Cell culture, transfection, and treatment
Human EC Ishikawa cell line (Beijing Union Cell Bank, China) were maintained in a basal medium (Dulbecco’s modified Eagle’s medium; Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA) in a 37°C humidified incubator with 5% CO2. The Arr2 full-length vector and the GFP vector were generous gifts from Dr. Gang Pei (Shanghai Institutes for Biological Sciences, China) [26].
Ishikawa cells (1 × 105) were seeded on 24-well plates for 48 h before transfection. Transfection was performed with 1 μg of either Arr2 full-length vector or GFP vector using Lipofectamine 2000 (Invitrogen Corporation, Carlsbad, CA, USA), according to the manufacturer’s instructions. Forty-eight hours later, the medium was replaced with the basal medium containing 1 μg of G418. After screening by G418 for 2 weeks, single clone was selected and seeded in 35-mm dishes. Stable transfection was verified by western blot, namely, the Ishikawa/Arr2+ cell line and the Ishikawa/GFP cell line.
Animals
Female BALB/c nude mice 6 to 8 weeks old were purchased from Shanghai Ling Chuang Biotechnology. Mice were housed under pathogen-free conditions with 12 h light/12 h dark, temperature of 20–25 °C and humidity of 40–70%. Mice had free access to complete nutritional palletized feedings and drinking water. Mice were subcutaneously injected in their right armpit with 0.1 ml cell suspension of 1 × 107/ml in vitro cultured Ishikawa cells to initiate tumor growth. In vivo experiments in our study were performed according to the Institutional Animal Care and Use Committee (IACUC) guidelines. All procedures were approved by the Peking University People’s Hospital Committee on Animal Care.
In vivo studies
Mice were randomly divided into two groups with 15 mice in each group. The control group were injected with Ishikawa/GFP cells and the Arr2+ group were injected with Ishikawa/Arr2+ cells. On Day14 when the size of tumor reached 75-100 mm3, each group of mice were randomly divided into three subgroups with 5 mice in each. On Day14 and Day21, the three subgroups of mice were intraperitoneally injected with normal saline (NS), 10 mg/kg paclitaxel and 20 mg/kg paclitaxel respectively. The xenografted tumor size was measured using vernier caliper every 2 to 3 days. Tumor volume (TV) was calculated by the formula 1/2 × A × B2, where A is the long diameter, B is the short diameter. Relative tumor volume (RTV) was calculated by Vt/V0 where V0 is the TV right before paclitaxel was first given, and Vt was the TV measure each time after that. To evaluate anti-tumor activity, relative tumor growth ratio T/C(%) and anti-tumor activity index were calculated by formula shown as follow:
$$ T/C\left(\%\right)=\frac{T_{RTV}}{C_{RTV}}\times 100\% $$
TRTV: RTV in treatment group, CRTV:RTV in control group.
All of the animals were euthanized on Day28 by CO2 inhalation and the xenograft were taken for the measurement of tumor weight. Tumor tissues were taken for further assessment of cell necrosis and expression of different molecules.
Hematoxylin- and-eosin (H&E) staining
All tissues were fixed in 4% neutralised formaldehyde, embedded in paraffin, cut into 4-Ìm sections and stained by hematoxylin- and-eosin (H&E) to confirm their histological diagnosis and other microscopic characteristics. Assessment criteria included tumor cell morphology, necrosis, angiogenesis, inflammatory cell infiltration and fibrous tissue formation. Necrotic scores from 0 to 4 were given according to the percentage of necrotic area. 12%~ 25% was given 1 point, 25%~ 50% was given 2 points, 50%~ 75% was given 3 points. The tissue with necrotic area above 75% were given 4 points and those less than 12% were given 0.5 points. If there wasn’t any necrosis in the observed area, 0 point was given. Three different visual areas were chosen randomly in each section and their average was used in the analysis.
