Advanced
H9 Induces Apoptosis via the Intrinsic Pathway in Non-Small-Cell Lung Cancer A549 Cells
H9 Induces Apoptosis via the Intrinsic Pathway in Non-Small-Cell Lung Cancer A549 Cells
Journal of Microbiology and Biotechnology. 2015. Mar, 25(3): 343-352
Copyright © 2015, The Korean Society For Microbiology And Biotechnology
  • Received : December 29, 2014
  • Accepted : January 06, 2015
  • Published : March 28, 2015
Download
PDF
e-PUB
PubReader
PPT
Export by style
Article
Author
Metrics
Cited by
TagCloud
About the Authors
Sae-Bom Kwon
Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Republic of Korea
Min-Je Kim
Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Republic of Korea
Ham Sun Young
Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Republic of Korea
Ga Wan Park
Genomic Informatics Center, Hankyong National University, Anseong 456-749, Republic of Korea
Kang-Duk Choi
Genomic Informatics Center, Hankyong National University, Anseong 456-749, Republic of Korea
Seung Hyun Jung
School of Oriental Medicine, Dongguk University, Ilsan 410-820, Republic of Korea
Yoon Do-Young
Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Republic of Korea
ydy4218@konkuk.ac.kr

Abstract
H9 is an ethanol extract prepared from nine traditional/medicinal herbs. This study was focused on the anticancer effect of H9 in non-small-cell lung cancer cells. The effects of H9 on cell viability, apoptosis, mitochondrial membrane potential (MMP; ∆ψ m ), and apoptosisrelated protein expression were investigated in A549 human lung cancer cells. In this study, H9-induced apoptosis was confirmed by propidium iodide staining, expression levels of mRNA were determined by reverse transcriptase polymerase chain reaction, protein expression levels were checked by western blot analysis, and MMP (∆ψ m ) was measured by JC-1 staining. Our results indicated that H9 decreased the viability of A549 cells and induced cell morphological changes in a dose-dependent manner. H9 also altered expression levels of molecules involved in the intrinsic signaling pathway. H9 inhibited Bcl-xL expression, whereas Bax expression was enhanced and cytochrome C was released. Furthermore, H9 treatment led to the activation of caspase-3/caspase-9 and proteolytic cleavage of poly(ADP-ribose) polymerase; the MMP was collapsed by H9. However, the expression levels of extrinsic pathway molecules such as Fas/FasL, TRAIL/TRAIL-R, DR5, and Fas-associated death receptor were downregulated by H9. These results indicated that H9 inhibited proliferation and induced apoptosis by activating intrinsic pathways but not extrinsic pathways in human lung cancer cells. Our results suggest that H9 can be used as an alternative remedy for human non-small-cell lung cancer.
Keywords
Introduction
The main characteristics of cancer are abnormal growth, invasion, and metastasis. Cancer cells move around the body through blood vessels and lymphatic ducts. Globally, lung cancer is a leading cause of cancer-related deaths [14] , and it can be categorized into two histological groups: nonsmall-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC). Roughly 85% of all lung cancers are categorized as NSCLC, which has a 5-year survival rate [15] . The high mortality of this cancer type is due to difficulties in early diagnosis and its great potential to invade locally and metastasize to distant organs [21] . Many treatments are available to extend patient life and remove the cancer, including surgery, radiation, and drugs [30] . However, these therapies often lead to diminished quality of life owing to side effects. Herbal extracts have been used as alternative remedies for various diseases because they have fewer side effects than commercial anticancer agents [24] . The traditional/medicinal herbal extracts consist of many metabolites, such as flavonoids, glycosides, polyphenols, and other secondary metabolites that offer a variety of benefits, including antibacterial, antiallergic, antioxidative, and anticancer effects [3 , 17] . As an alternative therapy, herbal extracts have been an important source of cancer treatments, some of which are currently used in clinical practice [8] . In recent years, natural herbal extracts have been extensively screened to develop potential anticancer agents [6 , 25 , 34] .
Among the molecular mechanisms, apoptosis is a highly controlled process of cell death. Apoptotic cells have distinguishable changes, such as chromatin condensation, nuclear fragmentation, and blebbing [23] . Apoptosis involves both intrinsic and extrinsic pathways that serve to eliminate heavily damaged cells. Over the years, apoptosis has been shown to be the major mechanism to eliminate cancer cells [9] . Traditional herbs have been confirmed to have anticancer benefits through apoptosis [1] .
