Acta Biochim Biophys Sin 2014, 46: 15 21 | The Author 2013.

3. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute
of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmt123.
Advance Access Publication 17 November 2013

Original Article

Casticin suppresses self-renewal and invasion of lung cancer stem-like cells

from A549 cells through down-regulation of pAkt
Fei Liu1, Xiaozheng Cao1, Zhihong Liu2, Hui Guo2, Kaiqun Ren1, Meifang Quan1, Yuan Zhou1, Honglin Xiang1,
and Jianguo Cao1 *
1

College of Medicine, Hunan Normal University, Changsha 410013, China

Department of pathology, Hunan Provincial Tumor Hospital, Changsha 410013, China

These authors contributed equally to this work.

*Correspondence address. Tel/Fax: 86-731-88912434; E-mail: caojianguo2005@126.com
2

Keywords
lung cancer; casticin; cancer stem cells; pAkt;
therapeutic action
Received: May 22, 2013

Accepted: September 2, 2013

Introduction
Lung cancer is the most common cause of cancer deaths
worldwide for its high incidence and mortality. Globally,
more than one million new lung cancer cases were reported
each year [1,2]. Lung cancer is generally subdivided into

small cell lung cancer and non-small cell lung cancer

(NSCLC). The 5-year survival for NSCLC is less than 9% in
developing countries [2], and no more than 15% in USA [3].
So it is necessary to explore the novel therapeutic approaches
for improving life quality of patients with lung cancer
including NSCLC.
Recently, more and more researchers are focusing on
cancer stem cells (CSCs), trying to find new breakthrough
point for curing cancer. The CSCs possess self-renewal capacity and differentiation potential, and can reconstruct the
phenotypic and histologic heterogeneity of its parent tumor
when transplanted in vivo [4]. The CSC theory presumes that
the tumor carcinogenesis, recurrence, and resistance are
caused by the existence of a small subpopulation of CSCs.
Recent studies are mainly involved in characterizing CSCs
from different tumor cell lines or solid tumor tissues including glioblastoma, melanoma, breast cancer, lung cancer,
etc., and their crucial signal molecules [58].
CSCs can be enriched or identified using cell surface
markers via flow cytometry and immunomagnetic beads
sorting system [9,10], or sphere-forming assay [4,11] in selective serum-free medium. In NSCLC, CSCs are commonly
characterized as a subpopulation of cells expressing CD133
[10], or aldehyde dehydrogenase (ALDH) [12,13] or as a
side population [14] of cells capable of effluxing Hoechst
dye. Identification of CSCs in tumor cells is important, but
studies on this area are just the beginning. What is more
important is to find the effective therapeutic strategies,
including screening of novel drugs targeting CSCs.
Casticin (5,30 -dihydroxy-3,6,7,40 -tetramethoxyflavone) is
a main active ingredient of Fructus Viticis Simplicifoliae,
which is often used as the traditional Chinese medicine for
the treatment of certain types of cancer. Studies have shown
that casticin possesses extensive anticancer bioactivity for
cancers including lung cancer [15], cervical cancer [16], leukemia [17,18], colon cancer [19], hepatocellular carcinoma
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A subpopulation of cancer stem cells is recognized as the cause

of tumorigenesis and spreading. To investigate the effects
of casticin (5,30 -dihydroxy-3,6,7,40 -tetramethoxyflavone),
derived from Fructus Viticis Simplicifoliae, on lung cancer
stem cells, we isolated and identified a subpopulation of
lung cancer stem-like cells (LCSLCs) from non-small-cell
lung carcinoma A549 cells with the features including selfrenewal capacity and high invasiveness in vitro, elevated
tumorigenic activity in vivo, and high expression of stemness markers CD133, CD44, and aldehyde dehydrogenase
1 (ALDH1), using serum-free suspension sphere-forming
culture method. We then found that casticin could suppress
the proliferation of LCSLCs in a concentration-dependent
manner with an IC50 value of 0.4 mmol/L, being much
stronger than that in parental A549 cells. In addition, casticin could suppress the self-renewal and invasion of
LCSLCs concomitant with decreased CD133, CD44, and
ALDH1 protein expression and reduced MMP-9 activity.
Further experiments showed that casticin suppressed selfrenewal and invasion at least partly through down-regulation
of Akt phosphorylation. In conclusion, casticin suppressed
the characteristics of LCSLCs, suggesting that casticin may
be a candidate compound for curing lung cancer via eliminating cancer stem cells.

