Molecular mechanisms of econazole-Molecular mechanisms of econazole-induced toxicity on human colon cancer cells: G0/G1 cell cycle arrest and caspase 8-independent apoptotic signaling pathways

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Molecular mechanisms of econazole-induced toxicity on human

colon cancer cells: G0/G1 cell cycle arrest and caspase

8-independent apoptotic signaling pathways

Yuan-Soon Ho

a

, Chih-Hsiung Wu

b

, Hsiao-Min Chou

c

, Ying-Jan Wang

d

, How Tseng

e

,

Chien-Ho Chen

f

, Li-Ching Chen

g

, Chia-Hwa Lee

h

, Shyr-Yi Lin

i,*

a

Graduate Institute of Biomedical Technology, Taipei Medical University, Taipei 110, Taiwan

b

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

c

d

Department of Environmental and Occupational Health, National Cheng Kung University Medical College, Tainan 701, Taiwan

e

Graduate Institute of Medical Sciences, Taipei Medical University, Taipei 110, Taiwan

f

g

Department of Nursing, TzuChi University, Hualien 970, Taiwan

h_{Graduate Institute of Biomedical Technology, Taipei Medical University, Taipei 110, Taiwan}

i_{Department of Internal Medicine, Taipei Medical University Hospital, Department of Internal Medicine, School of Medicine,}

Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan Received 9 September 2004; accepted 12 April 2005

Abstract

Econazole (Eco), a potent broad-spectrum anti-fungal agent, has been used in the treatment of superﬁcial mycosis. Eco is a store-operated Ca2+_{channel antagonist which induces cytotoxic cell death of leukemia. However, little is known about its cytotoxic eﬀect}

upon solid tumor cells. The purpose of this study is to investigate both the in vitro and in vivo molecular mechanisms of Eco-induced toxicity on colon cancer cells. We used COLO 205 cell line and nude mice xenograft model to investigate the cytotoxic effect of Eco. We demonstrated that lower doses Eco (5–20 lM) arrested human colon cancer cells at the G0/G1 phase of the cell cycle. The protein levels of p53, p21/Cip1, and p27/Kip1 were significantly elevated while CDK2 and CDK4 kinase activity were signif-icantly suppressed by Eco treatment in COLO 205 cells. At higher doses (40–60 lM), Eco induced COLO 205 cells apoptosis evi-denced by ladder formation in DNA fragmentation assay and sub-G1 peak in flow cytometry analysis. Western blot analysis showed that caspases 3, 9 but not 8 were activated by high dose Eco treatment to the COLO 205 cells accompanied with cytochrome c and apoptosis-inducing factor (AIF) translocation. Significant anti-tumorigenesis effect was further demonstrated in vivo by treating nude mice bearing COLO 205 tumor xenografts with Eco 50 mg/kg intraperitoneally. Our findings highlight the molecular mech-anisms underlying the Eco-induced toxicity on colon cancer cells.

Keywords: Econazle; Colon cancer; Apoptosis; Cytotoxicity; Cell cycle

Abbreviations: AIF, apoptosis-inducing factor; BCIP, 5-bromo-4-chloro-3-indolyl phosphate; DMSO, dimethylsulfoxide; Eco, Econazle; FCS, fetal calf serum; iNOS, nitric oxide synthase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; NBT, nitroblue tetrazolium; PARP, poly-ADP-ribose polymerase; PBS, phosphate-buﬀered saline; PCNA, proliferating cell nuclear antigen; VDAC, voltage-dependent anion channel.

* _{Corresponding author. Tel.: +886 2 27361661x3210; fax: +886 2 27393447.}

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

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1. Introduction

Econazole (Eco), an anti-fungal imidazole com-pound, has been used in the treatment of vulvovaginal candidiasis (Dellenbach et al., 2000; Osser et al., 1991) and superficial fungal infection (Aron-Brunetiere et al., 1977; Prajna et al., 2003; Rigoni et al., 1989). Eco has been shown to inhibit lipopolysaccharide-inducible ni-tric oxide synthase (iNOS) activity in rat aortic rings and cultured J774 murine macrophage cells (Bogle and Vallance, 1996) suggesting that it exerted a potential anti-inflammatory effect. Furthermore, Eco at 50 lM re-duced prostaglandin E2 production in rabbit platelets suggesting that it inhibited cyclooxygenase activity (Jiang et al., 1991). Eco at dose of 1 lg/ml was found to reduce cell numbers, viability and survival, and DNA synthesis in the NS1 myeloma line (Denyer et al., 1985). More importantly, Eco has been demon-strated to exert selective inhibition of leukemic and breast cancer cell lines than hemopoietic progenitors using clonogenicity assays. (Soboloff et al., 2002; Zhang et al., 2002). These findings suggested the potential anti-tumor effects of Eco.

