We purchased human MiaPaCa-2, Panc-1, and BxPC-3 tumor cells from the Chinese Academy of Sciences Cell Bank of Type Culture Collection (CBTCCCAS, Shanghai, China). The MiaPaCa-2 and Panc-1 cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA), and BxPC-3 was cultured in RPMI-1640 supplemented with 10% FBS and 1% penicillin-streptomycin. The pancreatic cancer cell lines were cultured under standard conditions at 37°C with a 5% CO₂ atmosphere. The antibodies against E-cadherin, N-cadherin, and vimentin were purchased from Cell Signaling Technology (Danvers, MA, USA); the antibody against β-actin was obtained from Sigma (St. Louis, MO, USA); and the antibodies against TGF-β and p-SMAD2/3 were obtained from Abcam (Cambridge, MA, USA). ITZ was initially dissolved in DMSO (dimethyl sulfoxide) at a stock concentration of 2 mM. Working dilutions of ITZ were made in culture medium immediately before use.

The viability of pancreatic cancer cells was measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) (Sigma) assays. Panc-1, BxPC-3 and MiaPaCa-2 cells were seeded onto 96-well plates at a density of 5,000 cells per well and incubated overnight in 10% FBS medium. After treatment with ITZ for 24, 48 and 72 h at 37°C and 5% CO₂, the inhibition rate of the pancreatic cancer cells was assessed. Briefly, 20 µl of MTT solution (5 mg/ml in distilled water) was added to each well, and the cells were incubated for 4 h at 37°C for the absorption of MTT; then, the medium was removed and replaced by 200 µl of DMSO, and the optical density (OD) was measured at 490 nm on a multifunctional microplate reader (POLARstar OPTIMA; BMG, Offenburg, Germany).

Panc-1 and BxPC-3 cells were seeded onto 35-mm petri dishes (1,000 cells per dish) and allowed to adhere overnight. Then, cells were treated with different concentrations of ITZ and further cultured for 2 weeks to allow colonies to form. At the indicated time point, colonies were fixed with 4% paraformaldehyde for 2 h, stained with 0.1% crystal violet solution and rinsed. The colonies >0.5 mm in diameter were counted using a microscope (Nikon Eclipse Ti-S; Nikon Instruments, Inc., Tokyo, Japan) at a magnification, ×400.

We assessed apoptosis of Panc-1 and BxPC-3 cells using an Annexin V-FITC/PI apoptosis detection kit (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer's instructions. Pancreatic cancer cells (10⁵ cells/well) were seeded onto 6-well plates and pretreated with FBS-free DMEM or RPMI-1640 for 8 h; then, the medium was replaced with fresh medium containing varying concentrations of ITZ. A total of 48 h later, the cancer cells were trypsinized, washed with PBS and then stained with Annexin V and propidium iodide in the dark. The percentage of apoptotic cells was quantified by flow cytometry.

Whole-cell lysates of Panc-1 and BxPC-3 cells were prepared using RIPA buffer (Beyotime, Guangzhou, China) according to the manufacturer's instructions. Protein concentration was determined using a BCA protein assay kit (Pierce, Rockford, IL, USA). The protein lysates were resolved on a 10% polyacrylamide gel with a 5% stacking gel. The proteins were subsequently blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked for 2 h in TBS containing 0.1% (vol/vol) Tween-20 and 10% (wt/vol) non-fat dry milk powder and then incubated with primary antibodies overnight at 4°C. Following incubation with secondary HRP-coupled antibodies for 2 h at room temperature, the membranes were washed with 0.1% TBS-Tween-20, and the immunocomplexes were detected using the enhanced chemiluminescence (ECL) kit and a Molecular Imager ChemiDoc XRS system (Bio-Rad Laboratories, Hercules, CA, USA). β-actin was used as an internal loading control.

The cells were fixed in 4% formaldehyde diluted in phosphate-buffered saline (PBS) for 20 min. Following permeabilization with 0.3% Triton X-100, the cells were treated with blocking buffer (5% BSA in PBS) for 1 h and then incubated with a primary antibody at 4°C overnight. Green conjugated secondary antibodies from Jackson ImmunoResearch Laboratories (West Grove, PA, USA) were applied at room temperature, and the nuclei were stained with 4′-6-diamidino-2-phenylindole (DAPI). Images were pseudocolored using a Zeiss Instruments confocal microscope.

Matrigel invasion assays were performed as previously described to assess the invasive viability of pancreatic cancer cells (24). In brief, the upper chambers of the wells were coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). The tumor cells (10⁵) were suspended in serum-free DMEM and seeded onto the upper chamber. The cells were allowed to invade toward the lower chamber, which contained DMEM with 10% FBS, for 48 h and were then fixed with 4% formaldehyde, followed by the removal of non-invasive cells in the upper chamber with a cotton swab. The invading cells were stained with 0.1% crystal violet. The number of invaded cells was quantified by counting the stained cells under a microscope.

The migration ability of the cancer cells was assessed by wound-healing assays. The cancer cells were pretreated as indicated, and then, the cells were scratched using a 10-µl pipette tip. Next, the cancer cells were washed 3 times with PBS and cultured in serum-starved medium. Images were obtained under a microscope (Nikon Instruments, Inc.).

LSL-Kras^G12D/+; p53^fl/+; Pdx1-Cre (KPC) mice were used to study the therapeutic effect of ITZ in vivo. The breeding of KPC mice was achieved by crossing the Pdx1-Cre mice with LSL-Kras^G12D mice and p53^fl/fl mice (purchased from the Nanjing Biomedical Research Institute of Nanjing University, Nanjing, China). All mice were housed under pathogen-free conditions and had free access to water and food. All experimental protocols were approved by the Ethics Committee of the First Affiliated Hospital of Medical College, Xi'an Jiaotong University (Xi'an, China).

