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H9 Inhibits Tumor Growth and Induces Apoptosis via Intrinsic and Extrinsic Signaling Pathway in Human Non-Small Cell Lung Cancer Xenografts
H9 Inhibits Tumor Growth and Induces Apoptosis via Intrinsic and Extrinsic Signaling Pathway in Human Non-Small Cell Lung Cancer Xenografts
Journal of Microbiology and Biotechnology. 2015. May, 25(5): 648-657
Copyright © 2015, The Korean Society For Microbiology And Biotechnology
  • Received : January 16, 2015
  • Accepted : January 28, 2015
  • Published : May 28, 2015
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About the Authors
Min-Je Kim
Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Republic of Korea
Sae-Bom Kwon
Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Republic of Korea
Seung Hoon Ham
Department of Laboratory Animal Medicine, Veterinary Science Research Institute, College of Veterinary Medicine, Konkuk University, Seoul 143-701, Republic of Korea
Eui-Suk Jeong
Department of Laboratory Animal Medicine, Veterinary Science Research Institute, College of Veterinary Medicine, Konkuk University, Seoul 143-701, Republic of Korea
Yang-Kyu Choi
Department of Laboratory Animal Medicine, Veterinary Science Research Institute, College of Veterinary Medicine, Konkuk University, Seoul 143-701, Republic of Korea
Kang Duk Choi
Graduate School of Future Convergence Technology, Genomic Informatics Center, Hankyong National University, Anseong 456-749, Republic of Korea
Jin Tae Hong
College of Pharmacy, Chungbuk Nation University, Chungbuk 361-763, Republic of Korea
Seung Hyun Jung
Oriental Medical Hospital, Dongguk University, Ilsan 780-714, Republic of Korea
Do-Young Yoon
Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Republic of Korea
ydy4218@konkuk.ac.kr

Abstract
H9, a novel herbal extract, demonstrated cytotoxicity in A549 non-small cell lung cancer (NSCLC) cell lines. In this study, we investigated whether H9, and/or co-treatment with an anticancer drug, pemetrexed (PEM), inhibited tumor growth in BALB/c nude mice models bearing A549 NSCLC cells. The mice were separated into groups and administered H9 and PEM for 2 weeks. Protein and mRNA levels were detected using western blotting and reverse transcription polymerase chain reaction, respectively; immunohistochemistry (IHC) was also performed on the tumor tissues. H9 and co-treatment with PEM induced the cleavage of proapoptotic factors, such as caspase-3, caspase-8, caspase-9, and poly(ADP)-ribose polymerase (PARP). Expression levels of cell-death receptors involving Fas/FasL, TNF-related apoptosisinducing ligands (TRAIL), and TRAIL receptors were increased by H9 and co-treatment with PEM. Furthermore, analysis of levels of cell-cycle modulating proteins indicated that tumor cells were arrested in the G1/S phase. In addition, the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt survival signaling pathways were inhibited by H9 and co-treatment with PEM. In conclusion, H9 and co-treatment with PEM inhibited tumor growth in BALB/c nude mice models bearing A549 NSCLC cells. These results indicate that H9 and co-treatment with PEM can be used as an anticancer therapy in NSCLC.
Keywords
Introduction
Lung cancer is a major cause of cancer deaths worldwide and is classified into two types: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) [23] . Accounting for approximately 85% of all cases, NSCLC is the primary type of lung cancer, and SCLC accounts for 15% [20] . Progress has been made in research for a cure to lung cancer, with the several studies focusing on inducing apoptosis in cancer cells. Among other phenomena, apoptosis involves chromatin condensation, DNA fragmentation, cytoplasm shrinkage, and formation of apoptotic bodies [17 , 19] . Apoptosis occurs via two mechanisms: the intrinsic pathway, which is mitochondria-dependent, and the extrinsic pathway, which is death-receptor-dependent [1 , 36] . The intrinsic apoptotic cascade is initiated by cellular death stimulation in the mitochondrial pathway. Released mitochondrial cytochrome C directly activates caspase-3 by apoptosome complex formation. Then, caspase-3 cleaves cellular targets to initiate programmed cell death [12 , 15] . The extrinsic apoptotic cascade is activated by cell-surface death receptors, such as the tumor necrosis factor receptor superfamily, including Fas, TRAIL receptor, and DR5. The extrinsic signal is triggered by death receptor trimerization. The Fasassociated death domain (FADD) recruits caspase-8 and forms the death-inducing signaling complex (DISC), triggering the extrinsic apoptosis cascade with downstream effectors [12 , 32] .
