Plasma membrane redox system activity in tumour cell lines of mammary origins with different proliferation rates

Size: px

Start display at page:

Download "Plasma membrane redox system activity in tumour cell lines of mammary origins with different proliferation rates"

Shona Parrish
5 years ago
Views:

1 Protoplasma (1998) 205: PROTOPLASMA 9 Springer-Verlag 1998 Printed in Austria Plasma membrane redox system activity in tumour cell lines of mammary origins with different proliferation rates A. E. del Valle*, A. del Castillo-Olivares, F. Sfinehez-Jim~nez, J. Mfirquez, I. Nfifiez de Castro, and M. A. Medina Laboratorio de Bioqufmica y Biologfa Molecular, Facultad de Ciencias, Universidad de Mfilaga, Mfilaga Received May 28, 1998 Accepted July 27, 1998 Summary. Plasma membrane redox systems seem to play a role in the control of cell growth. In fact, we have found that in mamlnary tumour cell lines the increase in the proliferation rate is accompanied by a decrease in the plasma membrane redox activity. The oxygen consumption rates, the glycolytic fluxes and other bioenergetic parameters have been studied in two cell strains of Ehrlich ascites tumour with different proliferation rates. In the more proliferative Ehrlich cell strain, the decrease in plasma membrane redox system activity is accompanied by decreased oxygen consumption mad glycolytic flux and to a generally less energised status. Keywords: Plasma membrane redox; Ehrlich ascites tumour cell; MCF-7 human breast cancer cell; MDA-MB231 human breast cancer cell; Glycolysis; Lactate. sible differences in PMRS activities among tumour cell lines with different proliferation rates have not been studied yet. In the present report, two human breast cancer cell lines with different proliferation rates and two experimental mammary tumour cell strains growing in mice at different rates are compared in regard to their respective PMRS activities. Furthermore, some bioenergetic parameters and glycolytic flux potential in both experimental murine mammary tumour cell strains are compared. Introduction Plasma membrane redox systems (PMRS) have been found in every living cell tested and they have been involved in several vital functions, including bioenergetics, defence, iron uptake, cell growth, and proliferation (Medina et al. 1997). There is experimental evidence connecting PMRS to the control of cell growth (Crane et al. 1985, Medina and N6fiez de Castro 1995). In fact, it has been shown that transformed cells have PMRS activity levels different from those exhibited by control nontransformed cells (Bruno et al. 1992; Bur6n et al. 1993; L6w et al. 1991; Sun et al. 1983, 1986). However, to our knowledge the pos- *Correspondence and reprints: Laboratorio de Bioqufmica y Biolog/a Molecular, Facultad de Ciencias, Universidad de Mfilaga, E M~laga, Spain. Material and methods Material Human breast cancer cell lines were obtained from ATCC. All reagents were of the highest degree of purity available and purchased from Bio-Whittaker (culture media, serum, and antibiotics), Boehringer Mannheim (enzymes), Fluka (ammonium hexamolibdate), or Sigma (the rest of the reagents). Cell cultures MCF-7 (estrogen-sensitive human breast cancer) and MDA-MB-231 (estrogen-insensitive human breast cancer) were grown in DMEM- Ham's F-12, and Leibovitz's L-15 medium, respectively, supplemented with 10% foetal calf serum (FCS), and antibiotics (penicillin, streptomycin, and amphotericin). MDA-MB-231 cells were grown at 37 ~ without a supplement of CO2. MCF-7 cells were grown at the same temperature under a 5% CO2 atmosphere. Ehrlich ascites tumour cells were maintained in mice as described (P6rez-Rodrfguez et al. 1987). Two different Ehrlich cell strains, hereafter called Roma and Cbm, were used. All the experiments were carried out in cells harvested 9-11 days after turnout inoculation.

