Assessment of antioxidant activity of natural compound by water- and lipid-soluble antioxidant factor

We evaluated the antioxidant activity of natural compounds in water-soluble and lipid-soluble phases and found that ferulic acid, quercetin and caffeic acid showed stronger activity in the water-soluble phase. Various fractions isolated from Bidens pilosa showed this activity mainly in the water-soluble phase. Antioxidant activity in the lipid-soluble phase of propolis depended on the lipophilic extraction.     

Alterations of antiproliferative effects of serum obtained from patients with acute cerebral infarction treated with a radical scavenger, edaravone, with or without amlodipine using an in vitro cultured basilar artery smooth muscle cells

The guinea-pig basilar artery smooth muscle cell (GBa-SM3) culture system in the Dulbecco's modified Eagle's medium for 3 days serves as a useful in vitro model for assessing antiproliferative effects of various therapeutic agents on vessels. With use of this system we studied whether human serum obtained from patients with acute cerebral infarction (n = 16) would have a proliferative effect on vessels and whether an administration of a free radical scavenger, edaravone, with or without amlodipine would elicit antiproliferative effects. The control serum was obtained from 3 healthy human subjects. Time courses of the cell growth and survival were measured colorimetrically by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrzolium bromide (MTT) test. The stimulatory effect on the proliferation of GBa-SM3 cells of patients' serum obtained immediately after infarction was significantly (p < 0.05) greater than those obtained from the same patients after the treatment of edaravone for 2 weeks. In addition, the serum obtained from the patients treated by edaravone and amlodipine (n = 7) showed a significantly (p < 0.05) greater antiproliferative effect than that obtained from those treated by edaravone (n = 9). In conclusion, edaravone may have a clinically beneficial antiproliferative effect on vascular smooth muscle cells. Co-administration of amlodipine, possessing an antioxidative calcium channel blocker, with edaravone may be a promising combination to patients with acute cerebral infarction. Further controlled clinical trials with a large number of patients should be warranted.     

Effects of angiotensin II receptor blockers, angiotensin converting enzyme inhibitors, 3-hydroxy-3-methyl glutaryl (HMG) CoA reductase inhibitors, amlodipine and epalrestat on cultured basilar artery smooth muscle cell proliferation

Proliferation of vascular smooth muscle cells (VSMC) stimulated by oxidative stresses and reactive oxygen species (ROS) may play a pivotal role in the pathogenesis of atherosclerosis. Antiatherosclerotic effects of angiotensin II receptor blockers, angiotensin converting enzyme inhibitors, HMG CoA reductase inhibitors, calcium channel blocker and epalrestat were studied with an in vitro guinea-pig basilar artery smooth muscle cell (GBa-SM3) culture system over 3 days incubated with 0 to 10% of fetal bovine serum. Results demonstrated that simvastatin (0.1 mM), fluvastatin (0.3 mM), amlodipine (0.2 mM) and epalrestat (1 mM) elicited significant (p < 0.05 or 0.01) antiproliferative effects, whereas losartan (1 mM), valsartan (1 mM), enalapril (0.1 mM), captopril (1 mM), trandolapril (0.01 mM), pravastatin (0.7 mM) did not. In conclusion, the present in vitro VSMC culture system may serve as a comprehensive screening method for pleiotropic effects of commonly used therapeutic agents.     

Japanese encephalitis subunit vaccine composed of virus-like envelope antigen particles purified from serum-free medium of a high-producer J12#26 cell clone

A stable cell clone, J12#26, which continuously secretes large amounts of the envelope (E) antigen of Japanese encephalitis (JE) virus (J. Virol. 77 (2003) 8745) was adapted to serum-free medium. The J12#26 antigen possessed hemagglutinating activity, as well as the viral E and M proteins. More than 10 and 1mg of the antigen quantified with the licensed JE vaccine (JE-VAX) as a standard by E-ELISA and protein determination, respectively, were recovered from 500 ml of serum-free medium by membrane ultrafiltration, Sephacryl S-300 chromatography, sucrose gradient centrifugation and Sephadex G-25 chromatography. SDS-PAGE and Western blot analyses confirmed the high yield and purity of the J12#26 E antigen, which was comprised of small spherical virus-like particles (VLP) of approximately 25 nm in diameter. This antigen induced in mice without adjuvant neutralizing antibody (NT Ab) titers, as high as or higher than the licensed JE vaccine, and complete protection against challenge with wild-type virus. These results suggest that the J12#26 antigen is a promising second-generation JE subunit vaccine.     

