Supplementary Materialsoncotarget-06-2235-s001. tumor growth and metastases in vivo. Our novel data demonstrate that III-tubulin is definitely a key player in promoting pancreatic malignancy growth and survival, and silencing its expression may be a potential therapeutic strategy to increase the long-term survival of pancreatic cancer patients. 1) decreases clonogenicity; 2) decreases anchorage-dependent and independent proliferation; 3) increases apoptosis and anoikis; and 4) increases sensitivity to chemotherapy drugs including gemcitabine and the TBAs paclitaxel and vincristine. AUY922 inhibitor Notably, we demonstrate the importance of III-tubulin in regulating tumor growth and metastases in a clinically-relevant orthotopic pancreatic cancer mouse model. RESULTS III-tubulin is expressed in human pancreatic tumor cells III-tubulin was expressed at high levels in pancreatic tumor cells, while absent in the acinar and normal ductal cells in PDA tissue (Figure ?(Figure1A).1A). To determine whether the expression pattern was specific to III-tubulin, we also AUY922 inhibitor examined the levels of another -tubulin isotype, II-tubulin, which has been shown to be differentially expressed in tumor cells [27-29]. It too was present at high levels in pancreatic tumor cells, however in contrast to III-tubulin, it was also present in acinar and normal ductal cells (Supplementary Figure 1). Next, we measured III-tubulin expression Proc by western blotting in cell lysates from 3 different pancreatic tumor cell lines produced from primary (MiaPaCa-2, Panc-1) and metastatic (HPAF-II) sites. III-tubulin levels were significantly higher in all 3 pancreatic cancer cell lines compared to normal non-tumorigenic human pancreatic ductal epithelial (HPDE) cells (Figure ?(Figure1B).1B). II-tubulin was also higher in 2/3 pancreatic cancer cell lines (MiaPaCa-2 and Panc-1) compared to HPDE cells (Figure ?(Figure1B).1B). Notably, I-tubulin, which is constitutively expressed in most tissues, was expressed at AUY922 inhibitor similar levels in the pancreatic cancer cell lines and the normal HPDE cells (Figure ?(Figure1B1B). Open in a separate window Figure 1 III-tubulin expression in PDA patient tissue and PDA cell linesA) Immunohistochemistry for III-tubulin in a representative human AUY922 inhibitor PDA tissue specimen. Panels show tissue stained with either isotype control antibody (i) or III-tubulin antibody (ii-iv). The isotype control was negative and tumor elements had strong immunoreactivity for III-tubulin. Panel iv demonstrates an absence of III-tubulin staining in normal acinar cells (region marked by dashed border) and normal ductal cells (arrow) away from the tumor. B) Western blot analysis for I-, II-and III-tubulin in protein extracts from pancreatic cancer cell lines (MIA Paca-2, Panc-1, HPAF-II) versus normal human non-tumorigenic pancreatic ductal epithelial cells (HPDE). GAPDH was used as a loading control. C) Densitometry analysis of III-tubulin expression normalized to GAPDH expression demonstrates that III-tubulin is significantly increased in all 3 pancreatic cancer cell lines compared to HPDE cells (*p 0.05; n=3). Potent and specific knockdown of III-tubulin in pancreatic cancer cells To examine whether III-tubulin could be suppressed in pancreatic cancer cells, we transfected two-independent pancreatic cancer cell lines (MiaPaCa-2 and HPAF-II) with III-tubulin siRNA. 48h and 72h post transfection, III-tubulin expression was measured. Knockdown of III-tubulin was observed at the gene level in both cell lines (MiaPaCa-2, 84.4 2.6% knock-down; HPAF-II, 76.8 1.1% knock-down relative to control-siRNA; 72h post-transfection) (Figure 2A and B). This correlated to knockdown ( 90%) of III-tubulin at the protein level (Figure 2A and B). Knockdown of II-tubulin was also observed when pancreatic cancer cells (MiaPaCa-2 and HPAF-II) were treated with II-tubulin siRNA (Supplementary Figure 2). Open in a separate window Figure 2 III-tubulin silencing in pancreatic cancer cell linesA) Top panel, Western blot analysis of III-tubulin silencing in proteins components from MiaPaCa-2 cells. Cell lysates were harvested from cells 72h or 48h after.