Elsevier

Journal of Proteomics

Volume 127, Part A, 8 September 2015, Pages 185-192
Journal of Proteomics

Differential regulation of urine proteins in urothelial neoplasm

https://doi.org/10.1016/j.jprot.2015.04.022Get rights and content

Highlights

  • Eight urine proteins are differentially regulated in urothelial neoplasm.

  • Seven up-regulated and one down-regulated

  • Proteomic levels vary with the severity of the disease.

  • Potential as noninvasive method for disease monitoring

Abstract

Urothelial neoplasm of the urinary bladder has a high rate of multifocality and recurrence. To understand this we first need to understand the changes in the molecular level that distinguishes a normal individual from a patient and also a low grade neoplasm from a high grade. In this work we aim to study the urine proteome of Indian patients with urothelial neoplasm categorised on the basis of their p53 immunohistochemistry. The urine samples of pre-operative patients were subjected to two dimensional gel electrophoresis followed by densitometric analysis and spot identification using MALDI mass spectrometry. Our study shows that few proteins such as albumin, alpha 1 antitrypsin, apolipoprotein A1, transferrin, transthyretin, haptoglobin and haemoglobin β chain were upregulated and inter alpha trypsin inhibitor heavy chain was downregulated in the disease samples. Further we have reported that some of these proteins show an association with disease severity. The present study marks the first step in the identification of new diagnostic markers as well as therapeutic targets.

Biological significance

Bladder carcinoma is the ninth most common cancer worldwide. It has gained attention within both clinicians and cancer biologists because of its recurrence and mortality rate. Identifying the prognostic factors of progression is a challenge, so that high risk patients who may be a candidate for a radical cystectomy may be identified. In this study we have attempted to study the changes observed in the urinary protein levels of urothelial neoplasm patients. The samples were graded based on p53 immunohistochemistry staining. We have reported eight (8) proteins, mostly highly abundant; those have exhibited differential regulation in case of diseased samples. This study is first of its kind that associates the changes in the urinary protein levels to that of the severity of the disease. We believe that the findings can be used as a stepping stone in the development of a noninvasive prognostic tool for the disease.

This article is part of a Special Issue entitled: Proteomics in India.

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.

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