Human cell line cultures and chemotherapeutic agents
Two human CCA cell lines, KKU-100 and KKU-M214, were developed from tumor tissues of CCA patients at the Srinagarind Hospital, Faculty of Medicine, Khon Kaen University. Liver Chang cells and normal bile duct epithelial cells, MMNK1, were also used in this study. CCA cells and normal cells were routinely cultured in Ham’s F12 media, supplemented with 4 mmol/L L-glutamine, 12.5 mmol/L N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES), at pH 7.4, 100 U/mL penicillin, 100 μg/mL streptomycin sulfate, and 10% fetal bovine serum (FBS) in a humidified atmosphere containing 5% CO2 at 37°C. The media was renewed every 2–3 days. After the cells became confluent, cells were trypsinized with 0.25% trypsin-EDTA and subcultured in the same media. Some aliquots of cells were transferred to freezing medium containing 10% DMSO and stored at -80°C for subsequent use.
Chemotherapeutic agents were selected on the basis of the frequent usage for CCA, gastrointestinal tract cancers and solid tumors. These included 5-fluorouracil (5-FU) dissolved in DMSO (100 mM), doxorubicin HCl (Boryung Pharm, Seoul, South Korea: Doxo) dissolved in DMSO (100 mM), and gemcitabine (Gemzar, Eli Lilly, IN, USA: Gem) dissolved in phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4). They were added to the culture media without FBS to make final concentrations indicated in the “Results” section and incubated for a designated period of time.
Transient transfection of NQO1 and/or p53 small interfering RNA (NQO1 and/or p53 siRNA)
Pre-designed NQO1 siRNA (siGENOME SMARTpool siRNA #M-005133-02-0020), p53 siRNA (siGENOME SMARTpool siRNA #M-003329-03-0005), and control siRNA (siGENOME non-targeting siRNA #D-001210-02-20) were purchased from Thermo Scientific. In this study, NQO1 siRNA and p53 siRNA were the pooled siRNAs, each is composed of four different sequences of siRNA, targeting for NQO1 and p53, respectively. For transfection of the siRNA, 1.5×105 KKU-100 cells were plated in 6-well plates and grown in Ham’s F12 medium supplemented with FBS, without antibiotics. The cells were transfected with 50 or 100 pmole of the siRNA for 6 hr using 0.4 or 2 μL of Lipofectamine™ 2000 reagent (Invitrogen, Calsbad, CA, USA) in 500 μL of Ham’s F12 medium without FBS and antibiotics. After transfection, the cells were added with 1.5 mL of Ham’s F12 medium supplemented with FBS, without antibiotics, and incubated further for 24-48 hr. The efficiency of the NQO1 knockdown by transient transfection was determined by gene expression with reverse transcription real-time polymerase chain reaction (RT-qPCR) using specific primers, NQO1 activity assay, and Western blotting analysis.
For cytotoxicity assay, CCA cells were seeded onto 96-well cultured plates with FBS, without antibiotics at a density of 5 × 103 cells/well for an overnight. The cells were transfected with 3 pmole of the siRNA for 6 hr using 0.06 μL of Lipofectamine™ 2000 reagent in 100 μL of Ham’s F12 medium without FBS and antibiotics. After 6 hr, the cells were added 100 μL of Ham’s F12 medium supplemented with FBS, without antibiotics, and incubated for 48 hr. The cells were then incubated with chemotherapeutic agents in serum free medium for additional 24 hr.
Transfection of NQO1 vector into CCA cells
A plasmid encoding human wild-type NQO1 in pCMV6-XL5 (4,707 bp) was purchased from Origene Technologies (#SC119599; Rockville, MD). The insert cDNA (1,120 bp) contained the complete NQO1 coding sequence (NM_000903.2). For transfection of the pCMV6-XL5-NQO1 or pCMV6-XL5, as a negative control vector, KKU-M214 at a density of 5×105 cells were plated in 6-well plates and grown overnight. At 70-80% confluent condition, cells were transfected with 2.5 μg of pCMV6-XL5-NQO1 or pCMV6-XL5 for 24 hr using Lipofectamine® LTX and Plus™ reagent (Invitrogen) protocol as directed by the manufacturer in 2 mL of Ham’s F12 medium without FBS and antibiotics. Then the cells were collected for Western blot analysis and enzymatic assay. The empty vector control was prepared by cutting the NQO1 insert site from pCMV6-XL5-NQO1 plasmid at the EcoRI and XbalI site. The bearing vector was ligated with oligonuclotide (non-coding sequence) and cloned into E. coli (JM109). The empty vector control was purified and the presence of vector was confirmed by restriction digestion and run it on 2% agarose gel.
