A recombinant MnSOD (rMnSOD) synthesized by specific cDNA clones derived from a liposarcoma cell line was shown to have the same sequence as the wild-type MnSOD expressed in the human myeloid leukaemia cell line U937, except for the presence of the leader peptide at the N-terminus. These results were fully confirmed by the molecular mass of rMnSOD as evaluated by ES/MS analysis (26662.7 Da) and the nucleotide sequence of the MnSOD cDNA. The role of the leader peptide in rMnSOD was investigated using a fluorescent and/or ⁶⁸Gallium-labelled synthetic peptide. The labelled peptide permeated MCF-7 cells and uptake could be inhibited in the presence of an excess of oestrogen. In vivo it was taken up by the tumour, suggesting that the molecule can be used for both therapy and diagnosis. The in vitro and in vivo pharmacology tests confirmed that rMnSOD is only oncotoxic for tumour cells expressing oestrogen receptors. Pharmacokinetic studies in animals performed with ¹²⁵I- and ¹³¹I-labelled proteins confirmed that, when administered systemically, rMnSOD selectively reached the tumour, where its presence was unambiguously demonstrated by scintigraphic and PET scans. PCR analysis revealed that Bax gene expression was increased and the Bcl2 gene was down regulated in MCF7 cells treated with rMnSOD, which suggests that the protein induces a pro-apoptotic mechanism. © 2008 Wiley-Liss, Inc.

