Cancer Investigation, I0(6), 505-5 1 1 (1992)
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Antitumor Activity of Tiazofurin in Human Colon Carcinoma HT-29 Weining Zhen, M.D.’,*, Hiremagalur N. Jayaram, Ph.D.I, and George Weber, M.D.I ’Laboratory for Experimental Oncology Walther Oncology Center Indiana University School of Medicine Indianapolis, Indiana 46202-5200
ABSTRACT Tiazofurin is effective in treating end-stage leukemic patients (Tricot et al., Cancer Res 49:3696-3701, 1989). In sensitive tumors, the active metabolite of tiazofurin, TAD, potently inhibits IMP dehydrogenase activity, resulting in reduced guanylate pools. To elucidate tiazofurin activity in human solid tumors, we examined its activity in human colon carcinoma HT-29. Tiazofurin exhibited an LCs0 of 35 p.44 in cultured HT-29 cells. Incubation of HT-29 cells with 100 p.44 tiazofurin for 2 h resulted in TAD formation (9.3 nmollg cells) and in a 64% decrease in GTP pools. For biochemical and chemotherapy studies, athymic nude mice were transplanted S.C. with HT-29 cells. Twenty-four days later, mice were injected i.p. with tiazofurin (500 mglkg); 6 h later, tumors were removed and analyzed. These tumors formed 17 nmollg of TAD with decreased GTP pools (56%). To study oncolytic activity, transplanted mice were treated 24 h later with tiazofurin (500 mglkg, once a day for I0 days). To examine the eflectiveness of tiazofurin in established tumors, the drug was administered to mice 14 days after tumor implantation (500 mglkg, once a day for 5 days, course repeated 4 times with a 10-day rest). Both treatment schedules resulted in significant antitumor activity. This study illustrates the potential usefulness of tiazofurin in treating human colon carcinoma.
INTRODUCTION
L1210 lymphoid leukemia, P388 leukemia, Lewis lung (2-~-D-ribofuranosylthiazole-4-carbox- carcinoma, and plutonium 239-induced osteosarcoma Tiazofurin (1-6). It has also shown promising activity in end-stage amide, NSC 286193) is a novel C-nucleoside with anleukemic patients (7-9). In sensitive tumors, tiazofurin titumor activity against several murine tumors, including 505
Copyright 0 1992 by Marcel Dekker, Inc.
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506
is anabolized to its active metabolite, TAD, which potently inhibits the activity of IMP dehydrogenase (EC 1. I . 1.205), the rate-limiting enzyme of de novo GTP biosynthesis, resulting in decreased guanylate concentration leading to the inhibition of tumor cell proliferation (10-14). We have demonstrated the importance of TAD as a predictor of sensitivity to tiazofurin in human leukemic cells in vitro and in vivo (18,19). Early phase I studies with tiazofurin in solid tumors described doselimiting toxicities (20-22). The present communication is aimed at examining the sensitivity of human solid tumor to tiazofurin. Since IMP dehydrogenase activity was markedly elevated in human colon carcinoma compared to normal human colon mucosa (23), IMP dehydrogenase could be a target for chemotherapy in human colon carcinoma. Tiazofurin was shown to be a selective inhibitor of IMP dehydrogenase with oncolytic property in treating end-stage leukemic patients (7-9). We have elucidated the activity of tiazofurin in human colon carcinoma HT-29 both in vitro and in vivo. This study might be helpful in reopening clinical trials in solid tumors with tiazofurin.
MATERIALS AND METHODS Materials Tiazofurin was provided by the Drug Synthesis and Chemistry Branch, Drug Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, Bethesda, Maryland. RCM Partisil 10-SAX column and HPLC apparatus consisting of an NEC computer (APC IV, Power Mate 2 mode), a 991 photodiode array detector, a 600E multisolvent delivery system, and a refrigerated 7 12-WISP autoinjector were purchased from Waters Associates, Milford, Massachusetts. Ammonium phosphate (monobasic, HPLC grade) was obtained from the Fisher Scientific Co., Itasca, Illinois.
Tumor System Male athymic nude mice (4-5 weeks old) were purchased from Harlan Sprague Dawley, Inc., Cumberland, Indiana. Animals were housed in individual cages, and food and water were available ad libitum. Human colon carcinoma HT-29 cells (2.2 X lo6)were S.C. implanted, and 24 h later, they were injected i.p. with sterile saline or tiazofurin (500 mg/kg, once a day for 10 days). Another group of tumor-transplanted mice were not treated until the average tumor weight had reached about 220 mg, which took about 14 days. These mice were treated
i.p. with sterile saline or tiazofurin (500 mg/kg, once a day for 5 days, rested for 10 days and the cycle was repeated 4 times). Control groups were injected i.p. with sterile saline instead of tiazofurin. The tumor weight (W) was measured 3 times a week and calculated by the following equation:
W = 1/2
X
A
X
B2
where A and B were the experimental measurements of tumor length and width in millimeters, respectively. The toxicity was monitored by measuring body weight 3 times a week.
Biological Studies Groups of athymic nude mice were S.C.transplanted with colon carcinoma HT-29 cells as detailed above. Twenty-four days later, when the tumor weight reached 450 mg, mice were injected i.p. either with saline or with tiazofurin (500 mg/kg). Mice were lightly anesthetized with ether 6 h later, and the tumors were rapidly excised and freeze-clamped within a second as described earlier (24,25). The freeze-clamped tumor tissues were ground under liquid nitrogen and then extracted with 10% TCA. The acid-soluble extracts were immediately neutralized with 0.5 M TOA in freon ( I :2 v / v ) (16,26). For the measurement of nucleotide concentration in tissue culture cells, logarithmically growing HT-29 cells in RPMI 1640 medium containing 10% fetal bovine serum were harvested after 2 h of exposure to tiazofurin (100 pM) or saline at 37°C; the cells were washed once with cold PBS and then extracted with 10% TCA followed by neutralization with TOA, as described above.
