Brief Communication: Preferential Cytotoxicity of 5-Thio-D-glucose Against Hypoxic Tumor Cells 1,
2
Chang W. Song, Jacob J. Clement, and Seymour H. Levitt 3 ,4 ABSTRACT-5-Thio-o-glucose effectively killed hypoxic mastocytoma cells of DBA/2J mice, whereas it merely suppressed the growth of oxic cells. This specific toxicity suggested that 5-thio-oglucose may be a useful adjuvant to radiotherapy by eliminating hypoxic protection.-J Natl Cancer Inst 57: 603-605, 1976.
Since hypoxic cells are more resistant to X-rays or gamma rays than are well-oxygenated cells, the presence of hypoxic foci in tumors is believed to be a major limiting factor in the local control of tumors by conventional radiotherapy (l). N oncycling hypoxic cells seem also to be resistant to some chemotherapeutic drugs (2). Hyperbaric oxygen (3), high linear energy transfer radiations, i.e., neutrons (4) or 1T'- mesons (5), and electron-affinic chemicals (6, 7), which specifically sensitize hypoxic cells to radiation, are being tested by many investigators for the elimination of hypoxic protection in radiotherapy. However, these methods are not yet applicable in clinical radiotherapy. We previously suggested (8) that hypoxic protection may be overcome by sterilization of hypoxic cells, before or after irradiation, with 5-SH-glu, which is specifically toxic to hypoxic cells. In this report, we describe our further observations that 5-SH-glu kills hypoxic tumor cells in vitto and suppresses tumor growth in vivo; thus 5-SH-glu may have a clinical application in radiotherapy and chemotherapy by eliminating hypoxic cells. MATERIALS AND METHODS
In vitro study.-P815-X2 mastocytoma ascites cells of DBA/2] mice were harvested 2-3 days before experiments and cultured in vitro with RPMI-1640 medium (Associated Biomedic Systems, Buffalo, N.Y.) supplemented with 100 U penicillin/ml, 100 /-tg streptomycin/ ml, and 10% fetal calf serum. Exponentially growing cells in culture were centrifuged for 10 minutes at 500 rpm and resuspended in medium alone or medium containing various concentrations of 5-SH-glu (mol wt, 196.3; Pfanstiehl Laboratories, Inc., Waukegan, I1l.). Cell suspensions containing 2 X 105 cells/mi were then subdivided for the following experiments: group 1culture under oxic conditions; group 2-culture under hypoxic conditions; group 3-culture under oxic conditions with 5-SH-glu; and group 4-culture under hypoxic conditions with 5-SH-glu. These lO-ml cell suspensions were pipetted into round glass chambers (6.5 em in diameter and 3.5 em in height) with two opposite sidearms (3 em long and I em wide) attached at a 45degree upward angle. The chambers containing the cell suspensions were incubated at 37° C with gassing as follows: Croups I and 3 were gassed with 5% CO 2 in air and groups 2 and 4 with a mixture of 5% CO 2 and 95% N2 through one of the sidearms with the use of glass tubes connected to a manifold. The gasses were humidified by being bubbled through water. The rate of gassing was about I liter/minute. The oxygen content in the VOL. 57, NO.3, SEPTEMBER 1976
603
mixture of CO 2 and N2 was 50-100 ppm, according to manufacturer's specification (Ohio Medical Products, Cleveland, Ohio). The other sidearm of each chamber was kept closed except when samples were taken. The cell suspensions were gently rocked for the first 11/2 hours of incubation and gassing. At various times, aliquots from each flask were removed through the sidearms, and the numbers of intact cells able to exclude trypan blue were counted with a hemacytometer. The cloning efficiency of the cells was studied by a method similar to that of Chu and Fischer (9). Cells were diluted in plastic tissue culture tubes (Falcon 3033; Falcon Plastics, Oxnard, Calif.) with RPMI-1640 medium supplemented with 20% fetal calf serum; they were then mixed with Noble agar in the same medium to the final agar concentration of 0.12%. Five replicates were made for the each sample. The tubes were kept in crushed ice, and, after a gel was obtained, they were incubated at 37° C in a mixture of 5% CO 2 and 95% air. Visible clones were counted 10 days later. The cloning efficiency of control tumor cells (group I) was 50-60%. From the dilution factors and the number of clones formed, the number of clonogenic or surviving cells in each culture was computed. In separate experiments, we determined the chronic toxicity of 5-SH-glu by culturing tumor cells in agar media with various concentrations of the compound. In vivo study.-The effect of 5-SH-glu on the growth of solid P815-X2 mastocytoma induced in flanks of DBA/2] mice was studied. After the hair on the right side flank was clipped, I X 105 tumor cells obtained from ascites were inoculated sc. Tumors became palpable within a few days. On day 13, when the tumors had grown to 8-9 mm in diameter, the animals were divided into two groups and the drinking water of one group of animals was replaced with 5-SH-glu water solution at 1 mg/ml. We studied the growth of tumors by measuring tumor diameters in two perpendicular directions with vernier calipers 2-3 times a week. RESULTS In Vitro Study
