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Reversal of multidrug resistance in cancer cells by Rhizoma Alismatis extract
ARTICLE IN PRESS
Phytomedicine 14 (2007) 160–165 www.elsevier.de/phymed
Reversal of multidrug resistance in cancer cells by Rhizoma Alismatis extract W.-F. Fong, C. Wang, G.-Y. Zhu, C.-H. Leung, M.-S. Yang, H.-Y. Cheung Bioactive Products Research Group, Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong SAR, China
Abstract Prolonged chemotherapy may lead to the selective proliferation of multidrug resistant (MDR) cancer cells. In MDR HepG2-DR and K562-DR cells that over-expressed P-glycoprotein (Pgp), the extract of the rhizomes of Alisma orientalis (Sam) Juzep. showed a synergistic growth inhibitory effect with cancer drugs that are Pgp substrates including actinomycin D, puromycin, paclitaxel, vinblastine and doxorubicin. At the same toxicity levels the herbal extract was more effective than verapamil, a standard Pgp inhibitor, in enhancing cellular doxorubicin accumulation and preventing the efflux of rhodamin-123 from the MDR cells. The extract restored the effect of vinblastine on the induction of G2/M arrest in MDR cells. Our data suggest that A. orientalis may contain components that are effective inhibitors of Pgp. r 2006 Elsevier GmbH. All rights reserved. Keywords: Multidrug resistance; P-glycoprotein; HepG2-DR cells; K562-DR cells; Rhizoma Alismatis
Introduction Prolonged cancer chemotherapy could lead to the selective survival of multidrug resistant (MDR) cells that exhibit simultaneous resistance to a wide spectrum of structurally and functionally unrelated chemotherapeutic agents. Molecular changes contributing to the development of MDR include the up-regulation or activation of transporter proteins, detoxification systems and target repair mechanisms, and the disregulation of cell death pathways (Coley, 2003). One of the better understood mechanisms leading to MDR is the over-expression of membrane transport proteins including the 170 kD P-glycoprotein (Pgp). Pgp is an ATP-dependent membrane transporter that Corresponding author. Tel.: +852 2788 9181; fax: +852 2788 7406.
E-mail address: [email protected] (W.-F. Fong). 0944-7113/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2006.03.004
extrudes a variety of hydrophobic anti-tumor drugs thus Pgp inhibitors may re-sensitize MDR cells. Examples are verapamil, reserpine, cephalosporin, gramicidin, and cyclosporin A (Castaing et al., 2000) but unfortunately their clinical usefulness has so far been limited because of severe side-effects (Coley, 2003). Plant materials have a long history of being used in treating cancer. In some countries traditional herbal medicines are often used together with Western chemotherapeutic agents. We have initiated an extensive screening program to identify medicinal herbs that may restore the sensitivity of MDR cancer cells to anticancer drugs. We report here that Rhizoma Alismatis (RA) extract may reverse MDR probably by inhibiting Pgp. RA is the underground part of a hardy aquatic plant, Alisma plantago orientale (Sam) Juzep. found commonly in southern China. For centuries it has been used for the treatment of urinary tract diseases, nephrolithiasis and
ARTICLE IN PRESS W.-F. Fong et al. / Phytomedicine 14 (2007) 160–165
atherosclerosis. Recent studies also showed anti-inflammatory, cardiovascular regulatory (Zhou and Shi, 1997), and calcium antagonistic (Gao, 1990) actions.
161
HepG2-DR and K562-DR selected for doxorubicin resistance, were provided by Dr. Judy Chan, Chinese University of Hong Kong (Chan et al., 2000). They were cultured at 37 1C, 5% CO2 in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 100 units/ml Antibiotic-antimycotic. To maintain the MDR phenotype, 1.2 and 0.1 mM doxorubicin was added to HepG2-DR and K562-DR cell cultures, respectively.
Materials and methods RA extract preparation One single batch of RA from Jiangxi province, China, was purchased through Guangdong Yue Xing Medicine Company, Hong Kong. The herbarium specimen (Accession No. MG002A) was deposited at the City University of Hong Kong. The herb was powdered and extracted with 95% ethanol (1 kg/l) at room temperature for 48 h trice. Combined extracts were dried and the residue was extracted by chloroform/water (1:1). The chloroform fraction was dried and the residue (E30 g) dissolved in dimethyl sulfoxide to a concentration of 100 mg/ml.
SRB growth inhibition assay Cell number was estimated by SRB protein staining (Skehan et al., 1990) in a 96-well format. Cells were seeded at 5 103 cells per 100 ml of medium per well and incubated overnight. Fresh medium with drugs was added and plates were incubated for a further 72 h. Cells were fixed for 1 h in ice-cold 50% trichloroacetic acid, stained with 0.4% SRB for 10 min, rinsed with 1% acetic acid and air-dried. Bound SRB was dissolved in 100 ml per well of 10 mM Tris base (pH 10.5) and the optical density at 515 nm, which is proportional to cell number, was determined. Each experiment was repeated for at least three times. Drug toxicity was expressed in terms of doses inhibiting 50% growth (ID50).