In these studies, we investigated the anticancer effect of H9 on human lung cancer cells. To this end, this study aimed to assess H9-mediated toxicity and apoptosis in A549 cells.
Materials and Methods
- Reagents and Antibodies
CellTiter 96 AQ ueous One Solution Cell Proliferation Assay Reagent (MTS; 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) was purchased from Promega (Madison, WI, USA). Propidium iodide (PI) and 4’,6-diamidino-2-phenylindole (DAPI) stain and were purchased from Sigma (St. Louis, MO, USA). NE-PER Nuclear and Cytoplasmic Extraction Reagents were purchased from Pierce (Rockford, IL, USA). Antibodies specific to PARP, caspase-3, caspase-9, p53, Bcl-2, Bcl-xL, Bax, proliferating cell nuclear antigen, and cytochrome C were purchased from Cell Signaling Technology (Beverly, MA, USA). The anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibody and anti-mouse IgG HRP-conjugated secondary antibody were purchased from Millipore (Billerica, MA, USA). Antibodies specific to cyclin D1, cyclin E, cyclin A, p27, p21, actin, and glyceraldehyde 3-phospahte dehydrogenase (GAPDH) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethyl benzimidazolycarbocyanine chloride) was purchased from Enzo (Farmingdale, NY, USA)
- Plant Materials
H9 consisted of nine oriental medicinal herbs ( Table 1 ). The main ingredients of H9 and their proportions (w/w) were determined as follows: 12% Psoraleae Semen, 8% Evodia Fruit, 12% Fennel, 12% Nutmeg, 20% Ginseng, 8% Alpiniae Officinarum Rhizome, 4% Sparganium Rhizome, 12% Curcuma Root, and 12% Cinnamon Bark. The herbal ingredients were obtained from the Oriental Medical Hospital, Dongguk University (Ilsan, Korea) and were authenticated by Dr. Seong-Hyun Jung (Department of Oriental Herbal Materials, Dongguk University).
H9 formula consisting of nine oriental medicinal herbs.
PPT Slide
Lager Image
H9 formula consisting of nine oriental medicinal herbs.
- Methods of Extraction
The ethanol extracts from the plants listed above were prepared as follows. The dried and pulverized medicinal herbs were mixed together. Materials of 8 kg were soaked with 40% ethanol, and then extracted for 3 h at 90-100℃. This extract was filtered through a 11 µm microfilter, extracted for 3 h at 90-100℃, vacuum evaporated at 60℃ using a rotary evaporator, lyophilized, and then reconstituted in distilled water for in vitro studies.
- Cell Culture
We purchased human NSCLC cell line A549 cells from the American Type Culture Collection (ATCC; Rockville, MD, USA). These cells were cultured in RPMI medium (Welgene Incorporation, Daegu, Korea) containing 10% (v/v) heat-inactivated fetal bovine serum (Hyclone Laboratories, Logan, UT, USA). The cells were incubated at 37℃ in saturating humidity of 5% CO 2 /95% air.
- Cell Viability Assays
Cell viability was observed by MTS dye-reduction assay, which measures the mitochondrial respiratory function. The A549 (1.0 × 10 4 ) cells were seeded in 100 µl of medium in 96-well plates, incubated overnight, and then treated with various concentrations of H9, as mentioned in the figure legends, for 24 h. Cell viability was calculated by MTS metabolism, as previously reported [1] . In brief, the effect of H9 on cell viability was assessed by an electroncoupling reagent containing MTS and PMS (99 to 1 ratio). These reagents mixed with medium were treated (100 µl to each well), and the cells were incubated for another 1 h. Optical density was measured at 492 nm using an ELISA reader (Apollo LB 9110; Bertholo, Technologies GmbH, Germany). The percentage of viable cells was estimated by comparison with the untreated controls. The viability assay was repeated three times.
- DAPI Staining
Apoptotic nuclear morphology was observed using DAPI staining. The A549 cells were seeded on coverslips in 6-well plates and treated with specified concentrations of H9 for 24 h. The coverslips in the 6-well plates were washed with phosphatebuffered saline (PBS). Then, the A549 cells were fixed with 4% paraformaldehyde and stained with DAPI staining solution. The coverslips were then washed with PBS, dried completely, and mounted on microscope slides with a mounting solution. The stained cells were observed using fluorescence microscopy (Olympus, Japan).
