Subjects
Under the approval by the local institutional Ethnics Committee, 68 OS patients undergoing treatment in our clinic centers between 2008 March and 2013 January were included in this study. All the patients were histologically diagnosed as OS. Baseline characteristics of the patients were collected from the medical record, including age, gender, tumor size, histologic differentiation, and Anneking Stages. Before the surgery, all the patients had received two cycles of neoadjuvant chemotherapy composed of DOX, methotrexate and cisplatin. Tumor tissues were collected from each patient during surgery. Good response to the chemotherapy was histologically defined as more than 90% necrosis rate in the tumor tissues. All patients signed written informed consent concerning the collection of blood and tissue samples.
Cell culture and establishment of DOX resistance
The human OS cell lines U2OS and HOS were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). All cell lines were cultured in Dulbecco’s modified Eagle’s medium (4.5 g/l glucose)/Ham F12 (1:1) (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (FCS). All cells were cultured at 37 °C in a humidified atmosphere of 5% CO2. DOX was purchased from Sigma (Buchs, Switzerland). DOX-resistant cell lines were induced by consecutive exposure of gradually increasing concentration of DOX to the parental cell lines for 6 months as previously reported. As a starting concentration, 0.015 µM DOX was applied and the concentration was gradually increased up to 0.12 µM in a period of 3 months. One month after exposure to 0.12 µM doxorubicin, the resistance stability was defined as not significantly altered IC50 of U2OS and HOS cells when incubated in DOX for a week.
Knockdown of ABCG2 in OS cells
The lentivirus shRNA specifically targeting ABCG2 (shABCG2) and the negative control scramble shRNA (shCtrl) were designed and constructed by Shanghai Genechem Company Ltd., China. Lentivirus particles were generated by co-transfecting recombined and packing vectors into 293T cells via Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). U2OS and HOS cells were then cultured in 6-well plates and transfected with shABCG2 or shCtrl. Both cells lines were cultured for 5 days. The knockdown efficiency of the target gene was further evaluated for both cell lines with qPCR analysis.
Cell proliferation assay
Cell proliferation was analyzed by the colorimetric water-soluble tetrazolium salt assay using a Cell Counting Kit-8 (CCK8) according to the manufacturer’s instructions (Dojindo, Tokyo, Japan). Cells were treated with the gradient concentration of DOX (0, 0.5, 1.0, 2.0, 4.0, 8.0 µM) for 24 h, and then cultured for another 48 h. CCK-8 reagent (10 µL) was added to each well, followed by incubation for 1 h in a humidified atmosphere (37 °C, 5% CO2). The number of viable cells was evaluated by the absorbance at 450 nm using a microplate reader (Tecan Spectra Fluor Plus, Crailsheim, Germany).
qPCR
Total RNA was isolated from the tumor tissues or tumor cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Reverse transcription into cDNA was performed with a SuperScript III Reverse Transcriptase kit (Invitrogen, Carlsbad, CA, USA). The quantitative gene expression level was measured using SYBR Master Mixture (TAKARA, Tokyo, Japan) on the LightCycler 480 (Roche Applied Science, Mannheim, Germany). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control. The sequences of the primers were as follows: ABCG2, 5′- GCCACAGAGATCATAGAGCCT − 3′, 5′- TCACCCCCGGAAAGTTGATG − 3′; GAPDH, 5’- GAGTCAACGGATTTGGTCGT − 3’, reverse 5’ -TTGATTTTGGAGGGATCTCG- 3’. Quantitative analysis normalized to GAPDH was performed using the 2−ΔΔCt method.
Immunohistochemical staining
Paraffin-embedded tissue samples were cut into 4-µm sections and de paraffinized for immunohistochemical staining (IHC). Slides were then incubated with primary anti ABCG2 antibody (1:400, Abcam) overnight at 4 °C, followed by incubation with secondary antibody for 30 minutes at 20 °C. All slides were stained by diaminobenzidine and then counterstained by hematoxylin. The IHC-stained tissue sections were scored by two pathologists. The signal intensity was scored as follows: score 0, no signal; score 1, weak; score 2, moderate; and score 3, marked. The staining distribution was scored as follows: score 0, no positive staining cells; score 1, 1–30% positive staining cells; score 2, 31–60% positive staining cells; score 3, 61–100% positive staining cells. The final score calculated by multiplying the score of staining distribution with the signal intensity score. Remarkably higher staining of ABCG2 was defined as the score more than 2.
Genotyping of functional variant
DNA was extracted using the DNA extraction kit (QIAGEN Inc., Tokyo, Japan) according to the protocol of the manufacturers. SNP rs2231142 (c.421C > A) was genotyped using TaqMan SNP Genotyping Assay. The genotyping assay was performed with ABI 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA).
Statistical analysis
The results were presented as mean ± standard deviation, and analyzed with SPSS software (version 19.0; SPSS Inc, Chicago, IL, USA). The data was shown as means ± SD for continuous variables. The relationship between ABCG2 expression and clinicopathological characteristics of the patients was analyzed using the Student’s t-test or the Chi-square test. The gene expression and the overall survival were compared among different genotypes of rs2231142 with One-way ANOVA test. Cumulative survival time was calculated by the Kaplan-Meier method and analyzed by the log-rank test. P < 0.05 was considered statistically significant.