Chemicals Cancer drugs, verapamil and sulforhodamin B (SRB) were purchased from Sigma Chemical (St. Louise, US); Actinomycin D from Acros Organics (New Jersey, US); and propidium iodide (PI) and rhodamine-123 (Rh-123) from Molecular Probes (Oregon, US). Materials for cell culture were purchased from GibcoBRL (New York, US). Nitrocellulose membranes and secondary antibody (horseradish–peroxidase-conjugated anti-rabbit IgG) were purchased from Bio-Rad (California, US) and the rabbit anti-Pgp antibody was from Calbiochem (California, US).
Drug synergism analysis by combination indices (CI) Drug interactions were studied by the determination of combination indices (CI) (Chou and Talalay, 1983, 1984; Lam et al., 1999). Cells were seeded at 5 103 per 100 ml medium per well and cultured overnight. Fresh medium containing drug mixtures at constant ratios equal to ratios of their respective IC50 values was added. After 72 h cell growth was evaluated by SRB assay. The CI-isobolograms were constructed by a computer software and CI44 was taken as an indication of antagonism, o0.8 synergism, and 0.8oCIo4 additive
Cells and cultures Human hepatocarcinoma cell line HepG2, leukemia cell line K562 and their respective MDR sublines,
0.10
AU
0.08 0.06 0.04 0.02
Peak 2
Peak A
Peak 9 Peak 8 Peak 7 Peak 11 Peak 6 Peak 12 Peak 5 Peak 1 Peak 13 Peak 3 Peak 14 Peak 4 Peak 10 Peak 15
Peak 16
0.00 0.00
5.00
10.00
15.00
20.00 Minutes
25.00
30.00
35.00
40.00
Fig. 1. HPLC analysis of RA extract. RA extract was separated by HPLC on a RP18 column eluted by methanol/water (75:25, v/v) at 1.0 ml/min and monitored by a Waters Refraction Index Detector at 203 nm. Alisol B 23-acetate (peak A) was identified by a standard marker.
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effect (Chou and Talalay, 1983, 1984; Giulio and Frances, 1997). All experiments were repeated for at least three times.
Table 1. Effects of RA extract and verapamil on cytotoxicity of cancer drugs
Actinomycin D +RAE +Verapamil Puromycin +RAE +Verapamil Paclitaxel +RAE +Verapamil Vinblastine +RAE +Verapamil Doxorubicin +RAE +Verapamil
IC50-HepG2DR
IC50-HepG2
Resistance factora
2.1970.50 0.2170.03 0.5870.06 311792.6 1.1870.40 6.4571.63 4.3070.23 0.4370.15 0.7570.05 0.4670.03 0.1270.02 0.1670.01 41.672.30 1.4270.38 1.4070.04
0.00270.0004 1095 0.00270.0002 105 0.00270.0008 290 0.3570.01 888 0.19670.013 6 0.29070.061 22 0.00370.0003 1433 0.00270.001 215 0.00270.001 375 0.000470.0002 1150 0.000370.0001 400 0.000370.0002 533 0.05570.004 756 0.04970.019 29 0.04670.018 30
Rh-123 retention and doxorubicin accumulation To monitor Rh-123 retention, 1 106 cells in 5 ml of medium were incubated with 5 mg/ml Rh-123 at 37 1C for 1 h. Rh-123 loaded cells were washed with ice-cold phosphate buffered saline (PBS), resuspended in Rh123-free medium containing Pgp inhibitors for 1 h at 37 1C to allow Rh-123 efflux (Ludescher et al., 1992). For doxorubicin accumulation studies cells grown in 96well plates as above were incubated with 10 mM doxorubicin and Pgp inhibitors at 37 1C for 1 h (Krishan and Ganapathi, 1979). Cellular drug concentrations were estimated by fluorescence flow cytometry (FACSCAN, Becton Dickinson Immunocytometry Systems, San Jose, CA) and data were analyzed with the Macintosh CellQuest software.
Cells were incubated with drugs with RA extract (25 mg/ml) or Verapamil (10 mM) for 72 h. IC50 values (in mM) were expressed as means7standard deviations of three experiments. a Resistance factor is the IC50 ratio of HepG2-DR to GepG2 cells.
Cell cycle effects of vinblastine Approximately 1 106 cells in 5 ml of medium were treated with vinblastine (300 ng/ml) and Pgp inhibitors
Actinomycin D + RAE / Verapamil
Vinblastine + RAE / Verapamil 1.5
RAE Verapamil
1.5 1
1
0.5
0.5
0
RAE Verapamil
0 0
0.2 0.4 0.6 0.8 Fractional inhibition fa
1
0
Combination Index
Puromycin + RAE / Verapamil 1.5
1
1
0.5
0.5
0
0
0.2 0.4 0.6 0.8 Fractional inhibition fa
1
Doxorubicin + RAE / Verapamil
RAE Verapamil
1.5
0.2 0.4 0.6 0.8 Fractional inhibition fa
1
0
RAE Verapamil
0
0.2 0.4 0.6 0.8 Fractional inhibition fa
1
Paclitaxel + RAE / Verapamil RAE Verapamil
1.5 1 0.5 0
0
0.2 0.4 0.6 Fractional inhibition fa
0.8
1
Fig. 2. Synergistic effect of RA extract with anticancer drugs. Effects of RA extract (solid curve) and verapamil (positive control, dash curve) on the growth of HepG2-DR cells treated with actinomycin D, puromycin, paclitaxel, vinblastine or doxorubicin were evaluated by SRB assay and combination index (CI). Data were averages of three separate experiments. Solid horizontal line; CI ¼ 1.0; dash horizontal lines: CI ¼ 0.8.
