INT. J. HYPERTHERMIA,
1992,
VOL.
8,
NO.
1, 139-146
Preferential killing of glucose-depleted HeLa cells by menadione and hyperthermia J. H. KIM?, S. H. KIM?, P. DUTTAS and J. PINTOS ?Henry Ford Hospital, Department of Radiation Oncology, 2799 W. Grand Boulevard, Detroit, MI 48202, USA $Memorial Sloan-Kettering Cancer Center, Department of Medicine, 1275 York Avenue, New York, NY 10021, USA Int J Hyperthermia Downloaded from informahealthcare.com by University of Calgary on 02/05/15 For personal use only.
(Received I7 May 1991; revised 23 August 1991; accepted 23 August 1991)
Energy deprivation of cancer cells increases sensitivity to killing by hyperthermia. Recent cell culture studies suggest that certain naphthoquinones, especially menadione (vitamin K,), have anti-tumour activity by interfering with the energy metabolism of cells, resulting in the inhibition of aerobic glycolysis. We therefore studied the cytotoxic effects of menadione in HeLa cells in combination with hyperthermia. The cell culture data show that the cytotoxicity is markedly increased in cells deprived of glucose in the medium at 37°C after exposure to menadione. When cells were exposed to menadione (20-40 VM) and hyperthermia (41-42"C), there was a dramatic potentiation of heatinduced cytotoxicity in cells deprived of glucose in the medium. These data suggest that glucose-deficient cancer cells could be selectively killed by the combined treatment of menadione and mild hypertherrnia, both of which can be readily achievable in humans. Key words: Menadione, hyperthermia, glucose deprivation
1. Introduction Temperature elevation to 42 "C produces cytotoxic effects both in cell cdlilre systems and in vivo; energy-impaired cells display great sensitivity to hyperthermia, as do cells under acidic media conditions which presumably place increased energy demands upon the cell (Kim, S. H. et al. 1980,, Lava1 and Michel 1982, Gerweck et al. 1984). The thermosensitivity of well-oxygenated cells could be significantly enhanced following glucose deprivation and inhibition of oxidative phosphorylation (Kim, J. H. et al. 1985, Kim, S. H. et al. 1985). On the other hand, inhibitors of glycolysis selectively enhance the thermosensitivity of hypoxic cells, since cells under hypoxia are heavily dependent on their source of energy via anaerobic glycolysis (Nagle et al. 1985, Kim, S . H. et al. 1978). In pursuing the cellular mechanism of thermosensitization by chemicals that impair energy metabolism, we undertook the present cell culture study to determine whether the cytotoxic effects of menadione, vitamin K,, could be enhanced by heat, since certain naphthoquinones, especially menadione, indirectly interfere with energy metabolism (Bellomo et al. 1982, Orrenius 1985). Further, menadione and its analogue, menadiol sodium diphosphate, have shown antitumour activity in experimental tumour systems (Chlebowski et al. 1984, Prasad et al. 1981), and the compound is currently undergoing clinical trials as an antiturnour agent (Akman et al. 1988). 2. Materials and methods Experiments were carried out with HeLa S-3 cells grown in Eagle's minimal essential medium supplemented with 10%foetal calf serum. Details of the cell culture procedures, including maintenance, trypsinization, and test for contamination with mycoplasma of cultures, are described elsewhere (Kim, S. H. et al. 1980, Kim, J. H. et al. 1985). Plated monolayer cells (5 x lo5 cells per plate) were heated to within 0.05"C of the 0265-6736/92 $3.00 01992 Taylor & Francis Ltd
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desired temperature by totally immersing plastic culture flasks in a water bath heated by a Haake Model 52 temperature circulator. Following drug exposure, the cells were washed with saline, trypsinized, enumerated, and appropriate dilutions seeded into 60 mm dishes. Surviving cells (colonies) were then fixed, stained, and counted 12-14 days later. The glucose-deprived medium was prepared by adding 10% dialysed foetal calf serum to the culture medium without glucose obtained from Grand Island Biological Co. the dialysed foetal calf serum contained less than 1 mg glucose/100 ml, so that the final concentration of glucose in the glucose-deprived medium was 0.001 mgiml. The method used to obtain hypoxic cells was the same as described previously (Kim, S. H. et al. 1980). The cellular concentration of ATP, ADP, and AMP was measured by high-performance liquid chromatography (HLPC). For determination of adenine nucleotides, samples were prepared as described by Bump et al. (1984). The samples were injected into HPLC instruments (Water Associates, Milford, MA) using a Partsphere 5 SAX column. The flow rate was 1 - 5 ml/min, and absorbance was measured at 260 nm. The retention times for AMP, ADP, and ATP were 2.5, 14, and 29 