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M arrest
Toxicology Letters 219 (2013) 107–115
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Oroxylin A reverses P-glycoprotein-mediated multidrug resistance of MCF7/ADR cells by G2/M arrest Litao Zhu a,1 , Li Zhao a,1 , Hu Wang a , Yu Wang a , Di Pan a , Jing Yao a , Zhiyu Li c , Guanzhong Wu b,∗ , Qinglong Guo a,∗ a State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, People’s Republic of China b Department of Pharmacology, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, People’s Republic of China c Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, People’s Republic of China
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
Oroxylin A induced cells G2/M arrest by increasing the expression of pChk2 (Thr68). The resistance was associated with the decreased expression of p-Chk2. Inhibiting p-Chk2 can decrease the reversal fold of oroxylin A. Oroxylin A reversed multidrug resistance by G2/M arrest.
a r t i c l e
i n f o
Article history: Received 3 December 2012 Received in revised form 21 January 2013 Accepted 22 January 2013 Available online 5 March 2013 Keywords: Oroxylin A MCF7/ADR G2/M P-gp Chk2
a b s t r a c t Oroxylin A is a naturally occurring monoflavonoid isolated from the root of Scutellaria baicalensis Georgi, which has been used in traditional Chinese medicine for its anti-tumor, anti-inflammatory and antibacterial properties. The purpose of this study is to investigate the reversal effect and the fundamental mechanisms of oroxylin A in MCF7/ADR cells. Data indicated that oroxylin A showed strong reversal potency in MCF7/ADR cells and the reversal fold (RF) reached 4.68. After treatment with oroxylin A, MCF7/ADR cells displayed reduced functional activity and expression of MDR1 at both the protein and mRNA levels. Meanwhile, oroxylin A induced cells G2/M arrest in a concentration-dependent manner by increasing the expression of p-Chk2 (Thr68). Moreover, western blot and EMSA assays were used to reveal the inhibition of NF-B in nucleus and the suppression of NF-B binding activity by oroxylin A. NSC 109555 ditosylate-Chk2 inhibitor partly dismissed G2/M arrest induced by oroxylin A, reversed the increased trend of p-Chk2 and p-P53 (Ser20), inhibited the decreasing effect of oroxylin A on the expression of P-gp and decreased the reversal fold of 90 M oroxylin A from 4.68 fold to 1.73 fold. In conclusion, we suggested that oroxylin A reversed MDR by G2/M arrest and the underlying mechanism attributed to the suppression of P-gp expression via Chk2/P53/NF-B signaling pathway. © 2013 Elsevier Ireland Ltd. All rights reserved.
Abbreviations: MCF7/ADR, Adriamycin resistant human breast cancer cell line; MDR, multidrug resistance; Chk2, checkpoint kinase 2; NF-B, nuclear factor-kappa B; PMSF, phenylmethylsulfonyl fluoride. ∗ Corresponding authors. Tel.: +86 25 83271055; fax: +86 25 83271055. E-mail addresses: [email protected] (G. Wu), anticancer [email protected] (Q. Guo). 1 These authors contributed equally to this article. 0378-4274/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2013.01.019
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1. Introduction Breast cancer is the most commonly occurring cancer in women, comprising almost one third of all malignancies in females. It comes in a second position after lung cancer as a cause of cancer mortality, and it is the leading cause of death for American women between the ages of 40 and 55 (Richie and Swanson, 2003). Despite the large repertoire of therapies available and the following efforts to incorporate new drugs into clinical practice, it is generally believed that there is a long way to control cancer. A major factor contributing to the failure of chemotherapy in breast cancer is the development of drug resistance (Schultz and Weber, 1999; Stockler et al., 2000). Adriamycin has significant antitumor activity and it is widely used for the treatment of breast cancer (Blum and Carter, 1974), however the high rate of resistance limit its effectiveness and thus its use. Therefore, it is urgently needed to develop new adjuvants that enhance the efficacy of Adriamycin-based chemotherapy and circumvent chemoresistance. Resistance to multiple categories of chemotherapy, termed multidrug resistance (MDR), is a complex hurdle in cancer therapy. The mechanisms behind MDR are still under active investigation, but members of the ATP binding cassette (ABC) transporter superfamily, that often act as cellular efflux pumps for a wide range of chemotherapeutic compounds, have well-established roles (Calcagno et al., 2007; Gottesman et al., 2002). There are at least 49 MDR genes described, of which the ABCB1 gene that encodes an efflux transporter P-glycoprotein (P-gp) or MDR1 which is probably the most well known (Bram et al., 2009). The prognostic significance of P-gp as an indicator for failure in chemotherapy has been demonstrated in a number of clinical studies (Chan et al., 1991; Holmes and West, 1994; Sauerbrey et al., 1994; Verrelle et al., 1991). Other drug substrates of P-gp include calcium channel blockers (e.g. verapamil), antiarrhythmics (e.g. quinidine), steroids (e.g. dexamethasone) and anti-parasitics (e.g. ivermectin), antidepressants and antiepileptic drugs to mention but a few (Ford et al., 1996; Schinkel and Jonker, 2003). Consequently, P-gp not only protects the body from harmful xenobiotics taken up with nutrition, but affects drug absorption, distribution and secretion. Therefore, the protein is of great importance in cancer treatment as well as in general pharmacology. Cell cycle de-regulation resulting in uncontrolled cell proliferation is the one of the most frequent alterations that occur during tumor development (Collins et al., 1997). For this reason, blockage of the cell cycle is regarded as an effective strategy for eliminating cancer cells (Buolamwini, 2000; Hajduch et al., 1999). Two major checkpoints, one at the G1/S transition and another at the G2/M transition, regulate the cell cycle. The G2/M checkpoint plays a key role in the maintenance of chromosomal integrity by allowing cells to repair DNA damage before entering mitosis. DNA damage signaling is mediated by the ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia related) protein kinases. Once activated, these kinases phosphorylate a wide range of target proteins, including the checkpoint kinases (Chk1 and Chk2), the tumor suppressor P53 and the histone variant H2AX (Jowsey et al., 2009). Chk2 is a mammalian homolog of the Saccharomyces cerevisiae Rad53 and Schizosaccharomyce pombe Cds1 checkpoint genes (Blasina et al., 1999; Chaturvedi et al., 1999; Matsuoka et al., 1998). These target proteins, along with many others, act to slow cell cycle progression, regulate transcription, enhance the DNA repair capacity of cells or direct cells to die via apoptosis if the damage encountered is too great. Scutellariae radix, the root of Scutellaria baicalensis, has been widely used in traditional Chinese medicine as an analgesic, antipyretic, anti-inflammation, anticancer, antiviral and antibacterial infections remedy (Sun et al., 2009). Oroxylin A (C16 H12 O5 , Fig. 1) is one of active flavonoids isolated from the root of
Fig. 1. Chemical structure of oroxylin A. The molecular formula of oroxylin A is C16 H12 O5 and the molecular weight is 284.
