Intracellular Ca2+ mediates the cytotoxicity induced by bepridil and benzamil in human brain tumor cells

CANCER LETTERS ELSEVIER

Cancer Letters 88 ( 1995)87-91

Intracellular Ca2+ mediates the cytotoxicity induced by bepridil and benzamil in human brain tumor cells Yong So0 Leea7b,Mohammed M. SayeedaTb, Robert D. Wurster*a’b aDepartment of Neurological Surgery, Loyola University Medical Center, 2160 S. First Ave., Maywood, IL 60153, USA bDepartment of Physiology, byola University Medical Center, 2160 S. First Ave., Maywood, IL 60153, USA

Received 2 November 1994;accepted 4 November 1994

Abstract

The effectsof bepridil and benzamil,known Na+-Ca*+ exchangeblockers,on the growth of humanbrain tumor cellswereevaluatedusingSK-N-MC humanneuroblastomaandU-373MG humanastrocytomacellsasmodelcellular systems.Thesedrugsinducedcytotoxicity in both cellsin a dose-dependent manner.Agonist (2%fetal bovineserum) aloneinduceda rapid increasedintracellular Ca*+concentrationand then it returnedto the basallevel.However,the pretreatmentsof thesedrugsresultedin a moresustainedhigh intracellular Ca*+concentrationmobilizedby an agonist. Moreover,BAPTA/AM, an intracellular Ca*’ chelator, significantly blockedthe cytotoxicity inducedby these drugs.Theseresultssuggestthat bepridil and benzamilact as effectiveinhibitors of in vitro growth of humanbrain tumor cellsand that intracellular Ca*+may be involved in the mechanismof actionsof theseagents. Keywords:Bepridil; Benzamil;Human brain tumors;Intracellular Ca*+

1. Introduction An increasing body of evidence suggests that intracellular Ca2+ has an important role in the processesof cell proliferation [l] and cell death [2]. Previously, we have also shown that intracellular Ca*+ signalling mechanisms are involved in the modulation of cell growth and cell death by a number of drugs in brain tumor cell lines [3,4]. Bepridil (BP), an antianginal agent, has heen shown to potentiate rnitoxantrone-induced cytotoxicity in human chronic myeloid leukemia cells * Corresponding author.

[5] and to reverse anthracycline resistance in cancer patients [6]. Although the mechanisms of these actions of BP are unknown, the cardiac effects of BP are attributed to the alteration of intracellular Ca2+ signalling mechanisms, including decreased Ca2+ influx through the plasma membrane Ca2+channels and inhibition of Na+Ca2+ exchange [7]. Considering the importance of the Ca2+ signals in cell growth and the modulation of the signals by BP, we hypothesized that BP may affect the growth of tumor cells. In order to test this hypothesis, in this study we investigated the effects of hepridil and henzamil (BZ), which is an amiloride

0304-3835/9X$09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3835(94)03619-T

88

Y.S. Lee el ui. / Cmcer Lellrrs XX (I945 i X7-91

derivative and sets as an inhibitor of Na+-Ca2+ exchange fl], on the growth of human brain tumor cells. We explored the possible mechanism of action of these drugs relating to intracellular Ca’+, using SK-N-MC human neuroblastoma and U-373 MG human astrocytoma cell lines as model cellular systems. 2. Mate&Is

ad methods

2.1. Materials

U-373 MG human astrocytoma and SK-N-MC human neuroblastoma cell lines were purchased from American Type Culture Collection (Rockville, MA). The powders for Eagle’s minimum essential medium (MEM) and Earle’s basal salt solution (EBSS), trypsin solution, trypan blue, sodium pyruvate and all salt powders were obtained from Sigma Chemical Co. (St. Louis, MO). Bepridil hydrochloride (BP) and benzamil hydrochloride (BZ) were from RBI (Natick, MA). 1-(2,5-Carboxyoxazol-2-yl-S-aminobenzfuran-Soxyl)-2-(2 ’ -amino-5 ‘-methylphenoxy)-ethane-N, N,N’,N’-tetraacetoxymethyl ester (Fura-2/AM) and bis-(o-aminophenoxy)-ethane-N,ZV,N’,N’tetraacetic acid/acetoxymethyl ester (BAPTA/AM) were obtained from Molecular Probes, Inc. (Eugene, OR). Fetal bovine serum (FBS) and antibiotics (penicilline and streptomycin mixture) were purchased from GIBCO (Grand Island, NY). Stock solutions of the drugs were made in distilled water (BZ), ethanol (BP) or DMSO (BAPTA/AM) and added to the culture medium. 2.2. Cell culture

