Introduction

Oral cancer accounts for approximately 8% of all malignancy which ranges from the 6th to the 9th most popular cancer all over the world [1]. About 90% of oral cancer is squamous cell carcinoma (OSCC) developed in the mucosal membranes of mouth, tongue, floors of mouths, gums, and hard palates [2,3]. High-risk factors of OSCC include tobacco or/and alcohol consumption, micronutrient inadequacy, human papillomavirus infection and ultra-violet radiation exposure. Despite the therapeutic advances, the relapse incidence of OSCC is still 40 to 60% and the survival rate for longer than three years is only 30 to 50% after treatment [1,4].

Octyl gallate (OG) and other members of gallate family have been well-known for various biological activities such as Helicobacter pylori inhibition, neurodegeneration improvement and tumorigenesis suppression [5]. Gallic acid and its ester derivatives were reported to have various therapeutic effects in anti-oxidation, anti-inflammation and anti-tumorigenesis [6,7]. Particularly, OG showed characteristic anticancer effects on various types of cancers with various molecular mechanisms. In human breast cancer cell lines of MCF-7 and MDA-MB-231, OG and gallic acid, isolated from Terminalia bellarica, inhibited cell cycle progression and induced apoptosis by downregulating positive cell cycle regulators of Cyclins and Cdks and upregulating negative regulators such as p21 and p27 [8]. In murine melanoma cell line, OG promoted oxidative stress and mitochondrial dysfunction to increase proapoptotic protein Bax while decreasing antiapoptotic protein Bcl-2, leading to caspase 3 activation and cell death [9]. From this perspective, we were motivated to investigate the OG’s effects on OSCC and its underlying molecular mechanism. In the present study, we demonstrated the effects of OG on cell proliferation and apoptosis induction in FaDu pharyngeal squamous carcinoma cells. OG significantly inhibited the cell proliferation by decreasing cell cycle proteins and induced caspasedependent apoptosis by the inhibition of the integrin family proteins and the activation of downstream mitogen-activated protein kinase (MAPK) pathway (extracellular signal-regulated kinases [ERK] and c-jun N-terminal kinases [JNK]).

Materials and Methods

1. Materials and reagents

Thiazolyl blue tetrazolium bromide (MTT), dimethylsulfoxide (DMSO), and OG were purchased from Sigma-Aldrich (St. Louis, MO, USA). Minimum essential medium (MEM), fetal bovine serum (FBS), trypsin-ethylenediaminetetraacetic acid (EDTA), and antibiotic-antimycotic solution were from Welgene (Daegu, Korea). The antibodies of Cyclin A, Cyclin B1, Cdk2, Cdk4 and precursor caspase 3 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and cleaved poly (ADP-ribose) polymerase (PARP) was obtained from Abcam (Seoul, Korea). Primary antibodies of integrin family (integrin α4, αv, β3, β4), Cleaved caspase 3, p-p42/p44 ERK1/2, p-p38 MAPK1/2, p- JNK, γ-H2AX were purchased from Cell Signaling (Danvers, MA, USA).

2. Cell culture

FaDu hypopharyngeal squamous cancer cells (Korean cell line bank) were grown in MEM media supplemented with 10% FBS and 100 units/mL penicillin/streptomycin and incubated in a humidified atmosphere (37℃ with 5% CO₂). For experiments, cells (2 × 10⁵ cells/mL) were seeded in MEM and incubated overnight to reach about 70 to 80% of confluency before treating with OG.

3. Cell viability assay

The cell viability was measured by an MTT assay. Briefly, cells (4 × 10⁴ cells/well) were seeded in 96-well plate for 24 hours. In 80% of confluence, cells were treated with various doses of OG. After the period of 24 or 48 hours, the media were replaced by MTT (0.5 mg/mL) solution and the plates were further incubated for 4 hours until violet crystals (formazan) were formed. MTT solution was aspirated and 200 μL of DMSO was added to dissolve formazan. The absorbance at 570 nm was measured by using Multiskan^TM FC Microplate Photometer (Thermo Fisher Scientific, Waltham, MA, USA).

