Activision Of MMP-2 And Expression Of MT1-MMP MRNA Correlated With Invasiveness In Thymic Epithelial Tumors

ObjectiveThymic epithelial tumors which include noninvasive ,invasive thymoma and thymic carcinoma are most popular in mediastinal tumors. The classification of thymic tumors was replete with misconceptions and misnomers for many years. In 2004 WHO classified thymomas into histological types as A, AB, B1, B2, B3. Conparing with in 1999 it separated thymic carcinoma from Thymic epithelial tumors. However, these histological subtypes so classified did not corelate consistently with their clinical behaviors. Although thymoma exhibits cytological-ly bland neoplastic epithelial cells, it occasionally shows invasive growth and pleural seeding, and rarely lymphogenous or hematogenous metastasis. The staging procedure predicted according to the degree of tumor invasion remains the best prognostic factor for thymoma such as Masaoka system with TNM syetem.There is increasing evidence that matrix metalloproteinases ( MMPs) and Tissue inhibitor of metalloproteinases (TIMPs) play important roles in tumor invasion and metastasis. MMP -2, with the descriptive name gelatinase A, is secreted as a 72 - kDa inactive pro - form, which is converted to a 67 - kDa active form by proteolytic processing. The activated MMP - 2 can degrade collagen types IV and V, constituents of basement membrane, and some of the interstitial tissue; thus, it is related to tumor progression. TIMP -2 is the specific inhibitor of MMP -2. Some researcher reported pro - MMP -2 is being activated can be achieved by MT1 - MMP and TIMP - 2 bound MT1 - MMP in other cancers. Membrane - type MMPs ( MT MMPs ) , a subset of MMPs, are not secreted but remain attached to the cell surface, it play an important role in this procession.In order to validate this hypothesis exist in thymoma, we examined the expressions of MMP - 2, MT1 - MMP and TIMP - 2 mRNA and the activation of pro -MMP -2 in noninvasive^invasive thymoma and thymic carcinoma, To.study the relationship between them.MATERIALS AND METHODS1. MATERIALSTumorous and thymic tissues were obtained from 16 thymic epithelial tumor patients who underwent surgery at the Second Department of Surgery, Kanazawa medical University hospital of Japan in 2002. twelve with thymoma,two with thymic carcinoma and two with thymic tissue. All samples were stored at -80t until use. HE section were diagnosed by two advanced pathologists.2. METHODS2. 1 Real - time RT - PCR(Taqman method)Total RNA was extracted using the AGPC ( acid guanidium - phenol -chloroform) method. Extracted RNA was quantified by measuring the each ab-sorbancy at 260nm, and its purity was confirmed by electrophoresis. After reverse - transcribed into GAPDH cDNA, real - time quantitative PCR analysis was performed by use of a PE Applied Biosystems 7700 Sequence Detector ( PE Applied Biosystems, Inc. , Foster, CA, USA). The relative mRNA expression levels (MMP-2,MT1 - MMP, TIMP - 2/GAPDH ) were calculated from each set of quantified data. Normalizer ratio was 1.2. 2 Gelatinolytic ZymographyFor gelatinolytic zymography, some sections store in - 80 T! were homogenized in SDS - PAGE sample buffer. Lysate was clarified by centrifugation. Samples were separated by electrophoresis on 10% polyacrylamide gel containing 0. 1% SDS and 2% gelatin as a substrate. Thereafter, gels were incubated in reaction buffer for 16 - 24 hrs at 37 X. Gels were stained for 60 min in Coom-assie Brilliant Blue G - 250 and then de - stained. The gelatinolytic activity of each collagenase was evident as a clear band against the blue background of stained gelatin. We quantified the intensity of bands (92 kDa, 72 kDa, and68kDa) by gelatinolytic zymography using NIH image analysis. The gelatinolytic activity ratio was calculated.Activity ratio of pro - MMP - 2 = MMP - 2 /pro - MMP - 2 + MMP - 2 o2.3 Film in situ zymography(FIZ)Frozen sections were cut to a thickness of 7um with a cryotome. Those sections were mounted on polyethylenetelephthalate film coated with gelatin (FIZ -GN). As a control, these sections were mounted on polyethylenetelephthalate film coated with gelatin and 1,10- phenanthroline as an MMP inhibitor ( FIZ -GI). HE staining was also carried out on frozen sections. Frozen sections on these films were incubated in a moist chamber at 37Xl for 8hr. After dry, Po-neau 3R stain 4 min, wash 10min,dry and observed by microscope. Result determinant : According to the intensity and area of stain, red symmetrical background is negative ( - ) ; sprinkle pink spot with red background is ( ± ) ; sprinkle pink spot account for 50% is ( + ) ; stain for rosiness to white is ( + + ).Statistical AnalysisAll values were indicated by the mean_SD. Statistical analysis was performed by Mann Whitney's U test and Scheffes test ,the relationship between protein activation and expression of mRNA using the Spearman rank correlation procedure, and a P - value below 0.05 was considered significant.Results1 Real - time RT - PCRThere were no significant difference in the expressions of MMP - 2, MT1 -MMP and TIMP - 2 mRNA between I and II stage or III and IV stage thymomas ( P > 0. 