Abstract
Background: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the world. While the incidence of HNSCC associated with tobacco and alcohol abuse is falling, the incidence of HNSCC associated with human papilloma virus (HPV) is rising. Proliferation, cell migration and formation of metastases are dependent on interactions between the tumor cells, tumor stromal cells and the extracellular matrix (ECM). Degradation of the ECM is a crucial step in the process of local tumor infiltration and formation of locoregional and distant metastases. Matrix metalloproteinases (MMPs) are a family of enzymes that are able to degrade the ECM. Locally advanced HNSCC with cervical node metastases are treated with docetaxel in induction chemotherapy (ICT) combined with platinum-based chemotherapy and 5-fluorouracil (5-FU) as standard clinical anti-neoplastic regimens. This study evaluated the expression of MMP-14 and MMP-2 in HPV-positive (CERV196) and HPV-negative squamous cell carcinoma (HNSCC 11A and 14C) and the alteration of expression levels after exposure to either docetaxel or 5-FU. Materials and Methods: Tumor cells were exposed to 5-FU or docetaxel in concentrations of 1.0 and 5.0 μmol/ml. MMP-protein expression was evaluated by enzyme-linked immunosorbent assay (ELISA) after 2, 3, 5, 8 and 10 days of incubation. Results: Docetaxel exposure significantly decreased MMP-14 expression in HNSCC 11A and especially 14C but not in CERV196 apart from an apoptotic process. 5-FU had no significant effect on MMP-14 expression independent of the HPV-status. Significant alterations of MMP-2 could be detected in HNSCC 11A only. Conclusion: Although neither of the applied drugs were selective inhibitors of MMP-expression, surprisingly docetaxel significantly decreased MMP-14 in HNSCC 14C and 11A in this study. Interestingly, HPV-positive CERV196 was not sensitive to decreased MMP-14 or -2 expression following incubation with 5-FU or docetaxel.
Abbreviations: 5-FU: 5-Fluorouracil; BSS: balanced salt solution; CAFs: cancer-associated fibroblasts; CIS: carcinoma in situ; DMEM: dulbecco's modified essential medium; DNA: deoxyribonucleic acid; DPD: dihydropyrimidine dehydrogenase; dTMP: deoxythymidine monophosphate; dUMP: deoxyuridine monophosphate; ECM: extracellular matrix; ELISA: enzyme-linked immunosorbent assay; FCS: fetal calf serum; HNSCC: head and neck squamous cell carcinoma; HPV: human papilloma virus; ICT: induction chemotherapy; MMPs: matrix metalloproteinases; MT-MMPs: membrane-type matrix metalloproteinases; NCCN: National Comprehensive Cancer Network; PBS: phosphate buffered saline; RNA: ribonucleic acid; SCC: squamous cell carcinoma; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; siRNA: small interfering ribonucleic acid; TIMPs: tissue inhibitors of metalloproteinases.
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide with a global incidence of approximately 631,000 cases and 352,000 associated deaths in 2008 (1). Local tumor size and the presence of cervical lymph node metastases are the most important prognostic factors. Despite advances in surgical techniques, radiation and chemotherapy, the survival rate has remained almost unchanged (2). Identification of new predictive markers is mandatory to improve treatment and outcome for patients. The incidence of HNSCC has decreased in recent years, which is attributed to a falling prevalence of tobacco abuse (3). Still, smoking and alcohol abuse are important risk factors and have synergistic effects in cancerogenesis (4, 5). In contrast, the incidence of oropharyngeal cancer, especially of the tonsils and tongue base, has increased in patients 20-44 years old (6).
Human papilloma virus (HPV) and its cancerogenic potential is well known for carcinomas of the uterine cervix. Risk factors for HPV infection are promiscuity and unsafe sex. Consequently, HPV is associated with anogenital and oropharyngeal cancer (7). The growing relevance of the viral etiology of HNSCC is illustrated by the rising incidence of HPV-associated oropharyngeal cancer (8, 9). HPV-negative and -positive HNSCC differ in molecular and clinical aspects (10-12). The viral oncogenes suppress inflammatory signaling as a mechanism of immune escape (13). It is assumed that HPV-positive HNSCCs have a higher sensitivity to radiation and chemotherapy and may be associated with a better prognosis (14, 15).
