In Vitro Modulation of MMP-2 and MMP-9 Secretion by Cytokines, Inducers, and Inhibitors in Head and Neck Squamous Carcinoma Cells (FaDu) and Tongue Carcinoma Cells (SCC-25)

C l i n M e d International Library Citation: M Waheed Roomi, Bhanap B, Niedzwiecki A, Rath M (2017) In Vitro Modulation of MMP-2 and MMP-9 Secretion by Cytokines, Inducers, and Inhibitors in Head and Neck Squamous Carcinoma Cells (FaDu) and Tongue Carcinoma Cells (SCC-25). J Otolaryngol Rhinol 3:029 Received: November 02, 2016: Accepted: February 18, 2017: Published: February 21, 2017 Copyright: © 2017 M Waheed Roomi, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. M Waheed Roomi et al. J Otolaryngol Rhinol 2017, 3:029 Volume 3 | Issue 1


Introduction
Head and neck squamous cell carcinoma (HNSCC) is a devastating disease with poor prognosis. In the United States, HNSCC is the sixth most commonly diagnosed malignancy and approximately 60,000 cases are diagnosed annually. These include cancers of the oral cavity, larynx, salivary glands, tongue, thyroid, and nose [1,2]. Men are most commonly affected over the age of 50. Head and neck cancers are known for their aggressive nature and propensity to metastasize. The greatest risk factors for HNSCC are tobacco and alcohol use. Eighty-five percent of head and neck cancer cases can be linked to tobacco use including smokeless tobacco [2,3]. Other risk factors for HNSCC include sun exposure, occupational exposure to nickel, radium, chromium, mustard gas, leather, wood dust, and radiation, Epstein Barr Virus infections, and DNA repair disorders such as Fanconi anemia. The resulting costs associated with the treatment of HNSCC in the United States alone add up to billions of dollars per year for surgery, radiation therapy, chemotherapy and post-treatment care [4].

Gelatinase zymography
Gelatinase zymography was performed in a 10% NOVEX Pre-Cast SDS Polyacrylamide Gel (Invitrogen Corporation) in the presence of a 0.1% gelatin under non-reducing conditions. Culture media (20 µL) were mixed with a sample buffer and loaded for SDS-PAGE with tris-glycine SDS buffer as suggested by the manufacturer (Novex). Samples were not boiled [6,7]. Chemotherapy and radiotherapy indiscriminately attack normal as well as diseased cells. Such treatments often lead to serious side effects like anemia, an impaired immune system predisposing the patient to infections, and bleeding and additional cancers caused by the highly toxic nature of chemotherapeutic agents or radiation damage to DNA from radiotherapy [8]. For HNSCC, even when detected and treated in its early stages, the radiation or chemotherapy used creates a 9%-23% incidence of secondary tumors, which decreased overall survival rates [7,8].
Degradation of the extracellular matrix (ECM) by matrix metalloproteinases (MMPs) play a critical role in the formation of tumors and metastasis. MMPs are proteolytic enzymes that digest collagen IV and other components of ECM and the cell basement membrane. In particular MMP-2 and MMP-9 play a key role in the degradation and a good correlation has been observed in increased incidence of invasion and metastasis with increased MMP-2 and MMP-9 [9][10][11]. Moreover, an increased MMP-9 secretion was noted to correlate with aggressiveness, relapse and shortened survival rates in head and neck cancers [12][13][14]. The essential role played by the MMPs in metastasis makes MMP-2 and MMP-9 inhibitors (MMPIs), logical targets for therapeutic intervention in cancers. The identification and synthesis of specific non-toxic MMP-2 and MMP-9 inhibitors has been the target of extensive research for several years [15][16][17][18]. However, to date the MMPIs that have been clinically tested have not been proven effective in Phase 3 clinical trials because of poor protocols. MMP activity is regulated by, and dependent upon, environmental influences from surrounding stroma cells, ECM proteins, cytokines, inducers and inhibitors, and other factors [19,20]. Furthermore, MMPs are regulated at multiple levels including transcription, modulation of messenger RNA half-life (translation), secretion, localization, activation, and inhibition [21].
In this study, we investigated the effects of selected cytokines, inducers, mitogens and natural and chemical inhibitors, affecting cancer cell metabolism on the regulation of MMP-2 and MMP-9 activities on HNSCC and SC-255 cell lines. We also studied the agents that affect transcription and translation of genes, cyclohexamide and actinomycin-D respectively. Here we also report the dose dependent MMP-2 and MMP-9 inhibitory effects of a non-toxic nutrient mixture (NM) consisting of vitamin C, L-lysine, L-proline, and green tea extract (EGCG) compared to other MMP-2 and MMP-9 inhibitors, cytokines, mitogens, and inducers.