Text-figure I shows the changes in numbers of surviving tumor cells after incubation with different concentrations of 5-SH-glu under aerobic or hypoxic conditions. The control tumor cells incubated under aerobic conditions in the absence of 5-SH-glu increased from 2x 105 to 3.2x 105/ml during the 8-hour incubation. The ABBREVIATION USED: 5-SH-glu=5-thio-D-glucose. Received January 2, 1976; accepted March 12, 1976. Supported by Public Health Service grants CA08832 and CAI5548 from the National Cancer Institute. 3 Department of Therapeutic Radiology, University of Minnesota Medical School, Minneapolis, Minn. 55455. 4 The excellent technical assistance of Irene F. Patrek is gratefully acknowledged. 1
2
J NATL CANCER INST
604
SONG, CLEMENT, AND LEVITT
106 , - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - .
were sufficiently diluted before being mixed with agar solution so that the concentration of 5.. SH-glu in agar medium was always less than 0.5 mM. Therefore, the decrease in survival of 5-SH-glu-treated hypoxic cells (text-fig. 1) was due to the toxicity of 5-SH-glu on hypoxic cells rather than to residual 5-SH-glu introduced into the agar medium with the cells.
E 10 5
.........
In Vivo Study
(/)
Text-figure 2 shows the growth of solid mastocytoma in control mice and mice treated with 5-SH-glu. Each mouse consumed about 2.5-3.5 mg 5-SH-glu daily during the early period of the experiment. The animals drank less water when the growing tumor made them weaker. As the growth curves indicate, treatment with 5-
Q)
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-
TABLE
0
.....
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l.--Changes in cloning efficiency by different concentrations of 5-SH-glu in agar-supplemented medium a
5-SH-glu concentration, mM
Cloning efficiency, b %
o
100 e 104 97
10 3
0.5 1
5 10
SO 71
Cells were cultured for 10 days in agar medium containing 5-SHglu under 5% CO 2 in air. b Average of four replicate tubes. e Absolute cloning efficiency with 0 mM was 57%. a
30,-------------------------~
Time I.-Changes in number of surviving PSI5-X2 mastocytoma cells in suspension cultures. Cells were incubated with various concentrations of 5-SH-glu under aerobic and hypoxic conditions. Only results for the S-hour incubation are shown for aerobic cells. Each data point represents separate experiments.
TEXT-FIGURE
growth of aerobic cells was not significantly affected by 5-SH-glu at 5 mM. However, the higher concentrations of 5-SH-glu, i.e., 10 and 25 mM, inhibited the growth of aerobic cells. Under hypoxic conditions without 5-SHglu, the cell number increased slightly for 4 hours and then decreased. The number of surviving cells was l.3x 105/ m l at the end of 8 hours' incubation. Unlike aerobic cells, hypoxic cells were sensitive to 5-SH-glu. At 5 mM of 5-SH-glu, the cell number began to decline 2 hours after incubation and gassing with 5% CO 2 and 95% N2 were begun. The number of surviving cells was l.7 X 104/ml at the end of 8 hours' incubation. Higher concentrations of 5-SH-glu resulted in a rapid decrease in the number of hypoxic cells after the 2-hour lag period. Incubation for 6 hours with 10 mM and 4 hours with 25 mM 5-SH-glu decreased the survivals to nearly 1 X 102 cells/ml, which was only 0.05% of the original cell number of 2x 105/ m !. Table 1 shows the cloning efficiency of the tumor cells in agar medium containing various concentrations of 5SH-glu. Chronic exposure to 5-SH-glu at a concentration of 1 mM or less in the agar medium did not reduce the cloning efficiency, though the size of clones tended to be smaller than in control cultures without the drug. In the short-term hypoxia studies, the cell suspensions
J
NATL CANCER INST
E E ~
o
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r-o
~
Q) Q)
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o
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10
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30
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Days 2.-Growth curves of solid PSI5-X2 mastocytoma of DBA/2] mice. e: control mice. 0: mice treated with 5-SH-glu solution (l20-ISO mg/kg/day). Each value represents the average of S tumors ± I SE.
