Tumor suppressive activity of a variant isoform of manganese superoxide dismutase released by a human liposarcoma cell line

Abstract

A cell line derived from a pleiomorphic liposarcoma, named LSA, was previously reported to secrete (a) factor(s) exhibiting oncotoxic properties. The present article describes the isolation, purification and sequence analysis of a protein released by LSA cells into conditioned culture medium. This protein proved to be a variant isoform of manganese superoxide dismutase (MnSOD), hence its designation as LSA-type-MnSOD. This LSA-type-SOD differed from conventional SODs in its secretion by producer cells, contrasting with the normal localization of SODs in the mitochondrial matrix. Interestingly, during the protein purification process, LSA-type-SOD cosegregated with a cytotoxic activity directed against a number of tumor cell lines, as determined under in vitro conditions. This cytopathic effect was most likely due to LSAtype-SOD, since it could be fully reproduced using recombinant SOD that was expressed from cDNA clones isolated from LSA cells mRNA preparations and henceforth designated L-rSOD. In addition to its manifestation in cell lines kept in tissue culture, the oncotoxicity of LSA-type-SOD was further reflected in a remarkable capacity of this protein for suppression of mammary tumors in Balb-C-FR III mice. Animals subcutaneously injected with L-rSOD in the tumor area showed a complete disruption of established mammary carcinomas, as monitored by nuclear magnetic resonance (NMR) scanning. Moreover, metastatic spreading, which was readily detected in the control group, was suppressed in the treated animals. Altogether these data suggest that LSA-type-SOD interferes with survival and spreading of neoplastically transformed cells and deserves to be future validated as a therapeutic agent against cancer, either alone or in combination with conventional treatments. ' 2006 Wiley-Liss, Inc.

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

“…14 The LSA-type MnSOD, in fact, is secreted by LSA cells whereas native MnSOD is located in the mitochondrial matrix. 15 The molecular weight of the LSAtype MnSOD is about 30 kDa, as estimated by SDS-PAGE, 10 which is significantly higher than the value of 24 kDa calculated for human native MnSOD. 16 Finally, and more importantly, when injected in vitro or in vivo, the LSA-type MnSOD permeates and selectively kills tumour cells, provided that they express oestrogen receptors and low levels of catalase.…”

“…Start material (LSA-CM) was prepared as previously described 10 and then concentrated 12-fold by ultrafiltration through Amicon filters to a cut-off point of 3,000 Da. Proteins were fractioned by discontinuous 12% SDS-PAGE and blotted onto nitrocellulose membranes according to Ref.…”

“…10 The LSA-secreted proteins were fractionated by SDS-PAGE and the protein band likely corresponding to the MnSOD was excised from the gel, digested in situ with trypsin and identified by using LC-MS/MS methods. 20 Once the exact electrophoretic mobility of LSA-type MnSOD was established, a peptide map of the secreted protein was constructed by tryptic digestion of the corresponding gel band.…”

“…9 Among these, a novel isoform of manganese superoxide dismutase (LSA-type MnSOD) was recently identified. 10 In addition to the enzymatic activity common to all SODs directed to transform free radicals into hydrogen peroxide, [11][12][13] the LSA-type MnSOD showed peculiar structural and functional properties that are different from the corresponding native MnSOD molecule expressed in the human myeloid leukaemia cell line U937 (Swiss Prot. Code P04179).…”

“…In contrast, normal cells are not affected by the protein, making the cytotoxic activity of LSAtype MnSOD specific for tumour cells. 10 Therefore, a recombinant form of LSA-type MnSOD (rMnSOD) was produced from specific cDNA clones and demonstrated to retain both the structural and oncotoxic properties of the native protein. 10 In this article we characterize the biophysical and biochemical properties of rMnSOD and report on its pharmacokinetic properties and biodistribution in vivo.…”