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Inhibition of human brain tumor cell growth by a receptor-operated Ca2+ channel blocker
Cancer Letters, 12 (1993) 77-81 Elsevier Scientific Publishers Ireland
77 Ltd.
Inhibition of human brain tumor cell growth by a receptor-operated Ca*+ channel blocker Yong Soo Lee, Mohammed M. Sayeed and Robert D. Wurster Department of Physiology, Loyola University Medical Center, 2160 S. First Ave. Maywood, IL 60153 (USA) (Received 30 March 1993) (Revision received 13 May 1993) (Accepted 14 May 1993)
Summary
SK&F 96365, a reported receptor-operated Ca2+ channel blocker, inhibited the growth of IJ-373 MG human astrocytoma and SK-N-MC human neuroblastoma cell lines in a dosedependent manner. Carbachol and serum which act as growth factcrs for these cells induced a rapid, transient increase of intracellular Ca2+ concentration without a sustained increase. SK&F 96365 also exerted a significant inhibition of carbachol or serum-induced intracellular Ca2+ mobilization. These results suggest that SK&F 96365 is a potent inhibitor of brain tumor cell growth and that its effect may be mediated by the inhibition of agonist-induced intracellular Ca2’ mobilization.
Keywords: SK&F 96,365; human Ca2+ mobilization
brain tumor;
Introduction
Recently the Ca’+ entry mechanism into the cells, e.g. receptor-mediated Ca2+ entry (RMCE), by occupation of membrane receptors has been described in many cellular systems, including tumor cells [l-4]. However, the function and mechanism of RMCE are less well understood. Correspondence to: Robert D. Wurster, Department of Physiology, Loyola University Medical Center, 2160 S. First Ave, Maywood. IL 60153. U.S.A. 0304-3835/93/$06.00 0 1993 Elsevier Scientific Printed and Published in Ireland
Publishers
Ireland
Merritt and associates reported that a novel compound, SK&F 96365, which is structually distinct from classic Ca2+ antagonists, effectively blocks RMCE [5]. Since SK&F 796365 has such a regulatory role in the Ca2+ signalling mechanisms, which are important in tumor cell growth [6-91, the effect of this drug on brain tumor cell growth was investigated. Materials and Methods Materials
U-373 MG human astrocytoma and SK-N-MC human neuroblastoma cell lines were purchased from the American Type Culture Collection (Rockville, Massachusetts). The powders for Eagle’s minimum essential medium (MEM) and Earle’s basal salt solution (EBSS), trypsin solution, trypan blue, sodium pyruvate, carbamylcholine chloride and all salt powders were obtained from Sigma Chemical Company (St. Louis, Missouri). Fetal bovine serum (FBS) and antibiotics (penicilline and streptomycin mixture) were purchased from GIBCO (Grand Island, New York). SK&F 96365 was generously supplied by Dr. Hills from Smith Kline Beecham Pharmaceuticals (Welwyn, Hertfordshire, England). Cell culture Cells were grown at 37°C in a humidified incubator under 5% CGz/95% air in a MEM supplemented with 10% FBS, 200 IU/ml penicillin, Ltd.
