Inhibit multidrug resistance and induce apoptosis by using glycocholic acid and epirubicin

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Inhibit multidrug resistance and induce apoptosis by using glycocholic acid and epirubicin Y.L. Lo a,∗ , C.T. Ho b , F.L. Tsai c a b c

Department of Biological Sciences and Technology, National University of Tainan, No. 33, Sec. 2, Su-lin Street, Tainan City 700, Taiwan Department of Information Management, Shih-Chien University, Kaohsiung, Taiwan Graduate Institute of Biotechnology, Chia-Nan University of Pharmacy and Science, Tainan, Taiwan

a r t i c l e

i n f o

a b s t r a c t

Article history:

Cancer-cell resistance to chemotherapy limits the efficacy of cancer treatment. The pri-

Received 4 March 2008

mary mechanisms of multidrug resistance (MDR) are “pump” and “non-pump” resistance.

Received in revised form

We evaluated the effects and mechanisms of glycocholic acid (GC), a bile acid, on inhibit-

26 May 2008

ing pump and non-pump resistance, and increasing the chemosensitivity of epirubicin

Accepted 5 June 2008

in human colon adenocarcinoma Caco-2 cells and rat intestine. GC increased the cyto-

Published on line 20 June 2008

toxicity of epirubicin, significantly increased the intracellular accumulation of epirubicin in Caco-2 cells and the absorption of epirubicin in rat small intestine, and intensified

Keywords:

epirubicin-induced apoptosis. GC and epirubicin significantly reduced mRNA expression

Multidrug resistance

levels of human intestinal MDR1, MDR-associated protein (MRP)1, and MRP2; downregu-

Apoptosis

lated the MDR1 promoter region; suppressed the mRNA expression of Bcl-2; induced the

Glycocholic acid

mRNA expression of Bax; and significantly increased the Bax-to-Bcl-2 ratio and the mRNA

Epirubicin

levels of p53, caspase-9 and -3. This suggests that GC- and epirubicin-induced apoptosis was mediated through the mitochondrial pathway. We conclude that simultaneous suppression of pump and non-pump resistance dramatically increased the chemosensitivity of epirubicin. A combination of anticancer drugs with GC can control MDR via a mechanism that involves modulating P-gp and MRPs as well as regulating apoptosis-related pathways. © 2008 Elsevier B.V. All rights reserved.

1.

Introduction

The intrinsic and acquired resistance of cancer cells to chemotherapy limits the efficacy of cancer treatment. Multidrug resistance (MDR) is resistance to structurally and functionally unrelated multiple anticancer drugs after treatment with a single agent. Multiple mechanisms contribute to chemoresistance and eventually lead to the failure of cancer chemotherapy. The primary mechanisms of MDR can be subdivided into two major classes: “pump” and “non-pump” resistance (Pakunlu et al., 2004; Wang et al., 2007). The efflux membrane proteins that pump out the anticancer agents from

∗

Corresponding author. Tel.: +886 6 260 6300; fax: +886 6 260 6153. E-mail address: [email protected] (Y.L. Lo). 0928-0987/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2008.06.003

cells or cell organelles decrease intracellular drug accumulation and efficacy, which causes pump resistance. The main mechanism of non-pump resistance is an activated cellular “anti-cell death” defense, which augments tumor cell survival and delays the apoptosis cascade (Minko et al., 2004). Other mechanisms may include changed target enzymes (e.g. mutated topoisomerase II), decreased drug activation, altered activity of cytochrome p-450 and glutathione S-transferases, subcellular redistribution, drug interaction, and enhanced DNA repair (Luqmani, 2005). Overexpressed P-glycoprotein (P-gp) and MDR-associated proteins (MRPs), such as MRP1 and MRP2, are the main causes

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of pump-related multidrug resistance in different cancer cells (Gottesman and Pastan, 1993; Lo, 2003). P-gp, MRP1, and MRP2 are all ATP-binding cassette (ABC) transporter proteins. The inhibition of P-gp and MRPs by MDR modulators should antagonize resistance and, therefore, lead to increased intracellular anticancer drug concentrations and more efficacious chemotherapy (Narasaki et al., 1997; Lo, 2003). Although inhibiting drug efflux pumps increased intracellular drug concentration, it did not lead to a substantial improvement in treating some cancers. The major reason for this failure is activation of an antiapoptotic defense, namely “non-pump resistance” (Minko et al., 2004; Wang et al., 2007). Upregulating the cellular survival defensive system is the primary activator of this antiapoptotic defense, and key proteins such as Bcl-2 and Bcl-xL are important for such survival mechanisms (Tsujimoto, 2003; Pommier et al., 2004; Yamanaka et al., 2006). Usually, Bcl-2 and Bcl-xL overexpression does not affect the uptake and efflux of anticancer drugs in tumor cells. Instead, Bcl-2 protein prevents mitochondria from releasing cytochrome c, which is necessary for starting the caspase apoptosis cascade. Therefore, suppressing antiapoptosis-related proteins such as Bcl-2 and Bcl-xL may allow apoptosis and increase the therapeutic efficacy of anticancer drugs (Pakunlu et al., 2004; Yamanaka et al., 2006). There is a close relationship between pump and nonpump resistance. Recent studies show that P-gp is involved in increasing tumor cell survival and delaying the apoptosis cascade (Pallis and Russell, 2000). However, Barcia et al. (2003) have found that P-gp function shows no correlation with apoptotic capability. Another study by Park et al. (2006) reveals that P-gp overexpression enhances TRAIL-triggered apoptosis in MDR cells. These contradictory results suggest that the correlation between P-gp expression and apoptotic capability needs further clarification (Smyth et al., 1998; Pallis and Russell, 2000; Igney and Krammer, 2002; Ruefli et al., 2002; Barcia et al., 2003; Park et al., 2006). The literature and our preliminary investigation suggest a direct link between modulating P-gp, MRP1, and MRP2 and regulating apoptosis through Bcl-2, Bcl-xL, Bax, caspases, p53, and cytochrome c (Smyth et al., 1998; Pallis and Russell, 2000; Igney and Krammer, 2002; Ruefli et al., 2002; Karwatsky et al., 2003). These facts led us to hypothesize that a novel combination of an anticancer drug, epirubicin, and a bile salt, sodium glycocholic acid (GC), targeted against pump resistance-related (e.g., MDR1-, MRP1-, MRP2-) and non-pump resistance-related (e.g., Bcl-2, Bax, caspases) pathways will achieve two major goals: (1) reverse efflux pumps in order to increase the anticancer drug concentration in cancer cells, decrease the need for high anticancer drug doses, and reduce adverse side effects to healthy tissue and organs and (2) modulate intracellular pathways of apoptosis in order to suppress the cellular antiapoptotic defense. Bile salts include the naturally occurring components of bile as well as their synthetic derivatives. Various bile salts are therapeutically important penetration enhancers. Bile salts combine with phospholipids to solubilize cholesterol and fatty acids as mixed micelles in the GI tract (Tolman, 2000). Bile salts are useful in dissolving gallstones and treating primary biliary