Endothelium, cancer-associated fibroblasts (CAFs), pericytes and the infiltrating inflammatory cells are part of the tumor stroma and are referred to as tumor stromal cells. They form the extracellular matrix (ECM) and are attributed with important functions during tumor progression (16). The ECM consists of various glycoproteins, hyaluronic acid, proteoglycans, collagen and elastic fibres. It is a physical barrier for cellular migration. The basement membrane is the most relevant structure concerning the depth of tumor infiltration. It connects the epithelium with the subepithelial connective tissue. The most important components of the basement membrane are type IV collagen and various glycoproteins, such as laminin, which interacts with integrins in the cell membrane of the epithelial cells. Tumor cell invasion of the basement membrane defines an invasive carcinoma and differentiates it from a carcinoma in situ. Without invasion of the basement membrane, the formation of lymphatic or hematogenous metastases is impossible because the epithelial layer is free of vessels (17).
Currently, matrix metalloproteinases (MMPs) are a group of over 20 zinc-dependent endopeptidases that exist as soluble and membrane-bound enzymes (18, 19). Tumor cells as well as the tumor stromal cells can synthesize MMPs (20, 21). A study by Bodnar et al. demonstrated that MMP-2 expression is higher in tumor stromal cells and MMP-14 expression is higher in HNSCC tumor cells (22). Jerome Gross and Charles Lapière first described MMPs in 1962 (23). MMPs are synthesized as inactive proenzymes and cleavage of the propeptide leads to activation of the enzyme. Classification of MMPs can be based on substrate specificity or cellular location. Known groups of MMPs are collagenases, gelatinases, stromelysins and membrane-type MMPs (MT-MMPs). The enzyme activity of MMPs is strictly regulated in healthy tissues since uncontrolled activity could result in tissue damage. Currently, four specific tissue inhibitors of metalloproteinases (TIMPs) are known (24). It has been shown that the expression of TIMP-2 in HNSCC, which is involved in the activation of MMP-2 by MMP-14, is associated with later lymph node and hematogenous metastases as well as poorer 5-year survival rate (25). Although the primary function of MMPs in the formation of metastases is the local degradation of the ECM including the basement membrane, they have a crucial role in the maintenance of the microenvironment of the tumor that supports tumor growth and angiogenesis (18).
MMP-14, also known as MT1-MMP, was the first membrane-type MMP to be discovered. Its potency to promote invasion and metastasis of cancer cells was demonstrated by various studies (26). Substrates of MMP-14 are collagen types I, II and III, fibrin, laminin and aggrecan. Additionally, MMP-14 activates other MMPs, such as MMP-2 and MMP-13, by cleavage of their propeptides. Thus, MMP-14 is said to initiate several enzymatic cascades. Besides promotion of cancer cell invasion and formation of metastasis, MMP-14 is involved in physiological processes, such as wound healing, skeletal development and angiogenesis (27-29). The latter emphasizes that MMPs not only support tumor growth and invasion by degradation of the ECM but also by supporting a tumor microenvironment. In the process of MMP-2 activation, MMP-14 builds a complex with TIMP-2 that on one hand abolishes the enzyme activity of MMP-14 and on the other hand works as a receptor for MMP-2 that brings MMP-2 into close contact with other unbound and active MMP-14 (30). An association of MMP-14 with TIMP-2 expression in particular was found in patients with lymph node metastasis of HNSCC (31). Zhang et al. demonstrated that MMP-14 expression increased significantly in advanced-stage HNSCC and lymph node metastasis compared to stage I or II HNSCC. Furthermore, elevated MMP-14 expression was associated with poorer prognosis (32).
Activation of MMP-2 is believed to be a crucial step during tumor invasion (33). MMP-2 is a secreted gelatinase that degrades gelatin, laminin and various types of collagen including collagen IV, a major component of the basement membrane (34). The active form of MMP-2 correlates with tumor invasiveness (35). Local degradation of the basement membrane is a mandatory step for tumor invasion and the formation of lymphatic or hematogenous metastases. Ruokolainen and colleagues showed that MMP-2 expression is strongly related to the formation of lymph node or distant metastases (25).
