INT. J . HYPERTHERMIA,
1991, VOL. 7,
NO.
5 , 763-722
Enhancement of sensitivity to hyperthermia by Lonidamine in human cancer cells G. P. RAAPHORST, M. M. FEELEY, L. MARTIN, C. E. DANJOUX, J. MAROUN, A. J. DESANCTIS and D. KO The Ontario Cancer Treatment and Research Foundation, 190 Melrose Avenue, Ottawa, Ontario, Canada
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(Received 23 July 1990; revised 3 December I990; accepted 28 January 1991)
Human glioma (87MG) and squamous cell carcinoma of the head and neck UMSCC-1 were shown to be sensitized to hyperthermia by Lonidamine treatment before and during hyperthermia. The degree of thermal sensitization increased with increasing heating times and temperatures. In addition, the thermal sensitization by Lonidamine as well as cellular thermal sensitivity were dependent on pH and increased with the more acidic pH. Even though plateau phase cells were more thermally resistant than exponentially growing cells, Lonidamine treatment caused thermal sensitization under both conditions. These data show that Lonidamine may hold potential to enhance the effectiveness of hyperthermia in cancer treatment and that especially in tumours with low pH an enhanced therapeutic gain may be achieved. Key words: hyperthermia, Lonidamine pH, human tumour cells
1. Introduction Lonidamine is being evaluated as an anticancer agent at the in vitro level on a host of different cell lines and in vivo on animal tumours and in the clinic on a range of different types of tumours. The in vitro studies have shown that Lonidamine can arrest the growth of cancer cells and enhance the radiation sensitivity of mammalian cells. It can also enhance the sensitivity of mammalian cells to various chemotherapeutic agents (Raaphorst et al. 1990a, 1990b, Hahn et al. 1984, Floridi et al. 1981, Silvestrini et al. 1984, De Martino et al. 1984, Kim et al. 1984a,b, 1986, 1989, Privitera et al. 1987, Magno et al. 1984, Carapella et al. 1984, Band et al. 1986, Rosbe et al. 1989). Lonidamine has been shown to have an effect on the cellular mitochondria and results in the inhibition of oxidative phosphorylation and aerobic glycolysis, the latter through the inhibition of the mitochondria1 bound hexokinase (Floridi et al. 1981, Silvestrini et al. 1984, De Martino et al. 1984, Szekely et al. 1989). It is well known that both these pathways lead to the production of 5 ’-triphosphate (ATP) and consequently their inhibition by Lonidamine leads to reduced energy levels, although this was not directly measured. However, one study showed that Lonidamine could inhibit adriamycin reduction in cells, which occurs through an energyrequiring active process (Floridi et al. 1988). It has also been shown that if the capacity for energy production in cells is impaired, thus reducing the energy status, cells become more sensitive to hyperthermia (Calderwood 1987, Haveman and Hahn, 1981, Kim et al. 1980). In fact, the work of Gerweck et al. (1984, and reviewed in Gerweck 1988) showed that the reduction of ATP through various oxygen and glucose concentrations resulted in an increased thermal sensitivity. It was shown, using nuclear magnetic resonance techniques, that hyperthermia could reduce energy status in vivo; however, it is not clear whether this was related to cell death and tissue destruction (Sijens et al. 1989, Evanochko et al. 1983). Others have shown that hyperthermia per se may not cause the cellular depletion of high energy phosphates (Henle et al. 1984). These findings suggest the potential 0256-6736/91 $3.00 81991 Taylor & Francis Ltd
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interaction of hyperthermia with Lonidamine in the treatment of human cancer, and several studies have indicated that there may be an increase in the effectiveness of low temperature hyperthermia when combined with Lonidamine in the treatment of cancer (Kim et al. 1984a,b, Cavaliere er al. 1984, Silvestrini et al. 1983, Chitnis et ul. 1986, Feeley et a / . 1988a,b). In v i m studies have shown that Lonidamine could enhance the thermosensitivity of human cells in culture and that this enhancement is dependent on pH (Kim et al. 1984a). Further studies with Lonidamine combined with hyperthermia showed that this combination inhibited DNA synthesis in chronic myeloid leukaemia cells (Chitnis et al. 1986). In addition, studies carried out in this laboratory have shown that Lonidamine could enhance the hyperthermia-induced inhibition of recovery from radiation damage in human glioma cells and that high levels of Lonidamine caused growth inhibition in glioma, squamous cell carcinoma and Chinese hamster cells cultured in vitro (Raaphorst et al. 1990a,b, Feeley ef al. 1988a.b). This inhibition was dependent on the cell line used. These data indicate that there is potential for the use of hyperthermia combined with Lonidamine in the treatment of cancer. However, very few studies that have been carried out evaluate the combination of Lonidaniine and hyperthermia in human cancer cells. In this study these combined treatments were evaluated in human glioma and squamous cell carcinoma cell lines. The effect of Lonidaniine combined with hyperthermia under various pH conditions was also studied, as pH is an effective modulator of sensitivity to both hyperthermia and Lonidamine (Kim et al. 1984, Freeman et ul. 1980).
2. Materials and methods The human glionia cell line (U87MG) was derived from a human malignant glioblastoina and was obtained from the human tumour bank of the American Tissue Type Culture Collection. The development of this cell line and its characterization are described elsewhere (Bigner et ul. 1981). The human squamous cell carcinoma cell line (UMSCC-I) was established from a human squamous cell carcinoma of the head and neck and was classified as resistant to radiation. Details concerning this cell line are described elsewhere (Krause ef ul. 1981). All cells were cultured in a medium of Dulbecco’s modified essential medium (DMEM), nutrient mixture S-12 ( I ; l), supplemented with IS % heat-inactivated fetal bovine serum (Hyclone). 1 % non-essential amino acids and 20 mM Hepes and 10 mM sodium hydrogen carbonate buffers. The cell cultures were incubated at 37°C in a humidified atmosphere of 2 % CO, in 98% air. For routine subculturing all cells were grown in tissue culture plasticware and resuspended for handling and subculturing by trypsinization (0.2 %). For experiments, cells were grown to exponential or plateau growth phase as indicated in Section 3. At the desired phase of cell growth, the cells were exposed to experimental conditions and after the experimental treatment the cells were collected via trypsinization, counted on an electronic cell counter and plated into 60 mm dishes at numbers estimated to produce SO- ISO viable colonies. The plating efficiencies of the cell lines ranged from 20 to 40% for the glioma cell line and from 20 to 40% for the squamous cell carcinoma cell line. For these experiments, Lonidamine was added either immediately before hypertherniia, 24 h before hyperthermia, or for prolonged periods before hyperthermia, as indicated in the tigure captions. Lonidamine was dissolved in dimethylsulphoxide (DMSO) and was diluted by a ratio of 500: 1 for the treatment of cell cultures. Appropriate DMSO controls were also evaluated under the various experimental conditions and no toxicity was found when the cells were exposed to the residual DMSO concentrations alone during the various hyperthermia and pH conditions. It should be noted that the previous work of Gerweck (1988) showed that DMSO concentrations as low as 0.086% caused a reduced oxygen
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uptake. The concentrations used here were 0 . 2 % and no effects on survival measurements were observed, which is in agreement with earlier work on FEL cells (Raaphorst et al. 1983). However, the effects on oxygen uptake were not measured and such changes, if they occurred at low levels, did not affect the thermal survival results. All Lonidamine treatments were carried out at 37°C and at the hyperthermia temperatures indicated in the figures. After treatment Lonidamine was removed by decanting the medium from the culture dishes and rinsing with fresh medium. For hyperthermia treatment the cells were plated into 25 cm2 tissue culture flasks which were sealed with Parafilm just before the hyperthermia treatment. All hyperthermia treatments were carried out in a temperaturecontrolled circulating water-bath accurate to f0.05"C. The hyperthermia treatment times are the total immersion times of the cultures in the temperature-controlled water-bath. After treatment the cell cultures were incubated for the colony-forming assay. After 8-12 days the tissue culture flasks o r dishes were rinsed, fixed and stained, and the colonies were counted. Only colonies with &50 cells were scored for the survival assay. Each experiment was repeated two or more times and the error bars on the figures indicate the standard error of the mean (S.E.M.) of six replicates per point. The pH in the cultures was set by titrating each culture with 0.1 M hydrochloric acid or 0.1 M sodium hydroxide and the pH remained constant during the hyperthermia exposure time.
