Trilinolein inhibits proliferation of human non-small cell lung carcinoma A549
through the modulation of PI3K/Akt pathway
Pei-Yu Choua, Guan-Jhong Huangb, Chun-Hsu Panc, Yi-Chung Chiena, Ying-Yi
Chenc, Chieh-Hsi Wuc, Ming-Jyh Sheuc*, Hsu-Chen Chenga*
a
Department of Life Science, National Chung Hsing University, 250 Kuo-Kuang Rd.,
Taichung, 402, Taiwan b
Institute of Chinese Pharmaceutical Science, China Medical University, 91,
Hsueh-Shih Road, Taichung, 404, Taiwan c
School of Pharmacy, China Medical University, 91, Hsueh-Shih Rd., Taichung, 404,
Taiwan
Running title: anticancer effects of trilinolein on A549
* Corresponding author:
Hsu-Chen Cheng, Ph.D,
Department of Life Science, National Chung Hsing University,
250 Kuo-Kuang Rd., Taichung, 402, Taiwan.
Tel.: +886 4 2205 3366 5158
Fax: +886 4 2287 4740
Email: soybean13mtdtw@gmail.com
Abstract: Trilinolein has been identified as one of the active constituents isolated from
Panax notoginseng used widely in traditional Chinese medicine. Protective actions of
Panax notoginseng against cerebral ischaemia, beneficial effects on the
cardiovascular system, and haemostatic, antioxidant, hypolipidaemic,
hepatoprotective, renoprotective and estrogen-like activities have been illustrated. In
the present study, the effects of trilinolein on the growth of non-small cell lung
carcinoma A549 were investigated. It was found that the exposure of A549 cells to
trilinolein resulted in growth inhibition and the induction of apoptosis in a dose- and
time- dependent manner. Trilinolein treatment induced the upregulation of
pro-apoptotic Bax, downregulation of anti-apoptotic Bcl-2 expression, which was
associated with the proteolytic activation of caspases and the concomitant degradation
of poly(ADP-ribose) polymerase (PARP) protein. Intracellular reactive oxygen
species seem to play a role in the trilinolein-induced apoptosis, since ROS were
produced early in the trilinolein treatment. Moreover, the activity of PI3K/Akt was
downregulated in trilinolein-treated cells. Our results demonstrated that the most
important regulators of trilinolein-induced apoptosis are Bcl-2 family and
caspase-3,which are associated with cytochrome c release and dephosphorylation on
the Akt signaling pathway.
Introduction
Lung cancer is among the leading causes of death and its incidence is
continuously increasing. Surgery, radiotherapy, and chemotherapy are currently the
major treatments used to reduce lung cancer mortality (Saba and Khuri, 2005),
however, these therapies have detrimental side effects on the normal healthy cells in
the body. Therefore, it is important to discover new agents to treat lung cancer safely
without affecting the body’s healthy cells. Deregulation of signaling pathways like
PI3K/Akt are often implicated in the pathogenesis of NSCLC (Li et al., 2010).
Therefore the need for accelerated development of effective NSCLC therapies is
critical. At present, major work is being stressed on designing new therapeutic
strategies targeting multiple signaling pathways for more effective disease
management in NSCLC. We aimed to investigate how trilinolein affects several
pathways including mitochondria-dependent, ROS, PI3k/Akt, and p53/p21 signals.
Certain Chinese herbs have been used as alternative therapeutic approaches, for
treating lung cancer patients in Chinese population (Lu et al., 2009; Sun et al., 2010).
