Tetrandrine and fangchinoline, bisbenzylisoquinoline alkaloids from Stephania tetrandra can reverse multidrug resistance by inhibiting P-glycoprotein activity in multidrug resistant human cancer cells.
Multidrug resistance (MDR) is considered as an important obstacle for an effective clinical cancer chemotherapy (Johnstone et al. 2000). MDR often emerges as a result of overexpression of ABC transporters, which obtain energy from ATP hydrolysis to actively export lipophilic drugs across extra- and intra-cellular membranes (Robey et al. 2008). Presently, 49 human ABC transporters with subfamilies A to G have been recorded in the human genome. ABC transporter can export xenobiotics which entered a cell by diffusion with broad substrate specificity (Leonard et al. 2003). ABC transporters decrease drug accumulation in multidrug-resistant cells which can thus mediate the development of resistance of cells to anticancer drugs. In particular, subfamily B member 1 [ABCB1/MDR1 gene; P-glycoprotein (P-gp)] was the first studied protein of ABC transporters (Lautier et al. 1996). P-gp is a polypeptide dimer (with 1280 amino acid residues); it represents a pore forming membrane protein that can export many types of drugs in MDR cells (Leselle et al. 2005).
A possibility to overcome the problem of ABC transporter-mediated MDR is the development of efficient inhibitors, which either down-regulate the expression of transporter proteins or have a synergistic effect with chemotherapeutic agents by inhibiting the efflux function of ABC transporters (Liu et al. 2013).
Medicinal plants are rich sources of secondary metabolites which can be used to treat human diseases and illnesses (Van Wyk and Wink 2004). Many natural compounds and phytopharmaceuticals have been discovered that can inhibit P-gp and re-sensitize resistant tumour cells in vitro (review in Wink et al. 2012; Wagner and Ulrich-Merzenich 2009). We have screened several natural compounds having a complementarity effect with chemotherapeutic agents used in treating cancer cells. Successful examples include the isolation of limonin and other secondary metabolites from Citrus species as P-gp inhibitors (EI-Readi et al. 2010).
Tetrandrine and fangchinoline are bisbenzylisoquinoline alkaloids, occurring in roots of the creeper Stephania tetrandra Moore (Wu et al. 2010). In China, the alkaloids are employed to decrease portal venous pressure and blood pressure (Kim et al. 1999). In recent years, tetrandrine and fangchinoline have been found to exert anti-inflammatory (Choi et al. 2000) and antitumor activities (Yang et al. 2007), inhibiting proliferation and inducing apoptosis of hepatoma, breast cancer, lung cancer and leukaemia cells (Chen et al. 2009). However, we are not aware on any report whether tetrandrine and fangchinoline have the ability to reverse ABC transporter-mediated MDR.
In this study a series of experiments were carried out to investigate the effect of tetrandrine and fangchinoline alone or in combination with doxorubicin to the reverse P-gp mediated MDR. The direct inhibition of P-gp by the alkaloids was studied in rhodamine 123 competition experiments. In addition, we could show that both alkaloids can down-regulate the expression of P-gP
Materials and methods
Chemicals and reagents
Doxorubicin, dimethyl sulfoxide (DMSO), rhodamine 123 (Rho123), verapamil hydrochloride and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoleum) were purchased from Sigma-Aldrich GmbH, Germany. The polyclonal antibody against MDR1/ABCB1 and HRP-conjugated rabbit IgG antibody were purchased from Boster Co. (China). RPMI-1640 and DMEM medium were products of Gibco, Karlsruhe, Germany. FBS (foetal bovine serum) was from BioChrom KG, Berlin, Germany.
Identification of terandrine and fangchinoline by LC-MS
Tetrandrine ([C.sub.38][H.sub.42][O.sub.6][N.sub.2]) and fangchinoline ([C.sub.37][H.sub.40][O.sub.6][N.sub.2]) were purchased from National Institute for Food and Drug Control, China. They were further identified using a reversed phase column (Luna 3u C18(2) 150 mm x 2mm, Phenomenex, Aschaffenburg, Germany) with a flow rate of 0.1 ml/min. Gradient elution was performed with [H.sub.2]0 and MeOH, both containing 0.1% formic acid: 0-60 min, 0-50% MeOH; 60-70 min, 50-100% MeOH; 70-84 min, 100% MeOH; 84-85 min, 100-0%; 85-105 min, 0% MeOH; MS spectra were recorded with positive ionization mode. Nitrogen was used as dry gas. Data analysis was performed using the Data-Analysis 5.1 software (Bruker Daltonics, Bremen, Germany).