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cirrhosis and fatty liver (Matsumoto et al., 2004). After synthesis by the liver and excretion into the digestive tract, the primary bile acids, such as cholic acid (CA) and chenodeoxycholic acid (CDCA), are metabolized by intestinal bacteria (e.g., removal of 7-alpha hydroxyl group) to produce secondary bile acids such as deoxycholic acid (DCA). Bile acids are conjugated to glycine or taurine, such as the formation of glycocholic acid (GC), to decrease their hydrophobicity and cytotoxicity (Hofmann, 1984; Powell et al., 2001). Bile acids then enter the enterohepatic circulation as endogenous bile salts (Yamamoto et al., 1992; Tolman, 2000). Interestingly, hydrophobicity of bile acid is correlated with apoptosis induction and/or growth arrest (Powell et al., 2001). Highly hydrophobic bile acids, such as CDCA and glycochenodeoxycholic acid (GCDCA), are able to trigger apoptosis rapidly (Powell et al., 2001) and cause hepacellular injury through the mechanisms including stimulation of lipid peroxidation and induction of mitochondrial dysfunction (Utanohara et al., 2005). Hydrophilic bile acids, such as GC, are less toxic in comparison with hydrophobic bile acids (Hofmann, 1984; Marin et al., 1998; Powell et al., 2001; Utanohara et al., 2005). GC is a main component of human bile and is used as a biological absorption enhancer. In addition, owing to the amphipathic nature of GC, GC has also been used to modify the solubility properties of drugs (Marin et al., 1998). Because our aim was to develop GC as an adjuvant to intensify the potency of epirubicin, we chose GC with low toxicity instead of hydrophobic bile acids for the combination study with epirubicin. Furthermore, in a previous study (Lo and Huang, 2000), we verified the increased absorption and MDR-modulating effects of sodium deoxycholate, another hydrophilic bile salt, on epirubicin in human colon adenocarcinoma Caco-2 cells. In the present study, we aimed to develop a formulation with epirubicin and GC to simultaneously modulate pump and non-pump resistance, and increase the chemosensitivity of epirubicin in Caco-2 cells and rat intestines.

2.

Materials and methods

2.1.

Materials

Epirubicin (Pharmorubicin) was purchased from Pfizer Inc. (New York, NY, USA). Sodium glycocholic acid (GC) was purchased from Sigma–Aldrich Co. (St. Louis, MO, USA). All cell culture medium and reagents were purchased from Gibco BRL (Grand Island, NY, USA). Most of the other chemical reagents were purchased from either Merck (Darmstadt, Germany) or Sigma–Aldrich.

2.2.

Cell culture

Caco-2 cells were obtained from the Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 0.1 mM of nonessential amino acids, and 10,000 units/ml of penicillin/streptomycin (Gibco BRL) at 37 ◦ C in a humidified atmosphere of 5% CO2 and 95% air.

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2.3.

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agarose gel, and amplified DNA was visualized using a DNA gel stain (SYBR Safe; Molecular Probes, Inc., Eugene, OR, USA) staining (Chearwae et al., 2004). The gels were digitally photographed and scanned using a gel documentation system (UVIdoc; UVItec Limited, Cambridge, UK) and commercial software (TotalLab; Nonlinear Dynamics Ltd., Newcastle upon Tyne, UK). Gene expression of MDR1, MRP1, MRP2, Bcl-2, Bax, caspase-3, caspase-8, caspase-9, and p53 was calculated as the ratio of mean band density of analyzed RT-PCR product to that of the internal standard (GAPDH). The expression values of different mRNAs treated with epirubicin or GC, or both, were compared to the expression values of the control (treated with medium only). The expression values of different mRNAs treated with epirubicin combined with GC were also compared with the expression values of different mRNAs treated with GC or epirubicin alone (Chearwae et al., 2004; Pakunlu et al., 2004). Means ± S.D. from four independent measurements are shown.

Cell growth inhibition assay

Cell viability was determined using an MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Caco-2 cells were trypsinized, disaggregated through a pipette, and counted with a hemacytometer. The cells were seeded in 96-well plates (104 cells/well), allowed to attach overnight, and then treated with different concentrations of epirubicin with or without GC. After 0, 24, 48, or 72-h incubation, 20 ␮l of MTT reagent at the concentration of 5 mg/ml was added to each well and the cells were incubated for an additional 4 h. The supernatant from each well was then carefully removed, and 100 ␮l of dimethylsulfoxide (DMSO) was added to each well and thoroughly mixed. The plate was read for optical density at 545 nm, using an MRX microplate reader (Dynatech Laboratories Inc., Chantilly, VA, USA). The absorbance value (OD545 ) was transformed to the cell number in each well of the 96-well plates. Cell viability (%) was calculated by dividing the number of cells incubated with epirubicin and with GC by the number of cells incubated with tissue culture medium only (control). The cell-growth inhibition potency of epirubicin and the combination of epirubicin and GC are expressed as IC50 values, defined as the concentration of a drug necessary to inhibit the growth of cells by 50%. Data are the means ± S.D. of four experiments (Pakunlu et al., 2006).

2.5.

Plasmid construction

The 159-bp human MDR1 (hMDR1) promoter fragment (residues −120 to +39) was amplified using PCR with primers containing the 5 -primer (5 -CGCAGTCTCTCGAGCAATCAGCATTAGTCAGTGC) and the 3 -primer (5 -GTCAAGCTTGAGCTTGTAAGAGCCGCTACTAGA). The gel-purified PCR product was digested using XhoI and HindIII and then cloned into the pGL3-basic firefly luciferase reporter vectors (Promega, Madison, WI, USA) using T4 DNA ligase (Promega) (Lania et al., 1997; Takane et al., 2004). The cloning of the hMDR1 promoter fragment into the pGL3-promoter vector was confirmed using restriction enzyme digestion and direct sequencing, which showed a similarity of 99.9%. All the resulting plasmids were amplified in E. coli and then isolated using a magnetic separation unit (MagneSil Magnetic Separation Unit; Promega).

