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Research Article
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Proliferative Inhibition and Apoptotic Mechanism on Human Non-small-cell
Lung Cancer (A549 Cells) of a Novel Cucurbitacin from Wilbrandia ebracteata
Cogn
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I.T. Silva,
Marina R. Teixeira,
Karen L. Lang,
Tatiana da R. Guimaraes,
Sabine E. Dudek,
Fernando J. Duran,
Stephan Ludwig,
Miguel S.B. Caro,
Eloir P. Schenkel
and
Claudia M.O. Simoes
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ABSTRACT
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Cancer represents a major public health problem from all over the world and
the lung carcinoma is the leading cause of cancer death. In this sense, the
aim of the present study was to examine the cytotoxic effects of a novel Cucurbitacin
(Cuc1) isolated from Wilbrandia ebracteata on human non-small-cell lung
cancer (A549). In order to achieve this aim, the cell proliferation was measured
by MTT assay and actin cytoskeleton was stained by rhodamine-phalloidin, whereas,
the cell cycle distribution and apoptosis induction were quantified using flow
cytometry. The signal transduction profiling of Cuc1 treated cells, as well
as the levels of apoptotic proteins were analyzed by Western blotting. Cuc1
significantly inhibited cell growth showing IC50 values of 13.5±1.8 and
3.8±0.4 μM for 48 and 72 h of treatment, respectively. Additionally,
Cuc1 arrested cell cycle at G2/M phase, disrupted the actin dynamics and induced
apoptosis since the amount of apoptotic cells increased from 5.18±0.585%
in the untreated cells to 73.82±0.545% in the treated cells. Detailed
analysis on the mechanism of action revealed that Cuc1 inhibited the phosphorylation
of Protein Kinase B (PKB/AKT) and Signal Transducer and Activator of Transcription
(STAT3) signaling pathways, down-regulating the expression of Bcl-2 and consequently
inducing cytochrome c release from the mitochondria to the cytosol. These
results suggest that the Cuc1 could be a potential candidate for cancer chemotherapy
agent.
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How
to cite this article:
I.T. Silva, Marina R. Teixeira, Karen L. Lang, Tatiana da R. Guimaraes, Sabine E. Dudek, Fernando J. Duran, Stephan Ludwig, Miguel S.B. Caro, Eloir P. Schenkel and Claudia M.O. Simoes, 2013. Proliferative Inhibition and Apoptotic Mechanism on Human Non-small-cell
Lung Cancer (A549 Cells) of a Novel Cucurbitacin from Wilbrandia ebracteata
Cogn. International Journal of Cancer Research, 9: 54-68. DOI: 10.3923/ijcr.2013.54.68 URL: https://scialert.net/abstract/?doi=ijcr.2013.54.68
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Received:
March 12, 2013; Accepted: April 18, 2013;
Published: October 28, 2013 |
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INTRODUCTION
Cancer can be considered a major public health problem all around the world.
In the United States, 25% of all deaths are related to cancer. Only in 2011,
were estimated 221,130 new cases and 156,940 deaths related to lung cancer specifically.
Worldwide, the non-small-cell lung carcinoma (NSCLC) is the leading cause of
cancer death in both men and women (Han and Roman, 2010;
Siegel et al., 2011). In spite of quick advances
in diagnostic and surgical techniques, lung cancer remains one of the hardest
treatable human cancers. Until today treatment modalities are ineffective and
new therapies are necessary to reduce the effects of the increasing incidence
in this type of cancer (Kim et al., 2003).
Apoptosis is a form of programmed cell death that permits to eliminate unwanted,
redundant, or mutated cells from organisms, without causing damage to the cellular
microenvironment (Russo et al., 2006). In cancer
cells, multiple genetic mutations and the overall cellular stresses of malignant
transformation are associated with substantial pro-apoptotic activity (Call
et al., 2008). However, cancer can persist and dysregulation of apoptosis
might be essential for the survival of many cancers providing inherent resistance
to chemotherapeutic agents (Tan and White, 2008; Sayers,
2011). Thus, a rational approach for treating cancer is the modulation of
apoptosis pathway by targeting components of the apoptotic mechanism and its
regulators in order to reestablish the apoptotic function (Nicholson,
2000; Call et al., 2008; Indran
et al., 2011).
