Recombinant cathepsin S propeptide attenuates cell invasion by inhibition of cathepsin L-like proteases in tumor microenvironment

Human cathepsin L along with cathepsin S, K, and V are collectively known as cathepsin L-like proteases due to their high homology. The overexpression and aberrant activity of each of these proteases has been implicated in tumorigenesis. These proteases contain propeptide domains that can potently inhibit both their cognate protease and other proteases within the cathepsin L-like subfamily. In this investigation, we have produced the cathepsin S propeptide recombinantly and have shown that it is a potent inhibitor of the peptidolytic, elastinolytic, and gelatinolytic activities of the cathepsin L-like proteases. In addition, we show that this peptide is capable of significantly attenuating tumor cell invasion in a panel of human cancer cell lines. Furthermore, fusion of an IgG Fc-domain to the COOH terminus of the propeptide resulted in a chimeric protein with significantly enhanced ability to block tumor cell invasion. This Fc fusion protein exhibited enhanced stability in cell-based assays in comparison with the unmodified propeptide species. This approach for the combined inhibition of the cathepsin L-like proteases may prove useful for the further study in cancer and other conditions where their aberrant activity has been implicated. Furthermore, this strategy for simultaneous inhibition of multiple cysteine cathepsins may represent the basis for novel therapeutics to attenuate tumorigenesis.

Isoform-specific inhibition of TRPC4 channel by phosphatidylinositol 4,5-bisphosphate.

Full-length transient receptor potential (TRP) cation channel TRPC4alpha and shorter TRPC4beta lacking 84 amino acids in the cytosolic C terminus are expressed in smooth muscle and endothelial cells where they regulate membrane potential and Ca(2+) influx. In common with other "classical" TRPCs, TRPC4 is activated by G(q)/phospholipase C-coupled receptors, but the underlying mechanism remains elusive. Little is also known about any isoform-specific channel regulation. Here we show that TRPC4alpha but not TRPC4beta was strongly inhibited by intracellularly applied phosphatidylinositol 4,5-bisphosphate (PIP(2)). In contrast, several other phosphoinositides (PI), including PI(3,4)P(2), PI(3,5)P(2), and PI(3,4,5)P(3), had no effect or even potentiated TRPC4alpha indicating that PIP(2) inhibits TRPC4alpha in a highly selective manner. We show that PIP(2) binds to the C terminus of TRPC4alpha but not that of TRPC4beta in vitro. Its inhibitory action was dependent on the association of TRPC4alpha with actin cytoskeleton as it was prevented by cytochalasin D treatment or by the deletion of the C-terminal PDZ-binding motif (Thr-Thr-Arg-Leu) that links TRPC4 to F-actin through the sodium-hydrogen exchanger regulatory factor and ezrin. PIP(2) breakdown appears to be a required step in TRPC4alpha channel activation as PIP(2) depletion alone was insufficient for channel opening, which additionally required Ca(2+) and pertussis toxin-sensitive G(i/o) proteins. Thus, TRPC4 channels integrate a variety of G-protein-dependent stimuli, including a PIP(2)/cytoskeleton dependence reminiscent of the TRPC4-like muscarinic agonist-activated cation channels in ileal myocytes.

Combined inhibition of FLIP and XIAP induces Bax-independent apoptosis in type II colorectal cancer cells.

Death receptors can directly (type I cells) or indirectly induce apoptosis by activating mitochondrial-regulated apoptosis (type II cells). The level of caspase 8 activation is thought to determine whether a cell is type I or II, with type II cells less efficient at activating this caspase following death receptor activation. FLICE-inhibitory protein (FLIP) blocks death receptor-mediated apoptosis by inhibiting caspase 8 activation; therefore, we assessed whether silencing FLIP could convert type II cells into type I. FLIP silencing-induced caspase 8 activation in Bax wild-type and null HCT116 colorectal cancer cells; however, complete caspase 3 processing and apoptosis were only observed in Bax wild-type cells. Bax-null cells were also more resistant to chemotherapy and tumor necrosis factor-related apoptosis inducing ligand and, unlike the Bax wild-type cells, were not sensitized to these agents by FLIP silencing. Further analyses indicated that release of second mitochondrial activator of caspases from mitochondria and subsequent inhibition of X-linked inhibitor of apoptosis protein (XIAP) was required to induce full caspase 3 processing and apoptosis following FLIP silencing. These results indicate that silencing FLIP does not necessarily bypass the requirement for mitochondrial involvement in type II cells. Furthermore, targeting FLIP and XIAP may represent a therapeutic strategy for the treatment of colorectal tumors with defects in mitochondrial-regulated apoptosis.