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ISSN : 1226-7155(Print)
ISSN : 2287-6618(Online)
International Journal of Oral Biology Vol.46 No.2 pp.85-93
DOI : https://doi.org/10.11620/IJOB.2021.46.2.85

Methanol extracts of Asarum sieboldii Miq. induces apoptosis via the caspase pathway in human FaDu hypopharynx squamous carcinoma cells

Seul Ah Lee1, Bo-Ram Park2, Chun Sung Kim1*
1Department of Oral Biochemistry, College of Dentistry, Chosun University, Gwangju 61452, Republic of Korea
2Department of Dental Hygiene, College of Health and Welfare, Kyungwoon University, Gumi 39160, Republic of Korea

†Seul Ah Lee and Bo-Ram Park contributed equally to this work.


*Correspondence to: Chun Sung Kim, E-mail: cskim2@chosun.ac.kr
April 7, 2021 June 10, 2021 June 15, 2021

Abstract


Asarum sieboldii Miq. (Aristolochiaceae) is a perennial herbaceous plant and has been used as traditional medicine for treating diseases, cold, fever, phlegm, allergies, chronic gastritis, and acute toothaches. Also, it has various biological activities, such as antiallergic, antiinflammatory, antinociceptive, and antifungal. However, the anticancer effect of A. sieboldii have been rarely reported, except anticancer effect on lung cancer cell (A549) of water extracts of A. sieboldii . This study investigated the anticancer activity of methanol extracts of A. sieboldii (MeAS) and the underlying mechanism in human FaDu hypopharyngeal squamous carcinoma cells. MeAS inhibited FaDu cells grown dose-dependently without affecting normal cells (L929), as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide and live and dead assay. In addition, concentration of MeAS without cytotoxicity (0.05 and 0.1 mg/mL) inhibited migration and colony formation. Moreover, MeAS treatment significantly induced apoptosis through the proteolytic cleavage of caspase-3, -7, -9, poly (ADP-ribose) polymerase, and downregulation of Bcl-2 and upregulation of Bax in FaDu cells, as determined by fluorescence-activated cell sorting analysis, 4`6-diamidino- 2-phenylindole stain, and western blotting. Altogether, these results suggest that MeAS exhibits strong anticancer effects by suppressing the growth of oral cancer cells and the migration and colony formation via caspase- and mitochondrial-dependent apoptotic pathways in human FaDu hypopharyngeal squamous carcinoma cells. Therefore, MeAS can serve as a natural chemotherapeutic for human oral cancer.


Asarum sieboldii Miq , Human FaDu hypopharynx squamous cancer cells , Apoptosis , Oral cancer ,

초록

    © The Korean Academy of Oral Biology. All rights reserved.

    This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/bync/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

    Introduction

    Asarum sieboldii Miq. (Aristolochiaceae) is a perennial berbaceous plant and is distributed throughout Republic of Korea, China, and Japan. Traditionally, A. sieboldii has long been used to treat all types of colds, fever, phlegm, allergies, chronic gastirits, and acute toothaches [1,2]. In addition, it has been scientifically proven that A. sieboldii has various biological activities, such as anti-allergic, anti-inflammatory, anti-nociceptive, anti-fungal, anti-cancer, and neuroprotective effect [3-9]. The main physiolgically active substances of A. sieboldii contain asarylketone, safrole, limonene, pellitorine, sesamine and aristolactam, in addition to the essential oil components methyleugenol and xanthoxylol [3,10]. Asiasarum heterotropoides (AR) belonging to the same Asarum genus has been reported to have anti-cancer effects on various cancer cells, such as colon cancer (HCT-116, HT29), ovarian cancer cells (SKOV3 and A2780) and melanoma (G361) cells [7-9,11,12]. However, with the exception of the report that water extract of A. sieboldii induces apoptosis of lung cancer cells (A549), the anti-cancer effect of A. sieboldii has not yet been reported [8].

