The effect of hyperthermia on the uptake and cytotoxicity of melphalan in chinese hamster ovary cells

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16. pp. 187-191

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0360-3016189 $3.00 + .OO 8 1989 Pergamon Press plc

0 Original Contribution THE EFFECT OF HYPERTHERMIA ON THE UPTAKE AND CYTOTOXICITY OF MELPHALAN IN CHINESE HAMSTER OVARY CELLS DIANA

A. BATES,

PH.D.

AND WILLIAM

J. MACIULLOP,

M.B.,

CH.B.

McGill Cancer Centre, McGill University, Montreal, Quebec, Canada The effect of temperature on the cytotoxicity of melphalan in CHO cells was studied in an in vitro clonogenic assay. The cytotoxicity of melphalan was significantly increased at elevated temperatures with a 4 fold increase in cytotoxicity at 42°C compared to 37°C. The effect of temperature on membrane permeability to melphalan was studied to determine whether the increase in cytotoxicity was due to increased intracellular drug levels. Melphalan influx and efflux rates both increase with increasing temperature. There is, however, a small net increase in steady state intracellular drug levels with increasing temperature with a 20% increase in intracellular drug levels at 42°C comuared to 37°C. The increase in drug- uptake observed is insufficient to explain the much greater increase in cytotoxicity with increasing temperature. Hyperthermia,

Melphalan, Thermochemotherapy.

peratures on membrane permeability to melphalan, and have considered dosimetric effects of heat on melphalan cytotoxicity in Chinese hamster ovary cells.

INTRODUCIION The interaction between hyperthermia and certain cytotoxic drugs has been shown to be supra-additive ( l-3, 10, 11). The combination of regional hyperthermia and systemic chemotherapy may, therefore, be useful in the treatment of human cancers since local heating could enhance cytotoxicity of chemotherapeutic agents within a defined target volume. Thermal enhancement of melphalan cytotoxicity has been reported in vivo in two murine tumors (13), and melphalan has been used in combination with hyperthermia for the treatment of human melanoma using limb perfusion ( 15, 16). There is, however, little information available about the interactions between melphalan and hyperthermia at the cellular level. We have previously reported thermal enhancement of melphalan cytotoxicity at temperatures from 38°C to 42”C, and a supra-additive interaction between hyperthermia and melphalan at temperatures from 43°C to 45°C in Chinese hamster ovary (CHO) cells (2). To obtain a better understanding of interactions between heat and melphalan, we have studied the effect of elevated tem-

METHODS

AND

MATERIALS

Tissue culture CHO cells (Aux Bl) (10) were grown in monolayer in 75 cm2 plastic tissue culture flasks* at 37°C under 5% CO* in minimum essential medium Alpha (MEM Alpha),? supplemented with 10% fetal bovine serum (FBS)? and 1% penicillin (50 Units/ml)-streptomycin (50 &ml).+ Studies were carried out using cells grown to confluence and incubated for 24 hr at 37°C with fresh culture medium. Cells were harvested with titrated phosphate-buffered saline (0.14 M NaCl, 0.0 1 M sodium phosphate, 0.0 15 M sodium citrate), washed by centrifugation and resuspended at lo6 cells/ml. Cytotoxicity experiments Melphalang was freshly prepared before each experiment and kept on ice at all times. It was dissolved in 50

CHO = Chinese hamster ovary; DPM = Disintegrations per minute; FBS = Fetal bovine serum; MEM Alpha = minimum essential medium Alpha; PBS = Phosphate-buffered saline. Accepted for publication 27 July 1988. * Falcon, Becton-Dickinson Canada Inc., Mississauga, Ontario. t Gibco Canada, Burlington, Ontario. $ Flow Laboratories, Mississauga, Ontario. Q Wellcome Medical Division, Burroughs Wellcome, Inc., Kirkland, Ontario.