The fungal agents have been shown to exert anti-tumor effect. For example, ketoconazole has been used in the treatment of hormone-dependent prostate cancer (Blagosklonny et al., 2000; Mahler and Denis, 1992; Trachtenberg and Pont, 1984). Ketoconazole inhibited hepatic metastasis from a human pancreatic adenocarci-noma (Tzanakakis et al., 1990) and reduce the incidence of pulmonary metastases in the nude mice melanoma model (Nardone et al., 1988). As in our previous studies, the oral anti-fungal agents griseofulvin and ketocona-zole have been demonstrated to suppress tumor cell growth in vitro and in vivo (Chen et al., 2000; Ho et al., 2001; Ho et al., 1998; Wang et al., 2002). Griseo-fulvin significantly potentiated the anti-cancer effect of nocodazole, leading to cessation of tumor growth. These results suggested that combined administration of anti-fungal agent and chemotherapeutic agents might pro-vide a novel therapy for colorectal cancer (Ho et al., 2001).

Colorectal cancer is the second leading cause of can-cer mortality in Western societies and one of the most common malignancies worldwide (Chinery et al., 1997). Although intensive research during the past few years has led to considerable improvement in the treat-ment and diagnosis at an early stage, yet for those pa-tients with advanced stage or recurrent disease, the prognosis is still not satisfactory (Courtney et al., 2004; Gill et al., 2003; Lieberman and Atkin, 2004). Therefore, experimental and clinical investigators con-tinue to search for new therapeutic strategies. One ap-proach, as pursued in this study, seeks to identify medicinal agents capable of retarding the cell cycle and/or activating the cellular apoptotic response in the

cancerous cells. We demonstrated that low dose Eco in-duced growth inhibition in colon cancer cells through G0/G1 cell cycle arrest. Furthermore, high dose Eco in-duced colon cancer cells apoptosis mediated via mito-chondrial pathway which was not depended on caspase 8 activation. The anti-tumor eﬀect was further examined in vivo by treating Eco to athymic mice bear-ing COLO 205 tumor xenografts. This study provides novel molecular mechanisms of Eco-induced cytotoxic-ity upon colon cancer which might have potential appli-cation for colon cancer treatment.

2. Materials and methods 2.1. Cell lines and cell culture

The COLO 205 cell line was isolated from human co-lon adenocarcinoma (American Type Culture Collection CCL-222). The cell line FCH, a homozygous familial hypercholesterolemia cell (CRL 1831; American Type Culture Collection), was derived from primary cultures of normal colon epithelial cells (Siddiqui and Chopra, 1984). The cells were grown in RPMI 1640 supple-mented with 10% fetal calf serum (FCS), penicillin (100 U/ml), streptomycin (100 mg/ml), and 0.3 mg/ml glutamine for COLO 205; grown in DulbeccoÕs Modified EagleÕs Medium/HamÕs Nutrient Mixture F-12, 1:1 with 2.5 mML-glutamine, 1.2 g/l sodium bicarbonate, 15 mM HEPES and 0.5 mM sodium pyruvate supplemented with 10% FCS, 10 ng/ml cholera toxin, 0.005 mg/ml insulin, 0.005 mg/ml transferrin, 100 ng/ml hydrocorti-sone and 10 mM HEPES for CRL 1831 in a humidified incubator (37C, 5% CO2). Eco (Sigma Chemical Co., St. Louis, MO) was added at the indicated doses in 0.05% dimethylsulfoxide (DMSO). For control speci-mens, the same volume of DMSO was added in a final concentration of 0.05% (v/v) without Eco.

2.2. Determination of cell viability

COLO 205 and CRL 1831 cells were treated with Eco (10–30 lM). Cell viability was determined at the indi-cated times based on MTT assay. Brieﬂy, cells were seeded in a 96-well plate at a density of 1· 104cells/well and allowed to adhere overnight. After removing the medium, 200 ll of fresh medium per well, containing 10 mmol/l Hepes (pH 7.4) was then added. Then, 50 ll of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazo-lium bromide (MTT) was added to the wells and the plate was incubated for 2–4 h at 37C in the dark. The medium was removed and 200 ll DMSO and 25 ll SorensensÕs glycine buﬀer was added to the wells. Absorbance was measured using an ELISA plate reader at 570 nm.

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2.3. Determination of cell growth curve COLO 205 (1· 105

), CRL 1831 (1· 105

) cells were plated in 35 mm petri dishes. The next day, the medium was changed and Eco (5–60 lM) was added. Control cells were treated with DMSO in a ﬁnal concentration of 0.05% (v/v). The incubation medium was renewed every day during the experiment. At the end of incuba-tion, cells were harvested for cell count with a hemocytometer.

