Nasopharyngeal Carcinoma Cell And Mmp9 Production Biology Essay

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The matrix metalloproteinase (MMP) is one of the potential candidates responsible for tumor metastasis and invasion. Previous study has shown that the expression level of MMP9 mRNA was significantly greater in NPC tissues than that in the non-cancerous nasopharyngeal tissues. It is also believed responsible for tumor metastasis and invasion of NPC. The purpose of this study is to examine the anti-metastasis effects of 2-Methoxyestradiol (2ME2) on NPC cells. 2ME2, an endogenous derivative of E2 formed by the hydroxylation and subsequent methylation at the 2-position, is used as a chemotherapeutic agent to inhibit the growth of different tumors by its anti-proliferative and anti-angiogenic activity. C666-1, an EBV positive NPC cell lines was used in this study. Results from gelatin zymography indicated that 2ME2 could reduce the activity of MMP-9, and hence MMP-9 might also mediate the antitumour activity via the suppression of migration of NPC cells.

Nasopharyngeal carcinoma (NPC) is a special kind of cancer that occurs in head and neck (Vokes et al., 1997), developing in the epithelial cells that cover the surface of nasopharynx (Brennan, 2006). Nasopharynx is a hollow tube situates behind the nose, reaches the region of soft palate comprising the roof of the mouth (Jeyakumar et al., 2006) as showed in Figure 1.1. The air we breathe is moisturized and filtered by the nasopharynx, furthermore, the ear pressure and balance are also equalized and maintained by it (Tortora & Grabowski, 2000).

1.1.1 Symptoms of NPC

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Due to the anatomic location of nasopharynx, the diagnosis of NPC is rather difficult. Potential noticeable symptoms of NPC include enlarged cervical lymph nodes without pain, nasal congestion and bleeding (epistaxis), diminished hearing and ringing in the ears (tinnitus), dysfunction of cranial nerve, perennial otitis media, headache and sore throat (Cho, 2007).

1.1.2 Histopathology of NPC

According to the World Health Organization (WHO) classification of NPC, three types of NPC were identified in 1978. Type I is a keratinizing squamous cell carcinoma. Type II is a non-keratinizing carcinoma is Type III is an undifferentiated carcinoma (Ou et al., 2007; Shanmugaratnam & Sobin, 1993).

In 1991, WHO classification merged Type II and Type III NPC into non-keratinizing carcinoma. Non-keratinizing carcinoma was then divided into 'differentiated' and 'undifferentiated' ones. The differentiated subtype substituted the original Type II and the undifferentiated subtype substitute the original Type III. The keratinizing squamous cell carcinoma still constituted the Type I NPC (Ou et al., 2007; Lo et al., 2004; Barnes et al., 2005).

1.1.3 Epidemiology of NPC

The geographic distribution of NPC was distinctive. NPC was uncommonly found in North America and Europe, with a comparatively small number of cases reported (Hirayama et al., 1980; Tai & Mould, 2001a; Tai & Mould, 2001b). People from Southeast Asia and southern China were mainly be affected by NPC (Fedder & Gonzalez, 1985). NPC is known as Cantonese Cancer since it was particularly prevalent in southern China, especially in Guangdong province (Cao et al., 2008) and Hong Kong (Lee et al., 2003). With reference to the statistics of Leading Cancer Site in Hong Kong in 2008 from Hong Kong Cancer Registry, Hospital Authority, NPC was the seventh most common cancer and cancer killer in Hong Kong. There were 926 new cases of NPC registered in 2008, males contributed to 679 cases and females contributed to 247 cases. The male: female ratio is 2.7:1.

1.1.4 Risk factors for NPC

1.1.4.1 Epstein-Barr virus (EBV)

Epstein-Barr virus (EBV) is a herpes virus. This virus with lifelong persistence infects over 90% of the population worldwide. Usually an individual was latently infected for life by primary infection (Crawford, 2001). Previous studies showed that EBV contributes to certain disease like Hodgkin's disease (HD), Burkitt's lymphoma(BL), T-cell lymphoma, stomach cancer and NPC (Crawford, 2001; Hannigan & Wilson, 2010). Evidence showed that NPC patients have board range and higher levels of anti-EBV antibodies, with increased levels of IgA antibodies (Henle & Henle, 1980). Development of NPC results from the increased levels of serum antibodies by certain years (Ho et al., 1978a; Lanier et al., 1980). EBNA1, EBER, LMP1 and LMP2 are latent genes expressed during EBV infection (Izumi, 2001; Kaye et al., 2003). LMP1 is responsible for tumor progression and invasion in human epithelial cells (Yoshizaki et al., 1998; Kim et al., 2000).