Cells were grown at 37°C in a humidified incubator under 5% CO,/95% air in MEM supplemented with 10% FBS, 200 II-J/ml penicilline, 200 &ml of streptomycin and 1 mM sodium pyruvate. Culture medium was replaced every other day. After attaining confluency the cells were subcultured following trypsinization. 2.3. Cytotoxicity

test

Cells from 4-5day-old cultures were seededin equal numbers in 35 x 10 mm culture dishes with a density of 2 x IO5 and 1.25 x 10’ cells/dish in SK-N-MC neuroblastoma and U-373 MG astro-

cytoma cells, respectively. The volume ofthe medium in the dishes was 2 ml. Drugs to be tested were added to cultures 1 day after seedingin order to insure uniform attachment of cells at the onset of the experiments. The cells were grown for an additional 2 days. Drugs and culture medium were replaced every day. In control experiments cells were grown in the same media containing drug-free vehicle. Cell viability was assessedby the trypan blue exclusion method. Total and viable cells which were stained with 0.2% trypan blue after trypsinization, were counted using a hemocytometer. The results are presented as percent change of the number of viable cells obtained in the absenceof the test drug. 2 4 Measurement of [ Ca2+].

’ Aliquots of the tumor &ls, cultured for 3-5 days, were washed in EBSS. Then, 2 PM Fura2lAM was ad&d, 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 min. Cells were resuspendedat a density of 2 x 106/ml in Ca2+-free Krebs-Ringer buffer (KRB) containing 125 mM NaCl, 5 mM KCl, 1.2 mM KHzP04, 1.2 mM MgS04, 5 mM NaHCOs, 25 mM HEPES and 6 mM glucose (pH 7.4), transferred to a quartz cuvette and stirred continuously. Fluorescence emission (510 nm) was monitored with the excitation wavelength cycling between 340 and 380 nm at 37°C 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 Ca2+ concentrations using Fura- Ca2+ binding constant (224 nM) and the formula described by Grynkiewicz et al. [9]. 2.5. Data analysis

Data are expressedas mean f standard error of the mean (S.E.M.), and were analysed using one way analysis of variance and the Student-Newman-Keul test for individual comparisons. P values less than 0.05 are considered to be statistically significant.

Y.S. Lee rl al. /Cancer

5160 g 140 0 120 5100 4 60 0 60 2 40 .$ 20 ‘s P

60 60 40 20

0 -6

-5

4

-6

-5

4

(DRUG1 (log Ml

Fig. I. BP and BZ induce cytotoxicity in SK-N-MC human neuroblastoma (A) and U-373 MG human astrocytoma cells (B). Results are expressedas percent change of the number of viable cells obtained in the absence of the drug. Data points represent the mean values of four replicates with bars indicating S.E.M.

3.Resdts As shown in Fig. l., BP induced dual effects on the growth of SK-N-MC human neuroblastoma and U-373 MG human astrocytoma cells. At 5 CM, BP significantly enhanced the growth of both

Lei1rrs

88 (19951

XY

87-YI

cells. At concentrations over 5 PM, BP induced a dose-dependent cytotoxicity in both cells. Halfmaximal concentrations (ICsO)of BP were about 8-9 PM and lo-20 FM for SK-N-MC and U-373 MG cell lines, respectively. However, BZ induced only cytotoxicity in a dose-related manner in both cells. I&, values of BZ were about 20-30 PM and 30-40 PM for SK-N-MC and U-373 MG cell lines, respectively. The effectsof BP and BZ on agonist-induced intracellular Ca*+ mobilization in SK-N-MC cells were examined, and the results are depicted in Fig. 2. In these experiments 2% FBS was used for mobilizing intracellular Ca*+ [3]. The data show that agonist alone induced a rapid increased intracellular Ca*+ concentration (first phase) and then it returned to the basal level (second phase) (Fig. 2A). The pretreatments of BP (9 PM) or BZ (20 PM) did not alter the first phase response to 2% FBS (Fig. 2B,C). However, the second phase response to the agonist was profoundly affected by thesedrugs. They blocked the decline of intracellular Ca*+ to basal levels, and resulted in a sustained increased intracellular Ca*+ concentration (Fig. 2B,C). Moreover, these drugs alone did not affect the basal levels of intracellular Ca2+.