Percutaneous ethanol and lipiodol injection therapy for hepatocellular carcinoma

Radiofrequency ablation (RFA) therapy is of great significance in the treatment of hepatocellular carcinoma (HCC) or metastatic liver tumors. RFA is able to achieve widely coagulated necrosis in a few sessions without major complications. However, HCC cases exist that are resistant to RFA therapy for several reasons. In the present study, we performed injection of the mixture of ethanol and lipiodol (percutaneous ethanol-lipiodol injection therapy: PELIT) for HCCs that lacked clear visuality of the entire shape of the tumor by ultrasonography (US) or computed tomography (CT), or that were difficult to treat with RFA alone due to their locations in the liver or due to severe liver dysfunction of the patients. Local recurrence rates of HCC treated with PELIT were shown to be low in patients followed up for at least 4 months. In all patients treated with PELIT, lipiodol was accumulated in the entire region of the tumor after several trials of PELIT and the accumulation was kept for many months. The biopsy examination from the tumor treated with PELIT showed that HCC cells were totally destroyed by the PELIT. Although RFA therapy serves as a central role for the treatment of HCCs, PELIT, considered to be milder therapy, is likely to be important as a supportive treatment for HCCs and useful for the treatment of HCCs that are difficult to treat with RFA.     

Subcellular localization of MUC1 recognized by a monoclonal antibody MY.1E12 correlates with postsurgical prognosis in differentiated-type gastric carcinomas of stage II and III

Because of the complex nature of the diverse histologic grade in gastric carcinomas a useful biomarker should be provided to scale the aggressiveness of the disease and to determine surgical strategy, especially for advanced carcinomas. Our previous study of MUC1 in gallbladder carcinoma using mAb MY.1E12 has revealed the stromal localization of MUC1 adjacent to the carcinoma was correlated with poor prognosis. In gastric carcinoma the biological significance of the localization of MUC1 recognized by mAb MY.1E12 has not been fully investigated. We performed immunohistochemical analysis to determine the correlations with the localization of mAb MY.1E12-reactive-MUC1 (MY.1E12-MUC1) and clinicopathological findings. A total of 91 consecutive patients with stage II, IIIA or IIIB gastric carcinoma after curative resection were reviewed retrospectively. The localization of MY.1E12-MUC1 was classified as negative, apical, cytoplasmic or stromal type based on the predominant subcellular localization. Immunostaining of MY.1E12-MUC1 was recognized in 84% of the 55 cases of differentiated-type carcinoma and in 53% of the 36 cases of undifferentiated-type carcinoma (P<0.01). In differentiated-type carcinoma, the proportion of stromal-type dominant localization of MY.1E12-MUC1 was increased at the deepest invading sites. Postsurgical liver metastasis was seen in 11 (30%) of 37 cases showing stromal or cytoplasmic-type localization-dominant group and in 1 (6%) of 18 cases showing apical-type localization-dominant group or negative staining group (P<0.05). The postsurgical survival was significantly poorer in the former group than in the latter (P=0.004). In differentiated-type gastric carcinoma, the presence of the cytoplasmic- or stromal-type localization of MY.1E12-MUC1 at the deepest invading sites correlates with aggressiveness of the disease, such as the tendency to form liver metastasis. This phenotype may serve as a unique biological feature associated with the malignant behavior of differentiated-type gastric carcinomas.     