min, respectively. The area under peak was calculated by the integrator to determine the quantity of each nucleotide and energy charge was calculated as [(ATP+ %ADP)/(AMP+ATP)]. Determinations of the intracellular non-protein thiol fraction which contains predominantly reduced glutathione was performed by the method of Beutler (1971). Briefly, aliquots of trypsinized cells were suspended in cold phosphate buffered saline, centrifuged, and the supernatant fractions were carefully removed by aspiration. The pellet containing packed cells (1 x lo") was treated with 0.3 ml of metaphosphoric acid solution (glacial metaphosphoric acid, EDTA, and sodium chloride) and centrifuged. The supernatant solution, which contained the acid-soluble thiol fraction, was mixed with 0.5 ml of 0 . 3 M sodium phosphate solution and the absorbance was determined against a reagent blank at 412 nm. A second absorbance reading was made after the addition of 5,5'-dithiobis2-nitrobenzoic acid (DTNB). Since this method measures only acid-soluble thiol, and not reduced glutathione directly, we compared this method in a small number of samples (n= 15) with the enzymatic cycling method of Tietze (1969), in which the rate of DTNB reduction is proportional to the amount of either GSH or GSSG present. Analyses indicate that 93-95 % of the acid-soluble thiol fraction contained reduced glutathione. The final concentration of GSH was reported as nmolimg protein. The glutathione reductase activity was also measured by the method of Beutler (1969). Aliquots of samples were preincubated in a phosphate-EDTA buffer containing oxidized glutathione (GSSG) in the presence and absence of exogenously added flavin adenine dinucleotide. After 15 min incubation at 37°C the reaction was started by the addition of NADPH, and measurements of the loss of absorbance at 340 nm were made in a thermostated spectrophotometer. GSH reductase was reported in pmol NADPH oxidized per minute per mg protein. Each experiment was repeated twice or more, and the error bars on the figures indicate the standard error of the mean of six replicates per point. The plating efficiency of HeLa cells was in the range of 50-60 % . Menadione (sodium bisulphite salt), catalase, and diethyldithiocarbamate were all purchased from the Sigma Chemical Company, St. Louis, MO.
3. Results 3.1. EfSect of menadione on cell survival at 37°C Prior to our hyperthermia study, experiments were carried out to determine the cytotoxicity of menadione on HeLa cells at 37°C. Acute exposure of cells to menadione on cell survival was determined in terms of colony formation of single plated cells. The
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cell survival curves show a minimal cytotoxicity to 40 PM after a 2-h exposure under normal cultural conditions. However, cells under glucose-depleted medium exhibited enhanced cytotoxicity to the drug (Figure 1). Figure 2 shows the effects of hypoxia and other oxygen-modifying agents on the menadione-induced cytotoxicity . It is apparent that hypoxia protected the cytotoxic effects of the drug, which was most pronounced at the highest concentration of the drug (80 WM). Exposure of oxic cells to catalase also reduced the cytotoxic effects of the drug, indicating the involvement of oxygen-free radicals. Similarly, exposure of oxic cells to the inhibitor of superoxide dismutase, diethyldithiocarbamate, increased the cytotoxicity of the drug, more than 100-fold. 3.2. Effect of menadione on cell survival following hyperthermia Figures 3 and 4 show cell survival curves of menadione as a function of exposure time at 41°C and 42°C under glucose-fed and glucose-deprived conditions. It is clear that menadione increases the cytotoxicity of hyperthermia. Although present at 41 "C, the potentiating effect of menadione on hyperthermia-induced cell killing is far more prononced at 42 "C and is particularly evident under glucose-deprived conditions.
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Oxic,Catalarr (t)
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Figure 2. Effects of hypoxia and oxygen-modifying agents on the menadione-induced cytotoxicity : 0 , no drug; 0, 20 p ~ A, ; 40 p ~ A, ; 80 p ~ Catalase: . 30 pg/ml; diethyldithiocarbamate: 60 VM.
3.4. Effect of menadione on glutathione metabolism in HeLa cells Menadione caused an immediate depletion of GSH, followed by a gradual recovery to the control level at 4 h. In contrast, glucose-deprived cells exposed to the drug were 41"
Glucose (+)
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Glucose (-)
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Figure 3. Percentage of cell survival as a function of time after exposure of cells to menadione no drug; at 41°C under glucose-fed and glucose-deprived conditions: 0; no drug; 0, 10 pM; 0, 20 pM; A, 40 VM.
