Grape seed proanthocyanidin extract induced mitochondria-associated apoptosis in human acute myeloid leukaemia 14.3D10 cells

The important aim in cancer treatment is the selective killing of cancer cells without damaging healthy cells and the most commonly used chemotherapy for eliminating cancer is achieved by activating the mitochondrial apoptotic pathway.1 Therefore, identification of new agents that can selectively kill cancer cells becomes crucial in cancer clinical treatment. Grape seed proanthocyanidin extract (GSPE), a flavonoid in fruits, has been shown in vitro and in vivo, to possess superior antioxidant performance to vitamins C and E and β-carotene.2 Recently, several studies have shown that GSPE has anticancerous effects on various human cancer cells including prostate, breast, lung, skin and gastrointestinal cancers.3–5 Further, interestingly, GSPE enhances the viability and growth of normal human gastric mucosal cells and murine macrophage cells.4,6 Apoptosis can proceed by either the extrinsic pathway (death receptor) or intrinsic pathway (mitochondria).7 Chemotherapeutic agents have been shown to induce apoptosis by the redistribution of mitochondrial intermembrane proteins, such as cytochrome C, and consequent mitochondrial membrane potential (Δ Ψm) collapse.8 To our knowledge, the effects of GSPE on human acute myeloid leukaemia (AML) cells and normal peripheral blood neutrophils and eosinophils have not been studied and the molecular mechanisms underlying anticancerous activities of GSPE remain largely unknown. Thus, we hypothesize that: first, low dose GSPE (50 μg/mL) displays selective cytotoxic activity in AML cells without affecting normal human peripheral blood cells; second, GSPE induces apoptosis in AML cells via mitochondrial death pathway and caspases cascade. Therefore, this study had three purposes. First, investigate the effects of low dose GSPE on AML cells; second, determine whether a low dose of GSPE damages human normal peripheral blood cells; third, fathom the molecular mechanisms of GSPE induced apoptosis in AML cells. METHODS Cell line and cell culture The AML14.3D10 cell is a subline of the parental AML cell line established from a patient with FAB M2 acute myeloid leukaemia. The cell line was generated and kindly provided by Drs. Paul CC. and Baumann MA.9 Human normal peripheral blood neutrophils and eosinophils were from normal volunteer donors. Highly purified neutrophils and eosinophils were isolated by a method modified from Hansel et al10 using StemSepTM (StemCell Technologies Inc. Vancouver, Canada). Chemicals and reagents GSPE was from ActiVin (InterHealth Nutraceuticals Inc., Benicia, CA USA). Caspase-2 inhibitor (Z-VDVAD- FMK), Caspase-3 inhibitor (Z-DQMD-FMK), Cas-pase-8 inhibitor (Z-IETD-FMK) and Caspase-9 inhibitor (Z-LEHD-FMK) were purchased from Calbiochem (La Jolla, CA, USA). Broad spectrum caspases inhibitor Quinoline-Val-Asp-CH2-O-Ph (Q-VD-OPh) was from Enzyme Syatems Products, Inc (USA). JC-1 (5, 5', 6, 6', -tetrachloro-1, 1', 3, 3'-tetraethyl-benzimidazoly-Icarbocyanine iodide) for measuring mitochondrial membrane potential, was purchased from Molecular Probes, Inc (USA). Induction of apoptotic cell death assay Cell death was determined by both trypan blue exclusion assay and flow cytometric analysis after hypotonic propidium iodide (PI) staining nuclei of the AML14.3D10, eosinophil and neutrophil cells. At least 10 000 nuclei were examined, by Becton Dickinson LSR II flow cytometry (USA), for each sample to determine percentage of apoptotic nuclei in sub G1 DNA content. Mitochondrial membrane potential analysis The change of mitochondrial membrane potential (Δ Ψm) was examined by staining the cells with mitochondrial specific, fluorescent cationic dye, JC-1 and the alterations in Δ Ψm were analysed by flow cytometry. The JC-1 monomer and J-aggregate can be detected separately in the FITC-A (FL-1 channel, green) and PE-A (FL-2 channel, red). A red to green shift in the emission spectrum of the JC-1 indicates a loss in Δ Ψm. Caspase activity assay Caspases (−2, −3, −8 