Anti-UV activity of lignin-carbohydrate complex and related compounds.

BACKGROUND: We recently reported that an alkaline extract of the leaves of Sasa senanensis Rehder (SE) and Lentinus edodes mycelia extract (LEM), exhibiting lignin-carbohydrate complex (LCC)-like activity, protected cells from UV-induced injury (referred to as anti-UV activity). We investigated whether LCC is the major active components responsible for anti-UV activity. MATERIALS AND METHODS: Human oral squamous cell carcinoma HSC-2 cells were exposed to short UV irradiation in phosphate-buffered saline, containing different concentrations of LCC. After culturing for 48 h in fresh culture medium, the viable cell number was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. From the dose-response curve, the 50% cytotoxic concentration (CC(50)) and the concentration that increased the viability of the UV-irradiated cells to 50% of the control value (EC(50)) were determined. The selectivity index (SI) was determined by the following equation: SI=CC(50)/EC(50). RESULTS: LCCs (Fr. VI) of pine cones and seed shell, and sulfated LCC exhibited relatively high anti-UV activity (SI=7.1-38), compared with that of SE and LEM. LCCs with lower lignin content (Fr. VII) exhibited anti-UV activity, approximately one half that of Fr. VI. However, polysaccharides (laminarin, pullulan, dextran) introduced with dimethylaminoethyl- or sulfate groups with different substitution ratios were totally inactive (SI<1). The introduction of a sulfate group to LCC did not enhance the anti-UV activity of LCC. Sodium ascorbate and vanillin were the most active (SI=65), whereas gallic acid (SI=5), epigallocatechin gallate (SI=2.6), ar-trumeron (SI<1), and turmeric extract (SI<1) were much less active. CONCLUSION: The prominent anti-UV activity of SE and LEM seems to be generated by LCCs present in the extract.

Anti-UV activity of Lentinus edodes mycelia extract (LEM).

BACKGROUND: Using our recently established simple method for evaluating protective activity from ultraviolet ray injury (referred to as 'anti-UV activity'), the effectiveness of various antioxidants and plant extracts was investigated. MATERIALS AND METHODS: HSC-2 human oral squamous cell carcinoma cells were exposed to UV irradiation (wavelength: 253.7 nm, 6 J/m²) in phosphate-buffered saline (PBS(-)) containing various concentrations of samples and then incubated for 48 hours in regular culture medium to determine the viable cell number by the MTT method. RESULTS: Among the representative antioxidants, sodium ascorbate showed the most potent anti-UV activity, whereas catalase and N-acetyl-L-cysteine were inactive. Lentinus edodes mycelia extract (LEM) showed comparable anti-UV activity to sodium ascorbate. Hot water extracts of green tea and coffee, and PET-bottled of green tea extract showed slightly less, but noticeable anti-UV activity. On the other hand, hot water extracts of black tea and Jasmine tea, and PET-bottled of oolong tea, barley tea and Kohki tea were inactive. LEM was separated by gel filtration chromatography into four fractions from high to low molecular weight: polysaccharide, large and small lignin-carbohydrate complexes, and sugars. Anti-UV activity was shown by the lignin-carbohydrate fractions, but not the polysaccharide and sugar fractions. LEM, at high concentration, slightly enhanced the anti-UV activity of sodium ascorbate. CONCLUSION: LEM may be applicable as a UV-protective agent.

Non-apoptotic cell death induced by nutritional starvation in J774.1 mouse macrophage-like cell line.

The growth and amino acid utilization of a mouse macrophage-like cell line J774.1 was investigated in two different culture media supplemented with 10% fetal bovine serum (FBS). The J774.1 cells grew faster, and consumed glutamine and serine at higher rates in DMEM than in RPMI1640 medium. The consumption of other amino acids was much less, while considerable quantities of alanine, glutamic acid and glycine were produced by the J774.1 cells. When the cells became confluent, serine, but not glutamine, was nearly depleted from the culture medium, followed by cell death characterized by smear DNA fragmentation, slight caspase-3 activation and structural damage of the mitochondria. Serine is required for the growth of mouse macrophage-like cell lines, and DMEM is superior to RPMI1640 for long-term cell culture.

Biological impact of contact with metals on cells.

In order to investigate the in vivo effect of metals used in dentistry, we investigated the effect of direct contact with metal plates (20 x 20 x 0.5 mm3) made of gold (Au), silver (Ag), copper (Cu) or palladium (Pd) on human promyelocytic leukemic HL-60 cells grown in RPMI1640 medium supplemented with 10% fetal bovine serum. When 0.5 mL of cell suspension was applied to the metal plates, cells were precipitated on the surface of the metal plate within 10 min. Contact with Cu induced a rapid decline of cell viability, the smear pattern of DNA fragmentation, and only minor activation of caspase-3. These effects were accompanied by a progressive decrease in the extracellular concentration of methionine, cysteine and histidine, with a corresponding increase in the concentration of methionine sulfoxide. Electron microscopy showed that contact with Cu induced vacuolization and cytoplasmic damage, prior to nuclear damage, without affecting the cell surface microvilli or mitochondrial integrity. Contact with the other metals did not induce such changes during the 3 h incubation, nor was any hormetic response (beneficial action at lower concentration) observed in the cells with any metals. Addition of N-acetyl-L-cysteine (4-5 mM) almost completely abrogated the Cu-induced cytotoxicity, whereas sodium ascorbate (0.1-0.5 mM) and catalase (6,000(1)-30,000 units/mL) were ineffective. Numerous serum proteins were adsorbed to the Ag plate, while bovine serum albumin was the major protein adsorbed to other metal plates. The present study suggests that direct contact with Cu induced non-apoptotic cell death by an oxidation-involved mechanism. The present model system may be applicable to the study of the interaction between cells and dental restorative materials.

