Samsoeum, a traditional herbal medicine, elicits apoptotic and autophagic cell death by inhibiting Akt/mTOR and activating the JNK pathway in cancer cells
© Kim et al.; licensee BioMed Central Ltd. 2013
Received: 26 April 2013
Accepted: 18 September 2013
Published: 23 September 2013
Samsoeum (SSE), a traditional herbal formula, has been widely used to treat cough, fever, congestion, and emesis for centuries. Recent studies have demonstrated that SSE retains potent pharmacological efficiency in anti-allergic and anti-inflammatory reactions. However, the anti-cancer activity of SSE and its underlying mechanisms have not been studied. Thus, the present study was designed to determine the effect of SSE on cell death and elucidate its detailed mechanism.
Following SSE treatment, cell growth and cell death were measured using an MTT assay and trypan blue exclusion assay, respectively. Cell cycle arrest and YO-PRO-1 uptake were assayed using flow cytometry, and LC3 redistribution was observed using confocal microscope. The mechanisms of anti-cancer effect of SSE were investigated through western blot analysis.
We initially found that SSE caused dose- and time-dependent cell death in cancer cells but not in normal primary hepatocytes. In addition, during early SSE treatment (6–12 h), cells were arrested in G2/M phase concomitant with up-regulation of p21 and p27 and down-regulation of cyclin D1 and cyclin B1, followed by an increase in apoptotic YO-PRO-1 (+) cells. SSE also induced autophagy via up-regulation of Beclin-1 expression, conversion of microtubule-associated protein light chain 3 (LC3) I to LC3-II, and re-distribution of LC3, indicating autophagosome formation. Moreover, the level of B-cell lymphoma 2 (Bcl-2), which is critical for cross-talk between apoptosis and autophagy, was significantly reduced in SSE-treated cells. Phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) was increased, followed by suppression of the protein kinase B/mammalian target of rapamycin (Akt/mTOR) pathway, and phosphorylation of mitogen-activated protein kinases (MAPKs) in response to SSE treatment. In particular, among MAPKs inhibitors, only the c-Jun N-terminal kinase (JNK)-specific inhibitor SP600125 nearly blocked SSE-induced increases in Beclin-1, LC3-II, and Bax expression and decreases in Bcl-2 expression, indicating that JNK activation plays critical role in cell death caused by SSE.
These findings suggest that SSE efficiently induces cancer cell death via apoptosis as well as autophagy through modification of the Akt/mTOR and JNK signaling pathways. SSE may be as a potent traditional herbal medicine for treating malignancies.
KeywordsSamsoeum Apoptosis Autophagy Cell cycle arrest Akt/mTOR JNK
In cancer cells, the balance of cell death with survival is frequently disturbed by the mutation of oncogenes or tumor suppressor genes and by the alteration of signaling pathways . Because perturbation of the cell death process is closely related to cancer progression and resistance to chemotherapy or radiotherapy, accumulating evidence has shown that agents targeting the programmed cell death (PCD) pathway without affecting normal cells play crucial roles as potential drug targets in cancer treatment . PCD, a cell suicide program, plays pivotal roles in the development, tissue homeostasis, and elimination of damaged cells as a basic biological phenomenon and can be classified according to morphological differences as apoptosis (type I), autophagy (type II), and programmed necrosis (type III) [3, 4].
Apoptosis, a major type of cell death that occurs when DNA damage is unrecoverable, is characterized by distinct morphological and biochemical changes such as cell shrinkage, membrane blebbing, chromatin condensation, and ultimately, fragmentation of cells into membrane-enclosed vesicles designated as apoptotic bodies, loss of adherence to extracellular matrix, activation of proteases, and externalization of phosphatidylserine . Autophagy is a physiological process that provides energy required for the turnover of cellular proteins and other macromolecules under certain stress conditions such as nutrient deprivation, hypoxia, and metabolic, genotoxic, and oxidative stress. Autophagy is regarded as a cell survival and protection mechanism; thus, it may play a negative role in cancer therapy and limit the therapeutic efficacy of chemotherapeutic agents . However, recent studies have reported that excessive and sustained autophagy by various anti-cancer therapies can eventually induce cell death in many kinds of cancer cells [7–11], supporting the notion that autophagy may act as either a guardian or an executor. In addition, especially in apoptosis-defective cells, autophagy triggered by cytotoxic drugs promotes cancer cell death via excessive engulfment of cytoplasmic cellular components within a vacuole (autophagosome) and delivery to the lysosome for degradation . In some circumstances, autophagy and apoptosis occur simultaneously in cancers and may be interconnected by certain upstream signaling pathways [13, 14]. Among them, the protein kinase B/mammalian target of rapamycin/p70S6K (Akt/mTOR/p70S6K) signaling pathway is coordinately regulated in both apoptosis and autophagy, and Beclin-1 is an integrator that regulates apoptotic and autophagic activities .
