Effects of receptor tyrosine kinase inhibitors on VEGF165a- and VEGF165b-stimulated gene transcription in HEK-293 cells expressing human VEGFR2

Background and Purpose

Receptor tyrosine kinase inhibitors (RTKIs) targeted at VEGF receptor 2 (VEGFR2) have proved to be attractive approaches to cancer therapy based on their ability to reduce angiogenesis. Here we have undertaken a quantitative analysis of the interaction of RTKIs and two VEGF splice variants, VEGF₁₆₅a and VEGF₁₆₅b, with VEGFR2 by studying nuclear factor of activated T-cells (NFAT) reporter gene activity in live HEK-293 cells.

Experimental Approach

HEK-293 cells expressing the human VEGFR2 and a firefly luciferase reporter gene regulated by an NFAT response element were used for quantitative analysis of the effect of RTKIs on VEGF₁₆₅a- and VEGF₁₆₅b-stimulated luciferase gene expression.

Key Results

VEGF₁₆₅a produced a concentration-dependent activation of the NFAT-luciferase reporter gene in living cells that was inhibited in a non-competitive fashion by four different RTKIs (cediranib, pazopanib, sorafenib and vandetanib). The potency obtained for each RTKI from this analysis was similar to those obtained in binding studies using purified VEGFR2 kinase domains. VEGF₁₆₅b was a lower-efficacy agonist of the NFAT-luciferase response when compared with VEGF₁₆₅a. Analysis of the concentration–response data using the operational model of agonism indicated that both VEGF₁₆₅ isoforms had similar affinity for VEGFR2.

Conclusions and Implications

Quantitative pharmacological analysis of the interaction of VEGF₁₆₅ isoforms and RTKIs with VEGFR2 in intact living cells has provided important insights into the relative affinity and efficacy of VEGF₁₆₅a and VEGF₁₆₅b for activation of the calcineurin- NFAT signalling pathway by this tyrosine kinase receptor.     

Calcitonin gene-related peptide stimulates BMP-2 expression and the differentiation of human osteoblast-like cells in vitro

Aim： To investigate whether bone morphogenic protein-2 （BMP-2） expression was involved in calcitonin gene-related peptide （CGRP）- induced osteogenesis in human osteoblast-like cells in vitro. Methods： MG-63 osteogenic human osteosarcoma cells were treated with CGRP （10.8 mol/L） for 48 h. Cell cycle phases were deter- mined using flow cytometry assay. The protein levels of BMP-2, ALP, Osteocalcin, Collal, CREB, and pCREB were measured with Western blotting, while the mRNA level of BMP-2 was measured with qR-T PCR. The expression of ALP in MG-63 cells was also studied using immunofluorescence staining. The level of cAMP was measured with ELISA assay. Results： CGRP treatment significantly stimulated proliferation of MG-63 cells, and increased the expression of BMP-2 and the osteo- genic proteins ALP, Osteocalcin and Collal. Pretreatment with the BMP signaling inhibitor Noggin （100 ng/mL） did not affect CGRP- stimulated proliferation and BMP-2 expression, but abolished the CGRP-induced increases of the osteogenic proteins ALP, Osteocalcin and Collal. Furthermore, CGRP treatment markedly increased cAMP level in MG-63 cells, whereas pretreatment with the cAMP path- way inhibitor H89 （5 pmol/L） abolished the CGRP-induced increases of cAMP level and BMP-2 expression. Conclusion： In MG-63 cells, the BMP pathway is involved in CGRP-induced osteogenic differentiation but not in proliferation, whereas the cAMP/pCREB pathway is involved in the expression of BMP-2.     

VEGF ameliorates tubulointerstitial fibrosis in unilateral ureteral obstruction mice via inhibition of epithelial-mesenchymal transition

Aim:

Vascular endothelial growth factor (VEGF) has been shown to be a survival factor for renal tubular epithelial cells. In the present study, we investigated whether administration of VEGF ameliorates tubulointerstitial fibrosis in a mouse model of unilateral ureteral obstruction (UUO).

