title: Multiplex zymography captures stage-specific activity profiles of cathepsins K, L, and S in human breast, lung, and cervical cancer.
abstract: BACKGROUND
  Cathepsins K, L, and S are cysteine proteases upregulated in cancer and proteolyze extracellular matrix to facilitate metastasis, but difficulty distinguishing specific cathepsin activity in complex tissue extracts confounds scientific studies and employing them for use in clinical diagnoses. Here, we have developed multiplex cathepsin zymography to profile cathepsins K, L, and S activity in 10 μg human breast, lung, and cervical tumors by exploiting unique electrophoretic mobility and renaturation properties.
  
  
  METHODS
  Frozen breast, lung, and cervix cancer tissue lysates and normal organ tissue lysates from the same human patients were obtained (28 breast tissues, 23 lung tissues, and 23 cervix tissues), minced and homogenized prior to loading for cathepsin gelatin zymography to determine enzymatic activity.
  
  
  RESULTS
  Cleared bands of cathepsin activity were identified and validated in tumor extracts and detected organ- and stage-specific differences in activity. Cathepsin K was unique compared to cathepsins L and S. It was significantly higher for all cancers even at the earliest stage tested (stage I for lung and cervix (n = 6, p < .05), and stage II for breast; n = 6, p < .0001). Interestingly, cervical and breast tumor cathepsin activity was highest at the earliest stage we tested, stages I and II, respectively, and then were significantly lower at the latest stages tested (III and IV, respectively) (n = 6, p < 0.01 and p < 0.05), but lung cathepsin activity increased from one stage to the next (n = 6, p < .05). Using cathepsin K as a diagnostic biomarker for breast cancer detected with multiplex zymography, yielded 100% sensitivity and specificity for 20 breast tissue samples tested (10 normal; 10 tumor) in part due to the consistent absence of cathepsin K in normal breast tissue across all patients.
  
  
  CONCLUSIONS
  To summarize, this sensitive assay provides quantitative outputs of cathepsins K, L, and S activities from mere micrograms of tissue and has potential use as a supplement to histological methods of clinical diagnoses of biopsied human tissue.
authors.affiliation: Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, GA 30332, Atlanta, USA.
authors.forename: Binbin
authors.initials: B
authors.lastname: Chen
authors.forename: Manu O
authors.initials: MO
authors.lastname: Platt
mesh_terms: D000328:Adult; D014408:Biomarkers, Tumor / Q000378:metabolism; D001943:Breast Neoplasms / Q000175:diagnosis / Q000201:enzymology / Q000473:pathology; D002403:Cathepsins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005260:Female; D006801:Humans; D008175:Lung Neoplasms / Q000201:enzymology / Q000473:pathology; D008297:Male; D008875:Middle Aged; D009367:Neoplasm Staging; D009369:Neoplasms / Q000201:enzymology* / Q000473:pathology*; D009928:Organ Specificity; D012016:Reference Values; D002583:Uterine Cervical Neoplasms / Q000201:enzymology / Q000473:pathology
chemical_list: D014408:Biomarkers, Tumor; D002403:Cathepsins