thermosensitization and modification of cytosolic calcium concentration by verapamil and diltiazem...

Int. J. Radiation Oncology Biol. Phys., Vol. 29, No. 3, pp. 51 l-517. 1994 Copyright 0 1994 Elsevier Science Ltd Printed in the LISA. All rights reserved

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??Basic Studies on Multimodality Cancer Treatment

THERMOSENSITIZATION AND MODIFICATION OF CYTOSOLIC CALCIUM CONCENTRATION BY VERAPAMIL AND DILTIAZEM

IN MOUSE MAMMARY CARCINOMA CELLS

TAKASHI KONDO, D.V.M., PH.D.,* YOSHIAKI HABARA, D.V.M., PH.D.,+ TOMIO KANNO, M.D., PH.D.+ AND EIICHI KANO, M.D., PH.D.$

*Department of Radiation Biophysics, Kobe University School of Medicine, Kusunoki-cho 7-5 1, Chuo-ku, Kobe 650; +Laboratory of Cellular Metabolism, Department of Molecular Physiology, National Institute for Physiological Sciences, Okazaki

444; *Department of Experimental Radiology and Health Physics, Fukui Medical School, Matsuoka, Fukui 9 IO- I 1, Japan

Purpose: The cardinal role of Ca2+ signaling in the development of heat damage at 42°C and 44°C and the effect of verapamil and diltiazem on the change of cytosolic Ca2+ concentration, [Ca’+],, were investigated. Methods and Materials: Hyperthermic treatment was performed by immersing the tubes containing the mouse mammary carcinoma FM3A cells in a water-bath set at 42.O”C or 44.0% The [Ca2’le of a single cell after hyperthermia was monitored by a digital image analyzing technique using Fura-2. Influx of Ca2’ was examined by the measurement of the radioactivity of 45Ca2+ incorporated into the cells for 60 min during or after hyperthermia. Results: While the [Ca2’lc of the cells treated at 37°C for 60 min was less than 230 nM, the percentage of the cells showing more than 230 nM increased after 42°C hyperthermia and that of the cells showing more than 300 nM increased after 44°C hyperthermia. When cellular uptake of 4sCa2+ during and after hyperthermia was examined, the increase in 4sCa2+ uptake was observed only for 44°C hyperthermia. Verapamil or diltiazem (100 wM) enhanced a delay of cell growth, an increase in (Ca2+]c and an increase in 4sCa2t influx for 42°C and 44°C hyperthermia. Conclusion: These results are compatible with the view that the increase in [Ca2’]P after 42°C hyperthermia results from intracellular release from calcium store sites whereas the rise in [Ca2+], after 44°C hyperthermia is mainly due to entry of extracellular Ca2+. Verapamil or diltiazem combined with hyperthermia increased [Ca2’10 which may play a cardinal role in thermosensitization by these agents.

Calcium, Hyperthermia, Verapamil, Diltiazem, Neoplastic cells, Cultured.

INTRODUCIION

Calcium ion is known to play a key regulatory role in cellular metabolism (15). A possible common mechanism for the hyperthermia, changes in cytosolic Ca*+ concentration, [Ca*+],, have been regarded as a major intracellular signal among various factors responsible for development of heat damage (S-14, 16). Recently, analysis of [Ca2’], at the single cell level by digital imaging microscopy was applied to HT-29 human colon cancer cells treated with hyperthermia (12). The advantage of this method is that it allows one to discriminate the cells showing different [Ca*‘], and quantitate it at a single cell level.

atures lower than 43°C and those higher than 43°C. An additional purpose was to find the therapeutic basis of the drugs which are called Ca*+ antagonists, verapamil and diltiazem, since it has been shown that verapamil and diltiazem enhanced hyperthermic cell killing (2).

In this study, a digital image analyzing technique using Fura- was used for monitoring the [Ca*‘], of a single mouse mammary carcinoma FM3A cell after 42°C or 44°C hyperthermia. The effects of verapamil and diltiazem on delay of cell growth, change in [Ca2+lo and influx of 45Ca2f induced by 42°C or 44°C hyperthermia were investigated.

