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  • br Cell culture and reagents

    2020-08-28


    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 SRS16-86 (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
    In order to investigate the clinical significance of the pretherapeutic serum IL-6 and IL-8 levels in patients diagnosed with primary advanced gastric cancer, 111 cases, undergoing two cycles neoadjuvant che-motherapy containing platinum-based drugs, were enrolled in this study, and all these patients were performed radical surgery after neoadjuvant chemotherapy. Overall, 48 patients obtained complete response or partial response (chemosensitive group), and 63 patients underwent stable or progressive disease (chemoresistant group) based on RECIST. As shown in Table 1 and Fig. 1A, the serum IL-8 level was associated with Lauren diffuse and mixed type (P = 0.000), advanced tumor invasion (P = 0.017) and poor response to platinum-based che-motherapy (P = 0.000); however, the serum IL-6 level had no effect on the clinicopathological features (P > 0.05).