Methods:

Thirty-six male CD-1 mice were randomly divided into three groups: sham-operation, UUO and UUO+VEGF group. VEGF (50 μg/kg) was subcutaneously injected twice daily from d 1 to d 14. Mice in each group were killed at d 3, 7, or 14 after the operation, and the tubulointerstitial fibrosis was histopathologically evaluated. Human proximal tubular epithelial cells (HK-2) were used for in vitro study. The expression levels of α-SMA, E-cadherin, TGF-β1, CTGF, and BMP-7 in the kidney were determined using Western blot and RT-PCR.

Results:

In the UUO mice, the degree of interstitial fibrosis was dramatically increased in a time-dependent manner. At d 3, 7, and 14, both the mRNA and protein expression levels for α-SMA, TGF-β1, and CTGF were significantly upregulated, whereas those for E-cadherin and BMP-7 were significantly downregulated. At d 3 and 7, VEGF treatment significantly reduced interstitial fibrosis and the expression levels for α-SMA, TGF-β1, and CTGF, while significantly increased the expression of E-cadherin and BMP-7, as compared with the UUO mice. At d 14 after operation, no significant differences were observed in the expression of the examined markers between VEGF-treated mice and UUO mice, with the exception of CTGF. In HK-2 cells, VEGF blocked TGF-β1-induced α-SMA and vimentin expression and restored E-cadherin expression in a dose-dependent manner.

Conclusion:

VEGF may ameliorate renal tubulointerstitial fibrosis at the early stage in UUO mice. This effect may be related to inhibition of VEGF on renal tubular epithelial-mesenchymal transition (EMT).     

Brain protection against ischemic stroke using choline as a new molecular bypass treatment

Aim:

To determine whether administration of choline could attenuate brain injury in a rat model of ischemic stroke and the underlying mechanisms.

Methods:

A rat model of ischemic stroke was established through permanent middle cerebral artery occlusion (pMCAO). After the surgery, the rats were treated with choline or choline plus the specific α7 nAChR antagonist methyllycaconitine (MLA), or with the control drug nimodipine for 10 days. The neurological deficits, brain-infarct volume, pial vessel density and the number of microvessels in the cortex were assessed. Rat brain microvascular endothelial cells (rBMECs) cultured under hypoxic conditions were used in in vitro experiments.

Results:

Oral administration of choline (100 or 200 mg·kg⁻¹·d⁻¹) or nimodipine (20 mg·kg⁻¹·d⁻¹) significantly improved neurological deficits, and reduced infarct volume and nerve cell loss in the ischemic cerebral cortices in pMCAO rats. Furthermore, oral administration of choline, but not nimodipine, promoted the pial arteriogenesis and cerebral-cortical capillary angiogenesis in the ischemic regions. Moreover, oral administration of choline significantly augmented pMCAO-induced increases in the expression levels of α7 nAChR, HIF-1α and VEGF in the ischemic cerebral cortices as well as in the serum levels of VEGF. Choline-induced protective effects were prevented by co-treatment with MLA (1 mg·kg⁻¹·d⁻¹, ip). Treatment of rBMECs cultured under hypoxic conditions in vitro with choline (1, 10 and 100 μmol/L) dose-dependently promoted the endothelial-cell proliferation, migration and tube formation, as well as VEGF secretion, which were prevented by co-treatment with MLA (1 μmol/L) or by transfection with HIF-1α siRNA.

Conclusion:

Choline effectively attenuates brain ischemic injury in pMCAO rats, possibly by facilitating pial arteriogenesis and cerebral-cortical capillary angiogenesis via upregulating α7 nAChR levels and inducing the expression of HIF-1α and VEGF.     

Adeno associated viral vector-delivered and hypoxia response element-regulated CD151 expression in ischemic rat heart

Aim:

The aim of this study was to improve the delivery efficacy and target specificity of the pro-angiogenic gene CD151 to the ischemic heart.