3. Results The thermal response of plateau phase human glioma cells is shown in Figure 1. Cells were heated to 41 or 43°C in the presence or absence of Lonidamine. At both temperatures Lonidamine caused thermosensitization. At 41 "C, the thermosensitization occurred after longer heating times, whereas at 43°C thermosensitization occurred after 1 and 2 h of heating. Such shorter heating times are achievable in the clinic and thus show the potential for hyperthermia as a sensitizer under clinically achievable hyperthermia treatments. Figure 2 shows the response of plateau phase human glioma cells to hyperthermia at 42°C f Lonidamine. Lonidamine concentrations of both 10 and 50 pg/ml resulted in small to moderate degrees of thermosensitization. The sensitization increased with heating time and for the 50 pg/ml treatment the sensitivity was enhanced by factors of 1. I , 1 - 4 and 1.3 for the 1, 3 and 5 h hyperthermia exposures, respectively. In the clinical protocol used at this institute, hyperthermia treatments are given for a duration of 1 h, and therefore the effect of Lonidamine on 1 h heat treatments over a temperature range 41-44°C were evaluated. This is shown in Figure 3 for human glioma cells. The 50 pgirnl Lonidamine dose caused thermosensitization at all temperatures; however, the thermosensitization at 42°C was relatively small and this sensitization increased with temperature. The thermosensitization ratios are 1.05, 1 * I , 1 - 7 and 2.2 for the 1 h heating interval at 41, 42, 43 and 44"C, respectively. The effect of reducing the pH to 6 . 8 on the thermal sensitization to Lonidamine is shown in Figure 4 for human glioma cells. When Lonidamine (50 pg/ml) was given in combination with hyperthermia at a low pH, a large degree of thermosensitization was achieved for heating at 42°C. The thermal enhancement ratios were 2 - 0 , 4.8 and 4.0 for the 1, 5 and 7 h heat treatments, respectively. These data show that even for the 1 h heating time Lonidamine could cause extensive thermosensitization when the pH was 6.8. To further evaluate the effect of pH, human glioma cells were tested under plateau and exponential phase conditions over a range of pH conditions from 6.6 to 7.4. The data in Figure 5 indicates that heating at 42°C for 2 h resulted in an increased thermosensitivity as the pH of the culture was reduced from 7 - 4 to 6 - 6 . This effect was observed to a greater extent in exponentially growing cells than in plateau phase cells. When
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87MG
2
''0
PLATEAU PHASE
4
-
6 8 p 1 2 3 HEATING TIME in ( h )
4
Figure 1. Survival of cells treated with Lonidamine for 24 h and then exposed to hyperthermia and Lonidamine for the times indicated. The glioma cell cultures were in plateau phase and the pH was maintained at 7 . 4 .
Lonidamine was present during the hyperthermia treatment, Lonidamine thermosensitization increased as a function of the reduction of pH. This occurred for both plateau and exponential phase cell cultures. The thermo-enhancement ratios for Lonidamine treatment for plateau phase cells were 1 . 1 (pH 7.4), 1-14 (pH 7.2), 1.2 (pH 7.0), 2 . 2 (pH 6.8), 1 a 9 (pH 6.7) and 7 -0(pH 6.6). For the exponential phase cultures the thermo-enhancement ratios were 1.04 (pH 7.4), 1.6 (pH 7.0), 2 . 3 and (pH 6.6). The data in Figure 5 also show that plateau phase cells were more resistant to hyperthermia than exponential phase cells. This effect was fwther evaluated and is shown in Figure 6. In the experiments presented in Figure 6, the cells were exposed for longer periods of time to Lonidamine, as indicated in the figure caption, to attempt to obtain
1 LL.
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8 7 .MG
PLATEAU PHASE
I 1
RR -r
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Figure 2. Survival of plateau phase glioma cells exposed to two different concentrations of Lonidamine at pH 7 . 4 for 24 h before heating and during a 1 h heat treatment at 42°C.
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87 M G PLATEAU PHASE 1 h HEATING L =50 wm/mi
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7
41.0
42.0
I.o
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Figure 3. Survival of cells treated for 24 h before heating with 50 pg/ml of Lonidamine. Cells were heated for 1 h at temperatures ranging from 41 to 44°C and Lonidamine was removed immediately after heating. The pH was maintained at 7.4.
a greater level of sensitizationby Lonidamine. These data show that the prolonged exposure of Lonidamine did not further increase the thermosensitization to 42°C compared to the 1 day exposures for the data presented in the previous figures. The data show that plateau phase cells are much more resistant than exponential phase cells to hyperthermia and that Lonidamine sensitization occurred under both plateau phase and exponential phase conditions. Figure 7 shows the effect of hyperthermia in the presence or absence of Lonidamine on human squamous cell carcinoma cells cultured in plateau phase. The data show that the thermosensitivity increases with heating time and heating temperature and that Lonidamine exposure increased the thermosensitivity at both 41 "C and 42°C. Two hours
I 8 7 MG PLATEAU PHASE pH 6.8 L=SOpgm/rnl
'IIlk 2 1
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s 40 I
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5.0
T
b 70
TIME AT 42.OoC ( h )
Figure 4. Survival of human glioma plateau phase cells held at pH 6.8 during Lonidamine and hyperthermia treatment. Lonidamine treatment was started 24 h before heating and maintained during heating. L indicates the survival data from cells exposed to Lonidamine.
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L = 50 p q d m l
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PLATEAU
87MG
PHASE
42.0OC ( 2 h) EXPONENTIAL M S t
II:I
7.0
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66
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74
6.6
Figure 5 . Survival of exponentially growing and plateau phase cells to a combination treatment of Lonidamine and hyperthermia. Lonidamine exposure was given 24 h before and during the hypcrthermia treatment. During this period the pH was varied from 7.4 to 6.6. L indicates the survival o f the glioma cells that received Lonidamine treatment; the blank bars indicatc only pH and hyperthermia exposure. Hyperthermia exposure was for 2 h at 42°C.