From the chromatogram (Fig. 1), both tetrandrine and fangchinoline showed good purity, and their molecular weight were 622.3 and 608.3, respectively. They were dissolved in DMSO as a 10 mM stock concentration, the stock solutions were further diluted in fresh medium for cell experiment.
Cell lines and cell culture
The Caco-2 cell line represents heterogeneous human epithelial colon adenocarcinoma cells, which simultaneously express ABC-transporters including P-gp (MDR1), MRP1 and BCRP. HCT-116 is a human colon cancer cell line. The human leukaemia lymphoblast cell line CEM/CCRF shows a low expression of ABC transporters whereas its derivative daughter cell line CEM/ADR5000 over-expresses P-gp. CEM/ADR5000 cells were 126-fold resistant to doxorubicin as compared with the parental CEM/CCRF cells.
The adherent Caco-2 and HCT-116 colon cancer cell lines were cultured in DMEM medium, supplemented with 10% FBS, 1% glutamine, 1% penicillin and streptomycin. In addition, 1% sodium pyruvate and 1% NEAA (non-essential amino acids) were added to the Caco-2 cell medium. The suspension cultured cells CEM/CCRF and CEM/ADR5000 were maintained in RPMI-1640 complete medium. In order to keep a high P-gp level, CEM/ADR5000 cells were weekly cultivated for 24 h in fresh culture medium with 2 [micro]g/ml doxorubicin. All cells were cultivated at 37 [degrees]C, 5% C02 and 95% humidity atmosphere and the experiments were performed with cells in the logarithmic growth phase.
Cell viability was determined using the conventional MU assay according to our previous study (Eid et al. 2012a,b). Briefly, Caco-2 (2 x [10.sub.4]) and HCT-116 (1 x [10.sub.4]) cells were seeded in 96-well plates overnight, various concentrations of tetrandrine or fangchinoline (0.1-200 [micro]M) were added, and incubated for 24 h. MU (0.5mg/ml) was pipetted to each well and incubated for 3h. The formazan crystals produced were dissolved in 100 [micro]l DMSO and the optical density was measured at 570 nm using a Tecan microplate reader (Crailsheim, Germany). For CCRF and CEM/ADR5000,3 x [10.sup.4] cells/well were treated with drugs for 48 h, and analyzed by the above mentioned MU assay. Each compound was analyzed independently for at least 3 times. [IC.sub.50] is defined as the drug concentration which produces 50% of the desired effect. [IC.sub.50] values were determined by SigmaPlot 11.0 software.
MDR reversal assay
The combinations of tetrandrine and fangchinoline with a single chemotherapeutical drug doxorubicin were investigated in MDR Caco-2, CEM/ADR5000 cells and in sensitive HCT-116, CEM/CCRF cells. Different concentrations of doxorubicin were applied in combination with a non-toxic concentration ([IC.sub.20]) of tetrandrine and fangchinoline. To calculate the [IC.sub.20] of tetrandrine and fangchinoline, the online-software of GraphPad (compute [EC.sub.anything] from [EC.sub.50]) was used. Drug interactions were evaluated using combination index (Cl), reversal ratio (Rr) calculations and isobologram representation according to our previous papers (Eid et al. 2012a,b).
(I) Cl analysis which can assess the interactions of combinations in a quantitative manner was calculated as:
CI = [C.sub.AX]/[IC.sub.A] + [C.sub.BX]/[IC.sub.B]
In which [C.sub.AX] and [C.sub.BX] are the concentration of drug A and drug B used in combination to reach an [IC.sub.50] value. [IC.sub.A] and [IC.sub.B] are the [IC.sub.50] values for single A and B drugs.
(II) The reversal ratio was calculated as follows:
Reversal ratio (Rr) = [IC.sub.50] value of doxorubicin/[IC.sub.50] value of doxorubicin in combination with MDR inhibitor
Rr values indicate the cytotoxicity enhancement ratio, which can be used to quantify the potential power a MDR reversal agent.
(III) The extent of the interaction between a chemosensitizer (A) and a chemotherapeutical agent (B) was illustrated using an isobologram. The [IC.sub.50] concentrations of drugs A and B are plotted on the x andy axis in a two-coordinate plot, corresponding to (0, [C.sub.A]) and (0, [C.sub.B]), respectively. The line connecting these two points represents additive interactions. The concentrations of the two drugs used in combination to provide the same effect, denoted as ([C.sub.A], [C.sub.b]), are placed in the same plot. When [C.sub.A] and [C.sub.B] are located below the line, synergy is present: if they are on the line, we consider this as additivity and above the line as antagonism (Chou and Talalay 1984).