Materials
The HNSCC (FaDu), tongue cell carcinoma (SCC-25) cell lines used were purchased from ATCC (American Type Culture Collection, Rockville, MD, USA), along 1D shows the densitometry analysis of their zymograms. As shown in figure 1A, FaDu cells expressed two faint bands, and TC cells expressed two moderate bands corresponding to MMP-2 (72 kDa) and MMP-9 (92 kDa) (figure not shown). On PMA stimulation TC still showed two bands corresponding to MMP-2 and MMP-9. PMA did not alter the pattern of MMP-2 secretion; however, PMA treatment showed a significant dose dependent increase in MMP-9 secretion. PMA had no stimulatory effect on MMP-2 in FaDu cells, and significant dose dependent stimulatory effect on MMP-9 secretion. These results were further confirmed by densitometry analysis (Trend analysis: MMP-2, R2 = 0.3456; MMP-9, R2 = 0.842 for FaDu and MMP-2, R2 = 0.4306; MMP-9, R2 = 0.5337 for TC cell line). TNF-α, IL-1β and LPS have a stimulatory effect on MMP-9 secretion in both FaDu and TC cells in dose dependent manner. TNF-α and IL-1β had a negligible effect on MMP-2 in either cell line, however LPS had a moderate effect on MMP-2 secretion in FaDu and no effect on TC cells.

Effect of inhibitors doxycycline and EGCG on MMP-2 and MMP-9 secretion in FaDu and TC cell lines:
The effects of doxycycline and EGCG on MMP-2 and MMP-9 secretion in FaDu and TC cells were investigated with and without PMA. As shown in table 2 quantitative densitometry analysis of gelatinase zymography shows a dose dependent decrease in MMP-2 and MMP-9 secretion with doxycycline and EGCG. When stimulated with PMA to express MMP-9, both MMP-2 and MMP-9 decreased in a dose dependent manner with EGCG in before electrophoresis. Following electrophoresis, the gels were washed twice in a 2.5% Triton X-100 for 30 minutes at room temperature to remove SDS. The gels were then incubated at 37 °C overnight in a substrate buffer containing 50 mM Tris-HCl and 10 mM CaCl 2 at pH 8.0 and stained with a 0.5% Coomassie Blue R250 in a 50% methanol and a 10% glacial acetic acid for 30 minutes and destained. Protein standards were run concurrently and approximate molecular weights were determined by plotting the relative mobilities of known proteins. Gelatinase zymograms were scanned using a Canoscan 9950F Canon scanner at 300 dpi. The intensity of the bands was evaluated using the pixel-based densitometer program Un-Scan-It, Version 5.1, 32-bit, by Silk Scientific Corporation (P.O. Box 533, Orem, UT 84059, USA), at a resolution of 1 Scanner Unit (1/100 of an inch for an image that was scanned at 100 dpi). Table 1 shows the quantitative densitometry results from the effect of inducers PMA, TNF-α, IL-1β and LPS on MMP-2 and MMP-9 secretion in FaDu and TC cell lines. Figure  1A and figure 1C show gelatinase zymography of MMP-2 and MMP-9 secretion in PMA (0-100 ng/ml) treated TC and FaDu cells, respectively, and figure 1B and figure Table 1: Effect of inducers, cytokines and mitogens on MMP-2 and MMP-9 secretion in laryngeal squamous cells carcinoma cells (FaDu) and tongue cancer cells (SCC-25).