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for 24 h. Cells were harvested, washed twice with icedcold PBS, fixed in 70% ethanol at 20 1C overnight, washed with PBS and incubated with 200 mg/ml RNase at 37 1C for 30 min. PI solution was added to a final concentration of 40 mg/ml. Analysis was performed immediately after PI staining by a FACSCAN flow cytometer.
Western blot analysis of Pgp expression Treated cells were incubated in ice-cold lysis buffer (50 mM Tris pH 7.4, 100 mM NaCl, 2 mM EDTA, 1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 2 mM PMSF, 1% aprotinin) for 30 min. Cellular proteins at 40 mg per lane were separated by 8% SDS/ PAGE and electro-transferred to nitrocellulose membranes. Membranes were blocked with 5% non-fat milk/ 0.1% Tween-20/TBS (10 mM Tris pH 7.5, 100 mM NaCl), incubated with anti-Pgp antibody for 1 h at room temperature, followed by horseradish–peroxidase-conjugated secondary antibody for another 1 h at room temperature. Protein bands were detected by the enhanced chemiluminescent (ECL) method.
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puromycin. Sensitivity to doxorubicin was raised by 30 folds and actinomycin D and paclitaxel 10-folds (Table 1). CI indicated clear synergistic effects (CIo0.8) between RA extract and individual anticancer drugs (Fig. 2). Synergism for actinomycin D or paclitaxel was observed over the entire killing rate (fa) range. In other cases synergism was observed at either high or low fa values. In drug-sensitive HepG2 cells RA extract and verapamil showed only additive effects with cancer drugs (Table 1 and data not shown).
RA extract increased doxorubicin accumulation in MDR cells Drug resistance in MDR cells has been attributed to decreased drug accumulation caused by the overexpression of Pgp (Tarasiuk et al., 2002; Chan et al., 2000). Cells were incubated with 10mM doxorubicin and Pgp inhibitors at 0.1–5 times of their respective IC50 at 37 1C for 1 h and cellular doxorubicin was studied by Celluar accumulation of DOX in HepG2-DR cell line 75
RAE Verapamil
Results HPLC profile of RA extracts
Cytotoxicity of the RA extract and anticancer drugs
* % Changes in cellular doxorubicin
RA extracts was separated by HPLC on a RP18 column (C-18, 5 mm, Alltech 02051566.1) eluted with methanol/water (75:25, v/v) at 1.0 ml/min and monitored by a refraction index detector (Water PDA 2695, 2996) at 203 nm. Fig. 1 shows a typical RA extract HPLC profile showing 17 identifiable peaks and one of the major components was identified by using standard marker as alisol B 23-acetate.
**
**
50 *
25
*
0 0.1xIC50 0.5xIC50
IC50
2.5xIC50
5xIC50
Celluar accumulation of DOX in K562-DR cell line 125
**
RAE Verapamil
100 75
HepG2 was highly sensitive to all the anticancer drugs tested including actinomycin D, puromycin, paclitaxel, vinblastine and doxorubicin. Resistance factors, calculated as the ratios of IC50 for MDR cells over that of IC50 for sensitive cells, showed that the Pgp-overexpressing HepG2-DR are 700–1400 times ‘‘more resistant’’ than the parental drug sensitive HepG2 cells. RA extract and verapamil were relatively non-toxic and showed similar levels of toxicity to both the drug sensitive and MDR cells.
RA extracts re-sensitized MDR cells to cancer drugs Comparing IC50 values, RA extract almost completely re-sensitized drug resistant HepG2-DR cells to
**
** 50
*
* *
25
*
0 0.1xIC50 0.5xIC50
IC50
2.5xIC50
5xIC50
Fig. 3. RA extract increased doxorubicin accumulation in MDR cells. HepG2-DR and K562-DR cells were incubated with 10 mM doxorubicin and various amount of RA extract or verapamil (in folds of IC50). Doxorubicin fluorescence was monitored by flow cytometry and shown as percent increase over untreated control (means7standard deviation of three independent experiments). Significantly different at *po0.05 or **po0.01 levels. Little change was observed in drug sensitive cells (data not shown).
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fluorescence flow cytometry. Drug resistant cells accumulated more doxorubicin in the presence of RA extract or verapamil (Fig. 3). Little change was observed in drug sensitive parental cells.
RA extract inhibited Rh-123 extrusion When drug-sensitive cells preloaded with Rh-123 were placed in RH-123-free medium, cellular fluorescence remained relatively unchanged (Fig. 4a,c). In MDR cells similarly preloaded with Rh-123, cellular fluorescence dropped dramatically in Rh123-free medium and the decrease could be inhibited by RA extract or verapamil (Fig. 4b,d).
RA extract restored vinblastine action in MDR cells
90 a. HepG2 80 70 60 50 40 30 20 10 0 101 100
RA extract did not alter Pgp protein expression First we confirmed that both MDR cell lines expressed higher levels of Pgp (Chan et al., 2000; Tarasiuk et al., 2002) (data not shown). Treatment with 50 mg/ml of RA extract for 12 h had no detectable effect on Pgp protein levels in these cells (data not shown).
