The apoptotic cysteine protease, caspase-3, is expressed in cells as an inactive 32-kDa precursor from which 17 kDa (p17) and 12 kDa (p12) subunits of the mature caspase-3 are proteolytically generated during apoptosis. Two amino acid sequences, ESMD2S (amino acids 25-29) and IETD2S (amino acids 172-176), in the precursor have been defined as the cleavage sites for the production of the p17 and p12 subunits. Using a cell-free assay system, we demonstrate that the caspase-3 precursor appears to be cleaved first at the IETD2S site, producing the p12 subunit and a 20-kDa (p20) peptide. Subsequently, the p20 is cleaved at the ESMD2S site, generating the mature p17 subunit. The cleavage at the IETD2S site required a protease activity that was selectively inhibited by the peptide, Ac-IETD-CHO (acetyl-IETD-aldehyde), and other protease inhibitors, such as the cowpox viral serine protease inhibitor, CrmA, and N-␣-tosyl-L-phenylalanine chloromethyl ketone. The protease that catalyzed the cleavage at the ESMD/S site was selectively inhibited by another peptide, Ac-ESMD-CHO (acetyl-ESMD-aldehyde). More interestingly, the caspase-3 inhibitor, Ac-DEVD-CHO, but not the caspase-1 inhibitor, Ac-YVAD-CHO, also selectively inhibited the protease activity that cleaves at the ESMD2S site. This indicated that the cleavage at the ESMD2S site was either autocatalytic or that it required a caspase-3-like activity. In summary, we demonstrate that production of the p17:p12 form of caspase-3 is a sequential two-step process and appears to require two distinct enzymatic activities.In recent years, evidence has accumulated that the degradation of certain proteins by members of the caspase family is a general biochemical event taking place in cells undergoing apoptosis (for reviews, see Refs. 1-3). One caspase family member, caspase-3 (4 -6), has been studied extensively. In particular, several of the cellular protein targets of caspase-3 have been identifed. These include the DNA repair enzyme poly-(ADP-ribose) polymerase (PARP) 1 (5, 7), the 70-kDa protein component of the U1 small nuclear ribonucleoprotein (8), and the catalytic subunit of DNA-dependent protein kinase (DNA-PK cs ) (9 -11). Interestingly, inhibition of caspase-3 or caspase-3-like proteases in various cells has been shown to block apoptosis (6,(12)(13)(14)(15)(16)(17). In addition, the functional inactivation of the caspase-3 gene in knock-out mice results in the profound absence of apoptosis in certain tissues and lethality shortly after birth (18). Therefore, caspase-3 appears to be an extremely biologically relevant apoptotic protease.All caspase family members are initially synthesized as inactive precursors and require proteolytic processing themselves to generate the two subunits that form the active protease. This suggests that the apoptotic machinery may be regulated, in part, by a proteolytic cascade (for review, see Ref. 19). In the case of caspase-3, the mature enzyme is formed from 17 kDa (p17) and 12 kDa (p12) subunits, which are produced from a 32-kDa precurs...
Purpose: The plant-derived compound curcumin has shown promising abilities as a cancer chemoprevention and chemotherapy agent in vitro and in vivo but exhibits poor bioavailability. Therefore, there is a need to investigate modified curcumin congeners for improved anticancer activity and pharmacokinetic properties. Experimental Design: The synthetic curcumin analogue dimethoxycurcumin was compared with curcumin for ability to inhibit proliferation and apoptosis of human HCT116 colon cancer cells in vitro by estimating the GI 50 and LC 50 values and detecting the extent of apoptosis by flow cytometry analysis of the cell cycle. Metabolic stability and/or identification of metabolites were evaluated by recently developed mass spectrometric approaches after incubation with mouse and human liver microsomes and cancer cells in vitro. Additionally, circulating levels of dimethoxycurcumin and curcumin were determined in mice following i.p. administration. Results: Dimethoxycurcumin is significantly more potent than curcumin in inhibiting proliferation and inducing apoptosis in HCT116 cells treated for 48 h. Nearly 100% of curcumin but <30% of dimethoxycurcumin was degraded in cells treated for 48 h, and incubation with liver microsomes confirmed the limited metabolism of dimethoxycurcumin. Both compounds were rapidly degraded in vivo but dimethoxycurcumin was more stable. Conclusions: Compared with curcumin, dimethoxycurcumin is (a) more stable in cultured cells, (b) more potent in the ability to kill cancer cells by apoptosis, (c) less extensively metabolized in microsomal systems, and (d) more stable in vivo. It is likely that the differential extent of apoptosis induced by curcumin and dimethoxycurcumin in vitro is associated with the metabolite profiling and/or the extent of stability.Curcumin (diferuloylmethane) is the active yellow pigment in turmeric, a popular plant-derived coloring spice and ingredient of many cosmetics and pharmaceuticals (see refs. 1 -5 for reviews on structural and biological aspects of curcumin). In general, curcumin has been associated with a large number of biological and cellular activities/events, including antioxidative, anti-inflammatory, anticarcinogenic, and hypocholesterolemic properties (1 -5), and a large number of studies have focused on the pathways by which curcumin acts as a chemoprotective agent (1, 2, 6, 7). Further, curcumin congeners have been reported to induce apoptosis, sensitize and overcome resistance to various agents that induce apoptosis in diverse human cancer cells (8 -16), and decrease the occurrence of cardiomyocytic apoptosis after global cardiac ischemia/reperfusion (17). In general, curcumin mediates its effects by modulating several important molecular targets, including transcription factors, enzymes, cell cycle proteins, cytokines, receptors, and cell surface adhesion molecules (reviewed in refs. 2 -4).With regard to its anticancer potential, it has been shown that curcumin congeners can inhibit proliferation and/or induce apoptosis of cancer c...
We demonstrate that the catalytic subunit of the DNAdependent protein kinase (DNA-PK cs ) is specifically, proteolytically cleaved in HL-60 cells treated with staurosporine (STS), a potent inducer of apoptosis. The proteolysis of DNA-PK cs correlated with or preceded apoptotic chromosomal DNA degradation. Cell-free extracts prepared from STS-treated HL-60 cells recapitulated the proteolysis of DNA-PK cs in an in vitro assay using purified DNA-PK as the substrate. Western blot analyses of the apoptotic cell extract showed that the 32-kDa precursor of CPP32 is expressed in HL-60 cells and processed following STS treatment. In addition, whereas the DNA-PK cs protease activity was not inhibitable by many conventional protease inhibitors, it was inhibitable by a highly selective peptide-derived inhibitor of CPP32. These data strongly suggest that CPP32, or a CPP32-like protease, is responsible for DNA-PK cs proteolysis. Finally, our results demonstrated that the cleavage of DNA-PK cs in vitro proceeded in the presence of Bcl-2, indicating that the function provided by Bcl-2 lies upstream the proteolysis of DNA-PK cs .
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