Augmentation of the inhibitory effect of blue light on the growth of B16 melanoma cells by riboflavin

We have demonstrated that blue light has anticancer effects in cultured cancer cells and tumor-bearing animals. Based on our experimental findings, in addition to cytostatic activity that suppresses the proliferation of B16 melanoma cells, blue light may exert cytocidal activity through interaction with vitamin(s) contained in the culture medium. The present study was undertaken to identify the specific vitamins with which blue light interacts and to investigate the factors responsible for its cytocidal activity. B16 melanoma cells were incubated in media supplemented with various vitamins and exposed to blue light for 10 min. Cell necrosis was observed only in media containing riboflavin (0.4 mg/l). The effects of other components of visible light on riboflavin were also studied. Riboflavin-containing media were exposed to light of each of the three primary colors (red, green and blue) and the effects on the colony-forming capacity of B16 melanoma cells were evaluated. Cell necrosis was induced only in media exposed to blue light. The effects of riboflavin increased in a concentration-dependent manner in the range from 0.3 to 1.0 mg/l in blue-light-exposed media and were antagonized by the presence of catalase (200 U/ml). These findings suggest that cell necrosis is probably induced by active oxygen species such as hydrogen peroxide formed by the reaction of riboflavin with blue light.     

High serum soluble E-selectin levels are associated with postoperative haematogenic recurrence in esophageal squamous cell carcinoma patients

E-selectin has been reported to be associated with haematogenic metastasis in various cancer patients. In order to evaluate the risk of postoperative haematogenic recurrence of esophageal squamous cell carcinoma (SCC) patients, we examined the preoperative serum levels of soluble E-selectin and clinicopathological data of the patients. Preoperative serum was obtained from 135 esophageal SCC patients who received esophagectomies from 1990 to 1998. Serum soluble E-selectin levels were measured by means of enzyme linked immunoreactive synthesis assay (ELISA). The expression of sialyl Lewis A and X antigens were evaluated in 58 out of 135 patients. Thirty-five patients (25.9%) had haematogenic recurrence in their postoperative course. Serum soluble E-selectin levels of the haematogenic recurrence group (mean 55.6 ng/ml) were significantly higher than those of the non-haematogenic recurrence group (mean 41.1 ng/ml). When the cut-off level of soluble E-selectin was 56 ng/ml, the logistic regression analysis showed that high serum soluble E-selectin levels, lymph node metastasis and intraepithelial spread were associated with postoperative haematogenic recurrence of the esophageal SCC patients (OR 2.99, p=0.047, OR 4.94, p=0.009 and OR 5.0, p=0.019. respectively). Univariate analysis revealed that the patients with a high serum soluble E-selectin level tended to have poor survival (p=0.078) and Cox multivariate analysis revealed that a high serum soluble E-selectin level was a prognostic factor in esophageal SCC patients (RR 1.84, p=0.065). The patients with a high serum soluble E-selectin concomitant with expression of sialyl Lewis antigens had a significant risk of postoperative haematogenic recurrence (p=0.005). These results indicated that preoperative high serum soluble E-selectin was a risk factor in the development of postoperative haematogenic recurrence and was a prognostic factor in esophageal SCC patients.     

Thyroxine-derivatives of lipopeptides: bifunctional dimerization inhibitors of human immunodeficiency virus-1 protease

The structure of new lipopeptides targeting the enzymic dimer interface have been rationally improved resulting in dimerization inhibitors of the human immunodeficiency virus 1 protease (K(id)=5nM for the best inhibitor). The contribution of each amino acid in inhibitory 3-mer lipopeptides was analyzed demonstrating that the C-terminal amino acid residue may preferably be replaced by thyroxine and thyronine. The negative charge of Glu is not essential. Lengthening of the peptidic chain may lead to a decrease of efficiency and a change in the mechanism (competitive inhibition instead of dimerization inhibition). The N-terminal blocking group can be replaced by 2-aminopalmitic acid. The mechanism of inhibition has been ascertained using Zhang's kinetic analysis combined with a physical method based on binding of 1-anilino-8-naphtalene sulfonate to enzyme. By targeting the hydrophobic pocket and the interface antiparallel beta-sheet found relatively free of mutations in contrary to the active site, these efficient dimerization inhibitors may provide a way of overcoming the drug resistances observed with therapeutic antiproteases that bind to the active site.     