Enhancement of Rho123 accumulation
Rhodamine 123 (Rho123), a fluorescent P-gp substrate, is readily effluxed in MDR overexpressing cancer cells: it is frequently employed to evaluate P-gp activity (Wan et al. 2006). The effect of tetrandrine and fangchinoline on the accumulation of Rho123 in P-gp were determined in P-gp overexpressing and sensitive cells. Generally, the cells were treated with various concentrations of tetrandrine and fangchinoline (1.6,6.5,12.5, and 25 [micro]M) for 90 min at 37[degrees]C. After washing two times with ice-cold 1 x PBS, Rho123 (10 [micro]M final concentration) was added and the cells were incubated for further 90 min. Then cells were collected and washed twice with ice-cold washing buffer (5mM EDTA and 1% FBS in 500 ml 1 x PBS) and kept on ice in the dark until measurement. A Tecan reader (Crailsheim, Germany) was used to measure the fluorescence intensity of the adherent cells. CEM/ADR5000 and CEM/CCRF suspension cells were analyzed by a FACS[R]Calibur instrument (Becton Dickinson). At least 10,000 cells per samples were counted and acquired through the FL1 channel. Cell Quest software was used for data analysis. Verapamil hydrochloride, a known P-gp inhibitor, was used as a reference compound. The relative fluorescence intensity (inhibitory efficiency) for each kind of treatment was calculated as follows:
=(fluorescence intensity of test compound/fluorescence intensity of verapamil) x 100
At the same time, the images of the P-gp overexpressing cancer cells accumulating Rho123 were taken by fluorescence microscopy (BioRevo BZ-9000, KEYENCE).
Caco-2 and CEM/ADR5000 cells were treated with different concentrations of tetrandrine and fangchinoline (1.6, 6.5, 12.5 and 25 [micro]M) for 48 h and lysed after washing two times with ice-cold PBS. The protein concentration of the lysate was quantified using the Bradford assay. Equal amounts of protein from the lysate were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in buffer including 5% non-fat dried milk and then incubated with the primary P-gp antibody at 4[degrees]C overnight. Then they were washed three times with buffer, incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibodies for 1 h at room temperature, and then washed again three times with buffer. The protein signals were observed by an enhanced chemiluminescence (ECL) detection system (Sun et al. 2013). The house keeping protein [beta-]-actin was used as a control for densitometry measurements of immunoblotting. Data are represented as histograms showing means [+ or -] S.D. of three independent experiments.
All assays were carried out three times. The data are expressed as mean [+ or -] standard deviation. Statistical analysis was performed by one-way analysis of variance (ANOVA) using GraphPad Prism software (GraphPad Prism 5.01, GraphPad Software, Inc., CA, USA). Graphs were drawn using SigmaPlot 11.0 software.
Cytotoxicity of tetrandrine and fangchinoline
The cytotoxicity of tetrandrine or fangchinoline was investigated using two pairs of resistant (Caco-2, CEM/ADR5000) and sensitive cell models (HCT-116, CEM/CCRF). [IC.sub.50] values of the tetrandrine and fangchinoline are documented in Table 1. The two compounds showed significant antitumor effects against human colon and leukaemia cell lines, but their cytotoxicities are lower than that of doxorubicin except against CEM/ADR5000 cells. HCT116 and CEM/CCRF cells are more sensitive for both alkaloids than MDR Caco-2 and CEM/ADR5000 cells indicating the involvement of P-gp as a resistance mechanism. [IC.sub.50] values of fangchinoline were lower than tetrandrine, indicating that its antitumor activities are superior to tetrandrine.
The MDR reversal assay
Based on MTT assay results, non-toxic concentrations ([IC.sub.20]) of tetrandrine and fangchinoline were combined with doxorubicin in the reversal assays. The resulting dose-dependent cytotoxicity curves are illustrated in Fig. 2. The cytotoxicity of doxorubicin was apparently decreased when combined with an [IC.sub.20] concentration of tetrandrine and fangchinoline. The cytotoxicity of doxorubicin in CEM/ADR5000 cells when treated with 6.5 [micro]M tetrandrine was enhanced 12.96 fold, whereas 3.25 [micro]M fangchinoline increased the [IC.sub.50] of doxorubicin 34.86 fold (Table 2).