87MG
2 4 TIME at 42.OoC f h )
I
6
Figure 6. Survival o f exponentially growing and plateau phase cells exposed to Lonidamine for 90 h for cxponentially growing cells, or 120 h for plateau phase cells before heating at 42°C. Lonidamine exposure was maintained during the heating period. the pH of these glioma cell culturcs was maintained at 7 . 4 during the experiment.
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42 OC
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cz
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'0'0
PLATEAU PHASE
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L=SOj.grn/snl
z
I
I
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1
3 2 4 6 HEATING TIME i n ( h )
8
Figure 7 . Survival of squarnous cell carcinoma cell cultures held in plateau phase and exposed to Lonidamine 24 h before and during the heating period. Hyperthermia was given at 41 and 42°C. Lonidamine concentration was 50 pg/ml and is indicated by L. The pH of the culture was maintained at 7 . 4 during treatment.
of heating at 41°C or 42°C resulted in substantial sensitization and is consistent with potential heating times achievable in a clinical environment. The data in Figure 8 show the thermosensitivity of the human squamous cell sarcinoma line after heating to 43°C. The data in this figure show that Lonidamine exposure increased the thermosensitivity of this cell line under plateau phase conditions and that thermosensitization was significant at heating times as short as 1 h, indicating potential clinical efficiency.
t-
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-
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--> >
U 3
.-
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I
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Figure 8. Survival of plateau phase squamous cell carcinoma cells after exposure to Lonidamine 24 h before and during the hyperthermia treatment at 43°C. Lonidamine treatment is indicated by L and the pH was maintained at 7 . 4 during the treatments.
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4. Discussion Earlier evaluations of the effect of hyperthermia on cell killing and cellular energy status indicate that the two may be related. In particular, a paper by Sijens et al. (1989) indicated that the level of high energy metabolites could be directly correlated to thermal dose. It was further indicated that stress or the depletion of cellular energy status could result in thermal sensitization (Calderwood 1987) and Gerweck et al. (1984) showed that reduced ATP levels caused thermal sensitization. In addition, it was shown that Lonidamine could inhibit glycolysis in mammalian cells and thus may reduce the energy status in malignant cells as these rely on high levels of glycolysis; however, energy status was not directly measured (Floridi et al. 1981, Silvestrini et a f . 1984, De Martino et al. 1984). Considering that hyperthermia and Lonidamine may modulate and affect the same potential targets within the cell, the application of these two treatments may result in an interdependent interaction in terms of cell toxicity. This was proven in a limited number of studies shown that hyperthermia could enhance the cytotoxicity of Lonidamine in human cells and murine tumours (Feeley et al. 1988a,b, Chitnis et al. 1986, Silvestrini el al. 1983, Cavaliere et al. 1984, Kim et al. 1984a,b). In addition, preliminary data showed that the application of hyperthermia with Lonidamine could also enhance cytotoxicity in human glioma cells (Feeley et al. 1988a,b). In this study the interaction of hyperthermia and Lonidamine under various pH conditions has been investigated to assess the potential of this combined treatment for cancer therapy. The data show that hyperthermia can cause extensive sensitization to Lonidamine treatment at concentration levels achievable clinically. Such thermal sensitization of Lonidamine toxicity was achievable in both exponentially growing and plateau phase cell cultures of glioma and squamous cell carcinoma cell lines. In addition, thermal sensitization could be achieved at 1-2 h hyperthermia exposure times, which are generally accepted to be clinically achievable. The data in these experiments show that the degree of Lonidamine sensitization by hyperthermia depends strongly on the pH during the treatment time. The degree of sensitization increased as the pH became more acidic and the thermal enhancement factors varied from 1 -1 (pH 7.4) to 7.0 (pH 6.6), with heating for 1 h at 42°C in cells that exposed to 50 pg/ml of Lonidamine. Stryker and Gerweck (1988) have shown that reduced pH causes Lonidamine to have a greater inhibition on the oxygen uptake and suggest that under low extracellular pH conditions, Lonidamine, a weak acid, may be more concentrated in the relatively more basic intracellular compartment. The results presented here support such an idea as the effect of Lonidamine increased as the pH decreased. A recent review (Wike-Hooley et at. 1984) showed that low pH exists in a range of human tumours and therefore the thermal enhancement of Lonidamine toxicity in such tumours might hold potential for enhanced response. Thus combined hyperthermia and Lonidamine treatment of tumours could result in a therapeutic gain compared to normal tissues at normal pH. In addition, these data have shown that plateau phase cells are much more resistant to hyperthermia than exponential phase cells. This is probably related to the distribution of cells within the cell cycle, as several studies have shown that S phase cells are much more sensitive to hyperthermia than G,/G, cells (Westra are Dewey 1971, Raaphorst et a f . 1985). The increased percentage of the S phase fraction in exponentially growing cells provides an explanation for the heightened sensitivity. However, these data show that both plateau phase and exponentially growing cells are sensitized to combinations of hyperthermia and Lonidamine, and therefore, in cancer treatment in tumour environments containing both quiescent and progressive cells, hyperthermia and Lonidamine may offer a potential clinical enhancement of the therapeutic response.