2.4. Semiquantitative RT-PCR of P-gp, MRP1, MRP2, Bcl-2, Bax, caspases, and p53 Caco-2 cells were pretreated for 72 h with 250 ␮M of GC with or without 10 ␮g/ml of epirubicin. RNA was isolated from the cells using a kit (Total RNA Miniprep System; Viogene, Taipei, Taiwan) according to the manufacturer’s instructions. RNA yield and purity were assessed using spectrophotometric analysis. Total RNA (1 ␮g) from each sample was subjected to RT-PCR with templates, dNTPs, AMV reverse transcriptase, Tfl DNA polymerase, and the corresponding 5 - and 3 -primers of MDR1, MRP1, MRP2, Bcl-2, Bax, caspase-3, caspase-8, caspase9, and p53 (Table 1) in 50 ␮l of the total reaction volume. The PCR cycles for these proteins are given in Table 2. Expression levels of MDR1, MRP1, MRP2, Bcl-2, Bax, caspase-3, caspase8, caspase-9, and p53 mRNA were evaluated by measuring RT-PCR products after amplification. An aliquot of each reaction mixture was analyzed using electrophoresis on a 2%

2.6.

Transfection and dual luciferase activity assay

Cells were plated at a density of 2 × 105 per well in six-well plates and allowed to attach overnight. We mixed 2 ␮g/well of the hMDR1 promoter-pGL3 firefly luciferase reporter gene constructs and 0.2 ␮g/well of the pRL-TK Renilla luciferase reporter gene (Promega) with 6 ␮l of lipofectin reagent (Invitrogen Corp., Carlsbad, CA, USA), and then incubated the cells at

Table 1 – Gene-specific PCR primers of ABC transporter proteins and apoptosis-related proteins in Caco-2 cells Proteins MDR1 (P-gp) MRP1 MRP2 (cMOAT) Bcl-2 Bax Caspase-3 Caspase-8 Caspase-9 p53

Forward primers 

Reverse primers 

5 -CTC ATC GTT TGT CTA CAG TTC-3 5 -CAT GAA GGC CAT CGG ACT CT-3 5 -GAC TAT GGG CTG ATA TCC AGT GT-3’ 5 -TGCACCTGAGCCCCTTCAC-3 5 -ACCAAGAAGCTGAGCGAGTGTC-3 5 -GAATACCCTGGACAACA-3 5 -GGATGCCTTGATGCTATTCC-3 5 -GCCATGGACGAAGCGGATCGGC-3 5 - GAAGACCCAGGTCCAGATGA-3

5’-GCTTTCTGTCTTGGGCTTGAGATCCACG-3 5’-CAG GTC CAC GTG CAG ACA-3 5’-AGG CAC TCC AGA AAT GTG CT-3’ 5 -CTGTTTGATTTCTCCTGGCT-3 5 -ACAAAGATGGTCACGGTCTG-3 5 -ACGCCATGTCATCATCAA-3 5 -TCCTTCAATTCTACTTTGTTCACATC-3 5 -GGCCTGGATGAAGAAGAGCTTGGG-3 5 - GGTAGGTTTTCTGGGAAGGG-3

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Table 2 – RT-PCR conditions Temperature (◦ C)

Time (min)

Temperature (◦ C)

Time (min)

Temperature (◦ C)

Time (min)

Temperature (◦ C)

Time (min)

Cycles

GAPDH

48

40 Second round

94 94

1 1

55 50

1 1

72 72

1 1

20 20

MDR1

48

40 Second round

95 95

1 1

52 55

72 1

72 72

1 1

20 20

MRP1

48

40 Second round

95 95

1 1

56 60

72 1

72 72

1 1

20 20

MRP2

48

40 Second round

95 95

1 1

57 60

72 1

72 72

1 1

20 20

Bcl-2

48

40 Second round

95 95

1 1

55 59

72 1

72 72

1 1

20 20

Bax

48

40 Second round

95 95

1 1

57 52

72 1

72 72

1 1

20 20

Caspase-3

48

40 Second round

95 95

1 1

45 46

72 1

72 72

1 1

20 20

Caspase-8

48

40 Second round

95 95

1 1

52 53

72 1

72 72

1 1

20 20

Caspase-9

48

40 Second round

95 95

1 1

66 61

72 1

72 72

1 1

20 20

p53

48

40 Second round

95 95

1 1

54 54

72 1

72 72

1 1

20 20

25 ◦ C for 15 min and then at 37 ◦ C for 15 h. Next, the cells were treated with 25 ␮M of rifampicin (a positive control), 10 ␮g/mL of epirubicin, 250 ␮M of GC, or a combination of epirubicin and GC for 72 h. After the incubation, luciferase reporter gene activity was evaluated with a dual luciferase reporter assay system (Promega). Briefly, the cells were washed twice with cold PBS and then lysed in 300 ␮l of reporter lysis buffer (Promega). After they had been incubated at 37 ◦ C for 15 min, the lysates were mixed in a vortex blender for 15 s and centrifuged at 4 ◦ C for 30 s. The luciferase reaction was then initiated by auto-injecting 100 ␮l of a reagent with luciferin (Luciferase Assay Reagent II; Promega) to 20 ␮l of lysate supernatants; the resulting luminescence was measured using a luminometer (Model LB9506; Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany) (Chen et al., 2004). After we quantified the firefly luminescence, we quenched the reaction and then initiated a Renilla luciferase reaction by simultaneously adding 100 ␮l of reagent (Stop & Glo; Promega) to the same tube. After completing a background correction (for activity in untreated control cells), we calculated the results as the level of hMDR1 promoter-pGL3 activity divided by pRL-TK activity. The total cellular protein concentration was determined using a protein assay (Bio-Rad, Hercules, CA, USA). Data are the means ± S.D. of four independent experiments.

