Oral Biol Res 2022; 46(4): 165-170  https://doi.org/10.21851/obr.46.04.202212.165
Effects of cimifugin on cell growth inhibition and cell apoptosis induction in fadu human pharyngeal squamous cell carcinoma
Jong-Hyun Park1 and Do Kyung Kim2*
1Ph.D. Student, Institute of Dental Science, Chosun University, Gwangju, Republic of Korea
2Professor, Institute of Dental Science, Chosun University, Gwangju, Republic of Korea
Correspondence to: Do Kyung Kim, Department of Oral Physiology, School of Dentistry, Chosun University, 309 Pilmun-daero, Dong-gu, Gwangju 61452, Republic of Korea.
Tel: +82-62-230-6893, Fax: +82-62-225-6861 E-mail: kdk@chosun.ac.kr
Received: November 14, 2022; Revised: November 21, 2022; Accepted: November 26, 2022; Published online: December 31, 2022.
© Oral Biology Research. 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/by-nc/4.0) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Cimifugin is an important component of chromones in the dry roots of Saposhnikovia divaricata and is used in treating inflammatory diseases. However, the effect of cimifugin on cancer is unclear. The aim of this study was to investigate the effects of cimifugin on cell apoptosis induction in FaDu human pharyngeal carcinoma cells using MTT assay, live/dead cell assay, and western blot. Cimifugin-induced FaDu cell death in a dose-dependent manner. Cimifugin-induced apoptosis in FaDu cells was mediated by the expression of Fas and activation of caspase-8, caspase-3, and poly (ADP-ribose) polymerase (PARP). Western blot results showed the downregulation of Bcl-2 and Bcl-xL but the upregulation of Bad and Bax by cimifugin in FaDu cells. These results suggest that cimifugin inhibits cell survival 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.
Keywords: Anticancer; Apoptosis; Cimifugin
Introduction

Head and neck cancers such as oral, pharyngeal, and laryngeal cancers are the most common cancers worldwide, especially in South America, Asia, and Europe [1,2]. Over the past few decades, the survival rate of head and neck cancer patients has not improved significantly despite modern medical techniques and treatments, including the use of anticancer drugs [3]. Clinical treatment for head and neck cancer can cause side effects associated with functional changes in speech, swallowing, or chewing ability [4]. Therefore, much attention is being paid to the development of natural anticancer drugs that can minimize side effects while maintaining the effect of anticancer drugs [5,6].

Most anticancer drugs act as chemotherapy agents for cancer by causing cell apoptosis and inhibiting cancer cell proliferation [6]. Therefore, apoptosis of cancer cells due to the use of these anticancer drugs has become an important indicator of cancer treatment results [7,8]. Apoptosis can occur among cancer cells through a death receptor-dependent extrinsic pathway or a mitochondria-dependent intrinsic pathway that can be induced by chemotherapy [9].

Cimifugin, a coumarin rich in Saposhikavia divaricata (S. divaricata) roots, exerts analgesic effects [10]. This effect was also reported for 4-O-glucopyranosyl-5-O-methylvisamminol and cimifugin β-D-gluco-pyranoside, as well as anti-platelet aggregation, antipyretic, and anti-inflammatory activities [11]. Cimifugin β-D-glucopyranoside and cimifugin appear to inhibit major inflammatory pathways, nuclear factor-κB, cAMP response element-binding protein, mitogen- activated protein kinases and nitric oxide production [12,13]. However, the possible effect of cimifugin on cancer have not been clearly established.

Therefore, in this study, the effects of cimifugin on cell growth and the mechanism of cell death elicited by cimifugin were examined in FaDu human pharyngeal carcinoma cells. Our results showed that cimifugin inhibited cell viability and induce apoptosis in a dose-dependent manner in FaDu human pharyngeal carcinoma cells.