1.1.4.2 Environmental factors

Consumption of Cantonese salted fish was suspected to be a cause of NPC development in southern Chinese in the early 1970s (Ho et al., 1978b). Further studies have shown that consumption of Cantonese salted fish in Guangxi and Guangdong in Southern China (Zheng et al., 1994; Yu et al., 1989a; Guo et al., 2009), Tianjin and Shanghai in Northern and Eastern China (Ning et al., 1990; Yuan et al., 2000), was highly correlated to the NPC development. Moreover, the consumption of salted fish in childhood (weaning) demonstrated to be at a higher risk of developing NPC than adulthood exposure (Chang & Adami, 2006). Animal studies showed that rats fed on salted fish developed nasal or nasopharyngeal carcinoma (Huang et al., 1978; Yu et al., 1989b). Besides salted fish, consumption of other preserved food like fermented bean curd, salted vegetables and salted shrimp paste was reported to be related to the increased NPC risk in Chinese (Yu et al., 1989a; Ning et al., 1990). Moreover, intake of alcoholic beverages and smoking would also increase the chance of getting NPC (Cheng et al., 1999). Exposing human to fumes and wood dust occupationally or domestically was also account for the development of NPC (Hildesheim et al., 2001).

1.1.4.3 Genetic factors

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Genetic factors also play a crucial role in the development of NPC. Previous studies indicated that human leukocyte antigens (HLA), a kind of MHC antigens of humans, associated with NPC mainly among the Chinese (Simons et al., 1974; Chan et al., 1983). Simons et al. showed that HLA antigens contribute to the determination of the risk of NPC and survival of NPC patient (Simons et al., 1978). Besides HLA, genetic polymorphisms of enzyme cytochrome P450 2E1 (CYP2E1) was also reported to be related to the NPC development. CYP2E1 is an enzyme responsible for the metabolic activation of nitrosamines (Smith et al., 1992; Patten et al., 1997; Camus et al., 1993). In Taiwan, former studies revealed that NPC development was related to this enzyme among Chinese (Hildesheim et al., 1995; Hildesheim et al., 1997).

1.1.5 Treatment of NPC

Due to the anatomic location of NPC, surgery is seldom considered (Brennan, 2006). The primary mode for NPC treatment is Radiotherapy (RT), and the secondary mode for NPC treatment is chemotherapy since NPC is also sensitive to certain chemotherapeutic drug (Agulnik & Siu, 2005). Chemotherapy can be added to radiotherapy in different modes, including neoadjuvant (given before RT), concurrent (given at the same time with RT), adjuvant (given after RT), neoadjuvant and adjuvant (given before and after RT), concomitant and adjuvant (given at the same time with RT and followed by another chemotherapy). Agulnik and Siu believed that novel treatments including intensity-modulated Radiotherapy, molecular targeted therapy and immunotherapy with EBV-specific cytotoxic T lymphocytes needed to be further explored (Agulnik & Siu, 2005).

1.2 Metastasis

Primary tumor growth is not lethal; however, metastasis increases the risk of death of cancer sufferers (Yoon et al., 2003). Tumor metastasis is the movement of a single or group of cancer cells from a primary site to secondary ones that requires many steps (Deryugina & Quigley, 2006) as follows: a single or group of cancer cells detach from primary tumour and evade from anoikis. Then, the cancer cells can move in the degraded extracellular matrix. When the tumor cells escape through the basement membrane, they would invade the lymph and blood vessels followed by a sequential immunological evasion and attach to the endothelial cells. Afterward, the cancer cells pass out of the vessels into surrounding tissues followed by certain proliferation and angiogenesis (Bohle & Kalthoff, 1999; Nash et al., 2002). Matrix metalloproteinase-9(MMP-9) is believed to play a crucial role in tumor metastasis (Liabakk et al., 1996).

