- Research article
- Open Access
- Open Peer Review
So-Cheong-Ryong-Tang induces apoptosis through activation of the intrinsic and extrinsic apoptosis pathways, and inhibition of the PI3K/Akt signaling pathway in non-small-cell lung cancer A549 cells
© Park et al.; licensee BioMed Central. 2015
- Received: 24 October 2014
- Accepted: 30 March 2015
- Published: 10 April 2015
So-Cheong-Ryong-Tang (SCRT), a traditional Korean medicine containing eight species of medicinal plant, has been used to treat patients with bronchial asthma and allergic rhinitis for hundreds of years; however, its anti-cancer potential is poorly understood. The present study was designed to evaluate the apoptotic effect of SCRT against human non-small-cell lung cancer (NSCLC) A549 cells.
The effects of SCRT on cell growth and viability were evaluated by trypan blue dye exclusion and 3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyl tetrazoliumbromide (MTT) assays, respectively. Apoptosis was detected using 4,6-diamidino-2-phenyllindile (DAPI) staining, agarose gel electrophoresis and flow cytometry. The protein levels were determined by Western blot analysis. Caspase activity was measured using a colorimetric assay.
SCRT treatment resulted in significantly decreased A549 cell growth and viability by induction of apoptosis. SCRT induced the translocation of pro-apoptotic Bax to the mitochondria, mitochondrial membrane permeabilization, cytochrome c release from mitochondria to cytosol, and activated caspase-9 and caspase-3. SCRT also increased death receptor-associated ligands and enhanced the activation of caspase-8 and cleavage of its substrate Bid. However, the pan-caspases inhibitor significantly blocked the SCRT-induced apoptosis, suggesting that it is a caspase-dependent pathway. In addition, SCRT suppressed the phosphorylation of phosphoinositide 3-kinase (PI3K) and Akt, and treatment with a potent inhibitor of PI3K further increased the apoptotic activity of SCRT.
These findings suggest that SCRT may play its anti-cancer actions partly through a suppression of the PI3K/Akt signal pathway in A549 cells, and further in vivo studies on the potential of SCRT for prevention and therapy of NSCLCs are warranted.
- A549 cells
Non-small-cell lung cancer (NSCLC), accounting for about 80% of human lung cancers, is one of the leading causes of cancer deaths worldwide. Therapeutic strategies for lung cancer treatment include surgery, radiotherapy, chemotherapy, and targeted and combined therapies. Despite advances in treatment, the poor prognosis is largely attributable to the inherent or acquired resistance in cancer cells against conventional chemotherapy [1,2]. Although, many studies have reported potential chemotherapeutic effects of novel compounds, novel agents with proven efficacy and minimal toxicity are urgently required for the treatment of NSCLC.
The progression of cancer is characteristically linked to a resistance to apoptosis, a major type of cell death that occurs when DNA damage is irreversible. Apoptosis is characterized by distinct morphological and biochemical changes such as cell shrinkage, membrane blebbing, chromatin condensation, activation of proteases, and externalization of phosphatidylserine [3,4]. Therefore, agents targeting the apoptosis pathway without affecting normal cells play crucial roles as potential drug targets in cancer treatment. In mammalian cells, apoptosis may be initiated in two ways, via an intrinsic (mitochondrial-mediated) or extrinsic (death receptor (DR)-mediated) pathway that involves the activation of caspases [5,6]. The former is activated by the release of pro-apoptotic factors such as cytochrome c from the mitochondrial matrix following the loss of inner mitochondrial membrane integrity and activation of caspase-9. The latter is initiated by the binding of extracellular death ligands to their cell-surface death receptors leading to the activation of caspase-8. The extrinsic pathway can crosstalk to the intrinsic pathway through the caspase-8-mediated cleavage of Bid, a member of the Bcl-2 family of proteins, which ultimately leads to apoptosis [7,8].
