Chalcone HTMC causes in vitro selective cytotoxicity, cell-cycle G 1 phase arrest through p53-dependent pathway in human lung adenocarcinoma A549 cells, and in vivo tumor growth suppression

Article abstract of DOI:10.1016/j.bmcl.2010.09.056

The present Letter identified 2′-hydroxy-2,3,4′,6′- tetramethoxychalcone (HTMC) as a potent in vitro cytotoxic agent with selective activity against cell lines derived from human lung cancer. In A549 lung adenocarcinoma cells, HTMC caused G1 phase cell-cycle arrest. HTMC treatment also led to an inhibition of cell-cycle regulatory proteins phosphorylation of cdc2 (Tyr¹⁵ and Tyr¹⁶¹) and Rb (Ser⁷⁹⁵ and Ser^807/811), which was accompanied by the accumulation of tumor suppresser genes p53 and p21. In addition, in vivo data demonstrated that HTMC act as a tumor growth suppressing agent.

Full text of DOI:10.1016/j.bmcl.2010.09.056

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6508–6512
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Chalcone HTMC causes in vitro selective cytotoxicity, cell-cycle G1 phase
arrest through p53-dependent pathway in human lung adenocarcinoma
A549 cells, and in vivo tumor growth suppression
Yerra Koteswara Rao a, Te-Yu Kao b, Jiunn-Liang Ko c,d, , Yew-Min Tzeng a,
⇑
⇑
a Institute of Biochemical Sciences and Technology, Chaoyang University of Technology, Wufeng, Taiwan, ROC
b Institute of Medical and Molecular Toxicology, Chung Shan Medical University, Taichung, Taiwan, ROC
c Department of Medical Oncology and Chest Medicine, Chung Shan Medical University Hospital, Taichung, Taiwan, ROC
d 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
The present Letter identified 20-hydroxy-2,3,40,60-tetramethoxychalcone (HTMC) as a potent in vitro cyto-
toxic agent with selective activity against cell lines derived from human lung cancer. In A549 lung ade-
nocarcinoma cells, HTMC caused G1 phase cell-cycle arrest. HTMC treatment also led to an inhibition of
cell-cycle regulatory proteins phosphorylation of cdc2 (Tyr15 and Tyr161) and Rb (Ser795 and Ser807/811),
which was accompanied by the accumulation of tumor suppresser genes p53 and p21. In addition,
in vivo data demonstrated that HTMC act as a tumor growth suppressing agent.
Article history:
Received 9 July 2010
Revised 25 August 2010
Accepted 10 September 2010
Available online 17 September 2010
Keywords:
Chalcones
Ó 2010 Elsevier Ltd. All rights reserved.
HTMC
Lung adenocarcinoma cells
Selective activity
p53-dependent pathway
In vivo tumor suppression
Lung cancer is the most common cause of cancer-related death
in men and women worldwide including Taiwan.1 In addition, the
incidence of adenocarcinoma, a subtype of non-small cell lung can-
cer is one of the most lethal cancers because of its high incidence
and mortality.2 The survival rate of patients with lung adenocarci-
noma is poor even when treatment is given in the early stage of the
disease, because most patients already have disease that has
metastasized.2 Novel chemotherapeutic agents have been devel-
oped against lung cancer, include camptothecin (CPT), however,
it is not effective against lung adenocarcinoma due to tolerance
of the cancer cells toward this drug.3 Several CPT derivatives
including irinotecan and topotecan have also been introduced to
lung cancer therapy.3 However, the response rates to these drugs
remain low, and the overall survival rate has not been much im-
proved. Furthermore, chemotherapy consisting of CPT derivatives
usually encounters serious side effects, which hampers the contin-
uous administration of these drugs to treat the disease.3 Therefore,
the development of novel chemotherapeutics with selective
activity and alternative mode of action to treat lung adenocarci-
noma is desired.
Chalcones are precursors of flavonoids and isoflavonoids, which
are abundant in edible plants. The chemical structure of chalcones
(1,3-diphenyl-2-propen-1-ones) consists of two aromatic rings
joined by a three-carbon
a,b-unsaturated carbonyl system. Previ-
ous studies have indicated that flavonoids, chalcones, and their
derivatives demonstrate anticancer activity in various tumor cells.4
However, the exact mechanisms by which chalcone compounds
exert their cytotoxic effects in cancer cells remains unclear.
Through screening of chalcone compounds that inhibit cancer cell
proliferation, we previously reported that 20-hydroxy-2,3,40,60-tet-
ramethoxychalcone (HTMC, Fig. 1), a natural chalcone from a
medicinal plant Caesalpinia pulcherrima,5 has significant cytotoxic
⇑
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).
Figure 1. Chemical structure of 20-hydroxy-2,3,40,60-tetramethoxychalcone (HTMC).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.056