Paclitaxel, the first known taxane, was extracted from the rare tree Taxus brevifolia. Docetaxel is a semi-synthetic analog of paclitaxel and well established as a cytostatic chemotherapeutic drug. Taxanes bind tubulin reversibly and prevent depolymerization of the microtubules. This arrests mitosis between metaphase and anaphase. Docetaxel does not block the start of mitosis. Further properties of docetaxel are induction of apoptosis, anti-angiogenic effects and stimulation of the immune system (36). Over 90% of advanced-stage human tumor grafts displayed docetaxel sensitivity in mice (37). Combinations of docetaxel with 5-fluorouracil (5-FU), cyclophosphamide, etoposide or anti-angiogenic drugs showed synergistic effects in vivo. Demonstrated side effects were hematological, gastrointestinal and neuromotoric toxicity (36, 37). At present, docetaxel is used for the treatment of breast, ovarian, prostate, and non-small cell lung cancer. For locally advanced HNSCC, docetaxel is recommended by the National Comprehensive Cancer Network (NCCN) for induction chemotherapy (ICT) in combination with platinum-based chemotherapeutic drugs and 5-FU (38, 39). Patients who received docetaxel in addition to ICT had significantly higher survival rates compared to patients who received cisplatin and 5-FU ICT plus radiotherapy (40, 41).
The pyrimidine analog 5-FU irreversibly binds to thymidylate synthase and inhibits the methylation of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP), leading to a deficit of dTMP. dTMP is a necessary nucleotide for DNA replication. Different cytotoxic metabolites of 5-FU can be incorporated into DNA as substitutes for dTMP. This causes abortion of the DNA strand replication, leading to cell cycle arrest and apoptosis. Disturbance of nuclear RNA maturation is possible by incorporation of 5-FU metabolites into RNA. However, inhibition of DNA synthesis is considered to be the main effect of 5-FU. Cellular resistance to 5-FU is gained by alterations of 5-FU metabolism, mutations of thymidylate synthase or altered kinetics in respect to nucleotides or folates. Biochemical modulation of 5-FU metabolism may allow to overpower resistance to 5-FU (42). 5-FU is applied mainly by intravenous infusion (43) and primarily eliminated by dihydropyrimidine dehydrogenase (DPD) in the liver. A partial DPD deficiency can be found in about 3% of patients, which may lead to increased 5-FU-related side-effects. Although multiple mechanisms may lead to 5-FU resistance, DPD activity in tumor cells is significantly related to 5-FU sensitivity. Lower DPD activity is attributed to increased 5-FU efficacy (44).
This study sought to evaluate the expression of MMP-14 and MMP-2 in HPV-positive and -negative squamous cell carcinoma cells in vitro and to assess alterations of expression under the influence of docetaxel or 5-FU.
Materials and Methods
Cell lines. The human cell lines HNSCC 11A and 14C were received from Dr T.E. Carey (University of Michigan, Ann Arbor, MI, USA) and originated from SCC (squamous cell carcinoma) of the oropharynx and larynx. They were kept in Dulbecco's modified essential medium (DMEM) (Fisher Scientific and Co., Pittsburgh, PA, USA) with 10% fetal calf serum (FCS) and antibiotics (Life Technologies Inc., Gaithersburg, MD, USA). A p16-positive SCC of the uterine cervix was the origin of the cell line CERV196 (CLS, Eppelheim, Germany) and was cultured in Eagle's minimum essential medium with 2 mM L-glutamine, 10% FCS and Earle's balanced salt solution (BSS) with 1.5 g/l sodium bicarbonate, 0.1 mM non-essential amino acids and 1.0 mM sodium pyruvate. Cell cultures were kept at 37°C in a fully humidified atmosphere with 5% CO2. Docetaxel and 5-FU were stored at 4°C and solved in sterile water at the time of use. Cell lines were incubated with either docetaxel or 5-FU at concentrations of 1.0 or 5.0 μmol/ml for 48, 72, 120, 192 or 240 hours. The drug concentrations were selected after performing the alamarBlue® (AbD Serotec, Oxford, UK) cell proliferation assay, measuring proliferation of SCC cells quantitatively and establishing the relative cytotoxicity of the chemotherapeutic drugs examined. After incubation, cells were lysed and centrifuged, and the supernatants were collected in sterile tubes and stored at −20°C until further analysis.
