PubMed · 9410958
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M Okuma. 1997-09-10. [Platelet dysfunction--from bedside clinical study to molecular study].. https://pubmed.ncbi.nlm.nih.gov/9410958/
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The platelet type 12 lipoxygenase (12-LOX) adds molecular oxygen to C-12 arachidonic acid to yield 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid. It has been suggested that 12-LOX and its metabolites play an important role in tumor angiogenesis. A hammerhead ribozyme (Rz) targeted to the first GUC site within the 12-LOX mRNA was designed and cloned into an in vitro transcriptional or mammalian expression vector. In vitro, the Rz was able to cleave its substrate efficiently in a time-dependent manner. Under multiple turnover conditions, the Rz performed well at 37 degrees C, with a further improvement at 50 degrees C. When cloned into a mammalian expression vector, pSV2neo, the Rz construct efficiently decreased the level of 12-LOX mRNA in stably transfected human erythroleukemia cells to levels that were undetectable by Northern blot analyses. 12-LOX enzyme activity assays showed that Rz significantly reduced the 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid production in human erythroleukemia cells; this effect was sustained for up to 6 months in cell culture. The Rz developed in this study may represent a powerful tool for potential applications, ranging from an understanding of tumor angiogenesis to cancer gene therapy.
Regulation of arachidonate metabolism in human epidermoid carcinoma A431 cells by phospholipid hydroperoxide glutathione peroxidase (PHGPx) and cytosolic glutathione peroxidase (GPx1) was studied. In order to study the effect of reduced glutathione (GSH) on the catalysis regulation of these oxygenation enzymes, diethyl maleate was used to deplete the intracellular GSH. In the presence of 13-hydroperoxyoctadecadienoic acid, the enzymatic catalysis of cyclooxygenase and 12-lipoxygenase was significantly increased in the GSH-depleted cells. In terms of the inhibitory effect on 12-lipoxygenase, PHGPx was more sensitive to GSH concentrations than GPx1. Inhibition of PHGPx activity by the treatment of cells with antisense oligonucleotide of PHGPx mRNA increased the enzymatic catalysis of both cyclooxygenase and 12-lipoxygenase. In conclusion, the results indicate that catalysis of cyclooxygenase and 12-lipoxygenase in A431 cells was regulated by redox-reaction, and PHGPx seems to play an important role in the controlling of these reactions.
The role of mitogen-activated protein kinase signaling and the transcription factor c-Jun in epidermal growth factor (EGF)-induced expression of 12-lipoxygenase in human epidermoid carcinoma A431 cells was studied. EGF increased the activation of extracellular signal-regulated kinase (ERK) and c-Jun amino terminal kinase (JNK) in a time-dependent manner. Treatment of the cells with an mitogen-activated protein kinase kinase inhibitor, PD098059 (30 microM), inhibited the EGF- and pSV2ras-induced expression of 12-lipoxygenase mRNA. Transfection of the cells with Ras, ERK2, Rac, JNK dominant negative mutants pMMrasDN, K52R ERK2, RacN17, and mJNK all inhibited the EGF-induced promoter activation of the 12-lipoxygenase gene. EGF induced the expression of c-Jun and the activity of transcription factor activator protein 1 in cells, and these effects were blocked by the treatment with K52R ERK2 and mJNK. Overexpression of c-Jun increased the expression of 12-lipoxygenase mRNA and enzyme activity. Furthermore, the Sp1-binding sites in the promoter region of the 12-lipoxygenase gene were requisite for c-Jun response, which was similar to that previously observed in EGF response. The results indicate that the EGF-induced expression of 12-lipoxygenase in A431 cells was mediated through the Ras-ERK and Ras-Rac-JNK signal pathways. Subsequent induction of c-Jun led by ERK and JNK activation was essential for this EGF response.