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The matrix metalloproteinase ( MMP ) is one of the possible campaigners responsible for tumour metastasis and invasion. Previous survey has shown that the look degree of MMP9 messenger RNA was significantly greater in NPC tissues than that in the non-cancerous nasopharyngeal tissues. It is besides believed responsible for tumour metastasis and invasion of NPC. The intent of this survey is to analyze the anti-metastasis effects of 2-Methoxyestradiol ( 2ME2 ) on NPC cells. 2ME2, an endogenous derived function of E2 formed by the hydroxylation and subsequent methylation at the 2-position, is used as a chemotherapeutic agent to suppress the growing of different tumours by its anti-proliferative and anti-angiogenic activity. C666-1, an EBV positive NPC cell lines was used in this survey. Consequences from gelatin zymography indicated that 2ME2 could cut down the activity of MMP-9, and therefore MMP-9 might besides intercede the anticancer activity via the suppression of migration of NPC cells.

Nasopharyngeal carcinoma ( NPC ) is a particular sort of malignant neoplastic disease that occurs in caput and cervix ( Vokes et al. , 1997 ) , developing in the epithelial cells that cover the surface of nasopharynx ( Brennan, 2006 ) . Nasopharynx is a hollow tubing situates behind the nose, reaches the part of soft roof of the mouth consisting the roof of the oral cavity ( Jeyakumar et al. , 2006 ) as showed in Figure 1.1. The air we breathe is moisturized and filtered by the nasopharynx, moreover, the ear force per unit area and balance are besides equalized and maintained by it ( Tortora & A ; Grabowski, 2000 ) .

1.1.1 Symptoms of NPC

Due to the anatomic location of nasopharynx, the diagnosing of NPC is instead hard. Potential noticeable symptoms of NPC include hypertrophied cervical lymph nodes without hurting, rhinal congestion and hemorrhage ( nosebleed ) , diminished hearing and tintinnabulation in the ears ( tinnitus ) , disfunction of cranial nervus, perennial otitis media, concern and sore pharynx ( Cho, 2007 ) .

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1.1.2 Histopathology of NPC

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

In 1991, WHO classification merged Type II and Type III NPC into non-keratinizing carcinoma. Non-keratinizing carcinoma was so divided into ‘differentiated ‘ and ‘undifferentiated ‘ 1s. The differentiated subtype substituted the original Type II and the uniform subtype substitute the original Type III. The keratinising 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 typical. NPC was uncommonly found in North America and Europe, with a relatively little figure of instances reported ( Hirayama et al. , 1980 ; Tai & A ; Mould, 2001a ; Tai & A ; Mould, 2001b ) . Peoples from Southeast Asia and southern China were chiefly be affected by NPC ( Fedder & A ; Gonzalez, 1985 ) . NPC is known as Cantonese Cancer since it was peculiarly prevailing in southern China, particularly in Guangdong state ( Cao et al. , 2008 ) and Hong Kong ( Lee et al. , 2003 ) . With mention to the statistics of Leading Cancer Site in Hong Kong in 2008 from Hong Kong Cancer Registry, Hospital Authority, NPC was the 7th most common malignant neoplastic disease and malignant neoplastic disease slayer in Hong Kong. There were 926 new instances of NPC registered in 2008, males contributed to 679 instances and females contributed to 247 instances. The male: female ratio is 2.7:1.