Role of Th2 responses in the development of allergen-induced airway remodelling in a murine model of allergic asthma

(1) To clarify the involvement of Th2 responses in the development of allergen-induced airway remodelling, we investigated the effect of anti-CD4 monoclonal antibody (mAb) and anti-CD8 mAb, and the responses of IL-4 gene-knockout (KO) mice in a murine model of allergic asthma. (2) Mice were immunized twice by intraperitoneal injections of ovalbumin (OA), and exposed to aeroallergen (OA, 1% w v(-1)) for 3 weeks. Twenty-four hours after the final challenge, airway responsiveness to acetylcholine was measured, and bronchoalveolar lavage (BAL) and histological examinations were carried out. (3) Anti-CD4 mAb (1 mg kg(-1)) clearly inhibited allergen-induced increases in airway responsiveness to acetylcholine, the number of eosinophils in BAL fluid, serum OA-specific IgE levels, IL-13 and transforming growth factor-beta1 levels in BAL fluid, and amount of hydroxyproline in the lung by 100, 99, 100, 100, 84, and 60%, respectively. Furthermore, the antibody (1 mg kg(-1)) also attenuated allergen-induced goblet cell hyperplasia in the epithelium and subepithelial fibrosis by 72 and 83%, respectively. In contrast, anti-CD8 mAb (1 mg kg(-1)) showed no effect on each parameter. Furthermore, all these parameters were attenuated in IL-4KO mice by 57, 93, 100, 45, 84 and 60%, and also 72 and 83%, respectively. (4) These findings suggest that Th2 responses play a critical role for the development of allergen-induced airway remodelling, and that the inhibition of Th2 responses, e.g. using anti-CD4 mAb, is a therapeutic approach for the treatment of airway remodelling in asthma.     

Protein transduction by poly-arginine

Protein transduction methods have been developed utilizing the delivery of peptides and proteins into eukaryotic cells by the protein transduction domain (PTD). Initially, the PTD domain was developed from the sequences from HIV-1 TAT, HSV VP-22 and antennapedia homeoprotein. Recently, several novel PTDs were developed and has been used as a valuable strategy for transduction of therapeutic protein. We developed a novel, high efficiency PTD (11 arginine) based on the TAT sequence and used 11R for the regulation of intracellular signal cascades. PTD can deliver proteins and other bioactive compounds and therefore serves as a very useful strategy for the development of therapeutic agents.     

Molecular biological researches of the lower urinary tract function

Adrenergic alpha1 and beta receptors are present in the target organs of sympathetic nerve and they participate in the signal transduction mechanism of the lower urinary tract. Adrenergic alpha1 receptors are present in urethral and prostatic smooth muscles, and contract these muscles. Among these receptor subtypes, the alpha1-A receptor has the most important role, and mRNA expression of the corresponding alpha1-a subtype is predominant. In the human urinary bladder detrusor smooth muscle, the expression of adrenergic beta3 receptor subtype mRNA is predominant, and relaxation of detrusor smooth muscle is mediated mainly via beta3 receptor. Afferent nerve with lower threshold can easily transmit bladder sensation and takes an important role in the pathophysiology of urge urinary incontinence. Successful molecular cloning of vanilloid receptors, which are present in these afferent nerves, revealed that vanilloid receptors are ion-channels, sensitive for heat and pH, and termed VR1 and VRL1. Among purinergic receptors, ion channel type P2X3 receptor is found in afferent nerve fibers and plays some roles in the signal transduction of bladder sensation. In the near future, agonist for the adrenergic beta3 receptor and selective antagonists for VR1, VRL1, or P2X3 will possibly become drugs for pollakisuria and urge urinary incontinence.     