To detect synergism or antagonism of drug interactions, an isobologram of combined drug effects was constructed. Fig. 3 illustrates the isobologram for tetrandrine and fangchinoline with doxorubicin; values below the straight line indicate that both alkaloids exhibit synergistic interactions in colon and leukaemia cancer cells (Didiodato and Sharom 1997). Table 3 summarizes the synergistic effects of tetrandrine and fangchinoline with doxorubicin. The different analyses (Cl and Rr values) of the corresponding combinations clearly indicate a synergistic effect and confirm the data obtained from dose dependent cytotoxicity studies. These results indicate that tetrandrine and fangchinoline have significant reversal properties on all tested cells.
Tetrandrine and fangchinoline increase Rho123 accumulation in Caco-2 and in CEM/ADR5000 cells
The effect of tetrandrine and fangchinoline on P-gp activity was assessed by measuring the intracellular accumulation of Rho123. As shown in Figs. 4 and 5, Caco-2 and CEM/ADR5000 cells when treated with tetrandrine and fangchinoline exhibited a notable increase of Rho123 fluorescence in a dose-dependent manner. As expected from the cytotoxicity data fangchinoline was more active than tetrandrine. In the presence of 12.5 and 25 [micro]M fangchinoline, the relative fluorescence intensity in CEM/ADR5000 cells was increased 6.8 and 15 fold, which is significantly higher than that of positive control verapamil (p < 0.001). Tetrandrine and fangchinoline could not enhance Rho123 accumulation in sensitive CEM/CCRF cells (Fig. 6A-C). As shown in Figs. 4B and 5D, the high intensity of red fluorescence from Rho123 was mainly located around cell membranes of Caco-2 cells, whereas the retained intracellular of Rho123 was significantly increased. These results suggest that tetrandrine and fangchinoline effectively inhibit the efflux pump function of P-gp.
Effect of tetrandrine and fangchinoline on the expression of P-gp
A Western blot analysis was carried to detect the expression levels of P-gp in experimental cells (Tran et al. 2013). In drug resistant Caco-2 and CEM/ADR5000 cells a marked P-gp protein expression was observed as compared with that of sensitive HCT-116 and CEM/CCRF cells. Especially, CEM/CCRF cells hardly expressed P-gp (Fig. 7A). After 48 h incubation of Caco-2 cells with increasing concentrations of tetrandrine and fangchinoline, the levels of P-gp were significantly decreased in a concentration-dependent manner (Fig. 7B). As shown in Fig. 7C, similar results were obtained for CEM/ADR5000 cells. However, when CEM/ADR5000 cells were treated with 12.5 [micro]M fangchinoline P-gp expression was significantly reduced (p < 0.001). These results indicate that tetrandrine and fangchinoline down-regulate P-gp expression in human MDR cancer cells after 48 h treatment, which might contribute to their potent MDR reversal activity.
The overexpression of ABC transporters is considered as a primary cause for the failure of cancer chemotherapy. Several MDR reversal agents from natural sources have been investigated, as many secondary metabolites and phytotherapeutics show low side effects and good tolerability (Wang et al. 2004; Wink et al. 2012). To date, several secondary metabolites have been found to strongly inhibit the function of ABC transporters (review in Wink et al. 2012). However, the potential of tetrandrine and fangchinoline to reverse ABC transporter activity could be demonstrated for the first time in this investigation. In our study, both tetrandrine and fangchinoline not only showed a remarkable reversal effect in P-gp overexpressing Caco-2 and CEM/ADR5000 cells but also in sensitive parental cells. They significantly and synergistically enhanced the cytotoxicity of doxorubicin. Importantly, the reversal effect of fangchinoline was always stronger than that of tetrandrine.
To understand the mechanism of the reversal activity, we investigated the effect of tetrandrine and fangchinoline on the accumulation of Rho123 in sensitive and MDR cells. The results clearly showed that tetrandrine and fangchinoline can strongly inhibit the intracellular efflux of Rho123 in MDR cells, whereas no effect was seen in the parental sensitive cells. In general, the reversal of P-gp can be achieved either by inhibiting the efflux ability of ABC-transporters or regulating P-gp expression (Tan et al. 2000). Based on the above results, further experiments were conducted to examine the effect of tetrandrine and fangchinoline on the expression of P-gp expression. Western blot analysis demonstrated that both alkaloids reduced P-gp protein levels in a dose-dependent manner in MDR cells after 48 h treatment. Thus, we suggest that the reversal effects of tetrandrine and fangchinoline in our experiments may due to two mechanisms: regulation of the efflux function of ABC-transporter activity and inhibition P-gp expression.