1.1.4 Hazard factors for NPC

1.1.4.1 Epstein-Barr virus ( EBV )

Epstein-Barr virus ( EBV ) is a herpes virus. This virus with womb-to-tomb continuity infects over 90 % of the population worldwide. Normally an person was latently infected for life by primary infection ( Crawford, 2001 ) . Previous surveies showed that EBV contributes to certain disease like Hodgkin ‘s disease ( HD ) , Burkitt ‘s lymphoma ( BL ) , T-cell lymphoma, tummy malignant neoplastic disease and NPC ( Crawford, 2001 ; Hannigan & A ; Wilson, 2010 ) . Evidence showed that NPC patients have board scope and higher degrees of anti-EBV antibodies, with increased degrees of IgA antibodies ( Henle & A ; Henle, 1980 ) . Development of NPC consequences from the increased degrees of serum antibodies by certain old ages ( Ho et al. , 1978a ; Lanier et al. , 1980 ) . EBNA1, EBER, LMP1 and LMP2 are latent cistrons expressed during EBV infection ( Izumi, 2001 ; Kaye et al. , 2003 ) . LMP1 is responsible for tumour patterned advance 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 surveies have shown that ingestion 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 extremely correlated to the NPC development. Furthermore, the ingestion of salt-cured fish in childhood ( ablactating ) demonstrated to be at a higher hazard of developing NPC than maturity exposure ( Chang & A ; Adami, 2006 ) . Animal surveies showed that rats fed on salt-cured fish developed nasal or nasopharyngeal carcinoma ( Huang et al. , 1978 ; Yu et al. , 1989b ) . Besides salt-cured fish, ingestion of other preserved nutrient like fermented bean curd, salted veggies and salted shrimp paste was reported to be related to the increased NPC hazard in Chinese ( Yu et al. , 1989a ; Ning et al. , 1990 ) . Furthermore, consumption of alcoholic drinks and smoke would besides increase the opportunity of acquiring NPC ( Cheng et al. , 1999 ) . Exposing human to exhausts and wood dust occupationally or domestically was besides history for the development of NPC ( Hildesheim et al. , 2001 ) .

1.1.4.3 Familial factors

Familial factors besides play a important function in the development of NPC. Previous surveies indicated that human leucocyte antigens ( HLA ) , a sort of MHC antigens of worlds, associated with NPC chiefly among the Chinese ( Simons et al. , 1974 ; Chan et al. , 1983 ) . Simons et Al. showed that HLA antigens contribute to the finding of the hazard of NPC and endurance of NPC patient ( Simons et al. , 1978 ) . Besides HLA, familial polymorphisms of enzyme cytochrome P450 2E1 ( CYP2E1 ) was besides 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 surveies 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 rarely considered ( Brennan, 2006 ) . The primary manner for NPC intervention is Radiotherapy ( RT ) , and the secondary manner for NPC intervention is chemotherapy since NPC is besides sensitive to certain chemotherapeutic drug ( Agulnik & A ; Siu, 2005 ) . Chemotherapy can be added to radiotherapy in different manners, including neoadjuvant ( given before RT ) , coincident ( given at the same clip with RT ) , accessory ( given after RT ) , neoadjuvant and accessory ( given before and after RT ) , attendant and accessory ( given at the same clip with RT and followed by another chemotherapy ) . Agulnik and Siu believed that fresh interventions including intensity-modulated Radiotherapy, molecular targeted therapy and immunotherapy with EBV-specific cytotoxic T lymphocytes needed to be farther explored ( Agulnik & A ; Siu, 2005 ) .

1.2 Metastasis

Primary tumour growing is non deadly ; nevertheless, metastasis increases the hazard of decease of malignant neoplastic disease sick persons ( Yoon et al. , 2003 ) . Tumor metastasis is the motion of a individual or group of malignant neoplastic disease cells from a primary site to secondary 1s that requires many stairss ( Deryugina & A ; Quigley, 2006 ) as follows: a individual or group of malignant neoplastic disease cells detach from primary tumors and evade from anoikis. Then, the malignant neoplastic disease cells can travel in the debauched extracellular matrix. When the tumour cells escape through the cellar membrane, they would occupy the lymph and blood vass followed by a consecutive immunological equivocation and attach to the endothelial cells. Afterward, the malignant neoplastic disease cells pass out of the vass into environing tissues followed by certain proliferation and angiogenesis ( Bohle & A ; Kalthoff, 1999 ; Nash et al. , 2002 ) . Matrix metalloproteinase-9 ( MMP-9 ) is believed to play a important function in tumour 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 household, is synthesized as a zymogen with a molecular mass of 92 kDa in human ( Atkinson & A ; Senior, 2003 ) . In human, the cistron of MMP-9 is located on chromosome 20q11.1-13.1 and MMP-9 lack is non likely happened ( Zhang et al. , 1999 ) . The booster part of MMP-9 cistron comprises adhering sites for AP-1, AP-2, SP-1, NF-I?B and Ets, with an repressive component of TGF-I? ( Atkinson & A ; Senior, 2003 ) . MMP-9 is a 92-kDa type IV collagenase, besides named as gelatinase B, affecting fibronectin type II-like repetitions for binding of gelatin and elastin ( Shipley et al. , 1996 ) . Previous surveies have revealed that the look degree of MMP-9 messenger RNA and protein were significantly greater in assorted malignant neoplastic diseases ( Roomi et al.,2010 ; Qin et al. , 2008 ; Zheng et al. , 2010 ; Liu et al. , 2010 ) . MMP-9 is believed to play a important function in tumour metastasis by advancing the extracellular matrix and cellar membrane debasement ( Liabakk et al. , 1996 ) .

