Identification of small molecule inhibitors of telomerase activity through transcriptional regulation of hTERT and calcium induction pathway in human lung adenocarcinoma A549 cells

Article abstract of DOI:10.1016/j.bmc.2010.08.021

High telomerase activity (TA) is detected in most cancer cells; and thus, TA inhibition by drug or dietary food components is a new strategy for cancer prevention. In this report, we examined the effects of fourteen natural or synthetic compounds on TA in human lung adenocarcinoma A549 cells. The results demonstrated that some of the tested compounds inhibited TA, being 2′-hydroxy-2,3,4′,6′-tetramethoxychalcone (HTMC) was the most potent among tested. In A549 cells, HTMC also inhibited the cell proliferation, decreased the expression of human telomerase reverse transcriptase (hTERT) and sequentially reduced the hTERT promoter. In soft agar assay HTMC treatment reduced the colony formation of A549 cells. Cellular fractionation and immunofluorescence assay demonstrated that there was no translocation of hTERT from nuclei to cytoplasm. Further studies revealed that the release of Ca ²⁺ was the underlying mechanism of suppressed TA and hTERT transcription in A549 cells exposed to HTMC. These in vitro data support the development of HTMC as a therapeutic agent for cancer complications.

Full text of DOI:10.1016/j.bmc.2010.08.021

Bioorganic & Medicinal Chemistry 18 (2010) 6987–6994
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Identification of small molecule inhibitors of telomerase activity through
transcriptional regulation of hTERT and calcium induction pathway
in human lung adenocarcinoma A549 cells
Yerra Koteswara Rao , Te-Yu Kao , Ming-Fang Wu c,d, Jiunn-Liang Ko c,e,*, Yew-Min Tzeng
a
b
a,*
a
Institute of Biochemical Sciences and Technology, Chaoyang University of Technology, Wufeng, Taiwan, ROC
Institute of Medical and Molecular Toxicology, Chung Shan Medical University, Taichung, Taiwan, ROC
Department of Medical Oncology and Chest Medicine, Chung Shan Medical University Hospital, Taichung, Taiwan, ROC
School of Medicine, Chung Shan Medical University, Taichung, Taiwan, ROC
b
c
d
e
Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 July 2010
Revised 9 August 2010
Accepted 10 August 2010
Available online 13 August 2010
High telomerase activity (TA) is detected in most cancer cells; and thus, TA inhibition by drug or dietary
food components is a new strategy for cancer prevention. In this report, we examined the effects of
fourteen natural or synthetic compounds on TA in human lung adenocarcinoma A549 cells. The results
0
0
0
demonstrated that some of the tested compounds inhibited TA, being 2 -hydroxy-2,3,4 ,6 -tetramethoxy-
chalcone (HTMC) was the most potent among tested. In A549 cells, HTMC also inhibited the cell prolifer-
ation, decreased the expression of human telomerase reverse transcriptase (hTERT) and sequentially
reduced the hTERT promoter. In soft agar assay HTMC treatment reduced the colony formation of A549
cells. Cellular fractionation and immunofluorescence assay demonstrated that there was no translocation
of hTERT from nuclei to cytoplasm. Further studies revealed that the release of Ca2 was the underlying
mechanism of suppressed TA and hTERT transcription in A549 cells exposed to HTMC. These in vitro data
support the development of HTMC as a therapeutic agent for cancer complications.
Keywords:
Small molecule
Telomerase activity
hTERT
hTERT promoter
Anchorage-independent growth
Calcium release
+
Ó 2010 Elsevier Ltd. All rights reserved.
1
. Introduction
Telomerase as an enzyme is responsible for the renewal of the
these, we are primarily interested in identification of compound
that induced down regulation of TA via suppression of hTERT
expression, because the hTERT is a core telomerase protein and
1
2
chromosomal ends, the so-called telomeres. Telomeres are essen-
tial units that prevent the loss of genetic information. In normal so-
matic cells, which show little or no telomerase activity (TA) to
synthesize new telomeres.1 In contrast, most cancer cells have
mechanisms that compensate for telomere shortening through
the activation of telomerase, allowing them to stably maintain their
its expression limited to germinal and cancer cells. It is well known
that the expression of dominant-negative hTERT or its siRNA re-
duces the lifespan of human cancer cells by completely inactivating
2
telomerase. Therefore, it is thought that suppression of hTERT
expression is an ideal strategy for the development of anticancer
1
,3
chemotherapeutics.
