Human Cancer Cells Retain Modest Levels of Enzymatically Active Matriptase Only in Extracellular Milieu following Induction of Zymogen Activation

Li-Ling Chu,Jie-Ru Yang,Chen-Yong Lin,Hue-Yu Wang,Michael D Johnson,Yuan Xu,Jehng-Kang Wang,Hong-Yu Lai,Chun-Che Tseng,Yi-An Hu,Hsiang-Hua Chang,Jian Cao

doi:10.1371/journal.pone.0092244

Abstract

The type 2 transmembrane serine protease matriptase is broadly expressed in human carcinomas and hematological cancers. The proteolytic activity of matriptase is a potential target of drugs and imaging probes. We assessed the fate of active matriptase following the induction of matriptase zymogen activation. Exposing eight human carcinoma cells to pH 6.0 buffer induced robust matriptase zymogen activation followed by rapid inhibition of the nascent active matriptase by hepatocyte growth factor activator inhibitor (HAI)-1. Consequently, no enzymatically active matriptase was detected in these cells. Some active matriptase is, however, rapidly shed to the extracellular milieu by these carcinoma cells. The lack of cell-associated active matriptase and the shedding of active matriptase were also observed in two hematological cancer lines. Matriptase shedding is correlated closely with the induction of matriptase activation, suggesting that matriptase activation and shedding are kinetically coupled. The coupling allows a proportion of active matriptase to survive HAI-1 inhibition by rapid shedding from cell surface. Our study suggests that cellular free, active matriptase is scarce and might not be an effective target for in vivo imaging and drug development.

Highlights

Proteases catalyze the breakdown of proteins by the hydrolysis of peptide bonds
In order to begin to explore whether matriptase proteolytic activity associated with breast cancer cells might be used as a target for the development of drugs and/or imaging probes, we measured the cleavage of the substrate Boc-Gln-Ala-Arg-Amido-4- methylcoumarin (AMC) by MCF7 breast cancer cells prior to and following the induction of robust matriptase zymogen activation (Fig. 1A)
Matriptase zymogen activation apparently occurs constitutively in MCF7 cells since the 120-kDa matriptase-hepatocyte growth factor activator inhibitor (HAI)-1 complex was readily detected in addition to the 70-kDa matriptase zymogen in cell lysates prior to the induction of matriptase zymogen activation (Fig. 1A lane 1)

Summary

Introduction

Proteases catalyze the breakdown of proteins by the hydrolysis of peptide bonds. Through the regulated cleavage of proteins, proteases are involved in many highly controlled physiological processes, such as DNA replication, cell-cycle progression, cell death, angiogenesis, blood coagulation, inflammation, neurogenesis and immunity. In spite of the success of some drugs and probes, targeting proteolytic activity for development of drug and biomarkers has not always been very satisfying As attractive as they are, proteases-inspired diagnostics and therapies have many inherent complexities and limitations that need to be taken into consideration before developing new drugs or probes targeting proteases and proteases activities. These limitations include the activational status of the proteases, the functional localization of the proteases, and endogenous proteases inhibitors, all of which affect protease activity and can in turn affect the effectiveness of the protease inhibitor and probes

Methods

Results

Conclusion