Tetrandrine and fangchinoline have a similar structure, but a major difference is that a phenolic hydroxyl group is replaced by a methoxy group in tetrandrine (Fig. 1). Although tetrandrine is more lipophilic than fangchinoline, fangchinoline was a stronger MDR reversal agent or Rho123 efflux inhibitor than tetrandrine under the same conditions. In addition, the effect of both alkaloids on Rho123 accumulation in Caco-2 and CEM/ADR5000 cells was also observed by fluorescent microscopy (Figs. 4B and 5D). A stronger Rho123 accumulation was observed when the cells were treated with fangchinoline than with tetrandrine.
In summary, this study demonstrates that the bisbenzylisoquinoline alkaloids tetrandrine and fangchinoline can reverse P-gp mediated MDR and P-gp activity (both protein expression and efflux activity), and may serve as promising MDR reversal agents and potential adjuvants for cancer therapy themselves or serve as leads for the development of more powerful semisynthetic derivatives.
Received 7 January 2014
Received in revised form 17 February 2014
Accepted 18 April 2014
Conflict of interest statement
The authors declare that there are no conflicts of interest. Acknowledgement
The authors thank the Germany Federal Ministry of Education and Research for providing a DAAD (German Academic Exchange Service) scholarship for Yan Fang Sun.
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Yan Fang Sun (a,b) *, Michael Wink (a), *
(a) Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany
(b) College of Science, Liaoning Technical University, Fuxin, Liaoning 123000, China
* Corresponding authors at: Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany. Tel.: +49 6221 54 4880; fax: +49 6221 544884.
E-mail addresses: firstname.lastname@example.org (Y.F. Sun), email@example.com (M. Wink).
Table 1 The cytotoxicity of tetrandrine, fangchinoline and doxorubicin in resistant and sensitive cancer cells. Data represent mean [+ or -] S.D. CACO-2 HCT-II6 [IC.sub.50] [IC.sub.50] Compounds value [[micro]/M] value [[micro]/M] Tetrandrine 19.38 [+ or -] 4.92 11.13 [+ or -] 2.20 Fangchinoline 14.70 [+ or -] 3.49 4.36 [+ or -] 0.51 Doxorubicin 5.74 [+ or -] 0.94 2.82 [+ or -] 1.25 CEM/ADR5000 CEM/CCRF [IC.sub.50] [IC.sub.50] Compounds value [[micro]/M] value [[micro]/M] Tetrandrine 24.98 [+ or -] 2.62 16.59 [+ or -] 1.61 Fangchinoline 13.00 [+ or -] 1.69 8.81 [+ or -] 1.61 Doxorubicin 43.74 [+ or -] 13.57 0.35 [+ or -] 0.23 Table 2 The cytotoxity of doxorubicin with and without non-toxic concentrations of tetrandrine ([IC.sub.20]) or fangchinoline ([IC.sub.20]) in resistant and sensitive cancer cells. Cells Doxorubicin +Tetrandrine [IC.sub.50] value [IC.sub.50] value [[micro]M] [[micro]M] Caco-2 5.74 [+ or -] 0.94 0.62 [+ or -] 0.10 HCT-116 2.82 [+ or -] 1.25 1.61 [+ or -] 0.57 CEM/ADR5000 43.74 [+ or -] 13.57 3.38 [+ or -] 0.31 CEM/CCRF 0.35 [+ or -] 0.23 0.18 [+ or -] 0.01 Cells +Fangchinoline [IC.sub.50] value [[micro]M] Caco-2 0.14 [+ or -] 0.06 HCT-116 0.99 [+ or -] 0.41 CEM/ADR5000 1.25 [+ or -] 0.05 CEM/CCRF 0.11 [+ or -] 0.13 Table 3 Effects of the combination of tetrandrine and fangchinoline with doxorubicin: combination index method (CI), reversal ratio (Rr) and isobologram (IB). Compounds Caco-2 HCT-116 CI IB Rr Cl IB Rr Tetrandrine 0.44 Syn 9.38 0.86 Syn 1.75 Fangchinoline 0.24 Syn 41.56 0.72 Syn 2.86 Compounds CEM/ADR5000 CEM/CCRF Cl IB Rr Cl IB Rr Tetrandrine 0.35 Syn 11.59 0.72 Syn 1.92 Fangchinoline 0.28 Syn 31.18 0.51 Syn 3.05 Note: Cl values were interpreted as follows: Cl < 1 synergism, Cl = 1 [right arrow] additive effect and Cl > 1 [right arrow] antagonism. Isobologram data (IB) were interpreted according to Fig. 3 and abbreviated as: Syn = synergism, Add = additive and Ant = antagonism. Rr values indicate the cytotoxicity enhancement ratio.
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|Author:||Sun, Yan Fang; Wink, Michael|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Jul 15, 2014|
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