Casticin suppresses lung cancer stem-like cells

cells [20], and so on [21,22]. We have found that casticin

could inhibit self-renewal of glioma stem-like cells [23].
However, the effects of casticin on lung cancer stem-like
cells (LCSLCs) and its mechanisms remain elusive.
In this study, we enriched and identified LCSLCs from
NSCLC A549 cells, and found that casticin could suppress
the self-renewal and invasion of LCSLCs, accompanied
with a reduced CD133, CD44, and ALDH expression. The
underlying mechanisms were associated with the decrease of
phosphorylation Akt, which was reported to play a crucial
role in the maintenance of CSC features.

Cell culture
Human lung cancer A549 cell line was purchased from
Chinese Academy of Sciences Cell Bank (Shanghai, China),
cultured in RPMI-1640 (HyClone, Logan, USA), supplemented with 10% fetal bovine serum (FBS) at 378C in 5%
CO2.

Matrigel invasion assay

Two thousands cells incubated in serum-free medium were
added on the top of the transwell chamber coated with fresh
Matrigel (Gibco). The medium supplemented with 10%
FBS, as the chemical inducing agent, was added to the
bottom of the transwell chamber. After incubation for 48 h,
the cells on top of the chamber were scraped off using a
cotton swab. And the cells in the bottom of the chamber
were fixed with methanol and stained with Wright staining
solution. The cells invading through the membrane were
counted under an optical microscope.

Sphere-forming and self-renewal assay

To obtain sphere cultures, cells were plated at a density of
5  103 cells/well in 6-well ultra-low plates (Corning, Acton,
USA) containing serum-free medium DMEM/F12 (Gibco,
Carlsbad, USA), supplemented with commercial hormone
mix B27 (Gibco), 20 ng/ml EGF (PeproTech, Rocky Hill,
USA), 10 ng/ml bFGF (PeproTech), 0.4% bovine serum
albumin (Gibco), 4 mg/ml insulin (Gibco), 100 U/ml penicillin and 100 U/ml streptomycin at 378C. After being cultured
for 6 days, the lung cancer spheres were collected, dissociated
into single cell suspension and resuspended in fresh medium
for serial subcultivation every 6 days.
To investigate the self-renewal capacity of lung cancer
sphere-forming cells (SFCs), single cell suspension prepared
from the lung cancer sphere was diluted to 500 cells/ml.
Two microliters of the single cell suspension was plated in
96-well ultra-low plates containing 150 ml serum-free
medium per well. Wells containing no cells or more than
one cell were excluded, and those with one cell were marked
and monitored daily under a microscope (Nikon Eclipse
TE2000-S, Nikon, Japan) for 6 days and the colonies were
counted. To investigate the effects of casticin on self-renewal
of SFCs, single cell suspension of SFCs was plated at a
density of 2  103 cells/ml in 6-well ultra-low plates.
Different concentrations of casticin (1.0, 5.0, and 10.0 mM)
were added to medium. After being cultured for 6 days, the
colonies were counted under a microscope.

MMP-9 activity assay

The SFCs were plated at a density of 1  106 cells/well in
6-well ultra-low plates containing serum-free medium. After
24 h incubation, the medium was replaced with serum-free
medium containing different concentrations of casticin (1.0,
5.0, 10.0 mM), cultured for additional 72 h. Then the
medium was collected and subject to MMP-9 activity assay
using human active MMP-9 fluorokine E kit (R&D
Systems, Minneapolis, USA) according to manufacturers
instructions. The activity unit was defined as ng/ml/106
cells.