    Oral squamous cell carcinoma (OSCC) is a main type of head and neck squamous cell carcinoma, and according to the World Health Organization, in 2018, the incidence of oral cancer is the fourth highest in the world [13]. OSCC originates from the mucosa of oral organ with the squamous initima and is charaterized by high potential invasion and lymph node metastasis [14]. Due to the high metastatic capacity of OSCC, the 5-year survival rate is less than 30% despite the development of comprehensive sequence therapy [13,15,16]. The risk factor for OSCC are related with viral infections and accumulation of genetic mutation as well as chronic exposure to carcinogens such as smoking, alcohol [13,17]. The main treatment of OSCC is surgery combined with radiotherapy or chemotherapy, but many side effects reduce the patient’s quality of life [18,19]. Therefore, the anti-cancer effect is being evaluated in natural products with relatively few side effects, and recently several candidate substances have been reported [20,21]. However, the A. sieboldii for oral cancer has not been clarified. Therefore, to aid understanding of the A. sieboldii anti-cancer activity, we investigated the effect of methanol extraction of A. sieboldii on human FaDu hypopharynx squamous carcinoma cells.

    Materials and Methods

    1. Preparation of methanol extracts of Asarum sieboldii (MeAS) Miq.

    In order to produce an extract having excellent pharmacological activity, it was extracted with methanol, which has a broader spectrum. A. sieboldii (10 g) of A. sieboldii was extracted in 40 volumes of methanol (v/w) at 37℃ for 24 hours, and then the extract was filtered through filter paper. The supernatant was concentrated in a rotary vacuum evaporator (Eyela, Tokyo, Japan) and lyophilized. The powder was dissolved in dimethyl sulfoxide (DMSO) at 100 mg/mL, and the solution was passed through a 0.45-μM syringe filter. The final extract was stored at –20℃ until use, but after dissolving it was stored at 4℃.

    2. Reagents

    Dried A. sieboldii purchased from Gangwon-do (Korea). Crystal violet, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 4`6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Live & Dead viability/cytotoxicity kit was purchased from Molecular Probe (Eugene, OR, USA). Cell scratcher and FITCAnnexin V apoptosis detection kit I were purchased from SPL Life Science (Pocheon, Korea), BD Bioscience (San Diego, CA, USA), respectively. Primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): cleaved caspase-9 (cat. no. 7237), cleaved caspase-3 (cat. no. 9661), cleaved caspase-7 (cat. no. 9491), poly (ADP-ribose) polymerase (PARP, cat. no. 9542), Bcl-2 (cat. no. 2872), Bax (cat. no. 2772) and anti-α-tubulin was purchased from Thermo Fisher Scientific (Rockford, IL, USA). Minimum essential medium Eagle (MEM) and a penicillin/streptomycin solution were purchased from Welgene (Daegu, Korea). Fetal bovine serum (FBS) was purchased from Atlas Biologicals (Fort Collins, CO, USA).

    3. Cell culture

    Human hypopharynx squamous carcinoma FaDu and mouse fibroblast normal L929 cells were purchased from Korean Cell Line Bank (Seoul, Korea). In a humidified 5% CO2 incubator at 37℃, FaDu cells were cultured in MEM medium containing 10% FBS and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin), and L929 cells were cultured in MEM medium containing 10% FBS and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin).

    4. Cytotoxicity

    FaDu and L929 cells were plated in 12-well plates at a density 2 × 105 cells/mL, and incubated for 16 hours. After incubation, cells were treated with varying concentration of MeAS (0, 0.05, 0.1, 0.2, and 0.4 mg/mL) for 24 hours. Cytotoxicity was determined by measuring formazan generated by mitochondrial-dependent redox reaction using tetrazolium salt solution (5 mg/mL). At the endpoint, 100 μL of MTT solution (5 mg/mL) was added and the formazan formed after 4 hours reaction was dissolved using DMSO, and then measured absorbance at 590 nm in microplate reader (Epoch; BioTek Instruments, Winooski, VT, USA). The results was expressed as the cell viability rate by setting the absorbance of untreated control cells to 100% and the latex-treated cells was calculated as the surviving percentages. The percentage of cell viability was calculated using the following formula:

