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ISSN : 1226-7155(Print)
ISSN : 2287-6618(Online)
International Journal of Oral Biology Vol.45 No.3 pp.107-114
DOI : https://doi.org/10.11620/IJOB.2020.45.3.107

Inhibition of cell growth and induction of apoptosis by acacetin in FaDu human pharyngeal carcinoma cells

Kyeong-Rok Kang1, Jae-Sung Kim1, Tae-Hyeon Kim1, Jeong-Yeon Seo1, Jong-Hyun Park1, Jin Woong Lim1, Sun-Kyoung Yu1, Heung-Joong Kim1, Sang Hun Shin1, Bo-Ram Park2, Chun Sung Kim1, Do Kyung Kim1*
1The Institute of Dental Science, Chosun University, Gwangju 61452, Republic of Korea
2Department of Dental Hygiene, College of Health and Welfare, Kyungwoon University, Gumi 39160, Republic of Korea
*Correspondence to: Do Kyung Kim, E-mail: kdk@chosun.ac.kr
June 29, 2020 August 20, 2020 August 21, 2020

Abstract


Acacetin, which is present in damiana (Turnera diffusa ) and black locust (Robinia pseudoacacia ), has several pharmacologic activities such as antioxidant, anti-inflammatory, and anti-proliferative effects on cancer cells. However, the effect of acacetin on head and neck cancers has not been clearly established. This study aimed to examine the effects of acacetin on cell growth and apoptosis induction in FaDu human pharyngeal carcinoma cells. These were investigated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, Live/Dead cell assay, 4′,6-diamidino-2-phenylindole dihydrochloride staining, caspase-3 and caspase-7 activation assay, and immunoblotting in FaDu cells. Acacetin induced FaDu cell death in a dose-dependent manner, with an estimated IC50 value of 41.9 µM, without affecting the viability of L-929 mouse fibroblasts as normal cells. Acacetin treatment resulted in nuclear condensation in the FaDu cells. It promoted the proteolytic cleavage of procaspase-3, -7, -8, and -9 with increasing amounts of the cleaved caspase isoforms in FaDu cells. Acacetin-induced apoptosis in FaDu cells was mediated by the expression of Fas and activation of caspase-8, caspase-3, and poly (ADP-ribose) polymerase. Immunoblotting showed downregulation of the anti-apoptotic mitochondrial proteins Bcl-2 and Bcl-xL, but upregulation of the mitochondria-dependent pro-apoptotic proteins Bax and Badin FaDu cells after acacetin treatment. These findings indicate that acacetin inhibits cell proliferation and induces apoptotic cell death in FaDu human pharyngeal carcinoma cells via both the death receptor-mediated extrinsic apoptotic pathway and the mitochondria-mediated intrinsic apoptotic pathway.


Acacetin , Cell death , Apoptosis , Anticancer effect

초록

    Chosun University
    © 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

    Head and neck cancers, including oral cancers, pharyngeal cancers and laryngeal cancers, is a common type of cancer worldwide and is particularly common in Asia, Europe and South America [1-3]. Also most of them are squamous cell carcinomas [4]. Despite treatment strategies such as modern surgical techniques and the use of anticancer drugs, the survival rate of head and neck cancers has not improved significantly over the past 30 years [3,5]. Clinical treatment for head and neck cancers can cause side effects associated with functional changes in the ability to chew, swallow or speak [3,6], and anticancer agent treatment can also cause side effects such as gastrointestinal complications, immune dysfunction and decreased bone marrow function [7,8]. Therefore, a lot of efforts have been made to develop anticancer drugs derived from natural substances that can minimize side effects while maintaining the effects of the anticancer drugs in an alternative way [9].

    Apoptosis is a major form of programmed cell death controlled by genes that play an important role in regulating tissue development and homeostasis in eukaryotes [10-12]. Most of the anti-cancer agents cause apoptosis and inhibit cancer cell proliferation, thereby acting as chemotherapy agents for cancer [13-15]. Thus, apoptosis of cancer cells caused by the use of these anti-cancer agents has become an important indicator of the results of cancer treatment [11,12]. Apoptosis can occur in cancer through the death receptor-dependent extrinsic pathway or mitochondria-dependent intrinsic pathway that can be induced by treatment with chemotherapeutic agents [16,17].

