Targeting Podoplanin for the Treatment of Osteosarcoma.

PURPOSE: Osteosarcoma, the most common bone malignancy in children, has a poor prognosis, especially when the tumor metastasizes to the lungs. Therefore, novel therapeutic strategies targeting both proliferation and metastasis of osteosarcoma are required. Podoplanin (PDPN) is expressed by various tumors and is associated with tumor-induced platelet activation via its interaction with C-type lectin-like receptor 2 (CLEC-2) on platelets. We previously found that PDPN contributed to osteosarcoma growth and metastasis through platelet activation; thus, in this study, we developed an anti-PDPN humanized antibody and evaluated its effect on osteosarcoma growth and metastasis. EXPERIMENTAL DESIGN: Nine osteosarcoma cell lines and two osteosarcoma patient-derived cells were collected, and we evaluated the efficacy of the anti-DPN-neutralizing antibody PG4D2 and the humanized anti-PDPN antibody AP201, which had IgG4 framework region. The antitumor and antimetastasis effect of PG4D2 and AP201 were examined in vitro and in vivo. In addition, growth signaling by the interaction between PDPN and CLEC-2 was analyzed using phospho-RTK (receptor tyrosine kinase) array, growth assay, or immunoblot analysis under the supression of RTKs by knockout and inhibitor treatment. RESULTS: We observed that PG4D2 treatment significantly suppressed tumor growth and pulmonary metastasis in osteosarcoma xenograft models highly expressing PDPN. The contribution of PDGFR activation by activated platelet releasates to osteosarcoma cell proliferation was confirmed, and the humanized antibody, AP201, suppressed in vivo osteosarcoma growth and metastasis without significant adverse events. CONCLUSIONS: Targeting PDPN with a neutralizing antibody against PDPN-CLEC-2 without antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity is a novel therapeutic strategy for PDPN-positive osteosarcoma.

Novel knock-in mouse model for the evaluation of the therapeutic efficacy and toxicity of human podoplanin-targeting agents.

Podoplanin is a key molecule for enhancing tumor-induced platelet aggregation. Podoplanin interacts with CLEC-2 on platelets via PLatelet Aggregation-inducing domains (PLAGs). Among our generated antibodies, those targeting the fourth PLAG domain (PLAG4) strongly suppress podoplanin-CLEC-2 binding and podoplanin-expressing tumor growth and metastasis. We previously performed a single-dose toxicity study of PLAG4-targeting anti-podoplanin-neutralizing antibodies and found no acute toxicity in cynomolgus monkeys. To confirm the therapeutic efficacy and toxicity of podoplanin-targeting antibodies, a syngeneic mouse model that enables repeated dose toxicity tests is needed. Replacement of mouse PLAG1-PLAG4 domains with human homologous domains drastically decreased the platelet-aggregating activity. Therefore, we searched the critical domain of the platelet-aggregating activity in mouse podoplanin and found that the mouse PLAG4 domain played a critical role in platelet aggregation, similar to the human PLAG4 domain. Human/mouse chimeric podoplanin, in which a limited region containing mouse PLAG4 was replaced with human homologous region, exhibited a similar platelet-aggregating activity to wild-type mouse podoplanin. Thus, we generated knock-in mice with human/mouse chimeric podoplanin expression (PdpnKI/KI mice). Our previously established PLAG4-targeting antibodies could suppress human/mouse chimeric podoplanin-mediated platelet aggregation and tumor growth in PdpnKI/KI mice. Repeated treatment of PdpnKI/KI mice with antibody-dependent cell-mediated cytotoxicity activity-possessing PG4D2 antibody did not result in toxicity or changes in hematological and biochemical parameters. Our results suggest that anti-podoplanin-neutralizing antibodies could be used safely as novel anti-tumor agents. Our generated PdpnKI/KI mice are useful for investigating the efficacy and toxicity of human podoplanin-targeting drugs.

A novel IRS-1-associated protein, DGKζ regulates GLUT4 translocation in 3T3-L1 adipocytes.

Insulin receptor substrates (IRSs) are major targets of insulin receptor tyrosine kinases. Here we identified diacylglycerol kinase zeta (DGKζ) as an IRS-1-associated protein, and examined roles of DGKζ in glucose transporter 4 (GLUT4) translocation to the plasma membrane. When DGKζ was knocked-down in 3T3-L1 adipocytes, insulin-induced GLUT4 translocation was inhibited without affecting other mediators of insulin-dependent signaling. Similarly, knockdown of phosphatidylinositol 4-phosphate 5-kinase 1α (PIP5K1α), which had been reported to interact with DGKζ, also inhibited insulin-induced GLUT4 translocation. Moreover, DGKζ interacted with IRS-1 without insulin stimulation, but insulin stimulation decreased this interaction. Over-expression of sDGKζ (short-form DGKζ), which competed out DGKζ from IRS-1, enhanced GLUT4 translocation without insulin stimulation. Taking these results together with the data showing that cellular PIP5K activity was correlated with GLUT4 translocation ability, we concluded that IRS-1-associated DGKζ prevents GLUT4 translocation in the absence of insulin and that the DGKζ dissociated from IRS-1 by insulin stimulation enhances GLUT4 translocation through PIP5K1α activity.

