Myc upregulates Ggct, γ-glutamylcyclotransferase to promote development of p53-deficient osteosarcoma.

Osteosarcoma (OS) in humans is characterized by alterations in the TP53 gene. In mice, loss of p53 triggers OS development, for which c-Myc (Myc) oncogenicity is indispensable. However, little is known about which genes are targeted by Myc to promote tumorigenesis. Here, we examined the role of γ-glutamylcyclotransferase (Ggct) which is a component enzyme of the γ-glutamyl cycle essential for glutathione homeostasis, in human and mouse OS development. We found that GGCT is a poor prognostic factor for human OS, and that deletion of Ggct suppresses p53-deficient osteosarcomagenesis in mice. Myc upregulates Ggct directly by binding to the Ggct promoter, and deletion of a Myc binding site therein by genome editing attenuated the tumorigenic potential of p53-deficient OS cells. Taken together, these results show a rationale that GGCT is widely upregulated in cancer cells and solidify its suitability as a target for anticancer drugs.

Development of an activity-based chemiluminogenic probe for γ-glutamylcyclotransferase.

While γ-glutamylcyclotransferase (GGCT) has been implicated in cancer-cell proliferation, the role of GGCT enzymatic activity in the regulation of cancer-cell growth remains unclear. Toward further understanding of GGCT in vivo, here we report a novel cell-permeable chemiluminogenic probe "MAM-LISA-103" that detects intracellular GGCT activity and apply it to in vivo imaging. We first developed a chemiluminogenic probe LISA-103, which simply and sensitively detects the enzymatic activity of recombinant GGCT through chemiluminescence. We then designed the cell-permeable GGCT probe MAM-LISA-103 and applied it to several biological experiments. MAM-LISA-103 successfully detected the intracellular GGCT activity in GGCT-overexpressing NIH-3T3 cells. Moreover, MAM-LISA-103 demonstrated tumor-imaging ability when administered to a xenograft model using immunocompromised mice inoculated with MCF7 cells.

Fluorizoline Blocks the Interaction between Prohibitin-2 and γ-Glutamylcyclotransferase and Induces p21Waf1/Cip1 Expression in MCF7 Breast Cancer Cells.

Prohibitin-2 (PHB2) is a scaffold protein that has pleiotropic functions, which include interacting with γ-glutamylcyclotransferase (GGCT) in the cytoplasm and repressing the transcriptional activities of the p21Waf1/Cip (p21) gene in the nucleus. The cytotoxic drug fluorizoline binds to PHB1/2 and exerts antiproliferative actions on cancer cells. However, the precise mechanism underlying the antiproliferative effects of fluorizoline is not fully elucidated. In the present study, we first show that fluorizoline induces p21 expression in several human cancer cell lines, including MCF7 breast cancer cells. Treatment of MCF7 cells with fluorizoline suppressed proliferation and prevented cells from entering into the DNA synthesis phase. Knockdown of p21 rescued the suppressed proliferation, indicating that fluorizoline inhibited MCF7 cell growth via the induction of p21. Overexpression of PHB2 in MCF7 cells prevented the induction of p21 expression by fluorizoline and restored the antiproliferative effects and blockade of cell cycle progression. Moreover, treatment of MCF7 cells with fluorizoline inhibited the interaction between endogenous PHB2 and GGCT proteins and reduced the level of nuclear localization of PHB2 proteins. These results indicate that targeting PHB2 with fluorizoline induces the expression of p21 and consequently blocks proliferation of cancer cells. SIGNIFICANCE STATEMENT: This study shows that fluorizoline may be a promising novel anticancer drug candidate that induces p21 expression and blocks cell-cycle progression in human cancer cell lines. In addition, we show that fluorizoline inhibits the interaction between PHB2 and GGCT and reduces the nuclear localization of PHB2 proteins.

JCI-20679 suppresses autophagy and enhances temozolomide-mediated growth inhibition of glioblastoma cells.

Glioblastoma, a type of brain cancer, is one of the most aggressive and lethal types of malignancy. The present study shows that JCI-20679, an originally synthesized mitochondrial complex I inhibitor, enhances the anti-proliferative effects of suboptimal concentrations of the clinically used chemotherapeutic drug temozolomide in glioblastoma cells. Analysis of the effects of temozolomide combined with JCI-20679 using isobologram and combination index methods demonstrated that the combination had synergistic effects in murine and human glioblastoma cells. We found that JCI-20679 inhibited the temozolomide-mediated induction of autophagy that facilitates cellular survival. The autophagy induced by temozolomide increased ATP production, which confers temozolomide resistance in glioblastoma cells. JCI-20679 blocked temozolomide-mediated increases in ATP levels and increased the AMP/ATP ratio. Furthermore, JCI-20679 enhanced the therapeutic effects of temozolomide in an orthotopic transplantation model of glioblastoma. These results indicate that JCI-20679 may be promising as a novel agent for enhancing the efficacy of temozolomide against glioblastoma.