Plants synthesize a wide variety of biologically active compounds and for that
reason there is a growing interest in their use as a source of new anticancer
drugs (Lee et al., 2011). Cucurbitacins, for
instance, are a group of diverse tetracyclic triterpenoid molecules, predominantly
found in different species of the Cucurbitaceae family that are highly bitter
and toxic. Their structural configuration compromises tetracyclic cucurbitane
nucleus skeleton, with a variety of oxygenation functionalities at different
positions (Chen et al., 2005). Plants containing
cucurbitacins have been used for centuries with ethnomedical purposes (Lee
et al., 2010) and several compounds of this group have shown antitumor,
anti-inflammatory and hepatoprotective effects (Jayaprakasam
et al., 2003; Rios et al., 2012).
They have also shown strong activity against tumor expansion (Sun
et al., 2010; Yasuda et al., 2010)
as demonstrated for cucurbitacins B, D, E and I that inhibited the growth of
several cancer cell lines in in vitro and in vivo models (Su
et al., 2008; Wakimoto et al., 2008;
Yin et al., 2008; Chen et
al., 2010; Dong et al., 2010; Sun
et al., 2010; Yasuda et al., 2010;
Zhang et al., 2010; Hsu
et al., 2011; Ishdorj et al., 2011).
Recently, novel cucurbitacins obtained from Wilbrandia ebracteata Cogn.
(Cucurbitaceae) have been described (Lang et al.,
2011) as well as some semi-synthetic derivatives (Lang
et al., 2012) that exhibited promising cytotoxic activity against
different human cancer cell lines. The present study examined the impact of
one of these new cucurbitacins (2β, 16α, 2R-trihydroxy-10α, 17α-cucurbit-5,
25-dien-3, 11, 22-trione, Cuc 1) (Fig. 1) on non-small-cell
lung cancer (A549 cells), including the evaluation of its effects on cell growth,
cell cycle distribution, apoptosis, morphological changes and expression of
regulatory proteins involved in such processes.
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Fig. 1: |
Structure of a novel cucurbitacin (Cuc 1) isolated from Wilbrandia
ebracteata roots |
MATERIALS AND METHODS
Isolation and identification of Cuc 1: Roots of Wilbrandia ebracteata
Cogn. were purchased from Lohmann Company Ltd. and authenticated by Prof. Dr.
Sergio A.L. Bordignon (Unilassale, Canoas, RS, Brazil). Dried and powderedroots
(1.5 kg) were extracted with dichloromethane (DCM) at room temperature for 72
h. The DCM extract (9 g) was subjected to vacuum liquid column chromatography
on silica gel using hexane with increasing amounts of ethyl acetate (20-100%)
to afford eight fractions (F1 to F8). The fractions F3 and F4 (500 mg) were
combined and subjected to column chromatography using silica gel as adsorbent
and hexane/ethyl acetate 40% as mobile phase providing Cuc 1 (10 mg) which was
identified by spectroscopic methods as described previously (Lang
et al., 2011).
Cell line: The human non-small-cell lung cancer (A549 cells) was kindly provided by Dr. Rosina Gironès from Microbiology Department of University of Barcelona, Spain. A549 cells (ATCC-CCL185) were grown in Minimal Essential Medium supplemented with 10% fetal bovine serum, 100 U mL-1 penicillin G and 100 μg mL-1 streptomycin in a humidified 5% CO2 atmosphere at 37°C.
Cell viability assay: The effect of Cuc 1 treatment on the viability
of A549 cells was determined by MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide] (Sigma, MO, USA) based on the ability of live cells to
cleave the tetrazolium ring to a molecule that absorb at 540 nm (Mosmann,
1983). Briefly, cells were plated in 96-well culture plates (1¯104
cells/well). After 24 h, seeding cells were treated with different concentrations
of Cuc 1. After 48 and 72 h of treatment at 37°C, the medium was replaced
by 50 μL of MTT reagent (1 mg mL-1) and cells were further incubated
at 37°C for 4 h. The MTT solution was removed, 100 μL of dimethyl sulfoxide
was added to each well, followed by reading on a scanning multiwell spectrophotometer
(Infinite 1200 TECAN, Grödje, Austria). The 50% inhibition concentration
(IC50) was defined as the concentration that inhibited cell proliferation
by 50% when compared to untreated controls (=untreated cells). Paclitaxel (Sigma,
MO, USA) (at 0 to 10 μM) was used as positive control. Final solvent concentration
showed no interference with cell growth.
