#ASO-2010-05-0746# (highlighted manuscript )
Induction of the mitochondria apoptosis pathway by
phytohemagglutinin erythroagglutinating in human lung
cancer cells
Wei-Ting Kuo
a, Yung-Jen Ho
b, Shyh-Ming Kuo
c, Feng-Huei Lin
d,
Fuu-Jen Tsai
e, Yueh-Sheng Chen
b,e†, Guo-Chung Dong
d†, Chun-Hsu
Yao
b,e*
†a
Graduate Institute of Clinical Medical Science, China Medical
University, Taichung 40402, Taiwan
b
Department of Biomedical Imaging and Radiological Science, China
Medical University, Taichung 40402, Taiwan
c
Department of Biomedical Engineering,
I-Shou University, Kaohsiung 84001, Taiwan
d
Division of Medical Engineering Research, National Health Research
Institutes, Miaoli 35053, Taiwan
e
School of Chinese Medicine, China Medical University, Taichung 40402,
Taiwan
* Correspondence and reprint request to:
Dr. Chun-Hsu Yao, Department of Biomedical Imaging and Radiological
Science, China Medical University, Taichung 40402, Taiwan;
Tel: 886-4-22053366 ext. 7806; Fax: 886-4-2205-4179;
Email: [email protected]; [email protected] †
Synopsis
In this paper, PHA-E was used to induce apoptosis of A-549 lung cancer cells, and the
possible signal transduction pathway was determined.and determine the possible
signal transduction pathway. The results show that PHA-E can induce growth
inhibition and cytotoxicity of lung cancer cells, which is mediated through an
Abstract
Background
De-regulation of apoptosis will influence the balance of cell proliferation and cell death, resulting in various fatal diseases that can include cancer. In prior research reports related to cancer therapy, phytohemagglutinin, a lectin extracted from red kidney beans, demonstrated the ability to inhibit the growth of human cancer cells. However, one of its isoforms, erythroagglutinating has yet to be evaluated on its anti-cancer effects.
Methods
PHA-E was used to induce apoptosis of A-549 lung cancer cells and the possible signal transduction pathway was elucidated,in order to determine the possible signal transduction pathway, as measured by the MTT assay, G6PD release assay, flow cytometry, and Western blot analysis.
Results
PHA-E treatment caused a dose-dependent increase of cell growth inhibition and cytotoxicity on A-549 cells. In Annexin V/PI and TUNEL/PI assay, we found that the rate of apoptotic cells was raised as the concentration of PHA-E increased. Treatment of A-549 cells with PHA-E resulted in enhancing the release of cytochrome c, which thus activated an increase in caspase 9 and caspase 3, the up-regulation of Bax and Bad, the down-regulation of Bcl-2 and phosphorylated Bad, and finally the inhibition of the epidermal growth factor receptor and its downstream signal pathway PI3K/Akt and MEK/ERK.
Conclusions
mitochondria apoptosis pathway. These results suggest that PHA-E can be developed into a new therapeutic treatment that can be applied as an effective anti-lung cancer drug in the near future.
Keywords: Apoptosis; Lung cancer; Phytohemagglutinin Erythroagglutinating; Epidermal growth factor receptor
Introduction
Lung cancer, generally divided into small cell lung cancer (SCLC) and non-small
cell lung cancer (NSCLC), is the leading cause of cancer-related deaths in both men
and women worldwide [1, 2]. NSCLC accounts for approximately 85% of lung cancer
cases, and is furthermore histologically subdivided into adenocarcinoma (50%),
squamous cell carcinoma (30%) and large cell carcinoma (20%) [3]. Therefore, it is
critical to study new and effective drug treatments, and develop novel therapeutic
strategies for the management of lung cancer. Past studies have revealed that both
growth inhibition and apoptosis induction are important determinants in response to
anti-cancer therapy [4].