Ex-vivo aortic root and coronary artery cast measurement to validate the accuracy of virtual imaging.

BACKGROUND AND AIM OF THE STUDY: To investigate the accuracy of two methods of measuring features in cardiac anatomy, using an objective standard cast model. METHODS: We made a silicone cast using a swine heart. Computerized tomography data of the solidified cast were processed through virtual reality (VR) software and through two-dimensional multiplanar-reconstruction (2D-MPR), and all measurements were compared against physical measurements of the cast. RESULTS: The cast perfectly demonstrated the fine detail of the aortic valve and the proximal parts of coronary arteries. Anatomical features were measured by 3D-VR, 2D-MPR, and directly on the cast. Measurement differences between 2D-MPR and the cast were on average at least 3.6 times larger than those between 3D-VR and the cast. CONCLUSIONS: Based on the observed accuracy, 3D-VR measurements seem considerably more accurate than the current standard 2D-MPR, and 3D-VR may be considered as the next gold standard for 3D measurement of cardiac anatomy in vivo.

[A Novel Virtual-reality Imaging System in Cardiovascular Surgery:a Potentially Promising Technology for Surgeons].

Adequate preoperative planning may facilitate successful procedures in cardiovascular surgery. We have developed a system named the Vesalius 3D suite, combining three-dimensional (3D) image-processing software with an optic-tracking spatial navigation, allowing quick, accessible 3D image interpretation for virtual reality (VR) exploration and measurement from one or more of a range of imaging modalities. We present a novel method of virtual imaging analysis for preoperative planning and simulation in cardiovascular surgery using this 3D-VR system. Based on unimodal or multimodal medical imaging data, digital imaging and communication in medicine (DICOM) data sets can be reconstructed for 3D visualization. Virtually reconstructed images can be viewed on flat-screen or stereoscopic display, revealing each patient's specific anatomy and the internal structures in exquisite detail. Highly accessible 3D interpretation promptly permits precise measurements of repair-relevant anatomical parameters including geometrically complex shapes. This technology may promote understanding of form and function in the cardiovascular system, and facilitate operative procedures in more challenging cases, and it seems especially valuable for any surgeon to gain experience in practicing for rarely-performed procedures or uncommon patient-specific preoperative surgical rehearsal.

Identification of U83836E as a γ-glutamylcyclotransferase inhibitor that suppresses MCF7 breast cancer xenograft growth.

γ-Glutamylcyclotransferase (GGCT) is involved in glutathione homeostasis, in which it catalyzes the reaction that generates 5-oxoproline and free amino acids from γ-glutamyl peptides. Increasing evidence shows that GGCT has oncogenic functions and is overexpressed in various cancer tissues, and that inhibition of GGCT activity exerts anticancer effects in vitro and in vivo. Here, we demonstrate that U83836E ((2R)-2-[[4-(2,6-dipyrrolidin-1-ylpyrimidin-4-yl)piperazin-1-yl]methyl]-3,4-dihydro-2,5,7,8,-tetramethyl-2H-1-benzopyran-6-ol, dihydrochloride), a lazaroid that inhibits lipid peroxidation, inhibits GGCT enzymatic activity. U83836E was identified from a high-throughput screen of low molecular weight compounds using a fluorochrome-conjugated GGCT probe. We directly quantified that U83836E specifically inhibited GGCT by measuring the product of a fluorochrome-conjugated GGCT substrate assay, and showed that U83836E inhibited GGCT activity in extracts of NIH3T3 cells overexpressing GGCT. Moreover, U83836E significantly inhibited tumor growth in a xenograft model that used immunodeficient mice orthotopically inoculated with MCF7 human breast cancer cells. These results indicate that U83836E may be a useful GGCT inhibitor for the development of potential cancer therapeutics.

Depletion of gamma-glutamylcyclotransferase inhibits cancer cell growth by activating the AMPK-FOXO3a-p21 axis.