    Flavonoids are part of our daily diet, and pharmacological properties for many diseases including cancer are being studied [18]. Acacetin (5,7-dihydroxy-4’-methoxyflavone, Fig. 1), a flavonoid compound, which is present in damiana (Turnera diffusa) and black locust (Robinia pseudoacacia) [18]. This phytochemical is known to show antioxidant, anti-inflammatory and anti-proliferative effects on cancer cells [3,18-20]. However, acacetin effects on head and neck cancers are not clearly established.

    In the current study, therefore, the effect of acacetin on cell growth and the mechanism of cell death elicited by acacetin were examined in FaDu human pharyngeal carcinoma cells. Our results showed that acacetin can inhibit cell viability and induce apoptosis in a dose-dependent manner in FaDu human pharyngeal carcinoma cells.

    Materials and Methods

    1. Materials

    Acacetin (Fig. 1), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) were supplied by Sigma (St. Louis, MO, USA). Anti-cleaved caspase-3, -8, -9, anti-Fas, anticleaved poly (ADP-ribose) polymerase (PARP), anti-Bcl-2, anti-Bcl-xL, anti-Bax and anti-Bad antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The Live/Dead cell viability assay kit was obtained from Thermo- Fisher Scientific, Inc. (Waltham, MA, USA). Other analytical reagents were purchased based on the analytical grade.

    2. Cell line and cell cultures

    The L-929 mouse normal fibroblasts and FaDu human pharyngeal carcinoma cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured as according to the cell culture instructions provided by ATCC. Briefly, the L-929 cells and FaDu cells were grown in Eagle’s minimum essential medium (EMEM, ATCC) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA). The cells were maintained as monolayers in plastic culture plate at 37℃ in a humidified atmosphere containing 5% CO2.

    3. Cell viability test (MTT assay)

    The L-929 cells and FaDu cells were seeded at a concentration of 5 × 103 cells/well in 24-well plates. After 24 hours growth, the cells were treated with acacetin at various concentrations for 24 hours. The cell viability test was evaluated using the MTT assay. At least 3 separate experiments were performed on each concentration combination.

    4. Live/Dead cell assay

    The L-929 cells and FaDu cells (1 × 105 cells/well) were cultured in an 8-well chamber slide (Sigma), and allowed to attach to the bottom of the chamber slide overnight. Thereafter, the cells were treated with 0, 12.5, 25 or 50 µM acacetin for 24 hours at 37℃ and stained using the Live/Dead cell viability assay kit. The cells were imaged using a fluorescence microscope (Eclipse TE2000; Nikon Instruments, Melville, NY, USA). The Live/Dead cell viability assay kit (ThermoFisher Scientific, Inc.) uses green calcein AM to stain the live cells (green fluorescence) and ethidium homodimer‑1 to stain the dead cells (red fluorescence).

    5. DAPI staining

    Nuclear staining with DAPI was performed and the level of apoptosis was examined. The L-929 cells and FaDu cells were cultured in 24-well plates at a seeding density of 5 × 103 cells/well. After 24 hours growth, the cells were treated with 0, 12.5, 25 or 50 µM acacetin for 24 hours. The cells were fixed with 1% paraformaldehyde for 30 minutes and washed twice with phosphate-buffered saline (PBS). The cells were permeated with ice-cold ethanol for 5 minutes at room temperature and washed twice with PBS. The fixed cells were stained with 300 nM DAPI for 5 minutes at room temperature in the dark and washed twice with PBS. The stained cells examined by fluorescent inverted microscopy (Eclipse TE2000; Nikon Instruments).

    6. Determination of caspase-3/-7 activation

    The activity of caspase-3/-7 was determined using the cell-permeable fluorogenic substrate PhiPhiLux-G1D2 (OncoImmunin, Inc., Gaithersburg, MD, USA), which was used according to the manufacturer’s instructions. The FaDu cells were treated with 0, 25 or 50 µM acacetin for 24 hours and incubated with PhiPhiLux-G1D2. The activity of caspase-3/-7 was visualized by fluorescence microscopy.

    7. Immunoblotting

    The FaDu cells were treated with 0, 25 or 50 µM acacetin for 24 hours. Immunoblotting was done according to the previously described method with minor modifications [21]. The anti-cleaved caspase-3, -8, -9, anti-Fas, anti-cleaved PARP, anti-Bcl-2, anti-Bcl-xL, anti-Bax or anti-Bad antibody was used as the primary antibody.

