Abstract
Aim: The present study evaluated the expression of α1 and β1 Na,K-ATPase, as well as the effects of digoxin (DGX) on oral squamous cell carcinomas (OSCCs). Patients and Methods: Immunohistochemical expression of α1 and β1 Na,K-ATPase were evaluated in 60 patients who underwent treatment at the São João de Deus Hospital. SCC-25 viability was assessed by the colorimetric assay. Chi-square or Fisher's exact tests were used to analyze the association of α1 and β1 Na,K-ATPase expression with the variables. Results: Immunoexpression of α1 and β1 Na,K-ATPase were observed in 28% and 55% of the tumors, however these proteins were not significant prognostic factors. Tobacco was significantly associated with α1 expression. SCC-25 viability decreased significantly after treatment with 1 μM DGX at 24 h. Conclusion: The smoking status of OSCC patients was significantly associated with α1 expression and DGX affected the SCC-25 viability in a dose- and duration-dependent manner.
Oral squamous cell carcinoma (OSCC) is one of the 10 most common malignant human tumours (1) and every year, around 263,000 cases occur worldwide and 127,000 die from this disease (2). Despite multimodal treatment, 50-60% of patients with stage III or IV disease relapse locoregionally. Out of these, most are not suitable for surgery or radiotherapy, or develop distant metastases (3).
Chemotherapy is accepted as standard therapy in the context of a metastatic or recurrent disease and, as adjuvant therapy in an attempt to eradicate the microscopic lesions presumed to remain after surgery, radiation therapy or both approaches (3). Although platinum-based chemotherapy resulted in higher response rates than monotherapy, combination regimens did not provide a survival benefit (4). It may be fair to conclude that is necessary to identify novel compounds that can result in higher rates of treatment response and improved survival of OSCC patients.
Digoxin (DGX), also known as cardiotonic steroid (CTS), is among the most ancient and effective therapies for congestive heart failure and arrhythmia (5, 6). It is a natural product characterized by the ability to inhibit membrane-bound sodium-potassium-activated ATPase (Na,K-ATPase) (7, 8). This pump, essential for cell homeostasis, consists of three transmembrane polypeptides: alpha, beta and gamma subunits. Four isoforms of the alpha subunit, three of the beta and two of the gamma subunit have been described, however α1 and β1 are expressed in most tissues, where gamma is found only in a few (9, 10).
Although the initial discovery and pharmacological development of CTSs are unrelated to oncology, interest in the links between CTSs and cancer began to rise in the late 1970s (8), owing to the discoveries that human cancer cells express a combination of Na,K-ATPase subunits that have a particularly high affinity for CTS (5, 8, 11).
Several studies had noted changes in expression of Na,K-ATPase in malignant tumors of the central nervous system, bladder, kidney, breast, colon, pancreas and lung (12-14). In medulloblastomas, overexpression of the α1 and α3 Na,K-ATPase subunits were correlated with better patient's prognosis (12). Additionally, expression of α and β Na,K-ATPase subunits were decreased in in situ and invasive bladder tumors and the authors suggested that this may be useful predictors of clinical outcomes, such as recurrence-free time (14).
Considering that little has been published on the effects of DGX against oral cancer cells and on the expression of the Na,K-ATPase in OSCC patients, the present study was undertaken.
Patients and Methods
Patients and patients' data. The study population consisted of 60 patients who underwent treatment for primary OSCC at the Oncologic Section of the São João de Deus Hospital/Fundação Fraldo Correia, Divinópolis, Minas Gerais, Brazil, from 2002 to 2009. The study was approved by the Institutional Ethics Committee (protocol 119 – 06-09-2010).
Clinical data of the patients were obtained from the medical records and included age, gender, tobacco and alcohol consumption, tumor location and Tumor, Node, Metastases (TNM) stage. Microscopic features of the OSCCs were evaluated using a 4-μm section stained by hematoxylin-eosin (HE) and included histopathological malignancy grading, vascular embolization, perineural infiltration and extracapsular nodal spread.
α1 and β1 Na,K-ATPase immunoexpression. A formalin-fixed 4-μm section of tumor tissue was taken from the pathology archive for immunohistochemistry analyses of α1 and β1 Na,K-ATPase expression.
After antigen retrieval using 1M citrate buffer, pH 6.0, in a microwave oven for 15min, endogenous peroxidase activity was blocked by incubation in 3% H2O2 for 60min. Antibodies (α1 and β1 Na,K-ATPase - Santa Cruz Biotechnology, Paso Robles, California, United States) diluted 1:100 was used as the primary antibody and the incubation period was 12 hours in a dark humid chamber at 4°C. Next, peroxidase activity was revealed using diaminobenzidine (DAB) (Dako, Glostrup, Hovedstaden, Denmark). For negative control, sections were processed in the absence of the primary antibodies. Specimens were counterstained in hematoxylin solution. The expression of each marker was evaluated independently by two observers.
