Figure 1.
Results of MTT and migration assays.
MTT assays (A) in Panc-1 (with activating point mutations in K-ras) and BXPC-3 (without ras mutations) cells in vitro. Exposure for 7 days to epinephrine (15 nM) significantly (p<0.001) increased the number of viable cell in both cell lines. The Cox-2 inhibitor celecoxib (1 µM) completely blocked this response to epinephrine (p<0.001) while additionally reducing (p<0.001) the number of viable cells in cells not pre-treated with epinephrine. The effects of identical treatments on cell migration are shown in Fig. B. In both cell lines migration was significantly more stimulated by chronic epinephrine than cell proliferation. Celecoxib significantly (p<0.001) reduced epinephrine-induced migration but did not completely block this response. BCPC-3 cells were slightly more responsive to epinephrine in both assays, but the differences between the two cell lines were not significant. Columns are mean values and standard deviations of 5 samples per treatment group.
Figure 2.
Cell proliferation and migration dose response-curves.
Dose response-curves for celecoxib in Panc-1 and BXPC-3 cells in vitro in the presence and absence of pretreatment for 7 days with epinephrine (15 nM). Figures A and B show cell proliferation responses in MTT assays while Figures C and D show cell migration responses. While both cellular responses were similar among the two cell lines, EC50 values for celecoxib in Figs. B, C and D were significantly (p<0.001) lower in the cells pretreated for 7 days with epinephrine. Data points are mean values and standard deviations (n = 5). EC50 values and curve fit were established by nonlinear regression analysis for sigmoidal dose-responses.
Figure 3.
Mean values and SE of xenograft volumes over time from the BXPC-3 cell line in groups of mice (n = 20).
Animals were exposed to social stress (SS) or not exposed to stress (NS) in the presence and absence of celecoxib (C) or celecoxib and GABA (C+G) treatment. The xenograft volume was significantly increased (p<0.0001) by social stress. This response was significantly inhibited by treatment with celecoxib alone (p<0.001) or by celecoxib and GABA (p<0.0001). Overall, the differences in tumor volumes were statistical significant (p<0.001) between treated and untreated groups. Photographs show representative examples of xenografts for each treatment group.
Figure 4.
ELISA immunoassays demonstrating levels of intracellular cAMP and PGE2.
cAMP was assessed in the cellular fraction of blood and in xenograft tissues (A) and levels of PGE2 in serum and xenograft tissues (B). Social stress increased the levels of both factors significantly (p<0.001), responses significantly (p<0.001) reduced by celecoxib. Combined celecoxib/GABA significantly (p<0.001) enhanced the inhibitory effects of celecoxib (p<0.001) on systemic and tumor levels of cAMP and PGE2 in stress free and social stress-exposed mice. Columns are mean values and SD of 5 randomly-selected samples per treatment group.
Figure 5.
Protein expression of p-ERK and p-CREB (A) or p-AKT and p-Src (B) in BxPC-3 xenografts of stress free (NS) and social stress (SS) exposed mice.
Western blots (A and B) illustrating induction of p-ERK, p-CREB, p-AKT and p-Src by social stress and inhibition of these responses by celcoxib alone or celecoxib + GABA. Quantitative assessment of these changes at the protein level were determined in the ELISA assays shown in Figure C and D. Induction of all investigated phosphorylated proteins by social stress (SS) was significant (p<0.001). Inhibition of these responses by celcoxib alone or the combination of celecoxib + GABA were significant (p<0.001) for all phosphorylated proteins. Columns in the graphs (C and D) are mean values and SD of quantitative ELISA immunoassays from five randomly-selected xenografts per treatment group.
Figure 6.
Protein expression of COX-2 and p-5-LOX.
Western blot (A) is representative of three independent blots prepared from three randomly-selected xenografts per treatment group. Semi-quantitative densitometry of the bands from three independent Western blots showed significant (p<0.0001) increases in protein expression by social stress. These responses in turn were significantly reduced by celecoxib treatment alone (p<0.001) or by celecoxib and GABA (p<0.0001). Columns in the graph (B) are mean values and SD of four densitometric readings per band adjusted for actin in independent blots prepared from three randomly-selected xenografts per treatment group.
Figure 7.
ELISA immunoassays demonstrating levels of VEGF in serum and xenograft tissues.
Social stress significantly (p<0.001) increased VEGF levels in both serum and xenografts, a response significantly reduced by celecoxib (C, p<0.001) while celecoxib and GABA (C+G) treatment completely blocked the stress-induced induction of VEGF in serum and tumor tissue. In addition, celecoxib plus GABA significantly reduced VEGF below basal levels in mice not exposed to stress (p<0.001). Columns are mean values and SD of five randomly-selected samples per treatment group.
Figure 8.
Simplified working model showing the effects of celecoxib and GABA.
Xenograft growth from human pancreatic cancer cell line BxPC-3 was induced in mice by psychological stress via the interaction of β-adrenergic and COX-2 signaling pathways. Adenylyl cyclase activation downstream of the β-ARs induces the cAMP-PKA-CREB pathway, transactivates EGFR, and induces the release of EGF and AA, which in turn enhances VEGF and PGE2 production. The inhibitory neurotransmitter GABA inhibits this signaling cascade by Gαi-mediated inhibition of adenylyl cyclase activation, whereas the reduction in PGE2 formation caused by celecoxib additionally reduced adenylyl cyclase activation downstream of the Gαs-coupled PGE2 receptors. The resulting simultaneous inhibition of VEGF, CREB, MAPK, Src, and AKT pathways contributed to the significant inhibition of tumor progression.