Cell Line and Cell Culture - Materials and Methods

2. Materials and Methods

2.2. Cell Line and Cell Culture

Human NSCLC PC-9 cells and the iressa-resistant cell line PC-9/IR were cultured with RPMI-1640 (GIBCO, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (GIBCO, Invitrogen) and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin, GIBCO, Invitrogen). Cells were maintained at 37℃ in humidified 5% CO2 atmosphere. For treatment, resveratrol and Iressa were diluted in medium and added to cultures to give the desired final concentrations. Untreated cultures received the same amount of the carrier solvent, DMSO.

2.3 Establishment of the Iressa-resistant PC-9 cells in vitro

In order to create a Iressa-resistant cell line, the PC-9 lung cancer cells were exposed to 1 μM Iressa for 48 hrs in PRMI-1640 plus 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin,

which was lower than the IC50 of PC-9/WT cells. After exposure to Iressa, cells were washed and cultured in drug-free medium until the surviving population of cells was grown to 80% confluence.

The surviving cells were continuously exposed to increasing dosages and finally to a concentration of 10 μM. The established resistant cell line (PC-9/IR) was maintained by culture in a medium containing 7.5 μM Iressa. For all the in vitro studies, the resistant cells were cultured in drug-free medium for 1 day to eliminate the effects of Iressa.

2.4 Cell cycle analysis

Flow cytometric assay was performed to determine the cell cycle profiles of PC-9 lung cancer cells in response to Iressa treatment. PC-9/WT and PC-9/IR cells were plated in 10-cm tissue culture dishes and treated with various concentration of Iressa (1, 5, 10 μM) for 48 hrs, and then the cells were washed twice with PBS, trypsinized and fixed in 70% ethanol overnight at 20℃. Cells were resuspended in PBS containing 0.1% Triton X-100 and RNase A (0.2 mg/ml) (Sigma-Aldrich).

The cell suspension (3 x 10⁵~5 x 10⁵cells/ml) was incubated at 37℃ for 30 min. Propidium iodide (Sigma-Aldrich) was added at a final concentration of 20 μg/ml and the cell suspension was kept in the dark at 4℃ for 1 hr. The cells were filtered and the cell cycle was analyzed by flow cytometry with the FACSCalibur flow cytometer (Becton Dickinson). Cellular debris was excluded from the analysis by raising the forward scatter threshold, and the DNA content of the intact nuclei was recorded on a linear scale. A minimum of 10000 events was collected on each sample.

2.5 Cell proliferation by MTS assay

Growth and inhibition of growth were assessed by the MTS assay according to the manufacturer's instructions (CellTiter 96^® AQueous One Solution Cell Proliferation Assay, Promega, Madison, WI, USA). This assay, a colorimetric method for determining the number of viable cells, is based on the bioreduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H- tetrazolium (MTS) by cells to a formazan product that is soluble in cell culture medium, can be detected spectrophotometrically. Briefly, the cells were seeded into 96-well plates at a density of 4 x 10³ cells per well, and treated with Iressa (0-10 μM) or resveratrol (0-10 μM).

After incubation for 48 hrs, the MTS reagent were added to each well and then incubated for a further 2 hrs (37℃ and 5% CO2). Absorbance was measured at 490 nm using a microplate reader (BioTek). Each combination of cell line and drug concentration was set up in 4 replicate wells and repeated at least 3 times. The percentage of cell viability is shown relative to untreated controls.

2.6 Western blot analysis

Western blot analysis was used for assaying the phospho-EGFR, EGFR, phosphor-AKT, AKT, phospho-extracellular signal-regulated kinase (ERK) 1/2, ERK, LDHA expression. Bio-Rad expression system was employed in this experiment. Briefly, the whole cells were harvested and washed by PBS. The process was first lysed in NETN lysis buffer (150 mM NaCl, 20 mM Tris-HCl pH 8.0, 0.5 % NP 40 and 1 Mm EDTA) containing protease inhibitor cocktail (Sigma-Aldrich) and centrifuged at 13000 rpm for 30 min. The supernatant was extracted and transferred to the acrylamide gel under manufacture’s recommendation (Amersharm, Arlington Heights, IL). Protein were heated in 4X sample buffer at 100℃ 10 min and then loaded into each well for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated protein was transferred to a PVDF member (Millipore) by all-wet method (Bio-Rad) under 300 mA for 3 hrs.

