Toxicity of diethylaminoreserpine to tumor cells: Effects of drug alone and in combination with radiation

Inr. J. Radiation Oncology Biol Phys.. Vol. Printed in the U.S.A. All rights reserved

0360.3016/82/03050545$03.00/0 Copyright 8 1982 Pergamon Press Ltd.

8. pp. 505-509

??Session II-Mechanisms

of Sensitization

and Sensitizers

TOXICITY OF DIETHYLAMINORESERPINE TO TUMOR CELLS: EFFECTS OF DRUG ALONE AND IN COMBINATION WITH RADIATION SHIRLEY

LEHNERT,

PH.D.

Department of Radiation Oncology, McGill University,

Montreal,

Canada

DL-152 is toxic to cells of two transplantable mouse tumors. Minimal numbers of surviving tumor cells are seen at 24 and 48 hours following administration of the drug to the tumor bearing mouse, the small fraction of cells surviving at this time (for doses as low as 5 mg/kg) indicates that both hypoxic and aerated cells are killed. For short (one hour) exposures in viva, however, both acutely and chronically hypoxic cells are more sensitive to DL-152 than are their aerated counterparts. For KHT cells growing as lung nodules, DL-152 toxicity is not demonstrable until 11-14 days after initiation of the lung tumor, coinciding with the time at which an hypoxic fraction develops in this model. In vitro experiments using KHT cells in suspension showed certain concentrations of DL-152 to he selectively toxic to hypoxic cells. At higher concentrations both hypoxic and aerated cells were killed by the drug. The combined effect of DG152 and irradiation has been investigated using, as an index of response, the time required for a transplantable tumor to regrow to a given volume after treatment. DL-152 was equally effective in prolonging the period of radiation-induced growth delay when it was given either shortly before or shortly after irradiation. Growth delay attributable to dlrug plus irradiation was greater than would be predicted if the effects of the two modalities were additive. It is concluded that DL-152 has a concentration-dependent specific toxicity for hypoxic cells and that the drug may require a hypoxic milieu to manifest toxicity. Diethylaminoreserpine,

Cytotoxicity,

Tumors, Radiation.

INTRODUCTION

natively in Balb/c mice and in tissue culture using Waymouth’s MB752/ 1 medium, supplemented with 15% fetal calf serum, penicillin and streptomycin. The colony-forming ability of KHT sarcoma cells was assayed by an in vitro agar plating technique.2 Cell suspensions were prepared from subcutaneous or intramuscular tumors or from lungs containing tumor nodules by the enzyme procedure described by Thomson and Rauth.” For assay of clonogenic survival of EMT6 cells, tumors were disaggregated using an enzyme mixture described by Howell and Koch.4 Total viable cell numbers were determined by hemocytometer counts of trypan-blue excluding cells, and corrected for the presence of host cells. Appropriate numbers of cells were inoculated into replicate flasks containing conditioned Waymouth’s medium and macroscopic colonies were fixed, stained and counted 12 days later. The surviving fraction of clonogenic cells per tumor was,calculated from the surviving fraction (SF) of clonogenie cells (i.e. the ratio of plating efficiencies treated/ control) and the ratios of treated to corresponding control values for tumor volume and cell yield per unit volume. Growth rate of intramuscular tumors implanted in the leg was determined from 3 times weekly measurements of

Diethylaminoreserpine (DL- 152) is a drug with hypotensive action like reserpine, but without the sedative properties of the parent compound.’ DL-152 has also been shown to be a potent inhibitor of cyclic AMP phosphodiesterase.3 During an investigation of the effect of several cyclic nucleotide plhosphodiesterase inhibitors on the response of animals to whole body irradiation, injection of DL-152 prior to irradiation was found to have some radioprotective effects for normal tissue in mice.6.* During an investigation of the radioprotective effect of DL-152, it was found that the drug was toxic to cells of two transplantable mouse tumors.’ Further investigations of the cytotoxic effect of DL-152 and the response of transplantable tumors to combined treatment with radiation and DL-152 are described in this paper. METHODS