2
telomeres and grow indefinitely. These observations suggest that
The inhibition of TA has been approached through different strat-
egies, ranging from anti-sense-based inhibitors to random screening
telomerase reactivation is a rate-limiting step in cellular immortal-
ity and carcinogenesis, and telomerase repression can act as a tu-
mor-suppressive mechanism.2 The key advantages of targeting
telomerase in comparison with most other cancer targets are its rel-
ative universality, criticality and specificity for cancer cells, includ-
ing the putative cancer stem cell.2 Telomere length in human is
primarily controlled by three major components including human
1
of synthetic and natural products. Among the latter, several com-
4
5
pounds have been reported include rubromycins, alterperylenol,
6
7–9
epigallocatechin gallate, anthraquinone derivatives,
plex ligands. In our previous studies, we found that loss of
telomerase activity can be potentially favorable prognostic marker
Weare currentlyundertakingthetherapeu-
tic assessment of medicinal mushroom Antrodia camphorata.
G-quadru-
1
0–12
1
3–16
inlung carcinomas.
17–20
telomerase RNA component (hTR), telomerase-associated protein
1
In
2
(TP1), and human telomerase reverse transcriptase (hTERT). Of
parallel, we sought to identify potent TA inhibitor compounds to
expand the scope of bioactive agents and to gain insight into their
anticancer mechanism of action. To this end, here, we report the
screeningof 14 naturalor syntheticcompoundson TA inhuman lung
adenocarcinoma cell line A549 cells.
*
Corresponding authors. Tel.: +886 4 24730022x11694; fax: +886 4 24751101
(
J.-L.K.); tel.: +886 4 23323000x4471; fax: +886 4 23395870 (Y.-M.T.).
E-mail addresses: jlko@csmu.edu.tw (J.-L. Ko), ymtzeng@cyut.edu.tw (Y.-M.
Tzeng).
0
968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.08.021

6
988
Y. K. Rao et al. / Bioorg. Med. Chem. 18 (2010) 6987–6994
2
2
2
. Results
ative normal cells. To determine whether the decreased levels of
hTERT in A549 cells was the result of decreased hTERT promoter
.1. Inhibition of telomerase activity (TA) in A549 cells
In an effort to identify potential candidate to inhibit TA, we have
activity, we dissected the full-length hTERT fragment (ꢀ548 to
+
50) into five regions p548 (ꢀ548 to +50), p212 (ꢀ212 to +50),
p196 (ꢀ196 to +50), p177 (ꢀ177 to +50) and p95 (ꢀ95 to +50)
selected 14 natural or synthetic compounds (Fig. 1), based on their
growth inhibitory potential against tumor cell proliferation and
(Fig. 4A). After transfection, A549 cells were treated with 25 lM
HTMC for 24 h and luciferase reporter activity was determined
by transient transfection assay. A plasmid expressing the bacterial
b-galactosidase gene was co-transfected in each experiment to
serve as internal control of transfection efficiency. The luciferase
activity in HTMC treated cells was clearly down-regulated in the
constructs p548, p212, p196, p177, and p95 in the range approxi-
mately from 25% to 70% as compared with untreated cells (Fig. 4B).