Cytotoxicity Studies For the determination of cytotoxicity, clonogenic assay was employed. Human colon carcinoma HT-29 cells were grown logarithmically at 37°C in RPMI 1640 medium supplemented with 10% fetal bovine serum in a humidified atmosphere of air with 5% C 0 2 . The cells were trypsinized, washed, and resuspended in the same medium, transferred into a 25-cm2flask (300 cells/flask), and incubated for 4 h to allow the cells to attach to the flask. The cells were then exposed to tiazofurin at various concentrations (1-100 pM) for 2 or 24 h . After tiazofurin treatment, the medium was aspirated, fresh medium was added, and the cells were incubated for 10 days in an atmosphere of air with 5% CO,. The colonies were then stained with crystal violet and counted. The surviving fraction of tiazofurin-treated cells was calculated as the
Therapy of Colon Tumor with Tiazofurin
507
percentage of colonies formed by the saline-treated cells. Lethal concentration of tiazofurin that results in 50% survival of cell colonies (LC,,) was calculated from a linear plot of tiazofurin concentration versus cytotoxicity.
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Determination of Nucleotide Pools Determination of nucleotide pools was carried out on HPLC as described earlier (19). Briefly, an aliquot ( 100200 pl) of the neutralized samples was loaded on a Waters Partisil 10-SAX column in an RCM-10 module preequilibrated with 5 mM ammonium phosphate buffer, pH 3.0. A gradient with 650 mM ammonium phosphate buffer, pH 3.7, was applied, and the gradient conditions for analysis were as described (19). Nucleotide and TAD concentrations were quantitated with a photodiode array detector that scanned and stored spectral data between 190 and 390 nm.
RESULTS
roi 0
1
20
40
80
80
100
Tiarofurln concentration (uM)
Figure 1 Cytotoxicity of tiazofurin in HT-29 cells. Logarithmically growing HT-29 cells were exposed to tiazofurin at various concentrations (1-100 p M ) for 2 or 24 h, as described in Materials and Methods. The colonies were stained and counted. Cell culture studies were carried out in triplicate
flasks.
Cytotoxicity of Tiazofurin In the clonogenic survival assay, 24-h exposure of human colon carcinoma HT-29 cells to tiazofurin resulted in an LCS0of 35 p M . However, 2-h incubation did not produce 50% inhibition of colony formation up to 100 p M concentration of tiazofurin (Fig. 1).
Biochemical Impact of Tiazofurin The biochemical effects of tiazofurin on HT-29 cells were examined both in vitro and in vivo. Human colon carcinoma HT-29 cells in culture were incubated with 100-300 p M tiazofurin or with saline for 2 h, and then TAD concentrations were assayed as detailed in Materials and Methods. Figure 2 shows that a significant concentration of TAD was formed in the cells and there was a linear relationship between tiazofurin concentration and TAD accumulation. To examine whether TAD accumulation in HT-29 cells was related to its biochemical effects, HT-29 cells in culture were incubated with saline or tiazofurin (100 p M ) for 2 h and then nucleotide pools were analyzed (Table 1). Tiazofurin incubation significantly decreased the guanylate pools, with a marked increase in IMP concentration (402%), but NAD and adenylate concentrations were not significantly perturbed. To examine whether in vitro effects observed with tiazofurin were applicable in vivo, HT-29 tumor-bearing mice were injected with tiazofurin (500 mg/kg) or saline.
Six hours later, tumors were removed and analyzed on HPLC as detailed in Materials and Methods (Table 2). The results showed that GTP concentration was reduced by 44%, with a concurrent increase in IMP pools to 512%. At the same time, 17 nmol/g of TAD had ac-
40 1
TR concentration (IN)
Figure 2 TAD formation in HT-29 cells. HT-29 cells growing in exponential phase of growth were incubated with tiazofurin (100-300 p M ) for 2 h, and cells were extracted with TCA followed by immediate neutralization with TOA. The acidsoluble extracts were analyzed on HPLC as detailed in Materials and Methods. Data expressed as mean k S.E. from triplicate
samples.
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Table 1
Table 2
Effect of Ticizofurin on the Nucleotide Pools of Human Colon Carcinoma HT-29 Cells in Culturea
Effect of Tiazofurin on the Nucleotide Pools of Human Colon Carcinoma HT-29 Cells I n Vivo"
Nucleotide
Control (nrnolig cells)
Tiazofurin treated (% of control)
ATP
2314.0 f 208.8
90
ADP
936.4
f
5 1.6
138
AMP
64.4
f
11.1
146
GTP
548.7 f 56.8
36"
GDP
124.1 2 5.3
72"
GMP
9.0 2 1.2
4"
IMP NAD CTP
* 4.2 433.1 * 35.2 163.7 * 13.5 15.4
402" 93 97
CDP
21.7
f
1.6
110
CMP
29.5
f
3.4
120
UTP
1113.8 k 116.9
91
UDP
113.2 2 12.0
18Sh
UMP
70.2
* 2.9
226"
'HT-29 cells growing in logarithmic phase were incubated with tiazofurin (100 pM) for 2 h. Cells were harvested, washed with PBS, extracted with 10% TCA, immediately neutralized with TOA, and analyzed using HPLC techniques as outlined in Materials and Methods. Values were calculated from triplicate samples and given as mean % S.E. hSignificantly different from control ( p < 0.0.5).
cumulated in the tumor. These studies demonstrate that tiazofurin exerted similar biochemical effects both in vitro and in vivo.