1.3 Matrix metalloproteinase-9 (MMP-9)

Matrix metalloproteinase-9 (MMP-9), one of the members of the matrix metalloproteinases family, is synthesized as a proenzyme with a molecular mass of 92 kDa in human (Atkinson & Senior, 2003). In human, the gene of MMP-9 is located on chromosome 20q11.1-13.1 and MMP-9 deficiency is not likely happened (Zhang et al., 1999). The promoter region of MMP-9 gene comprises binding sites for AP-1, AP-2, SP-1, NF-κB and Ets, with an inhibitory element of TGF-β (Atkinson & Senior, 2003). MMP-9 is a 92-kDa type IV collagenase, also named as gelatinase B, involving fibronectin type II-like repeats for binding of gelatin and elastin (Shipley et al., 1996). Previous studies have revealed that the expression level of MMP-9 mRNA and protein were significantly greater in various cancers (Roomi et al.,2010; Qin et al., 2008; Zheng et al., 2010; Liu et al., 2010). MMP-9 is believed to play a crucial role in tumor metastasis by promoting the extracellular matrix and basement membrane degradation (Liabakk et al., 1996).

1.4 2-methoxyestradiol (2ME2)

2-methoxyestradiol (2ME2), which is a naturally occurring and endogenous derivative of 17β-estradiol (E2), that formed by consecutive 2-hydroxylation and O-methylation (Gelbke & Knuppen, 1976). The chemical structure of 2ME2 is shown in figure 1.2. It is now accepted as a potent therapeutic agent for cancer with no toxicity although once disregard as an inert end-metabolic of estradiol (Zhu & Conney, 1998; Lui et al., 2000).

Different from 17β-estradiol (E2) which would induce proliferation of cancer cells depending on estrogen receptor, previous studies have reported that 2ME2 demonstrates anti-cancer character in various examples (Fotsis et al., 1994; Mukhopadhyay & Roth, 1997; Schumacher et al., 1999; Seegers et al., 1997; Pribluda et al.,2000; Zhu & Conney, 1998) and inhibits growth of different tumors by anti-angiogenic and anti-proliferative activity (Pribluda et al.,2000; Zhu & Conney, 1998; Mooberry, 2003). Certain mechanisms of action for these activities were demonstrated by 2ME2 such as regulating cell cycle kinases and arrest (Lottering et al., 1996; Zoubine et al., 1999; Attalla et al., 1996), inducing apoptosis in different tumors (Mooberry, 2003), affecting superoxide dismutase (Huang et al., 2000; Kachadourian et al.,2001), disrupting microtubule dynamics (D'Amato et al., 1994; Cushman et al., 1995), up-regulating p53 (Mukhopadhyay & Roth, 1997; Seegers et al., 1997). 2ME2 also possess anti-metastasis effect (Plum et al., 2009).

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Previous studies reveled that 2ME2 had limited oral bioavailability in capsule form (Dahut et al., 2006; Sweeney et al., 2005). Thus, 2ME2 is reformulated to Nanocrystal dispersion (NCD). Improvement of drug dissolution rate was achieved by diminishing the size of drug particles into nanometer-sized particles in NCD (Kawashima, 2001). Steady plasma levels of 2ME2 were achieved by 2ME2 NCD in certain preclinical studies, indicating that oral bioavailability of 2ME2 in humans could be improved by using 2ME2 NCD. A phase I study was then conducted by Tevaarwerk et al. in patients with solid malignancies by using 2ME2 NCD. Pharmacokinetics variables and a principal metabolite of 2ME2 were assessed in blood samples. At steady state, the minimum effective concentration attained by trough levels in all dose groups and the maximum tolerated dose of 1000 mg orally for every 6 hours was suggested for phase II regimen (Tevaarwerk et al., 2009).

1.5 Objective

Although the primary mode for NPC treatment is Radiotherapy (RT), NPC is also sensitive to certain chemotherapeutic drug. Chemotherapy can be added in different modes to aid radiotherapy. 2ME2, regarded as a potent therapeutic agent for cancer with no toxicity, demonstrates anti-cancer character in various examples and inhibits growth of different tumors by anti-angiogenic and anti-proliferative activity. However, the anti-metastasis effect of 2ME2 on NPC has not been fully studied. Thus, we would like to examine the anti-metastasis effects of 2ME2 on C666-1 NPC cells in this study. After confirming 2ME2 could inhibit the migration of C666-1 cells, we would like to investigate the effect of 2ME2 on MMP-9 activity and EGF-induced MMP-9 activity in C666-1 cells.