Enzyme-linked immunosorbent assay (ELISA) for total MMP-2 and MMP-14. After incubation with different concentrations of either docetaxel or 5-FU, the cells were washed with phosphate buffered saline (PBS). Afterwards, 350 μl of lysis buffer was added to each well. Lysed cells were stirred up with a vortex at 2-8°C for 30 min and then microcentrifuged at 14,000 g for 5 min. The supernatant was pipetted into a clean tube.
MMP-2 levels were determined with a sandwich ELISA (R&D Systems, Wiesbaden, Germany). The system used a solid-phase monoclonal antibody and an enzyme-linked polyclonal antibody against MMP-2 (binding both the proenzyme and the active form). The specificity of antibodies to MMP-2 used in the ELISA kit was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting. According to the manufacturer's directions, each ELISA was performed on 50 μl of supernatant. All analyses and calibrations were carried out twice. The calibrations on each microtiter plate included recombinant human MMP-2 standards that were provided in the kit. Optical density was determined using a microplate reader at a wavelength of 450 nm. Wavelength correction was set to 540 nm and concentrations were reported as pg/ml. The inter-assay coefficient of variation reported by the manufacturer varied from 5.4-9.8% and the range of detection was 289-50,000 pg/ml.
An ELISA kit for human MMP-14 (USCN Life Science Inc., Houston, TX, USA) provided a 96-well plate precoated with an antibody specific to human MMP-14. A serial dilution of an MMP-14 standard provided in the kit was prepared according to the manufacturer's instructions as well as the supernatant samples. Each ELISA was performed with 100 μl of supernatant. Optical density was measured using a microplate reader at 450 nm. The inter-assay coefficient of variation reported by the manufacturer was <10% and the range of detection for MMP-14 was 630-100,000 pg/ml. All analyses and calibrations were carried out twice.
Measuring total protein. Total protein levels were determined using DC Protein Assay (Bio-Rad, Hercules, CA, USA). Incubation, cell lysis and centrifugation were performed as previously described. A serial dilution of a protein standard was prepared according to the manufacturer's instructions. Measurement was carried out with 100 μl of protein standard or cell supernatant using a spectrophotometer set to 750 nm; concentrations were reported as μg/ml.
Statistical analysis. Statistical analysis was performed in cooperation with PD Dr. C. Weiss, Institute of Biomathematics, Faculty of Medicine, Mannheim, Germany. A p-value ≤0.05 was considered statistically significant. The two-coefficient variance test (SAS Statistics, Cary, NC, USA) and Dunnett's test were performed.
Results
Total protein assay. The total protein was measured in order to differentiate the decrease in protein expression of MMP-2 or MMP-14 from cytotoxicity (apoptosis) caused by the applied drugs. Total protein levels of the cell lysate were compared to levels of MMP expression. For the negative control and 5-FU, a minor fluctuation of the protein quotient (MMP expression level/total protein level) was determined but no increase of the protein quotient could be stated. Thus a measured alteration of MMP expression levels cannot be the result of apoptosis due to cytotoxicity of 5-FU. For docetaxel, an increase of the protein quotient could be detected in a time-dependent manner irrespective of the HPV-status. Consequently, a cytotoxic effect of docetaxel can be stated (data not shown).
ELISA for MMP-14 expression in HNSCC 11A, 14C and CERV196 cells. MMP-14 was expressed in all evaluated cell lines. Expression levels of MMP-14 in HNSCC 14C exceeded the expression levels in HNSCC 11A and CERV196 at least twofold. A trend towards decreased expression levels after incubation with chemotherapeutic drugs was observed, especially in HNSCC 14C but also in HNSCC 11A.
A statistically significant reduction of MMP-14 expression in HNSCC 14C cells was found with 5-FU after 3 and 5 days of incubation (p-value=0.002 and 0.014), which corresponds to an average reduction of 63.5%. 5-FU had no significant impact on MMP-14 expression in HNSCC 11A or CERV196.
