| Background and ObjectiveBladder cancer is one of the most common human malignant carcinomas, and is also the most common carcinoma of urinary system in our country. More than 90% of them are transitional cell carcinoma (BTCC). The superficial cancer accounts for 70%-85% of all primary BTCC. Near 70% of them recur after the local excisions which reserve the remaining bladder for the first time, and about 20% of them develop to invasive cancer. Currently, therapeutic efficacy of BTCC is not ideal, although it has been improved in the past few years due to the studies on the molecular biological mechanisms of tumorigenesis. The capacities of recurrence, invasion and matastasis of BTCC are major causes leading to poorer prognosis and death. Therefore, it is very important to inhibite tumorigenesis as well as the capacities of recurrence, invasion and metastasis, which has become a key point in studying the BTCC.In BTCC the process of invasion and metastasis is very complicated, which involves several stages, factors and genes. The most important in this process is the breakdown of basement membrane (BM) and the degradation of extracellular matrix (ECM), which is associated with many kinds of proteases. The matrix metalloproteinases (MMPs) are the most important proteases in the progress and play key roles in the progress of tumor invasion and metastasis. There are at least 26 kinds of MMPs. MMP-2 and MMP-9 can degrade Type IV collagen specifically, which is essential component of BM and ECM. Expression of MMP-2 and MMP-9 is relatively low in normal tissue, while in malignant tumor, such as BTCC, it is usually overexpressed. Thus, suppressing the activities of MMP-2 and MMP-9 might inhibit tumor invasion and metastasis. RECK (reversion-inducing-cysteine-rich protein with Kazal motifs) gene is a new MMP inhibitor, which is able to inhibit MMP-2, MMP-9 and MT1-MMP expression and suppress tumor invasion and metastasis.Recently, studies on the relationship between RECK and BTCC are increasing while the relationship between RECK and recurrence of BTCC has not been reported yet. In our study, we detected the expression of RECK, MMP-2 and MMP-9 in normal bladder tissue and BTCC by streptavidin-peroxidase (SP) immunohistochemistry, and analyzed all data including the expression levels of RECK, MMP-2 and MMP-9 and their relationships with the clinical data such as tumor differentiation grades, pathological stages and recurrence, to investigate what roles they play in the pathological processes of BTCC including tumorigenesis, tumor development, invasion, metastasis and recurrence, and assess the relationships among RECK, MMP-2 and MMP-9 as well as utilization as biological markers in predicting the disease. To this end, we expect to provide a new way and theoretical support to diagnose and treat BTCC early, predict prognosis and recurrence more accurately, even provide a new possible way for new anticancer agents.Materials and MethodsA total of 42 cases of human BTCC tissues were enrolled in this study. All cases were collected from the patients with BTCC who underwent open operation or endoscopic biopsy at the department of Urology of the First or Second affiliated hospital of Zhengzhou University from July 2005 to October 2006. All cases were diagnosed pathologically and all clinical data were kept completely. Among these cases, 30 were men and 12 were women. Ages ranged from 32 to 88 years old, mean 59.93±12.63 years old. 24 were primary, other 18 were recurring. According to the WHO criteria, 11 were G1, 16 were G2, 15 were G3. According to the UICC-TNM system, 23 were non-muscle-invasive (Tis, Ta, T1), 19 were muscle-invasive (T2-T4). A total of 13 cases of normal bladder tissues were collected as control group and collected from the patients with benign prostate hyperplasia (BPH) who underwent open operation and without history of any tumor. Using the S-P immunohistochemical staining method, the expression levels of RECK, MMP-2, and MMP-9 in BTCC and normal bladder tissues were detected respectively. We analyzed the relationship between the expression levels and the clinical data including age, gender, tumor differentiation grade, pathological stage and recurrence. All analyses were performed with the Statistical Package for Social Sciences software (version 13.0 for Windows). The statistical comparison between groups was performed using Chi-square test or Fisher's exact test (for small samples) and the correlation between factors was assessed by the Spearman rank correlation test. Two-tailed P values