MTT [3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide] assay
The SFCs or parental cells were plated at 5000 cells/well in
96-well ultra-low plates. The cells were first incubated with

Western blot analysis

The cells were lysed using lysis buffer (50 mM Tris-HCl,
pH 7.4, 150 mM NaCl, 0.2 mM EDTA, 0.2% NP-40, 10%
glycerol, 1 M b-Me, 1 mg/ml aprotinin, 0.5 mg/ml aprotinin

Acta Biochim Biophys Sin (2014) | Volume 46 | Issue 1 | Page 16

Tumorigenicity assay
The SFCs or parental cells were injected subcutaneously
into the back of 4-week-old BALB/C-nude mice (supplied
by the Shanghai Experimental Animal Center, Chinese
Academy of Sciences, Shanghai, China). The mice were
reared for 2 months, and the tumor growth and tumorigenic
time were examined visually. Tumor volumes were calculated in accordance with the formula: V (transplanted tumor
volume, mm3) L (longest diameter, mm)  W (minimum
diameter, mm)2  0.5. At the end of experiment, the mice
were sacrificed under deep anesthesia with pentobarbital.
The tumors were then dissected, weighed and fixed with
10% neutral formalin. Paraffin sections were prepared
following routine procedures for H&E staining.

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Materials and Methods

casticin at the indicated concentrations for 48 h, and then

incubated with 5 mg/ml MTT for 4 h. Lastly, 100 ml of
dimethyl sulfoxide (DMSO) was added to each well, and the
absorbance at 570 nm (A570) each well was detected on a
plate reader (Bio-Tek EXL-800, Winooski, USA). The relative cell proliferation inhibition rate (1 experimental
group A570 mean/control group A570 mean)  100%. IC50
(50% inhibiting concentration) was calculated by Prism
(GraphPad Software, San Diego, USA).

Casticin suppresses lung cancer stem-like cells

Statistical analysis
Data were presented as the mean + standard deviation. To
assess the statistical significance of differences, students
t test was performed. P , 0.05 was regarded as statistically
significant.

Results
Lung cancer SFCs derived from A549 cell lines have
the self-renewal capability
In order to enrich LCSLCs from human lung cancer A549
cell line, stem cell conditioned medium suspension culture
method was used. The results showed that A549 cells could
form non-adherent sphere which is called lung cancer SFCs
(Fig. 1A2). The results suggested that LCSLCs exist in
human lung cancer A549 cells.
We next performed the SFCs subculture and tumor sphere
formation test. It was found that the single cell of SFC from
lung cancer A549 was able to form a new sphere (Fig. 1B),
suggesting that the SFCs from A549 cells have the ability of
self-renewal.
Lung cancer SFCs overexpress stem cell markers
Furthermore, we investigated the expressions of CSC biomarker CD44, CD133, and aldehyde dehydrogenase 1
(ALDH1) in SFCs using western blotting. The results
demonstrated that, compared with human lung cancer parental cells, the CD44, CD133, and ALDH1 protein expression
levels were apparently increased in their SFCs (Fig. 1C).
Lung cancer SFCs possess higher tumorigenicity
In addition, the xenograft tumor model in nude mouse was
used to investigate the tumorigenicity of A549 cells and

Figure 1. Identification of LCSLCs from lung cancer A549

cells Parental cell A549 (A1) and its SFC enriched by stem cell
conditioned medium suspension culture method (A2). The single cell of
SFC formed a new sphere (B). The CD44, CD133 and ALDH1 protein
expression levels in SFC and their PCs (C). The tumorigenicity (D) and
immunohistochemical characteristics (E) of A549 cells and its SFC
xenograft tumor in Balb/c-nu immunodeficient mice. PC: Parental cell.

their SFCs in Balb/c-nu immunodeficient mice. The results

showed that 1  104 A549 SFCs were enough to form
tumors in vivo (Fig. 1D), and the tumors have similar immunohistochemical characteristics with its parental cell xenograft tumor (Fig. 1E). However, in the same model, at least
1  106 A549 parental cells were required to induce stable
tumor formation (Table 1). Our data indicate that the SFCs
from A549 cell line have higher tumorigenicity in vivo than
parental cells.
The above data confirmed that SFCs from human lung
cancer A549 cell line cells possessed characteristics of CSC.
Therefore, we can, at least, deduce that the lung cancer SFCs
prepared by us belong to LCSLCs, and the LCSCs were
possibly enriched in them.

