Curcumin Enhances the Effect of Chemotherapy against Colorectal Cancer Cells by Inhibition of NF-κB and Src Protein Kinase Signaling Pathways

Objective

Development of treatment resistance and adverse toxicity associated with classical chemotherapeutic agents highlights the need for safer and effective therapeutic approaches. Herein, we examined the effectiveness of a combination treatment regimen of 5-fluorouracil (5-FU) and curcumin in colorectal cancer (CRC) cells.

Methods

Wild type HCT116 cells and HCT116+ch3 cells (complemented with chromosome 3) were treated with curcumin and 5-FU in a time- and dose-dependent manner and evaluated by cell proliferation assays, DAPI staining, transmission electron microscopy, cell cycle analysis and immunoblotting for key signaling proteins.

Results

The individual IC₅₀ of curcumin and 5-FU were approximately 20 µM and 5 µM in HCT116 cells and 5 µM and 1 µM in HCT116+ch3 cells, respectively (p<0.05). Pretreatment with curcumin significantly reduced survival in both cells; HCT116+ch3 cells were considerably more sensitive to treatment with curcumin and/or 5-FU than wild-type HCT116 cells. The IC₅₀ values for combination treatment were approximately 5 µM and 1 µM in HCT116 and 5 µM and 0.1 µM in HCT116+ch3, respectively (p<0.05). Curcumin induced apoptosis in both cells by inducing mitochondrial degeneration and cytochrome c release. Cell cycle analysis revealed that the anti-proliferative effect of curcumin and/or 5-FU was preceded by accumulation of CRC cells in the S cell cycle phase and induction of apoptosis. Curcumin potentiated 5-FU-induced expression or cleavage of pro-apoptotic proteins (caspase-8, -9, -3, PARP and Bax), and down-regulated anti-apoptotic (Bcl-xL) and proliferative (cyclin D1) proteins. Although 5-FU activated NF-κB/PI-3K/Src pathway in CRC cells, this was down-regulated by curcumin treatment through inhibition of IκBα kinase activation and IκBα phosphorylation.

Conclusions

Combining curcumin with conventional chemotherapeutic agents such as 5-FU could provide more effective treatment strategies against chemoresistant colon cancer cells. The mechanisms involved may be mediated via NF-κB/PI-3K/Src pathways and NF-κB regulated gene products.     

Curcumin as a Potent and Selective Inhibitor of 11β-Hydroxysteroid Dehydrogenase 1: Improving Lipid Profiles in High-Fat-Diet-Treated Rats

Background

11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) activates glucocorticoid locally in liver and fat tissues to aggravate metabolic syndrome. 11β-HSD1 selective inhibitor can be used to treat metabolic syndrome. Curcumin and its derivatives as selective inhibitors of 11β-HSD1 have not been reported.

Methodology

Curcumin and its 12 derivatives were tested for their potencies of inhibitory effects on human and rat 11β-HSD1 with selectivity against 11β-HSD2. 200 mg/kg curcumin was gavaged to adult male Sprague-Dawley rats with high-fat-diet-induced metabolic syndrome for 2 months.

Results and Conclusions

Curcumin exhibited inhibitory potency against human and rat 11β-HSD1 in intact cells with IC₅₀ values of 2.29 and 5.79 µM, respectively, with selectivity against 11β-HSD2 (IC₅₀, 14.56 and 11.92 µM). Curcumin was a competitive inhibitor of human and rat 11β-HSD1. Curcumin reduced serum glucose, cholesterol, triglyceride, low density lipoprotein levels in high-fat-diet-induced obese rats. Four curcumin derivatives had much higher potencies for Inhibition of 11β-HSD1. One of them is (1E,4E)-1,5-bis(thiophen-2-yl) penta-1,4-dien-3-one (compound 6), which had IC₅₀ values of 93 and 184 nM for human and rat 11β-HSD1, respectively. Compound 6 did not inhibit human and rat kidney 11β-HSD2 at 100 µM. In conclusion, curcumin is effective for the treatment of metabolic syndrome and four novel curcumin derivatives had high potencies for inhibition of human 11β-HSD1 with selectivity against 11β-HSD2.     