    Cell viability (%) = Means Abs   ⋅   of the sample − Means Abs ⋅ of the blank Means Abs ⋅ of the control − Menas Abs ⋅ of the control × 100

    5. Live & Dead assay and DAPI staining

    FaDu cells were plated in a 4-well chamber slide at a density 1 × 105 cells/well, and incubated for 16 hours. After incubation, the cells were treated with varying concentration of MeAS (0, 0.1, 0.2, and 0.4 mg/mL) for 24 hours. For Live & Dead assay, the cells were washed with phosphate-buffered saline (PBS), and 100 μL of a working soultion (2 μM calcein-acetoxymethyl and 4 μM of ethidium homodimer-1 in PBS) was added to each well containing 100 μL PBS, followed by reaction in a 37℃ incubator for 20 minutes. For DAPI staining, the cells were washed with PBS, fixed with 4% paraformaldehyde for 10 minutes, and stained with 300 nM DAPI for 20 minutes. The stained cells were analyzed using a fluorescence microscope (Eclipse TE2000; Nikon Instruments, Melville, NY, USA).

    6. Flow cytometric analysis

    FaDu cells were plated in a 6-well plates at a density 2 × 105 cells/well, and incubated for 16 hours. After incubation, the cells were treated with varying concentration of MeAS (0 and 0.4 mg/mL) for 24 hours, after which they were harvested using 0.25% trypsin-EDTA and washed with pre-chilled PBS twice. The cells were re-suspended in a 200 μL binding buffer, and then stained with FITC-Annexin V/PI (BD Biosciences) according to the manufacturere’s protocol. Subsequently, the stained cells were analyzed using a Gallios flow cytometer (Beckman Coulter Life Science, Indianapolis, IN, USA). The quantitatively data was expressed as density plots using Kaluza analysis software (Beckman Coulter Life Science).

    7. Wound healing assay

    FaDu cells were plated in a 24-well plates at a density 4 × 103 cells/well, and incubated for 16 hours. A cell scratcher was used to generate a constant wound in each well. After washing with PBS, non-cytotoxic concentrations of MeAS against FaDu cells (0.025 and 0.05 mg/mL) and the lowest concentration inducing cytotoxicity (0.1 mg/mL) were added to each well, and reacted for two days. EVOS XL Core (Thermo Fisher Scientific, Waltham, MA, USA) was used for cell imaging.

    8. Colony formation

    FaDu cells were plated in a 6-well plates at a density 2 × 103 cells/well, and incubated for 16 hours. The non-cytotoxic concentration of MeAS against FaDu cells (0.025 and 0.05 mg/ mL) and the lowest concentration inducing cytotoxicity (0.1 mg/mL) were treated and reacted for 24 hours. After 24 hours, the cells were further cultured in growth medium containin no extracts for 10 days, and then crystal violet staining was performed for colony formation analysis. Culture medium was removed, lightly washed with PBS, and then fixed with 95% ethanol for 10 minutes. After completely drying, 0.1% crystal violet solution was added and stained for 10 minutes. Rinse lightly with deionized water to remove the unstained areas, the degree of staining was observed with canon G16.

