  
Shohei Tsuji ◽  
Shinsuke Nakamura ◽  
Yusuke Egashira ◽  
Masamitsu Shimazawa ◽  
...  
Keyword(s):  
Cell Lines ◽  
Dna Methyltransferase ◽  
Antitumor Effect ◽  
Metabotropic Glutamate Receptor ◽  
Migration And Invasion ◽  
Antitumor Effects ◽  
Mgmt Expression ◽  
Methylguanine Dna Methyltransferase

OBJECTIVEGlutamatergic signaling significantly promotes proliferation, migration, and invasion in glioblastoma (GBM). Riluzole, a metabotropic glutamate receptor 1 inhibitor, reportedly suppresses GBM growth. However, the effects of combining riluzole with the primary GBM chemotherapeutic agent, temozolomide (TMZ), are unknown. This study aimed to investigate the efficacy of combinatorial therapy with TMZ/riluzole for GBM in vitro and in vivo.METHODSThree GBM cell lines, T98G (human; O6-methylguanine DNA methyltransferase [MGMT] positive), U87MG (human; MGMT negative), and GL261 (murine; MGMT positive), were treated with TMZ, riluzole, or a combination of both. The authors performed cell viability assays, followed by isobologram analysis, to evaluate the effects of combinatorial treatment for each GBM cell line. They tested the effect of riluzole on MGMT, a DNA repair enzyme causing chemoresistance to TMZ, through quantitative real-time reverse transcription polymerase chain reaction in T98G cells. Furthermore, they evaluated the efficacy of combinatorial TMZ/riluzole treatment in an orthotopic mouse allograft model of MGMT-positive GBM using C57BL/6 J mice and GL261 cells.RESULTSRiluzole displayed significant time- and dose-dependent growth-inhibitory effects on all GBM cell lines assessed independently. Riluzole enhanced the antitumor effect of TMZ synergistically in MGMT-positive but not in MGMT-negative GBM cell lines. Riluzole singularly suppressed MGMT expression, and it significantly suppressed TMZ-induced MGMT upregulation (p < 0.01). Furthermore, combinatorial TMZ/riluzole treatment significantly suppressed tumor growth in the intracranial MGMT-positive GBM model (p < 0.05).CONCLUSIONSRiluzole attenuates TMZ-induced MGMT upregulation and enhances the antitumor effect of TMZ in MGMT-positive GBMs. Therefore, combinatorial TMZ/riluzole treatment is a potentially promising novel therapeutic regimen for MGMT-positive GBMs.

scholarly journals Role of O6-methylguanine-DNA methyltransferase and p53 in response of neuroblastoma cells to an alkylating agent temozolomide

2018 ◽  
Vol 99 (1) ◽  
pp. 47-53
Author(s):  
R R Khusnutdinov ◽  
S V Boychuk
Keyword(s):  
Dna Methyltransferase ◽  
Alkylating Agent ◽  
Neuroblastoma Cells ◽  
Methylguanine Dna Methyltransferase

Цель. Изучить роль белка р53 и О6-метилгуанин-ДНК-метилтрансферазы в чувствительности клеток нейробластомы к действию темозоломида. Методы. Исследование проводили на клеточной линии нейробластомы SK.N.SH, культивируемой в среде DMEM с добавлением эмбриональной телячьей сыворотки и антибиотиков пенициллина-стрептомицина в стандартных условиях (37 °C и 5% СО2). Клетки инкубировали с алкилирующим агентом темозоломидом в течение 48-72 ч. В ряде случаев осуществляли преинкубацию клеток в течение 2 ч с О6-бензилгуанином (ингибитором О6-метилгуанин-ДНК-метилтрансферазы) или нутлином-3а (реактиватором р53). Пролиферативную активность оценивали с помощью системы многопараметрического анализа клеточных культур (RTCA iCELLigence), а также колориметрического MTS-теста. Экспрессию белков определяли методом иммуноблоттинга с использованием соответствующих моноклональных антител. Результаты. Реактивация белка р53 приводила к значительному снижению скорости пролиферации клеток линии SK.N.SH. Цитотоксический эффект данного препарата более выражен по сравнению с темозоломидом, считающимся препаратом выбора при проведении химиотерапии пациентам с мультиформной глиобластомой и нейробластомой. Ингибирование О6-метилгуанин-ДНК-метилтрансферазы также приводило к усилению цитотоксического эффекта темозоломида, тем не менее, цитотоксический эффект химиопрепарата был менее выраженным по сравнению с действием темозоломида на фоне реактивации белка р53. Вывод. Для оценки чувствительности клеток нейробластомы к действию алкилирующего препарата темозоломида функциональное состояние белка р53 в опухолевых клетках служит более важным прогностическим критерием по сравнению с уровнем экспрессии О6-метилгуанин-ДНК-метилтрансферазы; кроме того, реактивация белка р53 приводит к снижению скорости пролиферации клеток нейробластомы линии SK.N.SH и их гибели по механизму апоптоза.

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