Cower Letters. 57 (1991) 199-202 Elsevier Scientific Publishers Ireland
Lowered superoxide melanoma cells
in highly metastatic
J.K. Kwee, E. Mitidieri and O.R. Affonso Centro
de Pesquisa Bcisica,
de CCIncer PraCa Cruz Vermelha, Rio de Janeiro (Brasil)
(Received 6 February 1991) (Accepted 11 February 1991)
Summary Superoxide dismutases (SOD), which are enzymes scavenging the superoxide radical, were studied in two variant lines of the B16 melanoma: B16Fl with low metastatic potential and B16FlO with high me&static potential. SOD activity was measured by a method utilizing reduction in the chemiluminescence of luminol. Using cell free extracts it was shown that the highly metastatic B16FlO cell line has a SOD activity lower (20.70 f 3.07) units/mg protein, n = 8, than that of the less metastatic B16Fl cell line (81.38 f 6.78) unitslmg protein, n = 8. Acrylamide gel electrophoresis suggested that Mn-SOD activity is higher in
erythrocytes: one contains both copper and zinc and the other contains manganese. The activities of these two types of the enzyme have been studied in various tumor systems in most cases they differed [3,4,6,8,12,151; from those in their normal cell counterparts. It is difficult to compare SOD activities from one system to another. Comparison between a cell line and the tissue of origin can be made only in a few cases. In this report we compared the SOD activities in two variant sublines of the B16 melanoma: B16Fl with low metastatic potential and B16FlO which produces significantly more pulmonary nodules. Materials
Keywords: superoxide cells; melanoma B16
Introduction Two types of superoxide dismutase (SOD) have been found in all mammalian cells except Correspondence Lo: O.R. Affonso. Centro de Pesquisa Bbsica. Instituto National de C$ncer Praqa Cruz Vermelha. 23, Rio de Janeiro
20 230 R. J., Brasil.
0304-3835/91/$03.50 Published and Printed
0 1991 Elsevier Scientific in Ireland
Xanthine, hypoxanthine, RPMI-1640 medium, nitrobluetetrazolium, phenazine methosulfate, streptomycin sulfate, 5-amino-2,3-dihydro- 1,4_phtalazinedione (luminol) ,2-amino2-hydroxy-methyl1,3-propanediol (Tris) , bovine serum albumin and Folin and Ciocalteu’s phenol reagent were products of Sigma Chemical Co. Ethlyenediaminetetraacetic acid (EDTA) acrylamide, N,N’-methyland N,N,N’,,N’-tetraene-bis-acrylamide methylethylenediamine were obtained from E. Merck Darmstadt. Penicillin-G potassium salt was purchased from Fontoura-Wyeth S.A. All Ireland Ltd
other chemicals were of highest available quality and all operations utilized water doubledistilled from glass vessels. Enzymes Copper-zinc superoxide dismutase was prepared from rat erythrocytes by an adaptation of the method of McCord and Fridovich  and stored frozen. Xanthine oxidase was purified from rat liver by a procedure which avoided exposure to proteolytic agents as described by Mitidieri and Aff onso [ 101, All preparations employed presented an (A,,,/A,,,) < 11.5. Cell culture
Tumor cells were grown in vitro in RPMI- 1640 medium supplemented with 2 mM L-glutamine, 10% (v/v) fetal bovine serum, penicillin (100 IU/ml) , streptomycin (100 pg/ml) and sodium bicarbonate (2 mg/ml). Cells were seeded at approximately 4 x 104 cells/cm*. Cell cultures were maintained in Falcon tissue flasks and were incubated at 37°C in a humidified atmosphere of 5% CO, in air. Tumor cells were detached from subconfluent monolayers by a brief exposure to 0.2% EDTA in phosphate buffered saline (PBS), washed several times with PBS, resuspended in RPM1 and counted. Cell viability was routinely greater than 95% as determined by Trypan blue exclusion. Under these conditions the population doubling time of exponential-phase cells was approximately 20 h for the B16Fl and 18 h for the B16FlO cells. For the enzymatic analyses cells were resusin 0.2 M pended (3 x lo6 cells/ml) phosphate buffer 7.8) and disrupted with an Ultra-Turrax homogenizer, for 30 s, on ice. After centrifugation at 10 000 X g for 15 min at 4°C the cell-free extracts were used for electrophoresis fractionation and the assays of the SOD activity and the protein content. Superoxide dismutase and protein assays The total SOD activity of each sample was determined by a method utilizing reduction in the chemiluminescence of luminol . This
method utilizes xanthine oxidase acting upon hypoxanthine as a source of 027 Measurements of light intensity were carried out on the 3H-channel of a Beckman scintillation spectrophotometer in the non-coincidence mode with one of the photomultiplier tubes disconnected. All counts were done in triplicate. Assays were counted for 12 s. The quantity of enzyme causing 50% inhibition was defined as 1 unit. Results were expressed as units/mg protein. Gel electropnoresis Disc electrophoresis was performed with 7 % acrylamide resolving gels with the use of Trisglycine buffer (pH 8.5) as described by Davis  at 4°C. SOD activities were localized on the gels as described in [l] with the modifications introduced by Salin and McCord . Copper-zinc superoxide dismutase was prepared from-rat erythrocytes by an adaptation of the method of McCord and Fridovich . Cyanide-sensitive SOD was differentiated from cyanide-insensitive SOD on polyacrylamide electrophoretograms by including 1 mM cyanide in the developing reagents to suppress the activity of the sensitive enzyme. Protein concentration was estimated by the method of Lowry et al.  using bovine serum albumin as the calibration standard. Statistical analysis Statistical significance of difference between mean values was assessed by Student’s t-test. Results
The total superoxide dismutase activity in the two variant sublines of the B16 melanoma is shown in Fig 1. The statistical analysis revealed that the SOD activity in B16Fl cells was significantly higher (P < 0.001) than in B16FlO cells. Acrylamide gel electrophoresis showed in both cases two bands of proteins with SOD activity. The more rapidly migrating bands of activity were suppressed by 1.0 mM cyanide and presented the same relative mobility as the
Z60 & F40
Fig.1. Left: Superoxide
dismutase activity in two variant sublines of the B16 melanoma (mean f standard deviation of 8 determinations for each subline). Right: Results of polyacrylamide gel electrophoretograms stained for SOD activity in the presence or absence of 10m3M cyanide. Gels are represented by drawing. Details for each gel: 1 - rat erythrocyte superoxide dismutase, 7 pg of protein (no cyanide); 2 - B16Fl homogenate, 16.5 pg protein (no cyanide); 3 - the same as 2 but stain developed in the presence of cyanide; 4 - B16FlO homogenate, 52.5 pg protein (no cyanide); 5 - the same as 4 but stain developed in the presence of cyanide.