Menadione and hyperthermia
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42"
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Glucose (+)
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Figure 4. Percentage of cell survival as a function of time after exposure of cells to menadione 10 pM; 0, 20 WM; A,40 pM. at 42°C: 0 , no drug;
unable to recover from the depressed GSH to the control level (Figure 5). The GSH reductase levels increased shortly after exposure to the drug, but they gradually returned to the control level by 4 h.
Discussion The data presented herein demonstrate that cytotoxicity is markedly increased in cells deprived of glucose in the medium at 37°C after exposure to menadione. When cells were exposed to menadione (40 PM) and hyperthermia (42"C), there was a dramatic potentiation of heat-induced cytotoxicity in cells deprived of glucose in the medium (Figures 3 and 4). Evidence has been obtained in the present study showing that the cytotoxicity of menadione is principally mediated by the activated oxygen species such as superoxide and hydrogen peroxide. Simultaneous exposure of cells to menadione and catalase abrogated 4.
Table 1.
Effects of menadione on cellular energy metabolism in HeLa cells. 2h
Treatment groupa Glucose alone ( 5 mM) Glucose + menadione (40 p M ) Glucose-deprived Glucose-deprived+menadione (40 pM)
ATP~ 100 81&3d 97*3 80&3
EC' 0.76&0.02d 0.63&0.03 0.76*0.06 0.85&0.04
4h
ATP EC 100 0.76 z t 0.02 95*4 0.75*0.03 92k3 0*76=t0*01 1 0 9 ~ 1 ~ 4 0.87*0.03
"Means of two separate experiments. bATP levels are expressed as percentage of controls. 'Multiple estimates of energy charge (EC) under control conditions are reproducible with a range of 0.75-0.82. dStandard deviation.
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Figure 5. Effects of menadione on glutathione metabolism at 37°C under glucose-fed and glucosedeprived conditions: 20 pM with glucose; 0, 40 p M with glucose; 40 p~ without glucose. the cytotoxicity of menadione, while the treatment of cells with diethyldithiocarbamate (DDC), an inhibitor of superoxide dismutase, significantly enhanced the cytotoxicity . Likewise, hypoxia substantially reduced the cytotoxicity of menadione (Figure 2). Lin et al. (1979) showed hyperthermic potentiation in exponentially growing DON cells by prior treatment with DDC. The present data are qualitatively similar with results obtained with mitochondrial inhibitors of oxidative phosphorylation (Lava1 and Michel 1982, Kim, J. H. et al. 1985). Using rhodamine- 123, a mitochondria1 binding agent, we have previously shown that there was a pronounced enhancement of cell kill in glucose-deprived cells following exposure of HeLa cells to the drug at 42°C. No enhanced effects of heat were seen in the glucosefed cells, unlike the present result with menadione. Although the above data were generally consistent with the concept that energy deprivation by means of oxidative phosphorylation increases sensitivity to killing by hyperthermia (Kim, S . H. et al. 1980, Gerweck et al. 1984), the present data on the ATP levels and energy charge of cells exposed to menadione failed to show an appreciable energy deprivation (Table 1). In fact, the principal effects of menadione metabolism result in a decrease in the intracellular glutathione level and NADPH oxidation (DiMonte et al. 1984). It is the oxidation of NADPH which is thought to affect calcium transport in mitochondria (Orrenius 1985). Menadione caused an immediate depletion of the cellular GSH, most pronounced in the glucose-deprived cells (Figure 5). The sustained depletion of the GSH in glucose-deprived cells may be in part responsible for the hyperthermic potentiation to menadione. There have been several cell culture studies showing that the GSH depletion by buthionine sulphoximine significantly enhances the thermosensitivity of cells (Mitchell and Russo 1983, Freeman et ul. 1985). Menadione and menadiol sodium diphosphates seem to be good candidates for clinical hyperthermic sensitizers. The drug is relatively non-toxic at 37°C. Menadiol sodium diphosphate has received some evaluation as a radiation sensitizer in the treatment of head
Menadione and hyperlhermia
145
and neck cancers (Krishanamurthi et al. 1971, Mitchell et al. 1965). Further, there is some evidence showing that menadiol sodium diphosphate is preferentially localized in human cancers (Mitchell et al. 1983). If menadione is found to have selective cytotoxic effects against tumour cells in vivo without undue normal tissue toxicity, it may have potential utility as a hyperthermic sensitizer, because menadione and mild hyperthermia (less than 42°C) can be readily achievable in humans.
