br Determination of IFN serum levels
2.8. Determination of IFN- γ serum levels by ELISA assay
ELISA assay was performed to determine interferon-γ (IFN-γ) serum level using mice IFN-γ ELISA kits obtained from Sun Red Biotechnology® (China), according to the manufacturer instructions. IFN-γ serum level was calculated from the generated standard ELISA curve.
2.9. Total RNA extraction and semi-quantitive real time RT-PCR analysis of
p53 relative gene expression
Frozen tumor tissues were disrupted and homogenized using TissueLyser II (Qiagen, Germany) with subsequent total RNA extraction using Simply P total RNA extraction kit (Cat # BSC52S1) (BioFlux®, China) following the manufacturer’s instructions. Total RNA con-centration and purity was determined using Nanodrop (Thermo Scientific™, USA). Complementary DNA was synthesized from 1 μg of total RNA by reverse transcription using Quantitect reverse transcrip-tion kit (Cat # 205,311) (Qiagen®, Germany). The reverse-transcription master mix was composed of 1 μl of Quantiscript reverse transcriptase, 4 μl of 5X Quantiscript RT buffer, 1 μl of RT primer mix and 14 μl of template RNA (after genomic DNA elimination reaction) in a total vo-lume of 14 μl. The tubes were transferred to thermo shaker (Biometra®, Germany) and incubated at 42 °C for 15 min. Then, the Quantiscript reverse transcriptase was inactivated by incubation at 95 °C for 3 min and chilled on ice. The synthesized cDNA concentration was calculated
Synthesized cDNA was used as a template for amplification of p53 using SYBR green-based real-time PCR and was detected by Piko real-time PCR instrument (Thermo Fisher Scientific Co., Finland). Gene Moniliformin of p53 was measured using SensiFAST™ SYBR No-ROX Kit (Cat # BIO-980050) (Bioline, Germany). P53 primer (Invitrogen, USA) sequences are: forward: 5′- GTTCCGAGAGCTGAATGAGG-3′, reverse: 5′- TTTTATGGCGGGACGTAGAC-3′. The thermal cycling conditions were as follows: intial polymerase enzyme activation at 95 °C for 2 s, then 40 cycles of 95 °C for 5 s, annealing at 55 °C for 10 s and extension at 72 °C for 20 s. The data represent the average of 3 independent ex-periments. The relative fold change in gene expression of p53 was calculated using Livak method (2− ΔΔCT method). Glyceraldehyde 3-phosphate dehydrogenase (GADPH) was used as a reference gene for mRNA level normalization. No-template sample was used as a negative control.
2.10. Statistical analysis
Statistical analyses were performed using Statistical Package for Social Science (SPSS) version 19. Results were expressed as mean ± SD. To determine significant differences between two groups, P values were calculated by unpaired student t-test. Comparison between 3 or more studied groups was performed with one-way ANOVA (F-testing). P < 0.05 was considered statistically significant.
3.1. Enrichment of tumor cells bearing stem cell like characteristics from parental EC cell line based on their chemotherapeutic drug resistance In order to develop a method to enrich chemotherapeutic drug re-sistant tumor cells
The vast majority of cells died with increasing doses of che-motherapeutic drugs.
EC showed relatively slow growth in low doses of applied che-motherapeutic agents and showed a significant change in cell mor-phology (Fig. 2a) and cell density (Fig. 2b). Alternatively, at higher doses of chemotherapeutic drugs, cancer cell growth greatly decreased and most of the cells died and floated in the culture medium within 72 h of exposure to the chemotherapeutic drugs. After 72 h of exposure to chemotherapeutic drugs, some cells were able to survive, grow and form resistant cells population termed as “drug surviving cells (DSCs)”. We assumed that these DSCs were cancer stem cells (CSCs). To verify this, DSCs were counted using trypan blue staining method and ana-lyzed for CSC surface markers (CD44+ /CD24−) using flow cytometry to select the concentration of one of the applied chemotherapeutic drugs that showed a significantly high percentage of CD44+ /CD24-cells to be used in the preparation of CSC-based DC vaccine.