78
200 pg/ml of streptomycin and 1 mM sodium pyruvate. Cell medium was replaced twice weekly. After attaining confluency, the cells were subcultured by trypsinization. Cell growth studies Cells from 4 to 5-day-old cultures were seeded in equal numbers in 35 x 10 mm culture dishes at the density of lo5 and 2 x lo5 cells per dish in neuroblastoma and astrocytoma cell lines, respectively. The volume of the medium in the dishes was 2 ml. The drug to be tested was added to cultures 1 day after seeding in order to ensure uniform attachment of cells at the beginning of the experiments. The cells were grown for an additional 2 days. Drug and culture medium were replaced every day. Assessment of cell growth was made by counting viable cells in a hemocytometer, using trypan blue exclusion after trypsinization. Measurement of [Ca2+li Aliquots of the tumor cells, cultured for 3-5 days, were washed in EBSS. Then 2 PM Fura-2AM was added, and the cells were incubated for 60 min at room temperature (22-23°C). Unloaded Fura-2-AM was removed by centrifugation at 150 x g for 3 minutes. Cells were resuspended at a density of 2 x 106/ml in Krebs-Ringer buffer (KRB) containing 125 mM NaCl, 5 mM KCl, 1.2 mM KH2P04, 1.2 mM MgS04, 5 mM NaHCOs, 1.3 mM CaC12, 25 mM HEPES and 6 mM glucose (pH 7.4), transferred to a quartz cuvette and stirred continuously. Fluorescence emission (5 10 nm) was monitored with the excitation wavelength cycling between 340 and 380 nm, using a Hitachi F2000 fluorimeter. At the end of an experiment, fluorescence maximum and minimum values at each excitation wavelength were obtained by first lysing the cells with 0.1% Triton X-100 (maximum) and then adding 10 mM EGTA (minimum). With the maximum and minimum values, the 340:380 nm fluorescence ratios were converted into free Ca*+ concentrations using FuraCa’+ binding constant (224 nM) and the formula described by Grynkiewicz et al. [lo]. Data analysis All experiments
were performed
at least four
times. All the data was displayed as a percentage of the control condition, which was taken as 100%. Data was expressed as mean f standard error of the mean (S.E.M.). The data was analyzed using one-way analysis of variance (ANOVA) and Student-Newman-Keul‘s test for individual comparisons. The data that has a P value less than 0.05 is considered to be statistically significant. Results As shown in Fig. 1, SK&F 96365 inhibited the in vitro growth of human brain tumor cells in a dose-dependent manner. The half-maximum concentrations for growth inhibition (ICsO) by the drug were between 5 and 10 PM and close to 1 PM for U-373 MG and SK-N-MC cell lines, respectively, which are significantly less than that (around 20 PM) for the inhibition of agonistinduced Mn*+ influx, an indicator of Ca2+ influx [5]. The discrepancy of IC50 values of these two effects implies that the mechanism of the drug in the inhibition of tumor cell growth may or may
I
0
100 -
6
80-
E $
60-
: U-373 MG : SK-N-MC
0 $
4020 -
-7
-6
SK&F
-5
96365
-4
-3
(log M)
Fig. 1. Effect of SK&F 96365 on the growth of U-373 MC human astrocytoma (0) and SK-N-MC human neuroblastoma ( v ) cell lines. The results were expressed as percentage change of the number of the cells obtained in the absence of the drug. The data points represent the mean value of four replicates, with bars indicating S.E.M.
79
not be related to RMCE. To ascertain the role of RMCE, the changes of intracellular Ca2+ concentrations induced by growth factors were measured using Fura- fluorescence. Fig. 2. shows that carbachol and serum, which act as growth factors in these cell lines, induced a rapid, transient rise of intracellular Ca2+ concentration without a sustained elevation. Furthermore Ca2+-free medium did not alter agonistevoked responses (Fig. 2). These results indicate that most of the increased intracellular Ca2+ induced by growth factors came from internal store release rather than Ca2+ influx. These results further suggest, therefore, that RMCE may not be involved in the growth of these brain tumor cells. Further, the effect ‘of SK&F 96365 on agonistinduced Ca2+ mobilization was also investigated to elucidate the mechanism of action of this drug in tumor cell growth. The results in Fig. 3 illustrate
A am-
intracellular Fig. 3. Effect of SK&F 96365 on agonist-induced Ca*’ mobilization in U-373 MG human astrocytoma (A) and SK-N-MC human neuroblastoma (B) cell lines incubated in Ca*+-free medium. Control cells (left traces) and cells pretreated with 20 pM SK&F 96365 for 3 min (right traces) were stimulated with 0.8 mM carbachol or 2% serum. Quantitative changes (C) were expressed as percentage change of the increased intracellular Ca*+ concentration induced by the drug compared with agonist alone. Each column represents mean f S.E.M. *P < 0.05 compared with agonist alone.