05 ). Prominent differences were shown in the three groups of I - II stage, HI - IV stage and thymic carcinomas ( P <0.05) and the three groups of AB - Bl (lymphocy - rich and mixed types ) , B2 - B3 ( cortical and predominantly polygonal cells types) and thymic carcinomas(P <0.05).2 Gelatinolytic ZymographyThe activations of pro — MMP - 2 in thymoma were I stage (0. 149 ± 0. 060) ,11 stage(0.097 ±0.097) ,111 stage(0.299 ±0. 102) ,IV stage(0.448 ±0.026) ,thymic carcinoma( 0.437 ±0.014). Prominent differences were shown in the three groups of I - II stage, HI - W stage and thymic carcinomas ( P < 0.05) , no significant difference between I and II stage, as well as III and IV stage.MT1 - MMP, TIMP -2 mRNA expression levels were correlated with pro -MMP-2 activation ratio (Spearman rank correlation; r =0.7235, r =0.7647 P < 0.005 ). No significant differences were found in the expressions of MMP - 9 in the thymomas and thymic carcinomas.FIZFIZ demonstrated the presence of gelatinase activity were negative in the thymic tissue and mostly noninvasive thymoma. Positive ratio were 0 and 33. 3% , while in invasive thymoma and thymic cancer, positive ratio were 83.3% and 100% . Gelatinolytic activities was localized on the invasing tumor cell nests and stroma just adjacent to them.DiscussionIt is well - known that Matrix metalloproteinases ( MMPs) and Tissue inhibitor of metalloproteinases (TIMPs) play important roles in most tumor invasion and metastasis, but the study about them was few in thymic epithelial tumor. MMP - 2 was the most popular Matrix metalloproteinases in the study of thymoma, This study tested the expressions of MMP - 2mRNA using real - time PCR for the first time. Result shows it be expressed in all kind of stages and types. And invasive thymoma (III and IV stages) and thymic Carcinomawere significantly higher than those of noninvasive and microinvasive thymomas (I and II stages) , and also between B2, B3 and AB, Bl. These results are similar as kondo and Sogawa and Takahashis reports.According to the new classified method of WHO in 2004, type B2 is cortical type; Type B3 thymoma is histologically characterized by a marked predominance of polygonal epithelial cells, a distinct epidermoid differentiation, and scant immature T - lymphocytes. This tumor shows an increased risk of being invasive stage despite a mild degree of cellular atypia and low mitotic counts, withvirtually no metastasis via lymphatics or blood vessels. Takahashi first reported expressions of MMPs in types of thymoma. They confirmed the expression of MMP-2 had prominent difference between type B3 and type A,AB,B1. Our results showed the MMP - 2 mRNA expression in type B2 and B3 markedly higher than type AB and Bl. It also shows that thymic epithelial cells, specially polygonal cells, have highly invasion.MMP2, are produced and secreted in an inactive form (pro - MMP - 2 ). The overexpression of pro - MMP - 2 does not mean that this enzymes function immediately, and tumor cells would need to activate the pro - MMP -2. So,the most important point is mechanism of the activation of proMMP -2. It was long been believed that TIMPs suppressed tumor invasion by inhibiting MMPs in experimental and clinical studies. However,a recent biochemical analysis demonstrated that TIMP -2 contributed to the activation of MMP -2. In the MMP -2 activation mechanism, pro - MMP - 2 secreted by fibroblasts or tumor cells binds to TIMP *- 2 complexed with membrane Type 1 MMP (MT1 - MMP) on the tumor cell surface, and the pro - MMP - 2/TIMP - 2/MT1 - MMP ternary complex is formed. Pro - MMP - 2 in the ternary complex is activated by MT1 -MMP that is free from TIMP - 2. Our results showed this hypothesis also existed in thymic epithelial tumor.Kondo showed elavation of the gelatinolytic activity of active MMP - 2 in an invasive region compared with a central region in invasive thymoma and thymic carcinoma. It suggested that in such invasive regions, tumor cells overexpressed MT1 -MMP, which induced MMP-2 activation, and invaded the ECM. Takahashi had demonstrated the expression of MMP - 2 mRNA in both the tumor cells and the stromal cells of thymomas using Immunohisto - chemical . We result also showd it both in the tumor and the stromal cells by FIZ method. These findings indicate that MMP - 2 is produced primarily in the stromal cells and then binds to the tumor cells, where this enzyme is activated in the process of tumor progression.On the other hand, in our study, elevation of the gelatinolytic activity of MMP -9 in the thymus was detected. There was no correlation between the in-vasiveness of thymic epithelial tumor and the gelatinolytic activity of MMP -9.