2 170 A. E. del Valle et al.: Plasma membrane redox system activity in tumour cell lines PMRS activity assays In Ehrlich ascites cells, PMRS activity was determined as ferricyanide reductase by the method previously described (Medina et al. 1988c). In short, ceils suspended at 6 X 107 cells per ml were incubated at 37 ~ with continuous shaking; aliquots of 1 ml were removed at different times, poured into microfuge tubes and centrifuged at 13,000 g for 2 rain. Ferricyanide reduction was followed by measuring the absorbance at 420 nm in the supematants. Ferricyanide absorption coefficient was taken as 1/mM 9 cm. In human breast cancer cell lines, PMRS activity was measured as inhibition of ascorbate autooxidation in Tris-buffer by a continuous recording method (Medina et al. 1992). In brief, 2 ml of 0.1 mm ascorbate in 0.1 M Tris-buffer (ph 7.4) with or without cells were placed in a quartz cuvette and the decrease in absorbance at 265 nm was followed at 37 ~ for 5 rain; PMRS activity was measured as the percentage of inhibition of ascorbate autooxidation in the presence of cells. Bioenergetic parameters and glycolytic flux Oxygen uptake was measured by the conventional manometric method with a Warburg respirometer. Cell concentration was cells per ml. Measurements were made every 5 rain. The respiratory rate was linear for both Ehrlich cell strains throughout 40 rain of incubation. Other determinations were carried out as follows: harvested cells (1.2 X 108 cells per ml) were suspended in phosphate-buffered saline and preincubated in 50 ml Erlenmeyer flasks under a 95% 02 and 5% CO2 atmosphere for 15 min. Suspensions were then diluted to cells per ml in flasks containing the incubation medium and substrates, and incubated without further gassing. The incubations were carried out at 37 ~ in a metabolic incubator with continuous shaking. At different times, 1 ml aliquots were taken and added to ml of cold 10% (v/v) HC104. Samples were centrifuged at 4,000 g for 5 rain, and supematants were neutralised with 40% (w/v) KOH. The precipitated KC104 was removed by an additional centrifugation step at 4,000 g for 3 min. The neutralised supernatants were used for the enzymatic determination of glucose and lactate, as previously described (Medina et al. 1988b). Metabolite compartmentation was carried out by the Zuurendonk and Tager (1974) method. Cell fractionation was performed in a 3 ml polypropylene centrifuge tube loaded with 0.5 ml of 10% (v/v) HC104, 1 ml of silicon oil consisting in a mixture of types AR200 and AR20 (2 : 1, w/w) and 0.5 ml of a medium containing 0.25 M mannitol, 3 mm EDTA, 20 mm morpholinopropanesulfonic acid and 1 mm purified digitonin. The values for whole cells were obtained by the same procedure, except the digitonin treatment. The values for the cytosolic compartment were calculated as the values obtained for digitonin-treated cells minus those obtained for whole cells. Digitonin purification was carried out as described by Janski and Cornell (1980). At 0 and 15 min, 0.5 ml aliquots of cell suspension were added to the upper layer of the two tubes; after 10 s, they were centrifuged at 3,000 g for 90 s. The upper and middle layers were carefully removed and the bottom layers were neutralised as described above and centrifuged. Neutralised supernatants were used for the determination of adenine nucleotide contents by enzymatic methods (Jaworek and Welsh 1985, Trautschold et al. 1985); the determination of inorganic phosphate contents was carried out by the very sensitive colorimetric method described by Lanzetta et al. (1979). Mitochondria and water spaces were determined by the radioactive method described previously (Martfnez et al. 1984). The translocation energy is defined as the increase across the inner mitochondrial membrane of the variation of free enthalpy of phosphorylation and can be calculated as A(AG') = RT In{ ([ATP]c [ADP]m [Palm) / ([ATP]~ [ADP]c [Pi]c) }. Results and discussion In different human neuroblastoma cell lines, we have observed a positive correlation between PMRS activity and malignancy (Medina et al. 1992). This observation agrees with the reported NADH oxidase activity of plasma membrane hepatoma, higher than that of plasma membranes from normal liver (Bruno et al. 1992). On the contrary, several reports indicate that transformed cells show lower PMRS activity than nontransformed cells. This is the case for pineal and 3T3 cells transformed with SV40 (L6w et al. 1991, Sun et al. 1986). As a complementary example, a transient increase in PMRS activity has been shown in TPA-differentiated HL 60 cells (Bur6n et al. 1993). PMRS activities of two cell lines of human breast cancer with different proliferation rates were compared. In our hands, proliferation rate of MDA- MB231 cells was 55% faster than that of MCF-7 cells over an incubation time of 4 days (results not shown). Table 1 shows that ascorbate-free-radical reductase activity was higher in the less proliferant MCF-7 cell line than in the more proliferant MDA-MB231 cell line. In fact, to obtain the same level of activity measured for MCF-7 cells it was necessary to use a suspension of MDA-MB231 cells with a cellular density increased 5-fold (that is, 2.5 X 105 cells). These results are in contrast with our previous observations in human neuroblastoma cells (Medina et al. 1992). A possible explanation of this different behaviour could rely on the fact that ascorbic acid at the used concentrations seems to be cytotoxic for neuro- Table 1. Oxidation rate of ascorbate in the presence of two human breast cancer cell lines with different proliferation rates Cell line Proliferation rate (%) Ascorbate oxidation (%) None MDA-MB MCF _+ 5 Proliferation rates of both cell lines were normalized, taking as 100% the increase in MCF-7 cell number after 4 days of culture. The oxidation rate of 0.1 mm ascorbate in the absence or presence of 5 x 104 cells was followed spectrophotometrically, as described in Material and methods. Data are percentages of ascorbate oxidation, taking that measured in the absence of cells as 100%. Data are means with SD of three different and independent experiments