Pharmacological effects of darifenacin on human isolated urinary bladder

Darifenacin [(S)-2--2,2-diphenylacetamide] is a novel antimuscarinic drug currently undergoing phase III trials for the treatment of overactive bladder. We investigated the functional antagonist potency of darifenacin, and the antimuscarinic drugs propiverine, oxybutynin and atropine, on human detrusor smooth muscle. Urinary bladder specimens were obtained from 20 patients who underwent total cystectomy for malignant bladder tumor. Using an organ-bath technique, the effects of the compounds on carbachol-, KCl-, CaCl(2)- or electrical field stimulation (EFS)-induced contractions of the tissues were evaluated. The order of antagonist potency (pA(2 )values) at the muscarinic M(3) receptors was: darifenacin (9.34) > atropine (9.26) > oxybutynin (7.74) > propiverine (7.68). Darifenacin and atropine, at concentrations up to 10(-6) mol/l, did not inhibit the KCl- and CaCl(2)-induced contractions (concentrations 80 and 5 mmol/l, respectively), while propiverine and oxybutynin (10(-5) mol/l) significantly inhibited these contractions. Pretreatment with darifenacin (10(-9)-10(-6) mol/l), propiverine (10(-8)- 10(-5) mol/l), oxybutynin (10(-8)-10(-5) mol/l) and atropine (10(-9)-10(-6) mol/l) significantly inhibited maximum EFS-induced contractions. Darifenacin inhibited contractions of human detrusor smooth muscle only through its antimuscarinic action, while propiverine and oxybutynin had both antimuscarinic and Ca(2+) channel antagonist actions. These findings indicate that darifenacin is a potent antagonist at the M(3) receptor and support its use as a treatment for overactive bladder.     

Effects of RS-601, a novel leukotriene D(4)/thromboxane A(2) dual receptor antagonist,on asthmatic responses in guinea pigs

The effects of 4-[4-[5,5,6,6,6-pentafluoro-1-(4-fluorobenzene-sulfonamido)hexyl]phenyl]butyric acid (RS-601), a novel leukotriene D(4) (LTD(4))/thromboxane A(2) (TxA(2)) dual receptor antagonist, on bronchial asthmatic responses in guinea pigs were examined. The effects were compared with those of pranlukast (LTD(4) receptor antagonist) and S-1452 (TxA(2) receptor antagonist). RS-601 inhibited the increase in airway resistance caused by LTD(4) and TxA(2) mimetic compound, U-46619, but not by histamine. RS-601 and pranlukast but not S-1452 inhibited an antigen-induced late asthmatic response. In addition, RS-601 inhibited an antigen-induced airway hyperresponsiveness (AHR), whereas pranlukast and S-1452 had no effect on the AHR. The antigen-induced increase in inflammatory cells in airway was not affected by all examined agents. Furthermore, bacterial lipopolysaccharide-induced AHR in guinea pigs was clearly suppressed by RS-601 but not by pranlukast and S-1452. The increase in airway inflammatory cells caused by lipopolysaccharide was not affected by all three drugs. These findings indicate that RS-601 has a potent antiasthmatic efficacy, especially on AHR, but does not affect accumulation of eosinophils in the airways.     

Ethnic difference in serology of Helicobacter pylori CagA between Japanese and non-Japanese Brazilians for non-cardia gastric cancer

The usefulness of serology against CagA of Helicobacter pylori as a biomarker to identify high-risk individuals for non-cardia gastric cancer (ncGC) remains unclear among several ethnic populations with a high prevalence of cagA-positive strains. We investigated ethnic differences of CagA serology in two sets of case-control subjects, Japanese-Brazilians (JB) and non-Japanese Brazilians (NJB). We performed a cross-sectional comparison of IgG antibody titers to CagA (CagA-Ab) and the combination of CagA-Ab with conventional surface antigen (Hp-Ab) in 80 JB and 178 NJB ncGC patients and their controls (160 JB and 178 NJB). The level of CagA-Ab titer in cancer cases was significantly higher in NJB than in JB. The strength of the association between CagA-Ab seropositivity (+) (>10 U/ml) and ncGC was almost 2-fold higher in NJB than in JB [odds ratio (OR) (95% confidence interval), 4.5 (2.6–7.8) and 2.1 (1.2–3.6), respectively]. However, in both JB and NJB, the OR was highest in CagA-Ab(+) subjects with low titer (10–29 U/ml), and decreased inversely with elevating CagA-Ab titer. In addition, the serological status of CagA-Ab(+) and Hp-Ab(-) showed a similar close association with ncGC between JB and NJB [5.4 (1.9–15.3) and 5.4 (2.0–15.0), respectively]. These results suggest that although the roles of CagA in the carcinogenic process of ncGC might be different between JB and NJB, the CagA-Ab could be a useful marker for ncGC, independently of ethnicity, particularly in high-risk individuals with the serological status of CagA-Ab(+) with low IgG titer or combined with Hp-Ab(-). (Cancer Sci 2003; 94: 64–69)