1.4 2-methoxyestradiol ( 2ME2 )

2-methoxyestradiol ( 2ME2 ) , which is a of course happening and endogenous derived function of 17I?-estradiol ( E2 ) , that formed by back-to-back 2-hydroxylation and O-methylation ( Gelbke & A ; Knuppen, 1976 ) . The chemical construction of 2ME2 is shown in figure 1.2. It is now accepted as a powerful curative agent for malignant neoplastic disease with no toxicity although one time disregard as an inert end-metabolic of estradiol ( Zhu & A ; Conney, 1998 ; Lui et al. , 2000 ) .

Different from 17I?-estradiol ( E2 ) which would bring on proliferation of malignant neoplastic disease cells depending on estrogen receptor, old surveies have reported that 2ME2 demonstrates anti-cancer character in assorted illustrations ( Fotsis et al. , 1994 ; Mukhopadhyay & A ; Roth, 1997 ; Schumacher et al. , 1999 ; Seegers et al. , 1997 ; Pribluda et al.,2000 ; Zhu & A ; Conney, 1998 ) and inhibits growing of different tumours by anti-angiogenic and anti-proliferative activity ( Pribluda et al.,2000 ; Zhu & A ; Conney, 1998 ; Mooberry, 2003 ) . Certain mechanisms of action for these activities were demonstrated by 2ME2 such as modulating cell rhythm kinases and apprehension ( Lottering et al. , 1996 ; Zoubine et al. , 1999 ; Attalla et al. , 1996 ) , bring oning programmed cell death in different tumours ( Mooberry, 2003 ) , impacting superoxide dismutase ( Huang et al. , 2000 ; Kachadourian et al.,2001 ) , interrupting microtubule kineticss ( D’Amato et al. , 1994 ; Cushman et al. , 1995 ) , up-regulating p53 ( Mukhopadhyay & A ; Roth, 1997 ; Seegers et al. , 1997 ) . 2ME2 besides possess anti-metastasis consequence ( Plum et al. , 2009 ) .

Previous surveies reveled that 2ME2 had limited unwritten bioavailability in capsule signifier ( Dahut et al. , 2006 ; Sweeney et al. , 2005 ) . Therefore, 2ME2 is reformulated to Nanocrystal scattering ( NCD ) . Improvement of drug disintegration rate was achieved by decreasing the size of drug atoms into nanometer-sized atoms in NCD ( Kawashima, 2001 ) . Steady plasma degrees of 2ME2 were achieved by 2ME2 NCD in certain presymptomatic surveies, bespeaking that unwritten bioavailability of 2ME2 in worlds could be improved by utilizing 2ME2 NCD. A stage I study was so conducted by Tevaarwerk et Al. in patients with solid malignances by utilizing 2ME2 NCD. Pharmacokinetics variables and a chief metabolite of 2ME2 were assessed in blood samples. At steady province, the minimal effectual concentration attained by trough degrees in all dose groups and the maximal tolerated dosage of 1000 milligram orally for every 6 hours was suggested for stage II regimen ( Tevaarwerk et al. , 2009 ) .

1.5 Objective

Although the primary manner for NPC intervention is Radiotherapy ( RT ) , NPC is besides sensitive to certain chemotherapeutic drug. Chemotherapy can be added in different manners to help radiation therapy. 2ME2, regarded as a powerful curative agent for malignant neoplastic disease with no toxicity, demonstrates anti-cancer character in assorted illustrations and inhibits growing of different tumours by anti-angiogenic and anti-proliferative activity. However, the anti-metastasis consequence of 2ME2 on NPC has non been to the full studied. Therefore, we would wish to analyze the anti-metastasis effects of 2ME2 on C666-1 NPC cells in this survey. After corroborating 2ME2 could suppress the migration of C666-1 cells, we would wish to look into the consequence of 2ME2 on MMP-9 activity and EGF-induced MMP-9 activity in C666-1 cells.

