Multiplex Zymography Captures Stage-specific Activity Profiles of Cathepsins K, L, and S in Human Breast, Lung, and Cervical Cancer

Binbin Chen,Manu O Platt

doi:10.1186/1479-5876-9-109

Abstract

BackgroundCathepsins 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.MethodsFrozen 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.ResultsCleared 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.ConclusionsTo 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.

Highlights

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
Multiplex cathepsin zymography detects mature cathepsins K, L, and S activity Mature cathepsins K, L, and S were loaded for cathepsin zymography and parallel samples were loaded for Western blotting to first determine if the zymographically active bands of cathepsins K, L, and S would appear at different electrophoretic migration distances
The immunodetected cathepsin K band is near 37 kDa, cathepsin S exhibited two bands near 25 kDa, and the cathepsin L protein was detected at three sizes, but only the smallest of the three immunodetected bands was zymographically active (Figure 1B)

Summary

Introduction

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. Migration, invasion and metastasis involves proteolytic activity, and the cathepsin family of cysteine proteases are proteases that have been implicated in each of these mechanisms, cathepsins B, K, L, and S [1,2]. Cathepsins K, L, and S are elastinolytic and collagenolytic cysteine proteases that share greater than 60% sequence homology [6], but the variable portions confer important differences in proteolytic activity and regulatory mechanisms. While cathepsins K and L prefer acidic environments for optimal activity, cathepsin S has the unique property of maintaining high elastinolytic activities at neutral pH and has been shown to be active in angiogenesis, lung cancer, and emphysema [16,17,18]

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