Cell lines and cell culture
Human ESCC cell lines TE-1, EC109 and KYSE150 were obtained from Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Human ESCC cell line KYSE510 was obtained from German Collection of Microorganisms and Cell Cultures (Germany). All cells were routinely cultured in RPMI-1640 or MEM medium supplemented with 10% fetal bovine serum (FBS) and maintained at 37°C in 5% CO2 and 100% humidity. For continuous treatment cells, the 1/2 and 1/4 IC50 concentrations of cisplatin were continuously given to cell lines for 2-3 weeks, and then cells were collected for next investigation.
Patientsand tissue specimens
A total of 59 ESCC patients who received platinum-based regimens were enrolled between Jan 2006 and Dec 2007 from Southwest Hospital (Chongqing, China). The enrolled patients met the following eligibility criteria: histological or cytological confirmation of esophageal squamous cell carcinoma, presence of measurable disease, no second malignancies, and availability of adequate diagnostic tumor tissues. The clinicopathologic features of these patients are summarized in Table S1. Overall survival (OS) was defined as the time between the onset of chemotherapy and the date of the last follow up or death from any cause. Ethical oversight and approval were obtained from the Institutional Review Board of Southwest Hospital, and written informed consent was obtained from all patients.
Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded ESCC sections (4 μm) using Dako REALTM EnVision™ detection System (Code K5007; Dako, Glostrup, Denmark) as previously described . Briefly, the sections were pretreated by 0.3% H2O2 and antigen retrieval was performed according to the manufacturer’s instructions. The slides were incubated with mouse anti-human Shh (1:100; Cell Signaling Technology, USA) or Sox2 (1:100; Cell Signaling Technology, USA) at 4 °C overnight. The secondary antibody (1:400; Jackson ImmunoResearch, USA) was added for incubation at 37 °C for 30 min. All slides were evaluated independently by two pathologists in a blind manner. The intensity of immunohistochemical staining and the proportion of positively stained tumor cells was evaluated as previously described . Briefly, the staining intensity was scored with “0” (no staining), “1” (weakly positive), “2” (moderately positive), and “3” (strongly positive). The staining average percentage of positive cells was scored as:0=0%, 1=1-25%, 2=26-50%, 3=51-75%, and 4=76-100%. The expression levels of detected proteins were reported by multiplication of staining density and average percentage of positive cells. It was defined as high expression if calculation of the scores more than 4 or 2 for Shh and Sox2, respectively, and the cut-off was derived from X-tile analysis, other tumor tissues were considered as low expression.
Compounds and reagents
Dihydroartemisinin (DHA) and cisplatin (CDDP) were obtained from Sigma, USA. These agents were dissolved in DMSO to 100 mM and stored at -20°C. Before treatment, the stock solution was diluted to different concentrations. The final concentration of DMSO in cultures was 0.1% (v/v) or less. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) was purchased from Sigma, USA. The primary antibodies against Shh, PTCH1, Gli1, Nanog, Sox2, Oct4, P-gp, ALDH1A1, Tubulin, and β-actin were listed in Table S2.
Cell viability assay
The in vitro cell viability was determined by MTT assay. The cells (/mL) were seeded into 96-well culture plates at 5×103 cells/well in 100 μL DMEM containing 10% FBS. After incubation overnight, the cells were treated with various concentrations of agents for 48 h. Then 10 μL MTT solution (2.5 mg/mL in PBS) was added to each well, and the plates were incubated for an additional 4 h at 37 oC. After centrifugation (2500 rpm, 10 min), the medium with MTT was aspirated, and replaced with 100 μL DMSO. The optical density of each well was measured at 570 nm with a Molecular Devices M5 Reader.
For transiently silencing Shh, the Shh Silencer Select Validated siRNA was purchased from Life Technologies, USA (Catalog #AM16708). For overexpression of Shh, human Shh full-length cDNA was cloned into the pCMV expression vector. The Shh siRNA and pCMV-Shh (2 μg/μL) was transiently transfected into TE-1 or KYSE-510 cells by Lipofactamine 2000 (Invitrogen) according to the manufacturer’s instructions. Transfection efficiency was verified by western blotting.
ALDEFLUOR assay and flow cytometry
An ALDEFLUOR kit (Stem Cell Technologies, Canada) was used to isolate tumor cells with ALDH enzymatic activity according to the manufacturer’s instruction. Briefly, ESCC cells were suspended in ALDEFLUOR assay buffer containing ALDH substrate, then treated with BODIPY-aminoacetaldehyde (BAAA) and incubated for 40 min at 37 °C. Diethylaminobenzaldehyde (DEAB), a specific ALDH inhibitor, was used as a negative control. 7-Amino-actinomycin D (7-AAD) staining solution (BD Pharmingen, USA) was used to exclude dead cells. Cells with intact plasma membranes were sorted for experiments.