2.7. Measuring the intracellular accumulation of epirubicin in Caco-2 cells using flow cytometry We measured intracellular epirubicin fluorescence as previously described (Lo and Huang, 2000; Lo, 2003). Cells (104

per well) were seeded into 24-well plates to permit confluence. The cells were then rinsed twice with PBS, pretreated with 250 ␮M of GC, and incubated at 37 ◦ C for 72 h. The experiment was then done by adding 10 ␮g/ml of epirubicin to the culture medium. After they had been incubated at 37 ◦ C for 3 h, the cells were washed twice with icecold PBS, trypsinized, centrifuged, and then resuspended in PBS. Flow cytometric analysis was then done using a flow cytometer (FACSort; Becton Dickinson, Mountain View, CA, USA) equipped with an argon ion laser (Spectra Physics) and operated at 488 nm and 15 mW. Red epirubicin fluorescence was measured through a 585/42 nm band pass filter. Data acquisition and analysis were done using commercial software (Lysis II; Becton Dickinson). Forward- and side-scatter signals were collected using linear scales, and fluorescence signals were collected on a logarithmic scale. At least 10,000 cells were analyzed in each sample. Within each experiment, determinations were done in quadruplicate.

2.8. Chromatin condensation detection using a fluorescence microscope After we had treated the cells with medium alone, GC, epirubicin, or GC plus epirubicin, we used centrifugation (200 g for 1 min) to collect 1 × 106 cells. The pellets were resuspended in 100 ␮l of PBS and 10 ␮l of 100 mg/ml acridine orange, and then visualized under an inverted microscope (Eclipse TS-100; Nikon Co., Tokyo, Japan) equipped with a fluorescence image capture device (C-SHG; Nikon) controlled with an Image-Pro Plus software (Media Cybernetics, Inc., Bethesda, MD, USA). The characteristics of fragmented nuclei and condensed chro-

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matin were observed and compared with those of the control (Kosmider et al., 2004).

2.9. Sub-G1 accumulation of DNA content detection using a flow cytometer After we had treated cells with medium alone, 10 ␮g/ml of epirubicin, 250 ␮M of GC, or a combination of epirubicin and GC for 24 h, we used centrifugation (1200 rpm for 5 min) to collect 105 cells. The pellets were resuspended in 200 ␮l of PBS, fixed with 800 ␮l of ice-cold absolute ethanol, and maintained at −20 ◦ C for 30 min. We used centrifugation (1200 rpm for 5 min) to collect cells, washed them twice with ice-cold PBS, resuspended them in 50 ␮g/ml of propidium iodide in PBS, and then incubated them for 40 min at 25 ◦ C in the dark. We analyzed the samples using a flow cytometer (FACSort; Becton Dickinson). Data acquisition and analysis were done using commercial software (CELLQuest; Becton Dickinson). Fluorescence signals were collected on a logarithmic scale. Each experiment was repeated between four and six times (Barcia et al., 2003; Wang et al., 2006; Yamanaka et al., 2006).

2.10.

2.12.

Statistical analysis

Results are given as means ± S.D. Statistical analysis was done using one-way analysis of variance (ANOVA) and Dunnett’s

Everted sacs of rat jejunum and ileum

Male Sprague–Dawley rats bred and housed in the animal center were used. The animal-use protocols were in accord with nationally approved guidelines. Everted sacs of rat jejunum and ileum were prepared using a method previously described (Lo and Huang, 2000; Lo, 2003). Rats weighing about 300 g were deprived of food for 1 day and given only double-distilled water until they were killed with carbon dioxide. The jejunum and distal ileum of the rat intestines (approximately 25 cm each) as well as the underlying mesenterium were removed. The segments were washed with iced saline before they were mounted in Tyrode’s solution. The sacs were everted, filled with 3 ml of Tyrode’s solution, and ligated at both ends. One of the two ends was ligated with a needle for the following sampling. The sacs were then incubated for 60 min with 50 ml of Tyrode’s solution with or without 250 ␮M of GC or verapamil. The solution was bubbled with air and maintained at 37 ◦ C throughout the experiment. At 0 min, 100 ␮g/ml of epirubicin was added in the mucosal side. In each study, 200 ␮l of the solution inside the sacs was taken every 10 min for 60 min, and was replaced with fresh Tyrode’s solution to keep the volume of the serosal solution constant. Each experiment was done in triplicate. The concentration of epirubicin in each sample was determined using high-performance liquid chromatography (HPLC) as described below.

2.11.

plus 0.5% acetic acid and 2.5 mM of sodium heptanesulfonic acid, run at a flow rate of 1.2 ml/min. The detection wavelength was 254 nm. The ratio of epirubicin to daunorubicin by peak height was calculated and compared with the calibration curve for quantitation. For consistency, we used Tyrode’s solution to prepare the standard curve for quantitation in HPLC.

Epirubicin concentration analyzed using HPLC

The analytical method for epirubicin was modified from previous reports (Lo and Huang, 2000; Lo, 2003). Daunorubicin was used as the internal standard. The HPLC system consisted of a pump (L7100; Hitachi, Tokyo, Japan) equipped with an automated injector (L2200), a 5-␮m LiChrospher column (25-cm long, 4-mm inside diameter; Merck & Co, Inc., Whitehouse Station, NJ) and a UV detector (L2400; Hitachi, Tokyo, Japan). The mobile phase included methanol and water (75:25, v/v)

Fig. 1 – (A) The effect of glycocholic acid (GC) at the concentrations of 0, 100, 250, 300, 400, and 500 ␮M after 0, 24, 48, and 72-h of incubation on the cell viability of Caco-2 cells. Data are means ± S.D. of four independent experiments. * P < 0.05 compared to the control (GC at 0 ␮M); † P < 0.05 compared to GC at 100 ␮M; ‡ P < 0.05 compared to GC at 250 ␮M; ¶ P < 0.05 compared to GC at 300 ␮M. (B) The effect of GC on the cytotoxicity of epirubicin (Epi) in Caco-2 cells: : Epi alone; 䊉: Epi plus GC (250 ␮M). IC50 values were calculated using non-linear analysis by the least-square method as described in Section 2. The mean IC50 values for the combination of Epi plus GC was significantly lower than that for Epi alone (8.08 ± 0.21 ␮g/ml versus 40.31 ± 1.11 ␮g/ml, P < 0.05, one-way ANOVA, n = 4 independent experiments).

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multiple comparison tests. Statistical significance was set at P < 0.05.

3.