Discussion Our conclusion that RA extract reverses MDR phenotype by inhibiting Pgp is supported by several lines of evidence: (1) In the first place RA extract affected only the MDR cell lines that differ from their parental cell lines primarily by expressing higher levels of Pgp. (2) The toxicity of RA extract was much the same on both the drug-sensitive and MDR cells. (3) RA 120 b. HepG2-DR 100
RAE
60
Loaded
Residual
40 20 102 FL1-H
103
0 100
104
100
101
102 FL1-H
103
104
120 c. K562
d. K562-DR 100
80
Verapamil
80
60
Counts
Counts
Verapamil
80 Counts
Counts
RA extract acted by mainly restoring actions of individual cancer drugs in MDR cells. For example vinblastine impedes mitotic spindle formation and induces G2/M arrest (Tashiro et al., 1998) and this was observed in drug-sensitive HepG2 but not in MDR cells. HepG2 cells normally showed a cell cycle distribution of approximately 65.0%:9.4%:21.1% (G0/G1:S:G2/M) (the percentage of subG1 ‘‘apoptotic’’ cells was not shown). Vinblastine treatment (300 ng/ml, 24 h) changed this to approximately 5.3%:4.6%:88.8% and RA extract (2.5
or 25 mg/ml) had no significant effect on the drug sensitive HepG2 cells with or without vinblastine. In drug resistant HepG2-DR cells similar vinblastine treatment only slightly increased the G2/M cells and changed the ratio to approximately 43.1%:9.7%:23.4%. The addition of RA extract to vinblastine treatment, however, drastically increased the G2/M fraction and the ratio became 14.3%:8.6%:71.4%.
40
Loaded
40
20 0 100
60
RAE Residual
20 101
102 FL1-H
103
104
0 100
101
102 FL1-H
103
104
Fig. 4. RA extract increased Rh-123 retention in MDR cells. Cells pre loaded with Rh-123 were incubated in Rh-123 free medium for 1 h. ‘‘Loaded’’ (thick solid line) and ‘‘residual’’ (light solid line) cellular fluorescence was measured by flow cytometry. Dash line: residual Rh-123 fluorescence in the presence of 25 mg/ml RA extract. Dotted line: residual Rh-123 fluorescence in the presence of 10 mM verapamil (positive control).
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extract showed a synergistic effect with all the tested anticancer drugs that are Pgp substrates in the Pgp overexpressing MDR cells. (4) RA extract restored vinblastine’s action on inducing G2/M arrest. (5) It increased cellular doxorubicin accumulation and slowed down Rh-123 efflux from Pgp over-expressing MDR cells. None of the above effects could be observed in the drug sensitive parental cell lines. (6) Lastly RA extract did not change the expression of Pgp nor did it affect intracellular ATP levels (data not shown) that is needed for Pgp activity. We noted that RA extract showed different levels of synergism when added with different drugs, from a 10fold increase of drug sensitivity to a complete reversal of resistance. This is expected since there exist different modes of binding of various structurally different ligands to Pgp and there are also multiple ligand binding sites on Pgp. There is strong evidence suggesting that one of the key factors influencing ligand-Pgp affinities could be ligand hydrophobicity (Yang et al., 1991). The three-dimensional atomic structure of Pgp is not yet known but enzymatic and functional studies suggest that there exist three substrate binding sites, an ATP hydrolysis site and an allosteric regulatory site (Pascaud et al., 1998). Finally, to unequivocally identify the mechanism of RA inhibition would first require the isolation and identification of pure active compounds from RA extract. These studies are being carried out in our laboratory.
Acknowledgement This work was supported in full by City University of Hong Kong Strategic Research Grant 7001110.
References Castaing, M., Brouant, P., Loiseau, A., Santelli-Rouvier, C., Santelli, M., Alibert-Franco, S., Mahamoud, A., Barbe, J., 2000. Membrane permeation by multidrug-resistance-modulators and non-modulators: effects of hydrophobicity and electric charge. J. Pharmaceut. Pharmacol. 52, 289–296. Chan, J.Y., Chu, A.C., Fung, K.P., 2000. Inhibition of Pglycoprotein expression and reversal of drug resistance of human hepatoma HepG2 cells by multidrug resistance gene (mdr1) antisense RNA. Life Sci 67, 2117–2124. Chou, T.C., Talalay, P., 1983. Analysis of combined drug effects: a new look at a very old problem. Trends Pharmacol. Sci. 4, 450–454. Chou, T.C., Talalay, P., 1984. Quantitative analysis of dose–effect relationships: the combined effects of multiple
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drugs or enzyme inhibitors. In: Weber, G. (Ed.), Advances in Enzyme Regulation, vol. 22. Pergamon Press, Oxford, pp. 27–55. Coley, T.H., 2003. Overcoming multidrug resistance in cancer: an update on the clinical strategy of inhibiting Pglycoprotein. Cancer Control 10, 159–165. Gao, X.J., 1990. Review on the explored calcium channel blockers in Chinese traditional medical herbs. Zhong Xi Yi Jie He Za Zhi (Chinese Journal of Integrated Traditional and Western Medicine) 10, 447. Giulio, D., Frances, J.S., 1997. Interaction of combinations of drugs, chemosensitizers, and peptides with the P-glycoprotein multidrug transporter. Biochem. Pharmacol. 53, 1789–1797. Krishan, A., Ganapathi, R., 1979. Laser flow cytometry and cancer chemotherapy: detection of intracellular antharcyclines by flow cytometry. J. Histochem. Cytochem. 