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γ-Glutamylcyclotransferase (GGCT) is up-regulated in a broad range of cancers, including breast cancer, and GGCT inhibition has been shown to be a promising strategy for therapy. Herein, we evaluated the efficacy and mechanism of action of pro-GA, a GGCT enzymatic inhibitor, in MCF7 breast cancer cells.Proliferation was evaluated by WST-8 and trypan blue dye exclusion assays. Western blot analysis was conducted to examine the expression of cyclin-dependent kinase inhibitors (CDKI), including p21, p27, and p16. Induction of senescence was assessed by senescence-associated β-galactosidase staining. Generation of mitochondrial superoxide reactive oxygen species (ROS) was assessed using flow cytometry. The effect of N-acetylcysteine (NAC) on pro-GA dependent inhibition of proliferation, ROS generation, and senescence was also studied. The efficacy of systemic administration of pro-GA was evaluated in a MCF7 xenograft mouse model.Treatment with pro-GA inhibited proliferation of MCF7 cells, increased CDKI expression and mitochondrial ROS, and induced cellular senescence. We found that cotreatment with NAC restored proliferation in pro-GA treated cells. NAC similarly suppressed CDKI expression, mitochondrial ROS generation, and senescence induced by pro-GA. Furthermore, the systemic administration of pro-GA in an MCF7 xenograft model had significant antitumor effects without toxicity.Pro-GA may be a promising therapeutic agent for the treatment of breast cancer.

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.

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 theDNA 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 that regulates p21 expression.

Fluorizoline is a cytotoxic trifluorothiazoline that targets the scaffold proteins prohibitins-1 and -2 (PHB1/2) to inhibit the kinase C-RAF and promote the expression of the cyclin-dependent kinase inhibitor p21 to induce cancer cell death. In melanocytes, fluorizoline also induces the synthesis of melanin. Herein we report the first structural requirement of fluorizoline analogues for these activities. We identified in particular some compounds that display enhanced anti-C-RAF and anti-MEK activities, and a higher cytotoxicity in HeLa cells compared to fluorizoline. These results provide a foundation for further optimization of PHB ligands for the treatment of cancers. We also discovered an analogue of fluorizoline that displays pharmacological effects opposed to those of fluorizoline and that can be used as a chemical tool to explore PHB signaling in cancers and other diseases.

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.

The prognosis of glioblastoma remains poor despite intensive research efforts. Glioblastoma stem cells (GSCs) contribute to tumorigenesis, invasive capacity, and therapy resistance. Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5), a stem cell marker, is involved in the maintenance of GSCs, although the properties of Lgr5-positive GSCs remain unclear. Here, the Sleeping-Beauty transposon-induced glioblastoma model was used in Lgr5-GFP knock-in mice identify GFP-positive cells in neurosphere cultures from mouse glioblastoma tissues. Global gene expression analysis showed that Gli2 was highly expressed in GFP-positive GSCs. Gli2 knockdown using lentiviral-mediated shRNA downregulated Hedgehog-related and Wnt signaling pathway-related genes, including Lgr5; suppressed tumor cell proliferation and invasion capacity; and induced apoptosis. Pharmacological Gli inhibition with GANT61 suppressed tumor cell proliferation. Silencing Gli2 suppressed the tumorigenicity of GSCs in an orthotopic transplantation model in vivo. These findings suggest that Gli2 affects the Hedgehog and Wnt pathways and plays an important role in GSC maintenance, suggesting Gli2 as a therapeutic target for glioblastoma treatment.

Metabolic reprogramming leading to aerobic glycolysis, termed the "Warburg effect," is a critical property of cancer cells. However, the precise mechanisms underlying this phenomenon are not fully understood. A growing body of evidence indicates that γ-glutamylcyclotransferase (GGCT), an enzyme involved in glutathione homeostasis that is highly expressed in many types of cancer, represents a promising therapeutic target. In this study, we identified GGCT as a novel regulator of hypoxia-inducible factor-1α (HIF-1α), a transcription factor that plays a role in hypoxia adaptation promoting aerobic glycolysis. In multiple human cancer cell lines, depletion of GGCT downregulated HIF-1α at the mRNA and protein levels. Conversely, in NIH3T3 mouse fibroblasts, overexpression of GGCT upregulated HIF-1α under normoxia. Moreover, depletion of GGCT downregulated HIF-1α downstream target genes involved in glycolysis, whereas overexpression of GGCT upregulated those genes. Metabolomic analysis revealed that modulation of GGCT expression induced a metabolic switch from the citric acid cycle to glycolysis under normoxia. In addition, we found that GGCT regulates expression of HIF-1α protein via the AMPK-mTORC1-4E-BP1 pathway in PC3 cells. Thus GGCT regulates the expression of HIF-1α in cancer cells, causing a switch to glycolysis.