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Refeences AKMAN, S . , CARRB., LEONG,L., MARGOLIN, K., ODUJINRIN, 0. and DOROSHOW, J., 1988, Phase I trial of menadiol sodium diphosphate in advanced cancer. Proceedings of the American Society of Clinical Oncology, 7 , 290. BELLOMO, G., JEWELL, S. A. and ORRENIUS, S . , 1982, The metabolism of menadione impairs the ability of rat liver mitochondria to take up and retain calcium. Journal of Biological Chemistry, 257, 11558-1 1562. BEUTLER,E., 1969, Effect of flavin compounds on glutathione reductase activity: in vivo and in vitro studies. Journal of Clinical Investigation, 48, 1957-1966. BEUTLER, E., 1971, Red Cell Metabolism: A Manual of Biochemical Methods (New York: Grune & Stratton ), pp. 103-105. S.K., SAWYER, J. M. and BROWN,J. M., 1984, Role of the adenylate BUMP,E. A., CALDERWOOD, energy charge in the response of Chinese hamster ovary cells to radiation. International Journal of Radiation Oncology, Biology and Physics, 10, 1411-1414. CHLEBOWSKI, R. T., DIETRICH, M. and BLOCK,J. B., 1984, Vitamin K, (menadione) inhibition of human tumor growth in the soft agar assay system. Proceedings of the American Society of Clinical Oncology, 3, 93. G., EKLOW,L. and ORRENIUS, S., 1984, Alterations in DIMONTE,D., Ross, D., BELLOMO, intracellular thiol homeostasis during the metabolism of menadione by isolated rat hepatocytes. Archives of Biochemistry and Biophysics, 235, 334-342. FREEMAN, M. L., MALCOLM,A. W. and MEREDITH,M. J., 1985, Role of glutathione in cell survival after hyperthermic treatment of Chinese hamster ovary cells. Cancer Research, 45, 6308-63 13. GERWECK, L. E., DAHLBERG, W. K., EPSTEIN,L. F. and SHIMM,D. S., 1984, Influence of nutrient and energy deprivation on cellular response to single and fractionated heat treatments. Radiation Research, 99, 573-581. KIM,J. H., KIM,S. H. and ALFIERI,A. A., 1985, Interaction of rhodamine 123 and hyperthermia in HeLa cells in culture. International Journal of Hyperthermia, 1, 247-253. KIM,S. H., KIM, J. H. and HAHN,E. W., 1978, Selective potentiation of hyperthermic killing of hypoxic cells by 5-thio-o-g~ucose.Cancer Research, 38, 2935-2938. KIM, S. H., KIM,J. H., HAHN,E. W. and ENSIGN,N. A , , 1980, Selective killing of glucose and oxygen-deprived HeLa cells by hyperthermia. Cancer Research, 40, 3459-3462. KIM, S. H., KIM,J. H., ALFIERI,A. A. and YOUNG, C. W., 1985, Gossypol, a hyperthermic sensitizer of HeLa cells. Cancer Research, 45, 6338-6340. KRISHANAMURTHI, S . , SHANTA,V. and SISTRI,N., 1971, Combined therapy in buccal mucosa cancers. Radiology, 99, 409-415. LAVAL, F. and MICHEL,S . , 1982, Enhancement of hyperthermia-induced cytotoxicity upon ATP deprivation. Cancer Letters, 15, 6 1-65. LIN,P. S., KWOCK,L. and BUTTERFIELD. C. E., 1979, Diethyldithiocarbamate enhancement of radiation and hyperthermia effects on Chinese hamster cells in vitro. Radiation Research, 77, 501-511. MITCHELL, J. B. and Russo, A,, 1983, Thiols, thiol depletion and thermosensitivity. Radiation Research, 95, 471-485. J. S., BROMBLEY, D. and HAYLITTLE, J. L., 1965, Clinical trial of radiosensitizers, MITCHELL, including Synkavite and oxygen inhaled at atmospheric pressure. Acta Radiologica Series 2: Oncology, Radiation Therapy, Physics and Biology, 3, 329-341. MITCHELL, J . S., BROWN,I. and CARPENTER, R. N., 1983, Attempts to develop radioactive anticancer drugs. Intemarional Journal ofRadiation Oncology, Biology and Physics, 9,57-59. NAGLE,W. A , , Moss, A. J. and HENLE,K. J., 1985, Sensitization of cultured Chinese hamster cells to 42°C hyperthermia by pentalenolactone, an inhibitor of glycolytic ATP synthesis. Internuiional Journal of Radiation Oncology, Biology and Physics, 48, 821-835.
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ORRENIUS, S., 1985, Biochemical mechanisms of cytotoxicity. Trends in Pharmacological Science, FEST Suppl., 515-520. PRASAD,K. N., EDWARDS-PRASAD, J. and SAKAMOTO, A., 1981, Vitamin K3 (menadione) inhibits the growth of mammalian tumor cells in culture. Life Science, 29, 1387-1392. TIETZE,F . , 1969, Enzymatic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Analytical Biochemistry, 27, 502-522.
