Biophysical and biochemical characterization of a liposarcoma-derived recombinant MnSOD protein acting as an anticancer agent.
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 (68)Gallium-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 (125)I- and (131)I-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.
Mancini
Aldo
A
Department of Molecular Biology and Biotherapy, National Cancer Institute G. Pascale Naples, Naples, Italy. aldo_mancini@tiscali.it
Borrelli
Antonella
A
Schiattarella
Antonella
A
Aloj
Luigi
L
Aurilio
Michela
M
Morelli
Franco
F
Pica
Alessandra
A
Occhiello
Antonella
A
Lorizio
Roberto
R
Mancini
Roberto
R
Sica
Alessandro
A
Mazzarella
Lelio
L
Sica
Filomena
F
Grieco
Paolo
P
Novellino
Ettore
E
Pagnozzi
Daniela
D
Pucci
Piero
P
Rommelaere
Jean
J
D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000627:therapeutic use*; D001483:Base Sequence; D001669:Biochemical Phenomena; D001671:Biochemistry; D055592:Biophysical Phenomena; D001703:Biophysics; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D004958:Estradiol / Q000494:pharmacology; D006262:Health; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D008080:Liposarcoma / Q000188:drug therapy / Q000201:enzymology* / Q000473:pathology*; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D017421:Sequence Analysis; D021241:Spectrometry, Mass, Electrospray Ionization; D013482:Superoxide Dismutase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D023041:Xenograft Model Antitumor Assays
D000970:Antineoplastic Agents; D011994:Recombinant Proteins; D004958:Estradiol; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase