dysfunction and apoptosis in HL60 cells

Involvement of proapoptotic Bcl-2 family members in terbinafine-induced mitochondrial

dysfunction and apoptosis in HL60 cells

Kuo-Ching Yang ^a,1 , Chi-Chen Wu ^b,1 , Chih-Hsiung Wu ^c , Jur-Hao Chen ^a,1 , Chien-Hwa Chu ^d , Chien-Ho Chen ^d , Yean-Hwei Chou ^e , Ying-Jan Wang ^f , Wen-Sen Lee ^g , How Tseng ^g , Shyr-Yi Lin ^h , Chia-Hwa Lee ^d , Yuan-Soon Ho ^d,*

a

Department of Internal Medicine, Division of Gastroenterology, Shin Kong Wu Ho-Su Memory Hospital, Taipei 112, Taiwan

b

Department of Anesthesiology, School of Medicine, Taipei Medical University and Hospital, Taipei 110, Taiwan

c

Department of Surgery, School of Medicine, Taipei Medical University and Hospital, Taipei 110, Taiwan

d

Graduate Institute of Biomedical Technology, Taipei Medical University, No. 250 Wu-Hsing Street, Taipei 110, Taiwan

e

Department of Surgery, Division of General Surgery, Shin Kong Wu Ho-Su Memory Hospital, Taipei 112, Taiwan

f

Department of Environmental and Occupational Health, National Cheng Kung University Medical College, Tainan 704, Taiwan

g

Graduate Institute of Medical Sciences, Department of Physiology, School of Medicine, Taipei Medical University, Taipei 110, Taiwan

h

Department of Internal Medicine, School of Medicine, Taipei Medical University, Taipei 110, Taiwan Received 1 February 2005; accepted 1 July 2005

Abstract

Terbinafine (TB, lamisil

), a promising world widely used oral-anti-fungal agent, has been used in the treatment of superficial mycosis. In this study, we found that apoptosis but not cell growth arrest was induced by TB (1 lM, for 24 h) in human promyelocytic leukemia (HL60) cells. The apoptotic effect induced by TB in the HL60 cell was not through the general differentiation mechanisms evidenced by evaluation of three recognized markers, including CD11b, CD33, and morphological features. In addition, our results also revealed that TB-induced apoptosis was not through the cellular surface CD 95 receptor-mediated signaling pathway. We found that the mitochondria membrane in the TB-treated HL60 cells was dissipated by decreasing of the electrochemical gradient (DW

) led to leakage of cytochrome c from mitochondria into cytosol. Such effects were completely blocked by in vitro transfection of the HL60 cells with Bcl-2 overexpression plasmid (HL60/Bcl-2). However, our data found that TB-mediated apoptosis could not be completely prevented in the Bcl-2 over expressed (HL60/Bcl-2) cells. Such results implied that additional mediators (such as caspase-9) other than mitochondria membrane permeability might contribute to the TB-induced cellular apoptosis signaling. This hypothesis was sup- ported by the evidence that administration of caspases-9 specific inhibitor (z-LEHD-fmk) blocked the TB-induced apoptosis. Our studies highlight the molecular mechanisms of TB-induced apoptosis in human promyelocytic leukemia (HL60) cells.

 2005 Elsevier Ltd. All rights reserved.

Keywords: Terbinafine; Apoptosis; HL60; Caspase-9; Mitochondria

0278-6915/$ - see front matter  2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.fct.2005.07.008

Abbreviations: Apaf-1, apoptotic protease activating factor 1; CCCP, carbonyl cyanide m-chlorophenylhydrazone; CHX, cycloheximide; DMEM, DulbeccoÕs modified EagleÕs medium; FACS, fluorescence-activated cell sorter; FCS, fetal calf serum; PBS, phosphate-buffered saline; PI, propidium iodide; PMSF, phenylmethyl sulfonyl fluoride; FITC, fluorescein isothiocyanate; RA, retinoic acid; SDS-PAGE, sodium dodesyl sulfate- polyacrylamide gel electrophoresis; TB, terbinafine; Val, valinomycin.

*

Corresponding author. Tel.: +886 2 27361661x3311; fax: +886 2 2739 3422.

E-mail address: [email protected] (Y.-S. Ho).

1

Contributed equally.

www.elsevier.com/locate/foodchemtox

1. Introduction

Terbinafine (TB) (lamisil

), a promising world widely used oral-anti-fungal agent, is a newly synthesized oral anti-mycotic drug in the allylamines class: a fungicidal agent that inhibits ergosterol synthesis at the stage of squalene epoxidation (Petranyi et al., 1984). TB shows a good safety profile and relatively few drug interactions (Abdel-Rahman and Nahata, 1997). The cream form and oral tablet of TB have been approved for clinical uses in the United States (Gupta and Shear, 1997).

Our recent studies have shown that a number of anti- fungal agents exert anti-proliferative and/or apoptotic activities in various malignant cells in vitro and in vivo (Chen et al., 2000; Ho et al., 1998, 2001). The anti-pro- liferative effect of TB has not been reported until our recent studies demonstrating that TB induced cell cycle arrest at the G0/G1 phase of the cell cycle in hepatoma and colon cancer cells (Lee et al., 2003). We also showed that TB at a range of concentrations (0–120 lM) dose- dependently decreased cell number in cultured human umbilical vascular endothelial cells (Ho et al., 2004).

These findings suggest the potential application of TB in anti-angiogenic therapy for clinical anti-cancer purpose.

Previously, we have demonstrated that TB at a range of concentrations (1–30 lM) induces apoptosis in cul- tured HL60 cells (Lee et al., 2003). However, the mech- anism underlying of TB-induced the occurrence of apoptosis is not clear. The effector caspases, including caspase-3, may be activated via mitochondria-indepen- dent or -dependent pathways (Shi, 2002). The mitochon- dria independent pathway is activated through binding of the ligands (the tumor necrosis-factor receptor family, such as Fas and TNF) to their receptors and requires the direct cleavage of caspase-3 by activated caspase-8 (Stennicke et al., 1998). On the other hand, caspase-3 can also be activated through the mitochondria-depen- dent signaling proteins by releasing cytochrome c from its intermembrane space into the cytoplasm (Reed, 1997). In the cytoplasm, cytochrome c, in the presence of ATP or dATP, associates with a complex of apoptotic protease activating factor 1 (Apaf-1) and caspase-9, and leads to autocatalytic cleavage and activation of cas- pase-9 in this complex (Qin et al., 1999; Saleh et al., 1999). Caspase-9 can directly cleave and activate the procaspase-3.

Recent studies have demonstrated that anti-tumor therapeutic agents-induced HL60 cells apoptosis was through the caspases 2, 3, 8, and 9 independent release of cytochrome c into the cytosol (Perchellet et al., 2004). Such results implied that accumulation of cyto- solic cytochrome c level may play some important roles in anti-cancer drug-induced apoptosis in the HL60 cells.

Although the mechanisms by which cytochrome c trans- located from mitochondria into the cytosol are not fully

understood, recent studies clearly demonstrated that members of the Bcl-2 family proteins regulate the mito- chondria membrane functions and control the release of cytochrome c (Wang et al., 2004). This family is subdi- vided into two classes: anti-apoptotic members, such as Bcl-2 and Bcl-xL, which protect cells from apoptosis, and proapoptotic members, such as Bax and Bak, which trigger or sensitize the cells for apoptosis (Borner, 2003).

Our recent studies have demonstrated that the anti- tumor effects of TB in the solid (COLO 205, p53 wild type) and leukemia (HL60, p53 null) cancer cells were through p53-dependent and p53–independent mecha- nisms (Ho et al., 2004; Lee et al., 2003). However, these cancer cells have different sensitivities in terms of the occurrence of apoptosis and cell cycle arrest in response to TB treatment. These findings promoted us to investi- gate the molecular mechanisms of apoptosis induced by TB. The human promyelocytic leukemia cell (HL60, with nulled p53) was served as a research model for investiga- tion of the mechanisms of TB-induced apoptosis. Our results show that TB induces apoptosis by altering mito- chondrial transmembrane potential, which causes the re- lease of cytochrome c into the cytosol, and leads to activation of the Apaf-1/caspase-9 apoptosome.

2. Materials and methods 2.1. Chemicals and reagents

Protease inhibitors (phenylmethyl sulfonyl fluoride (PMSF), pepstatin A, leupeptin, and aprotinin) were ac- quired from Sigma Chemical Company (Sigma Aldrich Chemie GmbH, Steinheim, Germany). Caspase-8 inhib- itor (zIETD-fmk), caspase-9 inhibitor (zLEHD-fmk), caspase-3 inhibitor (zDEVD-fmk), and the general cas- pase inhibitor (zVAD-fmk) were obtained from Alexis Corporation (Switzerland). DulbeccoÕs modified EagleÕs medium (DMEM), Fetal calf serum (FCS), penicillin/

streptomycin solution, and fungizone were purchased from Gibco-Life Technologies (Paisley, UK).

2.2. Antibodies

The following polyclonal or monoclonal antibodies

were obtained from various sources as indicated: anti-

caspase-8, anti-cytochrome c, anti-Bax, anti-Apaf-1,

and anti-Bcl-2 antibodies (Santa Cruz Biotechnology,

CA), anti-caspase-9, and anti-caspase-3 antibodies

(Stressgen Biotechnologies, Victoria, British Columbia,

Canada), anti-cytochrome c oxidase, (Research Diag-

nostics, Flanders, New Jersey, USA), and anti-actin

mAb (Sigma Aldrich Chemie GmbH, Steinheim, Ger-

many). CD11b leu

-15 antibody, and CD33 antibody

(rabbit anti-human) were purchased from Becton Dick-

inson (Cambridge, UK).

2.3. Transfection of Bcl-2 with expression plasmid HL60 cells were transfected with pBcl-2 plasmid (pcDNA3-Bcl-2; Science Reagent, El Cajon, CA) using the Lipofectin reagent (Life Technologies, Inc., Gai- thersburg, MD). Briefly, 0.8 ml of the cell suspension (3 · 10

cells/ml) was added to each well in six-well plates. Two micrograms of pBcl-2 plasmid DNA were diluted in 98 ll of OPTI-MEM1 medium, and 16 ll of Lipofectin reagent were diluted in 84 ll of OPTI- MEM1 medium. The vector plasmid without Bcl-2 gene was used as a negative control. After a 45-min incuba- tion at room temperature, the DNA and Lipofectin dil- uents were combined and incubated for 15 min at room temperature. Then, 200 ll of the DNA/Lipofectin mix- ture were added to each well, and cells were incubated for 12 h at 37 C. Cells were allowed to express Bcl-2 for 2 days. Bcl-2 expression was confirmed by Western blotting analysis.