Results

3.1. Combined GC and epirubicin treatment significantly increased the cytotoxicity of epirubicin We used the MTT dye reduction method to assay cell-growth inhibition. Fig. 1A shows the results of the viability assay after treatment of Caco-2 cells with 0, 100, 250, 300, 400, and 500 ␮M of GC after 0, 24, 48, and 72-h of incubation. The effect of GC on cell viability % as measured by MTT assay was time dependent with the most profound effects seen after 72 h. After incubation with 250 ␮M of GC, the viability was 80 ± 3% after 72-h incubation, whereas it was 96 ± 4% and 90 ± 4% after 24-h and 48-h incubation, respectively. We fixed the period of incubation at 72 h, which was consistent with the studies of Kosmider et al. (2004) and Budman et al. (2007). They found that the most profound effect of growth inhibition and apoptosis induction

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was after 72-h of incubation. Because our aim was to develop GC as an adjuvant to intensify the potency of epirubicin, we chose 250 ␮M of GC (
Fig. 2 – The effect of treatment with medium (control; C), glycocholic acid (GC), epirubicin (Epi) (10 ␮g/ml), and Epi plus GC for 72 h on the expression of (A) the MDR pump-related genes encoding P-glycoprotein (MDR1), MRP1, MRP2; (B) apoptosis-related genes encoding Bax, Bcl-2, caspase-3 (Casp3), caspase-8 (Casp8), caspase-9 (Casp9), and p53 in Caco-2 cells. Gene expression was calculated as a ratio of the band intensity of the studied gene to that in the internal standard (GAPDH). (C) The Bax:Bcl-2 expression ratios in Caco-2 cells treated with GC, Epi, and Epi plus GC are shown. Means ± S.D. from four independent measurements are shown. * P < 0.05 compared to the control; † P < 0.05 compared to GC; ‡ P < 0.05 compared to Epi.

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Fig. 2 – (Continued ).

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(8.08 ± 0.21 ␮g/ml versus 40.31 ± 1.11 ␮g/ml; P < 0.05; one-way ANOVA; values are the means of four independent experiments).

3.2. Semiquantitative RT-PCR of P-gp, MRP1, MRP2, Bcl-2, Bax, and caspases To analyze the effect of GC and epirubicin on pump and non-pump resistance-related proteins, the mRNA expression levels of MDR1, MRP1, MRP2, Bax, Bcl-2, caspase-3, caspase8, caspase-9, and p53 were evaluated using RT-PCR. GC and epirubicin, used together or separately for 72 h, significantly down regulated the corresponding mRNA levels of MDR1, MRP1, MRP2, and Bcl-2 (P < 0.05) (Figs. 2A and B). The same treatment for 72 h, however, significantly upregulated the corresponding mRNA levels of Bax, caspase-3, caspase-9, and p53 (P < 0.05). Combined GC and epirubicin treatment resulted in significantly (P < 0.05) greater down- and upregulation of MDR1, MRP1, MRP2, and Bax expression than did treatment with GC or epirubicin alone. GC and epirubicin, individually or combined, significantly increased the Bax-to-Bcl-2 ratio (Fig. 2C), but did not significantly affect caspase-8 expression in Caco-2 cells (Fig. 2B). GC combined with epirubicin did not increase the mRNA levels of p53, caspase-9, and caspase-3 more than epirubicin alone did (P > 0.05).

3.3. Combined GC and epirubicin treatment significantly decreased the luciferase activity of the hMDR1 promoter region To investigate the potential effect of GC and epirubicin on the transcriptional regulation of hMDR1, 159 bp (residues −120 to +39) of the hMDR1 promoter was cloned upstream of the firefly luciferase reporter gene in the pGL3-basic vector. These 159 bp of hMDR1 DNA elements consist of the proximal promoter region that encodes AP-1, CAAT, the GC box, and the Y-box necessary for efficient transcription of hMDR1. For normalization, a Renilla luciferase reporter gene of pRL-TK vector was co-transfected. The luminescent activity of the hMDR1 promoter-pGL3 and pRL-TK vector was subsequently measured using a dual luciferase assay system. The effects of GC, epirubicin, and GC combined with epirubicin on the activity of the hMDR1 regulatory promoter region of 159 bp elements in Caco-2 cells were then compared. GC and GC combined with epirubicin for 72 h significantly suppressed hMDR1 promoter activity (P < 0.05; Fig. 3). The positive control, rifampicin, significantly increased the luciferase activity (P < 0.001), which indicated that rifampicin had significantly induced hMDR1 promoter activity. GC combined with epirubicin inhibited hMDR1 promoter-related luciferase activity significantly more than did GC or epirubicin alone (P < 0.05). In a negative control experiment, the activity levels of the hMDR1 promoter-deficient pGL3-basic luciferase reporter vectors were not affected by GC, epirubicin, or GC combined with epirubicin, which suggested that there was no nonspecific direct interaction between the individual testing agents and the luciferase reporter vectors.

Fig. 3 – The effect of treatment with glycocholic acid (GC), epirubicin (Epi), and Epi plus GC for 72 h on hMDR1 promoter activity in Caco-2 cells. The MDR1 promoter fragment from residues −120 to +39 was cloned into the pGL3-basic luciferase reporter vectors. The reporter gene plasmid pRL-TK vector was also included in every transfection. Thus, the cells were treated respectively with the control, the positive control (rifampicin (Rif)), 10 ␮g/ml of Epi, 250 ␮M of GC, or Epi and GC for 72 h. The luciferase reaction was subsequently measured using a dual luciferase assay reagent with a luminometer. After background correction (activity in untreated control cells), results were expressed as the level of pGL3-promoter activity divided by pRL-TK activity. Data are means ± S.D. of four independent experiments. * P < 0.05 compared to the control; † P < 0.05 compared to Rif; ‡ P < 0.05 compared to Epi; ¶ P < 0.05 compared to GC.

3.4. GC significantly increased the intracellular accumulation of epirubicin in Caco-2 cells We used flow cytometry to determine whether combined GC and epirubicin treatment of Caco-2 cells increased the retention of epirubicin. In addition, the functional involvement of MDR-related proteins such as P-gp in the efflux of epirubicin was verified by the addition of verapamil. Verapamil, a calcium channel blocker, is a typical P-gp substrate and one of the most studied MDR modulators (Germann, 1996; Lo and Huang, 2000). Studies have suggested that verapamil competes with other substrates for binding to P-gp and thus reverses MDR (Gottesman and Pastan, 1993). Pretreatment with 25 ␮M of Ver (Ver25), 250 ␮M of Ver (Ver250), or 250 ␮M of GC (GC250) for 72 h significantly increased the intracellular accumulation of epirubicin at 180 min in Caco-2 cells (P < 0.05; Fig. 4). GC 250 exhibited significantly higher retention of epirubicin than Ver25 did (P < 0.05; Fig. 4). But GC 250 demonstrated significantly lower retention of epirubicin than Ver250 did (P < 0.05; Fig. 4). These results suggest that inhibiting P-gp and MRPs