Antitumor Activity of Tiazofurin Because tiazofurin exerted potent cytotoxic and biochemical action in human colon carcinoma HT-29 cells in vitro and in vivo, we hypothesized that it should also have significant antitumor activity. To test this hypothesis, antitumor activity of tiazofurin in mice bearing S.C. HT-29 tumor was examined under two different treatment protocols. In the first schedule, tiazofurin treatment was started 1 day after HT-29 tumor implantation. This protocol was designed to test the efficacy of tiazofurin on the early stages of colon tumor infiltration (Fig. 3). Ten
Tiazofurin treated
Control Nucleotide
(nrnol/g)
nrnol / g
ADP
* 98 426 * 77
AMP
287 f 20
GTP
336 f 23
GDP
96 f 1
* 48 187 * 3 61 * 7
GMP
44*6
18 f 2
CTP
92 -+ 6
CDP
19 f 5
CMP
88
ATP
1428
*3
UTP
378 2 17
UDP
83 rfr. 16
UMP
55 r 12
IMP NAD TAD
%
of control
* 44
102
401 +- 21
94
1463
397
100
* 10
22 f 1 65 456
*2 27
63 5 7
139 56h 64" 41" 109
I I6 74h 121 79
8 * 2
*7 41 * 6
513"
248 2 21
226 2 13
91
-
50
17
91
*3
"Nude mice were transplanted subcutaneously with HT-29 cells. Twenty-four days later, they were injected with either saline or tiazofurin (SO0 mgikg). Six hours later, tumors were quickly removed, freeze-clamped, extracted with cold 10% TCA, neutralized with 0.5 M TOA, and analyzed on HPLC as detailed in Materials and Methods. Values were expressed as mean % S.E. from 3 miceigroup. "Significantly different from control ( p < 0.0.5).
consecutive daily injections of tiazofurin (500 mg/kg/ day) were administered. This treatment slowed the tumor growth rate to 50% of control. The second protocol was to elucidate the efficacy of tiazofurin against established human colon HT-29 tumor. The treatment was started 2 weeks after tumor implantation (Fig. 4).By this time the average tumor weight had reached approximately 220 mg. Four cycles of treatment were given; each cycle included five consecutive i.p. injections at a dose of 500 mg/kg/day followed by a 10-day rest period between the cycles. The tumor growth rate in this group was reduced to 54% of control. In both treatment protocols, only a mild toxicity (less than 10% loss of body weight) was observed.
Therapy of Colon Tumor with Tiazofurin
509
Control
-
II
M
v
I
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P ,M t
Y
5
0
15
10
20
25
30
35
40
Time (day)
Figure 3 Twenty-four hours after S.C.implantation of HT-29 (2.2 X lo6)cells, nude mice (7 micelgroup) were treated i.p. with sterile saline or tiazofurin (500 mg/kg/day) for 10 consecutive days. Tumor volume was recorded 3 times a week and calculated as outlined in Materials and Methods.
Treatmcntq
I n
3
4 51
PM
'I
01 0
I
5
I
I
I
I
I
I
I
I
I
I
I
10 15 20 25 3 0 35 4 0 45 50 55 60
Time (day)
Figure 4 Nude athymic mice (7 mice/group) were S.C.trans-
planted with HT-29 cells (2.2 X 10"). Two weeks after inoculation of HT-29 cells, the average tumor weight reached 220 mg. Tumor-bearing nude mice were injected,i.p. with sterile saline or tiazofurin (500 mg/kg/day) for 5 consecutive days. Tiazofurin treatment was repeated for four cycles with a 10-day rest period between the cycles. Tumor and body weight were recorded 3 times a week.
DISCUSSION Currently a phase 1/11 clinical protocol to study the efficacy and toxicity of tiazofurin in ANLL, CGL-BC, secondary leukemia, and advanced stages of MDS is being tested at the Indiana University School of Medicine (7,8). Encouraging therapeutic responses were observed, especially in patients with CGL-BC. In the present study, we have examined the effects of tiazofurin on human colon carcinoma HT-29 in vitro and in vivo. Marked alterations in the enzyme pattern were reported in human primary colon carcinoma compared to colon mucosa (23). IMP dehydrogenase activity in primary human colon carcinoma was 575% higher than in normal human colon mucosa (23). Thus, IMP dehydrogenase could be a target for cancer chemotherapy in human colon carcinoma. It was of interest to examine whether human colon carcinoma HT-29 cells could anabolize tiazofurin to yield a sufficient concentration of TAD to inhibit de novo biosynthesis of guanylates leading to suppression of tumor cell proliferation. Our studies have shown that incubation of HT-29 cells with tiazofurin provided enough TAD in the tumor cells to inhibit IMP dehydrogenase activity, resulting in marked depletion of GTP pools with a concurrent accumulation of IMP. We have further demonstrated the antitumor activity of tiazofurin in nude mice transplanted with HT-29 tumor. Tiazofurin significantly reduced the growth rate of early-stage (newly implanted) and established solid tumors in the nude mouse model. However, tiazofurin in this protocol did not result in complete regression of the colon cancer in mice. This might be partly due to the activity of guanylate salvage pathway. In human colon tumor, not only de novo, but also salvage, enzyme activities were higher compared to those of normal mucosa (23,28), and thus salvage activity can partly circumvent the effect of tiazofurin. Therefore, combination chemotherapy with salvage pathway inhibitors such as dipyridamole, or with other antitumor agents that have different mechanisms of action, should be beneficial. Tiazofurin trial in end-stage leukemic patients indicated an effective dose of 2200 mg/m2 daily for 15 days (8). These recent results suggest that in the early colon cancer phase I1 trial, the tiazofurin dose was too low (1650 mg/m2) and duration of treatment was too short (5 days) (22). In the present study, the mice received an equivalent of 1500 mg/m' for 10 days; thus, they were given twice the tiazofurin dose as in the phase I1 trial (22). Therefore, our results and the need for adequate clinical tiazofurin dose and length of treatment should
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5 10
provide the basis for reexamination of the potential effectiveness of tiazofurin in patients with colon carcinoma. HT-29 cell line is used by NCl as one of the tumor panels for testing drugs against colon carcinoma. However, HT-29 or any other colon cell carcinoma lines may not necessarily predict the response of a particular colon cancer patient. Incubation of colon carcinoma cells with labeled tiazofurin and measuring TAD production should be helpful in attempting to predict sensitivity to this drug. Since our studies showed that human colon carcinoma cells can anabolize tiazofurin to the active metabolite, TAD, this also provides a biochemical rationale for reopening trials on solid tumors based on their capacity to synthesize high concentrations of TAD. Novel aspects of this study include: (1) evidence for the formation of the active metabolite, TAD, in human colon carcinoma cells; (2) demonstration for the first time of the sensitivity of human colon carcinoma HT-29 to tiazofurin; and (3) correlation of biochemical effects of tiazofurin in human colon carcinoma cells with antitumor activity in athymic nude mice. Abbreviations: CGL-BC, chronic granulocytic leukemia in blast crisis; i.p., intraperitoneal; s.c., subcutaneous; TR, tiazofurin; TAD, thiazole4-carboxamide adenine dinucleotide; TCA, trichloroacetic acid; TOA, tri-n-octylamine; HPLC, high-pressure liquid chromatography.