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References BAND,P. R., MAROUN, J., PRITCHARD, K., STEWART,D., COPPIN,C. M., WILSON,K., and EISENHAUER, E. A., 1986, Phase I1 study of Lonidamine in patients with metastatic breast cancer. Cancer Treatment Reports, 70, 1305-1310. B., MAHALEY, M. S., RVOSLAHTI, BIGNER,D. D., BIGNER,S. H., PONTEN,J., WESTERMARK, E., HERSHCHMAN, H., ENG,L. F., and WIKSTRAND, C. J., 1981, Heterogeneity of genotypic and phenotypic characteristics of 15 permanent cell lines derived from human glioma. Journal of Neuropathology and Experimental Neurology, 40, 20 1-229. S. K., 1987, Role of energy in cellular responses to heat. In: Temperature and CALDERWOOD, Animal Cells, edited by K. Bowler and B. J. Fuller, (Cambridge: Council of Biology), pp. 212-233. S. D., and RICCIO,A., 1984, Lonidamine in primary CARAPELLA, C. M., IANDOLO, B., CHIARE, malignant brain tumours. Oncology, 41, 82-85. A., CARLINI,S., CALABRO, A., ALOE, L., and CAVALIERE, R., DI FILIPPO,F., VARANESE, PIARuLLi, L., 1984, Lonidamine and hyperthermia: clinical experience in melanoma. Oncology, 41, 116-120. CHITNIS, M., JUVEKAR, A,, AWANKAR, M., and ADVANI,S., 1986, Inhibition of DNA synthesis by Lonidamine and hyperthermia in human chronic myeloid leukema cells. Anti Cancer Research, 6, 749-152. T. H., DURANT, J. R., EVANOCHKO, W. T., NG, T. C., LILLY,M. B., LAWSON, A. J., CORBETT, J. D., 1983, In vivo 3'P NMR study of the metabolism of murine 16/C and GLICKSON, adenocarcinoma and its response to chemotherapy, X-irradiation and hyperthermia. Pruceedings of the National Academy of Sciences, USA, 80, 334-338. B., and MARTIN, FEELEY,M. M., RAAPHORST, G. P., DANJOUX, C. E., MAROUN, M., FISCHER, L., 1988a, The response of human cells and rodent cells to Lonidamine radiation and hyperthermia. Clinical and Investigational Medicine, C96, 11. G. P., DANJOUX, C. E., MAROUN, J., FISCHER,B., and MARTIN, FEELEY,M . M., RAAPHORST, L., 1988b, The response of human cells and rodent cells to Lonidamine radiation and hyperthermia. Clinical and Investigational Medicine, C96, 1 1. FLORIDI, A., GAMBACURTA, A., BAGNATO, A., BIANCHI, C., PAGGI,M. G., SILVESTRINI, B., and CAPUTO,A., 1988, Modulation of Adriamycin uptake by Lonidamine in Ehrlich ascites tumour cells. Experimental and Molecular Pathology, 499, 421-43 1. FLORIDI, A,, PAGGI,M. G., MARCANTE, M. L., SILVESTRINI, B., CAPUTO, A., and DE MARTINO, C., 1981, Lonidamine a selective inhibitor of aerobic glycolysis of murine tumour cells. Journal of the National Cancer Institute, 66, 497-499. FREEMAN, M. L., RAAPHORST, G. P., HOPEWOOD, L. E., and DEWEY,W. C., 1980, The effect of pH on cell lethality induced by hyperthermic treatment for cancer. Cancer, 45, 61-70. GERWECK, L. E., DAHLBERG, W. C., EPSTEIN,L., 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. GERWECK, L. E., 1988, Modifiers of thermal effects: environmental factors. In: Hypenhermia and Oncology, vol. 1, edited by M. Urano and E. Douple, (Utrecht: VSP), pp. 83-98. B., Inhibition of the recovery from potentially HAHN,G. M., VAN KERSEN,I., and SILVESTRINNI, lethal damage by Lonidamine. British Journal of Cancer, 50, 657-660. J., and HAHN,G. M., 1981, The role of energy in hyperthermia induced mammalian HAVEMAN, cell inactivation. A study of the effects of glucose starvation and an uncoupler of oxidative phosphorylation. Journal of Cell Physiology, 108, 231-241. HENLE,K. J., NAGLE,W. A., MOSS, A. J., and HERMAN, T. S., 1984, Cellular ATP content of heated Chinese hamster ovary cells. Radiation Research, 97, 630-639. 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. G., 1984a, Lonidamine: KIM,J. H., KIM, S. H., ALFIERI,A., YOUNG,C. W., and SILVESTRINI, A hyperthermic sensitizer of HeLa cells in culture and of the Meth-A tumour in vivo. Oncology, 41, 30-35. B., 1984b, RadioKIM, J. H., ALFIERI,A. A., KIM, S . H., YOUNG, C. W., and SILVESTRINI, sensitization of Meth-A fibrosarcoma in mice by Lonidamine. Oncology, 41, 36-38. A. A., KIM,S. H., and YOUNG,C. W., 1986, Potentiation of radiation effects KIM,J. H., ALFIERI, on two murine tumours by Lonidamine. Cancer Research, 46, 1120-1 123.
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KIM, J. H., KIM, S. H., HE, S. Q., ALFIERI,A. A , , and YOUNG,C . W . , 1989, Potentiation of radiation effects on multicellular tuniour spheroids (MTS) of HeLa cells by Lonidamine. Journal of Radiation Oncology, 16, 1277-1280. KRAUSE,C . J., CAREY,T. E., OTT, R . W . , HURBIS,C . , CLATCHEY, M . C . , and REGEZI,K. Z., 198I , Human squamous cell carcinoma. Establishment and characterization of new permanent cell lines. Archives of Otolaryngology, 107, 703-7 10. MAGNO.L., TERRANEO, F., and CIOTTOLI, G. B., 1984, Lonidamine and radiotherapy in head and neck cancers. Oncology. 41, 113-1 15. DE MARTINO, C.. BATTEI.I.I.T., PAGGI,M . G., NISTA, A., MARCANTE, M . L., ATRI, S. D., MALORNI,W . , GALLO,M . , and FLORIDI, A , , 1984, Effects of Lonidamine on murine and human tumour cells in vitro. A morphological and biochemical study. Oncology, 41, 15-29. PRIVITERA, G., CIOTTOLI. G. B., PATAINE,C . , PALMUCCI, T., TAFURI,G., MARLETTA,F., DELUCA,B., MAGNANI, F.. DEGREGORIO, M.. and GRECO,S., 1987, Phase I1 double-blind randomized study of Lonidamine and radiotherapy in epidermoid carcinoma of the lung. Rudiotherapy and Oncology, 10, 285-290. RAAPHOKSL', G . P., AZZAM, E. I., BORSA,J., EINSPENNER, M . , and VADASZ, J., 1983, Hypertherniia in a differentiated murine erythroleukemia cell line 11. Effect of heat on heme induction by radiation. International Journal (.f Radiation Biology, 44, 343-352. RAAPHORST, G. P . , BROSKI,A. P., and AZZAM,E. I., 1985, Sensitivity to heat, radiation and hcat plus radiation of Chinese hamster cells, synchronized by mitotic selection, thymidine block or hydroxyurea. Journal .f Thermal Biology, 10, 177-181. RAAPHORST, G. P., FEELEY,M . M . , DANJOUX. C . E., MARTIN,L.. MAROUN, J., and DESANCTIS, A . J . , 1990a, The effect of Lonidamine on radiation and thermal responses of human and rodcnt cell lines. Internuiional Journul of Radiation Oncology, 20, 509-5 IS, 1991. RAAPHORST, G. P., FEBLEY, M . M . , HELLER.D. P . , DANJOUX, C . E., MARTIN,L., MAROON. J . , and DESANCTIS, A. J., 1990b, Lonidamine can enhance the cytotoxic effect of cisplatin in human tumour cells and rodent cells. Anticancer Research, 10, 923-928. ROSBE,K. W . , B R A N NT. , W.. HOLDEN,S. A , , TEICHER, B. A., and FREI,E. 111, 1989, Effect of Lonidaniinc on the cytotoxicity of four alkylating agents in vitro. Cancer Chemorhercips and Pharmacology, 25, 32-36. SIJENS,P. E., BOVEE,W . M . M. J . , KOOLE,P., and SCHIPPER, J.. 1989, Phosphorous NMR study of response of a murine tumour to hyperthermia as a function of treatment time and t e n perature. International Journal of Hyperthermia, 5 , 35 1-351. SILVESTRINI. B., HAHN,G. M., CIOLI, V., and DE MARTINO,C . , 1983, Effects of Lonidaniinc alone or combined with hyperthermia in some experimental cell and tumour systems. British Journal of Cuncer, 47, 221-235. SII.VESTRINI, B., PALAZZO, G . , and DEGREGORIO, M . , 1984, Lonidamine and related compounds. In: Progress in Medicinal Chemistry, edited by G . P. Ellis and C. P. West), vol. 2, pp. 1 11-135. STRYKER, J. A., and GERWECK,L. E., 1988, Lonidamine-induced pH dependent inhibition of cellular oxygen utilization. Radiation Research, 113, 356-361 1. SZEKELY, J . G . , LOHKEAU, A. U., DELANEY. S., RAAPHORST, G. P., and FEELEY,M . M . , 1989, Morphological effects of Lonidamine on human tumour cells. Scanning Electron Micmx-op-y. 3, 68 1-693. WESTKA,A., and DEWEY,W . C., 1971, Variation in sensitivity to heat shock during the cell cycle of Chinese hamster cells in virro. International Journal of Radiation Biology, 19, 461-477. WIKE-HOOLEY, J . L . , HAVGMAN, J., and REINHOLD,H. S., 1984, The relevance of tuniour pH to the treatment of malignant disease. Radiotherapy and Oncology, 2, 343-366.