4. Western blotting

After treated with different concentrations of OG for 24 hours, proteins were collected by using lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM disodium ethylenediaminetetraacetic acid [Na₂EDTA], 1 mM ethylene glycol tetraacetic acid, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, 1 μg/mL leupeptin, 1 mM phenylmethylsulfonyl fluoride [PMSF] and 1% phosphatase inhibitor cocktail) (Sigma-Aldrich). Total proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene fluoride membranes by using a Biorad wet blot system. The membrane was blocked by 5% skim milk for 1 hour at 4℃ and then incubated with primary antibody overnight at 4℃. Excessive antibody was removed by washing buffer (phosphate buffer saline [PBS] with 1% tween-20). Thereafter, the membrane was incubated with corresponding horseradish peroxidase (HPR)-conjugated secondary antibodies for 2 to 3 hours at room temperature. Finally, target proteins were detected by chemo-luminescence reagents and captured by Kodak Digital Science Image Station (Bruker BioSpin, Billerica, MA, USA). Quantification analysis was performed by using Carestream molecular imaging software (Carestream Health Inc., Rochester, NY, USA).

5. Immunofluorescence imaging

FaDu cells (1 × 10⁵ cells/well) were seeded on coverslips in 24-well plates for 24 hours. After reaching 90% confluence, cells were treated with various concentrations of OG for 24 hours or 48 hours. After washed with PBS, cells were fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature. Residual PFA was removed by washing with PBS (3×). Washing buffer (PBS with 1% goat serum and 0.1% Triton X-100) was used to block and permeabilize cell membranes in 30 minutes at 4℃. Thereafter, cells were treated with primary antibody at 4℃ overnight and then followed by secondary antibodies and 4,6-diamidino-2-phenylindole (DAPI) for 2 hours at room temperature. Finally, coverslips were mounted and images were captured by fluorescence microscopy (Carl Zeiss, Oberkochen, Germany).

6. Statistical analysis

All experiments were triplicated and the data were shown as the mean ± standard errors. The Student’s t-test was used to analyze statistical significance between two groups.

Results

1. Octyl gallate inhibited FaDu cell proliferation

To investigate the cell viability on FaDu cells, various concentrations of OG (0 to 200 μM) were treated with cells for 24 or 48 hours, and then an MTT assay was carried to identify the percent cell viability. OG inhibited the proliferation of FaDu cells in time and dose-dependent manners with half-maximal inhibitory concentration (IC50) of 121.1 μM at 24 hours and the cell viability was rapidly decreased when cells were exposed to OG longer (48 hours) (Fig. 1).

2. OG’s effect on cell cycle regulators

The expression of Cyclin B and Cdk2 were significantly suppressed in a dose-dependent manner while Cyclin A and Cdk4 were less affected in the OG range of 50 to 100 μM. At 150 μM of OG, Cyclin A and Cdk4 were also suppressed (Fig. 2).

3. OG induced caspase-dependent apoptosis

In Western blotting, OG significantly induced the cleavage of endoprotease caspase 3 and PARP. The appearance of executioner caspase 3 and its downstream PARP bands in the cleaved form demonstrated that OG-induced apoptosis was occurred through the caspase-dependent pathway (Fig. 3).

4. OG caused DNA damage on FaDu cells

To examine the effect of OG on DNA damage, a typical marker of double-strand DNA breaks (p-H2AX or γ-H2AX) was used in western blotting and immunofluorescence imaging. Under OG treatment, p-H2AX expression in Western blotting was increased and reached the maximum level at 150 μM of OG treatment (Fig. 4). Similarly, strong expression of p-H2AX was appeared dose-dependently in the fluorescence image.

5. OG suppressed the expression of integrin family proteins

OG suppressed the expression of various types of integrins (both alpha and beta subunits) on Fadu cells. The detachment of cell clumps was also observed under the OG treatment (data not shown). In western blotting results, the expression of integrin α4 was rapidly reduced even at low dose of OG while integrin αv, β3, and β5 were gradually decreased (Fig. 5). On SDS-PAGE, integrin α4 migrated as three different molecular sizes (mature proteins of 180 and 150 kDa and a precursor form of 140 kDa). At a low concentration of OG (50 μM), the expression of integrin α4 of mature forms (150 and 180 kDa proteins) were significantly suppressed while the expression of a precursor form (140 kDa) was only slightly decreased (Fig. 5).