ACKNOWLEDGMENTS Financial support for this project was provided by U.S. Public Health Service, National Cancer Institute Grant CA-51770 to HNJ and an Outstanding Investigator Grant CA-425 10 to GW. Address reprint requests to Hiremagalur N. Jayaram, Laboratory for Experimental Oncology, Indiana University School of Medicine, 702 Barnhill Drive, RR 337, Indianapolis, IN 46202-5200.
REFERENCES Robins RK, Srivastava PC, Narayanan VL, et al: 2-P-D-Ribofuranosylthiazole-4-carboxamide, a novel potential antitumor agent for lung tumors and metastases. J Med Chem 25:107-108, 1982. O’Dwyer PJ, Shoemaker DD, Jayaram HN, et al: Tiazofurin: a new antitumor agent. Invest New Drugs 2:79-84, 1984. Ahluwalia GS, Jayaram HN, Plowman JP, et al: Studies on the mechanism of action of 2-~-D-ribofuranosylthiazole-4-carboxamide. V. Factors governing the response of murine tumors to tiazofurin. Biochem Pharmacol 33:1195-1203, 1984.
4. Jayaram HN, Smith AL, Glazer R1, et al: Studies on the mechanism of action of 2-P-D-ribofuranosylthiazole-4-carboxamide (NSC 286193). 11. Relationship between dose level and biochemical effects in P388 leukemia in vivo. Biochem Pharmacol 3 1~3839-3845, 1982. 5. Earle MF, Glazer RI: Activity and metabolism of 2-P-D-ribofuranosylthiazole-4-carboxamidein human lymphoid tumor cells in culture. Cancer Res 43:133-137, 1983. 6. Carney DN, Ahluwalia GS, Jayaram HN, et al: Relationships between the cytotoxicity of tiazofurin and its metabolism by cultured human lung cancer cells. J Clin Invest 75:175-182, 1985. 7. Tricot GJ, Jayaram HN, Nichols CR, et al: Hematological and biochemical action of tiazofurin in a case of refractory acute myeloid leukemia. Cancer Res 47:4988-4991, 1987. 8. Tricot GJ, Jayaram HN, Lapis E, et al: Biochemically directed therapy of leukemia with tiazofurin, a selective blocker of inosine 5’-phosphate dehydrogenase activity. Cancer Res 49:3696-3701, 1989. 9. Weber G, Yamaji Y, Olah E, et al: Clinical and molecular impact of inhibition of IMP dehydrogenase activity by tiazofurin. Adv Enzyme Regul 28:335-356, 1989. 10. Cooney DA, Jayaram HN, Gebeyehu G, et al: The conversion to an analogue of of 2-P-D-ribofuranosylthiazole-4-carboxamide NAD with potent IMP dehydrogenase inhibitory properties. Biochem Pharmacol 31:2133-2136, 1982. I I . Cooney DA, Jayaram HN, Glazer RI, et al: Studies on the mechanism of action of tiazofurin metabolism to an analog of NAD with potent IMP dehydrogenase inhibitory activity. Adv Enzyme Regul 21:271-303, 1983. 12. Jayaram HN, Dion RL, Glazer RI, et al: Initial studies on the mechanism of action of a new oncolytic thiazole nucleoside, 2P-D-ribofuranosy Ithiazole-4-carboxamide (NSC 286193). Biochem Pharmacol 31:2371-2380, 1982. 13. Jayaram HN, Cooney DA, Glazer RI, et al: Mechanism of resistance to the oncolytic C-nucleoside, 2-PD-ribofuranosylthiazole-4-carboxamide (NSC 286193). Biochem Pharmacol 3 I : 2557-2560, 1982. 14. Lui MS, Faderan MA, Liepnieks JJ, et al: Modulation of IMP dehydrogenase activity and guanylate metabolism by tiazofurin (2-P-D-ribofuranosylthiazole-4-carboxamide).J Biol Chem 259:5078-5082, 1984. 15. Kuttan R, Robins RK, Saunders PP: Inhibition of inosinate dehydrogenase by metabolite of 2-P-D-ribofuranosylthiazole-4-carboxamide. Biochem Biophys Res Commun 1072362-868, 1982. 16. Jayaram HN: Biochemical mechanisms of resistance to tiazofurin. Adv Enzyme Regul 24:67-89, 1986. 17. Jayardm HN, Pillwein K, Lui MS, et al: Mechanism of resistance to tiazofurin in hepatoma 3924A. Biochem Pharmacol 35587593, 1986. 18. Jayaram HN, Pillwein K, Nichols CR, et al: Selective sensitivity to tiazofurin of human leukemic cells. Biochem Pharmacol 35:2029-2032, 1986. 19. Zhen W, Jayaram HN, Weber G: Determination of thiazole-4carboxamide adenine dinucleotide (TAD) levels in mononuclear cells of leukemic patients treated with tiazofurin. Biochem Pharmacol 41:281-286, 1991. 20. Trump DL, Tutsch KD, Koeller JM, et al: Phase I clinical study with pharrnacokinetic analysis of 2-~-D-ribofuranosylthiazole-4carboxamide (NSC 286193) administered as a five-day infusion. Cancer Res 45:2853-2858, 1985.
Therapy of Colon Tumor with Tiazofurin 21.