Cell culture. SCC-25 human oral squamous carcinoma cells (ATCC, Manassas, Virginia, United States) were grown in 25 cm2 flasks containing 5ml of DMEM/F12 medium (Invitrogen, Carlsbad, California, United States) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, California, United States), 400 ng/mL hydrocortisone and 100 μg/ml gentamycin and kanamycin at 37°C in a humidified atmosphere of 5% CO2, as previously described by Agostini et al. (15).
Cell viability assay. SCC-25 viability after exposure to the DGX was assessed by a colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), as described by Gomes et al. (16).
SCC-25 cells were seeded (2×105 cells per well) in two 96-well plates containing DMEM/F12 medium supplemented with 10% fetal bovine serum. After 24 hours to allow cell fixation to the substrate, the plates were washed twice with phosphate-buffered saline (PBS) prior to use. Negative control wells received 100 μl of complete medium whereas treated cells were incubated with 0.01 μM, 0.02 μM, 0.05 μM, 0.1 μM, 0.25 μM, 0.5 μM, 0.75 μM, 1 μM, 1.25 μM and 1.5 μM of DGX followed by incubation for 24 and 48 hours (one plate each).
At the end of incubation, the medium was removed from the wells, discarded and the cells were washed twice with PBS. Each well received 50 μl of MTT solution (Sigma–Aldrich Corp., Saint Louis, Missouri, United States - 0.5 mg/ml in PBS), including six wells without cells (blanks). The plates were incubated at 37°C for 4hours followed by the addition of 200 μl of dimethylsulfoxide (DMSO) and incubation with shaking at 37°C for 20 min. The resulting absorbances were read at 570 nm in a microplate reader (Spacemax M5e, Molecular Devices, Sunnyvale, CA, USA) and cell viability was calculated using the equation:
where A is the absorbance of the treated cells, B is the absorbance of the blank and C is the absorbance of the control. All experiments were done in triplicate.
Statistical analysis. All statistical analyses were performed using the R program version 2.13.0 (The R Foundation for Statistical Computing, Vienna, Austria). Chi-square or Fisher's exact tests was used to analyze the association of α1 and β1 Na,K-ATPase expression with the variables. Survival rates were calculated using the Kaplan–Meier method. The comparison of the survival curves were performed using the log rank test. The follow-up period was the time between surgery and the death or last patient information date. Concerning the cell viability assay, data were expressed as mean±standard deviation SD. Statistical comparisons of cell viability (MTT assay) were done using analysis of variance (ANOVA) followed by the Dunnett multiple comparisons test. Values of p≤0.05 indicated a significant difference.
Results
The analysis of 60 patients with OSCC with average age of 58.8 years revealed a male predominance (90.0%) over female (10.0%). Tobacco or alcohol consumption was documented in 78.3% and 71.7%, respectively.
The tongue was the primary site in 30 (50.0%) patients, floor of the mouth in 19 (31.7%), retromolar area in 6 (10.0%) and gingiva in 5 (8.3%). The majority of the patients (81.7%) had advanced tumors (stages III and IV). Thirty-three tumors (55.0%) were classified as moderately differentiated. Vascular embolization was detected in 23.3% of the OSCCs and perineural invasion in 16.7%.
Correlation of α1 Na,K-ATPase subunit expression with clinical and microscopic features in patients with oral squamous cell carcinoma.
Normal oral cavity epithelium expressed Na,K-ATPase in the membrane, whereas malignant cells presented diffuse expression in membrane and cytoplasm. Twenty-eight percent of the OSCC patients were Na,K-ATPase α1 positive and fifty-five percent β1 positive (Figure 1).
There were no statistically significant correlations regarding the clinical and pathological parameters and α1 and β1 expression (Tables I and II), but it was observed that the habit of smoking was significantly associated with the positivity of α1 (p=0.007).
The 5-year overall survival rate was 68%. There were no differences in the 5-year overall survival rate according to the histologic grade or α1 or β1 Na,K-ATPase subunit expression (Figure 2).
Correlation of β1 Na,K-ATPase subunit expression with clinical and microscopic features in patients with oral squamous cell carcinoma.
After 48 h of incubation with DGX, many cells showed cytoplasmic shrinkage and loss of normal nuclear architecture, detached from monolayer and found floating in the medium. As shown in Figure 3, DGX affected the viability of SCC-25 cells in a dose- and duration-dependent manner. The SCC-25 proliferation decreased significantly after being treated with 1 μM DGX at 24 h. Besides, at 48 h of exposure, the cell viability was reduced significantly when the SCC25 cell line was treated with 250 nM of DGX.
Discussion
In our series, the majority of the patients were male, smokers, with a median age of 59 years and advanced stage tumors. The main locations were the tongue and mouth floor, which is consistent with data from the literature (17-19).
Immunostaining for Na,K-ATPase pump subunits α1 (A, B and C) and β1 (D, E and F) in moderate-differentiated squamous cell carcinoma of oral cavity cancer. The sections C and F are positive controls for the immunohistochemistry assay with the subunit α1 and β1, respectively. The sections were counterstained with hematoxylin.