inflammatory mediators production in different type of cell lines
as we reported previously.2
1–26
The effects of these compounds
(Fig. 1) on TA were examined in A549 cells with a random concentra-
tion of 25
l
M, using modified telomeric repeat amplification proto-
0
col (TRAP) assay. We found that among tested, 2 -hydroxychalcone
derivatives, 2 -hydroxy-2,3,4 ,6 -tetramethoxychalcone (HTMC)
0
0
0
followed by compound 6 potentially inhibited TA with respect to
0
0
0
control (Fig. 2). Among the tested 3 ,4 ,5 -trimethoxychalcone
derivatives, compounds 07–01, 07–02, 07–03, and isoflavonoid,
isobonducellin (37) have moderate effect on TA (Fig. 2). In contrast,
the flavonol glycosides, kaempferitrin (20) and kaempferol 3-O-b-
2.4. Effect of HTMC on the anchorage-independence of A549
cells
We next examined the effect of HTMC on the colony formation
efficiency of A549 cells using anchorage-independence cell growth
assay. A549 cells are highly tumorigenic and readily form colonies
in soft agar medium. In this report, the results of soft agar assay
demonstrated that colonies formation efficiency of HTMC treated
A549 cells was drastically reduced as compared with untreated
cells. Additionally, the colonies observed in the HTMC-treated cul-
tures were smaller and more densely packed than the untreated
controls (Fig. 5A). The number of colonies formed in agar was re-
D
-glucopyranosyl-(1?4)-
a-L-rhamnopyranosyl-7-O-a-L-rhamno-
pyranoside (21); flavanones, 5,7-dimethoxyflavonone (31) and
7
4
-O-methylglabranin (32); lignan, phyllanthin (38); phenolic acid,
-methoxygallic acid (40); and 3 ,4 ,5 -trimethoxychalcone deriva-
0
0
0
tive 07–04 have no effect on TA at the tested concentration 25 lM
(Fig. 2). Furthermore, the potent compound HTMC also inhibited
the A549 cells TA in a dose (6.25, 12.5, 25.0 and 50.0 lM) and time
(
6 h, 12 h, and 24 h) dependent manner (see Supplementary data).
Among the tested, HTMC was almost completely inhibited the TA
of A549 cells at concentration of 25 M (Fig. 2). Subsequently, HTMC
duced by 23% when A549 cells were stimulated with 6.25
lM
l
HTMC. Stimulation of A549 cells with 12.5 and 25.0 M HTMC de-
l
was used to examine the TA associated cellular and molecular
events in A549 cells.
creased the number of colonies by 96% and 99%, respectively
(Fig. 5B) as compared with control cells. In general, increased the
concentration of HTMC pretreatment concomitantly reduced the
colony formation efficiency of A549 cells.
We next examined the HTMC effect on A549 cell proliferation
by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphe-
nyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. The results demon-
strated that HTMC treated for 24 h, A549 cells proliferation was
decreased in a concentration-dependent manner (data not shown).
2.5. HTMC suppressed TA and hTERT expression in A549 cells
through calcium induction
Particularly, treated with 50
cells proliferation by 59%.
lM HTMC for 24 h reduced the A549
Previous studies have shown that accumulation of wild-type
2+
proteins in endoplasmic reticulum leads to release of Ca from
2
+
2,14
organelles that can be blocked by Ca chelators.
To evaluate
2
.2. Effect of HTMC on the expression of hTERT in A549 cells
Although active telomerase requires the co-expression of hTR
the signaling pathways involved in HTMC telomerase regulation,
the intracellular calcium levels were analyzed in HTMC treated
A549 cells. As shown in Figure 6A, HTMC treated A549 cells re-
and hTERT components, the expression of hTERT correlates closely
with TA in most cancers.2 Therefore, we attempted to elucidate
whether the above observed HTMC inhibitory effect on TA was
due to the repression of hTERT expression in A549 cells. We used
RT-PCR, real-time RT-PCR (Taqman assay) and Western blot analy-
sis and examined the changes in hTERT expression following A549
cells treated with HTMC for 6 h. As indicated in Figure 3A, in HTMC
treated A549 cells the mRNA expression levels of hTERT decreased
in a dose-dependent manner. In contrast, hTR and GADD153 mRNA
expressions were not altered, demonstrated the specificity for
hTERT mRNA (Fig. 3A). To quantify the degree of the decreased
hTERT mRNA, real-time PCR was performed using total RNA from
A549 cells treated with vehicle or HTMC in the concentration range
2
+
leased the Ca in a concentration-dependent manner, which was
detected by calcium-sensitive indicator Fluo-3 AM. This was evi-
denced by increased green staining in A549 cells after treatment
with HTMC for 24 h (Fig. 6A). These results suggested that HTMC
2
+
induced the release of Ca in A549 cells. To examine the specific
2
+
role of HTMC in the released Ca , A549 cells were pretreated with
calcium chelator BAPTA-AM for 1 h and then with HTMC for 24 h.
