Hinokitiol suppressed pan-histone expression and cell growth in oral
squamous cell carcinoma cells.
Yin-Hua Shiha,b, Kuo-Wei Changa,c, Cheng-Chia Yub, Ming-Ching Kaod, Michael
Yuanchien Chene, Tong-Hong Wangf, Tzu-Yun Chig, Yi-Ling Cheng, Tzong-Ming Shiehg,h*
a. Institute of Oral Biology, School of Dentistry, National Yang-Ming University, No. 155, Sec. 2, Linong Street, Taipei 11217, Taiwan
b. Institute of Oral Science, School of Dentistry, Chung Shan Medical University, No. 110, Sec. 1, Jianguo N. Road, Taichung 40201, Taiwan
c. Department of Dentistry, Taipei Veterans General Hospital, No. 201, Sec. 2, Shipai Rd., Beitou District, Taipei 11217, Taiwan
d. Department of Biological Science and Technology, College of Life Sciences, China Medical University, No. 91 Hsueh-Shih Road, Taichung 40402, Taiwan
e. Department of Oral & Maxillofacial Surgery, China Medical University Hospital, Taichung, Taiwan, School of Dentistry, College of Medicine, China Medical University, No. 91 Hsueh-Shih Road, Taichung 40402, Taiwan
f. Tissue Bank, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan
g. Department of Dental Hygiene, College of Health Care, China Medical University, No. 91, Hsueh-Shih Road, Taichung 40402, Taiwan.
Phone: +8864-22053366-7707 Fax: +8864-22073556 E-mail: tmshieh@mail.cmu.edu.tw
h. Oral Biology Laboratory, China Medical University, No. 91, Hsueh-Shih Road, Taichung 40402, Taiwan
Abstract:
Hinokitiol is reported to inhibit oral squamous cell carcinoma cells growth but its mechanism of action remains unclear. Hinokitiol induced cell cycle arrest in G1 or G1/S phase and induced cell apoptosis in oral squamous cell carcinoma cells. The cDNA microarray data showed 6.25-12.5 μM hinokitiol suppressed pan-histone mRNA expression and quantitative RT-PCR and western blotting showed the reproducibility of the results. Hinokitiol impaired the chromatin folding in oral squamous cell carcinoma cells. In addition, the binding of FLICE-associated huge protein and nuclear protein of the ataxia telangiectasia mutated locus on histone H4/e promoter was decreased, and H4/e promoter activity was significantly decreased. The malignant phenotypes of oral squamous cell carcinoma cells were suppressed in low dose (0.75-6.25 μM hinokitiol) in vitro. Feeding 10 mg/kg hinokitiol to mice suppressed xenograft tumourigenecity of HSC-3 cells in vivo. Hinokitiol showed the efficacy against oral squamous cell carcinoma cells growth by pan-histone suppression. Keywords: FLICE-associated huge protein, hinokitiol, nuclear protein of the ataxia telangiectasia mutated locus, oral squamous cell carcinoma, pan-histone
1. Introduction
Oral cancer is the sixth most common cancer worldwide and an increase of 62% has been reported in the developing countries (Ramshankar & Krishnamurthy, 2014). The regions with the highest oral cancer incidence are Melanesia, South-Central Asia, and Central-Eastern Europe and the 5-year survival rate is low (Jemal et al., 2011; Petersen, 2009; Warnakulasuriya, 2009). Oral squamous cell carcinoma (OSCC) accounts for more than 90% of oral cancer cases (Markopoulos, 2012). Alcohol drinking, cigarette smoking, and betel nut chewing are the major risk factors of oral cancer that has been proposed to cause epigenetic abnormalities such as varying DNA methylation, histone modification, and microRNA expression to induce initiation and progression of OSCC.
Hinokitiol is a natural bioactive compound that is present in the wood of Cupressaceae. It is a well-known anti-microbial agent that was used as a remedy for
pulmonary gangrene, tuberculous fistula, chemotherapeutic resistant pulmonary tuberculosis, decubitus ulcers, and periodontal diseases in the 19th century. In the 20th century hinokitiol is applied more frequently in daily living products, such as cosmetics (Choi et al., 2006), mouth cleaning gel (Iha, Suzuki, Yoneda, Takeshita, & Hirofuji, 2013), and food additive. In addition, it is a functional food additive for preservation purpose, including food processing, extending shelf life of fruit (Saya
Hinokitiol 用於食物保存、抗 菌、防蟲各別描述與引用文獻
Okumura, 2011; Sholberg & Shimizu, 1991). , and there is no obvious toxic effect during long term oral feeding in rat (Cho et al., 2011; Ema, Harazono, Fujii, & Kawashima, 2004). In addition to anti-microbial activity, hinokitiol is reported to exhibit antiplatelete and anati-cancer effects (Eduardo Fuentes, 2014; Lin et al., 2013).
Hinokitiol has also been reported to exhibit anti-cancer properties in a wide range of tumour cells. The androgen/androgen receptor-mediated cell growth and androgen-stimulated DNA synthesis are inhibited by hinokitiol in prostate cancer cells (S. Liu & Yamauchi, 2006). Hinokitiol suppresses melanoma cell growth by inhibiting the S-phase kinase-associated protein 2 (SKP2) and pRb expression, and impairs cdk2 function to cause G1-phase cell cycle arrest ((S. Liu & Yamauchi, 2009). In colon cancer, hinokitiol activates p21 expression and suppresses cyclin A, cyclin E, and cdk2 expression to cause S-phase cell cycle arrest and induce cell apoptosis through the caspase 9 signaling cascade (Lee et al., 2013). Hinokitiol exhibits specific cytotoxicity to oral cancer cell lines compared to normal oral keratinocytes and fibroblasts, and exhibits anti-microbial activity to oral disease pathogens (Y.H. Shih et al., 2013; Yasumoto et al., 2004).
The oral cavity is a warm, humid, and hypoxic environment, in which many common pathogens flourish. The patient receiving chemotherapy for cancer is highly susceptible to infection, and infection accounts for approximately 70% of patient
fatalities (McElroy, 1984). In head and neck surgery, surgical site infection (SSI) is common in patients with OSCC because surgery is the main therapeutic procedure (Ma et al., 2012). Using antibiotic mouthwash is effective in improving postoperative complications after head and neck surgery (Jones, Kaulbach, Nichter, Edlich, & Cantrell, 1989). The application of hinokitiol in oral cancer therapy may exhibit both anti-cancer and anti-SSI effects.