Figure1.1 Diagram demoing parts of the throat.

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

Figure 1.2 The chemical construction of 2ME2.

Chapter 2

Materials and Methods

2.1 Materials

2.1.1 Cell Lines

C666-1, a subclone of C666, was derived from uniform 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 brooder at 37A°C with 5 % CO2. For the existent experiments, cell civilization medium has been replaced with RPMI medium.

2.1.2 Reagents for cell civilization

2.1.2.1 Fetal Bovine Serum ( FBS )

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

2.1.2.2 Penicillin and Streptomycin ( PS )

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

2.1.2.3 RPMI 1640 Medium

RPMI 1640 power ( Sigma, R6540 ) was dissolved in 800ml of MilliQ H2O with 2g Sodium carbonate. The pH of solution was adjusted to 7.3 by adding 2M NaOH and 5M HCl solutions, so the solution was eventually made up to 1 litre. The medium was so filtered and stored at 4A°C. 10 % of FBS and 2 % of Penicillin and Streptomycin ( PS ) were used to finish the RPMI medim. The cRPMI was stored at 4A°C until usage.

2.1.2.4 Dulbecco ‘s Phosphate Buffered Saline ( PBS )

PBS pulverization ( Sigma, D5652 ) was dissolved in 800ml of MilliQ H2O so made up to 1 litre. The solution was sterilized by filtrating 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 -20A°C until usage.

2.1.3 Reagent for cell numeration

2.1.3.1 Trypan blue stain 0.4 %

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

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 -80A°C until usage.

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 -20A°C until usage.

2.1.4.3 EGF

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

2.1.5 Reagent for Wound mending check

2.1.5.1 Fibronectin

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

2.1.6 Reagents for Transwell migration check

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 fade outing 250Aµl 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 4A°C until usage.

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 glycerin ( USB, US16347 ) , 5mg bromophenol blue ( Amersham Bioscince, US12370 ) and 1.2 milliliter MilliQ H2O made up the non-reducing sample buffer ( 5X ) . The buffer was aliquoted and stored at -20A°C until usage.

2.1.7.2 Reagents for Stacking and Resolving gel

1 ) 2 % gelatin

0.2g of gelatin pulverization ( Sigma, G1890 ) was dissolved in 10 milliliter of MilliQ H2O, and stored at 4A°C until usage.

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

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

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

36.34g of Tris-HCl pulverization ( USB, US75825 ) was dissolved in 100ml of MilliQ H2O. The pH of solution was adjusted to 8.8 by adding 2M NaOH and 5M HCl solutions, and so the solution was eventually made up to 200ml.

4 ) 0.5M Tris ( pH6.8 )

12.11g of Tris ( USB, US75825 ) was dissolved in 100ml of MilliQ H2O. The pH of solution was adjusted to 6.8 by adding 2M NaOH and 5M HCl solutions, so the solution was eventually made up to 200ml.

5 ) 10 % SDS

10g of SDS pulverization ( USB, US75819 ) was dissolved in 100ml of MilliQ H2O and stored at room temperature.

6 ) 10 % Ammonium Persulfate ( APS )

3g of APS pulverization ( USB, 76322 ) was dissolved in 30ml of MilliQ H2O and aliquoted to 1.5 milliliters eppendorfs and stored at -20A°C until usage.

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 H2O. The pH of solution was adjusted to 8.3 by adding 2M NaOH and 5M HCl solutions, so the solution was eventually made up to 1 litre.

2.1.7.4 2.5 % Triton X-100

2.5 % Triton X-100 ( USB, 22686 ) was prepared by fade outing 5ml of Triton X-100 in 195ml of MilliQ H2O 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 assorted with 180ml of MilliQ H2O 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 % methyl alcohol ( 80ml ) ( Merck, 1.060007.2500 ) and 50 % of MilliQ H2O ( 100ml ) .