Casticin inhibits the self-renewal capability of LCSLCs

The unlimited proliferation potential is an important feature
of CSCs [24]. Genistein is a natural polyphenolic compound
that can preferentially inhibit the proliferative activity of
the pancreatic CSCs than parent cells [23]. Our results
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aldehyde peptide, 0.1 mM Na3VO4, 0.5 mM 4 NPP,

0.5 mM NaF, and protease inhibitors), and the total cell
extracts were prepared. The protein content was measured
using the Bradford method. The proteins were separated on
10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride
(PVDF) membrane. After being blocked with 5% non-fat
milk in phosphate buffered saline containing Tween-20
(PBST), the PVDF membrane was incubated using the following mouse anti-human primary monoclonal antibodies:
anti-pAkt (Ser473) (1 : 500; Cell signaling, Beverly, USA),
anti-Akt (1 : 2000; cell signaling), anti-CD44 (1 : 500;
cell signaling), anti-CD133 (1 : 500; cell signaling), antiALDH1 (1 : 500; cell signaling), and anti-b-actin (1 : 200;
Sigma, St. Louis, USA), with slight shaking at 48C overnight.
Then the membrane was incubated with peroxidase-labeled
secondary antibody for 1 h, and the signal was detected using
an enhanced chemiluminescence kit (Amersham Biosciences,
Buckinghamshire, UK) according to manufactures protocols.

Casticin suppresses lung cancer stem-like cells

showed that casticin (0.1, 1, 3, 10, 30 mM) could inhibit the

proliferation of human lung cancer SFCs in a dosedependent manner (Fig. 2A), and the IC50 of casticin on
A549 cells and their SFCs were 14.3 and 0.4 mmol/L, respectively, suggesting that casticin is able to preferentially
suppress the proliferation activity of LCSLCs.
Furthermore, casticin (1, 5, 10 mM) could reduce the size
and number of SFCs from A549 cells in a dose-dependent
manner (Fig. 2B,C), suggesting that casticin can suppress
the self-renewal of LCSLCs.

Table 1. Xenotransplantation of human lung cancer A549 cells and its

SFCs into Balb/c-nu immunodeficient mice

Cell

Inoculum size Tumor incidence Latency period (d)

A549 cells 1  104

1  105
1  106
SFCs
2  103
1  104
1  105

0/4
0/4
2/4
2/4
4/4
4/4

31
29
19
11

Casticin reduces CD133, CD44, and ALDH1 expression

in LCSLCs
Western blot analysis showed that casticin (1, 5, 10 mM)
reduced CD133, CD44, and ALDH1 protein expression
in SFC from A549 cells, in a dose-dependent manner
(Fig. 3A). It suggests that casticin can decrease CSC marker
expression in LCSLCs.
Casticin inhibits the self-renewal of LCSLCs via
down-regulation of pAkt
Western blot analysis showed that the phosphorylation levels
of Akt in SFCs from A549 cells were higher than those in
A549 cells (Fig. 3B). Casticin could decrease phosphorylated Akt ( p-Akt) expression in the SFCs in a dosedependent manner (Fig. 3C). Akt inhibitor LY294002
(5, 10, 20 mM) could effectively reduce phosphorylation of

Figure 2. Effects of casticin on LCSLCs Casticin inhibited proliferation (A) and reduced the size (B) and number (C) of SFC from A549 cells. Casticin
suppressed the invasion of SFC from A549 cells (D) and (E). Casticin decreased MMP-9 activity (F). *P , 0.05 vs. 0.01% DMSO. PC, Parental cell.
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Casticin decreases invasion of LCSLCs

In addition, in view of the important role of CSCs in cancer
early metastasis, the Transwell invasion assay was used to

investigate the effects of casticin on invasion of LCSLCs.