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Fig. 4 – The effect of treatment with glycocholic acid (GC) and verapamil (Ver) for 72 h on the intracellular accumulation of fluorescent epirubicin (Epi) in Caco-2 cells. Cells were pretreated with 25 ␮M of Ver, 250 ␮M of Ver, or 250 ␮M of GC for 72 h, and incubated with 10 ␮g/mL of Epi for 180 min. (A) Three-dimensional view of cell number versus fluorescence intensity of epirubicin in Caco-2 cells. I: GC250 control; II: Epi control; III: Epi pretreated with Ver25; IV: Epi pretreated with Ver250; and V: Epi pretreated with GC250. (B) Mean fluorescence intensity of epirubicin control was normalized as 100%. Mean fluorescence intensity of Epi plus GC or Epi plus Ver was normalized relative to the Epi control. Data are means ± S.D. of four independent experiments. The mean fluorescence intensity of 250 ␮M of GC is also shown here to demonstrate that the auto-fluorescence of GC was negligible. * P < 0.05 compared to Epi; † P < 0.05 compared to Epi plus Ver (25 ␮M); ‡ P < 0.05 compared to Epi plus Ver (250 ␮M).

by using GC combined with epirubicin may account for the increase in the intracellular uptake of epirubicin in Caco-2 cells.

3.5. Combined GC and epirubicin treatment significantly induced chromatin condensation We used fluorescence microscopy and fluorescent DNAbinding dye AO staining to evaluate whether the cytotoxic effect of GC and epirubicin was related to their effect on apoptosis induction in Caco-2 cells. Viable cells had a uniform bright green nucleus (Fig. 5). Apoptotic cells displayed bright green areas of condensed or fragmented chromatin in the nucleus. No obvious morphological changes were seen in the

control group (Fig. 5A). However, Caco-2 cells exposed to GC, epirubicin, or GC combined with epirubicin for 72 h showed condensed chromatin in the nucleus and appearance of apoptotic bodies (Fig. 5B–D, white arrows). Based on Fig. 5B–D, it was found that more bright spots were observed in cells exposed to GC combined with Epi compared to Epi or GC alone (P < 0.05 in both cases), which is consistent with our other data.

3.6. Combined GC and epirubicin treatment significantly increased the sub-G1 accumulation of DNA content Caco-2 cells treated with GC alone, epirubicin alone, or both for 72 h, showed a pattern typical of DNA content that

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Fig. 5 – Nuclear chromatin condensation in Caco-2 cells treated with (A) medium, (B) 10 ␮g/mL of epirubicin (Epi), (C) 250 ␮M of glycocholic acid (GC), and (D) GC plus Epi for 72 h. Visualized using an inverted microscope (Eclipse TS-100) equipped with a fluorescence image capture device (C-SHG; Nikon) controlled with an Image-Pro Plus software.

reflected sub-G1, G0/G1, S, and G2/M phases of the cell cycle (Fig. 6). The percentages of the sub-G1 phase of Caco-2 cells, which correspond to the proportions of apoptotic cells, significantly increased after all three treatments. The percentage of the sub-G1 phase of cells treated with GC plus epirubicin (46.3%; P < 0.001) was significantly higher than the percentage treated with GC alone (26.8%) or epirubicin alone (20.6%).

3.7. The effect of GC on the absorption of epirubicin in everted gut sacs of rats Effect and mechanism(s) of modulators on intestinal absorption of drugs in MDR spectrum may differ depending on cell types, species, and physicochemical properties of substrates of efflux transporter proteins (Komarov et al., 1996; Lo, 2003). Whereas cell lines are efficient methods to study mechanistic interaction of anticancer drugs and MDR modulators at the cellular level, studies using other systems, such as ex vivo everted sac, in situ perfusion, and in vivo experiments, are necessary to assess the absorption/exsorption mechanism(s) of epirubicin in the presence and absence of GC or vaerapamil. Caco-2 cell monolayers have the advantage of human origin, but the system is static, gives very low rates of transport, and exaggerated enhancement of the paracellular route compared with small intestine (Barthe et al., 1999). Ex vivo

everted sac technique provides quantitative information on mechanisms of drug absorption from the mucosal side to the serosal side through testing the drug content in the intestinal sac, whiles P-gp and MRPs in the apical cell membrane may limit bioavailability by expelling drugs from the mucosal cells. This model maintains tissue viability, gives reliable data, and appears particularly useful for studying drug interactions with transporters, although it is a closed system (Barthe et al., 1999; van de Kerkhof et al., 2007). Permeability values for small hydrophilic molecules using the improved everted sac gives data close to those for humans, while values with Caco2 cells are orders of magnitude lower (Barthe et al., 1999). In situ rat intestinal perfusion is a reliable technique to investigate drug absorption potential in combination with intestinal metabolism. However, this system is time-consuming and therefore not suited for screening purposes (Bohets et al., 2001). It gives no information on events at the cellular level, and absorption may be reduced by anesthesia and surgical manipulation (Barthe et al., 1999). Finally, in vivo absorption in animals can provide valuable bioavailability data; however, it is even more time- and cost-consuming, so it is not practical for screening (Barthe et al., 1999; Bohets et al., 2001). Therefore, everted sac system is an appropriate ex vivo model to study epirubicin absorption. In addition, the regional difference of drug absorption in intestinal tissues has been reported (Makhey et al., 1998;

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and/or MRPs, thus causing a decrease in epirubicin efflux, an increase in epirubicin absorption, or both, for the combined treatment.

4.

Fig. 6 – Cell cycle analysis, using flow cytometry, of Caco-2 cells pretreated with glycocholic acid (GC) (250 ␮M), epirubicin (Epi) (10 ␮g/ml), and GC plus Epi for 72 h. M1 represents the ratio of the number of cells in the sub-G1 population to the number of total cells. Means ± S.D. from four independent measurements are shown. * P < 0.05 compared to the control; † P < 0.05 compared to GC; ‡ P < 0.05 compared to Epi.