- Cell Cycle and Apoptosis Analyses by Flow Cytometry
The cell cycle and apoptosis were analyzed by PI staining and flow cytometry. The A549 cells (1.0 × 10 5 cells/well) were seeded in 6-well plates and exposed to various concentrations of H9 for 24 h. The cells were harvested with trypsin-ethylenediamine tetra acetic acid (EDTA) and fixed with 80% ethanol. After fixation, the cells were washed twice with cold PBS, centrifuged, and the supernatants were eliminated. The pellet was resuspended and stained with PBS containing 50 µg/ml PI and 100 µg/ml RNase A, for 20 min in the dark. The DNA content was analyzed by flow cytometry using a FACSCalibur instrument and CellQuest software (BD Bioscience, San Jose, CA, USA).
- Reverse-Transcription Polymerase Chain Reaction (RT-PCR)
The cells treated with H9 were harvested. RNA was extracted using an easy-BLUE Total RNA Extraction Kit (iNtRon Biotechnology, SungNam, Korea) according to the manufacturer’s instructions, as previously reported [1] . The cDNA products were obtained using M-MuLV reverse transcriptase (New England Biolabs, Beverly, MA, USA). Using a PCR Thermal Cycler Dice instrument (TaKaRa, Otsu, Shiga, Japan), we performed a RT-PCR analysis with the following primer sets: Fas 5’-AGG GAT TGG AAT TGA GGA AG-3’ (forward), 5’-ATG GGC TTT GTC TGT GTA CT-3’ (reverse); FasL 5’-AGT CCA CCC CCT GAA AAA AA-3’ (forward), 5’-ATT CCA TAG GTG TCT TCC CA-3’ (reverse); FADD 5’-ACC TCT TCT CCA TGC TG-3’ (forward), 5’-CAC ACA GGT CTT CCC CA-3’ (reverse); DR5 5’-GTC TGC TCT GAT CAC CCA AC-3’ (forward), 5’-CTG CAA ACT GTG ACT CCT AT-3’ (reverse); TRAIL 5’-GTC TCT CTG TGT GGC TGT AA-3’ (forward), 5’-TGT TGC TTC TTC CTC TGG CT-3’ (reverse); and GAPDH 5’-GGC TGC TTT TAA CTC TGG TA-3’ (forward), 5’-TGG AAG ATG GTG ATG GGA TT-3’ (reverse). GAPDH was used as an internal control.
- Detection of Caspase-3/-9 Activity
A549 cells were plated at 1 × 10 6 cells/well in 60 ϕ plates. After H9 treatment for 24 h, control and treated cells were harvested with trypsin-EDTA. The activity of caspase-3 was measured using a Caspase-3 Colorimetric Assay kit (BioVision, Mountain View, CA, USA) in accordance with the manufacturer’s protocol. The absorbance was measured at 405nm, using a UV spectrophotometer (Biochrom WPA Bio-wave II, Cambridge, UK).
- Western Blot Analysis
Cells were treated with specified concentrations of H9 for 24 h and then harvested. The cells were washed with ice-cold PBS and recentrifuged (1,890 × g , 5 min, 4℃). The cell pellets that were obtained were resuspended in a lysis buffer containing 50 mM Tris (pH 7.4), 1.5 M sodium chloride, 1 mM EDTA, 1% NP-40, 0.25% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and a protease inhibitor cocktail. The cell lysates were incubated on ice for 1 h, and then clarified by centrifugation at 17,010 × g for 30 min at 4℃. The protein content was quantified using a Bradford assay (Bio-Rad, Hercules, CA, USA) and a UV spectrophotometer. The cell lysates were separated by 10-12% SDS polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred to polyvinylidene difluoride membranes (PVDF; Millipore). The membranes were blocked in 5% non-fat dried milk dissolved in Tris-buffered saline, containing Tween-20 (2.7 M NaCl, 53.65 mM KCl, 1 M Tris-HCl, pH 7.4, 0.1% Tween-20), for 1 h at room temperature. The membranes were incubated overnight at 4℃ with the specific primary antibodies. After washing three times, the secondary antibodies (HRP-conjugated anti-rabbit or antimouse IgG) were incubated with the membranes for 1 h at room temperature. After washing three times, the blots were analyzed using West-Zol plus a western blot detection system (iNtRON Biotechnology).
- Nuclear and Cytoplasmic Fractionation
The H9-treated cells were collected and fractionated using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific Inc, IL, USA), according to the manufacturer’s protocol. After fractionation, we calculated each extract concentration, performed a 15% Tris-glycine gel electrophoresis, and transferred them to a PVDF membrane. The membrane was blocked with 5% TBST skim milk and incubated with primary antibodies overnight. The membrane was washed with TBST and incubated with secondary antibodies for 1 h. After washing with TBST, we added a chemiluminescent substrate (Westzol; iNtRon Biotechnology).
