Figure 3.

SP promotes lung tumorigenesis by stimulating PI3K/AKT and NF-kB signaling pathways

(A) PAFR-expressing H460 and H1299 cells treated with SP were analyzed by Western blot to determine expression of PI3K, AKT, and NF-kB. The cancer cells incubated with SP had higher expression levels of PI3K, AKT, and NF-kB compared with cells treated with PBS after 48 h treatment. Band intensity was determined by using ImageJ, and the ratio of each band was normalized to the corresponding GAPDH and shown below each band. SP activated PI3K, AKT, and NF-kB in H460 and H1299 cells.

(B) siRNA was used to deplete PAFR in H460 and H1299. The cells were treated with SP. SP-induced activations of PI3K, AKT, and NF-kB were inhibited by the depletion of PAFR in the cells. Data presented as mean ± SEM (n = 3); ∗p < 0.01 by one-way ANOVA.

(C) siRNA was used to deplete PI3K, AKT, and NF-kB in H460 and H1299, respectively. The deletion of PI3K, AKT, or NF-kB decreased the SP-stimulated cell proliferation in the cancer cells. Data presented as mean ± SEM (n = 3); ∗p < 0.01 by one-way ANOVA.

(D) PCR array was used to analyze the inflammatory cytokine gene expression. SP activated pro-inflammatory cytokines (IL-1β, IL-4, IL-6, IL-8, IL-11, IL-12, TNF-α, and MCP-1) (Red). Expression levels of the cytokines in the cells treated with PBS were designated as “1” (Blue). The results are presented as the mean ± SD of three different experiments with triplicates. Data presented as mean ± SEM (n = 3); ∗p < 0.01 by one-way ANOVA.

(E) Forced expression of PAFR in H226 cells was done by using PAFR-overexpressing plasmid. Enforced PAFR expression in the cancer cells increased SP-stimulated activations of PI3K, AKT, and NF-kB determined by Western Blots.

(F) Enforced PAFR expression in H226 cells activated cytokines in H226 cancer cells (Red). Expression levels of the cytokines in the H226 with forced PAFR expression treated with PBS (Blue) were designated as “1”. Data presented as mean ± SEM (n = 3); ∗p < 0.01 by one-way ANOVA.