Englund et al., 2006). Therefore, effect of GC or verapamil on epirubicin absorption at separate intestinal segments, including jejunum and ileum was investigated. Epirubicin was transported from the mucosal side to the serosal side at different segments of the rats’ small intestines for 60 min (Fig. 7A and B). The epirubicin concentrations, measured in sacs pretreated with 250 ␮M of GC or verapamil, were significantly higher than those in the control groups in both the jejunum and the ileum (P < 0.01, n = 3 rats per group). Ileal epirubicin absorption in combination with GC or verapamil was significantly higher (P < 0.05) than that in jejunum. The enhancing effect of verapamil was higher than that of GC (P < 0.05 compared to Epi plus GC), which was consistent with the intracellular accumulation study. This implies that GC or verapamil exhibited an inhibitory effect on P-gp

Discussion

The development of MDR in cancer cells may contribute to decreased drug accumulation (pump-related resistance) changes in drug targets, and suppression of apoptotic signaling pathways (non-pump-related resistance). Therefore, simultaneously inhibiting the pump and non-pump resistance of tumors may substantially improve the efficacy of traditional anticancer drugs (Pakunlu et al., 2006). In the present study, combining epirubicin, an anticancer drug, and GC, a bile salt used as an MDR modulator, amplified the cytotoxic effect of epirubicin on Caco-2 cells. Various mechanisms are involved in the circumvention of MDR in Caco-2 cells. First, combining epirubicin with GC inhibited the drug efflux transporter proteins (reversal of pump-related resistance). Second, this combination also suppressed the antiapoptotic cellular defense (modulation of non-pump-related resistance). Epirubicin combined with GC inhibited the drug efflux transporter proteins P-gp, MRP1, and MRP2. MDR reversing agents, such as cyclosporine A, reserpine, verapamil, and trifluoperazine, regulate P-gp gene expression (Sonneveld and Wiemer, 1997; Furuya et al., 1997; Shin et al., 2006). Narasaki et al. (1997) and Baba et al. (1995) also showed that the reversing effect of MDR modulators was proportional to the level of MDR1 and MRP genes, which suggested that these modulators directly interacted with and inhibited MDR1 and MRP genes. Furthermore, MDR1 gene transcription is regulated by multiple transcription factors (Lania et al., 1997; Takane et al., 2004; Daschner et al., 1999), but details about the mechanisms used to activate and suppress MDR1 have not been fully elucidated. The hMDR1 promoter region includes a distinct GC box, an inverted CCAAT box (Y box), a CAAT site, and an AP-1 site (Lania et al., 1997; Daschner et al., 1999; Takane et al., 2004). The GC box binds to transcription factors such as Sp1 and the EGR family, and modulates the hMDR1 promoter region (Lania et al., 1997). YB-1 and NF-Y binding sites are in the Y-box and necessary for UV radiation to activate the hMDR1 promoter (Takane et al., 2004). The binding of c-fos and c-jun with the AP-1 element increases the expression of the hMDR1 gene (Daschner et al., 1999). Thus, in the present study, we studied regulation of hMDR1 promoter elements of 159 bp and used rifampicin to induce this promoter region as a positive control. Rifampicin has been reported to increase P-gp expression and decrease uptake of rhodamine 123 and doxorubicin in Caco-2 and LLC-PK1 cells (Collett et al., 2004; Magnarin et al., 2004). We found that the combination of GC and epirubicin significantly inhibited hMDR1 promoter expression in Caco-2 cells, which indicated that this combination might provide a new molecular mechanism for MDR antagonism. This transcriptional suppression is consistent with the inhibitory effect of GC combined with epirubicin on the mRNA expression levels of MDR1, and the increase in the intracellular accumulation of epirubicin. However, we could not rule out the possibility that a change in mRNA stabilization and its subsequent translation was also involved in suppressing hMDR1. It is likely

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Fig. 7 – (A) A diagram of everted gut sacs. The rat intestines (approximately 25 cm each) were everted, filled with 3 ml of Tyrode’s solution, and ligated at both ends. The sacs were then incubated for 60 min with 50 ml of Tyrode’s solution with or without 250 ␮M of GC or verapamil. The solution was bubbled with air at 37 ◦ C. At 0 min, 100 ␮g/ml of epirubicin was added in the mucosal side. In each study, 200 ␮l of the solution inside the sacs was taken every 10 min for 60 min. (B) Time profiles of epirubicin (Epi) concentrations inside everted sacs of (I) jejunum and (II) ileum of rats, with or without 250 ␮M of glycocholic acid (GC) or 250 ␮M of verapamil for 60 min. 䊉: Epi; : Epi plus GC; : Epi plus verapamil. Data are means ± S.D. of triplicate experiments. Multiple comparisons were done using one-way ANOVA and Dunnett’s test. * P < 0.05 compared to Epi; † P < 0.05 compared to Epi plus GC.

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that multiple mechanisms work either cooperatively or exclusively to regulate the MDR1 gene (Scotto, 2003). Moreover, the regulatory effects of GC and epirubicin on MRP genes require additional study. Epirubicin combined with GC also suppressed the antiapoptotic cellular defense (modulation of non-pump-related resistance). It increased drug-induced apoptosis in Caco-2 cells compared to epirubicin alone, modified the expression of genes related to apoptosis in Caco-2 cells, and significantly downregulated the mRNA expression levels of Bcl-2, an antiapoptotic gene that prevents programmed cell death. Treating the cells with epirubicin alone had a marginal effect on Bcl2 expression levels, however. Minko et al. (2005) found that treating cells with doxorubicin alone increased levels of Bcl-2 in resistant MCF-7 cells. Bcl-2 overexpression in tumor cells results in MDR to a broad spectrum of anticancer drugs and is correlated with a poor response to chemotherapy in various human cancers (Buchholz et al., 2003; Minko et al., 2005). A related study (Karwatsky et al., 2003) reported that verapamil treatment preferentially induced apoptosis in MDR cells and that Bcl-2 gene overexpression inhibited this apoptosis (Karwatsky et al., 2003). In the present study, epirubicin alone or combined with GC increased the expression levels of Bax, a proapoptotic gene. Combined GC and epirubicin treatment resulted in significantly (P < 0.05) greater upregulation of Bax expression and Bax-to-Bcl-2 ratio than did treatment with GC or epirubicin alone. The overexpression of Bax proteins sensitizes cancer cells to several chemotherapeutic agents (Miao et al., 2003). An increase in Bax/Bcl-2 ratio of the proapoptotic activity gene (Bax) and the antiapoptotic cellular defense (Bcl-2) may represent the activation of a proapoptotic mitochondria-derived signal. We found that treating Caco-2 cells with epirubicin alone increased the proapoptotic signal (Bax expression) but had no significant effect on antiapoptotic activity (Bcl-2 expression). Therefore, epirubicin alone moderately induced apoptosis. Treating cells with GC alone or with epirubicin combined with GC significantly increased the Bax-to-Bcl-2 ratio, which suggested that inhibiting antiapoptotic members (e.g., Bcl-2) or inducing proapoptotic members (e.g., Bax) of the Bcl-2 family proteins is a feasible strategy for reversing MDR. When cells are exposed to apoptotic stimuli, there are two major apoptosis signaling pathways, the intrinsic (mitochondrial) and extrinsic (receptor; transmembrane), both of which meet on caspases, a family of cysteine proteases. Caspases are divided into two groups: initiators and effectors. When initiator caspases bind to adaptor molecules, they are activated, and then they activate effector caspases. The initiator caspase for the mitochondrial pathway is caspase-9 (and possibly caspase-2), whereas the initiator caspases for the receptor pathway are caspases 8 and 10. Both pathways share the effector caspases (caspase-3, -6, and -7) (Shabbits et al., 2003; Pakunlu et al., 2006). In the present study, GC and epirubicin, used together or separately, significantly elevated the expression levels of caspase-3, caspase-9, and p53 (P < 0.05). Normal p53 proteins activated in response to different cell injuries result in cell cycle arrest or apoptosis (Bargonetti and Manfredi, 2002; Harada et al., 2003). p53 activates the transcription of the proapoptotic proteins Bax, Puma, and Noxa, whereas