The protective effects of bilberry and lingonberry extracts against UV light-induced retinal photoreceptor cell damage in vitro.

Bilberry extract (B-ext) and lingonberry extract (L-ext) are currently used as health supplements. We investigated the protective mechanisms of the B-ext and L-ext against ultraviolet A (UVA)-induced retinal photoreceptor cell damage. Cultured murine photoreceptor (661W) cells were exposed to UVA following treatment with B-ext and L-ext and their main constituents (cyanidin, delphinidin, malvidin, trans-resveratrol, and procyanidin). B-ext, L-ext, and constituents improved cell viability and suppressed ROS generation. Phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), c-Jun N-terminal kinase (JNK), and protein kinase B (Akt) were analyzed by Western blotting. B-ext and cyanidin inhibited phosphorylation of p38 MAPK, and B-ext also inhibited phosphorylation of JNK by UVA. L-ext, trans-resveratrol, and procyanidin alleviated the reduction of phosphorylated Akt levels by UVA. Finally, a cotreatment with B-ext and L-ext showed an additive effect on cell viability. Our findings suggest that both B-ext and L-ext endow protective effects against UVA-induced retinal damage.

The Effects of Brazilian Green Propolis against Excessive Light-Induced Cell Damage in Retina and Fibroblast Cells.

Background. We investigated the effects of Brazilian green propolis and its constituents against white light- or UVA-induced cell damage in mouse retinal cone-cell line 661W or human skin-derived fibroblast cells (NB1-RGB). Methods. Cell damage was induced by 3,000lx white light for 24 h or 4/10 J/cm(2) UVA exposure. Cell viability was assessed by Hoechst33342 and propidium iodide staining or by tetrazolium salt (WST-8) cell viability assay. The radical scavenging activity of propolis induced by UVA irradiation in NB1-RGB cells was measured using a reactive-oxygen-species- (ROS-) sensitive probe CM-H2DCFDA. Moreover, the effects of propolis on the UVA-induced activation of p38 and extracellular signal-regulated kinase (ERK) were examined by immunoblotting. Results. Treatment with propolis and two dicaffeoylquinic acids significantly inhibited the decrease in cell viability induced by white light in 661W. Propolis and its constituents inhibited the decrease in cell viability induced by UVA in NB1-RGB. Moreover, propolis suppressed the intracellular ROS production by UVA irradiation. Propolis also inhibited the levels of phosphorylated-p38 and ERK by UVA irradiation. Conclusion. Brazilian green propolis may become a major therapeutic candidate for the treatment of AMD and skin damage induced by UV irradiation.

Laxative effects and mechanism of action of Brazilian green propolis.

BACKGROUND: Brazilian green propolis is reported to have wide range of biological properties including antibacterial, anti-inflammatory, anti-influenza, and antioxidant activities. In the digestive system, a protective effect of propolis on gastric ulcer has been reported, but a laxative effect has not yet been reported. We investigated the effect and the mechanism of action of water and ethanol extracts of Brazilian green propolis. METHODS: We examined the laxative effect of propolis on stool frequency by administering orally an ethanol extract of propolis (EEP) or a water extract of propolis (WEP) at 10, 50, 100, or 500 mg/kg to normal mice. We then investigated the effects of propolis using constipation model mice induced by two types of drugs, loperamide (a µ opioid receptor agonist) and clonidine (an α-2 adrenergic receptor agonist). We also investigated the effects of WEP on gastrointestinal transit and contractional tension of the ileum to uncover the mechanism of action of WEP. RESULTS: Treatment with WEP, but not with EEP, significantly increased the weight of stools (p<0.01 at 500 mg/kg). WEP treatment significantly restored stool frequency and stool weight in clonidine-induced constipation model mice, but not in loperamide-induced constipation model mice. WEP treatment did not affect gastro-intestinal transit, but significantly increased the contractional tension of the isolated ileum of guinea pigs. This increase was inhibited by an acetylcholine receptor antagonist (atropine), but not by a 5-HT receptor antagonist (GR113808). CONCLUSION: These findings indicate that WEP has laxative effects both in normal mice and in clonidine-induced constipation model mice. The laxative effects of WEP might be mediated by increased contractional tension of the ileum exerted at least in part via activation of an acetylcholine receptor.