Docetaxel significantly reduced MMP-14 levels (30%) in HNSCC 11A after 3 days of incubation with a p-value of 0.043. For HNSCC 14C cells, there was a significant reduction in MMP-14 expression after 3, 5, 8 and 10 days of incubation with docetaxel (p-values were 0.004, <0.001, 0.03 and 0.024, respectively). On average, docetaxel reduced MMP-14 levels by 31%. Taking into account the increased protein quotient after incubation with docetaxel (see section on total protein assay), this reduction could be due to cytotoxicity and increased apoptosis, not a specific effect on MMP-14 expression. However, although the increase of protein quotient was detected in all three cell lines, a similar decrease of MMP-14 could not be found in HNSCC 11A and CERV196. Docetaxel did not significantly alter MMP-14 expression in CERV196 independent of the incubation time. Different drug concentrations had no statistically significant effect on the expression of MMP-14. For simplification, only the data for 5-FU and docetaxel at a concentration of 5.0 μmol/ml are shown (Table I, Figure 1).
Comparing the impact of 5-FU and docetaxel on the reduction of MMP-14 expression, there was a significant difference in HNSCC 14C cells after 5, 8 and 10 days of incubation (p-value=0.004, 0.014 and 0.001 respectively). For HNSCC 11A and CERV196, no significant differences between the effect of 5-FU and docetaxel could be detected.
ELISA for MMP-2 expression in HNSCC 11A, 14C and CERV196 cells. Expression of MMP-2 was detected in all studied cell lines. Expression levels in CERV196 were slightly lower compared to the HPV-negative SCC cell lines. With two exceptions, both 5-FU and docetaxel showed no significant alteration of MMP-2 expression. A statistically significant alteration of MMP-2 levels (47% reduction) could be observed only in HNSCC 11A after 8 days of incubation with 5-FU (p-value=0.005) and with docetaxel (49% reduction; p-value=0.003). No statistically significant alteration of MMP-2 expression could be observed in HNSCC 14C and CERV196 after incubation with 5-FU or docetaxel irrespective of the incubation time or drug concentration (Table II) (Figure 2).
Discussion
The aim of the present study was to evaluate the expression of MMP-14 and MMP-2 in HPV-positive and -negative SCC and to determine alterations of MMP expression after incubation of these tumor cells with 5-FU or docetaxel as standard chemotherapeutic drugs in clinical regimens.
Docetaxel and 5-FU are not direct inhibitors of MMPs. Docetaxel affects mitosis by inhibition of the spindle apparatus, while 5-FU disturbs DNA replication. Only a small number of studies have investigated the alteration of MMPs after therapy with 5-FU or docetaxel. Yamamoto et al. demonstrated suppression of lung micrometastasis progression after therapy with docetaxel in combination with an MMP-inhibitor (45). Further studies showed possible interactions of MMP-inhibition and chemotherapeutic drugs. For instance, a knockdown of MMP-7 expression by siRNA (small interfering ribonucleic acid) increased sensitivity to 5-FU and radiation in colon carcinoma (46).
Our study showed that MMP-2 and MMP-14 were expressed in all observed cell lines. MMP-2 expression levels were slightly lower in HPV-positive CERV196 compared to HPV-negative SCC cells and they were also lower compared to the expression levels of MMP-14 in all cell lines. As previously mentioned, Bodnar et al. demonstrated that MMP-2 expression is higher in tumor stromal cells and MMP-14 expression is higher in HNSCC tumor cells, which might explain our results and might indicate that epithelial cells are less sensitive to an alteration of MMP-2 expression following chemotherapy (22). Interestingly, MMP-14 levels in HNSCC 14C exceeded the expression levels in HNSCC 11A and CERV196 by at least two-fold. Higher levels of MMP expression can often be found in more advanced tumors or metastases (18, 32, 47).
We were able to demonstrate that docetaxel and 5-FU reduce the protein expression of MMP-14 and MMP-2 in HPV-negative SCC. As previously described, 5-FU appeared to be less effective in altering protein expression compared to docetaxel (48). However, in the case of a significant reduction of MMP levels following incubation with 5-FU, the reduction was equal to the effect of docetaxel. In fact, 5-FU revealed the highest levels of suppression, with 63.5% for MMP-14 expression in HNSCC 14C. As previously mentioned, 5-FU disturbs RNA synthesis as one possible mechanism of action. This inhibition of protein expression may include expression of MMPs, which may be a possible mechanism for MMP reduction after exposure to 5-FU.