    8. Data analysis

    All experiments were performed at least 3 times. The results were presented as mean ± standard deviation. The statistical significance was analyzed by using Student’s t-test for two groups and one way analysis of variance for multi-group comparisons. All statistical analyses were performed using SPSS version 12.0 (SPSS Inc., Chicago, IL, USA). A p-value ＜ 0.05 was considered statistically significant.

    Results

    1. Cytotoxic effect of acacetin in L-929 cells and FaDu cells

    To analyze the effect of acacetin on the viabilities of L-929 cells and FaDu cells, the cells were treated with acacetin at various concentrations for 24 hours, and then the MTT assay was performed. As shown in Fig. 2A, treatment with 0–100 µM acacetin did not significantly decrease the viability of L-929 mouse fibroblasts, which were used as normal cells; however, treatment with 200 µM acacetin decreased the viability of L-929 mouse fibroblasts to 74.6 ± 5.7% compared with the control (p ＜ 0.05). The relative cell viability was found to be 73.8 ± 5.9, 54.9 ± 5.7, 46.6 ± 5.3, 35.8 ± 4.7 and 22.7 ± 2.9% in FaDu cells treated with 12.5, 25, 50, 100 and 200 µM acacetin, respectively (Fig. 2B). The IC50 value of acacetin on the FaDu cell viability was approximately 41.9 µM. These data suggest that acacetin induces FaDu cell death in a dose-dependent manner without affecting the viability of L-929 mouse fibroblasts as normal cells. Hence, 25 and 50 µM acacetin was used to treat FaDu cells in the present study.

    2. Induction of apoptosis by acacetin in FaDu cells

    To confirm the viabilities of L-929 cells and FaDu cells with acacetin, a Live/Dead cell assay was performed, using green calcein AM and ethidium homodimer-1 to stain live and dead cells, respectively. As shown in Fig. 3A, almost all L-929 cells treated with acacetin emitted green fluorescence following staining with green calcein AM, which stains live cells. However, the FaDu cells exposed to acacetin emitted red fluorescence in a dose-dependent manner following staining with ethidium homodimer-1, which stains dead cells. Taken together, these results indicate that acacetin induces FaDu cell death without affecting the viability of L-929 normal fibroblasts (Fig. 3A).

    To determine whether acacetin-induced FaDu cell death is due to apoptosis, DAPI staining was performed to detect nuclear condensation, a typical feature of apoptosis. The nuclei of the L-929 cells had a normal regular and oval shape (Fig. 3B). As shown in Fig. 3B, the number of FaDu cells with condensed nuclei increased upon exposure to acacetin in a dosedependent manner, which are the characteristics of apoptosis.

    In addition, activation of caspase-3/-7 in acacetin treated FaDu cells was confirmed by fluorescence microscopy using a cell permeable fluorogenic substrate PhiPhiLux-G1D2. As shown in Fig. 3C, acacetin treatment led to activate the caspase- 3/-7 in the living FaDu cells. These results strongly suggest that acacetin induces apoptotic cell death in FaDu cells in a dose-dependent manner.

    3. Extrinsic death receptor-dependent and intrinsic mitochondria-dependent apoptotic signaling pathways induced by acacetin in FaDu cells

    To determine the cellular apoptotic pathways associated with acacetin-induced FaDu cell death, immunoblotting was performed. Fas, which is an apoptotic ligand that triggers the death receptor-dependent extrinsic apoptotic pathway in cancer cells [22,23], was induced significantly by acacetin (Fig. 4A). The level of cleaved caspase-8, the downstream target of pro-apoptotic factor Fas, increased following acacetin treatment (Fig. 4A). These results suggest the involvement of the extrinsic death receptor-mediated apoptosis pathway in acacetin-induced FaDu cell apoptosis.

    The expression levels of Bcl-2 and Bcl-xL, anti-apoptotic factors associated with the intrinsic mitochondria-dependent apoptosis pathway, were downregulated by acacetin, while those of mitochondria-dependent pro-apoptotic factors such as Bax and Bad were upregulated by acacetin in FaDu cells (Fig. 4B). And also, acacetin treatment increased the expression level of cleaved caspase-9 in FaDu cells (Fig. 4B). This result indicates that acacetin-induced FaDu cell death involves the intrinsic mitochondria-dependent apoptosis pathway.