Figure1.1 Diagram showing parts of the pharynx.

Adapted from © 1998-2011 Mayo Foundation for Medical Education and Research (MFMER).

Figure 1.2 The chemical structure of 2ME2.

Chapter 2

Materials and Methods

2.1 Materials

2.1.1 Cell Lines

C666-1, a subclone of C666, was derived from undifferentiated nasopharyngeal carcinoma (NPC), which is an EBV-positive NPC cell line (Cheung et al., 1999). C666-1 cells were cultured in RPMI and supplemented with 10% Fetal Bovine Serum (FBS), 1% Penicillin and Streptomycin. Cell lines were grown in a humidified incubator at 37°C with 5% CO2. For the actual experiments, cell culture medium has been replaced with RPMI medium.

2.1.2 Reagents for cell culture

2.1.2.1 Fetal Bovine Serum (FBS)

Fetal Bovine Serum (Clontech, cc4101) was stored at -20°C until use.

2.1.2.2 Penicillin and Streptomycin (PS)

Penicillin and Streptomycin antibiotics (5000 units/ml penicillin and 5000 µg/ml Streptomycin) (GIBCO, 15070-063) was stored at -20°C until use.

2.1.2.3 RPMI 1640 Medium

RPMI 1640 power (Sigma, R6540) was dissolved in 800ml of MilliQ water with 2g Sodium carbonate. The pH of solution was adjusted to 7.3 by adding 2M NaOH and 5M HCl solutions, then the solution was finally made up to 1 liter. The medium was then filtered and stored at 4°C. 10% of FBS and 2 % of Penicillin and Streptomycin (PS) were used to complete the RPMI medim. The cRPMI was stored at 4°C until use.

2.1.2.4 Dulbecco's Phosphate Buffered Saline (PBS)

PBS powder (Sigma, D5652) was dissolved in 800ml of MilliQ water then made up to 1 liter. The solution was sterilized by filtering through a filter. Filtered PBS was stored at room temperature.

2.1.2.5 Trypsin-EDTA (1X)

Trypsin-EDTA with 0.25% trypsin and 1mM EDTA (Invitrogen, 25200-072) was stored at -20°C until use.

2.1.3 Reagent for cell counting

2.1.3.1 Trypan blue stain 0.4%

Trypan blue stain 0.4% (Sigma, T-6146) was stored at 4°C until use.

2.1.4 Chemicals

2.1.4.1 Purified human Matrix metalloproteinase-9 (MMP-9)

Purified human MMP-9 (Chemicon, CA92590) with stock concentration of 0.1mg/ml was stored at -80°C until use.

2.1.4.2 2-Methoxyestradiol (2ME2)

2-Methoxyestradiol (2ME2) (Sigma, M6383) with stock concentration of 20mM was prepared. The 20mM 2-Methoxyestradiol was stored at -20°C until use.

2.1.4.3 EGF

EGF (Clontech, cc4017) with stock concentration of 2mg/ml was stored at -20°C until use.

2.1.5 Reagent for Wound healing assay

2.1.5.1 Fibronectin

Fibronectin (Millipore, 92509) with stock concentration of 1mg/ml was stored at 4°C until use.

2.1.6 Reagents for Transwell migration assay

2.1.6.1 1.5% Paraformaldehyde

35% Paraformaldehyde (Merck, 3688617) was added to PBS and stored at room temperature.

2.1.6.2 0.2% Triton X-100

0.2% Triton X-100 (USB, 22686) was prepared by dissolving 250µl of Triton X-100 in 9.75ml of PBS.

2.1.6.3 4',6-diamidino-2-phenylindole (DAPI)

4',6-diamidino-2-phenylindole (DAPI) (Sigma, D8417) with stock concentration of 1mg/ml was stored at 4°C until use.

2.1.7 Materials for Gelatin zymography

2.1.6.7 Non-reducing Sample buffer (5X)

Mixture of 5ml 1.5M Tris-HCl (pH6.8) (USB, 22676), 2.4g SDS (USB, US75819), 12ml glycerol (USB, US16347), 5mg bromophenol blue (Amersham Bioscince, US12370) and 1.2 ml MilliQ water made up the non-reducing sample buffer (5X). The buffer was aliquoted and stored at -20°C until use.

2.1.7.2 Reagents for Stacking and Resolving gel

1) 2% gelatin

0.2g of gelatin powder (Sigma, G1890) was dissolved in 10 ml of MilliQ water, and stored at 4°C until use.

