A small molecule inhibitor of Bcl-2, HA14-1, also inhibits ceramide glucosyltransferase

Article abstract of DOI:10.1016/j.bbrc.2013.02.052

HA14-1 is a Bcl-2 inhibitor that is widely used for studies of apoptosis. In the course of searching for a ceramide glucosyltransferase inhibitor that catalyzes the first glycosylation step of glycosphingolipid synthesis, we unexpectedly found that HA-14-1 also has the ability to inhibit ceramide glucosyltransferase. The IC₅₀ value of HA14-1 against ceramide glucosyltransferase is 4.5μM, which is lower than that reported for Bcl-2 in vitro. Kinetic analyses revealed that HA14-1 is a competitive and mixed-type inhibitor with respect to C₆-NBD-ceramide and UDP-glucose, respectively.

Full text of DOI:10.1016/j.bbrc.2013.02.052

Biochemical and Biophysical Research Communications 433 (2013) 170–174
Contents lists available at SciVerse ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
A small molecule inhibitor of Bcl-2, HA14-1, also inhibits
ceramide glucosyltransferase
a
b
c
a
a,
⇑
Satomi Niino , Yutaka Nakamura , Yoshio Hirabayashi , Michiyo Nagano-Ito , Shinichi Ichikawa
a
Laboratory for Animal Cell Engineering, Niigata University of Pharmacy and Applied Life Sciences (NUPALS), 265-1 Higashijima, Akiha-ku, Niigata-shi, Niigata 956-8603, Japan
Laboratory of Environmental Organic Chemistry, Niigata University of Pharmacy and Applied Life Sciences (NUPALS), 265-1 Higashijima, Akiha-ku, Niigata-shi, Niigata 956-8603,
b
Japan
c
Hirabayashi Research Unit, Brain Science Institute (BSI), The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi, Saitama 351-01, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 January 2013
Available online 26 February 2013
HA14-1 is a Bcl-2 inhibitor that is widely used for studies of apoptosis. In the course of searching for a
ceramide glucosyltransferase inhibitor that catalyzes the first glycosylation step of glycosphingolipid
synthesis, we unexpectedly found that HA-14-1 also has the ability to inhibit ceramide glucosyltransfer-
ase. The IC50 value of HA14-1 against ceramide glucosyltransferase is 4.5
reported for Bcl-2 in vitro. Kinetic analyses revealed that HA14-1 is a competitive and mixed-type inhib-
itor with respect to C -NBD-ceramide and UDP-glucose, respectively.
Ó 2013 Elsevier Inc. All rights reserved.
lM, which is lower than that
Keywords:
Glucosylceramide
Inhibitor
6
Bcl-2
Apoptosis
1
. Introduction
Ceramide glucosyltransferase (UDP-glucose: ceramide gluco-
mulation of GlcCer due to a deficiency of the GlcCer-degrading
enzyme glucocerebrosidase [9]. NB-DNJ decreases GlcCer levels
by inhibiting de novo synthesis of GlcCer.
syltransferase, GlcT-1. EC 2.4.1.80) catalyzes the first glycosyla-
tion step of GSL synthesis. The enzyme transfers glucose from
UDP-glucose to ceramide, forming GlcCer [1,2]. The lipid sub-
strate of GlcT-1, ceramide, serves as a second messenger of apop-
tosis [3], and GlcT-1 regulates intracellular ceramide content [4].
GlcT-1 has also been implicated in insulin resistance of lipid cells
Bcl-2 is a protein factor known to suppress apoptosis by binding
and inactivating pro-apoptotic proteins such as Bax and BH3-only
family proteins. The formation of homodimers or heterodimers
with other Bcl-2 family proteins is necessary for Bcl-2 to exert its
anti-apoptotic activity. High levels of Bcl-2 expression are found
in various cancers [10]. Similar to GlcT-1, overexpression of Bcl-2
confers cancer cell resistance against anti-cancer agents. HA-14 is
a small molecule Bcl-2 inhibitor originally developed by Wang
et al. [11]. HA14-1 mimics the BH3 domain required for homo-
and heterodimer formation and thus inhibits the binding of these
proteins to each other. Thus HA14-1 sensitizes the Bcl-2 over-
expressing cancer cells.