For cytotoxicity assay, KKU-M214 cells were seeded onto 96-well cultured plates at a density of 7.5 × 103 cells/well for an overnight, the cells were transfected with 100 ng of pCMV6-XL5-NQO1 or pCMV6-XL5 using Lipofectamine® LTX and Plus™ reagent for 24 hr. The cells were then incubated with chemotherapeutic agents in serum free medium for additional 24 hr (Doxo) or 48 hr (5-FU and Gem), since it was the optimal incubation time for each drug.
NQO1 enzyme activity assay
NQO1 assay was performed according to the method described previously . Cells were seeded at 7.5 × 103 cells/well in flat-bottomed 96-well cultured plates overnight. After cells were cultured for the designated time, cells were lysed with 50 μL solution containing 0.8% digitonin and agitated on a shaker at room temperature for 10 min. Twenty-five microliter of 0.55% dicoumarol was added into culture wells designated as baseline activity, while the corresponding paired wells were added with distilled water (DW) designated as the test activity wells. After that, all wells were added with 200 μL of reaction mixture (the following stock solution was prepared for each set of assay: 7.5 mL of 0.5 M Tris–HCl (pH 7.4), 100 mg of bovine serum albumin (BSA), 1 mL of 1.5% Tween-20 solution, 0.1 mL of 7.5 mM FAD, 1 mL of 150 mM glucose-6-phosphate, 100 μL of 50 mM β-NADP, 275 unit of yeast glucose-6-phosphate dehydrogenase, 45 mg of MTT, and DW to a final volume of 150 mL and menadione (1 μL of 50 mM menadione dissolved in acetonitrile per milliliter of reaction mixture) was added just before the mixture is dispensed into the microtiter plates. A blue color developed and the plates were placed into a microplate reader with filter wavelength of 620 nm and readings were made at 0.5 min interval for about 10 min. The rate of increase of the optical readings with times represents the activity of the reaction. Using the extinction coefficient of MTT formazan of 11,300 M-1 cm-1 at 610 nm and correction for the light path of the microplate, NQO1 activity was expressed as nmol/min/mg protein.
Cytotoxicity or SRB assay
Cytotoxicity testing is used to evaluate the effects of chemotherapeutic agents. In brief, CCA cells were seeded onto 96-well cultured plates at a density of 7.5 × 103 cells/well overnight, then media was renewed with fresh media containing test compound and further incubated for the indicated times. Assay was performed at the endpoint of treatment to determine amount of protein remaining in each well. Media was discarded and replaced with 100 μL of ice-cold 10% trichloroacetic acid (TCA) and placed in 4°C for at least 1 hr. Then TCA was removed and wells were carefully rinsed with deionized (DI) water for 5 times. After 10 min of air drying, 50 μL of 0.4% sulforhodamine B (SRB) in 1% acetic acid was added for 30 min. Cells were rinsed 3–4 times with 1% acetic acid and air dried for 1 hr at room temperature. Finally, adhered cells were solubilized with 200 μL of 10 mM Tris base and plates were shaken for 20 min before absorbance reading with a microplate reader with filter wavelength of 540 nm.