Scutellaria baicalensis Georgi (Gao et al., 2010). Studies have shown that oroxylin A induced G2/M arrest via inhibiting Cdk7-mediated expression of Cdc2/p34 in human gastric carcinoma BGC-823 cells (Yang et al., 2008), blocked NF-B binding and transcriptional activation associated with decreased p65 proteins in the nucleus induced by LPS (Chen et al., 2000), regulated gene expressions of thymidine synthetase (TS) and dihydropyrimidine dehydrogenase (DPD) and thus increased the sensitivity of human hepatocellular carcinoma HepG2 to 5-fluorouracil (5-FU) in vivo and in vitro (Zhao et al., 2010); furthermore, it reversed CAM-DR via inhibition of Integrin1 and by regulating PI3K/AKT pathway (Zhu et al., 2012). Meanwhile, oroxylin A reversed the multidrug resistance of human BEL7402/5-FU cells via down-regulation of P-gp expression by inhibiting NF-B signaling pathway (Yang et al., 2012). Considering the aforementioned factors, we hypothesized that oroxylin A potentially switch MDR activity via cell cycle arrest. From our results, we confirmed that oroxylin A reversed the resistance of MCF7/ADR cells and the underlying mechanisms are attributed to P-gp-mediated MDR and the activity of Chk2/P53/NFB pathway. 2. Materials and methods 2.1. Materials Oroxylin A was isolated from Scutellariae radix according to the protocols reported previously (Li and Chen, 2005). Oroxylin A was dissolved in dimethyl sulfoxide (DMSO) as a stock solution, stored at −20 ◦ C, and diluted with medium before experiment. The final DMSO concentration did not exceed 0.1% in all experiments. Paclitaxel, Adriamycin, Docetaxel, Propidium iodide (PI), Rhodamine 123, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma–Aldrich (St. Louis, Missouri, USA). NSC 109555 ditosylate, the Chk2 inhibitor, was obtained from Tocris Bioscience. It was dissolved in DMSO at a concentration of 10 mM and stored at −20 ◦ C. Primary antibodies of -actin (1:2000), LaminA (1:500), MDR1 (1:500), NF-B (1:500), p-P53 (1:500), Chk2 (1:500) were obtained from Santa Cruz (Santa Cruz, CA). p-Chk2 (1:800) was from Bioworld (OH, USA). IRDyeTM 800 conjugated secondary antibodies were obtained from Rockland Inc. (Philadelphia, PA). 2.2. Cell culture Parental human breast adenocarcinoma MCF7 cells and Adriamycin reduced drug-resistant MCF7/ADR cells were purchased from Cell Bank of Shanghai Institute of Biochemistry & Cell Biology, Chinese Academy of Sciences and Tumor Center of Beijing. Cells were cultured in RPMI-1640 medium (Gibco, Invitrogen Corporation, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (Sijiqing, Zhejiang, China), 100 U/ml benzyl penicillin, and 100 g/ml streptomycin in a humidified environment with 5% CO2 at 37 ◦ C. To maintain drug resistance, Adriamycin (5 g/ml) was supplied to MCF7/ADR cells at regular intervals. Cells could be harvested and used for experiments 2 weeks after removal of Adriamycin. 2.3. Determination of multidrug resistance To determine the multidrug resistance of MCF7/ADR cells to chemotherapeutic agents, MCF7/ADR cells and the parent MCF7 cells were seeded into 96-well plates and incubated with various concentrations of anticancer drugs (Adriamycin, Docetaxel, Paclitaxel) for 48 h. Each group consisted of five parallel wells. MTT assay was performed to determine the percentage of survived cells at each concentration. After plotting the dose–response curve, IC50 was calculated, from which reversal
L. Zhu et al. / Toxicology Letters 219 (2013) 107–115 index was calculated. The degree of resistance was calculated by resistance index (RI), which obtained from the data = IC50 of MCF7/ADR cells/IC50 of MCF7 cells (Yang et al., 2012). All assays were performed in triplicate. 2.4. Intrinsic cytotoxicity assay MTT colorimetric assay was performed to determine the cytotoxicity of oroxylin A in vitro. MCF7/ADR cells growing in log phase were seeded into 96-well plates at a density of 1 × 104 cells/well and then incubated for 24 h at 37 ◦ C under 5% CO2 . After being exposed to various concentrations of oroxylin A for 48 h, 20 l MTT dye (5 mg/ml) was added to each well and incubated for 4 h. The dye was solubilized with 100 l DMSO and plates were read at 570 nm on an automated microtiter plate reader. Blank wells containing only media and drug were also run as control in all experiments. For required concentrations of reversal multidrug agents, which are neither inhibitory nor toxic (Zheng et al., 2008), final concentrations of 30, 60 and 90 M were chosen. The inhibitory ratio was calculated using the following formula: inhibitory ratio (%) = 1 − average absorbance of treated group/average absorbance of control group × 100% (Zhu et al., 2012). The IC50 values were determined by GraphPad Software. All determinations were carried out in triplicate. 2.5. Reversal effect assay The ability of oroxylin A to reverse MDR in MCF7/ADR cells was measured by MTT method. MCF7/ADR cells were seeded in 96-well plates. Combinations of different concentrations of Adriamycin with oroxylin A (30, 60 and 90 M) or verapamil (20 M) diluted in RPMI1640 were then added. The cells were incubated in a humidified incubator in 5% CO2 at 37 ◦ C for 48 h, and the quantity of viable cells was determined using MTT method. Each group consisted of five parallel wells. The RF values, as the reversal potency, were obtained from fitting the data = IC50 of cytotoxic drug alone/IC50 of cytotoxic drug in the presence of the test drugs (Li et al., 2009).
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Table 1 Determination of IC50 of different anticancer drugs. IC50 values of anticancer drugs were determined in MCF7 and MCF7/ADR cells. Cells were treated with various concentrations of Adriamycin, Docetaxel and Paclitaxel for 48 h. IC50 value was calculated and the RI was evaluated. Data were expressed as means ± SEM of three independent experiments. Anticancer drugs
IC50 of MCF7 (M)
IC50 of MCF7/ ADR (M)
RI
Adriamycin Docetaxel Paclitaxel
0.55 ± 0.06 1.14 ± 0.11 1.7 ± 0.14
46.08 ± 5.26 7.38 ± 1.04 5.43 ± 0.85
83.78 6.47 3.19
the manufacturer’s instructions. Reverse transcription reaction was carried out by using the TaKaRa PrimeScript RT reagent Kit from TaKaRa Biotechnology Co. Ltd. (Dalian, China). Real-time PCR amplifications were undertaken in ABI Prism 7500 SDS (Applied Biosystems, Foster City, USA) using SYBR® Premix Ex TaqTM II (TaKaRa Biotechnology, Dalian, China). 2 l of cDNA, 0.8 l each forward and reverse primers (10 M), 6 l distilled/deionized water (ddH2 O), and 10 l TaKaRa SYBR Premix Ex TaqTM (2×) with 0.4 l Rox Reference Dye II (50×). The primers used were as follows: MDR1, forward primer, 5 -CAT TGG CGA GCC TGG TAG-3 , and reverse primer, 5 -TCG TAG GAG TGT CCG TGG AT-3 ; human -actin, forward primer, 5 -CTG TCC CTG TAT GCC TCT G-3 , and reverse primer, 5 -ATG TCA CGC ACG ATT TCC-3 . The optimal PCR condition for samples was 95 ◦ C for 30 s followed by 40 cycles of 95 ◦ C for 5 s, and 60 ◦ C for 34 s. Melt curve analysis was employed at the end of each PCR to confirm the specificity of the PCR product. Each measurement was performed in at least triplicate. Data was analyzed according to the comparative Ct method and the expression of target gene was normalized to -actin expression levels in each sample. 2.10. Electrophoretic mobility shift assay (EMSA) of NF-B
2.6. Enhancement of rhodamine-123 accumulation P-gp is one of the active efflux pumps. It is able to extrude a large number of chemotherapeutic drugs from the cells. 1 × 106 MCF7 or MCF7/ADR cells per well were seeded into 6-well plates and incubated in 5% CO2 at 37 ◦ C for 24 h. Oroxylin A at 30, 60 and 90 M was added in different wells for 48 h. Then plates were washed twice with PBS and 5 g/ml rhodamine 123—the fluorescent P-gp substrate was added. After incubation for 30 min, cells were placed in ice-water to cease the reaction followed by harvesting and twice-washing with ice-cold PBS. Then samples were analyzed by FACSCalibur (Becton-Dickinson). Excitation wavelength and emission wavelength are 488 and 530 nm, respectively. 2.7. Propidium iodide (PI) staining for cell cycle analysis Cell cycle distribution was assayed by determining DNA content treated with oroxylin A (30, 60, 90 M) for 48 h. MCF7/ADR cells were washed twice with PBS, and fixed in 70% ethanol overnight at 4 ◦ C. The fixed cells were then washed with PBS and resuspended in a staining solution containing RNase A (100 L) and propidium iodide (400 L). After 30 min, DNA content was determined using a FACScan laser flow cytometer (FACSCalibur, Becton Dickinson, USA). Data was analyzed using MODFIT and CELLQUEST software (Verity Software House, Topsham, Maine, USA). 2.8. Western blot analysis MCF7/ADR cells treated with oroxylin A (30, 60, 90 M) and verapamil (20 M) combined with 10 M NSC 109555 ditosylate for 48 h. Then cells were collected and lysed in lysis buffer (100 mM Tris–Cl, pH 6.8, 4% (m/v) SDS, 20% (v/v) glycerol, 200 mM b-mercaptoethanol, 1 mM PMSF, and 1 g/ml aprotinin) for 1 h on ice. Lysates were centrifuged at 12,000 × g for 30 min at 4 ◦ C. Meanwhile, after treatment with 90 M oroxylin A for 2, 4, 8 and 12 h, nuclear and cytoplasmic protein of MCF7/ADR cells were extracted using Nuclear and Cytoplasmic Protein Extraction Kit according to the manufacturer’s protocol. The concentration of proteins in the supernatants was detected using the BCA assay with a Varioskan multimode microplate spectrophotometer (Thermo, Waltham, MA, USA) at 562 nm. After which, equal amounts of proteins were separated with 10% or 8% SDS-PAGE gel and transferred onto the nitrocellulose membranes (Millipore, Billerica, MA). Immune complexes were formed by incubation of proteins including Chk2, p-Chk2, P53, p-P53, NF-B, P-gp, actin with primary antibodies overnight at 4 ◦ C followed by IRDyeTM 800 conjugated second antibody for 1 h at 37 ◦ C. Detection was performed by the Odyssey Infrared Imaging System (LI-COR Inc., Superior St. Lincoln, NE, USA). All blots were stripped and probed with polyclonal anti--actin or anti-LaminA antibody to ascertain equal loading of the proteins.
Cell nuclear proteins were extracted as described above. EMSA was performed with a non-radioactive (biotin label) gel shift assay according to the manufacturer’s protocol (Beyotime Institute of Biotechnology, Haimen, China). EMSA reactions were prepared by adding the following components: (a) 8 g of nuclear extract protein from MCF7/ADR cells treated with 90 M oroxylin A at 2, 4, 8, 12 h; (b) 20 l binding buffer; (c) 1 l of biotin-labeled oligonucleotide probe (0.2 M): NF-B (5 -AGT TGA GGG GAC TTT CCC AGG C-3 , 3 -TCAACT CCC CTG AAA GGG TCC G-5 ) consensus oligonucleotide probe was end labeled with biotin with terminal deoxynucleotidyl transferase; (d) wild-type unlabeled NF-B sequence; (e) mutated unlabeled NF-B sequence: NF-B (5 -AGT TGA GGC GAC TTT CCC AGG C-3 , 3 -TCA ACT CCG CTG AAAGGG TCC G-5 ) or AP-1 (5 -CGC TTG ATG ACT TGG CCG GAA-3 , 3 -GCG AAC TAC TGA ACC GGC CTT-5 ); and (f) antibodies of NF-B. The mixture was incubated for 20 min at room temperature. Following the addition of 5 l of sample buffer, the DNA–protein complexes were loaded on a 6% non-denaturing polyacrylamide gel in a 0.5× Tris–borate–EDTA buffer at 380 mA for 1 h and then transferred to positively charged nitrocellulose membrane and cross-linked in a Stratagene cross-linker. Finally, the gel shifts were visualized with Bio-Rad Infrared system by chemiluminescence using the Chemiluminescent EMSA Kit (Beyotime, China). 2.11. Statistical analysis All results shown represent means ± SEM from triplicate experiments performed in a parallel manner unless otherwise indicated. Statistically significant differences (analysis of variance (ANOVA) and Tukey’s tests) were undertaken using SPSS 16.0. See details of each statistical analysis used in the legends of figures.
