Cytotoxic activity of hydrolyzable tannins against human oral tumor cell lines--a possible mechanism.

Hydrolyzable tannins showed higher cytotoxic activity against human oral squamous cell carcinoma and salivary gland tumor cell lines than against normal human gingival fibroblasts, whereas gallic acid, a component unit of tannins, showed much weaker selective cytotoxicity. The cytotoxic activity of dimeric compounds was generally higher than that of monomeric compounds. Macrocyclic ellagitannin oligomers, such as oenothein B, woodfordin C and woodfordin D showed the greatest cytotoxic activity, and their activity (per given number of molecules) was one order higher than those of gallic acid and epigallocatechin gallate, a major component of green tea. These compounds induced apoptotic cell death characterized by DNA fragmentation (as demonstrated by the TUNEL method) and cleavage of cytokeratin 18 by activated caspase(s) (as demonstrated by M30 monoclonal antibody). ESR spectroscopy revealed that these macrocyclic compounds at higher concentrations produced their own radicals and significantly enhanced the radical intensity of sodium ascorbate, possibly by their prooxidant actions. Catalase failed to eliminate their apoptosis-inducing activity, reducing the possibility of the involvement of hydrogen peroxide production in the extracellular fraction. These observations suggested that the antitumor activity of macrocyclic ellagitannin oligomers reported previously might be explained by their apoptosis-inducing activity.

Induction of apoptosis by flavones, flavonols (3-hydroxyflavones) and isoprenoid-substituted flavonoids in human oral tumor cell lines.

Various flavones, flavonols (3-hydroxyflavones) and isoprenoid-substituted flavones (flavonols) were investigated for their cytotoxic activity. Most of these compounds were more cytotoxic against human oral squamous cell carcinoma and salivary gland tumor cell lines than human gingival fibroblasts. The cytotoxic activity of flavonoids was generally higher than that of tannin-related compounds. Flavonoids induced apoptotic cell death characterized by DNA fragmentation (as identified by TUNEL method) and activation of caspase(s) (as identified by degradation products of cytokeratin 18 with M30 monoclonal antibody). ESR spectroscopy revealed that higher concentrations of flavonoids produced radicals under alkaline conditions. However, not all of them enhanced the radical intensity of sodium ascorbate, suggesting that the redox potential of flavonoids differs considerably from samples to samples. Catalase failed to eliminate the cytotoxic activity of flavonoids, reducing the possibility of the involvement of hydrogen peroxide for the cytotoxicity induction by them.

Alkalization produced by high-dose carbachol in HSG cell line is independent of Ca2+.

OBJECTIVES: The aim of this investigation was to clarify the mechanism of alkalization induced by carbachol in HSG cells. MATERIALS AND METHODS: Cells of the HSG cell line derived from a human submandibular gland adenocarcinoma and those of the A-431 human epidermoid carcinoma cell line were loaded with a fluorescent pH indicator, BCECF/AM, and the change in the intracellular pH of adherent cells and suspended ones were measured following stimulation with various concentrations (10(-7) M to 10(-2) M) of neurotransmitters (carbachol, noradrenaline, and isoproterenol). RESULTS: Isoproterenol did not cause alkalization of either cell type, whereas, noradrenaline and carbachol alkalized both types over the concentration ranges of 10(-6) M to 3 x 10(-3) M (HSG cell by noradrenaline), 10(-7) M to 2 x 10(-4) M (A-431 cell by noradrenaline), and 7 x 10(-5) M to 10(-4) M (A-431 cell by carbachol). On the other hand, alkalization induced by carbachol in the HSG cells was recognized at concentrations higher than 6 x 10(-5) M, and it showed no upper limit in terms of carbachol concentration. This high-dose carbachol alkalization was not eliminated by preincubation with nifedipine (100 microM), a Ca2+ channel blocker, or with thapsigargin (100 microM), a microsomal Ca(2+)-ATPase inhibitor. CONCLUSIONS: The alkalization system induced by carbachol in the HSG cell was quite different from that in the A-431 cell, and that induced by high-dose carbachol in HSG cells appeared to be independent of intracellular Ca2+. These findings will be useful to clarify the mechanism of salivary secretion stimulated by neurotransmitters.

Ginkgo biloba extract protects brain neurons against oxidative stress induced by hydrogen peroxide.

Effect of Ginkgo biloba extract was examined on dissociated rat cerebellar neurons suffering from oxidative stress induced by hydrogen peroxide using a flow cytometer and ethidium bromide. Hydrogen peroxide at a concentration of 3 mM increased the number of neurons stained with ethidium (presumably dead neurons) in a time-dependent manner. Pretreatment of neurons with G. biloba extract (10 micrograms/ml) greatly delayed a time-dependent increase in number of dead neurons during exposure to hydrogen peroxide. It was true, but less effective, in the case of treatment with G. biloba extract immediately or 60 min after start of oxidative stress. Results implicate G. biloba extract as a potential agent in protecting the neurons suffering from oxidative stress induced by hydrogen peroxide.

Ca(2+)-induced increase in oxidative metabolism of dissociated mammalian brain neurons: effect of extract of ginkgo biloba leaves.

Effect of an extract of Ginkgo biloba leaves (EGb) on oxidative metabolism was studied using rat brain neurons and 2',7'-dichlorofluorescin fluorescence. Ionomycin (100 nM to 1 microM), a Ca(2+)-ionophore, dose-dependently augmented the 2',7'-dichlorofluorescin fluorescence in the presence of external Ca2+, but not under the external Ca(2+)-free condition. Preincubation of neurons with EGb (3 micrograms/ml) greatly reduced the ionomycin-induced increase in 2',7'-dichlorofluorescin fluorescence. Results suggest that EGb may reduce the Ca(2+)-induced increase in the oxidative metabolism of brain neurons.

Flow cytometric estimation of the effect of Ginkgo biloba extract on the content of hydrogen peroxide in dissociated mammalian brain neurons.

The effect of Ginkgo biloba extract (GBE) on the content of hydrogen peroxide was estimated in cerebellar neurons dissociated from rats, by means of a flow-cytometer and 2',7'-dichlorofluorescein (DCF) diacetate, a fluorescent dye for intracellular hydrogen peroxide. The GBE started to reduce the DCF fluorescence of the neuron at 0.1 microgram/ml to 0.3 microgram/ml. Further increases in the GBE concentration (up to 3 micrograms/ml) produced a dose-dependent decrease in the DCF fluorescence, suggesting that GBE reduces the content of hydrogen peroxide or suppresses the reactive oxygen species (ROS) formation of cerebellar neurons. The present technique may be useful for preliminary evaluations of agents affecting the ROS formation in mammalian brain neurons.