6. OG activated ERK and JNK in MAPK pathway

Under various doses of OG treatment for 24 hours, the expressions of phosphorylated ERK1/2 and JNKs were increased while p38 remained unchanged. Furthermore, phosphorylated c-jun (p-c-jun), a downstream transcription factor of JNKs, was also highly expressed under the high concentration of OG (150 μM) (Fig. 6).

Discussion

In this study we investigated the antitumor effect of OG on FaDu cells by tracking the expression of essential proteins involved in cell proliferation and apoptosis. OG inhibited cell proliferation dose-dependently with IC50 of 121.1 μM in 24 hours treatment. Under this condition, protein expression for cell cycle regulation (cyclin A, cyclin B, Cdk2, and Cdk4) was significantly decreased, and DNA was damaged which was confirmed by the detection of the phosphorylated γ-H2AX, a typical double-stranded DNA marker. FaDu cells exposed to OG showed shrunken morphology with low density in cell population (data not shown). Presumably chromosomal damage might be due to the reactive oxygen species or other stress mediators generated by OG interaction with cellular organelles. Thereby, cells delayed the cell-division to repair the damaged DNA by reducing the expression of cell cycle promoting proteins (Cyclins and Cdks). However, at high dose condition of OG (150 μM), cells were forced to suicide by activating the caspase cascade. Interestingly, the expression of integrins was downregulated dose-dependently in response to OG treatment although the degree of reduction was integrin subunit dependent. For example, integrin α4 and β5 were significantly downregulated under OG treatment while integrin αv and β3 were slightly inhibited. Particularly the expression of matured integrin α4 (150 kDa) was dramatically decreased even at low dose (50 μM) of OG treatment while the precursor form of integrin α4 (140 kDa) was decreased slightly only. Integrins, a family of transmembrane proteins, comprise of noncovalently associated α and β subunits and transmit signals inwards/ outwards through the cells to mediate cell adhesion, migration, cell proliferation, cell survival and even cell death via binding to extracellular matrix ligands such as collagen, fibronectin, laminin, or growth factors [10,11]. Integrin α4 was known to play significant roles in tumor development and metastasis and several integrin α4 antagonists were actually explored to develop the antitumor therapy [12,13]. In this context, the suppressed expression of integrin α4 and other integrin family (integrin αv, β3, β4) by OG treatment could be due to the antagonistic action of OG to integrins.

MAPK comprises three subgroups including c-jun N-ter- minal kinase 1/2 (JNK1/2), stress-activated protein kinases (p38) and extracellular signal-regulated kinase 1/2 (ERK1/2). MAPK plays an essential role in maintaining cellular homeostasis and regulating cell proliferation and apoptosis as well [14,15]. MAPK may function as either anti-apoptotic or proapoptotic effector depending on the stimulus and the cell circumstances. Especially ERK1/2 was the most characterized subfamily which promotes cell survival or induces apoptosis in certain circumstances [15]. For example, DNA damaging stimuli such as ionizing irradiation, ultraviolet irradiation, etoposide (an anti-cancer drug) activated the ERK1/2 pathway to induce apoptosis [16,17]. In our results, significant expression of p- ERK, p-JNK and its downstream transcription factor p-c-jun demonstrates that OG functioned as an exogenous stimulus to induce apoptosis on FaDu cells through the activation of ERK and JNK pathways. It is unclear whether there is any crosstalk between the OG-mediated inhibition of the integrin expression and the overexpression of p-ERK and p-JNK. The shortage of integrin expression by OG treatment may have triggered the overexpression of other growth factors for compensation, leading to the activation of downstream regulators (p-ERK1/2 and p-JNK p-c-jun), and eventually inducing the cell death via caspase activation [18]. Overall, we demonstrated that OG has antitumor activity on hypopharyngeal squamous carcinoma cells by suppressing cell proliferation and inducing apoptosis.