Western blot analysis
ESCC cells or FACS-sorted ALDHhigh cells were gathered after pre-treatment for the indicated time periods as described previously . Western blotting was performed as previously described . Briefly, equal amounts of total protein extracts from cultured cells or tissues were fractionated by 8-12% SDS-PAGE and then electrically transferred onto polyvinylidene difluoride (PVDF) membranes. Mouse or rabbit primary antibodies and appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies were used to detect the designated proteins. The bound secondary antibodies on the PVDF membrane were reacted with ECL detection reagents (Pierce; Rockford, USA) and detected by a ChemiDocXRS system (Bio-Rad). β-actin or Tubulin were used as loading control.
Quantitative RT-PCR analysis
Total RNA was isolated from cells using RNeasy Mini Kits (Qiagen) as described in the product insert. The RNA was reverse transcribed with RevertAid First Strand cDNA Synthesis Kits (Thermo) and PCR was done using iQ SYBR Green Supermix and the CFX96 Real-Time PCR Detection System (Bio-Rad). Primers used in this study were listed in Table S3. The expression of each gene was determined using the 2−ΔΔCT method. Results were normalized against GAPDH. All experiments were performed in triplicate, and results were plotted as the mean ± s.d.
Real-time cell analysis (RTCA)
The RTCA assay was performed as previously described . A chemotactic signal for movement was provided by inoculating 30,000–50,000 TE-1 cells in serum-free medium in the upper chamber and supplying RPMI-1640 with 10% FBS in the lower chamber. Cell index (electrical impedance) was monitored every 5 min for the duration of the experiment. The cell index represents the capacity of cell migration, and the slope of the curve was related to the migration velocity of tumor cells.
Colony formation assay
One hundred viable FACS-sorted ALDHhigh TE-1 cells per well were seeded in each well of 24-well plates and cultured in DMEM containing 10% FBS. After incubation for 2 weeks, the colonies were stained with crystal violet and the colonies containing more than 50 cells were counted.
Tumorsphere formation assay
FACS-sorted ALDHhigh cells were seeded in 6-well ultra-low attachment plates (Corning, NY) at 500 cells/well with or without the indicated treatments in serum-free DMEM/F-12 medium supplemented with B27 (1×, Invitrogen), 20 ng/mL human recombinant bFGF (PeproTech), 20 ng/mL EGF (PeproTech), 10 ng/mL leukemia inhibitory factor (Chemicon) and 4 U/L insulin (Sigma). After culture for two weeks, spheres were counted under a phase contrast microscope, and pictures were taken by AF6000 and DFC350FX (Leica).
Immunofluorescence confocal microscopy
FACS-sorted ALDHhigh cells were seeded on cover slides and cultured in DMEM containing 10% FBS for 7 days with or without treatment. The cells were then fixed and blocked by preimmune goat serum. Primary mouse anti-human Gli1 (1:100, Cell Signaling, USA) was added to the cells and incubated at 4 °C overnight. Secondary goat anti-mouse IgG conjugated with Cy5 (1:500, Cell Signaling, USA) was added to the cells and incubated at 37 °C for 30 min. DAPI was used to stain the nuclei. Cells were then observed under laser confocal microscopy (Zeiss, Germany).
Determination of combination index
TE-1 or KYSE-510 cells were treated with different concentrations of DHA alone, cisplatin alone, or the two agents in combination. The cell viability was measured by MTT assay. The nature of the drug interaction was analyzed by using the combination index (CI) according to the method of Chou and Talalay. A CI value lower than 0.90 indicates synergism; a CI value between 0.90 and 1.10 indicates an additive effect; and a CI value higher than 1.10 indicates antagonism. Data analysis was performed using Calcusyn software (Biosoft, Oxford, UK).
Chromatographic conditions of Cisplatin detection
The cells were treated as above mentioned and collected according to the previous reports. The HPLC analysis was used to detect cisplatin in cells and performed on an Agilent 1100 series HPLC system comprising degasser, binary pump, thermostatted column compartment and UV detector (Waldbronn, Germany). Chromatographic separations was achieved on a Kromasil C18(250mm×4.6mm, 5μm) with a mobile phases consisting of methanol -water (80:20, v/v) at the flow rate of 1.0 mL/min. The column temperature was kept at 25C and the UV detector was set at 254 nm.
Mouse xenograft tumor study
For tumorigenesis assessment, viable TE-1 cells (1×106/100 μL PBS per mouse), as confirmed by trypan blue staining, were subcutaneously injected into the right flank of 7- to 8-week-old female BALB/c mice. When the average tumor volume reached 100 mm3, the mice were randomly divided into four treatment groups, including control (saline only, n=4), DHA (25 mg/kg/5 days/week, i.p.; n=4), CDDP (4 mg/kg/week, i.p.; n=4), and the combination (n=4). Tumor size was measured once every three days with a caliper (calculated volume=shortest diameter2×longest diameter/2). The body weight was also measured once every three days to assess gross toxicity. After 35 days, the mice were sacrificed and the tumors were excised and stored at -80°C until western blot analysis. These studies were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Shenyang Pharmaceutical University.
Differences between experimental groups were evaluated by one-way ANOVA with Turkey’s post-hoc test using the SPSS11.5 software package for Windows (SPSS, Chicago, IL). Survival curves were constructed using the Kaplan–Meier method. Statistical significance was based on a P-value of 0.05 (P<0.05, two-tailed test).