Yasushi Ohno

Anti-tumor effect of gallic acid on LL-2 lung cancer cells transplanted in mice

We previously reported that gallic acid (3,4,5-trihydroxybenzoic acid), a naturally occurring plant phenol, can induce apoptosis in four kinds of human lung cancer cell lines in vitro. The present study further investigated the in vivo anti-tumor effects of orally administered gallic acid. Gallic acid reduced cell viability of LL-2 mouse lung cancer cells in vitro dose dependently, with a 50% inhibitory concentration (IC50) value of around 200 μM. C57Black mice were transplanted with LL-2 cells, and administered gallic acid (1 mg/ml in drinking water, ad libitum) and/or cisplatin (4 mg/kg i.p. injection, once a week). The average weight of the transplanted tumors, obtained at 29 days after transplantation, in the mice of control, gallic acid-treated cisplatin-treated and cisplatin plus gallic acid-treated groups was 4.02, 3.65, 3.19 and 1.72 g, respectively. The average tumor weight of the mice treated with cisplatin combined with gallic acid was significantly smaller than that of the control group (p<0.05). The amount of apoptotic cells in the tumor tissues of mice treated with gallic acid and/or cisplatin was significantly higher than those of the control mice. Combination of gallic acid and cisplatin increased the tumor cell apoptosis compared with the treatment with cisplatin alone. The present findings suggest that the combination of gallic acid with an anti-cancer drug, including cisplatin, may be an effective protocol for lung cancer therapy.

A Definitive Role of RhoC in Metastasis of Orthotopic Lung Cancer in Mice

Purpose: Lung cancer is a major cause of cancer death, and its incidence is increasing in the world. Conventional therapies remain less effective for metastases of lung cancer, leading to poor prognosis of this disorder. The present study investigates pathological roles of RhoC in metastasis of lung cancer using a clinically relevant mouse model of lung cancer. Experimental Design: RhoA, RhoC, dominant-negative Rho (dnRho) or green fluorescent protein gene was retrovirally transduced to murine lung cancer cells. For in vivo study, these transduced cells were intrapulmonary inoculated in syngeneic mice, and subsequently, growth and metastasis were analyzed. Migration and invasion activities were further investigated by in vitro chemotaxic chamber assays. Expression levels and activities of certain matrix metalloproteinases (MMPs) were explored by reverse transcription-PCR and gelatin zymography. Results: Metastasis of lung cancer in the animal model, as well as in vitro migration and invasion, were significantly enhanced or inhibited by overexpression of RhoC or dnRho, respectively, without affecting the growth of primary tumors. Expression levels of certain MMPs and the activity of MMP-2 were significantly enhanced or suppressed by overexpression of RhoC or dnRho, respectively. Conclusion: RhoC plays a crucial role in metastasis of lung cancer. RhoC does not affect tumor growth but enhances the metastatic nature of lung cancer by not only stimulating cell motility but also up-regulating certain MMPs. Attenuation of RhoC activity may be a potential target in the development of a novel strategy for treating metastasis of lung cancer.

Induction of apoptosis by gallic acid in lung cancer cells.

The apoptosis-inducing effect of gallic acid (3,4,5-trihydroxybenzoic acid) was investigated in four human lung cancer cell lines, SBC-3 (small cell carcinoma), EBC-1 (squamous cell carcinoma), A549 (adenocarcinoma) and SBC-3/CDDP (cisplatin-resistant subclone of SBC-3). Gallic acid induced apoptosis in a dose-dependent manner as evidenced by analyses of DNA fragmentation, changes in cell morphology and loss of viability. Fifty percent inhibitory concentration (IC50) values of gallic acid on the cell viability of SBC-3, EBC-1 and A549 were around 10, 20 and 60 microg/ml, respectively. The IC50 value for SBC-3/CDDP cells was almost the same as that of SBC-3, suggesting that susceptibility of cells to gallic acid-induced apoptosis is not altered by the acquisition of cisplatin resistance. The apoptotic process was effectively triggered by 30 min exposure to gallic acid. A caspase inhibitor and alpha-tocopherol effectively prevented the gallic acid-induced apoptosis, indicating the involvememt of caspase activation and oxidative processes during the course of apoptosis in gallic acid-treated cancer cells. These findings suggest the possible applicability of gallic acid in lung cancer therapy, especially to circumvent resistance to anti-cancer drugs.