2) 30% Acrylamide/Bis solution, 29:1 (3.3%C)

30% Acrylamide/Bis solution (Bio-Rad, 161-0156) was stored at 4°C until use.

3) 1.5M Tris-HCl (pH8.8)

36.34g of Tris-HCl powder (USB, US75825) was dissolved in 100ml of MilliQ water. The pH of solution was adjusted to 8.8 by adding 2M NaOH and 5M HCl solutions, and then the solution was finally made up to 200ml.

4) 0.5M Tris (pH6.8)

12.11g of Tris (USB, US75825) was dissolved in 100ml of MilliQ water. The pH of solution was adjusted to 6.8 by adding 2M NaOH and 5M HCl solutions, then the solution was finally made up to 200ml.

5) 10% SDS

10g of SDS powder (USB, US75819) was dissolved in 100ml of MilliQ water and stored at room temperature.

6) 10% Ammonium Persulfate (APS)

3g of APS powder (USB, 76322) was dissolved in 30ml of MilliQ water and aliquoted to 1.5 ml eppendorfs and stored at -20°C until use.

7) TEMED

TEMED (Amersham Bioscience, US76320) was stored at room temperature.

8) 2-propanol

2-propanol (Fluka, 59300) was stored at room temperature.

2.1.7.3 Electrode buffer

Mixture of 144.2g of glycine, 30g of Tris and 1g of SDS was dissolved in 800ml MilliQ water. The pH of solution was adjusted to 8.3 by adding 2M NaOH and 5M HCl solutions, then the solution was finally made up to 1 liter.

2.1.7.4 2.5% Triton X-100

2.5% Triton X-100 (USB, 22686) was prepared by dissolving 5ml of Triton X-100 in 195ml of MilliQ water and stored at room temperature.

2.1.7.5 Substrate buffer

4ml of 10mM CaCl2 (Sigma, C-3881), 6ml of 0.15 M NaCl (USB, US21618), 10ml of 50mM Tris-HCl (pH 8) (USB, 22676) and 0.02% Brij-35 were mixed with 180ml of MilliQ water and stored at room temperature.

2.1.7.6 0.5% Coomasie blue

1g of Coomassie Blue (Fluka, 27815) was dissolved in 10% acetic acid (20ml) (Sigma, 19516), 40% methanol (80ml) (Merck, 1.060007.2500) and 50% of MilliQ water (100ml).

2.1.7.7 Destaining solution

10% acetic acid (20ml) (Sigma, 19516), 40% methanol (80ml) (Merck, 1.060007.2500) and 50% of MilliQ water (100ml) were mixed.

2.2 Methods

2.2.1 Wound healing assay

Wound healing assay was used to study cell migration in vitro. For better cell attachment, each well in 96-well plate was coated with 50µl of 10µg/ml of fibronectin at 4°C overnight. After removing the excess fibronectin, 100µl of 8 Ã- 104 of C-6661 cells were seeded to 96-well plate for 24 hours. The floating cells were removed and pipette tips were used to wound the cell monolayers. Images of the wounded area were captured with a X10 objective immediately after wounding at 0 hour, 24 hours and 48 hours to see the migration of cells into the wounded area.

2.2.2 Transwell migration assay

C666-1 cells with cell density of 5 Ã- 105 were seeded to 35-mm petri dish and grown in a humidified incubator at 37°C with 5% CO2 for 72 hours. The cells were treated with 10mM of 2ME2 for 72 hours. After 72 hours, 100ul of 2 Ã- 105 of 2ME2-treated cells in serum-free RPMI medium were seeded to a membrane in the upper chamber of a transwell. The transwell was placed in a 24-well plate consisted of serum-free medium RPMI. The cells were allowed to migrate for 72 hours in RPMI. After 72 hours, the non-migrated cells in the upper chamber were removed by pipettement and cotton bud. At 37°C, cells that had migrated to the lower side of the membrane were fixed with 1.5% paraformaldehyde for 20 minutes, followed by 3 times PBS washing. Afterward, the cells were permeablized by 0.2% Triton X-100 for 10 minutes, followed by 3 times PBS washing. After the washing, the cells were subsequently stained with 4',6-diamidino-2-phenylindole (DAPI) for 10 minutes, then destained in PBS for 5 times. The transwell membrane was removed and mounted on a glass slide in an upside down position, immersed in a PBS and covered with a cover slip. The DAPI-stained cells were then visualized under a microscope. The C666-1 cells that had migrated through the membrane pore spontaneously were counted and expressed as the percentage of migration compared with the control.