For thousands of years, herbal medicines have been used seemingly safely and effectively to treat and alleviate various diseases in Asia, including Korea, China, and Japan. Recently, there has been increasing interest in the pharmacological activity of traditional medicines, and numerous studies support their potential clinical benefit for diseases that are difficult to treat [9,10]. Typical traditional Korean medicines derived from ancient Chinese herbal medicines and consist of at least four 4 components that are mixed to minimize side effects, maximize medical effects, and improve the patient’s quality of life. So-Cheong-Ryong-Tang (SCRT), also known as Xiao-Qing-Long-Tang in China and Sho-Seiru-To in Japan, is an aqueous polyherbal formulation, consisting of eight species of medicinal herb. SCRT has been used to treat bronchial asthma and allergic rhinitis for hundreds of years in Asian countries . Studies regarding its pharmacological action have shown an inhibition of histamine release and degranulation of mast cells , reduced serum IgE level in allergic rhinitis patients [13,14], decreased allergen-induced bronchial inflammation , the growth and differentiation of basophils  and passive cutaneous anaphylaxis . Recently, Byun et al.  demonstrated that SCRT improved the quality of life and reduced the symptoms of illness in common cold patients, and it was also found that SCRT exhibits immunomodulative and anti-inflammatory activities [19-21]. However, despite its valuable effects, little is known about the anti-cancer potential of SCRT. In this study, as a part of our search for novel biologically active substances for the prevention and treatment of cancer from traditional Korean medicine we evaluated whether SCRT could inhibit cell growth and trigger apoptosis in NSCLC A549 cells.
Reagents and antibodies
Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, and trypsine-ethylene diamine tetraacetic acid (EDTA) were purchased from Gibco-BRL (Gaithersburg, MD, USA). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), 4,6-diamidino-2-phenyllindile (DAPI), phenol:chloroform:isoamylalcohol, ethidium bromide (EtBr), RNase A, dithiothreitol (DTT), bovine serum albumin (BSA), propidium iodide (PI), paraformaldehyde and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1) were purchased from Sigma-Aldrich Chemicals (St. Louis, MO, USA). N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), a pan-caspase inhibitor, and LY294002, a selective inhibitor of phosphatidylinositol 3-kinase (PI3K), were obtained from Calbiochem (San Diego, CA, USA) and Cell Signaling Technology, Inc. (Danvers, MA, USA), respectively. Annexin V-fluorescein isothiocyanate (FITC) and caspase activity assay kits were purchased from R&D Systems (Minneapolis, MN, USA). DNA ladder size marker and enhanced chemiluminescence (ECL) kit were purchased from Invitrogen (Carlsbad, CA, USA) and Amersham Corp. (Arlington Heights, IL, USA), respectively. A mitochondrial fractionation kit was obtained from Active Motif (Carlsbad, CA, USA). Primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), Chemicon (Temecula, CA, USA) and Sigma-Aldrich. Peroxidase-labelled donkey anti-rabbit and sheep anti-mouse immunoglobulin were purchased from Amersham. All other chemicals not specifically mentioned here were purchased from Sigma-Aldrich.
Preparation of SCRT extract
Components of So-Cheong-Ryong-Tang (SCRT) extract granules
Herbal medicine (pharmacognostic nomenclature)
Raw material amount (g/%)
Ephedra sinica Stadf. (Ephedrae Herba)
Paeonia lactiflora Pall. (Paeoniae Radix)
Schizandra chinensis (Turcz.) Ball. (Schizandrae Fructus)
Pinellia ternata (Tenore et Breit. (Pinelliae Rhizoma)
Asiarum sieboldii F. Maekawa (Asiasari Radix)
Zingiber officinale Rosc. (Zingiberis Rhizoma)
Cinnamomum cassia Blume. (Cinnamomi Ramulus)
Glycyrrhiza glabra L. (Glycyrrhizae Radix)
The human NSCLC A549 cells and WI-38 human fetal lung fibroblasts were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in DMEM supplemented with 10% FBS, 1% L-glutamine and penicillin/streptomycin. The cells were cultured in an incubator with 5% CO2 at 37°C.
Cell growth and cell viability assay
Cell growth was assessed using the trypan blue dye exclusion assay. In brief, cells (2 × 104 cells/well) were seeded in 6-well plates. After treatment with the indicated concentrations of the SCRT for the indicated times, the cells were trypsinized and viable cells were counted by trypan blue dye exclusion using a hemocytometer under an inverted microscope (Carl Zeiss, Jena, Germany). Cell viability was determined using the MTT assay. In brief, cells (2 × 104 cells/well) were seeded in 24-well plates and exposed to the extracts of SCRT for the indicated times. After treatment, 5 mg/ml MTT solution was added, followed by 3 h incubation at 37°C in the dark, and the media was then removed. The formazan precipitate was dissolved in dimethyl sulfoxide, and absorbance of the formazan product was measured at a wavelength of 540 nm with an enzyme-linked immunosorbent assay (ELISA) reader (Molecular Devices, Sunnyvale, CA, USA). For the morphological study, cells were photographed directly using an inverted microscope (Carl Zeiss, Oberkochen, Germany).