Suppression of Experimental Choroidal Neovascularization by Curcumin in Mice

Purpose

To investigate the effects of curcumin on the development of experimental choroidal neovascularization (CNV) with underlying cellular and molecular mechanisms.

Methods

C57BL/6N mice were pretreated with intraperitoneal injections of curcumin daily for 3 days prior to laser-induced CNV, and the drug treatments were continued until the end of the study. The CNV area was analyzed by fluorescein-labeled dextran angiography of retinal pigment epithelium (RPE)-choroid flat mounts on day 7 and 14, and CNV leakage was evaluated by fluorescein angiography (FA) on day 14 after laser photocoagulation. The infiltration of F4/80 positive macrophages and GR-1 positive granulocytes were evaluated by immunohistochemistry on RPE-choroid flat mounts on day 3. Their expression in RPE-choroid complex was quantified by real-time PCR (F4/80) and Western blotting (GR-1) on day 3. RPE-choroid levels of vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF)-α, monocyte chemotactic protein (MCP)-1, and intercellular adhesion molecule (ICAM)-1 were examined by ELISA on day 3. Double immunostaining of F4/80 and VEGF was performed on cryo-sections of CNV lesions on day 3. The expression of nuclear factor (NF)-κB and hypoxia-inducible factor (HIF)−1α in the RPE-choroid was determined by Western blotting.

Results

Curcumin-treated mice had significantly less CNV area (P<0.05) and CNV leakage (P<0.001) than vehicle-treated mice. Curcumin treatment led to significant inhibition of F4/80 positive macrophages (P<0.05) and GR-1 positive granulocytes infiltration (P<0.05). VEGF mainly expressed in F4/80 positive macrophages in laser injury sites, which was suppressed by curcumin treatment (P<0.01). Curcumin inhibited the RPE-choroid levels of TNF-α (P<0.05), MCP-1 (P<0.05) and ICAM-1 (P<0.05), and suppressed the activation of NF-κB in nuclear extracts (P<0.05) and the activation of HIF−1α (P<0.05).

Conclusion

Curcumin treatment led to the suppression of CNV development together with inflammatory and angiogenic processes including NF-κB and HIF−1α activation, the up-regulation of inflammatory and angiogenic cytokines, and infiltrating macrophages and granulocytes. This provides molecular and cellular evidence of the validity of curcumin supplementation as a therapeutic strategy for the suppression of age-related macular degeneration (AMD)-associated CNV.     

Abundant Lipid and Protein Components of Drusen

Background

Drusen are extracellular lesions characteristic of aging and age-related maculopathy, a major retinal disease of the elderly. We determined the relative proportions of lipids and proteins in drusen capped with retinal pigment epithelium (RPE) and in RPE isolated from non-macular regions of 36 human retinas with grossly normal maculas obtained <6 hr after death.

Methodology/Principal Findings

Druse pellets were examined by light and electron microscopy. Component proteins were extracted using novel methods for preserved tissues, separated, subjected to tryptic digestion and LC-MS(MS)² analysis using an ion trap mass spectrometer, and identified with reference to databases. Lipid classes were separated using thin layer chromatography and quantified by densitometry. Major druse components were esterified cholesterol (EC), phosphatidylcholine (PC), and protein (37.5±13.7, 36.9±12.9, and 43.0±11.5 ng/druse, respectively). Lipid-containing particles (median diameter, 77 nm) occupied 37–44% of druse volume. Major proteins include vitronectin, complement component 9, apoE, and clusterin, previously seen in drusen, and ATP synthase subunit β, scavenger receptor B2, and retinol dehydrogenase 5, previously seen in RPE. Drusen and RPE had similar protein profiles, with higher intensities and greater variability in drusen. C8, part of the complement membrane attack complex, was localized in drusen by immunofluorescence.

Conclusions/Significance

At least 40% of druse content is comprised by lipids dominated by EC and PC, 2 components that are potentially accounted for by just one pathway, the secretion of lipoproteins by RPE. Manipulating genes encoding apolipoprotein pathways would be a fruitful approach to producing drusen with high EC content in laboratory animals. Therapies that directly mitigate drusen should prepare for the substantial volume of neutral lipids. The catalog of major druse proteins is nearing completion.     

