Background
Bladder cancer is the most common cancer in Egypt during the past 50 year [
1,
2]. The most common histopathological type of bladder cancer in Egypt is the squamous cell carcinoma (SCC), constituting from 59% to 81% between 1960–1981 [
3]. Chronic bladder infection with
schistosoma haematobium is the most important risk factor for bladder cancer [
4]. Oncologists and pathologists have suggested that the ratio between SCC: TCC (transitional cell carcinoma) types of bladder cancer was changed over the past 10–15 years. This is because the change in the etiology of bladder cancer [
5]. Definitive diagnosis of bladder cancer requires invasive cystoscopic examination. Despite urinary cytology is widely used for screening for bladder cancer, it is sometimes difficult to judge cytological specimens [
6]. Therefore, non invasive and sensitive method is required for screening, diagnosis or even for monitoring of the disease.
The extracellular matrix (ECM) has a central role in organizing tissue architecture in animals and humans, providing the structural scaffold for cells. However, ECM is not merely a passive framework, but rather a dynamic system that interacts with cells and influences their functions such as cell proliferation, growth, differentiation, migration, adhesion and survival. Conversely, cells are also able to influence and reorganize their surrounding ECM. ECM-cell interaction is essential to maintaining the physiological microenvironment and homeostasis. Any disturbance in this complex regulation have been implicated in several pathological processes including unregulated tumor growth, invasion and metastasis [
7]. Overexpression of MMPs in tumor tissues and stroma can increase level of MMPs activities in various body fluids. Multiple lines of evidence show that MMPs can be detected in urine samples procured from patients suffering prostate and bladder cancers and MMPs were independent predictor of disease status [
8,
9]. Several MMP species were detected in urine from cancer patients including MMP-2, MMP-9, MMP-9/NGAL. Several studies identified three high molecular weight gelatinases in urine of bladder cancer patients [
10,
11]. These MMPs were MMP-9 and its inhibitor TIMP-1 (140KDa), MMP-9 dimer (220KDa), and disintegrin and metalloproteinase with thrombospondin motifs-7 (ADAMTs-7) [
12].
The objective of the current study is to identify the different gelatinases activity in the urine samples collected from bilharzial bladder cancer patients (in particular HMW gelatinases) and to correlate their activities with different clinico-pathological parameters of the disease.
Discussion
Detection of certain MMPs profile in urine of patients could provide invaluable information for the tumor biology features. The presence of different MMPs including HMWs (MMP9-dimer, MMP9-TIMP-1, ADAMTS-7) was reported in the urine of a variety of cancer patients including bladder [
11,
12]. Increase of MMP activities serve to facilitate both tumor cell migration and development of new tumor-related blood vessels [
10]. The current study aimed to detect HMW-MMPs gelatinases in the urine of bladder cancer patients and not only to predict the disease onset but also to differentiate between bilharzial and non bilharzial bladder cancer. The activity of MMPs was detected in urine of 66 bladder cancer patients as well as 100 normal healthy controls using zymographic analysis technique. In our study, the activity of MMP-2 and MMP-9 recorded was 55% and 62%, respectively, and the higher activity of MMP-9 may be explained as the effect of transcriptional regulation by inflammatory cytokines such as TNFα and interlukins. MMP-2 is expressed by many cell types and most likely is regulated by the level of proenzyme activation [
13]. Different independent studies consistently reported elevated MMP-2 activity in urine samples from bladder cancer as compared to normal healthy subjects, which confirm our results and raise the hope HMW MMPS can be used as a diagnostic biomarker for this malignancy [
8,
14,
15]. On the other hand, the secretion of MMP-9 was enhanced by presence of inflammatory cells and so the presence of cystitis might lead to false positive results and decrease specificity [
16]. In addition, low sensitivity of urinary MMP-9 was detected in bladder cancer, which was approximately 40% in two independent studies [
14,
16] including our study (62%), showing a limited diagnostic value of MMP-9 in the urine of such type of malignancy. The higher activity of both MMP-2 and MMP-9 were detected in high grades and muscle invasion to prostate gland and this may indicate their role in the progression of disease. Also, higher levels of gelatinases (2 & 9) in bilharzial bladder cancer patients compared to their counterpart control might have clinical relevance in the detection of this specific type of bladder cancer. The non – significant association between MMPs and other clinical parameters probably came from the limited number of cases examined. Our study warrants further investigations to clarify this finding.