Cell cycle analysis by flow cytometry: A549 cells (5x105/six-well) were treated with Cuc 1 for 24 h and harvested by 0.25% trypsin. Then cells were washed twice with Phosphate-buffered Saline (PBS), centrifuged at 500x g for 5 min, fixed in 70% ethanol at -4°C. After fixation, the cells were treated with 50 μg mL-1 RNase and stained with 100 μg mL-1 Propidium Iodide (PI) in the dark. Flow cytometry analyses were carried out on a FACS CAnto II instrument (Becton Dickinson, USA). The population of cells in each cell-cycle phase was determined using FlowJo 8.6.3 software (Tree Star, Inc., Ashland, USA). Apoptosis analysis by flow cytometry: Phosphatidylserine (PS) exposed on the outside of apoptotic cells was detected by Annexin V-FITC and PI double-staining by using a detection kit purchased from Sigma (MO, USA). Briefly, adherent A549 cells (5x105/six-well) were treated with Cuc 1 for 12 h. Cells were harvested and rinsed twice with PBS (pH 7.4) followed by Annexin V-FITC and PI labeling according to the manufacturers instructions. The stained apoptotic cells were analyzed by flow cytometer (FACS CAnto II, Becton Dickinson, USA). Cytoskeletal and nuclei staining: F-actin detection was performed on A549 cells grown on coverslips placed into a six-well plate. Cells were treated with Cuc 1 for 12 h and fixed by 4% (w/v) paraformaldehyde in PBS for 15 min at room temperature, washed in PBS and permeabilized with 0.5% (v/v) Triton X-100 for 30 min at RT. After three-times washing, cells were incubated with TRITC-labeled-phalloidin (Invitrogen, Carlsbad, USA) for 40 min in the dark. Preparations were washed and stained with Hoechst (Invitrogen, Carlsbad, USA) for 5 min to detect nuclei, washed and finally mounted in mounting medium (80% glycerol in PBS). Confocal images were collected on a Leica DMI6000 B microscope (Leica Microsystems, Wetzlar, Germany). Caspase assay: Caspases -3, -8 and -9 protease activity was determined by using commercially available kits (Millipore, MA, USA) according to the manufacturers instructions. The tests are based on the spectrophotometric detection of the chromophore p-nitroanaline (pNA) after cleavage from the caspase substrate (a caspase-specific peptide conjugated to pNA). Briefly, adherent A549 cells (5x105/six-well) were treated with Cuc 1 for 12 h and harvested by 0.25% trypsin. The cells were resuspended in 50 μL of chilled cell lysis buffer, centrifuged for 1 min (10,000xg) and then the supernatants were transferred (cytosolic extract) to a fresh tube and put on ice for immediate assay. The cell lysates were tested for protease activity by the addition of a labelled caspase substrate (DEVD-pNA for caspase-3 activity, IETD-pNA for caspase-8 activity and LEHD-pNA for caspase-9) and incubated at 37°C for 2 h. pNA absorbance was quantified using a spectrophotometer (Infinite 1200 TECAN, Grödje, Austria) at a wavelength of 405 nm. Fold-increase in caspase-activity can be determined by comparing the OD reading from the induced apoptotic sample with the level of the uninduced control. Western blotting analysis: Adherent A549 cells (1x106/six-well) were treated with Cuc 1 for 12, 24 or 48 h. To evaluate the influence of Cuc 1 on Akt-, MAPKs- and NFκB- signaling pathways, TNFα stimulation (30 ng mL-1, 20 min) was used as a pathway inducer. Whole cell lysates were harvested after lysis with RIPA buffer containing proteases and phosphatases inhibitors and then centrifuged at 10,000x g for 10 min at 4°C. The supernatants were collected and the protein concentrations were determined using the Bradford assay. Equivalent amounts of protein were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by electrotransfer to a nitrocelullose membrane (Schleicher and Schuell, Dassel, Germany). After blocking, membranes were incubated overnight with appropriate primary antibodies. After incubation with the corresponding secondary antibodies conjugated to horseradish peroxidase, protein bands were revealed by using Pierce ECL substrate (Thermo Scientific, MA, USA), according to the manufacturers protocol. Cytosolic fraction was prepared for detecting cytochrome c release. For this, treated and untreated A549 cells were washed twice with cold PBS and lysed with 200 μL of extraction buffer, containing proteases inhibitors, for 10 min on ice. Next, cells were homogenized by 10 passages through a 26-gauge needle. Homogenates were centrifuged at 1,000x g for 5 min to remove unbroken cells and nuclei. The supernatant fraction was centrifuged at 12,000x g for 30 min at 4°C. The resulting supernatant containing the cytosolic fraction was collected and the protein concentrations were determined using the Bradford assay. Then equivalent amounts of protein were analyzed by Western/ECL analyses as described above. Protein bands were quantified using the Advanced Image Data Analyzer Software (AIDA, Raytest, GmbH, Straubenhardt, Germany). The total band densities were measured against the local background. Results were presented as normalized fold changes in relation to control. The antibodies against cleaved caspase-3, Akt, phospho-p38 MAPK (Thr180/Tyr182) (3D7), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), phospho-NFκB p65 (Ser536)(93H1) and NFκB p65 were all purchased from Cell Signaling Technology, MA, USA. The phospho-STAT3 and Bcl-2 antibodies were obtained from Millipore, MA, USA whereas anti-cytochrome c and anti-JNK/SAPK (pT183/pY185) were purchased from Becton Dickinson, NJ, USA. The anti-p38α, IκBα and JNK 1/3 were obtained from Santa Cruz Biotechnology, CA, USA and the phospho-Akt/PKB [pS473] antibody was acquired from Invitrogen, CA, USA. Antibodies against β-actin (Millipore, MA, USA) and α-tubulin (Sigma-Aldrich, MO, USA) served as controls for equal loading.