Apoptosis, programmed cell death, is a physiological mechanism that is required
for maintaining cell numbers and removing unnecessary cells [5, 6]. The two major
pathways that trigger apoptosis are an extrinsic pathway initiated by death receptors
and an intrinsic pathway that occurs through the mitochondria. The extrinsic pathway
is activated at the cell surface when a particular ligand binds to its respective death
receptors. The intrinsic pathway involves the release of cytochrome c from the
mitochondrial intermembrane space into the cytoplasm by the loss of mitochondrial
downstream caspases such as caspase 3, and finally results in apoptosis [7-11].
Additionally, apoptosis pathway of cells was also controlled by Bcl-2 family proteins
[12, 13]. Morphological and biochemical features of apoptosis include cell membrane
blebbing, cell shrinkage, chromatin condensation and DNA fragmentation, and the
final stage includes the engulfment by macrophages or neighboring cells [5].
De-regulation of apoptosis can affect the balance of cell proliferation and cell death,
resulting in various diseases that can include cancer [14, 15]. Thus, drugs devised to
re-stimulate apoptosis might be an effective method in fighting against cancer [16,
17].
Phytohemagglutinin (PHA) is the lectin derived from red kidney beans (Phaseolus vulgarus), and phytohemagglutinin erythroagglutinating (PHA-E or E4) belongs to the PHA isoform groups [18, 19]. The content of PHA in red kidney beans is reported to be 0.4-1.2% [20]. PHA consists of four subunits which are grouped into E- and L-subunits. E-subunit (for erythroagglutinating) appears to be involved in erythrocyte agglutination, and the other type L-subunit (for leucoagglutinin) is involved in lymphocyte agglutination. These subunits are synthesized alongside in the endoplasmatic reticulum and then randomly combined to form five isolectins that are classified the structures L4, L3E1, L2E2, L1E3, and E4. All of these isolectins are glycoproteins of approximately 125 kDa [21]. PHA is usually used for the activation
of normal T-cells [22], and has been demonstrated to exhibit antifungal, antiviral, and HIV-1 reverse transcriptase inhibitory activities [23, 24]. In prior literature related to cancer therapy, PHA was also reported to inhibit the growth of human cancer cells [25, 26]. Also, the PHA-L4 has also been reported to exert its therapeutic effects through the ability to exhibit direct antitumor cytotoxic effects, to enhance the antineoplastic effect of radiation and chemotherapy, and to decrease the liability to malignant transformation [27]. However, PHA-E has never been evaluated on its anti-cancer effects against lung cancer cells. In this study, we aimed at evaluating the ability of PHA-E to induce apoptosis and investigating its effects and on the signal transduction in lung cancer cells A-549.
Phytohemagglutinin (PHA), the lectin derived from red kidney beans (Phaseolus
vulgarus), has been demonstrated to inhibit the growth of human cancer cells [18].
PHA-E (erythroagglutinating) belongs to the PHA isoform groups [19]. This study
evaluated the ability of PHA-E to induce apoptosis and its stimulated effect on the
Materials and methods
Materials
PHA-E was purchased from Sigma (USA). The anti-bodies for α-Tubulin (B-7),
p-PI 3-kinase p85α (Tyr 508), p-MEK-1/2 (Ser 218/Ser 222), p-ERK (E-4), Bad (C-7),
Bax (P-19), Bcl-2 (C-2) and cytochrome c (7H8) were purchased from Santa Cruz
(USA). The anti-bodies for phospho-Akt (Ser473), phospho-Bad (Ser155) and
cleaved caspase 9 (Asp353) were purchased from Cell Signaling (USA). The
anti-body for caspase 3 was purchased from Millipore (USA). The anti-bodies for
EGFR and phospho-EGFR (Y1068 and Y1101) were purchased from Abcam (USA),
and EGF itself was purchased from Upstate (USA). The cytotoxicity assay kit,
Annexin V conjugates, APO-BrdU™ TUNEL assay kit, propidium iodide solid and
DAPI dilactate were all purchased from Invitrogen (USA).
Cell culture
A-549 (human lung adenocarcinoma) cells were obtained from BCRC (Taiwan).
