Enhancement of all-trans retinoic acid-induced HL-60 cell differentiation by thalidomide and its metabolites

Article abstract of DOI:10.1248/bpb.28.563

Thalidomide (Thal: 1) and its two metabolites, 5-hydroxythalidomide (5-HT: 2) and N-hydroxythalidomide (N-HT: 3), showed an enhancing effect on all-trans retinoic acid (ATRA)-induced HL-60 cell differentiation. 5-HT and N-HT showed tubulin polymerization-inhibiting activity, but thalidomide did not.

Full text of DOI:10.1248/bpb.28.563

March 2005
Communications to the Editor
Biol. Pharm. Bull. 28(3) 563—564 (2005)
563
Enhancement of All-trans Retinoic Acid- was condensed with O-benzylhydroxylamine in the presence
of dicyclohexylcarbodiimde (DCC) to give N-benzyl-
oxythalidomide, which was deprotected by hydrogenation
Induced HL-60 Cell Differentiation by
Thalidomide and Its Metabolites
(
H gas, Pd/C) to afford N-HT (3). The structures of the syn-
2
thesized compounds were confirmed by NMR and mass
spectroscopy and elemental analysis.
a
a
Tomomi NOGUCHI, Chihiro SHINJI,
a
b
Hisayoshi KOBAYASHI, Makoto MAKISHIMA,
First, we investigated the effect of the compounds on tubu-
a
,a
9)
Hiroyuki MIYACHI, and Yuichi HASHIMOTO*
lin polymerization using the method previously described.
a
Briefly, microtubulin was prepared from porcine brain and
tubulin polymerization was followed by turbidity measure-
ments at 37 °C in microtubule assemble buffer containing
Institute of Molecular & Cellular Biosciences, The University of
b
Tokyo; 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113–0032, Japan: and De-
partment of Biochemistry, Nihon University, School of Medicine;
3
9)
0
.5 mM MgCl and 1 mM GTP (pH 6.5). As already re-
0–1 Oyaguchi-kamicho, Itabashi-ku, Tokyo 173–8610, Japan.
2
Received January 13, 2005; accepted January 20, 2005; published ported, Thal (1) showed no effect on tubulin polymerization,
online January 24, 2005
and 5-HT (2) showed tubulin polymerization-inhibiting activ-
9)
ity with the IC50 value of approximately 50 mM (Fig. 2). N-
HT (3) was revealed to possess tubulin polymerization-in-
hibiting activity with similar potency with that of 5-HT (2),
as shown in Fig. 2.
Next, we investigated the effect of these compounds on
ATRA-induced HL-60 cell differentiation. ATRA was se-
lected because it is used clinically in differentiation-inducing
therapy of leukemia, especially for the treatment of acute
promyelocytic leukemia (APL), and is an endogenous active
Thalidomide (Thal: 1) and its two metabolites, 5-hydroxy-
thalidomide (5-HT: 2) and N-hydroxythalidomide (N-HT: 3),
showed an enhancing effect on all-trans retinoic acid (ATRA)-in-
duced HL-60 cell differentiation. 5-HT and N-HT showed tubu-
lin polymerization-inhibiting activity, but thalidomide did not.
Key words thalidomide; hydroxythalidomide; retinoic acid; differentia-
tion; tubulin
Thalidomide (Thal: 1), a sedative drug which was once form of vitamin A that is generally present in normal serum.
withdrawn from the market because of its teratogenicity, has Measurement of HL-60 cell differentiation was performed as
10)
been established to be effective for the treatment of various described previously. Briefly, HL-60 cells were incubated
diseases, including leprosy, multiple myeloma (MM), AIDS, in RPMI1640 medium in the presence or absence of a test
1—5)
and various cancers.
The anti-MM activity of Thal (1) is compound with 2 nM ATRA for 3 d. Treated HL-60 cells
of particular interest, because Thal (1) overcomes the resis- were mixed with phosphate-buffered saline (PBS) containing
6)
tance of human MM cells to conventional drug therapy. Al- 0.2% nitroblue tetrazolium (NBT) and 20 mM TPA in a 1 : 1
though various pharmacological effects elicited by Thal (1), (v/v) ratio and incubated at 37 °C for 20 min. NBT positivity
including tumor necrosis factor (TNF)-a production-regulat- was measured by counting 200—300 cells and the results
ing activity, anti-angiogenic activity and cyclooxygenase were expressed as the percentage of NBT-positive cells (Figs.
1
—5,7,8)
(
COX)-inhibiting activity,
have been reported, the 3a—c). The cell differentiation was also confirmed morpho-
logically by microscopy after Wright-Giemsa staining (Figs.
mechanism of its anti-MM activity is unclear.
During our structural development studies of Thal (1), we 3d—h). Of course, the percentage values differed from ex-
found that one of its major metabolites, 5-hydroxythalido- periment to experiment, but the results were basically repro-
mide (5-HT: 2), possesses tubulin polymerization-inhibiting ducible and a typical set of data is presented in Fig. 3. Under
activity, resulting in G1 arrest and apoptosis of myeloma our experimental conditions, the percentages of NBT-positive
9)
cells. We also previously reported that tubulin polymeriza- cells when HL-60 cells were treated with 2 nM, 5 nM and
tion inhibitors, including colchicine, vinblastine, rhizoxin, 10 nM ATRA for 3 d were typically approximately 2.5 % (al-
maytansine, and ustiloxin, and tubulin depolymerization in- most negligible, because non-treated HL-60 cells generally
hibitors, including taxol, enhance the chemically induced dif- contain 1—2% NBT-positive cells), 15% and 40%, respec-
10)
ferentiation of human leukemia cell lines. Though the rela- tively. Thal (1), 5-HT (2) and N-HT (3) showed no HL-60
tionship between cell differentiation induction and tubulin cell differentiation-inducing activity by themselves in the
polymerization/depolymerization inhibition is not clear, en- concentration range examined (Fig. 3).
hancement of chemically induced cell differentiation seems
to be one of general features of tubulin disruptors. These sess tubulin polymerization-inhibiting activity (Fig. 2),
As expected, 5-HT (2) and N-HT (3), both of which pos-
10)
findings led us to suspect that the enhancing effect of Thal showed an enhancing effect on the ATRA-induced HL-60
(1) and/or its metabolites plays a role in the anti-MM activity cell differentiation in a dose-dependent manner (Fig. 3, pan-
of the drug, at least in part. Thal (1) is known to be metaboli- els b and c). As mentioned above, HL-60 cell differentiation-
cally unstable, and many metabolites have been identified, in- inducing activity of ATRA was scarcely detected at the con-
cluding 5-HT (2) and N-hydroxythalidomide (N-HT: 3). In centration of 2 nM. But addition of 100 mM 5-HT (2) or N-HT
this paper, we deal with the enhancing effect on all-trans
retinoic acid (ATRA)-induced HL-60 cell differentiation and
the tubulin polymerization-inhibiting activity of Thal (1), 5-
HT (2) and N-HT (3).
Thal (1) and 5-HT (2) were prepared as described previ-
9
,11)
ously.
N-HT (3) was prepared by the method described by
12)
Robin et al. Briefly, N-phthaloylglutamic acid anhydride Fig. 1. Structures of Thal (1), 5-HT (2) and N-HT (3)
∗
To whom correspondence should be addressed. e-mail: hashimot@iam.u-tokyo.ac.jp
© 2005 Pharmaceutical Society of Japan