    9. Western blot analysis

    FaDu cells were plated in a 6-well plates at a density 2 × 105 cells/well, and incubated for 16 hours. After incubation, the cells were treated with varying concentration of MeAS (0, 0.2, and 0.4 mg/mL) for 24 hours, and cells were lysed with protein extraction reagent (iNtRON Biotechnology, Seongnam, Korea) for 30 minutes on ice, and then centrifuge at 12,000 rpm for 15 minutes at 4℃. The supernatant was transferred to a new 1.5 mL tube and protein was quantified using BCA protein assay (Pierce, Rockford, IL, USA) method. Protein (20 μg) was mixed with 5X sample buffer, denatured at 100℃ for 5 minutes, electrophoresed on 8%, 10% or 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis gels, and then transferred to a polyvinylidene fluoride membrane (Bio-Rad Laboratories, Hercules, CA, USA). Membrans was blcoked with 5% blocking solution (5% bovine serum albumin in Tris-bufferd saline containing 0.1% Tween-20 [TBS-T] for 30 minutes and the specific primary antibodies, cleaved caspase-9 (1:1,000), cleaved caspase-3 (1:500), cleaved caspase-7 (1:1,000), PARP (1:1,500), Bcl-2 (1:1,000), Bax (1:1,000) and α-tubulin (1:2,000), were incubated at 4℃ overnight. Thereafter, the membranes were washed with TBS-T three times for 15 minutes each, followed by incubated with a secondary antibody conjugated with horseredish peroxidase (1:5,000) for 1 hour at room temperature, and then washed with TBS-T three times for 15 minutes each. Protein were detected by Immobilon Western Chemiluminescent HRP Substrate (ECL; Millipore, Bedford, MA, USA) and visualized on a MicroChemi 4.2 device (DNR Bio Imaging Systems, Jerusalem, Israel).

    10. Statistical analysis

    All data are expressed as the means ± standard deviation. All data were derived from at least three independent experiments. Statistical significance was determined using one-way ANOVA followed by Turkey’s analyses in GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA). Statistical significance was set to *p ＜ 0.05.

    Results

    1. Cytotoxic effect of MeAS on FaDu and L929 cells

    To verify whether MeAS affects viability of FaDu cancer cells and L292 normal cells, MTT assay and Live & Dead assay were performed. As shown in Fig. 1, FaDu cell viability began to decrease and cell viability rate for each concentration was 95%, 87%, 70%, and 44% at 0.05, 0.1, 0.2, and 0.4 mg/mL, respectively. However, MeAS did not affect L929 cell viability. Since MeAS exhibited cytotoxicity at 0.1 mg/mL, further experiments were carried out at 0.1, 0.2, and 0.4 mg/mL. Furthermore, results of Live & Dead assay shown that in MeAStreated group, green-fluorescence indicating live cells was gradually reduced, but red-fluorescence indicating dead cells was gradually increased (Fig. 1C).

    2. Inhibition effect of MeAS on wound-healing and colony formation of FaDu cells

    To investigate whether MeAS affects the unlimited proliferation and wound-healing of cancer cells, colony formation and wound-healing assay were performed. As a result, despite the concentration not showing cytotoxicity, the wound-healing ability was significantly decreased in a concentration-dependent manner (Fig. 2A). Furthermore, 0.1 mg/mL, which had a cytotoxic effect about 10%, also showed an effect of significantly reducing colony formation in FaDu cancer cells (Fig. 2B). These results suggest that MeAS can delay canceration even at concentrations that do not show cytotoxicity.

    3. Analysis of nuclear morphology change by MeAS in FaDu cells

    To investigate whether the reduction in FaDu cell viability by MeAS was associated with apoptosis, nucleic morphology was analyzed using DAPI stain. As a result, the number of cells with apoptotic nuclear morphology such as chromatin condensation and chromatin atrophy significantly was increased in the MeAS-treated group, compared with control group (Fig. 3).

    4. Induction of apoptotic cells by MeAS in FaDu cells

    When apoptosis is induced, phosphatidylserine (PS) located on the inner cell membrane is turned over and directed outside the cells. Therefore, cells undergoing apoptosis can be observed through annexin-V staining, which has a strong affinity with PS. To quantify the apoptotic cell of FaDu by MeAS, flow cytometirc was performed using annexin-V and propidium iodide. As FACS results (Fig. 4), the early apoptosis rates were 3.3% and 5.1% at 0 and 0.4 mg/mL MeAS, respectively, and late apoptosis rates were 1.0% and 16.9% MeAS, respectively. The population of total apoptotic cells was increased to 18% at 0.4 mg/mL, respectively, compared to control group (4.1%) (Fig. 4). These results suggest that inhibition of cell viability by MeAS was associated with apoptosis in FaDu cells.