However, the mechanism of hinokitiol in suppressing OSCC cell growth remains unclear. The aim of this study was to investigate the mechanism of hinokitiol in suppressing OSCC cell growth. This study reported the efficacy of hinokitiol in suppressing OSCC cell growth by pan-histone suppression in vitro and in vivo, and exhibited low cytotoxicity to normal cells. Hinokitiol suppressed pan-histone transcription and caused defects in the chromatin assembly, caused G1/S-phase cell cycle arrest, and induced cell apoptosis in OSCC cells.
2 Materials and Methods 2.1 Cell lines and reagents
The normal gingival epithelium cell line (S-G) and OSCC cell lines (Ca9-22, HSC-3, OC3, OEC-M1, SAS and SCC4) culture method were described in previous studies (Chang et al., 2010; C. J. Liu et al., 2014). The medium contained 10% fetal bovine
serum (FBS) (
Gibco® | Life Technologies
, Waltham, MA,USAGibco) and 1% antibiotic-antibiotics ((Gibco® | Life Technologies, Waltham, MA, USAGibco). Normal human oral keratinocytes and fibroblasts (NHOK and NHOF) were primarily cultured from oral tissues, and the method was described in previous studies (Y.H. Shih et al., 2013) (Shih et al., 2014). The institutional review board approved sampling of normal oral tissues (DMR98IRB-158). All cells were cultured under standard conditions. Hinokitiol (β-thujaplicin) (PubChem CID:24870612) and chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).
2.2 Cell viability assay
A total of 1×104 cells were seeded in a 96-well plate per well and treated with hinokitiol for 24-h, and the dosage were 2 fold serial diluted from 800 µM to 6.25 µM.
The cells were washed with PBS then added 200 µL fresh prepared 1 mg/mL [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) in serum free medium. After reacting with MTT for 4-h, cells were washed with PBS, and added 50 μL DMSO to dissolve the blue crystal in cells. The signal detection performed at OD570 by VersaMaxTM ELISA microplate reader (Molecular Devices, Sunnyvale, CA, USA) (Y.
H. Shih et al., 2013). 2.3. Cell cycle detection
Cells were grown to 70% confluence in 60-mm dishes and were treated with hinokitiol for 24-h. The cells were then harvested and fixed with 70% ethanol at -20 oC overnight and stained with 1.0 mL of propidium iodide (PI)/Triton X-100 reagent at room temperature for 30 min. The PI/Triton X-100 reagent was composed of 0.1% Triton X-100, 0.2 mg/mL RNase A, and 20 μg/mL PI. The cells were filtered through 35 μm nylon mesh to remove clumps before analysis and the cell cycles were detected using a FACSCanto flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA).
2.4 Cell apoptosis detection
The apoptotic cells were labeled using the FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen™, Franklin Lakes, New Jersey, USA), and the procedure was performed according to the vendor's protocol (Hsia et al., 2015). Cells were grown to
The cells were then harvested and stained with 5 μL of PI and 5 μL of FITC Annexin V at room temperature for 15-min and filtered through 35-μm nylon mesh to remove clumps before analysis. The cell apoptosis percentage was determined using a FACSCanto flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA). 2.5 Microarray gene analysis and reverse transcription quantitative PCR
HSC-3 cells were grown to 70% confluence in 100-mm dishes and were treated with 6.25 and 12.5 µM hinokitiol for 24-h. The total RNA was extracted using TRI Reagent® (Molecular Research Center, Inc. Cincinnati, OH, USA). Total RNA reverse transcription was performed using random primer and the cDNA were screened using an Affymetrix GeneChip Human Gene 1.0 ST array. The reverse transcription-quantitative PCR (RT-qPCR) was performed using SYBR Green system to confirm the results of microarray, and GAPDH expression was used as the internal control. The fluorescence signal was detected using an Applied Biosystems 7900HT (Life TechnologiesTM, Waltham, MA, USA). The data were analyzed using SDS2.2 software (Life TechnologiesTM, Waltham, MA, USA). The PCR primer sequences are listed in the Supporting Information Table S1.
2.6 Western blot analysis
Western blot was performed using 50 µg of total protein and the procedure was used by a previous study (Y.H. Shih et al., 2013). Depending on protein size, protein was resolved by 7.5–12.5% polyacrylamide gel. GAPDH was used as the internal
control for total lysate and gamma-tubulin was used as the internal control for nuclear lysate. The nuclear protein was purified using the ProteoJET™ Cytoplasmic and Nuclear Protein Extraction Kit (Fermentas, Life TechnologiesTM, Waltham, MA, USA). The primary antibodies used in this study are listed in the Supporting Information Table S2. A 1:2000 dilution of mouse and rabbit secondary antibodies (GeneTex, Irvine, CA, USA) was used. The signal was detected using the SuperSignal West Femto Chemiluminescent Substrate (Thermo Scientific Life TechnologiesTM, Waltham, MA, USA) and Fusion Solo (Vilber Lourmat, Marne-la-Vallée Cedex, France).
2.7 Micrococcal nuclease assay
Micrococcal nuclease assay (MNase assay) was performed using the EZ nucleosomal DNA prep kit (Zymo Research, Irvine, CA, U.S.A.) and the procedure was performed according to the vendor's protocol. Cells were grown to 70% confluence in 100-mm dishes and were treated with hinokitiol for 24-h. The cells were harvested, and the chromatin extracted from 1×106 cells was digested with 0.5 U micrococcal nuclease (MNase) in 25 oC water bath for 5 to 20-min. The genomic DNA was used as a histone-free digestion control and the uncut chromatin was used as a loading control. The 800 μg DNA separated in 2% agarose gel, and stained with
ethidium bromide (EtBr). The DNA signals on the gel were visualized with UV light.
2.8 Chromatin immunoprecipitation PCR
Chromatin immunoprecipitation (CHIP) was performed using the MAGnifyTM Chromatin Immunoprecipitation System (InvitrogenTM, Life TechnologiesTM, Waltham, MA, USA) and the procedure was performed according to the vendor's protocol. Cells were grown to 70% confluence in 100-mm dishes, and were treated with hinokitiol for 24-h. The cell lysate were sonicated using a Misonix S-4000 sonicator (Cole-Parmer, Taipei, Taiwan) to obtain 200-500 bp of chromatin fragments. Immunoprecipitation was performed using anti-FLASH (Bethyl Laboratories, Inc. Montgomery, TX, USA), anti-NPAT ((Bethyl Laboratories, Inc. Montgomery, TX, USA), and rabbit IgG antibody. The amplification region and primer design were referred to previous study (Table S1) (Zhao et al., 2000), and the amplicons of human alfa satellite DNA was used as internal control. The amplicons were resolved in 2% agarose gel and and stained with EtBr. The DNA signals on the gel were visualized with UV light.
2.9 Promoter activity assay
The H2B/r (includes nucleotides −120 to +1 of the H2B/r promoter), H3/a (contains the sequence of 300 nucleotides upstream from the initiation ATG of histone H3/a), and H4/e (includes nucleotides −113 to +8 of the H4/e promoter) promoters were
constructed as in a previous study, and these promoters are regulated by FLASH and NPAT (Barcaroli et al., 2006; Zhao et al., 2000). Each promoter was cloned into basic plasmid between the XhoI and NcoI restriction sites to obtain pGL3-H2B/r, pGL3-H3/a, and pGL3-H4/e, respectively. The pGL4.73 plasmid was used as a control for achieving transfection efficiency. The transfection of the promoters to the cells was performed using Lipofectamine 2000 in antibiotic-free culture medium for 4-h, and replaced with regular culture medium for another 20-h. Plasmid-containing cells were cultured with hinokitiol containing 10% FBS medium for 24-h. Luciferase activity was detected using the Duo-Glo Luciferase Assay System kit (Promega Corporation, Madison, WI, USA) and SpectraMaxL (Molecular Devices, Sunnyvale, CA, USA). The procedure was performed according to the vendor's protocol.
2.10 Migration assay
OSCC cells were seeded in a 6-well plate and cultured with medium containing 10% FBS to 100% confluence. Using a sterile 200 μL plastic pipette tip to produce the gap. Floating cells were removed by washing twice with PBS. The culture medium was replaced by serum-free medium containing 0.75-6.25 μM hinokitiol. The cells were
cultured in a 37 oC incubator, observed under a 40
×
magnification microscope andmigrated to the wound area was expressed as the percentage of cell migration distance (Chen et al., 2011).
2.11 Colony formation assay
OSCC cells were seeded in a 6-well plate with 5×103 cells per well. The cells were treated with 0.75-6.25 μM hinokitiol for 24-h and replaced with the culture medium without hinokitiol every 3-d. On the fifth day, the colonies were fixed with 95% alcohol and stained with haematoxylin. The number of colonies was counted under a 40
×
X magnification microscope by using 5 random views. A colony was defined as a group of at least 30 cells.2.12 Anchorage-independent growth assay
Anchorage-independent growth assays were performed in accordance with the procedures used in a previous study (Shieh et al., 2007). OSCC cells were seeded in a 6-well soft agar plate with 5×104 cells per well, and treated with 0, 3.125, 6.25 μM hinokitiol. Another 200 μL fresh medium was supplied on third day. The colonies were harvested on the seventh day and stained with 0.5% crystal violet. The number of spheres was counted under a 40
×
X magnification microscope by choosing 5 random views. The spheres with a diameter of more than 50 μm were counted and the average number of colonies was subsequently calculated.The xenograft tumourigenesis was performed referred to affidavit of approval of animal use protocol (100-243-N). The experiment procedure was refer to previous study (Wu et al., 2014). BALB/cAnN-Foxn1 female mice (purchased from Taiwan National Laboratory Animal Center, 4-6 wk-old, and weighed 16-18 g were used. The mice were kept in a pathogen-free animal unit. The room temperature and relative humidity were controlled at 25 °C and 50%, respectively. The unit provided a 12-h light/dark cycle. Four mice were used for each experimental group. A total of 5×106 HSC-3 cells were suspended in 100 µL of sterile PBS and inoculated to the right flank of the mice. The control mice were fed with 5% alcohol and the test mice were fed with 10 mg/kg hinokitiol in 5% alcohol by gavage twice a week, and the tumour size and body weight were measured. The decision of feeding dosage was referred to previous studies (Ema, Harazono, Fujii, & Kawashima, 2004; S. Liu & Yamauchi, 2009). Tumour volume was calculated using the formula 0.5×a×b2, in which a and b are the long and short diameters of the tumour, respectively. The tumour weight was measured after sacrificing the mice.
2.14 Immunohistochemistry (IHC)
Standard operating procedures for total histone H3 (Cell Signaling Technology, Danvers, MA, USA), total histone H4 (Santa Cruz Biotechnology, Dallas, Texas,
USA), cleaved caspase3 (Cell Signaling, Danvers, MA, USA), and proliferating cell nuclear antigen (PCNA) (GeneTex, Irvine, CA, USA) were established using formalin-fixed paraffin-embedded sections (2μm thick) of mice xenograft tumour. Deparaffinization, rehydration, and antigen retrieval procedure were the same with previous study (Wang, Chang, Ho, Wu, & Chen, 2012). The slides were immersed in 3% hydrogen peroxide for 10 min to suppress endogenous peroxidase activity. After triple washing with 1×X PBS, sections were subsequently exposed to primary antibody for 1 hour at room temperature. The slides were washed 3 times with 1 ×X
PBS then incubated with biotinylated secondary antibody (Dako, Glostrup, Denmark), the peroxidase activity was detected with 3, 3-diaminobenzidine (DAB) substrate chromogen (Dako, Glostrup, Denmark), and counterstained with haematoxylin.
2.15 Statistical analysis
The statistical data was derived from 3 independent experiments. An unpaired t testwas conducted using Prism 5.0 (GraphPad Software, Inc. La Jolla, CA, USA) to examine the statistically significant difference. Differences between the variants were considered significant when P < 0.05.
3. Results and discussion
3.1 Hinokitiol exhibited specific cytotoxicity to OSCC cells.
The cell viability of first 24-h treatment significantly decreased in moderate or high malignant cells (Ca9-22, HSC-3, OEC-M1, SAS and SCC4) compared to low malignant cell (OC3) and normal cells (NHOK, NHOF, and S-G) (Fig. 1A). Extended hinokitiol treatment for 48-h and 72-h in NHOK, HSC-3, OEC-M1, and SAS, the cell viability were decreasing in time and dose dependent manner. The half maximal inhibitory concentration (IC50) of NHOK in first 24-h of treatment was more than 800
µM, the IC50 of HSC-3 was less than 100 µM, OEC-M1 and SAS were 200 µM (Fig.
1B). Hinokitiol exhibited specific cytotoxicity to OSCC cells than normal oral cells, and exhibited the most cytotoxicity to HSC-3 cells among OSCC cell lines.
3.2 Hinokitiol induced cell cycle arrest in G1 or G1/S phase.
Two doses lower than the IC50 of each cell line were used in cell cycle distribution
analysis. NHOK and NHOF were treated with 100 and 400 µM hinokitiol, HSC-3 was treated with 6.25 and 12.5 µM hinokitiol, and SAS were treated with 50 and 100 µM hinokitiol. The histograms of PI stained cell cycle distribution were shown in figure 2A., and the distribution percentage of each phase were shown in figure 2B. The data showed that hinokitiol induced G1 phase arrest in NHOK, NHOF, and SAS, and induced G1/S phase arrest in HSC-3 (Fig. 2A, 2B). In cell cycle checkpoint protein detection, NHOK was treated with 50 and 100 μM hinokitiol as a control of check
point protein expression. The G1/S checkpoint protein cyclin A, cyclin E, cdk2 were not decreased by increasing dose of hinokitiol in NHOK, HSC-3, and SAS. The p53 and p21 were increased in NHOK and SAS, but not in HSC-3. The total pRb were increased, and p-pRb (phosphorylation form of pRb) were decreased in NHOK, HSC-3, and SAS (Fig. 2C). We inferred that hinokitiol decreased the pRb phosphorylation to caused the cell cycle arrest in G1 or G1/S phase.