A2E Induces IL-1? Production in Retinal Pigment Epithelial Cells via the NLRP3 Inflammasome

Aims

With ageing extracellular material is deposited in Bruch’s membrane, as drusen. Lipofuscin is deposited in retinal pigment epithelial cells. Both of these changes are associated with age related macular degeneration, a disease now believed to involve chronic inflammation at the retinal-choroidal interface. We hypothesise that these molecules may act as danger signals, causing the production of inflammatory chemokines and cytokines by the retinal pigment epithelium, via activation of pattern recognition receptors.

Methods

ARPE-19 cells were stimulated in vitro with the following reported components of drusen: amyloid-ß (1-42), Carboxyethylpyrrole (CEP) modified proteins (CEP-HSA), Nε-(Carboxymethyl)lysine (CML) modified proteins and aggregated vitronectin. The cells were also stimulated with the major fluorophore of lipofuscin: N-retinylidene-N-retinylethanolamine (A2E). Inflammatory chemokine and cytokine production was assessed using Multiplex assays and ELISA. The mechanistic evaluation of the NLRP3 inflammasome pathway was assessed in a stepwise fashion.

Results

Of all the molecules tested only A2E induced inflammatory chemokine and cytokine production. 25 µM A2E induced the production of significantly increased levels of the chemokines IL-8, MCP-1, MCG and MIP-1α, the cytokines IL-1ß, IL-2, IL-6, and TNF-α, and the protein VEGF-A. The release of IL-1ß was studied further, and was determined to be due to NLRP3 inflammasome activation. The pathway of activation involved endocytosis of A2E, and the three inflammasome components NLRP3, ASC and activated caspase-1. Immunohistochemical staining of ABCA4 knockout mice, which show progressive accumulation of A2E levels with age, showed increased amounts of IL-1ß proximal to the retinal pigment epithelium.

Conclusions

A2E has the ability to stimulate inflammatory chemokine and cytokine production by RPE cells. The pattern recognition receptor NLRP3 is involved in this process. This provides further evidence for the link between A2E, inflammation, and the pathogenesis of AMD. It also supports the recent discovery of NLRP3 inflammasome activation in AMD.     

Retinal Thickness Normative Data in Wild-Type Mice Using Customized Miniature SD-OCT

Objective

To report normative data for retinal thickness in wild-type C57BL/6 mouse utilizing a miniature SD-OCT system.

Methods

Thirty adult mice (range: 3–5 months) were anesthetized and secured into the Bioptigen Spectral Domain Ophthalmic Imaging System. Right eye SD-OCT images were standardized by centralizing the optic nerve head (ONH) prior to image acquisition. Global and quadrant total retinal thickness (TRT) values were measured from retinal nerve fiber layer to retinal pigment epithelial layer. Posterior segment analyses also included the outer retinal layer (ORL) and inner retinal layer (IRL). Further sublayer analyses of four layers from the ORL and three layers comprising the IRL were also performed.

Results

The overall mean±SD global TRT in a C57BL/6 mouse model was 204.41±5.19 µm. Quadrant mean TRT values were 204.85±5.81 µm inferiorly, 204.97±6.71 µm nasally, 205.08±5.44 µm temporally, and 202.74±4.85 µm superiorly. Mean±SD thickness for ORL, and IRL were 126.37±10.01 µm, and 107.03±10.98 µm respectively. The mean±SD estimates for the four layers of the ORL were 18.23±2.73 µm, 26.04±4.21 µm, 63.8±6.23 µm, and 19.22±4.34 µm. Mean±SD values for the three IRL sublayers were 27.82±4.04 µm, 59.62±6.66 µm and 19.12±3.71 µm.

Conclusion

This study established normative values for the total retinal thickness and sublayer thickness for the wild-type C57BL/6 mice. Moreover, it provides a standard of retinal morphology, in a commonly used animal model, for evaluating therapeutic interventions and retinal disease pathophysiology.     