References

1 Bishop JM. The molecular genetics of cancer. Science 1987; 235: 305–11.
10.1126/science.3541204
CAS PubMed Web of Science® Google Scholar
2 Rabbitts TH. Chromosomal translocations in human cancer. Nature 1994; 372: 143–9.
10.1038/372143a0
CAS PubMed Web of Science® Google Scholar
3 Mitelman F,Mertens F,Johansson B. A breakpoint map of recurrent chromosomal rearrangements in human neoplasia. Nat Genet 1997; 15: 417–74.
10.1038/ng0497supp-417
CAS PubMed Web of Science® Google Scholar
4 Doll R,Peto R. The causes of cancer: quantitative estimates of avoidable risks of cancer in the United States today. J Natl Cancer Inst 1981; 66: 1191–308.
10.1093/jnci/66.6.1192
CAS PubMed Web of Science® Google Scholar
5 Sun J,Hemler ME. Regulation of MMP-1 and MMP-2 production through CD147/extracellular matrix metalloproteinase inducer interactions. Cancer Res 2001; 61: 2276–81.
CAS PubMed Web of Science® Google Scholar
6 Folkman J. Tumor angiogenesis: a possible control point in tumor growth. Ann Intern Med 1975; 82: 96–100.
10.7326/0003-4819-82-1-96
CAS PubMed Web of Science® Google Scholar
7 Konopka TE,Barker JE,Bamford TL,Guida E,Anderson RL,Stewart AG. Nitric oxide synthase II gene disruption: implications for tumor growth and vascular endothelial growth factor production. Cancer Res 2001; 61: 3182–7.
CAS PubMed Web of Science® Google Scholar
8 Arbiser JL,Petros J,Klafter R,Govindajaran B,McLaughlin ER,Brown LF,Cohen C,Moses M,Kilroy S,Arnold RS,Lambeth JD. Reactive oxygen generated by Nox1 trigger the angiogenic switch. Proc Natl Acad Sci USA 2002; 99: 715–20.
10.1073/pnas.022630199
CAS PubMed Web of Science® Google Scholar
9 Mancini A,Garbi C,D'Armiento FP, Borrelli A,Ambesi-Impiombato FS. Culture and cloning of an adipocytes cell line froma human liposarcoma. Bollettino Istituto dei Tumori di Napoli 1991; 38: 43–49.
Google Scholar
10 Mancini A,Borrelli A,Schiattarella A,Fasano S,Occhiello A,Pica A,Sehr P,Tommasino M,Nüesch JP,Rommelaere J. Tumor suppressive activity of a variant isoform of manganese superoxide dismutase released by a human liposarcoma cell line. Int J Cancer 2006; 119: 932–43.
10.1002/ijc.21904
CAS PubMed Web of Science® Google Scholar
11 McCord JM,Fridovich I. Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J Biol Chem 1969; 244: 6049–55.
10.1016/S0021-9258(18)63504-5
CAS PubMed Web of Science® Google Scholar
12 Folz RJ,Guan J,Seldin MF,Oury TD,Enghild JJ,Crapo JD. Mouse extracellular superoxide dismutase: primary structure, tissue-specific gene expression, chromosomal localization, and lung in situ hybridization. Am J Respir Cell Mol Biol 1997; 17: 393–403.
10.1165/ajrcmb.17.4.2826
CAS PubMed Web of Science® Google Scholar
13 Ookawara T,Kizaki T,Yakayama E,Imazeki N,Matsubara O,Ikeda Y,Suzuki K,Li Ji L,Tadakuma T,Taniguchi N,Ohno H. Nuclear translocation of extracellular superoxide dismutase. Biochem Biophys Res Comm 2002; 296: 54–61.
10.1016/S0006-291X(02)00804-5
CAS PubMed Web of Science® Google Scholar
14 Borgstahl GE,Parge HE,Hickey MJ,Beyer WF,Jr,Hallewell RA,Tainer JA. The structure of human mitochondrial manganese superoxide dismutase reveals a novel tetrameric interface of two 4-helix bundles. Cell 1992; 71: 107–18.
10.1016/0092-8674(92)90270-M
CAS PubMed Web of Science® Google Scholar
15 Davis CA,Hearn AS,Fletcher B,Bickford J,Garcia JE,Leveque V,Melendez JA,Silverman DN,Zucali J,Agarwal A,Nick HS. Potent anti-tumor effects of an active site mutant of human manganese-superoxide dismutase. Evolutionary conservation of product inhibition. J Biol Chem 2004; 279: 12769–76.
10.1074/jbc.M310623200
CAS PubMed Web of Science® Google Scholar
16 Wispe JR,Clark JC,Burhans MS,Kropp KE,Korfhagen TR,Whitsett JA. Synthesis and processing of the precursor for human mangano-superoxide dismutase. Biochim Biophys Acta 1989; 994: 30–6.
10.1016/0167-4838(89)90058-7
CAS PubMed Web of Science® Google Scholar
17 Corbau R,Salomon N,Rommeleare J,Nuesch JP. Phosphorylation of the viral non structural protein NS1 during MVMp infection of A9 cells. Virology 1999; 259: 402–15.
10.1006/viro.1999.9786
CAS PubMed Web of Science® Google Scholar
18 Holgate CS,Jackson P,Cowen PN,Bird CC. Immunogold-silver staining: new method of immunostaining with enhanced sensitivity. J Histochem Cytochem 1983; 31: 938–44.
10.1177/31.7.6189883
CAS PubMed Web of Science® Google Scholar
19 Stewart JM,Young JD. Solid phase peptide synthesis. Rockford, IL: Pierce Chemical, 1984.
Google Scholar
20 Matthias M,Hendrickson RC,Pandey RC. Analysis of proteins and proteosomes by mass spectrometry. Annu Rev Biochem 2001; 70: 437–73.