A.

B.

Control

BP(9uM)

C. BZ(20uM)

250 sec. Fig. 2. Effects of BP and BZ on agonist (2% FBS)-induced intracellular Ca*+ mobilization in SK-N-MC human neuroblastoma cells. The data represent intracelluiar Ca*+ changes with time. The arrows show the time points for the addition of 2% FBS. BP and BZ were applied 3 min before fluorescencemeasurements.

3

B.

A.

280

,120

,

; 60

100 80

a8 40

60

4 20

40

.g

20

z -8

0 BP

BZ

Ni

’

,

I

BP

BZ

Ni

Fig. 3. BAPTA/AM protects against the BP, BZ or Ni2+induced cytotoxicity in SK-N-MC human neuroblastoma (A) and U-373 MG human astrocytoma cells (B). 1.5 pM of BAPTAIAM was used in these experiments. BP (11, 14.5PM), BZ (22.5, 34 pM) and NiZ+ (300, 400 CM) were used in these experiments for (SK-N-MC,U-373 MG) cells, respectively. Results are expressedas percent change of the number of viable cells obtained in the absenceof the drug. Data points represent the mean values of four replicates with bars indicating S.E.M.

BAPTAIAM, an intracellular Ca*+ chelator [lo], significantly inhibited the cytotoxicity induced by BP, BZ or Ni2+ in both cells as shown in Fig. 3. The effects of Ni2+ ions were included in this study because they have been reported to inhibit the Na+-Ca*+ exchange [II] and to induce cytotoxicity in the brain tumor cells used in the present study [3]. 4. Ditmmh Previously, we have found that in both cell proliferation and cell death of human brain tumor cells, intracellular Ca*+ acts as an essential signal transducer [3,4]. The major finding of this study is that interference with intracellular Ca*+ homeostatic mechanism by Na+-Ca2+ exchange blockers results in the induction of cytotoxicity in human brain tumor cells. Although its physiological role is poorly understood, the presenceof Na+-Ca*+ exchanger in the brain tissues (neurons and astrocytes) has been demonstrated [ 12,131.In other excitable cells the exchangerplays a major role in the rapid exclusion of Ca2+during recovery from activation [ 141. Considering possible regulation of intracellular Ca2+ homeoststic mechanisms by Na+-Ca2+ ex-

changer, we investigated the effects of BP and BZ which are known blockers of Na+-Ca*+ exchanger [8], on the growth of human brain tumor cells using SK-N-MC human neuroblastoma and U-373 MG human astrocytoma cell lines as model tumor cell systems. In the present study we clearly showed that BP and BZ induced cytotoxicity in both human brain tumor cells in a dose-dependent manner (Fig. 1). Although BP has been previously shown to potentiate mitoxantrone-induced cytotoxicity in human chronic myeloid leukemia cells [5] and to reverse anthracycline resistance in cancer patients [6], in these studies the mechanism of action of BP has not been explored. In the present study, however, we examined the possible involvement of intracellular Ca*+ signals in these cytotoxic actions of BP and BZ. The pretreatments of BP and BZ blocked the decline to basal levels of intracellular Ca*+ mobilized by serum, resulting in a more sustained high intracellular Ca*+ concentration (Fig. 2). These results suggest that intracellular Ca2+ may be an important mediator of the actions of BP and BZ. In a number of studies sustained increased intracellular Ca*+ has been demonstrated to be a common mediator of chemical-induced cell death [2,10,15, 161.Blockade of the cytotoxicity by BAPTA/AM, an intracellular Ca*+ chelator (Fig. 3), further supports the role of increased intracellular Ca2+ in the cytotoxicity induced by BP and BZ. Previously, we have reported that Ni*+ ions, which are known to inhibit Na+-Ca*’ exchangers [ 111, also caused concentration-dependent cytotoxicity in human brain tumor cell lines [3]. Interestingly, the results of the present study show that BAPTA/AM also significantly blocked the Ni*+-induced cytotoxicity (Fig. 3). Although we did not directly measurethe activity of Na+-Ca*+ exchanger, these results suggest that the Na+-Ca2+ exchange mechanism may have an important involvement in the regulation of intracellular Ca*+ homeostasis. These results further suggestthat inhibition of the Na+-Ca*+ exchange mechanismby antagonists induce disruption of intracellular Ca*+ homeostasis, which in turn, induces cell cytotoxicity. In conclusion, these results suggestthat bepridil