Decreased VEGF-A and sustained PEDF expression in a human retinal pigment epithelium cell line cultured under hypothermia

Background

Previous reports have described a decrease in retinal temperature and clinical improvement of wet age-related macular degeneration (AMD) after vitrectomy. We hypothesized that the retinal temperature decrease after vitrectomy plays a part in the suppression of wet AMD development. To test this hypothesis, we evaluated the temperature dependence of the expression of vascular endothelial growth factor-A (VEGF-A) and in vitro angiogenesis in retinal pigment epithelium (RPE).

Results

We cultured ARPE-19 cells at 37, 35, 33 and 31°C and measured the expression of VEGF-A, VEGF-A splicing variants, and pigment epithelium–derived factor (PEDF). We performed an in vitro tube formation assay. The dehydrogenase activity was also evaluated at each temperature. Expression of VEGF-A significantly decreased with decreased temperature while PEDF expression did not. VEGF165 expression and in vitro angiogenesis also were temperature dependent. The dehydrogenase activity significantly decreased as the culture temperature decreased.

Conclusions

RPE cultured under hypothermia that decreased cellular metabolism also had decreased VEGF-A and sustained PEDF expression, creating an anti-angiogenic environment. This mechanism may be associated with a beneficial effect after vitrectomy in patients with wet AMD.     

Development of a Semi-Automatic Segmentation Method for Retinal OCT Images Tested in Patients with Diabetic Macular Edema

Purpose

To develop EdgeSelect, a semi-automatic method for the segmentation of retinal layers in spectral domain optical coherence tomography images, and to compare the segmentation results with a manual method.

Methods

SD-OCT (Heidelberg Spectralis) scans of 28 eyes (24 patients with diabetic macular edema and 4 normal subjects) were imported into a customized MATLAB application, and were manually segmented by three graders at the layers corresponding to the inner limiting membrane (ILM), the inner segment/ellipsoid interface (ISe), the retinal/retinal pigment epithelium interface (RPE), and the Bruch''s membrane (BM). The scans were then segmented independently by the same graders using EdgeSelect, a semi-automated method allowing the graders to guide/correct the layer segmentation interactively. The inter-grader reproducibility and agreement in locating the layer positions between the manual and EdgeSelect methods were assessed and compared using the Wilcoxon signed rank test.

Results

The inter-grader reproducibility using the EdgeSelect method for retinal layers varied from 0.15 to 1.21 µm, smaller than those using the manual method (3.36–6.43 µm). The Wilcoxon test indicated the EdgeSelect method had significantly better reproducibility than the manual method. The agreement between the manual and EdgeSelect methods in locating retinal layers ranged from 0.08 to 1.32 µm. There were small differences between the two methods in locating the ILM (p = 0.012) and BM layers (p<0.001), but these were statistically indistinguishable in locating the ISe (p = 0.896) and RPE layers (p = 0.771).

Conclusions

The EdgeSelect method resulted in better reproducibility and good agreement with a manual method in a set of eyes of normal subjects and with retinal disease, suggesting that this approach is feasible for OCT image analysis in clinical trials.     

Curcumin Alleviates Diabetic Cardiomyopathy in Experimental Diabetic Rats

Objectives

Diabetic cardiomyopathy (DCM), characterized by myocardial structural and functional changes, is an independent cardiomyopathy that develops in diabetic individuals. The present study was sought to investigate the effect of curcumin on modulating DCM and the mechanisms involved.

Methods

An experimental diabetic rat model was induced by low dose of streptozoticin(STZ) combined with high energy intake on rats. Curcumin was orally administrated at a dose of 100 or 200 mg·kg⁻¹·d⁻¹, respectively. Cardiac function was evaluated by serial echocardiography. Myocardial ultrastructure, fibrosis area and apoptosis were assessed by histopathologic analyses. Metabolic profiles, myocardial enzymes and oxidative stress were examined by biochemical tests. Inflammatory factors were detected by ELISA, and interrelated proteins were measured by western blot.