The main purpose of the present study was to establish the cardinal role of Ca*+ signaling in the development of heat damage and to address the question whether the source in [Ca”], is different in hyperthermia at temper-

METHODS AND MATERIALS

Mouse mammary carcinoma cells, FM3A, were ob- tained from the Japanese Cancer Research Resource

Presented at Eighth International Conference on Chemical Modifiers of Cancer Treatment, Kyoto, Japan, 16- 19 June 1993.

Reprint requests to: Dr.Takashi Kondo. Acknowledgements-The present research activity was in part supported by a Grant in Aid for Cancer Research from the Min-

511

istry of Education, Science and Culture, Japan. A part of this work was performed at the National Institute for Physiological Sciences at Okazaki with financial support.

Accepted for publication 22 October 1993.

512 1. J. Radiation Oncology 0 Biology 0 Physics Volume 29, Number 3, I994

Bank. The cells were grown in Eagle’s minimal essential medium (MEM) supplemented with 10% newborn calf serum. ’ Hyperthermic treatment was performed by immersing the tubes containing the cells in a water-bath set at 42.O”C or 44.O”C (+ O.OS”C). Temperature was monitored by a digital thermometer during experiments.

The cells were collected by centrifugation and washed with HEPES-buffered Ringer (HR) solution (/, pH 7.4). The composition of HR was: NaCl, 118 mM; KCl, 4.7 mM; CaC12, 2.5 mM; MgC12, 1.13 mM; Na2HP04, 1.0 mM; glucose, 5.5 mM and HEPES, 10 mM. The buffer was supplemented with 0.2% bovine serum albumin2 minimal Eagle’s essential amino acids3 and 2 mM L- glutamine. About 3 X lo5 cells in 3 ml HR solution were loaded with 10 PM Fura-2-AM4 for 30 min at 25°C and subsequently for 30 min at 37°C. The cells loaded with Fura-2-AM were washed once with HR and twice with the growth medium by centrifugation. After hyperthermia the cells were washed with HR and an aliquot of cell sus- pensions ( 10 ~1) was transferred onto a coated glass5 held in a chamber.6 After addition of HR to the chamber, digital imaging of Fura- fluorescence was carried out with an inverted microscope’ and a digital image processor’ as reported previously (6, 7). The fluorescence ratio (340/ 380 nm) at 5 10 nm was converted to [Ca2’], using the equation described by Grynkiewicz et al. (5).

different [Ca2+], is displayed in Figure 1. The number of cells showing higher [Ca2’], increased as the temperature increased. The appearance of the cells containing higher [Ca2’], was characteristic of the cells treated with hyperthermia, especially with 44°C hyperthermia. The number of cells showing more than 230 nM of [Ca”], (ratio 2.5) and more than 300 nM of [Ca2’], (ratio 3.0) was counted to evaluate the different effect of 42°C and 44°C hyperthermia on [Ca2’],. The percentage of cells showing more than 230 nM increased after 42°C and 44°C hyperthermia and the percentage of cells showing more than 300 nM increased after 44°C hyperthermia (Table 1).

The radioactivity of 45Ca2+ taken up by the cells during and after 42°C or 44°C hyperthermia was shown in Table 2. Although the radioactivities (dpm/5 X lo6 cells) of 45Ca2f incorporated during and after 42°C hyperthermia were not significantly different from those during and after 37°C incubation, the significant increase in 45Ca2+ uptake was observed only during and after 44°C hyperthermia. These results indicate that the increase of [Ca2’], induced by 44°C hyperthermia is mainly due to incorporation of Ca2+ from outside of cells.

kfects of verapamil and diltiazem

The cellular uptake of Ca2+ during hyperthermia was determined by incubating 5 ml of cell suspension ( lo6 cells/ml) at 37°C 42°C or 44°C for 60 min in the medium in the presence of 74 KBq/ml of 45CaC12.9 For measurement of 45Ca2+ uptake after hyperthermia, tubes containing the cells in the growth medium were immersed for 60 min in a water-bath set at 37°C 42°C and 44’C followed by 60 min-incubation at 37°C in the presence of 74 KBq/ml of 45CaC12. The washing procedure used in this study has been described in the papers previously reported (10, 11). Following the last wash, the cells were solubilized with sodium deoxycholate (0.1% in 0.1 N NaOH) and the radioactivity of 45Ca2+ was counted with a liquid scintillation counter. lo

When the cells were treated with 42°C and 44°C hyperthermia in the presence of verapamil or diltiazem at a concentration of 100 /IM, an enhancement of delay of the cell growth induced by both treatments was observed (Fig. 2). Significant enhancement of delay of growth by these chemicals was observed in the cells treated with 44°C hyperthermia, but the combined effect with 42°C hyperthermia was minimal.