Inhibition of gamma-glutamylcyclotransferase (GGCT), which is highly expressed in various cancer tissues, exerts anticancer effects both in vitro and in vivo. Previous studies have shown that depletion of GGCT blocks the growth of MCF7 breast cancer cells via upregulation of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21); in addition, induction of autophagy plays a role in the upregulation of p21 upon GGCT knockdown. However, the mechanisms underlying induction of p21 in cancer cells are not fully understood. Here, we show that GGCT knockdown in PC3 human prostate cancer and A172 glioblastoma cells upregulates the mRNA and nuclear protein levels of Forkhead box O transcription factor 3a (FOXO3a), a transcriptional factor involved in tumor suppression. Simultaneous knockdown of FOXO3a and GGCT in PC3 and A172 cells attenuated upregulation of p21, followed by growth inhibition and cell death. Furthermore, simultaneous knockdown of GGCT and AMP-activated protein kinase (AMPK) α, a metabolic stress sensor, in PC3 and A172 cells led to marked attenuation of cellular responses induced by GGCT knockdown, including an increase in FOXO3a phosphorylation at Ser413, upregulation of p21, growth inhibition, and cell death. These results indicate that the AMPK-FOXO3a-p21 axis plays an important role in inhibition of cancer cell growth by depletion of GGCT.

Multiple myeloma cells adapted to long-exposure of hypoxia exhibit stem cell characters with TGF-ß/Smad pathway activation.

The emergence of new molecular targeting agents has improved the prognosis of patients with multiple myeloma (MM). However, MM remains incurable because MM stem cells are likely resistant to these agents. Thus, it is important to further investigate the biology of MM stem cells, which reside in the hypoxic bone marrow niche. In this study, we established and investigated the characteristics of hypoxia-adapted MM (HA-MM) cells, which could proliferate for more than six months under hypoxic conditions (1% O2). The G0 fraction of HA-MM cells was larger than that of parental MM cells under normoxic conditions (20% O2). HA-MM cells possess enhanced tumorigenicity in primary and secondary transplantation studies. HA-MM cells also exhibited increased mRNA levels of stem cell markers and an enhanced self-renewal ability, and thus demonstrated characteristics of MM stem cells. These cells overexpressed phosphorylated Smad2, and treatment with a transforming growth factor (TGF)-ß/Smad signaling inhibitor decreased their clonogenicity in a replating assay. In conclusion, MM cells adapted to long-exposure of hypoxia exhibit stem cell characters with TGF-ß/Smad pathway activation.

Prohibitin-2 is a novel regulator of p21WAF1/CIP1 induced by depletion of γ-glutamylcyclotransferase.

Previous studies show that gamma-glutamylcyclotransferase (GGCT) is expressed at high levels in various cancer tissues and that its knockdown inhibits MCF7 cancer cell growth via upregulation of p21WAF1/CIP1 (p21). However, the detailed underlying mechanism is unclear. Here, we used yeast two-hybrid screening and co-immunoprecipitation to identify Prohibitin-2 (PHB2) as a novel protein that interacts with GGCT. We also show that nuclear expression of PHB2 in MCF7 cells falls upon GGCT knockdown, and that overexpression of PHB2 inhibits p21 upregulation. A chromatin immunoprecipitation assay revealed that nuclear PHB2 proteins bind to the p21 promoter, and that this interaction is abrogated by GGCT knockdown. Moreover, knockdown of PHB2 alone led to significant upregulation of p21 and mimicked the cellular events induced by GGCT depletion, including G0/G1 arrest, cellular senescence, and growth inhibition, in a p21 induction-dependent manner. Taken together, the results indicate that PHB2 plays a central role in p21 upregulation following GGCT knockdown and as such may promote deregulated proliferation of cancer cells by suppressing p21.

Mechanisms of Tumor Growth Inhibition by Depletion of γ-Glutamylcyclotransferase (GGCT): A Novel Molecular Target for Anticancer Therapy.

γ-Glutamylcyclotransferase (GGCT), which is one of the major enzymes involved in glutathione metabolism, is upregulated in a wide range of cancers-glioma, breast, lung, esophageal, gastric, colorectal, urinary bladder, prostate, cervical, ovarian cancers and osteosarcoma-and promotes cancer progression; its depletion leads to the suppression of proliferation, invasion, and migration of cancer cells. It has been demonstrated that the suppression or inhibition of GGCT has an antitumor effect in cancer-bearing xenograft mice. Based on these observations, GGCT is now recognized as a promising therapeutic target in various cancers. This review summarizes recent advances on the mechanisms of the antitumor activity of GGCT inhibition.

