Discovery of Apo-A1 as a potential bladder cancer biomarker by urine proteomics and analysis

https://doi.org/10.1016/j.bbrc.2014.03.053Get rights and content

Highlights

  • Urine proteomics is promising for biomarker identification of bladder cancer.

  • Urine Apo-A1 could be a potential biomarker of bladder cancer.

  • Apo-A1 level could be an auxiliary examination for exfoliative urinary cytology.

Abstract

Bladder cancer is clinically characterized by high recurrent rate and poor prognosis and thereby patients need regular re-examinations which are invasive, unpleasant, and expensive. A noninvasive and less expensive method for detecting and monitoring bladder cancer would thus be advantageous. In this study, by using the two-dimensional electrophoresis (2-DE) approach with subsequent mass spectrometry (MS), we demonstrated the increased expression of apolipoprotein-A1 (Apo-A1) in individual urine from patients with bladder cancer, which was confirmed by Western blot results. A further analysis of the urinary Apo-A1 levels by an enzyme-linked immunosorbent assay yielded results that were consistent with the Western blot, and suggested Apo-A1 could provide diagnostic utility to distinguish patients with bladder cancer from healthy controls at 19.21 ng/ml. Further validation assay in a larger number of urine samples (n = 379) showed that Apo-A1 could be used as a biomarker to diagnosis bladder cancer with a sensitivity and specificity of 89.2% and 84.6% respectively. Moreover, the application of exfoliative urinary cytology in combination with the urine Apo-A1 detection could significantly increased the sensitivity in detecting bladder cancer. Our data showed a significant relationship of expressed Apo-A1 was established between bladder cancer and normal controls. Apo-A1 could be a potential biomarker for the diagnosis of bladder cancer.

Introduction

Bladder cancer is the second most common urological cancer, clinically characterized by high recurrent rate and poor prognosis [1]. It is estimated that bladder cancer will account for 73,510 new cases of cancer and 14,880 cancer-related deaths in the United States during 2012 [2]. Therefore, bladder cancer remains a focus in cancer research. Generally, within 5–15 years after initial resection, the recurrence rate is as high as 60–85% [3]. Moreover, approximately 20% of the recurred bladder cancer cases can develop into muscle-invasive tumors [4].

At present, cystoscopy with cytology is the standard for diagnosing and monitoring bladder cancer. Cystoscopy is an invasive, relatively costly technique that may also be affected at times, particularly in cystitis cases [5], [6]. Cytology is specific and noninvasive for diagnosing bladder carcinoma but less sensitive, particularly for detecting low-grade disease, which has limited the clinical relevance [7]. The invasiveness of cystoscopy and the limitations of cytology for detecting bladder cancer have generated interest in other simple, noninvasive diagnostic tools.

Proteomic patterns in body fluids gain increasing importance as a new tool for the identification of novel, highly sensitive diagnostic markers for the detection of cancer [8], [9], [10]. Proteomics determines disease-associated biomarkers by observing the expression patterns of proteins thereby distinguishing between normal and dysregulated processes. Two-dimensional electrophoresis (2-DE) followed by mass spectrometry (MS) has been wildly applied in differential proteomic studies of urine specimens for biomarker discovery [11], [12]. One promising approach in the search for useful bladder cancer biomarkers is to study the urine proteome during the occurrence of the disease [13], [14]. Urine is in direct contact with bladder epithelia cells and the changes of the composition, quantity, and quality may reflect the information that could represent the generation, development, and prognosis of the urinary tumor. Compared with the collection of plasma and other body fluid samples, the collection of urine is convenient and noninvasive. In the past years, several proteins in urine were researched as biomarkers for bladder cancer, such as orosomucoid, zinc-alpha2-glycoprotein, fibrinogen β chain and α-1-antitrypsin [12], [15].

The development of proteomics has provided us new tools in determining biomarkers for the detection and follow-up of bladder cancer. In this study, by using the 2-DE approach with subsequent MS, we demonstrated the increased expression and secretion of apolipoprotein-A1 (Apo-A1) protein in individual urine from patients with bladder cancer, which was further confirmed by Western blot analyses and enzyme-linked immunosorbent assay (ELISA). Moreover, the application of exfoliative urinary cytology in combination with the urine Apo-A1 detection could significantly increased the sensitivity and accuracy in bladder cancer detecting. These findings strongly suggest Apo-A1 could be a potential biomarker for bladder cancer detection and diagnosis.

Section snippets

Study population

Patients with bladder cancer were enrolled at Second Hospital of Tianjin Medical University between 2011 and 2013. All the patients had histopathologically confirmed bladder cancer, and none had received chemotherapy or radiation before enrollment. Patients were confirmed without resent post medical history (PMH) and any symptom of kidney failure. Age-, sex-, and ethnicity-matched control subjects were healthy volunteers with no history or evidence of urological cancer who were recruited from

Apo-A1 express is increased in urine of patients with bladder cancer

Urine proteins from patients with bladder cancer and healthy controls were resolved by 2-DE individually. 2-DE images stained by silver revealed expression profiles of urinary proteins in the bladder cancer and normal control groups were similar but not identified (data not shown). Of those differentially expressed protein dots, two spots about 28 kDa in pH range 5–6 attracted our attention, which obviously expressed at an increased level in bladder cancer than in healthy control (Fig. 1) and

Discussion

Several biomarkers indicated changes in the expression of urinary proteins associated with increased risk have been identified for the detection of bladder cancer by using proteome analysis [9], [10], [11], [12], [17], [18]. In our previous study, we identified several urine proteins with increased or decreased levels between pooled urine samples from healthy controls and patients with bladder cancer by using the proteomics technology [11]. Among those identified proteins, a protein about 28 kDa

Acknowledgments

The authors thank the patients, health volunteers and their families for their involvement in this study. The authors acknowledge Dr. Shixin Wang for the assistance in 2-DE and MS analysis, and Mrs. Jin Wang for her help in urine sample collection and urinary cytology examination. This study was funded by Tianjin Natural Science Foundation to C.L. (13JCQNJC11500) and supported in part by Science and Technology Foundation of Tianjin Public Health Bureau of China to C.L. and T.Z. (2012KZ081,

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