Bong Jun Cho

Enhanced cytotoxic effect of radiation and temozolomide in malignant glioma cells: targeting PI3K-AKT-mTOR signaling, HSP90 and histone deacetylases

BackgroundDespite aggressive treatment with radiation therapy and concurrent adjuvant temozolomide (TMZ), glioblastoma multiform (GBM) still has a dismal prognosis. We aimed to identify strategies to improve the therapeutic outcome of combined radiotherapy and TMZ in GBM by targeting pro-survival signaling from the epidermal growth factor receptor (EGFR).MethodsGlioma cell lines U251, T98G were used. Colony formation, DNA damage repair, mode of cell death, invasion, migration and vasculogenic mimicry as well as protein expression were determined.ResultsU251 cells showing a low level of methyl guanine transferase (MGMT) were highly responsive to the radiosensitizing effect of TMZ compared to T98G cells having a high level of MGMT. Treatment with a dual inhibitor of Class I PI3K/mTOR, PI103; a HSP90 inhibitor, 17-DMAG; or a HDAC inhibitor, LBH589, further increased the cytotoxic effect of radiation therapy plus TMZ in U251 cells than in T98G cells. However, treatment with a mTOR inhibitor, rapamycin, did not discernibly potentiate the radiosensitizing effect of TMZ in either cell line. The mechanism of enhanced radiosensitizing effects of TMZ was multifactorial, involving impaired DNA damage repair, induction of autophagy or apoptosis, and reversion of EMT (epithelial-mesenchymal transition).ConclusionsOur results suggest possible strategies for counteracting the pro-survival signaling from EGFR to improve the therapeutic outcome of combined radiotherapy and TMZ for high-grade gliomas.

Effect of ginsam, a vinegar extract from Panax ginseng, on body weight and glucose homeostasis in an obese insulin-resistant rat model

Extracts of ginseng species show antihyperglycemic activity. We evaluated the antihyperglycemic and antiobesity effects of ginsam, a component of Panax ginseng produced by vinegar extraction, which is enriched in the ginsenoside Rg3. Otsuka Long-Evans Tokushima Fatty rats, an obese insulin-resistant rat model, were assigned into 1 of 3 groups (n = 8 each): controls (isotonic sodium chloride solution, 5 mL/d), rats given 300 mg/(kg d) ginsam, and rats given 500 mg/(kg d) ginsam. An intraperitoneal 2-hour glucose tolerance test was performed at the end of the 6-week treatment. After 8 weeks, body and liver weights, visceral fat measured by computed tomography, and fasting glucose and insulin concentrations and lipid profiles were recorded. Insulin-resistant rats treated with ginsam had lower fasting and postprandial glucose concentrations compared with vehicle-treated rats. Importantly, overall glucose excursion during the intraperitoneal 2-hour glucose tolerance test decreased by 21.5% (P < .01) in the treated rats, indicating improved glucose tolerance. Plasma insulin concentration was significantly lower in ginsam-treated rats. These changes may be related to increased glucose transporter 4 expression in skeletal muscle. Interestingly, when the data from both ginsam-treated groups were combined, body weight was 60% lower in the ginsam-treated rats than in the controls (P < .01). Liver weight and serum alanine aminotransferase concentrations were also lower in the ginsam-treated rats. These effects were associated with increased peroxisome proliferator-activated receptor gamma expression and adenosine monophosphate-activated protein kinase phosphorylation in liver and muscle. Our data suggest that ginsam has distinct beneficial effects on glucose metabolism and body weight control in an obese animal model of insulin resistance by changing the expression of genes involved in glucose and fatty acid metabolism.

Effect of a dipeptidyl peptidase-IV inhibitor, des-fluoro-sitagliptin, on neointimal formation after balloon injury in rats.