Western blotting
Tumor tissues were lysed in RIPA buffer (Kaiji Biotech, Nanjing, China) with protease inhibitors (Amresco, Solon, OH, USA). After centrifugation at 14,000 rpm for 15 min, the supernatants were collected. Proteins were quantified using the BCA assay. Protein samples (20–30 μg) were resolved by 10% SDS-PAGE under reducing conditions and subjected to western blot analysis. After protein transfer to a PVDF membrane (Amersham, GE Healthcare, Waukesha, WI, USA), the membrane was blocked overnight at 4 °C using 5% BSA blocking buffer. The antibodies used for western blotting were 1:500 human monoclonal anti-β-arrestin-2 (Abcam, Cambridge, MA, USA, Ab54790), 1:1000 human monoclonal anti-TLR2 (Abcam, Cambridge, MA, USA, Ab24192), and 1:500 human monoclonal AKT (Kaiji Biotech, Nanjing, China, KG21054), GSK3β(Kaiji Biotech, Nanjing, China, KG21002), NFkBp65(Kaiji Biotech, Nanjing, China, KGYM0474), caspase3(Kaiji Biotech, Nanjing, China, KG22205) and caspase9(Kaiji Biotech, Nanjing, China, KG22222) antibodies. The secondary antibody was 1:2000 horseradish peroxidase-conjugated antibody (Kaiji Biotech, Nanjing, China). Western blots were developed by ECL (Pierce Chemical, Dallas, TX, USA). The bands were quantified using the Quantity One V4.52 software (Bio-Rad, Hercules, CA, USA) and were normalized with the density of GADPH bands.
Immunofluorescence microscopy
Tumor tissues were fixed and incubated in a blocking buffer (0.5% Triton X-100, 1% BSA-PBS) for 1 h at room temperature followed by incubation with 1:200 human monoclonal anti-β-arrestin-2 (Abcam, Cambridge, MA, USA, Ab54790), 1:200 human monoclonal anti-TLR2 (Abcam, Cambridge, MA, USA, Ab24192) at 4 °C overnight. They were then incubated with antibody conjugated with fluorphores at room temperature for 1 h, next to 5 mg/ml DAPI for 10 min for nuclear staining. The fluorescence signals were observed under a fluorescence microscope (Olympus, Japan).
Real-time PCR
Total RNAs were extracted from tissues with TRIzol (Invitrogen) according to the manufacturer’s instructions. Reverse transcription of 2 μg of the purified RNA was performed using RevertAid™First Strand cDNA Synthesis Kit (Thermo Fisher, USA). Then quantification of the genes was performed by real-time PCR, using ABI Step one plus Real time-PCR system with Real time PCR Master Mix (SYBR Green) (TOYOBO, Japan). Expression values were normalized to those obtained with control GAPDH. The primer pairs are as follows:
GAPDH, Sense 5′-TATGTCGTGGAGTCTACTGGT-3′, Anti-sense 5′-GAGTTGTCATATTTCTCGTGG -3′;
IL6, Sense 5′-CAATGGCAATTCTGATTGTATG-3′, Anti-sense 5′-AGGACTCTGGCTTTGTCTTTC-3′.
IL8, Sense 5′-TGTTGAGCATGAAAAGCCTCTAT-3′, Anti-sense, 5′-AGGTCTCCCGAATTGGAAAGG-3′.
TNF-α, Sense 5′-CCTGTAGCCCACGTCGTAG-3′, Anti-sense, 5′-GGGAGTAGACAAGGTACAACCC-3′.
Statistical analysis
All statistical analyses were conducted using SPSS 25 (IBM, Armonk, NY, USA). Data was expressed as mean ± standard deviation (SD) from at least three independent experiments. The differences among groups were assessed using one-way analysis of variance (ANOVA) with the Bonferroni’s post hoc test. Two-sided P-values < 0.05 were considered statistically significant, while P-value < 0.01 were considered very significant.