Y. K. Rao et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6508–6512
6509
activity against Jurkat (human lymphocytic) and U937 (human
monocytic) cells,6 however, the molecular mechanisms of its
anti-proliferative action on malignant cell growth are not clear.
On the other hand, a polymethoxyphenyl moiety is frequently
met in the number of naturally occurring anticancer agents such
as colchicine and combretastatin A.7 In an effort to contribute to
develop potential anticancer agents, and as part of a subsequent
study to further explore the mechanism of HTMC anticancer activ-
ity,6 we disclose here its effects on human lung adenocarcinoma
cells.
HTMC (Fig. 1), was prepared by the Claisen–Schmidt condensa-
tion of 2-hydroxy-4,6-dimethoxyacetophenone with 2,3,-dime-
thoxybenzaldehyde in the presence of KOH (50%) following the
procedure reported previously.6,8 In this Letter, in an initial ap-
proach to analyze the effects of HTMC on human lung cancer cell
lines, we performed MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carb-
oxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] cytotoxic-
ity assay,8,9 using A549, H1299, and H1355 tumor cell lines in
comparison to normal cell lines MRC-5, SV-40 transfected Beas-
2B, and WI-38 cells. Cells seeded in a 96-well plate were treated
further examine the cell-specific anti-proliferation effect of HTMC,
the lung cancer cells A549 and another normal cell line WI-38 cells
were treated with HTMC for 24 h and subjected to apoptotic cell
morphology observation by phase contrast microscopy (Fig. 2B).
The apoptotic cells were dose-dependently increased in cancer cell
line A549 cells as compared with normal cell line WI-38 cells
(Fig. 2B). The arrows indicated the apoptotic cells induced by
HTMC in A549 cells (Fig. 2B, arrows). These results indicated that
HTMC apparently showed selective cytotoxicity against cancer-de-
rived cells. Among the three lungs cancer cell lines examined,
HTMC was more sensitive to A549 cells than H1299 and H1355,
subsequently led to further examination on the mechanism of
the anti-proliferative effect of HTMC in A549 cells.
Induction of apoptosis and/or inhibition of cell proliferation are
highly correlated with the activation of variety of intracellular sig-
naling pathways leading to arrest of cell cycle in the G1, S, or G2/M
phase.10 Cell-cycle regulation and its modulation by various plant-
derived agents are gaining widespread attention in recent years.11
A large number of phytochemicals have been shown to inhibit
cell-cycle progression of various cancer cells.12 To understand the
mechanism involved in the anti-proliferative effects of HTMC, we
performed the A549 cells cell-cycle phase distribution analysis.
A549 cells were exposed with HTMC in the concentration range
with HTMC at concentrations of 6.25, 12.5, 25.0, and 50.0 lM. Cells
treated with solvent (DMSO) were used as controls. After 24 h
treatment with HTMC, the proliferation of three lung cancer cell
lines were significantly reduced in a wide range of doses, with
A549 cells being slightly sensitive than H1299 and H1355 cells
6.25–50 lM for 24 h and stained with propidium iodide and ana-
lyzed by flow cytometry to determine the distribution of the total
cells population in the different phases (G1, S, and G2/M). As
shown in Fig. 3A, in the HTMC treated A549 cells the G1-phase
population maximally increased together with a decreased S phase
(Fig. 2A). A549 cells treated with 50
the proliferation by 59%, and the calculated IC50 values were 47,
48, and 76 M against A549, H1355, and H1299, respectively. In
contrast, normal cell lines MRC-5 and Beas-2B cells were not
affected with 50 M HTMC, the highest concentration tested. To
lM HTMC for 24 h reduced
l
population (p <0.05). After 24 h of 12.5
lM HTMC treatment, the
l
G1 population was increased from 62.9% to 83.4% and the S phase
Figure 2. (A) Cytotoxic effect of 20-hydroxy-2,3,40,60-tetramethoxychalcone (HTMC) in A549, H1299, H1355 lung cancer cells, and in normal cell MRC-5 and SV-40 transfected
Beas-2B. Cells were plated and incubated with indicated concentrations of HTMC (0, 6.25, 12.5, 25, and 50
measured by MTS assay. Data represent the means SEM of triplicate experiments ( ) indicated a p <0.05 with student t-test, as compared with DMSO treated cells. (B)
lM) or control DMSO, after 24 h of incubation the viable cells were
*
Morphological changes of A549 and WI-38 after treatment with HTMC for 24 h.