3.3 Hinokitiol induced apoptosis via extrinsic pathway in OSCC cell lines.
The cell shape of HSC-3 was getting larger and flatter after hinokitiol treatment for 24-h (Fig. S1A and S1B). The senescence, autophagy and DNA damage biomarkers: senescence-associated beta-galactosidase (SA-β-gal), LC3-II / LC3-I ratio, and γ-H2A.X were detected in hinokitiol-treated OSCC cells. The SA-β-gal was not expressed and the LC3-II / LC3-I ratio was not increased in hinokitiol-treated HSC-3 cells in 24-h of treatment (S-Fig. 1A and 1C). In DNA damage test, the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay showed negative results (Fig. S2A), and the γ-H2A.X was down-regulated in hinokitiol-treated SAS cells in 24-h of treatment (Fig. S2B). The possibility of hinokitiol induced senescence, autophagy, and DNA damage in the first 24-h of treatment were excluded. To address whether or not hinokitiol induce OSCC cells necrosis or program cell death, the
hinokitiol-treated OSCC cells were double stained with PI and annexin-V-FITC, and analyzed by flow cytometry. Figure 3A and 3B were dot plot and apoptotic cells percentage (Q2+Q4), respectively. After 6.25-25 µM hinokitiol treatments for 24-h and 48-h, the apoptosis percentage of HSC-3 revealed no significant difference (Fig. 3B left). Additional increasing 50 µM and 100 µM hinokitiol treatment for 24-h and 48-h in both HSC-3 and SAS, the population of apoptotic cells (Fig. 3A and 3B), cleaved caspase-8, cleaved caspase-3, and cleaved poly ADP-ribose polymerase (PARP) were increased (Fig. 3C). The cleaved caspase-9 was undetectable (data not shown). We inferred that hinokitiol effectively induced OSCC cell apoptosis via extrinsic pathway in doses of 50 and 100 μM in 48-h of treatment.
3.4 Hinokitiol down-regulated pan-histone mRNA and protein expression to impair the chromatin assembly in OSCC cells
Since hinokitiol exhibited more cytotoxicity to HSC-3, HSC-3 was treated with 6.25 and 12.5 µM hinokitiol, and performed the microarray analysis to screen the genes that regulated by hinokitiol. Twenty genes were identified, including 9 genes that exhibited an increase in expression by 2 fold, and 11 genes that exhibited a decrease in expression by 2 fold. Ten replication-dependent histone genes on chromosome 6 were included in the down regulation group (Fig. 4A). HISTH2BM and
HISTH3J mRNA expression were chosen to verify the reproducibility of the cDNA
microarray data. Both semi-quantitative RT-PCR (Fig. 4A up panel) and RT-qPCR (Fig. 4B) indicated that HISTH2BM and HISTH3J genes were down regulated and the results were consistent with the microarray data. Furthermore, the microarray raw data exhibited a trend of pan-histone mRNA suppression caused by hinokitiol (Supporting Information Table S3). The pan-histone protein expression (H1, H2A, H2B, H3, and H4) of HSC-3 and SAS were detected. Except for the H1 and H2A in both cell lines, hinokitiol suppressed total H2B, H3, and H4 histone protein expression in the OSCC cells after hinokitiol treatment (Fig. 4C).
Histone plays a crucial role in chromatin assembly especially for histone H4 N-terminal tail. We inferred that the pan-histone suppression that caused by hinokitiol could affect the chromatin to be more sensitive to micrococcal nuclease (MNase). We performed MNase assay to check the chromatin folding status in hinokitiol-treated HSC-3 and SAS cells. We use the genomic DNA and MNase free chromatin as histone free digestion control and loading control, respectively. Both chromatin of hinokitiol-treated HSC-3 and SAS cells were digested to small fragments by MNase and showed ladders on the gel. The increasing dosage of hinokitiol caused the chromatin of hinokitiol-treated OSCC cells were easy to be digested into small fragments, and the results were coordinated in two different durations of enzyme digestion. We inferred
that hinokitiol causes defects in chromatin assembly in OSCC cells (Fig. 4D).
3.5 Hinokitiol suppressed histone mRNA expression through down regulated transcription instead of posttranscriptional modification
FLASH and NPAT are histone gene transcription activators, and stem-loop binding protein (SLBP) is a stabilizer of histone mRNA. NPAT and FLASH but not SLBP expression were suppressed in hinokitiol-treated HSC-3 and SAS cells (Fig. 5A). The well-studied histone promoter regions, which are regulated by FLASH and NPAT were used to perform CHIP PCR assay and the promoter activity assay. The CHIP PCR assay showed the bindings of both NPAT and FLASH to the H2B/r promoter were decreased in 6.25µM, but not in 12.5 µM in hinokitiol-treated HSC-3. The binding of NPAT was decreased and FLASH was increased to H3/a promoter in 6.25µM hinokitiol-treated HSC-3 (Fig. 5B, top). The binding of NPAT to the H2B/r and H3/a promoters were not decreased in 50 µM and 100 µM hinokitiol-treated SAS cells, but the binding of FLASH to the H2B/r and H3/a promoters were clearly decreased after hinokitiol treatment (Fig. 5B, bottom). The bindings of both NPAT and FLASH to the H4/e promoter were decreased in both hinokitiol-treated HSC-3 and SAS (Figs. 5B). Hinokitiol slightly suppressed the H2B/r and H3/a promoter activities in HSC-3, but significantly suppressed the H2B/r, H3/a promoter activities in SAS,
and H4/e promoter activity in both HSC-3 and SAS cells (Fig. 5C).
According to these results, we inferred that hinokitiol suppressed pan-histone expression by decreasing NPAT and FLASH expression, then, the binding of NPAT or FLASH to the histone promoters were decreased. As a result, the histone promoter activities were down-regulated, and the histone mRNA and protein expressions were decreased in both hinokitiol-treated HSC-3 and SAS cells.
3.6 Hinokitiol suppressed the malignant phenotypes of OSCC cells in vitro
The hinokitiol dosage that retained over 98% cell viability was used in malignant phenotype assay to eliminate the cytotoxic effect in OSCC cells. HSC-3 cells were treated with 0.75 and 1.5 μM hinokitiol, and the SAS and OEC-M1 cells were treated with 3.125 and 6.25 μM hinokitiol. The data indicated that hinokitiol suppressed the spheres formation (Fig. 6A), migration distance percentage (Fig. 6B, 6D), and colonies formation (Fig. 6C, 6E) in a dose dependent manner. Thus we inferred that a relative low dose hinokitiol effectively inhibited the malignancy of OSCC cells.
3.7 Hinokitiol suppressed xenograft tumourigenicity in vivo
The highly malignant OSCC cell line, HSC-3 was used in xenograft tumourigenicity to investigate the anti-tumour growth ability of hinokitiol in vivo. No significant differences were observed in body weight between the control and feeding 10 mg/kg
hinokitiol group during the experiment (Fig. 7A). The average tumour size exhibited by the mice in the hinokitiol group was smaller than that of the mice in the control group from Day 9, and the average tumour size of the hinokitiol group was smaller than the control group (1.348±1.029 cm3 and 3.167±1.175 cm3, P=0.0586) on Day 33
(Fig. 7B). The mice were sacrificed, and the tumours were isolated from the flank of the mice on day 33. The tumour weight of the test group were smaller than control group (0.332±0.213 g and 0.916±0.443 g, P<0.05) (Fig. 7C). The IHC pictures were taken in the same area on tissue section. The xenograft tumours showed that the total histone H3, total histone H4, cleaved caspase 3, and PCNA were decreased both in cytoplasm and nucleus of hinokitiol group (Fig. 7D). Thus, we inferred that hinokitiol effectively suppressed xenograft tumour growth and malignancy in vivo.
4. Discussion
Hinokitiol could significantly induced annexin-V expression in 100 μM hinokitiol-treated SAS in 24-h of treatment (Fig. 3A bottom, Fig. 3B right). The result was double confirmed by TUNEL assay. However, the TUNEL assay showed negative result in 100 μM hinokitiol-treated SAS in 24-h of treatment (S Fig. 2A). Terminal deoxynucleotidyl transferase (TdT) catalyzing reaction needs cobalt, Mg2+, and Mn2+
Molloy, Steed, & Wright, 2001). It chelates with metal ions such as Zn2+ and Fe3+ (Ido
et al., 1999), and could inhibit the metalloprotease or polyphenol oxidase activity that needs metal ion as catalyzing cofactor (Inamori et al., 1999; Saya Okumura, 2011). We inferred that the negative result of TUNEL assay in 100 μM hinokitiol-treated SAS in 24-h of treatment might be due to that hinokitiol chelated the essential metal cofactors to inhibit the catalyzing reaction.
During the cell cycle, G1 checkpoint control mechanism ensures that everything is ready for DNA synthesis, and DNA replication occurs in S phase. pRb is a tumour suppressor protein that de-phosphorylated in G1-S phase and binds to E2F family to prevent gene transcription of damaged DNA (Gao et al., 2003; Su et al., 2004). In addition, cyclin A, cyclin E, CDK2 expression were critical for G1-S transition. According to previous studies, pan-histone repression causes defect in chromatin assembly and DNA damage without prolong inhibition the cyclin A, cyclin E, CDK2 expression (Dorigo, Schalch, Bystricky, & Richmond, 2003; Nelson et al., 2002; Simpson, 1976; Yu, Olsen, Zhang, Boeke, & Bi, 2011). In the present data, hinokitiol arrested cell cycle in G1 and S phase without suppressing cyclin A, cyclin E, CDK2 expression, and reduced pRB phosphorylation in cells. We inferred that cell cycle arrest and cell apoptosis in hinokitiol-treated OSCC cells were directly caused by the suppression of histone expression.
p53 is a tumour suppressor protein and it activates cell apoptosis, DNA damage repair and genomic stability maintenance (Sengupta & Harris, 2005; Ye et al., 2003). The mutation of p53 in the OSCC cells occurs frequently. The p53 TAAG insertion mutation occurs between codon 305 and 306 in the HSC-3 cells and a GAG to TAG point mutation at codon 336 occurs in the SAS cells (Yoko Kamiya, 2005). The hinokitiol-treated HSC-3 cells failed to activate the p53 and p21 signaling cascade that might cause HSC-3 to be more susceptible to hinokitiol (Fig. 2C).
The NPAT expression is suppressed by p53 and p21 signaling cascade activation, and the histone protein expression decreases during DNA damage (Su et al., 2004). In present data, p53 and p21 were not activated by hinokitiol in the HSC-3 cells (Fig. 2C), and the NPAT was down regulated (Fig. 5A). We inferred that the pan-histone suppression in hinokitiol-treated OSCC cells might be p53 and p21 cascade independent. In addition, NPAT expression was activated by E2F transcription factors, and pRb bound to the E2F family to prevent gene transcription from damaged DNA (Gao et al., 2003; Su et al., 2004). In the present study, it was suggested that hinokitiol suppressed pan-histone expression and increased pRb expression may reduce NPAT transcription, which accelerated the decrease of histone expression.
Replication-dependent histone genes are typically organized in clusters and the transcription of these genes is affected by distinct subtype-specific consensus
sequences (SSCSs)-a short consensus sequence in the proximal region of the histone promoter (Heintz, 1991; Osley, 1991). NPAT activates the transcription of several histone genes in the promoter region and the activation of H2B and H4 transcription is mediated through SSCSs. Histone H4 SSCSs are highly conserved among human histone H4 promoters (Barcaroli et al., 2006; Zhao et al., 2000). This explained that hinokitiol suppressed NPAT expression (Fig. 5A) cause dramatically decreasing of total histone H4 protein expression and promoter activity in HSC-3 and SAS (Fig. 4C). Cleaved caspase 3 activates the apoptotic cascade and has been characterized as direct link to cell apoptosis. It is present in cytosol and mitochondria in cells (Samali, Zhivotovsky, Jones, & Orrenius, 1998), and expressed higher in cytoplasm of less well-differentiated tumour (Hague et al., 2004). Caspase-3 has been identified in numerous benign or malignant neoplasms, including breast cancer (O'Donovan et al., 2003), gastric cancer (Sun et al., 2006), and head and neck cancer, and expressed more in head and neck malignant tumours (Chrysomali, Nikitakis, Tosios, Sauk, & Papanicolaou, 2003; Hague et al., 2004; Parenti et al., 2006), which is related to tumour progression and poor differentiation. It is conflicted with the idea that caspase 3 is reduced in malignancy. There are references of nonapoptotic roles of caspase 3 including stimulation of differentiation (Szymczyk, Freeman, Adams, Srinivas, & Steinbeck, 2006), dedifferentiation (Li et al., 2010), T cell proliferation (Kennedy,
Kataoka, Tschopp, & Budd, 1999), and is involved in tumour cell development and repopulation (Huang et al., 2011). Whether or not the caspase 3 play nonapoptotic role in head and neck malignant tumour is unknown. In the present study, hinokitiol reduced the xenograft tumour size, and the IHC data showed reduced cleaved caspase 3 expression in both cytoplasm and nucleus of hinokitiol group. It provides the evidence that hinokitiol effectively reduced the malignant phenotypes not only in vitro but also in vivo. We inferred that hinokitiol might be a potential compound of therapeutic approach for caspase 3 overexpression cancer.