3. Results 3.1. Determination of multidrug resistance RI was an important metric by which we evaluated the resistance of multidrug resistant cells to various anticancer drugs (Brooks et al., 2003). As shown in Table 1, compared with MCF7 cells, MCF7/ADR cells showed resistance to multiple anticancer drugs. The RI was 83.78 in Adriamycin group, 6.47 in Docetaxel group and 3.19 in Paclitaxel group, which indicated that MCF7/ADR cell line was multidrug resistant. 3.2. Reversal effect of oroxylin A on the MCF7/ADR cells
2.9. Real-time RT-PCR MCF7/ADR cells were treated with 30, 60 and 90 M oroxylin A for 48 h, the real-time RT-PCR was performed to determine mRNA level of P-gp. Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to
To obtain an indication of the potential therapeutic concentration as modulator, MTT method was used to test the toxicity of oroxylin A on MCF7/ADR cells. Oroxylin A inhibited the growth of
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Fig. 2. Reversal effect of oroxylin A in MCF7/ADR cells. MTT assay was used to detect MCF7/ADR cells viability and the effect of oroxylin A on the sensitivity of MCF7/ADR cells toward Adriamycin. (A) After treatment with oroxylin A at different concentrations of oroxylin A for 48 h, growth inhibition was assessed. (B) Cells were treated with various concentrations of Adriamycin in the presence of 30, 60 and 90 M oroxylin A for 48 h. IC50 values for Adriamycin were calculated. Data was expressed as means ± SEM of three independent experiments.
MCF7/ADR cells in a concentration-dependent manner (Fig. 2A). As determined by the concentration–effect curve, 30, 60 and 90 M oroxylin A were weakly cytotoxic (inhibition rate < 15%). So, concentrations of oroxylin A at 30, 60 and 90 M were chosen to assess the reversal effect on MCF7/ADR cells. Additionally, verapamil was used as positive control with a concentration that was 20 M (Ma and Wink, 2008). As shown in Fig. 2B, oroxylin A at concentrations of 30, 60 and 90 M enhanced the sensitivity of MCF7/ADR cells to Adriamycin by 1.16 fold, 2.04 fold and 4.68 fold respectively compared with negative control; the data was 1.71 fold in verapamil group. The reversal effect of the modulator, evaluated by RF, was scored as follows: RF > 1 indicates enhanced drug sensitivity in the presence of the modulator, RF = 1 indicates no effect and RF < 1 indicates decreased drug sensitivity; the greater the RF magnitude is, the more significant is the reversal effect (Yang et al., 2012). From the results, oroxylin A at a concentration of 90 M reversed the resistance of MCF7/ADR cells to Adriamycin markedly.
3.3. Inhibitory effect of oroxylin A on the functional activity and the expression of P-gp P-gp serves to actively transport various xenobiotics including cytotoxic drugs in multidrug resistant cells and mediates MDR to widely used anticancer drugs including anthracyclines, taxanes, Vinca alkaloids, mitoxantrone, and etoposide. The activity of the P-gp drug pump can be determined by the degree of intracellular accumulation of rhodamine-123, which in turn is gauged by the measurement of intracellular fluorescence (Anuchapreeda et al., 2002; Tan et al., 2000). The results demonstrated a 3.25 fold of rhodamine-123 accumulation in MCF7 cells compared to that in MCF7/ADR cells. Compared with negative group, oroxylin A enhanced the rhodamine-123 accumulation to 1.52 fold, 1.57 fold and 1.77 fold at concentrations of 30, 60 and 90 M respectively. Verapamil enhanced rhodamine-123 accumulation by 2.43 fold (Fig. 3A). These data revealed that oroxylin A increased the intracellular accumulation of rhodamine-123 through the inhibition of the cellular efflux function of P-gp in a concentration-dependent manner. P-gp was encoded by the gene MDR1; RT-PCR and western blot assays were used to assess the modulation by oroxylin A of MDR1 mRNA and P-gp expression. As shown in Fig. 3B and C, a high expression of P-gp was observed in MCF7/ADR cells compared with MCF7 cells. Meanwhile, expression of MDR1 mRNA was higher in MCF7/ADR cells (Fig. 3D). However, oroxylin A and verapamil
decreased the expression of P-gp and MDR1 mRNA in MCF7/ADR cells in a concentration dependent manner, suggesting that oroxylin A reversed MDR through down-regulation of P-gp and MDR1 mRNA expression. 3.4. Oroxylin A induced the G2/M arrest and regulated cell cycle related proteins in MCF7/ADR cells A previous study had shown that oroxylin A induced cell-cycle arrest at G2/M phase in human gastric carcinoma BGC-823 cells (Yang et al., 2008). To investigate the effect of oroxylin A on MCF7/ADR cell cycle, the synchronized cells treated with or without oroxylin A were analyzed by flow cytometric analysis. After exposure to oroxylin A for 48 h, MCF7/ADR cells showed a significant increase in the G2/M fraction accompanied by a decrease in the S fraction (Fig. 4A and B). Compared with the negative control group, the percentage of cells in G2/M phase increased by 1.76 fold, 3.30 fold and 4 fold at concentrations of 30, 60 and 90 M respectively. Chk2 is required for the maintenance of G2 arrest in response to DNA damage. After activation, Chk2 can phosphorylate and inhibit Cdc25C, contributing to the maintenance of G2 for cells in the S or G2 phase. Meanwhile, Chk2 phosphorylates P53 on Ser20 , which results in P53 stabilization (Hirao et al., 2000). Tumor suppressor gene, P53 is an upstream suppressor of NF-B-a nuclear transcription factor mediating inflammatory responses. With increased level of P53, it is believed that NF-B transcriptional activity was inhibited (Chen and deGraffenried, 2012). In order to demonstrate the underlying mechanism involved in oroxylin A on arresting cell cycle at G2/M, we monitored the expression of p-Chk2, Chk2, p-P53 and P53 in MCF7/ADR cells after treatment with oroxylin A for 2, 4, 8 and 12 h. Results from the western blot analysis showed that pChk2, P53 and p-P53 were up-regulated while Chk2 and P-gp were down-regulated. The significant change was discovered after drug treatment of 8 h (Fig. 4C and D). 3.5. Oroxylin A reduced NF-B level in nuclear fraction and inhibited the DNA binding activity of NF-B in MCF7/ADR cells Previous reports identified a consensus NF-B binding site in the first intron of the human MDR1 gene and discerned that NFB complexes could bind with this intronic site (Yang et al., 2012). Moreover, NF-B transactivated an MDR1 promoter luciferase construct (Bentires-Alj et al., 2003; Deng et al., 2001). EMSA analysis was used to examine whether the inhibitory effect of oroxylin A on MDR1 expression can be linked to NF-B activity. As shown
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Fig. 3. Inhibitory effect of oroxylin A on the activity and the expression of P-gp. MCF7/ADR cells were treated with oroxylin A or verapamil for 48 h. (A) Intracellular rhodamine-123 fluorescence was measured to assess the efflux pump function of P-gp. (B) Western blot assay was performed to examine the expression of P-gp. (C) Analysis of P-gp expression. (D) The level of MDR1 mRNA expression was determined by RT-PCR. Results represent mean values of three experiments ± SEM, *P < 0.05 compared with MCF7/ADR negative group, **P < 0.01 compared with MCF7/ADR negative group.
in Fig. 5A, NF-B binded effectively to the biotin-labeled oligonucleotide probe as indicated in lane 5. This binding was reduced by the addition of wild-type unlabeled probes, whereas it was not affected by the addition of mutated unlabeled probes. Supershifts are bands that result from the binding of each specific antibody. Treatment of oroxylin A inhibited NF-B binding activity dramatically and in a time-dependent manner. Accordingly, results from Fig. 5B and C showed that, without affecting cytoplasmic protein levels, oroxylin A decreased the expression of NF-B significantly in nuclear extracts and the most obvious decrease was observed at 12 h.
p-Chk2 and p-P53 were reversed by NSC 109555 ditosylate. Similarly, the decreased effect of oroxylin A on P-gp expression was inhibited by NSC 109555 ditosylate. To verify the important role of Chk2 on the reversal effect of oroxylin A in MCF7/ADR cells, MTT assay was used. Result indicated that NSC 109555 ditosylate could partly dismiss the reversal effect of oroxylin A in MCF7/ADR cells (Fig. 6E). Oroxylin A at concentration of 90 M enhanced the sensitivity of MCF7/ADR cells to Adriamycin by 4.68 fold. However, when cells were treated with combinations of oroxylin A and NSC 109555 ditosylate, the RF was only 1.73. To sum up, these data demonstrated that oroxylin A suppressed the P-gp expression by G2/M arrest via Chk2 activation.