Reprint requests to: Dr. W. J. Mackillop, Ontario Cancer Foundation, Kingston Regional Cancer Centre, King Street West, Kingston, Ontario K7L 2V7 Canada. Acknowledgements-This work was supported by a grant from the National Cancer Institute of Canada (W.J.M.). D.A.B. was the recipient of a research fellowship from the Cancer Research Society Inc. The assistance of J.H.T. Bates with computer analysis of the data is gratefully acknowledged, and the authors wish to thank Bryn Harris for expert assistance in the preparation of the manuscript. The abbreviations used are: BSA = Bovine serum albumin; 187

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ul ethanol (95%):HC1(2% w/v) solution and then diluted to the appropriate concentration in culture medium in screw-topped polystyrene tubes. The final concentrations of ethanol and HCl did not exceed 0.005% and 0.001% respectively, and did not affect the pH of the solution or contribute to cytotoxicity, in agreement with the findings of other authors (6). Cells were harvested as above and resuspended in MEM Alpha containing 10% FBS and 20 mM HEPES. 0.1 ml aliquots of cells were added to 0.9 mi of meiphaian soiution (prewarmed for 3 min at the incubation temperature). The tubes were incubated in a temperature controlled waterbath** at temperatures ranging from 33” to 45°C. Under these conditions 1 ml of aqueous solution reached a temperature within 0.1 “C of the waterbath temperature within 3 min. Tubes were removed from the waterbath at different time intervals and then wntrifllvd 13 min ItNXl ~,) 0) wa for 7 days. The plates were washed with PBS, fixed with 95% ethanol and stained with methylene blue before counting macroscopic colonies. Control plating efficiencies were greater than 80%. Percentage survival was expressed as the mean number of colonies obtained relative to the mean number of colonies obtained in the control. Two hundred cells per plate were seeded in the control, but where levels of cell survival were uncertain, cells were plated at more than one density to ensure that countable colonies would be obtained.

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the liquid scintillation cocktail Scinti Verse II$$ was added. The radioactivity was determined using an LKB model 12 18 Rackbeta liquid scintillation counter equipped with a dpm calculation program.$$ We measured no change in cell volume after 60 min at temperatures from 37°C to 45°C therefore melphalan uptake was not normalized with respect to cell volume at elevated temperatures (3).

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Freshly harvested CHO cells (lO’/ml) were pre-loaded with melphalan (5 pg/ml) for 15 min at 37°C in PBS- 1% BSA- 10 mM glucose at pH 7.4. The cells were centrifuged (2 min, 1000 g) and washed three times with ice-cold PBSBSA. For efflux measurements the cells were resuspended in ice-cold melphalan-free PBS-BSA-glucose and alifXl r_ rrl lnts ~1x99 tnhes nllnted ___ _-_- in __-cI_-__ _--_-. 0_._3 m-1 nfPBS-BS_A_1 _ _ ___ intn _____ 1 glucose (prewarmed at the incubation temperature) were added and the cells incubated for varying times at temperatures from 37” to 45°C. To stop efflux the cell sus-

Measurement of melphalan influx 14C-labeled melphalan (specific activity: 43.8 &i/mg) was a gift from SRI international, Ravenswood Ave., Menlow Park, California. Freshly harvested CHO cells were resuspended at 10’ cells/ml in PBS- 1% bovine serum albumin (BSA)- 10 mM glucose at room temperature. 100 ul aliquots were placed in glass tubes and preheated for 2 min in a circulating waterbath to allow them to reach the inmlhatinn L1.W..I”U”..CIVII

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phalan. The temperature of the cells was monitored with a 24 gauge hypodermic thermistor temperature probe?? and was found to reach the temperature of the waterbath within 30 sec. At time zero, 100 ul aliquots of freshly prepared melphalan solution, previously equilibrated at the incubation temperature for 3 min, were added to the cells and the suspension was mixed and incubated for the required time. To stop influx, 4 ml of ice-cold PBS-BSA buffer were added and the cells were centrifuged (1 min, 1000 g) and washed 3 times with ice-cold PBS-BSA. The final dry pellet of cells was solubilized with 1% SDS and