    Both cleaved caspase-8 and caspase-9, acting in the extrinsic death receptor-mediated and intrinsic mitochondriadependent apoptosis pathways, induced the expression of cleaved caspase-3 and PARP in FaDu cells following acacetin treatment (Fig. 4C). These findings indicate that acacetin‑induced FaDu cell death is coordinated by death receptor-mediated extrinsic and mitochondria-dependent intrinsic apoptosis through activation of the caspase cascade in FaDu cells.

    Discussion

    Acacetin, a flavonoid present in damiana (Turnera diffusa) and black locust (Robinia pseudoacacia), is known to have antioxidant effect, anti-inflammatory effect and inhibitory effect on cancer cell proliferation [3,18-20]. However, the effects of acacetin on cancer cells are not clearly established. In the present study, the cytotoxic activity and apoptotic activity of acacetin were examined in FaDu human pharyngeal carcinoma cells. The results of this study indicated that anti-proliferative activity of acacetin against pharyngeal carcinoma cells was due to its ability to induce cell apoptosis.

    In our cell viability test, 12.5–100 µM acacetin did not affect the viability of L-929 fibroblast cells used as normal cells (Fig. 2). Similar to this result, 12.5–50 µM acacetin did not affect the cell viability of L-929 cells in the Live/Dead cell assay (Fig. 3). Hence, these results indicate that it can be biologically safe for normal cells in the at least 12.5–50 µM acacetin dose range in this study. In cell viability test (Fig. 2) and in Live/Dead cell assay (Fig. 3), however, acacetin inhibited growth of FaDu cells in a concentration-dependent manner. These results speculated that the acacetin has cytotoxicity for pharyngeal carcinoma cells without affecting normal cells and potential value for anticancer drug discovery.

    Induction of apoptosis in the process of inhibiting cancer cell growth is a useful strategy for the development of herbal anticancer drugs [24]. Therefore, many studies have been conducted to induce apoptosis of cancer cells in various natural products, including herbal medicines [24]. In the present study, we examined the nuclear morphological changes with DAPI staining to confirm whether apoptosis is involved in the inhibition of FaDu cell growth by acacetin. The acacetin induced the formation of nuclear condensation in FaDu cells (Fig. 3), suggesting apoptotic cell death by acacetin. Caspase -3, -7, -8 and -9 may act as effector caspases of apoptotic cell death in mammalian cells [13,25-27]. They are synthesized as inactive proenzymes, which require proteolytic activation of enzymes cleaved by various stimuli [13,25-27]. In this study, the activity of caspase-3/-7 using the fluorogenic substrate PhiPhiLux- G1D2 was increased by acacetin in living FaDu cells compared to dimethyl sulfoxide treatment as a control (Fig. 3). In addition, the immunoblotting results show that low levels of cleaved capase-3, -8 and -9 were present in acacetin-untreated FaDu cells, and the amount of cleaved enzymes was increased after acacetin treatment in FaDu cells (Fig. 4). These results suggested that acacetin induce apoptotic cell death by the activation of caspases-3/-7/-8/-9 in FaDu cells.

    Fas, an important regulator of apoptosis, binds to the receptor FasR across the surface of the target cells and then initiates the death receptor-mediated extrinsic apoptotic pathway through activation of caspase-8, -3 and PARP [22,23]. In our study, the amount of Fas protein was significantly increased by acacetin in FaDu cells (Fig. 4). Subsequently, the Fas stimulated by acacetin triggered caspase cascade, which results in the activation of apoptotic factors including cleaved caspase-8 and -3 (Fig. 4) [22,23]. Finally, activated caspase-3 cleaved the major substrate, PARP, leading to apoptosis and cell death in FaDu cells (Fig. 4) [22,23]. Thus, these results indicate that acacetin-induced apoptosis in FaDu cells is mediated by the death receptor-mediated extrinsic apoptotic pathway through the Fas/PARP axis.

    Next, we examined the effect of acacetin on the expressions of Bax, Bad, Bcl-2 and Bcl-xL proteins in FaDu cells. The proapoptotic proteins such as Bax and Bad and the anti-apoptotic mitochondrial proteins such as Bcl-2 and Bcl-xL are important modulators of cytochrome c release from the mitochondria [27-29]. In the present study, acacetin treatment increased the levels of Bax and Bad protein expressions, but decreased the levels of Bcl-2 and Bcl-xL protein expressions in FaDu cells (Fig. 4). Changes in the levels of these anti- and proapoptotic factors associated with the mitochondria-dependent intrinsic pathway subsequently induced the activation cascade of caspase-9, caspase-3 and PARP in FaDu cells treated with acacetin (Fig. 4). Taken together, these findings indicate that acacetin induces apoptosis in FaDu cells involving the death receptor- and mitochondrial-signal transduction pathways. Meanwhile, the mechanism of acacetin-induced apoptosis in FaDu cells is not fully understood. Therefore, further research is needed to investigate the precise cellular and molecular mechanisms of acacetin-induced cellular apoptosis.

