Drastic effect of several caffeic acid derivatives on the induction of heme oxygenase-1 expression revealed by quantitative real-time RT-PCR.

Among antioxidative polyphenols, caffeic acid esters such as caffeic acid phenethyl ester (CAPE) and chlorogenic acid are contained in propolis, vegetables and coffee. In this study, we compared the efficacy of some polyphenols on the activation level of a cytoprotective heme oxygenase-1 (HO-1) gene in RAW264.7 mouse macrophage cells using quantitative real-time RT-PCR. The quantitative study revealed a variety of activation level of HO-1 gene by the chemicals. CAPE and caffeic acid ethyl ester (CAEE) at the final concentration of 2 M drastically activated the HO-1 gene to 39.2-fold and 20.1-fold, respectively. Curcumin, structurally related with caffeic acid and an element of turmeric, induced the HO-1 gene to 5.8-fold. In contrast, no activation was observed by other caffeic acid esters such as chlorogenic acid and rosmarinic acid,. Higher concentrations were necessary for the activation by an the antioxidant cysteamine and an electrophile diethyl maleate. Although the inducible activities of CAPE and chlorogenic acid were distinctly different, they showed similar reductive capacities when determined by cyclic voltammetry. These results show that the drastic activation of HO-1 gene by CAPE and CAEE is dependent upon their chemical structures, rather than the reductive activity of polyphenols, possibly reflecting the physiological effects of the nutritional elements

Drastic induction of heme oxygenase-1 expression by several caffeic acid derivatives revealed by quantitative real-time RT-PCR

Cytoprotective activities of polyphenols have been reported occasionally. Among the polyphenols, caffeic acid esters such as caffeic acid phenethyl ester and chlorogenic acid are contained in propolis, vegetables and coffee. In this study, we compared the efficacy of polyphenols on the activation level of heme oxygenase-1 (HO-1) gene, which is involved in the cellular protection system, in RAW264.7 cells using quantitative real-time RT-PCR. The quantitative study revealed that there is a variety in the activation level of HO-1 gene by the chemicals. Caffeic acid phenethyl ester (CAPE) and caffeic acid ethyl ester (CAEE) at the final concentration of 2 microM drastically activated the HO-1 gene 39.2-fold and 20.1-fold, respectively. Curcumin, structurally related with caffeic acid and an element of turmeric, induced HO-1 gene as 5.8-fold. In contrast, chlorogenic acid, another caffeic acid ester, induced HO-1 as a weak level (1.3-fold) at the same concentration. Higher concentrations were necessary for the activation by antioxidants such as cysteamine and resveratrol, and an electrophile diethyl maleate. When the content of HO-1 protein in cell lysates was assayed by enzyme immunoassay, it was confirmed that HO-1 protein accumulated remarkably in the CAPE-treated cells at 4 and 8 h. Although the inducible activities of CAPE and chlorogenic acid were distinctly different, they showed similar reductive capacities when determined by a cyclic voltammetry spectroscopy. These results show that the drastic activation of HO-1 gene by CAPE and CAEE is dependent upon their chemical structures, rather than the reductive activity of polyphenols, possibly reflecting the difference among the physiological effect of nutritional elements.20th IUBMB International Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress of Biochemistry and Molecular Biolog

Alteration of radioprotective effects of heat-killed Lactobacillus casei in X-irradiated C3H/He mouse related to blood level of proinflammatory cytokines by corticoids.

It is well known that a pre-administration of proinflammatory cytokines alters hematopoietic progenitor cells to promote an increase resistance against radiation and increases the survival rate in mice irradiated with lethal doses of radiation. Inflammation stimulators, such as some bacterial constituents, are also reported to have similar radioprotective action. We found that pre-administration of heat-killed Lactobacillus casei (HLC) to mice increases the level of interleukin (IL)-1 beta in circulation as well as the survival rate following lethal dose of radiation. Since HLC stimulates early immune responses, effects by drugs to modify inflammation were studied. The increase of both blood IL-1 beta levels and survival rates by HLC were simultaneously accelerated by coadministration of mineralocorticoid and inhibited by glucocorticoids or corticotropin. Neither parameter was modified by non-steroidal anti-inflammatory or anti-rheumatoid drugs. This suggests that both expected radioprotective action and unexpected systemic action, realized as an increase in plasma cytokines, by inflammation-related radioprotectors can be controlled by the coadministration of drugs at least in C3H/He mice, based on consideration of their pharmacological properties

Caffeic acid phenethyl ester and caffeic acid ethyl ester activate transcription of heme oxygenase-1 gene in RAW 264.7 mouse monocyte-macrophage cell line independently of redox-regulated pathway