Methods:

To achieve the inducible expression of adeno-associated viral (AAV)-delivered CD151 gene in only the ischemic myocardium, we generated an AAV construct in which CD151 expression can be controlled by the hypoxia response element (HRE) sequence from the human Enolase gene. The function of this vector was examined in rat H9C2 cardiac myoblasts and in ischemic rat myocardium. The expression of CD151 in the areas of ischemic myocardium was confirmed at the mRNA level by real-time PCR and on the protein level by Western blot, whereas the CD151 expression in the microvessels within the areas of ischemic myocardium was detected by immunohistochemistry.

Results:

HRE significantly enhances the expression of CD151 under hypoxic conditions or in the ischemic myocardium, and forced CD151 expression increases the number of microvessels in the ischemic myocardium.

Conclusion:

The AAV-mediated, HRE regulated delivery of the CD151 gene shows higher expression in the ischemic myocardium and more efficiently targets CD151 to the hypoxic regions after myocardial infarction.     

Boldine improves endothelial function in diabetic db/db mice through inhibition of angiotensin II-mediated BMP4-oxidative stress cascade

BACKGROUND AND PURPOSE

Boldine is a potent natural antioxidant present in the leaves and bark of the Chilean boldo tree. Here we assessed the protective effects of boldine on endothelium in a range of models of diabetes, ex vivo and in vitro.

EXPERIMENTAL APPROACH

Vascular reactivity was studied in mouse aortas from db/db diabetic and normal mice. Reactive oxygen species (ROS) production, angiotensin AT₁ receptor localization and protein expression of oxidative stress markers in the vascular wall were evaluated by dihydroethidium fluorescence, lucigenin enhanced-chemiluminescence, immunohistochemistry and Western blot respectively. Primary cultures of mouse aortic endothelial cells, exposed to high concentrations of glucose (30 mmol L⁻¹) were also used.

KEY RESULTS

Oral treatment (20 mg kg⁻¹day⁻¹, 7 days) or incubation in vitro with boldine (1 μmol L⁻¹, 12 h) enhanced endothelium-dependent aortic relaxations of db/db mice. Boldine reversed impaired relaxations induced by high glucose or angiotensin II (Ang II) in non-diabetic mouse aortas while it reduced the overproduction of ROS and increased phosphorylation of eNOS in db/db mouse aortas. Elevated expression of oxidative stress markers (bone morphogenic protein 4 (BMP4), nitrotyrosine and AT₁ receptors) were reduced in boldine-treated db/db mouse aortas. Ang II-stimulated BMP4 expression was inhibited by boldine, tempol, noggin or losartan. Boldine inhibited high glucose-stimulated ROS production and restored the decreased phosphorylation of eNOS in mouse aortic endothelial cells in culture.

CONCLUSIONS AND IMPLICATIONS

Boldine reduced oxidative stress and improved endothelium-dependent relaxation in aortas of diabetic mice largely through inhibiting ROS overproduction associated with Ang II-mediated BMP4-dependent mechanisms.     

Nicotine induction of theta frequency oscillations in rodent medial septal diagonal band in vitro

Aim:

This study aimed to examine the role of the nicotinic receptor (nAChR) in the generation of theta oscillations (4–12 Hz) in vitro.

Methods:

Electrophysiological studies were performed on medial septal diagonal band area (MSDB) slices to measure theta oscillation. Immunofluorescence and confocal microscopy studies were carried out to detect α4 nAChR and β2 nAChR subunits in perfused-fixed tissue from VGluT2-GFP and GAD67-GFP transgenic mice.

Results:

Application of nicotine to MSDB slices produced persistent theta oscillations in which area power increased in a dose-responsive manner. This activity was inhibited by GABA_A receptor antagonists and partially by ionotropic glutamate receptor antagonists, indicating the involvement of local GABAergic and glutamatergic neurons in the production of the rhythmic activity. The nicotine-induced theta activity was also inhibited selectively by non-α7*nAChR antagonists, suggesting the presence of these receptor types on GABAergic and glutamatergic neuron populations in the MSDB. This was confirmed by immunofluorescence and confocal microscopy studies in transgenic mice in which the GABAergic and glutamatergic neurons express green fluorescent protein (GFP), showing localisation of β2 nAChR and α4 nAChR subunits, the most common constituents of non-α7*nAChRs, in both cell types in the MSDB.