Statistical analysis: The results were expressed as the Mean±SD
of three independent experiments, unless otherwise stated. Statistical analyses
were performed by one-way ANOVA followed by Tukeys post hoc test (p<0.05).
Calculations were performed with GraphPad Prism software 5.1 version (GraphPad,
USA).
RESULTS Cuc 1 inhibits A549 cells growth in vitro: To evaluate the cytotoxicity of Cuc 1 on human non-small lung cancer cells, A549 cells were initially treated with different concentrations of Cuc 1 at different time points and cellular proliferation was evaluated using the MTT assay. Treatment with this compound inhibited cellular proliferation in a concentration and time-dependent manner (Fig. 2). Their IC50 values were 13.5±1.8 and 3.8±0.4 μM for 48 and 72 h, respectively.
Cuc 1 induces cell cycle arrest at G2/M phase: Most anticancer agents
exhibit their effects on tumor cell growth by inducing cell cycle arrest and
apoptosis. To gain insights into the mechanism by which cell proliferation is
reduced, the effects of Cuc 1 on distribution of cell cycle phases in a cell
population was investigated by Fluorescence-activated Cell Sorting (FACS) analysis
(Fig. 3a). The untreated control cells showed a typical distribution
in G0/G1, G2/M and S phase, but a 24 h exposure of A549 cells to 15 and 30 μM
of Cuc 1 caused a significant enrichment of cells in G2/M phase in a concentration-dependent
manner. An increase from 17.06-32.42 and 37.72% cells in G2/M phase was detected
after the treatment with 15 and 30 μM of Cuc 1, respectively (p<0.0001
vs. control).
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Fig. 2: |
Cell growth inhibition by Cuc 1. Human non-small cell lung
cancer cells (A549 cells) were treated with different concentrations of
the Cuc 1 for 48 and 72 h. The growth inhibition effects were determined
by MTT assay and the IC50 was calculated by Graph Pad Prism 5.1.
Values were averaged expressed as a percentage relative to the untreated
controls. Values indicate the Mean±SD in triplicate tests |
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Fig. 3(a-c): |
Effects of Cuc 1 on cell cycle distribution and apoptosis
of A549 cells. (a) A549 cells (5x105) were treated with 15 and
30 μM and analyzed after 24 h by DNA flow cytometry. The values indicate
the percentage of A549 cells in the indicated phases of the cell cycle (G0/G1,
S and G2/M). *p<0.01 and **p<0.0001 as compared with control, (b)
The cells were treated with 15 and 30 μM for 12 h, stained with Annexin
V/PI and submitted to flow cytometry for the analysis of apoptotic cells
proportion. The horizontal (FITC-A) and the vertical (PE-A) axes represent
labeling with Annexin V and PI, respectively, (c) Graphic representation
of data obtained from Annexin V and PI staining assay. **p<0.0001 as
compared with control. The values represented means of three independent
experiments and SD. The most representative results of three independent
experiments are shown here |
This was followed by a reduction in G0/G1 phase cells (72.14% cells in the
controls, decreasing to 61.77 and 52.48% cells after the treatment with 15 and
30 μM of Cuc 1, respectively). These results indicated that the compound
induces in vitro growth inhibition of A549 cells by causing G2/M cell cycle
arrest.
