Contributed equally
Honokiol (HK), a novel plant-derived natural product, is a physiologically activated compound with polyphenolic structure, and has been identified to function as an anticancer agent. It has been widely used in several diseases as a traditional medicine for a long time. We investigated whether HK could show anticancer effects on two oral squamous cell lines (OSCCs), HN-22 and HSC-4. We demonstrated that HK-treated cells showed dramatic reduction in cell growth and apoptotic cell morphologies. Intriguingly, the transcription factor specificity protein 1 (Sp1) was significantly inhibited by HK in a dose-dependent manner. Furthermore, we checked changes in cell cycle regulatory proteins and anti-apoptotic proteins at the molecular level, which are known as Sp1 target genes. The important key regulators in the cell cycle such as p27 and p21 were up-regulated by HK-mediated down-regulation of Sp1, whereas anti-apoptotic proteins including Mcl-1 and survivin were decreased, resulting in caspase-dependent apoptosis. Taken together, results from this study suggest that HK could modulate Sp1 transactivation and induce apoptotic cell death through the regulation of cell cycle and suppression of anti-apoptotic proteins. In addition, HK may be used in cancer prevention and therapies to improve the clinical outcome as an anticancer drug.
Oral squamous cell carcinoma (OSCC) is a common type of malignant tumor in the world. New cases of oral cancer occur at around 275,000 patients per year, OSCC cases comprise approximately >90% of diagnosed patients with oral cancer (
Natural products as sources of new drugs have been explored and expanded in anticancer drug development for the past several decades. In fact, 74.8% of all the anticancer drugs have been discovered and semi-modified from natural sources, for example, 20 small molecules were approved in 2010 (
Many researchers have paid attention to biological effects of HK in various cancer cells because it has many remarkable pharmacological abilities, such as anti-inflammatory, anti-thrombotic, anti-arrhythmic, anti-platelet and anti-oxidative effects, without appreciable toxicity (
Although the anticancer effects of HK have been well demonstrated against numerous cancer cell lines and models, little is known about the effect of HK on oral squamous cell carcinoma (OSCC). To characterize the effect of HK on OSCC, this study specifically examined the anticancer effect of HK on cell viability against two oral squamous cell carcinoma cell lines, HN-22 and HSC-4, and identified the regulated proteins by HK treatment in these cells. Interestingly, an important gene regulating protein specificity protein 1 (Sp1) in cell proliferation, cell cycle progression and oncogenesis was significantly regulated when cells were treated with HK (
The human oral squamous cancer cells, HN-22 and HSC-4, were generously provided by Dr Sung-Dae Cho (Chonbuk National University, Jeonju, Korea) and cultured in Hyclone Dulbecco’s modified Eagle’s medium (DMEM; Thermo Scientific, Logan, UT) containing 10% heat-inactivated fetal bovine serum and 100 U/ml each of penicillin and streptomycin (Thermo Scientific) at 37°C with 5% CO2 in humidified air. HK was purchased from Sigma-Aldrich (St. Louis, MO).
Cell viability was measured using the CellTiter 96™ AQueous assay kit (Promega, Madison, WI) according to the manufacturer’s protocol. Both HN-22 and HSC-4 cells were seeded on a 96-well microtiter plate (HN-22, 2×103 cells/well and HSC-4, 3×103 cells/well) and then cells were treated with different doses of 0, 2.5, 5 or 10
The endogenous Sp1 knockdown was induced via the transient transfection of siRNA. Knockdown of Sp1 was performed using a pool of four duplexes targeting Sp1 (TARGETplus SMARTpool siRNA, Thermo Scientific Dharmacon, Lafayette, CO). HN-22 and HSC-4 cells were seeded in 96-well plates and 100-mm culture dishes, and Sp1 targeting siRNA or non-targeting controls (Dharmacon) at a 50 nM were introduced using the DharmaFECT2 transfection reagent. After transfection, cells were subjected to MTS assay and western blot analysis.
The apoptotic events were visualized by terminal deoxynucleotidyltransferase UTP nick end labeling (TUNEL) assay using an
The cells were seeded over each sterilized glass coverslips on 6-well tissue culture plates for 24 h and incubated with HK for 48 h. The cells were fixed and permeabilized with cytofix/cytoperm solution for 30 min. For expression of Sp1, the cells were blocked with 1% BSA and then incubated with monoclonal Sp1 antibody at 4°C overnight. After washing with PBS containing 0.05% Tween-20 (PBST) solution, the Sp1 antibody was reacted with a Jackson 488-conjugated anti-mouse secondary antibody at room temperature for 1 h and mounted with Mountin solution-Vectashield mounting medium for fluorescence with DAPI (Vector Laboratories Inc., Burlingame, CA) onto the cells. The cells were visualized using a FluoView confocal laser microscope.
The total cell lysate were prepared using PRO-PREP™ protein Extraction Solution (iNtRON Biotechnology, Seoul, Korea) containing 1
Statistical significance was assessed using a Student’s t-test. A p-value of <0.05 compared with the non-treated cool was considered statistically significant.