Nuclear staining with DAPI
For the assessment of apoptosis, morphological changes of nuclei were visualized following DNA staining by the fluorescent dye, DAPI. The cells were fixed with 3.7% paraformaldehyde for 20 min at room temperature and washed with phosphate-buffered saline (PBS). Cells were then stained with 2.5 μg/ml DAPI solution for 10 min at room temperature. The cells were washed twice with PBS and stained nuclei were observed using a fluorescence microscope (Carl Zeiss).
DNA fragmentation assay
After treatment with SCRT, cells were lysed in a buffer containing 10 mM Tris (pH 7.4), 150 mM NaCl, 5 mM EDTA and 0.5% Triton X-100 for 30 min on ice. Lysates were vortexed and cleared by 19,000 g centrifugation for 30 min at 4°C. Fragmented DNA in the supernatant was extracted with an equal volume of neutral phenol:chloroform:isoamylalcohol (25:24:1, v/v/v), followed by electrophoretic analysis on 1.5% agarose gels containing 0.1 μg/ml EtBr.
Flow cytometric detection of apoptosis
To analyze cell cycle profiles, cells were harvested, washed twice with ice-cold PBS, fixed with 75% ethanol at 4°C for 30 min, and washed in PBS with 0.1% BSA. The cells were then incubated with 1 U/ml of RNase A (DNase free) and 10 μg/ml of PI overnight at room temperature in the dark. Cells were analyzed using a FACSCalibur flow cytometer (Becton Dickenson; San Jose, CA). The level of apoptotic cells containing sub-G1 DNA content was determined as a percentage of the total number of cells. The cells were also stained with 5 μl of annexin V-FITC and 5 μl of PI in each sample. After at 15 min incubation at room temperature in the dark, the degree of apoptosis was quantified as a percentage of the Annexin V-positive and PI-negative (annexin V+/PI− cells) cells by a flow cytometer .
Western blot analysis
Cells were harvested and lysed with lysis buffer (20 mM sucrose, 1 mM EDTA, 20 μM Tris-Cl, pH 7.2, 1 mM DTT, 10 mM KCl, 1.5 mM MgCl2, and 5 μg/ml aprotinin) for 30 min at 4°C. The mixtures were centrifuged at 12,000 g for 10 min at 4°C, and the supernatants were collected as whole-cell extracts. In a parallel experiment, the mitochondrial and cytosolic fractions were isolated using a mitochondrial fractionation kit according to the manufacturer’s instructions. The protein content was determined using the Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA, USA) and BSA as a standard according to the manufacturer’s instructions. Equal amounts of protein were separated electrophoretically using sodium dodecyl sulfate (SDS)-polyacrylamide gels transferred to nitrocellulose membranes (Amersham). The membranes were soaked in blocking buffer (5% skimmed milk) and incubated overnight with primary antibodies followed by horseradish peroxidase conjugated antibodies at room temperature. Detection of specific proteins was carried out with an ECL Western blotting kit according to the manufacturer’s instructions.
Determination of caspase activity
The activity of caspase-like protease was measured using a caspase activation kit according to manufacturer’s protocol. The kits utilize synthetic tetrapeptides labeled with p-nitroanilide (pNA) linked to the end of the caspase-specific substrate. To evaluate caspase activity, cell lysates were prepared after treatment with SCRT and incubated with the supplied reaction buffer and the colorimetric substrates at 37°C for 2 h in the dark. The cleavage of the peptide by the caspase releases the chromophore pNA, which can be quantified spectrophotometrically at a wavelength of 405 nm using an ELISA reader.
Measurement of mitochondrial membrane potential (MMP, Δψm)
The values of MMP were determined using the dual-emission potential-sensitive probe, JC-1, which is internalized and concentrated by respiring mitochondria and can therefore reflect changes in MMP in live cells. Cells were collected and incubated with 10 μM of JC-1 for 30 min at 37°C in the dark. After the JC-1 was removed, the cells were washed with PBS to remove any unbound dye and the amount of JC-1 retained by 10,000 cells per sample was measured at 488 nm and 575 nm using a flow cytometer.
The data are reported as the mean ± standard deviation (SD) of three independent experiments. A one-way ANOVA was performed to determine statistical significance. Significant differences were established at p <0.05.