Analysis of calcium fluorescence demonstrated that the influx of
calcium release was decreased, indicated that HTMC significantly
2
+
contributed in the released Ca in A549 cells (Fig. 6A). In addition,
2
+
the specific role of Ca released by HTMC on the regulation of TA
was also examined. For this, A549 cells were treated with 25
HTMC, or pretreated for 1 h with various cancer cells growth inhib-
itors such as MEK inhibitor U0126 (10 M), or ERK inhibitor
PD98059 (20 M), or MAPK inhibitor SB203580 (50 M), or intra-
cellular calcium chelator BAPTA-AM (20 M), or 26S proteasome
inhibitor MG132 (20 M), or nuclear export inhibitor leptomycin
B (20 nM), or PI3 K inhibitor LY294002 (50 M) and then treated
with 25 M HTMC for 24 h and assayed for TA using TRAP method.
Similar to the previous results, HTMC inhibited the TA; however,
the concomitant addition of 20 M BAPTA-AM completely abol-
lM
from 6.25
decreased in a dose-dependent manner and reached to 92% after
M HTMC treatment for 6 h. Furthermore, the expression levels
lM to 50 lM. As shown in Figure 3B, hTERT mRNA was
l
l
l
50 l
l
of hTERT were also shown to undergo a dose-dependent decrease
in whole cell extraction fraction following treatment with HTMC
as confirmed by Western blot analysis (Fig. 3C).
l
l
l
2
.3. Effect of HTMC on the hTERT promoter activity
l
ished the ability of HTMC to inhibit TA (Fig. 6B). Using TRAP and
RT-PCR assays, we found that BAPTA-AM completely abolished
the ability of HTMC to repress TA and hTERT mRNA levels in
The hTERT promoter activity has been closely associated with
TA and hTERT expression in cancer cells but not in telomerase-neg-

Y. K. Rao et al. / Bioorg. Med. Chem. 18 (2010) 6987–6994
6989
Figure 1. Chemical structures of natural or synthetic compounds tested on telomerase activity in human lung cancer cell line A549 cells.
A549 cells (Fig. 6C and D). In the absence of HTMC treatment, incu-
bation of A549 cells with BAPTA-AM alone did not significantly
change the levels of TA and hTERT as compared with controls
to cytoplasm (Fig. 6E). However, A549 cells pretreated for 1 h with
intracellular calcium chelator BAPTA-AM and then treated with
25 M HTMC for 24 h, partly restored the expression of hTERT in
l
(
Fig. 6C and D). Furthermore, immunofluorescence staining of
M HTMC treated A549 cells demonstrated a decreased expres-
sion of hTERT, and there was no translocation of hTERT from nuclei
the nuclei (Fig. 6E). Taken together, these results suggested that
HTMC suppressed telomerase activity and hTERT expression by
inducing calcium release in A549 cells.
25 l

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Y. K. Rao et al. / Bioorg. Med. Chem. 18 (2010) 6987–6994
Figure 2. Effect of various natural and synthetic compounds on the level of telomerase activity in human lung A549 cells. After (24 h) plating, cells were exposed to 25 lM of
each tested compound for 24 h. Cell pellets were collected and subjected to TRAP assay. The telomerase activity in untreated A549 cells was as control (C). N, negative control
used lysis buffer only.