Hinokitiol is used as a food additive for preservative purposes, and the suggested concentration is 125 ppm (762 µM) per day, which is higher than the dose used in the present study. No known toxic effect from chronic exposure to hinokitiol has been reported (Cho et al., 2011). In addition, the hinokitiol containing oral hygiene products have been used to prevent dental caries and periodontal inflammation. Direct role for hinokitiol suppressing pan-histone expression in OSCC cells has not been described before to our knowledge. Except being a remedy for oral cancer, combining the anti-microbial and anti-cancer activities, hinokitiol might be applied to reduce SSI effects and cancer recurrence in postoperative wound care.
In conclusion, hinokitiol showed the efficacy to suppress OSCC cells growth in
investigate the potential effect as a chemoprevention compound against OSCC pre-cancerous lesion.
3.1 Hinokitiol exhibited specific cytotoxicity to OSCC cells, and induced cell cycle arrest in G1 or G1/S phase.
The cell viability of first 24-h treatment significantly decreased in moderate or high malignant cells (Ca9-22, HSC-3, OEC-M1, SAS and SCC4) compared to low malignant cell (OC3) and normal cells (NHOK, NHOF, and S-G) (Fig. 1A). Extended hinokitiol treatment for 48-h and 72-h in NHOK, HSC-3, OEC-M1, and SAS, the cell viability were decreasing in time and dose dependent manner. The half maximal inhibitory concentration (IC50) of NHOK in first 24-h of treatment was more than 800
µM, the IC50 of HSC-3 was less than 100 µM, OEC-M1 and SAS were 200 µM (Fig.
1B). Hinokitiol exhibited specific cytotoxicity to OSCC cells than normal oral cells, and exhibited the most cytotoxicity to HSC-3 cells among OSCC cell lines.
Two doses lower than the IC50 of each cell line were used in cell cycle distribution
analysis. NHOK and NHOF were treated with 100 and 400 µM hinokitiol, HSC-3 was treated with 6.25 and 12.5 µM hinokitiol, and SAS were treated with 50 and 100 µM hinokitiol. The histograms of PI stained cell cycle distribution were shown in figure 1C., and the distribution percentage of each phase were shown in figure 1D. The data showed that hinokitiol induced G1 phase arrest in NHOK, NHOF, and SAS, and
induced G1/S phase arrest in HSC-3 (Fig. 1C, 1D). In cell cycle checkpoint protein detection, NHOK was treated with 50 and 100 μM hinokitiol as a control of check point protein expression. The G1/S checkpoint protein cyclin A, cyclin E, cdk2 were not decreased by increasing dose of hinokitiol in NHOK, HSC-3, and SAS. The p53 and p21 were increased in NHOK and SAS, but not in HSC-3. The total pRb were increased, and p-pRb (phosphorylation form of pRb) were decreased in NHOK, HSC-3, and SAS (Fig. 1E).
p53 is a tumour suppressor protein and it activates cell apoptosis, DNA damage repair and genomic stability maintenance (Sengupta & Harris, 2005; Ye et al., 2003). The mutation of p53 in the OSCC cells occurs frequently. The p53 TAAG insertion mutation occurs between codon 305 and 306 in the HSC-3 cells and a GAG to TAG point mutation at codon 336 occurs in the SAS cells (Yoko Kamiya, 2005). The hinokitiol-treated HSC-3 cells failed to activate the p53 and p21 signaling cascade that might cause HSC-3 to be more susceptible to hinokitiol (Fig. 1A, 1E).
During the cell cycle, G1 checkpoint control mechanism ensures that everything is ready for DNA synthesis, and then DNA replication occurs in S phase. pRb is a tumour suppressor protein that de-phosphorylated in G1-S phase and binds to E2F family to prevent gene transcription of damaged DNA (Gao et al., 2003; Su et al., 2004). In addition, cyclin A, cyclin E, CDK2 expression were critical for G1-S
transition. According to previous studies, pan-histone repression causes defect in chromatin assembly and DNA damage without prolong inhibition the cyclin A, cyclin E, CDK2 expression (Dorigo, Schalch, Bystricky, & Richmond, 2003; Nelson et al., 2002; Simpson, 1976; Yu, Olsen, Zhang, Boeke, & Bi, 2011). In the present data, hinokitiol arrested cell cycle in G1 and S phase without suppressing cyclin A, cyclin E, CDK2 expression, and reduced pRB phosphorylation in cells. We inferred that cell cycle arrest and cell apoptosis in hinokitiol-treated OSCC cells were directly caused by the suppression of histone expression.
3.2 Hinokitiol induced apoptosis via extrinsic pathway in OSCC cell lines.
The cell shape of HSC-3 was getting larger and flatter after hinokitiol treatment for 24-h (Fig. S1A and S1B). The senescence, autophagy and DNA damage biomarkers: senescence-associated beta-galactosidase (SA-β-gal), LC3-II / LC3-I ratio, and γ-H2A.X were detected in hinokitiol-treated OSCC cells. The SA-β-gal was not expressed and the LC3-II / LC3-I ratio was not increased in hinokitiol-treated HSC-3 cells in 24-h of treatment (S-Fig. 1A and 1C). In DNA damage test, the γ-H2A.X was down-regulated in hinokitiol-treated SAS cells in 24-h of treatment (Fig. S2B). The possibility of hinokitiol induced senescence, autophagy, and DNA damage in the first 24-h of treatment were excluded. To address whether or not hinokitiol induce OSCC cells necrosis or program cell death, the hinokitiol-treated OSCC cells were double
stained with PI and annexin-V-FITC, and analyzed by flow cytometry. Figure 2A and 2B were dot plot and apoptotic cells percentage (Q2+Q4), respectively. After 6.25-12.5 µM hinokitiol treatments for 24-h and 48-h, the apoptosis percentage of HSC-3 revealed no significant difference (Fig. 2B left). Additional increasing the dose to 50 µM and 100 µM hinokitiol and treatment for 24-h and 48-h in both HSC-3 and SAS, the population of apoptotic cells (Fig. 2A and 2B), cleaved 8, cleaved caspase-3, and cleaved poly ADP-ribose polymerase (PARP) were increased (Fig. 2C). The cleaved caspase-9 was undetectable (data not shown). We inferred that hinokitiol effectively induced OSCC cell apoptosis via extrinsic pathway in doses of 50 and 100 μM in 48-h of treatment.