2 mM L-glutamine, and 1.5 g/L sodium bicarbonate. The cells were maintained under
standard cell culture conditions at 37°C and 5% CO2 in a humid environment.
Typically, adherent cells were harvested with trypsin and re-suspended in a
serum-containing medium before use in the assays as described below.
MTT assay
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) MTT
(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed
to determine the effect of PHA-E on cell growth. This assay is based on the cleavage
of the yellow tetrazolium salt MTT to purple formazan crystal by mitochondrial
succinate dehydrogenase of living cells. This reduction occurs only when
mitochondrial reductase enzymes are active so that they can be directly related to the
number of viable cells. The crystals can be dissolved in DMSO, and the concentration
determined by microplate reader at 570 nm. The result is proportional to the cell
numbers.
Briefly, A-549 cells were seeded at 10,000 10000 cells/well in 24-well plates and
incubated for 24 h hr. The medium was then replaced with serum-free medium,
medium was then replaced with 0.5 mg/ml MTT medium and incubated for 4 h hr.
The MTT solution was removed from the wells and the formazan crystals were
dissolved in DMSO. Finally, the concentration was determined by a microplate reader.
G6PD release assay
Glucose-6-phosphate dehydrogenase (G6PD) G6PD release assay was performed
to determine the effect of PHA-E on cell death. The number of dead cells was
assessed by measuring the release of cytosolic enzyme G6PD glucose-6-phosphate
dehydrogenase (G6PD) from damaged cells into the surrounding medium, using a
cytotoxicity assay kit and following the manufacturer’s instructions. The assay
quantifies plasma membrane integrity as released G6PD, based on the
G6PD-dependent conversion of NADP+ to NADPH and subsequent reduction of
resazurin to resorufin, which emits fluorescence at 590 nm after excitation at 530 nm.
Briefly, A-549 cells were seeded at 10,000 10000 cells/well in 24-well plates and
incubated for 24 h hr. The medium was then replaced with a serum-free medium,
PHA-E (20-160 μg/ml), which was added and co-cultured for 48 h hr. After
homogenized, only 50 μl/well of the supernatant medium was transferred to 96-well
light-protected incubation for 20 min,. at 37°C, measurements were made by using the
fluorescence microplate reader.
Annexin V/PI assay
Annexin V assay was used to quantify the number of apoptotic cells by flow
cytometry. In apoptotic cells, phosphatidylserine (PS) is translocated from the inner to
the outer leaflet of the plasma membrane. Annexin V labeled with a fluorophore can identify apoptotic cells by binding to PS exposed on the outer leaflet.
Briefly, cells were harvested after treatment of PHA-E, washed in a cold PBS
and re-suspended in 100 μl binding buffer. Samples were incubated with 5 μl Annexin
V and 2 μl of the 100 μg/ml PI in a dark environment for 15 min at room temperature.
The volume was adjusted to 200 μl with a binding buffer, and fluorescence was then
measured with a flow cytometry cytometer.
TUNEL/PI assay
Terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL) TUNEL
identify cell apoptosis. In apoptotic cells, nuclei that are activated eventually degrade
their nuclear DNA into fragments. The TUNEL assay kit can detect the DNA
fragmentation of these apoptotic cells.
After treatment of PHA-E, the harvested cells were briefly fixed on ice for 15
min with 1% formaldehyde, then followed with an overnight permeabilization in 70%
ethanol at -20°C. The 70% ethanol was removed and the fixed cells were washed.
This process was followed by incubation with a DNA-labeling solution at 37°C
for 60 min. An antibody solution was subsequently added and the cells were treated in
a dark environment for 30 min at room temperature. Finally, a PI/RNase solution used
to detect their specific stage of cell cycle was added to the cells, which were
maintained in the dark environment for an additional 30 min at room temperature.
Fluorescence was measured with a flow cytometry cytometer.