of agonist-induced increase of intracelFig. 2. Independence lular Ca*+ concentration from extracellular Ca*+ concentration in U-373 MG human astrocytoma (A) and SK-N-MC human neuroblastoma (B) cell lines. Carbachol (0.8 mM) or serum (2%) were used as agonists. Aliquots of 2 x IO6 cells/ml were incubated with 2 PM of Fura-2-AM for 60 min at room temperature (22-23°C). The cells were washed, resuspended in buffer solution containing 1.3 mM Ca2+ (left traces) or Ca?+free medium (right traces) and transferred to a quartz cuvette for fluorescence measurements. The data represents intracellular Ca*+ changes with time. The arrows show the time points for the addition of agonists.
that SK&F 96365 significantly inhibited agonistinduced Ca2+ mobilization in both cell lines. Although this inhibition required a much higher concentration of the drug than did the inhibition of tumor cell growth, these results suggest that the growth-inhibitory effect of SK&F 96365 is at least partially due to the interference with agonistinduced intracellular Ca2+ mobilization. The exact mechanism of this action of SK&F 96365 remains to be determined.
80
Discussion
Classic Ca2+ channel antagonists, such as verapamil, nifedipine and diltiazem, have been shown to inhibit cell proliferation in many different cellular systems, including tumor cells [ 1l- 141. Furthermore, growth factors increase intracellular Ca2+ concentration [ 15- 171, and therefore the role of Ca2+ influx in cell proliferation has been stressed [ 18-201. However, most non-excitable cells appeared not to have voltagesensitive Ca2+ channels [21,22], and so the Ca2+ entry pathway is obscure. Recently the Ca2+ entry mechanism into the cells by occupation of membrane receptors through receptor-operated Ca2+ channels has been described in many cellular systems [l-4]. The purpose of the present study was to evaluate the role of Ca2+ influx through this pathway in tumor cell growth and to examine the effect of SK&F 96365 - a known blocker of receptor-operated Ca2+ channels [5] - on cell growth, using two human brain tumor cell lines as model cellular systems. In this study we have found that SK&F 96365 very effectively inhibited the growth of human brain tumor cells. To our knowledge this is the first report of growth-inhibitory effect of the drug on tumor cells. A sustained increased intracellular Ca2+ concentration following an initial transient rise has been shown to be induced by carbachol and other growth factors in many cellular systems [4,20,23,24]. The results of the present study, in which no sustained increase of intracellular Ca2+ was induced by the agonists, suggest cell-type specific action of these agonists without an RMCE in the tumor cell lines used in this study. These results further suggest that in these tumor cells, agonist-induced Ca2+ mobilization from internal stores is an important signalling mechanism in cell proliferation. Furthermore, since SK&F 96365 significantly inhibited agonist-induced Ca2+ mobilization, the growth-inhibitory effect of this drug may be at least partially attributed to the interference with agonist-induced intracellular Ca2+ mobilization. In conclusion, SK&F 96365, a receptoroperated Ca 2+ channel blocker, effectively inhibited brain tumor cell growth, and this effect may
be due to the inhibition of agonist-induced intracellular Ca2’ mobilization from internal stores. These results suggest a possible application of this drug to the management of brain tumor cell proliferation. Acknowledgements
This study was supported by the Mr. and Mrs. Barney Kahn Fund and NIH Grant GM 32288. References 1
2 3 4
5
6
7
8 9
10
11
12
13
14