0
0
0
Figure 3. Effect of 2 -hydroxy-2,3,4 ,6 -tetramethoxychalcone (HTMC) on the expression of hTERT, hTR and GADD153 mRNA and protein level in A549 cells. (A) After 6 h of
incubation with HTMC, total RNA was isolated, and RT-PCR was performed using indicated primers, the amplified products were run in 1.5% agarose gel and visualized by
ethidium bromide staining. b-Actin was used as an house-keeping control gene. (B) A549 cells were treated with indicated concentrations of HTMC for 6 h, relative hTERT
*
mRNA level was determined by real-time RT-PCR (Taqman Assay) and normalized to GAPDH mRNA. Representative data were from three independent experiments and
indicates p <0.05 as compared with untreated cells. (C) Western blot analysis of hTERT subunit A549 cells treated with indicated concentrations of HTMC for 24 h.
3
. Discussion
Other putative inhibitors derived from natural sources are curcu-
min, epigallocatechin gallate (EGCG), retinoic acids and a herbal
complex based on plant extracts from Hoelen, Angelicae, Scutellar-
The unique role of telomerase in particular in cancer cells intro-
1
duced a new area of research directed to the goal of telomerase
inhibition as smart approach for cancer therapy. Numerous stud-
riae, Glycyrrhizae radix and other herbs. Previous studies indicate
1
that EGCG reduce telomerase activity by decrease in hTERT pro-
moter methylation and increase in hTERT repressor E2F-1 binding
ies using various active ingredients were performed to inhibit TA.
Beside the G-quadruplex ligands,1
0–12
small synthetic and nucleic
27
at the promoter. Our previous report also demonstrated that cur-
acids based telomerase inhibitors, there is a multiplicity of com-
pounds that can cause a decrease in TA. Differentiating agents like
cumin inhibit telomerase activity and its transcription factors
including NFkB, AP1 and Sp1. Multiple mechanisms and several
1
16
all-trans retinoic acids, adriamycin and doxorubicin reduced TA
and hTERT mRNA expression. Another family of telomerase inhib-
transcription factors regulate telomerase ability at DNA and pro-
tein level. In this report, our efforts attempted to identify potential
compound that can modulates TA by up-regulating calcium con-
centration and repress hTERT expression at transcriptional level.
1
itors is the isothiazolone derivatives which could reduce the en-
1
zyme activity by interfering with a cysteine in its active center.

Y. K. Rao et al. / Bioorg. Med. Chem. 18 (2010) 6987–6994
6991
0
0
0
Figure 4. Effect of 2 -hydroxy-2,3,4 ,6 -tetramethoxychalcone (HTMC) on hTERT promoter activity. A549 cells were transfected with luciferase reporter plasmid containing
hTERT promoter deletion mutants p548 (ꢀ548 to +50), p212 (ꢀ212 to +50), p196 (ꢀ196 to +50), and p177 (ꢀ177 to +50), and were left untreated or treated with HTMC for
2
4 h followed by measurement of hTERT activity by luciferase reporter assay.
clear. To the best of our knowledge, this was the first report to eval-
uate the compounds tested here for their TA inhibitory assay.
hTERT is a structurally and functionally important component of
telomerase, which exerts its function of elongating telomere end re-
peats in the nuclei. Telomerase inhibition is sequence-dependent
and the primary mode of regulation of hTERT is proposed to be tran-
2
9
scriptional. However, recent studies indicate that another possi-
ble mechanism is the posttranscriptional regulation which
involved the translocation of hTERT from nuclei into cytoplasm.29
As shown in Figure 6E, immunofluorescence staining of HTMC trea-
ted A549 cells demonstrated that the expression of hTERT protein
decreased. In addition, although hTERT is detected in both nuclei
and cytoplasm, the majority of the signal is localized in the nuclei,
as the production of hTERT takes place in cytoplasm, therefore a
weaker hTERT staining can also be detected. These results indicated
that HTMC regulation of hTERT was transcriptional in A549 cells. In
addition, the ꢀ548 to +50 bp region proximal to the transcription
initiation site of the hTERT promoter is responsible for most of its
transcriptional activity. This region has several binding sites in-
clude two typical E-boxes and several GC-boxes for the transcrip-
2
tion factors c-myc and Sp1, respectively. The core promoter
p548, which is necessary for hTERT expression also contains Sp1/
2
Sp3 binding sites. Thus, investigation of mechanisms that modu-
late hTERT expression in tumor and normal cells has become an
area of intense interest in the study of human cancer.30 In this re-
port, the data from the luciferase assays showed that HTMC de-
creased the hTERT promoter activity sequentially (Fig. 4). These
results further indicated that HTMC treatment in A549 cells in-
duced the down regulation of TA with suppression of hTERT expres-
sion at transcriptional levels. As the exact hTERT promoter-binding
site of HTMC unknown, further studies were needed to elucidate
the effects of HTMC on the hTERT promoter activity.
