Identification of a Novel Putative Non-selenocysteine Containing Phospholipid Hydroperoxide Glutathione Peroxidase (NPGPx) Essential for Alleviating Oxidative Stress Generated from Polyunsaturated Fatty Acids in Breast Cancer Cells

A dramatic reduction in the expression of a novel phospholipid hydroperoxide glutathione peroxidase (PHGPx), which incorporates cysteine instead of selenocysteine in the conserved catalytic motif was observed in a microarray analysis using cDNAs amplified from mRNA of Brca1-null mouse embryonic fibroblasts. This non-selenocysteine PHGPx named NPGPx is a cytoplasmic protein with molecular mass of ∼22 kDa and has little detectable glutathione peroxidase activity in vitro. Ectopic expression of NPGPx in Brca1-null cells that were sensitive to oxidative stress induced by hydrogen peroxide conferred a similar resistance level to that of the wild-type cells, suggesting the importance of this protein in reducing oxidative stress. Expression of NPGPx was found in many tissues, including developing mammary gland. However, the majority of breast cancer cell lines studied (11 of 12) expressed very low or undetectable levels of NPGPx irrespective of BRCA1 status. Re-expression of NPGPx in breast cancer lines, MCF-7 and HCC1937, which have very little or no endogenous NPGPx, induced resistance to eicosapentaenoic acid (an omega-3 type of polyunsaturated fatty acid)-mediated cell death. Conversely, inhibition of the expression of NPGPx by the specific small interfering RNA in HS578T breast cancer cells that originally express substantial amounts of endogenous NPGPx increased their sensitivity to eicosapentaenoic acid-mediated cell death. Thus, NPGPx plays an essential role in breast cancer cells in alleviating oxidative stress generated from polyunsaturated fatty acid metabolism. A dramatic reduction in the expression of a novel phospholipid hydroperoxide glutathione peroxidase (PHGPx), which incorporates cysteine instead of selenocysteine in the conserved catalytic motif was observed in a microarray analysis using cDNAs amplified from mRNA of Brca1-null mouse embryonic fibroblasts. This non-selenocysteine PHGPx named NPGPx is a cytoplasmic protein with molecular mass of ∼22 kDa and has little detectable glutathione peroxidase activity in vitro. Ectopic expression of NPGPx in Brca1-null cells that were sensitive to oxidative stress induced by hydrogen peroxide conferred a similar resistance level to that of the wild-type cells, suggesting the importance of this protein in reducing oxidative stress. Expression of NPGPx was found in many tissues, including developing mammary gland. However, the majority of breast cancer cell lines studied (11 of 12) expressed very low or undetectable levels of NPGPx irrespective of BRCA1 status. Re-expression of NPGPx in breast cancer lines, MCF-7 and HCC1937, which have very little or no endogenous NPGPx, induced resistance to eicosapentaenoic acid (an omega-3 type of polyunsaturated fatty acid)-mediated cell death. Conversely, inhibition of the expression of NPGPx by the specific small interfering RNA in HS578T breast cancer cells that originally express substantial amounts of endogenous NPGPx increased their sensitivity to eicosapentaenoic acid-mediated cell death. Thus, NPGPx plays an essential role in breast cancer cells in alleviating oxidative stress generated from polyunsaturated fatty acid metabolism. The complex interplay among genetic susceptibility, hormones, environmental carcinogens, and dietary consumption contributes to the complexity of breast cancer development, treatment, and prevention (1Ambrosone C.B. Antioxid. Redox Signal. 2000; 2: 903-917Crossref PubMed Scopus (165) Google Scholar, 2Rose D.P. Connolly J.M. Med. Oncol. Tumor Pharmacother. 1990; 7: 121-130PubMed Google Scholar). Hormone metabolism and environmental genotoxic agents generate reactive oxygen species (ROS) 1The abbreviations used are: ROS, reactive oxygen species; GPx, glutathione peroxidase; PHGPx, phospholipid hydroperoxide glutathione peroxidase; BRCA1, breast cancer susceptibility gene 1; MEF, mouse embryonic fibroblast; EPA, eicosapentaenoic acid methyl ester; PUFA, polyunsaturated fatty acid; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2H-tetrazolium, inner salt); siRNA, small interfering RNA; FCS, fetal calf serum; RT, reverse transcriptase; EGFP, enhanced green fluorescent protein; NPGPx, non-selenocysteine containing phospholipid hydroperoxide glutathione peroxidase. that may introduce genetic mutation and aberrant signal transduction that can lead to uncontrolled cell proliferation (3Thannickal V.J. Fanburg B.L. Am. J. Physiol. 