Inhibition by parthenolide of phorbol ester-induced transcriptional activation of inducible nitric oxide synthase gene in a human monocyte cell line THP-1

Excessive nitric oxide production by inducible nitric oxide synthase (iNOS) in stimulated inflammatory cells is thought to be a causative factor of cellular injury in inflammatory disease states. Compounds inhibiting iNOS transcriptional activity in inflammatory cells are potentially anti-inflammatory. An assay method for estimating iNOS transcriptional activity in the human monocyte cell line THP-1 was established using a luciferase reporter gene system. In this study, we demonstrate that parthenolide, the predominant sesquiterpene lactone in European feverfew (Tanacetum parthenium), exerts potent inhibitory effects on the promoter activity of the iNOS gene in THP-1 cells. Parthenolide effectively suppressed iNOS promoter activity in a dose-dependent manner at concentrations higher than 2. 5 microM, with an IC(50) of about 10 microM. A tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), significantly increased the iNOS promoter-dependent reporter gene activity, and the TPA-induced increase in iNOS promoter activity was effectively suppressed by parthenolide, with an IC(50) of approximately 2 microM. The present findings may further explain the anti-inflammatory property of parthenolide.

Beneficial effects of autologous bone marrow mononuclear cell transplantation against elastase-induced emphysema in rabbits.

The authors investigated whether autologous bone marrow mononuclear cell (BMC) transplantation via the left and right main bronchi would mitigate elastase-induced pulmonary emphysema in rabbits. Four weeks after elastase administration, rabbits also receiving BMCs showed significantly better pulmonary function (FVC, FEV100, FEVPEF) and smaller alveolar airspaces, as indicated by a smaller mean linear intercept, than those receiving porcine pancreatic elastase (PPE) (200 U/kg) alone via the left and right main bronchi. BMCs also significantly reduced cell counts in bronchoalveolar lavage fluid, the incidence of apoptotic (TUNEL-positive) cells and matrix metalloproteinase (MMP)-2 expression, while increasing numbers of proliferative (Ki-67–positive) cells. Thus, BMCs may inhibit the progression to emphysema by attenuating inflammation, MMP-2 expression, and apoptosis, while enhancing alveolar cell proliferation.

Narirutin inhibits airway inflammation in an allergic mouse model

1 Flavonoids are naturally occurring compounds that possess anti‐allergic, anti‐inflammatory, antiproliferative and anti‐oxidant properties. In the present study, we investigated whether the flavonoid narirutin could reduce airway inflammation in ovalbumin (OVA)‐sensitized/challenged NC/Nga mice, a model of allergic eosinophilic airway inflammation. 2 Mice were initially immunized intraperitoneally with OVA on Days 0 and 7 and then challenged with inhaled OVA on Days 14, 15 and 16. In addition, some mice received narirutin orally at doses of 0.1, 1 or 10 mg/kg bodyweight daily on Days 7–16. 3 At 10 mg/kg, but not 0.1 or 1 mg/kg, narirutin significantly diminished OVA‐induced airway inflammation caused by infiltration of lung tissue with inflammatory and mucus‐producing cells, as well as reduced eosinophil counts in the peripheral blood and bronchoalveolar lavage fluid (BALF), interleukin (IL)‐4 levels in BALF and IgE levels in serum. 4 The mechanism of the anti‐inflammatory effect of narirutin are likely to be associated with a reduction in the OVA‐induced increases of IL‐4 and IgE in a murine model of allergic eosinophilic airway inflammation. These findings suggest that narirutin may be an effective new tool in the treatment of bronchial asthma.

Inhibition by costunolide of phorbol ester-induced transcriptional activation of inducible nitric oxide synthase gene in a human monocyte cell line THP-1.

Costunolide, the predominant sesquiterpene lactone in Saussureae radix, has been reported to exhibit potent chemopreventive effects on carcinogenesis. Effects of costunolide on cellular activation induced by a tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) were investigated using a reporter gene assay which was designed to reflect the promoter activity of the inducible nitric oxide synthase (iNOS) gene in a human monocyte cell line THP-1. iNOS promoter-dependent reporter gene activity was significantly increased by TPA, and the TPA-induced increase of the reporter gene activity was efficiently reduced by costunolide, with an IC50 of approximately 2 microM. The addition of sulfhydryl (SH) compounds effectively abrogated the inhibitory effects of costunolide, suggesting the involvement of its reactivity with SH groups of target proteins and/or thiol-depleting property. The present findings may further explain the cancer-preventive property of costunolide.

The effect of acupuncture in the treatment of chronic obstructive pulmonary disease.