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Y. K. Rao et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6508–6512
Figure 3. Effect of 20-hydroxy-2,3,40,60-tetramethoxychalcone (HTMC) on A549 cell-cycle distributions. (A) Exponentially growing A549 cells were left untreated or were
treated with indicated concentrations of HTMC (0, 6.25, 12.5, 25, and 50 M) for 24 h and subjected to flow cytometric cell-cycle analysis. (B) A549 cells were left untreated or
were treated with 12.5 M HTMC, and cell-cycle distributions analyzed as indicated time periods. Cell-cycle distributions are shown by histogram of DNA content and cell
number determined by flow cytometry. Data represent the means SEM of triplicate experiments and compared with DMSO treated cells (p <0.05).
l
l
population was decreased from 31.3% to 13.3%. The cell population
in the G2/M phase was not changed significantly by 12.5 M HTMC
indicated that the cell-cycle arrest at G1 phase was the cause of
HTMC anti-proliferation effect in A549 cells.
l
treatment (p <0.05). Noticeably, there was no significant effect
(p <0.05) on the cell-cycle population when the HTMC concentra-
The control of the cell cycle is a highly regulated process, which
involves a complex cascade of events. The modulation of cell-cycle
regulatory proteins, include the cyclins, cyclin-dependent kinases
(Cdk’s), and Cdk inhibitors (Cki’s) that drive cell-cycle progression
through the phosphorylation and dephosphorylation of cell-cycle
checkpoints at G1/S and G2/M.13 To understand the mechanism
underlying G1 arrest in HTMC-treated A549 cells, we next deter-
mined the effect of HTMC on cell-cycle regulatory molecules
including cell division cycle 2 kinase (cdc2) and retinoblastoma
protein (Rb). A549 cells were treated with different concentrations
tion increased from 12.5
l
M to 25 and 50
lM (Fig. 3A). Further-
more, A549 cells treated with 12.5
l
M HTMC for 12 h and 24 h
also increased the G1 phase population in a time-dependent fash-
ion with concomitant decrease in the S phase (Fig. 3B). Interest-
ingly, A549 cells treated with 12.5 lM HTMC for 6 h temporarily
led to decreased (p <0.05) G1 phase population with concomitant
increase in the S phase (Fig. 3B). This indicated that proliferation
and S-phase progression was adversely affected within 6 h of treat-
ment, while a block of cell-cycle progression in G1 in this non-syn-
chronized culture unfolded within 12 h of treatment. These results
(0, 6.25, 12.5, and 25
lM) of HTMC for 24 h and then harvested for
Western blot analysis.8 The cdc2 phosphorylation were examined

