Cell lines and cultures
ES2 and TOV21G cell lines were obtained from the American Type Culture Collection. All cells were maintained in a humidified atmosphere containing 5 % CO2 at 37 °C. ES-2 cells were grown in McCoy’s 5A medium with 10 % FBS, and TOV21G maintained in MCDB 105/medium 199 supplemented with 10 % heat-inactivated fetal bovine serum.
Establishment of chemo-resistant tumor cell lines
Paclitaxel-resistant ES-2 and TOV21G tumor cell lines were developed by continuous exposure to paclitaxel. Briefly, ES-2 and TOV21G cells were exposed to increasing concentrations of paclitaxel, with an initial concentration of 0.001 μM. When the tumor cells regained exponential growth after paclitaxel treatment, the concentration of paclitaxel was doubled until the concentration reached 0.2 μM. The resulting paclitaxel-resistant tumor cell lines were named ES2TR160 and TOV21GTR200. The ES2TR160 and TOV21GTR200 cells were passaged weekly and treated monthly with respective concentrations of paclitaxel to maintain their paclitaxel chemo-resistance.
Generation of HIN-1 over-expressing ES2 and ES2TR160 cell lines
ES2 and ES2TR160 tumor cells were transfected with the HIN-1 gene to generate HIN-1 over-expressing ES2 and ES2TR160 transfectants as described previously .
Reagents and antibodies
ECL Western blotting detection reagents were purchased from Perkin Elmer (Boston, MA). Antibodies recognizing HIN-1, mTOR, phospho-mTOR (Ser2448), AKT, phospho-AKT (Ser473) (Thr308), and GAPDH were purchased from Cell Signaling Technology (Beverly, MA). A Cell Titer 96-well proliferation assay kit was obtained from Promega (Madison, WI), and paclitaxel was obtained from Genetaxyl Cream Less Company.
MTT assays for cytotoxicity and proliferation assays
The sensitivities of various tumor cell lines to paclitaxel were first assessed by MTT assay. Briefly, cells (4000 cells/well) in a 96-well plate were exposed to paclitaxel at the indicated concentrations for 72 h at 37 °C. The cells exposed only to the culture medium served as controls. MTT at a final concentration of 0.5 mg/ml was added to the cells and incubated at 37 °C for 3 h. At the end of incubation, the cultured medium was removed and 200 μl of DMSO (Sigma) was added to dissolve the blue formazan crystals, and the optical density was measured at 490 nm using a universal microplate reader (Elx800, Bio-tek Instruments). IC50 values (the concentration that produced a 50 % reduction in absorbance) were analyzed and recorded. MTT assays were then performed for the proliferation of tumor cells treated with 5-aza-2-dC as described earlier. Briefly, ES2 and ESTR160 cells (1×104 cells/well in a 6-well plate) with or without 10 μM 5-aza-2-dC (in growth media for 1, 3, 7, 10 or 14 days with 5 % CO2 at 37 °C). 5 mg/ml of MTT solution was then added to each well and incubated at 37 °C for 3 h. The medium was then aspirated and replaced with solubilization solution (DMSO). The plates were read on a Micro Elisa reader (Anthos 2001) at 570 nm.
Cell cycle analysis of tumor cells treated with 5-aza-2-dC by flow cytometry analysis
To evaluate the influence of 5-aza-2-dC on the cell cycle of the tumor cells, the tumor cells were treated with or without 10 μM 5-aza-2-dC for 3 days, washed with PBS, and fixed with 70 % ethanol overnight at 4 °C. The cells were then stained with propidium iodide (50 μg/ml) and RNase A (100 μg/ml; Roth, Karlsruhe, Germany) for 20 min at 4 °C. The percentages of cells in the G0/G1 phase, S phase, and G2/M phase were determined using a FACScan flow cytometer and analyzed by Cell Quest software (Becton Dickinson, San Jose, CA).
Caspase-3/7 activity of the tumor cells treated with paclitaxel and/or 5-aza-2-dC
Caspase-3/7 activity of the tumor cells was determined quantitatively using a Caspase-Glo 3/7 assay kit (Promega) according to the manufacturer’s instructions. Briefly, the tumor cell lines were seeded and treated with paclitaxel or 5-aza-2-dC. After 24 h, the cells were lysed and luminogenic substrates specific for the caspase species were added. Light emission was measured in a luminometer (Berthold Technologies, Wildbad, Germany).
Genomic DNA and RNA extraction
Genomic DNA and RNA was extracted from the tumor cell lines using a QIAamp tissue kit (Qiagen, Valencia, CA) following the instructions of the manufacturer.