Panax notoginseng Burk. F.H. Chen (Araliaceae) (P. notoginseng) is a highly valuable
and important herb in oriental medicine for its therapeutic abilities. P. notoginseng has
been widely used for hemostasis and protection of the cardiovascular system (Chen et
serum total cholesterol and triglyceride levels (Joo et al., 2010). The extract of the
roots of P. notoginseng exhibited a significant anti-tumor-promoting activity on
two-stage carcinogenesis of mouse skin tumors (Konoshima et al., 1999). P.
notoginseng extract was reported effective on precancerouspatients (Yu, 1993). P.
notoginseng extract and ginsenoside Rb1 increased the sensitivity of KHT sarcoma to
ionizing radiation (Chen et al., 2001). P. notoginseng was cytotoxic for the treatment
of PC3 human prostate cancer cells (Chung et al., 2004). There is a report that the
serum of a dog fed with P. notoginseng extract inhibited proliferation of human
gastric mucosa epithelium GES-1 cells (Wang et al., 2004). A dammarane glycoside
derived from ginsenoside Rb3 showed toxicity against breast cancer cells (He et al.,
2005). Also, P. notoginseng powder protects a precancerous stomach lesion (Shi et al.,
2003). The effects of crude P. notoginseng extract on tumor cells suggested that
further purified or synthetic versions of P. notoginseng extract may be useful not only
in vascular-related diseases, but also cancer therapy (Chen et al., 2001).
The antitumor activity from P. notoginseng are mainly focused on its extract
(Konoshima, et al., 1999) and its constituent ginsenoside Rb1 (Chen et al., 2001).
Trilinolein has been focused on its antioxidant activity (Ng et al., 2004), however, its
anticancer activity has never been explored. Trilinolein is a candidate active
triacylglycerol, which carries two unsaturated bonds (C 18:2, MW = 890; Fig. 1A), at
all three esterified positions of glycerol (Hong et al., 1993). Trilinolein has been
reported to provide a number of beneficial effects including reducing thrombogenicity
(Chan et al., 2002), increasing erythrocyte deformability (Hong et al., 1993),
anti-ischemic (Chen et al., 2008), anti-arrhythmic (Chan et al., 1995), and displaying
antioxidant effects in various experimental models (Chan et al., 1997; Chan et al.,
2002). Additionally, trilinolein has been reported to reduce free radical damage
associated with atherogenesis, and myocardial damage caused by ischaemia and
reperfusion (Kritchevsky et al., 2000). The sum of these pharmacologic effects may
explain the benefits derived from treating circulatory disorders with the herb over the
centuries. Therefore, we aimed to investigate the effects of trilinolein on human
non-small cell lung cancer cells A549 and to explore the molecular mechanism
through which trilinolein induces cell death
Materials and methods
Materials
Trilinolein was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO,
USA). 3-(4,5-Dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), RNase A,
propidium iodide (PI), trypsin, BSA, Tween-20,-80 and DMSO were purchased from
F-12 and fetal bovine serum (FBS) were purchased from GIBCO BRL (Rockville,
MD, USA). Antibodies for pAkt, Akt, PIP3K, poly(ADPribose) polymerase (PARP),
caspases-3 and -9 were purchased from Cell Singnaling (Boston, MA, USA).
Antibody for cytochrome c was purchased from BioLegend (San Diego, CA, USA).
Antibodies against p53, p21, Bax and Bcl-2 were purchased from Santa Cruz (Santa
Cruz, CA, USA). Secondary antibodies were acquired from Santa Cruz (Santa Cruz,
CA, USA).
Cell Lines and Cultures
Human renal cell carcinoma cell line A498, gastric adenocarcinoma MKN-45,
and human NSCLC A549 cells were obtained from Food Industry Research and
Development Institute (Hsinchu, Taiwan). A498 and MKN-45 cells were cultured in
Dulbecco’s Modified Eagle Medium (DMEM; Gibco BRL, Rockville, MD, USA),
and A549 cell was grown in F-12 medium (Gibco BRL, Rockville, MD, USA)
containing 10% FBS (Gibco BRL, Rockville, MD, USA), 100 U/mL of penicillin, and
100 mg/mL streptomycin mixed antibiotics (Gibco BRL, Rockville, MD, USA) at
37oC in a humidified atmosphere comprised of 95% air and 5% CO2. In all of the
Cell proliferation assay
MTT assay was performed in the A498, MKN-45 and A549 cell lines to measure
the cytotoxicity of trilinolein. All cell lines were seeded in 96-well plates with 2×104
cells/well in culture medium. Trilinolein was dissolved in 0.8% (v/v) Tween 80 in
PBS and sterilized by filtration. Cells were treated with various concentrations of
trilinolein as indicated in each figure. After 24 h., the number of viable cells was
determined. Briefly, 5 mg/mL MTT was added to each well, and the plate was
incubated at 37oC for 4 h. The medium was removed, and a 50 µL aliquot of DMSO
was added, and the absorbance at 590 nm was measured for each well on ELISA
reader. Data are presented as the mean ± SE of three independent experiments.