Over the past few decades, several aspects pertaining to the biological mechanisms underlying NSCLC have been elucidated, which have consequently led to new cures. However, despite these findings, cytotoxic anticancer drugs and molecular targeted therapies are essential choices for the clinical management of cancer because of drug resistance and relapses [33] . To advance NSCLC therapy, there have been trials evaluating the synergistic effects of these therapies [14 , 26] . However, there are some studies on the synergistic effects of cancer therapy with pemetrexed (PEM) [1 , 14 , 18] . DNA-synthesis inhibition is another major anticancer mechanism, and PEM is an antifolate drug that limits dihydrofolate reductase and glycinamide ribonucleotide transformylase, which are related to DNA and RNA synthesis [34 - 35] , and thymidylate synthase, which has a crucial role in DNA replication and repair [2 , 9] .
Plants contain several flavonoids, glycosides, and polyphenols, which are secondary metabolites that have been isolated from fruits, vegetables, and even phytogenic beverages [13] . Moreover, herbal extracts have been used to cure diseases in Asia for centuries. Therefore, herbal extracts have now begun to garner attention as therapeutic agents for cancer treatment. Herbal extracts can inhibit tumor growth, metastasis, and angiogenesis, with only a few side effects. In addition, they have anti-inflammation, antibacterial, and antioxidative effects [5 , 24] . Combining chemotherapeutic drugs with herbal extracts can lower the concentration of chemotherapeutic drug required, with the same anticancer effects [27] . To overcome the side effects of chemotherapy, extracts from medicinal herbs may be used to provide the required additive or synergistic preventive effects for the inhibition of tumor growth [6] . H9 was extracted from nine ingredients, including Psoraleae Semen, Evodia Fruit, Foeniculi Fructus, Nutmeg, Ginseng Radix, Alpiniae Officinari Rhizoma, Sparganium Rhizome, Curcumae Radix, and Cinnamon Bark. In a recent study, we showed that H9 induces apoptosis via the intrinsic pathway in A549 NSCLC cells [25] . Here, we investigated the synergistic effects of PEM and H9 in tumor-bearing mouse models.
Materials and Methods
- Reagents
Pemetrexed disodium (Alimta) ( N -[4-[2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo [2, 3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid sodium salt; N -[4-[(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2, 3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid disodium) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). DAB (3,3-diaminobenzidine) Substrate Kit was purchased from Vector Laboratories (Burlingame, CA, USA). Eosin Y solutions (eosin Y, 0.5% (w/v) in acidified ethanol, 90% (v/v)) and hematoxylin stains (hematoxylin, 6 g/l; sodium iodate, 0.6 g/l; aluminum sulfate, 52.8 g/l, and stabilizers) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
- Antibodies
Antibodies against phospho-pRb, pRb, PARP, caspase-3, caspase-8, caspase-9, p53, p-p53, phospho-Akt1/2/3 (Ser-473), Bcl-2, Bcl-xL, Bax, proliferating cell nuclear antigen (PCNA), and cytochrome C were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-rabbit and anti-mouse IgG horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Millipore (Billerica, MA, USA). Antibodies against PI3K, Akt, p27, p21, cyclin D1, cyclin A, cyclin E, and GAPDH were purchased from Santa Cruz Biotechnology.