Sodium bisulfite treatment, sequencing, and methylation-specific polymerase chain reaction analysis
Genomic DNA of the tumor cells was isolated using a Genomic DNA kit (Geneaid Biotech, Bade City, Taiwan), converted with sodium bisulfite using a CpGenome DNA modification kit (Millipore, MA, USA), purified, and then amplified by a PCR with DNA polymerase (ThermoHotStart 2× Gold PCR Master mix; Applied Biosystems) and HIN-1-specific primers. The primer sequences for methylated HIN-1 were 5’-GAAGTTTCGTGGTTTTGTTCG-3’ (forward) and 5’-AAAACCTAAAATCCACGATCGAC-3’ (reverse), and the primer sets for unmethylated HIN-1 were 5’-TAAGAAGTTTTGTGGTTTTGTTTGG-3’ (forward) and 5’-AAAAAACCTAAAATCCACAATCAAC-3’ (reverse). Bisulfite-modified Sss I (New England Biolabs, MA)-treated normal lymphocyte DNA served as the methylated control, and bisulfite-treated normal lymphocyte DNA as the unmethylated control. PCR products were analyzed on 3 % agarose gels. A methylation specific-PCR in a final volume of 20 μl was performed under the following conditions: 95 °C for 10 min, followed by 40 cycles at 95 °C for 30 s, 62 °C for 30 s, and 72 °C for 40 s, with a final extension at 72 °C for 10 min and holding at 4 °C. The PCR products were purified and then directly sequenced using an Applied Biosystems ABI automated DNA sequencer.
Quantitative real-time RT-PCR (QRT RT-PCR)
QRT RT-PCR was used to measure the MDR1, NANOG, HIF-1α, HIF-2α, Snai2, TWIST1, and ABCG2 mRNA of the tumor cell lines. GAPDH was used as the internal control. The QRT RT-PCR was performed in an ABI Prism 7300 Sequence Detection System (Applied Biosystems) with a Taqman Gene Expression Assay (Hs00369360_g1) under the following conditions: 2 min at 50 °C, 10 min at 95 °C, and a two-step cycle at 95 °C for 15 s and 60 °C for 1 min for 40 cycles with an additional dissociation curve. The interpolated number (Ct) of cycles to reach a fixed threshold above background noise was used to quantify amplification.
5-aza-2-dC treatment and QRT RT-PCR of HIN-1 of the tumor cell lines
The tumor cell lines were treated with or without 5-aza-2-dC 10 μM, and renewed every 24 h. QRT RT-PCR was used to measure the mRNA of HIN-1 as described earlier.
Formalin-fixed, paraffin-embedded specimens were sliced by a microtome at a thickness of 3–5 um and placed on coated slides. The tissue slides were then incubated with purified goat anti-human UGRP2 (S-15) polyclonal Ab (Santa Cruz Biotechnology) using a Thermo Scientific Autostainer 360 (Thermo Fisher Scientific Inc., CA). The immuno-reactive HIN-1 was scored semi-quantitatively, and the expression was scored according to the intensity as 0 or 1, 2 or 3 indicating no or low, intermediate or strong immuno-reactivity, respectively. Tissues containing more than 10 % neoplastic cells with a score of 2–3 intensity were considered to be positive. The percentages of each score in the neoplastic tissues were also recorded. If less than 10 % of the neoplastic cells expressed HIN-1 the expression was defined as being weak, and if more than 10 % of the neoplastic cells expressed HIN-1 the expression was defined as being strong. A pathologist not involved in the present study evaluated the immunostaining under blinded conditions.
Western blot analysis
Tumor cell lines were first treated with paclitaxel or 5-aza-2-dC for 72 h. The cells were then collected and lysed in PBS containing 1 % Triton X-100 using an ultrasonic cell disruptor. The lysates were separated by SDS-PAGE (12.5 %) and transferred to a PVDF membrane. The membrane was blocked in blocking buffer (TBS containing 0.2 % Tween 20 and 1 % I-block (NEN)) and incubated with the polyclonal antibodies separately for 1 h. A purified rabbit anti-human GAPDH polyclonal Ab (Santa Cruz Biotechnology, Inc.) was also used at the same time to normalize the signals generated from anti-HIN-1, AKT, AKT p-Akt (Ser473), pAKT (Thr308), mTOR, and pMTOR (Cell Signaling). After washing, alkaline phosphatase-conjugated anti-rabbit Ab (Vector Laboratories) was applied. The membrane was washed and the bound Abs was visualized by developing with NBT/BCIP as chromogens.
In vivo animal experiments
NOD/SCID (NOD.CB17 Prkdc scid/Jnarl) mice were obtained from the National Animal Center (Taipei, Taiwan) and maintained in accordance with institutional policies. All of the experiments were approved by the Institutional Animal Care and Use Committee of Cathay General Hospital. Five to 7-week-old NOD/SCID mice (n = 4) were inoculated subcutaneously into the bilateral flank with 1 × 107 of tumor cells treated with or without 10 μM 5-aza-2-dC for 3 days before inoculation. Tumor growth was measured using calipers, and volumes were calculated based on the modified ellipsoid formula (L × W × W/2) at the indicated time points. All of the experiments were carried out in duplicate.
The median inhibitory concentrations (IC50) of paclitaxel were calculated using Sigma Plot 8.0 software (SPSS, Inc., Chicago, IL). All numerical data were expressed as the mean ± SD. Significance of the difference between two groups was determined with the Mann–Whitney U test. A p value less than 0.05 was considered to be statistically significant.