27, 1655–1666. Lam, W., Chan, H., Yang, M., Cheng, S., Fong, W.F., 1999. Synergism of energy starvation and dextranconjugated doxorubicin in the killing of multidrugresistant KB carcinoma cells. Anticancer Drugs 10, 171–178. Ludescher, C., Thaler, J., Drach, J., Spitale, M., Gattringer, C., Huber, H., Hofmann, J., 1992. Detection of activity of P-glycoprotein in human tumor samples using rhodamine 123. Br. J. Haematol. 2, 161–168. Pascaud, C., Garrigos, M., Orlowski, S., 1998. Multidrug resistance transporter P-glycoprotein has distinct but interacting binding sites for cytotoxic drugs and reversing agents. Biochem. J. 333, 351–358. Skenhan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, J., Vistica, D., Warren, J.T., Bokesch, H., Kenney, S., Boyd, M.R., 1990. New colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl. Cancer Inst. 82, 1107–1112. Tarasiuk, J., Stefanska, B., Plodzich, I., Tkaczyk-Gobis, K., Seksek, O., Martelli, S., Garnier-Suillerot, A., Borowski, E., 2002. Anthrapyridones, a novel group of antitumor non-cross resistant anthraquinone analogues, synthesis and molecular basis of the cytotoxic activity towards K562/ DOX cells. Br. J. Pharmacol. 135, 1513–1523. Tashiro, E., Simizu, S., Takada, M., Umezawa, K., Imoto, M., 1998. Caspase-3 activation is not responsible for vinblastine-induced Bcl-2 phosphorylation and G2/M arrest in human small cell lung carcinoma Ms-1 cells. Jpn. J. Cancer Res. 89, 940–946. Yang, C.P., Greenberger, L.M., Horwitz, S.B., 1991. Reversal of multidrug resistance in tumor cells. In: Chou, T.C., Rideout, D.C. (Eds.), Synergism and Antagonism in Chemotherapy. Academic Press, San Diego, USA, pp. 311–338. Zhou, T.S., Shi, D.W., 1997. Review on the chemistry and pharmacology property of Rhizoma Alismatis. Zhong Cao Yao (Chinese Traditional and Herbal Drugs) 28, 125–127.
Phytomedicine 14 (2007) 160–165 www.elsevier.de/phymed
Reversal of multidrug resistance in cancer cells by Rhizoma Alismatis extract W.-F. Fong, C. Wang, G.-Y. Zhu, C.-H. Leung, M.-S. Yang, H.-Y. Cheung Bioactive Products Research Group, Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong SAR, China
Abstract Prolonged chemotherapy may lead to the selective proliferation of multidrug resistant (MDR) cancer cells. In MDR HepG2-DR and K562-DR cells that over-expressed P-glycoprotein (Pgp), the extract of the rhizomes of Alisma orientalis (Sam) Juzep. showed a synergistic growth inhibitory effect with cancer drugs that are Pgp substrates including actinomycin D, puromycin, paclitaxel, vinblastine and doxorubicin. At the same toxicity levels the herbal extract was more effective than verapamil, a standard Pgp inhibitor, in enhancing cellular doxorubicin accumulation and preventing the efflux of rhodamin-123 from the MDR cells. The extract restored the effect of vinblastine on the induction of G2/M arrest in MDR cells. Our data suggest that A. orientalis may contain components that are effective inhibitors of Pgp. r 2006 Elsevier GmbH. All rights reserved. Keywords: Multidrug resistance; P-glycoprotein; HepG2-DR cells; K562-DR cells; Rhizoma Alismatis
Introduction Prolonged cancer chemotherapy could lead to the selective survival of multidrug resistant (MDR) cells that exhibit simultaneous resistance to a wide spectrum of structurally and functionally unrelated chemotherapeutic agents. Molecular changes contributing to the development of MDR include the up-regulation or activation of transporter proteins, detoxification systems and target repair mechanisms, and the disregulation of cell death pathways (Coley, 2003). One of the better understood mechanisms leading to MDR is the over-expression of membrane transport proteins including the 170 kD P-glycoprotein (Pgp). Pgp is an ATP-dependent membrane transporter that Corresponding author. Tel.: +852 2788 9181; fax: +852 2788 7406.
E-mail address: [email protected] (W.-F. Fong). 0944-7113/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2006.03.004
extrudes a variety of hydrophobic anti-tumor drugs thus Pgp inhibitors may re-sensitize MDR cells. Examples are verapamil, reserpine, cephalosporin, gramicidin, and cyclosporin A (Castaing et al., 2000) but unfortunately their clinical usefulness has so far been limited because of severe side-effects (Coley, 2003). Plant materials have a long history of being used in treating cancer. In some countries traditional herbal medicines are often used together with Western chemotherapeutic agents. We have initiated an extensive screening program to identify medicinal herbs that may restore the sensitivity of MDR cancer cells to anticancer drugs. We report here that Rhizoma Alismatis (RA) extract may reverse MDR probably by inhibiting Pgp. RA is the underground part of a hardy aquatic plant, Alisma plantago orientale (Sam) Juzep. found commonly in southern China. For centuries it has been used for the treatment of urinary tract diseases, nephrolithiasis and
ARTICLE IN PRESS W.-F. Fong et al. / Phytomedicine 14 (2007) 160–165
atherosclerosis. Recent studies also showed anti-inflammatory, cardiovascular regulatory (Zhou and Shi, 1997), and calcium antagonistic (Gao, 1990) actions.