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Enhancement of sensitivity to hyperthermia by Lonidamine in human cancer cells G. P. RAAPHORST, M. M. FEELEY, L. MARTIN, C. E. DANJOUX, J. MAROUN, A. J. DESANCTIS and D. KO The Ontario Cancer Treatment and Research Foundation, 190 Melrose Avenue, Ottawa, Ontario, Canada
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(Received 23 July 1990; revised 3 December I990; accepted 28 January 1991)
Human glioma (87MG) and squamous cell carcinoma of the head and neck UMSCC-1 were shown to be sensitized to hyperthermia by Lonidamine treatment before and during hyperthermia. The degree of thermal sensitization increased with increasing heating times and temperatures. In addition, the thermal sensitization by Lonidamine as well as cellular thermal sensitivity were dependent on pH and increased with the more acidic pH. Even though plateau phase cells were more thermally resistant than exponentially growing cells, Lonidamine treatment caused thermal sensitization under both conditions. These data show that Lonidamine may hold potential to enhance the effectiveness of hyperthermia in cancer treatment and that especially in tumours with low pH an enhanced therapeutic gain may be achieved. Key words: hyperthermia, Lonidamine pH, human tumour cells
1. Introduction Lonidamine is being evaluated as an anticancer agent at the in vitro level on a host of different cell lines and in vivo on animal tumours and in the clinic on a range of different types of tumours. The in vitro studies have shown that Lonidamine can arrest the growth of cancer cells and enhance the radiation sensitivity of mammalian cells. It can also enhance the sensitivity of mammalian cells to various chemotherapeutic agents (Raaphorst et al. 1990a, 1990b, Hahn et al. 1984, Floridi et al. 1981, Silvestrini et al. 1984, De Martino et al. 1984, Kim et al. 1984a,b, 1986, 1989, Privitera et al. 1987, Magno et al. 1984, Carapella et al. 1984, Band et al. 1986, Rosbe et al. 1989). Lonidamine has been shown to have an effect on the cellular mitochondria and results in the inhibition of oxidative phosphorylation and aerobic glycolysis, the latter through the inhibition of the mitochondria1 bound hexokinase (Floridi et al. 1981, Silvestrini et al. 1984, De Martino et al. 1984, Szekely et al. 1989). It is well known that both these pathways lead to the production of 5 ’-triphosphate (ATP) and consequently their inhibition by Lonidamine leads to reduced energy levels, although this was not directly measured. However, one study showed that Lonidamine could inhibit adriamycin reduction in cells, which occurs through an energyrequiring active process (Floridi et al. 1988). It has also been shown that if the capacity for energy production in cells is impaired, thus reducing the energy status, cells become more sensitive to hyperthermia (Calderwood 1987, Haveman and Hahn, 1981, Kim et al. 1980). In fact, the work of Gerweck et al. (1984, and reviewed in Gerweck 1988) showed that the reduction of ATP through various oxygen and glucose concentrations resulted in an increased thermal sensitivity. It was shown, using nuclear magnetic resonance techniques, that hyperthermia could reduce energy status in vivo; however, it is not clear whether this was related to cell death and tissue destruction (Sijens et al. 1989, Evanochko et al. 1983). Others have shown that hyperthermia per se may not cause the cellular depletion of high energy phosphates (Henle et al. 1984). These findings suggest the potential 0256-6736/91 $3.00 81991 Taylor & Francis Ltd
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interaction of hyperthermia with Lonidamine in the treatment of human cancer, and several studies have indicated that there may be an increase in the effectiveness of low temperature hyperthermia when combined with Lonidamine in the treatment of cancer (Kim et al. 1984a,b, Cavaliere er al. 1984, Silvestrini et al. 1983, Chitnis et ul. 1986, Feeley et a / . 1988a,b). In v i m studies have shown that Lonidamine could enhance the thermosensitivity of human cells in culture and that this enhancement is dependent on pH (Kim et al. 1984a). Further studies with Lonidamine combined with hyperthermia showed that this combination inhibited DNA synthesis in chronic myeloid leukaemia cells (Chitnis et al. 1986). In addition, studies carried out in this laboratory have shown that Lonidamine could enhance the hyperthermia-induced inhibition of recovery from radiation damage in human glioma cells and that high levels of Lonidamine caused growth inhibition in glioma, squamous cell carcinoma and Chinese hamster cells cultured in vitro (Raaphorst et al. 1990a,b, Feeley ef al. 1988a.b). This inhibition was dependent on the cell line used. These data indicate that there is potential for the use of hyperthermia combined with Lonidamine in the treatment of cancer. However, very few studies that have been carried out evaluate the combination of Lonidaniine and hyperthermia in human cancer cells. In this study these combined treatments were evaluated in human glioma and squamous cell carcinoma cell lines. The effect of Lonidaniine combined with hyperthermia under various pH conditions was also studied, as pH is an effective modulator of sensitivity to both hyperthermia and Lonidamine (Kim et al. 1984, Freeman et ul. 1980).
2. Materials and methods The human glionia cell line (U87MG) was derived from a human malignant glioblastoina and was obtained from the human tumour bank of the American Tissue Type Culture Collection. The development of this cell line and its characterization are described elsewhere (Bigner et ul. 1981). The human squamous cell carcinoma cell line (UMSCC-I) was established from a human squamous cell carcinoma of the head and neck and was classified as resistant to radiation. Details concerning this cell line are described elsewhere (Krause ef ul. 1981). All cells were cultured in a medium of Dulbecco’s modified essential medium (DMEM), nutrient mixture S-12 ( I ; l), supplemented with IS % heat-inactivated fetal bovine serum (Hyclone). 1 % non-essential amino acids and 20 mM Hepes and 10 mM sodium hydrogen carbonate buffers. The cell cultures were incubated at 37°C in a humidified atmosphere of 2 % CO, in 98% air. For routine subculturing all cells were grown in tissue culture plasticware and resuspended for handling and subculturing by trypsinization (0.2 %). For experiments, cells were grown to exponential or plateau growth phase as indicated in Section 3. At the desired phase of cell growth, the cells were exposed to experimental conditions and after the experimental treatment the cells were collected via trypsinization, counted on an electronic cell counter and plated into 60 mm dishes at numbers estimated to produce SO- ISO viable colonies. The plating efficiencies of the cell lines ranged from 20 to 40% for the glioma cell line and from 20 to 40% for the squamous cell carcinoma cell line. For these experiments, Lonidamine was added either immediately before hypertherniia, 24 h before hyperthermia, or for prolonged periods before hyperthermia, as indicated in the tigure captions. Lonidamine was dissolved in dimethylsulphoxide (DMSO) and was diluted by a ratio of 500: 1 for the treatment of cell cultures. Appropriate DMSO controls were also evaluated under the various experimental conditions and no toxicity was found when the cells were exposed to the residual DMSO concentrations alone during the various hyperthermia and pH conditions. It should be noted that the previous work of Gerweck (1988) showed that DMSO concentrations as low as 0.086% caused a reduced oxygen
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uptake. The concentrations used here were 0 . 2 % and no effects on survival measurements were observed, which is in agreement with earlier work on FEL cells (Raaphorst et al. 1983). However, the effects on oxygen uptake were not measured and such changes, if they occurred at low levels, did not affect the thermal survival results. All Lonidamine treatments were carried out at 37°C and at the hyperthermia temperatures indicated in the figures. After treatment Lonidamine was removed by decanting the medium from the culture dishes and rinsing with fresh medium. For hyperthermia treatment the cells were plated into 25 cm2 tissue culture flasks which were sealed with Parafilm just before the hyperthermia treatment. All hyperthermia treatments were carried out in a temperaturecontrolled circulating water-bath accurate to f0.05"C. The hyperthermia treatment times are the total immersion times of the cultures in the temperature-controlled water-bath. After treatment the cell cultures were incubated for the colony-forming assay. After 8-12 days the tissue culture flasks o r dishes were rinsed, fixed and stained, and the colonies were counted. Only colonies with &50 cells were scored for the survival assay. Each experiment was repeated two or more times and the error bars on the figures indicate the standard error of the mean (S.E.M.) of six replicates per point. The pH in the cultures was set by titrating each culture with 0.1 M hydrochloric acid or 0.1 M sodium hydroxide and the pH remained constant during the hyperthermia exposure time.