Figure 5. Effect of HTMC on anchorage-independent growth. (A) A549 cells were
treated with different concentrations of 2 -hydroxy-2,3,4 ,6 -tetramethoxychalcone
in 0.35% agarose containing 10% FBS-DMEM over 0.5% agarose containing 10% FBS-
0
0
0
DMEM, cell colonies were counted after 21-day incubation at 37 °C in 5% CO
microscopy. (B) Size >30 M were counted as a colony. Each bar represents
mean ± SD of triplicate experiments.
2
under
l
We found that compound HTMC was the most potent among
tested, and caused a dose-dependent decrease in TA and hTERT
mRNA expression without a marked alteration of hTR or GADD153
as supported by both RT-PCR and Western blot experiments (Figs.
Anchorage independence is an important feature that allows tu-
mor cells to survive under certain inappropriate host conditions,
3
1
such as during invasion and metastasis. In this report, A549 cells
lost their ability to form colonies in the presence of HTMC for three
weeks treatment (Fig. 5). One possible explanation to the observed
effects was that HTMC-induced telomerase inhibition may cause a
chronic DNA damage response of an uncapped telomere or a few
chromosome ends thus reduced the colony formation efficiency
2
and 3). It was interesting to note that HTMC, a natural chalcone
28
from a medicinal plant Caesalpinia pulcherrima, has significant
cytotoxic activity against Jurkat (human lymphocytic) and U937
(
human monocytic) cells,21 however, the molecular mechanisms
3
1
of its anti-proliferative action on malignant cell growth are not
of the A549 cells.

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Y. K. Rao et al. / Bioorg. Med. Chem. 18 (2010) 6987–6994
0
0
0
Figure 6. Involvement of intracellular calcium ion in 2 -hydroxy-2,3,4 ,6 -tetramethoxychalcone (HTMC)-induced inhibition of telomerase activity in A549 cells. (A) A549
M) for 24 h, and were treated with Fluo-3 AM for Ca2 staining. (B) 1 h prior to incubation with HTMC, A549 cells
+
cells were treated with HTMC (0, 6.25, 12.5, 25.0 and 50.0
were pretreated with U0126 (10 M), SB 203580 (50 M), BAPTA-AM (20
BAPTA-AM (20 and 40 M) was added to A549 cells 1 h prior to incubation with HTMC, after 24 h cells were harvested and subjected to TRAP assay. (D) Effect of BAPTA-AM
on HTMC-induced suppression of hTERT mRNA level in A549 cells. BAPTA-AM (20 M) was added to cells 1 h prior to incubation with HTMC and after 24 h total RNA was
l
l
l
l
M), MG132 (20 M), Leptomycin B (20 nM), LY294002 (50 M). (C) Intracellular calcium chelator
l
l
l
l
isolated and RT-PCR was performed using indicated primers. The amplified products were run in 1.5% agarose gel and visualized by ethidium bromide staining. b-Actin was
used as an house-keeping control gene. (E) Effect of HTMC on the distribution of hTERT protein in A549 cells. Immunofluorescence staining for hTERT using specific anti-
hTERT antibodies and TRITC goat anti-rabbit IgG (H+L) conjugate secondary antibodies in culture A549 cells after treatment with HTMC (25 lM) for 24 h.