Background Recently, it has been suggested that enhancement of incretin effect improves cardiac function. We investigated the effect of a DPP-IV inhibitor, des-fluoro-sitagliptin, in reducing occurrence of restenosis in carotid artery in response to balloon injury and the related mechanisms. Methods and Findings Otsuka Long-Evans Tokushima Fatty rats were grouped into four: control (normal saline) and sitagliptin 100, 250 and 500 mg/kg per day (n = 10 per group). Sitagliptin or normal saline were given orally from 1 week before to 2 weeks after carotid injury. After 3 weeks of treatment, sitagliptin treatment caused a significant and dose-dependent reduction in intima-media ratio (IMR) in obese diabetic rats. This effect was accompanied by improved glucose homeostasis, decreased circulating levels of high-sensitivity C-reactive protein (hsCRP) and increased adiponectin level. Moreover, decreased IMR was correlated significantly with reduced hsCRP, tumor necrosis factor-α and monocyte chemoattractant protein-1 levels and plasminogen activator inhibitor-1 activity. In vitro evidence with vascular smooth muscle cells (VSMCs) demonstrated that proliferation and migration were decreased significantly after sitagliptin treatment. In addition, sitagliptin increased caspase-3 activity and decreased monocyte adhesion and NFκB activation in VSMCs. Conclusions Sitagliptin has protective properties against restenosis after carotid injury and therapeutic implications for treating macrovascular complications of diabetes.

Serum fibroblast growth factor-21 concentration is associated with residual renal function and insulin resistance in end-stage renal disease patients receiving long-term peritoneal dialysis.

Fibroblast growth factor-21 (FGF-21) is a new metabolic regulator, which is related to antiobesity and insulin sensitivity in vivo. However, the clinical implication of FGF-21 is poorly understood. To investigate whether FGF-21 may play a role as a metabolic regulator in patients with end-stage renal disease, we measured serum concentrations of FGF-21, inflammatory markers, and metabolic parameters in healthy people (n = 63) and nondiabetic patients receiving peritoneal dialysis (PD, n = 72). The patients were treated with angiotensin receptor blocker for 6 months, and the changes in FGF-21 concentration and metabolic parameters were assessed. Compared with controls, serum FGF-21 concentration was 8 times higher in patients undergoing PD (754.2 ± 463.5 vs 86.9 ± 60.2 pg/mL, P < .001). In controls, only lipid parameters correlated positively with FGF-21 concentration. In contrast, inflammatory markers (interleukin-6, fibrinogen, high-sensitivity C-reactive protein) and homeostasis model assessment of insulin resistance (HOMA-IR) correlated positively and residual renal function correlated inversely with serum FGF-21 concentration in PD patients. In a multivariate analysis adjusting these factors, residual renal function, HOMA-IR, and fibrinogen concentration were independent determinants of serum FGF-21 concentration. After 6-month angiotensin receptor blocker treatment, serum FGF-21 concentration declined significantly by 13% and HOMA-IR and inflammatory markers improved in PD patients. These findings suggest that FGF-21 may play a role in insulin resistance in patients with end-stage renal disease.

EGb761, a Ginkgo Biloba Extract, Is Effective Against Atherosclerosis In Vitro, and in a Rat Model of Type 2 Diabetes

Background EGb761, a standardized Ginkgo biloba extract, has antioxidant and antiplatelet aggregation and thus might protect against atherosclerosis. However, molecular and functional properties of EGb761 and its major subcomponents have not been well characterized. We investigated the effect of EGb761 and its major subcomponents (bilobalide, kaemferol, and quercetin) on preventing atherosclerosis in vitro, and in a rat model of type 2 diabetes. Methods and Results EGb761 (100 and 200 mg/kg) or normal saline (control) were administered to Otsuka Long-Evans Tokushima Fatty rats, an obese insulin-resistant rat model, for 6 weeks (from 3 weeks before to 3 weeks after carotid artery injury). Immunohistochemical staining was performed to investigate cell proliferation and apoptosis in the injured arteries. Cell migration, caspase-3 activity and DNA fragmentation, monocyte adhesion, and ICAM-1/VCAM-1 levels were explored in vitro. Treatment with EGb761 dose-dependently reduced intima-media ratio, proliferation of vascular smooth muscle cells (VSMCs) and induced greater apoptosis than the controls. Proliferation and migration of VSMCs in vitro were also decreased by the treatment of EGb761. Glucose homeostasis and circulating adiponectin levels were improved, and plasma hsCRP concentrations were decreased in the treatment groups. Caspase-3 activity and DNA fragmentation increased while monocyte adhesion and ICAM-1/VCAM-1 levels decreased significantly. Among subcomponents of EGb761, kaemferol and quercetin reduced VSMC migration and increased caspase activity. Conclusions EGb761 has a protective role in the development of atherosclerosis and is a potential therapeutic agent for preventing atherosclerosis.