- Plant Materials
H9 consisted of nine oriental medicinal herbs. 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 Rhizoma, 4% Sparganium Rhizome, 12% Curcuma Root, and 12% Cinnamon Bark [25] . 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).
- Methods of Extraction
The ethanol extracts from the plants listed above were prepared as recently reported [25] . Briefly, 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.
- Cell Culture
We obtained the human NSCLC cell line A549 from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in RPMI medium (Welgene Incorporation, Daegu, Korea), supplemented with heat-inactivated 10% (v/v) fetal bovine serum (Hyclone Laboratories, Logan, UT, USA), and incubated in humidified conditions of 5% CO 2 and 37℃. Cells used in this study were subjected to no more than 20 cell passages.
- Trypan Blue Assay
A549 cell line was seeded in 1.5 ml of RPMI medium in 6-well plates and incubated overnight. After 20 h of growth, the cells were treated with various concentrations of H9, PEM, and cotreatment for 24 h. The cell viability was estimated using the trypan blue assay. Briefly, the medium was removed and washed using PBS, harvested using trypsin-EDTA, and then centrifuged at 100 × g for 5 min at 4℃. The supernatant was removed and the pellet was resuspended with PBS. After mixing the cell suspension in 0.4% trypan blue solution (1:1 (v/v)), the mixture was incubated for 2 min at room temperature, and then stained (nonviable) and unstained cells (viable) were counted using a hemocytometer.
- Experimental Xenograft Animal Model
The experimental protocols were approved by the Institutional Animal Care and Use Committee (Permission No. KU 14141) and were performed according to the Konkuk University Animal Experimentation regulations. Male BALB/c nude mice (5 weeks old) were purchased from Narabiotech (Seoul, Korea). After 1 week of acclimation, the mice were inoculated subcutaneously in the back with a suspension (5.0 × 10 6 cells/100 µl) of A549 cells, as previously described [18] .
- Tumor Growth Inhibition Assay
The length (a) and width (b) of the tumor masses were measured twice a week and the body weight was measured once a week; the tumor volume was calculated using tumor volume = (a × b 2 )/2. When the tumor volume reached 50 mm 3 , the mice were divided into four groups consisting of five mice per group. One group was injected with 100 µl of PBS intraperitoneally and administered 100 µl of water b y oral injection. The second group was injected with PEM intraperitoneally at 100 mg kg -1 week -1 , which is onethird of the maximum tolerated dose [18] . The third group was administered H9 at a concentration of 400 mg kg -1 day -1 by oral injection. The fourth group was injected intraperitoneally and orally with PEM and H9, respectively. During the drug treatments, we supplied a low-folate diet (Rodent NIH-31; Zeigler, PA, USA), because excess folate uptake is known to lead to PEM resistance [26] .
- Immunohistochemistry (IHC)
All of the livers, kidneys, and tumors were fixed in formalin and paraffin-embedded for histological examination. The tissues were sliced using a microtome into 3 µm thickness and attached to glass slides. We eliminated the paraffin in a 60℃ dry oven overnight. The tissue sections were stained with H&E (Hematoxylin & Eosin) and for IHC study, as previously reported [3] . Paraffin-embedded sections were deparaffinized, rehydrated, washed in distilled water, and then subjected to heat-mediated antigen retrieval treatments. Endogenous peroxidase activity was quenched by incubation in 2% hydrogen peroxide and methanol for 15 min and then cleared in PBS for 5 min. The sections were blocked for 30 min with 3% fetal bovine serum (Hyclone) diluted in PBS. The sections were then blotted and incubated with primary mouse proliferating cell nuclear antigens, Ki-67 monoclonal antibodies, at 1:200 dilution in PBS overnight at 4℃. The next day, the slides were washed three times for 5 min each in PBS and incubated in biotinylated anti-mouse and rabbit antibody for 2 h. The slides were washed in PBS, followed by formation of the avidin-biotinperoxidase complex (ABC; Vector Laboratories, Inc.). The slides were washed and the peroxidase reaction was developed with diaminobenzidine and peroxide; then, the slides were counterstained with hematoxylin, mounted in aqua-mount, and evaluated using a dark field microscope (200×; Nikon, Tokyo, Japan) and optical microscope (200×; Olympus, Tokyo, Japan).