2000; 279: L1005-L10028Crossref PubMed Google Scholar, 4Droge W. Physiol. Rev. 2002; 82: 47-95Crossref PubMed Scopus (7605) Google Scholar, 5Martindale J.L. Holbrook N.J. J. Cell. Physiol. 2002; 192: 1-15Crossref PubMed Scopus (1942) Google Scholar). Interestingly, many tumor susceptibility genes that have been linked to breast carcinogenesis such as p53, ATM, PTEN, BRCA1, and BRCA2 have either direct or indirect roles in cellular defense against oxidative stress as the result of excessive ROS production without concomitant up-regulation of antioxidants (6Barzilai A. Rotman G. Shiloh Y. DNA Repair (Amst.). 2002; 1: 3-25Crossref PubMed Scopus (316) Google Scholar, 7Tan M. Li S. Swaroop M. Guan K. Oberley L.W. Sun Y. J. Biol. Chem. 1999; 274: 12061-12066Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 8Lee S.R. Yang K.S. Kwon J. Lee C. Jeong W. Rhee S.G. J. Biol. Chem. 2002; 277: 20336-20342Abstract Full Text Full Text PDF PubMed Scopus (849) Google Scholar, 9Le Page F. Randrianarison V. Marot D. Cabannes J. Perricaudet M. Feunteun J. 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Omega-3 forms of PUFA such as eicosapentaenoic acid (EPA) and docosahexaenoic acid, which are highly enriched in fish oil, are known to inhibit proliferation of many different types of cancers (17Rose D.P. Connolly J.M. J. Natl. Cancer Inst. 1993; 85: 1743-1747Crossref PubMed Scopus (192) Google Scholar). Because EPA has been demonstrated to increase peroxisomal β-oxidation, which generates hydrogen peroxides (18Abdi-Dezfuli F. Froyland L. Thorsen T. Aakvaag A. Berge R.K. Breast Cancer Res. Treat. 1997; 45: 229-239Crossref PubMed Scopus (24) Google Scholar, 19Aarsland A. Lundquist M. Borretsen B. Berge R.K. Lipids. 1990; 25: 546-548Crossref PubMed Scopus (59) Google Scholar, 20Demoz A. Willumsen N. Berge R.K. Lipids. 1992; 27: 968-971Crossref PubMed Scopus (65) Google Scholar), the inhibitory effect of omega-3 PUFA has been attributed to the production of oxygen radicals and initiation of intracellular lipid peroxidation (21Stoll B.A. Br. J. 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It remains GPx contributes to the of oxidative stress generated from endogenous cellular and metabolism of have a novel GPx that a similar to The of cysteine instead of a conserved of the catalytic the non-selenocysteine phospholipid glutathione peroxidase or of NPGPx expression was found in the majority of breast cancer cell lines and of NPGPx expression in MCF-7 and breast cancer cells conferred resistance to cell death. Conversely, inhibition of the expression of NPGPx in HS578T breast cancer cells by the specific increased their sensitivity to eicosapentaenoic acid-mediated cell death. a role for NPGPx in alleviating oxidative stress induced by dietary consumption of fatty acids in breast cancer and and EPA were from EPA was in to yield a of were from and used to the mouse and NPGPx were from with and and to the cancer cell lines including and were from and were in with fetal calf for MCF-7 and HCC1937, which were in in A cell was in with embryonic Q. Chen C.F. Chen P.L. Lee W.H. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar) were in low with Expression and of NPGPx in NPGPx was in by with protein was from cellular by using a of the to was by the of by in the of the protein a by peroxidase activity was by using a as M. C. F. 1990; PubMed Scopus Google Scholar). were in the containing and glutathione and hydroperoxide or hydroperoxide the of containing or containing glutathione peroxidase glutathione peroxidase was used as a of NPGPx were with the and to a of The was by the of peroxidase activity was by the in using a with an of and microarray analysis was as by Xiao N. Wang W. L. J. R. K. R. R. Natl. S. A. 2002; PubMed Scopus Google Scholar). RNA was from and and used as the for cDNAs as for The microarray of genes Xiao N. Wang W. L. J. R. K. R. R. Natl. S. A. 2002; PubMed Scopus Google Scholar). was used to acid analysis was used to different of GPx analysis was used to the for fatty acid was from S. J.M. Cancer PubMed Scopus Google Scholar). breast cancer cells were into of for The was into for an The cells were in with different of EPA in and was by as the cells of mouse embryonic were into and with in The was for were and the of and cells is expressed as of of RNA for RNA was from either or cells as by the was as (12Zheng L. Pan H. Li S. Flesken-Nikitin A. Chen P.L. Boyer T.G. Lee W.H. Mol. Cell. 2000; 6: 757-768Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). The of used for specific gene is in for of of