2.1.7.7 Destaining solution

10 % acetic acid ( 20ml ) ( Sigma, 19516 ) , 40 % methyl alcohol ( 80ml ) ( Merck, 1.060007.2500 ) and 50 % of MilliQ H2O ( 100ml ) were assorted.

2.2 Methods

2.2.1 Wound healing check

Wound mending check was used to analyze cell migration in vitro. For better cell fond regard, each well in 96-well home base was coated with 50Aµl of 10Aµg/ml of fibronectin at 4A°C overnight. After taking the extra fibronectin, 100Aµl of 8 A- 104 of C-6661 cells were seeded to 96-well home base for 24 hours. The drifting cells were removed and pipette tips were used to injure the cell monolayers. Images of the hurt country were captured with a X10 aim instantly after injuring at 0 hr, 24 hours and 48 hours to see the migration of cells into the hurt country.

2.2.2 Transwell migration check

C666-1 cells with cell denseness of 5 A- 105 were seeded to 35-mm petri dish and grown in a humidified brooder at 37A°C with 5 % CO2 for 72 hours. The cells were treated with 10mM of 2ME2 for 72 hours. After 72 hours, 100ul of 2 A- 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 home base 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 37A°C, cells that had migrated to the lower side of the membrane were fixed with 1.5 % paraformaldehyde for 20 proceedingss, followed by 3 times PBS rinsing. Afterward, the cells were permeablized by 0.2 % Triton X-100 for 10 proceedingss, followed by 3 times PBS lavation. After the lavation, the cells were later stained with 4′,6-diamidino-2-phenylindole ( DAPI ) for 10 proceedingss, so destained in PBS for 5 times. The transwell membrane was removed and mounted on a glass slide in an upside down place, immersed in a PBS and covered with a screen faux pas. The DAPI-stained cells were so visualized under a microscope. The C666-1 cells that had migrated through the membrane pore spontaneously were counted and expressed as the per centum of migration compared with the control.

2.2.3 Gelatin zymography

Conditionss 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 denseness of 5 A- 105 were seeded to 35-mm petri dish and grown in a humidified brooder at 37A°C with 5 % CO2 for 72 hours. In experiments analyzing the consequence of 2ME2 on MMP-9 activity, the cells were treated with 10mM of 2ME2 for 72 hours. In experiment analyzing the consequence of EGF on MMP-9 activity, the cells were starved for 24 hours, and so treated with 0, 10, 50, 100, 200, and 400ng/ml of EGF for 72 hours. In experiment analyzing the co-treatment consequence 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 analyzing the pre-treatment consequence 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 so collected and centrifuged at 14000 rcf for 15 proceedingss. Afterwards, the learned media were denatured in the absence of cut downing agent and electrophoresed in a 7.5 % SDS-polyacrylamide gel incorporating 0.1 % ( w/v ) gelatin. The gels were so incubated at room temperature for 1 hr in the presence of 2.5 % Triton X-100 with soft agitation and afterwards at 37A°C for 45 proceedingss in a substrate buffer incorporating 10mM CaCl2, 0.15 M NaCl, 50 millimeter Tris-HCl ( pH 8 ) and 0.02 % Brij-35. Then the gels were incubated overnight at 37A°C in a new substrate buffer. Gels were stained with 0.5 % Coomassie Blue and destained with destaining solution until clear sets were observed. Clear bands over the dark background represent countries of enzymatic activity.

2.2.4 Statistical analyses

Datas are expressed as agencies A± SDs. Two-tailed Student ‘s t-test was performed for statistical analysis between control and intervention groups. Statistical significance ( P value ) was presented in the figure legends.

Chapter 3

Consequences

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

Consequences from wound mending check were shown in figure 3.1, no lesion closings were observed after injuring C666-1 cells for 24 hours and 48 hours. Therefore, it is found that lesion mending check is non a suited check to analyze the consequence of 2ME2 on the migration ability of C666-1 cells. Then, transwell migration check was used to the survey the consequence of 2ME2 on the migration ability of C666-1 cells. Consequence from transwell migration check was shown in figure 3.2. The consequence showed that 2ME2 inhibited the migration of C666-1 cells by cut downing the % of migration to 38 % of the control.