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Batist G, Klecker RW, Jayaram HN, et al: Phase I and phannacokinetic study of tiazofurin (TCAR, NSC 286193) administered by continuous infusion. Invest New Drugs 3:349-355, 1985. 22. Maroun JA, Eisenhauer E, Cripps C, et al: Phase I1 study of tiazofurin in colorectal cancer: a National Cancer Institute of Canada study. Cancer Treat Rep 71:1297-1298, 1987. 23. Natsumeda Y, Lui MS, Emrani J, et al: Purine enzymology of human colon carcinomas. Cancer Res 452556-2559, 1985. 24. Weber G, Stubbs M, Moms H P Metabolism of hepatomas of different growth rates in situ and during ischemia. Cancer Res 3 I :2177-21 83, 1971.
51 1 25.
Pillwein K, Jayaram HN, Weber G: Effect of ischemia on nucleosides and bases in rat liver and hepatoma 3924A. Cancer Res 47:3092-3096, 1987.
Khym JX: An analytical system for rapid separation of tissue nucleotides at low pressures on conventional anion exchanges. Clin Chem 21:1245-1252, 1975. 27. Weber G: Enzymology of cancer cells. N Engl J Med 296:486-
26.
493, 541-551, 1977. 28. Weber G: Biochemical strategy of cancer cells and the design of
chemotherapy. G. H. A. Clowes Memorial Lecture. Cancer Res 43 :3466- 3492, 1983.
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Antitumor Activity of Tiazofurin in Human Colon Carcinoma HT-29 Weining Zhen, M.D.’,*, Hiremagalur N. Jayaram, Ph.D.I, and George Weber, M.D.I ’Laboratory for Experimental Oncology Walther Oncology Center Indiana University School of Medicine Indianapolis, Indiana 46202-5200
ABSTRACT Tiazofurin is effective in treating end-stage leukemic patients (Tricot et al., Cancer Res 49:3696-3701, 1989). In sensitive tumors, the active metabolite of tiazofurin, TAD, potently inhibits IMP dehydrogenase activity, resulting in reduced guanylate pools. To elucidate tiazofurin activity in human solid tumors, we examined its activity in human colon carcinoma HT-29. Tiazofurin exhibited an LCs0 of 35 p.44 in cultured HT-29 cells. Incubation of HT-29 cells with 100 p.44 tiazofurin for 2 h resulted in TAD formation (9.3 nmollg cells) and in a 64% decrease in GTP pools. For biochemical and chemotherapy studies, athymic nude mice were transplanted S.C. with HT-29 cells. Twenty-four days later, mice were injected i.p. with tiazofurin (500 mglkg); 6 h later, tumors were removed and analyzed. These tumors formed 17 nmollg of TAD with decreased GTP pools (56%). To study oncolytic activity, transplanted mice were treated 24 h later with tiazofurin (500 mglkg, once a day for I0 days). To examine the eflectiveness of tiazofurin in established tumors, the drug was administered to mice 14 days after tumor implantation (500 mglkg, once a day for 5 days, course repeated 4 times with a 10-day rest). Both treatment schedules resulted in significant antitumor activity. This study illustrates the potential usefulness of tiazofurin in treating human colon carcinoma.
INTRODUCTION
L1210 lymphoid leukemia, P388 leukemia, Lewis lung (2-~-D-ribofuranosylthiazole-4-carbox- carcinoma, and plutonium 239-induced osteosarcoma Tiazofurin (1-6). It has also shown promising activity in end-stage amide, NSC 286193) is a novel C-nucleoside with anleukemic patients (7-9). In sensitive tumors, tiazofurin titumor activity against several murine tumors, including 505
Copyright 0 1992 by Marcel Dekker, Inc.
Zhen, Jayaram, and Weber
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506
is anabolized to its active metabolite, TAD, which potently inhibits the activity of IMP dehydrogenase (EC 1. I . 1.205), the rate-limiting enzyme of de novo GTP biosynthesis, resulting in decreased guanylate concentration leading to the inhibition of tumor cell proliferation (10-14). We have demonstrated the importance of TAD as a predictor of sensitivity to tiazofurin in human leukemic cells in vitro and in vivo (18,19). Early phase I studies with tiazofurin in solid tumors described doselimiting toxicities (20-22). The present communication is aimed at examining the sensitivity of human solid tumor to tiazofurin. Since IMP dehydrogenase activity was markedly elevated in human colon carcinoma compared to normal human colon mucosa (23), IMP dehydrogenase could be a target for chemotherapy in human colon carcinoma. Tiazofurin was shown to be a selective inhibitor of IMP dehydrogenase with oncolytic property in treating end-stage leukemic patients (7-9). We have elucidated the activity of tiazofurin in human colon carcinoma HT-29 both in vitro and in vivo. This study might be helpful in reopening clinical trials in solid tumors with tiazofurin.
MATERIALS AND METHODS Materials Tiazofurin was provided by the Drug Synthesis and Chemistry Branch, Drug Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, Bethesda, Maryland. RCM Partisil 10-SAX column and HPLC apparatus consisting of an NEC computer (APC IV, Power Mate 2 mode), a 991 photodiode array detector, a 600E multisolvent delivery system, and a refrigerated 7 12-WISP autoinjector were purchased from Waters Associates, Milford, Massachusetts. Ammonium phosphate (monobasic, HPLC grade) was obtained from the Fisher Scientific Co., Itasca, Illinois.
Tumor System Male athymic nude mice (4-5 weeks old) were purchased from Harlan Sprague Dawley, Inc., Cumberland, Indiana. Animals were housed in individual cages, and food and water were available ad libitum. Human colon carcinoma HT-29 cells (2.2 X lo6)were S.C. implanted, and 24 h later, they were injected i.p. with sterile saline or tiazofurin (500 mg/kg, once a day for 10 days). Another group of tumor-transplanted mice were not treated until the average tumor weight had reached about 220 mg, which took about 14 days. These mice were treated
i.p. with sterile saline or tiazofurin (500 mg/kg, once a day for 5 days, rested for 10 days and the cycle was repeated 4 times). Control groups were injected i.p. with sterile saline instead of tiazofurin. The tumor weight (W) was measured 3 times a week and calculated by the following equation:
W = 1/2
X
A
X
B2
where A and B were the experimental measurements of tumor length and width in millimeters, respectively. The toxicity was monitored by measuring body weight 3 times a week.