Results

Rats with DCM showed declined systolic myocardial performance associated with myocardial hypertrophy and fibrosis, which were accompanied with metabolism abnormalities, aberrant myocardial enzymes, increased AGEs (advanced glycation end products) accumulation and RAGE (receptor for AGEs) expression, elevated markers of oxidative stress (MDA, SOD, the ratio of NADP⁺/NADPH, Rac1 activity, NADPH oxidase subunits expression of gp91^phox and p47^phox ), raised inflammatory factor (TNF-α and IL-1β), enhanced apoptotic cell death (ratio of bax/bcl-2, caspase-3 activity and TUNEL), diminished Akt and GSK-3β phosphorylation. Remarkably, curcumin attenuated myocardial dysfunction, cardiac fibrosis, AGEs accumulation, oxidative stress, inflammation and apoptosis in the heart of diabetic rats. The inhibited phosphorylation of Akt and GSK-3β was also restored by curcumin treatment.

Conclusions

Taken together, these results suggest that curcumin may have great therapeutic potential in the treatment of DCM, and perhaps other cardiovascular disorders, by attenuating fibrosis, oxidative stress, inflammation and cell death. Furthermore, Akt/GSK-3β signaling pathway may be involved in mediating these effects.     

Mometasone and desloratadine additive effect on eosinophil survival and cytokine secretion from epithelial cells

Background

Although antihistamines and topical corticosteroids are used in combination to treat allergic rhinitis, their additive effect has not been yet demonstrated. The aim was investigate the antiinflammatory additive effect of mometasone and desloratadine on cytokine and sICAM-1 secretion by epithelial cells, and on eosinophil survival stimulated by human epithelial cells secretions from nasal mucosa and polyps.

Methods

Epithelial cells obtained from nasal mucosa or polyps were stimulated with 10% fetal bovine serum in presence of mometasone (10^-11M-10^-5M) with/without desloratadine (10^-5M). Cytokine and sICAM-1 concentrations in supernatants were measured by ELISA. Peripheral blood eosinophils were incubated during 4 days with epithelial cell secretions with (10^-11M-10^-5M) and/or desloratadine (10^-5M) and survival assessed by Trypan blue. Results are expressed as percentage (mean ± SEM) compared to control.

Results

Fetal bovine serum stimulated IL-6, IL-8, GM-CSF and sICAM-1 secretion. In mucosa and polyp epithelial cells, mometasone inhibited this induced secretion while desloratadine inhibited IL-6 and IL-8. The combination of 10^-5M desloratadine and 10^-9M mometasone reduced IL-6 secretion (48 ± 11%, p < 0.05) greater extent than mometasone alone (68 ± 10%) compared to control (100%). Epithelial cell secretions induced eosinophil survival from day 1 to 4, this effect being inhibited by mometasone. At day 4, the combination of mometasone (10^-11M) and desloratadine (10^-5M) provoked an increased inhibition of eosinophil survival induced by cell secretions (27 ± 5%, p < 0.01) than mometasone (44 ± 7%) or desloratadine (46 ± 7%) alone.

Conclusions

These results suggest that the combination of desloratadine and mometasone furoate have a greater antinflammatory effect in an in vitro model of eosinophil inflammation than those drugs administered alone.     

Retinal Arterioles Narrow with Increasing Duration of Anti-Retroviral Therapy in HIV Infection: A Novel Estimator of Vascular Risk in HIV?

Objectives

HIV infection is associated with an increased risk of age-related morbidity mediated by immune dysfunction, atherosclerosis and inflammation. Changes in retinal vessel calibre may reflect cumulative structural damage arising from these mechanisms. The relationship of retinal vessel calibre with clinical and demographic characteristics was investigated in a population of HIV-infected individuals in South Africa.

Methods

Case-control study of 491 adults ≥30 years, composed of 242 HIV-infected adults and 249 age- and gender-matched HIV-negative controls. Retinal vessel calibre was measured using computer-assisted techniques to determine mean arteriolar and venular diameters of each eye.