Cuc 1 induces apoptosis: As the treatment of A549 cells with Cuc 1 inhibited
the cell growth it was further analyzed whether this effect might be related
to apoptosis induction. One of the earliest events of apoptosis is the loss
of plasma membrane polarity accompanied by translocation of Phosphatidylserine
(PS) from the inner to outer membrane leaflets, thereby exposing PS to the external
environment. The phospholipids-binding protein Annexin V has a high affinity
to PS thereby labeling cells with externally exposed PS which correlates with
loss of membrane polarity during apoptosis, but precedes the complete loss of
membrane integrity that accompanies later stages of cell death resulting either
in apoptosis or necrosis (Van Genderen et al., 2006).
In contrast, PI can only enter into cells after complete loss of membrane integrity.
Thus, dual staining with Annexin V and PI allows clear discrimination between
healthy cells (low FITC fluorescence and low PI fluorescence, LL), early apoptotic
cells (high FITC fluorescence and low PI fluorescence, LR), late apoptotic cells
(high FITC fluorescence and high PI fluorescence, UR) and necrotic cells (low
FITC fluorescence and high PI fluorescence, UL). A 12 h exposure of A549 cells
to 15 and 30 μM of Cuc 1 increased the percentage of apoptotic cells from
5.18% (LR+UR) in the controls to 47.96 and 73.82% in treated cells, respectively
(p<0.0001 vs. control) (Fig. 3b and c).
Thus, treatment of A549 cells with Cuc 1 induces apoptosis.
Cuc 1 alters cell morphology and actin organization: Under light microscopy,
A549 cells showed drastic alterations in their overall morphology after exposure
to Cuc 1 including reduced cytoplasmatic volume and cell rounding (data not
shown). Some of these morphological changes could be explained by the disruption
of actin cytoskeleton homeostasis by cucurbitacins, as it has been previously
described (Tannin-Spitz et al., 2007; Wakimoto
et al., 2008; Lee et al., 2010).
Therefore, the effects of Cuc 1 treatment on the cytoskeletal network were assessed.
For this purpose, untreated and treated A549 cells were stained with TRITC-labeled-phalloidin
which binds selectively to F-actin. As shown in Fig. 4a, the
untreated cells did not show a prominent polymerized, filamentous actin. However,
12 h exposure to 15 and 30 μM of Cuc 1 induced accumulation of F-actin
into globular aggregates in the cytoplasm near to the nuclei as it was demonstrated
by the intense fluorescent accumulation of rhodamine-phalloidin staining (Fig.
4b and c). In addition, when cell nuclei were visualized
with Hoechst staining, further morphological alterations in Cuc 1 treated A549
cells were observed such as chromatin condensation, nuclear fragmentation and
appearance of multinucleated cells. These observations further underline the
potential of Cuc 1 to induce cell death by apoptosis.
Cuc 1 induces apoptosis via activated caspases-dependent pathway: Caspases
are well known executors of the apoptotic process through their ability to cleave
several cellular substrates. To evaluate the effects of Cuc 1 on the activation
of the caspase-3, -8 and -9 in A549 cells, the cytosolic extracts of treated
or untreated control cells were incubated with different caspase-specific substrates.
As shown in Fig. 5, Cuc 1 caused a significant increase in
the proteolytic activity of caspases -3 and -9 after 12 h of treatment with
the higher concentration (30 μM) compared to untreated control cells. Thus
the morphological apoptosis induction observed correlates well with caspase
activation.
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Fig. 4(a-c): |
Effects of Cuc 1 on F-actin organization and nuclear fragmentation
by confocal microscopic analysis. A549 cells were either untreated (a) or
treated with 15 μM (b) and 30 μM (c) of Cuc 1 for 12 h, fixed
and stained with Hoechst (left panels) and TRITC-labeled-phalloidin (middle
panels). Overlay images are shown on the right side |
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Fig. 5: |
Activation of various caspases in A549 cells treated with
Cuc 1. A549 cells were incubated with 15 and 30 μM for 12 h. Cytosolic
fraction of cells was analyzed for changes in the activity of caspase-3,
caspase-8 and caspase-9. The date represent Means±SD of three independent
experiments. **p<0.0001 as compared with control |
Cuc 1 suppresses STAT3 activation: Persistent STAT3 (signal transducer
and activator of transcription 3) activation in cancer cells seems to confer
their normal physiological function in controlling cell growth, survival, angiogenesis
and immune responses. Conversely, the blockage of STAT3 signaling pathway in
cancer cells has been shown to induce apoptosis, inhibit cell proliferation,
suppress angiogenesis and stimulate immune responses and for these reasons the
STAT proteins are emerging as ideal targets for cancer therapy (Yu
and Jove, 2004). Previous studies have shown that cucurbitacins suppress
tumor growth and induce apoptosis in A549 cells by inhibiting STAT3 signaling
(Sun et al., 2005). To determine the relationship
between the cytotoxic activity of Cuc 1 and STAT3 phosphorylation, the p-STAT3
expression in A549 cells treated with Cuc 1 was investigated by Western blotting
analysis. It was shown that 30 μM of Cuc 1 reduced the STAT3 phosphorylation
almost completely after 12 h of treatment compared to the untreated control
cells (Fig. 6a).