Biological Studies Groups of athymic nude mice were S.C.transplanted with colon carcinoma HT-29 cells as detailed above. Twenty-four days later, when the tumor weight reached 450 mg, mice were injected i.p. either with saline or with tiazofurin (500 mg/kg). Mice were lightly anesthetized with ether 6 h later, and the tumors were rapidly excised and freeze-clamped within a second as described earlier (24,25). The freeze-clamped tumor tissues were ground under liquid nitrogen and then extracted with 10% TCA. The acid-soluble extracts were immediately neutralized with 0.5 M TOA in freon ( I :2 v / v ) (16,26). For the measurement of nucleotide concentration in tissue culture cells, logarithmically growing HT-29 cells in RPMI 1640 medium containing 10% fetal bovine serum were harvested after 2 h of exposure to tiazofurin (100 pM) or saline at 37°C; the cells were washed once with cold PBS and then extracted with 10% TCA followed by neutralization with TOA, as described above.
Cytotoxicity Studies For the determination of cytotoxicity, clonogenic assay was employed. Human colon carcinoma HT-29 cells were grown logarithmically at 37°C in RPMI 1640 medium supplemented with 10% fetal bovine serum in a humidified atmosphere of air with 5% C 0 2 . The cells were trypsinized, washed, and resuspended in the same medium, transferred into a 25-cm2flask (300 cells/flask), and incubated for 4 h to allow the cells to attach to the flask. The cells were then exposed to tiazofurin at various concentrations (1-100 pM) for 2 or 24 h . After tiazofurin treatment, the medium was aspirated, fresh medium was added, and the cells were incubated for 10 days in an atmosphere of air with 5% CO,. The colonies were then stained with crystal violet and counted. The surviving fraction of tiazofurin-treated cells was calculated as the
Therapy of Colon Tumor with Tiazofurin
507
percentage of colonies formed by the saline-treated cells. Lethal concentration of tiazofurin that results in 50% survival of cell colonies (LC,,) was calculated from a linear plot of tiazofurin concentration versus cytotoxicity.
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Determination of Nucleotide Pools Determination of nucleotide pools was carried out on HPLC as described earlier (19). Briefly, an aliquot ( 100200 pl) of the neutralized samples was loaded on a Waters Partisil 10-SAX column in an RCM-10 module preequilibrated with 5 mM ammonium phosphate buffer, pH 3.0. A gradient with 650 mM ammonium phosphate buffer, pH 3.7, was applied, and the gradient conditions for analysis were as described (19). Nucleotide and TAD concentrations were quantitated with a photodiode array detector that scanned and stored spectral data between 190 and 390 nm.
RESULTS
roi 0
1
20
40
80
80
100
Tiarofurln concentration (uM)
Figure 1 Cytotoxicity of tiazofurin in HT-29 cells. Logarithmically growing HT-29 cells were exposed to tiazofurin at various concentrations (1-100 p M ) for 2 or 24 h, as described in Materials and Methods. The colonies were stained and counted. Cell culture studies were carried out in triplicate
flasks.
Cytotoxicity of Tiazofurin In the clonogenic survival assay, 24-h exposure of human colon carcinoma HT-29 cells to tiazofurin resulted in an LCS0of 35 p M . However, 2-h incubation did not produce 50% inhibition of colony formation up to 100 p M concentration of tiazofurin (Fig. 1).
Biochemical Impact of Tiazofurin The biochemical effects of tiazofurin on HT-29 cells were examined both in vitro and in vivo. Human colon carcinoma HT-29 cells in culture were incubated with 100-300 p M tiazofurin or with saline for 2 h, and then TAD concentrations were assayed as detailed in Materials and Methods. Figure 2 shows that a significant concentration of TAD was formed in the cells and there was a linear relationship between tiazofurin concentration and TAD accumulation. To examine whether TAD accumulation in HT-29 cells was related to its biochemical effects, HT-29 cells in culture were incubated with saline or tiazofurin (100 p M ) for 2 h and then nucleotide pools were analyzed (Table 1). Tiazofurin incubation significantly decreased the guanylate pools, with a marked increase in IMP concentration (402%), but NAD and adenylate concentrations were not significantly perturbed. To examine whether in vitro effects observed with tiazofurin were applicable in vivo, HT-29 tumor-bearing mice were injected with tiazofurin (500 mg/kg) or saline.
Six hours later, tumors were removed and analyzed on HPLC as detailed in Materials and Methods (Table 2). The results showed that GTP concentration was reduced by 44%, with a concurrent increase in IMP pools to 512%. At the same time, 17 nmol/g of TAD had ac-
40 1
TR concentration (IN)
Figure 2 TAD formation in HT-29 cells. HT-29 cells growing in exponential phase of growth were incubated with tiazofurin (100-300 p M ) for 2 h, and cells were extracted with TCA followed by immediate neutralization with TOA. The acidsoluble extracts were analyzed on HPLC as detailed in Materials and Methods. Data expressed as mean k S.E. from triplicate
samples.
Zhen, Jayararn, and Weber
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508
Table 1
Table 2
Effect of Ticizofurin on the Nucleotide Pools of Human Colon Carcinoma HT-29 Cells in Culturea
Effect of Tiazofurin on the Nucleotide Pools of Human Colon Carcinoma HT-29 Cells I n Vivo"
Nucleotide
Control (nrnolig cells)
Tiazofurin treated (% of control)
ATP
2314.0 f 208.8
90
ADP
936.4
f
5 1.6
138
AMP
64.4
f
11.1
146
GTP
548.7 f 56.8
36"
GDP
124.1 2 5.3
72"
GMP
9.0 2 1.2
4"
IMP NAD CTP
* 4.2 433.1 * 35.2 163.7 * 13.5 15.4
402" 93 97
CDP
21.7
f
1.6
110
CMP
29.5
f
3.4
120
UTP
1113.8 k 116.9
91
UDP
113.2 2 12.0
18Sh
UMP
70.2
* 2.9
226"
'HT-29 cells growing in logarithmic phase were incubated with tiazofurin (100 pM) for 2 h. Cells were harvested, washed with PBS, extracted with 10% TCA, immediately neutralized with TOA, and analyzed using HPLC techniques as outlined in Materials and Methods. Values were calculated from triplicate samples and given as mean % S.E. hSignificantly different from control ( p < 0.0.5).
cumulated in the tumor. These studies demonstrate that tiazofurin exerted similar biochemical effects both in vitro and in vivo.