2.4. Cell synchronization, drug treatment, and ﬂow cytometry analysis

At 24 h after plating of cells, cells were washed three times with phosphate-buffered saline (PBS) and then incubated with medium containing 0.04% FCS for addi-tional 24 h. Under such conditions, cells were arrested in G0/G1 as determined by flow cytometry analysis (Lee et al., 2003). After serum starvation, the low-serum (0.04% FCS) medium was removed and the cells were then challenged by addition of medium containing 10% FCS. Eco solutions were prepared in a final concen-tration of 0.05% (v/v) DMSO. The cell cycle stages in the Eco and DMSO-treated groups were measured by flow cytometry analysis. Cells were harvested and stained with propidium iodide (50 lg/ml) (Sigma Chemical Co., St. Louis, MO), and DNA content was measured using a FACScan laser flow cytometer analysis system (Becton–Dickinson, San Jose, CA); and 15,000 events were analyzed for each sample.

2.5. Western analysis

Western blotting analysis was performed as described previously (Ho et al., 2001). Brieﬂy, cell lysates were prepared, electrotransferred, immunoblotted with anti-bodies, and then visualized by incubating with the colorigenic substrates (nitroblue tetrazolium, NBT and 5-bromo-4-chloro-3-indolyl phosphate, BCIP) (Sigma Chemical Co., St. Louis, MO). The expression of GAPDHwas used as the control for equal protein loading.

2.6. Immunoprecipitation, CDK2, and CDK4 kinase activity assay

The p21/Cip1-associated CDK-2 kinase activity was determined as described previously (Wu et al., 1996). Briefly, using 2 lg anti-CDK2 antibody and 20 ll pro-tein A agarose beads, the propro-tein complexes were precip-itated from 200 lg of protein lysates per sample as described above. Beads were washed twice with lysis buffer and then once with kinase assay buffer (50 mM Tris–HCl, pH 7.4, 10 mM MgCl2, and 1 mM DTT). Phosphorylation of the histone H1 was measured by

incubating the beads with 40 ll of ‘‘hot’’ kinase solution [0.25 ll (2.5 lg) of histone H1, 0.5 ll of [c-32P]ATP, 0.5 ll of 0.1 mM ATP, and 38.75 ll of kinase buffer] for 30 min at 37C. The reaction was stopped by boiling the samples in SDS sample buffer for 5 min. The samples were analyzed using 12% SDS–PAGE, and the gel was dried and subjected to autoradiography. Similarly, the CDK4 kinase activity was determined as described by previous report (Wu et al., 1996) with some modifica-tions. Briefly, Eco-treated cells were lysed in Rb lysis buffer (50 mM Hepes–KOH, pH 7.5, containing 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 1 mM DTT, 0.1% Tween 20, 10% glycerol, 80 mM b-glycero-phosphate, 1 mM sodium fluoride, 0.1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 10 lg/ml leupetin and aprotinin) and immunopre-cipitated with 2 lg anti-CDK4 antibody. The protein complexes in beads were washed twice with Rb lysis buf-fer and then once with Rb kinase assay buffer (50 mM Hepes–KOH, pH 7.5, containing 2.5 mM EGTA, 10 mM b-glycerophosphate, 1 mM sodium fluoride, 0.1 mM sodium orthovanadate, 10 mM MgCl2, and 1 mM DTT). Phosphorylation of Rb was measured by incubating the beads with 40 ll of hot Rb kinase solu-tion [0.25 ll (2 lg) Rb–GST fusion protein, 0.5 ll of [c-32P]ATP, 0.5 ll of 0.1 mM ATP, and 38.75 ll of Rb kinase buffer] for 30 min at 37C. The reaction was stopped by boiling the samples in SDS sample buffer for 5 min. The samples were analyzed using 12% SDS– PAGE, and the gel was dried and subjected to autoradiography.

2.7. 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 de-scribed (Ho et al., 2003; Lin et al., 2001). Briefly, COLO 205 cells were lysed by addition of cell lysis buffer and protein concentration was measured. Caspase activity was assayed at 37C in 100 ll of assay buffer containing 50 lg (for caspase 3) or 30 lg (for caspase 9) of the indi-cated colorimetric peptide. Caspase activity was mea-sured 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.

2.8. Treatment of COLO 205-derived xenografts in vivo COLO 205 cells were grown in RPMI 1640 supple-mented with 10% FCS as described above. Cells were harvested through two consecutive trypsinizations, cen-trifuged at 300g for 5 min, washed twice, and resus-pended in sterile PBS. Cells (5· 106) in 0.2 ml were injected subcutaneously between the scapulae of each

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nude mouse (purchased from National Science Council Animal Center, Taipei, Taiwan). After transplantation, tumor size was measured using calipers and the tumor volume was estimated according to the formula tumor volume (mm3) = L· W2

/2, where L is the length and W is the width (Ho et al., 2001). Once tumors reached a mean size of 200 mm3, animals received intraperito-neal injections of either 25 ll DMSO or 50 mg/kg Eco three times per week for 6 weeks.

2.9. Analysis of DNA fragmentation

Analysis of DNA fragmentation was performed as previously described (Ho et al., 1996). Brieﬂy, the Eco and DMSO-treated cells were seeded on 100 mm dishes. The DNA was extracted twice with equal volumes of phenol and once with chloroform-isoamyl alcohol (24:1 v:v), then precipitated with 0.1 volume of sodium acetate, pH4.8, and 2.5 volumes of ethanol at 20C overnight, and ﬁnally centrifuged at 13,000g for 1 h. Genomic DNA was quantitated, and equal amounts of DNA sample in each lane were electrophoresed in a 2% agarose gel. The DNA was visualized by ethidium bromide staining.

