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A serine protease activity in C3H/10T1/2 cells that is inhibited by anticarcinogenic protease inhibitors
Abstract:
Several different protease inhibitors have the ability to suppress transformation in vitro and carcinogenesis in vivo. The mechanism(s) by which protease inhibitors suppress carcinogenesis, however, is not fully understood. Presumably, these agents inhibit one or more intracellular proteases whose functions are essential for the induction and/or expression of the transformed phenotype. We have isolated an endopeptidase activity capable of hydrolyzing the substrate Boc-Val-Pro-Arg-MCA (Boc = butoxycarbonyl; MCA = 7-amino-4-methylcoumarin) from C3H/10T1/2 mouse embryo fibroblast cells. This intracellular protease was inhibited by the soybean-derived Bowman-Birk inhibitor (BBI), chymostatin, and L-1-tosylamido-2-phenylethyl chloromethyl ketone, all of which have anticarcinogenic activity, but was unaffected by soybean trypsin inhibitor, which lacks anticarcinogenic activity. Other protease inhibitors affected the proteolytic activity to an extent that correlates with their relative ability to suppress transformation in vitro. The enzyme has a mass of about 70 kDa, contains a single subunit, and exhibits maximal activity at pH 7.0. Diisopropyl fluorophosphate covalently binds to this enzyme and blocks its activity, indicating that the enzyme is a serine protease. We have previously demonstrated that several protease inhibitors are effective suppressors of radiation-induced transformation of C3H/10T1/2 cells. Since these agents reduce the Boc-Val-Pro-Arg-MCA-hydrolyzing activity to an extent that correlates with their ability to inhibit malignant transformation in vitro, this endopeptidase activity may be a cellular target of the anticarcinogenic protease inhibitors.
Insights
Protease inhibitors suppress cancer by targeting intracellular proteases. Researchers identified a specific serine protease in mouse cells inhibited by cancer-suppressing agents, suggesting it
Area of Science:
- Biochemistry
- Cell Biology
- Oncology
Background:
- Protease inhibitors demonstrate in vitro and in vivo anticarcinogenic properties.
- The precise mechanisms by which protease inhibitors suppress carcinogenesis remain incompletely understood.
- Intracellular proteases are hypothesized to be key targets for protease inhibitor action in suppressing the transformed phenotype.
Purpose of the Study:
- To isolate and characterize an intracellular protease activity in C3H/10T1/2 mouse embryo fibroblast cells.
- To investigate the correlation between the inhibition of this protease activity and the anticarcinogenic effects of various protease inhibitors.
- To determine if this endopeptidase activity represents a cellular target for anticarcinogenic protease inhibitors.
Main Methods:
- Isolation of endopeptidase activity using the substrate Boc-Val-Pro-Arg-MCA.
- Testing the inhibitory effects of known anticarcinogenic protease inhibitors (e.g., Bowman-Birk inhibitor, chymostatin) and non-anticarcinogenic inhibitors (e.g., soybean trypsin inhibitor).
- Characterization of the enzyme's properties, including mass, subunit composition, optimal pH, and reactivity with diisopropyl fluorophosphate to identify it as a serine protease.
Main Results:
- An endopeptidase activity hydrolyzing Boc-Val-Pro-Arg-MCA was isolated from C3H/10T1/2 cells.
- This activity was inhibited by anticarcinogenic protease inhibitors (BBI, chymostatin) but not by soybean trypsin inhibitor.
- The enzyme is a ~70 kDa serine protease, optimally active at pH 7.0, and its inhibition by various agents correlated with their ability to suppress malignant transformation in vitro.
Conclusions:
- The identified endopeptidase activity is likely a cellular target of anticarcinogenic protease inhibitors.
- Inhibition of this specific serine protease may mediate the suppression of malignant transformation and carcinogenesis.
- Further research into this protease could elucidate novel therapeutic strategies for cancer prevention and treatment.