Western blot analysis
After treating the cells with PHA-E, they were then rinsed twice with PBS. The
cells were then directly solubilized in a lysis buffer (0.5 M Tris-HCl, pH 7.4, 1.5 M
NaCl, 2.5% deoxycholic acid, 10% NP-40, 10 mM EDTA) containing a protease
collected in a eppendorf tube. The lysate was cleared by centrifugation at
12,00012000g for 30 min. at 4°C, and the protein concentration in the supernatant
was determined by the Bradford method (Bio-Rad protein assay).
For Western blotting, equal amounts of proteins were resolved over 10-12%
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE
(sodium dodecyl sulfate polyacrylamide gel electrophoresis) and transferred onto a
polyvinylidene fluoride (PVDF) PVDF (polyvinylidene fluoride) membrane. The
nonspecific sites on blots were blocked by incubating them in a blocking buffer (5%
nonfat dry milk/TBS, pH 7.4) for 1 h hr at room temperature. Incubation was
performed with an appropriate monoclonal primary antibody in TBS overnight at 4°C,
and then incubated with a horseradish peroxidase (HRP) conjugated secondary
antibody for 1 h hr at room temperature. Immunoreactive bands were visualized using
an enhanced chemiluminescence system.
Statistical analysis
Numerical data were presented as mean standard deviation. Statistical
differences among samples were evaluated by one-way analysis of variance (ANOVA)
Results
PHA-E results in growth inhibition and cell death
In this study, we first evaluated the cell viability of E-PHA on A-549 cells by
MTT assay. As shown in Figure 1a, PHA-E treatment of A-549 cells resulted in cell
growth inhibition by 13, 27, 41 and 49% at the doses of 20–160 μg/ml. To investigate
cytotoxicity, we used an assay that measured the leakage of the enzyme G6PD
glucose 6-phosphate dehydrogenase (G6PD) from the cytoplasm into the culture
medium. PHA-E treatment (20-160 μg/ml; 48 h hr) caused a dose-dependent increase
of fluorescence in A-549 cells (Fig. 1b). Because the resulting fluorescence signal is
proportional to the amount of G6PD release, PHA-E treatment results in cytotoxicity
of A-549 cells.
PHA-E induces apoptosis
We next confirmed whether PHA-E induced growth inhibition and cytotoxicity of
A-549 cells is mediated through the induction of apoptosis, and . We quantified cell
analyzed by Annexin V/PI assay. Early apoptotic cells were the Annexin V-positive
but PI-negative cells, while late apoptosis were the Annexin V-positive and
PI-positive cells. The apoptotic cells were the sum of early apoptosis and late
apoptosis. Dead cells were the PI-positive but Annexin V-negative cells, live cells
were the PI- negative and Annexin V-negative cells (Fig. 2a). As shown in Table 1,
after treatment with PHA-E at 20, 40, 80 and 160 μg/mL for 48 h hr,, the apoptotic
cells were 7.4±2.7%, 18.2±2.5%, 22.6±4.4% and 35.6±1.3%5.9%, 15.8%, 20.2% and
36.5%, respectively, which were higher than the control at 2.6±0.3%2.9%. Dead cells
were a dose-dependent increase and live cells were a reduction. In addition, the same
trend of apoptosis rate was determined by TUNEL/PI assay. PHA-E treatment of
A-549 cells resulted in 6.4%, 7.1%, 8.9% and 14.6% of apoptotic cells at the doses a
dose of 20, 40, 80 and 160 μg/mL, respectively (Fig. 2b).
PHA-E increases the expression level of cytochrome c, cleaved caspase 9 and cleaved
caspase 3
The mitochondria play an important role in apoptosis by releasing apoptogenic
factors such as cytochrome c. The release of cytochrome c can activate caspase 9 and
cleaved caspase 9 and cleaved caspase 3. As shown in Figure 3, cytochrome c was
increased after treatment with PHA-E by Western western blot analysis. The
expression level of cleaved caspase 9 and cleaved caspase 3 were also enhanced when
the PHA-E dose was raised.
