SPRi Biosensor for Simultaneous Determination of HIF-1α, Angiopoietin-2, and Interleukin-1ß in Blood Plasma.

A new analytical method, based on SPRi biosensors, has been developed for the simultaneous determination of the pro-angiogenic factors HIF-1α, angiopoietin-2 (ANG-2), and interleukin-1ß (IL-1ß) in biological fluids. These proteins take part in the process of angiogenesis, i.e., the creation of new blood vessels, which is a key stage of cancer development and metastasis. A separate validation process was carried out for each individual compound, indicating that the method can also be used to study one selected protein. Low values of the limit of detection (LOD) and quantification (LOQ) indicate that the developed method enables the determination of very low concentrations, in the order of pg/mL. The LOD values obtained for HIF-1α, ANG-2, and IL-1ß were 0.09, 0.01, and 0.01 pg/mL, respectively. The LOQ values were 0.27, 0.039, and 0.02 pg/mL, and the response ranges of the biosensor were 5.00-100.00, 1.00-20.00, and 1.00-15.00 pg/mL. Moreover, determining the appropriate validation parameters confirmed that the design offers high precision, accuracy, and sensitivity. To prove the usefulness of the biosensor in practice, determinations were made in plasma samples from a control group and from a study group consisting of patients with diagnosed bladder cancer. The preliminary results obtained indicate that this biosensor can be used for broader analyses of bladder cancer. Each of the potential biomarkers, HIF-1α, ANG-2, and IL-1ß, produced higher concentrations in the study group than in the control group. These are preliminary studies that serve to develop hypotheses, and their confirmation requires the analysis of a larger number of samples. However, the constructed biosensor is characterized by its ease and speed of measurement, and the method does not require special preparation of samples. SPRi biosensors can be used as a sensitive and highly selective method for determining potential blood biomarkers, which in the future may become part of the routine diagnosis of cancers.

Application of surface plasmon resonance imaging biosensors for determination of fibronectin, laminin-5 and type IV collagen in serum of transitional bladder cancer patients.

The urothelial basement membrane (UBM) contains type IV collagen, laminin-5, and fibronectin. Urothelial neoplasm can break through the UBM underlying the urothelium to invade the lamina propria. Conceptually, all bladder cancer staging over Ta (T1-T4) may demonstrate disturbances in the UBM structure, as well as alterations in the serum concentrations of the studied components. The aim of this study was to determine the blood serum concentration of collagen IV, laminin-5 and fibronectin in bladder cancer patients. Quantification of their concentrations and correlation with various clinicopathological parameters may be useful for making more accurate predictions and identifying high-risk patients. The study included 96 patients with bladder cancer confirmed by transurethral resection or cystectomy and 26 patients with diagnosed cystitis chronica or BPH (benign prostate hyperplasia). Collagen IV, laminin-5 and fibronectin were detected using Surface Plasmon Resonance Imaging biosensors. Significant differences in blood serum concentrations of the studied biomarkers were observed between the control group and bladder cancer patients, as well as between nonmuscle-invasive and muscle-invasive groups. ROC analysis gave satisfactory results for differentiation between the control group and bladder cancer group (AUC 0.92-0.99), with lower values only for collagen IV between nonmuscle-invasive and muscle-invasive patients (AUC 0.71), and a statistically insignificant difference for laminin-5. Laminin-5 concentration was more closely correlated to tumour grade, size and recurrence rate; fibronectin to tumour stage, size and morphology; and collagen IV to tumour stage, grade and recurrence rate. The relations between serum concentrations of the presented biomarkers of the urothelial basement membrane may be useful for bladder cancer detection and for determination of the tumour stage, hence simplifying the making of therapeutic decisions.

Plasma aromatase as a sensitive and selective potential biomarker of bladder cancer and its role in tumorigenesis.