2.4. Cells and culture conditions

The COLO 205 cell line was isolated from human co- lon adenocarcinoma (CCL-222: American Type Culture Collection, ATCC). The HL60 cell line was derived from human myeloid leukemia cells (59170: ATCC). The cell line #76 KhGH (CRL 8858; ATCC) was composed of keratinocytes derived from normal human epidermis (Lee et al., 2003). The cell lines were grown in RPMI 1640 (for COLO 205 and HL60 cells) supplemented with 10% FCS, 50 lg/ml gentamycin and 0.3 mg/ml gluta- mine in a humidified incubator (37 C, 5% CO

). A 3:1 mixture of HamÕs F12 medium and DMEM medium (for #76 KhGH cells) supplemented with 10% FCS, 40 ng/ml hydrocortisone, 0.01 mg/ml cholera toxin, 0.005 mg/ml insulin, and 10 ng/ml epidermal growth factor. HL60 cells were differentiated into mature neu- trophil-like cells by treating with 1 lM retinoic acid (RA) for 5 days.

For the caspase inhibitors studies, the HL60 cells were seeded onto plastic six-well culture plates or 100-mm cul- ture dishes at 5 · 10

cells/ml and allowed to grow for 2–

3 days. Following this, they were incubated with various concentrations (10–60 lM) of inhibitors specific to cas- pase-8 (zIETD-fmk), or caspase-9 (zLEHD-fmk) or with 50 lM of the general inhibitor of caspases (zVAD-fmk) for 4 h. The cells were then exposed to TB at a concentra- tion of 30 lM for 24 h. Following the treatment, cells were harvested by centrifugation at 500·g for 5 min, washed with PBS, and subsequently used for various bio- chemical investigations.

2.5. Determination of cell viability

Cell viability was determined based on the trypan blue exclusion method as described previously (Ho

et al., 2001; Lee et al., 2003). The viability percentage was calculated based on the percentage of unstained cells.

2.6. Wright–Giemsa stain

For Wright–Giemsa staining to monitor the extent of cellular differentiation, cells were cytospun onto etha- nol-cleaned SuperFrost glass slides as described previ- ously (Rice et al., 2004), fixed with methanol (RT, 15 min), air-dried, and stained with Wright–Giemsa stain solution (Sigma Chemical Co., St. Louis, MO), fol- lowing the manufacturerÕs instructions.

2.7. Flow cytometry analysis

The cell cycle stages in the TB- or DMSO-treated groups were determined by flow cytometry analysis (Ho et al., 2001, 2004; Lee et al., 2003). Nuclear DNA was stained with a reagent containing propidium iodine (50 lg/ml) and DNase-free RNase (2 U/ml) and mea- sured using a fluorescence-activated cell sorter (FACS).

The population of nuclei in each phase of the cell cycle was determined using established CellFIT DNA analysis software (Becton Dickenson, San Jose, CA).

2.8. Protein extraction, immunoprecipitation and Western blotting analysis

The HL60 cells treated with DMSO or TB were har- vested, washed twice with PBS, lysed, and electroblotted onto a PVDF membrane (Millipore) using standard techniques as described in our recent publications (Ho et al., 2004; Lee et al., 2003). The PVDF membrane were blocked by incubation for 2 h in PBS buffer containing 5% nonfat dry milk and 0.1% Tween-20, and then probed with antibodies:anti-caspase-8 (1:1000), anti-cas- pase-9 (1:3000), anti-caspase-3 (1:2000), anti-cyto- chrome c (1:2000), anti-Bcl-2 (1:1000), anti-Bax (1:

500), or anti-cytochrome c oxidase (1:500). The blots were then incubated with horseradish peroxidase-conju- gated secondary antibodies (1:2000 dilution) (N20;

Santa Cruz, California, USA). The antigen–antibody

complex was detected by SuperSignal chemilumines-

cence kit as described in the manufacturerÕs protocol

(Pierce Biotechnology, Rockford, IL) and visualized

by autoradiography. To confirm equal loading of pro-

teins, the blots were also immunoprobed with a rabbit

polyclonal antibody against the cytoskeletal protein b-

actin (1:2500 dilution). The consistent equal signals of

b-actin from the different extracts also indicate that

TB and the caspases inhibitors do not interfere with pro-

tein synthesis in HL60 cells (data not shown). Immuno-

precipitation was performed as described in our studies

(Ho et al., 2004; Lee et al., 2003). Equal amounts of pro-

tein were immunoprecipitated with saturating amounts

of anti-caspase-9 antibody. The caspase-9-immunopre- cipitated Apaf-1 protein was then measured by Western blot analysis. Isolation of mitochondria and cytosolic fractions of cell lysates were performed as described pre- viously (Ashktorab et al., 2004).