Y. K. Rao et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6508–6512
6511
Figure 4. Effect of 20-hydroxy-2,3,40,60-tetramethoxychalcone (HTMC) on cell-cycle regulatory proteins. (A) Western blot analysis of phosphorylation status of cdc2 and Rb in
24 h HTMC treated A549 cells. (B) Expression of p53 and p21 protein in A549 cells after treatment with HTMC for 24 h.
by immunoblot cellular homogenates with anti-phospho cdc2
polyclonal antibodies that detect cdc2 when it is phosphorylated
at Thr161 or Tyr15, two sites known to be important for full activa-
tion of the kinase.13 As shown in Fig. 4A, HTMC caused a dose-
dependent de-phosphorylation of cdc2 at Thr161 and at Tyr15. On
the other hand, the protein Rb acts as a critical regulator for the
G1–S phase progression of the cell cycle by trapping E2F family,
an essential transcriptional factor required for the expression of
cell proliferation-associated genes.14 We subsequently determined
the phosphorylation status of Rb following exposure of exponen-
tially growing A549 cells to HTMC by immunoblotting using spe-
cific antibodies against the phosphorylation sites in Rb.8 As
shown in Fig. 4A, the degrees of Rb phosphorylation at Ser795 and
Ser807/811 were down-regulated by HTMC in a dose-dependent
manner. It is known that interaction between pRb and E2F family
controls over G1–S transition of the cell cycle. In early G1, pRb is
phosphorylated to a limited extent by cdk4 and cdk6 in association
with D-type cyclins, this hypophosphorylated pRb forms a complex
with E2F transcription family and preventing E2F from DNA, there-
fore causing cell cycle to arrest in G1.14 As reported previously,
upregulation of p21, p53, and the downregulation of phospho-
cdc2 and phosphor-Rb may be one of the molecular mechanisms
by which HTMC inhibited A549 cells growth and induced G1
cell-cycle arrest.
We also examined the in vivo antitumor activity of HTMC.8 For
this purpose, 1 ꢀ 107 A549 cells injected sc into the flank of Balb/c
mice rapidly gave rise to exponentially growing tumors. Small so-
lid tumors were observed 7 days following cell inoculation. The tu-
mor volumes were monitored every 3 days. As shown in Figure 5,
the results showed that after 30 days the mean volume of tumors
in mice treated with HTMC (1 mg/kg body weight) was reduced
by 33% as compared with the vehicle-treated mice (control group
mean, 1654.4 124.5 mm3; HTMC group mean, 1234.0
204.7 mm3; p <0.05). The mean volume of tumors in positive con-
trol sesame oil treated mice was reduced to 731.8 140.3 mm3
(Fig. 5). HTMC was well tolerated by the mice, and the animals
did not show any differences in the body weights and health or
behavioral problems. To our knowledge, studies described in this
Letter represent the first systematic characterization of both
in vitro and in vivo anticancer activities of HTMC.
phosphorylation of Ser795
,
Ser807/811 initiates the process of
In summary, this study demonstrated for the first time that
HTMC was a selective cytotoxic agent and induced G1 cell-cycle ar-
rest in human lung cancer cells, and the upregulation of p21, p53
and the downregulation of cdc2, Rb may be one of its molecular
mechanisms. In addition, in vivo data demonstrated that HTMC
act as a tumor growth suppressing agent. Therefore, the potential
cdk-mediated pRb inactivation and the deliberation of E2F.14 In
this letter, Western blot analysis of pRb expression showed phos-
phorylation of pRb was inhibited by HTMC (Fig. 4A), in combina-
tion with flow cytometry analysis in Figure 3A showed treatment
of HTMC increased G1 arrest by 20% at 12.5
ther time-course treatment of HTMC (12.5
l
l
M. In Figure 3B, a fur-
M) yielded the same
result that cells arrest in G1 increased by 20%, indicating an inhib-
itory effect of HTMC on A549 cell growth by decreased pRb phos-
phorylation at Ser795 and Ser807/811. These results indicated that
reduced expression of phospho-cdc2 and phosphor-Rb may be
involved in HTMC-induced G1 phase arrest and led to A549 cell
growth inhibition.
Tumor suppressor gene p53 is a key element in the induction of
cell-cycle arrest and apoptosis following DNA damage or cellular
stress in human cells.15 The p53-dependent cell-cycle arrest re-
quires trans-activation of p21WAF1/Cip1 or other cell-cycle-related
factors.15 The induction of p21WAF1/Cip1 causes subsequent arrest
in the G1 or G2/M phase of the cell cycle via its ability to inhibit
cdc2.16 We therefore, next determined the effect of HTMC on the
cytoplasmic levels of p21 and p53. As shown by immunoblot anal-
ysis in Fig. 4B, HTMC dose-dependently upregulated the expression
of tumor suppressor protein p53 and its downstream target mole-
cule p21. The induction of p21 protein expression was p53-depen-
dent, because incubation of A549 cells with HTMC did not cause
any significant change in p21 protein expression (data not shown).
It is unclear at the present time whether HTMC could also cause
DNA damage and triggers p53 activation and accumulation of
p21 and remain to be further elucidated. Taken together, the
Figure 5. Effect of HTMC on tumor growth in vivo. Approximately 1 ꢀ 107 A549
cells were sc injected into each flank of the studied mice to initiate tumor growth.
Twenty-four hours after cell implantation, the group
1 continued to receive
sterilized PBS, whereas animals in groups 2 and 3 received sesame oil and HTMC
(1 mg/kg body weight, N = 5, respectively). Sesame oil and HTMC were ip injected
every week. Once tumors started to grow, their sizes were measured every 3 days
and the tumor volume was calculated. The quantitative data are shown as
means SD of average tumor volume for each treatment group.

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Acknowledgment
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This study was supported by National Science Council of Taiwan
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Supplementary data (experimental procedure for synthesis of
HTMC and its 1H, 13C NMR and ESI-MS spectral data; and experi-
mental procedures for biological testing are included) associated
with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2010.09.056.
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