3. Results The thermal response of plateau phase human glioma cells is shown in Figure 1. Cells were heated to 41 or 43°C in the presence or absence of Lonidamine. At both temperatures Lonidamine caused thermosensitization. At 41 "C, the thermosensitization occurred after longer heating times, whereas at 43°C thermosensitization occurred after 1 and 2 h of heating. Such shorter heating times are achievable in the clinic and thus show the potential for hyperthermia as a sensitizer under clinically achievable hyperthermia treatments. Figure 2 shows the response of plateau phase human glioma cells to hyperthermia at 42°C f Lonidamine. Lonidamine concentrations of both 10 and 50 pg/ml resulted in small to moderate degrees of thermosensitization. The sensitization increased with heating time and for the 50 pg/ml treatment the sensitivity was enhanced by factors of 1. I , 1 - 4 and 1.3 for the 1, 3 and 5 h hyperthermia exposures, respectively. In the clinical protocol used at this institute, hyperthermia treatments are given for a duration of 1 h, and therefore the effect of Lonidamine on 1 h heat treatments over a temperature range 41-44°C were evaluated. This is shown in Figure 3 for human glioma cells. The 50 pgirnl Lonidamine dose caused thermosensitization at all temperatures; however, the thermosensitization at 42°C was relatively small and this sensitization increased with temperature. The thermosensitization ratios are 1.05, 1 * I , 1 - 7 and 2.2 for the 1 h heating interval at 41, 42, 43 and 44"C, respectively. The effect of reducing the pH to 6 . 8 on the thermal sensitization to Lonidamine is shown in Figure 4 for human glioma cells. When Lonidamine (50 pg/ml) was given in combination with hyperthermia at a low pH, a large degree of thermosensitization was achieved for heating at 42°C. The thermal enhancement ratios were 2 - 0 , 4.8 and 4.0 for the 1, 5 and 7 h heat treatments, respectively. These data show that even for the 1 h heating time Lonidamine could cause extensive thermosensitization when the pH was 6.8. To further evaluate the effect of pH, human glioma cells were tested under plateau and exponential phase conditions over a range of pH conditions from 6.6 to 7.4. The data in Figure 5 indicates that heating at 42°C for 2 h resulted in an increased thermosensitivity as the pH of the culture was reduced from 7 - 4 to 6 - 6 . This effect was observed to a greater extent in exponentially growing cells than in plateau phase cells. When
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Figure 1. Survival of cells treated with Lonidamine for 24 h and then exposed to hyperthermia and Lonidamine for the times indicated. The glioma cell cultures were in plateau phase and the pH was maintained at 7 . 4 .
Lonidamine was present during the hyperthermia treatment, Lonidamine thermosensitization increased as a function of the reduction of pH. This occurred for both plateau and exponential phase cell cultures. The thermo-enhancement ratios for Lonidamine treatment for plateau phase cells were 1 . 1 (pH 7.4), 1-14 (pH 7.2), 1.2 (pH 7.0), 2 . 2 (pH 6.8), 1 a 9 (pH 6.7) and 7 -0(pH 6.6). For the exponential phase cultures the thermo-enhancement ratios were 1.04 (pH 7.4), 1.6 (pH 7.0), 2 . 3 and (pH 6.6). The data in Figure 5 also show that plateau phase cells were more resistant to hyperthermia than exponential phase cells. This effect was fwther evaluated and is shown in Figure 6. In the experiments presented in Figure 6, the cells were exposed for longer periods of time to Lonidamine, as indicated in the figure caption, to attempt to obtain
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Figure 2. Survival of plateau phase glioma cells exposed to two different concentrations of Lonidamine at pH 7 . 4 for 24 h before heating and during a 1 h heat treatment at 42°C.
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87 M G PLATEAU PHASE 1 h HEATING L =50 wm/mi
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Figure 3. Survival of cells treated for 24 h before heating with 50 pg/ml of Lonidamine. Cells were heated for 1 h at temperatures ranging from 41 to 44°C and Lonidamine was removed immediately after heating. The pH was maintained at 7.4.
a greater level of sensitizationby Lonidamine. These data show that the prolonged exposure of Lonidamine did not further increase the thermosensitization to 42°C compared to the 1 day exposures for the data presented in the previous figures. The data show that plateau phase cells are much more resistant than exponential phase cells to hyperthermia and that Lonidamine sensitization occurred under both plateau phase and exponential phase conditions. Figure 7 shows the effect of hyperthermia in the presence or absence of Lonidamine on human squamous cell carcinoma cells cultured in plateau phase. The data show that the thermosensitivity increases with heating time and heating temperature and that Lonidamine exposure increased the thermosensitivity at both 41 "C and 42°C. Two hours
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Figure 4. Survival of human glioma plateau phase cells held at pH 6.8 during Lonidamine and hyperthermia treatment. Lonidamine treatment was started 24 h before heating and maintained during heating. L indicates the survival data from cells exposed to Lonidamine.
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Figure 5 . Survival of exponentially growing and plateau phase cells to a combination treatment of Lonidamine and hyperthermia. Lonidamine exposure was given 24 h before and during the hypcrthermia treatment. During this period the pH was varied from 7.4 to 6.6. L indicates the survival o f the glioma cells that received Lonidamine treatment; the blank bars indicatc only pH and hyperthermia exposure. Hyperthermia exposure was for 2 h at 42°C.
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Figure 6. Survival o f exponentially growing and plateau phase cells exposed to Lonidamine for 90 h for cxponentially growing cells, or 120 h for plateau phase cells before heating at 42°C. Lonidamine exposure was maintained during the heating period. the pH of these glioma cell culturcs was maintained at 7 . 4 during the experiment.
