Imidazo [4,5f][1,10] phenanthroline derivatives as inhibitor of c-myc gene expression in A549 cells via NF-κB pathway

Article abstract of DOI:10.1016/j.bmcl.2011.11.063

1,10-Phenanthroline has been shown to exhibit anticancer activity. Here, a series of imidazo [4,5f][1,10] phenanthroline derivatives 1-10 were synthesized and their biological activities were further elucidated. We found that 2-(4-Brominephenyl)-imidazo [

Full text of DOI:10.1016/j.bmcl.2011.11.063

Bioorganic & Medicinal Chemistry Letters 22 (2012) 102–105
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Bioorganic & Medicinal Chemistry Letters
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Imidazo [4,5f][1,10] phenanthroline derivatives as inhibitor of c-myc gene
expression in A549 cells via NF-jB pathway
Dong-dong Sun a,b, Wei-zhang Wang c, Jian-wen Mao c, Wen-jie Mei a,b, , Jie Liu b,
⇑
⇑
a School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, PR China
b Department of Chemistry, Jinan University, Guangzhou 510632, PR China
c School of Basic Courses, Guangdong Pharmaceutical University, Guangzhou 510006, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 July 2011
Revised 16 November 2011
Accepted 18 November 2011
Available online 23 November 2011
1,10-Phenanthroline has been shown to exhibit anticancer activity. Here, a series of imidazo [4,5f][1,10]
phenanthroline derivatives 1ꢀ10 were synthesized and their biological activities were further elucidated.
We found that 2-(4-Brominephenyl)-imidazo [4,5f][1,10] phenanthroline (compound 3) possessed potent
antiproliferation activities again a variety of tumor cell lines using 3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphenyl tetrazolium bromide (MTT) assay. Flow cytometric analysis revealed that compound 3 induced
both through apoptosis and necrosis in human lung adenocarcinoma cell line, A549. Moreover, com-
Keywords:
Imidazo [4,5f][1,10] phenanthroline
derivatives
NF-jB pathway
Apoptosis
pound 3 treatment led to up-regulation of I
Taken together, these results suggested that compound 3 inhibited cell proliferation by suppression of
NF- B activity and down-regulation of c-myc gene expression and may be a candidate for further eval-
jBa and down-regulation of p65 and c-myc in A549 cells.
j
uation as a chemopreventive and chemotherapeutic agent for human cancers, especially for lung cancer.
Ó 2011 Elsevier Ltd. All rights reserved.
Anticancer
Lung cancer is one of the leading causes of cancer death in the
Most of the apoptosis-related genes are involved in two signal-
ing pathways leading to apoptosis, such as the nuclear factor
(NF- B) or the mitochondrial-mediated signaling pathway. The
NF-
world. Cancer death is mainly caused by metastasis and an in-
creased resistance to chemotherapy. However, lung cancer is often
presented at stages beyond surgical respectability. Novel therapies
are necessary to reduce the increasing incidence in pulmonary
neoplasm because current treatment modalities are inadequate.
Classic chelates based on 1,10-phenanthroline and a number of
its derivatives have attracted particular attention in the past few
decades because of their unique properties as chelating agents.
1,10-Phenanthroline and substituted derivatives play a key role
in the performance of a wide variety of biological systems in tran-
sition metal complexes, relying on the availability of multifunc-
tional ligands.1–4 Most of the investigations of their applications
have been reported in chemistry, physics, and material, as well
as biological, sciences.5,6 Several mechanisms have been described
to elucidate the anticancer activities of 1,10-phenanthroline based
on metal-chelating, such as the inhibition of DNA synthesis7,8 and
mitochondria-mediated apoptosis.9 However, the underlying
mechanism of the action of 1,10-phenanthroline derivatives in
lung cancer cells has remained largely unknown.
jB
j
j
B family of transcriptional factors play an important role in
cancer, immunology, and inflammation,10,11 and regulate the tran-
scription of many genes that are involved in tumor promotion,
angiogenesis, metastasis, and increased cell survival.12–14 It has
five cellular members: p105/p50 (NF-
p65 (RelA), RelB, and c-Rel. In almost all cell types, NF-
plexes are typically localized in the cytoplasm, where they bind
to I B inhibitory proteins, including I , I Bb, and I . Upon
stimulation, I B proteins are rapidly phosphorylated by I-
kinases and b (IKK and—b) and degraded via the ubiquitin–pro-
teasome pathway. Most important, the activation of NF- B is
jB1), p100/p52 (NF-
jB2),
j
B com-
j
j
Ba
j
jBe
j
jB
a
a
j
linked to increased resistance of tumors to chemotherapeutic
drugs and radiation therapy.15 Many classes of compounds have
been reported to inhibit NF-
zine analogs,16 glucocorticoids,17 and nonsteroidal anti-inflamma-
tory agents.18,19 Thus, NF-
B inhibition is a viable strategy for both
chemotherapy and chemoprevention. However, the mechanisms of
1,10-phenanthroline derivatives to inhibit NF- B activation are not
jB activation, such as phenyl-pipera-
j
j
described. In the present study, the antiproliferative activity of
1,10-phenanthroline derivatives was determined, and the effect
of these compounds on apoptosis in the A549 human lung cancer
cell line was examined. Furthermore, to establish the anticancer
Abbreviations: NF-jB, nuclear factor-kappa B; MTT, 3-(4,5-dimethyl-2-thiazol-
yl)-2,5-diphenyl tetrazolium bromide; Compound 3, 2-(4-Brominephenyl)-imidazo
[4,5f][1,10] phenanthroline; A549, human lung adenocarcinoma.
⇑
mechanism of 1,10-phenanthroline derivatives, the levels of I-
jB-
Corresponding authors. Tel.: +86 2039352122; fax: +86 2039352129.
E-mail addresses: wenjiemei@126.com (W. Mei), tliuliu@jnu.edu.cn (J. Liu).
a
, NF- B P65, and c-myc, which are strongly associated with the
j
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.063

