HaA4

Background and objective: Both tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and some monoclonal agonistic antibodies against TRAIL receptors have antitumor activity. We have previously prepared a novel monoclonal agonistic antibody against human death receptor 5 (DR5) and designated it as mDRA-6. This study was to explore the Caspase-dependent molecular mechanisms of mDRA-6 inducing apoptosis of human leukemia Jurkat cells. Methods: After exposure to different doses of mDRA-6, DNA fragmentation of Jurkat cells was detected by agarose gel electrophoresis, cell proliferation was detected by MTT assay, and cell apoptosis was detected by flow cytometry after Annexin V-FITC/PI double staining. Jurkat cells were further treated with the inhibitors for Caspase-10, -9, -8 and -3. The active cleavage products of Caspase-10, -9, -8, -3 and poly ADP-ribose polymerase (PARP), BH3 interacting domain death agonist (Bid), truncated Bid (tBid) and cytochrome c (Cyto c), were analyzed by western blot. Results: After mDRA-6 treatment, DNA fragmentation was detected in Jurkat cells. mDRA-6 inhibited cell proliferation in a dose-dependent manner. When treated with 2.0 microg/mL mDRA-6, the apoptosis rates of Jurkat cells were 16.2% at 0.25 h, 28.3% at 0.5 h, 69.2% at 1 h and 78.2% at 2 h. Interestingly, the mDRA-6-induced apoptosis was repressed by 77.9% by Caspase-8 inhibitor ZIF, 54.2% by Caspase-3 inhibitor ZDF, and 8.7% by Caspase-9 inhibitor ZLF, but was not repressed by Caspase-10 inhibitor ZAF. After mDRA-6 exposure, the proenzymes of Caspase-8, -9 and -3 were reduced and their active cleavage products were increased along with the increase of exposure time, the cleavage products of PARP were also increased, Bid was degraded to tBid, and an abundance of Cyto c was released from mitochondria, but the proenzyme of Caspase-10 showed no change and no cleavage products of Caspase-10 were detectable. Conclusion: mDRA-6 can induce apoptosis of Jurkat cells via the Caspase-dependent and mitochondrial pathways.

Purpose: Apicidin, a histone deacetylase inhibitor, is a novel cyclic tetrapeptide that exhibits potent antiproliferative activity against various cancer cell lines. The aim of this study was to examine the potential of apicidin to induce apoptosis in human Bcr-Abl-positive leukemia cells and to assess the mechanism of apicidin-induced apoptosis. Experimental design: Cells were exposed to various concentrations of apicidin for 2-72 h, after which the levels of apoptosis, histone acetylation, mitochondrial damage, caspase activation, and Bcr-Abl expression were assessed. Results: Apicidin induced apoptosis in K562 cells in a concentration- and time-dependent manner. Similarly, apicidin notably induced the apoptosis in the primary leukemic blasts obtained from chronic myelogenous leukemia patients in blast crisis. The acetylated histone H4 levels increased in a concentration-dependent manner in the K562 cells. However, the timing of cell death caused by apicidin did not exactly correlate with the histone deacetylase inhibitory effect. The disruption of the mitochondrial membrane potential, cytochrome c release into the cytosol, and the mitochondrial Bax translocation were notably demonstrated after the apicidin treatment. Apicidin induced the proteolytic cleavage of procaspase-9, -3, -8, and poly(ADP-ribose) polymerase. Pretreatment of the K562 cells with the caspase-3 inhibitor, DEVD-CHO, completely inhibited the apicidin-induced apoptosis, suggesting that apicidin-induced apoptosis was caspase-dependent. The Fas/Fas ligand death receptor pathway was not involved in the apicidin-mediated apoptosis in K562 cells. Pretreatment of the cells with the caspase-9 inhibitor LEHD-fmk abrogated the apicidin- induced cleavage of procaspase-3, -8, and poly(ADP-ribose) polymerase. The p210 Bcr-Abl protein levels were notably decreased after the apicidin treatment, with near complete loss after 48 h. Reverse transcription-PCR assay demonstrated that the Bcr-Abl mRNA level was also remarkably decreased in a time-dependent manner. Conclusions: These results indicate that apicidin effectively induces the apoptosis of Bcr-Abl-positive leukemia cells through the activation of the mitochondrial pathway-dependent caspase cascades. The down-regulation of Bcr-Abl mRNA might also be one of the mechanisms implicated in the apicidin-mediated apoptosis in the K562 cells. This study provides the rationale to additionally investigate apicidin as a potential therapeutic agent for the drug-resistant Bcr-Abl-positive leukemia cells.

Ethnopharmacological relevance: The dried spikes of Prunella vulgaris var. lilacina (Labiatae) have been used for traditional herbal medicine to treat fever, inflammation, dropsy, gonorrhea and cancer in Korea, Japan and China. The present study evaluated the apoptotic effect of 2α,3α-dihydroxyurs-12-en-28-oic acid (DHURS), purified from the dried spikes on human acute leukemia Jurkat T cells. Materials and methods: Cell viability was assessed by MTT assay. Mitochondrial membrane potential (Δψm) loss, apoptotic change of the cell cycle, and apoptotic cells were measured by flow cytometric analysis. Mitochondrial cytochrome c release and activation of caspase cascade were determined by Western blot analysis. Caspase-12 activity and caspase-3 activity were assayed using the Fluorometric Assay Kit and the Colorimetric Assay Kit, respectively. Results: Treatment of Jurkat T cells with DHURS (20-25 μg/ml) caused cytotoxicity and apoptotic DNA fragmentation along with Δψm loss, mitochondrial cytochrome c release, activation of caspase-9, -7, -3, and -8, and PARP degradation. However, these apoptotic events were abrogated by overexpression of Bcl-2. Pretreatment of the cells with the pan-caspase inhibitor (z-VAD-fmk), the caspase-9 inhibitor (z-LEHD-fmk) or the caspase-3 inhibitor (z-DEVD-fmk) to prevent DHURS-induced apoptosis could block the activation of caspase-7 and -8, and PARP degradation, but not the Δψm loss, activation of caspase-9 and -3. Both FADD- and caspase-8-positive wild-type Jurkat clone A3, FADD-deficient Jurkat clone I2.1, and caspase-8-deficient Jurkat clone I9.2 exhibited similar susceptibilities to the cytotoxicity of DHURS, excluding an involvement of Fas/FasL system in triggering the apoptosis. The IC(50) value for Jurkat T cells was ~22 μg/ml, whereas that for human peripheral T cells was 25 μg/ml. Conclusions: These results indicate that DHURS-induced apoptogenic activity in Jurkat T cells, which was less potent in normal peripheral T cells, was mediated by Δψm loss, mitochondrial cytochrome c release, and subsequent activation of caspase-9 and -3, leading to activation of caspase-7 and -8, which could be regulated by Bcl-2.