[Effects of estrogen and keishibukuryogan on hot flash-like symptoms induced by yohimbine in ovariectomized rats].

Hot flash (HF) is the most common phenomenon in climacteric symptoms which often develop concomitantly with a decrease in estrogen in postmenopausal women. The onset mechanism of the hot flash is complicated and remains unclear. To date, some animal models of postmenopausal HF have been devised, but they are not fully available because of the difficulty in producing them. It is thought that hyperactivity of the central α-adrenergic system with a decrease in estrogen participates in the onset of postmenopausal HF. Therefore, in the present study, we examined whether a HF model could be easily produced by administering yohimbine (YOH), a presynaptic α2-adrenoceptor antagonist which promotes norepinephrine release, to female rats. HF-like symptoms such as a rise in tail skin temperature and a fall in rectal temperature were shown in the rats who received YOH (3 mg/kg) subcutaneously seven days after the ovariectomy (OVX). Such symptoms following YOH administration were observed in sham rats as well, but were much more clearly noted in OVX rats. We next examined the effects of various drugs, which are clinically effective against postmenopausal HF, on HF-like symptoms in YOH-treated OVX rats: clonidine, a presynaptic α2-adrenoceptor agonist which inhibits norepinephrine release; ß-estradiol as an estrogen; and Keishibukuryogan, a Kampo medicine. These drugs inhibited HF-like symptoms in YOH-treated OVX rats. These results suggest that the activity of the α-adrenergic system is enhanced with a decrease in estrogen in OVX rats whereby YOH causes HF-like symptoms more conspicuously than in sham rats. Therefore, it is thought that YOH-treated OVX rats will be a novel and simple model of postmenopausal HF.

CCL21 promotes the migration and adhesion of highly lymph node metastatic human non-small cell lung cancer Lu-99 in vitro.

To develop new therapy strategies for lung cancer, we established an animal model, which reflects the clinical features of mediastinal lymph node metastasis of lung cancer. This study was designed to determine whether CCL21 induced biological functions associated with the metastasis of highly lymph node metastatic human non-small cell lung cancer (NSCLC) selected by our model. Orthotopic intrapulmonary implantation of human NSCLC (Lu-99 and A549) was performed to analyze the metastatic characteristics of these cells. The expression of CCR7, which is a receptor of CCL21, was detected using CCL19 [also called EBI1-ligand chemokine (ELC)]-Fc chimera by flow cytometric analysis. The effects of CCL21 on the migration, adhesion and growth of human NSCLC were investigated. After orthotopic implantation of human NSCLC cell lines, Lu-99, but not A549, metastasized to mediastinal lymph nodes, forming large size nodules, and expressed CCR7 on the surface. Accordingly, its ligand CCL21 induced chemotactic migration and alpha4beta1-mediated adhesion to VCAM-1 of Lu-99. The expression of CCR7 and vigorous responses to its ligand CCL21 potentially account for lymph node metastasis of a human NSCLC line Lu-99.

Aminopeptidase N (APN/CD13) is selectively expressed in vascular endothelial cells and plays multiple roles in angiogenesis.

Proteolytic enzyme-mediated degradation of the extracellular matrix (ECM) is crucial for the formation of both tumor metastasis and angiogenesis. Recently, several reports have suggested that aminopeptidases are involved in this process, but precisely how is largely unknown. We found here that aminopeptidase N (APN/CD13) was selectively expressed in vascular endothelial cells including human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells (HAEC), and was not detectable in a majority of normal cells and tumor cell lines we examined. RNA interference (RNAi) of APN resulted in the inhibition of capillary tube formation of HUVEC on Matrigel. APN siRNA suppressed the migration of HUVEC through a fibronectin-coated Transwell membrane, and reduced the cellular adhesion to Matrigel and various adhesion molecules including type IV collagen, type I collagen and fibronectin. These findings suggest that APN is a multifunctional protein with important roles in vascular endothelial morphogenesis during angiogenesis.

Antitumor effects of synthetic VEGF-receptor binding antagonist, VGA1155.

Vascular endothelial growth factor (VEGF) plays key roles in tumor angiogenesis. Therefore, VEGF and its receptors are considered to be primary targets for antiangiogenic strategy during cancer chemotherapy. Our previous study reported that VGA1155, a low-molecular-weight inhibitor of the binding of VEGF, inhibited VEGF binding to KDR/Flk-1 receptor-overexpressing cells. In the present study, the antitumor effects and antimetastatic effect of VGA1155 were examined in vivo. VGA1155 suppressed the growth of human lung, breast, colon and epidermoid cancers (LC-6, HT29, MX-1, Col-1 and A431) in the nude mouse xenograft model, and pulmonary metastasis of melanoma in the spontaneous metastasis model. These results suggest that VGA1155 has antitumor effects in vivo through the inhibition of VEGF binding to its receptors.

