We first confirmed the sensitivity of the KP3 and BxPC3 pancreatic cancer cell lines with different tumorigenicity to Notch inhibition using GSI IX. Cell proliferation assays showed GSI IX treatment reduced the number of viable KP3 and BxPC3 cells in a dose and time dependent manner (Fig. 1C), consistent with prior studies of Notch inhibition in PDAC. As expected, Western blot and RT-PCR showed that GSI IX treatment strongly suppressed expression of the Notch downstream target Hes1 at 96 hrs, whereas expression was moderately decreased at 48 h time point (Fig. 1A, Fig. S1A?B, 2C). We found that GSI IX treatment induced significant levels of apoptosis in these pancreatic cancer cell lines, with levels increasing with drug dose (Fig. S3A). Finally, GSI IX strongly inhibited the ability of KP3 cells to form colonies in soft agar compared to DMSO treated cells (Fig. S2A). These studies suggest that Notch signaling is necessary for the proliferation, survival, and anchorage independent clonogenic growth of these pancreatic cancer cell lines.(Fig. 4A). The data from Western blot analysis also demonstrated that after 48 h GSI treatment the deregulation of EMT was minor compared to 96 h GSI treatment results (Fig. S1C). Therefore, these data show that GSI treatment could selectively inhibit the EMT phenotype in human pancreatic cancer cell lines.
c-secretase inhibitor IX (GSI) attenuates cell proliferation and mesenchymal program of sorted pancreatic tumor initiating CD44+/EpCAM+ cells
It has been shown in many studies that Notch signaling plays a role in the stem cell renewal and cell fate determination in neural, hematopoietic as well as in embryonic stem cells [41]. In PDAC, it is reported that CSCs are highly tumorigenic and contain high levels of Notch1 and Notch2 [16,37]. We hypothesized that the deregulation of the Notch signalling pathway by GSI could be a useful approach to treat human pancreatic cancer. The pancreatic cancer cell line KP3 expressed 98% CD44 positive and 34% of EpCAM positive cells, but when the markers were sorted together only 19% of cells were positive for both of the markers (Fig. 5A). To further determine the effect of GSI on sorted tumor initiating CD44+/EpCAM+ cells, we analyzed the effect of GSI by cell proliferation assay. As shown in Figure 5B GSI, inhibited the cell proliferation in a dose and time dependent manner. These data reveal the anti-proliferative effects of GSI on tumor initiating CD44+/EpCAM+ cells. We also performed apoptotic assay in these sorted tumor initiating CD44+/EpCAM+ cells in order to study the mechanism by which GSI IX treatment affects cell growth. The results indicated that GSI IX treatment induced significant levels of apoptosis at 10 mM concentration (Fig. S4). Furthermore we studied the effect of GSI treatment on Notch downstream target Hes1 and tumor initiating cell markers CD44 and EpCAM in sorted CD44+/EpCAM+ cells. The results from the Western Blots showed a down regulation of Hes1, CD44 and EpCAM in a dose dependent manner (Fig. 5D). These data suggest that down regulation of Notch may thus contribute to the inhibition of tumor initiating cells or CSC. We further observed the effect of GSI on regulation of EMT process in sorted CD44+/EpCAM+ cells. E-cadherin expression was stable and had no change independent of GSI dosage (Fig. 5E). Next we analyzed mesenchymal markers N-cadherin, Vimentin and Slug where we found that they are also down regulated in dose dependent manner (Fig. 5E). These data suggest that GSI inhibition can be a promising therapeutic intervention to eliminate pancreatic tumor initiating CD44+/EpCAM+ cells and partially prevent EMT phenotype.
Targeting of Notch signalling results in an inhibition of human pancreatic cancer cell migration and invasion
Notch signalling has been implicated in the invasive growth of a number of cancer types. Thus, we sought to explore the impact GSI IX on this process in human pancreatic cancer cell lines. First, we examined cell motility by employing wound healing assays in the presence or absence of GSI IX. Twenty four hours after the scratch, cell migration into the wound was captured under the microscope at 106 magnification (Fig. 2A). For both KP3 and BxPC3 cells, significant inhibition of wound closure was seen with treatment of 5?0 mM GSI IX (P,0.05). In contrast, 80?0% wound healing was seen after 24 h in all untreated cells. Thus, GSI can effectively inhibit the migration of pancreatic cancer cells. Next, we tested cell invasion with transwell chambers. Pancreatic cancer cells were plated in wells of an invasion chamber in the presence of different drug concentrations and experiments were conducted as described in Material and Methods. As shown in Fig. 3A, the invasion of pancreatic cancer cells was significantly reduced (P,0.05) upon treatment with GSI IX, with reductions of up to 75%?0% in the number of invading cells compared to the control group. Thus, treatment with GSI IX has an anti-invasive effect on human pancreatic cancer cell lines.
Intraperitoneal treatment with c-secretase inhibitor IX (GSI) effectively inhibits the growth of CD44+/EpCAM+ xenograft tumors and targets EMT
Given the ability of GSI to inhibit pancreatic tumor initiating CD44+/EpCAM+ cells in vitro, we further tested the in vivo role of GSI in a Xenograft nude mouse (NMRI-nu/nu) model. Sorted CD44+/EpCAM+ cells were subcutaneously injected into the left and right flank. Intraperitoneal treatment with either GSI or vehicle started when the tumor volume reached a size of 10 mm3. All mice were treated for 5 weeks, using an established 3 days on and 4 days off intermittent dose schedule [29].Xenograft tumors grew continuously in vehicle-treated animals, whereas GSI treatment significantly inhibited tumor growth (Fig. 6A�B). Histological analysis of explanted xenograft tumors from GSI or vehicle treated mice did not show any morphological differences (Fig. 6C).
c-secretase inhibitor IX (GSI IX) prevents mesenchymal transition of pancreatic cancer cell lines
Activation of Notch signalling can contribute to the acquisition of EMT, a process associated with invasion and metastasis [39,40]. In order to further examine whether GSI IX can attenuate EMT in human pancreatic cancer cell lines we treated KP3 and BxPC3 cells with different GSI concentrations (2.5 mM, 5 mM, 10 mM) for 48 and 96 h. Pharmacological treatment resulted in a dosedependent decreased expression of mesenchymal markers like Ncadherin, Vimentin and transcriptional factor Slug as assessed by Western Blot (Fig. 4A). The epithelial cell marker E-cadherin had no change irrespective of the dosage in both the cell linesFigure 1. GSI IX inhibits cell proliferation in human pancreatic cancer cell lines and down regulates the Notch pathway downstream target Hes1. (A) KP3 and (B) BxPC3 cells were treated with GSI (2.5 mM, 5 mM, 10 mM) and control (DMSO) for 96 hrs showed a down regulation of Hes1 protein by Western Blot analysis. GSI treatment resulted in a shift in the growth curves. The cell proliferation of (C) KP3 and (D) BxPC3 was measured by cell proliferation assay, GSI inhibited cell proliferation in a dose- and time-dependent manner. Note that these results reveal the antiproliferative effects of GSI on human pancreatic cancer cells. Light microscopic pictures (106magnification) were taken at 96 h to show the effect of GSI on cell proliferation of (E) KP3 and (F) BxPC3.