PHA-E results in up-regulation of Bad and Bax and down-regulation of phospho-Bad
and Bcl2
Because Bcl-2 family members are the major regulators of apoptosis, we next
determined the effect of PHA-E on the protein levels of Bad, phospho-Bad, Bcl-2 and
Bax in A-549 cells. The Western blot analysis exhibited an increase in the expression
of Bad and Bax concomitant with a decrease in the expression of phospho-Bad and
Bcl-2 in a dose-dependent fashion (Fig. 4), further confirming the induction of the
apoptotic process.
PHA-E inhibits the expression level of EGFR and phosphorylation of EGFR, PI3K,
Akt, MEK and ERK
cancers, and is associated with lower rates of survival. We assessed the effect of
PHA-E on EGFR and its specific phosphorylation sites Y1068 and Y1101. Treatment
of A-549 cells with PHA-E resulted in a dose-dependent decrease in the specific
phosphorylation sites Y1068 and Y1101, and the expression level of EGFR also
reduced (Fig. 5a). Downstream signaling pathways of EGFR include the PI3K/AKT
and MEK/ERK pathways pathway. We next determined the effect of PHA-E
treatment to A-549 cells on the expression of phosphorylation of PI3K, Akt, MEK and
ERK, and we found that these molecules investigated were all inhibited We found that
Discussion
As a result of its poor prognosis, lung cancer is the leading cause of cancer-related
deaths in the world. Therefore, the development of novel anti-cancer drugs that can
provide effective drug treatments is necessary to enhance the quality of life. It is
important to develop treatments that can effectively trigger cell death in tumor cells
during cancer therapy. Previous studies have demonstrated that effective treatment
concerning anti-cancer drugs is largely dependent on their ability to inhibit growth
and induce apoptosis in cancer cells [20].
PHA has been reported to possess anti-cancer effects on several human cancer cells [18], but one of its isoforms, PHA-E PHA- E, had not been thoroughly explored until this study.
As a result of its poor prognosis, lung cancer is the leading cause of cancer-related deaths in the world. Therefore, an early diagnosis and aggressive therapy are important to maintaining a patient’s quality of life. Once diagnosed, a patient’s anti-lung cancer therapy can usually consist of surgery, radiotherapy and chemotherapy. As scientific research develops, a significant progress in the development of targeted therapy drugs in conjunction with radiochemotherapy can have a better outcome on anti-cancer treatment [28]. It is important to channel this research towards the development of treatments that can effectively trigger cell death in tumor cells during cancer therapy. The reason being is that previous studies have
successfully demonstrated that effective anti-cancer drugs are largely dependent on their ability to inhibit growth and induce apoptosis in cancer cells [29].
PHA has been reported to possess anti-cancer effects on several different forms of human cancer cells [18]. One of its isoforms, PHE-L, has been used to recognize breast carcinoma metastasis by binding β1,6-branched oligosaccharides [30]. Another isoform, PHA-E, has been proved to bind to the bisecting GlcNAc in pediatric brain tumors which could be a diagnostic tumor marker [31], but it had not been thoroughly explored on lung cancer treatment prior to this study. Our project examined the effects
of PHA-E on lung cancer cell A-549. According to our research, the data presented
here suggests that PHA-E resulted in a dose-dependent increase in growth inhibition
and cell death of A-549 cells (Fig. 1). Apoptosis is associated with cell number
reduction and cytotoxicity enhancement, therefore our main objective was to
determine whether apoptosis was involved in PHA-E-induced cell death. We used two
assays to quantify cell apoptosis by flow cytometry. The apoptotic cells were the sum
of early apoptosis (Annexin V-positive and PI-negative) and late apoptosis (Annexin
V-positive and PI-positive). Our results suggest that as the concentration of PHA-E
was increased, the proportion rate of apoptotic cells was also raised (Fig. 2a, Table 1).