Hinokitiol could significantly induced annexin-V expression in 100 μM hinokitiol-treated SAS in 24-h of treatment (Fig. 2A bottom, Fig. 2B right). The result was double confirmed by TUNEL assay. However, the TUNEL assay showed negative result in 100 μM hinokitiol-treated SAS in 24-h of treatment (S Fig. 2A). Terminal deoxynucleotidyl transferase (TdT) catalyzing reaction needs cobalt, Mg2+
, and Mn2+
administration in vitro. Hinokitiol is a well known metal chelator (Barret, Mahon, Molloy, Steed, & Wright, 2001). It chelates with metal ions such as Zn2+
and Fe3+
(Ido et al., 1999), and could inhibit the metalloprotease or polyphenol oxidase activity that needs metal ion as catalyzing cofactor (Inamori et al., 1999; Saya Okumura, 2011). We
inferred that the negative result of TUNEL assay in 100 μM hinokitiol-treated SAS in 24-h of treatment might be due to that hinokitiol chelated the essential metal cofactors to inhibit the catalyzing reaction.
3.3 Hinokitiol down-regulated pan-histone mRNA and protein expression to impair the chromatin assembly in OSCC cells
Since hinokitiol exhibited more cytotoxicity to HSC-3, HSC-3 was treated with 6.25 and 12.5 µM hinokitiol, and performed the microarray analysis to screen the genes that regulated by hinokitiol. Twenty genes were identified, including 9 genes that exhibited an increase in expression by 2 fold, and 11 genes that exhibited a decrease in expression by 2 fold. Ten replication-dependent histone genes on chromosome 6 were included in the down regulation group (Fig. 3A). HISTH2BM and
HISTH3J mRNA expression were chosen to verify the reproducibility of the cDNA
microarray data. Both semi-quantitative RT-PCR (Fig. 3A up panel) and RT-qPCR (Fig. 3B) indicated that HISTH2BM and HISTH3J genes were down regulated and the results were consistent with the microarray data. Furthermore, the microarray raw data exhibited a trend of pan-histone mRNA suppression caused by hinokitiol (Supporting Information Table S3). The pan-histone protein expression (H1, H2A, H2B, H3, and H4) of HSC-3 and SAS were detected. Except for the H1 and H2A in both cell lines, hinokitiol suppressed total H2B, H3, and H4 histone protein expression in the OSCC
cells after hinokitiol treatment (Fig. 3C).
Histone plays a crucial role in chromatin assembly especially for histone H4 N-terminal tail. We inferred that the pan-histone suppression that caused by hinokitiol could affect the chromatin to be more sensitive to micrococcal nuclease (MNase). We performed MNase assay to check the chromatin folding status in hinokitiol-treated HSC-3 and SAS cells. We use the genomic DNA and MNase free chromatin as histone free digestion control and loading control, respectively. Both chromatin of hinokitiol-treated HSC-3 and SAS cells were digested to small fragments by MNase and showed ladders on the gel. The increasing dosage of hinokitiol caused the chromatin of hinokitiol-treated OSCC cells were easy to be digested into small fragments, and the results were coordinated in two different durations of enzyme digestion. These data showed that hinokitiol causes defects in chromatin assembly in OSCC cells (Fig. 3D).
3.4 Hinokitiol suppressed histone mRNA expression through down regulated transcription instead of posttranscriptional modification
FLASH and NPAT are histone gene transcription activators, and stem-loop binding protein (SLBP) is a stabilizer of histone mRNA. NPAT and FLASH but not SLBP expression were suppressed in hinokitiol-treated HSC-3 and SAS cells (Fig. 4A). The well-studied histone promoter regions, which are regulated by FLASH and
CHIP PCR assay showed the bindings of both NPAT and FLASH to the H2B/r promoter were decreased in 6.25µM, but not in 12.5 µM in hinokitiol-treated HSC-3. The binding of NPAT was decreased and FLASH was increased to H3/a promoter in 6.25µM hinokitiol-treated HSC-3 (Fig. 4B, top). The binding of NPAT to the H2B/r and H3/a promoters were not decreased in 50 µM and 100 µM hinokitiol-treated SAS cells, but the binding of FLASH to the H2B/r and H3/a promoters were clearly decreased after hinokitiol treatment (Fig. 4B, bottom). The bindings of both NPAT and FLASH to the H4/e promoter were decreased in both hinokitiol-treated HSC-3 and SAS (Fig. 4B). Hinokitiol slightly suppressed the H2B/r and H3/a promoter activities in HSC-3, but significantly suppressed the H2B/r, H3/a promoter activities in SAS, and H4/e promoter activity in both HSC-3 and SAS cells (Fig. 4C).
The NPAT expression is suppressed by p53 and p21 signaling cascade activation, and the histone protein expression decreases during DNA damage (Su et al., 2004). In present data, p53 and p21 were not activated by hinokitiol in the HSC-3 cells (Fig. 1E), and the NPAT was down regulated (Fig. 4A). We inferred that the pan-histone suppression in hinokitiol-treated OSCC cells might be p53 and p21 cascade independent. In addition, NPAT expression was activated by E2F transcription factors, and pRb bound to the E2F family to prevent gene transcription from damaged DNA (Gao et al., 2003; Su et al., 2004). In the present study, it was suggested that hinokitiol
increased pRb expression may reduce NPAT transcription, which accelerated the decrease of histone expression.
Replication-dependent histone genes are typically organized in clusters and the transcription of these genes is affected by distinct subtype-specific consensus sequences (SSCSs)-a short consensus sequence in the proximal region of the histone promoter (Heintz, 1991; Osley, 1991). NPAT activates the transcription of several histone genes in the promoter region and the activation of H2B and H4 transcription is mediated through SSCSs. Histone H4 SSCSs are highly conserved among human histone H4 promoters (Barcaroli et al., 2006; Zhao et al., 2000). This explained that hinokitiol suppressed NPAT expression (Fig. 4A) cause dramatically decreasing of total histone H4 protein expression and promoter activity in HSC-3 and SAS (Fig. 4C).
3.5 Hinokitiol suppressed the malignant phenotypes of OSCC cells in vitro
The hinokitiol dosage that retained over 98% cell viability was used in malignant phenotype assay to eliminate the cytotoxic effect in OSCC cells. HSC-3 cells were treated with 0.75 and 1.5 μM hinokitiol, and the SAS and OEC-M1 cells were treated with 3.125 and 6.25 μM hinokitiol. The data indicated that hinokitiol suppressed the spheres formation of OEC-M1 and SAS (Fig. 5A), migration distance percentage of SAS (Fig. 5B), and colonies formation of HSC-3, OEC-M1, and SAS (Fig. 5C) in a dose dependent manner. Thus we inferred that a relative low dose hinokitiol effectively
inhibited the malignancy of OSCC cells.
