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ISSN : 1226-7155(Print)
ISSN : 2287-6618(Online)
International Journal of Oral Biology Vol.41 No.1 pp.1-8
DOI : https://doi.org/10.11620/IJOB.2016.41.1.001

Anticancer Properties of Icariside II in Human Oral Squamous Cell Carcinoma Cells

In-Ryoung Kim, Young-Seok Kim, Su-Bin Yu, Hae-Mi Kang, Hyun-Ho Kwak, Bong-Soo Park
Department of Oral Anatomy, School of Dentistry, Pusan National University
Bong-Soo Park, Department of Oral Anatomy,School of Dentistry, Pusan National University, Yangsan, 626-870,Korea. Tel.: +82-51-510-8242, Fax: +82-51-510-8241 E-mail: parkbs@pusan.ac.kr ORCID: 0000-0002-9799-5627
February 3, 2016 February 25, 2016 February 26, 2016

Abstract

OSCC is currently the most common malignancy of thehead and neck, affecting tens of thousands of patients per year worldwide. Natural flavonoids from plants are potential sources for novel anti-cancer drugs. Icariin is the active ingredient of flavonol glycoside, which is derived from the medical plant Herba Epimedii. A metabolite of icariin, icariside II exhibits a variety of pharmacological actions, including anti-rheumatic, anti-depressant, cardiovascular protective, and immunomodulatory functions. However, the exact mechanism causing the apoptosis-inducing effect of icariside II in OSCC is still not fully understood. In the present study, we assessed the anti-cancer effect of icariside II in OSCC cell lines by measuring its effect on cell viability, cell proliferation, and mitochondria membrane potential (MMP). Icariside II treatment of OSCC cells resulted in a dose- and time-dependent decrease in cell viability. Hoechst staining indicated apoptosis in icariside II-treated HSC cells. Icariside II inhibited cell proliferation and induced apoptosis in HSC cells, with significant increases in all present parameters in HSC-4 cells. The results clearly suggested that icariside II induced apoptosis via activation of intrinsic pathways and caspase cascades in HSC-4 cell lines. The collective findings of the study suggested that Icariside II is a potential treatment for OSCC; in addition, the data could provide a basis for the development of a novel anti-cancer strategy.


초록


    Pusan National University
    International Journal of Oral Biology

    This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

    Introduction

    Oral cancer and, most commonly, oral squamous cellcarcinoma (OSCC) is currently the most common human malignancy of the head and neck, affecting over 500,000 patients per year worldwide [1, 2]. OSCC accounts for approximately 3% of all cases of cancer. Until recently, the five-year survival rate for OSCC was below 50%. Common cancer treatments, including surgery, radiation therapy, and chemotherapy, are often used to treat OSCC. However, there are still high death rates associated with these therapies [3, 4, 5]. Therefore, new therapeutic approaches using natural agents for the treatment of OSCC are currently being investigated. The use of natural agents seems to be one of the most promising anti-cancer treatments currently in development.

    Apoptosis is the physiological process of controlledelimination of unhealthy or damaged cells [6]. When a cell undergoes apoptosis, it shows nuclear condensation, DNA fragmentation, and membrane blebbing through various pathway signals [7, 8]. The mitochondria signaling pathway is one of the apoptosis pathways. Mitochondria play an important role in the regulation of cell apoptosis. Changes in the mitochondria membrane potential (MMP) are considered an early apoptosis event, and many pro-apoptotic proteins can be released from the mitochondria into the cytoplasm when the MMP is damaged [9]. Recent studies have reported that apoptosis might play an important role in various diseases. Cancer, in particular, seems to be closely related to apoptosis. Thus, in order to develop anti-cancer drugs with less side effects than are associated with the current cancer treatment options, it is important to study the anti-cancer capacities of natural compounds [10].

    Natural products and herbal medicines derived from plantsmay be potential sources for new anti-cancer drugs [11]. Flavonoids are polyphenols found in many foods, such as vegetables, fruit, red wine, and tea. These polyphenols are well known for their antioxidant, anti-inflammatory, anticancer, and antibacterial properties [12, 13]. Icariin is the active ingredient of the flavonol glycoside derived from the medical plant Herba Epimedii [14]. Icaritin and icariside II are metabolites of icariin [15, 16]. These products have been previously demonstrated to exhibit a variety of pharmacological activities, including anti-rheumatic, antidepressant, cardiovascular protection and immunomodulatory activities [16, 17]. The anti-tumor properties of icariin and its glycosides have recently attracted a great deal of attention from medical researchers [18, 19, 20]. However, the exact mechanism of the apoptosis-inducing effect of icariin and its glycosides in human OSCC is still not fully understood. In the present study, we demonstrate that icariin, icaritin, and icariside II are potent agents against OSCC, and that their anti-cancer functions derive from the signaling of apoptosis through the mitochondrial pathway.

