Catalase contents in cells determine sensitivity to the apoptosis inducer gallic acid.

Gallic acid (3,4,5-trihydroxybenzoic acid, GA) is known to induce apoptosis in cancer cells at lower IC50 values compared with values for normal cells. Apoptosis is inhibited completely by the addition of conditioned medium from cultured hepatocytes, whereas it is not prevented by conditioned media from tumor cells. We therefore studied the reason for the different response to GA-induced apoposis. GA-induced dRLh-84 cell death was completely abolished by the addition of peroxisome or cytosol as well as conditioned medium from primary cultured rat hepatocyte. As GA-induced cell death is known to be mediated by reactive oxygen species (ROS) and intracellular Ca2+, we determined the type of ROS generated by GA and found that GA generated hydrogen peroxide in culture medium. The addition of hydrogen peroxide generated by GA induced cell death in dRLh-84 cells. These results suggest that GA-induced cell death is mediated by hydrogen peroxide. On the other hand, the inhibitory activity of hepatocyte medium on GA-induced cell death was completely abolished by anti-catalase antibody. When the amount of catalase antigen was determined by Western blotting analysis, conditioned medium and the cytoplasm of hepatocytes contained high concentrations of catalase. Conditioned media from various tumor cell lines did not contain catalase, and the cytoplasm contained only low levels of catalase. These results show that GA-sensitive cells, including various tumor cells, produce only small amounts of catalase and secreted little enzyme into media, suggesting a lack of protective machinery against GA. In contrast, GA-insensitive cells, including hepatocytes, produce large amounts of catalase and release it in medium, resulting in the development of insensitivity to GA. In conclusion, catalase contents in cells determine different sensitivity to GA.

Ca2+-Dependent caspase activation by gallic acid derivatives.

Gallic acid (GA) derivatives, 3,4-methylenedioxyphenyl 3,4,5-trihydroxybenzoate (GD-1) and S-(3,4-methylenedioxyphenyl)3,4,5-trihydroxythiobenzoate (GD-3), were previously reported to induce apoptosis in tumor cells with IC50s of 14.5 microm and 3.9 microm, respectively. To elucidate the mechanism by which these gallic acid derivatives (GDs) induce apoptosis, we studied whether GD-1 and GD-3 can activate caspases. When promyelocytic leukemia HL-60RG cells were treated with GD-1 and GD-3, poly(ADP-ribose)polymerase (PARP), a substrate of caspase-3, was cleaved into 85 kDa of degradative product with increasing incubation time. GA also activated PARP cleavage, which was inhibited by catalase, N-acetyl-L-cysteine (NAC), and intracellular Ca2+ chelator 1,2-bis(2-aminophenoxyethane)-N,N,N,N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM), in addition to a caspase inhibitor, Z-VAD-FMK. Its inhibitory pattern was identical with that of hypoxanthine/xanthine oxidase. On the other hand, GD-1- and GD3-induced PARP cleavage was not suppressed by catalase or NAC, but by BAPTA-AM. This suggested that the GD-elicited signaling pathway is different from GA's. Taken together, GDs activated caspase-3 following intracellular Ca2+ elevation independent of reactive oxygen species. Thus, it became evident that the signaling pathway leading to apoptosis was regulated by GDs in a different manner from GA.

Different generation of inhibitors against gallic acid-induced apoptosis produces different sensitivity to gallic acid.

Gallic acid (3,4,5-trihydroxybenzoic acid), a naturally occurring plant phenol, showed selective cytotoxicity against tumor cells with higher sensitivity than normal cells such as hepatocytes and keratinocytes. To elucidate the difference in sensitivity between normal and tumor cells to gallic acid, we studied whether the inhibitor of gallic acid-induced apoptosis existed or not. A serum-free conditioned medium, prepared from high density rat primary cultured hepatocytes and cytoplasm of hepatocytes, prevented gallic acid-induced apoptosis. In contrast, hepatomas and hepatic cell lines such as dRLh-84, PLC/PRF/5, HLE, and HUH and two other kinds of tumor cell, HeLa and KB, scarcely generated such an inhibitor in either their conditioned medium or their cells. Biochemical characterization of the inhibitors revealed that the inhibitor in the hepatocyte conditioned medium was completely inactivated by heating at 65 degrees C for 10 min. Its molecular weight was estimated at 150-250 kDa by gel filtration column chromatography, indicating that the inhibitor may be a protein-like substance. These results suggest that the generation of a large amount of the inhibitor may endow hepatocytes with insensitivity to gallic acid. In conclusion, the difference in the amount of the inhibitors generated by hepatocytes and tumor cells should contribute to the underlying mechanism in the difference in sensitivity of cells to gallic acid.

Role of reactive oxygen species in gallic acid-induced apoptosis.

We earlier demonstrated that gallic acid (3,4,5-trihydroxybenzoic acid) induced apoptosis in promyelocytic leukemia HL-60RG cells, which was inhibited by catalase and intracellular Ca2+ chelator. In this study, we further studied the involvement of reactive oxygen species (ROS) and intracellular Ca2+ in gallic acid-induced apoptosis. The enhancement of intracellular ROS in HL-60RG cells was detected dose-dependently as early as 5 min after stimulation with gallic acid by using 5,6-carboxy-2',7'-dichlorofluorescin diacetate (DCFH-DA). Further studies that used various antioxidants and ROS scavengers showed that the intracellular peroxide level was well correlated with the potency to induce apoptosis and that the increased intracellular peroxides after gallic acid treatment seemed likely to result from the influx of H2O2 derived from superoxide which were generated extracellularly. In addition, gallic acid, HX/XO, and H2O2-induced apoptosis was completely inhibited by pretreatment with intracellular Ca2+ chelator 1,2-bis(2-aminophenoxyethane)-N,N,N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM), but increase of intracellular peroxide levels by gallic acid were suppressed only slightly. It is suggested that intracellular ROS induced by gallic acid plays an important role in eliciting an early signal in apoptosis. Especially, H2O, which is derived from superoxide anion generated extracellularly may increase intracellular Ca2+ levels or cooperate with intracellular Ca2+, thus resulting in apoptosis induction.