Regarding Na,K-ATPase immunoexpression, normal oral cavity epithelium expressed this protein in the membrane, whereas malignant epithelium presented diffuse expression in membrane and cytoplasm. Previous studies (20, 21) demonstrated predominantly membrane expression, but others described cytoplasmic expression. According to Newman et al. (22), the activated Na,K-ATPase induces its translocation to the nucleus via endocytosis, which could justify the cytoplasmatic localization.
Seventy-one and forty-five percent of the tumor cells did not express α1 and β1 subunits, respectively. This was a surprising result given that virtually all cells, including malignant cells, do express Na,K-ATPase to some degree, as it is an essential enzyme for cellular homeostasis. One possible explanation is that Na,K-ATPase expression is low in squamous tissue and can thus only be assessed by gene expression. Decreased expression of the β1 and α1 subunits may be associated with low activity of Na,K-ATPase from a loss of functional efficiency of this enzyme during carcinogenesis (14, 21).
Overall survival curve according to α1 Na,K-ATPase expression.
Effect of digoxin on SCC-25 viability measured by MTT metabolism. Data are expressed as mean±SD (normalized to control mean in the absence of digoxin) of three experiments.
There were no statistically significant correlations regarding the clinicopathological parameters and α1 and β1 expression; however, tobacco use was significantly associated with the α1 subunit expression (p=0.007). Tobacco participates in the early phase of the carcinogenic process leading to genetic and epigenetic cellular changes that result in AKT signaling pathway activation (23). Liu et al. (24) described a possible association between the AKT and Na,K-ATPase pathways, through interactions of ouabain with Na,K-ATPase, thereby inducing phosphoinositide 3-kinase (PI3K) to activate AKT.
Na,K-ATPase α1 and β1 expression were not significantly associated with the overall survival. Nevertheless, α1 negative tumors had a trend towards worse survival (Figure 2). These data agree with the studies of renal and bladder neoplasias (20, 21), in which higher α1 expression and lower β1 expression were associated with worse prognoses.
The MTT assay showed that DGX was cytotoxic on SCC25 cells after 24 h of exposure (1-1.5 μM) and after 48 h of exposure (0.25-1.5 μM), reducing the cell viability significantly when compared with the negative control groups. Our results are similar to those published by Hallbook et al. (25) and Felth et al. (7), who reported that DGX was cytotoxic on normal peripheral blood, leukemia and colon cancer cells lineages in concentrations around 0.02 and 1 μM.
The effects of CTSs have been studied in a wide variety of cancer cell lines, however, little or nothing has been published on the cytotoxic effects of the CTSs against oral cancer cells. According to Schoner and Scheiner-Bobis (26), DGX and other digitalis may influence cell proliferation, differentiation, and, eventually, cell death via the Na,K-ATPase signalosome pathways. However, these authors affirm that in vitro studies show that other mechanisms can be related to cellular responses after exposure to CTSs. For instance, bufalin, a hydrofobic CTS, down-regulates the expression of cyclin A, Bcl-2 and Bcl-XL and up-regulates the expression of p21 and Bax in ovarian endometrial cyst stromal cells affecting the cell cycle progression and inducing apoptosis (27). DGX, at low concentrations (<10 nM) prevents apoptosis in HeLa cells but at concentrations higher than 10 nM the apoptotic process is triggered in a cytochrome-c release dependent pathway (28).
There are several studies describing the relationship between the cisplatin effect and Na,K-ATPase. Cell lines resistant to the action of cisplatin presented lower expression of Na,K-ATPase than susceptible lineages, and the expression of Na,K-ATPase was related with the susceptibility or not of the cell to cisplatin (29). Kishimoto et al. (30) showed that cisplatin-sensitive cell lines presented a high expression of the α1 subunit of Na,K-ATPase after cisplatin treatment, which can be correlated with their sensitivity to the drug.
Cisplatin is the most frequently used chemotherapeutic drug for the treatment of head and neck squamous cell carcinoma (HNSCC). However, the overall survival of patients with head and neck cancer has not improved significantly for the last 30 years, despite the extensive use of platinum-based therapies for the last two decades (31). These recent data justify all the efforts to find novel compounds like DGX that could be used in combination with conventional agents, contributing to a more efficient strategy in the treatment of patients with OSCC.
Conclusion
Our results demonstrated that Na,K-ATPase is expressed in a variable degree in OSCC, however it was not associated with the overall survival rate. Significant correlation was noted between the smoking status of patients and α1 immunoexpression. In addition, DGX affected SCC-25 viability in a dose- and duration-dependent manner.
Acknowledgements
This work was supported by FAPEMIG (Fundação de Amparo a Pesquisa do Estado de Minas Gerais), CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico - grant # 472394/2012-6).
- Received June 9, 2014.
- Revision received July 10, 2014.
- Accepted July 11, 2014.
- Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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