D. Sun et al. / Bioorg. Med. Chem. Lett. 22 (2012) 102–105
103
signal transduction pathway of apoptosis and known to affect the
compound 3 ( M)
chemosensitivity of tumor cells to anticancer agents, were assayed.
The cytotoxic and antitumor effects of compounds 1–10 were
studied using MTT assay, and the inhibitory activities (IC50) of
these complexes are listed in Table S3. These compounds exhibited
high activity against human lung adenocarcinoma (A549) cell line,
Control
5
1
2.5
10.81%
50.94%
35.26%43.26%
43.26%
with inhibitory activities IC50 ranging from 0.85 to 35.5
high cytotoxic activities of a few compounds were even beyond
that of cisplatin (15.94 M). The IC50 value for compound 3 was
(0.85 0.06) M, confirming that this compound is more effective
lM. The
l
l
in inhibiting A549 cell growth. This result suggests that com-
pounds that contain a pendant halogen functional group might
be advantageous for inhibitory activities.
60
50
40
30
20
10
0
The following observations can be derived from Table S3: (1)
Although belonging to the same family of halogen substituted
compounds, the complexes might show some differences in activ-
ity. Cytotoxic activities increase as electronegativity decreases; (2)
The same positions related to the electron-withdrawing group
were fluorine, chlorine, and bromine, whereas those related to
electron donor were methoxy, ethoxy, and propoxy. The latter also
showed higher activities; and (3) The difference in results might
involve the steric hindrance of substituents, and small group sub-
stitutes in para-positions could possess appreciable alteration in
their properties.
control
1μΜ
2.5μΜ
5μΜ
According to the results of the MTT assay (Table S3), compound
3 is more active than the other compounds in inhibiting A549 cell
growth. Thus, this compound was used to investigate further the
underlying mechanisms. The IC50 values for compound 3 against
human melanoma cells A375, human hepatoma cells HepG2, hu-
man ovarian carcinoma cells HO8910, human hepatoma cells
Bel-7402, nasopharyngeal carcinoma cell CNE-1 were 0.98, 4.3,
Figure 1. FCM scatter of the apoptosis of A549 cell treated with compound 3 after
24 h. The effect of 1, 2.5 and of compound on A549 cell apoptosis
5
l
M
3
determined by flow cytometry. Untreated (control) cells or cells treated for 24 h
were harvested, fixed, stained with Annexin V/PI, and assessed for cell apoptosis
distribution by flow cytometric analysis. Percentages of apoptotic cells were
measured by the annexin V/PI flow cytometry analysis, as described in Section 2.
The y-axis plots the sum of early and late apoptotic cells as the mean SD of three
independent experiments.
1.56, 5.8 and 1.2 lM, respectively, (Table 1). These data indicate
the high activity of compound 3 against these cell lines, not only
against the A549 cell line. Compound 3 also exhibited higher activ-
ity compared with cisplatin.
Apoptosis is a physiological process that functions as an essen-
tial mechanism of tissue homeostasis and is regarded as the pre-
ferred way to eliminate unwanted cells. Most of the available
cytotoxic anticancer drugs mediate their effects via induction of
apoptosis in cancer cells.20 Given that compound 3 had the lowest
of NF-
in Figure 2A, compound 3 treatment of A549 cells for 24 h evi-
dently induced I protein expression in a dose-dependent man-
ner. Meanwhile, the protein levels of c-myc and p65 were
decreased after compound 3 exposure in a dose—and/or time-
dependent manner (Fig. 2B and C). These data suggest that com-
jB signal pathway, were assayed by Western blot. As shown
jBa
pound 3 inhibited NF-jB signal pathway by upregulation of IjBa
IC50 value of 0.85 lM (Table S3), this compound was used to sub-
stantiate further the mechanistic role of inhibition of A549 cancer
cell proliferation. The flow cytometric (FCM) analysis (determined
and downregulation of p65, thus suppressing of c-myc expression
and in turn inhibiting proliferation and inducing apoptosis in A549
cell.
The 2ꢀ44ct method was used to calculate relative changes in
gene expression that were determined from real-time quantitative
PCR experiments.21 To investigate whether the induction of apop-
tosis by compound 3 is due to the downregulation of the c-myc
gene, quantitative real-time PCR was conducted in compound 3-
treated A549 lung cancer cells. As shown in Figure 3, compound
3 had a markedly suppressing effect on the c-myc expression level
in a dose-dependent manner. Therefore, compound 3 might be a
potent agent for the treatment of A549 human lung cancer.
24 h post drug treatment) of compound 3 at 1, 2.5, and 5
showed a significant increase in the cell apoptosis at 24 h. The ratio
of cell apoptosis reached 50.94% for 5 M compound 3 (Fig. 1). The
lM
l
result of control (10.81%) was approximately fivefold lower than
that of compound 3, indicating that the latter induced A549 cell
apoptosis.
In view of the important role of NF-
proliferation, the effect of compound 3 upon the protein levels of
p65, I and c-myc, a well-documented downstream target gene
jB signal pathway in the cell
jBa
Table 1
Chemical structure of compound 3 and inhibitory activity (IC50 values) of compound 3 against human cancer cellsa
Compd
IC50(lM)
A549
A375
Hep G2
Ho8910
Bel-7402
CNE-1
3
DDP
0.85 0.06
15.94 1.02
0.98 0.07
7.3 0.34
4.3 0.21
8.3 0.37
1.56 0.09
4.4 0.22
5.8 0.26
15.3 1.05
1.2 0.08
6.5 0.05
a
IC50 values are given in lM. Compd, compound, DDP, cisplatin. The values are expressed as the mean SD (triplicates).