less than 0.05 were considered statistically significant.Results1. The positive expression rate of RECK protein in the normal bladder tissue was 84.62%(11/13) and was significantly higher than that in the BTCC (52.38%, 22/42) (P<0.05). While the positive expression rate of MMP-2 protein in the normal bladder tissue was 23.08%(3/13) and was lower than that in the BTCC (73.81%, 31/42) (P<0.05). The positive expression rate of MMP-9 protein in the normal bladder tissue was 30.77%(4/13) and was lower than that in the BTCC (85.71 %, 36/42) (P<0.05).2. No significant difference was observed between the positive expression rates of RECK protein in different ages or genders (P>0.05). The positive expression rate of RECK protein in G1,G2,G3 descended gradually ,which was 63.64%(7/11), 56.25 %(9/16) and 40.00%(6/15) respectively. But there was no significant difference between the positive rates of well-differentiated group (G1) and poor-differentiated group (G2+G3) (P>0.05). In comparison with clinical pathological stages, the positive expression rate of RECK protein descended obviously, which was 69.57%(16/23) and 31.58%(6/19) in the non-muscle-invasive group (Tis,Ta,T1) and muscle-invasive group (T2-T4) respectively (P<0.05). Significant difference of positive expression rates of RECK protein also was observed between recur group (27.78%, 5/18) and primary group (70.83%, 17/24) (P<0.05).3. No significant difference was observed between the positive expression rates of MMP-2 in different ages or genders (P>0.05). The positive expression rate of MMP-2 protein was 45.45%(5/11), 81.25%(13/16) and 86.67%(13/15) in G1,G2, G3 respectively. There was significant difference between the positive rates of well-differentiated group (G1) and poor-differentiated group (G2+G3) (P<0.05). The positive expression rate of MMP-2 protein was 56.52% (13/23) and 94.74% (18/19) in the non-muscle-invasive group (Tis,Ta,Tl) and muscle-invasive group (T2-T4) respectively (P<0.05). Significant difference of positive expression rates of MMP-2 protein also was observed between recur group (94.44%, 17/18) and primary group (58.33%, 14/24) (P<0.05).4. No significant difference was observed between the positive expression rates of MMP-9 in different ages or genders (P>0.05). The positive expression rate of MMP-9 protein was 36.36%(4/11), 87.50%(14/16) and 93.33%(14/15) in G1,G2,G3 respectively. And significant difference was observed between the positive rates of well-differentiated group (G1) and poor-differentiated group (G2+G3) (P<0.05). The positive expression rate of MMP-9 protein was 60.87%(14/23) and 94.74% (18/19) in the non-muscle-invasive group (Tis,Ta,Tl) and muscle-invasive group (T2-T4) respectively (P<0.05). Significant difference of positive expression rates of MMP-9 protein also was observed between recur group (94.44%, 17/18) and primary group (62.50%, 15/24) (P<0.05).5. In all cases of BTCC, a negative correlation was observed between expression of RECK and MMP-2 (r = - 0.460, P<0.05), as well as between RECK and MMP-9 (r = - 0.309, P<0.05). However, expression of MMP-2 and MMP-9 showed a positive correlation (r = 0.557, P<0.05).Conclusions1. Expression level of RECK in BTCC tissue descends significantly, while expression of MMP-2 and MMP-9 increases markedly. All of them play important roles in the pathogenesis of BTCC.2. The expression level of RECK in BTCC descends obviously along with the progression of tumor differentiation grade and pathological stage and tumor recurrence. These findings suggest that the loss of RECK expression may enhance abilities of BTCC cell invasion and associate with the invasion, metastasis and recurrence. Moreover, it may also provide a new anticancer target. In addition, the expression levels of MMP-2 and MMP-9 upregulate obviously along with the progression of tumor differentiation grade and pathological stage, as well as with rumor recurrence, suggesting that RECK, MMP-2 and MMP-9 may be valuable to predict the degree of malignant, potency of invasion and recurrence.3. There is a positive correlation between the expressions of MMP-2 and MMP-9 in BTCC, which suggests that they may act synergistically in the pathogenesis of BTCC. Meanwhile, a negative correlation is seen between the expressions of RECK and MMP-2 or MMP-9 in BTCC, indicating that these molecules can be used to evaluate the pathogenesis, development and prognosis of BTCC, and that they may contribute to the exploitation of therapeutic strategies and new anticancer agents.
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