- Analysis of Mitochondrial Transmembrane Potential (MTP)
The MTP (∆ψ m ) was evaluated by JC-1 staining and flow cytometry. JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethyl-benzimidazolylcarbocyanine chloride) was purchased from Enzo. The A549 cells were seeded in 6-well plates (1.0 × 10 5 cells/well) and treated with various concentrations of H9. Cells were harvested with trypsinEDTA and transferred to 1.5 ml tubes. JC-1 (5 µg/ml) was added to the cells, mixed until completely dissolved, and incubated in the dark for 10 min in a 37℃ incubator. The cells were centrifuged (300 × g , 5min, 4℃), washed twice with PBS, and resuspended in 200 µl of PBS. The solutions were divided using a FACSCalibur instrument and analyzed by CellQuest software (BD Bioscience). The entire protocol was performed in minimal light.
- Statistical Analysis
Data are presented as the mean ± SEM from at least three independent experiments. Statistical significance was assessed with the Student’s t -test. * p < 0.05 or ** p < 0.005 was considered statistically significant.
Results
- Chemical Content of H9
H9 contains ethanol extracts from nine oriental medicinal herbs, as described in the Materials and Methods section ( Table 1 ). We identified the potential medicinal components of the H9 extract using gas chromatography mass spectrometry ( Fig. 1 ). Compounds were identified by comparing with the compounds in the Wiley 6th edition of the MS spectra library (search program hits that were >90% probable were viewed as likely hits). Table 2 lists the major components in the H9 extract, such as coumarin, isoeugenol, isoelemicin, and angelicin. It has been reported that coumarin and angelicin have anticancer properties [11 , 35] . However, there are no reports regarding the effects of the other compounds, such as isoeugenol and isoelemicin, on cancer cells ( Table 2 ).
PPT Slide
Lager Image
GC-MS analysis of H9, ethanol extracts of medicinal herbs. H9 was analyzed by gas chromatography mass spectrometry and results are summarized in Table 2. Among the volatile compounds in H9, coumarin and angelicin are major components. Coumarin and angelicin have anticancer properties.
Volatile compounds identified in H9.
PPT Slide
Lager Image
Volatile compounds identified in H9.
- Effect of H9 on Cell Viability
The cell viability of A549 cells with H9 treatment was analyzed by MTS assay. We evaluated the cell viability of H9-treated cells compared with untreated control cells. A549 cells were treated with H9 in various concentrations for 24 h. Compared with the other concentrations, a distinct decrease was not shown at concentrations lower than 50 µg/ml. Compared with the other concentrations, the cell viability was increased in concentrations of 50-200 µg/ml ( Fig. 2 A). A549 cells were treated for 24 h with up to 200 µg/ml of H9 for optical observation by phase-contrast microscopy. As the concentration of H9 increased, the cell density gradually decreased. In addition, cell shapes were changed into shrunken forms ( Fig. 2 B). DAPI staining revealed that nuclear condensation was significantly increased in H9-treated A549 cells, suggesting an inhibition of proliferation or progression of cell death ( Fig. 2 C).
PPT Slide
Lager Image
Antiproliferative and pro-apoptosis effects of H9 on A549 cells. (A) The cell viability was calculated by MTS assay. A549 cells were treated for 24 h with various concentrations of H9. Data are expressed as the mean ± SEM; *p < 0.05, **p < 0.005, compared with the untreated control. (B) Morphological changes were imaged by phase-contrast microscopy. A549 cells were treated with H9 for 24 h. (C) Apoptotic nuclei after treatment with H9, as detected by fluorescence micrographs. A549 cells were treated with H9 for 24 h and stained with 4,6-diamidino-2-phenylindole.
- H9-Induced Apoptosis in A549 Cells
PI staining was performed to determine whether H9-mediated cell death was due to cell cycle arrest or apoptosis ( Fig. 3 A). PI staining revealed that H9 enhanced cell numbers at the sub-G 1 phase, where hypodiploid (≤2N) fragmented DNA is a marker of apoptosis [18] . However, H9 inhibited the G 0 /G 1 cell population ( Fig. 3 B). In order to check the modulators involved in the early cell cycle, such as the sub-G 1 and G 0 /G 1 phases, western blot analysis was performed to detect the cell-cycle-dependent kinases (CDKs) and the cell-cycle-dependent kinase inhibitors (CDKIs) ( Fig. 3 C). H9 enhanced the expression levels of p53 and its target CDKIs (p21/p27) and suppressed cyclin D; these findings suggested that H9 inhibited the progress of the G 0 /G 1 phase and resulted in enhanced cell numbers at the sub-G 1 phase ( Figs. 3 A and 3 B).