    Materials and Methods

    Reagents

    The following reagents were obtained commercially:3-[4,5-dimethylthiazol-2-yl]2, 5-diphenyl tetrazolium bromide (MTT), and acridine orange were purchased from Sigma (St. Louis, MO, USA). Antibodies against the caspase-3 cleaved form of caspase-3 and PARP were purchased from Cell Signaling Technology (Beverly, MA, USA). The AIF, Bcl-2, BAK, cytochrome c, caspase-9, ICAD, GAPDH mouse anti-rabbit IgG antibody, and rabbit anti-mouse IgG antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals and reagents were purchased from Sigma, unless otherwise specified.

    Cell Culture

    HSC-2, -3, -4 cells were kindly supplied by Prof. Sung-DaeCho of Chonbuk National University, Jeonju, Korea. The cells were cultured in Minimum Essential Medium/Earle's Balanced Salt Solution (MEM/EBSS) supplemented with 10% FBS and 1% penicillin streptomycin (GIBCO-BRL, Rockville, MD, USA) under CO2 humidified at 5%.

    Treatment of Icariin, Icaritin, and Icariside II

    The original medium was removed and icariin, icaritin, andicariside II (100 mM) stock solution were added to the medium to obtain 10 to 100 μM of these three supplements. Icariin, icaritin, and icariside II (100 mM each) were prepared in DMSO and stored in a frozen state at -20°C. When needed, the icariin, icaritin, and icariside II stock solutions were diluted to the indicated concentration with MEM/EBSS.

    Cell Viability Assay

    The cells were seeded on a 96-well microtiter plate (1×104cells/well) and then treated with different concentrations of icariin, icaritin, and icariside II (10 to 100 μM). The cells were then incubated for different time periods. Next, the existing medium was removed and 100 μl of MTT solution (500 μg/mL) was added to each well. The cells were incubated for 4 h at 37°C. The reaction was stopped by the addition of DMSO (150 μl/well) and constantly shook for 10 min. The cell viability was monitored on an ELISA reader (Tecan, Mänedorf, Switzerland) at an excitatory emission wavelength of 620 nm.

    Hoechst Staining for Morphological Aspects of Apoptosis

    The number of cells undergoing apoptosis followingtreatment with icariside II was quantified by Hoechst staining. Different concentrations of icariside II (10, 25, and 50 μM) were treated in HSC-2, -3, and -4 cells for 24 h. After treatment, the HSC-2, -3, and -4 cells were harvested and cytocentrifuged onto clean, fat-free glass slides using a cytocentrifuge. The cells were stained with Hoechst 33342 (1 μg/ml) for 10 min at 37°C. After being washed with phosphate-buffered saline (PBS) and mounted with glycerol, the cells were observed using an inverted fluorescence microscope (Carl Zeiss, Goettingen, Germany). The untreated cell nuclei showed dispersion and uniform fluorescence, while the apoptotic cells were characterized by nuclear shrinkage, condensation, and fragmentation.

    Measuring DNA Contents and MMP Using FACS

    The DNA contents of HSC-2, -3, and -4 were analyzedusing FACS. The cells were seeded in 100 mm culture dishes (2×106cells/dishes) and incubated for 24 h. After the icariside II treatments, the cells were harvested and centrifuged at 3000 rpm for 5 min. Next, they were fixed overnight in 95% ice-cold ethanol with 0.5% Tween 20, then washed in 1% bovine serum albumin PBS solution and re-suspended in PBS containing 50 μg/mL RNase A. Finally, they were incubated at 4°C for 30 min. The cells were stained with propidium iodide (50 μg/ml), and the stained cells were measured using a CYTOMICS FC500 flow cytometer system. The data was analyzed using Multi Cycle software, which allowed a simultaneous estimation of cell-cycle parameters and apoptosis.