Quantification of polyphenols and pharmacological analysis of water and ethanol-based extracts of cultivated agarwood leaves.

Mangiferin (3) and genkwanin 5-O-ß-primeveroside (5) are the two major bioactive polyphenols with laxative property present in the extracts of agarwood (Aquilaria sinensis) leaves (AL). Here we developed an HPLC method to determine these bioactive components and four other major polyphenols in AL extracts and evaluated the pharmacological equivalence of organic and water extracts. Using mobile phase gradient conditions combined with UV detection at 330 nm, all six compounds were separated and we determined the relative extraction ratios of the six compounds present in A. sinensis extracts that were prepared under different conditions and compared the contents of the two laxative polyphenols present in the 60% ethanol extracts of A. sinensis and A. crassna. The polyphenols present in water extracts of 13 commercially cultivated A. crassna plants have also been analyzed. The laxative properties of 60% ethanol and four water extracts of A. crassna were evaluated by the frequency and weight of stools in loperamide-induced constipation model mice. The pharmacological equivalence of 60% ethanol extract and hot water (95°C) extract was identified in mice.

Ligation of the pterygopalatine and external carotid arteries induces ischemic damage in the murine retina.

PURPOSE: This study aimed to characterize the functional and morphologic changes in a murine model of ocular ischemic disease caused by vascular occlusion. METHODS: Retinal ischemia was induced by unilateral ligation of the pterygopalatine artery (PPA) and the external carotid artery (ECA) in anesthetized mice. Changes in ocular blood flow and retinal circulation were evaluated by three different methods: laser speckle blood flow imaging, fundus imaging, and fluorescein isothiocyanate angiography. Five days after reperfusion following 3- or 5-hour ischemia, an electroretinogram (ERG) was recorded, and the retinal histology was examined and quantified. The effects of a free radical scavenger, edaravone, using the model were evaluated by ERG and histologic analysis. RESULTS: The ligation of both the PPA and the ECA significantly reduced ocular blood flow and narrowed the blood vessels. Five hours of ischemia reduced the a-wave, b-wave, and oscillatory potential amplitudes of the ERG. The number of cells in the ganglion cell layer and the thickness of both the inner plexiform layer and the inner nuclear layer were reduced in the ischemic group. Retinal ischemia caused an increase in terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells in the inner layer after 21-hour reperfusion following 3-hour ischemia and 19-hour reperfusion following 5-hour ischemia. Edaravone (1 mg/kg, administered intraperitoneally) significantly reduced the retinal ischemic damage. CONCLUSIONS: These findings indicate that the murine model in which both the PPA and the ECA are ligated may be useful to clarify the pathologic mechanisms of retinal ischemic diseases and to evaluate neuroprotective drugs that target retinal ischemic injury.

Blockade of phosphodiesterase-III protects against oxygen-glucose deprivation in endothelial cells by upregulation of VE-cadherin.

We recently reported that a phosphodiesterase-III inhibitor, cilostazol, prevented the hemorrhagic transformation induced by focal cerebral ischemia in mice treated with tissue plasminogen activator (tPA) and that it reversed tPA-induced cell damage by protecting the neurovascular unit, particularly endothelial cells. However, the mechanisms of cilostazol action are still not clearly defined. The adheren junction (AJ) protein, VE-cadherin, is a known mediator of endothelial barrier sealing and maintenance. Therefore, we tested whether cilostazol might promote expression of adhesion molecules in endothelial cells, thereby preventing deterioration of endothelial barrier functions. Human brain microvascular endothelial cells were exposed to 6-h oxygen-glucose deprivation (OGD). We compared cilostazol with aspirin treatments and examined 2 representative AJ proteins: VE-cadherin and platelet endothelial cell adhesion molecule-1 (PECAM-1). A protein kinase A (PKA) inhibitor, LY294002 (a PI3-K inhibitor), db-cAMP, and RP-cAMPS were used to assess the roles of cAMP, PKA, and PI3-K signaling, respectively, in cilostazol-induced responses. Cilostazol and db-cAMP prevented OGD-stress injury in endothelial cells by promoting VE-cadherin expression, but not PECAM-1. Aspirin did not prevent cell damage. P13-K inhibition by LY294002 had no influence on the effects of cilostazol, but inhibition of cAMP/PKA with PKA inhibitor and Rp-cAMPS suppressed cilostazol-induced inhibition of cell damage and promotion of VE-cadherin expression. In contrast, OGD stress had no detectable effects on VEGF, VEGF receptor, or angiopoietin-1 levels. Cilostazol promotes VE-cadherin expression through cAMP/PKA-dependent pathways in brain endothelial cells; thus, cilostazol effects on adhesion molecule signaling may provide protection against OGD stress in endothelial cells.