Suppression of MMP-14 expression after incubation with docetaxel in HNSCC 11A was rather sparse compared to the broad and significant reduction of MMP-14 in HNSCC 14C, which also showed higher expression levels than HNSCC 11A in negative controls. Comparison of the total protein levels with MMP expression as the target variable in a protein quotient (MMP expression level/total protein level) revealed an increasing protein quotient after incubation with docetaxel in an incubation time-dependent manner due to a decrease of the denominator (total protein level). This can be interpreted as a sign of a cytotoxic effect of docetaxel. Cytotoxicity is known to be higher in docetaxel than 5-FU because docetaxel directly affects mitosis and the spindle fibers. However, the protein quotient increased under docetaxel incubation in all three cell lines. Still, a similar decrease of the target proteins MMP-14 and MMP-2 was not present in HNSCC 11A and CERV196. Therefore, the alterations of MMP expression under docetaxel cannot only be explained as a cytotoxic effect and/or increase of apoptosis.
A statistically significant alteration of MMP-2 expression was observed in HNSCC 11A after 8 days of incubation with docetaxel and with 5-FU, but no differences in HNSCC 14C could be detected. HPV-positive CERV196 cells showed no significant differences in MMP-14 or MMP-2 expression after exposure to docetaxel or 5-FU, which was shown in a previously published study (48). This contradicts the hypothesis that p16-positive HNSCCs have increased sensitivity to chemotherapy.
MMPs are said to support tumor cell promotion in invasive carcinoma, primary tumor growth, angiogenesis, invasion, intravasation, extravasation, local migration and sustained tumor growth (18). MMP-2 and MMP-14 are important for the local degradation of the ECM, which is crucial for tumor cell invasion (especially for an early-stage tumor) and the formation of metastasis. Most clinical studies that investigated the effectiveness of MMP-Inhibitors included patients with advanced-stage tumors. Prolonged progression-free survival in combination with standard antitumor therapy was observed, but overall survival did not improve with MMP-Inhibitors. Furthermore, MMP-Inhibitors revealed several side effects, such as nausea, fatigue, fevers, abdominal pain, elevation of hepatic enzymes, thrombocytopenia, arthralgia, myalgia and maculopapular rash (49-51). In advanced-stage tumors, the processes of local degradation of the ECM, neoangiogenesis, generation of a tumor microenvironment and the formation of metastases have occurred already. Therefore, inhibition of this process cannot be expected to make a significant therapeutic impact apart from delayed local tumor progress. Advanced-stage HNSCCs also tend to express a wide set and higher levels of MMPs (47). It is not surprising that MMP-Inhibitors are insufficient for therapy of advanced-stage tumors (52-55). Local degradation of the ECM is most relevant in early-stage tumors. It is reasonable to suggest that MMP-Inhibition would be most effective before cancer cells infiltrate the basement membrane, but a carcinoma in situ (CIS) is either not detected or can be successfully treated by local excision.
Branca et al. showed that expression of MMP-2 increases with the grade of an intraepithelial neoplasia. Increased MMP-2 expression was not associated with high-risk HPV, which is consistent with our findings (56). In contrast, Shiau and colleagues detected that HPV-16 up-regulates the expression of MMP-2 and -9 via Interleukin 8 in adenocarcinomas of the lung (57). Our results did not reveal HPV-status dependent differences of MMP-2 and MMP-14 expression. This contradicts the findings of da Silva et al. who demonstrated an HPV-16-associated increase of MMP-2 and -14 expression (58). Furthermore, CERV196 appeared to be less sensitive to reduced MMP expression following incubation with either 5-FU or docetaxel. Our study evaluated the MMP expression in tumor cells only. As previously mentioned, MMP-2 is said to be expressed mainly by stromal cells. As tumor stromal cells are not present in cell cultures, the relevance of stromal expression of MMPs could not be evaluated in this study.
Further studies are planned to investigate the relevance of MMPs in the progression of HNSCC with eventual examination in an animal model.
Acknowledgements
We, the authors, would like to thank Petra Prohaska for her outstanding technical assistance and PD Dr. C. Weiss for her distinguished advice on statistical analysis.
- Received May 11, 2014.
- Revision received June 25, 2014.
- Accepted June 26, 2014.
- Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved