ABSTRACT
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.
ABSTRACT
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.
Subject(s)
Down Syndrome , Mice , Animals , Humans , Down Syndrome/genetics , Down Syndrome/pathology , Disease Models, AnimalABSTRACT
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.
Subject(s)
gamma-Glutamylcyclotransferase , Animals , Humans , Mice , gamma-Glutamylcyclotransferase/chemistry , MCF-7 Cells , Fluorescent Dyes/chemistryABSTRACT
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.
Subject(s)
Prostatic Neoplasms , Retinal Neoplasms , Retinoblastoma , Humans , Male , Animals , Mice , AMP-Activated Protein Kinases , gamma-Glutamylcyclotransferase , Disease Models, AnimalABSTRACT
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.
Subject(s)
Glioblastoma , Humans , Glioblastoma/drug therapy , Glioblastoma/pathology , AMP-Activated Protein Kinases , Cell Line, Tumor , Thiophenes/pharmacology , Thiophenes/therapeutic use , Cell Proliferation , Ethylene Glycols/pharmacology , Ethylene Glycols/therapeutic useABSTRACT
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.
Subject(s)
Glioblastoma , Adult , Humans , Animals , Mice , Brain , Apoptosis , Cell Proliferation , Stem Cells , STAT5 Transcription FactorABSTRACT
BACKGROUND/AIM: 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. MATERIALS AND METHODS: 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 ß-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. RESULTS: 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. CONCLUSION: Pro-GA may be a promising therapeutic agent for the treatment of breast cancer.
Subject(s)
Breast Neoplasms , gamma-Glutamylcyclotransferase , Acetylcysteine/pharmacology , Animals , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Proliferation , Enzyme Inhibitors/pharmacology , Female , Humans , MCF-7 Cells , Mice , Mitochondria/metabolism , Reactive Oxygen Species/metabolismABSTRACT
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.
Subject(s)
Apoptosis , Prohibitins , Cyclin-Dependent Kinase Inhibitor p21/metabolism , HeLa Cells , Humans , Ligands , Melanins/metabolism , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-raf/metabolism , Proto-Oncogene Proteins c-raf/pharmacology , Repressor Proteins , Transcription Factors/metabolismABSTRACT
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.
Subject(s)
Surgeons , Virtual Reality , Computer Simulation , Humans , Imaging, Three-Dimensional/methods , TechnologyABSTRACT
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.
Subject(s)
Glioblastoma , AMP-Activated Protein Kinases/metabolism , Animals , Cell Proliferation , Glioblastoma/drug therapy , Glioblastoma/genetics , Glioblastoma/metabolism , Mice , Neoplastic Stem Cells/metabolism , Trypan Blue/metabolism , Trypan Blue/therapeutic useABSTRACT
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.
Subject(s)
Aortic Valve , Coronary Vessels , Animals , Aorta/diagnostic imaging , Aortic Valve/diagnostic imaging , Aortic Valve/surgery , Coronary Vessels/diagnostic imaging , Imaging, Three-Dimensional , Swine , Tomography, X-Ray ComputedABSTRACT
Glioblastoma (GBM) is the most common and malignant type of brain cancer in adults with poor prognosis. GBM stem cells (GSCs) reside within niches in GBM tissues and contribute to recurrence and therapy resistance. Previous studies have shown that expression of leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5), a Wnt pathway-related stem cell marker, correlates with a poor prognosis in GBM, and its knockdown in GSCs induces apoptosis accompanied with downregulation of signal transducer and activator of transcription 5b (Stat5b). Here, we show that Stat5b co-localizes with Lgr5 in hypoxia-inducible factor 2α (Hif2α)-positive regions in GBM tissues. Functional analyses using GSCs derived from a murine de novo GBM model induced by oncogenic genes transduction using the Sleeping-Beauty transposon system revealed that expression of Stat5b was induced by culturing under hypoxia together with Lgr5, repressed by Hif2α knockdown, and reduced by Lgr5 knockdown or a Wnt/ß-catenin signaling inhibitor ICG-001 treatment. Stat5b inhibition in the GSCs induced apoptosis and caused downregulation of Cyclin E2 resulted in blockade of entry into S-phase in the cell cycle. Disruption of Stat5b in an orthotopic transplantation model significantly prolongs event-free survival. These results suggest that Stat5b, regulated by hypoxia and the Wnt pathway, plays an important role in the maintenance and tumorigenicity of GSCs and may be a promising therapeutic molecular target to attack GSCs.
