Dorsomorphin br Cell culture and reagents br Human gastric adenocarcinoma

2.2. Cell culture and reagents
Human gastric adenocarcinoma cell lines, AGS (ATCC, VA, USA) and MGC-803 (CBTCCCAS, Shanghai, China), and human stomach
fibroblat line Hs738 (ATCC, VA, USA) were cultured in the complete DMEM (ATCC, VA, USA) with 10% fetal calf serum. Cisplatin and human IL-8 were obtained from Sigma-Aldrich (USA).
2.3. Isolation and culture of primary fibroblasts
Primary CAFs were isolated from gastric carcinoma tissue samples, and primary normal fibroblasts (NFs) were isolated from the  Cancer Letters 454 (2019) 37–43
noncancerous mucosa tissues at least 5 cm from the outer tumor margin in the same patient according to reported protocol [24]. Briefly, fresh samples were washed with serum-free DMEM, cut into small pieces, and were transferred to a 0.15% collagenase IV solution, followed by in-cubation at 37 °C for 40 min. Digested cells were filtered through a 40-
mm cell strainer (Milex-GP) and centrifuged at 1500 rpm for 10 min. The single-cell suspension was incubated in a Fibroblast Medium Kit (Cat. No. P60108, Innoprot) for 24 h, allowing fibroblasts to attach on culture plates. Unattached cells were removed after 24 h incubation, and the adherent cells were further cultivated for experiments. Cultured CAFs and NFs less than five passages were used for our experiments.
The enzyme-linked immunosorbent assay (ELISA) (Invitrogen, CA, USA) was used to detect IL-6 and IL-8 in the patient serums and in the cell culture supernatants. Each experiment was repeated at least three times.
2.5. Cell proliferation assay
Cell proliferation was analyzed using a Cell Counting Kit-8 (CCK-8) assay (Dojindo, Japan) according to the manufacturer's protocol. The results were plotted as mean ± standard error (SE) of three separate experiments for each experimental condition.
2.6. Immunohistochemistry
The expression of α-smooth muscle Dorsomorphin (α-SMA) and IL-8 in gas-tric tissue specimens were detected by immunohistochemistry (IHC). A mouse monoclonal anti-IL-8 antibody (Abcam, Cambridge, UK), and a mouse monoclonal anti-α-SMA antibody (Cell Signaling Technology, MA, USA) were used. IHC was performed on paraffin-embedded for-malin-fixed tissues according to standard protocols.
2.7. Western blotting
Protein expression levels of the indicated molecules were detected using the western blotting [25]. The antibodies used for the analyses were as following: rabbit monoclonal anti-α-SMA antibody, rabbit monoclonal anti-PI3 Kinase antibody, rabbit monoclonal anti-AKT an-tibody, rabbit monoclonal anti-p-AKT antibody, mouse monoclonal anti-IKKα antibody, rabbit monoclonal anti-p-IKKα/β antibody, mouse monoclonal anti-IkBα antibody, rabbit monoclonal anti-p-IkBα anti-body, rabbit monoclonal anti-p65 antibody, rabbit monoclonal anti-p-p65 antibody, rabbit monoclonal anti-ABCB1 antibody, rabbit mono-clonal anti-β-actin antibody, rabbit monoclonal anti-Tubulin antibody (Cell Signaling Technology, MA, USA). The relative levels were quan-tified and normalized against β-actin or Tubulin in the same sample with a densitometric analysis.
2.8. Statistical analysis
Data are expressed as mean ± standard error. In experiments in-volving protein expression, the data were representative of three in-dependent experiments. The associations between the protein levels and various clinicopathological parameters were analyzed with the Pearson's χ2 test. Quantitative data were compared between the control and treatment groups by analysis of variance. All analyses were per-formed with SPSS software (version 19.0; SPSS Inc., Chicago, IL). Values of P < 0.05 were considered to indicate statistical significance.
Fig. 4. IL-8 mediates chemoresistance to cisplatin in human gastric cancer cells via NF-κB activation and ABCB1 up-reg-
3. Results
3.1. High serum IL-8 level in gastric cancer patients is associated with poor chemotherapy response