Previously, it has been reported that HK inhibits cell proliferation and tumor growth of various cell lines derived from different cancers (
The transcription factor of Sp1 highly overexpressed in various cancer-derived cell lines including human glioblastoma, lung and pancreatic cancers etc., and has regulated transcriptional activity on differentiation, growth and oncogenesis genes (e.g. cyclins,
It has been reported that transcriptional activity of Sp1 has an important role in oncogenesis. Indeed, many cancer-derived cells showed enforced expression levels of Sp1 in comparison to normal cells. Moreover, the level of Sp1 was shown to affect the fate of cancer cells through modulating the genes involved in cell cycle progression, growth and apoptosis (
To determine the regulatory role of HK, we focused on the expression levels of the Sp1 downstream targets and pro-apoptotic proteins. We found that cell cycle arrest proteins, such as p27 and p21, were markedly enhanced in a dose-dependent manner by HK, whereas cell proliferation and survival associated proteins, such as cyclin D1, Mcl-1 and survivin, were decreased by HK treatment (
HK is a physiologically activated natural product, which has been widely used in China and Japan as a traditional herbal medicine for treatment of stroke, fever, anxiety and nervous disturbance (
In this study, we extensively explored the apoptotic effects of HK in OSCCs, since oral cancer is one of serious diseases in many parts of the world. Oral cancer, which is the cancer of the oral and pharyngeal cavities, is ranked as the sixth most commonly occurring cancer in the world. Oral squamous cell carcinomas (OSCCs) account for over 90% of oral cancers (
Transcription factor, Sp1 is known to be ubiquitously expressed and closely associated in various cellular processes through its anti-tumor activity and regulation of signal transductions (
Another cell cycle involving protein, cyclin D1, was also regulated by HK treatement. It has been reported that cyclin D1 is indispensable for cell cycle progression because it promotes G1/S phase transition via interaction with cyclin dependent kinases. Thus, its expression level is closely associated with tumorigenesis and cell maintenance. A study showed that cyclin D1 was induced by oncogenes including Ras, Src and β-catenin, when cells were stimulated by oncogenic signals (
Unlike the cell cycle arrest proteins such as p21 and p27, we observed that pro-survival proteins were significantly reduced by HK in OSCCs. It is known that survivin is a member of inhibitor of apoptosis protein family and its expression level has been considered to play an important role in oncogenesis (
In this study, we investigated the cancer chemoprevention effect of HK on OSCCs. Our results indicate that HK has cell growth inhibitory activity and induces apoptosis in OSCCs through inhibition of Sp1 expression, followed by transcriptional regulation of the cell cycle regulating and anti-apoptotic proteins. Taken together, HK might be a promising therapeutic agent in the treatment of oral cancers. However, molecular mechanisms and clinical studies for HK are necessary to elucidate its unexpected potential toxicity and its clinical applications.
3-(4,5-dimethylthiazol–2-yl)-5-(3-carboxyme thoxyphenyl)-2-(4-sulfophenyl)-2H–tetrazolium;
phenazine methosulfate;
4′-6-diamidino-2-phenylindole;
terminal deoxynucleotidyltransferase UTP nick end labeling
This research was supported by Basic Science Research program through the National Research Foundation Korea (NRF) Funded by the Ministry of Education, Science and Technology (2011-0008463 and 2010-0021532) and the Cooperative Research Program for Agriculture Science and Technology Development (PJ007963), Rural Development Administration, Republic of Korea.
The effect of honokiol (HK) on cell viability of oral cancer cells. (A) Chemical structure of HK. (B) The cell viability effect of HK on HN-22 and HSC-4 cells. HN-22 cells (2×103 cells/well) and HSC-4 (3×103 cells/well) cells were seeded in 96-well plates and incubated for 24 and 48 h with increasing concentration of HK in 10% FBS-DMEM. Cell viability was estimated using an MTS assay kit, as described in Materials and methods. Results are indicated as cell viability relative to the HK untreated, and data are the means ± SD from three independent experiments. The asterisk indicates a significant difference compared to the negative control (untreated cells) (*p<0.05). (C) TUNEL analyses were performed using a FluoView confocal laser microscope. The morphological changes observed in the HK treated (2.5, 5 and 10
The effect of honokiol (HK) on specificity protein 1 (Sp1) protein expression in HN-22 and HSC-4 cells. The HN-22 and HSC-4 cells were incubated with different concentrations of HK for 48 h. The cells were harvested and prepared for western blot analysis as described in Materials and methods. (A and B) Protein expression levels of Sp1 were detected using a specific antibody against Sp1 and its levels were quantified after actin normalization. The HK-treated cells were compared with the untreated cells, and data are the means ± SD of three independent experiments. The asterisk indicates a significant difference compared with the negative control (untreated cells) (*p<0.05). Time-dependent effects of HK on Sp1 and PARP expressions were performed in (C) HN-22 and (D) HSC-4 cells for 48 h with 12-h intervals. (E) Immunocytochemistry analysis was performed in HK treated HN-22 and HSC-4 cells. HN-22 and HSC-4 cells were treated with different concentrations of HK for 48 h and cells were immunostained with Sp1 specific antibody, and then signals were detected with Jackson 488-conjugated anti-mouse secondary antibody. DAPI was used for nucleus staining.
Knockdown of specificity protein 1 (Sp1) for functional study on cell proliferation of oral cancer cells. Sp1 specific (siSp1) or non-target siRNA (siCon) were transfected into the (A) HN-22 and (B) HSC-4 cells. Cell proliferation in siCon and siSp1-transfected (A) HN-22 and (B) HSC-4 cells was quantified by an MTS assay at 24, 48, 72 and 96 h post-transfection. Data are the means ± SD of three independent experiments. The asterisk indicates a significant difference in the siSp1 transfected cells compared with the siCon transfected cells (*p<0.05). The siCon or siSp1 transfected (C) HN-22 and (D) HSC-4 cell lysates were determined by western blot analysis using anti-Sp1, anti-PARP and anti-cleaved-caspase-3. The values measured by Image J densitometry are representative of three independent experiments. Actin was used as a loading control.
The effect of honokiol (HK) on downstream target protein of specificity protein 1 (Sp1). (A) HN-22 and (B) HSC-4 cells were incubated with HK (2.5, 5 and 10
The effect of honokiol (HK) on apoptosis of OSCCs. (A) HN-22 and (B) HSC-4 cells were treated with or without the indicated concentrations of HK (0, 2.5, 5 and 10