and −9) activities were detected using standard fluorogenic substrates by BD ApoAlertTM Caspases Assay Plates (Becton Dickinson, Palo Alto, CA, USA) following manufacture's protocol. Western blot analysis Protein concentration was determined using the Bradford assay (Bio-Rad, Hercules, CA, USA). The cell lysates were separated by 12% SDS-PAGE minigels electrophoresis and transferred onto nitrocellulose membrane using a semidry electrophoretic transfer system (Bio-Rad). Immunoblotting was performed according to the ECL Western blotting protocol for detection of cleavage caspase-3. The results were visualized by exposing blots to autoradiographic film (Image Reader, LAS-3000, Fujifilm, Japan). Statistical analysis Values are presented by mean ± standard error (SE). One way analysis of variance (ANOVA) and two tailed, unpaired t test was performed to assess the statistical significance of the difference between control and each treatment group. P<0.05 was considered statistically significant. RESULTS GSPE caused dose dependant, apoptotic cell death in AML14.3D10 Cells AML14.3D10 cells were treated with DMSO (0.1% final concentration) alone or various doses (25 to 100) μg/ml of GSPE. GSPE induced dose dependant, apoptotic cell death in AML14.3D10 cells for 12, 18, 24 and 48 hours (Fig. 1, n=3, P<0.05; Data not shown for 12 and 18 hours).Fig. 1.: GSPE induced dose- and time-dependant apoptotic cell death in AML14.3D10 Cells. AML14.3D10 cells were treated with (DMSO, 0.1% final concentration) alone or various doses of GSPE. GSPE induced dose dependant apoptotic cell death in AML14.3D10 cells and approximately 32.85%, 48.96% and 61.82% at 25, 50 and 100 μg/ml of GSPE compared with control showing 12.26% apoptotic cell death were detected respectively in the cells after 24 hours (n=3, * P<0.05 compared with control). Data are presented as mean ± SE of percentage of apoptotic nuclei of the cell and display a hypoploid DNA (Sub-G1) population.GSPE did not induce apoptotic cell death in normal human peripheral blood neutrophils and eosinophils Interestingly, apoptotic cell death, induced by low dose of 50 μg/ml of GSPE in AML14.3D10 cells, did not occur in normal peripheral blood neutrophils and eosinophils while GSPE could prevent the normal peripheral blood cell from apoptosis. Even at high doses (100 − 200 μg/ml), GSPE did not induce significant cell death in normal peripheral blood cells for 12 and 24 hours (Data not shown). GSPE induced apoptotic cell death is mediated via caspases activation in AML14.3D10 cells The activities of caspases (−2, −3, −8 and −9) were measured using a standard fluorogenic substrate assay. Activation of caspases (−2, −3 and −8) were detected following 6 hours of treatment by low dose of 50 μg/ml GSPE; while significant caspase-9 activity in AML14.3D10 cells was not detected until 12 hours following treatment (Data not shown). These caspases activities were significantly increased compared with control (DMSO, 0.1% final concentration). Activation of caspase-3 in AML14.3D10 cells was detected by Western blot analysis after 24 hours with 50 to 200 μg/ml of GSPE (Fig. 2A). Pan-caspases inhibitor blocked activated caspase-3 with GSPE 50 μg/ml for 24 hours in AML14.3D10 Cells (Fig. 2B). The low dose of 50 μg/ml of GSPE was completely inhibited by pan-caspases inhibitor (OPH) and was partially blocked by specific caspase (−2, −3, −8 and −9) inhibitors for 24 hours (Fig. 3, a, b, n=4, P<0.05).Fig. 2.: Pan-caspases inhibitor blocked activated caspase-3 by GSPE in AML14.3D10 cells. Cleaved fragments of caspases-3 detected after treatment with 50-200 μg/ml of GSPE for 24 hours by Western blot analysis in AML14.3D10 cells (A). Pan- caspases inhibitor reversed activated caspase-3 on 50 μg/ml GSPE for 24 hours in AML14.3D10 cells (B).Fig. 3.: GSPE induced cell death was blocked by caspases inhibitors in AML14.3D10 cells. Low dose (50 μg/mL) GSPE induced cell death was completely inhibited by pan-caspases inhibitor and was partly blocked by specific caspases (−2, −3, −8 and −9) inhibitors for 24 hours [n=4, * P<0.05 compared