PPT Slide
Lager Image
Apoptotic effects of H9 on A549 cells. (A) Effect of H9 on cell cycle proportions in A549 cells. A549 cells were analyzed by flow cytometry. (B) Percentage of sub-G1 population summarized as a bar graph. Data are expressed as the mean ± SEM; *p < 0.05, **p < 0.005, compared with the untreated cells used as a control. (C) The expression levels of cell-cycle-related proteins, such as p53, p27, p21, cyclin E, and cyclin D, were detected by western blot analysis. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control.
- H9-Treated A549 Cells Evaded Extrinsic Pathways
In order to investigate whether H9 would induce apoptosis mediated via the extrinsic signaling pathways, we revealed cell surface death receptors and their interacting factors. PCR analysis revealed that H9 treatment downregulated the expression levels of Fas/FasL, FADD, DR5, and TRAIL/TRAIL receptors ( Fig. 4 A). These results suggested that A549 lung cancer cells might suppress or evade extrinsic pathways by downregulating cell surface death receptors.
PPT Slide
Lager Image
Effects of H9 on the apoptosis signaling pathway. (A) The expression levels of TRAIL, TRAIL-receptor, FAS, FAS ligand, Fas-associated death receptor (FADD), and DR5 were detected by RT-PCR. (B) The expression levels of intrinsic-pathway-related proteins, such as caspase-9, caspase-3, and poly(ADP-ribose) polymerase (PARP), were detected by western blot analysis. GAPDH and actin were used as internal controls. (C) Caspase-3 and caspase-9 activities were determined by a colorimetric assay.
- H9 Induced Apoptosis Through Intrinsic Pathways
Since extrinsic pathways were not involved in the H9-induced apoptotic process, we focused on the intrinsic signaling pathways. H9 treatment led to the processing of caspase-3, caspase-9, and PARP in a dose-dependent manner ( Fig. 4 B). H9 treatment also increased the activation of caspase-3 and caspase-9 ( Fig. 4 C). Caspase-3 cleaved PARP, which resulted in cell death [20] . In order to elucidate whether H9 activates the mitochondrial pathway, we examined the expression levels of factors related to the intrinsic pathways. We examined the valence between expressions of pro-apoptosis and pro-survival proteins by western blot analysis. The pro-apoptotic factor Bax expression level was increased by H9, while H9 decreased the anti-apoptotic factor Bcl-xL expression level ( Fig. 5 A). To elucidate the pathway underlying apoptosis, we observed mitochondrial cytochrome C release into the cytosol following H9 treatment on A549 cells for 24 h. As shown in Fig. 5 A, cytochrome C was released into the cytosol in a dosedependent manner. The mitochondrial outer membrane permeabilization is essential to cytochrome C release [31] . Released cytochrome C triggers executor caspase activation [31] . For that reason, we examined the mitochondrial membrane potential collapse and cytochrome C release. We performed JC-1 staining. In apoptotic cells, JC-1 dye exists as a monomer, but JC-1 aggregation occurs in viable cells.
PPT Slide
Lager Image
Effects of H9 on mitochondrial membrane potential. (A) The expression levels of mitochondrial-membrane-related proteins, such as Bax, Bcl-xL, Bcl-2, and cytochrome C, were detected by western blot analysis. (B) H9 induced mitochondrial membrane potential collapse. The difference in JC-1 colors was analyzed by flow cytometry.
JC-1 is a lipophilic cationic dye that can enter and accumulate in the mitochondrial matrix. Healthy mitochondria have a negative charge. In apoptotic cells, the mitochondrial membrane potential collapses. The orange fluorescence of JC-1 shows intact mitochondria, whereas green fluorescence shows monomeric JC-1 that remain unprocessed owing to the collapse of the mitochondrial membrane potential. The condensational form of JC-1 emits orange fluorescence at 590 nm (FL-2) and the monomer emits green fluorescence at 525 nm (FL-1) [29] . To measure the mitochondrial potential change by H9 treatment, we conducted JC-1 staining and FACS analysis using the green and orange channels. As shown in Fig. 5 B, JC-1 staining revealed that JC-1 aggregation was reduced by H9 treatment and the peaks were shifted to the left. This result suggested that the mitochondrial potential loss was triggered at a higher concentration of H9 compared with the control.
