Up-Regulation of Bax and Endonuclease G, and Down-Modulation of Bcl-XL Involved in Cardiotoxin III-Induced Apoptosis in K562 Cells

EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 38, No. 4, 435-444, August 2006 Up-regulation of Bax and endonuclease G, and down-modulation of Bcl-XL involved in cardiotoxin III-induced apoptosis in K562 cells Sheng-Huei Yang1, Ching-Ming Chien1, Keywords: apoptosis; bcl-2-associated X protein; car- Mei-Chin Lu2, Yi-Hsiung Lin1, diotoxin III, Naja naja atra; caspase; endonuclease G; Xiu-Wei Hu1 and Shinne-Ren Lin1,3 K562 cells 1Faculty of Medicinal and Applied Chemistry 2 Introduction Graduate Institute of Natural Products Kaohsiung Medical University Many therapeutic and chemopreventive agents Kaohsiung 807, Taiwan, ROC 3Corresponding author: Tel, 886-7-3121101#2219; eliminate cancerous cells by inducing programmed Fax, 886-7-3123443; E-mail, [email protected] cell death (apoptosis) (Kaufmann et al., 2000; Ro- bertson et al., 2000). Apoptosis is an important Accepted 24 July 2006 cellular process for destruction of undesirable cells during development or homeostasis in multi-cellular Abbreviations: CTX III, Carditoxin III; MTT, 3-(4,5-dimethylthia- organisms. This process is characterized by distinct zol-2-yl)-2,5-diphenyltetrazolium bromide; PARP, poly (ADP-ribo- morphological changes including plasma membrane se) polymerase; Z-VAD-FMK, benzyloxycarbonyl-Val-Ala-Asp- bleb, cell shrinkage, depolarization of mitochondria, fluoromethylketone chromatin condensation, and DNA fragmentation (Kaufmann et al., 2001; Reed, 2001). Caspases are essential for the execution of cell death by various Abstract apoptotic stimuli (Cohen, 1997; Shi, 2002). Caspase activation is often regulated by various cellular pro- Cardiotoxin III (CTX III), a basic polypeptide with 60 teins including members of the IAP (Deveraux and amino acid residues isolated from Naja naja atra Reed, 1999) and Bcl-2 families (Adams and Cory, venom, has been reported to have anticancer activity. 1998; Antonsson and Martinou, 2000). Previous CTX III-induced K562 cell apoptosis was confirmed reports have demonstrated that some Bcl-2 family by DNA fragmentation (DNA ladder, sub-G1 for- members that are located on the mitochondrial mation) and phosphatidylserine (PS) externalization membrane can alter the permeability of the mito- with an IC value of 1.7 µg/ml at 48 h. A mechanistic chondrial membrane and trigger the release of 50 cytochrome c (Adams and Cory, 1998; Antonsson analysis demonstrated that CTX III-induced apop- and Martinou, 2000) or caspases (Salvesen and totic cell death was accompanied by up-regulation of Dixit, 1997), then activate the post-mitochondrial both Bax and endonuclease G (Endo G), and caspase cascade leading to apoptotic cell death. downregulation of Bcl-XL. CTX III had no effect on the Apoptosis induced by anticancer or chemopreven- levels of Bcl-2, Bid, XIAP survivin, and AIF proteins. tive agents can be mediated by additional proteins or CTX III treatment caused loss of the mitochondrial pathways including release of apoptogenic factors, membrane potential ( Ψm), release of mitochon- such as endonuclease G (endo G) and apoptosis- drial cytochrome c to the cytosol, and activation of inducing factor (AIF), from the mitochondrial inter- both caspase-9 and -3. CTX III-induced apoptosis was membrane space into the cytosol (Green and Reed, significantly blocked by the broad-spectrum cas- 1998; Ravagnan et al., 2002), or by oxidative stress, pase inhibitor Z-VAD-FMK. However, CTX III did not such as reactive oxygen species (ROS) (Fleury et generate reactive oxygen species (ROS) and al., 2000; Simon et al., 2000). antioxidants, including N-acetylcysteine and ca- The aim of this study was to identify the mecha- talase, did not block CTX III-induced apoptosis in nisms of CTX III-induced apoptosis. We show that K562 cells. Modulation of Bax, Bcl-XL, and the Endo CTX III-induced apoptosis in human leukemia G proteins, release of mitochondrial cytochome c, cancer cells is caspase-3 and -9 dependent, is and activation of caspase-3 and -9 all are involved in blocked by a pan-caspase inhibitor, and is accom- the CTX III-triggered apoptotic process in human panied by up-regulation of Bax and Endo G proteins, leukemia K562 cells. down-regulation of Bcl-XL, and an increase in the release of mitochondrial cytochrome c to cytosol. 436 Exp. Mol. Med. Vol. 38(4), 435-444, 2006 Materials and Methods well (1.2 mg/ml) and incubated for 4 h. This reaction results in mitochondrial dehydrogenases of viable Materials cells producing a purple formazan product. The RPMI 1640 medium, fetal calf serum (FCS), trypan amount of MTT-formazan product dissolved in blue, penicillin G, and streptomycin were obtained DMSO was estimated by measuring the absorbance from GIBCO BRL (Gaithersburg, MD). 3-(4,5-dime- at 570 nm in an ELISA plate reader. thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethylsulfoxide (DMSO), ribonuclease (RNase), Assessment of apoptosis propidium iodide (PI), rhodamine123, N-acetyl-L- Apoptosis was also evaluated by examining the cysteine (NAC), and catalase were purchased from characteristic pattern of DNA laddering generated in Sigma-Aldrich (St. Louis, MO) and 2',7'-dichlorodi- apoptotic cells using gel electrophoresis. Briefly, hydrofluorescein diacetate (H DCF-DA) was obtain- 6 2 cells were seeded at a density of 1×10 cells onto ed from Molecular Probes, Inc (Eugene, OR). Anti- 10-cm dishes 24 h before CTX III treatment. Then bodies were obtained from the following sources: cells were treated with various concentrations of cytochrome c (PharMingen, San Diego, CA), Bax, CTX III for 12 h and control cultures were treated Bcl-2, Bcl-X , Bid, XIAP, AIF, Survivin, Endo G, and L with PBS. Adherent and floating cells were collected -actin (Santa Cruz Biotechnology, Santa Cruz, CA), and lysed in 400 l of ice-cold lysis buffer (con- anti-mouse and anti-rabbit IgG peroxidase-containing 50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 0.3% jugated secondary antibody (Pierce, Rockford, IL). Triton X-100), incubated on ice for 30 min, and then Hybond ehanced chemiluminescence (ECL) trans- centrifuged. RNAase (100 g/ml) was added to the fer membrane and ECL Western blotting detection o supernatant, which was then incubated at 50 C for kit were obtained from Amersham Life Science 30 min, followed by addition of 200 g/ml of (Buckinghamshire, UK). The colorigenic synthetic o proteinase K and further incubation at 37 C for 1 h. peptide substrate, Ac-DEVD-pNA, and Ac-LEHD- Fragmented DNA was extracted with phenol /chloro- pNA, as well as the protease inhibitor for Z- form and then precipitated with ethanol. The DNA VAD-FMK were purchased from Calbiochem (San fragments were electrophoresed on a 2% agarose Diego, CA). An annexin V-FLOUS Staining Kit was a gel containing 0.1 g/ml of ethidium bromide. product of Roche Molecular Biochemicals (Mann- The accumulation of the sub G1 population in heim, Germany). K562 cells was also determined by flow cytometry. CTX III was purified from the venom of Naja naja Cells were seeded onto 6 cm dishes and treated atra (Taiwan cobra) by chromatogaraphy on Se- with or without the indicated CTX III for 24 h. Cells phadex G-50 and SP-Sephadex C-25, as previously were then washed twice with ice-cold PBS and described by Lin et al., (2002). Solutions of CTX III o collected by centrifugation at 200 g for 5 min at 4 C. were prepared in phosphate buffered saline (PBS) o Cells were fixed in 70% (v/v) ethanol at 4 C for 30 and sterilized by filtration. min. After fixation, cells were treated with 0.2 ml of DNA extraction buffer (0.2 M Na2HPO4 and 0.1 M Cell culture citric acid buffer, pH 7.8) for 30 min, centrifuged, and Human leukemia K562 cells were obtained from the resuspended in 1 ml of propidium iodide staining buffer (0.1% TritonX-100, 100 g/ml RNase A, 500 American Type Culture Collection (ATCC, Manas- o sas, VA) and were maintained in RPMI 1640 g/ml of propidium iodide in PBS) at 37 C for 30 medium supplemented with 10% fetal calf serum, 2 min. Cytometric analyses were performed using a mM glutamine, and antibiotics (100 U/ml of penicillin flow cytometer (FACS Calibur, Becton Dickinson) and 100 g/ml of streptomycin) at 37oC in a hu- and CellQuest software. Approximately 10,000 cells were counted for each determination. midified atmosphere of 5% CO2. The externalization of phosphatidylserine (PS) and membrane integrity were quantified using an Cell viability and cytotoxicity Annexin V-FLOUS staining kit. In brief, 106 cells Cell viability was determined by the Trypan blue dye were grown in 35 mm diameter plates and were exclusion method and cytotoxicity was assessed by labeled with Annexin V-FLOUS (10 g/ml) and PI an MTT assay. Exponentially growing cells (1 × 105) (20 g/ml) prior to harvesting. After labeling, all were plated in 96-well plates and, after 24 h of plates were washed with binding buffer and growth, were treated with a series of different harvested by scraping. Cells were resuspended in 5 concentrations of CTX III dissolved in PBS. Cells binding buffer at a concentration of 2 × 10 cells/ml exposed to 0.2% Trypan Blue were counted in a before analysis by flow cytometry. hemocytometer. MTT solution was added to each Apoptosis of K562 cells by CTX III 437 Western blot analysis fugation for 10 min at 750 × g. The supernatant was Cells were treated with CTX III for the indicated time then centrifuged at 100,000 × g for 15 min. This periods. After incubation, cells were lysed in mo- pellet, representing the mitochondrial fraction, was dified protein lysis buffer (50 mM Tris-HCl, pH 7.4, resuspended in buffer A. The supernatant was again 150 mM NaCl, 1 mM EDTA, 5% 2-mercaptoethanol, centrifuged at 100,000 × g for 1 h.

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