Materials and Methods

Materials

Cimifugin (Fig. 1) and 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) were purchased from Sigma Aldrich (St. Louis, MO, USA). Anti-cleaved caspase-3, -8, -9, anti-Fas, anti-cleaved poly (ADP-ribose) polymerase (PARP), anti-Bcl-2, anti-Bcl-xL, anti-Bax, anti-Bad, and anti-β-actin antibodies were supplied by Cell Signaling Technology, Inc. (Danvers, MA, USA). The Live/Dead cell viability assay kit was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA).

Fig. 1. Chemical structure of cimifugin.

Cell line and cell cultures

The FaDu human pharyngeal carcinoma cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). FaDu cells were grown in minimum essential medium (MEM; WelGene, Daegu, Korea) containing 10% fetal bovine serum (FBS; WelGene) at 37°C in an atmosphere containing 5% CO2.

Cell viability test (MTT assay)

The FaDu cells were seeded at a concentration of 1×104 cells/well in 48-well plates. After 24 hr growth, cells were treated with cimifugin various concentrations for 24 hr. Cell viability was evaluated using the MTT assay. At least 4 separate experiments were performed for each combination of concentration.

Live/Dead cell assay

FaDu cells (1×104 cells/well) were cultured overnight in an 8-well chamber slide, and allowed to attach to the bottom of the chamber slide. Thereafter, the cells were treated with 0, 75, or 150 µM cimifugin for 24 hr 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 uses green calcein AM to stain live cells (green fluorescence) and ethidium homodimer1 to stain dead cells (red fluorescence).

Immunoblotting

The FaDu cells were treated with 0, 75, or 150 µM of cimifugin for 24 hr. Immunoblotting was performed according to a previously described method with minor modifications [14]. Anti-cleaved caspase-3, -8, -9, anti-Fas, anti-cleaved PARP, anti-Bcl-2, anti-Bcl-xL, anti-Bax, anti-Bad, and anti-β-actin antibodies were used as the primary antibody.

Data analysis

All experiments were performed at least four times. The results are presented as mean±SEM. Statistical significance was analyzed using the 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

Cytotoxic effect on cimifugin in FaDu human pharyngeal carcinoma cell

To analyze the effect of cimifugin on the viability of FaDu cells, the cells were treated with cimifugin at various concentrations of cimifugin for 24 hr. Treatment with 10–160 µM cimifugin decreased the viability of FaDu cells compared to that of the control in a dose-dependent manner (Fig. 2A). The IC50 value of cimifugin on the FaDu cell viability was approximately 155.04 µM. These result suggests that cimifugin induces FaDu cell death in a dose-dependent manner.

Fig. 2. Effects of cimifugin on cell viability in FaDu pharyngeal carcinoma cells. (A) The FaDu cells were treated with various concentrations of cimifugin or without cimifugin for 24 hr. The cell viabilities were determined by the MTT assays. The percentage of cell viability was calculated as a ratio of A570nms of cimifugin treated cells and untreated control cells. Each data point represents the mean±SEM of four experiments. **p<0.01 vs. control and ***p<0.001 vs. control (the control cells were measured in the absence of cimifugin). (B) The cells were treated with 0, 75, or 150 µM cimifugin for 24 hr. Cimifugin induced the death of FaDu cells in a 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.

Induction of apoptosis by cimifugin in FaDu cells

To confirm the viability of FaDu cells treated with cimifugin, a Live/Dead cell assay was performed. FaDu cells exposed to cimifugin emitted red fluorescence in a dose-dependent manner after staining with ethidium homodimer-1, which stains dead cells (Fig. 2B). These results indicate that cimifugin induced apoptotic cell death in FaDu cells.

Extrinsic death receptor-mediated apoptosis induced by cimifugin in FaDu cells

Immunoblotting was performed to determine the cellular apoptotic pathways associated with cimifugin-induced FaDu cell death. Fas, an apoptotic ligand that triggers the death receptor-dependent extrinsic apoptotic pathway in cancer cells [15,16], was significantly induced by cimifugin in FaDu cells (Fig. 3). As shown in Fig. 3, the level of cleaved caspase-8, the downstream target of the pro-apoptotic factor Fas, increased following cimifugin treatment. These data indicate the involvement of the extrinsic death receptor-mediated apoptosis pathway in cimifugin-induced FaDu cell apoptosis.