3.6 Hinokitiol suppressed xenograft tumourigenicity in vivo
The highly malignant OSCC cell line, HSC-3 was used in xenograft tumourigenicity to investigate the anti-tumour growth ability of hinokitiol in vivo. No significant differences were observed in body weight between the control and feeding 10 mg/kg hinokitiol group during the experiment (Fig. 6A). The average tumour size exhibited by the mice in the hinokitiol group was smaller than that of the mice in the control group from Day 9, and the average tumour size of the hinokitiol group was smaller than the control group (1.348±1.029 cm3
and 3.167±1.175 cm3
, P=0.0586) on Day 33 (Fig. 6B). The mice were sacrificed, and the tumours were isolated from the flank of the mice on day 33. The tumour weight of the hinokitiol group were smaller than control group (0.332±0.213 g and 0.916±0.443 g, P<0.05) (Fig. 6C). The IHC pictures were taken in the same area on tissue section. The xenograft tumours showed that the total histone H3, total histone H4, cleaved caspase 3, and PCNA were decreased both in cytoplasm and nucleus of hinokitiol group (Fig. 6D).
Cleaved caspase 3 activates the apoptotic cascade and has been characterized as direct link to cell apoptosis. It is present in cytosol and mitochondria in cells (Samali, Zhivotovsky, Jones, & Orrenius, 1998), and expressed higher in cytoplasm of less well-differentiated tumour (Hague et al., 2004). Caspase-3 has been identified in
numerous benign or malignant neoplasm, including breast cancer (O'Donovan et al., 2003), gastric cancer (Sun et al., 2006), and head and neck cancer, and expressed more in head and neck malignant tumour (Chrysomali, Nikitakis, Tosios, Sauk, & Papanicolaou, 2003; Hague et al., 2004; Parenti et al., 2006), which is related to tumour progression and poor differentiation. It is conflicted with the idea that caspase 3 is reduced in malignancy. There are references of nonapoptotic roles of caspase 3 including stimulation of differentiation (Szymczyk, Freeman, Adams, Srinivas, & Steinbeck, 2006), dedifferentiation (Li et al., 2010), T cell proliferation (Kennedy, Kataoka, Tschopp, & Budd, 1999), and is involved in tumour cell development and repopulation (Huang et al., 2011). Whether or not the caspase 3 play nonapoptotic role in head and neck malignant tumour is unknown. In the present study, hinokitiol reduced the xenograft tumour size, and the IHC data showed reduced cleaved caspase 3 expression in both cytoplasm and nucleus of hinokitiol group. It provides the evidence that hinokitiol effectively reduced the malignant phenotypes not only in vitro but also in vivo. We inferred that hinokitiol might be a potential compound of therapeutic approach for caspase 3 overexpression cancer.
Conclusions
expression, then, the binding of NPAT or FLASH to the histone promoters were decreased. As a result, the histone promoter activities were down-regulated, and the histone mRNA and protein expressions were decreased in both hinokitiol-treated HSC-3 and SAS cells. A relative low dose hinokitiol effectively inhibited the malignancy of OSCC cells in vitro, and showed a positive result of suppressing xenograft tumour growth in vivo.
Hinokitiol is used as a food additive for preservative purposes, and the suggested concentration is 125 ppm (762 µM) per day, which is higher than the dose used in the present study. No known toxic effect from chronic exposure to hinokitiol has been reported (Cho et al., 2011). In addition, the hinokitiol containing oral hygiene products have been used to prevent dental caries and periodontal inflammation. Except being a remedy for oral cancer, combining the anti-microbial and anti-cancer activities, hinokitiol might be applied to reduce SSI effects and cancer recurrence in postoperative wound care. Hinokitiol exhibited specific cytotoxicity to OSCC cells than normal oral cells. Direct role for hinokitiol suppressing pan-histone expression in OSCC cells has not been described before to our knowledge. Future work should investigate the potential effect as a chemoprevention compound against OSCC pre-cancerous lesion.
This study was supported by grants from China Medical University (CMU98-N1-30, CMU100-S-30), the National Science Council, Taiwan (NSC99-2314-B-039-023-MY3, and NSC102-2628-B-040-001-MY3), and Ministry of Science and Technology, Taiwan (MOST103-2811-B-040-007).
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7. Titles and legends to Figures
Figure 1. Hinokitiol exhibited specific cytotoxicity to OSCC cells, induced G1 phase arrest in NHOK, NHOF, and SAS, induced G1/S phase arrest in HSC-3, and reduced the pRb phosphorylation.
(A) The cell viability of three normal oral cells (NHOK, NHOF, S-G) and six OSCC cell lines (Ca9-22, HSC-3, OC3, OEC-M1, SAS, SCC4) after hinokitiol treatment for
24-h. Hinokitiol exhibited the most cytotoxicity to HSC-3 among OSCC cell lines. (B) The cell viability of OK, HSC-3, OEC-M1, and SAS after hinokitiol treatment for 24-h, 48-24-h, and 72-h. The experiments were repeated twice, each in triplicate. Data shown are mean ± SE.
Figure 2. Hinokitiol induced G1 phase arrest in NHOK, NHOF, and SAS, and induced G1/S phase arrest in HSC-3, and reduced the pRb phosphorylation. (AC) The histograms of PI stained cell cycle distribution. The cell cycle distribution of NHOK, NHOF, HSC-3 and SAS after hinokitiol treatment for 24-h. The vertical axis represented the cell number; the horizontal axis represented the PI intensity; the dose of hinokitiol listed under each graph. (BD) The cell cycle distribution percentage of each phase in figure 2A. (CE) The G1/S transition and check point protein cyclin A, cyclin E, cdk2, phospho-pRb, pRb, p53, p21 expression of NHOK, HSC-3, and SAS after hinokitiol treatment for 24-h.
Figure 32. Hinokitiol induced apoptosis via extrinsic pathway in OSCC cell lines. (A) The apoptosis dot plot of hinokitiol-treated HSC-3 and SAS cells in 24-h and 48-h of treatment. (B) The cell percentage of annexin V positive cells (early and late apoptosis cells) in figure 3A. Data shown are mean ± SE. *P<0.05; **P<0.01; ***P<0.001; unpaired t test. (C) The caspase 3, caspase 8, and PARP expression of hinokitiol-treated HSC-3 and SAS cells in 48-h of treatment.