10.1146/annurev.biochem.70.1.437
PubMed Web of Science® Google Scholar
21 Wagner UG,Pattridge KA,Ludwig ML,Stallings WC,Werber MM,Oefner C,Frolow F,Sussman JL. Comparison of the crystal structures of genetically engineered human manganese superoxide dismutase and manganese superoxide dismutase from Thermus thermophilus: differences in dimer-dimer interaction. Protein Sci 1993; 2: 814–25.
10.1002/pro.5560020511
CAS PubMed Web of Science® Google Scholar
22 Mizuno K,Whittaker MM,Bächinger HP,Whittaker JW. Calorimetric studies on the tight binding metal interactions of Escherichia coli manganese superoxide dismutase. J Biol Chem 2004; 279: 27339–44.
10.1074/jbc.M400813200
CAS PubMed Web of Science® Google Scholar
23 Hsieh Y,Guan Y,Tu C,Bratt PJ,Angerhofer A,Lepock JR,Hickey MJ,Tainer JA,Nick HS,Silverman DN. Probing the active site of human manganese superoxide dismutase: the role of glutamine 143. Biochemistry 1998; 37: 4731–9.
10.1021/bi972395d
CAS PubMed Web of Science® Google Scholar
24 Mashiba H,Matsunaga K. Device for intracellular increase of oxygen free radicals and inhibition of MethA tumour cell proliferation: in vitro and in vivo studies. Int J Tissue React 1988; 10: 273–80.
CAS PubMed Web of Science® Google Scholar
25 Schimmel M,Bauer G. Proapoptotic and redox state-related signaling of reactive oxygen species generated by transformed fibroblasts. Oncogene 2002; 21: 5886–96.
10.1038/sj.onc.1205740
CAS PubMed Web of Science® Google Scholar
26 Mainous AG,III,Wells BJ,Koopman RJ,Everett CJ,Gill JM. Iron, lipids, and risk of cancer in the Framingham Offspring cohort Am J Epidemiol 2005; 161: 1115–22.
10.1093/aje/kwi131
PubMed Web of Science® Google Scholar
27 Wheeler MD,Smutney OM,Samulski RJ. Secretion of extracellular superoxide dismutase from muscle transduced with recombinant adenovirus inhibits the growth of B16 melanomas in mice. Mol Cancer Res 2003; 1: 871–81.
CAS PubMed Web of Science® Google Scholar
28 Mondola P,Annella T,Santillo M,Santangelo F,Mondola P,Annella T,Santillo M,Santangelo F. Evidence for secretion of cytosolic CuZn superoxide dismutase by Hep G2 cells and human fibroblasts. Int J Biochem Cell Biol 1996; 28: 677–81.
10.1016/1357-2725(96)00004-0
CAS PubMed Web of Science® Google Scholar
29 Janssen AM,Bosman CB,Kruidenier L,Griffioen G,Lamers CB,van Krieken JH,van de Velde CJ,Verspaget HW. Superoxide dismutases in the human colorectal cancer sequence. J Cancer Res Clin Oncol 1999; 125: 327–35.
10.1007/s004320050282
CAS PubMed Web of Science® Google Scholar
30 Janssen AM,Bosman CB,van Duijn W,Oostendorp-van de Ruit MM,Kubben FJ,Griffioen G,Lamers CB,van Krieken JH,van de Velde CJ,Verspaget HW. Superoxide dismutases in gastric and esophageal cancer and the prognostic impact in gastric cancer. Clin Cancer Res 2000; 6: 3183–92.
CAS PubMed Web of Science® Google Scholar
31 Punnonen K,Ahotupa M,Asaishi K,Hyöty M,Kudo R,Punnonen R. Antioxidant enzyme activities and oxidative stress in human breast cancer. J Cancer Res Clin Oncol 1994; 120: 374–7.
10.1007/BF01247464
CAS PubMed Web of Science® Google Scholar
32 Nishiura T,Suzuki K,Kawaguchi T,Nakao H,Kawamura N,Taniguchi M,Kanayama Y,Yonezawa T,Lizuka S,Taniguchi N. Elevated serum manganese superoxide dismutase in acute leukemias. Cancer Lett 1992; 62: 211–5.
10.1016/0304-3835(92)90098-G
CAS PubMed Web of Science® Google Scholar
33 Church SL,Grant JW,Ridnour RA,Oberlay LW,Swanson PE,Meltzer PS,Trent JM. Increased manganese superoxide dismutase expression suppresses the malignant phenotype of human melanoma cells. Proc Natl Acad Sci USA 1993; 90: 3113–17.
10.1073/pnas.90.7.3113
CAS PubMed Web of Science® Google Scholar
34 Hu Y,Rosen DG,Zhou Y,Feng L,Yang G,Liu J,Huang P. Mitochondrial manganese-superoxide dismutase expression in ovarian cancer: role in cell proliferation and response to oxidative stress. J Biol Chem 2005; 280: 39485–92.
10.1074/jbc.M503296200
CAS PubMed Web of Science® Google Scholar

Citing Literature

Volume123, Issue11

1 December 2008

Pages 2684-2695

Biophysical and biochemical characterization of a liposarcoma-derived recombinant MnSOD protein acting as an anticancer agent

Abstract

References

Citing Literature

References

Information

About Wiley Online Library

Help & Support

Opportunities

Connect with Wiley

Biophysical and biochemical characterization of a liposarcoma-derived recombinant MnSOD protein acting as an anticancer agent

Abstract

References

Citing Literature

References

Related

Information