Discussion
Traditional/medicinal herbal extracts have been used in Asian countries. These extracts can overcome the adverse effects of chemotherapy. Medicinal herbal extracts may be used to give additive or synergistic preventive effects for the inhibition of tumor growth [4] . H9 is composed of ethanol extracts from mixtures of Psoraleae Semen, Evodia Fruit, Fennel, Nutmeg, Ginseng, Alpiniae Officinarum Rhizome, Sparganium Rhizome, Curcuma Root, and Cinnamon Bark. There have been several reports on the subject of these materials [16 , 19 , 33] , but this study is the first to test the effect of this mixed extract, brown-colored H9. Table 2 shows that the major components in H9 extract are coumarin, isoeugenol, isoelemicin, and angelicin. The cell viability of A549 cells treated with H9 was analyzed by MTS assay. However, it seems that results of the cell viability assay did not match with microscopic data, specifically at 200 µg/ml concentration ( Figs. 2 A and 2 B). These results suggest that, as shown in Table 2 , H9 has many phytochemicals or compounds such as coumarin, isoeugenol, isoelemicin, and angelicin that can affect the electron-coupling reaction between MTS and PMS reagent, which results in the mismatch between MTS and microscopic data.
Among the compounds of H9, coumarin and angelicin have antitumor activities. Coumarin is widely distributed as a plant. Coumarin has been examined for anticoagulation, antiviral, anti-inflammatory, antibacterial, and anticancer activities [5 , 7 , 12 , 13] . Angelicin has been isolated from diverse plants. It is being used for its antimicrobial and central inhibitory activities, plus its inhibitory role in cell proliferation [11 , 26 , 28] . Angelicin increases cytotoxicity and induces apoptosis in human SH-SY5Y neuroblastoma cells; this effect is mediated by the activation of the intrinsic apoptotic pathway and indicates that angelicin activates the caspase-3-dependent apoptosis via the mitochondriamediated apoptotic pathway. However, angelicin treatment did not induce an increase in the levels of Fas/FasL activation and caspase-8 [27] . In other words, angelicininduced apoptosis is caspase-dependent, rather than being dependent on the Fas-receptor-mediated signaling pathways. H9-induced apoptosis was also caspase-dependent rather than being dependent on death-receptor-mediated signaling pathways ( Fig. 4 ).
Apoptosis is triggered by two major pathways that play essential roles in cell survival, growth, development, and tumorigenesis [32] . One is the extrinsic pathway and the other is the intrinsic pathway. The extrinsic pathway is begun by the death receptor located on the cell membrane [10] . The intrinsic pathway is generated by mitochondrial dysfunction and mitochondrial membrane potential collapse [2] .
A schematic summary of H9-induced cell death is presented in Fig. 6 . H9 treatment induces p53, a wellknown tumor suppressor protein that controls the cell cycle and initiates apoptosis in cancer cells [22] . In our case, H9 induced the intrinsic apoptotic pathway, not the extrinsic pathway. Well-known death receptors/ligands and FADD were downregulated in a dose-dependent manner by H9 treatment, suggesting that H9-treated A549 cells evaded the extrinsic pathway. On the other hand, H9 activated the intrinsic apoptotic pathway. H9 treatment altered the expression levels of Bax and Bcl-xL proteins embedded in the mitochondrial membrane; it also induced mitochondrial potential loss, induced mitochondrial cytochrome C release into the cytosol, activated caspase-9 and caspase-3 and the processing of PARP in A549 cells, and led to the successful induction of apoptosis.
PPT Slide
Lager Image
Schematic diagram of the effect of H9-induced apoptosis on A549 cells. The arrows symbolize activation of the signal pathways; the closed lines symbolize repression of the signals.
In summary, our study demonstrated that H9 has antiproliferative and anticancer effects on A549 non-small-cell lung cancer cells. H9 can function as a potential anticancer agent.
Acknowledgements
This study was supported by a grant (B110053) from the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea. D.Y. was partially supported by a program (2012-0006686) from the National Research Foundation of Korea (NRF).