1992,
VOL.
8,
NO.
1, 139-146
Preferential killing of glucose-depleted HeLa cells by menadione and hyperthermia J. H. KIM?, S. H. KIM?, P. DUTTAS and J. PINTOS ?Henry Ford Hospital, Department of Radiation Oncology, 2799 W. Grand Boulevard, Detroit, MI 48202, USA $Memorial Sloan-Kettering Cancer Center, Department of Medicine, 1275 York Avenue, New York, NY 10021, USA Int J Hyperthermia Downloaded from informahealthcare.com by University of Calgary on 02/05/15 For personal use only.
(Received I7 May 1991; revised 23 August 1991; accepted 23 August 1991)
Energy deprivation of cancer cells increases sensitivity to killing by hyperthermia. Recent cell culture studies suggest that certain naphthoquinones, especially menadione (vitamin K,), have anti-tumour activity by interfering with the energy metabolism of cells, resulting in the inhibition of aerobic glycolysis. We therefore studied the cytotoxic effects of menadione in HeLa cells in combination with hyperthermia. The cell culture data show that the cytotoxicity is markedly increased in cells deprived of glucose in the medium at 37°C after exposure to menadione. When cells were exposed to menadione (20-40 VM) and hyperthermia (41-42"C), there was a dramatic potentiation of heatinduced cytotoxicity in cells deprived of glucose in the medium. These data suggest that glucose-deficient cancer cells could be selectively killed by the combined treatment of menadione and mild hypertherrnia, both of which can be readily achievable in humans. Key words: Menadione, hyperthermia, glucose deprivation
1. Introduction Temperature elevation to 42 "C produces cytotoxic effects both in cell cdlilre systems and in vivo; energy-impaired cells display great sensitivity to hyperthermia, as do cells under acidic media conditions which presumably place increased energy demands upon the cell (Kim, S. H. et al. 1980,, Lava1 and Michel 1982, Gerweck et al. 1984). The thermosensitivity of well-oxygenated cells could be significantly enhanced following glucose deprivation and inhibition of oxidative phosphorylation (Kim, J. H. et al. 1985, Kim, S. H. et al. 1985). On the other hand, inhibitors of glycolysis selectively enhance the thermosensitivity of hypoxic cells, since cells under hypoxia are heavily dependent on their source of energy via anaerobic glycolysis (Nagle et al. 1985, Kim, S . H. et al. 1978). In pursuing the cellular mechanism of thermosensitization by chemicals that impair energy metabolism, we undertook the present cell culture study to determine whether the cytotoxic effects of menadione, vitamin K,, could be enhanced by heat, since certain naphthoquinones, especially menadione, indirectly interfere with energy metabolism (Bellomo et al. 1982, Orrenius 1985). Further, menadione and its analogue, menadiol sodium diphosphate, have shown antitumour activity in experimental tumour systems (Chlebowski et al. 1984, Prasad et al. 1981), and the compound is currently undergoing clinical trials as an antiturnour agent (Akman et al. 1988). 2. Materials and methods Experiments were carried out with HeLa S-3 cells grown in Eagle's minimal essential medium supplemented with 10%foetal calf serum. Details of the cell culture procedures, including maintenance, trypsinization, and test for contamination with mycoplasma of cultures, are described elsewhere (Kim, S. H. et al. 1980, Kim, J. H. et al. 1985). Plated monolayer cells (5 x lo5 cells per plate) were heated to within 0.05"C of the 0265-6736/92 $3.00 01992 Taylor & Francis Ltd
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desired temperature by totally immersing plastic culture flasks in a water bath heated by a Haake Model 52 temperature circulator. Following drug exposure, the cells were washed with saline, trypsinized, enumerated, and appropriate dilutions seeded into 60 mm dishes. Surviving cells (colonies) were then fixed, stained, and counted 12-14 days later. The glucose-deprived medium was prepared by adding 10% dialysed foetal calf serum to the culture medium without glucose obtained from Grand Island Biological Co. the dialysed foetal calf serum contained less than 1 mg glucose/100 ml, so that the final concentration of glucose in the glucose-deprived medium was 0.001 mgiml. The method used to obtain hypoxic cells was the same as described previously (Kim, S. H. et al. 1980). The cellular concentration of ATP, ADP, and AMP was measured by high-performance liquid chromatography (HLPC). For determination of adenine nucleotides, samples were prepared as described by Bump et al. (1984). The samples were injected into HPLC instruments (Water Associates, Milford, MA) using a Partsphere 5 SAX column. The flow rate was 1 - 5 ml/min, and absorbance was measured at 260 nm. The retention times for AMP, ADP, and ATP were 2.5, 14, and 29 min, respectively. The area under peak was calculated by the integrator to determine the quantity of each nucleotide and energy charge was calculated as [(ATP+ %ADP)/(AMP+ATP)]. Determinations of the intracellular non-protein thiol fraction which contains predominantly reduced glutathione was performed by the method of Beutler (1971). Briefly, aliquots of trypsinized cells were suspended in cold phosphate buffered saline, centrifuged, and the supernatant fractions were carefully removed by aspiration. The pellet containing packed cells (1 x lo") was treated with 0.3 ml of metaphosphoric acid solution (glacial metaphosphoric acid, EDTA, and sodium chloride) and centrifuged. The supernatant solution, which contained the acid-soluble thiol fraction, was mixed with 0.5 ml of 0 . 