    In order to measure the MMP, the HSC-4 cells wereseeded onto 60 mm dishes and incubated for one day. After being treated with icariside II for 24 h, the collected cells and DIOC6 were stained directly onto the cell culture medium (final concentration=1 μM) and incubated for 30 min. The MMP was analyzed with a flowcytometry system (Beckman Coulter, CA, USA).

    Western Blot Analysis

    The cells were harvested and washed in ice-cold PBS, thenre-suspended in 200 μl of ice-cold solubilizing buffer (300 mM NaCl, 50 mM Tris-Cl [pH 7.6], 0.5% Triton X-100, 2 mM PMSF, 2 μl/ml aprotinin, and 2 μl/ml leupeptin). The lysates were centrifuged at 13,200 rpm for 30 min at 4°C. The protein concentrations of the cell lysates were determined using a Bradford protein assay (Bio-Rad, Richmond, CA, USA). The lysates resolved on sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) were transferred, and the membrane was blocked with 5% skim milk for 2 h at room temperature. After the blocking was complete, the membranes were incubated overnight at 4°C with their respective primary antibodies. The membranes were washed 6 times for 1 h, and they reactivated secondary antibodies for 2 h at room temperature. After six washes, the membranes were detected using a Super Signal West Femto (Pierce, Rockford, IL, USA) enhanced chemiluminescence substrate before being detected with an Alpha Imager HP (AlphaInnotech, SantaClara, USA).

    Fluorescence Microscopy

    The cells were grown on coverslips and treated withicariside II. After 24 h, the cells were incubated with a Mitotracker at 37°C for 30 min before being fixed with 4% paraformaldehyde at room temperature for 10 min. The cells were permeabilized with 0.1% Triton X-100 in PBS at room temperature for 10 min. The coverslips were blocked with 3% BSA (in PBS) and incubated with the primary antibody (1:200) at room temperature for 2 h. The coverslips were then washed extensively with PBS and incubated with a FITC-conjugated secondary (1:200) antibody for 2 h at room temperature. They were then washed again in PBS and mounted with 90% glycerol. Finally, the coverslips were sealed. The cells were then visualized by laser scanning confocal microscopy. The cellular fluorescence changes were obtained using a confocal microscope LSM 700 (Carl Zeiss, Germany).

    Statistical Analysis

    The statistical analysis data were expressed as ± SD fromat least three independent experiments. A one-way ANOVA test was used to analyze the data regarding cell viability, DNA contents, and nuclear condensation ratio on a GraphPad Prism 5.0 (GraphPad Prism Software, San Diego, CA, USA). P values less than 0.05 were considered to be statistically significant.

    Results

    Cytotoxic Effects of Icariin, Icaritin, and Icariside II on OSCC Cell Lines

    The MTT assay was used to measure the effect of icariin,icaritin, and icariside II on the viability of OSCC cell lines (HSC-2, HSC-3, and HSC-4). First, the effects of icariin, icaritin, and icariside II on the OSCC cell lines were investigated. Next, various concentrations of icariin, icaritin, and icariside II (10 to 100 μM) were cultured for 24 h. As shown in Fig. 1, icaritin, and icariside II reduced the cell viabilities of all OSCC cells excepting icariin. Icariside II, in particular, significantly decreased the cell viability depending on the dose and time (Fig. 2). For this reason, icariside II was used to conduct the next experiment.