2.9. Analysis of apoptosis

Apoptosis in the HL60 cells subjected to various treatments was determined by using the Annexin V- FITC (fluorescein isothiocyanate) staining kit from BD Biosciences (Franklin Lakes, NJ). PI was used to differ- entiate apoptotic cells with preserved membrane integ- rity (Annexin

, PI ) from necrotic cells that lost membrane integrity (Annexin , PI

). The assay was per- formed following the manufacturerÕs procedure. After staining, the percentage of apoptotic cells under various treatments was analyzed by flow cytometry (FACS Cal- liber, BectonDickinson) as described previously (Tseng et al., 2002). Analysis of DNA fragmentation was per- formed as previously described (Ho et al., 1996).

2.10. Caspase activity assay

Caspase activity was measured by using caspases 3 (Promega, Madison, WI) and 9 (Chemicon, Temecula CA) colorimetric activity assay kits as previous described (Ho et al., 2003; Lin et al., 2001). Briefly, HL60 cells were lysed by addition of cell lysis buffer and protein concentration was measured. Caspase activ- ity was assayed at 37 C in 100 ll of assay buffer con- taining 50 lg (for caspase 3) or 30 lg (for caspase 9) of the indicated colorimetric peptide. Caspase activity was measured by the release of p-nitroaniline (pNA) from the labeled substrates Ac-DEVD-pNA and Ac- LEHD-pNA for caspase 3 and 9, respectively, and the free pNA was quantified at 405 nm.

2.11. Quantification of cell-surface antigen expression The expression of CD11b and CD33 antigen on the surface of differentiating HL60 cells was measured by flow cytometry. Cells (1 · 10

)/100 ll RPMI were trea- ted with 10 ll anti-CD11b/CD33 antibody and incu- bated at 4 C for 20 min. The cells were washed twice with 400 ll cold PBS at 1100 rpm for 10 min, and finally resuspended in 400 ll Isoton II solution on ice before they were analyzed by using flow cytometric analysis.

2.12. Mitochondrial transmembrane potential assay To assess the mitochondrial transmembrane potential (DW

), HL60 cells (1 · 10

) were seeded in a 6-well plate washed twice with PBS and then loaded with the cationic lipophilic fluorochrome JC-1 (5 lg/ml) for 10 min at 37 C. Cells were washed twice with PBS and submitted

to FACS-analysis. The red fluorescence of JC-1 aggre- gates corresponds to the mitochondrial membrane poten- tial whereas the green fluorescence of JC-1 monomers is indicative for the mitochondrial mass. Active mitochon- dria with high DW

accumulate JC-1 aggregates, which are red, whereas, in the mitochondria with low DW

, JC-1 stays in a monomeric, green form. This renders the red/green ratio, a sensitive indicator of the mitochondrial DW

changes. In addition, carbonyl cyanide m-chloro- phenylhydrazone (CCCP; Calbiochem) or valinomycin (Val, Sigma) were dissolved in 100% acetone and diluted in complete medium; the acetone concentration in the medium did not exceed 1%. Both of the CCCP and Val were added at a final concentration of 200 lM as a posi- tive control, and the fluorescence was assessed for each time point, a red/green fluorescence ratio was then calcu- lated. The mean red fluorescence of drug-treated cells was measured at 0, 2, 4, 6, 8, 10, and 12 h after TB treatment, and presented as a ratio of the absorbance in 590/535 nm (Maianski et al., 2004a,b).

2.13. Statistics

Statistical analysis was carried out using analysis of variance (ANOVA)—one way analysis of variance with Student–Newman correction, and the StudentÕs t-test.

Significance was assumed for values of P < 0.05.

3. Results

3.1. Cytotoxic effects of TB in human cancer cells To investigate the cellular regulatory mechanisms of apoptosis induced by TB, human HL60 cells were selected as a research model. As shown in Fig. 1, the via- bility of HL60 cells was dose-dependently decreased at 24 h after exposure to various concentrations of TB (0–30 lM), but was not affected by DMSO (0.05%, v/v) treatment. Fig. 1A and C showed that the human HL60 cell was the most susceptible to TB-induced cyto- toxic effects as compared to human untransformed keratinocytes and colon cancer (COLO 205) cells.

3.2. TB-induced apoptosis in human HL60 cells

DNA fragmentation was observed in the HL60 cells

treated with 1 lM TB for 24 h (Fig. 2A, left), whereas

a concentration higher than 90 lM TB was required

for induction of DNA laddering fragmentation in the

COLO205 cells (Fig. 2A, right). In consistent to

Fig. 2A, a significant sub-G1 peak determined by flow

cytometric analysis was observed in the HL60 cells after

TB treatment for 24 h (Fig. 2B, left). In the same condi-

tions, the COLO 205 cells were arrested at the G0/G1

phase (Fig. 2C, left).

3.3. TB-induced the occurrence of apoptosis in HL60 cells was not through differentiation signaling pathways

To further confirm whether TB-induced apoptosis was through differentiation processes as described previ- ously (Martin et al., 1990; Olins et al., 2000), three rec- ognized markers including the CD11b, CD33, and morphological changes were assessed (Stabellini et al., 2004). As shown in Fig. 3A, characteristic segmented nuclei and morphological features characteristic of blas- tic leukemic cell like was observed in the RA (1 lM, 48 h)-treated HL60 cells (Fig. 3A, middle, arrow). How- ever, apoptotic but not differentiated cell like morphol- ogy was observed in TB (1 lM, 48 h)-treated HL60 cells (Fig. 3A, right, arrow). Consistently, RA treatment resulted in an increase in the percentage of cells express- ing CD11b (Fig. 3B), and a time-dependent decrease in the percentage of CD33 expression (Fig. 3C).

Flow cytometric analysis of DNA content revealed that TB (1 lM) treatment resulted in a well-character- ized and time-dependent increase in the percentage of apoptotic cells as early as after 24 h treatment (Fig. 3D). In contrast, as shown in Fig. 3D, apoptotic cells were not detected in the HL60 cells until 3 days after RA (1 lM) treatment.

3.4. TB-induced HL60 cells apoptosis was not through protein synthesis and CD95 receptors signal pathways

Preincubation of HL60 with cycloheximide (CHX, 1 lg/ml, 1 h), a protein synthesis inhibitor (Maianski et al., 2004a,b; Mezzanzanica et al., 2004), had no influ- ence on TB-mediated cell death (data not shown), indi- cating that protein synthesis was not pre-requested for TB-induced apoptosis. We then examine whether a po- tential signaling of TB-induced apoptosis in the HL60 cells was via the cell surface CD95/Fas death receptor.

Preincubation of the HL60 cells with ZB4 (1 lg/ml), a neutralizing anti-CD95 antibody (Woo et al., 2004), showed a significant reduction in apoptosis induced by soluble CD95/FasL (100 ng/ml) when compared with the cells treated with CD95/FasL only (Fig. 4A, lanes 5 and 6). In contrast, ZB4 was unable to reduce TB (1 lM, 24 h)-triggered apoptosis (Fig. 4A, lane 4).

3.5. TB treatment caused the changes of mitochondria membrane permeability in HL60 cells

Since the CD95 death receptor seems to be not required for TB-induced apoptosis in HL60 cells (Fig. 4A), we then examined whether cytochrome c re- lease from mitochondria into the cytosol and dissipation of the electrochemical gradient (DW

) was involved in the TB-mediated apoptosis. A real-time plate reader assay showed that DW

stayed relatively stable in un- treated HL60 cells, while it was rapidly (within 6 h) dis- sipated by 1 lM TB treatment (Fig. 4B). The DW

was rapidly (within 2 h) dissipated by the uncoupled CCCP or by the K

ionophore Val and served as a positive control (Fig. 4B). As shown in Fig. 4B and C, TB (1 lM) increased outer (cytochrome c release) and inner (loss of DW

) mitochondria membrane permeability.

The release of cytochrome c (Fig. 4C) kinetically paral- leled a decreased of mitochondria membrane potential (Fig. 4B). These observations suggest that TB-induced apoptosis may be initiated with early alterations in mito- chondrial membrane stability.

3.6. Bcl-2 protein plays an important role in protection of TB-induced apoptosis in HL60 cells

We further investigated whether the observed dys- function of mitochondria is responsible for the TB-trig- gered apoptosis in HL60 cells. We found that the Bcl-2

0 6 12 24 48 (Hrs)

Viability (%)

0 20 40 60 80 100 A

B

C

Control 0.01 µM 0.1 µM 1 µM 10 µM 30 µM

0 6 12 24 48 (Hrs)

Viability (%)

0 20 40 60 80 100

0 6 12 24 48 (Hrs)

Viability (%)

0 20 40 60 80 100

Keratinocyte

HL 60

COLO 205

Fig. 1. Cytotoxic effects of TB in human normal and cancer cells. (A)

Human normal keratinocyte (CCD 922SK), (B) HL60, or (C) COLO

205 cells were treated with various concentrations of TB (0.01 to

30 lM). The cell viability was determined by trypan blue exclusion

assay at the indicated time points after exposure to TB. Results are the

means ± SEM of three independent experiments.

protein level was significantly decreased in the HL60 cells at 6 h after treatment with 10 lM TB (Fig. 5A).

To examine whether cytochrome c release was biologi- cally functioning in initiating apoptosome assembly, immunoprecipitation was performed with cytosolic preparation from TB-treated cells by using antibody specifically against the cytochrome c. As shown in Fig. 5A, a significant increase of Apaf-1 protein level in cytochrome c co-precipitates was detected from HL60 cells treated with TB for 12 h.

We therefore treated the Bcl-2 over-expressed HL60 cells with TB (0.1–30 lM) for 24 h (Fig. 5B). In response to lower dose (0.1–1 lM) of TB, the HL60/Bcl-2 cells, but not control (HL60/PcDNA3) cells, were prevented from the occurrence of apoptosis (Fig. 5B), suggesting that down-regulation of Bcl-2 protein might be involved in the TB-induced apoptosis through a mitochondria- dependent pathway. However, the apoptosis was not completely prevented in the HL60/Bcl-2 cells treated

with higher dose TB (>10 lM) (Fig. 5B). As shown in Fig. 5C, over-expression of Bcl-2 protein completely inhibited the TB-induced release of cytochrome c from mitochondria into cytosol in the HL60/Bcl-2 cells (Fig. 5C, lanes 2 and 3). However, the results from the time-dependent experiments revealed that higher dose (10 lM) TB-induced apoptosis could not be completely prevented in the HL60/Bcl-2 cells (Fig. 5D). These find- ings suggest that the results from cytochrome c release assay do not always correlate with the results from An- nexin V staining. Additional signaling proteins other than Bcl-2 might be involved in the TB-induced the occurrence of apoptosis in HL60 cells.