Flow cytometry analysis
2×105 A549 cells were seaded into each well of a 12-well plate (TPP; Techno
Plastic Products AG, Trasadingen, Switz) 24 h before treatment with various
concentrations of trilinolein for different time periods (0, 2, 12, 24, and 48 h). Cells
were harvested with trysin-EDTA, washed twice with 10 ml ice-cold PBS, fixed in
70% (v/v) ethanol, and kept at 4 oC prior to propidium iodide (PI) staining [100
µg/mL PI, 0.2% (v/v) Nondiet P-40, and 1 mg/mL RNase A (DNase-free) in PBS lacking Ca2+ and Mg2+; at a 1:1:1 ratio by volume] and analyzing DNA contents with
fluorescence was linearly amplified and both the area and width of the fluorescence
pulse were measured. Ten thousand events were acquired, and the percentage of
hypodiploid (apoptosis, sub-G1) events and percentages of cells in G0/G1, S and
G2/M phases were determined using the DNA analysis software ModFitL T, version
2.0 (Verity Software, Topsham, ME, USA).
Measurement of intracellular ROS generation
A549 cells were incubated for 4 h in the presence of
trilinolein (50, 75 and 100 µg/ml). In time course study, 75 µg/ml of trilinolein were
incubated for 1, 2 and 4 h. Intracellular ROS production was measured by using a
fluorescent dye, 2′,7′-dichlorodihydrofluorescein diacetate (H2-DCF-DA) (Molecular
Probes, Eugene, OR, USA), which can be converted to 2′,7′-dichlorofluorescein (DCF)
by esterases when taken up. DCF reacts with ROS to generate a new highly
fluorescent compound, dichlorofluorescein, which can be analyzed with FACS. The
treated cells were incubated by H2-DCF-DA (10 µM) at 37 °C for 30 min, washed
twice with PBS, and then measured with FACS.
Western blotting analysis
A549 were plated in 10-cm dishes at a density of 3×106 cells and incubated with
75 µg/mL of trilinolein in F-12 containing 1% (v/v) FBS for 0, 2, 6, 12, 24, and 48 h.
for 5 min. Total proteins were separated using SDS-PAGE before being transferred to
PVDF membranes, blocked with 5% (v/v) nonfat dry milk in PBS-Tween 20 and
probed with the desired antibody (pAkt, Akt, PI3K, p53, p21, Bax, Bcl-2, caspase-3,
caspase-9, cleaved PARP and cytochrome c) (dilution ratio = 1:1000) overnight at
4oC. The blots were then incubated with horseradish peroxidase-linked secondary
antibody for 1 h followed by development with the electrochemoluminsence (ECL)
reagent and exposure to Hyperfilm (Amersham, Arlington Height, IL, USA). The data
were analyzed by Gel-Logic 200 Imaging Systems, Molecular Imaging Software.
Statistical analysis
Values are presented as mean±SE relative to those of the control. Statistically
significant differences from the control group were identified by one-way
ANOVA for the data. p<0.05 was considered significant for all tests.
Results
Cytotoxic effect of trilinolein on A549 cells
In order to determine if trilinolein decreases cancer cell viability, the A549,
MKN-45 and A498 cells were stimulated with various concentrations of trilinolein for
24 h and the cell viability was measured using the MTT assay. Trilinolein treatment
significantly inhibited the cell viability of three cell lines in a concentration-dependent
properties against A549 cells (after 24 h treatment at 75 µg/mL, trilinolein decreased
the A549 cell viability by ~ 52.8%, compared with control). (Fig. 1B).