3 A. E. del Valle et al.: Plasma membrane redox system activity in tumour cell lines t71 Table 2. Significant biological differences between two Ehrlich ascites tumour cell strains Ehrlich cell Relative Cellular Host life strain growth (%) density a (cells/ml) span b (days) Cbm Roma _ acellular density was determined in the ascitic fluid harvested from five inoculated mice on the 10th day of tumour growth. Data are means with SD bhost mice life span was determined with five inoculated mice for each tumour cell strain. Data are means with SD Table 4. Oxygen consumption, lactate content, and lactate consumption by two Ehrlich ascites tumour cell strains with different proliferation rates Ehrlich cell 02 consumption a Lactate strain content b consumption b Cbm Roma 1.7 +_ adata are given as nmol/min per 106 cells and are means with SEM of, at least, six different experiments bdata are given as nmol/106 cells and are means with SEM of, at least, six different experiments blastoma cells, but not for breast cancer cells (Garcfa de Veas et al. 1995, and unpubl, data). In our laboratory, we maintain two Ehrlich cell strains with different proliferation rates. In fact, as shown in Table 2, Roma strain is not only more proliferative but also much more aggressive than Cbm strain. We have used previously this model system to carry out some comparative studies concerning glutamine and energy metabolism in these two Ehrlich cell strains (Sfinchez-Jim6nez et al. 1985; Medina et al. 1988b, c, 1990; Luque et al. 1990). Ehrlich ascites tumour cells are easily maintained in the peritoneal cavity of mice yielding very crowded suspensions in a few days. Thus, hundred of millions of cells can be harvested from each inoculated mouse. The availability of such a high number of cells makes it possible to carry out determinations of PMRS activity with ferricyanide as a substrate, as previously described (Medina et al. 1988c). Table 3 shows ferricyanide reductase activities of the Ehrlich ascites tumour cell strains Roma and Cbm. At all the incubation times tested, the less proliferative Table 3. Ferricyanide reduction by two Ehrlich ascites mmour cell strains with different proliferation rates Ehrlich cell strain Ferricyanide reductase activity Incubation time (min) Cbm _ _ Roma _ Ratio Roma/Cbm Experiments were carried out in the presence or absence of cells as described in Material and methods. Data are means with SD of, at least, two independent experiments and the units are nmol of ferricyanide reduced per 106 ceils Ehrlich cell strain (Cbm) reduced more ferricyanide than the more proliferative Ehrlich cell strain (Roma). In fact, ferricyanide reductase activity in Cbm cells was approximately 60% higher than in Roma cells. To test whether the differences in ferricyanide reductase activity observed in Cbm and Roma cells are a manifestation of intrinsic differences in their respective metabolic status, we determined several energetic parameters in both cell lines incubated in the absence or presence of 5 mm glucose. The results obtained are summarised in Tables 4 and 5 and Fig. 1. Some years ago, our group firstly described that the presence of lactate, an immediate source of cytoplasmic NADH, contributes to maintain a higher PMRS activity than in control situation (Medina etal. 1988c). As it is shown in Table 4, the endogenous content of lactate was much higher in Cbm cells than 125- "~ E t- O O d ; 1'0 2'0 3'0 Time (min) Fig. 1. Lactate contents in two Ehrlich ascites tumour cell strains incubated in the presence of 5 mm glucose: O Roma, 9 Cbm cells. Data are means with SEM of, at least, six different experiments T