CG13250, a novel bromodomain inhibitor, suppresses proliferation of multiple myeloma cells in an orthotopic mouse model.

Multiple myeloma (MM) is characterized by the clonal proliferation of neoplastic plasma cells. Despite a stream of new molecular targets based on better understanding of the disease, MM remains incurable. Epigenomic abnormalities contribute to the pathogenesis of MM. bromodomain 4 (BRD4), a member of the bromodomain and extraterminal (BET) family, binds to acetylated histones during M/G1 transition in the cell cycle promoting progression to S phase. In this study, we investigated the effects of a novel BET inhibitor CG13250 on MM cells. CG13250 inhibited ligand binding to BRD4 in a dose-dependent manner and with an IC50 value of 1.1 µM. It inhibited MM proliferation in a dose-dependent manner and arrested cells in G1, resulting in the induction of apoptosis through caspase activation. CG13250 inhibited the binding of BRD4 to c-MYC promoter regions suppressing the transcription of the c-MYC gene. Administered in vivo, CG13250 significantly prolonged survival of an orthotopic MM-bearing mice. In conclusion, CG13250 is a novel bromodomain inhibitor that is a promising molecular targeting agent against MM.

Depletion of γ-glutamylcyclotransferase inhibits breast cancer cell growth via cellular senescence induction mediated by CDK inhibitor upregulation.

BACKGROUND: Chromosome 7 open reading frame 24 (C7orf24) was originally identified as a highly expressed protein in various types of cancer, and later shown to be a γ-glutamylcyclotransferase (GGCT). GGCT depletion in cancer cells has anti-proliferative effects in vitro and in vivo, and it is therefore considered a promising candidate as a therapeutic target. However, the cellular events induced by GGCT depletion remain unclear. METHODS: GGCT was depleted by siRNA in MCF7, MDA-MB-231, PC3, A172, Hela, and LNCaP cells. Induction of cellular senescence was evaluated with senescence-associated ß-galactosidase (SA-ß-Gal) staining. Expression levels of p21WAF1/CIP1 and p16INK4A were assessed by qRT-PCR and Western blotting. Effects of simultaneous double knockdown of p21WAF1/CIP1 and p16INK4A together with GGCT on cell cycle regulation and cell growth was measured by flow cytometry, and trypan blue dye exclusion test. RESULTS: We found that GGCT knockdown induces significant cellular senescence in various cancer cells. Cyclin dependent kinase inhibitor p21WAF1/CIP1 and/or p16INK4A were upregulated in all cell lines tested. Simultaneous knockdown of p21WAF1/CIP1 recovered the cell cycle arrest, attenuated cellular senescence induction, and rescued the subsequent growth inhibition in GGCT-silenced MCF7 breast cancer cells. In contrast, in GGCT silenced MDA-MB-231 breast cancer cells, GGCT depletion upregulated p16INK4A, which played a regulatory role in senescence induction, instead of p21WAF1/CIP1. CONCLUSIONS: Our findings demonstrate that induction of cellular senescence mediated by the upregulation of cyclin-dependent kinase inhibitors is a major event underlying the anti-proliferative effect of GGCT depletion in breast cancer cells, highlighting the potential of GGCT blockade as a therapeutic strategy to induce cellular senescence.

Ring1B promotes hepatic stem/progenitor cell expansion through simultaneous suppression of Cdkn1a and Cdkn2a in mice.

UNLABELLED: Polycomb-group (PcG) proteins play crucial roles in self-renewal of stem cells by suppressing a host of genes through histone modifications. Identification of the downstream genes of PcG proteins is essential for elucidation of the molecular mechanisms of stem cell self-renewal. However, little is known about the PcG target genes in tissue stem cells. We found that the PcG protein, Ring1B, which regulates expression of various genes through monoubiquitination of histone H2AK119, is essential for expansion of hepatic stem/progenitor cells. In mouse embryos with a conditional knockout of Ring1B, we found that the lack of Ring1B inhibited proliferation and differentiation of hepatic stem/progenitor cells and thereby inhibited hepatic organogenesis. These events were characterized by derepression of cyclin-dependent kinase inhibitors (CDKIs) Cdkn1a and Cdkn2a, known negative regulators of cell proliferation. We conducted clonal culture experiments with hepatic stem/progenitor cells to investigate the individual genetic functions of Ring1B, Cdkn1a, and Cdkn2a. The data showed that the cell-cycle inhibition caused by Ring1B depletion was reversed when Cdkn1a and Cdkn2a were suppressed simultaneously, but not when they were suppressed individually. CONCLUSION: Our results show that expansion of hepatic stem/progenitor cells requires Ring1B-mediated epigenetic silencing of Cdkn1a and Cdkn2a, demonstrating that Ring1B simultaneously regulates multiple CDKIs in tissue stem/progenitor cells.