3. Results

3.1. Eco-induced cytotoxicity in COLO 205 cells

To investigate whether Eco inhibited colon cancer cell growth, COLO 205 and human normal colon epi-thelial cells (CRL 1831) were treated with different doses (5–60 lM) of Eco for 1–5 days and the cell growth num-bers were then determined (Fig. 1). Eco at lower doses (5–10 lM) suppressed COLO 205 cell proliferation while at higher dose (>20 lM) induced cell death (Fig. 1A). Interestingly, such effect was less profound on nor-mal human colon epithelial cells treated with Eco (Fig. 1B). We further demonstrated that the viability of COLO 205 was significantly decreased when exposed to more than 10 lM Eco for 24 h (Fig. 2A). In contrast, less significant influence of viability was observed in nor-mal colon epithelial cells treated with same dose of Eco (Fig. 2B). These results indicated that colon cancer cells were more susceptible to Eco treatment than normal hu-man normal colon epithelial cells.

3.2. Eco induced G0/G1 cell cycle arrest and apoptosis in COLO 205 cells

In order to determine the action of Eco on a speciﬁc phase of cell cycle, the COLO 205 cells were synchro-nized by switching them to media with 0.04% FCS for

Fig. 1. Cell growth inhibitory eﬀects of Eco on colon cancer cells and normal human colon epithelial cells. (A) Human colon cancer (COLO 205) cells and (B) Human normal colon epithelial cells (CRL 1831) were treated with various concentrations of Eco (5–60 lM) for 1–5 days. Dose-dependent suppression of cell growth was observed in COLO 205 cells and normal colon epithelial cells. Media with or without Eco were changed everyday until cell counting. Three samples were analyzed in each group and the results were presented as means ± SE.

Fig. 2. Dose and time-dependent response of viability of human cells treated with Eco. (A) COLO 205 cell and (B) Normal colon epithelial cells viability was determined after exposure to various concentrations of Eco (10–30 lM) for the indicated time points. Three samples were analyzed in each group and the results were presented as means ± SE.

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24 h to render them quiescent. Cells were then returned to culture media containing 10% FCS which stimulated cell proliferation with or without 20 lM Eco and, at var-ious times thereafter, they were harvested for ﬂow

cytometry analysis. Fig. 3A (lower panel) showed that 20 lM Eco induced a signiﬁcant accumulation (>85%) of cells in the G0/G1 phase of the cell cycle compared to 0.05% DMSO control (upper panel), suggesting that

Fig. 3. Eﬀect of Eco on cell cycle regulation of COLO 205 cells. Time-dependent eﬀects of (A) FACS analysis of DNA content was conducted after COLO 205 cells were released for quiescence by incubation in culture media supplement with 10% FBS containing 0.05% DMSO (upper panel) or Eco (20 lM in 0.05% DMSO, lower panel). (B) Dose dependent response of Eco on cell cycle regulation. COLO 205 cells were quiescent and released by incubation in culture media supplement with 10% FBS and Eco (1–60 lM) treatment for 24 h. Percentage of cells in sub-G1, G0/G1, S and G2/M phases of the cell cycle were determined using established CellFIT DNA analysis software. Three samples were analyzed in each group, and values were presented as means ± SE and (C) DNA fragmentation assay in COLO 205 cells. Cells were treated with Eco (5–60 lM) or DMSO (0.05%) as control and DNA fragmentation was examined 24 h later. M is the molecular weight marker.

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the growth inhibitory effect of Eco was due to an arrest of DNA replication in the cell cycle.Fig. 3B demonstrated the dose-dependent effect of Eco on the cell cycle regula-tion. The results demonstrate that Eco (20 lM) induced an accumulation (up to 85%) of the COLO 205 cells at the G0/G1 phase of the cell cycle. However, higher dose (40–60 lM) of Eco induced COLO 205 cell death as evi-denced by sub-G1 peak in the flow cytometry analysis. DNA fragmentation assay also demonstrated that DNA ladder formation was observed at 24 h after Eco (40–60 lM) treatment to colon cancer cells (Fig. 3C). 3.3. p53, p21/Cip1 and p27/Kip1 were the key

regulators in the Eco-induced G0/G1 arrest in COLO 205 cells

As shown inFig. 4, treatment of COLO 205 cells with lower doses (5–20 lM) Eco for 24 h induced increased of p53, p21/Cip1, p27/Kip1, cyclin E and decreased of phospho-CDK2 and phospho-Rb protein expression in a dose-dependent manner. In order to determine whether the changes of the levels of cyclins and CDKs were also involved in the Eco-induced G0/G1 arrest, we then examined cyclins and CDKs protein levels in response to Eco treatment. Eco at the concentrations of 5–20 lM did not induce any signiﬁcant changes of the levels of cyclin A, D1, D3, CDK4, and proliferating cell nuclear antigen (PCNA). However, in the higher doses (40–60 lM)-treated groups, the phospho-CDK2, CDK4, cyclins A2, D1, and D3 protein levels were sig-niﬁcantly down regulated in the COLO 205 cells. Such results might be due to cellular toxicity occurred in the COLO 205 cells treated with higher dose (40–60 lM) Eco (Fig. 4).