The autophagy gene Beclin-1, as part of the class III phosphatidylinositol 3 kinase (PI3K) complexes, participates in the formation of autophagic vesicles and is critical in the mediation of localization of other autophagic proteins to pre-autophagosomal membranes. Overexpression of Beclin-1 in human cervical cancer cells has been reported to induce massive autophagic cell death and inhibit cancer cell growth . Some anti-apoptotic B-cell lymphoma 2 (Bcl-2) family members including Bcl-2 and Bcl-xL can interact with Beclin-1, and the Beclin-1/Bcl-2 complex functions as an inhibitor of autophagy-induced cell death. Thus, dissociation of Beclin-1 from Bcl-2 is required for Beclin-1-dependent autophagy induction . Similarly, the constitutively activated class I PI3K/Akt pathway inhibits both apoptosis and autophagy because it acts as a positive regulator of the mTOR signaling pathway and plays a crucial role in cancer cell survival. Thus, disruption of the class I PI3K/Akt pathway by anti-cancer agents induces autophagy [18, 19].
Samsoeum (SSE, Shensuyin in Chinese, Jinsoin in Japanese), a traditional herbal medicine, was first described during the Song Dynasty of China and has been widely used as a remedy for headache, cough, rhinorrhea, and fever. SSE also has been used to treat congestion with phlegm, tidal fever, and emesis. Recent studies have reported the pharmacological efficacy of SSE in allergic and asthma reactions and pulmonary damage from ozone . SSE modulates allergic and inflammatory reactions via inhibition of the expression of cyclooxygenase 2 (COX-2) and inflammatory cytokines and suppression of nuclear factor-κB (NF-κB) activation . However, the anti-cancer effect of SSE and its exact mechanism of action remain to be examined. Therefore, the present study aimed to elucidate the effect of SSE on the cell growth and cell death in cancer cells and investigate the detailed mechanism of its anti-cancer activity.
The human gastric carcinoma AGS cell line, human fibrosarcoma HT1080 cell line, human epidermoid carcinoma A431 cell line, and murine melanoma B16F10 cell line were purchased from American Type Culture Collection (ATCC, Manassas, VA). Each cell line was maintained as a monolayer culture in Roswell Park Memorial Institute (RPMI) 1640 or Dulbecco’s Modified Eagle Medium (DMEM; Lonza, Walkersville, MD) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; GIBCO/Invitrogen, Carlsbad, CA), 100 units/mL penicillin, and 100 μg/mL streptomycin (Welgene) at 37°C in a humidified 5% CO2 incubator. Murine hepatocytes were isolated from 6–8 weeks old female ICR mouse purchased from Nara Bio animal center (Nara Biotech, Korea). Mice were housed under standard conditions at a temperature of 24 ± 1°C and humidity of 55 ± 5%, and experimental procedures were approved by Korea Institute of Oriental Medicine Care and Use Committee with a reference number 12–122. Mice were cared for in accordance with the dictates of the National Animal Welfare Law of Korea and experiments were carried out in accordance with the Korea Institute of Oriental Medicine Care Committee Guidelines. Murine hepatocytes were isolated using a perfusion system with some modification . After suspending in the William’s E medium containing 10% FBS, 100 IU/mL insulin, 2 mM L-glutamine, 15 mM HEPES, 100 units/mL penicillin, and 100 μg/mL streptomycin, hepatocytes were seeded on the culture plate coated with 10% gelatin/phosphate buffered saline (PBS), and incubated at 37°C in a humidified 5% CO2 incubator.
Antibodies and reagents
Propidium iodide (PI), Ribonuclease A (RNase A) from bovine pancreas, and 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St Louis, MO, USA). Antibodies against Cyclin D1, Cyclin B1, Cdc25, and α-tubulin were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Anti-p21Waf1/Cip1, anti-p27Kip1, anti-caspase-3, poly (ADP-ribose) polymerase (PARP), anti-p38, anti-phospho-p38 (Thr180/Tyr182), anti-extracellular signal-related kinase1/2 (ERK), anti-phospho-ERK (Thr202/Tyr204), anti-c-Jun-N-terminal kinase (JNK), anti-phopsho-JNK (Thr183/Tyr185), anti-Akt, anti-phopho-Akt (Ser473), anti-mTOR, anti-phospho-mTOR (Ser2481), anti-adenosine monophosphate activated-activated protein kinase (AMPK), anti-phospho-AMPK (Thr172), anti-Bcl-2, anti-Bax, and anti-Beclin-1 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-microtubule-associated protein light chain 3 (LC3) and anti-cleaved caspase-3 antibodies were from Sigma Chemical Co. and Abcam (Cambridge, UK), respectively. All of the other chemicals and solvents used were analytical grade.
