Journal of Biomedical Science

, Volume 9, Issue 5, pp 453–459 | Cite as

Inhibition of arachidonate metabolism in human epidermoid carcinoma A431 cells overexpressing phospholipid hydroperoxide glutathione peroxidase

  • Ching-Jiunn Chen
  • Huei-Sheng Huang
  • Wen-Chang Chang
Original Paper

Abstract

Phospholipid hydroperoxide glutathione peroxidase (PHGPx), a selenium-dependent glutathione peroxidase, can interact with lipophilic substrates, including phospholipid hydroperoxides, fatty acid hydroperoxides and cholesterol hydroperoxides, and can reduce them to hydroxide compounds. It also seems to be a major regulator of lipid oxygenation in human epidermoid carcinoma A431 cells. In order to study the functional role of PHGPx in the regulation of 12-lipoxygenase and cyclooxygenase, cDNA of PHGPx was inserted into pcDNA3.1/His, and a plasmid designated as S4 with the His-tag sequence inserted between PHGPx and its 3′-untranslated region was constructed. A number of stable transfectants of A431 cells that could express the tag-PHGPx were generated using plasmid S4. Using an intact cell assay system, the metabolism of arachidonic acid to prostaglandin E2 significantly decreased in stable transfectants of overexpressing PHGPx compared to that in a vector control cell line. If the intact cell assay was carried out in the presence of 13-hydroperoxyoctadecadienoic acid as a stimulator of lipid peroxidation, formation of 12-hydroxyeicosatetraenoic acid from arachidonic acid also significantly decreased in stable transfectants of overexpressing PHGPx compared to that in a vector control cell line, indicating that PHGPx could downregulate the 12-lipoxygenase activity in cells. These results support the hypothesis that PHGPx plays a pivotal role in the regulation of arachidonate metabolism in A431 cells.

Key Words

Phospholipid hydroperoxide glutathione peroxidase Cyclooxygenase 12-Lipoxygenase A431 cells 

