Preparation of the extracts for ZJW
Rhizoma Coptidis and Evodia were purchased from Lei-Yun-Shang Pharmaceutical Group (Shanghai) and identified as the coptis of ranunculaceae and the fruit of Tetradium, respectively. They were formally identified by Professor DZ Wu (The School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai, China).
ZJW was formulated by Rhizoma Coptidis and Evodia (in a ratio of 6:1). All the herbs were purchased from ShuGuang Hospital herb store and were done as described previously [1]. Briefly, the mixture (70 g) was extracted twice for 1 h each time by refluxing in ethanol (1:8, v/v). The filtrates were concentrated and dried in vacuum at 60°C. The concentrated extract was then dried by lyophilization to obtain the ZJW extract at a yield of dried powder of 24.4%. The extract was stored at 4°C, and its preparations were standardized, regulated and quality controlled according to the guidelines defined by Chinese State Food and Drug Administration (SFDA).
Cell culture and reagents
Human colorectal cancer HCT116 cells were purchased from the Shanghai Cell Collection (Shanghai, China). The HCT116/L-OHP MDR cell lines were established and maintained in our laboratory. Cells were grown in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal calf serum, 2 mM glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin (Invitrogen, Carlsbad, CA) at 37°C in a 5% CO2 humidified atmosphere. HCT116/L-OHP cells were routinely maintained in a medium containing 5,000 ng/mL oxaliplatin (L-OHP). Monoclonal antibodies against P-gp, MRP-2, LRP, IκB, Akt, p65, and GAPDH were purchased from Cell Signaling Technology (Beverly, MA, USA).
Cell viability assays
Cell proliferation was determined using the cell counting kit, CCK-8. Briefly, cells were seeded in 96-well plates at 1 × 104 cells/well. When the cells reached 60% confluence, the medium was removed and replaced with fresh medium containing varying concentrations of ZJW or its mixture with anti-tumor drug (L-OHP) and incubated for 48 h. The CCK-8 assay was then performed: after 2 h of incubation with culture medium containing the CCK-8 reagent, the absorbance was read at 450 nm using a microplate assay reader (Labsystems Dragon, Wellscan). Relative inhibitory rate of cell growth was calculated according to the formula: R = (A2 − A1)/A2 × 100% and P = A1/A2 × 100%, in which R was relative inhibitory rate, P was relative proliferation ratio of cell growth, A1 is the mean absorbance of transfected cells, and A2 is the mean absorbance value of untransfected control cells without drug treatment. All experiments were done with five wells per experiment in triplicate.
Apoptosis and cell-cycle assay
Cells were seeded in 6-well plates (4 × 105 cells/well). After 12 h, three dose concentrations of ZJW were added. Flow cytometry was used to detect apoptosis by determining the relative amount of AnnexinV-FITC-positive-PI-negative cells, as previously described [1]. Unstained cells, cells stained with Annexin V-FITC alone, and cells stained with propidium iodide alone were used as controls. Singly stained cells were used to adjust electronic compensation on the FL1 and FL2 channels. After apoptosis assay, cell cycle distributions were analyzed with the ModFit program (BD, San Diego, CA, USA). All samples were assayed in triplicate, and the fraction of each cell cycle phase was calculated.
Western blot analysis
Whole cell lysate for SDS-PAGE and western blot analysis for P-gp, MRP-2, LRP, IκB, Akt, p65, and phosphorylation of Akt and IκB expression was prepared as previously reported [1]. The lysate was incubated on ice in immunoprecipitation assay buffer for 2 h before being homogenized using a mortar and pestle. The homogenized sample was centrifuged, and the supernatant was collected and stored at −80°C. Equal loading was confirmed with GAPDH. Densitometric analysis was done using the Scion Imaging software (Scion Corporation), with GAPDH as internal control.
Quantitative RT-PCR analysis
Tumor cells were homogenized and suspended with an RNAspin Mini Kit (GE Healthcare, Waukesha, WI, USA) for RNA isolation according to the manufacturer’s instruction. For cDNA synthesis, 1 μg of total RNA was reverse-transcribed using oligo-dT primers and the Superscript Amplification System (Life Technologies, Carlsbad, CA, USA). Quantitative RT-PCR was carried out using SYBR Green PCR Master Mix (Life Technologies). The PCR parameters consisted of initial polymerase activation at 94°C for 5 min, followed as previously described [1]. Amplification of GAPDH RNA, a relatively invariant internal reference was performed in parallel, and cDNA amounts were normalized to equivalent GAPDH mRNA levels. Oligonucleotide primers for ABCB1 and GAPDH were as follows: Oligonucleotide sequence of ABCB1 (379 bp), F: 5′-TAATGCGACAGGAGATAGG-3′, R: 5′-TGCCATTGACTGAAAGAAC-3′, Oligonucleotide sequence of GAPDH (306 bp), F: 5′-ACCCACTCCTCCACCTTTGA-3′, R: 5′-CTGTTGCTGTAGCCAAATTCGT-3′.
ABCB1 promoter activity by vector transient transfection and dual luciferase assay
Transfection procedures were performed according to manufacturers’ instructions, with Lipofectamin 2000 as transfection reagent (Invitrogen). Briefly, 2 × 103 cells were plated in each well of a 96-well plate and incubated overnight. A mixture of Lipofectamine 2000 (10 nM) with ABCB1 promoter recombinant vector pGL3-Basic-ABCB1 promoter (0.8 μg/well) was added, followed by a 48 h incubation in regular medium. Then cells were harvested and analyzed using a dual-luciferase assay kit (Shanghai Lai’an Biotech. Co., Ltd, Shanghai, China) as previously reported [1].
Chromatin immunoprecipitation
Chromatin immunoprecipitation (ChIP) was done as described previously [16]. N-ethylmaleimide was added to the cell lysis buffer at a final concentration of 10 mM to preserve poly-ubiquitinated protein conjugates.
Statistical analysis
All experimental data are expressed as mean ± standard deviation of at least three independent experiments performed in duplicate. Significance was determined by a one-way analysis of variance (ANOVA) and Holm’s multiple-comparison test. Statistical significance was set at a P-value of less than 0.05. All analyses were carried out using SPSS13.0 (SPSS, Chicago, IL, USA).