Differential regulation of urine proteins in urothelial neoplasm☆
Graphical abstract
Introduction
Urothelial neoplasms of the upper urinary tract account for approximately 5% of all epithelial tumours of the urinary tract only, whereas urinary bladder tumours are the most common and are the fourth most common form of cancer. This disease is mainly diagnosed in the elders, approximately at the age of 65 years and is more common in men than in women [1]. Roychowdhury and co-workers showed that, p53 protein expression is associated with high grade urothelial neoplasm and advanced stage of the disease [2].
Mutations of p53 are found to be present in 40–45% of cancers, including all sites combined. Indeed, p53 mutation is the most frequent genetic event demonstrated to date [3]. These point mutations lead to the loss of its tumour suppressing function. The wild-type p53 protein has a short half-life of 15 to 30 min, whereas the mutated p53 gene results in a protein with a prolonged half-life, which is the basis of its nuclear accumulation that is detectable by immunohistochemistry (IHC). The accumulated p53 has been associated with the progression of bladder cancer and might play a role in the evolution of the tumours to a higher grade, shown in earlier studies [2], [4].
Urothelial neoplasm occurs at different sites in the urinary bladder with varying frequency. Vigilant monitoring of patients after definitive treatment for urothelial neoplasm is essential owing to the high rate of multifocality and recurrence. However, there are no clear cut ways of predicting which urothelial carcinomas would subsequently recur or progress or which muscle invasive tumours would progress following treatment. In this work, for the first time, we have tried to establish a correlation between different tumour grades and changes in the protein profile of urine samples of urothelial neoplasm patients. Urine being the body waste is easily obtainable for monitoring at various stages of the disease and hence will help in establishing a non-invasive disease and post-treatment monitoring method. Results indicate differential expression of proteins like inter alpha trypsin inhibitor heavy chain, apolipoprotein A1, haptoglobin and transferrin which could be easily monitored at various stages of the disease in a non invasive method.
Section snippets
Material
Amicon ultra centrifugal filter units with 5 kDa cut off membrane and PVDF membrane were obtained from Millipore. Ethanol from Merck, 2D rehydration buffer, 17 cm pH 3–10 IPG strips, Isoelectric focussing system, two dimensional gel electrophoresis (2-DE) system and western blot transfer setup were purchased from Bio-Rad. Colloidal Coomassie from Sigma and sypro ruby stain from Invitrogen. Sequence grade trypsin was purchased from Promega, in-gel tryptic digestion kit from Pierce Biotechnologies
Results
From the 2D gels 44 spots were analysed out of which 8 proteins were found to exhibit differential expression in the urine samples of the patients with respect to normal and control samples (analysis details given in supplementary material 1). These proteins are the ones showing more than 1.5 fold change in the mean ppm spot volume in any of the three categories compared to normal. These proteins were identified as albumin, transferrin, alpha 1-antitrypsin, apolipoprotein A1, haemoglobin β
Discussion
No enrichment technique was used for protein samples and hence, most of the proteins showing changes in urine levels are the highly abundant proteins e.g. albumin, transferrin, alpha 1 antitrypsin, apolipoprotein A1, transthyretin and haptoglobin. The differentially regulated proteins are mainly catalytic, enzymatic or transporters.
Albuminuria has been reported in different types of cancer such as lungs, breast, renal and colon/rectal. It has been suggested to be a nonspecific marker of
Conclusion
The most common prognostic marker used to monitor cancer progression is p53 immunohistochemistry, which is an invasive method. In this study we have been able to report urinary proteins which show differential regulation in case of the disease with varying severity.
We believe that these proteins should be studied in more details and with a wider and bigger sample pool as they have the potential to become diagnostic markers or therapeutic targets which could be easily collected and provide a
Conflict of interest
The authors declare no conflict of interest.
Acknowledgement
We would like to acknowledge funding from IBOP project, Department of Atomic Energy (DAE), India (15/3(7)2012/SINP/R&D-II/6221). We acknowledge Mr. Avik Basu for critical reading of the manuscript.
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This article is part of a Special Issue entitled: Proteomics in India.