3 M sodium phosphate solution and the absorbance was determined against a reagent blank at 412 nm. A second absorbance reading was made after the addition of 5,5'-dithiobis2-nitrobenzoic acid (DTNB). Since this method measures only acid-soluble thiol, and not reduced glutathione directly, we compared this method in a small number of samples (n= 15) with the enzymatic cycling method of Tietze (1969), in which the rate of DTNB reduction is proportional to the amount of either GSH or GSSG present. Analyses indicate that 93-95 % of the acid-soluble thiol fraction contained reduced glutathione. The final concentration of GSH was reported as nmolimg protein. The glutathione reductase activity was also measured by the method of Beutler (1969). Aliquots of samples were preincubated in a phosphate-EDTA buffer containing oxidized glutathione (GSSG) in the presence and absence of exogenously added flavin adenine dinucleotide. After 15 min incubation at 37°C the reaction was started by the addition of NADPH, and measurements of the loss of absorbance at 340 nm were made in a thermostated spectrophotometer. GSH reductase was reported in pmol NADPH oxidized per minute per mg protein. Each experiment was repeated twice or more, and the error bars on the figures indicate the standard error of the mean of six replicates per point. The plating efficiency of HeLa cells was in the range of 50-60 % . Menadione (sodium bisulphite salt), catalase, and diethyldithiocarbamate were all purchased from the Sigma Chemical Company, St. Louis, MO.
3. Results 3.1. EfSect of menadione on cell survival at 37°C Prior to our hyperthermia study, experiments were carried out to determine the cytotoxicity of menadione on HeLa cells at 37°C. Acute exposure of cells to menadione on cell survival was determined in terms of colony formation of single plated cells. The
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cell survival curves show a minimal cytotoxicity to 40 PM after a 2-h exposure under normal cultural conditions. However, cells under glucose-depleted medium exhibited enhanced cytotoxicity to the drug (Figure 1). Figure 2 shows the effects of hypoxia and other oxygen-modifying agents on the menadione-induced cytotoxicity . It is apparent that hypoxia protected the cytotoxic effects of the drug, which was most pronounced at the highest concentration of the drug (80 WM). Exposure of oxic cells to catalase also reduced the cytotoxic effects of the drug, indicating the involvement of oxygen-free radicals. Similarly, exposure of oxic cells to the inhibitor of superoxide dismutase, diethyldithiocarbamate, increased the cytotoxicity of the drug, more than 100-fold. 3.2. Effect of menadione on cell survival following hyperthermia Figures 3 and 4 show cell survival curves of menadione as a function of exposure time at 41°C and 42°C under glucose-fed and glucose-deprived conditions. It is clear that menadione increases the cytotoxicity of hyperthermia. Although present at 41 "C, the potentiating effect of menadione on hyperthermia-induced cell killing is far more prononced at 42 "C and is particularly evident under glucose-deprived conditions.
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Figure 2. Effects of hypoxia and oxygen-modifying agents on the menadione-induced cytotoxicity : 0 , no drug; 0, 20 p ~ A, ; 40 p ~ A, ; 80 p ~ Catalase: . 30 pg/ml; diethyldithiocarbamate: 60 VM.
3.4. Effect of menadione on glutathione metabolism in HeLa cells Menadione caused an immediate depletion of GSH, followed by a gradual recovery to the control level at 4 h. In contrast, glucose-deprived cells exposed to the drug were 41"
Glucose (+)
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Figure 3. Percentage of cell survival as a function of time after exposure of cells to menadione no drug; at 41°C under glucose-fed and glucose-deprived conditions: 0; no drug; 0, 10 pM; 0, 20 pM; A, 40 VM.
Menadione and hyperthermia
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Glucose (+)
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Figure 4. Percentage of cell survival as a function of time after exposure of cells to menadione 10 pM; 0, 20 WM; A,40 pM. at 42°C: 0 , no drug;
unable to recover from the depressed GSH to the control level (Figure 5). The GSH reductase levels increased shortly after exposure to the drug, but they gradually returned to the control level by 4 h.