TEXT-FIGURE
VOL. 57, NO.3, SEPTEMBER 1976
CYTOTOXICITY OF 5-THIO-O-GLUCOSE AGAINST HYPOXIC TUMOR CELLS
SH-glu significantly suppressed tumor growth. The effect of the drug became apparent from the fifth day of treatment. The median survival time of the untreated mice was 23 days, whereas that of the treated animals was 36 days from the time treatment was started, which was 13 days after tumor inoculation. DISCUSSION
5-SH-glu is preferentially toxic toward hypoxic cells. However, cell death by 5-SH-glu does not begin immediately (text-fig. 1). This lag in drug response probably results, at least in part, from the presence of oxygen in the medium during the early part of incubation. Mohindra and Rauth (personal communication) observed that removal of oxygen from aqueous medium by flushing with nitrogen gas takes 11/2-2 hours. The temperature of the cell suspension increases from room temperature to 37° C in about 20 minutes when it is transferred from the hood to the incubator. Thus low metabolic rate due to suboptimal temperature during the early period of incubation may be another factor responsible for the lack of cell death during the first 2 hours. It is probable, therefore, that cell death would begin earlier if the cells were at 37° C and hypoxic from the beginning of the incubation. About 15-25% of the viable cell population in all animal tumors studied are hypoxic (10) due to the limit of oxygen diffusion length. The solid mastocytoma we used in the present study probably also contains a considerable hypoxic cell population, and the retardation of growth by 5-SH-glu treatment (text-fig. 2) may be the result of death of hypoxic cells. The possibility of cytostatic effect of 5-SH -glu oh- oxic cells, however, could not be ruled out completely. Iii this connection, a question arises as to whether the 5-SH-glu can reach the hypoxic area. The diffusion length of -glucose is significantly longer than that of oxygen in tlimors, and hypoxic cells supposedly metabolize glucose by anaerobic glycolysis (11). In view of the similarity in structure between o-glucose and 5-SH-glu, it seems reasonable that 5-SH-glu can reach hypoxic areas in solid tumors, as can o-glucose, and kill the hypoxic cells. Concentration of 5-SH-glu in the blood reaches its highest level in 2 hours and is eliminated almost completely in 6 hours after ip injection in mice (Whistler RL: Personal communication). In our in vivo study, the animals were treated with the drug dissolved in drinking water at the dosage of 120-180 mg/kg/day. Therefore, the drug was continuously present in the body, though the concentration was probably far less than 120-180 mg/kg at any time. Since this treatment suppresses tumor growth significantly (text-fig. 2), a prolonged exposure even to low concentrations of 5-SHglu may be effective. 5-SH-glu was first synthesized by Zysk et al. (12), Chen and Whistler (13), and Maugh (14). This com-
VOL. 57, No.3, SEPTEMBER 1976
605
pound is almost nontoxic, with a mean lethal dose of 14 g/kg in mice, and effectively inhibits spermatogenesis in mice at 50 mg/kg/day (12-14). Chen and Whistler (13) attributed this suppressed spermatogenesis to a reduced energy production as a result of a competitive inhibition of glucose transport and metabolism by 5-SH-glu and by phosphate derivatives of 5-SH-glu. Energy production is diminished under hypoxic conditions, since glucose is utilized inefficiently by anaerobic glycolysis. Further reduction in energy production by blocking of glucose metabolism with 5-SH-glu may be lethal to hypoxic cells. This would explain why, as demonstrated in the present study, the hypoxic cells are more vulnerable to 5-SH-glu than are oxic cells. The low toxicity and preferential cytotoxicity of 5-SHglu toward hypoxic cells strongly suggest that this compound may be a useful adjuvant to radiotherapy and chemotherapy. Further investigations are in progress in our laboratory. REFERENCES