2.2.3 Gelatin zymography

Conditions for gelatin zymography were optimized. The enzymatic activity of MMP-9 in C666-1 cells was determined by a gelatinolytic activity assay, SDS-PAGE gelatin zymography. C666-1 cells with cell density of 5 Ã- 105 were seeded to 35-mm petri dish and grown in a humidified incubator at 37°C with 5% CO2 for 72 hours. In experiments studying the effect of 2ME2 on MMP-9 activity, the cells were treated with 10mM of 2ME2 for 72 hours. In experiment studying the effect of EGF on MMP-9 activity, the cells were starved for 24 hours, and then treated with 0, 10, 50, 100, 200, and 400ng/ml of EGF for 72 hours. In experiment studying the co-treatment effect of 2ME2 on EGF-induced MMP-9 activity, the cells were starved for 24 hours, and the control set was treated with 400ng/ml of EGF for 72 hours, while the experimental set was co-treated with 400ng/ml of EGF and 10mM of 2ME2 for 72 hours. In experiment studying the pre-treatment effect of 2ME2 on EGF-induced MMP-9 activity, the cells were starved for 24 hours, the experimental set was treated with 10mM of 2ME2 for 6 hours. Then both the control and experimental sets were treated with 400 ng/ml of EGF for another 66 hours. Conditioned media were then collected and centrifuged at 14000 rcf for 15 minutes. Afterwards, the conditioned media were denatured in the absence of reducing agent and electrophoresed in a 7.5% SDS-polyacrylamide gel containing 0.1% (w/v) gelatin. The gels were then incubated at room temperature for 1 hour in the presence of 2.5% Triton X-100 with gentle agitation and afterwards at 37°C for 45 minutes in a substrate buffer containing 10mM CaCl2, 0.15 M NaCl, 50 mM Tris-HCl (pH 8) and 0.02% Brij-35. Then the gels were incubated overnight at 37°C in a new substrate buffer. Gels were stained with 0.5% Coomassie Blue and destained with destaining solution until clear bands were observed. Clear bands over the dark background represent areas of enzymatic activity.

2.2.4 Statistical analyses

Data are expressed as means ± SDs. Two-tailed Student's t-test was performed for statistical analysis between control and treatment groups. Statistical significance (P value) was presented in the figure legends.

Chapter 3

Results

3.1 Effect of 2ME2 on the migration ability of C666-1 cells

Results from wound healing assay were shown in figure 3.1, no wound closures were observed after wounding C666-1 cells for 24 hours and 48 hours. Thus, it is found that wound healing assay is not a suitable assay to study the effect of 2ME2 on the migration ability of C666-1 cells. Then, transwell migration assay was used to the study the effect of 2ME2 on the migration ability of C666-1 cells. Result from transwell migration assay was shown in figure 3.2. The result showed that 2ME2 inhibited the migration of C666-1 cells by reducing the % of migration to 38 % of the control.

3.2 Effect of 2ME2 on MMP-9 activity in C666-1 cells

After confirming 2ME2 could inhibit the migration of C666-1 cells, the effect of 2ME2 on MMP-9 activity in C666-1 cells was studied. In figure 3.3, it is shown that MMP-9 is expressed in C666-1 cells by gelatin zymography. When the cells were treated with 2ME2, reduction in the MMP-9 activity was observed.

3.3 Effect of EGF on MMP-9 activity in C666-1 cells

The result from gelatin zymography showed that EGF treatment caused an increase in MMP-9 activity in C666-1 cells (Figure 3.4). It revealed that 400ng/ml of EGF caused an obvious increase in MMP-9 activity.

3.4 Effect of 2ME2 on EGF-induced MMP-9 activity in C666-1 cells

After confirming EGF could induce the activity of MMP-9 in C666-1 cells, the effect of 2ME2 on EGF-induced MMP-9 activity in C666-1 cells was investigated. When C666-1 cells were co-treated and pre-treated with 2ME2 to the treatment of 400 ng/ml of EGF, no changes in the MMP-9 activity were observed (Figure 3.5 & Figure 3.6). 2ME2 co-treatment and pre-treatment did not inhibit EGF-induced MMP-9 activity in C666-1 cells.