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Figure 7 . Survival of squarnous cell carcinoma cell cultures held in plateau phase and exposed to Lonidamine 24 h before and during the heating period. Hyperthermia was given at 41 and 42°C. Lonidamine concentration was 50 pg/ml and is indicated by L. The pH of the culture was maintained at 7 . 4 during treatment.
of heating at 41°C or 42°C resulted in substantial sensitization and is consistent with potential heating times achievable in a clinical environment. The data in Figure 8 show the thermosensitivity of the human squamous cell sarcinoma line after heating to 43°C. The data in this figure show that Lonidamine exposure increased the thermosensitivity of this cell line under plateau phase conditions and that thermosensitization was significant at heating times as short as 1 h, indicating potential clinical efficiency.
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Figure 8. Survival of plateau phase squamous cell carcinoma cells after exposure to Lonidamine 24 h before and during the hyperthermia treatment at 43°C. Lonidamine treatment is indicated by L and the pH was maintained at 7 . 4 during the treatments.
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4. Discussion Earlier evaluations of the effect of hyperthermia on cell killing and cellular energy status indicate that the two may be related. In particular, a paper by Sijens et al. (1989) indicated that the level of high energy metabolites could be directly correlated to thermal dose. It was further indicated that stress or the depletion of cellular energy status could result in thermal sensitization (Calderwood 1987) and Gerweck et al. (1984) showed that reduced ATP levels caused thermal sensitization. In addition, it was shown that Lonidamine could inhibit glycolysis in mammalian cells and thus may reduce the energy status in malignant cells as these rely on high levels of glycolysis; however, energy status was not directly measured (Floridi et al. 1981, Silvestrini et a f . 1984, De Martino et al. 1984). Considering that hyperthermia and Lonidamine may modulate and affect the same potential targets within the cell, the application of these two treatments may result in an interdependent interaction in terms of cell toxicity. This was proven in a limited number of studies shown that hyperthermia could enhance the cytotoxicity of Lonidamine in human cells and murine tumours (Feeley et al. 1988a,b, Chitnis et al. 1986, Silvestrini el al. 1983, Cavaliere et al. 1984, Kim et al. 1984a,b). In addition, preliminary data showed that the application of hyperthermia with Lonidamine could also enhance cytotoxicity in human glioma cells (Feeley et al. 1988a,b). In this study the interaction of hyperthermia and Lonidamine under various pH conditions has been investigated to assess the potential of this combined treatment for cancer therapy. The data show that hyperthermia can cause extensive sensitization to Lonidamine treatment at concentration levels achievable clinically. Such thermal sensitization of Lonidamine toxicity was achievable in both exponentially growing and plateau phase cell cultures of glioma and squamous cell carcinoma cell lines. In addition, thermal sensitization could be achieved at 1-2 h hyperthermia exposure times, which are generally accepted to be clinically achievable. The data in these experiments show that the degree of Lonidamine sensitization by hyperthermia depends strongly on the pH during the treatment time. The degree of sensitization increased as the pH became more acidic and the thermal enhancement factors varied from 1 -1 (pH 7.4) to 7.0 (pH 6.6), with heating for 1 h at 42°C in cells that exposed to 50 pg/ml of Lonidamine. Stryker and Gerweck (1988) have shown that reduced pH causes Lonidamine to have a greater inhibition on the oxygen uptake and suggest that under low extracellular pH conditions, Lonidamine, a weak acid, may be more concentrated in the relatively more basic intracellular compartment. The results presented here support such an idea as the effect of Lonidamine increased as the pH decreased. A recent review (Wike-Hooley et at. 1984) showed that low pH exists in a range of human tumours and therefore the thermal enhancement of Lonidamine toxicity in such tumours might hold potential for enhanced response. Thus combined hyperthermia and Lonidamine treatment of tumours could result in a therapeutic gain compared to normal tissues at normal pH. In addition, these data have shown that plateau phase cells are much more resistant to hyperthermia than exponential phase cells. This is probably related to the distribution of cells within the cell cycle, as several studies have shown that S phase cells are much more sensitive to hyperthermia than G,/G, cells (Westra are Dewey 1971, Raaphorst et a f . 1985). The increased percentage of the S phase fraction in exponentially growing cells provides an explanation for the heightened sensitivity. However, these data show that both plateau phase and exponentially growing cells are sensitized to combinations of hyperthermia and Lonidamine, and therefore, in cancer treatment in tumour environments containing both quiescent and progressive cells, hyperthermia and Lonidamine may offer a potential clinical enhancement of the therapeutic response.
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References BAND,P. R., MAROUN, J., PRITCHARD, K., STEWART,D., COPPIN,C. M., WILSON,K., and EISENHAUER, E. A., 1986, Phase I1 study of Lonidamine in patients with metastatic breast cancer. Cancer Treatment Reports, 70, 1305-1310. B., MAHALEY, M. S., RVOSLAHTI, BIGNER,D. D., BIGNER,S. H., PONTEN,J., WESTERMARK, E., HERSHCHMAN, H., ENG,L. F., and WIKSTRAND, C. J., 1981, Heterogeneity of genotypic and phenotypic characteristics of 15 permanent cell lines derived from human glioma. Journal of Neuropathology and Experimental Neurology, 40, 20 1-229. S. K., 1987, Role of energy in cellular responses to heat. In: Temperature and CALDERWOOD, Animal Cells, edited by K. Bowler and B. J. Fuller, (Cambridge: Council of Biology), pp. 212-233. S. D., and RICCIO,A., 1984, Lonidamine in primary CARAPELLA, C. M., IANDOLO, B., CHIARE, malignant brain tumours. Oncology, 41, 82-85. A., CARLINI,S., CALABRO, A., ALOE, L., and CAVALIERE, R., DI FILIPPO,F., VARANESE, PIARuLLi, L., 1984, Lonidamine and hyperthermia: clinical experience in melanoma. Oncology, 41, 116-120. CHITNIS, M., JUVEKAR, A,, AWANKAR, M., and ADVANI,S., 1986, Inhibition of DNA synthesis by Lonidamine and hyperthermia in human chronic myeloid leukema cells. Anti Cancer Research, 6, 