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

After corroborating 2ME2 could suppress the migration of C666-1 cells, the consequence 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, decrease in the MMP-9 activity was observed.

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

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

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

After corroborating EGF could bring on the activity of MMP-9 in C666-1 cells, the consequence 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 intervention of 400 ng/ml of EGF, no alterations in the MMP-9 activity were observed ( Figure 3.5 & A ; Figure 3.6 ) . 2ME2 co-treatment and pre-treatment did non suppress EGF-induced MMP-9 activity in C666-1 cells.

( A )

0hour

( B )

24hour

( C )

48hour

Figure 3.1 Images demoing cells migration of C666-1 cells into the hurt country.

Wound mending checks were performed in C666-1 cells ( 8x 104cells per good coated with fibronectin ) . Representative images captured with a X10 aim at the clip of injuring ( A ) 0 hr, so ( B ) 24 hours and ( C ) 48hours after injuring in C666-1 cells.

**

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

C666-1 cells ( 5 A- 105 cells in 1 ml serum-free RPMI per 35mm dish ) were treated with 10mM of 2ME2 for 72 hours. 100ul of 2 A- 105 of 2ME2-treated cells in serum-free RPMI medium were so seeded to a transwell. The migration of cells was determined by Transwell migration check. Cells that had migrated through the membrane pore spontaneously were counted and expressed as the per centum of migration compared with the control. Bar graph was expressed as average A± SD and two-tailed Student ‘s t-tests were performed ( **P & lt ; 0.01 ) .

Lane 1 2

2ME2 – +

MMP-9 ( 92kDa ) a†’

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

C666-1 cells ( 5 A- 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 intervention. The learned media were collected for finding of MMP-9 activity by gelatin zymography.

EGF ( ng/ml )

0 10 50 100 200 400

MMP-9 ( 92kDa ) a†’

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

C666-1 cells ( 5 A- 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 learned media were collected for finding of MMP-9 activity by gelatin zymography.

Lane 1 2 3

EGF – + +

2ME2 – – +

MMP-9 ( 92kDa ) a†’

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

C666-1 cells ( 5×105 cells in 1 ml serum-free RPMI per 35mm dish ) were starved for 24 hours. Lane 1: control without intervention. 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 learned media were collected for finding of MMP-9 activity by gelatin zymography.

Lane 1 2 3

EGF – + +

2ME2 – – +

MMP-9 ( 92kDa ) a†’

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

C666-1 cells in ( 5×105 cells in 1 ml serum-free RPMI per 35mm dish ) were starved for 24 hours. Lane 1: control without intervention. 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 learned media were collected for finding of MMP-9 activity by gelatin zymography.

Chapter 4

Discussion

The consequence of 2ME2 inhibits the migration of C666-1 cells is consistent with the consequence of 2ME2 reduces transwell migration of transformed murine pre-B-cell line and chronic myelogenous leukaemia cells in old survey by Sattler et Al. ( Sattler et al. , 2003 ) . 2ME2 may lend to cut down metastasis and invasion of C666-1 cells.

Degradation of the extracellular matrix is a important measure for tumour metastasis and invasion ( Liabakk et al. , 1996 ) . Matrix metalloproteinase-9 ( MMP-9 ) is believed to play a important function in tumour metastasis ( Liabakk et al. , 1996 ) . Previous surveies have revealed that the look degree of MMP-9 messenger RNA and protein were significantly greater in certain malignant neoplastic diseases like ovarian malignant neoplastic disease ( Roomi et al. , 2010 ) , breast malignant neoplastic disease ( Qin et al. , 2008 ) , lung malignant neoplastic disease ( Zheng et al. , 2010 ) and besides in NPC tissues ( Liu et al. , 2010 ) . Liu et Al. suggested that the comparatively higher MMP-9 protein look was related to NPC patterned advance and hapless forecast ( Liu et al. , 2010 ) . This survey showed that MMP-9 is expressed in C666-1 cells, which is consistent with the consequence of Liu et Al. that MMP-9 was significantly increased in NPC ( Liu et al. , 2010 ) . The anti-metastasis consequence of 2ME2 was so examined through the MMP-9 activity in C666-1 cells. This survey 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 lend to the reduced transwell migration. The mechanism of 2ME2 to impact the activity of MMP-9 was further studied.