Non-specific blocking was performed by immersing the membrane in 5 % milk in Tris-buffered saline-Tween (TBS-T) (10 ml of 2 M Tris-HCl pH 7.4, 100 ml of 5 M NaCl, 0.5 ml of 100 % Tween-20, distilled water 890 ml) for 1 hr at room temperature. Followed by various antibody;

phospho-EGFR (Tyr 1173), EGFR, phospho-AKT (Ser 473; Ser 308), AKT, phosphor-ERK, ERK, LDHA (Cell signaling) and β-actin (Sigma-Aldrich). All antibodies were diluted to a recognition level (1:1000) and incubated at room temperature for 2 hrs or overnight as condition needed.

Washing the membrane with TBST three times (10 min/time), and HRP labeled second antibody (Santa Cruz, CA, USA) for enhanced chemiluminescence (ECL) marker were diluted at a 1:5000 concentration. The membrane was incubated at room temperature for 1 hr, and wash with TBST three times (10 min/times) for removed exceed antibody. The ECL detection kit (Millipore) was added to the membrane and incubated for 1 min without agitation. The membrane was wrapped in

SaranWarp and placed in film cassette with film (Kodak) in the dark room for optimal exposure condition before final film development.

2.7 Oligo gene expression microarray analysis (Human Whole Genome OneArray ™ Microarray, Phalanx)

PC-9/WT and PC-9/IR cells were harvested and total RNA were purified using the TRIzol^® Reagent (Invitrogen) according to the manufacturer's instructions. Assays were performed in triplicate and total RNA from triplicates were pooled. All RNA samples were of the high purity with 260/280 absorbant ratio of 1.8-2.2 measured by NanoDrop ND-1000 (NanoDrop), well intact with RIN score of above 7 determined by Agilent RNA 6000 Nano Assay (Agilent), and no DNA contamination checked by agarose gel electrophoresis. The aminoallyl-RNA (aRNA) probes were amplified using the MessageAmp aRNA kit (Ambion) and labeled with the NHS-Cy5 (GE). The Cy5-labeled aRNA probes were also purified and quantified. The labeled probes were hybridized at 50°C for 16 hrs to the human Oligo Microarray (Phalanx Human Whole Genome OneArray ^TM Version 4.3, Phalanx Biotech), which contains 30,968 well-characterized genes. After washing 3 step (Wash ,Ⅰ 42°C, 5 min；Wash Ⅱ, 42°C, 5 min；Wash Ⅲ, 25°C, 5 min), the array was exposed to a phosphorimaging cassette and then scanned with the Axon 4000B Scanner (Molecular Devices) as well as analyzed with Genepix software (Molecular Devices).

Tests were at least performed twice (including technical and biological replicates;

reproducibility >0.95). The standard value to exclude “noise” or background was the signal intensity value of median-background >0, the GenePix flag score =0, and the signal/noise ratio (SNR) 3. The quali≧ fied probes must be present in at least 50% of the biological samples in order to be selected for normalization and fold change calculation. All data were processed by Global scaling normalization. Statistic P-value could be calculated using pair t-test of Microsoft Excel function.

2.8 MicroRNA microarray hybridization (NCode™ miRNA Labeling System,

Invitrogen)