Sage, 0.. Mahaut-Smith. M.P. and Rink, T.J. (1992) Calcium entry in nonexcitable ceils: lessons from human platelets. News Physiol. Sci., 7, 108-I 13. Putney, J.W. (1991) Receptor-regulated calcium entry. Pharmac. Ther., 48, 427-434. Rink, T.J. (1990) Receptor-mediated calcium entry. FEBS, 268, 381-385. Fischer, H., Illek, B., Negulescu, P.A., Clauss. W. and Machen, T.E. (1992) Carbachol-activated calcium entry into HT-29 cells is regulated by both membrane potential and cell volume. Proc. Nat. Acad. Sci. USA, 89. 1438-1442. Merritt. J.E., Armstrong, W.P., Benham. C.D., Hallam, T.J.. Jacob, R., Jaxa-Chamiec, A., Leigh, B.K., McCarthy, S.A.. Moores, K.E. and Rink, T.J. (1990) SK&F 96365, a novel inhibitor of receptor-mediated calcium entry. Biochem. J., 271. 515-522. Veigl, M.L., Vanaman, T.C. and Sedwick, W.D. (1984) Calcium and calmodulin in cell growth and transformation. Biochim. Biophys. Acta, 738, 21-48. Whitlield, J.F.. Boynton, A.I., MacManus. J.P., Rixon. R.H., Sikorska, M., Tsang, B.K. and Walker, P.R. (1980) The roles of calcium and cyclic AMP in cell proliferation. Ann. NY Acad. Sci.. 339. 216-240. Whitfield, J.F. (1992) Calcium signals and cancer. Critical Rev. Oncogenesis, 3, 55-90. Metcalfe, J.C., Moore, J.P.. Smith, G.A. and Hesketh, T.R. (1986) Calcium and cell proliferation. Br. Med. Bull.. 42, 405-412. Grynkiewicz, G.. Poene, M. and Tsien. R.Y. (1985) A new generation of Ca ” indicators with greatly improved fluorescence properties. J. Biol. Chem.. 260, 3440-3450. Taylor, J.M. and Simpson. R.U. (1992) Inhibition of cancer cell growth by calcium channel antagonists in the athymic mouse. Cancer Res., 52, 2413-2418. Shultz, P.J. and Raij, L. (1990) Inhibition of human mesangial cell proliferation by calcium channel blockers. Hypertension 15 (suppl. I), I-76-1-80. Birx, D.L., Berger, M. and Fleisher. T.A. (1984) The interference of T cell activation by calcium channel blocking agents. J. Immunol., 133, 2904-2909. Schuller, H.M.. Orloff, M. and Reznik, G.K. (1991) Anti-
81
15
16
17
18
19
proliferative effects Iof the Ca*+/calmodulin antagonist B859-35 and the C,a*+/channel blocker verapamil on human lung cancel- cell lines. Carcinogenesis, 12, 2301-2303. Moolenaar, W.H., Tsertoolen, L.G.J. and de Laat, S.W. (1984) Growth factors immediately raise cytoplasmic free Ca2+ in human fibroblasts. J. Biol. Chem.. 259, 8066-8069. Nishimoto, I., Ghkuni, Y., Ogata, E. and Kojima, I. (1987) Insulin-like growth factor II increases cytoplasmic free calcium in competent Balbic 3T3 cells treated with epidennal growth factor. Biochem. Biophys. Res. Commun., 142, 275-286. Bonventre, J.V., Skorecki. K.L., Kreisberg. J.I. and Cheung, J.Y. (1986) Vasopressin increases cytosolic free calcium concentration in glomerulosa mesangial cells. Am. J. Physiol., 20, :F94-F102. Diliberto, P.A., Hubbert, T. and Herman, B. (1991) Early PDGF-induced alterations in cytosohc free calcium are required for mitogenesis. Res. Commun. Chem. Pathol. Pharmacol., 72, 3-12. Olsen, R., Seewald, M. and Powis, G. (1989) Contribution of external and internal Ca*+ to changes in intracellular
20
21
22
23
24
free Ca*+ produced by mitogens in Swiss 3T3 fibroblasts: the role of dihydropyridine sensitive Ca*+ channels. B&hem. Biophys. Res. Cormnun., 162, 448-455. Tuker, R.W., Chang, D.T. and Maede-Coburn, K. (1989) Effect of platelet-derived growth factor and fibroblast growth factor on free intracellular calcium and mitogenesis. J. Cell. Biochem., 39, 139-151. Wang, S.Y., Castle, N.A. and Wang, G.K. (1992) Identification of RBKl potassium channels in C6 astrocytoma cells. Glia, 5, 146-153. Mozhayeva, G.N., Naumov, A.P. and Kuryshev, Y.A. (1991) Variety of Ca*+-permeable channels in human carcinoma A431 cells. J. Membrane Biol., 124, 113-126. Felder, C.C., Poulter, M.O. and Wess, J. (1992) Muscarinic receptor-operated Ca*+ influx in transfected tibroblast cells is independent of inositol phosphates and release of intracellular Ca*+. Proc. Nat. Acad. Sci., 89, 509-513. Lambert, D.G., Whitham, E.M., Baird, J.G. and Nahorski, S.R. (1990) Different mechanisms of Ca*’ entry induced by depolarization and muscarinic receptor stimulation in SH-SYSY human neuroblastoma cells. Mol. Brain Res., 8, 263-266.