- Western Blotting
The tumors of xenograft mice were harvested and pieces of the tumor were homogenized in 1 ml of lysis buffer containing 50 mM Tris (pH 7.4), 1 mM ethylenediaminetetraacetic acid, 1% NP-40, 1,500 mM sodium chloride, 0.1% sodium dodecyl sulfate, 0.25% sodium deoxycholate, and a protease inhibitor cocktail. Lysates were clarified by centrifugation at 17,010 × g for 30 min at 4℃. The protein concentrations were estimated using the Bradford assay (Bio-Rad, Berkeley, CA, USA) with a UV/VIS spectrophotometer (Bio-wave, Biochrom, Cambridge, UK). Equal amounts of tissue lysates were loaded onto 10-15% sodium dodecyl sulfate polyacrylamide gel electrophoresis gels, and the protein b ands were transferred to polyvinylidene difluoride membranes (Millipore). The membranes were blocked with Tris-buffered saline with Tween-20 (TBST) (2.7 M NaCl, 53.65 mM KCl, 1 M Tris-HCl (pH 7.4), 0.1% Tween-20) containing 5% non-fat dried milk for 1 h at room temperature. The membranes were incubated overnight at 4℃ with primary antibodies specific to each target protein. After washing with TBST three times, HRP-conjugated anti-rabbit or anti-mouse IgG secondary antibodies were incubated with the membranes for 1 h at room temperature. After washing three times with TBST, blots were exposed by the WEST-ZOL (plus) Western Blot Detection System (iNtRON Biotechnology, SeongNam, Korea).
- Reverse Transcription Polymerase Chain Reaction
The tumors of xenograft mice were harvested and homogenized in 1 ml of the easy-BLUE Total RNA Extraction Kit (iNtRON Biotechnology), using a homogenizer. The RNA was isolated according to the manufacturer’s instructions. Oligo(dT)-primed RNA (5 µg) was reverse transcribed with M-MuLV reverse transcriptase (New England Biolabs, Ipswich, MA, USA). A reverse transcription polymerase chain reaction (RT-PCR) analysis was performed using a PCR thermal cycler Dice instrument (TaKaRa, Otsu, Shiga, Japan) 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); GAPDH: 5’-GGC TGC TTT TAA CTC TGG TA-3’ (forward), 5’-TGG AAG ATG GTG ATG GGA TT-3’ (reverse); TRAIL: 5’-GTC TCT CTG TGT GGC TGT AA-3’ (forward), 5’-TGT TGC TTC TTC CTC TGG CT-3’ (reverse); TRAIL Receptor: 5’-AAG TTT GTC GTC GTC GGG GT-3’ (forward), 5’-TGG TGC AGG GAC TTC TCT CT-3’ (reverse); CIAP-1: 5’-CAG GTC CCT CGT ATC AAA AC-3’ (forward), 5’-TAA AAA CCA GCA CGA GCA AG-3’ (reverse); and cFLIP L : 5’-CGA GCA CCG AGA CTA CGA CA-3’ (forward), 5’-GCC CTC TGA CAC CAC ATA GT-3’ (reverse).
Results
- H9 and Co-Treatment with PEM Suppressed Cell Viability
We optimized the concentrations of the single treatment of H9, PEM, and co-treatment. We administered various concentrations of H9 and PEM to the A549 cells and estimated cell viability using the trypan blue assay. As shown in Fig. 1 , cell viability decreased the most at 4-8 µM in PEM-treated cells; similar effects of PEM on cell viability were shown at high concentrations, above 4 µM. The viability of H9-treated cells decreased the most at 200 µg/µl. Based on these results, we treated the cells with PEM and H9 and estimated the viability. As a result, there was an additive effect with 4 µM of PEM and 50 µg/ml of H9. However, there was no change with 50 µg/ml of H9 in a single treatment ( Fig. 1 A). In addition, compared with the other conditions, a greater amount of cell death and a decrease in cell population was observed with H9 and PEM co-treatment ( Fig. 1 B).