[
5] and drug resistance of cancer cells [6]. GlcT-1 demonstrates its
anti-apoptotic effect by metabolizing and inactivating ceramide,
which direct apoptosis. In fact, certain types of drug resistant can-
cer cells have demonstrated enhanced GlcT-1 activity, and inhibi-
tion of the enzyme re-sensitized the cells against drugs [6]. Thus,
inhibitors of GlcT-1 have potential for therapeutic use to treat
these diseases.
Two lines of GlcT-1 inhibitors are widely used for basic studies.
One line of inhibitor includes PDMP and its derivatives, which are
analogs of ceramide and compete with it [7]. The other line of
inhibitor includes an imino sugar N-butyldeoxynojirimycin (NB-
DNJ), and its derivatives compete with UDP-glucose [8]. NB-DNJ
has been used to treat Gaucher’s disease, which is caused by accu-
Here, we report that the widely used Bcl-2 inhibitor HA14-1
also inhibits GlcT-1 activity.
2
. Materials and methods
2.1. Materials
A fluorescent substrate for GlcT-1, 6-{((N-7-Nitrobenz-2-oxa-
1
,3-diazol-4yl)amino)caproyl}sphingosine
(C
6
-NBD-Cer)
was
Abbreviations: GlcCer, glucosylceramide; LacCer, lactosylceramide; GSL, glyco-
sphingolipid; SM, sphingomyelin; IPTG, isopropylthio-b-galactoside; HA14-1, ethyl-
purchased from Invitrogen (CA, USA). Ceramide from bovine
brain was obtained from Sigma (Tokyo, Japan) Ethyl-2-amino-6-
bromo-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate
(HA14-1) was purchased from Wako (Tokyo, Japan) or prepared by
2
-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate.
⇑
Corresponding author. Fax: +81 25025 5115.
E-mail address: shin@nupals.ac.jp (S. Ichikawa).
0
006-291X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bbrc.2013.02.052

S. Niino et al. / Biochemical and Biophysical Research Communications 433 (2013) 170–174
171
following an established procedure [12]. In short, 5-bromosalicyl-
aldehyde was treated with ethyl cyanoacetate in the presence of
MS3A in dry ethanol at room temperature to produce HA14-1 with
a 55% yield. Synthesized HA14-1 had inhibitory activity compara-
ble with commercial HA14-1. A SCADS inhibitor kit containing
was incubated at 30 °C for 1 h. For kinetic analysis experiments,
reaction mixtures contained NP-40 and C -NBD-Cer, instead of lip-
osomes, to avoid C -NBD-sphingomyelin (C -NBD-SM) formation.
After the incubation, lipids were extracted with CHCl /CH OH,
2:1 (vol/vol) and evaporated to dryness. The lipids were then re-
dissolved in a small volume of CHCl /CH OH, 2:1 (vol/vol) and
chromatographed on silica gel 60 TLC plates (Merck, Darmstadt,
Germany) in CHCl /CH OH/H O, 65:25:4 (vol/vol/vol). NBD lipids
6
6
6
3
3
3
25 small molecule bioactive compounds was obtained from the
3
3
Screening Committee of Anticancer Drugs (Tokyo, Japan). All other
reagents used were of analytical grade.
3
3
2
were visualized by UV-B illumination. In some experiments, cell ly-
sates from Escherichia coli (E. coli) expressing human GlcT-1 were
used as described previously [14]. The LacCer synthase reaction
was performed using the method of Nishie et al. with slight mod-
2
.2. Cell line and culture conditions
Cell lines were obtained from RIKEN cell bank (Tsukuba, Japan).
The cells were grown in humidified 5% CO
ium containing 10% fetal bovine serum.
