Cell culture
The T24 human bladder cancer cells were supplied by the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. The HCV-29 normal bladder epithelial cells and J82 human bladder cancer cells were provided by the Department of Cell Biology, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine. All cell lines were cultured in RPMI 1640 medium (Invitrogen Corp, Carlsbad, CA, USA) supplemented with 10 % fetal bovine serum at 37 °C in a humidified atmosphere containing 5 % CO2. Emodin was obtained from Sigma (St. Louis, MO, USA). Glutathione (GSH) assay kit was purchased from Jiancheng Bioengineering Institute (Nan Jing, China). Cisplatin was bought from Qilu Pharmaceutical Co., Ltd. (Nan Jing, China). N-acetylcysteine (NAC), the precursor of GSH, was provided by Sigma (St. Louis, MO, USA). For experiments of 2.3, 2.4, 2.5, 2.6, and 2.7, T24 and HCV-29 cells were treated with emodin (20 μM), cisplatin (1.5 μg/ml), or emodin/cisplatin co-treatment, respectively. J82 cells were treated with emodin (15 μM), cisplatin (1 μg/ml), or emodin/cisplatin co-treatment, respectively.
Cell viability and apoptosis analysis
Cells were seeded in 96-well plates with 2.0 × 104 cells per well. The cells were incubated with emodin for 24 h at different concentrations (0, 5, 10, 20, 30, 40, 50, 60, 70 μM) and chose the critical concentration (20 μM) treated with cells for 0, 6, 12, 24, 48, 72, 96 h. The cells were incubated with cisplatin for 24 h at different concentrations (0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 μg/ml). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MO, USA) assay was used to analyze the cell viability as previously described [22]. Cells were treated with drugs for 24 h and apoptotic rates were assessed with flow cytometry using AnnexinV-fluorescein isothiocyanate (AnnexinV-FITC)/propidium iodide (PI) kit (BD Pharmingen, San Diego, CA, USA). Samples were prepared according to the manufacturer’s instruction and analyzed by a flow cytometry (FCM) Calibur (Becton Dickson, San Diego, CA, USA).
ROS measurement and GSH detection
2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA) method was used for intercellular ROS accumulation [13]. After cells were treated with different regimens, cells were further incubated with 10 mM DCFH-DA for 15mins at 37 °C, with re-incubation of NAC (5 mM) for 4 h, if used. After washed once with ice-cold phosphate buffer saline (PBS), cells were harvested and kept on ice for an immediate detection by FCM. GSH measurement was conducted according to the instruction of assay kit (Jiancheng Bioengineering Institute, Nan Jing, China). The GSH content of the samples was detected as described by Wang et al [19].
Western blotting
T24 cells were plated in 6 well plates and treated with different regimens for 24 h before lysed in 100 μl of sample solution as previously used by Huang et al [16]. Equal amounts of proteins were electrophoresed on 12 % SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was incubated for 1 h in blocking buffer (5 % low-fat milk powder in blocking buffer containing) and then incubated with the mouse antibody against human MDR1, MRP1, MRP2 and ABCG2 (Abcam, Cambridge, UK) at 4 °C for overnight and horseradish peroxidase-conjugated goat anti-mouse immunoglobulin (Sigma, St. Louis, MO, USA) for 1 h before detected by an enhanced chemiluminescence system. The details of antibodies used in this study were shown in Additional file 1: Table S1.
qPCR and RT-PCR analysis
Total mRNA was extracted from treated cells using trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the instruction of the manufacturer. The cDNA was reverse-transcribed from 2 μg total RNA. β-actin was used as an internal control. For qPCR, detection of PCR products was performed on a Light Cycler system (Roche Applied Science, Basel, Switzerland) using the SYBR Green I kit (TaKaRa Biotechnology, Dalian, China), according to the manufacturer’s instructions. Each sample was done in triplicate. The expression levels of transporters were normalized to β-actin mRNA expression. The RT-PCR was performed as follows: denaturation for 5min at 95 °C, 30cycles of 95 °C for 30s, 55 °C for 45s and 72 °C for 30s, then extended for 10 min at 72 °C. The sequences for β-actin sense and antisense primers were shown in Additional file 2: Table S2.
MRP1 siRNA transfection
MRP1 siRNA oligonucleotides were transiently transfected, using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions with modifications as previously described [23, 24]. In brief, cells were 50 % confluent at the time of transfection. Oligomer-Lipofectamine® 2000 complexes were added to each well containing cells and medium. Mix gently by rocking the plate back and forth. After that, cells were incubated at 37 °C in a CO2 incubator for 48 h and medium were changed after 4–6 h. A nonspecific siRNA was transfected as control, which was randomly synthesized and did not correspond to any known gene in the genome database. Forty-eight hours later, cells were lysed for RT-PCR to verify the efficiency of silencing. After that, T24 cells were treated by cisplatin and the rate of cell apoptosis was detected by FCM described above. The siRNA sequences for MRP1 and nonspecific control were shown in Additional file 2: Table S2.
In vivo study in tumor-bearing mice
All the animal experiments were conducted according to institutional guidelines for animal welfare and animal ethics were approved for all the experiments from the animal committee of the Second Military Medical University. 3 × 106 T24 cells were harvested, washed, and resuspended in serum-free optimum medium and then injected subcutaneously into 6-week old BALB/c-nu/nu mice (n = 8 mice per group, purchased from Shanghai Experimental Animal Center, Shanghai, China). Three days after inoculation, the mice were intraperitoneally administered with PBS, emodin (50mg/kg), cisplatin (1mg/kg), or emodin/cisplatin every two days. On day 18, every mouse was sacrificed. After body weight measurement, tumors were isolated, weighted and fixed in 4 % paraformaldehyde (PFA). Hearts, livers and kidneys were stained with Hematoxylin & Eosin to determine the systemic toxicity as described by Li et al [22]. Terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick end label (TUNEL) assay (Intergen, NY, USA) was performed on paraformaldehyde-fixed and paraffin-embedded tumor sections, using the methods described previously [19].
Immunohistochemistry
MRP1 expression in tumor tissues was detected via immunohistochemistry. Briefly, all tumors were fixed in 4 % PFA, embedded in paraffin, and then cut into 5-μm paraffin sections for immunohistochemistry. Deparaffinized sections were dehydrated with alcohol series, then incubated with a monoclonal antibody, (Abcam, Cambridge, UK) at 4 °C overnight to detect MRP1 protein. The protein expression was defined as those showing cytomembrane or/and cytoplasm brown staining. Slides were then mounted using an aqueous solution and photographed.
Statistical analysis
Statistical analyses were performed using SPSS Statistics 17.0 software (SPSS Inc., Chicago, IL, USA) and results were considered statistically significant at p < 0.05 (two tailed). Data were shown as mean values ± S.D., and some of the data were displayed in the form of chart. The corresponding experimental figures were drawn using GraphPad Prism v 5.0 software (Graphpad Software Inc, La Jolla, CA, USA).