Real-time polymerase chain reaction (real-time PCR or qPCR)
CCA cells were seeded in 6-well plates at the density of 1.5×105 cells/well. Total RNA was extracted from CCA cell lines using TRIzol® reagent following the manufacturer’s instructions (Invitrogen). Total RNA was isolated using a previously described method . Total RNA (1 μg) was reverse transcribed in a 20 μL reaction mixture, containing 0.5 μg of oligo(dT)15 primer, 20 U of RNasin® ribonuclease inhibitor, and 200 U of ImProm-II™ reverse transcriptase in 1× PCR buffer, 3 mmol/L MgCl2, and 1 mmol/L dNTPs. The first-strand cDNA was synthesized at conditions of 42°C for 60 min. The reverse transcription products served as templates for real-time PCR. PCR amplification was performed using specific primers for the NQO1, wild type p53 and the internal control using β-actin. The primer sequences were as follows: 1) NQO1 (NM_000903.2): forward primer 5’-GGCAGAAGAGCACTGATCGTA-3’ and reverse primer 5’-TGATGGGATTGAAGTTCATGGC-3’; 2) wild type p53 (NM_005256778.1) : forward primer 5’-ATGGAGGAGCCGCAGTCAGATCC-3’ and reverse primer 5’-TTCTGTCTTCCCGGACTGAGTCTGACT-3’; 3) β-actin: forward primer 5’-TGCCATCCTAAAAGCCAC-3’ and reverse primer 5’-TCAACTGGTCTCAAGTCAGTG-3’. The real-time fluorescence PCR, based on EvaGreen® dye, was carried out in a final volume of 20 μL containing 1x SsoFast™ EvaGreen® supermix (#172-5200; Bio-Rad, CA, USA), 0.5 μmol/L of each NQO1 or wild type p53, and 0.25 μmol/L of β-actin primer. Thermal cycling was performed for each gene in duplicate on cDNA samples in 96-well reaction plates using the ABI 7500 Sequence Detection system (Applied Biosystems). A negative control was also included in the experimental runs. The negative control was set up by substituting the template with DI water. Real-time PCR was conducted with the following cycling conditions: 95°C for 3 min, followed by 40 cycles of 95°C for 15 s and 60°C for 31 s. To verify the purity of the products, a melting curve analysis was performed after each run. Upon completion of 40 PCR amplification cycles, there was a dissociation step of ramping temperature from 60°C to 95°C steadily for 20 min, while the fluorescence signal was continually monitored for melting curve analysis. The concentration of PCR product was calculated on the basis of established standard curve derived from serial dilutions of the positive control for NQO1, wild type p53 and β-actin in the CCA cell lines.
Western blot analysis
After treatment with chemotherapeutic agents, CCA cells were washed with PBS, collected, and lysed at 4°C with 1x cell lysis buffer with 1 mmol/L dithiothreitol and 0.1 mmol/L phenylmethylsulfonyl fluoride (PMSF) with vigorous shaking. After centrifugation at 12,000 g for 30 min, supernatant was collected and stored at -80°C until use. Thirty microgram of the protein samples were mixed with 5x loading-dye buffer, heated at 90°C for 10 min, and proteins were then separated by electrophoresis in 10% SDS-polyacrylamide gel. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes at 180 mA for 1 hr. The PVDF membranes were blocked for 1 hr at room temperature with 5% (w/v) skim milk powder in PBS with 0.1% Tween-20. PVDF membrane was incubated overnight at 4°C with primary antibodies diluted with PBS/Tween-20. The antibodies purchased from Santa Cruz BioTechnology, Inc. (California, USA) were: rabbit polyclonal IgG Bax (1:2500) (#sc-493), rabbit polyclonal IgG cyclin D1 (1:1000) (#sc-718), rabbit polyclonal IgG p21 (1:500) (#sc-56335), mouse polyclonal IgG p53 (1:500) (#sc-98), and mouse monoclonal IgG β-actin (1:2500) (#sc-1616). The rabbit polyclonal IgG NQO1 (1:2500) (#ab34173) was purchased from Abcam (Cambridge, MA, USA). The primary antibody was then removed and the blots were extensively washed with PBS/Tween-20. Blots were then incubated for 2 hr at room temperature with the secondary antibody horseradish peroxidase-labeled goat anti-mouse IgG (#sc-2005) or goat anti-rabbit IgG (#sc-2004) at 1:5000 dilutions in PBS. After removal of the secondary antibody and extensive washing in PBS/Tween-20, the blots were incubated in the ECL substrate solution (Amersham™ ECL™ prime Western Blotting detection reagent; GE Healthcare, Piscataway, NJ, USA). Densities of the specific bands of Bax, cyclin D1, p21, p53, NQO1 and β-actin were visualized and captured by ImageQuant™ LAS4000 (GE Healthcare).
Data were expressed as mean ± SEM of triplicate assays from three independent experiments. An analysis of variance with repeated measurement was used to determine significant differences between each experimental group. The level of significance was set at p < 0.05.