3.7. TB-induced apoptosis in HL60 cells is through activation of caspases-3 and -9, but not caspase-8

The HL60 cells were treated with various concentra- tions of TB (0.1–30 lM) for 24 h. TB at a lower dose

Fig. 2. Effects of TB on DNA fragmentation in human HL60 and COLO 205 cancer cells. (A) Human HL60 (left) and COLO 205 cells (right) were

treated with various doses of TB for 24 h and then assayed for DNA fragmentation. Effect of TB on cell cycle in human HL60 (B) and COLO 205 (C)

cells determined by flow cytometric analysis. The cells were treated with various concentrations of TB for 24 h. Percentage of cell population in the

different phase of the cell cycle were determined using established CellFIT DNA analysis software. Three samples were analyzed in each group, and

values represent the mean ± SEM.

(1 lM) caused activation of the caspase-3 and degrada- tion of the poly-ADP–ribose polymerase, the substrate for caspase-3 (Fig. 6A). To further elucidate the apopto- tic pathways involved in the activation of caspase-3, we examined the changes of the protein levels of caspases 8 and 9 in the TB-treated HL60 cells. Treatment of HL60 cells with TB (>1 lM) activated caspase-9, but not cas- pase 8, evidenced by degradation of the procaspases 9 as well as the appearance of its cleavage product (Fig. 6A).

To confirm that the absence of caspase-8 activation was not due to technical problem, TNFa (20 lM)-treated HL60 cells showing the cleavage of procaspase-8 as well as cleavage of its substrate, Bid protein, was served as a positive control (Fig. 6B). Caspase activity assays showed that treatment of HL60 cells with high dose TB (30 lM) significantly increased caspases-3 (7.8-fold) and -9 (5.6-fold) activity as early as 12 h after drug treat- ment as compared with DMSO-treated group, while the caspase 8 activity was not changed significantly even at a long-term (24 h) TB treatment (Fig. 6C). To further con-

firm these findings, the HL60 cells were pre-incubated for 4 h with or without the caspase-8-specific inhibitor z-IETD-fmk, caspase-9-specific inhibitor z-LEHD-fmk, or the broad range inhibitor of caspases z-VAD-fmk, followed by TB (30 lM) treatment for 24 h. The percentages of apoptotic cells were analyzed by flow cytometric assay. As shown in Fig. 7, both the cas- pase-9-specific inhibitor (z-LEHD-fmk) and the caspase general inhibitor (zVAD-fmk) reduced the TB-induced apoptosis to a same extent. In the absence of caspase inhibitors, TB caused a 59.6% apoptotic cell death. In the presence of z-LEHD-fmk (60 lM) and zVAD-fmk (50 lM), however, the percentage of TB-induced apop- totic cell death was decreased to 6.5% and 8.3%, respec- tively. In contrast, the caspase-8 inhibitor (z-IETD-fmk) at a concentration of 60 lM had no effect on the TB-in- duced apoptosis (62.1%). The caspase-8 inhibitor at a concentration as high as 100 lM still did not cause any decrease in TB-induced apoptosis (data not shown).

The TNF-a- and CD95/FasL-induced apoptosis was

Fig. 3. Effects of RA and TB on the differentiation and apoptosis of HL60 cells. (A) HL60 cells were treated with RA (1 lM, middle) or TB (1 lM,

right) for 48 h. Cells were cytospun, and then stained by Wright–Giemsa method. Magnification: 500·; scale bar: 20 lM. (B–C) HL60 cell

differentiation was determined at the indicated time points by monitoring of the CD11b and CD33 expression on the cell surface after exposure to TB

or RA (1 lM). (D) For apoptosis assay, the cells were harvested, stained with Annexin V-FITC and PI, and analyzed by flow cytometric analysis as

described in Section 2.

P < 0.05 versus day 0.

completely suppressed by caspase-8 inhibitor, suggesting that z-IETD-fmk at a concentration of 60 lM is suffi-

cient to inhibit caspase-8 activity. The specificity of these inhibitors was demonstrated by showing that vehicle

Fig. 4. Involvement of the CD95 receptor in TB-induced apoptosis. (A) The HL60 cells were pre-incubated for 1 h in the presence of PBS, antagonistic anti-Fas ZB4 mAb (10 lg/ml) or agonistic CD95L (1 lg/ml), supplemented with either vehicle (control) or TB (1 lM), and then incubated for an additional 24 h. The apoptotic cells were determined by annexin V staining and analyzed by flow cytometry. The number of the apoptotic cells was expressed as a percentage of total cells. The values are means ± SEM of three independent experiments. (B) The top insets represent the microscopic observations of the immunofluoresencent staining in HL60 cells treated with TB (1lM). Mitochondria membrane depolarization in TB-treated HL60 cells was measured by JC-1 staining. In the lower panel, HL60 cells were treated with TB (1 lM), RA (1 lM), Valinomycin (Val, 200 lM), or CCCP (200 lM) for the indicated time points. After drug treatment, the HL60 cells were stained with JC-1 (1 lg/ml).

Results were expressed as a change in the ratio between red JC-1 fluorescence (Em 590 nm), and green JC-1 fluorescence (Em 535 nm) over time.