amplified and and and mouse mouse in a NPGPx was to a glutathione from acid to The protein was expressed and from for as to generate mouse the NPGPx protein was used as to generate the Y. Chen C.F. Chen P.L. Lee W.H. Cancer Res. Google Scholar, T. D. Lee W.H. J. Biol. 1994; PubMed Scopus Google Scholar). were used in P.L. Wang Lee W.H. Cell. Full Text PDF PubMed Scopus Google Scholar), and the as Y. Chen C.F. Chen P.L. Lee W.H. Cancer Res. Google Scholar, T. D. Lee W.H. J. Biol. 1994; PubMed Scopus Google Scholar). of the for NPGPx RNA was by in of an R.K. M. J. PubMed Google Scholar). The by the as a for NPGPx was and of The expression was with into cells, which expressed proteins and as A. K. T. Natl. S. A. 1994; PubMed Scopus Google Scholar). of NPGPx from to from the A of the initiation of the NPGPx was for the as J. W. A. K. T. 2001; PubMed Scopus Google Scholar, J. A. W. T. J. 2001; PubMed Scopus Google Scholar). of RNA were HS578T breast cancer cells were into for and with of NPGPx or using to the cells were the and cell were to by using mouse NPGPx cells were into of the of in the of different of EPA for the as of for genes by BRCA1, a microarray analysis using a containing genes with cDNAs amplified from the mRNA of and The that the expression of genes K. Cancer Res. 2002; Google Scholar), C. Y. H. A. Biol. Med. 2002; PubMed Scopus Google Scholar), Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar), and glutathione or were in Brca1-null Because of wild-type of has been to sensitivity to oxidative stress (11Gowen L.C. Avrutskaya A.V. Latour A.M. Koller B.H. Leadon S.A. Science. 1998; 281: 1009-1012Crossref PubMed Scopus (453) Google Scholar, Avrutskaya A.V. Leadon S.A. V. Koller B.H. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar), was further A of acid of this with other GPx family members to cytoplasmic and to GPx4 Three of glutathione peroxidase are conserved in including the of and as the catalytic of the However, incorporates cysteine instead of selenocysteine by an the conserved in the catalytic of GPx family members R. Free Radic. Biol. Med. 1999; 27: 951-965Crossref PubMed Scopus (883) Google Scholar). acid against GPx family members the of that are conserved among PHGPx from many species F. M. R. A. D. L. PubMed Scopus Google Scholar, A.M. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). is the reported PHGPx that cysteine instead of selenocysteine in the catalytic and is to as NPGPx containing phospholipid hydroperoxide glutathione to to the of NPGPx that the NPGPx is to and is on NPGPx is conserved and to acid and acid with NPGPx has may an signal of NPGPx and the NPGPx as for in and a with kDa was that the protein is from the generated specific using from either glutathione or NPGPx proteins expressed in for The in NPGPx protein can by the specific against NPGPx, not by The not endogenous cellular NPGPx in by analysis a similar of the protein was by direct analysis by the of the endogenous NPGPx is which the molecular mass of NPGPx different from the in The in the in and the endogenous NPGPx that NPGPx may in Because NPGPx is the PHGPx that cysteine instead of selenocysteine in the catalytic it remains to whether the protein has glutathione peroxidase NPGPx to by using a and glutathione peroxidase activity or no peroxidase activity was found this this result is with many that have been H. Y. Free Radic. Biol. Med. PubMed Scopus Google Scholar), an and further the cellular of NPGPx in was by with either or was in the with little or undetectable signal in the suggesting that NPGPx is a cytoplasmic Expression of GPx in Brca1-null the from the microarray analysis and to the expression of other GPx family on RNA from and in was a up-regulation of NPGPx mRNA expression in in to wild-type However, NPGPx mRNA was in Brca1-null the of other members of the GPx family such as and GPx4 were not with the the NPGPx protein expression was in wild-type and not in Brca1-null cells the in the acid among GPx family was highly specific and which were expressed in was a and NPGPx in found no to a direct role for in the expression of NPGPx not and the reduction of NPGPx expression in the Brca1-null may not a direct of Ectopic Expression of NPGPx of Brca1-null a to the of NPGPx very little or no endogenous have been to sensitive to Avrutskaya A.V. Leadon S.A. V. Koller B.H. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar). It is likely that of NPGPx into Brca1-null induced express NPGPx in used types of either or with NPGPx expression in Brca1-null with NPGPx expressed the protein a level with that of wild-type or Brca1-null cells with not express detectable NPGPx The expressed NPGPx the endogenous NPGPx in cytoplasmic as and the Brca1-null cells with little NPGPx expression not specific NPGPx were on cells to the effect of on cell of and wild-type with of Brca1-null cells or with the other resistance to was observed in Brca1-null cells with NPGPx suggesting that NPGPx is for reducing the effect of However, it was that the resistance to of NPGPx Brca1-null cells is not that of the wild-type or the level of NPGPx and in members of the glutathione have been and their have a distinct of R. Free Radic. Biol. Med. 1999; 27: 951-965Crossref PubMed Scopus (883) Google Scholar). the expression of NPGPx, using specific against and GPx4 and are the that express levels of and R. Free Radic. Biol. Med. 1999; 27: 951-965Crossref PubMed Scopus (883) Google Scholar). the other the expression of the NPGPx mRNA was in and mammary NPGPx protein was expressed in including the mammary gland. the expression of NPGPx in and mammary Interestingly, reduction of NPGPx mRNA and protein expression was in and mammary in to However, the level of GPx4 mRNA not mammary development, the level of mRNA was increased in that NPGPx is expressed and its expression level may mammary of NPGPx Expression in Breast Cancer is that an oxidative stress and breast carcinogenesis (1Ambrosone C.B. Antioxid. Redox Signal. 2000; 2: 903-917Crossref PubMed Scopus (165) Google Scholar). Breast cancers have been to a glutathione peroxidation M. T. A. N. M. D. Cancer 2000; PubMed Scopus Google Scholar, M. T. A. N. M. D. J. Cancer Res. Oncol. 2001; PubMed Scopus Google Scholar). a of breast cancer cell lines for the expression of in of the breast cancer cell lines of their BRCA1 expressed little or no NPGPx protein NPGPx was in mammary cells by expressed not of the breast cancer cell lines studied expressed and not NPGPx, were expressed in the majority of breast cancer cell lines as by analysis with that breast cancer cell lines that expressed undetectable levels of NPGPx a signal NPGPx was in the of HS578T This result that breast cancer cells may sensitive to lipid peroxidation and NPGPx may have roles in breast cancer NPGPx for the of in Breast Cancer of the of the motif the NPGPx and PHGPx it is likely that NPGPx may essential for reducing the oxidative stress generated by specific polyunsaturated fatty this breast cancer cell lines levels of NPGPx, which wild-type BRCA1, and HCC1937, which BRCA1 were with or NPGPx cells with NPGPx NPGPx protein which was not in cells or cells with the NPGPx and and MCF-7 cells with eicosapentaenoic acid, an omega-3 fatty acid (17Rose D.P. Connolly J.M. J. Natl. Cancer Inst. 1993; 85: 1743-1747Crossref PubMed Scopus (192) Google Scholar, B.A. Br. J. Nutr. 2002; 87: 193-198Crossref PubMed Google Scholar). in expression of NPGPx in and MCF-7 cells conferred increased resistance to the effects of eicosapentaenoic acid that of cells without This result the that NPGPx is essential for reducing the oxidative stress generated by specific polyunsaturated fatty further this HS578T breast cancer cells that expressed detectable amounts of endogenous NPGPx with NPGPx Expression of NPGPx protein was by analysis using in the the expression of NPGPx protein for the gene little or no The cells were with of the expression of NPGPx in HS578T breast cancer cells by the specific NPGPx increased its sensitivity to eicosapentaenoic cell the no effect that expression of NPGPx in breast cancer cells plays a role in mediating the effect by a novel type of PHGPx, named NPGPx, which was microarray studies gene in and mouse embryonic fibroblasts. NPGPx is the of non-selenocysteine PHGPx and is Interestingly, a majority of breast cancer cells not express Expression of NPGPx effects of hydroperoxide as as effects of omega-3 type of that NPGPx may a gene of is a of gene and of to S.G. 2000; PubMed Scopus Google Scholar, G. Mol. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). However, expression of wild-type in Brca1-null by not result in the of NPGPx mRNA and Brca1-null either mouse or not detectable expression of expression of not the activity of the NPGPx of a and of linked to a gene in a not the of detectable NPGPx expression in Brca1-null cells may attributed to the indirect effect of PHGPx is a specific of GPx which is to complex lipid peroxides in to hydroperoxide substrates R. Free Radic. Biol. Med. 1999; 27: 951-965Crossref PubMed Scopus (883) Google Scholar). The PHGPx was GPx4 R.S. K. (Amst.). 1994; PubMed Scopus Google Scholar, H. D. 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