For NCode™ miRNA Labeling System experiments, RNA was prepared by TRIzol^® (Invitrogen) extraction. Total RNA was poly(A) tailed using poly(A) polymerase. A DNA polymer with either Alexa Fluor^® 3 or Alexa Fluor^® 5 dye molecules was ligated to the poly(A)-tailed RNA via T4 DNA ligase and an oligo(dT) bridge consisting of bases complementary to the dye-labeled DNA Alexa Fluor^® polymer and bases complementary to the poly(A) tail on each tailed RNA molecule. The ligation and subsequent steps were protected from light. Alexa Fluor^® 3– and Alexa Fluor^® 5–labeled samples were combined and their volume reduced to half by a SpeedVac^® Concentrator (Thermo Savant). Bovine serum albumin and 2X hybridization buffer were added to each sample. Samples were heated to 65°C for 10 min. The hybridization mix was applied to an NCode™ Multispecies Microar-ray V2 and incubated at 52°C for 8–16 hrs. Microarrays were washed for 15 min in three successive wash buffers: 2X SSC and 0.2% SDS at 52°C, 2X SSC at room temperature, and 0.2X SSC at room temperature. Microarrays were dried by centrifugation in a tabletop centrifuge and scanned using a GenePix^® 4000B Array Scanner (Molecular Devices).

GenePix Pro 5.0 software will be used for image acquisition, normalization, and data analysis.

2.9 Two-dimensional gel electrophoresis analysis, liquid chromatography and tandem mass spectrometry (LC–MS/MS)

The different expression proteins between PC-9/WT and PC-9/IR were investigated by two-dimensional gel electrophoresis serviced by Mission Biotech. Briefly, the cells were harvested and each sample of 1 mg protein were placed in a rehydration solution containing 7 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT, 0.2% Bio-Lyte (pH 3 to 10), and a trace of bromophenol blue and applied to linear IPG Readystrips (24 cm; pH 3–10; Bio-Rad) by in-gel rehydration for 12 hrs at 20°C. Isoelectric focusing (IEF) was performed using a protein IEF cell (Bio-Rad) under the following conditions: 20°C, 250 Vhr for 30 min; 1 kVhr for 2 hrs; 10 kVhr for 5 hrs; and 10 kVhr until 70 kVhr was achieved. Once the IEF was completed, the individual strips were equilibrated for 15 min in an equilibration buffer (6 M urea, 2% SDS, 0.375 M Tris–HCl (pH 8.8), 20% glycerol, and 2% DTT), and then for another 15 min in the same buffer except that the DTT was replaced by

2.5% iodoacetamide. After equilibration, the proteins were separated in the second dimension by vertical 12% SDS-PAGE with the IPG strips mounted on the top of the gels. SDS-PAGE was run with a constant voltage of 200 V at 16 °C until the bromophenol blue front reached the bottom of the gel. Gels were then stained using an improved commasie blue-staining method. The image analysis was by ImageMaster™ 2D Platinum (Swiss Institute of Bioinformatics). Differentially expressed protein spots were manually cut from the commasie blue-stained gels, transferred into eppendorf tubes and digested. Liquid chromatography and tandem mass spectrometry (LC–MS/MS) analysis was provided by GRC Mass Spectrometry facility (Genomics Research Center, Academia Sinica). Data processing for protein identification and quantification was performed using SWISS-PROT and IPI databases.

2.10 RNA isolation and reverse transcription

Cultured cells were washed twice with PBS, and total RNA was extracted using the TRIzol^® Reagent (Invitrogen). Reverse transcription reactions contained 1 μl of RNA, 50 nM stem-stoop RT primer, 0.25 mM each dNTP, 50 units of moloney murine leukemia virus reverse transcriptase (MMLV-RT; Invitrogen), 1X reverse transcription buffer, 10 mM DTT, and 4 units of RNase inhibitor. The stem-loop RT primers were designed according to Chen C (87). The sequences of mature miRNAs were obtained from the Sanger Center miRNA Registry (http://microrna.sanger.ac.uk/sequences/) and were as follows: miR-20a RT primer (5’- GTT GGC TCT GGT GCA GGG TCC GAG GTA TTC GCA CCA GAG CCA ACC TAC CTG -3’); miR-21 RT primer (5’- GTT GGC TCT GGT GCA GGG TCC GAG GTA TTC GCA CCA GAG CCA ACT CAA CAT-3’); miR-23a RT primer (5’- GTT GGC TCT GGT GCA GGG TCC GAG GTA TTC GCA CCA GAG CCA ACG GAA AT-3’); miR-200c RT primer (5’- GGT TGG CTC TGG TGC AGG GTC CGA GGT ATT CGC ACC AGA GCC AAC TCC ATC A-3’); miR-574-5p RT primer (5’- GTT GGC TCT GGT GCA GGG TCC GAG GTA TTC GCA CCA GAG CCA ACA CAC ACT-3’); and miR-U47 RT primer (5’-GTT GGC TCT GGT GCA GGG TCC GAG GTA TTC GCA CCA GAG CCA ACA CCT CAG-3’).

