Apoptogenic activity of 2α,3α-dihydroxyurs-12-ene-28-oic acid from Prunella vulgaris var. lilacina is mediated via mitochondria-dependent activation of caspase cascade regulated by Bcl-2 in human acute leukemia Jurkat T cells

Abstract

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. 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. 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. 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.