Human non-small cell carcinoma of the lung cancer A549 and H460 cell lines were obtained from the Cancer Institute of Southern Medical University (Guangzhou, China). The cells were maintained in a humidified atmosphere of 5% CO2 at 37 °C in DMEM culture medium (Hyclone) with 10% fetal bovine serum (Gibco). The lung CSCs which express CD133 were isolated by the BD MACs and identified by flow cytometry (FACS). CD133-positive cells were cultured in MEBM basal medium (CBM, New Jersey, USA) to maintain the characteristics of stem cells.
Cell viability assay
Cells in logarithmic phase were seeded into 96-well plates at a cell density of 1 × 104/well, and then different concentrations of SFN were added into each well and incubated together for 48h. The final concentrations of SFN were 0, 2, 4, 6, 8, 10, 12μmol/L, respectively. Then, 10 μL MTT reagent of concentration 5 μg/L was added into the cell medium of each well and incubated for 4 h at 37 °C. Following the removal of the supernatant, 150µL dimethyl sulfoxide(DMSO) was added to dissolve formazan. The absorbance at 490 nm was measured with a microplate reader(Beckman Coulter, Brea, CA, USA). Each reaction was performed in triplicate. At the same time, changes in cell density were observed by optical microscope.
Isolation and identification of lung cancer stem cells
CD133-positive cells were obtained from A549 and H460 cells using CD133 Microbeads by MiniMACS separator(MiltenyiBiotec, Bergish Gladbach, Germany). A549 and H460 cells were collected separately by centrifugation. Different groups of cells (1×108 cells/sample) were resuspended in 500μl of the degassed buffer, respectively. Then the cell suspension was added onto the prepared column. The unlabeled cells (CD133-negative) were collected as they passed through the columns. MS columns were washed with degassed buffer. Then the column was removed from the separator and placed on a new suitable collection tube. Buffer was pipetted onto the column, and a fraction was immediately flushed out with the magnetically labeled cells （CD133-positive）by firmly applying the plunger supplied with the column.
Flow cytometry analysis
Cells were collected separately by centrifugation, then the cells were washed twice with PBS solution, and up to 1×106 cells were resuspended in 500μl of PBS, respectively. The cells were then incubated with PE mouse anti-human CD133 for 30 min at room temperature in the dark. The positive cells were detected using a flow cytometer (BD Biosciences, San Jose, CA, USA) and were analyzed by FlowJo 9.1 software.
TumorSphere formation assay
Cells were placed in 6-well ultralow attachment plates (Corning Inc.) at a density of 1,000 cells/mL in tumorsphere culture medium DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 1% N2 supplement, 2% B27 supplement, and 100ng/mL epidermal growth factor at 37°C in a humidified atmosphere of 95% air and 5% CO2. These cells were then treated with different concentrations of SFN at the same time. Primary spheroids were collected following 14 days of culture, and tumorspheres were measured using an inverted microscope system (magnification, Eclipse Ti‑s, Nikon, Tokyo, Japan).
Reverse transcription and QPCR analysis
The total RNA was extracted from cells with Trizol reagents. cDNA was synthesized from 1μg of mRNA with a high capacity cDNA reverse transcription kit according to the manufacturer’s instructions. Subsequently, cDNA was amplified by qPCR with the SYBR Premix Ex Taq kit according to the manufacturer's instructions using the ABI7300 Sequence Detection System. The following gene-specific primers were used: Shh(forward)5’-CGC ACC TGC TCT TTG TGG-3’,(reverse)5’-GGA GCG GTT AGG GCT ACT CT-3’; Smo(forward) 5’-TCG CTA CCC TGC TGT TAT TC-3’, (reverse)5’-GAC GCA GGA CAG AGT CTC AT-3’;Gli1(forward) 5’-CTG GAT CGG ATA GGT GGT CT-3’, (reverse)5’-CAG AGG TTG GGA GGT AAG GA-3’; PHC3(forward)5’-AGT GGG GAG AGG AGA AGA-3’,(reverse) 5’-GGT GGT GGA ACA GAA ACA-3’. The housekeeping gene Beta-actin was used as a loading control. PCR conditions were as follows: one cycle at 95oC for 3 min, followed by 40 cycles at 95oC for 30s, 55oC for 30s, and 72oC for 1 min. All assays were performed in triplicate and were calculated on the basis of ∆∆Ct method. The n-fold change in mRNAs expression was determined according to the method of 2-∆∆CT.
Protein sample was extracted from cells with an ice-cold SDS protein lysis buffer. Protein concentration was measured by a Micro BCA Protein Assay Reagent kit. Then protein sample was separated by 10% SDS-PAGE electrophoresis and transferred onto 0.45mm PVDF membranes. The membranes were blocked with 5% non-fat milk in TBST buffer for 1 h, incubated overnight with primary antibody at 4°C and then incubated with secondary antibody for 1 h at room temperature. The antigen-antibody complexes were visualized using the ECL detection system. The analysis of the bands was conducted by the Image J software. The following antibodies were purchased from commercial sources including anti-Shh (ab53281), anti-Smo (ab32575), anti-Gli1(ab134906), anti-PHC3 (GTX32785 ) and anti-GAPDH (CWBIO).
siRNAs for SHH were purchased from Invitrogen and the following sequences were used. No.1,SHH-s:5’-ACAGGCUGAUGACUCAGAGGUGUAA-3’,SHH-as:5’-UUACACCUCUGAGUCAUCAGCCUGU-3’;No.2,SHH-s: 5’-GGUGUACUACGAGUCCAAGGCACAU-3’, SHH-as: 5’-GACUCGUAGUACACC-3’; No.3,SHH-s: 5’-CCGACAUCAUAUUUAAGGAUGAAGA-3’, SHH-as: 5’-UCUUCAUCCUUAAAUAUGAUGUCGG-3. Cells were seeded in 6-well plates at a cell density of 3×105 cells/well in 10% serum medium without antibiotics. After 24h, cells were transfected in Opti-MEM using LipofectamineRNAiMAX(Invitrogen) and the 20 nmole siRNA was resuspended according to manufacturers instructions. The cells treated with Stealth negative control med component of kit (#12935300, Invitrogen) were served as controls (si-control).siRNA transfection efficiency in cells was assessed by Western-blotting.
Statistical analysis was performed using SPSS16.0 software. Data presented were mean ± SD from three different experiments. Statistical significance between different groups was determined using Students t-test. A value of P<0.05 was considered statistically significant.