Calcium is an intracellular second messenger, which regulates a
variety of biological processes.32 In most cases calcium does not act
directly, but uses calcium-binding proteins as mediators of its sig-
nals then restored to basal level once signal is transmitted. Calcium
binds to these calcium-binding proteins and induces conforma-
tional changes, thus exposing initially inaccessible protein binding

Y. K. Rao et al. / Bioorg. Med. Chem. 18 (2010) 6987–6994
6993
sites and allowing their interaction with specific target proteins.32
Previously Rosenberger et al. (2007) suggest that calcium play a
functional role in the inhibition of TA in epidermal keratinocytes.
and substrate antibodies were purchased from Santa Cruz Biotech-
nology (Santa Cruz, CA). Antibody against b-actin was obtained
from Sigma. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymeth-
oxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) was purchased
from Promega (Madison, WI). Horseradish peroxidase-conjugated
goat anti-rabbit secondary antibody (Cell Signaling, MA) and en-
hanced chemiluminescence reagent (ECL) and Hyperfilm-ECL were
purchased from Amersham (Little Chalfont). All other reagents
used were of the analytical grade and obtained from Sigma.
3
3
These authors also reported that calcium-mediated inhibition of
telomerase activity by S100A8/S100A9 complex, in which calcium
is bound to S100A9 and subsequently releases S100A8 from the
complex, immediately S100A8 binds to telomerase, thus reducing
3
3
telomerase activity. In our study TRAP assay indicated that telo-
merase activity is reduced in a dose-dependent and time-depen-
dent manner (see Supplementary data Fig. 1). TA was
significantly reduced at 25
l
M HTMC, further analysis with this
5.2. Cell line and culture
concentration at different treatment periods we found that telome-
rase activity declined with longer treatment periods (see Supple-
mentary data Fig. 1), this suggested once HTMC was taken in by
cells, its telomerase inhibiting effect was a continuous process, be-
sides its ability of reducing hTERT expression in RNA and protein
level (as shown in Fig. 3), it may also caused a direct inhibitory ef-
fect by up-regulating calcium concentration. Therefore an in-
creased calcium concentration can still be observed after 24 h
treatment of HTMC. On the other hand, as shown in Figure 6A,
treatment with HTMC increased calcium concentration in the nu-
clei, this result suggested that calcium might play a role in the
mechanism of telomerase inhibition by HTMC. Previous report
indicate that a direct inhibition of telomerase activity by adding
calcium chloride in the cell-free extract by TRAP assay,3 and sup-
ported our finding that focal calcium stimulation in nuclei by
HTMC may inhibited TA via similar manner. BAPTA-AM is a cell-
membrane permeable calcium chelator, which widely used as an
intracellular calcium sponge. As shown in Figure 6A, calcium con-
centration was increased within or around the nuclei area, whereas
in A549 cells pretreated with BAPTA-AM, calcium stimulations
were seen in condensed, scattered dots outside the nuclei. We
hypothesize that calcium induced by HTMC was chelated by BAP-
TA-AM which resulted restored the TA in cells treated with HTMC.
These results further supported by the data from Figure 6B and C,
in which, along with various other inhibitors, TA was only restored
in cells treated with BAPTA-AM, and supported our hypothesis that
calcium participated in the mechanism of TA inhibition by HTMC.
Taken together, these results demonstrated that HTMC inhibited
telomerase activity through transcriptional regulation of hTERT
Human lung adenocarcinoma cell line A549 was obtained from
the American Type Culture Collection (Manassas, VA). Cells were
maintained at 37 °C in a 5% CO -humidified atmosphere on
2
Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Rockville,
MD)and Basal medium Eagle (BME) (Sigma, Saint Louis, MI) medium
containing 10% fetal bovine serum (FBS) and 100 ng/mL each of pen-
icillin and streptomycin (Life Technologies, Inc., Rockville, MD).
5
.3. Telomere repeat amplification protocol (TRAP) assay
The TRAP assay was performed as we described previously.13
.4. Cell proliferation assay
3
5
7
Cells were seeded at 1 ꢁ 10 cells/mL and then treated with the
indicated concentrations of HTMC. After 24 h incubation, the cell
number and viability were determined by MTS assay.13
5
.5. Isolation of RNA and RT-PCR
The detailed steps of RNA isolation and RT-PCR were performed
13
by the method as we described previously. The primer sequences
and PCR conditions for hTERT, hTR, GADD153 and b-actin were also
13
performed as we described previously. The products were visual-
ized via electrophoresis on 1.5% agarose gel and stained with ethi-
dium bromide. We confirmed the quality of cellular mRNA
according to the intensity of b-actin.