104
D. Sun et al. / Bioorg. Med. Chem. Lett. 22 (2012) 102–105
Figure 2. Effect of compound 3 on apoptosis-related proteins. Total cell lysates from cells treated for various concentrations with 1, 2.5 and 5
lM of compound 3 were
separated electrophoretically on 10% polyacrylamide gels and immunoblotted with an antibody against each protein, b-actin and GAPDH as an internal control, respectively.
(A) Effect of compound 3 on the expression of IjBa in A549 after 24 h; (B) Effect of compound 3 on the expression of c-myc in A549 after 24 h and 48 h, respectively; (C) Effect
of compound 3 on the expression of p65 in A549 after 24 h. The values are expressed as the mean SD (triplicates), and statistical significance is indicated by ⁄P <0.05, ⁄⁄P
<0.01.
In conclusion, a novel class of potent NF-
was discovered through the present study. Further studies by Wes-
tern blot and PCR assay have shown that compound 3 significantly
j
B signaling inhibitors
modulation toward an anticancer therapy. Further studies must
be conducted to gain an in-depth comprehension of these preli-
minary findings on the activity of these promising wide-spectrum
anticancer agents.
inhibited the expression levels of NF-
line in a time-dependent manner. The results show that compound
3 impaired the activity of NF- B to inhibit A549 cell proliferation
jB proteins in the A549 cell
j
Acknowledgements
by suppressing the c-myc expression. This class of compounds
can be beneficial in the treatment of lung cancer, autoimmune dis-
eases, anti-viral therapy, and interferon co-therapy. These com-
pounds could lead to a novel concept of host cell pathway
This work was supported by the National Natural Science Foun-
dation of China (20871056 and 81100369), the Planned Item of Sci-
ence and Technology of Guangdong Province (20070327029,
c1011220800060), the Research Foundation for Young Teacher of
Guangdong Pharmaceutical University, 211 project grant of Jinan
University and the Fundamental Research Funds for the Central
Universities.
4.5
4.0
24h
48h
3.5
Supplementary data
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Supplementary data (biological methods, experimental proce-
dures and the complete characterization data of the synthesized
compounds are provided.) associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.2011.11.063.
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