In fact, MMP-9 is regulated by several regulators included cuticular growing factor ( EGF ) ( Qiu et al. , 2004 ) , fibroblast growing factor ( FGF ) ( Lungu et al. , 2008 ) , interleukin-8 ( IL-8 ) ( Chakrabarti and Patel, 2005 ) , interferon beta ( IFN-I? ) ( Kurzepa & A ; Stryjecka-Zimmer, 2007 ) , nerve growing factor ( NGF ) ( Khan et al. , 2002 ) , tumour mortification factor ( TNF ) ( Scott et al. , 2004 ) , vascular endothelial growing factor ( VEGF ) ( Hollborn et al. , 2007 ) , hypoxia inducible factor-1 alpha ( HIF-1I± ) ( Schelter et al. , 2010 ) . Among those regulators, EGF, a sort of growing factor, binds to its receptor-epidermal growing factor receptor ( EGFR ) to modulate the cell growing, proliferation, and distinction ( Herbst, 2004 ) . EGF-induced MMP-9 secernment is considered to help tumour invasion and metastasis in certain malignant neoplastic diseases like vesica malignant neoplastic disease ( Nutt et al. , 2003 ) , lung malignant neoplastic disease ( Cox et al. , 2000 ) , ovarian malignant neoplastic disease ( Ellerbroek et al. , 1998 ) , caput and cervix squamous cell ( Charoenrat et al. , 2000 ) . Similar to the consequences from old surveies for other malignant neoplastic diseases, EGF was found to do an addition in MMP-9 activity in C666-1 cells in this survey.

Both 2ME2 pre-treatment and co-treatment did non suppress EGF-induced MMP-9 activity in C666-1 cells. It is suggested that 2ME2 can non wholly inhibits the EGF signaling tracts that are involved in MMP-9 initiation or 2ME2 inhibits other signaling tracts that are of import for MMP-9 initiation through other regulators. Furthermore, the deficiency of consequence of 2ME2 on EGF may lend to the uncomplete inhibitory consequence on MMP-9 activity in C666-1 cells.

Chapter 5

Decision and Further probe

5.1 Decision

2ME2 could suppress 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 intervention caused an addition in MMP-9 activity in C666-1 cells. However, 2ME2 did non suppress EGF-induced MMP-9 activity in C666-1 cells.

5.2 Further probe

Qiu et Al. showed that EGF induced activity of MMP-9 through PI3K/Akt and MAPK/ERK signalling tracts in trophoblast cell lines ( Qiu et al. , 2004 ) and Moulik et Al. showed that EGF induced activity of MMP-9 through NF-I?B and PI3K signalling tracts in human chest malignant neoplastic disease cells lines ( Moulik et al.,2008 ) . EGF induced look of MMP-9 in C666-1 cells may be related to the above tracts. The signalling tracts involved in EGF-induced MMP-9 activity in C666-1 cells need to be farther studied.

The mechanism of 2ME2 to impact the activity of MMP-9 in C666-1 cells through regulators other than EGF should be farther investigated. HIF-1I± overexpression was found to hold close relationship with the mortality of malignant neoplastic disease sick persons with cervical ( Burri et al. , 2003 ) , chest ( Schindl et al. , 2002 ) , ovarian ( Birner et al. , 2001 ) , oropharyngeal ( Aebersold et al. , 2001 ) and encephalon ( Birner et al. , 2001 ) malignant neoplastic diseases severally. Schelter et Al. suggested that the ordinance of invasion of malignant neoplastic disease cells into the liver by HIF-1I± is leastwise in portion by advancing the MMP-9 look ( Schelter et al. , 2010 ) . A former survey has shown that 2ME2 inhibits HIF-1I± activity in prostate and chest malignant neoplastic disease ( Mabjeesh et al. , 2003 ) . Furthermore, 2ME2 can suppress MMP-9 look by barricading the map of HIF-1I± in a survey of synapse loss after traumatic encephalon hurt ( TBI ) ( Dinging et al. , 2009 ) . Above all, HIF-1I± should be a possible campaigner for analyzing the mechanism of 2ME2 to impact the activity of MMP-9 in C666-1 cells.