Each point represents the mean ± SEM from three experiments. (C) The HL60 cells were treated with TB (1 lM) for the indicated time points. The

protein levels of the cytochrome c released from mitochondria into cytosol and the percentage of apoptotic cells were then determined. Annexin V-

stained cells were counted as apoptotic cells and expressed as a percentage of total cells. The values are means ± SEM of three independent

experiments. Top inset: the representative bands of cytochrome c detected by Western blot analysis.

(DMSO) treatment had no effect on TB-stimulated apoptosis. Taken together, our results suggest that TB-induced apoptosis is dependent on caspase-9 activation.

4. Discussion

The ability of chemotherapeutic agents to initiate apoptosis plays an important determinant of their ther-

Fig. 5. Effects of TB on the expression levels of mitochondria Bcl-2, Bax, and the caspase-9-associated Apaf-1 proteins. (A) The HL60 cells were treated with 10 lM TB at the indicated time points for 24 h. Protein extracts from the cytosolic and mitochondrial fractions were isolated from the TB-treated HL60 cells followed by immunoblotting analysis. Expression of the Bcl-2 and the caspase-9-associated Apaf-1 protein were determined by Western blot analysis, and the b-actin expression was served as a protein loading control. (B–D) Protection effect of Bcl-2 overexpression on the TB- induced apoptosis in the HL60 cells. (B) The inset shows a representative Western blot result of Bcl-2 protein in HL60 cells transfected with Bcl-2 (pBcl-2) or control (pcDNA3) plasmid. The HL60/Bcl-2 and HL60/pcDNA3 were then treated with TB in a dose-dependent (0.1–30 lM) manner for 24 h. Annexin V-stained cells were counted as apoptotic cells and expressed as a percentage of total cells. The values are means ± SEM of three independent experiments. Significant results were compared to the control group by statistic analysis described in Section 2 as indicated as

P < 0.05.

(C) The Bcl-2 overexpressed (HL60/Bcl-2) and the control (HL60/pcDNA3) cells were treated with 10 lM TB for 24 h, and the protein levels of the

cytochrome c in cytosolic and mitochondria fractions were determined by Western blot analysis. The b-actin protein level was determined and

represented as a protein loading control. (D) In vitro study of the effect of Bcl-2 overexpression on TB-induced apoptosis. Apoptotic cells were

quantified by Annexin V-FITC/PI stain and analyzed by flow cytometry as described in Section 2. Data points, the mean ± SEM of three

independent experiments.

apeutic response. However, significant toxicity at high doses has precluded the use of chemotherapeutic agents as a monotherapy for cancers. Combination therapy is one potential method to help in reducing a compound with undesirable toxic effects but still maintain or en- hance its anti-tumor efficacy. Recently, we have demon- strated that griseofulvin, an oral anti-fungal agent, potentiates the anti-cancer activities of nocodazole (ND) (Ho et al., 2001). Moreover, we showed an enhancement of TB on the ND-induced colon cancer cells apoptosis (Lee et al., 2003). TB has been used as an orally active broad-spectrum anti-fungal drug, espe- cially active in patients with histoplasmosis or nonmen- ingeal cryptococcosis (Caceres-Rios et al., 2000;

Rademaker and Havill, 1998). A previous study has demonstrated that approximately 70% of TB is ab- sorbed after an oral dose (250 mg) (Jensen, 1989) and the maximum plasma concentrations of 0.5–1.5 lg/ml are reached within 2 h (Humbert et al., 1995; Kovarik et al., 1992, 1995). Another report in a human study

showed that the plasma level of TB after daily oral receiving of 250 mg TB for 4 weeks was 1.7 ± 0.77 lg/

ml (5.83 lM) (Kovarik et al., 1995). Here, we showed that administration of TB at a concentration as low as 1 lM for 24 h induced significant apoptosis in the HL60 cells (Figs. 3D and 4A and C). We further demon- strated that the TB-induced the occurrence of apoptosis in the HL60 cells was not mediated through differentia- tion process (Fig. 3A). Such results implied that admin- istration of lower dose (1 lM) TB could reach the therapeutic concentrations in plasma. Importantly, cytotoxicity analysis showed that TB at the doses (0.01–30 lM) used in our in vitro studies was not cyto- toxic for the cultured untransformed human keratino- cyte. Moreover, the dose (50 mg/kg body weight) used in our previous in vivo study performed in the nude mice model was not cytotoxic for the vital organs (Lee et al., 2003).

The caspase 8/FADD (extrinsic) and mitochondrial (intrinsic) pathway are the two major signal pathways regulating apoptosis process. Both of the apoptosis routes were activated during erythroid cell differention (Testa, 2004) and cancer chemotherapy (Hajra and Liu, 2004). Recent studies have demonstrated that both CD95- and B cell receptor (BCR)-mediated apoptosis depend on Bax activation and cytochrome c release, although the timing and caspase-dependence of mito- chondrial membrane depolarization differed consider- ably after CD95- or BCR-triggering (Mackus et al., 2002). However, some other death receptors in the TNF receptor family (such as TNFR1, DR3/Apo3, DR4/DR5, etc.) have been reported to be a mediator in response to cancer chemotherapy. For example, it has been shown that clinically applied anti-cancer drug, cisplatin, induced apoptosis of solid tumor cells through the CD95 and DR5-dependent pathways (Han et al., 2003; Lacour et al., 2004). Another study revealed that camptothecin or etoposide (VP-16) in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) substantially accelerated kinetics of apoptosis in human leukemia (HL60) cells. The authors suggested that DR4 aggregation mediated by camptothecin or VP- 16 could represent as an important mediator that accel- erates TRAIL-induced apoptosis (Bergeron et al., 2004).