Discussion
Numerous studies have shown that most cancerous cells secrete massive quantities of matrix metalloproteinases, particularly MMP-2 and MMP-9, which remodel     MMP secretion in different types of tumor cells [31,32]. There are not many studies conducted on HNSCC and TC referring to cytokine and growth factor assays.
In this study, we compared MMP secretion patterns by cytokines (TNF-α, Il-β), inducers (PMA), and mitogenes (LPS) in HNSCC and TC cell lines that express MMP-2 and MMP-9 to different extents. Gelatinase zymography was utilized because of its high sensitivity to gelatinolytic enzymatic activity and ability to detect both pro and active forms of MMP-2 and MMP-9. Among various MMPs, MMP-2 and MMP-9 play a critical role in tumor cell invasion and metastasis [7,33]. MMP-2 (molecular weight 72 kD) is cleaved by other MMPs to yeild the activated forms of 68, 58, and 54 kD and MMP-9 (mol. Wt. 94 kD) into 92 kD. Upon renaturation of the enzyme, the gelatinases digest the gelatin in the gel and reveal clear bands against an intensely stained background. In addition, we also investigated the effect of inhibitors such as doxycycline, dexamethasone, retinoic acid and agents that affect transcription and translation levels such as actinomycin-D and cyclohexamide. The mechanism of doxycycline to inhibit MMP enzymes has been used in various diseases including wound healing, the development of effective matrix metalloproteinase inhibitors (MMPI) is a particularly attractive strategy for chemotherapeutic agents. An effective MMPI would not harm healthy cells at clinically relevant concentrations, would have no significant side effects, would target specific cancer cells, and completely suppress the secretion of MMP-2 and MMP-9, thus halting the metastatic process [15,23,24]. Theoretically, the specificity of MMP inhibitors would avoid collateral damage to healthy tissue caused by both chemotherapy and radiotherapy thereby increasing the long-term survival prospects and reducing or eliminating many of the debilitating side effects of current methods of HNSCC therapies [16,17,25].
Experimental and clinical studies have shown a correlation between increased MMPs and tumor progression and metastasis [7]. Moreover, MMP-2 and -9 are identified as useful phenotypic indicators to establish the invasive potential of oral cancers [26][27][28][29][30]. Thus, knowledge of MMP regulation is of importance for developing therapeutic strategies. MMP secretion is regulated at both pre-and post-transcriptional levels. Extracellular factors, including cytokines, growth factors, inducers and inhibitors, have been implicated in the regulation of cally inhibits overproduction of MMP-2 and MMP-9 in HNSCC. Finally, at a concentration of 500 µg/mL and 1000 µg/mL, the clinically relevant dosages, the NM completely suppressed the secretion MMP-2 and MMP-9 in both cell lines. Furthermore, the NM has been tested in other in vitro studies to prove its safety on vital organs such as, heart, liver and kidney or on the associated serum enzymes [43].
Our results show that inducers, cytokines, and mitogens show downregulation of MMP-2 and MMP-9 in HNSCC and TC. In conclusion, our results showed that cytokines, mitogens, and inhibitors modulated MMP-2 and MMP-9 secretion in FaDu and SCC-25 cells, suggesting the clinical use of MMP inhibitors, especially such potent and non-toxic ones as the nutrient mixture and its component EGCG, in management of head and neck cancers. cardiovascular diseases to anticancer properties [34][35][36]. Similarly, EGCG is shown to inhibit invasion and migration of oral cancer cells by decreased production of MMP-2, MMP-9 and uPA [37,38]. Retinoic acid has been researched as a potential agent for its use in treating different forms of cancer not only in prevention but also in treatment [39,40]. Dexamethasone has been shown to suppress the tumor invasion by targeting NF-κB especially in squamous cell carcinoma [41]. Therefore, we used these agents as comparison. Furthermore, we tested a nutrition mixture that had inhibitory effects on MMP-2 and MMP-9 secretion.
We found that while the FaDu cell line expressed two faint bands the TC cells expressed moderate bands of both MMP-2 and MMP-9. PMA, TNF-α, IL-1β and LPS treatment of both the cell lines strongly increased the secretion of MMP-9 in a dose-dependent manner but had negligible effect on MMP-2. TNF-α had a slight stimulatory effect on MMP-2 while a strong stimulatory effect was observed on MMP-9 in FaDu cells. However, TNF-α had a dose dependent decrease in MMP-9 secretion of TC cells. LPS showed a slight inhibition of MMP-2 and MMP-9 in HNSCC and a slight inhibition of MMP-2 in TC cells. Doxycycline and EGCG inhibited MMP-2 and MMP-9 secretion in a dose-dependent fashion in both the cell lines tested. Similarly, dexamethasone, Actinomycin-D, and cyclohexamide had an inhibitory effect on MMP-2 and MMP-9.
We have developed strategies to inhibit cancer development and its spread using naturally occurring nutrients such as lysine, proline, ascorbic acid, and green tea extract in a nutrient mixture (NM). The NM was designed by focusing on physiological targets in cancer progression and metastasis as documented in clinical and experimental studies. The nutrient mixture was formulated by selecting nutrients that act on critical physiological targets in cancer progression and metastasis as documented in both clinical and experimental studies. Combining these micronutrients expands metabolic targets, maximizing biological impact with lower doses of components. A previous study of the comparative effects of NM, green tea extract (GTE) and EGCG on inhibition of MMP-2 and MMP-9 secretion of different cancer cell lines with varying MMP secretion patterns, revealed the superior potency of NM over GTE and EGCG at equivalent doses [42]. These results can be understood from the more comprehensive treatment offered by the combination of nutrients in NM over individual components of NM since MMP-2 and MMP-9 are mediated by differential pathways.
Our current study demonstrates that the specific mixture of nutrients tested is as effective as the inhibitors doxycycline, dexamethasone, Actinomycin-D, cyclohexamide, and retinoic acid in stopping MMP-2 and MMP-9 secretion in the HNSCC (FaDu) and TC (SCC-25) cell lines. The NM does not harm healthy cells and specifi-