2
+
and induction of Ca -dependent pathway.
5
.6. Reporter gene assay
4
. Conclusions
The hTERT promoter deletion mutants p548 (ꢀ548 to +50),
p212 (ꢀ212 to +50), p196 (ꢀ196 to +50), and p177 (ꢀ177 to
In summary, this report identified a potent telomerase inhibitor
+
50), cloned upstream of the firefly luciferase reporter in the
HTMC. In addition, HTMC decreased the expression of hTERT, and
sequentially reduced the hTERT promoter in A549 cells. HTMC
treatment also reduced the colony formation efficiency of A549
cells. Additionally, the results of this study demonstrated that the
release of Ca was the underlying mechanism of suppressed telo-
merase activity and hTERT transcription. Therefore, the potential of
HTMC to inhibit telomerase activity and its associated molecular
events, and the chemical nature of this low-molecular-mass com-
pound that translates to lower cost of synthesis compared to nucle-
otide-based drugs suggested that it has potential to develop new
therapeutic drug for cancer therapy.
pGL3-Basic vector (Promega Corp.) by following the protocol de-
1
3
4
scribed previously. For luciferase assay, cells (7.5 ꢁ 10 ) were
seeded onto 24-well plates, cultured overnight, and transfected
with the plasmids described above (1
lg/well) with DEAE-dextran
2
+
(
Amersham-Pharmacia plc, Little Chalfont, Bucks, UK). After 24 h
incubation, the medium was carefully removed and fresh medium
containing various concentrations of tested compound was added
to the wells. The cells were treated continuously with HTMC for
2
4 h. Cells were collected and transcriptional activity was assayed
with Luciferase Assay System (Promega). A plasmid expressing the
bacterial b-galactosidase gene was co-transfected in each experi-
ment to serve as internal control of transfection efficiency.
5
5
. Experimental
5
.7. Western blot analysis
.1. Reagents and materials
Anti-hTERT (ab32020, abcam) and anti-b-actin (AC-40, Sigma)
Specialized chemicals used were obtained from Sigma Chemical
were used for the detection of hTERT, and b-actin, respectively.
Company (Saint Louis, MO). The preparation and structure identi-
The complete protocol for Western blot analysis has been de-
scribed in our previous report. Blots were developed with an en-
hanced luminol chemiluminescence (ECL) reagent (NEN, Boston).
1
3
fication details of tested compounds are reported in our previous
communications.2
1–26
Antibodies against hTERT, hTR, GADD153,

6
994
Y. K. Rao et al. / Bioorg. Med. Chem. 18 (2010) 6987–6994
5
.8. Soft agar colony formation assay
Anchorage-independent growth assay of A549 cells was per-
Acknowledgement
This study was supported by National Science Council of Taiwan
(NSC 98-2811-M-324-001 and NSC 99-2811-M-324-003).
15
formed by the method as we described previously.
5
.9. Fluorescence immunocytochemistry and confocal
microscopy
Supplementary data
4
A549 cells were seeded (1 ꢁ 10 cells per chamber) onto an
eight-chamber slide (Nunc 177402, Naperville, IL). Cells were ren-
dered quiescent, followed by treatment with HTMC for the indi-
cated concentration. Cells were fixed in 4% paraformaldehyde for
2
2
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.08.021.
0 min and permeabilized with 0.3% Triton X-100 in PBS for
0 min. Washed cells was incubated in 1% BSA in PBS for 1 h. For
co-immunostaining, cells were first incubated with an antibody
against hTERT (rabbit, 1:250; 1 h, room temperature; Rockland,
Gilbertsville, PA), and tetramethylrhodamine isothiocyanate goat
anti-rabbit IgG (H+L) conjugate was used as a secondary antibody
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6126.
multiple-comparison test. The symbol ( ) indicates p <0.05 when
compared with untreated controls.