AND MATERIALS

The KHT sarcoma5 was grown in male C3H mice. Subcutaneous or intramuscular tumors were produced by injection of 2.5 x 10’ cells at the appropriate site. Lung nodules were produced by intravenous injection of 250 viable tumor cells plus I .5 x lo6 heavily-irradiated cells. The EMT6 mammary carcinoma” was carried alterThis investigation

was supported in part by the National

Cancer Institute of Canada. Reprint requests to S. Lehnert,

Ph.D., Department

tion Oncology, Montreal General Hospital, 1650 Cedar Montreal, H3G lA4, Quebec, Canada. Accepted for publication 5 November I98 1.

of Radia505

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Fig. 1.A. Surviving fraction of clonogenic cells for EMT6 tumors (H) and KHT tumor (m) at various times B. Surviving fraction of clonogenic KHT tumor cells at 48 after injection of DL- 152, 320 mg/kg, intraperitoneal. hours aiter injection of various d&s bf DL- 152.

leg diameters which were converted to tumor weights by means of a previously determined calibration curve relating leg diameter and tumor weight. For determination of in vitro cytotoxicity of DL-152, a single cell suspension of KHT cells was stirred continuously in an atmosphere of 95% N,, 5% CO, (hypoxic) or 95% Air, 5% CO, (aerated) in the presence of various concentrations of DL-152.* Periodic samples were withdrawn for count of viable cells, dilution and plating. RESULTS Toxicity of DL-152

to cells offlank

and lung tumors

Changes in the surviving fraction of clonogenic tumor cells during the first 48 hours following DL-152 injection are shown in Figure 1A for KHT and EMT6 tumors, volume 0.2-0.4 cm3 at the time of treatment. There is a fall in the surviving fraction of clonogenic cells for both tumors, but a larger reduction is seen for the KHT tumor. In these experiments the dose of DL-152 used was 320 mg/kg given intraperitoneally, which is approximately two thirds of the mean lethal dose for mice.’ Figure 1B shows results of an experiment in which various doses of DL-152 were injected into tumor-bearing mice and the surviving fraction of clonogenic KHT cells assayed 48 hours latei. Significant reduction in surviving clonogenic tumor cells was found following doses as low as 5 mg/kg. The extent of the reduction in surviving fraction following DL-152 injection indicates that under these conditions, both hypoxic and aerated cells are killed by the drug. However, the following findings suggest that under some conditions, hypoxic cells are more sensitive to

*DL-152 was obtained subsequently France.

from

initially Laboratoires

from Clin Byla (Paris), and Lefrancq, Romainville,

DL- 152 than are aerated cells. a) The surviving fraction of clonogenic KHT cells is reduced by 50% at one hour following DL-152 injection, and by a further 50% if the tumor cells are hypoxic (as a result of nitrogen asphyxiation of the mouse) for 10 minutes prior to excision of the tumor. b) A dose of 1500 rad to tumor bearing mice will result in a surviving tumor cell population which consists almost entirely of hypoxic (radioresistant) cells. The number of tumor cells surviving 1500 rad was reduced by DL-152 injection, the surviving fraction relative to the number from tumors of irradiated saline-injected mice being 0.23. This smaller surviving fraction is found one hour after DL- 152 injection into unirradiated air-breathing mice and is similar to the value found for nonirradiated mice killed by nitrogen asphyxiation. c) The hypoxic fraction for flank tumors of volume 0.4-0.5 cm3 is reduced from 14% in controls to 7% at 60 minutes after DL-152 injection. (The size of the hypoxic fraction in each case was determined by comparison of survival curves for air-breathing and nitrogen asphyxiated mice.) Experiments were also done on the effect of DL- 152 on survival of KHT cells growing as lung colonies. In Figure 2A, the fraction of lung tumor cells surviving at 24 hours after DL-152 injection is plotted against time for which tumor cells have been growing in the lungs. First indication of toxicity of DL- 152 to lung colony cells develops at between 11 and 14 days after injection and the fraction of clonogenic cells killed becomes greater with increasing time after injection of tumor cells, as the lung nodules increase in size. Figure 2B shows the results of an experiment in which mice bearing 10, 14 or 21 day old