    5. Activation of apoptosis mechanism by MeAS in FaDu cells

    Caspases (cystein-aspartic acid-protease) are signaling transduction factors that are very closely related to apoptosis. Most caspases are cascade-type cystein proteases and cleave the aspartic acid residue of the substrate protein [22]. Therefore, we evaluated whether MeAS-induced apoptosis was related caspase activity using western blot analysis. Fig. 5 shown that cleaved caspase-9, -3 and -7 were dramatically increased by MeAS in a concentration-dependent manner, as a result, PARP, which downstream apoptotic indicator, was cleaved. In addition, MeAS significantly increased the expression of the anti-apoptosis protein Bcl-2 protein and decreased the expression of the pro-apoptosis protein Bax. These results suggest that MeAS-induced apoptosis can be induced by cas- pase- and mitochondrial apoptosis signaling in FaDu cells.

    Discussion

    A. sieblodii belongs to the Aristolochiaceae family and is a typical medicinal plant that has been used to treat various diseases, such as fever, phlegm, allergies, and acute toothaches [1,2]. The major bioactive substances of A. sieboldii are essential oil such as methyleugenol and xanthoxylol, which have been reported to inhibit cough, anti-allergic effects, antibacterial activity, protect brain cells and prevent dementia [3-9]. Various biological activities of A. sieboldii are expected to have high anticancer effects, but unfortunately, only a few studies on anticancer effects have been reported on lung cancer cells (A549). Therefore, in this study, we investigated the biological activity of methanol extract of A. sieboldii on oral cancer cells (FaDu) through evaluation of its ability to suppress cell growth and induce apoptosis.

    Actuality, the biggest side effect of anticancer drugs is that they affect the growth of normal cells as well as cancer cells. Therefore, anticancer substances that inhibit the growth of cancer cells without affecting the growth of normal cells can be recommended as strong candidates for the development of future anticancer therapeutic agents [18]. We confirmed that MeAS exerts remarkable cytotoxicity on cancer cells, FaDu, without affecting the growth of normal cells, L929. These results are consistent with the anti-growth effect of A. sieboldii in previous papers, water extracts of A. sieboldii inhibited the growth of A549 lung cancer cells, by 58% at 4 mg/mL [8]. In addition, ethanol extracts of A. heterotropoides, which belong to the same Asarum genus as A. sieboldii, had IC50 values of 19.89 ± 4.20 and 118.47 ± 19.78 μg/mL for ovarian cancer cells A2780 and SKOV3, respectively, and IC50 value for colon cancer cells (HCT-116) is 15.8 ± 0.7 μg/mL [9,11]. As can be seen from the above results, the growth inhibitory activity of A. sieboldii on some cancer cells was demonstrated, but the sensitivity to each cancer cells cannnot be evaluated because both the extraction amount (water: 100 g; methanol: 10 g) and the method (water: boling for 3 hours in 10 volumes; methanol: 37℃ for 24 hours in 40 volumes) are different [8]. Therefore, in order for A. sieboldii to be a strong candidate for anticancer drug development, future research to compare and analyze the anti-cancer effects on various cancer cells using several solvents extracted in the same amount will must be conducted.