77 Ltd.
Inhibition of human brain tumor cell growth by a receptor-operated Ca*+ channel blocker Yong Soo Lee, Mohammed M. Sayeed and Robert D. Wurster Department of Physiology, Loyola University Medical Center, 2160 S. First Ave. Maywood, IL 60153 (USA) (Received 30 March 1993) (Revision received 13 May 1993) (Accepted 14 May 1993)
Summary
SK&F 96365, a reported receptor-operated Ca2+ channel blocker, inhibited the growth of IJ-373 MG human astrocytoma and SK-N-MC human neuroblastoma cell lines in a dosedependent manner. Carbachol and serum which act as growth factcrs for these cells induced a rapid, transient increase of intracellular Ca2+ concentration without a sustained increase. SK&F 96365 also exerted a significant inhibition of carbachol or serum-induced intracellular Ca2+ mobilization. These results suggest that SK&F 96365 is a potent inhibitor of brain tumor cell growth and that its effect may be mediated by the inhibition of agonist-induced intracellular Ca2’ mobilization.
Keywords: SK&F 96,365; human Ca2+ mobilization
brain tumor;
Introduction
Recently the Ca’+ entry mechanism into the cells, e.g. receptor-mediated Ca2+ entry (RMCE), by occupation of membrane receptors has been described in many cellular systems, including tumor cells [l-4]. However, the function and mechanism of RMCE are less well understood. Correspondence to: Robert D. Wurster, Department of Physiology, Loyola University Medical Center, 2160 S. First Ave, Maywood. IL 60153. U.S.A. 0304-3835/93/$06.00 0 1993 Elsevier Scientific Printed and Published in Ireland
Publishers
Ireland
Merritt and associates reported that a novel compound, SK&F 96365, which is structually distinct from classic Ca2+ antagonists, effectively blocks RMCE [5]. Since SK&F 796365 has such a regulatory role in the Ca2+ signalling mechanisms, which are important in tumor cell growth [6-91, the effect of this drug on brain tumor cell growth was investigated. Materials and Methods Materials
U-373 MG human astrocytoma and SK-N-MC human neuroblastoma cell lines were purchased from the American Type Culture Collection (Rockville, Massachusetts). The powders for Eagle’s minimum essential medium (MEM) and Earle’s basal salt solution (EBSS), trypsin solution, trypan blue, sodium pyruvate, carbamylcholine chloride and all salt powders were obtained from Sigma Chemical Company (St. Louis, Missouri). Fetal bovine serum (FBS) and antibiotics (penicilline and streptomycin mixture) were purchased from GIBCO (Grand Island, New York). SK&F 96365 was generously supplied by Dr. Hills from Smith Kline Beecham Pharmaceuticals (Welwyn, Hertfordshire, England). Cell culture Cells were grown at 37°C in a humidified incubator under 5% CGz/95% air in a MEM supplemented with 10% FBS, 200 IU/ml penicillin, Ltd.