2
at 37 °C in DMEM med-
ifications [15]. A reaction mixture (50
HEPES buffer (pH 7.5), 200 M UDP-galactose, 5 mM MgCl
mM MnCl , 10 L liposomes (C -NBD-GlcCer (0.5 g) and lecithin
(5 g) in water) and 60 g cell protein was incubated at 30 °C for
2 h.
lL) composed of 50 mM
l
6
2
,
5
2
l
l
2.3. Enzyme assay
l
l
1
GlcT-1 activity was measured according to Lipsky and Pagano
13] with slight modifications [14]. To prepare cell lysates as an en-
[
2.4. Inhibition of HA14-1 of intracellular GlcT-1 activity
zyme source, B16 or SK-Mel-28 cells were harvested at semi-con-
fluency, washed with PBS, suspended in water, and lysed by
freezing and thawing. The cell lysate was used as an enzyme
source. For kinetic analysis with respect to UDP-glucose, the cell ly-
sate was dialyzed against 50 mM Tris–HCl (pH 7.5) buffer contain-
Inhibition of intracellular GlcT-1 activity was examined as fol-
lows. B16 cells were plated on a 10-cm dish at a density of
6
1 Â 10 cells/dish (10 ml) and cultured overnight. The next day,
HA14-1 was added to a final concentration of 50 lM. Fifteen min-
ing 25 mM KCl, 25
g/ml leupeptin hemisulfate, and 1
ride. A reaction mixture (50 L) composed of 10 mM Tris–HCl
M UDP-glucose, 10 L of liposomes (C -NBD-Cer
g) and lecithin (5 g) in water) and 30 g of cell protein
l
M amidinophenylmethanesulfonyl fluoride,
utes after the addition, 50
6
lL of liposomes (C -NBD-Cer (0.5 lg)
1
l
lg/ml antipain hydrochlo-
and lecithin (5 g) in water) was added to the medium and incu-
l
l
bated for another 30 min. After the incubation, cells were har-
vested. NBD lipids were extracted and analyzed as described in
the ‘‘enzyme assay’’ section.
(pH 7.5), 500
0.5
l
l
6
(
l
l
l
Fig. 1. Effect of HA14-1 on enzymatic activities. (A) Effect of HA14-1 on GlcT-1 and SM synthase activities. C
synthase activities, respectively, in cell lysates were separated by TLC. Cell lysates (30 g protein) from melanoma cells were used as the enzyme source. The enzyme
reactions were performed in the absence or presence of HA14-1. A(a) B16 melanoma cells. Lane G, C -NBD-GlcCer standard; lane C, C -NBD-Cer standard; lane 1, untreated
control; lane 2, HA14-1 treated cells. A(b) SK-Mel-28 melanoma cells. Lane G, C -NBD-GlcCer standard; lane C, C -NBD-Cer standard; lane 1, untreated control; lane 2, HA14-1
treated cells. (B) Dose dependent inhibition of HA14-1 on mouse GlcT-1 activity in B16 cell lysates. (C) HA14-1 had no effect on LacCer synthase activity. TLC analysis of the
LacCer synthase product catalyzed by cell lysates from B16 cells. Cell lysate (60 g protein) was used as the enzyme source. Lane L, C -NBD-LacCer standard, lane G, C -NBD-
GlcCer standard; lane C, C -NBD-Cer standard; lane 1, untreated control; lane 2, HA14-1 treated cells. LacCer synthase activity is comprised of activities of b-1,4-
galactosyltransferase V and b-1,4-galactosyltransferase VI.
6 6
-NBD-GlcCer and C -NBD-SM generated by GlcT-1 and SM
l
6
6
6
6
l
6
6
6

1
72
S. Niino et al. / Biochemical and Biophysical Research Communications 433 (2013) 170–174
purified GlcT-1 enzyme for assays, it is a membrane protein and
is thus difficult to purify. To eliminate the first alternative possibil-
ity, we used a cell lysate from E. coli that expressed human GlcT-1.