Trilinolein induces apoptosis in A549 cells
Further experiments using flow cytometry analysis were carried out to
determine if the anti-proliferative effects of trilinolein is the result of apoptotic cell
death. 75 µg/mL trilinolein-treated cell demonstrate higher percentage of hypldiploid
cells than in control cells (Fig. 2A). Also, 75 µg/mL trilinolein-treated cell showed
time-dependent manner (Fig. 2B). This results indicate the the cytotoxic effects
observed in response to trilinolein are correlated with the induction of apoptosis.
Modulation of PI3K/ Akt and activation of p53/p21 protein expression by trilinolein in
A549 cells
The PI3k and phosphorylation status of Akt in A549 cells after trilinolein
treatment was explored to determine if trilinolein-induced apoptosis is correlated with
the Akt signal, which is a downstream effector of PI3K for survival signaling. The
levels of PI3K and phosphorylation Akt were significantly decreased in a
time-dependent manner, and demonstrated significant decrease at 12 and 24 h,
respectively (Fig. 3A). Our results showed that the expression of p53 was markedly
increased at earlier time period with trilinolein treatment and also in time-dependent
trilinolein-treated A549 cells exhibited an increase in p21 expression after 24 h of
treatment.
Modulation of the expression of Bcl-2 family proteins by trilinolein in A549 cells
The expression of the pro-apoptotic factor Bax was significantly increased in
A549 cells after 6 h incubation with 75 µg/mL trilinolein (Fig. 3A). Bcl-2
significantly decreased after 12 h incubation with 75 µg/mL trilinolein treatment in
the A549 cancer cell lines. The Bax/Bcl-2 ratio was significantly elevated after 6 h
treatment (Fig. 3C).
Activates caspases and degradation of PARP by trilinolein in A549 cells
In order to determine if trilinolein-induced apoptosis is associated with the
activation of caspases, the protein expressions of caspase-3 and -9 in A549 cells were
measured. Our results demonstrated that the expression of caspase-3 significantly
increased after trilinolein treatment at 24 h. (Fig. 3B). Moreover, Western blotting
studies suggest that apoptosis induction occurs via the intrinsic pathway because
trilinolein induced the release of cytochrome c from mitochondria and stimulated
the cleavage of inactive pro-caspase-9, resulting in 35-37 kDa active fragments (Fig.
3B). We also analyzed the effect of trilinolein on hydrolysis of the zymogen by
significantly increased in trilinolein-treated cells (Fig. 3B) and PARP, a known
substitute for caspase-3, was effectively hydrolyzed to the 85 kDa fragment.
Trilinolein increased intracellular ROS levels in A549 cells
Production of intracellular ROS in trilinolein-treated A549 cell was monitored by
the oxidation-sensitive fluorescent dye DCFH-DA. An increase in DCFH
fluorescence was detected in trilinolein-treated cells (Fig. 4A, 4B). A rapid production
of ROS was detected at 1 h after treatment although the highest levels were not
reached until 4 h (Fig. 4B). These findings suggest that ROS generation maybe crucial
for trilinolein-induced cell death.
Discussion
Search for new chemopreventive and antitumor agents that are more effective but
less toxic has great interest in phytochemicals. This is the first study to evaluate the
cytotoxic properties of trilinolein in human non-small cell lung carcinoma A549 cells.
A549 was more sensitive to trilinolein cytotoxicity (Fig. 1B). There are no normal
cells or cell lines as controls in the present study. However, previous related studies
indicated that 0.1~10 uM trilinolein have been shown protective effects in astrocytes
and cardiomyocytes (Chiu et al., 1999; Yang et al., 2005).
Our results indicated that trilinolein can cause the accumulation of cells in the
demonstrated that trilinolein was effective in inhibiting the growth of A549 cells in a
dose- and time-dependent manner. Therefore, we investigated the biochemical
mechanism underlying the pro-apoptotic activity of trilinolein in A549 cells.
Trilinolein treatment was shown to induce release of mitochondria c and apoptosis in
A549 cells through modulation of Bax and Bcl-2 proteins and activation of caspase-3.
Moreover, p53-dependent downregulation of Akt may promote an apoptotic cell
death.