The reactions were incubated at 37℃ for 50 min, 16℃ for 30 min, followed by pulsed RT of 60 cycles at 20℃ for 30 sec, 42℃ for 30 sec and 50℃ for 1 sec. Reactions were terminated by incubating at 85℃ for 5 min to inactivate the reverse transcriptase.

2.11 Real-time PCR quantification

Real-time PCR will be performed using a Roche LightCycler 480 Real-Time PCR system.

PCR reactions contained 0.5 μM of each forward and reverse primer, 0.1 μM Universal ProbeLibrary Probe #21 (Roche), 1X LightCycler TaqMan Master, and 2 μl of cDNA.

Amplification curves will be generated with an initial denaturing step at 95℃ for 10 min, followed by 50 cycles of 95℃ for 5 sec, 60℃ for 10 sec, and 72℃ for 1 sec. The U47 small nuclear RNAs will be used as an internal control. The forward primers were for miR-20a (5’- CGG CGG ATA AAG TGC TTA TGT -3’), miR-21 (5’- CGG CGG TAG CTT ATC AGA CTG -3’), miR-23a (5’- CGG CGG ATC ACA TTG CCA GGG -3’), miR-200c (5’- CGG CGG TAA TAC TGC CGG GTA A -3’), miR-574-5p (5’- CGG CGG TGA GTG TGT GTG TGT -3’) and U47 (5’-CGG CGG TAA TGA TTC TGC CAA A-3’). The reverse primer was 5’-GTG CAG GGT CCG AGG T-3’.

2.12 Reverse transcriptase-polymerase chain reaction (RT-PCR)

Total RNA was isolated using TRIzol^® reagent (Invitrogen) and reverse transcribed into single-stranded cDNA with MMLV-RT (Invitrogen) as manufacturer’s instructions. The primer sequences for IGFBP7, ASNS, SLC47A2, KISS1, NKX2-5, MAGEC2, EFEMP1, PSAT1, PCK2, GAS6, FN1, STC2, SLC1A4, MT1E, S100A4, S100A2, SPRRIB and GAPDH used were shown in Table 1. The reaction mixture was first denatured at 95℃ for 5 min. For these candidate genes, the PCR condition was 95℃ for 30 sec, 50℃ for 30 sec and 72℃ for 30 sec for 30 cycles; for GAPDH was 94℃ for 30 sec, 55℃ for 30 sec and 72℃ for 30 sec for 22 cycles followed by 72℃ for 10 min.

2.13 Construction and production of shRNA in lentiviral vector

shHNRNPA2B1 (TRCN0000001058), shCPT2 (TRCN0000003091), shACAA1 (TRCN0000036072), shAPOBEC3C (TRCN0000052102), shLDHA (TRCN0000164922),

pLKO.1-shLuc vector (shRNA against luciferase, act as a control), pMD.G plasmid and pCMVdeltaR8.91 plasmid were obtained from National RNAi Core Facility at the Genomics Research Center (Academia Sinica, Taipei, Taiwan). Recombinant lentiviruses were produced by co-transfecting 293T cells with the lentivirus expression plasmid, the lentivirus packaging vector pCMVdeltaR8.91, and the vesicular stomatitis virus G glycoprotein (VSVG) expression vector pMD.G using the Lipofectamine ^TMLTX according to manufacture’s instructions. The viruses were collected from the culture supernatants on 2 days post-transfection and filtered by 0.45 filter.

Cultured cells were incubated with lentivirus containing 8 g/ml polybrene for 24 hrs, replaced medium and incubated for another 2 days. For stable clone, cells were then selected with 6 g/ml puromycin for 1 week.

