Vol. 180, No. 2, 1991 October 31, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 933-938
CYTOTOXICITY OF TIAZOFURIN AND ITS ARABINOSE AND XYIDSE ANAIZ)GUES IN K562 CELLS Weining Zhen 1, Hiremagalur N. Jayaram 1., Victor E. Marquez 2, Barry M. Goldstein 3, David A. Cooney 2 and George Weber / /Laboratory for Experimental Oncology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5200 2 Laboratory
of Medicinal Chemistry, National Cancer Institute, Bethesda, Maryland 20892
3Department of Biophysics, University of Rochester Medical Center, Rochester, New York 14642 Received September 17, 1991
SUMMARY: 2-1~-D-Arabinofuranosylthiazole-4-carboxamide and 2-f~-D-xylofuranosylthiazole-4-carboxamide are sugar modified analogues of tiazofurin, a C-glycosyl nucleoside which after anabolism to the dinucleotide, TAD (thiazole-4-carboxamide adenine dinucleotide), exhibits antitumor activity. However, ara-T and xylo-T did not exhibit cytotoxicity. Compared to tiazofurin, only 12.5% of the ara-T and 8.8% of the xylo-T were metabolized to TAD derivatives by human myelogenous leukemia K562 cells. This was reflected in the finding that guanylate pools were not depressed after treatment with either tiazofurin derivative. These results provide evidence that the ribose moiety is essential for the metabolism and cytotoxicity of tiazofurin. This investigation should be helpful in the design of new analogues of tiazofurin for future clinical trials. © 1991
Academic
Press,
Inc.
Tiazofurin, 2-B-D-ribofuranosylthiazole-4-carboxamide,
exhibits antitumor activity
against
several murine tumors (1-3) and currently is undergoing
humans
for the treatment of end-stage
anabolized
I/IItriais in
leukemia (4). In sensitive cells, tiazofurin is
to its active metabolite, TAD, a dinucleotide
activity of IMP dehydrogenase
phase
which potently inhibits the
(EC 1.1.1.205), the rate-limiting enzyme of de novo
*To whom correspondence should be addressed. Abbreviations: Ara-T, 2-1~-D-arabinofuranosylthiazole-4-carboxamide; HPLC, high pressure liquid chromatography; IC5o, the concentration of drug that causes a 50% reduction in cell proliferation; TAD,thiazole-4-carboxamide adenine dinucleotide; Xylo-T, 2-f~-D-xylofuranosylthiazole-4-carboxamide. 0006-291X/91 $1.50 933
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
o
HO
o
S. ~N
HO
7.S
OH OH
S. ~N
o
HO
S..~.N
OH
A
OH
B
C
FIG. 1. Structures of tiazofurin (A), ara-T (B) and xylo-T (C).
guanylate biosynthesis.
Consequently,
there ensues
a decrease
in guanylate pools
which leads to an inhibition of tumor cell proliferation (5-7). The tiazofurin derivatives, ara-T
and xylo-T, are analogues
been
replaced
by arabinosyl
of tiazofurin in which the ribofuranosyl moiety has
and xylosyl groups
(Fig.l). Despite
their structural
similarity to tiazofurin, ara-T and xylo-T are not cytotoxic to murine lymphoma P388 cells in culture (8). This result may be attributable to poor transport of these derivatives (9) or to restricted conformational properties corresponding
analogues
which interfere with their conversion to the
of NAD (10). In the present studies, we have observed that
although ara-T and xylo-T are converted to analogues
of NAD, compared to tiazofurin
this conversion occurs to only 9-13% in K562 cells.
MATERIALS AND METHODS Materials. Tiazofurin, NSC 286193, was provided by Dr. Ven Narayanan, National Cancer Institute, Bethesda, MD. [8-]4C]Adenine (53 mCi/mmol) was purchased from Amersham Co., Arlington Heights, IL. RCM Partisil 10-SAX columns and an 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 712WISP autoinjector were purchased from Waters Associates, Milford, MA. The Foxy fraction collector was from Isco Inc., Lincoln, NE. Ammonium phosphate (monobasic, HPLC grade) was from the Fisher Scientific Co., Itasca, IL. Cytotoxicity studies. Human myelogenous leukemia K562 cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum and 2 mM L-glutamine in an atmosphere of air with 5% CO 2 at 37°C. For cytotoxicity studies, cells growing exponentially (5 ml, lx105 cells/ml) were continuously exposed to various concentrations of tiazofurin (10-30 ~M), ara-T (50-300 #~M)and xylo-T (50-300 ~zM) for 24 or 48 hr. The cell number was determined in a Coulter counter. The IC5o was calculated from a linear plot of concentration versus percent inhibition of growth. 934
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Metabolites of TR, ara-T and xylo-T. To examine the formation of the adenine dinucleotide of each analogue, K562 cells growing in exponential phase (106 cells/ml; 25 ml/flask) were incubated with [8-14C]adenine (10 /~Ci/flask; 0.19 /~M)and with tiazofurin, ara-T or xylo-T for 2 hr at 37°C. Cells were then harvested by centrifugation, washed once with PBS and then extracted with 300 #l of cold 10% trichloroacetic acid followed by immediate neutralization with 0.5 M tri-n-octylamine in freon (1:2 v/v). An aliquot of the cell extract was analyzed on a Partisil 10-SAX column under the conditions reported earlier (11). One-min fractions were collected; each fraction was then transferred to a scintillation vial, 10 ml of scintillation liquid (ScindVerse II)was added and radioactivity counted in a Beckman scintillation counter. All three radiolabeled NAD analogues eluted at 21 rain. In addition, all three dinucleotides were quantitatively decomposed by venom phosphodiesterase. Total nucleotide pools were quantitated by HPLC. RESULTS Tiazofurin exhibits significant cytotoxicity to K562 cells, with an
IC5oof 5 #M.