Cuc 1 alters the expression of apoptosis-related proteins: In order to confirm the ability of Cuc 1 to activate caspase-3, as previously demonstrated by the cleavage of a caspase-3-specific substrate, the accumulation of the active cleaved form in A549 cells was investigated by Western blotting analysis. As shown in Fig. 6b, the treatment of A549 cells with both concentrations of Cuc 1 for 12 h increased the amount of active caspase-3 compared to untreated control cells. The active caspase-3 proteolytically cleaves and activates other caspases, as well as relevant targets in the apoptotic cells. Taken together, these results confirm the involvement of this enzyme in the mechanism of cell death mediated by Cuc 1.
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Fig. 6: |
Expression of apoptosis-related proteins and down-regulation
of the Akt signaling pathway in unstimulated (TNFα-) and stimulated
(TNFα+) A549 cells. (a and b) Western blotting analysis of cytochrome
C, p-STAT3, Bcl-2, caspase 3 (cleaved form) and survivin in A549 cells untreated
(lane 1) or treated with 15 μM (lane 2) and 30 μM (lane 3) of
Cuc 1 for 12 h. β-actin or α-tubulin was used as the loading control.
(c) Western blotting analysis of phospho-Akt in A549 cells untreated (lanes
1 and 2) or treated with 15 μM (lanes 3 and 4) and 30 μM (lanes
5 and 6) of Cuc 1 for 24 and 48 h. Total amount of Akt was used as the loading
control. Data were representative of three independent experiments |
Cytochrome c release is one of the limiting factors in caspase-9 activation and represents a coordinating control step in the apoptotic process. Since Cuc 1 induced caspase-9 activity (Fig. 5), its ability to trigger cytochrome c release in A549 cells was analyzed. Indeed, after 12 h of treatment with Cuc 1, release of mitochondrial cytochrome c into the cytosol of A549 cells was detected (Fig. 6a). In addition, the treatment with 30 μM of Cuc 1 reduced the expression of the anti-apoptotic protein Bcl-2 in A549 cells (Fig. 6a).
Akt, also known as Protein Kinase B (PKB), is a key player in regulating cellular
growth and survival. In the past decade the role of Akt in cancer has increased
enormously and it is now evident that activation of Akt is one of the most common
molecular alterations in human malignancy. Therefore, Akt has become an increasingly
important target of drug development efforts and several inhibitors are now
entering clinical trials (Altomare and Testa, 2005;
Radisavljevic, 2008). It has been known that disruption
of actin cytoskeleton network reduces Akt signaling, leading to a reduced expression
of anti-apoptotic proteins such as survivin which ends up in both G2/M arrest
and apoptosis (Mosmann, 1983; Liang
et al., 2003). Since cell transition to a round shape morphology
and disorganization of actin filamentous was observed after Cuc 1 treatment
(Fig. 4), the activation state or expression of Akt and survivin
was analyzed. Indeed, Cuc 1 treatment for 24 and 48 h caused a significant reduction
in Akt activation detected by its phosphorylation on Ser273 in both pre-activated
(stimulated) and unstimulated cells, whereas the total amount of Akt remained
unaffected (Fig. 6b). In addition, a 24 h treatment with Cuc
1 also led to a reduced survivin expression (Fig. 6c). However,
when analyzing the activation state of proteins of other signaling pathways
such as ERK, p38, JNK, p65 as well as the degradation of IκBα, no
significant changes were observed after treatment with Cuc 1 (data not shown).
Taken together, these data demonstrate that Cuc 1 induces apoptosis in A549
cells via a reduced activation of Akt signaling pathway.
DISCUSSION
Natural products provide a rich source of chemopreventive and chemotherapeutic
agents (Shukla and Singh, 2011). Cucurbitacins refer
to a group of tetracyclic triterpenoids initially identified in the plant family
of Cucurbitaceae. In traditional medicine cucurbitacin-containing plants have
been used against skin affections, as purgative and emetic and to treat inflammatory
conditions (Lewis and Elvin-Lewis, 1977).
This study evaluated a new cytotoxic cucurbitacin named Cuc 1 (Fig.
1), isolated for the first time by Lang et al.