Y.S. Lee et ul. /Cancer

and benzamil act as effective inhibitors of in vitro growth of human brain tumor cells. In addition, sustained increased intracellular Ca*+ levels may be the mechanismof the cytotoxic actions of these agents. Acknowledgements This work was supported by the J. Richard Wolfe Foundation and the Mr and Mrs Barney Kahn Fund. References [l] Villemal, M.L. and Byron, K.L. (1992) Calcium signals in growth factor signal transduction. Rev. Physiol. B&hem. Pharmacol., 119, 67-121. [2] Orrenius, S., McConkey, D.J., Bellomo, G. and Nicotera, P. (1989) Role of Ca2+ in toxic cell killing. Trends Pharmacol. Sci., 10, 281-285. [3] Lee, Y.S., Sayeed, M.M. and Wurster, R.D. (1994) Inhibition of cell growth and intracellular Ca2+ mobilization in human brain tumor cells by CaZ+ channel antagonists. Mol. Chem. Neuropathol., 22, 81-95. [4] Lee, Y.S. and Wurster, R.D. (in press) Dual effects of estrogen and antiestrogens on the growth of SK-N-MC human neuroblaatoma cells. Cancer Lett. (in press). [S] Parekh, H.K., Shallom, J.M., Advani, S.H. and Chitnis, M.P. (1991) Responseof human chronic myeloid leukemia cells to mitoxantrone cytotoxicity: Potentiation by bepridil, a calcium channel antagonist. Neoplasm, 36, 575-581. [6] van Kallen, C.K., van der Hoeven, J.J.M., de Jong, J., Giaccone, G., Schuurhuis, G.J., Maessen, P.A., Blokhuis, W.M.D., van der Vijgh, W.J.F. and Pinedo, H.M. (1991)Bepridil in combination with anthracyclines

Letters

88 (1995)

87-91

91

to reverse anthracychne resistance in cancer patients. Eur. J. Cancer, 27, 739-744. [71 Gill, A., Flaim, S.F., Damiano, B.P., Sit, S.P. and Brannan, M.D. (1992)Pharmacology of bepridil. Am. J. Cardiol., 69, 1lD-16D. 181Kaczorowski, G.J., Slaughter, R.S., King, V.F. and Garcia, M.L. (1989)Inhibitors of sodium-calcium exchange: Identification and development of probes of transport activity. B&hem. Biophys. Acta, 988, 287-302. [91 Grynkiewicz, G., Poene, M. and Tsien, R.Y. (1985) A new generation of Ca2+ indicators with greatly improved fluorescenceproperties. J. Biof. Chem.. 260, 3440-3450. 1101Jiang, S., Chow, S.C., Nicotera, P. and Grrenius, S. (1994) Intracellular Ca2+ signals activate apoptosis in thymocytes: Studies using the Ca2+-ATPase inhibitor thapsigargin. Exp. Cell Res., 212, 84-92. 1111Coetzee,W.A., Ichikawa, H. and Hearse, D.J. (1994)Oxidant stressinhibits Na-Ca-exchange current in cardiac myocytes: mediation by sulthydryl groups? Am. J. Physiol., 266, H909-H919. [121Kofuji, P., Hadley, R.W., Kieval, R., Lederer, W.J. and Schulze,D.H. (1992) Expression of the Na/Ca exchanger in diverse tissues:a study using the cloned human cardiac Na/Ca exchanger. Am. J. Physiol., 263, C1241-C1249. iI31 Goldman, W.F., Yarowsky, P.J., Juhaszova, M., Krueger, B.K. and Blaustein, M.P.(1994) Sodium/calcimn exchangein rat cortical astrocytes. J. Neurosci., 14, 5834-5843. (141 Blaustein, M.P., Goldman, W.F., Fontana, G., Krueger, B.K., Santiago, E., Steel, T.D., Weiss, D.N. and Yarowsky, P.J. (1991) Physiological roles of the sodium-calcium exchanger in nerve and muscle. Ann. N.Y. Acad. Sci., 639, 254-274. 1151 Schanne, F., Kane, A.B., Young, E.E. and Farber, J.L. (1979) Calcium dependenceof toxic cell death. Science, 206, 700-702. [I61 Duke, R.C., Witter, R.Z., Nash, P.B., Young, J.D.-E. and Ojcius, D.M. (1994) Cytolysis mediated by ionophores and pore-forming agents: Role of intracellular calcium in apoptosis. FASEB J., 8, 237-246.