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References

  1. 1.
    Chang WC, Nakao J, Orimo H, Murota S. Effects of reduced glutathione on the 12-lipoxygenase pathways in rat platelets. Biochem J 202:771–776;1982.Google Scholar
  2. 2.
    Chang WC, Ning CC, Lin MT, Huang JD. Epidermal growth factor enhances a microsomal 12-lipoxygenase activity in A431 cells. J Biol Chem 267:3657–3666;1992.Google Scholar
  3. 3.
    Chen CJ, Huang HS, Lee YT, Yang CY, Chang WC. Characterization and purification of a lipoxygenase inhibitor in human epidermoid carcinoma A431 cells. Biochem J 327:193–198;1997.Google Scholar
  4. 4.
    Chen CJ, Huang HS, Lin SB, Chang WC. Regulation of cyclooxygenase and 12-lipoxygenase catalysis by phospholipid hydroperoxide glutathione peroxidase in A431 cells. Prostaglandins Leukot Essent Fatty Acids 62:261–268;2000.Google Scholar
  5. 5.
    Chu FF, Doroshow JH, Esworthy RS. Expression, characterization, and tissue distribution of a new cellular selenium-dependent glutathione peroxidase, GSHPx-GI. J Biol Chem 268:2571–2576;1993.Google Scholar
  6. 6.
    Esworthy RS, Doan K, Doroshow JH, Chu FF. Cloning and sequencing of the cDNA encoding a human testis phospholipid hydroperoxide glutathione peroxidase. Gene 144:317–318;1994.Google Scholar
  7. 7.
    Ghyselinck NB, Dufaure JP. A mouse cDNA sequence for epididymal androgen-regulated proteins related to glutathione peroxidase. Nucleic Acids Res 18:7144;1990.Google Scholar
  8. 8.
    Huang HS, Chen CJ, Lu HS, Chang WC. Identification of a lipoxygenase inhibitor in A431 cells as a phospholipid hydroperoxide glutathione peroxidase. FEBS Lett 424:22–26;1998.Google Scholar
  9. 9.
    Imai H, Narashima K, Arai M, Sakamoto H, Chiba N, Nakagawa Y. Suppression of leukotriene formation in RBL-2H3 cells that overexpressed phospholipid hydroperoxide glutathione peroxidase. J Biol Chem 273:1990–1997;1998.Google Scholar
  10. 10.
    Imai H, Sumi D, Hanamoto A, Arai M, Sugiyama A. Molecular cloning and functional expression of a cDNA for rat phospholipid hydroperoxide glutathione peroxidase: 3′-Untranslated region of the gene is necessary for functional expression. J Biochem 118:1061–1067;1995.Google Scholar
  11. 11.
    Kozak M. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44:283–292;1986.Google Scholar
  12. 12.
    Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275;1951.Google Scholar
  13. 13.
    Maddipati KR, Gasparski C, Marnett LJ. Characterization of the hydroperoxide-reducing activity of human plasma. Arch Biochem Biophys 254:9–17;1987.Google Scholar
  14. 14.
    Maiorino M, Gregolin C, Ursini F. Phospholipid hydroperoxide glutathione peroxidase. Methods Enzymol 186:448–457;1990.Google Scholar
  15. 15.
    Marshall PJ, Kulmacz RJ, Lands WE. Constraints on prostaglandin biosynthesis in tissues. J Biol Chem 262:3510–3517;1987.Google Scholar
  16. 16.
    Nelson MJ, Chase DB, Seitz SP. Photolysis of ‘purple’ lipoxygenase: Implications for the structure of the chromophore. Biochemistry 34:6159–6163;1995.Google Scholar
  17. 17.
    Pushpa-Rekha TR, Burdsall AL, Oleksa LM, Chisolm GM, Driscoll DM. Rat phospholipid-hydroperoxide glutathione peroxidase. cDNA cloning and identification of multiple transcription and translation start sites. J Biol Chem 270:26993–26999;1995.Google Scholar
  18. 18.
    Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra WG. Selenium: Biochemical role as a component of glutathione peroxidase. Science 179:588–590;1973.Google Scholar
  19. 19.
    Sakamoto H, Imai H, Nakagawa Y. Involvement of phospholipid hydroperoxide glutathione peroxidase in the modulation of prostaglandin D2 synthesis. J Biol Chem 275:40028–40035;2000.Google Scholar
  20. 20.
    Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, ed 2. Cold Spring Harbor, Cold Spring Harbor Laboratory, 1989.Google Scholar
  21. 21.
    Schnurr K, Belkner J, Ursini F, Schewe T, Kuhn H. The selenoenzyme phospholipid hydroperoxide glutathione peroxidase controls the activity of the 15-lipoxygenase with complex substrates and preserves the specificity of the oxygenation products. J Biol Chem 271:4653–4658;1996.Google Scholar
  22. 22.
    Schnurr K, Borchert A, Gerth C, Anton M, Kuhn H. Bacterial and nonbacterial expression of wild-type and mutant human phospholipid hydroperoxide glutathione peroxidase and purification of the mutant enzyme in the milligram scale. Protein Expr Purif 19:403–410;2000.Google Scholar
  23. 23.
    Thomas JP, Maiorino M, Ursini F, Girotti AW. Protective action of phospholipid hydroperoxide glutathione peroxidase against membrane-damaging lipid peroxidation. In situ reduction of phospholipid and cholesterol hydroperoxides. J Biol Chem 265:454–461;1990.Google Scholar
  24. 24.
    Ursini F, Maiorino M, Gregolin C. The selenoenzyme phospholipid hydroperoxide glutathione peroxidase. Biochim Biophys Acta 839:62–70;1985.Google Scholar
  25. 25.
    Yamamoto S. ‘Enzymatic’ lipid peroxidation: Reactions of mammalian lipoxygenases. Free Radic Biol Med 10:149–159;1991.Google Scholar

Copyright information

© National Science Council 2002

Authors and Affiliations

  • Ching-Jiunn Chen
    • 2
  • Huei-Sheng Huang
    • 1
  • Wen-Chang Chang
    • 2
  1. 1.Department Medical Technology, College of MedicineNational Cheng Kung UniversityTainanTaiwan, ROC
  2. 2.Department of Pharmacology College of MedicineNational Cheng Kung UniversityTainanTaiwan (ROC)

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