Bladder cancer (BCa) is the ninth most common cancer in the world and its early detection is crucial for successful therapy. Unfortunately, there are no satisfactory tools to detect BCa at early stages and BCa's confirmation muscle-invasive. The search for a suitable biomarker is therefore necessary and aromatase is a potential candidate. The purpose of the current study was to determine if aromatase serves as a biomarker of BCa. A Surface Plasmon Resonance Imaging biosensor was applied for the quantification and determination of aromatase. A total of 3 µl blood plasma was used for a single measurement. The results revealed that the aromatase concentration in the plasma of patients with BCa (n=78) ranged from 17.41-57.44 ng/ml. The range determined in healthy donors (n=18) was 2.59-7.74 ng/ml. Additionally, it was revealed that muscle invasive BCa samples exhibited elevated, statistically significant (P=0.01) average aromatase concentrations in blood plasma (38.64 ng/ml) when compared with non-muscle invasive samples (29.83 ng/ml). The results demonstrated that plasma aromatase may serve as an excellent bimarker of BCa with 100% sensitivity, 100% selectivity and an area under the curve value of the reciever operating characteristic curve equal to 1.0. Furthermore, the marker differenciated between muscle-invasive and non muscle-invasive BCa with a sensitivity of 60% and a specificity of 81%. In conclusion, aromatase may serve a role in bladder tumorigenesis.

Utility of cystatin C as a potential bladder tumour biomarker confirmed by surface plasmon resonance technique.

BACKGROUND & OBJECTIVES: The determination of cystatin C (cysC) may be helpful in diagnosis and monitoring of cancer because the pathogenesis of cancer is linked with an increased activity of cysteine peptidases (cathepsins) and a decrease of cysC concentration. This study was aimed to examine the utility of cysC as a marker of bladder cancer (BCa) to be used in the diagnosis. METHODS: This study was conducted with 90 patients with BCa and 27 healthy people. Patients with other cancers, inflammation process and impaired renal function were excluded from the study. The concentrations of cysC in the plasma and urine were measured by surface plasmon resonance imaging technique. RESULTS: The concentration of cysC in the serum taken from the patients with BCa [0.35±0.02 µg/ml (range: 0.20-0.78 µg/ml)] was significantly (P<0.001) lower than the serum cysC concentration of the healthy people [0.68±0.05 µg/ml (range: 0.52-0.89 µg/ml)]. The urinary cysC concentration of the BCa patients [0.19±0.01 µg/ml (range: 0.09-0.34 µg/ml)] was not significantly different from the urinary cysC concentration of the healthy people [0.24±0.02 µg/ml (range: 0.16-0.33 µg/ml)]. Receiver operating characteristic (ROC) curve showed that BCa patients with cysC concentration <0.54 µg/ml [sensitivity: 87%; specificity: 92%; area under the curve (AUC) of ROC: 0.927; P=0.02] could be optimally separated from healthy people. The ROC curve further showed that superficial low-grade patients with cysC concentration lower than 0.36 µg/ml (sensitivity: 0.63%; specificity: 0.58%; AUC of ROC: 0.635; P=0.08) could not be optimally separated from high-risk tumour patients. INTERPRETATION & CONCLUSIONS: BCa patients have lower serum cysC concentration than the control group. Serum cysC may be considered as a potential marker of BCa but not its aggressiveness.

Bladder cancer detection using a peptide substrate of the 20S proteasome.

The 20S catalytic core of the human 26S proteasome can be secreted from cells, and high levels of extracellular 20S proteasome have been linked to many types of cancers and autoimmune diseases. Several diagnostic approaches have been developed that detect 20S proteasome activity in plasma, but these suffer from problems with efficiency and sensitivity. In this report, we describe the optimization and synthesis of an internally quenched fluorescent substrate of the 20S proteasome, and investigate its use as a potential diagnostic test in bladder cancer. This peptide, 2-aminobenzoic acid (ABZ)-Val-Val-Ser-Tyr-Ala-Met-Gly-Tyr(3-NO2 )-NH2 , is cleaved by the chymotrypsin 20S proteasome subunit and displays an excellent specificity constant value (9.7 × 10(5)  m(-1) ·s(-1) ) and a high kcat (8 s(-1) ). Using this peptide, we identified chymotrypsin-like proteasome activity in the majority of urine samples obtained from patients with bladder cancer, whereas the proteasome activity in urine samples from healthy volunteers was below the detection limit (0.5 pm). These findings were confirmed by an inhibitory study and immunochemistry methods.

