OBJECTIVEThis study measures the glutaredoxin (Grx) gene and protein expression in umbilical vein endothelial cells upon exposure to Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS). The involvement of the Akt-signaling pathway is also determined.

METHODSEA-hy926 cells were pretreated with 1,000 ng · mL⁻¹ P. gingivalis LPS for 4, 12, 18, and 24 h, and then real-time reverse transcription polymerase chain reaction was employed to detect Grx1 expression. The effect of Grx on Akt activity was investigated using Western blot for the control, LPS (1,000 ng · mL⁻¹ LPS), and carmus- tine (BCNU) groups (1,000 ng · mL⁻¹ LPS, and the EA-hy926 cells were pretreated with 25 μmol · ml⁻¹ BCNU for 30 min).

RESULTSGene expression of Grx1 significantly increased in LPS group compared with that in the control group. The Grx1 expression reached the peak level in 12 h, and the variation between the expression in 4 and 12 h was significant (P < 0.05). After 12 h, the protein levels of Grx and phosphorylated-Akt (p-Akt) significantly increased in the LPS group (P < 0.05), whereas the BCNU group showed a considerable decrease in both Grx and p-Akt expression levels (P < 0.05). Moreover, a slight difference was observed in the total Akt protein levels in the three groups (P > 0.05).

CONCLUSIONGrx expression increased upon exposure of EA-hy926 cells to the LPS. Akt activity could be inhibited by BCNU (a Grx inhibitor), which indicated that Akt might act as a downstream regulator of Grx.

Endothelial Cells ; Glutaredoxins ; genetics ; Humans ; Lipopolysaccharides ; pharmacology ; Oxidative Stress ; drug effects ; Phosphorylation ; Porphyromonas gingivalis ; pathogenicity ; Proto-Oncogene Proteins c-akt ; drug effects ; Signal Transduction ; drug effects ; Umbilical Veins

Holo glutaredoxin (Grx) is a homo-dimer that bridges a [2Fe-2S] cluster with two glutathione (GSH) ligands. In this study, both monothiol and dithiol holo Grxs are found capable of transferring their iron-sulfur (FeS) cluster to an apo ferredoxin (Fdx) through direct interaction, regardless of FeS cluster stability in holo Grxs. The ligand GSH molecules in holo Grxs are unstable and can be exchanged with free GSH, which inhibits the FeS cluster transfer from holo Grxs to apo Fdx. This phenomenon suggests a novel role of GSH in FeS cluster trafficking.
Circular Dichroism ; Dimerization ; Ferredoxins ; chemistry ; metabolism ; Glutaredoxins ; chemistry ; metabolism ; Glutathione ; metabolism ; Iron ; chemistry ; Ligands ; Magnetic Resonance Spectroscopy ; Sulfhydryl Compounds ; chemistry ; Sulfur ; chemistry ; Toluene ; analogs & derivatives ; chemistry

BACKGROUND: Reactive oxygen species are involved in multi -stage process of carcinogenesis. The most of cancer cell lines and cancer cells in tumor tissue produce reactive oxygen species and on the other hand, the activities of catalase, Mn - and CuZn-superoxide dismutase in tumor cells are usually low. These persistent oxidative stress in tumor tissue facilitates tumor invasion and metastasis. 12- kDa thioredoxin, which regulate the intracellular redox potential with glutathione and glutaredoxin is involved in cell activation, proliferation, differentiation and re dox- mediated apoptosis. It is also purified as 14 -kDa and 10- kDa eosinophilic cytotoxic enhancing factor(ECEF) from human histiocytic cell(U937) and 10 -kDa ECEF has more than 20 times eosinophilic stimulation activity than 14 - kDa ECEF. It has been reported that adult T-cell leukemia, squamous cell carcinoma of uterine cervix, and hepatocellular carcinoma show increased amounts of human thioredoxin and thioredoxin mRNA is increased in lung cancer. In this study, we investigated the expression of conventional antioxidant enzymes such as catalase, CuZn-SOD, and glutathione peroxidase and thioredoxin in lung cancer tissue compared to adjacent normal lung tissue and the induction of thioredoxin in macrophage cells after treatment of oxidative stress and endotoxin . METHODS: We measured the amount of conventional antioxidant enzymes such as catalase, CuZn-SOD, and glutathione peroxidase and thioredoxin in lung cancer tissue compared to adjacent normal lung tissue by immunoblot analysis and the induction of thioredo xin in mouse monocyte - macrophage cells(RAW 264.7) by treatment of 5 microM menadione and 1 microgram/ml endotoxin. RESULTS: On immunoblot analysis, the expression of 12 -kDa thioredoxin was increased in lung cancer tissue compared to paired normal lung tissue. but th e expression of catalase and CuZn-SOD were decreased in lung cancer tissue compared to paired normal tissue and the expression of glutathione peroxidase in lung cancer was variable. The expression of truncated thioredoxin was also increased in lung cancer. When mouse monocyte - macrophage cells were treated with 5 microM menadione and 1 microG/ml endotoxin, the expression of thioredoxin was peaked at 12 hrs and sustained to 48 hrs. CONCLUSION: In contrast with other conventional antioxidants, the expression of 12-kDa and truncated thioredoxin in lung cancer were increased and it is closely associated with persistent oxidative stress in tumor microenvironment. Considering especially the biological functions of truncated thioredoxin, the increased amount of truncated thioredoxin has significant role in tumor growth through cell proliferation.
Animals ; Antioxidants ; Apoptosis ; Carcinogenesis ; Carcinoma, Hepatocellular ; Carcinoma, Squamous Cell ; Catalase ; Cell Line ; Cell Proliferation ; Cervix Uteri ; Eosinophils ; Female ; Glutaredoxins ; Glutathione ; Glutathione Peroxidase ; Hand ; Humans ; Leukemia-Lymphoma, Adult T-Cell ; Lung Neoplasms* ; Lung* ; Macrophages ; Mice ; Monocytes ; Neoplasm Metastasis ; Oxidation-Reduction ; Oxidative Stress* ; Reactive Oxygen Species ; RNA, Messenger ; Thioredoxins* ; Tumor Microenvironment ; Vitamin K 3