Fig. 3. Extrinsic apoptotic signaling pathways induced by cimifugin in FaDu cells. The FaDu cells were treated with 0, 75, or 150 µM cimifugin for 24 hr. The cell lysate was prepared and analyzed by immunoblotting as described in “Materials and Methods”. Cimifugin 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.

Intrinsic mitochondria-dependent apoptosis induced by cimifugin in FaDu cells

The expression levels of Bcl-2 and Bcl-xL, anti-apoptotic factors associated with the intrinsic mitochondria-dependent apoptosis pathway, were downregulated by cimifugin in FaDu cells, wheres those of mitochondria-dependent pro-apoptotic factors such as Bad and Bax were upregulated by cimifugin in FaDu cells (Fig. 4). Cimifugin treatment increased the expression of cleaved caspase-9 in FaDu cells (Fig. 4). These data indicate that cimifugin-induced FaDu cell death involves an intrinsic mitochondria-dependent apoptosis pathway.

Fig. 4. Intrinsic mitochondria-dependent apoptotic signaling pathways induced by cimifugin in FaDu cells. Cimifugin 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.

Apoptosis mediated via PARP by cimifugin in FaDu cells

Both cleaved caspase-8 and caspase-9, which act in the extrinsic death receptor-mediated and intrinsic mitochondria-dependent apoptosis pathways, induced the expression of cleaved caspase-3 and PARP in FaDu cells following cimifugin treatment (Fig. 5). These results indicate that cimifugininduces FaDu cell death is coordinated by death receptor-mediated extrinsic and mitochondria-dependent intrinsic apoptosis through activation of the caspase cascade in FaDu cells.

Fig. 5. Extrinsic death receptor-mediated and intrinsic mitochondria-dependent apoptosis signaling pathways via the activation of caspase-3 and poly (ADP-ribose) polymerase (PARP) induced by cimifugin.
Discussion

In this study, the cytotoxic and apoptotic activities of cimifugin were examined in FaDu human pharyngeal carcinoma cells. The results of this study indicated that the anti-proliferative activity of cimifugin against human pharyngeal carcinoma cells was due to its ability to induce apoptosis.

In our cell viability test (Fig. 2A) and Live/Dead cell assay (Fig. 2B), cimifugin inhibited FaDu cell growth in a concentration-dependent manner. These results suggest that cimifugin is cytotoxicity to human pharyngeal carcinoma cells and potential value for anti-cancer drug discovery.

In this study, we examined that Caspase-3, -7, -8, and -9 can act as effector caspases of apoptotic cell death in eukaryotic cells [17,18]. The immunoblotting results showed that low levels of cleaved capase-3, -8, and -9 were present in cimifugin-untreated FaDu cells, and the amount of cleaved enzymes was increased after cimifugin treatment in FaDu cells (Fig. 4, 5). These results suggest that cimifugin induce apoptotic cell death by activation caspases-3/-7/-8/, and -9 in FaDu cells.

Fas, an important regulator of apoptosis, binds to the receptor FasR across the surface of the target cell, and then initiates the death receptor-mediated extrinsic apoptotic pathway through the activation of caspase-8, -3, and PARP [15,16]. In our study, the amount of Fas protein was significantly increased by cimifugin treatment in FaDu cells (Fig. 3). In succession, the Fas stimulated by cimifugin triggered the caspase cascade, which resulted in the activation of apoptotic factors including cleaved caspase-8 and -3 (Fig. 3-5). Finally, caspase-3 activation by cimifugin cleaved, PARP, leading to apoptosis of FaDu cells (Fig. 5). These results indicate that cimifugin-induced apoptosis in FaDu cells is mediated by the death receptor-mediated extrinsic apoptotic pathway via the Fas/PARP axis.