Podoplanin serum and urine concentration in transitional bladder cancer.

BACKGROUND: Podoplanin (PDP) is a mucin - a type of transmembrane protein expressed in numerous tissues during ontogeny and in adult animals, including the brain, heart, kidney, osteoblasts and lymphoid organs. OBJECTIVE: The aim of this study was to determine podoplanin concentration in the blood serum and urine of patients with bladder cancer. Quantifying podoplanin concentration and its correlation with various clinicopathological parameters may be useful for more accurate predictions and identifying high-risk patients. METHODS: The present study included 82 patients with bladder cancer confirmed by transurethral resection or cystectomy and 27 healthy volunteers. The Surface Plasmon Resonance Imaging biosensor was applied for the detection of podoplanin in the serum and urine samples. RESULTS: Significant differences in serum and urine podoplanin concentration levels were observed between bladder cancer patients. The statistically significant higher values of PDP were detected in serum of patients with invasive, more aggressive, larger, multifocal tumors. CONCLUSIONS: The association between podoplanin concentration and clinicopathological features indicates that it might be useful while making therapeutic decisions.

[Cathepsin D and B activity in the serum of patients with urothelial bladder cancer]. / Aktywnosc katepsyn D i B w osoczu chorych na raka przejsciowokomórkowego pecherza moczowego.

UNLABELLED: The key role of cathepsin D and B is intralysosomal digestion of used cellular proteins and other proteins that enter cells through endocytosis. Under pathological conditions like cancer formation and growth, cathepsins from lysosomes are released. The aim of the study was to determin of cathepsin D and B activities in serum of patients with urothelial bladder cancer depending on disease severity and determination of its' changes after transurethral resection of tumor. MATERIAL AND METHODS: Experiment involved 50 patients. Blood samples were obtained from 18 healthy volunteers and 32 urothelial bladder cancer patients. Samples from people with suspected urothelial bladder cancer were collected three times: before the surgery, 2 weeks and 6 weeks after the surgical treatment. RESULTS: Our research showed that cathepsin D activity, measured as the level increment of acid soluble tyrosine, is the highest before the surgery in muscle invasive bladder tumor (pT2) (57,9 nmol/ml). 2 weeks and 6 weeks after the surgical treatment, cathepsin D activity is decreased. In case of cathepsin B activity, measured as the level of released p-nitroaniline, decreased, 2 weeks and 6 weeks after the surgical treatment in both cases of disease severity. CONCLUSION: Cathepsin D and B activities in the serum of patients with urothelial bladder cancer are directly proportional to disease severity and significantly higher compared with control group. Transuretral resection of the tumor leads to diminution of their activities in second and 6th week after the procedure.

Cathepsin D serum and urine concentration in superficial and invasive transitional bladder cancer as determined by surface plasmon resonance imaging.

Determination of cathepsin D (Cat D) concentration in serum and urine may be useful in the diagnosis of bladder cancer. The present study included 54 healthy patients and 68 patients with bladder cancer, confirmed by transurethral resection or cystectomy. Cat D concentration was determined using a surface plasmon resonance imaging biosensor. Cat D concentration in the serum of bladder cancer patients was within the range of 1.3-5.59 ng/ml, while for healthy donors it was within the range of 0.28-0.52 ng/ml. In urine, the Cat D concentration of bladder cancer patients was within the range of 1.35-7.14 ng/ml, while for healthy donors it was within the range of 0.32-0.68 ng/ml. Cat D concentration may represent an efficient tumor marker, as its concentration in the serum and urine of transitional cell carcinoma patients is extremely high when compared with healthy subjects.