RESULTS

Efects of 42” C and 44°C hyperthermia

The [Ca2’], measured for the cells treated at 37°C for 60 min was 170.8 _+ 14.8 nM (mean + SD) (Fig. 3). After treatment of 42°C or 44’C, the [Ca2’], increased to 230.6 -t26.2nM(134.9%)or265.8+32.5nM(155.5%).Inthe presence of 100 PM verapamil, the [Ca”], of the cells increased from 178.9 & 12.5 nM at 37°C to 255.8 f 31.5 nM (142.9%) after 42°C hyperthermia and to 276.3 f 50.7 nM (154.4%) after 44°C hyperthermia. In the presence of 100 PM diltiazem, the [Ca*‘], also increased from 208.8 k 16.6 nM to 226.2 * 20.4 nM (108.3%) after 42°C hyperthermia and to 289.8 -t 4 1.1 nM (138.8%) after 44°C hyperthermia (Fig. 3).

The survival of the cells estimated by a colony-forming The percentage of the cells containing more than 230 assay in soft agar was 60.6 + 7.5% (mean f SD) for 42°C nM of [Ca”], increased from 45.0 * 13.4% (control with- hyperthermia (60 min) and < 0.1% for 44°C hyperthermia out drugs) to 67.3 f 6.2% (with verapamil) and to 62.3 (60 min). The [Ca”], in individual cells after 42°C or +- 12.3% (with diltiazem) after 42°C hyperthermia. The 44°C hypetthermia for 60 min was measured by digital increase of the percentage after 44°C hyperthermia was imaging microscopy. After analyzing the 100 randomly minimal. The percentage of the cells containing more than selected cells, the histogram of number of cells showing 300 nM of [Ca2’], increased from 3.5 _t 2.2% to 11.0 ?

’ Gibco, USA. ’ Sigma, USA. 3 Flow Laboratories, USA. 4 Dojindo Laboratories, Japan. 5 Cell-Tak, Collaborative Research, USA.

6 Sykus-Moore Chamber. ’ TMD-2, Nikon, Japan. ’ Argus- I OO/Ca, Hamamatsu Photonics, Japan. 9 Amersham, UK. lo Scintisol EX-H, Dojindo Laboratories, Japan.

Hyperthermia and calcium 0 T. KONW et al. 513

20 2.5 3.0 3.5 4.0 4.5

Ratlo (F34O/F380)

I 1 I I

100 200 300 400 500

Ca concentration (nM)

Hyperthermia (42 “C, 1 h)

1.5 20 25 3.0 3.5 4.0 4.5

Ratio (F34O/F380)

200 300 400 5&J


.C Hyperthermia (44 “C, 1 h)

0’ .-.

1.5 20 2.5 3.0 3.5 4.0 4.5

Ratio (F34O/F380)

I

100 1 I I 8

2ocl 300 400 500


Fig. 1. Histogram analysis of the effects of hyperthermia on [Ca2+lc in mouse mammary carcinoma FM3A cells. For measurement of [Ca*+], in a single cell, the square area (14 X 14 pixels, 49 km*) within a cell was manually selected and the average [Ca*‘], in the area was calculated in 100 randomly selected cells after 37°C (A), 42°C (B) and 44°C (C) hyperthermia. Arrows in (A) and in (B) indicate 230 nM (ratio 2.5) and 300 nM (ratio 3.0) of [Ca*+]_ respectively.

514 1. J. Radiation Oncology 0 Biology 0 Physics Volume 29, Number 3, 1994

Table 1. Percentage of cells containing more than 230 and 300 nM of [Ca*‘], after 42°C and 44°C hyperthermia

Condition Control With verapamil With diltiazem

> 230 nM 37°C 5.0 + 6.1 5.3 f 5.4 13.7 k 7.0 42°C 45.0 + 13.4 67.3 +- 6.2 62.3 + 12.3 44°C 71.3 f 20.1 72.5 f 7.9 83.6 f 5.1

> 300 nM 37°C 0 0 0 42°C 3.5 + 2.2 11.0 f 6.5 7.3 + 6.5 44°C 21.3 + 10.3 33.5 k 10.6 31.3 + 12.3

Concentration of verapamil and diltiazem was 100 PM. Values are means k SD of three independent experiments.