Discussion The data presented herein demonstrate that cytotoxicity is markedly increased in cells deprived of glucose in the medium at 37°C after exposure to menadione. When cells were exposed to menadione (40 PM) and hyperthermia (42"C), there was a dramatic potentiation of heat-induced cytotoxicity in cells deprived of glucose in the medium (Figures 3 and 4). Evidence has been obtained in the present study showing that the cytotoxicity of menadione is principally mediated by the activated oxygen species such as superoxide and hydrogen peroxide. Simultaneous exposure of cells to menadione and catalase abrogated 4.
Table 1.
Effects of menadione on cellular energy metabolism in HeLa cells. 2h
Treatment groupa Glucose alone ( 5 mM) Glucose + menadione (40 p M ) Glucose-deprived Glucose-deprived+menadione (40 pM)
ATP~ 100 81&3d 97*3 80&3
EC' 0.76&0.02d 0.63&0.03 0.76*0.06 0.85&0.04
4h
ATP EC 100 0.76 z t 0.02 95*4 0.75*0.03 92k3 0*76=t0*01 1 0 9 ~ 1 ~ 4 0.87*0.03
"Means of two separate experiments. bATP levels are expressed as percentage of controls. 'Multiple estimates of energy charge (EC) under control conditions are reproducible with a range of 0.75-0.82. dStandard deviation.
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1
Time 2 (hr)
3
4
.,
Figure 5. Effects of menadione on glutathione metabolism at 37°C under glucose-fed and glucosedeprived conditions: 20 pM with glucose; 0, 40 p M with glucose; 40 p~ without glucose. the cytotoxicity of menadione, while the treatment of cells with diethyldithiocarbamate (DDC), an inhibitor of superoxide dismutase, significantly enhanced the cytotoxicity . Likewise, hypoxia substantially reduced the cytotoxicity of menadione (Figure 2). Lin et al. (1979) showed hyperthermic potentiation in exponentially growing DON cells by prior treatment with DDC. The present data are qualitatively similar with results obtained with mitochondrial inhibitors of oxidative phosphorylation (Lava1 and Michel 1982, Kim, J. H. et al. 1985). Using rhodamine- 123, a mitochondria1 binding agent, we have previously shown that there was a pronounced enhancement of cell kill in glucose-deprived cells following exposure of HeLa cells to the drug at 42°C. No enhanced effects of heat were seen in the glucosefed cells, unlike the present result with menadione. Although the above data were generally consistent with the concept that energy deprivation by means of oxidative phosphorylation increases sensitivity to killing by hyperthermia (Kim, S . H. et al. 1980, Gerweck et al. 1984), the present data on the ATP levels and energy charge of cells exposed to menadione failed to show an appreciable energy deprivation (Table 1). In fact, the principal effects of menadione metabolism result in a decrease in the intracellular glutathione level and NADPH oxidation (DiMonte et al. 1984). It is the oxidation of NADPH which is thought to affect calcium transport in mitochondria (Orrenius 1985). Menadione caused an immediate depletion of the cellular GSH, most pronounced in the glucose-deprived cells (Figure 5). The sustained depletion of the GSH in glucose-deprived cells may be in part responsible for the hyperthermic potentiation to menadione. There have been several cell culture studies showing that the GSH depletion by buthionine sulphoximine significantly enhances the thermosensitivity of cells (Mitchell and Russo 1983, Freeman et ul. 1985). Menadione and menadiol sodium diphosphates seem to be good candidates for clinical hyperthermic sensitizers. The drug is relatively non-toxic at 37°C. Menadiol sodium diphosphate has received some evaluation as a radiation sensitizer in the treatment of head
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and neck cancers (Krishanamurthi et al. 1971, Mitchell et al. 1965). Further, there is some evidence showing that menadiol sodium diphosphate is preferentially localized in human cancers (Mitchell et al. 1983). If menadione is found to have selective cytotoxic effects against tumour cells in vivo without undue normal tissue toxicity, it may have potential utility as a hyperthermic sensitizer, because menadione and mild hyperthermia (less than 42°C) can be readily achievable in humans.