(2011) from Wilbrandia ebracteata Cogn. roots. Therefore,
the antiproliferative effects of Cuc 1 on human non-small-cell lung cancer cells
(A549 cells) as well as the molecular mechanism of its cytotoxic properties
were investigated.
MTT analysis showed significant inhibition of A549 cells viability in vitro
in a concentration and time-dependent manner (Fig. 2). Cuc
1 treatment inhibited cell growth showing IC50 values of 13.5±1.8
and 3.8±0.4 μM for 48 and 72 h, respectively. In addition, this
compound showed higher selectivity towards A549 cells, when compared to healthy
cells (human gingival fibroblasts) presenting an IC50 value of 132.95±24.11
μM after 48 h exposure to these cells (data not shown). It is well known
that most anticancer agents exhibit their inhibitory effects on tumor cell growth
by inducing cell cycle arrest and apoptosis. In order to better characterize
the mechanism underlying the observed inhibitory activity of Cuc 1 on A549 cells,
a set of experiments on cell cycle distribution and apoptosis detection by flow
cytometry was performed. Here A549 cells treated with Cuc 1 were arrested at
the G2/M phase of cell cycle with decreased cell population in G0/G1 (Fig.
3a), suggesting that this new compound inhibits cell growth via blocking
the division cell cycle at the G2/M phase. The results of the present study
are in agreement with recent reports which have demonstrated that this group
of natural compounds induces G2/M arrest and apoptosis in other human cancer
cell lines in vitro (Tannin-Spitz et al.,
2007; Li et al., 2010; Yasuda
et al., 2010).
Phosphatidylserine externalization is a hallmark of early steps in apoptosis.
As Cuc 1 was shown to induce cell death it was further analyzed whether this
special event is induced upon Cuc 1 treatment. By flow cytometry analysis with
Annexin V/PI staining was confirmed that it induced apoptosis in a concentration-dependent
manner (Fig. 3b and 3c).
Confocal microscopy analyses with rhodamine-phalloidin and Hoechst fluorescent
staining revealed that Cuc 1 exposure led to the development of morphologically
altered and multinucleated A549 cells at both tested concentrations (Fig.
4). These morphological changes are typical for apoptosis. One of the most
obvious morphological effects of Cuc 1 on A549 cells was the disruption of F-actin
cytoskeleton as showed by the altered appearance of rhodamine-phalloidin stainable
material. This is in complete consistence with the current knowledge of cucurbitacins.
Some scholars have reported that cucurbitacins can directly modulate the actin
cytoskeleton. Duncan et al. (1996) demonstrated
that cucurbitacin E acts as a potent disruptor of cytoskeletal integrity by
increasing the filamentous or polymerized actin fraction in prostate carcinoma
cells. Other studies carried out with cucurbitacin B also showed the aggregation
of F-actin in various human cancer cell lines (Haritunians
et al., 2008; Wakimoto et al., 2008;
Yin et al., 2008).
Multinucleation is a consistently reported morphological alteration in cancer
cell cultures that are treated with cucurbitacins (Siqueira
et al., 2009; Lee et al., 2010).
The final step in cell division, in which the cytoplasm is divided to form two
daughter cells, is known as cytokinesis. Since cytokinesis involves the assembly
and disassembly of actin filaments, compounds that interfere with actin polymerization
or its spatial organization will block this process and generate multinucleated
cells by uncoupling nuclear and cytoplasmic division. Therefore, molecules acting
on the actin cytoskeleton of tumor cells and thus inhibiting cell division and
cell proliferation, may be of high therapeutic value. Currently, growing evidence
indicates that cytoskeletal components are involved in the apoptotic cascade
thereby regulating cell survival (Cabado et al.,
2004). In a previous study, it was found that cytochalasin B which causes
cytoskeletal disruption, also inhibited cell proliferation, leading to arrest
in G2/M phase and finally apoptosis induction (Liang et
al., 2003). These effects are similar to those observed in this study
for Cuc 1. Thus, one can propose that the observed effects are mediated, at
least partially, by a modification of the cytoskeleton network causing changes
in cell morphology, leading to reduction of Akt phosphorylation, G2/M arrest
and apoptosis induction.
In the regulation of apoptosis, several caspases play important roles (Hengartner,
2000). They are organized into initiator or effector caspases, due to the
role they play in apoptosis induction. It is important to state that activation
of caspases is a hallmark of promoting apoptosis in response to death inducing
signals originated mainly from cell surface receptors or mitochondria. It has
also been shown that caspase-3 activation, the major effector caspase, requires
the activation of initiator caspases such as caspase-8 or -9 in response to
the different pro-apoptotic signals (Budihardjo et al.,
1999). Herein, the activation of the effector caspases-3 was observed in
response to the initiator caspase-9 in A549 cells treated with Cuc 1. This suggests
that the intrinsic pathway might be involved in apoptosis induced by this novel
compound.