Toxicity of diethylaminoreserpine

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Fig. 2.A. Surviving fraction of clonogenic KHT cells recoverable from the lungs of mice 24 hours after injection of DL-152. Groups of mice (4 DL treated, 4 saline treated) were injected at various times after intravenous injection of tumor cells, and killed at 24 hours after treatment. Surviving fraction of DL- 152 treated mice was the ratio of the mean number of clonogenic cells recoverable from the lungs of drug treated mice to the mean number recoverable from saline treated mice. B. Surviving fraction of clonogenic KHT cells recoverable from lungs of mice at various times afte:r injection of DL-152 (320 mg/kg) A-A mice treated 10 ‘days after injection of tumor cells. O---O mice treated 14 days after injection of tumor cells M mice treated 2 1 days after injection of tumor cells. lung nodules were injected with DL-152 and killed at intervals after injection Iof the drug. Maximum toxicity of DL-152 to ceils of 14 and 21 day old lung nodules occurs at approximately the same time as for flank tumors, 24 to 48 hours after injection of the drug.

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Fig. 3. Survival curves for clonogenic KHT cells incubated in suspension with various concentrations of DL- 152. Left panel, incubated under 95% N, 5% CO,. Right panel incubated under 95% 0,. 5% CO*. m no additions M IO-’ M DL-152. v-v 10m4 M DL-152, A-A lo-’ M DL-152.

The most effective protocol was found to be DL-I 52 injected shortly before or after irradiation. The reiationship of duration of growth delay to dose of DL- 152 when the latter is given immediately after a given dose (1350 rad) of irradiation is shown in Figure 4B. Figure 4C shows the effect on tumor growth delay of various radiation doses in combination with 320 mg/kg DL-152 injected immediately after irradiation. DISCUSSION

In vitro studies Data for KHT cells .in suspension, exposed to various concentrations of DL-lfi2, are shown in Figure 3. 10m3 M of DL-152 was toxic to both hypoxic and aerated cells, whereas for lower concentrations hypoxic cells showed a greater sensitivity. Aerated KHT ceils were completely resistant to 10m4 M DL-152 over a 12 hour incubation period. Incubation of hypoxic ceils in the same concentration of drug, however, reduced the surviving fraction to less than IO-‘, while a concentration of 10m5 M DL-152 was sufficient to kill 90% of hypoxic KHT ceils over the same period. Combined eflect of DL-152 and radiation The use of DL-152 in conjunction with radiation has been studied using intramuscular KHT tumors and growth delay as an index of response. Fig. 4A shows results of experiments in which sequence and timing of DL-152 injection with respect to irradiation was varied.

The results demonstrate DL- 152 to be toxic to cells of two transplantable tumors. The extent of ceil kill indicates that following prolonged exposure, the drug is toxic to both aerated and hypoxic cells. Results of other in vivo experiments however suggest that for a one hour exposure period, DL-152 exerts selective toxicity against hypoxic ceils. The in vitro experimehts are in agreement with in vivo observations in that while hypoxic ceils are killed by lower concentrations of DL- 152 than are aerated cells, at concentrations sufficient to kill both aerated and hypoxic cells, the extent of the shoulder on the aerated survival curve is greater than that for the hypoxic curve. Thus for the same concentration of drug a shorter exposure is required under hypoxic conditions for a given number of ceils to be killed. In experiments with lung tumors, it was found that for at least the first 11 days of tumor growth, tumor ceils were resistant to the drug. By 14 days of growth reduction in surviving fraction was observed in lung tumors follow-

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Fig. 4.A. Days to reach 4 x tumor treatment volume for mice treated with 1350 rad y rays (X) 320 mg/kg DL- 152 (DL) or both agents in varying timing and sequence. Numbers on abcissa indicate time (hours) between two treatments. B. Days to reach 4 x tumor treatment volume for mice given various doses of DL- 152 immediately after irradiation (1350 rad). C. Days to reach 4 x tumor treatment volume for mice given DL-152 (320 mg/kg) immediately after irradiation with various doses of X rays.