(1) THOMLINSON RH, GRAY LH: The histological structure of some human lung cancers and the possible implication for radiotherapy. Br J Cancer 9:539-549, 1955 (2) VALERIOTE F, VAN PUTTEN L: Proliferation-dependent cytotoxicity of anticancer agents: A review. Cancer Res 35:2619-2630, 1975 (3) VAN DEN BRENK HA: Hyperbaric oxygen in radiation therapy. Am J Roentgenol Radium Ther Nucl Med 102:8-26, 1968 (4) ELKIND MM: Summary of general discussion on radiobiological aspects of fast neutrons in radiotherapy. Eur J Cancer 7:249257, 1971 (5) RAJU MR, RICHMAN C: Physical and radiobiological aspects of negative pions with reference to radiotherapy. GANN Monogr 9: 105-121, 1970 (6) ADAMS GE: Chemical radiosensitization of hypoxic cells. Br Med Bull 29:48-53, 1973 (7) DENEKAMP J, HARRIS SR: Tests of two electron-affinic radiosensitizers in vivo using regrowth of experimental carcinoma. Radiat Res 61:191-203,1975 (8) SONG CW, LEVITT SH: The use of 5-thio-D-glucose in radiotherapy: Its preferential effects against hypoxic cells. In Abstract Book of the 17th Annual Meeting of American Society of Therapeutic Radiologists, No. 33. San Francisco, 1975 (9) CHU MY, FISCHER GA: The incorporation of 3H-cytosine arabinoside and its effect on murine leukemic cells. Biochem Pharmacol 17:753-767, 1968 (10) KALLMAN RF: The phenomenon of reoxygenation and its implication for fractionated radiotherapy. Radiology 105:135-142, 1972 (11) TANNOCK IF: The relation between cell proliferation and the vascular system in a transplantable mouse mammary tumour. Br J Cancer 22:258-273, 1968 (12) ZYSKJR, WHISTLER RL, BUSHWAY AA, et al: Inhibition of sperm cell development in mice by 5-thio-D-glucose. In Abstract Book of the 169th National Meeting of American Chemical Society, No. 22. Atlantic City, 1974 (13) CHEN M, WHISTLER RL: Action of 5-thio-D-glucose and its 1phosphate with glycogen enzymes. In Abstract Book of 170th National Meeting of American Chemical Society, No. 143. Chicago, 1975 (14) MAUGH TH: 5-Thio-D-glucose: A unique male contraceptive. Science 186:431, 1974
J NATL CANCER INST
2
Chang W. Song, Jacob J. Clement, and Seymour H. Levitt 3 ,4 ABSTRACT-5-Thio-o-glucose effectively killed hypoxic mastocytoma cells of DBA/2J mice, whereas it merely suppressed the growth of oxic cells. This specific toxicity suggested that 5-thio-oglucose may be a useful adjuvant to radiotherapy by eliminating hypoxic protection.-J Natl Cancer Inst 57: 603-605, 1976.
Since hypoxic cells are more resistant to X-rays or gamma rays than are well-oxygenated cells, the presence of hypoxic foci in tumors is believed to be a major limiting factor in the local control of tumors by conventional radiotherapy (l). N oncycling hypoxic cells seem also to be resistant to some chemotherapeutic drugs (2). Hyperbaric oxygen (3), high linear energy transfer radiations, i.e., neutrons (4) or 1T'- mesons (5), and electron-affinic chemicals (6, 7), which specifically sensitize hypoxic cells to radiation, are being tested by many investigators for the elimination of hypoxic protection in radiotherapy. However, these methods are not yet applicable in clinical radiotherapy. We previously suggested (8) that hypoxic protection may be overcome by sterilization of hypoxic cells, before or after irradiation, with 5-SH-glu, which is specifically toxic to hypoxic cells. In this report, we describe our further observations that 5-SH-glu kills hypoxic tumor cells in vitto and suppresses tumor growth in vivo; thus 5-SH-glu may have a clinical application in radiotherapy and chemotherapy by eliminating hypoxic cells. MATERIALS AND METHODS
In vitro study.-P815-X2 mastocytoma ascites cells of DBA/2] mice were harvested 2-3 days before experiments and cultured in vitro with RPMI-1640 medium (Associated Biomedic Systems, Buffalo, N.Y.) supplemented with 100 U penicillin/ml, 100 /-tg streptomycin/ ml, and 10% fetal calf serum. Exponentially growing cells in culture were centrifuged for 10 minutes at 500 rpm and resuspended in medium alone or medium containing various concentrations of 5-SH-glu (mol wt, 196.3; Pfanstiehl Laboratories, Inc., Waukegan, I1l.). Cell suspensions containing 2 X 105 cells/mi were then subdivided for the following experiments: group 1culture under oxic conditions; group 2-culture under hypoxic conditions; group 3-culture under oxic conditions with 5-SH-glu; and group 4-culture under hypoxic conditions with 5-SH-glu. These lO-ml cell suspensions were pipetted into round glass chambers (6.5 em in diameter and 3.5 em in height) with two opposite sidearms (3 em long and I em wide) attached at a 45degree upward angle. The chambers containing the cell suspensions were incubated at 37° C with gassing as follows: Croups I and 3 were gassed with 5% CO 2 in air and groups 2 and 4 with a mixture of 5% CO 2 and 95% N2 through one of the sidearms with the use of glass tubes connected to a manifold. The gasses were humidified by being bubbled through water. The rate of gassing was about I liter/minute. The oxygen content in the VOL. 57, NO.3, SEPTEMBER 1976
603
mixture of CO 2 and N2 was 50-100 ppm, according to manufacturer's specification (Ohio Medical Products, Cleveland, Ohio). The other sidearm of each chamber was kept closed except when samples were taken. The cell suspensions were gently rocked for the first 11/2 hours of incubation and gassing. At various times, aliquots from each flask were removed through the sidearms, and the numbers of intact cells able to exclude trypan blue were counted with a hemacytometer. The cloning efficiency of the cells was studied by a method similar to that of Chu and Fischer (9). Cells were diluted in plastic tissue culture tubes (Falcon 3033; Falcon Plastics, Oxnard, Calif.) with RPMI-1640 medium supplemented with 20% fetal calf serum; they were then mixed with Noble agar in the same medium to the final agar concentration of 0.12%. Five replicates were made for the each sample. The tubes were kept in crushed ice, and, after a gel was obtained, they were incubated at 37° C in a mixture of 5% CO 2 and 95% air. Visible clones were counted 10 days later. The cloning efficiency of control tumor cells (group I) was 50-60%. From the dilution factors and the number of clones formed, the number of clonogenic or surviving cells in each culture was computed. In separate experiments, we determined the chronic toxicity of 5-SH-glu by culturing tumor cells in agar media with various concentrations of the compound. In vivo study.-The effect of 5-SH-glu on the growth of solid P815-X2 mastocytoma induced in flanks of DBA/2] mice was studied. After the hair on the right side flank was clipped, I X 105 tumor cells obtained from ascites were inoculated sc. Tumors became palpable within a few days. On day 13, when the tumors had grown to 8-9 mm in diameter, the animals were divided into two groups and the drinking water of one group of animals was replaced with 5-SH-glu water solution at 1 mg/ml. We studied the growth of tumors by measuring tumor diameters in two perpendicular directions with vernier calipers 2-3 times a week. RESULTS In Vitro Study
Text-figure I shows the changes in numbers of surviving tumor cells after incubation with different concentrations of 5-SH-glu under aerobic or hypoxic conditions. The control tumor cells incubated under aerobic conditions in the absence of 5-SH-glu increased from 2x 105 to 3.2x 105/ml during the 8-hour incubation. The ABBREVIATION USED: 5-SH-glu=5-thio-D-glucose. Received January 2, 1976; accepted March 12, 1976. Supported by Public Health Service grants CA08832 and CAI5548 from the National Cancer Institute. 3 Department of Therapeutic Radiology, University of Minnesota Medical School, Minneapolis, Minn. 55455. 4 The excellent technical assistance of Irene F. Patrek is gratefully acknowledged. 1
2
J NATL CANCER INST
604
SONG, CLEMENT, AND LEVITT
106 , - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - .
were sufficiently diluted before being mixed with agar solution so that the concentration of 5.. SH-glu in agar medium was always less than 0.5 mM. Therefore, the decrease in survival of 5-SH-glu-treated hypoxic cells (text-fig. 1) was due to the toxicity of 5-SH-glu on hypoxic cells rather than to residual 5-SH-glu introduced into the agar medium with the cells.
E 10 5
.........
In Vivo Study
(/)
Text-figure 2 shows the growth of solid mastocytoma in control mice and mice treated with 5-SH-glu. Each mouse consumed about 2.5-3.5 mg 5-SH-glu daily during the early period of the experiment. The animals drank less water when the growing tumor made them weaker. As the growth curves indicate, treatment with 5-
Q)
U C'I C
> > ..... 104
::l CJ)
-
TABLE
0
.....