78
200 pg/ml of streptomycin and 1 mM sodium pyruvate. Cell medium was replaced twice weekly. After attaining confluency, the cells were subcultured by trypsinization. Cell growth studies Cells from 4 to 5-day-old cultures were seeded in equal numbers in 35 x 10 mm culture dishes at the density of lo5 and 2 x lo5 cells per dish in neuroblastoma and astrocytoma cell lines, respectively. The volume of the medium in the dishes was 2 ml. The drug to be tested was added to cultures 1 day after seeding in order to ensure uniform attachment of cells at the beginning of the experiments. The cells were grown for an additional 2 days. Drug and culture medium were replaced every day. Assessment of cell growth was made by counting viable cells in a hemocytometer, using trypan blue exclusion after trypsinization. Measurement of [Ca2+li Aliquots of the tumor cells, cultured for 3-5 days, were washed in EBSS. Then 2 PM Fura-2AM was added, and the cells were incubated for 60 min at room temperature (22-23°C). Unloaded Fura-2-AM was removed by centrifugation at 150 x g for 3 minutes. Cells were resuspended at a density of 2 x 106/ml in Krebs-Ringer buffer (KRB) containing 125 mM NaCl, 5 mM KCl, 1.2 mM KH2P04, 1.2 mM MgS04, 5 mM NaHCOs, 1.3 mM CaC12, 25 mM HEPES and 6 mM glucose (pH 7.4), transferred to a quartz cuvette and stirred continuously. Fluorescence emission (5 10 nm) was monitored with the excitation wavelength cycling between 340 and 380 nm, using a Hitachi F2000 fluorimeter. At the end of an experiment, fluorescence maximum and minimum values at each excitation wavelength were obtained by first lysing the cells with 0.1% Triton X-100 (maximum) and then adding 10 mM EGTA (minimum). With the maximum and minimum values, the 340:380 nm fluorescence ratios were converted into free Ca*+ concentrations using FuraCa’+ binding constant (224 nM) and the formula described by Grynkiewicz et al. [lo]. Data analysis All experiments
were performed
at least four
times. All the data was displayed as a percentage of the control condition, which was taken as 100%. Data was expressed as mean f standard error of the mean (S.E.M.). The data was analyzed using one-way analysis of variance (ANOVA) and Student-Newman-Keul‘s test for individual comparisons. The data that has a P value less than 0.05 is considered to be statistically significant. Results As shown in Fig. 1, SK&F 96365 inhibited the in vitro growth of human brain tumor cells in a dose-dependent manner. The half-maximum concentrations for growth inhibition (ICsO) by the drug were between 5 and 10 PM and close to 1 PM for U-373 MG and SK-N-MC cell lines, respectively, which are significantly less than that (around 20 PM) for the inhibition of agonistinduced Mn*+ influx, an indicator of Ca2+ influx [5]. The discrepancy of IC50 values of these two effects implies that the mechanism of the drug in the inhibition of tumor cell growth may or may
I
0
100 -
6
80-
E $
60-
: U-373 MG : SK-N-MC
0 $
4020 -
-7
-6
SK&F
-5
96365
-4
-3
(log M)
Fig. 1. Effect of SK&F 96365 on the growth of U-373 MC human astrocytoma (0) and SK-N-MC human neuroblastoma ( v ) cell lines. The results were expressed as percentage change of the number of the cells obtained in the absence of the drug. The data points represent the mean value of four replicates, with bars indicating S.E.M.
79
not be related to RMCE. To ascertain the role of RMCE, the changes of intracellular Ca2+ concentrations induced by growth factors were measured using Fura- fluorescence. Fig. 2. shows that carbachol and serum, which act as growth factors in these cell lines, induced a rapid, transient rise of intracellular Ca2+ concentration without a sustained elevation. Furthermore Ca2+-free medium did not alter agonistevoked responses (Fig. 2). These results indicate that most of the increased intracellular Ca2+ induced by growth factors came from internal store release rather than Ca2+ influx. These results further suggest, therefore, that RMCE may not be involved in the growth of these brain tumor cells. Further, the effect ‘of SK&F 96365 on agonistinduced Ca2+ mobilization was also investigated to elucidate the mechanism of action of this drug in tumor cell growth. The results in Fig. 3 illustrate
A am-
intracellular Fig. 3. Effect of SK&F 96365 on agonist-induced Ca*’ mobilization in U-373 MG human astrocytoma (A) and SK-N-MC human neuroblastoma (B) cell lines incubated in Ca*+-free medium. Control cells (left traces) and cells pretreated with 20 pM SK&F 96365 for 3 min (right traces) were stimulated with 0.8 mM carbachol or 2% serum. Quantitative changes (C) were expressed as percentage change of the increased intracellular Ca*+ concentration induced by the drug compared with agonist alone. Each column represents mean f S.E.M. *P < 0.05 compared with agonist alone.