161
HepG2-DR and K562-DR selected for doxorubicin resistance, were provided by Dr. Judy Chan, Chinese University of Hong Kong (Chan et al., 2000). They were cultured at 37 1C, 5% CO2 in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 100 units/ml Antibiotic-antimycotic. To maintain the MDR phenotype, 1.2 and 0.1 mM doxorubicin was added to HepG2-DR and K562-DR cell cultures, respectively.
Materials and methods RA extract preparation One single batch of RA from Jiangxi province, China, was purchased through Guangdong Yue Xing Medicine Company, Hong Kong. The herbarium specimen (Accession No. MG002A) was deposited at the City University of Hong Kong. The herb was powdered and extracted with 95% ethanol (1 kg/l) at room temperature for 48 h trice. Combined extracts were dried and the residue was extracted by chloroform/water (1:1). The chloroform fraction was dried and the residue (E30 g) dissolved in dimethyl sulfoxide to a concentration of 100 mg/ml.
SRB growth inhibition assay Cell number was estimated by SRB protein staining (Skehan et al., 1990) in a 96-well format. Cells were seeded at 5 103 cells per 100 ml of medium per well and incubated overnight. Fresh medium with drugs was added and plates were incubated for a further 72 h. Cells were fixed for 1 h in ice-cold 50% trichloroacetic acid, stained with 0.4% SRB for 10 min, rinsed with 1% acetic acid and air-dried. Bound SRB was dissolved in 100 ml per well of 10 mM Tris base (pH 10.5) and the optical density at 515 nm, which is proportional to cell number, was determined. Each experiment was repeated for at least three times. Drug toxicity was expressed in terms of doses inhibiting 50% growth (ID50).
Chemicals Cancer drugs, verapamil and sulforhodamin B (SRB) were purchased from Sigma Chemical (St. Louise, US); Actinomycin D from Acros Organics (New Jersey, US); and propidium iodide (PI) and rhodamine-123 (Rh-123) from Molecular Probes (Oregon, US). Materials for cell culture were purchased from GibcoBRL (New York, US). Nitrocellulose membranes and secondary antibody (horseradish–peroxidase-conjugated anti-rabbit IgG) were purchased from Bio-Rad (California, US) and the rabbit anti-Pgp antibody was from Calbiochem (California, US).
Drug synergism analysis by combination indices (CI) Drug interactions were studied by the determination of combination indices (CI) (Chou and Talalay, 1983, 1984; Lam et al., 1999). Cells were seeded at 5 103 per 100 ml medium per well and cultured overnight. Fresh medium containing drug mixtures at constant ratios equal to ratios of their respective IC50 values was added. After 72 h cell growth was evaluated by SRB assay. The CI-isobolograms were constructed by a computer software and CI44 was taken as an indication of antagonism, o0.8 synergism, and 0.8oCIo4 additive
Cells and cultures Human hepatocarcinoma cell line HepG2, leukemia cell line K562 and their respective MDR sublines,
0.10
AU
0.08 0.06 0.04 0.02
Peak 2
Peak A
Peak 9 Peak 8 Peak 7 Peak 11 Peak 6 Peak 12 Peak 5 Peak 1 Peak 13 Peak 3 Peak 14 Peak 4 Peak 10 Peak 15
Peak 16
0.00 0.00
5.00
10.00
15.00
20.00 Minutes
25.00
30.00
35.00
40.00
Fig. 1. HPLC analysis of RA extract. RA extract was separated by HPLC on a RP18 column eluted by methanol/water (75:25, v/v) at 1.0 ml/min and monitored by a Waters Refraction Index Detector at 203 nm. Alisol B 23-acetate (peak A) was identified by a standard marker.
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effect (Chou and Talalay, 1983, 1984; Giulio and Frances, 1997). All experiments were repeated for at least three times.
Table 1. Effects of RA extract and verapamil on cytotoxicity of cancer drugs
Actinomycin D +RAE +Verapamil Puromycin +RAE +Verapamil Paclitaxel +RAE +Verapamil Vinblastine +RAE +Verapamil Doxorubicin +RAE +Verapamil
IC50-HepG2DR
IC50-HepG2
Resistance factora
2.1970.50 0.2170.03 0.5870.06 311792.6 1.1870.40 6.4571.63 4.3070.23 0.4370.15 0.7570.05 0.4670.03 0.1270.02 0.1670.01 41.672.30 1.4270.38 1.4070.04
0.00270.0004 1095 0.00270.0002 105 0.00270.0008 290 0.3570.01 888 0.19670.013 6 0.29070.061 22 0.00370.0003 1433 0.00270.001 215 0.00270.001 375 0.000470.0002 1150 0.000370.0001 400 0.000370.0002 533 0.05570.004 756 0.04970.019 29 0.04670.018 30
Rh-123 retention and doxorubicin accumulation To monitor Rh-123 retention, 1 106 cells in 5 ml of medium were incubated with 5 mg/ml Rh-123 at 37 1C for 1 h. Rh-123 loaded cells were washed with ice-cold phosphate buffered saline (PBS), resuspended in Rh123-free medium containing Pgp inhibitors for 1 h at 37 1C to allow Rh-123 efflux (Ludescher et al., 1992). For doxorubicin accumulation studies cells grown in 96well plates as above were incubated with 10 mM doxorubicin and Pgp inhibitors at 37 1C for 1 h (Krishan and Ganapathi, 1979). Cellular drug concentrations were estimated by fluorescence flow cytometry (FACSCAN, Becton Dickinson Immunocytometry Systems, San Jose, CA) and data were analyzed with the Macintosh CellQuest software.
