Statistical analysis of microarray data and survival
Gene expression data from the lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD) dataset of The Cancer Genome Atlas (TCGA) were obtained from http://ualcan.path.uab.edu/index.html or http://xena.ucsc.edu. Pearson’s correlation coefficient was used to determine P values for co-expression. Prognostic significance of ABCB6 expression in lung cancer was analyzed using the Kmplot (www.kmplot.com) [18]. Briefly, gene was entered into the online database to obtain Kaplan–Meier survival plot. The log rank P were calculated and displayed on the webpage. The survival rates were investigated with median cut-off (Low represents patients with indicated gene mRNA levels less than the median. High represents patients with indicated gene mRNA levels greater than the median.)
Clinical and specimens
Patients diagnosed with advanced NSCLC were enrolled in the Cancer Centre of the First Affiliated Hospital (Southwest Hospital) of Army Medical University (Chongqing, China) from January 2010 to December 2014. The clinical and pathologic characteristics of the patients are summarized in Table 1. All patients received routine gemcitabine-platinum chemotherapy (completed 2–4 cycles). Forty-one of specimens were selected from patients with chemotherapy failure (including progressive disease, PD) after gemcitabine-based chemotherapy, and constituted the gemcitabine-resistant group. Forty of specimens were selected from patients who had positive response after gemcitabine therapy including partial response (PR), which constituted the gemcitabine-sensitive group. Adjacent normal specimens were collected from 10 NSCLC cancer patients who underwent surgery in Southwest Hospital. The histomorphology of all specimens had been confirmed by the Department of Pathology. Clinical investigation complied with the ethical standards codified in the 1964 Declaration of Helsinki. The protocol of immunohistochemistry for patient tissues was approved by the Ethics Committee of the First Affiliated Hospital (Southwest Hospital), Army Medical University, and all patients or family members involved provided written informed consent. All slides were prepared from stored pretreatment paraffin-embedded tissue blocks from lung cancer patients who underwent surgery at Southwest Hospital. All specimens were confirmed by pathological examination and were made into tissue microarrays as described previously [19].
Cell culture and drug treatment
All human NSCLC cell lines were purchased from the ATCC (Manassas, VA, USA) and cultured in DMEM (A549) or RPMI 1640 medium (NCI-H1703) supplemented with 10% fetal bovine serum (GIBCO), penicillin (100 U/ml), and streptomycin (0.1 mg/ml) (Beyotime Institute of Biotechnology, China). Cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. The gemcitabine-resistant cell lines (A549-GR and H1703-GR) was established as follow: A549 or H1703 parental cells were exposed to gradually increasing concentrations of gemcitabine (Sigma-Aldrich) from 10 nM initially up to 10 μM over a 7-month period. For hypoxic exposure, cells were placed in a modular incubator chamber that was flushed with a 1% O2/5% CO2/94% N2 gas mixture and sealed.
Lentiviruses transduction
The pLKO.1-puro lentiviral vectors encoding shRNA targeting HIF-1α (sh1α-1, Clone ID: NM_001530.x-2671s1c1; sh1α-2, Clone ID: NM_001530.x-1048s1c1), ABCB6 (shABCB6-1, Clone ID: NM_005689.1-1581s1c1; shABCB6-2, Clone ID: NM_005689.1-2814s21c1), and NTC were purchased from Sigma. Lentiviruses were packaged, and cells were transduced and subjected to puromycin selection as described [20].
RT-qPCR
Total cellular RNA was extracted using TRIzol (Invitrogen), precipitated with isopropanol, treated with DNase I (Ambion), and reverse transcribed with the iScript cDNA Synthesis kit (Bio-Rad). qPCR analysis was performed using SYBR Green (Bio-Rad) with the iCycler Real-time PCR Detection System (BioRad). The 2-ΔΔCt method was used to calculate the relative gene expression as described [21]. The nucleotide sequence of the primers is as follows: 18S rRNA forward primer: 5′-, GAGGATGAGGTGGAACGTGT-3′, reverse primer: 5′-AGAAGTGACGCAGCCCTCTA-3′; ABCB6 forward primer: 5′-TGCTGTTTCGCTTCTACG-3′, reverse primer: 5′-CCTCCACCTCATCATTCC-3′.