Effect of a peroxisome proliferator-activated receptor γ sumoylation mutant on neointimal formation after balloon injury in rats

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor regulating inflammation, atherosclerosis, insulin sensitivity and adipogenesis. Recently, it has been discovered that modification by the small ubiquitin-like modifier (SUMO) plays an important role in PPARgamma activity. In the present study, we investigated the effect of sumoylation on the antiatherogenic property of PPARgamma. PPARgamma-K107R sumoylation mutant, PPARgamma-wild type (WT) and control genes were transfected on vascular smooth muscle cells (VSMCs) to compare their effect on the proliferation and migration. Adenoviral vectors expressing the PPARgamma-K107R, PPARgamma-WT or control gene were delivered into the carotid arteries of rats after balloon injury. The PPARgamma-K107R increased the transcriptional activity of peroxisome proliferator response element (PPRE) and had a more potent transcriptional repression activity on the inducible nitric oxide synthase (iNOS) promoter as compared to the other sumoylation mutants or WT. PPARgamma-K107R or WT gene transfer inhibited VSMCs proliferation and migration to a greater extent than the control. The PPARgamma-K107R had more potent activity than PPARgamma-WT in this regard. PPARgamma-K107R or WT transfer showed a significantly lower intima-media ratio (IMR) than the control after balloon injury in rats. Again, the delivery of the PPARgamma-K107R decreased IMR further compared to PPARgamma-WT. In addition, the PPARgamma-K107R transfer showed a lower proliferation index and a higher apoptotic index than PPARgamma-WT. In conclusion, the PPARgamma sumoylation mutant K107R strongly inhibited VSMCs proliferation and migration, sustained apoptosis, and reduced neointimal formation after balloon injury. These results indicate that desumoylation at K107 in PPARgamma might play an important role against atherosclerosis.

Differential effects of trimetazidine on vascular smooth muscle cell and endothelial cell in response to carotid artery balloon injury in diabetic rats

BACKGROUND Treatment with trimetazidine (TMZ), 1-[2,3,4-trimethoxybenzyl] piperazine, dihydrochloride, improves cardiac function and ameliorates endothelial dysfunction. However, its potential efficacy against restenosis after balloon injury has not been addressed. We investigated the effect of TMZ on reducing the occurrence of restenosis in the carotid artery in response to balloon injury and explored potential mechanisms for the effects. MATERIAL AND METHODS Streptozotocin (40 mg/kg)-injected Sprague-Dawley rats and Otsuka Long-Evans Tokushima Fatty rats were used for type 1 and type 2 diabetes models, respectively. Both types of rats were divided into three groups: control and TMZ treatment 10 and 20mg/kg per day (n=10 per group). TMZ or normal saline was given orally from 2 weeks before to 2 weeks after carotid injury. RESULTS Four weeks of TMZ treatment resulted in a significant and dose-dependent reduction in the intima-media ratio in diabetic rats. This effect was accompanied by decreased proliferation of vascular smooth muscle cells (VSMCs) and accelerated re-endothelialization after carotid balloon injury. In vitro study with VSMCs decreased proliferation and migration, while human umbilical vein endothelial cells (HUVECs) increased proliferation and decreased apoptosis after TMZ treatment. Antioxidative effects of TMZ were observed in both VSMCs and HUVECs. CONCLUSIONS Reduction of restenosis by TMZ treatment involved changes in antioxidative and anti-inflammatory properties, which are cell-specific effects on either survival or apoptosis. The specific actions and physiological effects of TMZ may contribute to better understanding of the pathogenesis of atherosclerosis, which is a major complication of diabetes mellitus.