erythrocytes cuprozinc superoxide dismutase. Extracts of B16Fl showed the cyanideinsensitive superoxide dismutase band larger than that of the B16FlO extracts suggesting that mangano superoxide dismutase activity is higher in B16Fl cells. The gels with B16Fl extract presented the two bands with approximately the same width, i.e., the ratio CuZn-SOD:Mn-SOD is approximately 1 in B16Fl cells. On the other hand gels with B16FlO extract presented the cyanidesensitive band (CuZn-SOD) larger, approximately twice, than the cyanide-insensitive one (Mn-SOD), i.e., the ratio CuZn-SOD:MnSOD is approximately 2 in B16FlO cells. These results are better visualized with the diagram in Fig. 1. Oberley and Buettner [ll] observed that mitochondria from several malignant animal tumors are deficient in SOD activity and proposed that the decrease in this enzyme
together with increased radical generation might explain many of the properties of cancer cells. Shinkai et al.  presented results indicating that superoxide radical generated extracellularly by hypoxanthine and xanthine oxidase system increased in vitro the invasive capacity of rat ascites hepatoma cells and it was hypothesized that generation of superoxide radicals by host phagocytic cells may also potentiate tumor invasiveness. The present results showed not only a significant reduction of the SOD activity in the highly metastatic B16FlO cells but a drastic diminution of the manganese-containing enzyme in comparison to that of the less metastatic B16Fl cells. The rate of production of O,? by tumor cells remains unclear. If this rate is maintained and the level of SOD is lower than the flux of O,? the result will increase, and we may speculate about the consequences of this. These data suggest that further studies on this field may be warranted. Acknowledgement This study was supported by grants from Conselho National de Desenvolvimento Cientifico e Tecnologico (CNPq). References Beauchamp,
C. and Fridovich. I. (1971)
Cancer Res., 40, 3686-3693.
A., Mavelli, I., Finavi
against reactive oxygen
B. and Rotilio. G. (1976) derivatives.
tumor ceils. Mol. Cell. Biochem..
Bensinger, R.E. and Johnson, C.M. for
assays and an assay applicable
to human serum proteins.
11-16. II. Method
N. Y. Acad.
Sci. 121, 404-427. Dionisi, (1975)
tion in mitochondria Biochim.
radicals and hydrogen peroxide formafrom normal and neoplastic tissues.
Biophys. Acta, 403.
Lowry, O.H., Rosebrough, N. J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. Marklund, S.L., Westman, N.G., Lundgren, E. and Roos, G. (1982) Copperand zinc-containing superoxide dismutase, manganese-containing superoxide dismutase. catalase and glutathione peroxidase in normal and neoplastic human cell lines and normal human tissues. Cancer Res., 42, 1955- 1961. McCord. J.M. and Fridovich, I. (1969) Superoxide dismutase - An enzymic function for erythrocuprein (hemocuprein). J. Biol. Chem. 244, 6049-6055. Mitidieri, E. and Affonso, O.R. (1968) The relationship of phosphate and lipids to xanthine dehydrogenase. Experientia, 24, 330-331.
Oboerley, L.W. and Buettner, superoxide dismutase in cancer: 39, 1141-1149.
Peskin. A.V., Koen, Y.M., Zbarsky, I.B. and Konstantinov, A.A. (1977) Superoxide dismutase and glutathione peroxidase activities in tumors. FEBS Lett., 78. 41-45 Salin, M.L. and McCord, J.M (1974) Superoxide
G.R. (1979) Role of a review. Cancer Res.,
dismutases in polymorphonuclear leukocytes. J. Clin. Invest., 53, 1197-1201. Shinkai, K.. Mukai, M. and Akedo, H. (1986) Superoxide radical potentiates invasive capacity of rat ascites hepatoma cells in vitro. Cancer Lett., 32, 7-13. Van Balgooy, J.N.A. and Roberts, E. (1979) Superoxide dismutase in normal and malignant tissues in different species. Coomp. Biochem. Physiol. 62B, 263-268.