OBJECTIVE Many claims have been made regarding the therapeutic efficacy of acupuncture. However, most controlled clinical studies have been limited to treatment of pain-related disorders and do not provide objective, quantifiable data for analysis. Traditional acupuncture has been applied to chronic obstructive pulmonary disease (COPD). However, only a few studies have been performed to determine the efficacy of this treatment. This study was conducted to determine whether a combination of traditional acupuncture treatment and conservative treatment for COPD improves dyspnea on exercise. METHODS This was a prospective trial with matched-pair parallel groups of patients from the departments of respiratory internal medicine of Gifu University of Medicine, Meiji University of Oriental Medicine, and Gifu Red Cross Hospital, Japan. Thirty patients were divided into the acupuncture group (n = 15) and the control group (n = 15). The control group received conservative treatment with medication only. The acupuncture group received acupuncture treatments once a week for 10 weeks, in addition to conservative treatment with medication. The main outcome measure was the Modified Borg dyspnea scale after the 6-minute walk test. RESULTS The acupuncture group had significantly better results on the Borg scale than the control group after 10 weeks (2.2 +/- 2.7 versus 6.4 +/- 3.4, p = 0.0001, 95% confidence interval, -5.10 to -2.35, paired t-test). The 6-minute walk distance and oxygen saturation at the minimum rate improved significantly in the acupuncture group compared with the control group. CONCLUSIONS This study demonstrated that acupuncture contributed to the reduction of COPD-related dyspnea on exercise in 15 matched-pair parallel subjects.

Dynamic process of apoptosis in adult rat cardiomyocytes analyzed using 48-hour videomicroscopy and electron microscopy: beating and rate are associated with the apoptotic process.

Dynamic process of apoptosis has not been elucidated in adult rat cardiomyocytes. Soluble Fas ligand (0.1 microg/ml) in the presence of actinomycin D (0.05 microg/ml) induced apoptosis in cultured adult rat cardiomyocytes, as documented by activated caspase-3, DNA fragmentation, and apoptotic ultrastructure. In the present model, we observed 60 adult cardiomyocytes with a normal rod shape under a real-time videomicroscope continuously for 48 hours. Seventeen cells (28%) were unchanged and 7 cells (12%) showed oncosis (so-called necrosis) in which no beating was evident. In the remaining 36 cells (apoptosis, 60%), a slow beating (17 +/- 3/min) was initiated 16 +/- 1 hours later. Approximately 1 hour later, the rod cells showed long-axial shortening as bone- or club-like, or square-shaped, accompanied with faster beating rates (35 +/- 7/min). In 29 cells (type A1 and A2), marked shrinkage occurred; the cellular shape became almost completely round with a smooth surface and the beating ceased 3.0 +/- 0.4 hours later. Then, smooth budding appeared 0.6 +/- 0.2 hours later. Apoptotic bodies were found in 8 cells 10 +/- 4 hours later (type A1, 13%) but not in 21 cells (type A2, 35%). In the other 7 cells (type A3, 12%), the cell surface became rough 8 +/- 3 hours later and the beating ceased. Maximal beating rate was greatest in type A1 (72 +/- 26/min) and greater in type A2 (29 +/- 5/min) than in type A3 (10 +/- 2/min). Electron microscopy confirmed apoptotic ultrastructure even in the cardiomyocytes with bone-, club-like, or square shapes, suggesting that type A3 as well as A1 and A2 is also under apoptotic process. A caspase inhibitor, zVAD.fmk, blocked beating, apoptotic morphology, and DNA fragmentation, indicating these depended on caspase activation. In the caspase-dependent apoptotic process of cultured adult cardiomyocytes, beating and the following deformity of the cellular edges were the initial signs and the rate of beating was related with the subsequent three different processes of apoptosis.

Use of multidetector row CT to evaluate the need for bronchial arterial embolization in hemoptysis patients.

Background: Bronchial artery (BA) embolization (BAE) is recommended as a minimally invasive therapy for hemoptysis, though some patients recover after only conservative treatment. Objectives: The purpose of our study was to assess the characteristics of BAs using multidetector row computed tomography (MDCT) and identify BAs requiring BAE without BA angiography (BAG). Methods: We retrospectively studied 41 patients and classified the visualized BAs into groups based on their BAE and bleeding statuses. Patients presenting with massive hemoptysis requiring emergency BAE were excluded. Patients presenting with persistent hemoptysis that was resistant to conservative treatment received BAE. Radiologists measured BA diameters at the ostium, bronchial bifurcation and pulmonary hilum, and also evaluated the degree of vascularization. Results: MDCT enabled visualization of 102 ostia and 96 traceable BAs. Among the participating patients, 13 had at least one ectopic origin. We obtained a good correlation between BAG and MDCT diameters (r = 0.709, p < 0.001). The diameters of BAs responsible for bleeding and receiving BAE were apparently larger in each measured segment than those that were not (p < 0.05). Moreover, the diameters of arteries receiving BAE remained largely unchanged from the origin to the hilum and through the mediastinum. BAs with low MDCT scores were significantly less likely to required BAE than those with high scores (p = 0.004), and in multiple logistic regression analysis, ostium diameter and bleeding status were independent predictive factors for BAE. Conclusions: Evaluation of BAs on MDCT could be useful for identifying the anatomical characteristics of bleeding-related BAs and determining whether BAE is indicated or whether conservative treatment is sufficient.