    The term apoptosis was first used in the current classic paper by Kerr, Wyllie and Currie in 1972 to describe the morphologically distinct type of cell death, and many apoptosis review papers are still published today [22,23-25]. Apoptosis is a programmed cell death triggered by physiological conditions, which maintains the homeostasis of cell populations in tissue and act as a defense against external invasion [22]. However, if these apoptosis is not properly regulated, abnormal cells proliferate excessively, which is one of the most representative characteristics of cancer cells. Therefore, inhibition of cancer cell growth as well as induction of apoptosis are very important indicators in the development of anti-cancer drugs [18,22,26]. Among caspases, which are major factor regulating apoptosis, caspase-3, -7, -8, and -9 are known as protein that induce apoptosis, and their activity is triggered through proteolytic cleavage [22,26,27]. PARP, a protein that plays a role in repairing damaged DNA, is hydrolyzed by cleaved caspase-3 and is known as the ultimate indicator of apoptosis [26-29]. Our results showed that cleaved caspase-3, -7, and -9 significantly increased and cleavage of native PARP (116 kDa) into its small fragment PARP (89 kDa) increased accordingly. In addition, the Bcl-2 protein (anti-apoptotic factor) expression significantly decreased and Bax protein (anti-survival factor) expression increased by MeAS in FaDu cells. These results are consistent with previous studies, A. sieboldii and A. heterotropoides were reported that apoptosis was induced through caspase-3, -8, -9, and Bcl-2 family in ovarian, colon, lung cancer cells [8,9,11,12].

    Collectively, these results suggest that methanol extract of A. sieboldii exhibits a potent anticancer effects by inhibiting the growth, migration and colony formation of FaDu cells via apoptosis mediated by the activation of caspase and Bcl-2 family signaling pathways.

    In conclusion, our study suggest that the anti-cancer effect of methanol extract of A. sieboldii inhibits the cell growth and induced cell apoptosis in FaDu cells through extrinsic death receptor and intrinsic mitochondrial-dependent apoptotic signaling pathway. Although these advantage have been found, in order to clarify the anticancer efficacy of A. sieboldii , the mechanism analysis should be performed in various cancer cells and experimental animal under the same extraction conditions.

    Acknowledgements

    This study was supported by a research fund from Chosun University Dental Hospital, 2020.

    Figure

    IJOB-46-2-85_F1.gif

    Effect of methanol extracts of Asarum sieboldii (MeAS) on FaDu and L929 cell viability. Cells are treated with various concentration of MeAS (0–0.4 mg/mL) for 24 hours. Cell viability are determined using MTT assay (A, B) and Live & Dead assay (C). Results are expressed as a percentage of the control. Data are expressed as means ± standard deviation of three independent experiments. ×20 magnification.

    Conc., concentration.

    **p < 0.01, ***p < 0.001 compared with the control group.

    IJOB-46-2-85_F2.gif

    Inhibition of wound-healing and colony formation by methanol extracts of Asarum sieboldii (MeAS) in FaDu cells. Cells are treated with various concentration of MeAS (0–0.4 mg/mL) for 24 hours. (A) After wounding, culture is performed in a medium mixed with extracts for 2-days, and then woundhealing is analyzed. (B) The degree of cancer cells colony formation is measured by colony formation.

    IJOB-46-2-85_F3.gif

    Observation of nuclear change by methanol extracts of Asarum sieboldii (MeAS) in FaDu cells. Cells are treated with various concentration of MeAS (0–0.4 mg/mL) for 24 hours, and then 4`6-diamidino-2-phenylindole (DAPI, 300 nM) staining are performed. ×20 magnification.

    IJOB-46-2-85_F4.gif

    Induction of apoptosis by methanol extracts of Asarum sieboldii (MeAS) in FaDu cells. Cells are treated with various concentration of MeAS (0, 0.2, and 0.4 mg/mL) for 24 hours. (A) The cells stained with Annexin-V/ propidium iodide (PI), and apoptotic cells are analyzed by fluorescence-activated cell sorting anaylsis. The percentage of apoptosis and live sections are shown in (B).

    IJOB-46-2-85_F5.gif

    The expression levels of apoptosisrelated proteins in FaDu cells treated with methanol extracts of Asarum sieboldii (MeAS). Cells are treated with various concentrations of MeAS (0, 0.2, and 0.4 mg/mL) for 24 hours. (A, B) After MeAS treatment, the expression of apoptotic-related protein (cleaved caspase-9, cleaved caspase-3, cleaved caspase- 7, PARP, Bcl-2, and Bax) is assessed by western blotting, and α-tubulin is used as the loading control.

    Table

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