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TEMPERATURE

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Fig. 1. Melphalan cytotoxicity versus temperature. One ml reaction mixtures containing i0’ CHO ceiis and 0 (ij or 3 (@j fig/ml melphalan in MEM Alpha, 10% FBS and 20 mM HEPES were incubated for 20 min at the temperatures shown. Mean and SD are given for 4 estimations (with drug) and lo-20 estimations (without drug) from 2- 10 separate experiments.

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0 D. A. BATES AND W. J. MACKILLOP

pensions were centrifuged after addition of 3.4 ml icecold PBS-BSA, and the radioactivity was determined in the cell pellet. RESULTS

Figure 1 shows cell survival after 20 min exposures to temperatures from 37’C to 45°C in the presence and in the absence of melphalan (3 pg/ml). Melphalan cytotoxicity was enhanced at non-lethal temperatures below 43°C and also at lethal temperatures from 43°C to 45°C. Figure 2a shows the relationship between melphalan concentration and cytotoxicity at a range of temperatures between 33°C and 42°C and confirms that the cytotoxicity of melphalan increases with temperature from 33°C to 42°C. Figure 2b shows the melphalan survival curves at 37°C 40°C and 42°C with 95% confidence intervals, obtained as previously described (5). There is no overlap of these relatively narrow confidence intervals for the individual curves, indicating that the differences are significant. Figure 3 shows time courses for the uptake of i4C-labelled melphalan at a range of temperatures between 37°C and 45°C. At 37°C melphalan uptake increased rapidly during the first 15 min and by 30 min had reached a plateau. At temperatures between 37°C and 45°C both melphalan uptake after short times such as 5 min and equilibrium levels of melphalan in the cells after 30 to 45 min increased with increasing temperature. The effect of temperature on initial rates of melphalan uptake is better described by Figure 4, which shows melphalan uptake after 5 min as a function of melphalan concentrations at a range of temperatures between 37’C and 45°C. These

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Fig. 3. Time courses for melphalan uptake at elevated temperatures. Uptake of 14C-labelled melphalan was measured for varying times up to 60 min at 37°C (A), 41°C (0), 43°C (0) and 45°C (A)in reaction mixtures containing lo6 cells and melphalan (5 pg/ml) in 0.2 ml PBS-BSA-glucose at pH 7.4. Mean and SD are given for 3 estimations.

data confirm that initial rates of influx of melphalan increase with increasing temperature. Figure 5 shows the effect of elevated temperatures on efflux of melphalan from CHO cells. The cells were preloaded with melphalan (5 pg/mml), washed and efflux measured in the absence of extracellular melphalan. Efflux occurred most rapidly during the first 10 min and then continued at a much slower rate up to 60 min. The initial rate of efflux increased with temperature from 37’C to 45“C. In the absence of significant melphalan influx, the

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Malphalan concentration

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Fig. 2. Melphalan cytotoxicity versus drug concentration at temperatures from 33°C to 42°C. Reaction mixtures containing 10’ CHO cells and melphalan concentrations from 0 to 6 r/ml in 1 ml of MEM Alpha plus 10% FBS and 20 mM HEPES were heated for 60 min at 33°C (+), 37°C (A), 39°C (Cl), 40°C (O), and 42°C (0). Means are given for 6-12 estimations at 37°C and for 2-4 estimations at the other temperatures, from 3 separate experiments. Cell survival curves are shown with a) lines of best-fit and b) confidence intervals determined as described previously ( 12).

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Fig. 4. Concentration gradients for melphalan uptake at elevated temperatures. Melphalan uptake was measured for 5 min at 37°C (A),40°C (O), 43T (0) and 45T (A), in reaction mixtures containing lo6 cells and melphalan (0.5-30 pg/ml) in 0.2 ml PBS-BSA-glucose. Mean and SD are given for 3 estimations.