3.4. Eco-induced G0/G1 arrest was through inhibition of CDK2 and CDK4 kinase activity

It has been demonstrated that the progression of cell cycle activity is associated with coordinated successive activation of certain CDKs occurred late in the G1 phase and is instrumental in the transition from the G1 to the S phase (Hunter and Pines, 1994; Morgan, 1995). As described previously (Sherr and Roberts, 1995), the p21/Cip1 and p27/Kip1 were potent inhibitors of CDK2 and CDK4 which regulated the cells entering the restriction point in the G0/G1 phase of the cell cycle. Based on the flow cytometry analysis in the COLO 205 cells (Fig. 3A), the time points at the 0, 15, 18 and 24 h after released from quiescence represents the G0/G1, S, G2/M and the second G0/G1 phases of the cell cycle, respectively. Accordingly, these time points were se-lected for Western blot analysis to examine the effects of Eco on the expression of cell cycle regulatory pro-teins. We demonstrated that low dose Eco (20 lM) sig-nificantly suppressed CDK2 and CDK4 kinase activity

(Fig. 5A) with concomitant increased p21/Cip1 and p27/Kip1 protein expression as compared to DMSO control groups (Fig. 5B). These results suggested that the G0/G1 arrest induced by Eco was due to decreased of the CDK2 and CDK4 kinase activity mediated by an induction of p21/Cip1 and p27/Kip1 expression. 3.5. Eco activated the caspase 3, 9 but not caspase 8

Since it has been demonstrated that the occurrence of apoptosis requires the activation of caspases (Thornberry and Lazebnik, 1998), we investigated the involvement of

Fig. 4. Effect of Eco on the expression of G0/G1 phase regulatory proteins in COLO 205 cells. The cells were rendered quiescent by incubation for 24 h in the cultured media containing 0.04% FCS. After 15 h released from quiescence by incubation in culture media supple-ment with 10% FCS and 0.05% DMSO with or without Eco (5– 60 lM), the cells were harvested and protein extracts (100 lg per lane) were separate by SDS-PAGE. After electrophoresis, proteins were transferred onto Immobilion-P membranes, probed with proper dilution of specific antibodies, and then detected by using the NBT/ BCIP system. Membrane was also probed with anti-GADPHantibody to correct for difference in protein loading. M is the molecular weight marker.

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caspase activation in the Eco-induced apoptosis in the COLO 205 cells by Western blot analyses. Eco at higher dose (40–60 lM) activated the caspase 3 evidenced by a decreased in the protein level of procaspase 3 and degra-dation of the poly-ADP-ribose polymerase (PARP), the substrate for caspase 3 in both dose-dependent and time course studies (Figs. 6A and B). To further elucidate the apoptotic pathways involved in the activation of cas-pase 3, we examined the changes of cascas-pases 8 and 9 pro-tein levels in the Eco-treated COLO 205 cells. Treatment of COLO205 cells with high dose (40–60 lM) Eco acti-vated caspase 9 evidenced by degradation of the procasp-ases 9 as well as the appearance of its cleavage product but not caspase 8 (Figs. 6A and B). To further conﬁrm the Western blot results, we performed caspase activity assays. As shown inFig. 6C, treatment of COLO 205 cells with 60 lM Eco induced caspase 3 activity about 6 folds and 13.5 folds at 9 and 12 h compared to control, respec-tively, while induced caspase 9 activity about 2 folds and 5.5 folds at 9 and 12 h compared to control, respectively. To further conﬁrm that caspase 8 was truly not acti-vated, Western blot analysis was performed in the COLO 205 cells treated with high dose (60 lM) Eco in a time dependent study (Fig. 6D). Consistent results were shown inFig. 6E, high dose (60 lM) Eco induced COLO 205 cells apoptosis started after 6–12 h of treatment as evidenced by DNA laddering formation. However, un-der the same condition, caspase 8 activation was not de-tected even in a prolonged (48 h) Eco treatment in the COLO 205 cells detected by Western blot analysis (Fig. 6D). In contrast, TNFa (100 ng/ml)-treated COLO 205 cells which acted as a positive control demonstrated cas-pase 8 activation by 24 h as well as cleavage of its

sub-strate, bid protein, suggesting that caspase 8 was not involved in Eco-induced apoptosis in the COLO 205 cells (Fig. 6D). We also tested whether Fas/FasL pathways were involved in the caspase 8 activation in COLO 205 cells. Treatment with 100 ng/ml FasL to COLO 205 cells for 24 h failed to activate caspase 8 and bid protein (Fig. 6D). These results were consistent with a previous report that COLO 205 cells were resistant to FasL-mediated apoptosis (Herbeuval et al., 2003).