ABSTRACT
BACKGROUND/AIM: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: GGCT-ASOs appear to be promising as novel cancer therapeutic agents.
Subject(s)
Antineoplastic Agents/pharmacology , Cell Proliferation/drug effects , Lung Neoplasms/drug therapy , Oligonucleotides, Antisense/pharmacology , gamma-Glutamylcyclotransferase/metabolism , A549 Cells , Animals , Apoptosis , Caspases/metabolism , Cell Cycle Proteins/metabolism , Cycloheximide/analogs & derivatives , Cycloheximide/metabolism , Gene Expression Regulation, Neoplastic , Humans , Lung Neoplasms/enzymology , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Male , Mice, SCID , Signal Transduction , Tumor Burden , Xenograft Model Antitumor Assays , gamma-Glutamylcyclotransferase/geneticsABSTRACT
PURPOSE: To investigate a virtual reality imaging system in terms of visualization accuracy and appropriate orientation when displaying cardiac anatomy, we used an ex vivo model enabling direct comparison between reconstructed 3-dimensional visualization of intracardiac structures and real-time visual images. DESCRIPTION: We established a systole-diastole platform using a swine heart activated by an external mechanical pump and reservoir, allowing simultaneous acquisition of endoscopic visual and computed tomography images of the aortic valve. Virtual images were processed from computed tomography data using 3-dimensional software (the Vesalius 3D suite; PS Medtech, Amsterdam, Netherlands) and compared with visual images seen through a fiberoptic scope. EVALUATION: An endoscope gave a fine view of the aortic valve, whereas the virtual images elucidated the valve structures. Superimposition of the images from the 2 different modalities showed the virtual reality images precisely matching the visual images in both systole and diastole, confirming the validity of this virtual reality application. CONCLUSION: In view of this demonstrated fidelity of virtual imaging, this technology may be of sufficiently high quality to be considered a gold standard for cardiac anatomy.
Subject(s)
Aortic Valve , Tomography, X-Ray Computed , Animals , Aortic Valve/diagnostic imaging , Diastole , Humans , Imaging, Three-Dimensional , Netherlands , Swine , SystoleABSTRACT
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.
Subject(s)
Autophagy , Glioblastoma , Temozolomide , Animals , Humans , Adenosine Triphosphate/metabolism , Autophagy/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Synergism , Glioblastoma/pathology , Mice, SCID , Temozolomide/pharmacologyABSTRACT
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.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/metabolism , Cyclin-Dependent Kinase Inhibitor p21/biosynthesis , Gene Expression Regulation, Neoplastic/physiology , Prohibitins/metabolism , gamma-Glutamylcyclotransferase/metabolism , Antineoplastic Agents/chemical synthesis , Breast Neoplasms/genetics , Cell Proliferation/drug effects , Cell Proliferation/physiology , Cyclin-Dependent Kinase Inhibitor p21/genetics , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , MCF-7 Cells , Prohibitins/antagonists & inhibitors , gamma-Glutamylcyclotransferase/antagonists & inhibitorsABSTRACT
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.
Subject(s)
Citric Acid Cycle , gamma-Glutamylcyclotransferase , Animals , Cell Line, Tumor , Glycolysis/genetics , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Mice , NIH 3T3 Cells , gamma-Glutamylcyclotransferase/geneticsABSTRACT
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.
Subject(s)
Breast Neoplasms/pathology , Chromans/pharmacology , Enzyme Inhibitors/pharmacology , Piperazines/pharmacology , gamma-Glutamylcyclotransferase/antagonists & inhibitors , Animals , Cell Proliferation/drug effects , Cell Survival/drug effects , Female , Humans , MCF-7 Cells , Mice , Mice, SCID , NIH 3T3 Cells , Xenograft Model Antitumor Assays , gamma-Glutamylcyclotransferase/metabolismABSTRACT
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.
Subject(s)
Brain Neoplasms/genetics , Gene Expression Profiling/methods , Glioblastoma/genetics , Zinc Finger Protein Gli2/metabolism , Animals , Brain Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation , Disease Models, Animal , Glioblastoma/pathology , Humans , Mice , PrognosisABSTRACT
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.