Galectin-3 in urine of cancer patients: stage and tissue specificity

Abstract

Purpose: Galectin-3 has been implicated in advanced stage of cancer disease. In the current study we examined the possibility of urinary galectin-3 levels to stage cancer disease and to follow up therapy.

Experimental design: Urine was collected from all types of cancer patients at different stages including patients undergoing radio/chemotherapy. Galectin-3 level was determined by anti-galectin-3 based ELISA and agglutination assays. Immunoblotting and purification on lactosyl affinity column further confirmed the presence of galectin-3.

Results: Cancer samples exhibited stage dependent expression of galectin-3 approx. ranging from 1.0 to 3.3, 4.4 to 5.4, 5.4 to 24.7, 13.1 to 31.9, 13.9 to 32.9 ng/mg C (creatinine) for stage I-V, respectively, at P approximately <0.05 level. Galectin-3 levels were decreased by approx. threefolds after 5th day of therapy.

Conclusions: Sample collection being simple and non-invasive, urinary galectin-3 may be used as a potential diagnostic tool for monitoring or follow up of the stage of cancer disease.

Fig. 1

Calibration curve of standard galectin-3. Standard galectin-3 obtained from Sigma chemical co., USA was used as standard at the range of 0.02–0.1 μg/100 μL and detected employing ELISA using monoclonal anti-galectin-3 antibody as described under materials and methods. >5-fold difference between the noise to signal ratio was observed. Values are the mean value taken as average of triplicates with three individual measurements, hence expressing as mean data with standard deviation. Data validates the standard curve which showed a linearity with a correlation coefficient—r of ~1.0, when analyzed using origin 6.1 version of SPSS-10 programme

Fig. 2

Urine galectin-3 concentrations in carcinomas before and after treatment with chemotherapy. Urine galectin-3 concentrations were assayed by ELISA in random cancer patients in oesophagus, cervical and non-Hodgkin’s lymphoma cancer patients before and after 2–5 days of chemotherapy. Decrease in galectin-3 levels noted were plotted. Reduction in galectin-3 levels after therapy suggests that galectin-3 can be a potent urinary marker for staging, diagnosing and follow up of the disease. All the data are mean ± SD of three replicates

Fig. 3

Correlation between the stage of the disease and galectin-3 levels. Regression analysis was performed individually on each type of cancers between galectin-3 levels as determined by ELISA with the stage of the disease and regression coefficient-R and the level of significance was obtained employing SPSS programme of Origin 6.1 statistical programme. Gradational increase in galectin-3 levels was observed with the advancement of the stage of the disease irrespective of type of cancer. R value calculated separately for different types of samples in the similar programme is as follows: cervical—R −0.93, P ~ 0.019; oesophagus—R −0.957, P ~ 0.042; breast—R −0.925, P ~ 0.247; ovary—R −0.934, P ~ 0.012 and other types of cancers—R −0.952, P ~ 0.01. All the data are mean ± SD of three replicates

Fig. 4

Protein profile in urine samples of cancer and normal subjects. 12% SDS-PAGE was performed loading cancer patient’s urine samples. Lane 1 C1 cervical-stage IV, Lane 2 C2 oesophagus-stage V, Lane 3 C3 stomach-stage V, Lane 4 C4 cervical-stage 111, Lane 5 C5 Breast-stage V and those of normal urine sample in Lane 6 (N). After electrophoresis, gels were stained using silver nitrate staining reagent for proteins. Lane 7 (M) shows the profile of molecular weight markers. Negligible proteins were found in normal urine samples; while differential protein profiles were observed in the urine of advanced cancer patients

Fig. 5

Western blot analysis of galectin-3 in urine samples of cancer patients. 12% SDS-PAGE was performed loading cancer patient’s urine samples. Lane 1 C1 cervical-stage IV, Lane 2 C2 oesophagus-stage V, Lane 3 C3 stomach-stage V, Lane 4 C4 cervical-stage 111, Lane 5 C5 breast-stage V and those of normal urine sample in Lane 6 (N). After electrophoresis, proteins were transferred to a nitrocellulose membrane. Blots were probed with anti-human galectin-3 antibody followed by alkaline phosphatase conjugated goat anti-mouse IgG. BCIP (5-bromo-4-chloro-3-indolyl phosphate) was used to detect alkaline phosphatase in western blot analysis. Bands were visualized by enzyme substrate reaction as described under materials and methods and the molecular size was determined comparing to molecular markers which were stained by silver nitrate staining. Gel documentation was performed to understand differences in profile, in cancer samples