References
Bak Y , Ham S , Baatartsogt O , Jung SH , Choi KD , Han TY 2013 A1E inhibits proliferation and induces apoptosis in NCI-H460 lung cancer cells via extrinsic and intrinsic pathways. Mol. Biol. Rep. 40 4507 - 4519    DOI : 10.1007/s11033-013-2544-0
Bucur O , Ray S , Bucur MC , Almasan A 2006 APO2 ligand/tumor necrosis factor-related apoptosis-inducing ligand in prostate cancer therapy. Front. Biosci. 11 1549 - 1568    DOI : 10.2741/1903
Butt MS , Sultan MT 2009 Green tea: nature's defense against malignancies. Crit. Rev. Food Sci. Nutr. 49 463 - 473    DOI : 10.1080/10408390802145310
Cai Y , Luo Q , Sun M , Corke H 2004 Antioxidant activity and phenolic compounds of 112 traditional Chinese medicinal plants associated with anticancer. Life Sci. 74 2157 - 2184    DOI : 10.1016/j.lfs.2003.09.047
Chilin A , Battistutta R , Bortolato A , Cozza G , Zanatta S , Poletto G 2008 Coumarin as attractive casein kinase 2 (CK2) inhibitor scaffold: an integrate approach to elucidate the putative binding motif and explain structure-activity relationships. J. Med. Chem. 51 752 - 759    DOI : 10.1021/jm070909t
Cho SH , Chung KS , Choi JH , Kim DH , Lee KT 2009 Compound K, a metabolite of ginseng saponin, induces apoptosis via caspase-8-dependent pathway in HL-60 human leukemia cells. BMC Cancer 9 449 -    DOI : 10.1186/1471-2407-9-449
Donnelly AC , Mays JR , Burlison JA , Nelson JT , Vielhauer G , Holzbeierlein J , Blagg BS 2008 The design, synthesis, and evaluation of coumarin ring derivatives of the novobiocin scaffold that exhibit antiproliferative activity. J. Org. Chem. 73 8901 - 8920    DOI : 10.1021/jo801312r
Feng Y , Wang N , Zhu M , Li H , Tsao S 2011 Recent progress on anticancer candidates in patents of herbal medicinal products. Recent Pat. Food Nutr. Agric. 3 30 - 48    DOI : 10.2174/2212798411103010030
Fesik SW 2005 Promoting apoptosis as a strategy for cancer drug discovery. Nat. Rev. Cancer. 5 876 - 885    DOI : 10.1038/nrc1736
Fulda S , Debatin KM 2005 Resveratrol-mediated sensitisation to TRAIL-induced apoptosis depends on death receptor and mitochondrial signalling. Eur. J. Cancer 41 786 - 798    DOI : 10.1016/j.ejca.2004.12.020
Guo J , Wu H , Weng X , Yan J , Bi K 2003 [Studies on extraction and isolation of active constituents fromPsoralen corylifoliaL. and the antitumor effect of the constituentsin vitro]. Zhong Yao Cai 26 185 - 187
Hwu JR , Lin SY , Tsay SC , De Clercq E , Leyssen P , Neyts J 2011 Coumarin-purine ribofuranoside conjugates as new agents against hepatitis C virus. J. Med. Chem. 54 2114 - 2126    DOI : 10.1021/jm101337v
Hwu JR , Singha R , Hong SC , Chang YH , Das AR , Vliegen I 2008 Synthesis of new benzimidazole-coumarin conjugates as anti-hepatitis C virus agents. Antiviral Res. 77 157 - 162    DOI : 10.1016/j.antiviral.2007.09.003
Jemal A , Siegel R , Ward E , Murray T , Xu J , Thun MJ 2007 Cancer statistics, 2007. CA Cancer J. Clin. 57 43 - 66    DOI : 10.3322/canjclin.57.1.43
Jemal A , Siegel R , Xu J , Ward E 2010 Cancer statistics, 2010. CA Cancer J. Clin. 60 277 - 300    DOI : 10.3322/caac.20073
Ji Y , Rao Z , Cui J , Bao H , Chen C , Shu C , Gong JR 2012 Ginsenosides extracted from nanoscale Chinese white ginseng enhances anticancer effect. J. Nanosci. Nanotechnol. 12 6163 - 6167    DOI : 10.1166/jnn.2012.6443
Kandaswami C , Lee LT , Lee PP , Hwang JJ , Ke FC , Huang YT , Lee MT 2005 The antitumor activities of flavonoids. In Vivo 19 895 - 909
Karna P , Gundala SR , Gupta MV , Shamsi SA , Pace RD , Yates C 2011 Polyphenol-rich sweet potato greens extract inhibits proliferation and induces apoptosis in prostate cancer cellsin vitroandin vivo. Carcinogenesis 32 1872 - 1880    DOI : 10.1093/carcin/bgr215