    In conclusion, these results suggest that acacetin inhibits cell proliferation and induces apoptotic cell death in FaDu human pharyngeal carcinoma cells through both the death receptormediated extrinsic apoptotic pathway and the mitochondriamediated intrinsic apoptotic pathway (Fig. 5). In addition, the results of current study suggest that acacetin may provide a strategy to prevent and treat carcinoma cells.

    Acknowledgements

    This study was supported by research fund from Chosun University (2020).

    Figure

    IJOB-45-3-107_F1.gif

    Chemical structure of acacetin.

    IJOB-45-3-107_F2.gif

    Effects of acacetin on cell viability in L-929 mouse normal fibroblasts and FaDu human pharyngeal carcinoma cells. (A) Effects of acacetin in L-929 cells. (B) Effects of acacetin in FaDu cells.The L-929 fibroblasts and FaDu cells were treated with various concentrations of acacetin or without acacetin for 24 hours. The cell viabilities were determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assays. The percentage of cell viability was calculated as a ratio of A570nms of acacetin treated cells and untreated control cells. Each data point represents the mean ± standard error of the mean of four experiments.

    *p < 0.05 vs. control, **p < 0.01 vs. control and ***p < 0.001 vs. control (the control cells measured in the absence of acacetin).

    IJOB-45-3-107_F3.gif

    Induction of cell apoptosis by acacetin. (A) FaDu cell death by acacetin. The cells were treated with 0, 12.5, 25 or 50 µM acacetin for 24 hours. Acacetin induced the death of FaDu cells in the dose-dependent manner. Cells emitting green fluorescence are live cells stained by green calcein AM, whereas cells emitting red fluorescence are dead cells stained by ethidium homodimer-1. (B) Changes in nuclear morphology by acacetin. The cells were treated with 0, 12.5, 25 or 50 µM acacetin for 24 hours. Representative fluorescence photomicrographs show the nuclei morphology of cells. DAPI staining revealed that the number of FaDu cells with nuclear condensation was increased by acacetin. (C) Activation of caspase-3/-7 by acacetin treatment in living FaDu cells. The FaDu cells were treated with 0, 25 or 50 µM acacetin for 24 hours and added specific cell-permeable substrate PhiPhiLux-G1D2. Active of caspase-3/-7 was visualized by fluorescence microscopy. Acacetin increased the activity of caspase-3/-7, pro-apoptotic factor, in FaDu cells.

    IJOB-45-3-107_F4.gif

    Intrinsic mitochondria-dependent and extrinsic death receptor-mediated apoptotic signaling pathways induced by acacetin in FaDu cells. The FaDu cells were treated with 0, 25 or 50 µM acacetin for 24 hours. The cell lysate was prepared and analyzed by immunoblotting as described in “Materials and Methods”. (A) Extrinsic death receptor-mediated apoptotic signaling pathway induced by acacetin. Acacetin upregulated the expression level of the death receptor ligand Fas and subsequently activated the extrinsic death receptor-mediated apoptotic signaling pathway through the cleavage of caspase-8 in FaDu cells. (B) Intrinsic mitochondria-dependent apoptotic signaling pathway induced by acacetin. Acacetin downregulated anti-apoptotic factors Bcl-2 and Bcl-xL associated with the intrinsic mitochondria-dependent apoptotic pathway and upregulated the mitochondria-dependent pro-apoptotic factors Bax and Bad in FaDu cells. (C) Extrinsic death receptor-mediated and intrinsic mitochondria-dependent apoptosis signaling pathways via the activation of caspase-3/-7 and poly (ADP-ribose) polymerase (PARP) induced by acacetin. Cleaved caspase-8 and cleaved caspase-9 induced the activation of caspase-3 and PARP in FaDu cells treated with acacetin.

    IJOB-45-3-107_F5.gif

    Apoptotic signaling pathway induced by acacetin in FaDu carcinoma cells.

    PARP, poly (ADP-ribose) polymerase.

    Table

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