with 50 μg/ml GSPE (GSPE 50)]. OPH: pan-caspase inhibiter; G:GSPE; Cap 2: Caspase 2 inhibitor; Cap8: Caspase 8 inhibitor; Cap 3: Caspase 3 inhibitor; Cap 9: Caspase 9 inhibitor.GSPE disturbed mitochondrial membrane potential in AML14.3D10 cells The mitochondrial membrane potential (Δ Ψm) was examined by staining the cells with fluorescent dye JC-1. GSPE causes a dose response loss of Δ Ψm at 25—100 μg/ml for 24 hours (Data not shown). GSPE at 50 μg/ml induced a significant loss of Δ Ψm from 6 hours to 24 hours in AML 14.3D10 cells (Data not shown). We firstly treated separate groups of AML14.3D10 cells with pan-caspases inhibitor, a specific caspase −2, −3, −8 or −9 inhibitor or control (no inhibitor) for 1 hour. GSPE, 50 μg/ml, was then added to each group. The loss of Δ Ψm caused by this small dose of GSPE was completely inhibited by pan-caspases inhibitor and partially blocked by selective caspase −2, −3 and −8 inhibitors. In contrast, caspase-9 inhibitor did not block the loss of Δ Ψm in 24 hours (Data not shown). DISCUSSION This study, to our knowledge, is the first report that low dose GSPE induces apoptotic cell death in AML 14.3D10 cells without affecting normal peripheral blood eosinophils and neutrophils. Moreover, we present evidence that the mechanism of GSPE induced apoptosis in AML 14.3D10 cells is mitochondrial associated and caspase dependent. There are two pathways that initiate apoptosis: one is mediated by death receptors, also called “extrinsic pathway”; the other is mediated by mitochondria, referred to as “intrinsic pathway”.7 In both pathways, caspases, a family of cysteine aspartyl proteases that cleave cellular substrates and lead to the biochemical and morphological changes characteristic of apoptosis, are activated. Most of the cytotoxic drugs mediate cell death by activating caspases and initiating the mitochondrial pathway directly.7 Therefore, GSPE may involve a mitochondrial oxidative pathway to induce apoptosis in AML 14.3D10 cells. The induction of apoptosis has been associated with opening of the mitochondrial permeability transition pores and mitochondrial membrane potential (Δ Ψm) alterations characteristic of apoptosis.8 Δ Ψm loss can lead to release of cytochrome c from mitochondria into the cytosol and this triggers caspases activation. Our data showed that low dose GSPE not only caused activation of caspases (−2, −3, −8 and −9), but also induced a significant drop in Δ Ψm during apoptotic cell death in AML 14.3D10 cells. Prior treatment with pan-caspases inhibitor, Q-VD-OPH, completely abolished the GSPE induced apoptosis, loss of Δ Ψm and activity of caspase-3, but selective caspase (−2, −3, −8 or −9) inhibitors only partially reduced GSPE induced cell deaths. Caspase (−2, −3 and −8) inhibitors partially inhibited GSPE induced drop in Δ Ψm, but caspase-9 inhibitor increased the drop in Δ Ψm. These data demonstrate that low dose GSPE could induce AML 14.3D10 cell apoptosis via mitochondrial associated and caspase dependent pathway. Interestingly, a low dose of GSPE affects AML 14.3D10 cells, but has no toxicity towards normal human peripheral blood eosinophils and neutrophils. Part of the explanation may involve cell type specificity. A recent study of tumour cells reported that DADPH oxidase mediated generation of reactive oxygen species (ROS) is required for survival of undifferentiated cells in human promyelocytic leukaemia cell line HL-60.11 Some human tumour cells show increased ROS production compared to normal cells.12 However, in human neutrophils, electron microscopic studies identified hardly any mitochondria in neutrophils.13 Therefore, GSPE does not induce apoptosis in neutrophils because GSPE, as a mitochondrial antioxidant, had no effects on neutrophils. These results show that GSPE induced apoptosis may be cancer specific in AML 14.3D10 cells. Acknowledgements The authors thank Dr. Hamann KL for providing data analysis and kind support. We also thank Catherine G. Long, Susanna L. Chenette, Kim Wojcik, SHAO Zuo-hui, ZHU Xiang-dong and Jonathan Learoyd for their kind assistance.