Conclusion:

Theta activity in the MSDB may be generated by tonic stimulation of non-α7*nAChRs.     

Argonaute 2 promotes angiogenesis via the PTEN/VEGF signaling pathway in human hepatocellular carcinoma

Aim:

Argonaute2 (AGO2) protein is the active part of RNA-induced silencing complex, cleaving the target mRNA strand complementary to their bound siRNA. An increasing number of miRNAs has been identified as essential to angiogenesis of hepatocellular carcinoma (HCC). In this study we investigated how AGO2 affected HCC angiogenesis.

Methods:

Human HCC cell lines HepG2, Hep3B, Huh7, SMMC-7721, Bel-7404, MHCC97-H and LM-3, and human umbilical vein endothelial cells (HUVEC) were tested. The expression of AGO2 in HCC cells was knocked down with siRNA and restored using recombinant adenovirus expressing Ago2. The levels of relevant mRNAs and proteins were examined using RT-PCR, Western blot and EILSA. Nude mice were implanted with Huh7 or SMMC-7721 cells, and tumor volumes were measured. After the mice were euthanized, the xenograft tumors were used for immunohistological analysis.

Results:

In 6 HCC cell lines, AGO2 protein expression was significantly correlated with VEGF expression (r=+0.79), and with VEGF secretion (r=+0.852). Knockdown of Ago2 in Huh7 cells and SMMC-7721 cells substantially decreased VEGF expression, whereas the restoration of AGO2 reversed both VEGF expression and secretion. Furthermore, knockdown of Ago2 significantly up-regulated the expression of PTEN (a tumor suppressor involved in the inhibition of HCC angiogenesis), and vice versa. Moreover, the specific PTEN inhibitor bisperoxovanadate (7, 14, 28 nmol/L) dose-dependently restored the expression of VEGF and the capacity of HCC cells to induce HUVECs to form capillary tubule structures. In the xenograft nude mice, knockdown of Ago2 markedly suppressed the tumor growth and decreased PTEN expression and CD31-positive microvascular in the xenograft tumors.

Conclusion:

A direct relationship exists between the miRNA processing machinery AGO2 and HCC angiogenesis that is mediated by the AGO2/PTEN/VEGF signaling pathway. The results suggest the high value of Ago2 knockdown in anti-angiogenesis therapy for HCC.     

Dauricine inhibits insulin-like growth factor-I-induced hypoxia inducible factor 1α protein accumulation and vascular endothelial growth factor expression in human breast cancer cells

Aim:

To investigate the effects of dauricine (Dau) on insulin-like growth factor-I (IGF-I)-induced hypoxia inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) expression in human breast cancer cells (MCF-7).

Methods:

Serum-starved MCF-7 cells were pretreated for 1 h with different concentrations of Dau, followed by incubation with IGF-I for 6 h. HIF-1α and VEGF protein expression levels were analyzed by Western blotting and ELISA, respectively. HIF-1α and VEGF mRNA levels were determined by real-time PCR. In vitro angiogenesis was observed via the human umbilical vein endothelial cell (HUVEC) tube formation assay. An in vitro invasion assay on HUVECs was performed.

Results:

Dau significantly inhibited IGF-I-induced HIF-1α protein expression but had no effect on HIF-1α mRNA expression. However, Dau remarkably suppressed VEGF expression at both protein and mRNA levels in response to IGF-I. Mechanistically, Dau suppressed IGF-I-induced HIF-1α and VEGF protein expression mainly by blocking the activation of PI-3K/AKT/mTOR signaling pathway. In addition, Dau reduced IGF-I-induced HIF-1α protein accumulation by inhibiting its synthesis as well as by promoting its degradation. Functionally, Dau inhibited angiogenesis in vitro. Moreover, Dau had a direct effect on IGF-I-induced invasion of HUVECs.