2.14 Statistical analysis

Data are presented as mean ± SE. Two-tailed Student’s t test was used to analyze the difference between the means of the treatment and the control groups. Differences with a p value of less than 0.05 were considered statistically significant.

3. 結果 (Results)

3.1 The acquisition of Iressa resistance in NSCLC PC-9 cells

Lung cancer cell that have acquired resistance to Iressa may complicate future treatment. In order to define the effects of resveratrol on target therapy of lung cancer, we set up a EGFR tyrosine kinase inhibitor-resistant cell model. The Iressa-resistant cells were established and that were derived from the parental sensitive PC-9 cell line. These Iressa-resistant cells were selected by stepwise increasing the concentrations of Iressa. The concentrations of Iressa were started at 1 μM and achieved 10 μM. Several reports have been shown that the antitumor activity of EGFR tyrosine kinase inhibitor was associated with G1 phase arrest in cancer cells (88-90). In order to investigate the characters of Iress- resistant cells different from parental PC-9/WT cells, PC-9/WT and PC-9/IR cells were cultured in the absence (control) or presence of various concentration (1, 5, 10 μM) of Iressa for 48 hrs. After treatment with Iressa, cells were stained with propidium iodide (PI) and subjected to DNA profile analysis by flow cytometry. Treatment with Iressa induced G1 phase arrest of PC-9/WT cells, and increased the percentage of cells with sub-G1 DNA content, which means the cell death. However, we did not find alteration of cell cycle or increasing of sub-G1 cell population in PC-9/IR cells under the same experimental condition (Figure 1). To further confirm the EGFR tyrosine kinase inhibitor resistance, we performed MTS assay to analysis the inhibition of cell viability by Iressa on PC-9 lung cancer cells. Results showed that PC-9/IR cells were significantly resistant to Iressa than PC-9/WT cells in a concentration-dependent manner (Figure 2).

The IC50 of Iressa in PC-9/IR cells (>10 μM) was at least 10-fold higher then parental PC-9/WT cells (IC50, <1 μM). After that, we further evaluated the different expression of EGFR and downstream signaling proteins in PC-9/WT and PC-9/IR cells. Figure 3a showed that the expression of EGFR had no difference between PC-9/WT and PC-9/IR cells. Nevertheless, the protein expression of EGFR and downstream molecules were similar in parental PC-9 cells and Iressa-resistant cells (Figure 3B). In order to investigate the effects of resveratrol on cell viability in

lung cancer cells, PC-9/IR and PC-9/WT cells were treated with resveratrol for 48 hrs in indicated dosages (1.25, 2.5, 5, 10 μM). We found that resveratrol shows similar toxicity in both PC-9/WT and PC-9/IR cells (Figure 4). Treatment with resveratrol, even to 10 μM, did not cause significant reduction of cell viability in PC-9/WT and PC-9/IR cells. According to above data, we successfully established an Iressa-resistant lung cancer cell line, PC-9/IR, as the later study model.

3.2 Resveratrol-induced de-resistance of EGFR tyrosine kinase inhibitor in lung cancer cells

Recently, considerable attention has been also focused on resveratrol (3,5,4′-trans-trihydroxystilbene), a well-known natural polyphenol found in large amount in grapes (91), that has been reported to exert multiple biological activities (92) including anti-inflammatory (93), anti-oxidant (94), inhibition of platelet aggregation (95), antitumor (96), and induction of apoptosis (97). Remarkably, the cancer chemopreventive activity; Aggarwal et al. and Jang et al. of 2 represents an important add value and it seems to be strictly connected to the antitumor, and the proapoptotic effects (98-100). In 2010, Fukui and his research team reported that resveratrol sensitizes a number of cancer cell lines to several anti-cancer drugs, including paclitaxel (85).

EGFR tyrosine kinase inhibitor, such as Iressa, has provided dramatic clinical response for EGFR mutation lung cancer patients. However, its efficiency is limited by the development of drug resistance.