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Refeences AKMAN, S . , CARRB., LEONG,L., MARGOLIN, K., ODUJINRIN, 0. and DOROSHOW, J., 1988, Phase I trial of menadiol sodium diphosphate in advanced cancer. Proceedings of the American Society of Clinical Oncology, 7 , 290. BELLOMO, G., JEWELL, S. A. and ORRENIUS, S . , 1982, The metabolism of menadione impairs the ability of rat liver mitochondria to take up and retain calcium. Journal of Biological Chemistry, 257, 11558-1 1562. BEUTLER,E., 1969, Effect of flavin compounds on glutathione reductase activity: in vivo and in vitro studies. Journal of Clinical Investigation, 48, 1957-1966. BEUTLER, E., 1971, Red Cell Metabolism: A Manual of Biochemical Methods (New York: Grune & Stratton ), pp. 103-105. S.K., SAWYER, J. M. and BROWN,J. M., 1984, Role of the adenylate BUMP,E. A., CALDERWOOD, energy charge in the response of Chinese hamster ovary cells to radiation. International Journal of Radiation Oncology, Biology and Physics, 10, 1411-1414. CHLEBOWSKI, R. T., DIETRICH, M. and BLOCK,J. B., 1984, Vitamin K, (menadione) inhibition of human tumor growth in the soft agar assay system. Proceedings of the American Society of Clinical Oncology, 3, 93. G., EKLOW,L. and ORRENIUS, S., 1984, Alterations in DIMONTE,D., Ross, D., BELLOMO, intracellular thiol homeostasis during the metabolism of menadione by isolated rat hepatocytes. Archives of Biochemistry and Biophysics, 235, 334-342. FREEMAN, M. L., MALCOLM,A. W. and MEREDITH,M. J., 1985, Role of glutathione in cell survival after hyperthermic treatment of Chinese hamster ovary cells. Cancer Research, 45, 6308-63 13. GERWECK, L. E., DAHLBERG, W. K., EPSTEIN,L. F. and SHIMM,D. S., 1984, Influence of nutrient and energy deprivation on cellular response to single and fractionated heat treatments. Radiation Research, 99, 573-581. KIM,J. H., KIM,S. H. and ALFIERI,A. A., 1985, Interaction of rhodamine 123 and hyperthermia in HeLa cells in culture. International Journal of Hyperthermia, 1, 247-253. KIM,S. H., KIM, J. H. and HAHN,E. W., 1978, Selective potentiation of hyperthermic killing of hypoxic cells by 5-thio-o-g~ucose.Cancer Research, 38, 2935-2938. KIM, S. H., KIM,J. H., HAHN,E. W. and ENSIGN,N. A , , 1980, Selective killing of glucose and oxygen-deprived HeLa cells by hyperthermia. Cancer Research, 40, 3459-3462. KIM, S. H., KIM,J. H., ALFIERI,A. A. and YOUNG, C. W., 1985, Gossypol, a hyperthermic sensitizer of HeLa cells. Cancer Research, 45, 6338-6340. KRISHANAMURTHI, S . , SHANTA,V. and SISTRI,N., 1971, Combined therapy in buccal mucosa cancers. Radiology, 99, 409-415. LAVAL, F. and MICHEL,S . , 1982, Enhancement of hyperthermia-induced cytotoxicity upon ATP deprivation. Cancer Letters, 15, 6 1-65. LIN,P. S., KWOCK,L. and BUTTERFIELD. C. E., 1979, Diethyldithiocarbamate enhancement of radiation and hyperthermia effects on Chinese hamster cells in vitro. Radiation Research, 77, 501-511. MITCHELL, J. B. and Russo, A,, 1983, Thiols, thiol depletion and thermosensitivity. Radiation Research, 95, 471-485. J. S., BROMBLEY, D. and HAYLITTLE, J. L., 1965, Clinical trial of radiosensitizers, MITCHELL, including Synkavite and oxygen inhaled at atmospheric pressure. Acta Radiologica Series 2: Oncology, Radiation Therapy, Physics and Biology, 3, 329-341. MITCHELL, J . S., BROWN,I. and CARPENTER, R. N., 1983, Attempts to develop radioactive anticancer drugs. Intemarional Journal ofRadiation Oncology, Biology and Physics, 9,57-59. NAGLE,W. A , , Moss, A. J. and HENLE,K. J., 1985, Sensitization of cultured Chinese hamster cells to 42°C hyperthermia by pentalenolactone, an inhibitor of glycolytic ATP synthesis. Internuiional Journal of Radiation Oncology, Biology and Physics, 48, 821-835.
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ORRENIUS, S., 1985, Biochemical mechanisms of cytotoxicity. Trends in Pharmacological Science, FEST Suppl., 515-520. PRASAD,K. N., EDWARDS-PRASAD, J. and SAKAMOTO, A., 1981, Vitamin K3 (menadione) inhibits the growth of mammalian tumor cells in culture. Life Science, 29, 1387-1392. TIETZE,F . , 1969, Enzymatic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Analytical Biochemistry, 27, 502-522.