Conclusion:

Dau inhibits human breast cancer angiogenesis by suppressing HIF-1α protein accumulation and VEGF expression, which may provide a novel potential mechanism for the anticancer activities of Dau in human breast cancer.     

YL529, a novel,orally available multikinase inhibitor,potently inhibits angiogenesis and tumour growth in preclinical models

Background and Purpose

Targeted chemotherapy using small-molecule inhibitors of angiogenesis and proliferation is a promising strategy for cancer therapy.

Experimental Approach

YL529 was developed via computer-aided drug design, de novo synthesis and high-throughput screening. The biochemical, pharmacodynamic and toxicological profiles of YL529 were investigated using kinase and cell viability assays, a mouse tumour cell-containing alginate bead model, a zebrafish angiogenesis model and several human tumour xenograft models in athymic mice.

Key Results

In vitro, YL529 selectively inhibited the activities of VEGFR2/VEGFR3 and serine/threonine kinase RAF kinase. YL529 inhibited VEGF₁₆₅-induced phosphorylation of VEGFR2, as well as the proliferation, migration, invasion and tube formation of human umbilical vascular endothelial cells. It also significantly blocked vascular formation and angiogenesis in the zebrafish model. Moreover, YL529 strongly attenuated the proliferation of A549 cells by disrupting the RAF/mitogen-activated protein (MAP) or extracellular signal-regulated kinase (Erk) kinase (MEK) kinase kinase/MAPK pathway. Oral administration of YL529 (37.5–150 mg⁻¹·kg⁻¹·day⁻¹) to nude mice bearing established tumour xenografts significantly prevented the growth (60–80%) of A549, SPC-A1, A375, OS-RC-2 and HCT116 tumours without detectable toxicity. YL529 markedly reduced microvessel density and increased tumour cell apoptosis in the tumours formed in mice inoculated with the lung cancer cells, SPC-A1 and A549, and the colon carcinoma cells, HCT116.

Conclusions and Implications

YL529, an orally active multikinase inhibitor, shows therapeutic potential for solid tumours, and warrants further investigation as a possible anticancer agent.     

Ferulic acid inhibits nitric oxide-induced apoptosis by enhancing GABA81 receptor expression in transient focal cerebral ischemia in rats

Aim:

Ferulic acid (4-hydroxy-3-methoxycinnamic acid, FA) provides neuroprotection against apoptosis in a transient middle cerebral artery occlusion (MCAo) model. This study was to further investigate the anti-apoptotic effect of FA during reperfusion after cerebral ischemia.

Methods:

Rats were subjected to 90 min of cerebral ischemia followed by 3 or 24 h of reperfusion after which they were sacrificed.

Results:

Intravenous FA (100 mg/kg) administered immediately after middle cerebral artery occlusion (MCAo) or 2 h after reperfusion effectively abrogated the elevation of postsynaptic density-95 (PSD-95), neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS), nitrotyrosine, and cleaved caspase-3 levels as well as apoptosis in the ischemic cortex at 24 h of reperfusion. FA further inhibited Bax translocation, cytochrome c release, and p38 mitogen-activated protein (MAP) kinase phosphorylation. Moreover, FA enhanced the expression of gamma-aminobutyric acid type B receptor subunit 1 (GABA_B1) in the ischemic cortex at 3 and 24 h of reperfusion. In addition, nitrotyrosine-positive cells colocalized with cleaved caspase-3-positive cells, and phospho-p38 MAP kinase-positive cells colocalized with nitrotyrosine- and Bax-positive cells, indicating a positive relationship among the expression of nitrotyrosine, phospho-p38 MAP kinase, Bax, and cleaved caspase-3. The mutually exclusive expression of GABA_B1 and nitrotyrosine revealed that there is a negative correlation between GABA_B1 and nitrotyrosine expression profiles. Additionally, pretreatment with saclofen, a GABA_B receptor antagonist, abolished the neuroprotection of FA against nitric oxide (NO)-induced apoptosis.