ing DL- 152 injection, and at later times lung tumor cells showed increasing sensitivity with increasing tumor size. Reports from several sources indicate the presence of a demonstrable hypoxic fraction in these lung nodules by 14 days after injection of tumor cells. Siemann and Kochanski” have reported values of 6% and 20% for the hypoxic function of lung nodules at 14 days and 20 days of lung tumor growth, respectively, and preliminary results from this laboratory have confirmed this observation. One explanation of these findings with lung tumors could be that development of cytotoxicity is dependent on the presence of hypoxic cells, but that cell killing is not confined to hypoxic cells, possibly because DL-152 is metabolized under hypoxia to a product toxic to surrounding cells to which it is able to diffuse. DL-152 cytotoxicity, however, cannot be solely dependent on

reduction of the drug under hypoxic conditions since as demonstrated in the in vitro experiments, the drug is also toxic to fully aerated cells provided the concentration is sufficiently high. The optimum effect of DL-152 combined with radiation was observed when the drug was injected shortly before or shortly after irradiation. At these times, the growth delay attributable to drug plus irradiation is greater than if the effect of the two modalities were simply additive. The fact that DL-I 52 is as effective in prolonging growth delay when given after irradiation as it is when given before, indicates that the effect is not attributable to radiosensitization by the drug, but more probably to the toxicity of DL-152 to a radioresistant subpopulation of tumor cells.

REFERENCES Garattini, S.L., Lamesta, L., Mortari, A., Valzelli, L.: Pharmacological and biochemical effects of some reserpine derivatives. J. Pharm. Pharmacol. 13: 548-553, 196 1. Hill, R.P., Ng, R., Warren, B.F., Bush, R.S.: Effects of intercellular contact on the radiation sensitivity of KHT sarcoma cells. Radiat. Res. 77: 182-I 92, 1979. Honda, F., Imamura, H.: Inhibition of cyclic 3’5’nucleotide phosphodiesterase by phenothiazine and reserpine derivatives. Biochem. Biophys. Acta 161: 267-269, 1968. Howell, R.L., Koch, C.J.: The disaggregation, separation

and identification of cells from irradiated and unirradiated EMT6 mouse tumors. Int. .I. Rudiut. Oncol. Bio. Phys. 6: 31 I-318,

1980.

5. Kallman, R.F., Silini, G., van Patten, L.M.: Factors influencing the quantitative estimation of the in vivo survival of cells from solid tumors. /. Nutl. Cancer Inst. 39: 539-549,

1967.

6. Lehnert, S.: Radioprotection of mouse intestine by inhibitors of cyclic AMP phosphodiesterase. Int. J. Rudiut. Oncol. Biol. Phys. 5: 825-833,

1980.

Toxicity of diethylaminoreserpine

7. Lehnert, S.: Toxicity to tumour cells of diethylaminoreserpine. Br. J. Cancer 41. (Suppl IV): 222-225, 1980. 8. Lehnert, S., Fisher, G., Methot, G.: Radioprotection of normal and malignant tissue in the mouse by diethylaminoreserpine. Int. J. Rad. Biol. 40: 63-73, 198 1. 9. The Merck Index, 9th edition. Rahway, N.J., Merck and Co. Inc. 1977, p. 1235. IO. Rockwell, S.C., Kalhnan, R.F., Fajardo, L.F.: Characteristics of a serially transplanted mouse mammary tumor and

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its tissue culture adapted 49: 7355749,1972.

derivative.

J. Natl. Cancer Inst.

I I. Siemann,

D., Kochanski, K.: Combination of radiation and Misonidazole: The effect on lung tumor therapy in a mouse model system (Abstract). Radiat. Res. 83: 400, 1980.

12. Thomson, J.E., Rauth, A.M.: An in vitro assay to measure the viability of KHT tumor cells not previously exposed to culture conditions. Radiat. Res. 58: 262-276, 1974.