In addition, dead cells (PI-positive and Annexin V-negative) and live cells
increase and decrease according to the results of the G6PD release assay and MTT
assay. Another evaluation method, TUNEL/PI assay, determined the same trend of
apoptosis induced. Our data suggests that PHA-E induced growth inhibition and
cytotoxicity of A-549 cells is mediated through an induction of apoptosis.
The mitochondrial apoptotic pathway is one of the major pathways to initiate
apoptosis, which releases pro-apoptotic proteins such as cytochrome c from the
mitochondria to cytosol. Cytochrome c then combines with an apoptotic
protease-activating factor 1 (Apaf-1) and induces the formation of a complex known
as apoptosome. Apoptosome possesses a caspase recruitment domain (CARD), which
allows it to bind and process the crucial initiator, caspase 9. Activated caspase 9 then
cleaves and activates the downstream effector effectors of apoptotic cell death known
as caspase 3 [21][32]. Our objective was to identify whether PHA-E induced
apoptosis through an activation of the mitochondrial apoptotic pathway, therefore our
study assessed the expression levels of cytochrome c, cleaved caspase 9 and cleaved
caspase 3 (Fig. 3). Our data suggests that treatment with PHA-E could promote the
release of cytochrome c, which as a result can activate caspase 9 and caspase 3,
inducing apoptosis through the mitochondrial apoptotic pathway. Cytochrome c can
be released from a remarkable event called mitochondrial outer membrane
[33].
Bcl-2 family members are grouped into the following three classes: one class that
inhibits apoptosis such as Bcl-2, a second class that promotes apoptosis proteins such
as Bax, and a third divergent class of BH3-only proteins such as Bad [23][34].
Although Bcl-2 can prevent cytochrome c release and apoptosis, Bax is shown to
trigger cytochrome c release in cells [24][35]. Bad is activated by a loss of
phosphorylation [25][36], and it has a conserved BH3 domain that can bind and
regulate the anti-apoptotic protein Bcl-2 to promote apoptosis [26][37]. Recent
evidence indicates that Bad can affect Bax by directly binding and inhibiting Bcl-2
[27][38]. The present study suggests that treatment with PHA-E caused
de-phosphorylation and activation of Bad, which resulted in the activation of Bax and
an inactivation Bcl-2, triggering apoptosis (Fig. 4). Activated PI3K/Akt and
MEK/ERK pathways have been reported to promote survival signaling by
phosphorylating Bad. These phosphorylated residues result in the binding of Bad to
14-3-3 proteins, which subsequently inhibit Bad to bind with Bcl-2 [28, 29][39, 40].
However, PI3K/Akt and MEK/ERK pathways can be activated by the epidermal
growth factor receptor (EGFR) [30][41].
In literature, dysregulation of human epidermal growth factor receptor (EGFR) pathways by over-expression or constitutive activation can promote tumor processes
including angiogenesis and metastasis and is associated with poor prognosis in many human malignancies [42]. Clinically, EGFR and v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation analysis is the most reliable predictor of the lung carcinoma response to EGFR-targeted therapies [43]. In lung cancer, some papers have shown that EGFR was commonly overexpressed, especially in the non-small cell lung cancers [44, 45] but the detail mechanism is not well known. In addition, the activation of its downstream-activated proteins, such as p-Akt and p-STAT3, were correlated with well-differentiated tumor. In this study, we investigated the effects of PHA-E on activated-EGFR and overexpression of p-Akt in lung cancer cells. As we expected, PHA-E could effectively reverse the activation of p-Akt, which is consistent with the previous findings. EGFR is a member of ErbB
family of tyrosine kinase receptors (RTK) [31], and it is a transmembrance
glycoprotein comprised of extracellular ligand-binding domain, transmembrane
domain, intracellular protein tyrosine kinase domain and C-terminal domain.
