Lectin-based proteomic studies of urinary proteins from patients with endometrial, ovarian and cervical cancer / Alan Mu Kang Wai

Endometrial (ECa), ovarian (OCa) and cervical (CCa) cancers are among ten of the most common cancers affecting women worldwide. Cancers are known to cause some proteins to be differentially glycosylated or aberrantly excreted in the urine, which can be used as biomarkers. Since ECa, OCa and CCa are difficult to diagnose at the early stage, the aim of the present study was to identify a panel of new complementary biomarkers for early detection of the cancers. Identification of early biomarkers which are specific and efficient can increase the survival rate of the patients. To screen for potential biomarkers for ECa, separation of urinary proteins of control subjects and patients with ECa by 2-DE and densitometry analysis were performed. The analysis of 2-DE urinary profiles of controls and patients with ECa demonstrated significant differential expression of zinc-alpha 2 glycoprotein (ZAG), acid alpha-1 glycoprotein (AAG) and CD59. When similar 2-DE analysis was performed using CGB lectin-Western blotting approach to detect O-glycosylated urinary proteins, the level of nebulin (NEB) was detected to be reduced in the ECa patients compared to the controls. Using similar analysis with CGB lectin, the altered levels of kininogen (KNG), clusterin (CLU) and leucine rich alpha-2 glycoprotein (LRG) were also demonstrated in the patients with OCa. However, the expression levels of all O-glycosylated proteins appeared to be comparable in patients with CCa. A significant increase in the levels of urinary ZAG and AAG was detected in ECa patients when the 2-DE profiles were transferred onto membrane and probed with CMB lectin to exclusively capture N-glycosylated urinary proteins. Similar comparative analysis of the N-glycosylated protein profiles performed on OCa patients demonstrated overexpression of AAG, ZAG, LRG, CLU and HPβ chain but decreased in the levels of KNG. Comparable expression of N-glycosylated urinary proteins was detected between the controls and CCa patients. Analysis of bound fractions obtained from immoblilized CGB lectin affinity chromatography followed by the labeled-free quantification showed differential levels of LRG and NEB in patients with OCa and ECa, respectively. This is comparable with the earlier data obtained from CGB lectin probed Western blot. Similarly, spectral counting of the N-glycosylated proteins isolated by immobilized CMB lectin affinity chromatography demonstrated altered levels of AAG and ZAG for patients with ECa and OCa and differential expression of KNG and HPβ chain for OCa patients, which are also compatible with the findings using CMB lectin blot. In addition, the LC-MS/MS analysis of the CMB lectin bound fractions detected CD59 peptides only in the controls but not in patients with ECa, OCa and CCa. When SELDI-TOF and Biomarker Wizard analyses were carried out on the CMB lectin captured urinary glycopeptides from control subjects and patients with ECa, OCa and CCa, peaks m/z 1201 and 1449 were detected as potential group discriminators. Similarly, the analysis performed on the CGB lectin captured urinary glycopeptides from control subjects and the three groups of cancer patients predicted peaks m/z 2138 and 3656 as potential group discriminators. Taken together, the findings of this study suggest urinary proteins such as ZAG, AAG, NEB, KNG, LRG, CLU, HPβ chain and CD59 may serve as potential complementary biomarkers for the early detection of ECa, OCa and CCa although this requires further extensive validation on clinically representative populations