In contrast, tumors resistant to cytotoxic drugs may oc- cur through altered expression of death receptors DR4 and DR5 (Zhang and Fang, 2005), resistance to TRAIL-mediated apoptosis (Zhang and Fang, 2005), or sequestration of Fas/caspase-8 signaling pathways (Barnhart et al., 2005; Kim et al., 2001). In this study, although only one death receptor CD95/Fas has been examined to explore the eventual effect of TB on the extrinsic (nonmitochondrial) pathway of apoptosis.

These findings suggested that TB might be a useful sal- vage agent in the management of chemotherapy resis- tant cancer.

Fig. 6. Dose-dependent activation of caspases in TB-induced apopto- sis in HL60 cells. (A) HL60 cells were treated with TB (0.1–30 lM) for 24 h and the expression of caspase-associated proteins were detected by Western blot analysis. The expression of b-actin protein was examined and served as a loading control. (B) HL60 cells were treated with 20 lM TNFa for 48 h. The protein levels of the caspase and Bid were detected by Western blot analysis. (C) TB-induced caspase activities in COLO 205 cells. Cells were treated with 30 lM of TB for various time periods. Caspase activities were measured as described in Section 2.

Data represent means ± SEM for three determinations.

We have previously demonstrated that TB induced cell growth arrest at the G0/G1 phase through a p53- dependent signaling pathway (Ho et al., 2004; Lee et al., 2003). The TB-induced apoptosis, however, was through a p53-independent signaling pathway. Our data suggest that intracellular regulatory proteins other than p53 may be involved in TB-induced apoptosis. Accord- ingly, we investigated the p53-independent mechanisms in TB-induced apoptosis. Our results showed that prein- cubation of the HL60 (p53-null) cells with CHX (1 lg/

ml, 1 h) had no influence on TB-mediated cell death (data not shown), suggesting that de novo protein syn- thesis is not a prerequisite for TB-induced apoptosis.

In turn, decrease of Bcl-2 protein expression may re- sult in excess Bax homodimers, which will be translo- cated to the mitochondrial outer membrane (Fig. 5A), and then led to leakage of cytochrome c through its pore-forming activity (Wei et al., 2001). Our study pro- vide evidences showed that Bcl-2 protein significantly

prevented cytochrome c release, but cannot completely prevent the TB-treated HL60/Bcl-2 cells undergoing apoptosis (Fig. 5B and D), suggesting that additional apoptotic factors other than Bcl-2/Bax family proteins are involved in the TB-induced the occurrence of apop- tosis in HL60.

In this study, our results first demonstrated that TB induced promyelocytic (HL60) cell apoptosis via a sig- naling pathway independent of cell growth arrest. We further examined the sequence of the molecular events involved in the activation of mitochondria-mediated sig- naling pathways during the process of TB-induced apoptosis. Our results indicate that the leakage of cyto- chrome c was preceded by the translocation of Bax to mitochondria. Our data suggest that translocation of Bax to mitochondria, which lead to release of cyto- chrome c, is dependent on amplification of the specific caspase cascade and entry of the cell into the execution phase of apoptosis. This hypothesis is supported by our

Fig. 7. Effects of caspase activation on mitochondria-mediated apoptosis in the TB-treated HL60 cells. For the caspase inhibitors studies, the HL60

cells were incubated with 50 lM of inhibitors specific to caspase-8 (zIETD-fmk), caspase-9 (zLEHD-fmk), or the general inhibitor of caspases

(zVAD-fmk) for 4 h as indicated at the top of each profile. The HL60 cells treated with TNFa (20 lM) and FasL (100 ng/ml) in the presence of

zIETD-fmk were performed as a positive control. The cells were then exposed to TB at a concentration of 30 lM for an additional 24 h. The control

cells were incubated with DMSO without TB or caspase-specific inhibitors. The DNA contents were monitored by flow cytometry as described in

Section 2.

results showing that translocation of the Bax to mito- chondria, release of cytochrome c into cytosol, and the occurrence of cell apoptosis were clearly inhibited by the caspase-9-specific inhibitor (Z-LEHD-fmk) (Fig. 7). The caspase-9 might therefore play an impor- tant role in mitochondria signaling pathways for TB-in- duced apoptosis. Similar results described by previous report showed that activation of caspase-9 usually occurs downstream of cytochrome c release from mito- chondria (Saleh et al., 1999). Assembly of the apopto- some complex might represent the initiating step for the TB-mediated caspase cascade activation (Saelens et al., 2004). However, whether this apoptosome directly causes the release of the different mitochondrial apopto- genic factors simultaneously is currently unknown (Martin et al., 2004). Further studies are required to understand how the compositions of the apoptosome active Bax translocation to the mitochondria and trigger the mitochondrial release of cytochrome c.

Acknowledgment

This study was supported by the National Science Council grant NSC 93-2314-B-038-051- to Dr. Ho, NSC 92-2320-B-038-018 to Dr. Lee and NSC 93-2314- B-038-050- to Dr. Wu and by the Shin Kong Wu Ho-Su Memorial Hospital (SKH-TMU-92-07).

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