Activation of EGFR occurs when a ligand such as EGF binds to the extracellular
domain and induces dimerization of EGFR, leading directly to an activation of
tyrosine kinase domain, ensuing autophosphorylation of C-terminal tyrosine residues,
and activation of the downstream signaling pathway [32, 33]. Disruption of EGFR
been reported to possess inhibition of EGFR auto-phosphorylation [35]. Our study
suggests that treatment with PHA-E not only caused inhibition of EGFR, but also
resulted in blocking the PI3K/Akt and MEK/ERK signaling pathways that promoted
Conclusions
In this paper, we have found that PHA-E effectively inhibited the growth of A-549 cells and induced their apoptosis. As for apoptosis induction, PHA-E not only caused inhibition of EGFR, but resulted in blocking the PI3K/Akt and MEK/ERK signaling pathways that promoted activation of Bad, which can enhance the release of cytochrome c and activate caspase-9 and caspase-3. However, we also found that PHA-E suppressed tumor growth but the detail mechanisms need further investigation. The multiple approached evidences suggested that PHA-E could be developed into an effective anti-lung cancer drug in the near future, while the animal feeding model, xenograph assays and drug toxicity evaluation are needed before its clinical trials.
This study has demonstrated that PHA-E can induce growth inhibition and
cytotoxicity of lung cancer cells, which is mediated through the activation of the
mitochondria apoptosis pathway. We conducted a thorough MTT assay, G6PD release
assay, flow cytometry, and Western blot analysis in order to determine which specific
signal transduction pathway was responsible for cell growth inhibition and
cytotoxicity on A-549 cells. Our aforementioned research suggests that PHA-E can
directly inhibit lung cancer cell growth by triggering the apoptosis process through the
developed into an effective anti-lung cancer drug in the near future.
Acknowledgments
The authors would like to thank the China Medical University, for financially supporting this research under Contract No. CMU97-230.
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Figure legends
Figure 1: The effects of PHA-E on cell viability and cytotoxicity. A-549 cells were treated with PHA-E (20-160 μg/ml) for 48 h hr. (a) The cell viability was determined by MTT assay. (b) Cell cytotoxicity was determined by G6PD release assay. The data shown are mean SD for n = 6. *p < 0.05; **p < 0.01 vs. control.
Figure 2: The effects of PHA-E on cell apoptosis by flow cytometry. A-549 cells were treated with PHA-E (20-160 μg/ml) for 48 h hr. (a) The percentage of apoptotic cells was detected using Annexin V/PI assay. Q2 quadrant represented late apoptosis, while Q4 quadrant represented an early phase of apoptosis. (b) Apoptosis rate (P2) was considered using TUNEL/PI assay.
Figure 3: The effects of PHA-E on cytochrome c, cleaved caspase 9 and cleaved caspase 3 expression. A-549 cells were treated with PHA-E (20-160 μg/ml) for 6 h hr. Equal protein loading was confirmed by probing for α-tubulin.
Figure 4: The effects of PHA-E on the expression of Bcl-2 family members. A-549 cells were treated with PHA-E (20-160 μg/ml) for 6 h hr. Expressions of Bad, phospho-Bad, Bcl-2 and Bax. Equal protein loading was confirmed by probing for α-tubulin.
Figure 5: The effect of PHA-E on EGFR and phosphorylation of EGFR, PI3K, Akt, MEK and ERK. The A-549 cells were serum-starved for 24 h hr, co-cultured with PHA-E for 3 h hr, then treated with 50 ng/ml EGF for 3 h hr. (a) The effects of PHA-E on EGFR and its specific phosphorylation sites Y1068 and Y1101on. (b) The effects of PHA-E on the phosphorylation of PI3K, Akt, MEK and ERK. Equal protein loading was confirmed by probing for α-tubulin.
Figure 6: This model exhibits the PHA-E-induced apoptotic pathway in human lung cancer cells A-549. Treatment with PHA-E causes inhibition of EGFR, and results in the blockage of PI3K/Akt and MEK/ERK pathways. Dephosphorylation of Bad promotes the inhibition of Bcl-2 and the activation of Bax. This process releases cytochrome c, which activates caspase 9 and caspase 3, leading to apoptosis.