p53 represses antiapoptotic proteins such as Bcl-2 and Bcl-XL in many cell cycle-regulating and apoptosis-related processes (Bargonetti and Manfredi, 2002). A related study (Harada et al., 2003) reported that p53 expression is correlated with the response to chemotherapy. Thus, in the present study, incubating Caco-2 cells with GC alone or combined with epirubicin induced the central cell death signal by overexpressing p53, which subsequently activated a caspase-dependent pathway of apoptosis by overexpressing caspases-9 and caspases-3. This intrinsic apoptosis pathway is a mitochondria-involved signaling. In the presence of ATP, procaspase-9 is associated with cytochrome c and the adaptor molecule apoptotic protease-activating factor 1 (Apaf-1) to form cytochrome c/Apaf-1/caspase-9-containing apoptosome complex, and initiates the activation of caspase-9, which then triggers caspase-3 activation (Pommier et al., 2004). In contrast, incubating Caco-2 cells with GC alone, epirubicin alone, or GC combined with epirubicin did not change the expression levels of caspase-8. This suggested that GC and epirubicin induce apoptosis through the mitochondrial instead of the receptor pathway. However, there are contradictory results regarding the correlation between P-gp expression and apoptotic capability. In addition to efflux pump activity, P-gp and MRPs are also involved in augmenting tumor cell survival by preventing drug-resistant cells from anticancer drug-, Fas ligand-, TNF-␣-, and UV irradiation-mediated activation of caspase-dependent apoptosis (Ruefli et al., 2002). Accordingly, upregulating P-gp is related to the overexpression of the antiapoptotic Bcl family proteins (Campone et al., 2001) and inhibition of caspase-8 and caspase-3 (Ruefli et al., 2002). Nevertheless, Barcia et al. (2003) have found that cells with high and low P-gp function show no correlation with apoptotic capability. Another study by Park et al. (2006) reveals that P-gp overexpression enhances TRAIL-triggered apoptosis in neoplastic MDR cells. This TRAIL-induced, P-gp-potentiated apoptosis was associated with activation of caspase-6, -7, -8, and -9 (Park et al., 2006). Their study supports that P-gp expression enhances apoptosis. Conversely, our study found that epirubicin and/or GC inhibited P-gp, MRP1 and MRP2, but induced caspase-9 and caspase-3 in Caco-2 cells. Thus, our study supports that P-gp inhibits the mitochondrial pathway of apoptosis by suppressing caspase-9 and caspase-3, but P-gp does not affect the caspase-8-dependent receptor pathway. We suggest that inhibition of P-gp, MRP1 and MRP2 by epirubicin and/or GC is at least partially involved in the induction of caspase-9 and caspase-3. Our previous study (Lo and Huang, 2000) suggested that sodium deoxycholate, a bile salt, increased the transepithelial transport of epirubicin, an amphiphilic compound, via paracellular and transcellular routes. Sodium deoxycholate affects the transcellular route by creating membrane perturbation, which results from its interaction with membrane lipids and proteins. This bile salt also affects the paracellular route by opening tight junctions (Sakai et al., 1997). In addition, inhibiting P-gp function using multidrug-resistance reversing agents that act via substrate competition, ATP-depletion, or membrane perturbation may antagonize MDR (KvackajovaKisucka et al., 2001; Lo et al., 2003). Amphiphilic bile salts such as sodium deoxycholate usually have a membrane per-

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turbation property that changes the fluidity of Caco-2 cell membranes and rat colon epithelium, and thus modulates the efflux function of membrane-spanning proteins such as P-gp and MRPs (Sawada et al., 1991; Sakai et al., 1997). We could not rule out the possibility that GC has a similar membrane perturbing effect and thus the potential for increasing absorption and decreasing epirubicin efflux. We further hypothesize that the inhibitory effect of GC on pump-related resistance contributes, at least in part, to inhibiting P-gp and MRPs, as shown in our RT-PCR and MDR1 promoter activity studies. Furthermore, in the present study, GC combined with epirubicin induced caspases-3 and caspases-9 through the mitochondrial pathway. Pro-apoptotic stimuli induced by anticancer drugs may need a mitochondrion-dependent process involving permeabilization of the outer mitochondrial membrane and the release of mitochondrial proteins normally located in the intermembrane space (Petit et al., 1997; Minko et al., 2005). Based on the possible membrane perturbation characteristics of GC, we hypothesize that GC incorporates into mitochondrial membranes, changes the fluidity of mitochondrial membranes, and modulates the essential processes, such as the release of cytochrome c and proapoptotic proteins, and thus induces the mitochondrial pathway in apoptosis. However, the detailed mechanism requires further investigation.

5.

Conclusion

Given the inhibiting effects of GC combined with epirubicin on antiapoptotic- and MDR-related proteins, we hypothesize that there is a connection between pump and non-pump resistance. Simultaneously inhibiting pump and non-pump resistance with GC combined with epirubicin may be a novel strategy for reversing MDR. A combination of anticancer drugs with GC can control MDR via a mechanism that involves modulating P-gp and MRPs as well as regulating apoptosis-related pathways.

Acknowledgments This work was supported by grants NSC 95-2320-B-024-001 and NSC 96-2320-B-024-002 from the National Science Council, Taiwan.

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