Skip to main content
. 2017 Jul 13;8(7):e2932. doi: 10.1038/cddis.2017.319

Figure 2.

Figure 2

Chemotherapeutic drug stimulates breast cancer cells to secret inflammatory cytokines to activate inflammatory-related pathways. (a) qPCR analysis of the gene expression of various cytokines and chemokines in the cells after treatment for 4 days with vehicle or paclitaxel (15 nM) followed by culture in fresh medium for additional 4 days. At day 8, after 4-day paclitaxel withdrawal, cells were harvested for qPCR analysis (the culture media were collected as supernatants analyzed by human cytokine arrays in B or applied to fresh cells in consequent experiments). Data from vehicle-treated cells were set as 1 and GAPDH mRNA was used to normalize variability in template loading. Data represent the means±S.D., n=4; *P<0.05, **P<0.001. (b) Human cytokine arrays for protein concentrations of IFN-γ, TNF-α, CCL2, IL6, IL8 and IL1β in the supernatants as described in a. Analyses were performed blindly by Eve Technologies using human cytokine multiplex assays. Data represent means±S.D., n=3; *P<0.05, **P<0.001. Veh-SNs: supernatants (SNs) derived from vehicle (Veh)- pretreated cells; Pa-SNs: derived from paclitaxel (Pa, 15 nM)-pretreated cells. (c) Western blotting analysis of the phosphorylation of NF-κB, IκBα and Stat3 in SUM190 and SUM149 cells at 2 and 4 days after exposure to the vehicle (Veh)-derived or paclitaxel (Pa, 15 nM)-derived supernatants (SNs). β-actin as an internal loading control. (d) Flow cytometric analysis of the 7xTCF-eGFP reporter activity in Wnt reporter subline 7xTCF-SUM190 in the presence of vehicle (Veh)-derived or paclitaxel (Pa)-derived supernatants (SNs) for 4 days. Mock transduced SUM190 cells (non7xTCF) and 7xTCF-SUM190 subline generated by lentiviral transduction, and denoted as non7xTCF-Veh-SNs, 7xTCF-Veh-SNs and 7xTCF-Pa-SNs. Live cells were pre-gated based on 7-AAD-negative. The TCF-eGFP reporter activity was measured by the percentage of GFP-positive cells after different treatments. Data represent the means±S.D., n=3, *P<0.05. (e) qPCR analysis of the expression of NF-κB target gene IKBA and Wnt target gene AXIN2 in SUM190, SUM149 and MDA-MB-231 cells after exposure to vehicle (Veh)-derived or paclitaxel (Pa, 15 nM)-derived supernatants (SNs) for 4 days. Data represent means±S.D., n=3; *P<0.05. (f) Kaplan–Meier survival analysis of overall survival and disease-free survival time in TNBC patients treated with chemotherapeutic drugs. Poor survival rate and shorter disease-free survival time were observed if patients’ tumor samples expressed high levels of Wnt and NF-kB target genes (cBioPortal, TCGA, Nature Communications 2016, mRNA microarray, z-score±2.00, patient set: 165 TNBC patients treated with chemotherapy)