Q)
..0
E ::l
Z
l.--Changes in cloning efficiency by different concentrations of 5-SH-glu in agar-supplemented medium a
5-SH-glu concentration, mM
Cloning efficiency, b %
o
100 e 104 97
10 3
0.5 1
5 10
SO 71
Cells were cultured for 10 days in agar medium containing 5-SHglu under 5% CO 2 in air. b Average of four replicate tubes. e Absolute cloning efficiency with 0 mM was 57%. a
30,-------------------------~
Time I.-Changes in number of surviving PSI5-X2 mastocytoma cells in suspension cultures. Cells were incubated with various concentrations of 5-SH-glu under aerobic and hypoxic conditions. Only results for the S-hour incubation are shown for aerobic cells. Each data point represents separate experiments.
TEXT-FIGURE
growth of aerobic cells was not significantly affected by 5-SH-glu at 5 mM. However, the higher concentrations of 5-SH-glu, i.e., 10 and 25 mM, inhibited the growth of aerobic cells. Under hypoxic conditions without 5-SHglu, the cell number increased slightly for 4 hours and then decreased. The number of surviving cells was l.3x 105/ m l at the end of 8 hours' incubation. Unlike aerobic cells, hypoxic cells were sensitive to 5-SH-glu. At 5 mM of 5-SH-glu, the cell number began to decline 2 hours after incubation and gassing with 5% CO 2 and 95% N2 were begun. The number of surviving cells was l.7 X 104/ml at the end of 8 hours' incubation. Higher concentrations of 5-SH-glu resulted in a rapid decrease in the number of hypoxic cells after the 2-hour lag period. Incubation for 6 hours with 10 mM and 4 hours with 25 mM 5-SH-glu decreased the survivals to nearly 1 X 102 cells/ml, which was only 0.05% of the original cell number of 2x 105/ m !. Table 1 shows the cloning efficiency of the tumor cells in agar medium containing various concentrations of 5SH-glu. Chronic exposure to 5-SH-glu at a concentration of 1 mM or less in the agar medium did not reduce the cloning efficiency, though the size of clones tended to be smaller than in control cultures without the drug. In the short-term hypoxia studies, the cell suspensions
J
NATL CANCER INST
E E ~
o
E :J
r-o
~
Q) Q)
E
c
o
o
5
10
15
20
25
30
35
Days 2.-Growth curves of solid PSI5-X2 mastocytoma of DBA/2] mice. e: control mice. 0: mice treated with 5-SH-glu solution (l20-ISO mg/kg/day). Each value represents the average of S tumors ± I SE.
TEXT-FIGURE
VOL. 57, NO.3, SEPTEMBER 1976
CYTOTOXICITY OF 5-THIO-O-GLUCOSE AGAINST HYPOXIC TUMOR CELLS
SH-glu significantly suppressed tumor growth. The effect of the drug became apparent from the fifth day of treatment. The median survival time of the untreated mice was 23 days, whereas that of the treated animals was 36 days from the time treatment was started, which was 13 days after tumor inoculation. DISCUSSION
5-SH-glu is preferentially toxic toward hypoxic cells. However, cell death by 5-SH-glu does not begin immediately (text-fig. 1). This lag in drug response probably results, at least in part, from the presence of oxygen in the medium during the early part of incubation. Mohindra and Rauth (personal communication) observed that removal of oxygen from aqueous medium by flushing with nitrogen gas takes 11/2-2 hours. The temperature of the cell suspension increases from room temperature to 37° C in about 20 minutes when it is transferred from the hood to the incubator. Thus low metabolic rate due to suboptimal temperature during the early period of incubation may be another factor responsible for the lack of cell death during the first 2 hours. It is probable, therefore, that cell death would begin earlier if the cells were at 37° C and hypoxic from the beginning of the incubation. About 15-25% of the viable cell population in all animal tumors studied are hypoxic (10) due to the limit of oxygen diffusion length. The solid mastocytoma we used in the present study probably also contains a considerable hypoxic cell population, and the retardation of growth by 5-SH-glu treatment (text-fig. 2) may be the result of death of hypoxic cells. The possibility of cytostatic effect of 5-SH -glu oh- oxic cells, however, could not be ruled out completely. Iii this connection, a question arises as to whether the 5-SH-glu can reach