Signal transducer and activator of transcription protein 3 (STAT3) is a latent
cytosolic transcription factor that transfers signals from the cell membrane
directly to the nucleus. STAT3 is constitutively activated in multiple human
cancers including ovarian, breast, prostate and lung cancer. It plays a key
transcriptional role in cancer cell progression, differentiation and survival
by up-regulating several genes, including those that encode for anti-apoptotic
proteins and some cell cycle regulators (Yu and Jove, 2004;
Fletcher et al., 2009). Thus, since most of
the chemotherapeutic strategies aim to initiate apoptosis, it is now generally
accepted that STAT3 represents a valid target for novel anticancer drug design
(Al Zaid Siddiquee and Turkson, 2008). Although initial
interest in cucurbitacins as potential anticancer drugs declined for decades,
recent discoveries showing that they are strong STAT3 inhibitors have regained
the attention of the pharmaceutical industry one more time. Several studies
have suggested that cucurbitacins exert inhibitory effects against many human
cancer cell lines via suppression of STAT3 phosphorylation (Sun
et al., 2005; Thoennissen et al., 2009;
Chan et al., 2010; Liu et
al., 2010; Sun et al., 2010). In this
study, the evaluation of the phosphorylation state of STAT3 in A549 cells by
using Western blotting analysis demonstrated the anti-STAT3 activity of Cuc
1 (15 and 30 μM), as shown in Fig. 6a. These findings
suggest that the inhibition of STAT3 phosphorylation may be associated to the
induction of apoptosis by Cuc 1.
To determine the expression changes of STAT3 target genes involved in cell apoptosis, the expression of Bcl-2 was analyzed showing that Cuc 1 treatment decreased Bcl-2 expression in a concentration-dependent manner (Fig. 6a).
The Bcl-2 family members also play a critical role in the regulation of apoptosis,
comprising both pro-apoptotic molecules (Bax, Bcl-Xs, Bak, Bid, Bad, Bim, Bik)
and anti-apoptotic molecules (Bcl-2, Bcl-XL, Bcl-W, Mcl-1, A1). These molecules
control the release of mitochondrial cytochrome c by modulating the permeability
of the outer mitochondrial membrane (Lessene et al.,
2008). Herein, Cuc 1 treatment induced cytochrome c release from mitochondria
to the cytosol (Fig. 6a) and it is well known that cytochrome
c release is necessary for the activation of caspase-9 that initiates
the caspases cascade. These results also suggest that the mitochondrial pathway
plays an important role in the apoptosis of A549 cells induced by Cuc 1. In
addition, activated Akt is a well-established survival factor exerting anti-apoptotic
activity, in part by preventing the release of cytochrome c from the mitochondria
and inactivating pro-apoptotic factors such as Bad and procaspase-9 by phosphorylating
them (Cardone et al., 1998; Altomare
and Testa, 2005). Indeed, after the treatment with Cuc 1, Akt phosphorylation
was down regulated in A549 cells indicating an inhibition of this signaling
pathway by Cuc 1 (Fig. 6c). This effect might contribute to
apoptosis induction.
CONCLUSION In conclusion, it can be postulated that Cuc 1 induces apoptosis in A549 cells. This is mediated by G2/M phase cell cycle arrest and actin cytoskeleton disruption. Furthermore, Cuc 1 inhibits the activation of STAT-3 and Akt signaling pathways to down-regulate the expression of Bcl-2. This prompts cytochrome c to be released from the mitochondria to the cytosol which is an essential step for caspases -9 and -3 activation. Therefore, Cuc 1 can be taken to mean a new potent compound with a high anticancer potential due to its cytotoxic and apoptosis inducing effects on malignant lung cells. ACKNOWLEDGMENTS The authors acknowledge the financial support received from Brazilian funding agencies: CNPq/MCTI (grant number 472979/2011-6) and PRONEX/FAPESC (grant number 2671/2012-9) as well as the first one for their research fellowships. The study sponsors had no involvement in the design, collection, analysis and interpretation of the data and the decision to submit the manuscript for publication in International Journal of Cancer Research. We also would like to thank Rafael Matielo, Jadel Kratz and Annelise de Carvalho for their proficient editorial assistance.
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