    Icariside II Increased the Apoptosis of OSCC Cells

    To determine whether the cytotoxicity of icariside II occurredby apoptosis, morphological changes were conducted and the DNA contents of the nucleus were withdrawn using hoechst staining and FACS. The percentage of apoptosis occurring in the OSCC (HSC-2,-3,-4) cells was measured following treatment of the cells with increasing concentrations (0–50 μM) of icariside II. Treatment with icariside II resulted in an increased apoptotic ratio of dose dependency (Fig. 3). Hoechst staining proved that icariside II induced a change in the nuclear morphology. Untreated control cells had typical round nuclei, whereas those treated with 50 μM of icariside II for 24 h displayed condensed and fragmented nuclei in all of the OSCC cells (Fig. 4). This was especially true in the HSC-4 cells, where over 25 μM of icariside II resulted in more apoptotic cells with fragmented and cleaved nuclei (Fig. 3 and Fig. 4). To elucidate the molecular mechanisms of icariside II-induced apoptosis, the expression levels of the apoptosis-associated proteins, such as caspase-9, caspase-3, cleaved caspase-3, ICAD, and PARP, were further examined using western blot analysis. The expression levels of caspase-9, caspase-3, cleaved caspase-3, ICAD, and PARP proteins decreased at the 50 μM icariside II. In particular, cleaved caspase-3 only increased in HSC-4 cells at levels of 50 μM icariside II (Fig. 5). The results of the MMP measurement verified that a change occurred in MMP in icariside II-induced apoptosis. This occurred due to DIOC6 staining, and it wasmeasured by flowcytometry. Icariside II-treated HSC-4 cells showed a loss of MMP that changed depending on the dose (Fig. 6A). The pro-apoptotic protein, Bak, was decreased, while the anti-apoptotic protein, Bcl-2, was increased by icariside II in a dose-dependent manner (Fig. 6B). Mitochondrial AIF and cytochrome c were released from the mitochondria and were detected by confocal microscopy. After icariside II treatment, cytochrome c was released from the mitochondria into the cytosol, and the AIF was translocated from the mitochondria into the nuclei (Fig. 7). These results clearly suggest that icariside II-induced apoptosis is involved in the intrinsic pathway and caspase cascades.

    Discussion

    Many studies have reported that plant-derived flavonoid haspotent biological advantages against various types of cancer cells [21]. Icariside II has received significant attention because of its molecular biological advantages, such as its anti-inflammatory, anti-bacterial, and anti-cancer properties [22]. However, the biological mechanism behind icariside II-induced cell death against OSCC cell lines has not been well studied. OSCC is a type of malignant tumor; it is the most common oral cancer in the world. The age of onset for OSCC tends to be younger than that of other tumors (approximately 30~50 years of age) [23]. In this study, we provided emerging proof that icariin, icaritin, and icariside II have anti-cancer properties against HSC-2, -3, and -4 cells. Based on our preliminary experiments, we found that icariside II inhibits cell viability and proliferation more than icariin and icaritin in OSCC cell lines (Fig. 1). The present study investigated the effects of icariside II on the viability of HSC-2, -3, and -4 cells, revealing that icariside II produces a dose- and time-dependent reduction, particularly in the viability of HSC-4 cells. These data indicate that icariside II exerts a specific cytotoxic effect on HSC-4 cells.

    During apoptosis, the cells show specific morphologicaland biochemical changes, including cell shrinkage, DNA fragmentation, nuclear condensation, plasma membrane blebbing, and loss of adhesion to neighboring cells or to the extracellular matrix [24, 25]. For the purpose of Hoechst staining in the present study, the cells were treated with 0 to 50 μM icariside II for 24 h, then stained with Hoechst 33324 and analyzed under a fluorescence microscope (Fig. 3). In this study, we investigated the possible mechanisms of icariside II-induced apoptosis in HSC cells. We also assessed the DNA contents that performed the flow cytometry with PI staining in the HSC cells. Treatment with icariside II resulted in an increased apoptotic ratio in HSC-2, -3, and -4 cells that was dose dependent (Fig. 4). As a result, when they were compared with the control groups, the HSC cells were consistent with the enhanced effect of icariside II on the induction of apoptosis.

    In present study, we investigated the icariside II-inducedapoptosis occurring via the mitochondrial signaling pathway. We found that icariside II down-regulated MMP and released mitochondrial AIF and cytochrome c from the mitochondria. A number of studies have demonstrated that flavonoids induce apoptosis through stimulation of the mitochondrial signaling pathway [18, 26, 27]. Two principal apoptosis pathways exist: the intrinsic pathway emerges from the mitochondria, while the extrinsic pathway is activated by the ligation of the death receptor [28, 29]. The intrinsic pathway leads to apoptosis under the control of the mitochondrial [30]. The cells are initiated by either extracellular stimuli or intracellular signals, after which the outer mitochondrial membranes become permeable to the internal cytochrome c. This is then released into the cytosol. Cytochrome c recruits Apaf–1 and caspase-9 to compose the apoptosome, which downstream triggers a caspase-signaling cascade, culminating in apoptosis [31]. Thus, the anti-cancer activities of icariside II are induced by the stimulation of the mitochondrial signaling pathway.