Conclusion:

FA significantly enhances GABA_B1 receptor expression at early reperfusion and thereby provides neuroprotection against p38 MAP kinase-mediated NO-induced apoptosis at 24 h of reperfusion.     

WIN55,212-2 protects oligodendrocyte precursor cells in stroke penumbra following permanent focal cerebral ischemia in rats

Aim:

To explore whether the synthetic cannabinoid receptor agonist WIN55,212-2 could protect oligodendrocyte precursor cells (OPCs) in stroke penumbra, thereby providing neuroprotection following permanent focal cerebral ischemia in rats.

Methods:

Adult male SD rats were subjected to permanent middle cerebral artery occlusion (p-MCAO). The animals were administered WIN55,212-2 at 2 h, and sacrificed at 24 h after the ischemic insult. The infarct volumes and brain swelling were assessed. The expression of cannabinoid receptor type 1 (CB1) in the stroke penumbra was examined using Western blot assay. The pathological changes and proliferation of neural glial antigen 2-positive OPCs (NG2⁺ cells) in the stroke penumbra were studied using immunohistochemistry staining.

Results:

p-MCAO significantly increased the expression of CB1 within the stroke penumbra with the highest level appearing at 2 h following the ischemic insult. Administration of WIN55,212-2 (9 mg/kg, iv) significantly attenuated the brain swelling, and reduced the infarct volume as well as the number of tau-immunoreactive NG2⁺ cells (tau-1⁺/NG2⁺ cells) in the stroke penumbra. Moreover, WIN55,212-2 significantly promoted the proliferation of NG2⁺ cells in the stroke penumbra and in the ipsilateral subventricular zone at 24 h following the ischemic insult. Administration of the selective CB1 antagonist rimonabant (1 mg/kg, iv) partially blocked the effects caused by WIN55,212-2.

Conclusion:

Tau-1 is expressed in NG2⁺ cells following permanent focal cerebral ischemic injury. Treatment with WIN55,212-2 reduces the number of tau-1⁺/NG2⁺ cells and promotes NG2⁺ cell proliferation in the stroke penumbra, which are mediated partially via CB1 and may contribute to its neuroprotective effects.     

Attenuated Salmonella typhimurium carrying shRNA-expressing vectors elicit RNA interference in murine bladder tumors

Aim:

To examine whether attenuated Salmonella typhimurium (S typhimurium) could be used as an anti-cancer agent or a tumor-targeting vehicle for delivering shRNA-expressing pDNA into cancer cells in a mouse tumor model.

Methods:

Mouse bladder transitional cancer cell line (BTT-T739) expressing GFP was used, in which the GFP expression level served as an indicator of RNA interference (RNAi). BTT-T739-GFP tumor-bearing mice (4–6 weeks) were treated with S typhimurium carrying plasmids encoding shRNA against gfp or scrambled shRNA. The mRNA and protein expression levels of GFP were assessed 5 d after the bacteria administration, and the antitumor effects of S typhimurium were evaluated.

Results:

In BTT-T739-GFP tumor-bearing mice, S typhimurium (1×10⁹ cfu, po) preferentially accumulated within tumors for as long as 40 d, and formed a tumor-to-normal tissue ratio that exceeded 1000/1. S typhimurium carrying plasmids encoding shRNA against gfp inhibited the expression of GFP in tumor cells by 73.4%. Orally delivered S typhimurium significantly delayed tumor growth and prolonged the survival of tumor-bearing mice.

Conclusion:

This study demonstrates that attenuated S typhimurium can be used for both delivering shRNA-expressing vectors into tumor cells and eliciting RNAi, thus exerting anti-tumor activity, which may represent a new strategy for the treatment of solid tumors.     