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Lager Image
Cytotoxic effects of H9, PEM, and co-treatment on A549 cell lines. (A) A549 cells treated with the indicated concentrations of H9, PEM, and H9+PEM for 24 h. (B) Micrographs of A549 cells treated with the indicated concentrations of H9, PEM, and H9 + PEM. The photographs were taken by phase-contrast microscopy at a magnification of ×100. Data are presented as the mean ± standard deviation (n = 3). *p < 0.05 and **p < 0.005 versus control cells.
- H9 Inhibited Tumor Growth in thein vivoXenograft Model
The antitumor effects of H9 and co-treatment with PEM were investigated using xenograft mice. A549 cells were inoculated subcutaneously into mice (BALB/c-nu/nu). We evaluated the efficacy of H9 and co-treatments on tumor growth. Tumor growth was measured twice a week until the experiments were concluded. Antitumor effects were shown in every drug-treated group. The H9 single treatment was most effective, followed by the co-treatment with a slight difference. The PEM-treated group showed less effect than the H9 and co-treated groups. These results indicated that H9 and the co-treatment had more efficacy than PEM ( Fig. 2 A). In addition, the tumor image and tumor weight also showed the most effectiveness in the H9 and cotreatment groups ( Figs. 2 B and 2 C).
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Lager Image
Effects of H9, PEM, and co-treatment on tumor growth. (A) Tumor volume was calculated by tumor volume = (a × b2)/2, where a = length, b = width. The a and b values were measured twice a week using digital Vernier calipers. (B) After isolating the tumor, macroscopic images were taken using a digital camera (Cannon). (C) Tumor tissues were weighed immediately after mice sacrifice.
- H9 and PEM Did Not Show Toxicity in thein vivoExperiment
We measured the body weight every week, because the effects of toxicity are easily shown in weight loss. All of the groups’ body weights increased by up to 22–23g ( Fig. 3 A). Furthermore, to investigate injury to the organs and tissues by drug toxicity, we harvested the livers and kidneys of the mice; the organs were stained using the H&E staining method and indicated no differences between the control and the drug-treated groups ( Fig. 3 B).
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Lager Image
Effects of H9, PEM, and co-treatment on body weight. (A) Body weights were measured once a week. (B) Liver and kidney tissue were fixed in formalin immediately after mice sacrifice and analyzed by H&E staining (×200, Olympus).
- H9 Induced Intrinsic Pathway MediatedviaCaspase Activation
To determine the antitumor effect of H9 and co-treatment, we investigated the expression of apoptotic factors. First, we analyzed the cleavage of capase-3 and -9, and PARP, the downstream substrate of active capase-3. As shown in Fig. 4 , PARP (116 kDa) was divided into the cleaved form (89 kDa) in the co-treatment group. Moreover, caspase-3 and caspase-9 were cleaved in the H9 and co-treatment groups ( Fig. 4 A). To elucidate whether the apoptosis would be mediated via mitochondrial activation, we evaluated the protein levels of anti-/pro-apoptotic proteins. Bcl-2 and Bcl-xL were significantly decreased in the H9 and cotreated groups. STAT3 plays an anti-apoptotic role in the intrinsic pathway by modulating Bcl-xL [37] . As shown in Fig. 4 B, STAT3 was downregulated. However, Bax expression levels were not altered ( Fig. 4 B). Caspase-8 was slightly activated in the H9 and co-treatment groups ( Fig. 4 C). Hence, we performed RT-PCR to investigate whether death receptors were involved. Fas/FasL and TRAIL receptors were enhanced in the H9 and co-treatment groups. In addition, TRAIL expression was slightly increased, whereas cIAP-1, a cellular inhibitor of apoptosis, was decreased the most in the co-treatment group. FLIP L , which limits caspase-8 activity, was decreased in the co-treated group ( Fig. 4 D).