4 172 A. E. del Valle et al.: Plasma membrane redox system activity in tumour cell lines Table 5. Metabolic data and energetic parameters for two Ehrlich ascites tumour cell strains with different proliferation rates incubated in the presence of 5 mm glucose for 15 rain Parameter Ehrlich cell strain Cbm Roma Glucose consumption a 43 % 52% Glucose transformed into lactate b 66% 54% A(ATP/ADP)cytosolic c A(ATP/ADP)rnitochondrial c A(Translocation energy) d +3.5 kj/mol -0.2 kj/mol adata are given as percentages of the total amount of glucose added to medium bdata are given as the percentage of the carbon skeletons of glucose absorbed by the cells and transformed into lactate ~ compartmentation and the determination of ATP and ADP contents were carried out as described in Material and methods. Data are given as adimensional numbers obtained by subtracting the values of the ratios ATP/ADP at times 15 and 0 rain ametabolic compartmentation and the determination of ATP, ADP, and inorganic phosphate contents were carried out as described in Material and methods. Data are obtained by subtracting the values of the translocation energies at times 15 and 0 rain in Roma cells. Moreover, the consumption rate of this endogenous lactate in Cbm cells was approximately 60% higher than in Roma cells, despite the fact that lactate dehydrogenase activity of both cell strains is very similar (S~inchez-Jim6nez et al. 1985). It is noteworthy that the difference observed in lactate consumption rates is the same as that observed in ferricyanide reduction rates. The endogenous respiratory rate of Cbm cells was one third higher than that of Roma cells (Table 4). This is an expected result because it is well known that the efficiency of the mitochondrial respiratory chain declines with increasing malignancy during tumour progression. Glucose is the main metabolic substrate for those tumour cells showing very high rates of aerobic glycolysis (Medina and Ndfiez de Castro 1990). Thus, we decided to study the glycolytic flux and the changes in some energetic parameters in Cbm and Roma cells incubated in the presence of 5 mm glucose. As depicted in Fig. 1, the glycolytic flux was higher in Cbm cells than in Roma cells. In fact, Cbm cells used glucose at a lower rate but transformed a greater percentage of the used glucose into lactate than Roma cells (see Table 5). These results could reflect the fact that the more proliferative Roma cell strain has greater biosynthetic requirements. However, it can also be concluded that Roma cells waste a higher percentage of energy metabolites in a dissipative manner than Cbm cells. In fact, the glucose aerobically used by Roma cells was not able to increase the cytosolic ATP/ADP ratio. On the contrary, this ratio significantly decreased after 15 min of incubation. Furthermore, the glucose-derived skeletons respired by Roma cells did not increase the mitochondrial ATP/ADP ratio. The translocation energy for adenine nucleotides and inorganic phosphate across the inner mitochondrial membrane decreased after 15 min of incubation in the presence of 5 mm glucose. On the other hand, in Cbm cells the metabolised glucose was in part used to energise both the cytosolic and the mitochondrial compartments, as reflected by the increased values of ATP/ADP ratios in both compartments and by the increase in the translocation energy after 15 min of incubation in the presence of 5 mm glucose. In conclusion, it seems that PMRS activity decreases with increasing proliferation rates, at least in mammary tumours. Our studies on two Ehrlich cell strains show a parallelism between PMRS activity and the bioenergetic status of the cells. Tumour cells in their natural environment frequently are under hypoxic conditions. In this situation, aerobic metabolism is substituted by extremely high glycolytic fluxes. Since glycolysis increases the cytosolic levels of NADH, those metabolic pathways using NADH and able to function under hypoxia could contribute to maintain, at least in part, the high glycolytic fluxes required; this could be the case for PMRS. The present results agree with this hypothesis, because PMRS activity is higher in that Ehrlich cell strain with a higher glycolytic flux. On the other hand, the very aggressive and malignant Roma strain has an impaired, defective and dissipative energy metabolism and this correlates with the decreased redox activity. Acknowledgements Partially supported by grant SAF 98/0150 and funds from research groups CVI-0114 and CVI-0179 (PAI, Andalusian Government). References Bruno M, Brightman An, Lawrence J, Werderitsh D, Mort6 DM, Morre DJ (1992) Stimulation of NADH oxidase activity from rat liver plasma membranes by growth factors and hormones is decreased or absent with hepatoma plasma membranes. Biochem J 284: Burdn MI, Rodrfguez-Aguilera JC, Alcafn FJ, Navas P (1993) Transplasma membrane redox system in HL-60 cells is modulated during TPA-induced differentiation. Biochem Biophys Res Commun 192: Crane FL, Sun IL, Clark MG, Grebing C, L6w H (1985) Transplas-