Laxative effects of agarwood on low-fiber diet-induced constipation in rats.

BACKGROUND: Agarwood (Aquilaria sinensis), well known as incense in Southeast Asia, has been used as a digestive in traditional medicine. We investigated the laxative effects of an ethanol extract of agarwood leaves (EEA) in a rat model of low-fiber diet-induced constipation. METHODS: A set of rats was bred on a normal diet while another set was placed on a low-fiber diet to induce constipation. The laxative effect of agarwood was then investigated on both sets of rats. RESULTS: Pretreatment of normal rats with single dose of EEA (600 mg/kg, p.o.) significantly increased frequency and weight of stools. Also, treatments with EEA (300 and 600 mg/kg, p.o.) for 14 days caused a significant increase in stool frequency and weight. Feeding of the animals with a low-fiber diet resulted in a decrease in stool weight, frequency, and water content and also delayed carmine egestion. A single treatment with EEA (600 mg/kg) or senna (150 and 300 mg/kg) significantly increased stool frequency, weight, and water content and also accelerated carmine egestion in the model rats. Once daily administrations of EEA (150 mg/kg), for 14 days, caused a significant increase in water content of stools. The higher doses of EEA (300 and 600 mg/kg) significantly increased frequency, weight, and water content of the stools while accelerating carmine egestion in the constipated rats. Senna (150 and 300 mg/kg) produced similar effect as the higher doses of EEA but, in addition, induced severe diarrhea. CONCLUSION: These findings indicate that EEA has a laxative effect, without causing diarrhea, in a rat model of low-fiber diet-induced constipation. These findings suggest that EEA may be highly effective on constipation as a complementary medicine in humans suffering from life style-induced constipation.

Protective effects of a gastrointestinal agent containing Korean red ginseng on gastric ulcer models in mice.

BACKGROUND: Korean red ginseng (KRG) is a ginseng that has been cultivated and aged for 4-6 years or more, and goes through an extensive cleaning, steaming and drying process. KRG contains more than 30 kinds of saponin components and has been reported as having various biological properties, such as anti-fatigue action, immune restoration, and neurovegetative effect. The purpose of this study was to assess the effects of a KRG-containing drug (KRGCD) on gastric ulcer models in mice. METHODS: Stomach ulcers were induced by oral ingestion of hydrochloride (HCl)/ethanol or indomethacin. Treatment with KRGCD (30, 100, and 300 mg/kg, p.o.) occurred 1 hr before the ulcer induction. Effect of KRGCD on anti-oxidant activity and gastric mucosal blood flow with a laser Doppler flowmeter in mice stomach tissue was evaluated. RESULTS: KRGCD (100 and 300 mg/kg, p.o.) significantly decreased ethanol- and indomethacin-induced gastric ulcer compared with the vehicle-treated (control) group. KRGCD (100 and 300 mg/kg) also decreased the level of thiobarbituric acid reactive substance (TBARS) and increased gastric mucosal blood flow compared with the control group. CONCLUSIONS: These results suggest that the gastroprotective effects of KRGCD on mice ulcer models can be attributed to its ameliorating effect on oxidative damage and improving effect of gastric mucosal blood flow.

Agarwood induced laxative effects via acetylcholine receptors on loperamide-induced constipation in mice.

Agarwood (Aquilaria sinensis, Aquilaria crasna) is well known as an incense in the oriental region such as Thailand, Taiwan, and Cambodia, and is used as a digestive in traditional medicine. We investigated the laxative effects and mechanism of agarwood leaves extracted with ethanol (EEA-1, Aquilaria sinensis; EEA-2, Aquilaria crasna). EEA-1, EEA-2, the main constituents of EEAs (mangiferin, and genkwanin-5-O-primeveroside), and senna increased the frequency and weight of stools in loperamide-induced constipation model mice. EEA-1 and EEA-2 did not induce diarrhea as a side effect, but senna induced severe diarrhea. EEA-1 and senna increased gastro-intestinal (GI) transit in the model mice. EEA-1, but not senna, also increased the intestinal tension of isolated jejunum and ileum in guinea pigs, and the tension increase was blocked by atropine, a muscarinic receptor antagonist, but not by other inhibitors (granicetron, pyrilamine, or bradykinin-antagonist peptide). Furthermore, the increase in frequency and weight of stools induced by EEA-1 were blocked by pre-administration of atropine in the model mice. These findings indicate that EEAs exerted a laxative effect via acetylcholine receptors in the mouse constipation model.