Results

The median age was 40 years (IQR: 35–48 years). Among HIV-infected adults, 87.1% were receiving highly active antiretroviral therapy (HAART) (median duration, 58 months), their median CD4 count was 468 cells/µL, and 84.3% had undetectable plasma viral load. Unadjusted mean retinal arteriolar diameters were 163.67±17.69 µm in cases and 161.34±17.38 µm in controls (p = 0.15). Unadjusted mean venular diameters were 267.77±18.21 µm in cases and 270.81±18.98 µm in controls (p = 0.07). Age modified the effect of retinal arteriolar and venular diameters in relation to HIV status, with a tendency towards narrower retinal diameters in HIV cases but not in controls. Among cases, retinal arteriolar diameters narrowed with increasing duration of HAART, independently of age (167.83 µm <3 years of HAART vs. 158.89 µm >6 years, p-trend = 0.02), and with a HIV viral load >10,000 copies/mL while on HAART (p = 0.05). HIV-related venular changes were not detected.

Conclusions

Narrowing of retinal arteriolar diameters is associated with HAART duration and viral load, and may reflect heightened inflammatory and pro-atherogenic states of the systemic vasculature. Measurement of retinal vascular calibre could be an innovative non-invasive method of estimating vascular risk in HIV-infected individuals.     

Neuroprotective effect of novel cognitive enhancer noopept on AD-related cellular model involves the attenuation of apoptosis and tau hyperphosphorylation

Background

Noopept (N-phenyl-acetyl-L-prolylglycine ethyl ester) was constructed as a dipeptide analog of the standard cognition enhancer, piracetam. Our previous experiments have demonstrated the cognition restoring effect of noopept in several animal models of Alzheimer disease (AD). Noopept was also shown to prevent ionic disbalance, excitotoxicity, free radicals and pro-inflammatory cytokines accumulation, and neurotrophine deficit typical for different kinds of brain damages, including AD. In this study, we investigated the neuroprotective action of noopept on cellular model of AD, Aβ_25–35-induced toxicity in PC12 cells and revealed the underlying mechanisms.

Results

The neuroprotective effect of noopept (added to the medium at 10 μM concentration, 72 hours before Аβ_25–35) was studied on Аβ_25–35-induced injury (5 μM for 24 h) in PC12 cells. The ability of drug to protect the impairments of cell viability, calcium homeostasis, ROS level, mitochondrial function, tau phosphorylation and neurite outgrowth caused by Аβ_25–35 were evaluated.Following the exposure of PC12 cells to Аβ_25–35 an increase of the level of ROS, intracellular calcium, and tau phosphorylation at Ser396 were observed; these changes were accompanied by a decrease in cell viability and an increase of apoptosis. Noopept treatment before the amyloid-beta exposure improved PC12 cells viability, reduced the number of early and late apoptotic cells, the levels of intracellular reactive oxygen species and calcium and enhanced the mitochondrial membrane potential. In addition, pretreatment of PC12 cell with noopept significantly attenuated tau hyperphosphorylation at Ser396 and ameliorated the alterations of neurite outgrowth evoked by Аβ_25–35.

Conclusions

Taken together, these data provide evidence that novel cognitive enhancer noopept protects PC12 cell against deleterious actions of Aβ through inhibiting the oxidative damage and calcium overload as well as suppressing the mitochondrial apoptotic pathway. Moreover, neuroprotective properties of noopept likely include its ability to decrease tau phosphorylation and to restore the altered morphology of PC12 cells. Therefore, this nootropic dipeptide is able to positively affect not only common pathogenic pathways but also disease-specific mechanisms underlying Aβ-related pathology.     

Visible Light Is a Better Co-Inducer of Apoptosis for Curcumin-Treated Human Melanoma Cells than UVA

Curcumin attracts worldwide scientific interest due to its anti-proliferative and apoptosis inducing effects on different tumor cells at concentrations ranging from 10 to 150 µM (3.7–55 µg/ml). Unfortunately, because of a low oral bioavailability, only low and pharmacologically ineffective serum levels are achievable. In this study, an alternative treatment concept consisting of low concentration curcumin (0.2–5 µg/ml) and irradiation with UVA or visible light (VL) has been tested. The experimental results show clearly that this treatment decreases the proliferation and the viability of human melanoma cells while the cell membrane integrity remains intact. We identified the onset of apoptosis characterized by typical markers such as active caspases 8, 9 and 3 as well as DNA fragmentation accompanied by the loss of cell adhesion. The mitochondrial apoptosis signaling pathway is predominant due to an early activation of caspase-9. The present data indicate a higher efficacy of a combination of curcumin and VL than curcumin and UVA. Reduced effects as a result of light absorption by heavily pigmented skin are unlikely if VL is used. These results indicate that a combination of curcumin and light irradiation may be a useful additional therapy in the treatment of malignant disease.     