Regulation of angiotensin-(1-7) and angiotensin II type 1 receptor by telmisartan and losartan in adriamycin-induced rat heart failure

Aim:

To investigate the possible effects of telmisartan and losartan on cardiac function in adriamycin (ADR)-induced heart failure in rats, and to explore the changes in plasma level of angiotensin-(1–7)[Ang-(1–7)] and myocardial expression of angiotensin II type 1/2 receptors (AT1R / AT2R) and Mas receptor caused by the two drugs.

Methods:

Male Sprague-Dawley rats were randomly divided into 4 groups: the control group, ADR-treated heart failure group (ADR-HF), telmisartan plus ADR-treated group (Tel+ADR) and losartan plus ADR-treated group (Los+ADR). ADR was administrated (2.5 mg/kg, ip, 6 times in 2 weeks). The rats in the Tel+ADR and Los+ADR groups were treated orally with telmisartan (10 mg/kg daily po) and losartan (30 mg/kg daily), respectively, for 6 weeks. The plasma level of Ang-(1–7) was determined using ELISA. The mRNA and protein expression of myocardial Mas receptor, AT₁R and AT₂R were measured using RT-PCR and Western blotting, respectively.

Results:

ADR significantly reduced the plasma level of Ang-(1–7) and the expression of myocardial Mas receptor and myocardial AT₂R, while significantly increased the expression of myocardial AT1R. Treatment with telmisartan and losartan effectively increased the plasma level of Ang-(1–7) and suppressed myocardial AT1R expression, but did not influence the expression of Mas receptor and AT₂R.

Conclusion:

The protective effects of telmisartan and losartan in ADR-induced heart failure may be partially due to regulation of circulating Ang-(1–7) and myocardial AT1R expression.     

Curcumin enhanced antiproliferative effect of mitomycin C in human breast cancer MCF-7 cells in vitro and in vivo

Aim:

To investigate the efficacy of mitomycin C (MMC) in combination with curcumin in suppressing human breast cancer in vitro and in vivo.

Methods:

Human breast cancer MCF-7 cells were used. Cell viability was measured using MTT assay. The cell cycle phase was detected with flow cytometric analysis. Cell cycle-associated proteins were examined using Western blot analysis. MCF-7 breast cancer xenografts were established to monitor tumor growth and cell cycle-associated protein expression.

Results:

Curcumin inhibited MCF-7 breast cancer cell viability in a concentration-dependent manner (IC₅₀ value=40 μmol/L). Similarly, MMC inhibited the cell viability with an IC₅₀ value of 5 μmol/L. Combined treatment of MMC and curcumin showed a synergistic antiproliferative effect. In the presence of curcumin (40 μmol/L), the IC₅₀ value of MMC was reduced to 5 μmol/L. In MCF-7 xenografts, combined administration of curcumin (100 mg/kg) and MMC (1-2 mg/kg) for 4 weeks produced significantly greater inhibition on tumor growth than either treatment alone. The combined treatment resulted in significantly greater G₁ arrest than MMC or curcumin alone. Moreover, the cell cycle arrest was associated with inhibition of cyclin D1, cyclin E, cyclin A, cyclin-dependent kinase 2 (CDK2) and CDK4, along with the induction of the cell cycle inhibitor p21 and p27 both in MCF-7 cells and in MCF-7 xenografts. These proteins were regulated through p38 MAPK pathway.

Conclusion:

The results suggest that the combination of MMC and curcumin inhibits MCF-7 cell proliferation and cell cycle progression in vitro and in vivo via the p38 MAPK pathway.     

Combination of fluvastatin and losartan relieves atherosclerosis and macrophage infiltration in atherosclerotic plaques in rabbits

Aim:

To investigate whether the combination of fluvastatin and losartan synergistically relieve atherosclerosis and plaque inflammation induced by a high-cholesterol diet in rabbits.

Methods:

Atherosclerosis was induced with a high-cholesterol diet for 3 months in 36 New Zealand white rabbits. The animals were randomly divided into model group, fluvastatin (10 mg·kg^-1·d^-1) group, losartan (25 mg·kg^-1·d^-1) group, and fluvastatin plus losartan group. After the 16-week treatments, the blood samples the animals were collected, and the thoracic aortas were examined immunohistochemically. The mRNA and protein expression levels of monocyte chemotactic protein-1 (MCP-1) were measured using RT-PCR and Western blot.