Antitumor Activity of Tiazofurin Because tiazofurin exerted potent cytotoxic and biochemical action in human colon carcinoma HT-29 cells in vitro and in vivo, we hypothesized that it should also have significant antitumor activity. To test this hypothesis, antitumor activity of tiazofurin in mice bearing S.C. HT-29 tumor was examined under two different treatment protocols. In the first schedule, tiazofurin treatment was started 1 day after HT-29 tumor implantation. This protocol was designed to test the efficacy of tiazofurin on the early stages of colon tumor infiltration (Fig. 3). Ten
Tiazofurin treated
Control Nucleotide
(nrnol/g)
nrnol / g
ADP
* 98 426 * 77
AMP
287 f 20
GTP
336 f 23
GDP
96 f 1
* 48 187 * 3 61 * 7
GMP
44*6
18 f 2
CTP
92 -+ 6
CDP
19 f 5
CMP
88
ATP
1428
*3
UTP
378 2 17
UDP
83 rfr. 16
UMP
55 r 12
IMP NAD TAD
%
of control
* 44
102
401 +- 21
94
1463
397
100
* 10
22 f 1 65 456
*2 27
63 5 7
139 56h 64" 41" 109
I I6 74h 121 79
8 * 2
*7 41 * 6
513"
248 2 21
226 2 13
91
-
50
17
91
*3
"Nude mice were transplanted subcutaneously with HT-29 cells. Twenty-four days later, they were injected with either saline or tiazofurin (SO0 mgikg). Six hours later, tumors were quickly removed, freeze-clamped, extracted with cold 10% TCA, neutralized with 0.5 M TOA, and analyzed on HPLC as detailed in Materials and Methods. Values were expressed as mean % S.E. from 3 miceigroup. "Significantly different from control ( p < 0.0.5).
consecutive daily injections of tiazofurin (500 mg/kg/ day) were administered. This treatment slowed the tumor growth rate to 50% of control. The second protocol was to elucidate the efficacy of tiazofurin against established human colon HT-29 tumor. The treatment was started 2 weeks after tumor implantation (Fig. 4).By this time the average tumor weight had reached approximately 220 mg. Four cycles of treatment were given; each cycle included five consecutive i.p. injections at a dose of 500 mg/kg/day followed by a 10-day rest period between the cycles. The tumor growth rate in this group was reduced to 54% of control. In both treatment protocols, only a mild toxicity (less than 10% loss of body weight) was observed.
Therapy of Colon Tumor with Tiazofurin
509
Control
-
II
M
v
I
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P ,M t
Y
5
0
15
10
20
25
30
35
40
Time (day)
Figure 3 Twenty-four hours after S.C.implantation of HT-29 (2.2 X lo6)cells, nude mice (7 micelgroup) were treated i.p. with sterile saline or tiazofurin (500 mg/kg/day) for 10 consecutive days. Tumor volume was recorded 3 times a week and calculated as outlined in Materials and Methods.
Treatmcntq
I n
3
4 51
PM
'I
01 0
I
5
I
I
I
I
I
I
I
I
I
I
I
10 15 20 25 3 0 35 4 0 45 50 55 60
Time (day)
Figure 4 Nude athymic mice (7 mice/group) were S.C.trans-
planted with HT-29 cells (2.2 X 10"). Two weeks after inoculation of HT-29 cells, the average tumor weight reached 220 mg. Tumor-bearing nude mice were injected,i.p. with sterile saline or tiazofurin (500 mg/kg/day) for 5 consecutive days. Tiazofurin treatment was repeated for four cycles with a 10-day rest period between the cycles. Tumor and body weight were recorded 3 times a week.
DISCUSSION Currently a phase 1/11 clinical protocol to study the efficacy and toxicity of tiazofurin in ANLL, CGL-BC, secondary leukemia, and advanced stages of MDS is being tested at the Indiana University School of Medicine (7,8). Encouraging therapeutic responses were observed, especially in patients with CGL-BC. In the present study, we have examined the effects of tiazofurin on human colon carcinoma HT-29 in vitro and in vivo. Marked alterations in the enzyme pattern were reported in human primary colon carcinoma compared to colon mucosa (23). IMP dehydrogenase activity in primary human colon carcinoma was 575% higher than in normal human colon mucosa (23). Thus, IMP dehydrogenase could be a target for cancer chemotherapy in human colon carcinoma. It was of interest to examine whether human colon carcinoma HT-29 cells could anabolize tiazofurin to yield a sufficient concentration of TAD to inhibit de novo biosynthesis of guanylates leading to suppression of tumor cell proliferation. Our studies have shown that incubation of HT-29 cells with tiazofurin provided enough TAD in the tumor cells to inhibit IMP dehydrogenase activity, resulting in marked depletion of GTP pools with a concurrent accumulation of IMP. We have further demonstrated the antitumor activity of tiazofurin in nude mice transplanted with HT-29 tumor. Tiazofurin significantly reduced the growth rate of early-stage (newly implanted) and established solid tumors in the nude mouse model. However, tiazofurin in this protocol did not result in complete regression of the colon cancer in mice. This might be partly due to the activity of guanylate salvage pathway. In human colon tumor, not only de novo, but also salvage, enzyme activities were higher compared to those of normal mucosa (23,28), and thus salvage activity can partly circumvent the effect of tiazofurin. Therefore, combination chemotherapy with salvage pathway inhibitors such as dipyridamole, or with other antitumor agents that have different mechanisms of action, should be beneficial. Tiazofurin trial in end-stage leukemic patients indicated an effective dose of 2200 mg/m2 daily for 15 days (8). These recent results suggest that in the early colon cancer phase I1 trial, the tiazofurin dose was too low (1650 mg/m2) and duration of treatment was too short (5 days) (22). In the present study, the mice received an equivalent of 1500 mg/m' for 10 days; thus, they were given twice the tiazofurin dose as in the phase I1 trial (22). Therefore, our results and the need for adequate clinical tiazofurin dose and length of treatment should
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5 10
provide the basis for reexamination of the potential effectiveness of tiazofurin in patients with colon carcinoma. HT-29 cell line is used by NCl as one of the tumor panels for testing drugs against colon carcinoma. However, HT-29 or any other colon cell carcinoma lines may not necessarily predict the response of a particular colon cancer patient. Incubation of colon carcinoma cells with labeled tiazofurin and measuring TAD production should be helpful in attempting to predict sensitivity to this drug. Since our studies showed that human colon carcinoma cells can anabolize tiazofurin to the active metabolite, TAD, this also provides a biochemical rationale for reopening trials on solid tumors based on their capacity to synthesize high concentrations of TAD. Novel aspects of this study include: (1) evidence for the formation of the active metabolite, TAD, in human colon carcinoma cells; (2) demonstration for the first time of the sensitivity of human colon carcinoma HT-29 to tiazofurin; and (3) correlation of biochemical effects of tiazofurin in human colon carcinoma cells with antitumor activity in athymic nude mice. Abbreviations: CGL-BC, chronic granulocytic leukemia in blast crisis; i.p., intraperitoneal; s.c., subcutaneous; TR, tiazofurin; TAD, thiazole4-carboxamide adenine dinucleotide; TCA, trichloroacetic acid; TOA, tri-n-octylamine; HPLC, high-pressure liquid chromatography.