Effects of SCRT on A549 cell growth and viability
To measure apoptotic cell death upon SCRT treatment, we stained cells with PI and cycle profiles were analyzed using a flow cytometer. The results after PI staining showed that SCRT increased the percentage of cells in the apoptotic hypodiploid sub-G1 phase in a dose-dependent manner (Figure 2C). Annexin V-FITC/PI double staining was also performed to assess the rate of apoptotic cell death, and a significant degree of apoptosis was detected in the SCRT-treated A549 cells, as shown by an increased phosphatidyl exposure using annexin V-FITC (Figure 2D). The results we obtained corresponded very well with our cell morphology observations and DNA fragmentation analysis, indicating the cytotoxic effects observed in response to SCRT are associated with the induction of apoptosis in A549 cells.
Activation of caspases by SCRT in A549 cells
Mitochondrial translocation of Bax and loss of MMP by SCRT in A549 cells
The release of cytochrome c often involves changes in MMP, and the loss of MMP in cells is one of the early critical events in the intrinsic apoptosis pathway, eventually causing the initiation and activation of apoptotic cascades [28,29]. Therefore, we sought to determine whether SCRT treatment had any effect on the MMP in A549 cells using fluorescent dye JC-1 staining. Figure 5C shows that treatment of A549 cells with SCRT resulted in a dose-dependent increase in the proportion of green fluorescence-positive cells (indicating a loss of mitochondrial membrane potential), confirming that SCRT induced the loss of MMP. These results indicated that SCRT could induce the mitochondrial translocation of Bax and loss of MMP resulting in mitochondrial dysfunction, release of cytochrome c to the cytosol, and ultimately, apoptosis induction.
Effects of SCRT on the expression of cell death receptor (DR)-related proteins and Bid by SCRT in A549 cells
Effects of SCRT on the PI3K/Akt pathway in A549 cells
In the present study, we investigated the effects of SCRT on NSCLC A549 cells and demonstrated that SCRT inhibits A549 cell growth, an effect correlated with apoptosis induction. In our study, chromatin condensation of the nuclei, DNA fragmentation, and an increase of sub-G1 phase, and annexin V-FITC-stained cells demonstrated the apoptosis induction (Figures 1 and 2). We then examined various aspects of the mechanisms of apoptosis induction by SCRT in A549 cells and found SCRT-activated caspase-9 -8, and −3, which are associated with the loss of MMP and degradation of PARP and PLC-γ1. SCRT also down-regulated IAP family proteins, which reportedly block apoptosis directly by binding to and inhibiting several caspases (Figure 3). However, blocking caspase activity by pretreating the cells with a pan-caspase inhibitor significantly prevented SCRT-induced apoptosis and growth inhibition (Figure 4). Therefore, the data support a finding that SCRT-induced apoptosis in A549 cells is caspase-dependent, and both the intrinsic and the extrinsic pathways are activated by SCRT.
Apoptosis, as a programmed cell death, is a rigorous, active, and orderly process of cell death, regulated by numerous gene products to maintain the stability of the intracellular environment. Among them, cytochrome c is a small heme protein found loosely associated with the inner membrane of the mitochondrion. The release of cytochrome c from the mitochondria into the cytoplasm through an opening of the mitochondrial permeability transition pore (MPTP) followed by a swelling-induced rupture of the mitochondrial outer membrane is an important component in the intrinsic pathway. The protein binds apoptotic protease activating factor-1 (Apaf-1) and activates caspase-9 in the cytoplasm. Once initiated, caspase-9 can go on to activate effector caspases such as caspase-3 and −7, which then cleave many important cellular substrates and causes cell death [5,34,35].
The Bcl-2 protein family is a major regulator of apoptosis, as the balance of pro-apoptotic and anti-apoptotic Bcl-2 family proteins decide the fate of the cell. The proteins are also important regulators of cytochrome c release from the mitochondria through the regulation of MPT. For example, the pro-apoptotic protein Bax facilitates the openness of the MPTP and the subsequent cytochrome c release, which in turn activates the caspase cascade reaction, eventually resulting in apoptosis. Conversely, the anti-apoptotic protein Bcl-2 protects mitochondria against the loss of function during apoptosis by inhibiting the MPTP’s opening, the Bcl-2-Apaf1-caspase-9 complex, and apotosome, resulting in an anti-apoptosis effect [34,35]. Interestingly, the present data revealed that SCRT did not affect the total expression levels of Bax as well as Bcl-2; however, SCRT treatment resulted in a dose-dependent increase in mitochondrial levels of Bax with a concomitant decrease in the cytoplasmic Bax levels. Our data also clearly showed that SCRT treatment resulted in a dose-dependent increase in the release of cytochrome c (Figure 5). Because the release of cytochrome c requires mitochondrial membrane insertion and the oligomerization of Bax [22,28,29], the translocation of pro-apoptotic Bax proteins from the cytosol to mitochondria represents a key event for the activation of the intrinsic pathway. Therefore, our data indicated that SCRT induces Bax translocation to mitochondria from the cytosol, leading to the release of cytochrome c, apoptosome formation, and finally, induction of apoptosis in A549 cells.