Agrobacterium-mediated transformation of monocot and dicot plants using the NCR promoter derived from soybean chlorotic mottle virus.

The NCR promoter (PNCR) from soybean chlorotic mottle virus (SoyCMV) was used to express the selectable marker, neomycin phosphotransferase (nptII) gene, in Agrobacterium-mediated transformation of both monocot (rice) and dicot (tobacco) plants. A multi-cloning site for insertion of a gene of interest into the binary vector pTN is located proximal to the right border region of T-DNA. When chimeric genes under the control of other strong promoters were located in a head-to-head orientation to the PNCR-nptII gene, kanamycin-resistant tobacco shoots were generated more efficiently than when using the original pTN vectors. This suggests that the enhancer-like sequences in the promoters adjacent to PNCR may promote expression of the PNCR-nptII gene.

Cell death-inducing activity by gallic acid derivatives.

In this study, the cytotoxic activity of gallic acid derivatives (GDs) was studied using some cancer cell lines. Among them, 3,4-methylenedioxyphenyl 3,4,5-trihydroxybenzoate (GD-1) and S-(3,4-methylenedioxyphenyl)-3,4,5-trihydroxy-thiobenzoate (GD-3) were found to induce cell death in cancer cell lines with IC50s ranging from 2.9 to 114.4 microM, a concentration comparable with or lower than that of gallic acid. On the other hand, although gallic acid did not show any cytotoxicity against primary cultured rat hepatocytes and human keratinocytes, GD-1 and -3 showed slightly higher sensitivity against such normal cells, when compared with gallic acid. The cell death induced by gallic acid and GD-1 was accompanied by internucleosomal DNA fragmentation characteristic of apoptosis, whereas only smear DNA degradation was detected following GD-3 treatment. When the mechanism by which GD-1 and -3 caused cell death in HL-60RG cells was examined, GD-1 and -3-induced cell death was inhibited by the intracellular Ca2+ chelator, bis-(o-aminophenoxy)-N,N,N,N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM), calmodulin inhibitor, W-7, and the Ca2+/Mg2+ -dependent endonuclease inhibitor zinc sulfate. In contrast, catalase, N-acetylcysteine (NAC), and ascorbic acid inhibited gallic acid-induced apoptosis in HL-60RG cells, whereas they had no effect on GD-1- and -3-induced cell death. This result suggests that GD-1 and -3 induced cell death in a different manner to gallic acid. In conclusion, esterification of gallic acid with a 3,4-methylenedioxyphenyl group yielded potent agents to treat cancer with a different signaling pathway from gallic acid, although selectivity was lost.

Metabolic fate of gallic acid orally administered to rats.

The metabolic behavior of orally administered gallic acid was investigated by HPLC and 4-O-methyl gallic acid was found to be the main metabolite in rat peripheral blood and urine. After oral administration of gallic acid, maximum concentration in portal vein and inferior vena cava occurred at 15 and 30 min, respectively. In portal vein, gallic acid was preferentially detected relative to 4-O-methyl gallic acid, whereas gallic acid and 4-0-methyl gallic acid were equally detected in inferior vena cava. On the other hand, 4-O-methyl gallic acid but not gallic acid was found in liver. The contents of gallic acid and 4-O-methyl gallic acid in urine were nearly 100 times higher than those in blood. The ratio of 4-O-methyl gallic acid to total gallic acid metabolites in urine was from 0.55 to 0.76, indicating that a considerable amount of gallic acid was excreted without being metabolized. In this study we found that gallic acid administered orally existed in the blood for 6 h at most, and more than half was metabolized to 4-O-methyl gallic acid, followed by excretion into urine.

Interrelations between Carbon Dioxide and Ethylene on the Stimulation of Cocklebur Seed Germination.

Interrelations between CO(2) and C(2)H(4) on promotion of seed germination were examined in more detail at 23 degrees C with presoaked upper seeds of Xanthium pennsylvanicum Wallr. The germination-promoting effect of C(2)H(4) decreased gradually as its application time was delayed during a soaking period, whereas CO(2) was most promotive in application at 5 days of soaking, then its effect declined. CO(2) and C(2)H(4) were additive in earlier soaking periods and synergistic in later periods. Such changes in germination behavior in response to CO(2) and/or C(2)H(4) during a soaking period were closely associated with growth responsiveness of the axial tissues, but not of the cotyledonary ones. Growth responsiveness of axial tissues to CO(2) or C(2)H(4) disappeared finally during a soaking period, but their extinct responsiveness to any one of these gases was almost fully restored in the simultaneous presence of the other. The extinct responsiveness to CO(2) was partially recovered by a preexposure to C(2)H(4). This suggests that in the later period of soaking, unlike the case in a very early period of soaking, the C(2)H(4)-sensitive phase for seed germination precedes the CO(2)-sensitive phase in which CO(2) potentiated axial growth. The restoration of CO(2) responsiveness in axial growth occurred not only after C(2)H(4) treatment but also after exposure to 8 or 33 degrees C or after KCN treatment. Thus, secondarily dormant Xanthium seeds could germinate in response to CO(2) alone, when they were previously exposed for shortterms not only to C(2)H(4) but also 8 degrees C, 33 degrees C, or KCN.