5
64
Vol. 28, No. 3
Fig. 2. Time Course Curves of Inhibition of Tubulin Poly-
merization Monitored in Terms of Turbidity (Absorbance at
400 nm)
A tubulin preparation was incubated at 37 °C in the presence or ab-
sence (control) of test compound. The test concentration was 50 mM for
Thal (1), 5-HT (2) and N-HT (3), and 10 mM for rhizoxin.
Fig. 3. Effects of Thal (1), 5-HT (2) and N-HT (3) on ATRA-Induced HL-60 Cell Dif-
ferentiation
Panels a—c: percentage of NBT-positive cells treated with the indicated concentration of Thal (1),
5-HT (2), or N-HT (3) in the presence or absence of 2 nM ATRA. Panels d—h: typical morphology of
HL-60 cells treated with the compound(s) indicated.
(3) increased the amount of NBT-positive cells to 22% and metabolites, 5-HT (2) and N-HT (3), and the cell differentia-
32 %, respectively (Fig. 3), which exceeds the HL-60 cell tion-enhancing activity of Thal (1), 5-HT (2) and N-HT (3),
differentiation-inducing effect elicited by 5 nM ATRA (15% (ii) application of thalidomide to ATRA differentiation-in-
NBT-positive cells, vide supra). Differentiation-enhancing ducing therapy should make it possible to lower the neces-
activity could be observed at 1 mM 5-HT (2) and N-HT (3). sary dose of ATRA, and (iii) Thal (1) therapy might result in
Although differentiation-inducing activity of 5-HT (2) and cell differentiation induction, because normal serum contains
N-HT (3) had been expected on the basis of their tubulin nanomolar order of ATRA, at which concentration Thal (1),
polymerization-inhibiting activity, the much higher activity 5-HT (2) and N-HT (3) would increase ATRA-induced cell
of N-HT (3) than that of 5-HT (2) cannot be interpreted in differentiation to the level induced by pharmacological doses
terms of their similar potency of tubulin polymerization-in- of ATRA.
hibiting activity (Fig. 2). Moreover, unexpectedly, Thal (1),
which does not possess tubulin polymerization-inhibiting ac-
Acknowledgements The work described in this paper
tivity, also showed an enhancing effect on ATRA-induced was partially supported by Grants-in-Aid for Scientific Re-
HL-60 cell differentiation (Fig. 3, panel a). The results indi- search from The Ministry of Education, Culture, Sports, Sci-
cate that the tested compounds elicit differentiation-enhanc- ence and Technology, Japan, and the Japan Society for the
ing activity through a mechanism other than tubulin poly- Promotion of Science.
merization-inhibiting activity, even though this activity might
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