The effects of chronic exercise on the inflammatory cytokines interleukin-6 and tumor necrosis factor-α are different with age

Aging is associated with chronic low-grade inflammation, and interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) are key mediators of the inflammatory process. IL-6, especially muscle-derived IL-6, is expected to mediate the beneficial metabolic effects of exercise. There was no report that directly compares the effects of chronic endurance exercise on cytokine responses between old and young subjects in the same situation. Therefore, we compared the effects of endurance exercise on the expression of IL-6 and TNF-α in old and young rats. Young (3-month-old) and old (20-month-old) male Fisher rats were trained for 12 weeks on the treadmill. We measured serum TNF-α and IL-6 concentrations by enzyme-linked immunosorbent assay and examined mRNA expression of TNF-α and IL-6 in muscle, liver, and white adipose tissue using reverse transcription - polymerase chain reaction. We found that old rats had higher basal IL-6 levels in the liver, as well as in the serum and the muscle. After chronic endurance exercise, young rats exhibited significant decreases in serum TNF-α levels and hepatic IL-6 expression. However, old rats exhibited no significant changes in either serum or tissue cytokine levels after endurance exercise. These findings suggest that chronic endurance exercise could influence the inflammatory response of hepatic tissues, as well as muscle, and that the effects of chronic endurance exercise on inflammatory cytokine levels are different between old and young rats and an exercise program tailored for old subjects will be needed to obtain beneficial anti-inflammatory effects from exercise.

Inhibition of STAT3 enhances the radiosensitizing effect of temozolomide in glioblastoma cells in vitro and in vivo.

Even with aggressive treatment involving radiation therapy plus temozolomide (TMZ), the prognosis for glioblastoma remains poor. We investigated the potential for targeting signal transducer and activator of transcription-3 (STAT3) to improve the therapeutic outcome in glioblastoma. We evaluated the preclinical potential of a STAT3 inhibitor, Cpd188, combined with temozolomide and radiation using in vitro assays with two established glioblastoma cell lines (U251 and U87) and two patient-derived glioblastoma cell lines (GBL12 and GBL28) as well as in vivo studies with nude mice bearing intracranial U251 xenografts. Cpd188 potentiated the radiosensitizing effect of TMZ in U251 cells, which have high p-STAT3 expression levels. The enhanced radiosensitizing effects of TMZ were associated with impaired DNA damage repair, apoptosis and reversion of the epithelial-mesenchymal transition (EMT). Cpd188 delayed in vivo tumor growth alone and in combination with radiation and TMZ. We also confirmed the radiosensitizing effect of Cpd188 in GBL28 cells, which were originated from a patient with a high level of STAT3 expression and unmethylated MGMT. Targeting STAT3 using Cpd188 could be a viable therapeutic approach for improving the outcome of current standard therapy in glioblastoma patients with high p-STAT3 expression.

The Effect of Chemoradiotherapy with SRC Tyrosine Kinase Inhibitor, PP2 and Temozolomide on Malignant Glioma Cells In Vitro and In Vivo

Purpose We investigated the effect of chemoradiotherapy with PP2 and temozolomide (TMZ) on malignant glioma cells using clonogenic assays and in vivo brain tumor model. Materials and Methods The effect of PP2 on radiosensitivity of U251 and T98G cells was investigated using clonogenic assays. The expression of E-cadherin, matrix metalloproteinases 2 (MMP2), Ephrin type-A receptor 2 (EphA2), and vascular endothelial growth factor (VEGF) was measured by Western blotting and an accumulation of γH2AX foci 6 hours after radiotherapy was measured after PP2 treatment. The effect of PP2 on migration, invasion, and vasculogenic mimicry formation (VMF) of U251 cells was evaluated. In an orthotopical brain tumor model with U251 cells, PP2 was injected intraperitoneally with or without oral TMZ before, during and after whole brain radiotherapy. Bioluminescence images were taken to visualize in vivo tumors and immunohistochemical staining of VEGF, CD31, EphA2, and hypoxia-inducible factor 1a was performed. Results PP2 increased radiosensitivity of U251 and T98G cells without decreasing survival of normal human astrocytes. Chemoradiotherapy with PP2 and TMZ resulted in increased accumulation of γH2AX foci. PP2 induced overexpression of E-cadherin and suppression of MMP2, VEGF, and EphA2. PP2 also compromised invasion, migration, and VMF of U251 cells. In brain tumors, chemoradiotherapy with PP2 and TMZ decreased tumor volume best, but not statistically significantly compared with chemoradiotherapy with TMZ. The expression of VEGF and CD31 was suppressed in PP2-treated tumors. Conclusion PP2 enhances radiosensitivity of malignant glioma cells and suppresses invasion and migration of U251 cells. Chemoradiotherapy with PP2 and TMZ resulted in non-significant tumor volume decrease.