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Fig. 5. Semilog plot of intracellular melphalan versus time in melphalan free medium at elevated temperatures. Cell suspensions containing lo6 cells preloaded with melphalan were allowed to efflux in a volume of 0.4 ml of melphalan-free PBS-BSAglucose at 37°C (A), 40°C (0) 43°C (0) and 45°C (0). Mean and SD are given for 3 estimations.

melphalan content in cells after 60 min was very slightly lower in the heated cells than in the control cells at 37°C. Having observed an increase in both the initial rates of melphalan influx and in melphalan efflux at elevated temperatures, we determined the effect of elevated temperatures on intracellular drug concentrations in cells under approximately steady state conditions. The content of melphalan in CHO cells after 20 min drug exposure was measured at one degree intervals from 30°C to 45°C (Fig. 6). The intracellular melphalan concentration increased gradually with temperature, and at 45°C was 30% higher than at 37°C.

DISCUSSION We have previously shown that the cytotoxic effects of melphalan are enhanced at elevated but non-lethal temperatures and that the effects of heat and melphalan are supra-additive at lethal temperatures. In an attempt to explain the mechanism of the thermal enhancement of melphalan cytotoxicity, we studied the effect of elevated temperatures on membrane permeability to melphalan. Both the initial rates of melphalan uptake and the rates of drug efflux are increased at elevated temperatures but there is a net increase in intracellular drug concentrations with increasing temperature. The small increase in intracellular melphalan concentrations observed at elevated temperatures is insufficient to explain the much larger increase in melphalan cytotoxicity. We have previously shown that membrane permeability to adriamycin also increases with increasing temperature but the increase in intracellular adriamycin concentration was also insufficient to explain the much larger increases in adriamycin cytotoxicity (3). Others have reported a similar effect of temperature on the transport kinetics of methotrexate (12).

January 1989, Volume

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We have not attempted here to correlate intracellular drug concentrations and cytotoxicity because we have previously shown that melphalan is rapidly inactivated during heating, and in our uptake studies we were unable to distinguish the active drug from its inactive metabolite(s) (2). The primary metabolite of melphalan is probably a hydrolysis product, in which the chloride groups at the end of the side-chains have been replaced by hydroxyl groups (7, 14). In the radiolabelled melphalan used here it is the four carbon atoms of the side-chains which are replaced by 14C, and these are not lost during hydrolysis. Thus the intracellular counts measured in our uptake experiments may represent either active melphalan or its inactive metabolite. Melphalan is thought to enter cells by facilitated diffusion via two separate amino acid transport systems (8, 9). Melphalan uptake therefore may be expected to show biphasic Michaelis-Menten kinetics, and other authors have attempted to determine the kinetic parameters Vmax (maximum reaction velocity) and Km (binding constant) for each of the processes involved (9). We had intended to determine Km, and Km2 and Vmax, and Vmaxz for melphalan transport and to describe changes in these parameters as a function of temperature. A preliminary analysis of the data fitting problem, however, showed that even near perfect experimental data would produce the errors in the Km and Vmax parameters which are enormous and that this approach to the data has no value (4). In this study we have therefore simply drawn smooth curves to the data for ease of visualization. Our data, however, clearly show that the increase in melphalan toxicity observed at elevated temperatures was not adequately explained by alteration in membrane permeability. Further studies will be required to elucidate the mechanism of thermal enhancement of melphalan cytotoxicity.

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TEMPERATURE (‘C) Fig. 6. Intracellular melphalan concentration versus temperature. Melphalan concentration is given after 20 min incubation at the temperatures shown in reaction mixtures containing lo6 cells and 5 pg melphalan/ml in a total volume of 0.2 ml in PBS, 1% BSA, 10 mM glucose at pH 7.4. Mean and SD are given for 3 to 9 estimations from 3 separate experiments.

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