Cell cycle analysis revealed that trilinolein caused a significant cell cycle arrest
at the G0/G1 phase (Fig. 2B), accompanied by an increase in sub-G1 (Fig. 2B),
indicating cell death. Downstream target of p53 (e.g. p21) is known to play a role in
cell cycle control by inducing G1 or G2 arrest in response to DNA damage (Yu et al.,
1999) or apoptosis associated with up-regulation of endogenous p21WAF (Kannan et
al., 2001). Our results showed that the level of p21 increased significantly in A549
cells when treated with trilinolein for 24 h following the increase of p53 (Fig. 3A).
This suggests that p21 is involved in a p53-dependent pathway and plays a specific
role in trilinolein-induced G0/G1 cell cycle arrest in A549 cells.
Activation of PI3K/Akt plays an important role in carcinogenesis by maintaining
cancer cell proliferation, preventing apoptosis, and supporting the process of
al., 2010), recent efforts have focused on developing novel antitumor agents targeting
this pathway. It is targeted by genomic aberrations including mutation, amplification
and rearrangement more frequently than any other pathway in human cancer.
Therefore, we studied the effects of trilinolein treatment on the PI3K/Akt signaling
pathway. Trilinolein has been shown to inhibit PI3K and pAkt and upregulate p53
expressions. Our finding suggests that PI3K pathway may have been demonstrated as
the critical mediator in p53 activation in response to trilinolein (Fig. 3A). Our results
demonstrated that if efficient p53-dependent Akt cleavage is triggered, the
Akt-mediated survival signals will be aborted and will not able to block
p53-dependent apoptosis (Gottlieb et al., 2002).
It has been suggested that apoptosis requires the activation of caspases in many
cases (Ashkenazi and Dixit, 1998), we investigated the involvement of caspase
activation in trilinolein-induced apoptosis in A549 cells. Treatment with trilinolein
stimulated a time-dependent cleavage activation of procaspase 3 and PARP. To
elucidate the mechanism of activation of caspase 3 by trilinolein, we examined the
activation of its upstream activator, caspase 9. The activation of caspase 9 was
evidenced by the degradation of its proenzyme. Considering the crucial role of the
mitochondrial pathway in apoptosis, we examined changes in the levels of
increased of cytosolic cytochrome c appeared earlier than activation of caspases (Fig.
3B), we think trilinolein may target to disrupt Bax/Bcl-2 ratio rather than directly
damaging mitochondria integrity (Fig. 3C). When A549 cells were treated with
trilinolein, a decrease in the level of pAkt was observed before caspase-3 activation
(Fig. 3), indicating that Akt inhibition is an upstream event of caspase-3 activation in
trilinolein-induced apoptosis.
ROS are persistently produced during the metabolic process. Under
physiological conditions, the maintenance of an appropriate level of intracellular ROS
is important in keeping redox balance and cell proliferation (Martin and Barrett, 2002).