At
30/~M, tiazofurin reduced cell growth to 22% after 24 hr exposure and to 13% of control after 48 hr (Fig.2). However, the two tiazofurin derivatives produced concentrations
no cytotoxicity at
as high as 300 #M.
Since radiolabeled
ara-T and xylo-T were not available for metabolic studies, we
labeled K562 cells in culture with [14C]adenine, of any NAD analogues
that might be formed.
as a precursor to the adenylate moiety This method
provided a means
48-h exDosure
24-h exposure
130 -I
C B
100 ~ A
9 80 70
~
,~'~
0
-
-.
~
C
B
6O
50 40 .J
X
30
n¢n .J
A 20
..I
LM O
A 10
I
I
f IJIIl|
5
I
10
~
I IIIlll
50 100
I
I
300
I
I
F IIIIll
5
I
10
I
I IIIlll
50 100
I
I
300
DRUG C O N C E N T R A T I O N (IJM)
FIG.2. Cytotoxicity of tiazofurin, ara-T and xylo-T. Human myelogenous leukemia K562 cells in culture were continuously exposed to various concentrations of tiazofurin (A) (10-30 /~M), ara-T (B) (50-300 #M), and xylo-T (C) (50-300 #M) for 24 or 48 hr. The cell number was determined by a Coulter counter. 935
of
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
x 103
12
'~
100%
10 W
8
.Jo
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 933-938
CYTOTOXICITY OF TIAZOFURIN AND ITS ARABINOSE AND XYIDSE ANAIZ)GUES IN K562 CELLS Weining Zhen 1, Hiremagalur N. Jayaram 1., Victor E. Marquez 2, Barry M. Goldstein 3, David A. Cooney 2 and George Weber / /Laboratory for Experimental Oncology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5200 2 Laboratory
of Medicinal Chemistry, National Cancer Institute, Bethesda, Maryland 20892
3Department of Biophysics, University of Rochester Medical Center, Rochester, New York 14642 Received September 17, 1991
SUMMARY: 2-1~-D-Arabinofuranosylthiazole-4-carboxamide and 2-f~-D-xylofuranosylthiazole-4-carboxamide are sugar modified analogues of tiazofurin, a C-glycosyl nucleoside which after anabolism to the dinucleotide, TAD (thiazole-4-carboxamide adenine dinucleotide), exhibits antitumor activity. However, ara-T and xylo-T did not exhibit cytotoxicity. Compared to tiazofurin, only 12.5% of the ara-T and 8.8% of the xylo-T were metabolized to TAD derivatives by human myelogenous leukemia K562 cells. This was reflected in the finding that guanylate pools were not depressed after treatment with either tiazofurin derivative. These results provide evidence that the ribose moiety is essential for the metabolism and cytotoxicity of tiazofurin. This investigation should be helpful in the design of new analogues of tiazofurin for future clinical trials. © 1991
Academic
Press,
Inc.
Tiazofurin, 2-B-D-ribofuranosylthiazole-4-carboxamide,
exhibits antitumor activity
against
several murine tumors (1-3) and currently is undergoing
humans
for the treatment of end-stage
anabolized
I/IItriais in
leukemia (4). In sensitive cells, tiazofurin is
to its active metabolite, TAD, a dinucleotide
activity of IMP dehydrogenase
phase
which potently inhibits the
(EC 1.1.1.205), the rate-limiting enzyme of de novo
*To whom correspondence should be addressed. Abbreviations: Ara-T, 2-1~-D-arabinofuranosylthiazole-4-carboxamide; HPLC, high pressure liquid chromatography; IC5o, the concentration of drug that causes a 50% reduction in cell proliferation; TAD,thiazole-4-carboxamide adenine dinucleotide; Xylo-T, 2-f~-D-xylofuranosylthiazole-4-carboxamide. 0006-291X/91 $1.50 933
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
o
HO
o
S. ~N
HO
7.S
OH OH
S. ~N
o
HO
S..~.N
OH
A
OH
B
C
FIG. 1. Structures of tiazofurin (A), ara-T (B) and xylo-T (C).
guanylate biosynthesis.