Preparation of herbal extract, Samsoeum (SSE)
The prescription of Samsoeum (SSE)
Relative amount (%)
Angelicae Decursivae Radix
Autantii Fructus Immaturus
Glycyrrhizae Radix et Rhizoma
Citri Unshius Pericarpium
Zingiberis Rhizoma Crudus
Cell viability and cell death assay
Cells were seeded at a density of 5 × 103 cells/well in 96-well culture plates, and then incubated with concentrations of SSE between 10 to 250 μg/mL. Untreated ‘control’ cells were incubated with DMSO at final concentration of 0.01%. After 24 h of treatment, cells were incubated with 10 μL of MTT solution (5 mg/mL in PBS) for additional 4 h, formazan precipitates were dissolved by dimethyl sulfoxide (DMSO) and then absorbance was measured at 570 nm with Infinite® M200 microplate reader (TECAN Group Ltd. Switzerland). For cell death evaluation, SSE-treated cells were stained in 0.4% trypan blue solution (GIBCO) and then counted using a hemacytometer (Neubauer Improved, Marienfeld, Germany) under inverted microscope (Olympus CKX41SF; Olympus Optical Co. LTD, Tokyo, Japan). In the experiment with inhibitors, cells were treated with indicated concentrations of SSE for 24 h with or without a 1 h pretreatment with 10 μM SP600125 (Calbiochem, San Diego, CA), 10 μM SB203580 (Calbiochem), 10 μM PD98059 (Calbiochem), 100 μM 3-methyladenine (3-MA; Sigma), or 10 μM z-VAD-fmk (Calbiochem).
Cell cycle analysis
Cells were seeded on 60 mm culture dishes at a density of 5 × 105 cells/dish and allowed to adhere overnight. After incubation with 50 μg/mL of SSE for 6, 12, and 24 h, cells were harvested, washed twice with PBS, and fixed with ice-cold 70% ethanol at −20°C for 24 h. Subsequently, cells were centrifuged, washed once with PBS, and then intracellular DNA was labeled with 0.5 mL of cold propidium iodide (PI) solution (0.1% Triton X-100, 0.1 mM EDTA, 50 μg/mL RNase A, 50 μg/mL PI in PBS) on ice for 30 min in the dark. Cell cycle distribution was measured with FACSCalibur flow cytometry using CellQuest software (BD Biosciences, San Jose, CA) and analyzed using WinMDI 2.8 software (J. Trotter, Scripps Research Institute, La Jolla, CA).
Detection of YO-PRO-1 uptake and nuclear staining with DAPI
For the detection of apoptosis, cells seeded on 60 mm culture dishes were treated with 50 μg/mL of SSE for 6, 12, and 24 h, harvested, and then incubated with apoptosis-specific dye, YO-PRO-1 (Molecular Probes, Eugene, OR) at 10 μM for 5 min. YO-PRO-1 uptake was determined with FACSCalibur flow cytometry using CellQuest software and analyzed using WinMDI 2.8 software. In addition, SSE-treated cells (1 × 104 cells/0.2 mL PBS) were spun onto glass slides by cytospin centrifuge (Cellspin; Hanil, Korea) at 400 × g for 4 min, fixed with 4% paraformaldehyde for 10 min at 37°C, stained with DAPI solution for 10 min, and then observed under the fluorescence microscope (Olympus TH4-200; Olympus Optical Co. LTD).
Fluorescence analysis of LC3 distribution
Cells (5 × 104) grown on the coverslips in 24-well culture plates were transiently transfected with RFP-LC3 plasmid using TransIT-2020 (Mirus, Madison, WI), treated with 50 μg/mL SSE for 12 h, and then appearance of RFP-LC3 puncta was visualized on a confocal laser scanning microscope (FV10i-W; Olympus Optical Co. LTD) after mounting the coverslips onto glass slides with Vectashield (mounting medium with DAPI, Vector Laboratories, Burlingame, CA).
Western blot analysis
After washing cells twice with PBS, whole cell lysates were extracted in M-PER Mammalian Protein extraction Reagent (Thermo Scientific, Rockford, IL) by centrifugation (12000 g × 15 min, 4°C). Equal amount of protein (20–40 μg) was separated by electrophoresis on 8-15% SDS-polyacrylamide gels, and transferred to Immobilon®-P PVDF transfer membrane (Millipore, Bedford, MA). After immunoblotting, proteins were visualized using a PowerOpti-ECL Western blotting Detection reagent (Animal Gentetics, Inc. Korea) and an ImageQuant LAS 4000 mini (GE Healthcare, Piscataway, NJ). Band intensities were quantified using ImageJ software (National Institutes of Health, USA).