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Lager Image
Effects of H9 and co-treatment on the intrinsic and extrinsic signaling pathways in A549 cells. (A) Cleavage of pro-apoptotic factors of the intrinsic pathway, (B) mitochondrial membrane permeability modulating factors, and (C) cleavage of caspase-8, an extrinsic apoptotic pathway factor, were detected by western blotting. (D) The expression levels of death receptors and antiapoptotic factors were analyzed by RT-PCR.
- H9 Enhanced PEM-Induced Cell Cycle Arrest
To demonstrate that the tumor suppressor proteins would inhibit tumor growth, we performed a western blot analysis to identify the cell-cycle regulating protein p53. The expression level of phospho-p53, an activated tumor suppressor form, was increased in the co-treated group. Moreover, expression levels of p21 and p27, which modulate cyclin D and cyclin E, were upregulated, while p-pRb was inhibited ( Fig. 5 A). As shown in Fig. 5 B, the protein-level expression of cyclin E and cyclin A was decreased. On the contrary, cyclin D was not altered ( Fig. 5 B). To confirm the downregulation of proliferation in tumor tissues, we performed H&E and IHC staining. As shown in Fig. 5 C, the H&E staining revealed that there was little evidence of tumor growth arrest in the PEM group, whereas the other groups did not show histomorphological alteration. Next, staining of PCNA and Ki-67, which are cellular markers of proliferation, revealed that the expression of proliferation factors was decreased in tumor cells. The greatest effectiveness was shown in the co-treatment group ( Fig. 5 C).
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Lager Image
Effect of H9 and co-treatment on the expression of cell-cycle regulating factors and cell proliferation markers. (A) Expression levels of p53/p-p53/p21/p27/pRb and (B) cyclin A/cyclin E/cyclin D1 were detected in A549 tumors by western blot analyses. (C) Expression levels of PCNA and Ki-67 were detected by IHC.
- H9 Inhibited PI3K/Akt Cell Survival Factors
Because PI3K and its downstream substrate Akt are key factors for survival, we investigated the expression of PI3K and Akt. As shown in Fig. 6 , the expression levels of PI3K and phospho-Akt were significantly decreased in the cotreated group.
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Lager Image
Effects of H9 and co-treatment on the PI3K/Akt survival signaling pathway. Expression levels of PI3K and Akt/p-Akt were detected in A549 tumors by western blot analyses.
Discussion
H9 contains ethanol extracts from nine oriental medicinal herbs, as described in a recent report [25] . We identified the potential medicinal components of the H9 extract using gas chromatography-mass spectrometry [25] . Compounds were identified by comparing with the compounds in the Wiley 6th edition MS Spectra Library (search program hits that were >90% probable were viewed as likely hits). The major components in H9 extract were coumarin, isoeugenol, isoelemicin, and angelicin [25] . Coumarin has anticoagulation, antiviral, anti-inflammatory, antibacterial, and anticancer activities [7 , 11 , 21 , 22] . Angelicin has been isolated from diverse plants and increasingly used for its antimicrobial and inhibitory roles in cell proliferation [16 , 29 , 31] . Angelicin increases cytotoxicity and induces apoptosis in human SH-SY5Y neuroblastoma cells, mediated via the activation of the intrinsic and extrinsic apoptotic pathways [30] . However, there are no reports regarding the effects of other compounds, such as isoeugenol and isoelemicin, on cancer cells.