Above literatures lead us come out a hypothesis: whether resveratrol sensitize EGFR tyrosine kinase inhibitor and has the de-resistance effect on Iressa-resistance PC-9/IR cells. To this end, we selected the Iressa dose (2.5 μM) base on the finding from figure 2. Both of PC-9/WT and PC-9/IR cells were pretreated with vehicle or various dosages of resveratrol (1.25, 2.5, 5, 10 μM) for 4 hrs in serum-free medium. Thereafter, the cells were incubated with 2.5 μM of Iressa for another 48 hrs.

At the end of the incubation period, cell viability was analyzed by MTS assay. The results show that pre-treatment with non-toxic dosages of resveratrol increased Iressa-induced cell death in PC-9/IR cells (8.76% ± 1.56% cell death in vehicle + Iressa group; 48.71% ± 3.12% cell death in resveratrol

+ Iressa group, p<0.0001). The cell death of combined treatment with reveratrol and Iressa was increased at less 5-fold than Iressa treatment only in PC-9/IR cells (Figure 5).

These data suggested that resveratrol may sensitize and de-resistance of EGFR tyrosine kinase inhibitor in Iressa-resistant lung cancer cells, also provide a potential treatment strategy of EGFR tyrosine kinase inhibitor-resistant cancer patients.

3.3 The potential target molecular may involve in the resistance of lung cancer cells to Iressa

Resistance to anticancer drugs, not only chemotherapy drugs but also target therapeutic drugs is widely observed in lung cancer patients. This limitation of therapeutic potential provides a powerful stimulus for developing new therapeutic approaches. In our previous results, we showed that pre-treatment with resveratrol increased Iressa-induced cell death in Iressa-resistant lung cnacer cells. It suggested that resveratrol may sensitize EGFR tyrosine kinase inhibitor in Iressa-resistant lung cancer cells. Therefore, it is critical and timely to further evaluate the molecular mechanisms involved in resveratrol-induced de-resistance of EGFR tyrosine kinase inhibitor in lung cancer cells.

To this end, we used non-biased experiments to evaluate the potential targets may involves in resveratrol-mediated de-resistance of EGFR tyrosine kinase inhibitor in Iressa-resistant lung cancer cells. We performed two-dimensional gel electrophoresis analysis, oligo gene expression microarray analysis and microRNA microarray assay to analyze the differential expression of proteins, genes and microRNAs in PC-9/WT and PC-9/IR cells. We found some potential downstream targets may involve in the resistance of Iressa of lung cancer cells.

The different expression of proteins between PC-9/WT and PC-9/IR were shown in figure 6, table 2 and table 3. In figure 6, it showed the two-dimensional protein maps of the PC-9/WT cells and PC-9/IR cells. The proteins are separated by different isoelectric point and molecular weight. To identify the differential expression proteins from two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), we punch out the target spots and then send to liquid chromatography and tandem mass spectrometry (LC–MS/MS) assay (cooperate with Genomics Research Center,

Academia Sinica). There were 15 candidate proteins significantly increase in PC-9/IR cells compared with PC-9/WT cells (Table 2). We also found 9 candidate proteins were dramatically decreased in PC-9/IR cells compared with PC-9/WT cells (Table 3). Through the prediction of these candidate proteins by IPI and SWISS-PROT databases, we selected several candidate proteins, such as CPT2 (Carnitine O-palmitoyltransferase 2), ACAA1 (Acetyl-CoA acetyltransferase), LDHA (L-lactate dehydrogenase A), HNRNPA2B1 (Heterogeneous nuclear ribonucleoproteins A2/B1) and APBEC3C (Probable DNA dC->dU-editing enzyme APOBEC-3C), to investigate whether these candidate proteins involve in Iressa resistance and resveratrol-mediated de-resistance of Iressa of lung cancer cells. PC-9/IR cells were infected with shLuc, shLDHA, shHNRNPA2B1, shACAA1, shAPOBOC3C and shCPT2 as described under “materials and methods” and were subjected to analysis the knockdown efficiency by real-time PCR quantification (Figure 7A). Data from western blot confirmed the decreased the expression of LDHA by shLDHA infection (Figure 7B). To further

在文檔中中國醫藥大學機構典藏 China Medical University Repository, Taiwan:Item 310903500/32577 (頁 11-0)