IL-6 stimulates STAT3 and Pim-1 kinase in pancreatic cancer cell lines

Abstract

Objectives: We investigated the signaling pathways activated in response to interleukin 6 (IL-6) in pancreatic cell lines, with a focus on signal transducer and activator of transcription 3 (STAT3) and proto-oncogene serine/threonine-protein (Pim-1) kinase.

Methods: Interleukin 6 receptor (IL-6R) expression and IL-6-induced cell signaling was measured by Western blotting in human pancreatic cell lines. Cucurbitacin I was used as a pharmacological tool to investigate the role of STAT3 in Pim-1 activation. Stably overexpressing Pim-1 kinase cell lines were characterized for their response to IL-6 in vitro and for their growth rate as flank tumors in scid mice.

Results: Interleukin 6 receptor was expressed across multiple cancer cell lines. In Panc-1 cells, IL-6 treatment increased expression of phosphorylation of signal transducer and activator of transcription 3 and Pim-1 kinase. Cucurbitacin I treatment alone increased pErk1/2 expression in wild-type and Pim-1-overexpressing cell lines and resulted in exaggerated Pim-1 kinase protein levels in control and IL-6-stimulated cells, suggesting that up-regulation of Pim-1 may be partially STAT3 independent. Pim-1 overexpression did not significantly affect growth rate in vitro or in vivo in Panc-1 or MiaPaCa2 cell lines.

Conclusions: Interleukin 6 activates STAT3 and stimulates Pim-1 kinase in pancreatic cell line models. The regulation and consequence of Pim-1 expression seems to be highly context dependent.

Figure 1

A. Western blot analysis of total cell lysate prepared from pancreatic cancer cell lines. The blot was probed for IL-6Rα and stripped and re-probed for actin as a loading control. Densitometry analysis was performed and quantitation displayed as fold change from the HPD-EV cells. B. Western blot analysis of total cell lysates prepared from pancreatic cell lines which were serum starved for 24 hr prior to treatment with IL-6 for the times indicated. Blots were probed using an antibody specific for STAT3 phosphorylated at tyrosine 705. Blots were stripped and re-probed for total STAT3 as a loading control. Bands were subjected to densitometric analysis and quantitation displayed as fold change from the mock treated cells.

Figure 2

A. Western blot analysis of whole cell lysates prepared from pancreatic cell lines. Blots were probed for Pim-1, Pim-2, Pim-3 and actin as a loading control. B. Pim-1 message expression in a panel of pancreatic cell lines measured by quantitative RT-PCR and normalized to β2 Microglobulin (β2M). Cells were not treated with IL-6. These results are the mean value of samples run in duplicate. Similar results were obtained in three separate studies.

Figure 3

A. Western blot analysis of total cell lysates from cells that were serum starved and treated with either IL-6 or EGF for the times indicated. Blots were probed for Pim-1 with tubulin used as a loading control. A separate blot was also probed for phosphorylated STAT5 A/B with total STAT5 used as a loading control. B. Western blot analysis of total cell lysates from Panc-1 cells which were serum starved for 24 hr followed by a 4 hr pre-incubation with cucurbitacin I prior to a 1 hr incubation with IL-6. Blots were probed for Pim-1 and P-STAT3 with tubulin and total STAT3, respectively, for loading controls. C. Analysis of cell proliferation measured using an MTT assay. Panc-1(■) and MiaPaCa2 (▲) cells were grown for 72 hr and exposed to increasing levels of cucurbitacin I (N=6).

Figure 4

A. Western blot analysis of whole cell lysates prepared from Panc-1 and MiaPaCa2 cells including wild-type, negative control, and Pim-1 stably transfected cells. The blot was probed for Pim-1 and actin as a loading control. B. Western blot analysis of whole cell lysate prepared from wild-type and Pim-1 transfected Panc-1 cells which were serum starved for 24 hr and then treated with IL-6 for the times indicated. C. Relative Pim-1 message expression measured by qRT-PCR in wild-type Panc-1 cells that were serum starved 24 hr prior to treatment with IL-6 (100ng/mL) for the times indicated. Data shown is representative of the observed trend in three separate experiments. D. Western blot analysis of total cell lysates from wild type, negative control, and stably over-expressing Pim-1 kinase MiaPaCA2 and Panc-1 cells. Cells were serum starved 24 hr followed by a 4 hr pre-incubation with cucurbitacin I (5 μM) prior to a 1 hr incubation with IL-6. Blots were probed for Pim-1 and P-STAT3 with tubulin and total STAT3, respectively, for loading controls. E. Western blot analysis of total cell lysates from cells serum starved for 24 hours and treated with vehicle alone, or cucurbitacin (5 μM) for 4 hours. Blots were probed for Pim-1, tubulin as a loading control, and pErk1/2, and total Erk1/2.

Figure 5

A. AnnexinV/PI staining in the Panc-1 panel (wild-type, negative control, and Pim-1 over-expressing Pim-1 kinase cells) following 24 hr of serum starvation. Data shown is from one experimental replicate that represents the trend observed in three separate experiments. B. BrdU staining in the Panc-1 panel following 24 hr of serum starvation. C. Cell number counted every 24 hr for 4 days (96 hr) in the Panc-1 panel. D. Growth of the Panc-1 panel as flank xenografts in scid mice (N=4 mice/group). E. VEGF-A concentration in cell culture media from the Panc-1 panel of cells collected 48 hr following IL-6 treatment (N=3). E. MTT analysis of Panc-1 cells which were treated with IL-6 for 72 hours (following serum starvation for 24 hours). Each assay represents an N=6.