5 - - Schweigerer - SNlchez-Jim~nez A. E. del Valle et al.: Plasma membrane redox system activity in tumour cell lines 173 ma-membrane redox systems in growth and development. Biochim Biophys Acta 81 I: Garcfa de Veas R, Schweigerer L, Medina MA (1995) Why is ascorbate toxic to neuroblastoma cell lines and why does this toxicity increase with cell Iine malignancy? Redox Rep 1: Janski AM, Cornell NW (1980) Subcellular distribution of enzymes determined by rapid digitonin fractionation of isolated hepatocytes. Biochem J 186:423~-29 Jaworek D, Welsch J (1985) ADP, AMP: UV-method. In: Bergmeyer HU (ed) Methods of enzymatic analysis, vol 7. Verlag Chemie, Weinheim, pp Lanzetta PA, Alvarez LJ, Reinach PS, Candia OA (I979) An improved assay for nanomole amounts of inorganic phosphate. Anal Biochem 100:95-97 LOw H, Crane FL, Grebing C, Isaksson M, Lindgren A, Sun IL (1991) Modification of transplasma membrane oxidoreduction by SV40 transformation of 3T3 cells. J Bioenerg Biomembr 23: Luque P, Paredes S, Segura JA, Ndfiez de Castro I, Medina MA (1990) Mutual effect of glucose and glutarnine on their utilization by tumour cells. Biochem Int 21:9-15 Martlnez P, Carrascosa JM, Sfinchez-Jim6nez F, Otavarria JS, Ndfiez de Castro I (1984) Cellular compartmentation of Ehrlich ascites tumor cells. Rev Esp Fisiol 40: Medina MA, Ndfiez de Castro I (1990) Glutaminolysis and glycolysis interactions in proliferant cells. Int J Biochem 22: (1995) Plasma membrane redox systems in tumor cells. Protoplasma 184: Del Castillo-Olivares A, Nfifiez de Castro I (1997) Multifunctional plasma membrane redox systems. BioEssays 19: L (1992) Plasma membrane redox activity correlates with N-myc expression in neuroblastoma cells. FEBS Lett 311: F, Nffiez de Castro I (1990) Subcellular distri- bution of adenine nucleotides in two Ehrlich cell lines metabolizing glucose. Biol Chem Hoppe Seyler 371: Quesada AR, Mfirquez J, Sfinchez-Jim6nez F, Nffiez de Castro I (1988a) Inorganic phosphate and energy charge compartmentation in Ehrlich ascites tumour cells in the presence of glucose and/or glutamine. B iochem Int 16: S~inchez-Jim6nez F, Mfirquez J, Pdrez-Rodrfguez J, Quesada AR, Nffiez de Castro I (1988b) Glntamine and glucose as energy substrates for Ehrlich ascites tumour cells. Biochem Int 16: Segura JA, Sgmchez-Jim~nez E, Nfifiez de Castro I (1988c) Transmembrane ferricyanide reductase activity in Ehrlich ascites tumor cells. Biochim Biophys Acta 946:1-4 P6rez-Rodrfguez J, Sfinchez-Jim6nez F, Mgtrquez J, Medina MA, Quesada AR, Nfifiez de Castro I (1987) Malate-citrate cycle during glycolysis and glutaminolysis in Ehrlich ascites tumor cells. Biochimie 69: S~inchez-Jim6nez F, Martinez P, Nfifiez de Castro I, Olavarrfa JS (1985) The function of redox shuttles during aerobic glycolysis in two strains of Ehrlich ascites tumor cells. Biochimie 67: Sun IL, Crane FL, Chou JY (1986) Modification of transmembrane electron transport activity in plasma membranes of Simian virus 40 transformed pineal cells. Biochim Biophys Acta 886: L6w H, Grebing C (1983) Transformed liver cells have modified transplasma redox activity which is sensitive to adriamycin. Biochem Biophys Res Commun 116: Trautschold I, Lamprecht W, Schweitzer G (1985) ATP: UV-method with hexokinase and glucose-6-phosphate dehydrogenase. In: Bergmeyer HU (ed) Methods of enzymatic analysis, vol 7. Verlag Chemie, Weinheim, pp Zuurendonk PF, Tager JM (1974) Rapid separation of particulate components and soluble cytoplasm or isolated rat liver cells. Biochim Biophys Acta 333: Verleger: Springer-Verlag KG, Sachsenplatz 4-6, A-1201 Wien. Herausgeber: Prof. B. E. S. Gunning, Research School of Biological Sciences, The Australian National University, Canberra City, ACT 2601, Australien. -- Redaktion: Sachsenplatz 4-6, A-1201 Wien. -- Hersteller: Adolf Holzhausens Nachfolger, Kandlgasse 19 21, A-1070 Wien. -- Verlagsort: Wien. -- Herstellungsort: Wien. -- Printed in Austria

Trans-plasma membrane electron transport in muscle cells. Shannon Kelly

Trans-plasma membrane electron transport in muscle cells Shannon Kelly Introduction Trans-plasma membrane electron transport (tpmet) Shuttle-based electron transfer Enzyme-mediated electron transfer Intracellular