Results:

Compared to the treatment with losartan or fluvastatin alone, the combined treatment did not produce higher efficacy in reduction of blood cholesterol level. However, the combination did synergistically decrease the intimal and media thickness of thoracic aortas with significantly reduced macrophage infiltration and MCP-1 expression in the plaques.

Conclusion:

The combined treatment with losartan and fluvastatin significantly inhibited atherosclerotic progress and reduced inflammation associated with atherosclerotic plaques.     

Protective effects of ginsenoside Rb3 on oxygen and glucose deprivation-induced ischemic injury in PC12 cells

Aim:

To investigate the protective effects of ginsenoside Rb₃, a triterpenoid saponin isolated from the leaves of Panax notoginseng, on ischemic and reperfusion injury model of PC12 cells and elucidate the related mechanisms.

Methods:

PC12 cells exposed to oxygen and glucose deprivation (OGD) and restoration (OGD-Rep) were used as an in vitro model of ischemia and reperfusion. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) leakage were used to evaluate the protective effects of ginsenoside Rb₃. Cellular apoptosis and mitochondrial membrane potential (MMP) were analyzed using flow cytometry. Intracellular calcium ion concentration ([Ca²⁺]_i) was detected using fluorophotometer system. Caspase-3, -8, and -9 activities were measured using assay kits with an ELISA reader. Western blotting assay was used to evaluate the release of cytochrome c and expression of caspase-3, Bcl-2 and Bax proteins.

Results:

It was shown that ginsenoside Rb₃ (0.1–10 μmol/L) significantly increased cell viability and inhibited LDH release in a dose-dependent manner on the ischemic model. In addition, ginsenoside Rb₃ also significantly inhibited ischemic injury-induced apoptosis, [Ca²⁺]_i elevation, and decrease of MMP. Meanwhile, pretreatment with ginsenoside Rb₃ significantly induced an increase of Bcl-2 protein expression and a decrease of cytosolic cytochrome c, cleaved-caspase 3 and Bax protein expression, the caspase-3, -8, and -9 activity were also inhibited.

Conclusion:

The results indicated that ginsenoside Rb₃ could markedly protected OGD-Rep induced ischemic injury and the mechanisms maybe related to its suppression of the intracellular Ca²⁺ elevation and inhibition of apoptosis and caspase activity. Ginsenoside Rb₃ could be a promising candidate in the development of a novel class of anti-ischemic agent.     

Baicalin attenuates oxygen-glucose deprivation- induced injury by inhibiting oxidative stress-mediated 5-lipoxygenase activation in PC12 cells

Aim:

To determine whether the flavonoid baicalin attenuates oxygen-glucose deprivation (OGD)-induced injury by inhibiting oxidative stress-mediated 5-lipoxygenase (5-LOX) activation in PC12 cells.

Methods:

The effects of baicalin and the 5-LOX inhibitor zileuton on the changes induced by OGD/recovery or H₂O₂ (an exogenous reactive oxygen species [ROS]) in green fluorescent protein-5-LOX-transfected PC12 cells were compared.

Results:

Both baicalin and zileuton attenuated OGD/recovery- and H₂O₂-induced injury and inhibited OGD/recovery-induced production of 5-LOX metabolites (cysteinyl leukotrienes) in a concentration-dependent manner. However, baicalin did not reduce baseline cysteinyl leukotriene levels. Baicalin also reduced OGD/recovery-induced ROS production and inhibited 5-LOX translocation to the nuclear envelope and p38 phosphorylation induced by OGD/recovery and H₂O₂. In contrast, zileuton did not show these effects.

Conclusion:

Baicalin can inhibit 5-LOX activation after ischemic injury, which may partly result from inhibition of the ROS/p38 mitogen-activated protein kinase pathway.