We used BALB/c nude mice for the in vivo experiments. These animals lack a thymus; therefore, they are unable to produce T cells, causing the mice to be immunodeficient. Subcutaneously injected cancer cells are commonly eliminated by the immune system in normal animals. However, immunodeficient animals cannot eliminate the injected cancer cells. During the in vivo experiment, 3weeks after injecting cancer cells, we administered the drugs to the mice. We measured the tumor volume twice a week. The H9 and co-treatment groups showed that the H9-treated group efficiently suppressed tumor growth compared with the other groups ( Fig. 2 ). Weight loss is usually an indicator of toxicity. We therefore measured the body weight of the mice every week. No differences in body weights were observed between the control and drug-treated groups ( Fig. 3 A), suggesting that the H9, PEM, and combined treatments were non-toxic. To further validate these findings, we performed H&E staining of the liver and kidney to evaluate toxicity-induced tissue damage ( Fig 3 B). H&E staining revealed that there were no differences between the groups, demonstrating that the H9, PEM, and combined treatments did not induce tissue damage.
Apoptosis is a kind of cell-death mechanism frequently seen in oncogenesis. Caspase cascades are regulated via both the intrinsic and extrinsic apoptosis pathways. Intrinsic apoptosis often involves an increase in mitochondrial membrane permeability, and the extrinsic pathway involves death receptor oligomerization and Fas-associated death domain recruitment [15] . Here, to characterize the apoptotic mechanisms induced by H9 and the co-treatment, we investigated the expression levels of pro-apoptotic and anti-apoptotic proteins. H9 and treatments combined with PEM did not alter the Bax expression, whereas the FLIP L , cIAP-1, Bcl-2, STAT3, and Bcl-xL expression levels were inhibited ( Fig. 4 B). In addition, H9 and the combined treatment induced cleavage of caspase-3, caspase-9, and caspase-8 and upregulated the death receptors. Based on these results, we suggested that H9 and co-treatment promoted apoptosis via the intrinsic and extrinsic pathways in tumors.
We also evaluated the expression levels of cell-cycle modulating proteins. Combined treatment of H9 and PEM enhanced p-p53, regulating p21 and inhibiting cyclin E. Combined treatment increased cyclin-dependent kinase inhibitors such as p21 and p27, and inhibited cyclin E ( Fig. 5 ). These results suggested that tumor cells were arrested at the G1 phase. Moreover, H9 and combined treatment enhanced the expression of pRb, which inhibits E2F release from pRb, inhibiting progression to the S phase ( Fig. 5 ). In addition, cyclin A expression was also suppressed, suggesting that tumor cells were arrested at the S phase. Next, we detected the markers of proliferation in cancer. The H9 and combined treatments suppressed the expression of proliferation markers, Ki-67 and PCNA, in cancer cells. However, cyclin D, which modulates the overall cell cycle, was not altered. In summary, we suggest that H9 and cotreatment have the potential to suppress tumor proliferation.
The PI3K/Akt signaling pathway plays an essential role in the regulation of cell survival via activation of antiapoptotic downstream effectors [10] , phosphorylationdependent inhibition of pro-apoptotic signals, such as BAD protein, caspase-9, and the family of forkhead transcription factors [4 , 8 , 28] . To confirm the effects of H9 and cotreatment, we detected the expression of Akt, p-Akt, and PI3K. The expression of p-Akt and PI3K was inhibited by H9 and co-treatment ( Fig. 6 ). These results suggest that H9 and co-treatment downregulated PI3K/Akt survival factors and BcL-2/Bcl-xL anti-apoptotic effectors. Co-treatment with anticancer drugs and herbal extracts could lower the chemotherapeutic drug doses required to obtain the same anticancer effects [27] . To overcome the side effects of chemotherapy, extracts from medicinal herbs could be used to mediate the inhibition of tumor growth [6] .
In summary, our study showed the synergistic effects of PEM with H9 on tumor growth and apoptosis in an in vivo experiment using biochemical and molecular biological methods. Even though the medicinal herb extracts that comprise H9 have additive effects, H9 had low toxicity and suppressed tumor growth via various pathways. Based on these findings, H9 can be used as a potential adjuvant with chemotherapeutic drugs.
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).
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