3.6. Investigation of the relationship between Chk2, P53 and P-gp 4. Discussion To probe the role of Chk2 on cell cycle, we used a specific inhibitor of Chk2 kinase activity-NSC 109555 ditosylate (Gogineni et al., 2011). MCF7/ADR cells treated with oroxylin A combined with NSC 109555 ditosylate showed a significant lower peak in the G2/M population along with a higher one in the S population than that in oroxylin A treated group (Fig. 6A and B). To further establish the influence of Chk2 on downstream effectors, we carried out western blot assay. As shown in Fig. 6C and D, apparent increase in p-Chk2 and p-P53 were observed in the oroxylin A-treated MCF7/ADR cells with decreased P-gp expression. However, the increased trend of
Mechanisms involved in MDR include expressing the ATPbinding cassette (ABC) transporters (decreased drug uptake and/or increased drug efflux), increasing DNA repair, the cytochrome P450 oxidases in cancer cells, diminishing drug target interaction, altering cell-cycle regulation and so on (Desoize and Jardillier, 2000; Perez-Tomas, 2006). Among them, transmembrane transporter P-gp is the most important and thoroughly studied mechanism. Decreasing P-gp can help to circumvent P-gp-mediated MDR, and has been found to be a common mechanism for various known
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Fig. 4. Oroxylin A induced the G2/M phase arrest and regulated cell cycle related proteins in MCF7/ADR cells. Cells were treated with 30, 60 and 90 M oroxylin A for 48 h. (A) PI staining was used to analyze the cell cycle distribution. (B) Histogram of cell cycle distribution. (C) Western blot assay was performed to detect the expression of cell cycle regulatory proteins extracted from cells treated with 90 M oroxylin A for 2, 4, 8 and 12 h, including p-Chk2, Chk2, p-P53, P53 and P-gp. (D) Analysis of protein expression. Results represent mean values of three experiments ± SEM, *P < 0.05 compared with control, **P < 0.01 compared with control.
Fig. 5. Oroxylin A reduced the expression and DNA binding activity of NF-B in MCF7/ADR cells. Oroxylin A reduced the DNA binding activity of NF-B in MCF7/ADR cells. (A) Nuclear extracts were prepared and assayed for NF-B DNA binding activity by EMSA assay. Assay utilized a labeled probe containing the area of NF-B binding site, and the bolt was representative of three experiments. (B) Nuclear and cytoplasmic extracts were analyzed by western blot assay with anti-NF-B antibodies. (C) Analysis of NF-B expression. Data represent mean values of three experiments ± SEM, *P < 0.05 compared with 0 h group, **P < 0.01 compared with 0 h group.
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Fig. 6. Investigation of the relationship between Chk2, P53 and P-gp. MCF7/ADR cells were pretreated with 10 M/L NSC 109555 ditosylate and then incubated with 90 M oroxylin A for 48 h. PI staining for cell cycle analysis and western blotting on protein extracts were performed. (A) Cell cycle distribution was analyzed using PI staining. (B) Histogram of cell cycle distribution. (C) Western blotting was performed to assay the expression of proteins. (D) Analysis of protein expressions. (E) Cell viability was determined using MTT assay after the incubation of MCF7/ADR cells with various concentrations of Adriamycin in the presence of 90 M oroxylin A and 10 M NSC 109555 ditosylate for 48 h. Results represent mean values of three experiments ± SEM. *P < 0.05 compared with control, **P < 0.01 compared with control. # P < 0.05 compared with oroxylin A group, ## P < 0.01 compared with oroxylin A group.
P-gp inhibitors. It was reported that verapamil significantly reduced P-gp expression in MCF-7/ADR cells (Donmez et al., 2011). The intent of selective inhibition of P-gp is to reduce drug efflux and increase drug concentrations to pharmacologically effective levels in tumors. Thus, the development of specific inhibitors is
of major interest. During recent years, the industrial reservoirs of chemical lead structures have begun to dwindle and increasing attention has been paid to natural products from traditional Asian medicines (Efferth et al., 2007) to find new chemical structures for cancer therapy. Keeping in mind that most of the established
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Scheme 1. Schematic representation of the molecular mechanisms proposed in the reversal effect of oroxylin A in MCF7/ADR cells by G2/M arrest. Oroxylin A-induced DNA damage increased the expression of p-Chk2 and p-P53, resulting in P53 stabilization. With the increased level of p-P53, NF-B was decreased obviously in nucleus and the binding activity was inhibited as well as the expression of P-gp.
resistance-modifying agents (RMA) are too toxic at the required doses, the search for P-gp inhibitors from the field of natural products may be more promising, as many natural products and phytotherapeutics are appreciated for their low side effects and good tolerability. Oroxylin A, a naturally occurring monoflavonoid, shows effective anticancer activities and low toxicities both in vivo and in vitro in previous studies (Liu et al., 2009; Zhao et al., 2010). It can reverse CAM-DR of HepG2 cells by suppressing Integrin1 and its related PI3K/AKT pathway, increase the sensitivity of human hepatoma BEL7402/5-FU cells (Yang et al., 2012; Zhu et al., 2012). Some articles reported that Wogonin, another active flavonoids isolated from the root of Scutellaria baicalensis Georgi, reverses multi-drug resistance of cancer cells via P-gp and other mechanisms (Cheng et al., 2012; Lee et al., 2009; Wang et al., 2007). Our data showed that oroxylin A reversed MDR with more than 4 fold in vitro, increased accumulation of rhodamine 123 in MCF7/ADR cells notably by a concentration-dependent manner, decreased the MDR1 expression both in protein and mRNA level. These results suggested that the molecular mechanism was potentially attributed to the reducing transport activity and the expression of P-gp. Based on previous reports indicating that oroxylin A induced G2/M phase cell-cycle arrest via inhibiting Cdk7-mediated expression of Cdc2/p34 in human gastric carcinoma BGC-823 (Yang et al., 2008), and that the sensitivity of human hepatoma BEL7402/5-FU cells could be increased via down-regulation of P-gp expression by inhibiting NF-B pathway (Yang et al., 2012), we addressed that oroxylin A reversed the MDR of MCF7/ADR cells via G2/M arrest. Further experiments revealed that p-Chk2 played an important role in the oroxylin A inducing G2/M arrest in MCF7/ADR cells. Chk2 can phosphorylate and inhibit Cdc25C, contributing to the maintenance of G2 for cells in S or G2 phases, and it can phosphorylate P53 on Ser20 , which results in P53 stabilization (Hirao et al., 2000). P53 used different mechanism to activate NF-B (Ryan et al., 2000). After treatment of oroxylin A, p-Chk2, p-P53 and P53 were increased substantially, while Chk2 decreased significantly. The results suggested oroxylin A induced G2/M arrest by up-regulating p-Chk2 in MCF7/ADR cells. Previous studies revealed NF-B complexes could bind the first intron of the human MDR1 gene site, the inhibition of NF-B by oroxylin A could down regulate NF-B-dependent transcriptional activity, resulting in suppression of MDR1 activity (Yang et al., 2012). In our study, EMSA assay was carried out to examine the NF-B binding activity. Results showed that oroxylin A inhibited NF-B binding activity in a time-dependent manner in MCF7/ADR cells. To further explore the relationship between p-Chk2 and the reversal effect of oroxylin A on MCF7/ADR cells,
NSC 109555 ditosylate—a Chk2 inhibitor was used. As anticipated, 10 M of NSC 109555 ditosylate could partly dismiss the 90 M oroxylin A-induced G2/M arrest, as well as the reversal resistance of 90 M oroxylin A in MCF7/ADR cells. Meanwhile, p-Chk2 and p-P53 were up-regulated and P-gp was down-regulated after the exposure of MCF7/ADR cells to 90 M oroxylin A at 2 h, 4 h, 8 h and 12 h; again, NSC 109555 ditosylate reversed the trend. As a result, NSC 109555 ditosylate partly dismissed the reversal effect of oroxylin A. These data suggested that p-Chk2 played an important role on the reversal effect of oroxylin A in MCF7/ADR cells. In this study, we revealed that oroxylin A arrested Adriamycin resistant human breast cancer cell line MCF7/ADR in G2/M phase primarily through the up-regulation of p-Chk2, which phosphorylated P53 on Ser20 and resulted in P53 stabilization. With the increased level of p-P53, NF-B was decreased obviously in nucleus and the binding activity was inhibited as well as the expression of P-gp (Scheme 1). Based on the above results, we concluded that oroxylin A reversed MDR of MCF7/ADR cells via inhibition of P-gp and Chk2/P53/NF-B pathway. In summary, our data demonstrated that oroxylin A reversed Pgp-mediated MDR of MCF7/ADR cells via cell cycle arrest at weakly toxic concentrations. Oroxylin A suppressed the expression of P-gp by up-regulating the expression of p-Chk2, p-P53 and reducing the binding activity and the protein level of NF-B in nucleus. Oroxylin A may serve as a promising MDR reversal agent and a potential adjunctive agent for malignant cancer treatment. Conflict of interest statement The authors declare that there is no conflict of interest. Acknowledgments This work was supported by the Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University (No. JKGZ201101), the National Natural Science Foundation of China (Nos. 30973556, 81001452, 91029744, and 81173086), Natural Science Foundation of Jiangsu Province (No. BK2010432), the National Science & Technology Major Project (Nos. 2012ZX09304001, 2012ZX09103101-050), and Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT-IRT1193). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.toxlet. 2013.01.019.