Excessive ROS accumulation, however, can lead to cellular injury (Mallis et al.,
2001). Recent evidence indicates that accumulated ROS causes sustained JNK
activation and leads toapoptosis (van den Berg et al., 2001). Cancer cells normally
produce more ROS than do normal cells and addition of an agent that increases ROS
may push a tumor cell beyond the breaking point (Schumacker, 2006). So, cancer
cells might be vulnerable to damage by additional ROS stress, either through
inhibiting ROS elimination or by adding exogenous ROS (Huang et al., 2000). The
cell-damaging property of ROS and the increased ROS generation in cancer cells may
provide an opportunity to develop the cell killing potential of ROS by using
the threshold that triggers cell death. We attempted to measure changes in ROS levels
in trilinolein-treated cells. The antiproliferative effect of trilinolein included in this
study is associated with an increase in the intracellular level of ROS which was
detectable at 1 h of treatment and remained elevated for at least 4 h (Fig. 4B). It was
shown that ROS is decreased after trilinolein treatment in cardiomyocytes (Yang et al.,
2005;Chen et al., 2005), however the mechanism responsible for the increase in ROS
generation in trilinolein-treated A549 lung cancer cells is largely unclear. Oncogenic
signals, mitochondrial dysfunction, and active metabolism are likely factors
contributing to the increased production of ROS in cancer cells (Trachootham et al.,
2006). It is plausible that trilinolein could be involved in the regulation with the
abovementioned signals. Other study indicated that elevated accumulation of
resveratrol leads to increased intracellular ROS levels, which then subsequently
induces glioma cell apoptosis (Shao et al., 2009). The pro-oxidant property possibly
results from the generation of phenoxyl radicals of resveratrol by the peroxidase-H2O2
system, which co-oxidizes cellular glutathione or NADH, accompanied by O2 uptake
to form ROS (Galati et al., 2002). Thus, resveratrol probably acts as a pro-oxidant,
disrupting intracellular redox balance and leading to apoptosis, which is the common
postulated mechanism to explain resveratrol’s anti-cancer effect. Further study should
that several mitogen-activated protein kinase (MAPKs) including c-Jun N-terminal
kinase/stress activated protein kinase (JNK/SPK1/2) and p38MAPK play important
roles in triggering apoptosis in response to oxidative stress (Tobiume et al., 2001). We
should further investigate whether MAPKs play a role in trilinolein-induced apoptosis
on A549 cells.
In conclusion, our results suggested that trilinolein induced apoptosis in human
lung carcinoma cells. The pro-apoptotic response was correlated with the increase of
Bax, decrease of Bcl-2, cytochrome c release, caspase-3 activation and PARP
degradation. Furthermore, the inactivation of Akt may play an important role in
trilinolein-induced apoptosis. ROS could be another factor involved in the cell
apoptosis. These results provide the possible mechanisms for the apoptotic activity of
trilinolein.
Acknowledgements
We would like to thank Dr. Jeffery Conrad for critical reading of the manuscript.
Special thanks to Ryan Szynkarek and Matt Szynkarek for editing this paper. This
work was supported by Grants from the China Medical University (CMU97-141 and
CMU98-S-08). This study is supported in part by Taiwan Department of Health
Clinical Trial and Research Center of Excellence (DOH100-TD-B-111-004 and
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Figure legends:
Fig 1. (A) Chemical structure of trilinolein (B) Cytotoxic effects of trilinolein on
A498, MKN-45 and A549 cells. These cancer cells were treated with various
concentrations (0, 3.12, 6.25, 12.5, 25, 50, 75, and 100 µg/mL) of trilinolein for 24 h.
Data are the mean ± SD of three independent experiments.
Fig 2. Determination of the proportion of sub-G1 and G0/G1 cells following
trilinolein treatment of A549 cells, as determined by flow cytometry. (A) Distribution
of cell cycle phase in A459 cells after treatment with various concentrations of
trilinolein (0, 25, 50, 75, and 100µg/mL) for 24 h (B) Distribution of cell cycle
phase in A459 cells after treatment with 75 µg/mL trilinolein for 0, 2, 12, 24 and 48h
Fig 3. (A) Effects of trilinolein on the expression of PI3K, pAkt. Akt, p53 and p21
proteins in A549 Cells as determined using western blotting. Cells were treated with
75 µg/mL trilinolein for the times indicated. (B) Effect of trilinolein on the activity of
apoptosis-associated proteins. A549 cells treated with 75 µg/ml trilinolein and
proteins expression of Bax, Bcl-2, cytochrome c, poly(ADP-ribose) polymerase
Trilinolein induced cleavage of procaspases 9 and 3 in the cytosol. β-Actin was used
as an internal control. (C) The ratio of Bax/Bcl-2 prptein expression at 0, 6, 12, 24 and
48 h. Data are the mean ± SD of three independent experiments. *P ＜ 0.05
compared with control.
Fig 4. Trilinolein-induced ROS generation. (A) A549 cells were incubated for 4 h in
the presence of trilinolein (50, 75 and 100 µg/ml) or (B) with 75 µg/ml of trilinolein
for 1, 2 and 4 h. The fluorescence of oxidized DCF was determined by flow