of agonist-induced increase of intracelFig. 2. Independence lular Ca*+ concentration from extracellular Ca*+ concentration in U-373 MG human astrocytoma (A) and SK-N-MC human neuroblastoma (B) cell lines. Carbachol (0.8 mM) or serum (2%) were used as agonists. Aliquots of 2 x IO6 cells/ml were incubated with 2 PM of Fura-2-AM for 60 min at room temperature (22-23°C). The cells were washed, resuspended in buffer solution containing 1.3 mM Ca2+ (left traces) or Ca?+free medium (right traces) and transferred to a quartz cuvette for fluorescence measurements. The data represents intracellular Ca*+ changes with time. The arrows show the time points for the addition of agonists.
that SK&F 96365 significantly inhibited agonistinduced Ca2+ mobilization in both cell lines. Although this inhibition required a much higher concentration of the drug than did the inhibition of tumor cell growth, these results suggest that the growth-inhibitory effect of SK&F 96365 is at least partially due to the interference with agonistinduced intracellular Ca2+ mobilization. The exact mechanism of this action of SK&F 96365 remains to be determined.
80
Discussion
Classic Ca2+ channel antagonists, such as verapamil, nifedipine and diltiazem, have been shown to inhibit cell proliferation in many different cellular systems, including tumor cells [ 1l- 141. Furthermore, growth factors increase intracellular Ca2+ concentration [ 15- 171, and therefore the role of Ca2+ influx in cell proliferation has been stressed [ 18-201. However, most non-excitable cells appeared not to have voltagesensitive Ca2+ channels [21,22], and so the Ca2+ entry pathway is obscure. Recently the Ca2+ entry mechanism into the cells by occupation of membrane receptors through receptor-operated Ca2+ channels has been described in many cellular systems [l-4]. The purpose of the present study was to evaluate the role of Ca2+ influx through this pathway in tumor cell growth and to examine the effect of SK&F 96365 - a known blocker of receptor-operated Ca2+ channels [5] - on cell growth, using two human brain tumor cell lines as model cellular systems. In this study we have found that SK&F 96365 very effectively inhibited the growth of human brain tumor cells. To our knowledge this is the first report of growth-inhibitory effect of the drug on tumor cells. A sustained increased intracellular Ca2+ concentration following an initial transient rise has been shown to be induced by carbachol and other growth factors in many cellular systems [4,20,23,24]. The results of the present study, in which no sustained increase of intracellular Ca2+ was induced by the agonists, suggest cell-type specific action of these agonists without an RMCE in the tumor cell lines used in this study. These results further suggest that in these tumor cells, agonist-induced Ca2+ mobilization from internal stores is an important signalling mechanism in cell proliferation. Furthermore, since SK&F 96365 significantly inhibited agonist-induced Ca2+ mobilization, the growth-inhibitory effect of this drug may be at least partially attributed to the interference with agonist-induced intracellular Ca2+ mobilization. In conclusion, SK&F 96365, a receptoroperated Ca 2+ channel blocker, effectively inhibited brain tumor cell growth, and this effect may