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Oroxylin A reverses P-glycoprotein-mediated multidrug resistance of MCF7/ADR cells by G2/M arrest Litao Zhu a,1 , Li Zhao a,1 , Hu Wang a , Yu Wang a , Di Pan a , Jing Yao a , Zhiyu Li c , Guanzhong Wu b,∗ , Qinglong Guo a,∗ a State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, People’s Republic of China b Department of Pharmacology, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, People’s Republic of China c Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, People’s Republic of China
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
Oroxylin A induced cells G2/M arrest by increasing the expression of pChk2 (Thr68). The resistance was associated with the decreased expression of p-Chk2. Inhibiting p-Chk2 can decrease the reversal fold of oroxylin A. Oroxylin A reversed multidrug resistance by G2/M arrest.
a r t i c l e
i n f o
Article history: Received 3 December 2012 Received in revised form 21 January 2013 Accepted 22 January 2013 Available online 5 March 2013 Keywords: Oroxylin A MCF7/ADR G2/M P-gp Chk2
a b s t r a c t Oroxylin A is a naturally occurring monoflavonoid isolated from the root of Scutellaria baicalensis Georgi, which has been used in traditional Chinese medicine for its anti-tumor, anti-inflammatory and antibacterial properties. The purpose of this study is to investigate the reversal effect and the fundamental mechanisms of oroxylin A in MCF7/ADR cells. Data indicated that oroxylin A showed strong reversal potency in MCF7/ADR cells and the reversal fold (RF) reached 4.68. After treatment with oroxylin A, MCF7/ADR cells displayed reduced functional activity and expression of MDR1 at both the protein and mRNA levels. Meanwhile, oroxylin A induced cells G2/M arrest in a concentration-dependent manner by increasing the expression of p-Chk2 (Thr68). Moreover, western blot and EMSA assays were used to reveal the inhibition of NF-B in nucleus and the suppression of NF-B binding activity by oroxylin A. NSC 109555 ditosylate-Chk2 inhibitor partly dismissed G2/M arrest induced by oroxylin A, reversed the increased trend of p-Chk2 and p-P53 (Ser20), inhibited the decreasing effect of oroxylin A on the expression of P-gp and decreased the reversal fold of 90 M oroxylin A from 4.68 fold to 1.73 fold. In conclusion, we suggested that oroxylin A reversed MDR by G2/M arrest and the underlying mechanism attributed to the suppression of P-gp expression via Chk2/P53/NF-B signaling pathway. © 2013 Elsevier Ireland Ltd. All rights reserved.
Abbreviations: MCF7/ADR, Adriamycin resistant human breast cancer cell line; MDR, multidrug resistance; Chk2, checkpoint kinase 2; NF-B, nuclear factor-kappa B; PMSF, phenylmethylsulfonyl fluoride. ∗ Corresponding authors. Tel.: +86 25 83271055; fax: +86 25 83271055. E-mail addresses: [email protected] (G. Wu), anticancer [email protected] (Q. Guo). 1 These authors contributed equally to this article. 0378-4274/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2013.01.019
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1. Introduction Breast cancer is the most commonly occurring cancer in women, comprising almost one third of all malignancies in females. It comes in a second position after lung cancer as a cause of cancer mortality, and it is the leading cause of death for American women between the ages of 40 and 55 (Richie and Swanson, 2003). Despite the large repertoire of therapies available and the following efforts to incorporate new drugs into clinical practice, it is generally believed that there is a long way to control cancer. A major factor contributing to the failure of chemotherapy in breast cancer is the development of drug resistance (Schultz and Weber, 1999; Stockler et al., 2000). Adriamycin has significant antitumor activity and it is widely used for the treatment of breast cancer (Blum and Carter, 1974), however the high rate of resistance limit its effectiveness and thus its use. Therefore, it is urgently needed to develop new adjuvants that enhance the efficacy of Adriamycin-based chemotherapy and circumvent chemoresistance. Resistance to multiple categories of chemotherapy, termed multidrug resistance (MDR), is a complex hurdle in cancer therapy. The mechanisms behind MDR are still under active investigation, but members of the ATP binding cassette (ABC) transporter superfamily, that often act as cellular efflux pumps for a wide range of chemotherapeutic compounds, have well-established roles (Calcagno et al., 2007; Gottesman et al., 2002). There are at least 49 MDR genes described, of which the ABCB1 gene that encodes an efflux transporter P-glycoprotein (P-gp) or MDR1 which is probably the most well known (Bram et al., 2009). The prognostic significance of P-gp as an indicator for failure in chemotherapy has been demonstrated in a number of clinical studies (Chan et al., 1991; Holmes and West, 1994; Sauerbrey et al., 1994; Verrelle et al., 1991). Other drug substrates of P-gp include calcium channel blockers (e.g. verapamil), antiarrhythmics (e.g. quinidine), steroids (e.g. dexamethasone) and anti-parasitics (e.g. ivermectin), antidepressants and antiepileptic drugs to mention but a few (Ford et al., 1996; Schinkel and Jonker, 2003). Consequently, P-gp not only protects the body from harmful xenobiotics taken up with nutrition, but affects drug absorption, distribution and secretion. Therefore, the protein is of great importance in cancer treatment as well as in general pharmacology. Cell cycle de-regulation resulting in uncontrolled cell proliferation is the one of the most frequent alterations that occur during tumor development (Collins et al., 1997). For this reason, blockage of the cell cycle is regarded as an effective strategy for eliminating cancer cells (Buolamwini, 2000; Hajduch et al., 1999). Two major checkpoints, one at the G1/S transition and another at the G2/M transition, regulate the cell cycle. The G2/M checkpoint plays a key role in the maintenance of chromosomal integrity by allowing cells to repair DNA damage before entering mitosis. DNA damage signaling is mediated by the ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia related) protein kinases. Once activated, these kinases phosphorylate a wide range of target proteins, including the checkpoint kinases (Chk1 and Chk2), the tumor suppressor P53 and the histone variant H2AX (Jowsey et al., 2009). Chk2 is a mammalian homolog of the Saccharomyces cerevisiae Rad53 and Schizosaccharomyce pombe Cds1 checkpoint genes (Blasina et al., 1999; Chaturvedi et al., 1999; Matsuoka et al., 1998). These target proteins, along with many others, act to slow cell cycle progression, regulate transcription, enhance the DNA repair capacity of cells or direct cells to die via apoptosis if the damage encountered is too great. Scutellariae radix, the root of Scutellaria baicalensis, has been widely used in traditional Chinese medicine as an analgesic, antipyretic, anti-inflammation, anticancer, antiviral and antibacterial infections remedy (Sun et al., 2009). Oroxylin A (C16 H12 O5 , Fig. 1) is one of active flavonoids isolated from the root of
Fig. 1. Chemical structure of oroxylin A. The molecular formula of oroxylin A is C16 H12 O5 and the molecular weight is 284.