Melittin Suppresses HIF-1α/VEGF Expression through Inhibition of ERK and mTOR/p70S6K Pathway in Human Cervical Carcinoma Cells

Objective

Melittin (MEL), a major component of bee venom, has been associated with various diseases including arthritis, rheumatism and various cancers. In this study, the anti-angiogenic effects of MEL in CaSki cells that were responsive to the epidermal growth factor (EGF) were examined.

Methodology/Principal Findings

MEL decreased the EGF-induced hypoxia-inducible factor-1α (HIF-1α) protein and significantly regulated angiogenesis and tumor progression. We found that inhibition of the HIF-1α protein level is due to the shortened half-life by MEL. Mechanistically, MEL specifically inhibited the EGF-induced HIF-1α expression by suppressing the phosphorylation of ERK, mTOR and p70S6K. It also blocked the EGF-induced DNA binding activity of HIF-1α and the secretion of the vascular endothelial growth factor (VEGF). Furthermore, the chromatin immunoprecipitation (ChIP) assay revealed that MEL reduced the binding of HIF-1α to the VEGF promoter HRE region. The anti-angiogenesis effects of MEL were confirmed through a matrigel plus assay.

Conclusions

MEL specifically suppressed EGF-induced VEGF secretion and new blood vessel formation by inhibiting HIF-1α. These results suggest that MEL may inhibit human cervical cancer progression and angiogenesis by inhibiting HIF-1α and VEGF expression.     

Acute Simvastatin Inhibits KATP Channels of Porcine Coronary Artery Myocytes

Background

Statins (3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors) consumption provides beneficial effects on cardiovascular systems. However, effects of statins on vascular K_ATP channel gatings are unknown.

Methods

Pig left anterior descending coronary artery and human left internal mammary artery were isolated and endothelium-denuded for tension measurements and Western immunoblots. Enzymatically-dissociated/cultured arterial myocytes were used for patch-clamp electrophysiological studies and for [Ca²⁺]_i, [ATP]_i and [glucose]_o uptake measurements.

Results

The cromakalim (10 nM to 10 µM)- and pinacidil (10 nM to 10 µM)-induced concentration-dependent relaxation of porcine coronary artery was inhibited by simvastatin (3 and 10 µM). Simvastatin (1, 3 and 10 µM) suppressed (in okadaic acid (10 nM)-sensitive manner) cromakalim (10 µM)- and pinacidil (10 µM)-mediated opening of whole-cell K_ATP channels of arterial myocytes. Simvastatin (10 µM) and AICAR (1 mM) elicited a time-dependent, compound C (1 µM)-sensitive [³H]-2-deoxy-glucose uptake and an increase in [ATP]_i levels. A time (2–30 min)- and concentration (0.1–10 µM)-dependent increase by simvastatin of p-AMPKα-Thr¹⁷² and p-PP2A-Tyr³⁰⁷ expression was observed. The enhanced p-AMPKα-Thr¹⁷² expression was inhibited by compound C, ryanodine (100 µM) and KN93 (10 µM). Simvastatin-induced p-PP2A-Tyr³⁰⁷ expression was suppressed by okadaic acid, compound C, ryanodine, KN93, phloridzin (1 mM), ouabain (10 µM), and in [glucose]_o-free or [Na⁺]_o-free conditions.

Conclusions

Simvastatin causes ryanodine-sensitive Ca²⁺ release which is important for AMPKα-Thr¹⁷² phosphorylation via Ca²⁺/CaMK II. AMPKα-Thr¹⁷² phosphorylation causes [glucose]_o uptake (and an [ATP]_i increase), closure of K_ATP channels, and phosphorylation of AMPKα-Thr¹⁷² and PP2A-Tyr³⁰⁷ resulted. Phosphorylation of PP2A-Tyr³⁰⁷ occurs at a site downstream of AMPKα-Thr¹⁷² phosphorylation.