the hypoxic area. The diffusion length of -glucose is significantly longer than that of oxygen in tlimors, and hypoxic cells supposedly metabolize glucose by anaerobic glycolysis (11). In view of the similarity in structure between o-glucose and 5-SH-glu, it seems reasonable that 5-SH-glu can reach hypoxic areas in solid tumors, as can o-glucose, and kill the hypoxic cells. Concentration of 5-SH-glu in the blood reaches its highest level in 2 hours and is eliminated almost completely in 6 hours after ip injection in mice (Whistler RL: Personal communication). In our in vivo study, the animals were treated with the drug dissolved in drinking water at the dosage of 120-180 mg/kg/day. Therefore, the drug was continuously present in the body, though the concentration was probably far less than 120-180 mg/kg at any time. Since this treatment suppresses tumor growth significantly (text-fig. 2), a prolonged exposure even to low concentrations of 5-SHglu may be effective. 5-SH-glu was first synthesized by Zysk et al. (12), Chen and Whistler (13), and Maugh (14). This com-
VOL. 57, No.3, SEPTEMBER 1976
605
pound is almost nontoxic, with a mean lethal dose of 14 g/kg in mice, and effectively inhibits spermatogenesis in mice at 50 mg/kg/day (12-14). Chen and Whistler (13) attributed this suppressed spermatogenesis to a reduced energy production as a result of a competitive inhibition of glucose transport and metabolism by 5-SH-glu and by phosphate derivatives of 5-SH-glu. Energy production is diminished under hypoxic conditions, since glucose is utilized inefficiently by anaerobic glycolysis. Further reduction in energy production by blocking of glucose metabolism with 5-SH-glu may be lethal to hypoxic cells. This would explain why, as demonstrated in the present study, the hypoxic cells are more vulnerable to 5-SH-glu than are oxic cells. The low toxicity and preferential cytotoxicity of 5-SHglu toward hypoxic cells strongly suggest that this compound may be a useful adjuvant to radiotherapy and chemotherapy. Further investigations are in progress in our laboratory. REFERENCES
(1) THOMLINSON RH, GRAY LH: The histological structure of some human lung cancers and the possible implication for radiotherapy. Br J Cancer 9:539-549, 1955 (2) VALERIOTE F, VAN PUTTEN L: Proliferation-dependent cytotoxicity of anticancer agents: A review. Cancer Res 35:2619-2630, 1975 (3) VAN DEN BRENK HA: Hyperbaric oxygen in radiation therapy. Am J Roentgenol Radium Ther Nucl Med 102:8-26, 1968 (4) ELKIND MM: Summary of general discussion on radiobiological aspects of fast neutrons in radiotherapy. Eur J Cancer 7:249257, 1971 (5) RAJU MR, RICHMAN C: Physical and radiobiological aspects of negative pions with reference to radiotherapy. GANN Monogr 9: 105-121, 1970 (6) ADAMS GE: Chemical radiosensitization of hypoxic cells. Br Med Bull 29:48-53, 1973 (7) DENEKAMP J, HARRIS SR: Tests of two electron-affinic radiosensitizers in vivo using regrowth of experimental carcinoma. Radiat Res 61:191-203,1975 (8) SONG CW, LEVITT SH: The use of 5-thio-D-glucose in radiotherapy: Its preferential effects against hypoxic cells. In Abstract Book of the 17th Annual Meeting of American Society of Therapeutic Radiologists, No. 33. San Francisco, 1975 (9) CHU MY, FISCHER GA: The incorporation of 3H-cytosine arabinoside and its effect on murine leukemic cells. Biochem Pharmacol 17:753-767, 1968 (10) KALLMAN RF: The phenomenon of reoxygenation and its implication for fractionated radiotherapy. Radiology 105:135-142, 1972 (11) TANNOCK IF: The relation between cell proliferation and the vascular system in a transplantable mouse mammary tumour. Br J Cancer 22:258-273, 1968 (12) ZYSKJR, WHISTLER RL, BUSHWAY AA, et al: Inhibition of sperm cell development in mice by 5-thio-D-glucose. In Abstract Book of the 169th National Meeting of American Chemical Society, No. 22. Atlantic City, 1974 (13) CHEN M, WHISTLER RL: Action of 5-thio-D-glucose and its 1phosphate with glycogen enzymes. In Abstract Book of 170th National Meeting of American Chemical Society, No. 143. Chicago, 1975 (14) MAUGH TH: 5-Thio-D-glucose: A unique male contraceptive. Science 186:431, 1974
J NATL CANCER INST