E. coli lysates would not be expected to contain GlcT-1 regulatory
proteins. In addition, any enzymes that might modify HA14-1
would be different from those in animal cells. Human GlcT-1 ex-
pressed in E. coli was inhibited by HA14-1 to the same extent as
in B16 and SK-Mel-28 cell lysates, indicating that HA14-1 had a di-
rect effect on GlcT-1 (Fig. 2). The second alternative possibility was
ruled out by time course experiments, in which no increase of
inhibitory activity was observed even after prolonged incubation
times (data not shown).
3.2. Selectivity of H14-1 Inhibition
HA14-1 might have a broad specificity and inhibit many en-
zymes and proteins other than GlcT-1 and Bcl-2. However, the
inhibitory effect of HA14-1 appeared to be rather selective. In
our GlcT-1 assay system, C -NBD-Cer was supplied as a liposome
6
formulation containing phosphatidylcholine. In this system, SM
synthase in cell lysates transfers phosphocholine from phosphati-
Fig. 2. Effect of HA14-1 on recombinant human GlcT-1 expressed in E. coli. pET-CG-
plasmid carries human GlcT-1 cDNA in a pET3a E. coli expression vector. Human
GlcT-1 can be induced by IPTG addition. The plasmid was transformed in E. coli
1
BL21 (DE3). Lanes: 1, pET-CG-1/BL21 (DE3) with IPTG addition; 2, pET-CG-1/BL21
(
DE3) without IPTG addition; 3, BL21 (DE3); 4, pET-CG-1/BL21 (DE3) with IPTG and
HA14-1 additions; 5, pET-CG-1/BL21 (DE3) without IPTG addition and with HA14-1
addition; 6, BL21 (DE3) with HA14-1; G, C
standard.
6 6
-NBD-GlcCer standard; C, C -NBD-Cer
6 6
dylcholine to C -NBD-Cer, forming C -NBD-SM (Fig. 1A (a) and A
(
b)). As shown in the figures, SM synthase was not inhibited by
addition of HA14-1. We also examined the effect of H14-1 on an-
other glycosyltransferase, LacCer synthase, which transfers galact-
ose from UDP-galactose to GlcCer, forming LacCer. Again, H14-1
had no effect on LacCer synthase activity (Fig. 1C).
2
.5. Lipid analyses
Total lipids were extracted from the cells with CHCl
:1 (vol/vol) and evaporated to dryness. The lipids were then re-
/CH OH, 2:1 (vol/vol), and
3 3
/CH OH,
2
dissolved in a small volume of CHCl
3
3
3.3. Kinetic analyses
chromatographed on silica gel 60 TLC plates (Merck, Darmstadt,
Germany). Glucosylceramide and ceramide were analyzed as de-
scribed previously [16].
The kinetics of inhibition of GlcT-1 by HA14-1 have been inves-
tigated using Lineweaver–Burk plots. The results revealed that
HA14-1 was a competitive and mixed type inhibitor with respect
6
to C -NBD-Cer and UDP-glucose, respectively (Fig. 3). Interestingly,
HA-14 does not possess structural homology with the substrates
UDP-glucose and ceramide.
2
.6. Protein assay
Total protein was assayed using the BCA protein assay reagent
kit according to the manufacturer’s instructions (PIERCE, Rockford,
IL USA).
3.4. Inhibitory effect of HA14-1 on intracellular GlcT-1 activity
B16 cells were next used to examine whether HA14-1 could in-
3
. Results
6
hibit intracellular GlcT-1 activity. C -NBD-Cer, which can penetrate
into cells, was added to the medium as a liposome formulation in
the presence of HA14-1. Thirteen minutes afterwards, the cells
3.1. Inhibitory effect of HA14-1 against GlcT-1
6
were collected and the lipids were analyzed. The amount of C -
A screen of 325 bioactive small-molecules identified HA14-1 as
NBD-GlcCer synthesized in this system was decreased compared
to control experiments (Fig. 4A). The authentic sphingolipid con-
tents were further examined in B16 cells treated with 50 lM
HA14-1. Rounding of the cells began 4 h after addition of HA14-1
and most of the cells died within 24 h. The cells were thus har-
vested 4 h after addition of HA14-1 and their lipid contents were
analyzed. The GlcCer content was not altered (Fig. 4B and C (b))
at this point. In contrast, an increase in ceramide content (Fig. 4B
and C (a)) was observed.