Scutellaria baicalensis Georgi (Gao et al., 2010). Studies have shown that oroxylin A induced G2/M arrest via inhibiting Cdk7-mediated expression of Cdc2/p34 in human gastric carcinoma BGC-823 cells (Yang et al., 2008), blocked NF-B binding and transcriptional activation associated with decreased p65 proteins in the nucleus induced by LPS (Chen et al., 2000), regulated gene expressions of thymidine synthetase (TS) and dihydropyrimidine dehydrogenase (DPD) and thus increased the sensitivity of human hepatocellular carcinoma HepG2 to 5-fluorouracil (5-FU) in vivo and in vitro (Zhao et al., 2010); furthermore, it reversed CAM-DR via inhibition of Integrin1 and by regulating PI3K/AKT pathway (Zhu et al., 2012). Meanwhile, oroxylin A reversed the multidrug resistance of human BEL7402/5-FU cells via down-regulation of P-gp expression by inhibiting NF-B signaling pathway (Yang et al., 2012). Considering the aforementioned factors, we hypothesized that oroxylin A potentially switch MDR activity via cell cycle arrest. From our results, we confirmed that oroxylin A reversed the resistance of MCF7/ADR cells and the underlying mechanisms are attributed to P-gp-mediated MDR and the activity of Chk2/P53/NFB pathway. 2. Materials and methods 2.1. Materials Oroxylin A was isolated from Scutellariae radix according to the protocols reported previously (Li and Chen, 2005). Oroxylin A was dissolved in dimethyl sulfoxide (DMSO) as a stock solution, stored at −20 ◦ C, and diluted with medium before experiment. The final DMSO concentration did not exceed 0.1% in all experiments. Paclitaxel, Adriamycin, Docetaxel, Propidium iodide (PI), Rhodamine 123, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma–Aldrich (St. Louis, Missouri, USA). NSC 109555 ditosylate, the Chk2 inhibitor, was obtained from Tocris Bioscience. It was dissolved in DMSO at a concentration of 10 mM and stored at −20 ◦ C. Primary antibodies of -actin (1:2000), LaminA (1:500), MDR1 (1:500), NF-B (1:500), p-P53 (1:500), Chk2 (1:500) were obtained from Santa Cruz (Santa Cruz, CA). p-Chk2 (1:800) was from Bioworld (OH, USA). IRDyeTM 800 conjugated secondary antibodies were obtained from Rockland Inc. (Philadelphia, PA). 2.2. Cell culture Parental human breast adenocarcinoma MCF7 cells and Adriamycin reduced drug-resistant MCF7/ADR cells were purchased from Cell Bank of Shanghai Institute of Biochemistry & Cell Biology, Chinese Academy of Sciences and Tumor Center of Beijing. Cells were cultured in RPMI-1640 medium (Gibco, Invitrogen Corporation, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (Sijiqing, Zhejiang, China), 100 U/ml benzyl penicillin, and 100 g/ml streptomycin in a humidified environment with 5% CO2 at 37 ◦ C. To maintain drug resistance, Adriamycin (5 g/ml) was supplied to MCF7/ADR cells at regular intervals. Cells could be harvested and used for experiments 2 weeks after removal of Adriamycin. 2.3. Determination of multidrug resistance To determine the multidrug resistance of MCF7/ADR cells to chemotherapeutic agents, MCF7/ADR cells and the parent MCF7 cells were seeded into 96-well plates and incubated with various concentrations of anticancer drugs (Adriamycin, Docetaxel, Paclitaxel) for 48 h. Each group consisted of five parallel wells. MTT assay was performed to determine the percentage of survived cells at each concentration. After plotting the dose–response curve, IC50 was calculated, from which reversal
L. Zhu et al. / Toxicology Letters 219 (2013) 107–115 index was calculated. The degree of resistance was calculated by resistance index (RI), which obtained from the data = IC50 of MCF7/ADR cells/IC50 of MCF7 cells (Yang et al., 2012). All assays were performed in triplicate. 2.4. Intrinsic cytotoxicity assay MTT colorimetric assay was performed to determine the cytotoxicity of oroxylin A in vitro. MCF7/ADR cells growing in log phase were seeded into 96-well plates at a density of 1 × 104 cells/well and then incubated for 24 h at 37 ◦ C under 5% CO2 . After being exposed to various concentrations of oroxylin A for 48 h, 20 l MTT dye (5 mg/ml) was added to each well and incubated for 4 h. The dye was solubilized with 100 l DMSO and plates were read at 570 nm on an automated microtiter plate reader. Blank wells containing only media and drug were also run as control in all experiments. For required concentrations of reversal multidrug agents, which are neither inhibitory nor toxic (Zheng et al., 2008), final concentrations of 30, 60 and 90 M were chosen. The inhibitory ratio was calculated using the following formula: inhibitory ratio (%) = 1 − average absorbance of treated group/average absorbance of control group × 100% (Zhu et al., 2012). The IC50 values were determined by GraphPad Software. All determinations were carried out in triplicate. 2.5. Reversal effect assay The ability of oroxylin A to reverse MDR in MCF7/ADR cells was measured by MTT method. MCF7/ADR cells were seeded in 96-well plates. Combinations of different concentrations of Adriamycin with oroxylin A (30, 60 and 90 M) or verapamil (20 M) diluted in RPMI1640 were then added. The cells were incubated in a humidified incubator in 5% CO2 at 37 ◦ C for 48 h, and the quantity of viable cells was determined using MTT method. Each group consisted of five parallel wells. The RF values, as the reversal potency, were obtained from fitting the data = IC50 of cytotoxic drug alone/IC50 of cytotoxic drug in the presence of the test drugs (Li et al., 2009).
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Table 1 Determination of IC50 of different anticancer drugs. IC50 values of anticancer drugs were determined in MCF7 and MCF7/ADR cells. Cells were treated with various concentrations of Adriamycin, Docetaxel and Paclitaxel for 48 h. IC50 value was calculated and the RI was evaluated. Data were expressed as means ± SEM of three independent experiments. Anticancer drugs
IC50 of MCF7 (M)
IC50 of MCF7/ ADR (M)
RI
Adriamycin Docetaxel Paclitaxel
0.55 ± 0.06 1.14 ± 0.11 1.7 ± 0.14
46.08 ± 5.26 7.38 ± 1.04 5.43 ± 0.85
83.78 6.47 3.19
the manufacturer’s instructions. Reverse transcription reaction was carried out by using the TaKaRa PrimeScript RT reagent Kit from TaKaRa Biotechnology Co. Ltd. (Dalian, China). Real-time PCR amplifications were undertaken in ABI Prism 7500 SDS (Applied Biosystems, Foster City, USA) using SYBR® Premix Ex TaqTM II (TaKaRa Biotechnology, Dalian, China). 2 l of cDNA, 0.8 l each forward and reverse primers (10 M), 6 l distilled/deionized water (ddH2 O), and 10 l TaKaRa SYBR Premix Ex TaqTM (2×) with 0.4 l Rox Reference Dye II (50×). The primers used were as follows: MDR1, forward primer, 5 -CAT TGG CGA GCC TGG TAG-3 , and reverse primer, 5 -TCG TAG GAG TGT CCG TGG AT-3 ; human -actin, forward primer, 5 -CTG TCC CTG TAT GCC TCT G-3 , and reverse primer, 5 -ATG TCA CGC ACG ATT TCC-3 . The optimal PCR condition for samples was 95 ◦ C for 30 s followed by 40 cycles of 95 ◦ C for 5 s, and 60 ◦ C for 34 s. Melt curve analysis was employed at the end of each PCR to confirm the specificity of the PCR product. Each measurement was performed in at least triplicate. Data was analyzed according to the comparative Ct method and the expression of target gene was normalized to -actin expression levels in each sample. 2.10. Electrophoretic mobility shift assay (EMSA) of NF-B
2.6. Enhancement of rhodamine-123 accumulation P-gp is one of the active efflux pumps. It is able to extrude a large number of chemotherapeutic drugs from the cells. 1 × 106 MCF7 or MCF7/ADR cells per well were seeded into 6-well plates and incubated in 5% CO2 at 37 ◦ C for 24 h. Oroxylin A at 30, 60 and 90 M was added in different wells for 48 h. Then plates were washed twice with PBS and 5 g/ml rhodamine 123—the fluorescent P-gp substrate was added. After incubation for 30 min, cells were placed in ice-water to cease the reaction followed by harvesting and twice-washing with ice-cold PBS. Then samples were analyzed by FACSCalibur (Becton-Dickinson). Excitation wavelength and emission wavelength are 488 and 530 nm, respectively. 2.7. Propidium iodide (PI) staining for cell cycle analysis Cell cycle distribution was assayed by determining DNA content treated with oroxylin A (30, 60, 90 M) for 48 h. MCF7/ADR cells were washed twice with PBS, and fixed in 70% ethanol overnight at 4 ◦ C. The fixed cells were then washed with PBS and resuspended in a staining solution containing RNase A (100 L) and propidium iodide (400 L). After 30 min, DNA content was determined using a FACScan laser flow cytometer (FACSCalibur, Becton Dickinson, USA). Data was analyzed using MODFIT and CELLQUEST software (Verity Software House, Topsham, Maine, USA). 2.8. Western blot analysis MCF7/ADR cells treated with oroxylin A (30, 60, 90 M) and verapamil (20 M) combined with 10 M NSC 109555 ditosylate for 48 h. Then cells were collected and lysed in lysis buffer (100 mM Tris–Cl, pH 6.8, 4% (m/v) SDS, 20% (v/v) glycerol, 200 mM b-mercaptoethanol, 1 mM PMSF, and 1 g/ml aprotinin) for 1 h on ice. Lysates were centrifuged at 12,000 × g for 30 min at 4 ◦ C. Meanwhile, after treatment with 90 M oroxylin A for 2, 4, 8 and 12 h, nuclear and cytoplasmic protein of MCF7/ADR cells were extracted using Nuclear and Cytoplasmic Protein Extraction Kit according to the manufacturer’s protocol. The concentration of proteins in the supernatants was detected using the BCA assay with a Varioskan multimode microplate spectrophotometer (Thermo, Waltham, MA, USA) at 562 nm. After which, equal amounts of proteins were separated with 10% or 8% SDS-PAGE gel and transferred onto the nitrocellulose membranes (Millipore, Billerica, MA). Immune complexes were formed by incubation of proteins including Chk2, p-Chk2, P53, p-P53, NF-B, P-gp, actin with primary antibodies overnight at 4 ◦ C followed by IRDyeTM 800 conjugated second antibody for 1 h at 37 ◦ C. Detection was performed by the Odyssey Infrared Imaging System (LI-COR Inc., Superior St. Lincoln, NE, USA). All blots were stripped and probed with polyclonal anti--actin or anti-LaminA antibody to ascertain equal loading of the proteins.
Cell nuclear proteins were extracted as described above. EMSA was performed with a non-radioactive (biotin label) gel shift assay according to the manufacturer’s protocol (Beyotime Institute of Biotechnology, Haimen, China). EMSA reactions were prepared by adding the following components: (a) 8 g of nuclear extract protein from MCF7/ADR cells treated with 90 M oroxylin A at 2, 4, 8, 12 h; (b) 20 l binding buffer; (c) 1 l of biotin-labeled oligonucleotide probe (0.2 M): NF-B (5 -AGT TGA GGG GAC TTT CCC AGG C-3 , 3 -TCAACT CCC CTG AAA GGG TCC G-5 ) consensus oligonucleotide probe was end labeled with biotin with terminal deoxynucleotidyl transferase; (d) wild-type unlabeled NF-B sequence; (e) mutated unlabeled NF-B sequence: NF-B (5 -AGT TGA GGC GAC TTT CCC AGG C-3 , 3 -TCA ACT CCG CTG AAAGGG TCC G-5 ) or AP-1 (5 -CGC TTG ATG ACT TGG CCG GAA-3 , 3 -GCG AAC TAC TGA ACC GGC CTT-5 ); and (f) antibodies of NF-B. The mixture was incubated for 20 min at room temperature. Following the addition of 5 l of sample buffer, the DNA–protein complexes were loaded on a 6% non-denaturing polyacrylamide gel in a 0.5× Tris–borate–EDTA buffer at 380 mA for 1 h and then transferred to positively charged nitrocellulose membrane and cross-linked in a Stratagene cross-linker. Finally, the gel shifts were visualized with Bio-Rad Infrared system by chemiluminescence using the Chemiluminescent EMSA Kit (Beyotime, China). 2.11. Statistical analysis All results shown represent means ± SEM from triplicate experiments performed in a parallel manner unless otherwise indicated. Statistically significant differences (analysis of variance (ANOVA) and Tukey’s tests) were undertaken using SPSS 16.0. See details of each statistical analysis used in the legends of figures.