6.5% (with verapamil) and 7.3 + 6.5% (with diltiazem) after 42°C hyperthermia and from 21.3 f 10.3% to 33.5 f 10.6% (with verapamil) and 31.3 +- 12.3% (with diltiazem) after 44°C hyperthermia (Table 1).

The radioactivity of 45Ca2+ taken up by the cells during and after hyperthermia with or without verapamil and diltiazem is shown in Table 2. When the cells were treated with 44°C hyperthermia in the presence of verapamil and diltiazem at a concentration of 100 PM, 45Ca2f uptake was enhanced during and after hyperthermia. A slight enhancement was also observed in the cells after 42°C hyperthermia in the presence of verapamil and diltiazem. However, insignificant and minimal increase in 45Ca2+ uptake during 42°C hyperthermia was observed (Table 2). These results demonstrated that verapamil and diltiazem accelerate the incorporation of Ca*+ from outside of cells after 42°C hyperthermia, as well as after 44°C hyperthermia.

DISCUSSION

The present study provided direct evidence that the [Ca*‘], in mouse mammary carcinoma FM3A cells increased after 42°C and 44°C hyperthermia. The digital image analyzing technique using Fura- was applied for the measurement of [Ca”], in a single cell of FM3A cells

after 42°C and 44°C hyperthermia and could discriminate between cells containing different [Ca*‘],. The different patterns of change in [Ca2+lc induced by 42°C and 44°C hyperthermia reflected the different mechanism of increase in [Ca*‘],. The [Ca*‘], of most cells treated at 37°C for 60 min was less than 230 nM and the percentage of the cells showing more than 230 nM of [Ca*‘], increased after 42°C hyperthermia. In contrast, the percentage of cells showing more than 300 nM increased after 44°C hyperthermia. When uptake of 45Ca2+ by the cells during and after hyperthermia was examined, the increase in 45Ca2+ uptake was observed only for 44°C hyperthermia. These results suggest that the increase in [Ca2’], induced by 42°C hyperthermia is due to release of Ca*+ from intracellular store sites and the increase in [Ca”], induced by 44°C hyperthermia is mainly due to the influx of extracellular Ca*+.

The present study demonstrated that verapamil and diltiazem, which are called calcium antagonists, enhanced the increase in [Ca*‘], and the delay of the cell growth induced by hyperthermia. These agents also potentiated the uptake of 45Ca *+ The increase in mean values of . [Ca*‘], induced by 42°C and 44°C hyperthermia in the presence of verapamil and diltiazem was similar to or slightly less than those induced by 42°C and 44°C hyperthermia without drugs. However, the increase of the

Table 2. Radioactivity of 45Ca2+ incorporated in mouse mammary carcinoma FM3A cells during and after hyperthermia

Condition Control With verapamil With diltiazem

During hyperthermia 37°C 42°C 44°C

After hyperthermia 37°C 42°C 44°C

853 + 25 1042 + 150 1001 t 128 835 k 38 970 f 73* 913 f 123

1517 + 119 1910 k 124+ 2322 + 124t

906 k 101 987 + 95 916 -e 105 900 + 108 1299 k 161* 1213 t 121+

1853 -t 211 2315 + 194* 2357 k 257*

Radioactivity is expressed as dpm/5 X lo6 cells. Concentration of verapamil and diltiazem was 100 PM. Values are mean +- SD of 3-5 experiments. Statistical significance was examined by Student’s t-test. *(p < 0.05) and +(a < 0.01) compared with control values without drug.