The prognosis of glioblastoma, which is the most frequent type of adult‑onset malignant brain tumor, is extremely poor. Therefore, novel therapeutic strategies are needed. Previous studies report that JCI‑20679, 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 JCI‑20679 on glioblastoma stem cells (GSCs) is unknown. The present study demonstrated that JCI‑20679 inhibited the growth of GSCs derived from a transposon system‑mediated murine glioblastoma model more efficiently compared with the growth of differentiation‑induced adherent cells, as determined by a trypan blue staining dye exclusion test. The inhibition of proliferation was accompanied by the blockade of cell‑cycle entry into the S‑phase, as assessed by a BrdU incorporation assay. JCI‑20679 decreased the mitochondrial membrane potential, suppressed the oxygen consumption rate and increased mitochondrial reactive oxygen species generation, indicating that JCI‑20679 inhibited mitochondrial activity. The mitochondrial inhibition was revealed to increase phosphorylated (phospho)‑AMPKα levels and decrease nuclear factor of activated T‑cells 2 (NFATc2) expression, and was accompanied by a decrease in calcineurin phosphatase activity. Depletion of phospho‑AMPKα by knockdown of AMPKβ recovered the JCI‑20679‑mediated decrease in NFATc2 expression levels, as determined by western blotting and reverse transcription‑quantitative PCR analysis. Overexpression of NFATc2 recovered the JCI‑20679‑mediated suppression of proliferation, as determined by a trypan blue staining dye exclusion test. These results suggest that JCI‑20679 inhibited mitochondrial oxidative phosphorylation, which activated AMPK and reduced NFATc2 expression levels. Moreover, systemic administration of JCI‑20679 extended the event‑free survival rate in a mouse model transplanted with GSCs. Overall, these results suggested that JCI‑20679 is a potential novel therapeutic agent against glioblastoma.

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.

Background/aim To examine the dynamics of circulating tumour cells (CTCs) in pancreatic cancer (PC), new mouse CTC models from human PC xenografts were developed. Materials and methods Orthotopic (pancreas) and heterotopic (subcutaneous) transplantation models using GFP-tagged SUIT-2 PC cells were prepared. Using a cytology-based CTC detection platform, CTCs and metastasis were compared. Results The two types of orthotopic models, including the surgical transplantation model and the intraperitoneal injection model, showed a similar pattern of initial pancreatic tumour formation and subsequent development of peritoneal and hematogenous lung metastases. In the heterotopic model, only hematogenous lung metastasis was observed, and the number of CTCs and lung metastases was higher than that of the orthotopic model. Furthermore, KRAS mutation (G12D) was detected in CTCs. Conclusion These orthotopic and heterotopic models clearly differ in terms of the pattern of metastasis and CTCs and therefore, would be useful PC models to investigate the effect of drug-therapy on CTCs and the role of KRAS mutation.

Background/aim Based on the cytotoxic agent (-)-zampanolide, N,N'-(arylmethylene)bisamides were designed and synthesized as candidate anti-cancer agents. Among them, N,N'-[(3,4-dimethoxyphenyl)methylene]biscinnamide (DPMBC) was identified as the most potent cytotoxic analog against cancer cells. In this study, we investigated the mechanisms underlying DPMBC-induced cell death in HL-60 human promyelocytic leukemia and PC-3 human prostate cancer cells. Materials and methods Cell growth was assessed by the WST-8 assay. Induction of apoptosis was assessed by nuclear morphology, DNA ladder formation, and flow cytometry using Annexin V staining. Activation of factors in the apoptotic signaling pathway was assessed by western blot analyses. Knockdown of death receptor 5 (DR5) was performed using siRNA. Results DPMBC up-regulated expression levels of DR5 protein and induced apoptosis through the extrinsic apoptotic pathway mediated by DR5 and caspases. Conclusion DPMBC is an extrinsic apoptosis inducer, which has potential as a therapeutic agent for cancer therapy.

Amyloid-β (Aβ) accumulation in the brain triggers the onset of Alzheimer's disease (AD), and its prevention and elimination are high priorities for anti-AD therapeutic strategies. Microglia, the resident immune cells in the brain, promote Aβ clearance by phagocytosis. Previously, we demonstrated that injection of primary cultured rat microglia and mouse bone marrow-derived microglia-like cells into the brain decreases the level of Aβ and that intrahippocampal injection of these cells ameliorates cognitive impairment in a mouse model of AD. To advance this cell therapeutic strategy to the clinical stage, less invasive ways of preparing autologous microglia-like cells from elderly patients are required. In this study, we demonstrated that hematopoietic stem cells mobilized from the bone marrow to peripheral blood by administering granulocyte colony-stimulating factor and a CXCR4 antagonist to mice differentiated into microglia-like cells upon stimulation with colony-stimulating factor 1 and interleukin-34. The peripheral blood-derived microglia-like (PBDML) cells expressed microglial markers and engaged in Aβ phagocytosis. Although PBDML cells were in an anti-inflammatory state under nonstimulated conditions, they expressed mRNAs encoding proinflammatory cytokines following lipopolysaccharide treatment. PBDML cells injected into the hippocampi of a mouse AD model survived for at least 36 days while phagocytosing Aβ, contributed to a reduction in brain Aβ burden, and ameliorated cognitive impairment in the mice. These results strongly suggest that PBDML cells are a promising source for the development of a novel cell therapy against AD.

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.