ACKNOWLEDGMENTS Financial support for this project was provided by U.S. Public Health Service, National Cancer Institute Grant CA-51770 to HNJ and an Outstanding Investigator Grant CA-425 10 to GW. Address reprint requests to Hiremagalur N. Jayaram, Laboratory for Experimental Oncology, Indiana University School of Medicine, 702 Barnhill Drive, RR 337, Indianapolis, IN 46202-5200.
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4. Jayaram HN, Smith AL, Glazer R1, et al: Studies on the mechanism of action of 2-P-D-ribofuranosylthiazole-4-carboxamide (NSC 286193). 11. Relationship between dose level and biochemical effects in P388 leukemia in vivo. Biochem Pharmacol 3 1~3839-3845, 1982. 5. Earle MF, Glazer RI: Activity and metabolism of 2-P-D-ribofuranosylthiazole-4-carboxamidein human lymphoid tumor cells in culture. Cancer Res 43:133-137, 1983. 6. Carney DN, Ahluwalia GS, Jayaram HN, et al: Relationships between the cytotoxicity of tiazofurin and its metabolism by cultured human lung cancer cells. J Clin Invest 75:175-182, 1985. 7. Tricot GJ, Jayaram HN, Nichols CR, et al: Hematological and biochemical action of tiazofurin in a case of refractory acute myeloid leukemia. Cancer Res 47:4988-4991, 1987. 8. Tricot GJ, Jayaram HN, Lapis E, et al: Biochemically directed therapy of leukemia with tiazofurin, a selective blocker of inosine 5’-phosphate dehydrogenase activity. Cancer Res 49:3696-3701, 1989. 9. Weber G, Yamaji Y, Olah E, et al: Clinical and molecular impact of inhibition of IMP dehydrogenase activity by tiazofurin. Adv Enzyme Regul 28:335-356, 1989. 10. Cooney DA, Jayaram HN, Gebeyehu G, et al: The conversion to an analogue of of 2-P-D-ribofuranosylthiazole-4-carboxamide NAD with potent IMP dehydrogenase inhibitory properties. Biochem Pharmacol 31:2133-2136, 1982. I I . Cooney DA, Jayaram HN, Glazer RI, et al: Studies on the mechanism of action of tiazofurin metabolism to an analog of NAD with potent IMP dehydrogenase inhibitory activity. Adv Enzyme Regul 21:271-303, 1983. 12. Jayaram HN, Dion RL, Glazer RI, et al: Initial studies on the mechanism of action of a new oncolytic thiazole nucleoside, 2P-D-ribofuranosy Ithiazole-4-carboxamide (NSC 286193). Biochem Pharmacol 31:2371-2380, 1982. 13. Jayaram HN, Cooney DA, Glazer RI, et al: Mechanism of resistance to the oncolytic C-nucleoside, 2-PD-ribofuranosylthiazole-4-carboxamide (NSC 286193). Biochem Pharmacol 3 I : 2557-2560, 1982. 14. Lui MS, Faderan MA, Liepnieks JJ, et al: Modulation of IMP dehydrogenase activity and guanylate metabolism by tiazofurin (2-P-D-ribofuranosylthiazole-4-carboxamide).J Biol Chem 259:5078-5082, 1984. 15. Kuttan R, Robins RK, Saunders PP: Inhibition of inosinate dehydrogenase by metabolite of 2-P-D-ribofuranosylthiazole-4-carboxamide. Biochem Biophys Res Commun 1072362-868, 1982. 16. Jayaram HN: Biochemical mechanisms of resistance to tiazofurin. Adv Enzyme Regul 24:67-89, 1986. 17. Jayardm HN, Pillwein K, Lui MS, et al: Mechanism of resistance to tiazofurin in hepatoma 3924A. Biochem Pharmacol 35587593, 1986. 18. Jayaram HN, Pillwein K, Nichols CR, et al: Selective sensitivity to tiazofurin of human leukemic cells. Biochem Pharmacol 35:2029-2032, 1986. 19. Zhen W, Jayaram HN, Weber G: Determination of thiazole-4carboxamide adenine dinucleotide (TAD) levels in mononuclear cells of leukemic patients treated with tiazofurin. Biochem Pharmacol 41:281-286, 1991. 20. Trump DL, Tutsch KD, Koeller JM, et al: Phase I clinical study with pharrnacokinetic analysis of 2-~-D-ribofuranosylthiazole-4carboxamide (NSC 286193) administered as a five-day infusion. Cancer Res 45:2853-2858, 1985.
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