The results showed that casticin (1, 5, 10 mM) could inhibit
the invasion of SFCs from A549 cells in a dose-dependent
manner (Fig. 2D,E). It is reported that the MMP-9 is highly
expressed in LCSLCs from A549 cells than in parent cells
[25]. In this study, the MMP-9 activity in SFCs from A549
cells was found to be decreased gradually with the increase
of casticin concentration (Fig. 2F). These data indicate that
casticin can suppress the invasion of LCSLCs, possibly via
reducing MMP-9 activity.

Casticin suppresses lung cancer stem-like cells

Akt and the expression of stem cell markers CD44, CD133,

and ALDH1 in SFCs from A549 cells (Fig. 3D), and significantly decrease the number of SFCs (Fig. 3E). Moreover,
LY294002 could augment the down-regulation of p-Akt
(Fig. 3F) and the number reduction of SFCs (Fig. 3G)
induced by casticin. These results suggest that Akt plays an
important role in maintaining stem cell features of LCSLCs,
and the mechanisms underlying casticin suppressing
LCSLCs are partly via the down-regulation of pAkt.

Discussion
We isolated the SFC subpopulation from human NCLSC
A549 cells using a sphere-forming enrichment culture
method with selective serum-free medium. The results
showed that the SFCs could proliferate and form a new
sphere, confirming that the SFC subpopulation possessed
self-renewal capacity. Then we further discovered that the
expression levels of CSC markers CD133, CD44, and
ALDH1 in SFCs were much higher than that in parental
cells. CD133, CD44, and ALDH1 are important CSC

markers in sorting and identification of various types of

CSCs, especially in lung cancer.
CD133 often serves as a stemness biomarker for CD133
CSCs. CD133 cells are related to the maintenance, metastasis and drug-resistance of lung cancer [9,26,27]. CD133
cells from primary NSCLC tissue have higher tumorigenic
potential in vivo than the CD1332 counterpart [28]. CD44 is
also an important stemness biomarker in lung cancer. Recent
studies have shown that CD44 cells from NSCLC have differentiation potential in vivo, express the pluripotency genes
OCT4/POU5F1, NANOG, and SOX2, and are more resistant
to cisplatin treatment than CD442 cells [29]. It has been
demonstrated that the CD133CD44 cell subpopulation in
A549 cells possesses the continuous proliferative capacity
and differentiation potential [30]. ALDH1 has been reported
to be over-expressed in NSCLC stem-like cells [12], and the
ALDH1A1 lung cancer cells are more resistant to EGFR
tyrosine kinase inhibitor gefitinib than the ALDH1A12
cells [13].
Our results showed that the sorted SFCs from A549 cells
expressing higher levels of CD133, CD44, and ALDH1, had
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Figure 3. Casticin inhibits the self-renewal of LCSLCs via down-regulation of pAkt Casticin reduced CD133, CD44 and ALDH1 expression in SFC
from A549 cells (A). The pAkt was highly expressed in SFC (B). Casticin decreased pAkt level in the SFC (C). Akt inhibitor LY294002 reduced pAkt,
CD44, CD133 and ALDH1 expression in SFC (D), and decreased the number of SFC (E). LY294002 augmented down-regulation of pAkt (F) and reduction
of the number of SFC (G) induced by casticin. *P , 0.05 vs. 0.01% DMSO or the indicated group.

Casticin suppresses lung cancer stem-like cells

Acta Biochim Biophys Sin (2014) | Volume 46 | Issue 1 | Page 20

suggest that casticin is a candidate compound for curing

lung cancer via eliminating CSCs.

Acknowledgement
We thank Dr. Xiyun Deng from Hunan Normal University
for reviewing the manuscript.

Funding
This work was supported by the Project of Scientific
Research of Hunan Province the Administration Bureau of
Traditional Chinese Medicine (201269), the Youth Fund of
Hunan Normal University (110637), and the Hunan province Science and Technology Project (2011FJ4144).

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