Suppression of Sleeping Beauty-induced Gliomagenicity in Ts1Cje Mice, a Model of Down Syndrome.

BACKGROUND/AIM: Individuals with Down syndrome (DS), attributed to triplication of human chromosome 21 (Hsa21), exhibit a reduced incidence of solid tumors. However, the prevalence of glioblastoma among individuals with DS remains a contentious issue in epidemiological studies. Therefore, this study examined the gliomagenicity in Ts1Cje mice, a murine model of DS. MATERIALS AND METHODS: We employed the Sleeping Beauty transposon system for the integration of human oncogenes into cells of the subventricular zone of neonatal mice. RESULTS: Notably, Sleeping Beauty-mediated de novo murine gliomagenesis was significantly suppressed in Ts1Cje mice compared to wild-type mice. In glioblastomas of Ts1je mice, we observed an augmented presence of M1-polarized tumor-associated macrophages and microglia, known for their anti-tumor efficacy in the early stage of tumor development. CONCLUSION: Our findings in a mouse model of DS offer novel perspectives on the diminished gliomagenicity observed in individuals with DS.

Myb Repression Mediates Stat5b-knockdown-induced Apoptosis and Inhibits Proliferation of Glioblastoma Stem Cells.

BACKGROUND/AIM: Glioblastoma is the most common and aggressive malignant brain tumor in adults, and glioblastoma stem cells (GSCs) contribute to treatment resistance and recurrence. Inhibition of Stat5b in GSCs suppresses cell proliferation and induces apoptosis. Herein, we investigated the mechanisms of growth inhibition by Stat5b knockdown (KD) in GSCs. MATERIALS AND METHODS: GSCs were established from a murine glioblastoma model in which shRNA-p53 and EGFR/Ras mutants were induced in vivo using a Sleeping Beauty transposon system. Microarray analyses were performed on Stat5b-KD GSCs to identify genes that are differentially expressed downstream of Stat5b. RT-qPCR and western blot analyses were used to determine Myb levels in GSCs. Myb-overexpressing GSCs were induced by electroporation. Proliferation and apoptosis were evaluated by a trypan blue dye exclusion test and annexin-V staining, respectively. RESULTS: MYB, which is involved in the Wnt pathway, was identified as a novel gene whose expression was down-regulated by Stat5b-KD in GSCs. Both MYB mRNA and protein levels were down-regulated by Stat5b-KD. Overexpression of Myb rescued cell proliferation that was suppressed by Stat5b-KD. Furthermore, Stat5b-KD-induced apoptosis in GSCs was significantly inhibited by Myb overexpression. CONCLUSION: Down-regulation of Myb mediates Stat5b-KD-induced inhibition of proliferation and induction of apoptosis in GSCs. This may represent a promising novel therapeutic strategy against glioblastoma.

Identification of c-Met as a novel target of γ-glutamylcyclotransferase.

γ-Glutamylcyclotransferase (GGCT) is highly expressed in multiple types of cancer tissues and its knockdown suppresses the growth of cancer cells in vitro and in vivo. Although GGCT is a promising target for cancer therapy, the mechanisms underlying the antitumor effects remain unclear. The knockdown of GGCT inhibited the MEK-ERK pathway, and activated the tumor suppressor retinoblastoma gene (RB) at the protein level in cancer cell lines. c-Met was down-regulated by the knockdown of GGCT in cancer cells and its overexpression attenuated the dephosphorylation of RB and cell cycle arrest induced by the knockdown of GGCT in lung cancer A549 cells. STAT3 is a transcription factor that induces c-Met expression. STAT3 phosphorylation and its nuclear expression level were decreased in GGCT-depleted A549 and prostate cancer PC3 cells. The simultaneous knockdown of AMPK and GGCT restored the down-regulated expression of c-Met, and attenuated the dephosphorylation of STAT3 and MEK-ERK-RB induced by the knockdown of GGCT in PC3 cells. An intraperitoneal injection of a GGCT inhibitor decreased c-Met protein expression in a mouse xenograft model of PC3 cells. These results suggest that the knockdown of GGCT activates the RB protein by inhibiting the STAT3-c-Met-MEK-ERK pathway via AMPK activation.