To the next, we examined the effect of cimifugin on the expressions of Bax, Bad, Bcl-2, and Bcl-xL in FaDu cells. Pro-apoptotic proteins such as Bax and Bad, and anti-apoptotic mitochondrial proteins such as Bcl-2 and Bcl-xL are important regulators of cytochrome c release in mitochondria [19-21]. In our study, cimifugin treatment increased the levels of Bax and Bad protein expression, 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 pro-apoptotic factors associated with the mitochondria-dependent intrinsic pathway subsequently induced the activation cascade of caspase-9, -3 and PARP in FaDu cells treated with cimifugin (Fig. 4, 5). These results indicate that cimifugin induces apoptosis in FaDu cells via death receptor- and mitochondrial-signal transduction pathways.

In conclusion, these results suggest that cimifugin inhibits cell proliferation and induces apoptotic cell death in human pharyngeal carcinoma cells through both the death receptor-mediated extrinsic apoptotic pathway and the mitochondria-mediated intrinsic apoptotic pathway (Fig. 3-5). Additionally, our findings suggest that cimifugin may provide a strategy for treating human pharyngeal carcinoma.

Funding

This study was supported by research fund from the Chosun University (2021).

Conflicts of Interest

The authors declare that they have no competing interests.

References
  1. Warnakulasuriya S. Global epidemiology of oral and oropharyngeal cancer. Oral Oncol 2009;45:309-316. doi: 10.1016/j.oraloncology.2008.06.002.
    Pubmed CrossRef
  2. Kalavrezos N, Scully C. Mouth cancer for clinicians. Part 1: cancer. Dent Update 2015;42:250-252, 255-256, 259-260. doi: 10.12968/denu.2015.42.3.250.
    Pubmed CrossRef
  3. Pezzuto F, Buonaguro L, Caponigro F, Ionna F, Starita N, Annunziata C, Buonaguro FM, Tornesello ML. Update on head and neck cancer: current knowledge on epidemiology, risk factors, molecular features and novel therapies. Oncology 2015;89:125-136. doi: 10.1159/000381717.
    Pubmed CrossRef
  4. Nestor MV. Targeted radionuclide therapy in head and neck cancer. Head Neck 2010;32:666-678. doi: 10.1002/hed.21243.
    Pubmed CrossRef
  5. Le Tourneau C, Faivre S, Siu LL. Molecular targeted therapy of head and neck cancer: review and clinical development challenges. Eur J Cancer 2007;43:2457-2466. doi: 10.1016/j.ejca.2007.08.016.
    Pubmed CrossRef
  6. Caponigro F, Milano A, Basile M, Ionna F, Iaffaioli RV. Recent advances in head and neck cancer therapy: the role of new cytotoxic and molecular-targeted agents. Curr Opin Oncol 2006;18:247-252. doi: 10.1097/01.cco.0000219253.53091.fb.
    Pubmed CrossRef
  7. Yanumula A, Cusick JK. Biochemistry, Extrinsic Pathway of Apoptosis. [Updated 2022 Aug 1]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022.
  8. Hengartner MO. The biochemistry of apoptosis. Nature 2000;407:770-776. doi: 10.1038/35037710.
    Pubmed CrossRef
  9. Liu D, Zhou H, Wu J, Liu W, Li Y, Shi G, Yue X, Sun X, Zhao Y, Hu X, Wang T, Zhang X. Infection by Cx43 adenovirus increased chemotherapy sensitivity in human gastric cancer BGC-823 cells: not involving in induction of cell apoptosis. Gene 2015;574:217-224. doi: 10.1016/j.gene.2015.08.052.