(A)

0hour

(B)

24hour

(C)

48hour

Figure 3.1 Images showing cells migration of C666-1 cells into the wounded area.

Wound healing assays were performed in C666-1 cells (8x 104cells per well coated with fibronectin). Representative images captured with a X10 objective at the time of wounding (A) 0 hour, then (B) 24 hours and (C) 48hours after wounding in C666-1 cells.

**

Figure 3.2 Effect of 2ME2 on the migration of C666-1 cells.

C666-1 cells (5 Ã- 105 cells in 1 ml serum-free RPMI per 35mm dish) were treated with 10mM of 2ME2 for 72 hours. 100ul of 2 Ã- 105 of 2ME2-treated cells in serum-free RPMI medium were then seeded to a transwell. The migration of cells was determined by Transwell migration assay. Cells that had migrated through the membrane pore spontaneously were counted and expressed as the percentage of migration compared with the control. Bar graph was expressed as mean ± SD and two-tailed Student's t-tests were performed (**P < 0.01).

Lane 1 2

2ME2 - +

MMP-9 (92kDa)→

Figure 3.3 Effect of 2ME2 on the MMP-9 activity in C666-1 cells.

C666-1 cells (5 Ã- 105 cells in 1 ml serum-free RPMI per 35mm dish) were treated with 10mM 2ME2 for 72 hours in Lane 2. Lane 1: control without treatment. The conditioned media were collected for determination of MMP-9 activity by gelatin zymography.

EGF (ng/ml)

0 10 50 100 200 400

MMP-9 (92kDa)→

Figure 3.4 Concentration effect of EGF on MMP-9 activity in C666-1 cells.

C666-1 cells (5 Ã- 105 cells in 1 ml serum-free RPMI per 35mm dish) were treated with 0, 10, 50, 100, 200, and 400ng/ml of EGF for 72 hours. The conditioned media were collected for determination of MMP-9 activity by gelatin zymography.

Lane 1 2 3

EGF - + +

2ME2 - - +

MMP-9 (92kDa)→

Figure 3.5 Effect of pre-treated 2ME2 in EGF-induced activity of MMP-9 in C666-1 cells.

C666-1 cells (5x105 cells in 1 ml serum-free RPMI per 35mm dish) were starved for 24 hours. Lane 1: control without treatment. Lane 2: cells were treated with 400ng/ml of EGF for 72 hours. Lane 3: cells were co-treated with 10mM of 2ME2 with 400ng/ml of EGF for 72 hours. The conditioned media were collected for determination of MMP-9 activity by gelatin zymography.

Lane 1 2 3

EGF - + +

2ME2 - - +

MMP-9 (92kDa)→

Figure 3.6 Effect of pre-treated 2ME2 in EGF-induced activity of MMP-9 in C666-1 cells.

C666-1 cells in (5x105 cells in 1 ml serum-free RPMI per 35mm dish) were starved for 24 hours. Lane 1: control without treatment. Lane 2: cells were incubated with RPMI for 6 hours and treated with 400 ng/ml of EGF for another 66 hours. Lane 3: cells were pre-treated with 10mM of 2ME2 for 6 hours and treated with 400 ng/ml of EGF for another 66 hours. The conditioned media were collected for determination of MMP-9 activity by gelatin zymography.

Chapter 4

Discussion

The result of 2ME2 inhibits the migration of C666-1 cells is consistent with the result of 2ME2 reduces transwell migration of transformed murine pre-B-cell line and chronic myelogenous leukemia cells in previous study by Sattler et al. (Sattler et al., 2003). 2ME2 may contribute to reduced metastasis and invasion of C666-1 cells.