3.6. Eco upregulated p53 and Bax protein and translocated cytochrome c from mitochondria to cytosol and AIF from mitochondrial to nucleus

Our study demonstrated that in the COLO 205 cells treated with higher doses (40–60 lM) of Eco, the Bax protein was induced at a signiﬁcantly higher level than the control cells, whereas Bcl-2 and Bid expression re-mained the same as control (Fig. 7A). We further found that higher doses (40–60 lM) of Eco dose-dependently increased translocation of cytochrome c from mitochon-dria to cytosol in the COLO 205 cells (Fig. 7A). To examine whether cytochrome c release was biologically functioning in initiating apoptosome assembly, immu-noprecipitation was performed with cytosolic prepara-tions from Eco-treated cells using cytochrome c antibody. As shown in Fig. 7A, Western blotting de-tected a signiﬁcantly increase of Apaf-1 in cytochrome c coprecipitates from COLO 205 cells.

In this study, extremely high dose (60 lM) of Eco in-duced signiﬁcant apoptosis in COLO 205 cells as evi-denced by DNA laddering occurred at 6–12 h (Fig. 6E). According to the previous study that the tumor

Fig. 5. (A) Time course study of Eco on the CDK2 and CDK4 kinase activity. The cells were rendered quiescent by incubation for 24 h in the cultured media containing 0.04% FCS. The cells then were released from quiescence by incubation in culture media supplement with 10% FCS and 0.05% DMSO with or without Eco (20 lM), the cells were harvested and 200 lg protein per lane was used for the CDK2 and CDK4 kinase activity assay as described in the materials and methods and (B) Time course study of Eco on the expression of G0/G1 phase regulatory proteins in COLO 205 cells. The cells were prepared as (A) and protein extracts (100 lg per lane) were separate by SDS-PAGE. After electrophoresis, proteins were transferred onto Immobilion-P membranes, probed with proper dilution of speciﬁc antibodies, and then detected by using the NBT/BCIP system. Membrane was also probed with anti-GADPHantibody to correct for diﬀerence in protein loading. M is the molecular weight marker.

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suppressor p53 exerts its anti-neoplastic activity primar-ily through the induction of apoptosis (Schmitt et al., 2002). Recent study further demonstrated that the cyto-solic p53 directly activated the proapoptotic Bax protein in the absence of other proteins to permeabilize mito-chondria and engaged the apoptotic program (Chipuk et al., 2004). In our study, time-dependent experiment demonstrated that Eco induced p53 and Bax protein expression concurrently as early as 6–9 h (Fig. 6A). Sim-ilar responses of Eco-induced increased of the p53 (Fig. 4) and Bax (Fig. 7A) protein levels were found in the dose-dependent experiments.

Our data revealed that increased amounts of cyto-chrome c and Apaf-1 proteins were detected in the cytosolic fractions of the Eco-treated cells (Fig. 7B). The release of mitochondrial factors such as cytochrome cand Smac/DIABLO is considered a prerequisite for caspase 9 activation in apoptosis (Du et al., 2000; Li et al., 1997; Liu et al., 1996; Verhagen et al., 2000). In this study, presence of cytosolic cytochrome c was de-tected at 6–12 h after 60 lM Eco treatment leads to

Apaf-1-mediated apoptosome assembly and rapid acti-vation of caspase 9 (Fig. 6A). On the other hand, the Smac/DIABLO which translocated from mitochondria to cytosol was observed as early as 3–6 h (Fig. 7B) which then interacts with the XIAP (Du et al., 2000; Verhagen et al., 2000), and blocks its inhibitory eﬀects on activated caspases 9 and 3. Translocation of AIF from mitochon-drial to nucleus has been shown to activate apoptosis. Our results demonstrated that Eco at high dose induced AIF translocation from mitochondria to nucleus evi-denced by Western blot analysis (Fig. 7B).

3.7. Eco inhibited colon tumor growth in vivo

We further examined the anti-tumor eﬀect of Eco in vivo by treating athymic mice bearing COLO 205 tumor xenografts. After established palpable tumors (mean tu-mor volume, 200 mm3), animals received Eco at dosage of 50 mg/kg or DMSO (control) three times a week. The tumor volume was signiﬁcant smaller in the Eco-treated group than the DMSO-Eco-treated control started

Fig. 6. Effect of Eco on the expression of apoptosis regulatory proteins in COLO 205 cells. The cells were rendered quiescent by incubation for 24 h in the cultured media containing 0.04% FCS. The cells then were released from quiescence by incubation in culture media supplement with 10% FCS and 0.05% DMSO with or without Eco. (A) Time course. (B) Dose-dependent study of Western blotting were performed. M is the molecular weight marker. Membrane was also probed with anti-actin antibody to correct for difference in protein loading. (C) Eco-induced caspase activities in COLO 205 cells. Cells were treated with 60 lM of Eco for various time periods. Caspase activities were measured as described in Materials and Methods. Data represent means ± SE for three determinations. (D) Caspase 8 is not involved in the Eco-induced apoptosis. Western blot analysis of caspase 8 and bid proteins time course study treated with high dose (60 lM) Eco, 100 ng/ml TNFa, and 100 ng/ml FasL to COLO 205 cells. Membrane was also probed with anti-actin antibody to correct for difference in protein loading and (E) High dose (60 lM) Eco caused DNA ladder formation starting after 6 h of treatment. M is the molecular weight marker.