Kwon HK , Hwang JS , So JS , Lee CG , Sahoo A , Ryu JH 2010 Cinnamon extract induces tumor cell death through inhibition of NFkappaB and AP1. BMC Cancer 10 392 -    DOI : 10.1186/1471-2407-10-392
Lazebnik YA , Kaufmann SH , Desnoyers S , Poirier GG , Earnshaw WC 1994 Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE. Nature 371 346 - 347    DOI : 10.1038/371346a0
Lee ER , Kang YJ , Choi HY , Kang GH , Kim JH , Kim BW 2007 Induction of apoptotic cell death by synthetic naringenin derivatives in human lung epithelial carcinoma A549 cells. Biol. Pharm. Bull. 30 2394 - 2398    DOI : 10.1248/bpb.30.2394
May P , May E 1999 Twenty years of p53 research: structural and functional aspects of the p53 protein. Oncogene 18 7621 - 7636    DOI : 10.1038/sj.onc.1203285
Nagata S 2000 Apoptotic DNA fragmentation. Exp. Cell Res. 256 12 - 18    DOI : 10.1006/excr.2000.4834
Newman DJ , Cragg GM , Snader KM 2000 The influence of natural products upon drug discovery. Nat. Prod. Rep. 17 215 - 234    DOI : 10.1039/a902202c
Park HS , Park KI , Lee DH , Kang SR , Nagappan A , Kim JA 2012 Polyphenolic extract isolated from KoreanLonicera japonicaThunb. induce G2/M cell cycle arrest and apoptosis in HepG2 cells: involvements of PI3K/Akt and MAPKs. Food Chem. Toxicol. 50 2407 - 2416    DOI : 10.1016/j.fct.2012.04.034
Patnaik GK , Banaudha KK , Khan KA , Shoeb A , Dhawan BN 1987 Spasmolytic activity of angelicin: a coumarin fromHeracleum thomsoni. Planta Med. 53 517 - 520    DOI : 10.1055/s-2006-962799
Rahman MA , Kim NH , Yang H , Huh SO 2012 Angelicin induces apoptosis through intrinsic caspase-dependent pathway in human SH-SY5Y neuroblastoma cells. Mol. Cell Biochem. 369 95 - 104    DOI : 10.1007/s11010-012-1372-1
Sardari S , Mori Y , Horita K , Micetich RG , Nishibe S , Daneshtalab M 1999 Synthesis and antifungal activity of coumarins and angular furanocoumarins. Bioorg. Med. Chem. 7 1933 - 1940    DOI : 10.1016/S0968-0896(99)00138-8
Smiley ST , Reers M , Mottola-Hartshorn C , Lin M , Chen A , Smith TW 1991 Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregateforming lipophilic cation JC-1. Proc. Natl. Acad. Sci. USA 88 3671 - 3675    DOI : 10.1073/pnas.88.9.3671
Smith HO , Tiffany MF , Qualls CR , Key CR 2000 The rising incidence of adenocarcinoma relative to squamous cell carcinoma of the uterine cervix in the United States - a 24-year population-based study. Gynecol. Oncol. 78 97 - 105    DOI : 10.1006/gyno.2000.5826
Tait SW , Green DR 2010 Mitochondria and cell death: outer membrane permeabilization and beyond. Nat. Rev. Mol. Cell Biol. 11 621 - 632    DOI : 10.1038/nrm2952
Tan ML , Ooi JP , Ismail N , Moad AI , Muhammad TS 2009 Programmed cell death pathways and current antitumor targets. Pharm. Res. 26 1547 - 1560    DOI : 10.1007/s11095-009-9895-1
Yang HL , Chen SC , Chen CS , Wang SY , Hseu YC 2008 Alpinia priceirhizome extracts induce apoptosis of human carcinoma KB cellsviaa mitochondria-dependent apoptotic pathway. Food Chem. Toxicol. 46 3318 - 3324    DOI : 10.1016/j.fct.2008.08.003
Yang ZG , Sun HX , Ye YP 2006 Ginsenoside Rd fromPanax notoginsengis cytotoxic towards HeLa cancer cells and induces apoptosis. Chem. Biodivers. 3 187 - 197    DOI : 10.1002/cbdv.200690022
Zhang W , Li Z , Zhou M , Wu F , Hou X , Luo H 2014 Synthesis and biological evaluation of 4-(1,2,3-triazol-1-yl)coumarin derivatives as potential antitumor agents. Bioorg. Med. Chem. Lett. 24 799 - 807    DOI : 10.1016/j.bmcl.2013.12.095