Activation of the transcription of HO-1 gene by elements from foods was examined in a mouse monocyte-macrophage cell line RAW264.7 by measuring relative levels of HO-1 mRNA by real-time reverse transcription-polymerase chain reaction, and it was confirmed that caffeic acid phenethyl ester (CAPE), a component of propolis, had a distinguished potential to induce HO-1 gene expression in the macrophage tumor cell. When RAW cells were incubated with 2 mM of CAPE, the level of the mRNA of HO-1 gene was enhanced significantly even at 1 h, and reached a peak at 4 h leading to an drastic increase of 25 times, then was down-regulated at 6 h. A similar induction was observed in another mouse macrophage cell line, J774.1, but not in the human hepatocarcinoma cell line HepG2, indicating that the activation is common and specific to macrophages. Caffeic acid ethyl ester (CAEE) also induced transcription of HO-1 mRNA in RAW 264.7 cells, leading to 20 times activation. However, chlorogenic acid exhibited a little activation of HO-1 as 1.3 times although it is also an ester of caffeic acid. 2 mM of curcumin and ethyl ferulate increased HO-1 mRNA by 6.4 and 7.2 times, respectively. Resveratrol also elevated HO-1 mRNA 3.4 times, though at a 66 mM. When a non-phenolic antioxidant, cysteamine, was used, 10 mM of cysteamine was required to activate HO-1 gene as 2.3 times. To investigate whether this activation is dependent upon the reductive capacity of the chemicals, the one-electron oxidation potentials of these phenolic compounds were determined by the second-harmonic alternative current voltammetry. The E0ox values of CAPE (0.48), CAEE (0.49), and chlorogenic acid (0.48) determined from the intersection of the voltammograms were almost the same. However, the value of curcumin (0.42) was smaller than those three caffeic acid esters. Thus, the reductive capacity of curcumin was the most potent, and those of CAPE, CAEE, and chlorogenic acid were almost the same and were lower than that of curcumin. However, chlorogenic acid exhibited a very little activation of HO-1 gene, and curcumin was less potent than CAPE. From these results we suggest that the transcriptional activation of HO-1 gene by CAPE and CAEE is not dependent on a redox-regulated mechanism, but on a structure-specific interaction mediated by receptors.HO Conference 2005. 4th International Congress

Circadian Transitions in Radiation Dose-Dependent Augmentation of mRNA Levels for DNA Damage-induced Genes Elicited by Accurate real-time RT-PCR Quantification.

Molecular mechanisms of intracellular response after DNA-damage by exposure to ionizing radiation have been studied. In the case of cells isolated from living body of human and experimental animals, alteration of the responsiveness by physiological oscillation such as circadian rhythm must be considered. To examine the circadian variation in the response of p53-responsible genes p21, mdm2, bax, and puma, we established a method to quantitate their mRNA levels with high reproducibility and accuracy based on real-time RT-PCR and compared the levels of responsiveness in mouse hemocytes after diurnal irradiation to that after nocturnal irradiation. Augmentations of p21 and mdm2 mRNA levels with growth-arrest and of puma mRNA before apoptosis were confirmed by time-course experiment in RAW264.7, and dose-dependent increases in the peak levels of all the RNA were shown. Similarly, the relative RNA levels of p21, mdm2, bax, and puma per GAPDH also increased dose-dependently in peripheral blood and bone marrow cells isolated from whole-body-irradiated mice. Induction levels of all messages reduced by half after nighttime irradiation as compared with daytime irradiation in blood cells. In marrow cells, nighttime irradiation enhanced the p21 and mdm2 mRNA levels than daytime irradiation. No significant difference in bax or puma mRNA levels was observed between nighttime and daytime irradiation in marrow cells. This suggests that early-stage cellular responsiveness in DNA damage-induced genes is modulated between diurnal and nocturnal irradiation

Acceleration of Regeneration of Mucosa in Small Intestine Damaged by Ionizing Radiation using Anabolic Steroids.

Damage to intestine is a serious problem after accidental radiation. To examine substances to ameliorate damage by post-irradiation administration, we focused on the regeneration process following irradiation to intestine. Using experimental systems to examine the regeneration, effects of clinically used sex hormones were compared. An anabolic steroid, nandrolone (19-nortestosterone), stimulated proliferation in IEC-6 epithelial cells. Single injection of 19-nortestosterone ester with prolonged action to mice 24 h after abdominal irradiation at lethal dose of 15.7Gy showed significant life-saving effect. Regeneration indicators as microcolonies of BrdU-incorporated cells at Day 5 and c-myb mRNA expression levels at Day 4 were enhanced by 19-nortestosterone administration. In contrast, high concentration of estradiol inhibited growth of IEC-6. Introduction of estradiol ester to abdominally irradiated mice decreased levels of regeneration indicators and survival rate. These results suggest the effectiveness of anabolic steroid as well as the importance of manipulation of steroid receptors in the recovery of mucosa damaged by radiation