Hyperthermia and calcium 0 T. KOND~ et al. 515

+ 37c

+ 37 C + Verapamil

---C--- 42 “C

--S- 42 -C + Verapamil

106

I J 40 60 80 100

Time (hrs)

103 1 1 I 0 20 40 60 60 100

Time (hrs)

I u 37 c C --O-- 37 C + Diltiazem

U 42 C

t --)- 42 C + Diltiazem -

0 20 40 60 80

Time (hrs)

--o-- 37 c

--C 37 C + Diltiazem

---u-- 44c 106 --W- 44 C + Diltiazem

20 40 60

Time (hrs)

80 100

Fig. 2. Growth curve of mouse mammary carcinoma FM3A cells after hyperthermic treatments for 60 min. (A) 42°C hyperthermia with verapamil, (B) 44°C hyperthermia with verapamil, (C) 42°C hyperthermia with diltiazem, (D) 44°C hyperthermia with diltiazem. Drug concentration is 100 PM.

percentage of cells showing more than 230 nM was 1.49 The present results showing the delay of cell growth times (with verapamil), and 1.38 times (with diltiazem) supported previous findings that verapamil and diltiazem after 42°C hyperthermia, and that of the percentage of enhanced hyperthermic cell killing of Chinese hamster the cells containing more than 300 nM was 1.57 times ovary cells (2). Thermosensitization by these agents ap- (with verapamil), and 1.46 times (with diltiazem) after pears to be responsible for increase in [Ca*+],, since it was 44°C hyperthermia. These results indicate that the in- found that both agents enhanced the influx of 45Ca2+ dur- crease in [Ca*‘], in a single cell was not reflected in the ing and after 44°C hyperthermia, and even after 42°C corresponding increase in the mean value of [Ca*‘],. The hyperthermia. Verapamil has been shown to inhibit both increase in [Ca2’], induced by hyperthermia in the pres- Ca*+ sequestration by and release from sarcoplasmic re- ence of verapamil or diltiazem was due to heterogeneous ticulum (1). The present study indicated that verapamil increase in the fraction of cells showing higher [Ca*‘],. and diltiazem did not act as calcium channel blocker or Therefore the measurement and analysis of [Ca2+lc at a inhibitor of release from intracellular store site under hy- single cell level is essential for evaluating changes in [Ca”], perthermic condition. Various actions of verapamil have induced by hyperthermia and/or combination with been reported including inhibition of calmodulin-insen- chemicals. sitive and -sensitive cyclic nucleotide phosphodiesterase,

516 I. J. Radiation Oncology 0 Biology 0 Physics Volume 29, Number 3, 1994

37 “C + Verapamil

1.5 20 25 a.0 3.5 1.0 ..*

Ratio (F34O/F380)

0 v.5 ..O 20 25 3.0 3.5

Ratio (F34O/F380)

I -- (00 200 300 0 500

ca concentration (IN)

Irn 200 Yy) do sm

Cd concentration (nM)

42 ‘C + Verapamll

1.5 *o 21 a.0 3.5 40 4s

Ratlo (F34O/F380)

100 200 300 100 500


44 ‘C + Verapamll

15 20 2s 3.0 a.5 1.0 a

Ratio (F34O/F380)

r loo zlo aon 400 son


42 “C + Diltiazem

20 2.5 3.0 35 .o .5

Ratio (F34O/F380)

100 200 wo 400 SW


-1 zo 2.b 3.0 3.5 ,.o .5

Ratio (F34O/F380)

100 rm 300 m so0


Fig. 3. Histogram analysis of the effects of hyperthermia on [Ca”], in mouse mammary carcinoma FM3A cells. Measurement of [Ca2’], in 100 randomly selected cells was recorded after 42°C and 44°C hyperthermia in the presence of verapamil and diltiazem at a concentration of 100 PM. 37°C (A), 42°C (B) and 44°C (C) for 60 min in growth medium containing verapamil. 37°C (D), 42°C (E) and 44°C (F) for 60 min in growth medium containing diltiazem.

antagonism of calmodulin activation of the phosphodiesterase, and binding to calmodulin (3). Compromising action on calmodulin-activated processes and/or reducing effect on the ability of the endoplasmic reticulum or mi- tochondria which regulate intracellular free Ca2+ levels has been proposed as a possible mechanism of increase in [Ca2’], in the cells treated with hyperthermia (2). The detailed mechanism of verapamil and diltiazem to en-

hance the increase in [Ca2’], induced by hyperthermia is not fully understood and remains for future study. Mod- ifiers of calcium metabolism may be worthy of study in vivo as local thermosensitizers, since enhancement of hyperthermic cell killing by calmodulin antagonists has been reported (4). Hyperthermia appears to be a useful tool to study new pharmacological actions of drugs in cancer therapy.

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Hyperthermia and calcium 0 T. KONDO et al. 517

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