γ-Glutamyl cyclotransferase (GGCT) is up-regulated in various cancer types, including lung cancer. In this study, we evaluated efficacy of gapmer-type antisense oligonucleotides (ASOs) targeting GGCT in an A549 lung cancer xenograft mouse model and studied their mechanisms of action.GGCT was inhibited using GGCT-ASOs and cell proliferation was evaluated by dye exclusion test. Western blot analysis was conducted to measure expression of GGCT, p21, p16 and p27, phosphorylation of AMP-activated protein kinase, and caspase activation in A549 cells. Induction of apoptosis and up-regulation of reactive oxygen species were assessed by flow cytometry using annexin V staining and 2',7'-dichlorodihydrofluorescein diacetate dye, respectively.GGCT-ASOs suppressed GGCT expression in A549 cells, inhibited proliferation, and induced apoptosis with activation of caspases. GGCT-ASOs also increased expression of cell-cycle regulating proteins, phospho-AMPK and ROS levels. Systemic administration of GGCT-ASOs to animals bearing A549 lung cancer xenografts showed significant antitumor effects without evident toxicity.GGCT-ASOs appear to be promising as novel cancer therapeutic agents.

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.

Background/aim DJ-1, an oncogenic molecule, helps to maintain somatic stem cells by reducing the intracellular level of reactive oxygen species (ROS). This study investigated the role of DJ-1 in glioma stem cells (GSCs). Materials and methods U87-MG (U87) and U251-MG (U251) glioblastoma cell lines that express wild-type and mutant p53, respectively, were used. These were cultured with DJ-1-targeting siRNA and subjected to a variety of in vitro experiments or intracranial transplantation into nude mice. Results Knockdown of DJ-1 reduced clonogenicity only in U87 cells, which was rescued by p53 depletion. ROS accumulated in DJ-1-depleted cells, although treatment with N-acetyl cysteine, which quenches ROS, did not affect exhaustion of CSCs among U87 cells by DJ-1 knockdown. In a serial transplantation study, DJ-1 knockdown prolonged the survival of mice in secondary transplantation. Conclusion DJ-1 plays a pivotal role in maintenance of stem cell self-renewal in the U87 cell line.

Background/aim Trastuzumab is the only clinically approved targeted therapy for HER2 gene-amplified gastric cancer at present. However, the clinical significance of multi-targeting tyrosine kinase inhibitors (TKIs) in HER2-positive gastric cancer remains unclear. Materials and methods We examined the anti-tumor activity of lapatinib and afatinib, that are reversible and irreversible TKIs, in HER2 gene-amplified trastuzumab-sensitive and - resistant gastric cancer cells (GLM-1 and GLM-1HerR2) in vitro and in vivo. Results Afatinib inhibited the growth of GLM-1 and GLM-1HerR2 cells in vitro more efficiently than lapatinib by inducing G1 cell-cycle arrest and apoptosis. Preclinical studies in mice revealed that afatinib inhibited growth of intraperitoneal GLM-1 and subcutaneous GLM-1HerR2 tumor more strongly than lapatinib. Afatinib was more effective than lapatinib in blocking PI3K/Akt and MAPK signaling in both GLM-1 and GLM-1HerR2 cells. Conclusion Afatinib could be a potential new molecular-targeted therapy for trastuzumab-sensitive and trastuzumab-resistant HER2 gene-amplified gastric cancers.

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.

Background/aim γ-Glutamylcyclotransferase (GGCT) is highly expressed in many forms of cancer, and is a promising therapeutic target. The present study investigated whether inhibition of GGCT enhanced the antiproliferative effects of the drug docetaxel in prostate cancer cells. Materials and methods Immunohistochemistry and western blot analysis were conducted to measure GGCT expression in prostate cancer tissue samples and cell lines. GGCT was inhibited using RNAi and a novel enzymatic inhibitor, pro-GA, and cell proliferation was evaluated with single and combination treatments of GGCT inhibitors and docetaxel. Results GGCT was highly expressed in cultured prostate cancer cells and patient samples. GGCT inhibition alone inhibited prostate cancer cell line proliferation and induced cellular senescence. GGCT inhibition in combination with apoptosis-inducing docetaxel had more potent antiproliferative effects than either drug used alone. Conclusion GGCT inhibition may potentiate anticancer drug efficacy.

Background/aim γ-Glutamylcyclotransferase (GGCT), a key enzyme involved in glutathione metabolism, catalyzes a specific reaction that generates 5-oxoproline and free amino acids from the γ-glutamyl peptide. Inhibition of GGCT is a promising therapeutic strategy for the treatment of various cancers. Materials and methods Immuno-histochemistry was used to evaluate GGCT expression in bladder tumors. The growth inhibitory effect of pro-GA, a novel GGCT inhibitor, in the presence or absence of mitomycin C (MMC) was assessed in three distinct bladder cancer cell lines. Results Over half of the clinical bladder tumor samples overexpressed GGCT. Pro-GA reduced the growth of all bladder cancer cell lines in a dose-dependent manner, and increased the anti-tumor effect of MMC. Conclusion Inhibition of GGCT using pro-GA provides a novel therapeutic strategy for the treatment of bladder cancers.