Cytotoxicity and radical modulating activity of Moxa smoke.

The biological activities of Moxa, used as moxibustion, have not been well documented. We investigated here Moxa smoke for its tumor-specific cytotoxicity, anti-HIV activity, radical intensity and radical scavenging activity, in comparison with previously published data of Moxa extract. Moxa smoke showed slightly higher cytotoxicity against human tumor cell lines (oral squamous cell carcinoma HSC-2, HSC-3, promyelocytic leukemia HL-60) than against normal oral cells (gingival fibroblast HGF, pulp cell HPC, periodontal ligament fibroblast HPLF), yielding a tumor specificity index of 1.29. Moxa smoke dose-dependently induced internucleosomal DNA fragmentation, activation of caspases 3, 8 and 9, and slightly modified the expression of apoptosis-related proteins (Bcl-2, Bad, Bax) in HL-60 cells, but to much lesser extents than attained by positive controls (UV irradiation, actinomycin D treatment). ESR spectroscopy showed that Moxa smoke generated semiquinone-type radicals under alkaline conditions, and scavenged O2(-), hydroxyl radical, singlet oxygen and NO. All Moxa smoke preparations showed no apparent anti-HIV activity. These data demonstrate the antitumor potential of Moxa smoke.

Inhibition by Moxa smoke of NO production and iNOS expression in mouse macrophage-like cells Raw 264.7.

The biological activities of Moxa, used as moxibustion, have not been well documented. We investigated the effect of Moxa smoke on nitric oxide (NO) production by mouse macrophage-like Raw 264.7 cells. Moxa smoke failed to stimulate the Raw 264.7 cells to produce detectable amounts of NO, but rather inhibited the NO production by lipopolysaccharide (LPS)-activated Raw 264.7 cells. The 50% inhibitory concentration (IC50) of NO production by Moxa smoke (0.16%) was one order lower than the 50% cytotoxic concentration (CC50) (4.67%). Western blot and RT-PCR analyses demonstrated that a slightly higher concentration of Moxa is required to reduce the iNOS expression at protein and mRNA levels (IC50 = 0.99 and 2.03%, respectively). The inhibition of NO production by Moxa smoke is, thus, probably due to both the inhibition of iNOS expression and radical-scavenging activity. The present data suggest the possible anti-inflammatory effect of Moxa smoke.

A recombinant P4 protein of Haemophilus influenzae induces specific immune responses biologically active against nasopharyngeal colonization in mice after intranasal immunization.

Outer membrane protein P4, together with P6, is highly conserved among all typeable and nontypeable strains of Haemophilus influenzae (H. influenzae). Thus, the protein is an attractive antigen for the inclusion in a vaccine against nontypeable H. influenzae (NTHi). However, the ability of P4 to induce antibodies protective against NTHi infections is still controversial. In this study, we investigated the specific mucosal immune responses against NTHi induced by intranasal immunization with the lipidated form of recombinant P4 protein (rP4) and non-fatty acylated recombinant P6 protein (rP6) with or without cholera toxin (CT) in BALB/c mice model. Intranasal immunization with either rP4+CT, a mixture of rP4 and rP6+CT, or rP4 and rP6 without CT elicited anti-rP4 specific IgG antibody in serum of mice. Intranasal immunization with either rP4+CT or a mixture of rP4, rP6+CT elicited anti-rP4 specific IgA antibody in nasopharyngeal washing (NPW), while intranasal immunization with rP4 and rP6 without CT did not induced anti-rP4 specific IgA antibody responses in NPWs. Sera from mice intranasally immunized with rP4+CT and a mixture of rP4, rP6+CT also showed bactericidal activity. Significant clearance of NTHi in nasopharynx was seen 3 days after the inoculation of live NTHi in mice intranasally immunized with rP4+CT. The current findings suggested that P4 would be a useful antigen as the component of the vaccine to induce protective immune responses against NTHi. The use of an intranasal vaccine composed of the different surface protein antigens is an attractive strategy for the development of a vaccine against NTHi.

Retinoids in liver fibrosis and cancer.

Pathobiological functions and metabolism of retinoids (vitamin A and its derivatives) in liver fibrosis and hepatocellular carcinoma (HCC) are discussed in the present review. Retinoic acid (RA, active metabolite) exacerbates liver fibrosis that is not accompanied by hepatic necroinflammation, in which RA acts directly on hepatic stellate cells (HSCs); RA enhances plasminogen activator/plasmin levels and thereby induces proteolytic activation of latent transforming growth factor-beta (TGF-beta), a strong fibrogenic cytokine, resulting in enhanced collagen production. We have developed a protease inhibitor, camostat mesilate, that suppresses TGF-beta activation and thereby inhibits the transformation of HSCs, leading to reduced matrix production by the cells. The compound is effective not only in preventing but also in reducing hepatic fibrosis in rats when administered orally. HCC is refractory to RA due to its local depletion in the tumors and also due to malfunction of its nuclear receptor, retinoid X receptor-alpha (RXRalpha) Oral supplementation of a synthetic retinoid named acyclic retinoid led to the disappearance of serum lectin-reactive alpha-fetoprotein (AFP-L3) and subsequently suppressed posttherapeutic recurrence of HCC in cirrhotic patients. These results suggest eradication of AFP-L3-producing latent malignant clones from the liver by the retinoid. We propose the concept of "clonal deletion" therapy for cancer chemoprevention, a new category of cancer chemotherapy.