When MMP-9 present in excess amount relative to its endogenous inhibitor TIMP, it can form dimmers. MMP-9 dimer has been identified in a variety of MMP-9 producing cells including neutrophils and normal breast epithelial cells [
17,
18] and is a component of normal plasma [
19]. Enzymatic activity of the monomeric and dimeric forms of MMP-9 does not differ; however, the dimer can be activated by stromelysin with much lower efficiency (10 fold less) than monomer [
19]. The existence of more stable, slow activating MMP-9 dimer might serve as a regulatory mechanism during extracellular matrix degradation [
19]. In our study, MMP-9 was the most detectable gelatinases in the urine of bladder cancer patients which means a relative excess level of MMP-9 relative to TIMP-1 produced by the primary tumor and surrounding stroma. This in turn resulted in elevated levels of both monomeric and diametric forms of this protease in urine [
12].
It was previously demonstrated that the ~125 kDa MMP activity in the urine of cancer patients is a complex of MMP-9 and NGAL [
11], which may represent a new biomarker for the prediction of cancer disease [
20]. Several studies reported an elevation of this enzyme activity in the urine of variety of cancer including bladder cancer [
20,
21]. The formation of NGAL-MMP-9 complex has the ability to protecting MMP-9 from autodegradation. MMP-9/NGAL complex was detected more frequently in the urine of metastatic cancer than other types of disease. It is believed that invasive capacity of MMP-9 is enhanced when it is bound to NGAL and that is associated with metastasis [
11]. In the current study, MMP-9/NGAL enzyme activity was detected in about 60% of the urine specimens of the bladder cancer patients but not in any of the urine control healthy cases (p = 0.001). However, no marked difference was recorded in the ration of NGAL within bilharzial and non bilharzial bladder cancer patients or even between the TCC and SCC types. Likewise, MMP-2 and 9 had no significant difference in high grade and muscle invasion bladder cancer cases which confirm their role in the tumor progression.
Here, we report that the 190 kDa gelatinase (ADAMTS-7) was detected in the urine of a little number of bladder cancer patients (26%). To date, ADAMTS-7 has yet to be associated with human cancers except with those reported by Roy et al. [
12]. ADAMTS-7 is a family of disintegrin zinc – dependent proteases that have at least one thrombospondin type I motif [
12]. A non significant correlation was recorded between the enzymes activities with different clinico- pathological parameters.
MMP-9/TIMP-1 complex was reported to be serum marker for fibrosis in children with chronic hepatitis B [
22]. However, very little is known about its correlation with cancer. In this study, we found that urine from bladder cancer patients indicated the presence of 140 kDa MMP- 9/TIMP-1 complex but it was unfrequented in urine of those patients.
Taken together, our study detected many species of gelatinases in the urine of bladder cancer patients with primary tumor (both bilharzial and non bilharzial) including MMP-2, MMP-9, MMP-9 dimer, MMP-9/NGAL, MMP-9/TIMP-1 and ADAMTS-7. Each MMP was detected at significantly higher rate in urine from cancer patients compared to control subjects except in MMP-9/TIMP-1 and ADAMTS-7. The activity of urinary MMPs is a promising diagnostic tool and can be used as biomarkers for predicting bladder cancer. The study also revealed that the detection of urinary HMW gelatinases was not able to differentiate between bilharzial and non bilharzial bladder cancer subtypes.
Competing interests
All authors declared that they have no competing interests.
Authors’ contribution
MAM: conceived the design of the study, participated in the practical part and interpretation of data, helped to draft, revise the manuscript, has involved in the final approval of the version for publication. MFS: carried out most of the practical part, performed the statistical analysis and interpretation of data. MSA: participated in design of the study, participated in the data analysis and interpretation of data, has involved in the final approval of the version for publication. HMS: participated in design of the study, participated in the data analysis and interpretation of data, has involved in the final approval of the version for publication. AAA: participated in the practical part and interpretation of data, writing and drafting the manuscript, has involved in the final approval of the version for publication. All authors read and approved the final manuscript.