3. Results 3.1. Determination of multidrug resistance RI was an important metric by which we evaluated the resistance of multidrug resistant cells to various anticancer drugs (Brooks et al., 2003). As shown in Table 1, compared with MCF7 cells, MCF7/ADR cells showed resistance to multiple anticancer drugs. The RI was 83.78 in Adriamycin group, 6.47 in Docetaxel group and 3.19 in Paclitaxel group, which indicated that MCF7/ADR cell line was multidrug resistant. 3.2. Reversal effect of oroxylin A on the MCF7/ADR cells
2.9. Real-time RT-PCR MCF7/ADR cells were treated with 30, 60 and 90 M oroxylin A for 48 h, the real-time RT-PCR was performed to determine mRNA level of P-gp. Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to
To obtain an indication of the potential therapeutic concentration as modulator, MTT method was used to test the toxicity of oroxylin A on MCF7/ADR cells. Oroxylin A inhibited the growth of
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Fig. 2. Reversal effect of oroxylin A in MCF7/ADR cells. MTT assay was used to detect MCF7/ADR cells viability and the effect of oroxylin A on the sensitivity of MCF7/ADR cells toward Adriamycin. (A) After treatment with oroxylin A at different concentrations of oroxylin A for 48 h, growth inhibition was assessed. (B) Cells were treated with various concentrations of Adriamycin in the presence of 30, 60 and 90 M oroxylin A for 48 h. IC50 values for Adriamycin were calculated. Data was expressed as means ± SEM of three independent experiments.
MCF7/ADR cells in a concentration-dependent manner (Fig. 2A). As determined by the concentration–effect curve, 30, 60 and 90 M oroxylin A were weakly cytotoxic (inhibition rate < 15%). So, concentrations of oroxylin A at 30, 60 and 90 M were chosen to assess the reversal effect on MCF7/ADR cells. Additionally, verapamil was used as positive control with a concentration that was 20 M (Ma and Wink, 2008). As shown in Fig. 2B, oroxylin A at concentrations of 30, 60 and 90 M enhanced the sensitivity of MCF7/ADR cells to Adriamycin by 1.16 fold, 2.04 fold and 4.68 fold respectively compared with negative control; the data was 1.71 fold in verapamil group. The reversal effect of the modulator, evaluated by RF, was scored as follows: RF > 1 indicates enhanced drug sensitivity in the presence of the modulator, RF = 1 indicates no effect and RF < 1 indicates decreased drug sensitivity; the greater the RF magnitude is, the more significant is the reversal effect (Yang et al., 2012). From the results, oroxylin A at a concentration of 90 M reversed the resistance of MCF7/ADR cells to Adriamycin markedly.
3.3. Inhibitory effect of oroxylin A on the functional activity and the expression of P-gp P-gp serves to actively transport various xenobiotics including cytotoxic drugs in multidrug resistant cells and mediates MDR to widely used anticancer drugs including anthracyclines, taxanes, Vinca alkaloids, mitoxantrone, and etoposide. The activity of the P-gp drug pump can be determined by the degree of intracellular accumulation of rhodamine-123, which in turn is gauged by the measurement of intracellular fluorescence (Anuchapreeda et al., 2002; Tan et al., 2000). The results demonstrated a 3.25 fold of rhodamine-123 accumulation in MCF7 cells compared to that in MCF7/ADR cells. Compared with negative group, oroxylin A enhanced the rhodamine-123 accumulation to 1.52 fold, 1.57 fold and 1.77 fold at concentrations of 30, 60 and 90 M respectively. Verapamil enhanced rhodamine-123 accumulation by 2.43 fold (Fig. 3A). These data revealed that oroxylin A increased the intracellular accumulation of rhodamine-123 through the inhibition of the cellular efflux function of P-gp in a concentration-dependent manner. P-gp was encoded by the gene MDR1; RT-PCR and western blot assays were used to assess the modulation by oroxylin A of MDR1 mRNA and P-gp expression. As shown in Fig. 3B and C, a high expression of P-gp was observed in MCF7/ADR cells compared with MCF7 cells. Meanwhile, expression of MDR1 mRNA was higher in MCF7/ADR cells (Fig. 3D). However, oroxylin A and verapamil
decreased the expression of P-gp and MDR1 mRNA in MCF7/ADR cells in a concentration dependent manner, suggesting that oroxylin A reversed MDR through down-regulation of P-gp and MDR1 mRNA expression. 3.4. Oroxylin A induced the G2/M arrest and regulated cell cycle related proteins in MCF7/ADR cells A previous study had shown that oroxylin A induced cell-cycle arrest at G2/M phase in human gastric carcinoma BGC-823 cells (Yang et al., 2008). To investigate the effect of oroxylin A on MCF7/ADR cell cycle, the synchronized cells treated with or without oroxylin A were analyzed by flow cytometric analysis. After exposure to oroxylin A for 48 h, MCF7/ADR cells showed a significant increase in the G2/M fraction accompanied by a decrease in the S fraction (Fig. 4A and B). Compared with the negative control group, the percentage of cells in G2/M phase increased by 1.76 fold, 3.30 fold and 4 fold at concentrations of 30, 60 and 90 M respectively. Chk2 is required for the maintenance of G2 arrest in response to DNA damage. After activation, Chk2 can phosphorylate and inhibit Cdc25C, contributing to the maintenance of G2 for cells in the S or G2 phase. Meanwhile, Chk2 phosphorylates P53 on Ser20 , which results in P53 stabilization (Hirao et al., 2000). Tumor suppressor gene, P53 is an upstream suppressor of NF-B-a nuclear transcription factor mediating inflammatory responses. With increased level of P53, it is believed that NF-B transcriptional activity was inhibited (Chen and deGraffenried, 2012). In order to demonstrate the underlying mechanism involved in oroxylin A on arresting cell cycle at G2/M, we monitored the expression of p-Chk2, Chk2, p-P53 and P53 in MCF7/ADR cells after treatment with oroxylin A for 2, 4, 8 and 12 h. Results from the western blot analysis showed that pChk2, P53 and p-P53 were up-regulated while Chk2 and P-gp were down-regulated. The significant change was discovered after drug treatment of 8 h (Fig. 4C and D). 3.5. Oroxylin A reduced NF-B level in nuclear fraction and inhibited the DNA binding activity of NF-B in MCF7/ADR cells Previous reports identified a consensus NF-B binding site in the first intron of the human MDR1 gene and discerned that NFB complexes could bind with this intronic site (Yang et al., 2012). Moreover, NF-B transactivated an MDR1 promoter luciferase construct (Bentires-Alj et al., 2003; Deng et al., 2001). EMSA analysis was used to examine whether the inhibitory effect of oroxylin A on MDR1 expression can be linked to NF-B activity. As shown
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Fig. 3. Inhibitory effect of oroxylin A on the activity and the expression of P-gp. MCF7/ADR cells were treated with oroxylin A or verapamil for 48 h. (A) Intracellular rhodamine-123 fluorescence was measured to assess the efflux pump function of P-gp. (B) Western blot assay was performed to examine the expression of P-gp. (C) Analysis of P-gp expression. (D) The level of MDR1 mRNA expression was determined by RT-PCR. Results represent mean values of three experiments ± SEM, *P < 0.05 compared with MCF7/ADR negative group, **P < 0.01 compared with MCF7/ADR negative group.
in Fig. 5A, NF-B binded effectively to the biotin-labeled oligonucleotide probe as indicated in lane 5. This binding was reduced by the addition of wild-type unlabeled probes, whereas it was not affected by the addition of mutated unlabeled probes. Supershifts are bands that result from the binding of each specific antibody. Treatment of oroxylin A inhibited NF-B binding activity dramatically and in a time-dependent manner. Accordingly, results from Fig. 5B and C showed that, without affecting cytoplasmic protein levels, oroxylin A decreased the expression of NF-B significantly in nuclear extracts and the most obvious decrease was observed at 12 h.
p-Chk2 and p-P53 were reversed by NSC 109555 ditosylate. Similarly, the decreased effect of oroxylin A on P-gp expression was inhibited by NSC 109555 ditosylate. To verify the important role of Chk2 on the reversal effect of oroxylin A in MCF7/ADR cells, MTT assay was used. Result indicated that NSC 109555 ditosylate could partly dismiss the reversal effect of oroxylin A in MCF7/ADR cells (Fig. 6E). Oroxylin A at concentration of 90 M enhanced the sensitivity of MCF7/ADR cells to Adriamycin by 4.68 fold. However, when cells were treated with combinations of oroxylin A and NSC 109555 ditosylate, the RF was only 1.73. To sum up, these data demonstrated that oroxylin A suppressed the P-gp expression by G2/M arrest via Chk2 activation.