ChIP assay
ChIP assays were performed as previously described [19]. A549 and H1703 cells were cross-linked in 3.7% formaldehyde for 10 min and lysed with SDS lysis buffer. Chromatin was sheared by sonication and lysates were pre-cleared with salmon sperm DNA/protein A-agarose slurry (Millipore) and incubated with IgG (Novus Biologicals) or antibodies against the following proteins: HIF-1α (Santa Cruz), HIF-2α (Cell signaling), and HIF-1β (Novus Biologicals). Salmon sperm DNA/protein A-agarose slurry was added, and the agarose beads were washed sequentially with: low- and high-salt immune complex wash buffers; LiCl immune complex wash buffer; and twice with TE buffer (10 mM Tris-HCl/1 mM EDTA). DNA was eluted in 1% SDS with 0.1 M NaHCO3, and crosslinks were reversed by addition of 0.2 M NaCl. DNA was purified by phenol-chloroform extraction and ethanol precipitation, suspended in 50 μl TE buffer, and a 2-μl aliquot was used for qPCR. Primer sequences are listed as follows: ABCB6-HRE-site 1 forward primer: 5′-CTGGTCGTCAGATTTCCCG-3′, reverse primer: 5′-CCATTGGCCAAATGTCTCCG-3′; ABCB6-HRE-site 2 forward primer: 5′-CAGTTGGCAGGAGGGTCC-3′, reverse primer: 5′-ACGTGGACCAGGCCTCA-3′.
Western blot assay
Immunoblot assays were performed as previously described [20]. Aliquots of whole-cell lysates were prepared in RIPA lysis buffer, and proteins were separated by SDS/PAGE, blotted onto nitrocellulose membranes, and further probed with primary antibodies followed by HRP-conjugated secondary antibodies (GE Healthcare). The chemiluminescent signal was developed using ECL Plus (GE Healthcare). Antibodies used in immunoblot assays were: HIF-1α (BD Transduction Laboratory); ABCB6 (Novus Biologicals); Catalase (Novus Biologicals); β-actin (Santa Cruz).
Heme detection
Heme content in whole cells was determined by the fluorometric method [22]. Briefly, cells were washed and homogenized in
phosphate buffered saline (PBS) buffer and the protein content was determined by using the Bio-Rad protein assay system (Bio-Rad, Munchen, Germany). Ten mg of protein samples were incubated with 500 μl of 2 M Oxalic Acid (Sigma-Aldrich) at 100°C for 30 min. Samples were subsequently centrifuged at 14000 rpm for 10 min. The amount of heme in the supernatant was determined by fluorometric detection using a F4500 fluorescence spectrophotometer (Hitachi). Excitation and emission wavelengths were set at 405 and 662 nm, respectively. The background was evaluated by measuring fluorescence in non-boiled samples.
Catalase activity
Cells were seeded into 6-well plates at a density of 105 cells/well. Cells were harvested and homogenized in 50 ml of 0.1 M PBS buffer. The mixture was centrifuged at 3000 rpm for 5 minutes at 4°C. The supernatant was separated and assayed for protein content (Bradford assay, Bio-Rad). Catalase activity was determined calorimetrically in 10 μg protein using the Catalase Assay Kit (No. CAT-100, Sigma-Aldrich) according to the manufacturer’s instructions by spectrophotometry. Briefly, all working solutions were prepared from the reagents provided in the kit. The sample (10 µl), mixed with 750 µl of 1X assay buffer and 25 µl colorimetric assay substrate solution, was incubated for 5 minutes. The reaction was stopped using 900 µl of stop solution and the tubes were kept inverted. Within 15 minutes, after the enzymatic reaction, 10 µl aliquot of the reaction mixture was transferred to 1 ml of the color reagent. After the incubation of 15 minutes, the absorbance of the reaction mixture was measured at 520 nm. Specific catalase activity is reported as μmol H2O2 consumed min-1 μg protein-1.
Colony formation assay
For detection of clonogenic ability of the cells with drug treatment, cells were trypsinized and seeded into 6-well plates at a density of 1000 cells per well. The culture medium, which containing gemcitabine (concentrations indicated) for 72 hours and was refreshed every 3 days. After 15 days, cells were fixed with 4% paraformaldehyde for 10 min and then stained by using 0.1% crystal violet in ddH2O for 10 min and imaged using a digital camera to record the results.
Cell viability assay
1 x 104 cells/well were seeded into 96-well plates and cultured at 37°C overnight. Cells were then treated with various concentrations (0.001 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM, 100 μM) of gemcitabine for 72 h. Cell viability was determined using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS (3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium); Promega, USA) following manufacturer’s instructions. All experiments were done in triplicates and was repeated more than three times.
Soft agar assay
Cells were suspended in 1 ml top agar made of 0.35% agarose in medium supplemented with 10% FBS, and the mixture was seeded in
6-well plates containing a basal layer of 0.6% agarose at 5000 cells/well. After 3 weeks, colonies were stained with 0.05% crystal violet for 1 h,
and the colony size and colony number were measured by pictomicrography. Viable colonies larger than 0.1 mm in diameter were counted.