Our results also demonstrate that SCRT activates the death receptor-mediated extrinsic pathway. The extrinsic pathway can be triggered to undergo oligomerization when proper ligands bind their respective death receptors, ultimately recruiting adaptor molecules and caspase-8 into a complex resulting in activated caspase-8. In addition, caspase-8 connects to the intrinsic pathway via cleavage of the Bcl-2 family member Bid to form tBid. Under normal conditions, Bid is located in the cytoplasm in an inactive state; however, tBid can translocate the mitochondrial membrane where it interacts with Bax proteins. This interaction promotes the fusion of Bax and the mitochondria, which results in changes to the configuration of Bax proteins. These changes enhance mitochondrial dysfunction, resulting in the formation of membrane pores and permitting the release of cytochrome c to the cytosol, followed by the activation of caspase-9 [28,30]. This event leads to activation of caspase-3 for the induction of apoptosis via a release of cytochrome c to the cytosol [4,8]. Moreover, tBid directly binds to Bcl-2 and thereby inhibits its anti-apoptosis effect [30,31]. Although the levels of death receptors such as DR4, DR5, and Fas remained unchanged in SCRT-treated A549 cells, SCRT notably increased the levels of their ligands including TRAIL and FasL, while a decline of intact Bid occurred concurrently with a pronounced increase of tBid (Figure 6).
On the other hand, PI3K plays a critical role in cell survival and proliferation in collaboration with its major downstream effector Akt. Notably, the aberrant activation of the PI3K/Akt pathway is implicated in the initiation and progression of several types of human malignancies, including NSCLC . Furthermore, deregulation of the PI3K/Akt pathway is associated with resistance to chemotherapeutic agents [32,37]. Meanwhile, anti-apoptotic proteins such as survivin and Bcl-2, and caspase-3 are the downstream target of Akt, and are related to proliferation and apoptosis [38,39]. Many previous studies also have indicated that the pro-apoptotic potential of some anti-cancer agents is highly correlated with the inactivation of the PI3K/Akt pathway [33,40-43]. These observations indicate that the inhibition of the PI3K/Akt signaling cascade can serve as an effective strategy for the treatment of cancers. In the present study, we further studied the possible role of the PI3K/Akt pathway in the SCRT-induced apoptosis of A549 cells and found the levels of phosphorylated PI3K and Akt, were markedly decreased after SCRT treatment (Figure 7A). The total levels of PI3K and Akt were unchanged with SCRT treatment. In addition, there was a significant increase in apoptotic activity when A549 cells were treated with a combination of SCRT and LY294002, a PI3K inhibitor, whereas treatment with LY294002 alone did not affect the growth of A549 cells (Figure 7B-D). This result provides another piece of evidence that SCRT can induce apoptosis by reducing PI3K/Akt pathway activation in A549 cells.
In conclusion, we described how SCRT significantly inhibits the growth of NSCLC A549 cells and induces cell apoptosis in vitro. SCRT treatment increases the loss of MMP and translocalization of Bax to the mitochondria, and leads to cytochrome c release to the cytosol and activation of caspase-9 and −3, resulting in the activation of the intrinsic apoptosis pathway. SCRT also activates the extrinsic apoptosis pathway by increasing both TRAIL and FasL, resulting in the activation of caspase-8 and Bid truncation. Thereby the mechanism of action of SCRT is related to the activation of the extrinsic as well as the intrinsic apoptosis pathways, amplifying mitochondrial signaling. In addition, co-treatment of LY294002, a PI3K inhibitor, significantly increased SCRT-induced-apoptosis indicating that the PI3K/AKT pathway might have a survival role in SCRT-induced A549 cell apoptosis. To explore this rule, the pathway must be blocked while the cells are treated with SCRT. Although in vivo anti-cancer activity and deep molecular mechanisms need further investigation, based on the results of this study, we believe SCRT should continue to be developed as a possible new therapeutic treatment for NSCLC.
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) grant funded by the Korea government (No. 2012046358).
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