Cells were incubated with drugs with RA extract (25 mg/ml) or Verapamil (10 mM) for 72 h. IC50 values (in mM) were expressed as means7standard deviations of three experiments. a Resistance factor is the IC50 ratio of HepG2-DR to GepG2 cells.
Cell cycle effects of vinblastine Approximately 1 106 cells in 5 ml of medium were treated with vinblastine (300 ng/ml) and Pgp inhibitors
Actinomycin D + RAE / Verapamil
Vinblastine + RAE / Verapamil 1.5
RAE Verapamil
1.5 1
1
0.5
0.5
0
RAE Verapamil
0 0
0.2 0.4 0.6 0.8 Fractional inhibition fa
1
0
Combination Index
Puromycin + RAE / Verapamil 1.5
1
1
0.5
0.5
0
0
0.2 0.4 0.6 0.8 Fractional inhibition fa
1
Doxorubicin + RAE / Verapamil
RAE Verapamil
1.5
0.2 0.4 0.6 0.8 Fractional inhibition fa
1
0
RAE Verapamil
0
0.2 0.4 0.6 0.8 Fractional inhibition fa
1
Paclitaxel + RAE / Verapamil RAE Verapamil
1.5 1 0.5 0
0
0.2 0.4 0.6 Fractional inhibition fa
0.8
1
Fig. 2. Synergistic effect of RA extract with anticancer drugs. Effects of RA extract (solid curve) and verapamil (positive control, dash curve) on the growth of HepG2-DR cells treated with actinomycin D, puromycin, paclitaxel, vinblastine or doxorubicin were evaluated by SRB assay and combination index (CI). Data were averages of three separate experiments. Solid horizontal line; CI ¼ 1.0; dash horizontal lines: CI ¼ 0.8.
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for 24 h. Cells were harvested, washed twice with icedcold PBS, fixed in 70% ethanol at 20 1C overnight, washed with PBS and incubated with 200 mg/ml RNase at 37 1C for 30 min. PI solution was added to a final concentration of 40 mg/ml. Analysis was performed immediately after PI staining by a FACSCAN flow cytometer.
Western blot analysis of Pgp expression Treated cells were incubated in ice-cold lysis buffer (50 mM Tris pH 7.4, 100 mM NaCl, 2 mM EDTA, 1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 2 mM PMSF, 1% aprotinin) for 30 min. Cellular proteins at 40 mg per lane were separated by 8% SDS/ PAGE and electro-transferred to nitrocellulose membranes. Membranes were blocked with 5% non-fat milk/ 0.1% Tween-20/TBS (10 mM Tris pH 7.5, 100 mM NaCl), incubated with anti-Pgp antibody for 1 h at room temperature, followed by horseradish–peroxidase-conjugated secondary antibody for another 1 h at room temperature. Protein bands were detected by the enhanced chemiluminescent (ECL) method.
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puromycin. Sensitivity to doxorubicin was raised by 30 folds and actinomycin D and paclitaxel 10-folds (Table 1). CI indicated clear synergistic effects (CIo0.8) between RA extract and individual anticancer drugs (Fig. 2). Synergism for actinomycin D or paclitaxel was observed over the entire killing rate (fa) range. In other cases synergism was observed at either high or low fa values. In drug-sensitive HepG2 cells RA extract and verapamil showed only additive effects with cancer drugs (Table 1 and data not shown).
RA extract increased doxorubicin accumulation in MDR cells Drug resistance in MDR cells has been attributed to decreased drug accumulation caused by the overexpression of Pgp (Tarasiuk et al., 2002; Chan et al., 2000). Cells were incubated with 10mM doxorubicin and Pgp inhibitors at 0.1–5 times of their respective IC50 at 37 1C for 1 h and cellular doxorubicin was studied by Celluar accumulation of DOX in HepG2-DR cell line 75
RAE Verapamil
Results HPLC profile of RA extracts
Cytotoxicity of the RA extract and anticancer drugs
* % Changes in cellular doxorubicin
RA extracts was separated by HPLC on a RP18 column (C-18, 5 mm, Alltech 02051566.1) eluted with methanol/water (75:25, v/v) at 1.0 ml/min and monitored by a refraction index detector (Water PDA 2695, 2996) at 203 nm. Fig. 1 shows a typical RA extract HPLC profile showing 17 identifiable peaks and one of the major components was identified by using standard marker as alisol B 23-acetate.
**
**
50 *
25
*
0 0.1xIC50 0.5xIC50
IC50
2.5xIC50
5xIC50
Celluar accumulation of DOX in K562-DR cell line 125
**
RAE Verapamil
100 75
HepG2 was highly sensitive to all the anticancer drugs tested including actinomycin D, puromycin, paclitaxel, vinblastine and doxorubicin. Resistance factors, calculated as the ratios of IC50 for MDR cells over that of IC50 for sensitive cells, showed that the Pgp-overexpressing HepG2-DR are 700–1400 times ‘‘more resistant’’ than the parental drug sensitive HepG2 cells. RA extract and verapamil were relatively non-toxic and showed similar levels of toxicity to both the drug sensitive and MDR cells.