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from the second to the sixth week (Fig. 8A). In mice receiving these treatment regimens, no gross signs of toxicity were observed including body weight (Fig. 8B) and visible inspection of general appearance, and microscopic examination of individual organs such as intestinal epithelium histology (data not shown). Addi-tionally, the tumor weight and the tumor/body weight ratio were strongly inhibited in the Eco-treated group (Figs. 8C and D).

4. Discussion

Eco has been used to treat superﬁcial fungal infections and vaginal candidiasis. Successful treatment using Eco

in a case of middle aged women with opportunistic pul-monary aspergillosis had been reported (Hantschke et al., 1978). Toxicity and teratogenicity studies in gui-nea-pigs and rats indicated that Eco was well tolerated (Thienpont et al., 1975). However, little is known about its use in oral or intravenous applications (Heel et al., 1978; Midgley et al., 1981). The present study was under-taken to examine the cytotoxic effect of Eco on human colon cancer cells. Our in vitro studies demonstrated that Eco at concentrations of 5–20 lM inhibited growth rate of COLO 205 cells in a dose-dependent manner, but less significant in normal human colon epithelial cells. These results were due to an inhibitory effect of econzole on the mechanisms for cell division in the subcultured cancer cells. Furthermore, when Eco concentration was in-creased to 40–60 lM, apoptosis was observed in COLO 205 cells. In vivo studies show that intraperitoneal administration of Eco caused a significant suppression of the COLO 205 tumor mass. To our knowledge, this is the first demonstration that Eco induces toxicity of co-lon cancer cells both in vitro and in vivo.

By ﬂow cytometry analysis, we demonstrated that Eco at lower concentrations (5–20 lM) induced G0/G1 cell cycle arrest of COLO 205. Cell cycle progression is regulated by successive, coordinated activation of CDKs (Hunter and Pines, 1994; Morgan, 1995), whose activity is controlled by their association with a series of regula-tory subunits called cyclins and a group of CDK-inhib-itory proteins designated CKIs (Sherr and Roberts, 1995). Among these CKIs are two known as p21/Cip1 and p27/Kip1. The p21/Cip1 is induced by p53 tumor suppressor gene on DNA damage (Gartel et al., 1996). Transcriptional activation of p21/Cip1 associated with and inactivated the CDKs which resulted in G0/G1 ar-rest (el-Deiry et al., 1994; el-Deiry et al., 1993). The hu-man papilloma virus E7 oncoprotein interacted with p21/Cip1 protein and abrogated p21/Cip1-mediated inhibition of CDK2 activity (Funk et al., 1997). Kerati-nocyte from p21/Cip1-null mice are more tumorigenic than the wild type counterpart following expression of an activated ras allele (Missero et al., 1996) suggesting that p21/Cip1 is a candidate tumor suppressor gene. Our data demonstrated that p53 protein induction oc-curred in advance of p21/Cip1 suggesting that p53 might transcriptionally activate p21/Cip1 which in turn medi-ated Eco-induced G0/G1 arrest in COLO 205 cells.

p27/Kip1, a cell cycle regulatory protein, controls G1/ S transition checkpoint by inhibiting the activity of dif-ferent kinds of cyclin/CDK complexes in vitro and in vivo, including CDK2 and CDK4 complexes (Polyak et al., 1994). Homozygous deletion of p27/Kip1 alleles in mice results in enhanced growth, hyperplasia of vari-ous organs, and the spontanevari-ous formation of pituitary tumors (Kiyokawa et al., 1996; Nakayama et al., 1996). Overexpression of p27/Kip1 leads to apoptotic cell death in mammalian cells suggesting a potential therapeutic

Fig. 7. Eﬀect of Eco on the expression of apoptosis associated proteins. M is the molecular weight marker. (A) Dose-dependent study of Western blotting was performed.. Membrane was also probed with anti-actin antibody to correct for diﬀerence in protein loading. (B) Translocation of apoptosis associated factors in COLO 205 cells. Western blot analysis of time course study treated with high dose (60 lM) Eco. Upper panel, mitochondrial voltage-dependent anion channel (VDAC) and actin were used as loading controls for mitochondrial and cytosolic proteins, respectively. Lower panel, VDAC and PCNA were used as loading controls for mitochondrial and nuclear proteins, respectively. M is the molecular weight marker.