Thiophene Carboxamide Analogs with Long Alkyl Chains Comprising Ethylene Glycol Units Inhibit Glioblastoma Cell Proliferation by Activating AMPK.

Glioblastoma is a refractory malignant tumor that requires novel therapeutic strategies for effective treatment. We have previously reported that JCI-20679 (1), an analog of annonaceous acetogenins, shows potent antitumor activity against glioblastomas. However, the synthesis of 1 requires 23 steps, including 16 steps for the preparation of a tetrahydrofuran (THF) moiety. This study reports the design and synthesis of 11 analogs with a triethylene glycol moiety in place of the THF moiety in 1. Among these, the analog 2k with an n-decyl chain exhibited potent inhibitory activity against the growth of glioblastoma stem cells by inhibiting mitochondrial function and synergistically enhancing the effect of temozolomide (TMZ). Furthermore, 2k significantly suppressed tumor growth without critical toxicity in vivo. Hence, this study presents novel potential anticancer agents and a strategy for the development of these agents that can be produced easily.

Aclarubicin enhances tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis through death receptor 5 upregulation.

Anthracycline drugs are potent anti-tumor agents. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a death ligand with promising anti-cancer effects. However, some tumor types develop resistance to TRAIL. We examined the effect of aclarubicin (ACR), an anthracycline, in combination with TRAIL. The combination of TRAIL and ACR synergistically induced apoptosis in human acute lymphoblastic leukemia Jurkat cells and human lung cancer A549 cells. In contrast, another anthracycline, doxorubicin (DOX), only slightly sensitized Jurkat cells and A549 cells to TRAIL-induced apoptosis, with weaker enhancement of death receptor 5 (DR5) expression than ACR. The RNase protection assay, real time RT-PCR and western blot demonstrated that ACR upregulated the expression of a TRAIL receptor, DR5. Caspase inhibitors and dominant negative DR5 efficiently reduced the apoptotic response to the treatment with ACR and TRAIL, indicating that the combined effect depends on caspase activities and the interaction between TRAIL and its receptor. ACR but not DOX increased the activity of the DR5 gene promoter in Jurkat cells carrying a mutation in the p53 gene, suggesting that ACR upregulates DR5 expression through p53-independent transcription. These results suggest the combination of TRAIL and ACR to be a promising treatment for malignant tumors.

JCI­20679 suppresses the proliferation of glioblastoma stem cells by activating AMPK and decreasing NFATc2 expression levels.

The prognosis of glioblastoma, which is the most frequent type of adult­onset malignant brain tumor, is extremely poor. Therefore, novel therapeutic strategies are needed. Previous studies report that JCI­20679, which is synthesized based on the structure of naturally occurring acetogenin, inhibits mitochondrial complex I and suppresses the growth of various types of cancer cells. However, the efficacy of JCI­20679 on glioblastoma stem cells (GSCs) is unknown. The present study demonstrated that JCI­20679 inhibited the growth of GSCs derived from a transposon system­mediated murine glioblastoma model more efficiently compared with the growth of differentiation­induced adherent cells, as determined by a trypan blue staining dye exclusion test. The inhibition of proliferation was accompanied by the blockade of cell­cycle entry into the S­phase, as assessed by a BrdU incorporation assay. JCI­20679 decreased the mitochondrial membrane potential, suppressed the oxygen consumption rate and increased mitochondrial reactive oxygen species generation, indicating that JCI­20679 inhibited mitochondrial activity. The mitochondrial inhibition was revealed to increase phosphorylated (phospho)­AMPKα levels and decrease nuclear factor of activated T­cells 2 (NFATc2) expression, and was accompanied by a decrease in calcineurin phosphatase activity. Depletion of phospho­AMPKα by knockdown of AMPKß recovered the JCI­20679­mediated decrease in NFATc2 expression levels, as determined by western blotting and reverse transcription­quantitative PCR analysis. Overexpression of NFATc2 recovered the JCI­20679­mediated suppression of proliferation, as determined by a trypan blue staining dye exclusion test. These results suggest that JCI­20679 inhibited mitochondrial oxidative phosphorylation, which activated AMPK and reduced NFATc2 expression levels. Moreover, systemic administration of JCI­20679 extended the event­free survival rate in a mouse model transplanted with GSCs. Overall, these results suggested that JCI­20679 is a potential novel therapeutic agent against glioblastoma.