    Poly (ADP-ribose) polymerase (PARP), a DNA repairenzyme, plays a particularly well-known role in base excision repair [32]. PARP is part of the caspase-dependent pathway of apoptosis. Cleaved PARP increases during apoptosis and is an important apoptotic marker [30, 33]. The DNA fragmentation factor, which comprises a caspase-3-activated DNase (CAD) and its inhibitor (ICAD), may influence the rate of cell death by generating PARP-activating DNA fragmentation [34, 35].

    We clarified the activation of the caspases ICAD andPARP, which are associated with apoptosis by icariside II. The expression levels of caspase-9, caspase-3, ICAD, and PARP proteins activated and cleaved caspase-3, causing significant increases in the HSC-4 cells. These findings clearly suggest that icariside II-induced apoptosis via activation of the intrinsic pathway and caspase cascades in HSC-4 cell lines. Icariside II could be a potential treatment for OSCC, and it could help provide valuable data for the development of a novel anti-cancer strategy.

    Acknowledgements

    This work was supported by a 2-Year Research Grant of Pusan National University

    Figure

    321_F1.jpg

    Effects of icariin, icaritin, and icariside II treatment onthe viability of OSCC cells. (A) Icariin, (B) icaritin, and (C) icariside II (10–100 μM) were used as treatments in three types of OSCC cells (HSC-2, -3 and 4) for 24 h. The icariside II treatment group (C) showed dramatically decreased cell viability. The cell viability was analyzed using the MTT assay. Each value represents the mean of three independent experiments ± SD (n=6).

    321_F2.jpg

    The icariside II treatment clearly inhibited the cell viability of HSC-4. Icariside II (10, 25, and 50 μM) was used as treatment in three types of OSCC cells (HSC-2, -3, -4) for 24 to 72 h. Icariside II clearly inhibited the HSC-4 cell viability from 24 h to 72 h. The cell viability was analyzed using the MTT assay. Each value represents the mean of three independent experiments ± SD (n=6). *p<0.05, **p<0.01, ***p<0.001 when compared with the control group.

    321_F3.jpg

    Icariside II increased apoptotic cell death in the OSCC cells. The cells (HSC-2, -3, -4) were treated with Icariside II (10, 25, and 50 μM) for 24 h. (A) and (B): The ratio of apoptotic cells was determined by the FACS. Each value represents the mean of three independent experiments ± SD (n=3). *p<0.05 when compared with the control group.

    321_F4.jpg

    The icariside II promoted morphological change of the nuclei of the OSCC cells. The cells (HSC-2, -3, and -4) were treated with icariside II (10, 25, and 50 μM) for 24 h. After treatment, the cells were stained with hoechst and observed under a fluorescence microscope. The HSC-4 cells showed a larger amount of cell shrinkage, volume reduction, apoptotic body formation, and cell blebbing than the other cell lines. Each value represents the mean of three independent experiments ± SD (n=3). *p<0.05, **p<0.01 when compared with the control group.

    321_F5.jpg

    The icariside II activated the apoptosis-related molecules (caspase-9, caspase-3, cleaved caspase-3, ICAD, and PARP) in the OSCC cells. The cells (HSC-2, -3, and -4) were treated with icariside II (10, 25, and 50 μM) for 24 h, and the levels of caspase-9, caspase-3, cleaved caspase-3, ICAD, and PARP were measured using western blot analysis. After the treatment, these proteins were decreased. Cleaved caspase-3 was only increased in HSC-4 cells at a level of 50 μM icariside II. The levels of GAPDH were used as an internal standard.

    321_F6.jpg

    Icariside II reduced the MMP and changed the expressions of the anti- and pro-apoptotic proteins. The cells (HSC-2, -3, and -4) were treated with icariside II (10, 25, and 50 μM) for 24 h. (A) The MMP (△⍦m) was reduced in a dose-dependent manner in the HSC-4 cells; (B) The pro-apoptotic molecule, Bax, was significantly increased, whereas the anti-apoptotic factor, Bcl-2, was decreased in a dose-dependent manner. The levels of GAPDH were used as an internal standard.

    321_F7.jpg

    The icariside II induced the translocation of the mitochondrial cytochrome c and AIF. Confocal immunofluorescence microscopy staining of the cytochrome c was conducted, and the AIF was in the HSC-4 cells. After the icariside II treatment: (A) cytochrome c was released from the mitochondria into the cytosol, and (B), the AIF was translocated from the mitochondria into the nuclei.

    Table

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