TUNEL assay
To evaluate the cell apoptosis in tumor tissue, we performed terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick-end labeling (TUNEL) technique, to formalin-fixed tumor samples in paraffin blocks, using the one-step TUNEL apoptosis assay kit (Beyotime Institute of Biotechnology, China). The sections (4–5 μm) mounted on glass slides were deparaffinized, rehydrated thoursough graded alcohols to water, treated with 20 μg/ml proteinase K (37 °C, 20 min) and then washed in PBS buffer. TUNEL assay was then performed according to the manufacturer’s instructions. The images were acquired using a confocal microscope (Leica TSC-SP5, Germany). Ten fields were randomly selected in each tumor slide.
ROS detection
Total intracellular ROS was determined by staining cells with dichlorofluorescin diacetate (DCFH-DA, Beyotime Institute of Biotechnology) following the manufacturer's instruction. Briefly, cells were washed with PBS and incubated in the dark with 10 μM DCFH-DA at 37°C for 30 min. Cell treated with Rosup (agent provided in ROS assay kit) was considered as a positive control. Cells were then washed twice with PBS and then ROS levels were determined by fluorospectrophotometer (excitation wavelength 488 nm and emission wavelength 535 nm).
Immunohistochemistry
Tumors and adjacent normal tissue were fixed in 10% formalin and paraffin embedded. Tissue microarrays were dewaxed in xylene, hydrated with graded ethanol, followed by antigen retrieval using citrate buffer (pH 6.1). The instantaneous SP supersensitive kit (SP-9000, Beijing Zhongshan Jinqiao Biotechnology Co., Ltd) was used with antibodies against ABCB6 (Novus Biologicals). Sections were counterstained with Mayer's hematoxylin (Sigma). For quantitative measurement of ABCB6 positive staining on clinical samples (adjacent normal lung tissue, Gemcitabine resistant tumors and Gemcitabine sensitive tumors), immunohistochemical staining of ABCB6 was quantified by Image J software (NIH) as described previously [23]. For detail, we used average optical density (AOD) to evaluate the intensity of the IHC reaction in order to compare expression levels between tumor and peritumoral fields of different markers [24]. AOD scores were calculated from the optical density of 5 spots selected randomly on each sample under the microscope (×200).
Criteria for assessing IHC results
For each sample, five random fields were selected for scoring and a mean score of each slide was calculated in final analysis. The percentage of stained cells was scored as follows: 0 (no positive cells), 1 (<10% positive cells), 2 (10%–40% positive cells), 3 (40%–70% positive cells), and 4 (>70% positive cells). The intensity of positive staining was scored as follows: 0 (negative staining), 1 (weak staining exhibited as light yellow), 2 (moderate staining exhibited as yellow brown), and 3 (strong staining exhibited as brown). The proportion and intensity scores are added to obtain a total score (TS range: 0, 2-7). Samples were divided into two categories depending on the IHC score: Category low corresponded to IHC score less than 4; Category high corresponded to IHC score greater than or equal to 4. Slides were examined and scored independently using GOG criteria [25] by two histopathologists (F Wu and Y Ling) blinded to other pathological information.
Animal studies
Animal experiments were approved by the Institutional Animal Care and Use Committee of the Army Medical University (Chongqing, China). Six to eight-week-old male SCID mice were purchased from the Institute of Experimental Animal of the Army Medical University. A549 and H1703 subclones were harvested by trypsinization, resuspended at 107 cells/ml in a 50: 50 mix of PBS: Matrigel (BD Biosciences), and 2×106 cells were injected subcutaneously into the inguinal area of mice (12 mice per group). Tumor volume (mm3) was calculated as 0.52 × L × W × T (L: Length, W: width, T: thickness). Tumor volumes and body weights were monitored twice weekly. After palpable tumors formed (8 days after tumor implantation), mice received intraperitoneal injection of gemcitabine (20 mg/kg) or saline (250 µl), which was administrated twice per week. After 42 days, tumors were excised and weighed (n = 6). Xenografts were harvested to be subjected to TUNEL assay. To assess survival, the remaining animals (n = 10) were monitored for 100 days until being euthanized. The distribution of survival percentages over time was estimated using the Kaplan-Meier method. The log-rank test was used to determine the p value.
Statistical Analysis
All data were expressed as mean ± SEM and were analyzed using an unpaired two-tailed Student’s t-test for two groups or ANOVA followed by Bonferroni posttest for multiple groups. Survival data were analyzed
using Kaplan Meier survival plots and p values were calculated using the Wilcoxon rank sum test. A p value of < 0.05 was considered statistically significant.