    Pubmed CrossRef
  10. Okuyama E, Hasegawa T, Matsushita T, Fujimoto H, Ishibashi M, Yamazaki M. Analgesic components of saposhnikovia root (Saposhnikovia divaricata). Chem Pharm Bull (Tokyo) 2001;49:154-160. doi: 10.1248/cpb.49.154.
    Pubmed CrossRef
  11. Xue BY, Li W, Li L, Xiao YQ. [A pharmacodynamic research on chromone glucosides of fangfeng]. Zhongguo Zhong Yao Za Zhi 2000;25:297-299. Chinese.
  12. Kreiner J, Pang E, Lenon GB, Yang AWH. Saposhnikoviae divaricata: a phytochemical, pharmacological, and pharmacokinetic review. Chin J Nat Med 2017;15:255-264. doi: 10.1016/S1875-5364(17)30042-0.
    Pubmed CrossRef
  13. Matusiewicz M, Bączek KB, Kosieradzka I, Niemiec T, Grodzik M, Szczepaniak J, Orlińska S, Węglarz Z. Effect of juice and extracts from Saposhnikovia divaricata root on the colon cancer cells Caco-2. Int J Mol Sci 2019;20:4526. doi: 10.3390/ijms20184526.
    Pubmed KoreaMed CrossRef
  14. Park MG, Kim JS, Park SY, Lee SA, Kim HJ, Kim CS, Kim JS, Chun HS, Park JC, Kim DK. MicroRNA-27 promotes the differentiation of odontoblastic cell by targeting APC and activating Wnt/β-catenin signaling. Gene 2014;538:266-272. doi: 10.1016/j.gene.2014.01.045.
    Pubmed CrossRef
  15. Herrnring C, Reimer T, Jeschke U, Makovitzky J, Krüger K, Gerber B, Kabelitz D, Friese K. Expression of the apoptosis-inducing ligands FasL and TRAIL in malignant and benign human breast tumors. Histochem Cell Biol 2000;113:189-194. doi: 10.1007/s004180050438.
    Pubmed CrossRef
  16. Li HJ, Wang CY, Mi Y, Du CG, Cao GF, Sun XC, Liu DJ, Shorgan B. FasL-induced apoptosis in bovine oocytes via the Bax signal. Theriogenology 2013;80:248-255. doi: 10.1016/j.theriogenology.2013.04.002.
    Pubmed CrossRef
  17. Datta R, Kojima H, Yoshida K, Kufe D. Caspase-3-mediated cleavage of protein kinase C theta in induction of apoptosis. J Biol Chem 1997;272:20317-20320. doi: 10.1074/jbc.272.33.20317.
    Pubmed CrossRef
  18. Yang SJ, Lee SA, Park MG, Kim JS, Yu SK, Kim CS, Kim JS, Kim SG, Oh JS, Kim HJ, Chun HS, Kim YH, Kim DK. Induction of apoptosis by diphenyldifluoroketone in osteogenic sarcoma cells is associated with activation of caspases. Oncol Rep 2014;31:2286-2292. doi: 10.3892/or.2014.3066.
    Pubmed CrossRef
  19. Yuan H, Williams SD, Adachi S, Oltersdorf T, Gottlieb RA. Cytochrome c dissociation and release from mitochondria by truncated Bid and ceramide. Mitochondrion 2003;2:237-244. doi: 10.1016/S1567-7249(02)00106-X.
    Pubmed CrossRef
  20. Clayton R, Clark JB, Sharpe M. Cytochrome c release from rat brain mitochondria is proportional to the mitochondrial functional deficit: implications for apoptosis and neurodegenerative disease. J Neurochem 2005;92:840-849. doi: 10.1111/j.1471-4159.2004.02918.x.
    Pubmed CrossRef
  21. Odinokova IV, Sung KF, Mareninova OA, Hermann K, Evtodienko Y, Andreyev A, Gukovsky I, Gukovskaya AS. Mechanisms regulating cytochrome c release in pancreatic mitochondria. Gut 2009;58:431-442. doi: 10.1136/gut.2007.147207.
    Pubmed KoreaMed CrossRef


This Article


Funding Information

Services
Social Network Service

e-submission

Archives