3.6. Investigation of the relationship between Chk2, P53 and P-gp 4. Discussion To probe the role of Chk2 on cell cycle, we used a specific inhibitor of Chk2 kinase activity-NSC 109555 ditosylate (Gogineni et al., 2011). MCF7/ADR cells treated with oroxylin A combined with NSC 109555 ditosylate showed a significant lower peak in the G2/M population along with a higher one in the S population than that in oroxylin A treated group (Fig. 6A and B). To further establish the influence of Chk2 on downstream effectors, we carried out western blot assay. As shown in Fig. 6C and D, apparent increase in p-Chk2 and p-P53 were observed in the oroxylin A-treated MCF7/ADR cells with decreased P-gp expression. However, the increased trend of
Mechanisms involved in MDR include expressing the ATPbinding cassette (ABC) transporters (decreased drug uptake and/or increased drug efflux), increasing DNA repair, the cytochrome P450 oxidases in cancer cells, diminishing drug target interaction, altering cell-cycle regulation and so on (Desoize and Jardillier, 2000; Perez-Tomas, 2006). Among them, transmembrane transporter P-gp is the most important and thoroughly studied mechanism. Decreasing P-gp can help to circumvent P-gp-mediated MDR, and has been found to be a common mechanism for various known
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Fig. 4. Oroxylin A induced the G2/M phase arrest and regulated cell cycle related proteins in MCF7/ADR cells. Cells were treated with 30, 60 and 90 M oroxylin A for 48 h. (A) PI staining was used to analyze the cell cycle distribution. (B) Histogram of cell cycle distribution. (C) Western blot assay was performed to detect the expression of cell cycle regulatory proteins extracted from cells treated with 90 M oroxylin A for 2, 4, 8 and 12 h, including p-Chk2, Chk2, p-P53, P53 and P-gp. (D) Analysis of protein expression. Results represent mean values of three experiments ± SEM, *P < 0.05 compared with control, **P < 0.01 compared with control.
Fig. 5. Oroxylin A reduced the expression and DNA binding activity of NF-B in MCF7/ADR cells. Oroxylin A reduced the DNA binding activity of NF-B in MCF7/ADR cells. (A) Nuclear extracts were prepared and assayed for NF-B DNA binding activity by EMSA assay. Assay utilized a labeled probe containing the area of NF-B binding site, and the bolt was representative of three experiments. (B) Nuclear and cytoplasmic extracts were analyzed by western blot assay with anti-NF-B antibodies. (C) Analysis of NF-B expression. Data represent mean values of three experiments ± SEM, *P < 0.05 compared with 0 h group, **P < 0.01 compared with 0 h group.
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Fig. 6. Investigation of the relationship between Chk2, P53 and P-gp. MCF7/ADR cells were pretreated with 10 M/L NSC 109555 ditosylate and then incubated with 90 M oroxylin A for 48 h. PI staining for cell cycle analysis and western blotting on protein extracts were performed. (A) Cell cycle distribution was analyzed using PI staining. (B) Histogram of cell cycle distribution. (C) Western blotting was performed to assay the expression of proteins. (D) Analysis of protein expressions. (E) Cell viability was determined using MTT assay after the incubation of MCF7/ADR cells with various concentrations of Adriamycin in the presence of 90 M oroxylin A and 10 M NSC 109555 ditosylate for 48 h. Results represent mean values of three experiments ± SEM. *P < 0.05 compared with control, **P < 0.01 compared with control. # P < 0.05 compared with oroxylin A group, ## P < 0.01 compared with oroxylin A group.
P-gp inhibitors. It was reported that verapamil significantly reduced P-gp expression in MCF-7/ADR cells (Donmez et al., 2011). The intent of selective inhibition of P-gp is to reduce drug efflux and increase drug concentrations to pharmacologically effective levels in tumors. Thus, the development of specific inhibitors is
of major interest. During recent years, the industrial reservoirs of chemical lead structures have begun to dwindle and increasing attention has been paid to natural products from traditional Asian medicines (Efferth et al., 2007) to find new chemical structures for cancer therapy. Keeping in mind that most of the established
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Scheme 1. Schematic representation of the molecular mechanisms proposed in the reversal effect of oroxylin A in MCF7/ADR cells by G2/M arrest. Oroxylin A-induced DNA damage increased the expression of p-Chk2 and p-P53, resulting in P53 stabilization. With the increased level of p-P53, NF-B was decreased obviously in nucleus and the binding activity was inhibited as well as the expression of P-gp.
resistance-modifying agents (RMA) are too toxic at the required doses, the search for P-gp inhibitors from the field of natural products may be more promising, as many natural products and phytotherapeutics are appreciated for their low side effects and good tolerability. Oroxylin A, a naturally occurring monoflavonoid, shows effective anticancer activities and low toxicities both in vivo and in vitro in previous studies (Liu et al., 2009; Zhao et al., 2010). It can reverse CAM-DR of HepG2 cells by suppressing Integrin1 and its related PI3K/AKT pathway, increase the sensitivity of human hepatoma BEL7402/5-FU cells (Yang et al., 2012; Zhu et al., 2012). Some articles reported that Wogonin, another active flavonoids isolated from the root of Scutellaria baicalensis Georgi, reverses multi-drug resistance of cancer cells via P-gp and other mechanisms (Cheng et al., 2012; Lee et al., 2009; Wang et al., 2007). Our data showed that oroxylin A reversed MDR with more than 4 fold in vitro, increased accumulation of rhodamine 123 in MCF7/ADR cells notably by a concentration-dependent manner, decreased the MDR1 expression both in protein and mRNA level. These results suggested that the molecular mechanism was potentially attributed to the reducing transport activity and the expression of P-gp. Based on previous reports indicating that oroxylin A induced G2/M phase cell-cycle arrest via inhibiting Cdk7-mediated expression of Cdc2/p34 in human gastric carcinoma BGC-823 (Yang et al., 2008), and that the sensitivity of human hepatoma BEL7402/5-FU cells could be increased via down-regulation of P-gp expression by inhibiting NF-B pathway (Yang et al., 2012), we addressed that oroxylin A reversed the MDR of MCF7/ADR cells via G2/M arrest. Further experiments revealed that p-Chk2 played an important role in the oroxylin A inducing G2/M arrest in MCF7/ADR cells. Chk2 can phosphorylate and inhibit Cdc25C, contributing to the maintenance of G2 for cells in S or G2 phases, and it can phosphorylate P53 on Ser20 , which results in P53 stabilization (Hirao et al., 2000). P53 used different mechanism to activate NF-B (Ryan et al., 2000). After treatment of oroxylin A, p-Chk2, p-P53 and P53 were increased substantially, while Chk2 decreased significantly. The results suggested oroxylin A induced G2/M arrest by up-regulating p-Chk2 in MCF7/ADR cells. Previous studies revealed NF-B complexes could bind the first intron of the human MDR1 gene site, the inhibition of NF-B by oroxylin A could down regulate NF-B-dependent transcriptional activity, resulting in suppression of MDR1 activity (Yang et al., 2012). In our study, EMSA assay was carried out to examine the NF-B binding activity. Results showed that oroxylin A inhibited NF-B binding activity in a time-dependent manner in MCF7/ADR cells. To further explore the relationship between p-Chk2 and the reversal effect of oroxylin A on MCF7/ADR cells,
NSC 109555 ditosylate—a Chk2 inhibitor was used. As anticipated, 10 M of NSC 109555 ditosylate could partly dismiss the 90 M oroxylin A-induced G2/M arrest, as well as the reversal resistance of 90 M oroxylin A in MCF7/ADR cells. Meanwhile, p-Chk2 and p-P53 were up-regulated and P-gp was down-regulated after the exposure of MCF7/ADR cells to 90 M oroxylin A at 2 h, 4 h, 8 h and 12 h; again, NSC 109555 ditosylate reversed the trend. As a result, NSC 109555 ditosylate partly dismissed the reversal effect of oroxylin A. These data suggested that p-Chk2 played an important role on the reversal effect of oroxylin A in MCF7/ADR cells. In this study, we revealed that oroxylin A arrested Adriamycin resistant human breast cancer cell line MCF7/ADR in G2/M phase primarily through the up-regulation of p-Chk2, which phosphorylated P53 on Ser20 and resulted in P53 stabilization. With the increased level of p-P53, NF-B was decreased obviously in nucleus and the binding activity was inhibited as well as the expression of P-gp (Scheme 1). Based on the above results, we concluded that oroxylin A reversed MDR of MCF7/ADR cells via inhibition of P-gp and Chk2/P53/NF-B pathway. In summary, our data demonstrated that oroxylin A reversed Pgp-mediated MDR of MCF7/ADR cells via cell cycle arrest at weakly toxic concentrations. Oroxylin A suppressed the expression of P-gp by up-regulating the expression of p-Chk2, p-P53 and reducing the binding activity and the protein level of NF-B in nucleus. Oroxylin A may serve as a promising MDR reversal agent and a potential adjunctive agent for malignant cancer treatment. Conflict of interest statement The authors declare that there is no conflict of interest. Acknowledgments This work was supported by the Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University (No. JKGZ201101), the National Natural Science Foundation of China (Nos. 30973556, 81001452, 91029744, and 81173086), Natural Science Foundation of Jiangsu Province (No. BK2010432), the National Science & Technology Major Project (Nos. 2012ZX09304001, 2012ZX09103101-050), and Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT-IRT1193). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.toxlet. 2013.01.019.
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