RA extracts re-sensitized MDR cells to cancer drugs Comparing IC50 values, RA extract almost completely re-sensitized drug resistant HepG2-DR cells to
**
** 50
*
* *
25
*
0 0.1xIC50 0.5xIC50
IC50
2.5xIC50
5xIC50
Fig. 3. RA extract increased doxorubicin accumulation in MDR cells. HepG2-DR and K562-DR cells were incubated with 10 mM doxorubicin and various amount of RA extract or verapamil (in folds of IC50). Doxorubicin fluorescence was monitored by flow cytometry and shown as percent increase over untreated control (means7standard deviation of three independent experiments). Significantly different at *po0.05 or **po0.01 levels. Little change was observed in drug sensitive cells (data not shown).
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fluorescence flow cytometry. Drug resistant cells accumulated more doxorubicin in the presence of RA extract or verapamil (Fig. 3). Little change was observed in drug sensitive parental cells.
RA extract inhibited Rh-123 extrusion When drug-sensitive cells preloaded with Rh-123 were placed in RH-123-free medium, cellular fluorescence remained relatively unchanged (Fig. 4a,c). In MDR cells similarly preloaded with Rh-123, cellular fluorescence dropped dramatically in Rh123-free medium and the decrease could be inhibited by RA extract or verapamil (Fig. 4b,d).
RA extract restored vinblastine action in MDR cells
90 a. HepG2 80 70 60 50 40 30 20 10 0 101 100
RA extract did not alter Pgp protein expression First we confirmed that both MDR cell lines expressed higher levels of Pgp (Chan et al., 2000; Tarasiuk et al., 2002) (data not shown). Treatment with 50 mg/ml of RA extract for 12 h had no detectable effect on Pgp protein levels in these cells (data not shown).
Discussion Our conclusion that RA extract reverses MDR phenotype by inhibiting Pgp is supported by several lines of evidence: (1) In the first place RA extract affected only the MDR cell lines that differ from their parental cell lines primarily by expressing higher levels of Pgp. (2) The toxicity of RA extract was much the same on both the drug-sensitive and MDR cells. (3) RA 120 b. HepG2-DR 100
RAE
60
Loaded
Residual
40 20 102 FL1-H
103
0 100
104
100
101
102 FL1-H
103
104
120 c. K562
d. K562-DR 100
80
Verapamil
80
60
Counts
Counts
Verapamil
80 Counts
Counts
RA extract acted by mainly restoring actions of individual cancer drugs in MDR cells. For example vinblastine impedes mitotic spindle formation and induces G2/M arrest (Tashiro et al., 1998) and this was observed in drug-sensitive HepG2 but not in MDR cells. HepG2 cells normally showed a cell cycle distribution of approximately 65.0%:9.4%:21.1% (G0/G1:S:G2/M) (the percentage of subG1 ‘‘apoptotic’’ cells was not shown). Vinblastine treatment (300 ng/ml, 24 h) changed this to approximately 5.3%:4.6%:88.8% and RA extract (2.5
or 25 mg/ml) had no significant effect on the drug sensitive HepG2 cells with or without vinblastine. In drug resistant HepG2-DR cells similar vinblastine treatment only slightly increased the G2/M cells and changed the ratio to approximately 43.1%:9.7%:23.4%. The addition of RA extract to vinblastine treatment, however, drastically increased the G2/M fraction and the ratio became 14.3%:8.6%:71.4%.
40
Loaded
40
20 0 100
60
RAE Residual
20 101
102 FL1-H
103
104
0 100
101
102 FL1-H
103
104
Fig. 4. RA extract increased Rh-123 retention in MDR cells. Cells pre loaded with Rh-123 were incubated in Rh-123 free medium for 1 h. ‘‘Loaded’’ (thick solid line) and ‘‘residual’’ (light solid line) cellular fluorescence was measured by flow cytometry. Dash line: residual Rh-123 fluorescence in the presence of 25 mg/ml RA extract. Dotted line: residual Rh-123 fluorescence in the presence of 10 mM verapamil (positive control).
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extract showed a synergistic effect with all the tested anticancer drugs that are Pgp substrates in the Pgp overexpressing MDR cells. (4) RA extract restored vinblastine’s action on inducing G2/M arrest. (5) It increased cellular doxorubicin accumulation and slowed down Rh-123 efflux from Pgp over-expressing MDR cells. None of the above effects could be observed in the drug sensitive parental cell lines. (6) Lastly RA extract did not change the expression of Pgp nor did it affect intracellular ATP levels (data not shown) that is needed for Pgp activity. We noted that RA extract showed different levels of synergism when added with different drugs, from a 10fold increase of drug sensitivity to a complete reversal of resistance. This is expected since there exist different modes of binding of various structurally different ligands to Pgp and there are also multiple ligand binding sites on Pgp. There is strong evidence suggesting that one of the key factors influencing ligand-Pgp affinities could be ligand hydrophobicity (Yang et al., 1991). The three-dimensional atomic structure of Pgp is not yet known but enzymatic and functional studies suggest that there exist three substrate binding sites, an ATP hydrolysis site and an allosteric regulatory site (Pascaud et al., 1998). Finally, to unequivocally identify the mechanism of RA inhibition would first require the isolation and identification of pure active compounds from RA extract. These studies are being carried out in our laboratory.
Acknowledgement This work was supported in full by City University of Hong Kong Strategic Research Grant 7001110.
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