be due to the inhibition of agonist-induced intracellular Ca2’ mobilization from internal stores. These results suggest a possible application of this drug to the management of brain tumor cell proliferation. Acknowledgements
This study was supported by the Mr. and Mrs. Barney Kahn Fund and NIH Grant GM 32288. References 1
2 3 4
5
6
7
8 9
10
11
12
13
14
Sage, 0.. Mahaut-Smith. M.P. and Rink, T.J. (1992) Calcium entry in nonexcitable ceils: lessons from human platelets. News Physiol. Sci., 7, 108-I 13. Putney, J.W. (1991) Receptor-regulated calcium entry. Pharmac. Ther., 48, 427-434. Rink, T.J. (1990) Receptor-mediated calcium entry. FEBS, 268, 381-385. Fischer, H., Illek, B., Negulescu, P.A., Clauss. W. and Machen, T.E. (1992) Carbachol-activated calcium entry into HT-29 cells is regulated by both membrane potential and cell volume. Proc. Nat. Acad. Sci. USA, 89. 1438-1442. Merritt. J.E., Armstrong, W.P., Benham. C.D., Hallam, T.J.. Jacob, R., Jaxa-Chamiec, A., Leigh, B.K., McCarthy, S.A.. Moores, K.E. and Rink, T.J. (1990) SK&F 96365, a novel inhibitor of receptor-mediated calcium entry. Biochem. J., 271. 515-522. Veigl, M.L., Vanaman, T.C. and Sedwick, W.D. (1984) Calcium and calmodulin in cell growth and transformation. Biochim. Biophys. Acta, 738, 21-48. Whitlield, J.F.. Boynton, A.I., MacManus. J.P., Rixon. R.H., Sikorska, M., Tsang, B.K. and Walker, P.R. (1980) The roles of calcium and cyclic AMP in cell proliferation. Ann. NY Acad. Sci.. 339. 216-240. Whitfield, J.F. (1992) Calcium signals and cancer. Critical Rev. Oncogenesis, 3, 55-90. Metcalfe, J.C., Moore, J.P.. Smith, G.A. and Hesketh, T.R. (1986) Calcium and cell proliferation. Br. Med. Bull.. 42, 405-412. Grynkiewicz, G.. Poene, M. and Tsien. R.Y. (1985) A new generation of Ca ” indicators with greatly improved fluorescence properties. J. Biol. Chem.. 260, 3440-3450. Taylor, J.M. and Simpson. R.U. (1992) Inhibition of cancer cell growth by calcium channel antagonists in the athymic mouse. Cancer Res., 52, 2413-2418. Shultz, P.J. and Raij, L. (1990) Inhibition of human mesangial cell proliferation by calcium channel blockers. Hypertension 15 (suppl. I), I-76-1-80. Birx, D.L., Berger, M. and Fleisher. T.A. (1984) The interference of T cell activation by calcium channel blocking agents. J. Immunol., 133, 2904-2909. Schuller, H.M.. Orloff, M. and Reznik, G.K. (1991) Anti-
81
15
16
17
18
19
proliferative effects Iof the Ca*+/calmodulin antagonist B859-35 and the C,a*+/channel blocker verapamil on human lung cancel- cell lines. Carcinogenesis, 12, 2301-2303. Moolenaar, W.H., Tsertoolen, L.G.J. and de Laat, S.W. (1984) Growth factors immediately raise cytoplasmic free Ca2+ in human fibroblasts. J. Biol. Chem.. 259, 8066-8069. Nishimoto, I., Ghkuni, Y., Ogata, E. and Kojima, I. (1987) Insulin-like growth factor II increases cytoplasmic free calcium in competent Balbic 3T3 cells treated with epidennal growth factor. Biochem. Biophys. Res. Commun., 142, 275-286. Bonventre, J.V., Skorecki. K.L., Kreisberg. J.I. and Cheung, J.Y. (1986) Vasopressin increases cytosolic free calcium concentration in glomerulosa mesangial cells. Am. J. Physiol., 20, :F94-F102. Diliberto, P.A., Hubbert, T. and Herman, B. (1991) Early PDGF-induced alterations in cytosohc free calcium are required for mitogenesis. Res. Commun. Chem. Pathol. Pharmacol., 72, 3-12. Olsen, R., Seewald, M. and Powis, G. (1989) Contribution of external and internal Ca*+ to changes in intracellular
20
21
22
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