an inhibitor of GlcT-1. HA14-1 is a compound originally developed
as an inhibitor of Bcl-2 and is widely used in basic research of
apoptosis (11). We used a cell lysate from mouse melanoma B16
cells for the GlcT-1 assay because of its high enzymatic activity.
As shown in Fig. 1A (a), GlcT-1 activity was inhibited by HA14-1.
These studies also indicated dose-dependent inhibitory activity
of HA14-1 against GlcT-1, with an IC50 value of 4.5 lM (Fig. 1B).
Since mouse GlcT-1 was used for the assay, we next examined
the effect of HA14-1 against human GlcT-1. A cell lysate from a hu-
man melanoma cell line SK-Mel-28 was used as a source of GlcT-1,
and the effect of HA14-1 was examined. Fig. 1A (b) shows the
inhibitory effect of HA14-1 on human GlcT-1 activity, which was
similar to that found using the mouse enzyme. Since crude cell ly-
sates were used as enzyme sources, there was a possibility that the
inhibitory effect of HA14-1 was not directly due to GlcT-1 itself,
but was the result of indirect effects on proteins that regulated
GlcT-1 activity. For instance, if Bcl-2 binds to GlcT-1 and enhances
GlcT-1 activity, HA14-1 might inhibit GlcT-1 activity by interfering
with the binding of GlcT-1 to Bcl-2. It is also possible that modified
derivatives of HA14-1 generated by enzymes in the cell lysate can
inhibit GlcT-1 activity. Although it would be desirable to use
4. Discussion
Cell death along apoptotic pathways is regulated by various sig-
naling events. HA14-1 is widely used as a Bcl-2 inhibitor in order to
analyze apoptosis. Bcl-2 family proteins are anti-apoptotic or pro-
apoptotic mitochondrial proteins. Among them, Bcl-2 has an anti-
apoptotic effect and inhibition of this protein induces apoptosis in
various cell lines, especially cancer cells that overexpress Bcl-2.
In the present study, we identified HA14-1 as a novel GlcT-1
inhibitor with a new structure. It should be noted that HA14-1
has two asymmetric carbon atoms in its structure, indicating the
presence of four stereoisomers. It is desirable to isolate and test

S. Niino et al. / Biochemical and Biophysical Research Communications 433 (2013) 170–174
173
Fig. 3. Kinetics of inhibition of GlcT-1 by HA14-1. GlcT-1 activities were measured in the presence of 0, 4, or 8
lM HA14-1. (A) Lineweaver–Burk plot of ceramide in the
absence and presence of HA14-1. (B) Lineweaver–Burk plot of UDP-glucose in the absence and presence of HA14-1.
A
B
C -NBD-Cer
6
Cer
C -NBD-GlcCer
6
GlcCer
Origin
C
6
-NBD-SM
1
2 3 4 C G
Origin
C G 1 2
C(a)
C(b)
0
.8
.6
3
2
1
.5
.5
.5
0
0
0
.4
.2
0
0
1
2
1
2
Fig. 4. Effect of HA14-1 on GlcT-1 activity and sphingolipid contents in HA14-1 treated B16 cells. (A) Effect on intracellular GlcT-1 activity. C
substrate, was added to the medium as a liposome formulation. Incorporated C -NBD-Cer was metabolized to C -NBD-GlcCer. The generation of C
in the presence of 50 M HA14-1. Lane C, C -NBD-Cer standard; lane G, C -NBD-GlcCer standard; lane 1, untreated control; lane 2, HA14-1 treated cells. Details are described
in Section 2. (B) Effect of HA14-1 on ceramide and GlcCer contents: Total lipid from 2 Â 10 cells was used for the analysis. The cells were treated with 50
After separation by TLC, lipids were visualized with cupric acetate reagent. Lane C, ceramide standard; lane G, GlcCer standard; lane 1, untreated control; lane 2, HA14-1
treated cells; lane 3 ISP-1 (50 M) treated cells; lane 4, HA14-1 and ISP-1 (50 M) treated cells. ISP-1 is an inhibitor of serine palmitoyltransferase that catalyzes the first step
6
-NBD-Cer, a cell permeable
6
6
6
-NBD-GlcCer was inhibited
l
6
6
6
lM HA14-1 for 4 h.