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approach aimed at elevating p27/Kip1 expression for treatment of human cancers (Wang et al., 1997). In our study, low dose Eco induced p21/Cip1 and p27/ Kip1 protein expression, suppressed the CDK2 and CDK4 kinase activity and induced G0/G1 arrest while there were no significant changes of cyclins or CDKs expression suggesting that p21/Cip1 and p27/Kip1 are the main regulators of Eco-induced cell cycle inhibition. By flow cytometry and DNA fragmentation analyses, we also demonstrated that high dose Eco (40–60 lM) caused apoptosis in COLO 205 cells. The extrinsic, cas-pase 8/FADD pathway and intrinsic, mitochondrial pathway are the two major signal pathways regulated apoptosis process. Activaton of bid by caspase 8 links the extrinsic to the intrinsic apoptotic pathway through mitochondrial damages (Li et al., 1998). It has been shown that clinically applied anti-cancer drugs such as cisplatin, topotecan, and gemcitabine induced apoptosis of solid tumor cells through caspase 8 dependent path-ways (Ferreira et al., 2000). Tumor resistance to cyto-toxic drugs may occur through altered expression of caspase 8, upregulation of caspase 8 inhibitors or seques-tration of caspase 8 (Kim et al., 2001). Furthermore, hy-poxia is known to be associated with interference to chemotherapy or radiotherapy and hypoxia-induced apoptosis mainly relies mitochondrial pathways ( Wein-mann et al., 2004). Our results demonstrated that Eco-induced apoptosis in colon cancer cell death was primar-ily through mitochondrial pathway which was not de-pended on caspase 8 and bid activation. These findings

suggested that Eco might be a useful salvage agent in the management of chemotherapy resistant colon cancer. The bax gene promoter region contains four motifs with homology to consensus p53-binding sites suggest-ing that bax is a p53 primary-response gene, involved in a p53 transcriptionally regulated induction of apopto-sis (Miyashita and Reed, 1995). However, recent study further demonstrated that the cytosolic p53 directly acti-vated the proapoptotic Bax protein (Chipuk et al., 2004). Our study demonstrated that Eco induced p53 and Bax protein expression concurrently. Based on these ﬁndings, we propose that when p53 accumulates in the cytosol, it can function analogously to the BH3-only subset of proapoptotic Bcl-2 proteins to activate Bax and trigger the apoptosis mediated by mitochondria-sig-naling processes.

It has been discovered that in response to apoptotic stimuli, mitochondria can also release caspase-indepen-dent cell death effector such as AIF (Lorenzo et al., 1999). AIF is a phylogenetically ancient mitochondrial intermembrane flavoprotein endowed with the unique capacity to induce caspase-independent peripheral chro-matin condensation and large-scale DNA fragmentation when added to purified nuclei (Cande et al., 2002). In our study, high dose Eco treatment induced transloca-tion of AIF from mitochondria to nucleus suggesting a peculiar molecular mechanism in the Eco-induced cyto-toxicity toward colon cancer.

Based on the results of our present study, we proposed a model for the molecular mechanisms of Body Weight xg 0 5 10 15 20 DMSO Econazole Weeks of Treatment 0 1 2 3 4 5 6 7 T um o r Volu m e (m m 3) 0 500 1000 1500 2000 2500 3000 DMSO Econazole DMSO Econazole Tumor Weight x g 0 1 2 3 4 Tumor/Body Weight % 0 10 20 30 40 DMSO Econazole * * * * * * * (A) (C) (B) (D)

Fig. 8. The growth of COLO 205 tumor xenografts in nude mice was suppressed by Eco treatment. Athymic nude mice injected with COLO 205 cells into subcutaneous tissue of inter-scapular area. Once tumor volume reached approximately 200 mm3, the animal received treatment of 50 mg/kg Eco or DMSO intraperitoneally three times per week for 6 weeks. (A) Average tumor volume of DMSO-treated (circle, n = 5) versus Eco-treated (n = 5) nude mice. (B) Body weight. (C) Tumor weight and (D) Tumor/body weight ratio were measured at the end of the experiment. Five samples were analyzed in each group, and values represent the means ± SE. Comparisons were subjected to StudentÕs t test.*Signiﬁcant diﬀerence at p < 0.05.

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Eco-induced cytotoxic effect on COLO 205 cells, as shown in Fig. 9 (Ashkenazi and Dixit, 1998; Green and Reed, 1998). Our experimental findings introduce our basic observations that this anti-fugal agent-Eco can specifically inhibit cell proliferation and induce apoptosis in colon cancer cells both in cultures and also in colon cancer bearing nude mice. The results from the present in vitro and in vivo studies highlight the molec-ular mechanism of Eco-induced cytotoxicity upon colon cancer which might have potential application for the disease.

Acknowledgement

Supported by the National Science Council of Tai-wan; grant NSC B-038-029 and NSC 92-2314-B-038-045.

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