Degradation of the extracellular matrix is a crucial step for tumor metastasis and invasion (Liabakk et al., 1996). Matrix metalloproteinase-9 (MMP-9) is believed to play a crucial role in tumor metastasis (Liabakk et al., 1996). Previous studies have revealed that the expression level of MMP-9 mRNA and protein were significantly greater in certain cancers like ovarian cancer (Roomi et al., 2010), breast cancer (Qin et al., 2008), lung cancer (Zheng et al., 2010) and also in NPC tissues (Liu et al., 2010). Liu et al. suggested that the relatively higher MMP-9 protein expression was related to NPC progression and poor prognosis (Liu et al., 2010). This study showed that MMP-9 is expressed in C666-1 cells, which is consistent with the result of Liu et al. that MMP-9 was significantly increased in NPC (Liu et al., 2010). The anti-metastasis effect of 2ME2 was then examined through the MMP-9 activity in C666-1 cells. This study demonstrated that MMP-9 activity in C666-1 cells is reduced by 2ME2. Inhibition of MMP-9 activity in C666-1 cells by 2ME2 may contribute to the reduced transwell migration. The mechanism of 2ME2 to affect the activity of MMP-9 was further studied.

In fact, MMP-9 is regulated by several regulators included epidermal growth factor (EGF) (Qiu et al., 2004), fibroblast growth factor (FGF) (Lungu et al., 2008), interleukin-8 (IL-8) (Chakrabarti and Patel, 2005), interferon beta (IFN-β) (Kurzepa & Stryjecka-Zimmer, 2007), nerve growth factor (NGF) (Khan et al., 2002), tumour necrosis factor (TNF) (Scott et al., 2004), vascular endothelial growth factor (VEGF) (Hollborn et al., 2007), hypoxia inducible factor-1 alpha (HIF-1α) (Schelter et al., 2010). Among those regulators, EGF, a kind of growth factor, binds to its receptor-epidermal growth factor receptor(EGFR) to regulate the cell growth, proliferation, and differentiation (Herbst, 2004). EGF-induced MMP-9 secretion is considered to assist tumour invasion and metastasis in certain cancers like bladder cancer (Nutt et al., 2003), lung cancer (Cox et al., 2000), ovarian cancer (Ellerbroek et al., 1998), head and neck squamous cell (Charoenrat et al., 2000). Similar to the results from previous studies for other cancers, EGF was found to cause an increase in MMP-9 activity in C666-1 cells in this study.

Both 2ME2 pre-treatment and co-treatment did not inhibit EGF-induced MMP-9 activity in C666-1 cells. It is suggested that 2ME2 cannot completely inhibits the EGF signaling pathways that are involved in MMP-9 induction or 2ME2 inhibits other signaling pathways that are important for MMP-9 induction through other regulators. Moreover, the lack of effect of 2ME2 on EGF may contribute to the incomplete inhibitory effect on MMP-9 activity in C666-1 cells.

Chapter 5

Conclusion and Further investigation

5.1 Conclusion

2ME2 could inhibit the migration of C666-1 cells. It is found that MMP-9 is expressed in C666-1 cells and MMP-9 activity in C666-1 cells is reduced by 2ME2. EGF treatment caused an increase in MMP-9 activity in C666-1 cells. However, 2ME2 did not inhibit EGF-induced MMP-9 activity in C666-1 cells.

5.2 Further investigation

Qiu et al. showed that EGF induced activity of MMP-9 through PI3K/Akt and MAPK/ERK signalling pathways in trophoblast cell lines (Qiu et al., 2004) and Moulik et al. showed that EGF induced activity of MMP-9 through NF-κB and PI3K signalling pathways in human breast cancer cells lines (Moulik et al.,2008). EGF induced expression of MMP-9 in C666-1 cells may be related to the above pathways. The signalling pathways involved in EGF-induced MMP-9 activity in C666-1 cells need to be further studied.

The mechanism of 2ME2 to affect the activity of MMP-9 in C666-1 cells through regulators other than EGF should be further investigated. HIF-1α overexpression was found to have close relationship with the mortality of cancer sufferers with cervical (Burri et al., 2003), breast (Schindl et al., 2002), ovarian (Birner et al., 2001), oropharyngeal (Aebersold et al., 2001) and brain (Birner et al., 2001) cancers respectively. Schelter et al. suggested that the regulation of invasion of cancer cells into the liver by HIF-1α is leastwise in part by promoting the MMP-9 expression (Schelter et al., 2010). A former study has shown that 2ME2 inhibits HIF-1α activity in prostate and breast cancer (Mabjeesh et al., 2003). Moreover, 2ME2 can inhibit MMP-9 expression by blocking the function of HIF-1α in a study of synapse loss after traumatic brain injury (TBI) (Ding et al., 2009). Above all, HIF-1α should be a potential candidate for studying the mechanism of 2ME2 to affect the activity of MMP-9 in C666-1 cells.