l
l
in GSL synthesis. Thus, GlcCer and ceramide bands are very weak in lanes 3 and 4. C(a) Ceramide was quantitated after TLC separation. Column 1, HA14-1 treated cells;
column 2, untreated control. Data represent means from four experiments and bars indicate SD. C(b) GlcCer was quantitated after TLC separation. Column 1, HA14-1 treated
cells; column 2, untreated control. Data represent means from four experiments and bars indicate SD.
each isomer, since a distinct isomer might possess the inhibitory
activity on GlcT-1 or/and Bcl-2. However, analyses with HPLC
and NMR revealed that these isomers are unstable and rapidly
interconvert with each other (data not shown).
Although no amino acid sequence homology between GlcT-1
and Bcl-2 family proteins was detected, these two proteins might
have similar three-dimensional structures. Competitive and
mixed-type inhibition of substrates suggested a structural homol-
ogy between the GlcT-1 active site and the Bcl-2 BH3 domain,
where HA14-1 binds. The inhibitory activities also suggested
possible binding of GlcT-1 to Bcl-2, since the BH3 domain is
responsible for dimerization of Bcl-2 family proteins.
6
Inhibition of intracellular GlcT-1 activity estimated by C -NBD-
GlcCer formation indicates that HA14-1 can permeate cells
(Fig. 4A). Unexpectedly, the content of authentic GlcCer was not
decreased in HA14-1 treated cells (Fig. 4B and C (b)). This is pre-
sumably due to the slow degradation rate of the GSLs in B16 cells.
On the other hand, an elevated level of ceramide as a result of
blocking GlcCer synthesis was detected (Fig. 4B and C (a)).
Separovic et al. have demonstrated that the microsome fraction
from HA14-1 (20 lM) treated Jurkat cells had decreased GlcT-1 as
well as SM synthase activity [17]. However, they presumably ob-
served different phenomena from those described herein. In their
experiments, the decreased GlcT-1 activity was presumably not

1
74
S. Niino et al. / Biochemical and Biophysical Research Communications 433 (2013) 170–174
due to a direct effect on the enzyme, because HA14-1 is a reversible
inhibitor and must be diluted to under the effective concentration
during preparation of microsome fractions. In addition, HA14-1 did
not inhibit SM synthase under our experimental conditions.
Our results also indicate that the use of HA14-1 as a Bcl-2 inhib-
itor in studies of apoptosis might produce misleading results, be-
cause HA14-1 could also affect a ceramide-mediated cell death
pathway by inhibiting GlcT-1. From a different point of view, how-
ever, derivatives of HA14-1 could provide unique lead compounds
for cancer treatment, because this compound can induce death in
cancer cells, especially multi-drug resistant cells, via Bcl-2 inacti-
vation and ceramide accumulation.
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Acknowledgments
We thank the Screening Committee of Anticancer Drugs sup-
ported by Grant-in-Aid for Scientific Research on Priority Area
‘
‘Cancer’’ from The Ministry of Education, Culture, Sports, Science
and Technology, Japan for providing us SCADS inhibitor kit.
This study was supported by a Grant from the Strategic Re-
search Foundation Grant-aided Project for Private Universities
from the Ministry of Education, Culture, Sport, Science, and Tech-
nology, Japan, 2010-2012 (S1001030).
[
[
[
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