Consequently,
there ensues
a decrease
in guanylate pools
which leads to an inhibition of tumor cell proliferation (5-7). The tiazofurin derivatives, ara-T
and xylo-T, are analogues
been
replaced
by arabinosyl
of tiazofurin in which the ribofuranosyl moiety has
and xylosyl groups
(Fig.l). Despite
their structural
similarity to tiazofurin, ara-T and xylo-T are not cytotoxic to murine lymphoma P388 cells in culture (8). This result may be attributable to poor transport of these derivatives (9) or to restricted conformational properties corresponding
analogues
which interfere with their conversion to the
of NAD (10). In the present studies, we have observed that
although ara-T and xylo-T are converted to analogues
of NAD, compared to tiazofurin
this conversion occurs to only 9-13% in K562 cells.
MATERIALS AND METHODS Materials. Tiazofurin, NSC 286193, was provided by Dr. Ven Narayanan, National Cancer Institute, Bethesda, MD. [8-]4C]Adenine (53 mCi/mmol) was purchased from Amersham Co., Arlington Heights, IL. RCM Partisil 10-SAX columns and an 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 712WISP autoinjector were purchased from Waters Associates, Milford, MA. The Foxy fraction collector was from Isco Inc., Lincoln, NE. Ammonium phosphate (monobasic, HPLC grade) was from the Fisher Scientific Co., Itasca, IL. Cytotoxicity studies. Human myelogenous leukemia K562 cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum and 2 mM L-glutamine in an atmosphere of air with 5% CO 2 at 37°C. For cytotoxicity studies, cells growing exponentially (5 ml, lx105 cells/ml) were continuously exposed to various concentrations of tiazofurin (10-30 ~M), ara-T (50-300 #~M)and xylo-T (50-300 ~zM) for 24 or 48 hr. The cell number was determined in a Coulter counter. The IC5o was calculated from a linear plot of concentration versus percent inhibition of growth. 934
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Metabolites of TR, ara-T and xylo-T. To examine the formation of the adenine dinucleotide of each analogue, K562 cells growing in exponential phase (106 cells/ml; 25 ml/flask) were incubated with [8-14C]adenine (10 /~Ci/flask; 0.19 /~M)and with tiazofurin, ara-T or xylo-T for 2 hr at 37°C. Cells were then harvested by centrifugation, washed once with PBS and then extracted with 300 #l of cold 10% trichloroacetic acid followed by immediate neutralization with 0.5 M tri-n-octylamine in freon (1:2 v/v). An aliquot of the cell extract was analyzed on a Partisil 10-SAX column under the conditions reported earlier (11). One-min fractions were collected; each fraction was then transferred to a scintillation vial, 10 ml of scintillation liquid (ScindVerse II)was added and radioactivity counted in a Beckman scintillation counter. All three radiolabeled NAD analogues eluted at 21 rain. In addition, all three dinucleotides were quantitatively decomposed by venom phosphodiesterase. Total nucleotide pools were quantitated by HPLC. RESULTS Tiazofurin exhibits significant cytotoxicity to K562 cells, with an
IC5oof 5 #M.
At
30/~M, tiazofurin reduced cell growth to 22% after 24 hr exposure and to 13% of control after 48 hr (Fig.2). However, the two tiazofurin derivatives produced concentrations
no cytotoxicity at
as high as 300 #M.
Since radiolabeled
ara-T and xylo-T were not available for metabolic studies, we
labeled K562 cells in culture with [14C]adenine, of any NAD analogues
that might be formed.
as a precursor to the adenylate moiety This method
provided a means
48-h exDosure
24-h exposure
130 -I
C B
100 ~ A
9 80 70
~
,~'~
0
-
-.
~
C
B
6O
50 40 .J
X
30
n¢n .J
A 20
..I
LM O
A 10
I
I
f IJIIl|
5
I
10
~
I IIIlll
50 100
I
I
300
I
I
F IIIIll
5
I
10
I
I IIIlll
50 100
I
I
300
DRUG C O N C E N T R A T I O N (IJM)
FIG.2. Cytotoxicity of tiazofurin, ara-T and xylo-T. Human myelogenous leukemia K562 cells in culture were continuously exposed to various concentrations of tiazofurin (A) (10-30 /~M), ara-T (B) (50-300 #M), and xylo-T (C) (50-300 #M) for 24 or 48 hr. The cell number was determined by a Coulter counter. 935
of
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
x 103
12
'~
100%
10 W
8
.Jo