Preparation of standard and sample
The standard solutions of seven components, puerarin, daidzin, liquiritin, naringin, hesperidin, neohesperidin, and glycyrrhizin were prepared by dissolving 2 mg of each compound in methanol at the concentration to 200 ppm. The SSE powder was dissolved in water at the concentration of 50 mg/mL, and then filtered through a 0.45 μm PVDF membrane filter (ADVANTEC, Japan) before analysis.
RP-HPLC-DAD operating conditions for analysis of Samsoeum (SSE)
Waters 2695 and 966 photodiode array detector
Waters Empower 1.0 software system
RS tech C18 column (5 μm, 4.6 mm × 250 mm)
Water with 0.1% trifluoroacetic acid (TFA)(A) and Acetonitrile (B)
254 and 280 nm
0.1% TFA (A)
Data are presented as the mean ± S.D. values of at least 3 independent experiments, unless otherwise specified. Statistical significance was analyzed by the two-tailed Student’s t-test in Sigma Plot 8.0 software (SPSS Inc., Chicago, IL) and a P value of less than 0.05 was considered statistically significant.
Results and discussion
SSE treatment induces concentration- and time-dependent cell death and G2/M arrest in cancer cells
SSE induces both apoptosis and autophagy in AGS and B16F10 cells
To gain further insight into the mechanism by which SSE induces cell death, we examined the effect of SSE treatment on the expression of apoptosis- and autophagy-related proteins using western blot analysis (Figure 3D). The protein levels of Beclin-1, which initiates autophagosome formation during autophagy, were gradually increased in AGS and B16F10 cells after SSE treatment. Moreover, the ratio of LC3-II to LC3-I was significantly increased in SSE-treated AGS and B16F10 cells. In addition, SSE treatment significantly inhibited anti-apoptotic Bcl-2 expression, enhanced pro-apoptotic Bax expression, and resulted in the cleavage of caspase-3 and PARP, a downstream target of activated caspase-3. Bcl-2 family proteins including Bcl-2 and Bcl-xL are frequently overexpressed in cancers and inhibit apoptosis by binding to Bax or Bak . Moreover, Bcl-2 and Bcl-xL suppress autophagy by binding to the BH3 domain (amino acids 114–223) of the Beclin-1 protein and sequestering Beclin-1 from hVps34, which is a significant regulator in the initial steps of autophagy, indicating that Bcl-2 and Bcl-xL play essential roles in the crosstalk between autophagy and apoptosis . These data suggest that SSE treatment efficiently induces both autophagy and apoptosis, which partner to induce cell death cooperatively by modifying Beclin-1 and Bcl-2 expression.
SSE suppresses the PI3K/Akt/mTOR pathway via activation of AMPK and activates the mitogen-activated protein kinase (MAPK) pathway
JNK activation is required for the up-regulation of Beclin-1, LC3-II, and Bax and down-regulation of Bcl-2 expression in response to SSE
Identification of seven main components in SSE by RP-HPLC-DAD system
In summary, our finding clearly demonstrated that SSE has anti-cancer activity via suppression of the Akt/mTOR signaling pathway through AMPK activation, which resulted in the down-regulation of Bcl-2 and up-regulation of Beclin-1. SSE treatment activated MAPKs including p38, ERK, and JNK; however, only SP600125, a specific inhibitor for JNK activation, nearly prevented SSE-induced cell death by blocking changes in the level of key proteins such as Bcl-2, Bax, Beclin-1, and LC3-II. In particular, SSE causes both apoptosis and autophagy, and these PCD processes are indispensable for the induction of cell death. Collectively, these results provide new insight into the pharmacological action of SSE as a potent herbal medicine for the treatment of malignant tumors.
Programmed cell death
Mammalian target of rapamycin
Phosphatidylinositol 3 kinase
B-cell lymphoma 2
Adenosine monophosphate activated-activated protein kinase
Microtubule-associated protein light chain 3
Reverse phase high-performance liquid chromatography-photoiodide array detector.
This work was supported by the a Grant K12050 from the Korea Institute of Oriental Medicine, Ministry of Education, Science and Technology (MEST), Republic of Korea. We would like to thank Dr. Kyum-Yil Kwon (KAIST, Korea) and Prof. Jongkyeong Chung (Seoul National University, Korea) for providing the RFP-LC3 plasmid.
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