749-152. T. H., DURANT, J. R., EVANOCHKO, W. T., NG, T. C., LILLY,M. B., LAWSON, A. J., CORBETT, J. D., 1983, In vivo 3'P NMR study of the metabolism of murine 16/C and GLICKSON, adenocarcinoma and its response to chemotherapy, X-irradiation and hyperthermia. Pruceedings of the National Academy of Sciences, USA, 80, 334-338. B., and MARTIN, FEELEY,M. M., RAAPHORST, G. P., DANJOUX, C. E., MAROUN, M., FISCHER, L., 1988a, The response of human cells and rodent cells to Lonidamine radiation and hyperthermia. Clinical and Investigational Medicine, C96, 11. G. P., DANJOUX, C. E., MAROUN, J., FISCHER,B., and MARTIN, FEELEY,M . M., RAAPHORST, L., 1988b, The response of human cells and rodent cells to Lonidamine radiation and hyperthermia. Clinical and Investigational Medicine, C96, 1 1. FLORIDI, A., GAMBACURTA, A., BAGNATO, A., BIANCHI, C., PAGGI,M. G., SILVESTRINI, B., and CAPUTO,A., 1988, Modulation of Adriamycin uptake by Lonidamine in Ehrlich ascites tumour cells. Experimental and Molecular Pathology, 499, 421-43 1. FLORIDI, A,, PAGGI,M. G., MARCANTE, M. L., SILVESTRINI, B., CAPUTO, A., and DE MARTINO, C., 1981, Lonidamine a selective inhibitor of aerobic glycolysis of murine tumour cells. Journal of the National Cancer Institute, 66, 497-499. FREEMAN, M. L., RAAPHORST, G. P., HOPEWOOD, L. E., and DEWEY,W. C., 1980, The effect of pH on cell lethality induced by hyperthermic treatment for cancer. Cancer, 45, 61-70. GERWECK, L. E., DAHLBERG, W. C., EPSTEIN,L., 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. GERWECK, L. E., 1988, Modifiers of thermal effects: environmental factors. In: Hypenhermia and Oncology, vol. 1, edited by M. Urano and E. Douple, (Utrecht: VSP), pp. 83-98. B., Inhibition of the recovery from potentially HAHN,G. M., VAN KERSEN,I., and SILVESTRINNI, lethal damage by Lonidamine. British Journal of Cancer, 50, 657-660. J., and HAHN,G. M., 1981, The role of energy in hyperthermia induced mammalian HAVEMAN, cell inactivation. A study of the effects of glucose starvation and an uncoupler of oxidative phosphorylation. Journal of Cell Physiology, 108, 231-241. HENLE,K. J., NAGLE,W. A., MOSS, A. J., and HERMAN, T. S., 1984, Cellular ATP content of heated Chinese hamster ovary cells. Radiation Research, 97, 630-639. 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. G., 1984a, Lonidamine: KIM,J. H., KIM, S. H., ALFIERI,A., YOUNG,C. W., and SILVESTRINI, A hyperthermic sensitizer of HeLa cells in culture and of the Meth-A tumour in vivo. Oncology, 41, 30-35. B., 1984b, RadioKIM, J. H., ALFIERI,A. A., KIM, S . H., YOUNG, C. W., and SILVESTRINI, sensitization of Meth-A fibrosarcoma in mice by Lonidamine. Oncology, 41, 36-38. A. A., KIM,S. H., and YOUNG,C. W., 1986, Potentiation of radiation effects KIM,J. H., ALFIERI, on two murine tumours by Lonidamine. Cancer Research, 46, 1120-1 123.
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KIM, J. H., KIM, S. H., HE, S. Q., ALFIERI,A. A , , and YOUNG,C . W . , 1989, Potentiation of radiation effects on multicellular tuniour spheroids (MTS) of HeLa cells by Lonidamine. Journal of Radiation Oncology, 16, 1277-1280. KRAUSE,C . J., CAREY,T. E., OTT, R . W . , HURBIS,C . , CLATCHEY, M . C . , and REGEZI,K. Z., 198I , Human squamous cell carcinoma. Establishment and characterization of new permanent cell lines. Archives of Otolaryngology, 107, 703-7 10. MAGNO.L., TERRANEO, F., and CIOTTOLI, G. B., 1984, Lonidamine and radiotherapy in head and neck cancers. Oncology. 41, 113-1 15. DE MARTINO, C.. BATTEI.I.I.T., PAGGI,M . G., NISTA, A., MARCANTE, M . L., ATRI, S. D., MALORNI,W . , GALLO,M . , and FLORIDI, A , , 1984, Effects of Lonidamine on murine and human tumour cells in vitro. A morphological and biochemical study. Oncology, 41, 15-29. PRIVITERA, G., CIOTTOLI. G. B., PATAINE,C . , PALMUCCI, T., TAFURI,G., MARLETTA,F., DELUCA,B., MAGNANI, F.. DEGREGORIO, M.. and GRECO,S., 1987, Phase I1 double-blind randomized study of Lonidamine and radiotherapy in epidermoid carcinoma of the lung. Rudiotherapy and Oncology, 10, 285-290. RAAPHOKSL', G . P., AZZAM, E. I., BORSA,J., EINSPENNER, M . , and VADASZ, J., 1983, Hypertherniia in a differentiated murine erythroleukemia cell line 11. Effect of heat on heme induction by radiation. International Journal (.f Radiation Biology, 44, 343-352. RAAPHORST, G. P . , BROSKI,A. P., and AZZAM,E. I., 1985, Sensitivity to heat, radiation and hcat plus radiation of Chinese hamster cells, synchronized by mitotic selection, thymidine block or hydroxyurea. Journal .f Thermal Biology, 10, 177-181. RAAPHORST, G. P., FEELEY,M . M . , DANJOUX. C . E., MARTIN,L.. MAROUN, J., and DESANCTIS, A . J . , 1990a, The effect of Lonidamine on radiation and thermal responses of human and rodcnt cell lines. Internuiional Journul of Radiation Oncology, 20, 509-5 IS, 1991. RAAPHORST, G. P., FEBLEY, M . M . , HELLER.D. P . , DANJOUX, C . E., MARTIN,L., MAROON. J . , and DESANCTIS, A. J., 1990b, Lonidamine can enhance the cytotoxic effect of cisplatin in human tumour cells and rodent cells. Anticancer Research, 10, 923-928. ROSBE,K. W . , B R A N NT. , W.. HOLDEN,S. A , , TEICHER, B. A., and FREI,E. 111, 1989, Effect of Lonidaniinc on the cytotoxicity of four alkylating agents in vitro. Cancer Chemorhercips and Pharmacology, 25, 32-36. SIJENS,P. E., BOVEE,W . M . M. J . , KOOLE,P., and SCHIPPER, J.. 1989, Phosphorous NMR study of response of a murine tumour to hyperthermia as a function of treatment time and t e n perature. International Journal of Hyperthermia, 5 , 35 1-351. SILVESTRINI. B., HAHN,G. M., CIOLI, V., and DE MARTINO,C . , 1983, Effects of Lonidaniinc alone or combined with hyperthermia in some experimental cell and tumour systems. British Journal of Cuncer, 47, 221-235. SII.VESTRINI, B., PALAZZO, G . , and DEGREGORIO, M . , 1984, Lonidamine and related compounds. In: Progress in Medicinal Chemistry, edited by G . P. Ellis and C. P. West), vol. 2, pp. 1 11-135. STRYKER, J. A., and GERWECK,L. E., 1988, Lonidamine-induced pH dependent inhibition of cellular oxygen utilization. Radiation Research, 113, 356-361 1. SZEKELY, J . G . , LOHKEAU, A. U., DELANEY. S., RAAPHORST, G. P., and FEELEY,M . M . , 1989, Morphological effects of Lonidamine on human tumour cells. Scanning Electron Micmx-op-y. 3, 68 1-693. WESTKA,A., and DEWEY,W . C., 1971, Variation in sensitivity to heat shock during the cell cycle of Chinese hamster cells in virro. International Journal of Radiation Biology, 19, 461-477. WIKE-HOOLEY, J . L . , HAVGMAN, J., and REINHOLD,H. S., 1984, The relevance of tuniour pH to the treatment of malignant disease. Radiotherapy and Oncology, 2, 343-366.