Urinary trypsin inhibitor suppresses surgical stress-facilitated lung metastasis of murine colon 26-L5 carcinoma cells.

Recently, we have reported that surgical stress promoted the metastasis of murine colon carcinoma cells to the lung by inducing the expression of proteases such as matrix metalloprotease-9 (MMP-9) in lung tissue. Urinary trypsin inhibitor (UTI) is a serine protease inhibitor frequently used to treat pancreatitis and to improve the microcirculatory environment. The purpose of this study was to investigate the anti-metastatic properties of UTI in an animal model of surgical stress-induced cancer metastasis. The intraperitoneal administration of UTI after the intravenous injection of colon 26-L5 carcinoma (colon 26-L5) cells into mice subjected to surgical stress suppressed the enhancement of lung metastasis (p<0.05). Furthermore, we investigated the effect of UTI on tumor growth, adhesion to fibronectin, migration, invasion and enzymatic degradation in colon 26-L5. UTI reduced the invasive ability and the degradation by MMP-9 of gelatin substrate in colon 26-L5 cells. UTI may improve therapeutic efficacy in cancer patients after major surgery.

Vanillin suppresses in vitro invasion and in vivo metastasis of mouse breast cancer cells.

Vanillin, a food flavoring agent, has been reported to show anti-mutagenic activity and to inhibit chemical carcinogenesis. In this study, we examined the effect of vanillin on the growth and metastasis of 4T1 mammary adenocarcinoma cells in BALB/c mice. Mice orally administered with vanillin showed significantly reduced numbers of lung metastasized colonies compared to controls. In vitro studies revealed that vanillin, at concentrations that were not cytotoxic, inhibited invasion and migration of cancer cells and inhibited enzymatic activity of MMP-9 secreted by the cancer cells. Vanillin also showed growth inhibitory effect towards cancer cells in vitro. However, vanillic acid, a major metabolic product of vanillin in human and rat, was not active in these in vitro activity assays. Our findings suggest that vanillin has anti-metastatic potential by decreasing invasiveness of cancer cells. Since vanillin is generally regarded as safe, it may be of value in the development of anti-metastatic drugs for cancer treatment.

VGA1155, a novel binding antagonist of VEGF, inhibits angiogenesis in vitro and in vivo.

The process of angiogenesis involves the formation of new blood vessels from established vasculature and is essential for progressive tumor growth and metastasis. Since vascular endothelial growth factor (VEGF) plays a pivotal role in tumor angiogenesis, it is reasonable to expect that antagonizing VEGF binding to its receptor may be effective in cancer therapy. Our previous study found that a novel low molecular weight compound, VGA1155, inhibited binding between radioisotope-labelled VEGF and cells overexpressing its two receptors, Flt-1 and KDR/Flk-1, that is, NIH3T3-Flt-1 and NIH3T3-KDR, respectively. In the present study, we investigated the anti-angiogenic effects of VGA1155 based on VEGF inhibition. VGA1155 inhibited VEGF-induced DNA synthesis of human umbilical vein endothelial cells (HUVEC) and human retinal endothelial cells (HREC) in a concentration-dependent manner. VGA1155 also inhibited VEGF-induced tube formation of HUVEC in vitro and tumor angiogenesis toward B16-BL6 melanoma after orthotopic implantation into the skin of the back. On the other hand, VGA 1155 did not affect the proliferation of human epidermoid carcinoma (KB) cells and mouse mammary carcinoma (MM2) cells. It also had no effect on the activity of several cytosolic kinases such as p55fyn and p56lck. These findings suggest that VGA1155 inhibits endothelial cell growth and angiogenesis by inhibiting VEGF function but not non-specific cytotoxicity. VGA1155 thus exhibits promise as an antiangiogenic or anti-tumor agent with fewer side-effects.

Anti-tumor angiogenesis effect of aminopeptidase inhibitor bestatin against B16-BL6 melanoma cells orthotopically implanted into syngeneic mice.

We investigated the effect of bestatin, an inhibitor of aminopeptidase N (APN)/CD13 and aminopeptidase B, on the angiogenesis induced by B16-BL6 melanoma cells. Oral administration of bestatin (100-200 mg/kg/day) was found to significantly inhibit the melanoma cell-induced angiogenesis in a mouse dorsal air sac assay. Additionally, anti-APN/CD13 mAb (WM15), which neutralizes the aminopeptidase activity in tumor cells, as well as bestatin inhibited the tube-like formation of human umbilical vein endothelial cells (HUVECs) in vitro. Furthermore, the intraperitoneal administration of bestatin (50-100 mg/kg/day) after the orthotopic implantation of B16-BL6 melanoma cells into mice reduced the number of vessels oriented towards the established primary tumor mass on the dorsal side of mice. These findings suggest that bestatin is an active anti-angiogenic agent that may inhibit tumor angiogenesis in vivo and tube-like formation of endothelial cells in vitro through its inhibition of APN/CD13 activity.