For the purpose of this study, we refer to these miRNAs as “resistance-relevant”. Namely, we selected miR-16, miR-21, miR-23a,

miR-24, miR-26a, miR-106, miR-141, miR-155, miR-196a, miR-200a, miR-200b, miR-200c, miR-221, miR-222, miR-296-5p, miR-376a, miR-429 and let-7i for this study. The miScript PCR system (Qiagen, Germany) was then used to analyze miRNA expression of the resistance relevant miRNA candidates after PPI treatment (LD50). miScript assays were performed according to the manufacturer’s instructions. Briefly, for each sample, 500 ng of DNase pre-treated RNA was used for reverse transcription into cDNA. Following the manufacturer’s protocol, we utilized 4 μl miScript 5X RT Buffer, 1 μl Reverse Transcriptase and 5 μl nuclease-free water. PND-1186 purchase Incubation of reagents was performed in Sotrastaurin manufacturer a thermocycler (protocol: 60 minutes at 37°C, 5 minutes at 95°C, then a hold

at 4°C). For real-time PCR, 2 μl of cDNA was mixed with 10 μl QuantiTect SYBR, 2 μl 10X miScript Universal Primer, 2 μl gene specific 10X miScript Primer Napabucasin supplier Assay, and 4 μl nuclease-free water. All samples were assayed in triplicate reactions using a BioRad CFX 384 Real-Time System (Hercules/California USA). Quantitative analysis was performed using Bio-Rad CFX Manager 2.1. MiRNA expression data were normalized to the expression levels of SNORD25, SNORD44 and SNORD68, which displayed comparable expression across the different groups (data not shown). Statistical analysis All data are means ± standard deviation unless otherwise stated. The relative cell survival why after PPI treatment (viability assay) and after treatment with anticancer drugs was calculated by normalizing

the mean corrected absorbance of the treated cells to the corresponding untreated controls (given in%). For assessment of the effect of PPI treatment on sensitivity to chemotherapy, the relative survival of the negative controls was then be set to “0”, and the effect of pre-treatment was presented as relative survival of treated cells compared to negative controls (given in%). Data were assessed for statistical significance using parametric (Student’s t-test for equal and unequal variances) tests as appropriate. P <0.05 was considered to be statistically significant. All analyses were performed using SPSS 20.0 (SPSS, Chicago, IL). Results Esomeprazole inhibits survival of esophageal cancer cell lines At first, we aimed to assess if esomeprazole impacts on survival of esophageal cancer cell lines. Figure 1 presents an overview of the dose–response curves of PPI treatment with esomeprazole at various doses in SCC (A) and EAC (B) cell lines. In both tumour subtypes, increasing esomeprazole doses were dose-dependently associated with decreasinging cell survival with increasing esomeprazole doses, thus providing evidence for a negative impact of PPI treatment on tumour cell survival.