Cell culture
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 (Gibco, Carlsbad, CA, USA) with 10% FBS (fetal bovine serum).
The lung cancer stem cells 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. CD133-negative cells were cultured
in the RPMI-1640 culture medium.
Cell viability assay
The A549 and H460 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 and cell cycle
were observed by optical microscope and flow cytometry.
Isolation and identification of lung cancer stem cells
Lung cancer stem 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 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
A549 and H460 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 the CellQuest Analysis 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(Invitrogen, Carlsbad, CA, USA) supplemented
with DMEM 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 the cancer cells and cancer stem 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.
Western blotting
Protein sample was extracted from 5 ×106 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 peroxidase-conjugated goat anti-rabbit
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 (Polyclonal Antibody ,ab53281), anti-Smo (Polyclonal Antibody, ab32575), anti-Gli1(Polyclonal Antibody, ab134906), anti-SOX2(Polyclonal Antibody, ab92494), anti-PHC3 (Polyclonal Antibody, GTX32785 ) and anti-GAPDH (polyclonal, CWBIO).
RNA interference
siRNAs for SHH were purchased from Invitrogen and the following sequences were used.
SHH-s:5’-ACAGGCUGAUGACUCAGAGGUGUAA-3’,SHH-as:5’-UUACACCUCUGAGUCAUCAGCCUGU-3’;SHH-s:
5’-GGUGUACUACGAGUCCAAGGCACAU-3’, SHH-as:5’- GACUCGUAGUACACC-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 Lipofectamine RNAi MAX (Invitrogen) and the 20 nmole siRNA was resuspended
according to manufacturers instructions. siRNA transfection efficiency in cells was
assessed by using a commercially available kit (Blockitalexafluor red oligo, Invitrogen).
Statistical Analysis
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.