Intratumoral injection of IL-2-activated NK cells enhances the antitumor effect of intradermally injected paraformaldehyde-fixed tumor vaccine in a rat intracranial brain tumor model

Combined therapy with a fixed-tumor cell vaccine and intratumoral injection of NK cells induced strong tumor regression of rat glioma. Rat 9L glioma cells were inoculated into syngeneic male rats at the flank (subcutaneous tumor model) or at the basal ganglia of the right hemisphere (intracranial tumor model). Rats were intradermally injected three times with vaccine comprising fixed 9L cells, IL-2- and GMCSF-microparticles, and tuberculin prior to (protective studies) or after (therapeutic studies) challenge with live 9L cells. In the protective studies, the vaccine alone achieved significant tumor growth inhibition and elongation of mean life span in both the subcutaneous and intracranial tumor models. No therapeutic effect was observed in the intracranial tumor model with the vaccine alone. However, intratumoral injection of rat NK cells strongly assisted the therapeutic effect of the vaccine in the brain tumor model and resulted in a statistically significant elongation of life span. We propose that intratumoral injection of NK cells may not only kill brain tumor cells directly, but also trigger a strong immune response in the focal lesion of the brain after vaccination. (Cancer Sci 2004; 95: 98–103)     

DE-310, a novel macromolecular carrier system for the camptothecin analog DX-8951f: potent antitumor activities in various murine tumor models

DE-310 is a novel macromolecular conjugate composed of DX-8951f, a camptothecin analog, and a carboxymethyldextran polyalcohol carrier, which are covalently linked via a peptidyl spacer. In a murine Meth A (fibrosarcoma) solid tumor model, once daily×5 treatments (qd×5) with DX-8951f at the maximum tolerated dose (MTD) were required to shrink the tumor, and DX-8951f (qd×5) at 1/4 MTD was required to inhibit tumor growth. A single treatment (qd×1) with DE-310 at the MTD or 1/4 MTD shrank the tumor, with no body weight loss occurring at 1/4 MTD. Even at 1/16 MTD, DE-310 inhibited tumor growth. In a long-term assay, Meth A solid tumors disappeared in mice treated with DE-310 (qd×1) at the MTD and 1/2 MTD, and all 6 mice remained tumor-free on the 60th day after administration. Repeated injection (4 times) on schedules of every 3 days, 7 days or 14 days demonstrated that multiple treatment with DE-310 produced greater tumor growth delay than a single treatment with DE-310. Against 5 human tumor (colon and lung cancer) xenografts in mice, DE-310 (qd×1) was as effective as DX-8951f administered once every 4 days, 4 times. The life-prolonging activity of DE-310 was assessed in lung (3LL, Lewis lung carcinoma) and liver (M5076, histiocytoma) metastasis models. Against 3LL, DE-310 (qdx1) at the MTD to 1/3 MTD significantly prolonged survival, with an increase in life span (ILS) of 4.8- to 1.6-fold, respectively, over that in untreated control mice. Also, DE-310 (qd×1) significantly prolonged survival in the liver metastasis model of M5076. These results demonstrate that DE-310 is a promising agent for the treatment of cancer.     

Expression and distribution of c-kit receptor and its ligand in human CNS germ cell tumors: A useful histological marker for the diagnosis of germinoma

We previously reported the expression of protooncogenec-kit in CNS germ cell tumors and suggested that the soluble form of c-kit (s-kit) may represent a specific clinical marker for germinoma-containing tumors. Here we investigated the expression of stem cell factor (SCF), a specific ligand of c-kit, in CNS germ cell tumor samples from 16 patients, using immunohistochemical methods to assay the expression of c-kit and SCF protein. The immunostaining patterns of c-kit and SCF were almost identical. In all germinoma-containing tumors, c-kit and SCF were diffusely expressed on the surface of germinoma cells; lymphocytes and interstitial cells were negatively stained. In immature teratomas, only some mature components, e.g., cartilage and gland, were immunoreactive for c-kit and SCF. Syncytiotrophoblastic giant cells (STGCs) were negative for both SCF and c-kit, suggesting that germinoma cells primarily coexpress SCF and c-kit. The coexpression of c-kit and SCF may be an important immunohistochemical marker for the diagnosis of CNS germinoma, and the SCF/c-kit pathway may be an alternative molecular target for the treatment of human CNS germinomas.