Establishment and Characterization of Multidrug-resistant Gastric Cancer Cell Lines

Materials and Methods

Anti-cancer drugs. Five anti-cancer drugs, 5FU (Kyowa Hakko, Tokyo, Japan), PTX (Bristol-Myers, Wallingford, Connecticut), OXA (Yakult, Tokyo, Japan), irinotecan active metabolite SN38 (Yakult) and GEM (Eli Lilly, Kobe, Japan), were used in this study. All reagents were used as recommended by their suppliers.

Establishment of anti-cancer drugs resistant cancer cell lines. The human gastric cancer cell line, OCUM-2M, was used in this study. OCUM-2M was established from a diffuse type of primary gastric cancer obtained during a total gastrectomy (11). No chemotherapy was performed before the specimen was resected. OCUM-2M cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Nikken Biomedical Laboratory, Kyoto, Japan). The media were supplemented with 10% fetal bovine serum, 100 IU/ml penicillin (ICN Biomedical, Costa Mesa, CA), 100 μg/ml streptomycin (ICN Biomedical), and 0.5 mM sodium pyruvate (Cambrex, Walkersville, MD, USA). The cells were cultured at 37°C in a humidified atmosphere of 5% CO₂ in air. Anticancer-drugs resistant cells were established by exposure to increasing concentrations of 5 anticancer drugs, 5FU, PTX, OXA, SN38 or GEM similar to the previously described method (12). OCUM-2M cells were initially cultured in DMEM containing 5FU, PTX, OXA, SN38 or GEM each drug alone at the concentration of 1/160 of their 50% growth inhibition (IC₅₀), and then the cells were subcultured every 2 weeks in DMEM with increased concentrations of anticancer drugs, a 25% increase each time. Finally, the resultant cell lines that grew exponentially in the presence of high concentrations were designated as drug resistant gastric cancer cell lines, and named OCUM-2M/5FU, OCUM-2M/PTX, OCUM-2M/OXA, OCUM-2M/SN38 and OCUM-2M/GEM respectively. The final drug concentrations that were tolerated by each cell line are shown in Table I. Each cell line proliferated when exposed to an anticancer drug at a concentration between 0.2 and 2 times of their IC₅₀. In all resistant cell lines, experiments were performed after culturing for at least 4 weeks in the absence of the anti-cancer drugs.

Growth-inhibition assay. The effect of anti-cancer drugs on OCUM-2M cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Wako, Osaka, Japan) colorimetric assay. Cancer cells (2.5×10⁴/ml) were seeded into a 96-well plate in DMEM containing different concentrations of each anti-cancer drug. After incubation for 72 hr at 37°C, 20 μl of MTT (5 mg/ml in PBS) were added to each well and the plates were incubated at 37°C for 3 hr, and then 200 ml of dimethyl sulfoxide (Wako) was added. The formazan product of MTT was measured as the absorbance at 550 nm using a microtiter plate reader (Model 550; Bio-Rad Laboratories, Tokyo, Japan). The percentage of cell viability was determined as the ratio of the absorbance of the sample versus the control. The IC₅₀ was determined as the drug concentration showing 50% cell growth inhibition in comparison to the control cell growth. The resistance index (RI) was determined as the ratio of the IC₅₀ of the drug resistant cell line/IC₅₀ of OCUM-2M. Chemo-resistant cancer cell line was determined when RI of cancer cells showed 3 or more. Six replicate wells were used for each drug concentration, and the testing was carried out 3 times independently.

Detection of cross-resistance to anti-cancer drugs. Either OCUM-2M or the 5 drug resistant sublines were seeded into 96-well plates with or without the addition of each of the anticancer drugs at concentration of their IC₅₀ of OCUM-2M, and the plates were incubated for 72 hr at 37°C. The suppression of cell proliferation was examined using the MTT assay. The IC₅₀ of the 5 sublines to each drug was calculated using the methods described above. Six replicate wells were used for each drug concentration, and the testing was carried out 3 times independently.

Apoptosis assay. Apoptosis was detected using flow cytometry by staining cells with annexin V-FITC and propidium iodide (Medical & Biological Laboratories CO., LTD, Nagoya, Japan). Cancer cells were seeded at a density of 1.0×10⁵ cells/ml in a 6-well plate. With or without anti-cancer drugs at the concentration of IC₅₀, the plates were incubated for 72 h at 37°C. Using Apoptosis Kit, cells were stained with Annexin V-FITC and propidium iodide according to the instructions of the supplier, incubated for 15 min at room temperature in the dark, and immediately analyzed by FACScan flow cytometry (Becton Dickinson, Mountain View, CA).

Cell cycle test. Cancer cells (2×10⁴ cells) were harvested and managed according to instructions of the Cycle TEST PLUS DNA reagent kit protocol (Becton Dickinson, Mountain View, CA, USA), then incubated with ribonuclease A for 10 min at room temperature, and with propidium iodide for 30 min in the dark on ice. The sub-G₀/G₁, S, and G₂/M phase fractions of 2×10⁴ cells were determined by flow cytometry using a FACScaliber (Becton Dickinson). The results were analyzed using the Modofit software program (Beckton Dickinson).

Reverse transcription PCR. The mRNA expression of genes, including MDR1, MRP, DAPK1, DAPK2, DAPK3, DPD, TK1, TS, TP, MAD2L1, SLC28A3, HMGB1, RRM1, UMPK, TRAG3, Caspase3, and P53, by reverse transcription PCR (RT-PCR). Total cellular RNA was extracted from OCUM-2M and the 5 drug resistant cell lines using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. After the genomic DNA was removed by DNase, cDNA was prepared from 1 μg of RNA with Maloney mouse leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA) using random primers (Invitrogen). The relevant cDNA were amplified by PCR using specific primer pairs (Table II) with Taq DNA polymerase (Invitrogen) in a thermal cycler. The PCR conditions were as follows: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 1 min with 35 cycles of the three repeated steps, and final incubation at 72°C for 10 min. The PCR products were separated by electrophoresis on a 2% agarose gel. The mRNA expression of the each gene was normalized to that of the housekeeper GAPDH gene.

Statistical methods. The values of different groups were compared using Student's t-test. Probability values of p<0.05 were regarded as statistically significant. All statistical tests were two-sided.