Each experiment was performed at least three times. The data are presented as the mean ± standard deviation. Comparisons between groups were analyzed by Student's t-test using SPSS (version 15.0; SPSS, Inc., Chicago, IL, USA). P-values <0.05 were considered to be statistically significant.

We first investigated the effect of ITZ on the viability of pancreatic cancer cells. Pancreatic cancer cell lines Panc-1, BxPC-3 and MiaPaCa-2 were treated with increasing concentrations of ITZ (0, 0.5, 1, 2, 5 and 10 µM). The viability of pancreatic cancer cell lines was assessed by MTT assays at the indicated time points (24, 48 and 72 h). Our results showed that ITZ inhibited the viability of pancreatic cancer cell lines in a dose- and time-dependent manner (Fig. 1). We observed that ITZ at a concentration of 2 µM began to suppress the viability of pancreatic cancer, and 5 µM ITZ showed a sufficient effect. A previous study that involved thirty healthy men revealed that successive intake of ITZ every 12 h for 14 days produced a steady plasma concentration of 2.67 µM (25). According to the results of the clinical study and the MTT assay, the concentrations of 2 and 5 µM were chosen for further experiments.

To further investigate the therapeutic effect of ITZ in pancreatic cancer, we set out to detect the effect of ITZ on the clone formation capability of Panc-1 and BxPC-3 cell lines. Panc-1 and BxPC-3 cells were seeded onto 35-mm Petri dishes and allowed to adhere overnight; then, the cells were treated with 2 or 5 µM ITZ for 48 h. Cancer cells were cultured for two weeks, and the number of colonies were calculated. As shown in Fig. 2A, we found that ITZ at a concentration of 2 µM significantly suppressed colony formation in the Panc-1 and BxPC-3 cell lines. In the dishes that were treated with 5 µM of ITZ, few colonies were observed (Fig. 2C).

Next, flow cytometric analyses were performed to evaluate whether ITZ could induce apoptosis in pancreatic cancer cells. Panc-1 and BxPC-3 cells were pretreated with FBS-free DMEM or FBS-free RPMI-1640 for 12 h and then treated with 2 or 5 µM ITZ for 48 h. We found that compared to the vehicle-treated cells, cells treated with ITZ showed an increased percentage of apoptotic cells (Fig. 2B and D). These results revealed the role of ITZ in inhibiting colony formation and inducing pancreatic cancer cell apoptosis.

Invasion and metastasis are important characteristics of pancreatic cancer, and most patients with pancreatic cancer have already developed to advanced stages with complications involving distant metastasis at the time of diagnosis (5). Thus, we explored the effect of ITZ on the invasion and migration of pancreatic cancer cells. After starvation in FBS-free DMEM or FBS-free RPMI-1640 for 12 h, Panc-1 and BxPC-3 cells were treated with 2 µM or 5 µM ITZ for 48 h. The wound healing assays were performed under serum-free conditions to evaluate the migration of cancer cells. We found that the migration abilities of Panc-1 and BxPC-3 cells were impaired by ITZ intervention compared to vehicle-treated cells (Fig. 3A and B).

To investigate the invasive ability of cancer cells after ITZ intervention, Matrigel invasion assays were conducted. We found that ITZ at the concentration of 2 µM significantly inhibited the invasion of Panc-1 cells; at the concentration of 5 µM, the invasive abilities of Panc-1 and BxPC-3 were both suppressed (Fig. 3C and D). These findings suggest that ITZ inhibits the invasion and migration capacities of pancreatic cancer cells in vitro.

EMT is a complicated program that plays an important role in the invasion and metastasis of cancer (26,27). We then investigated whether ITZ could affect the EMT process. We found that in Panc-1 cells, ITZ treatment (2 or 5 µM) decreased the expression of the mesenchymal markers, N-cadherin and vimentin. However, expression of the epithelial marker, E-cadherin, was elevated (Fig. 4A). A similar effect was also observed regarding EMT in BxPC-3 cells (Fig. 4B).

Given previous evidence that TGF-β is involved in EMT as well as the invasion and migration of cancer cells (28,29), we set out to investigate the effect of ITZ on TGF-β/SMAD2/3 signaling. Western blotting was performed to detect the expression of TGF-β and p-SMAD2/3. As expected, we observed high expression of TGF-β and p-SMAD2/3 in Panc-1 and BxPC-1 cells. However, compared to vehicle-treated cells, the expression of TGF-β was downregulated after ITZ treatment. Additionally, the level of p-SMAD2/3 was also decreased (Fig. 4A and B). Binding of TGF-β with its membrane-bound receptor leads to phosphorylation of the SMAD family. The latter, in turn, translocates into the nucleus and acts as transcriptional factors to exert their functions (30). Then, we conducted immunofluorescence assays to study the effect of ITZ on TGF-β/SMAD2/3 signaling. We observed that ITZ treatment decreased the level of TGF-β and suppressed the nuclear accumulation of SMAD2/3 (Fig. 4C and D). These data suggest that ITZ treatment inhibits the EMT process and TGF-β/SMAD2/3 signaling in pancreatic cancer.

To investigate whether the effect of ITZ on EMT is mediated by impaired TGF-β/SMAD2/3 signaling, we reassessed the effect of TGF-β/SMAD2/3 signaling on EMT in pancreatic cancer cells. After starvation in FBS-free medium for 8 h, Panc-1 and BxPC-3 cells were treated with recombinant TGF-β1 (5 ng/ml) for 24 h, and then, the EMT markers were detected by western blot assays. Recombinant TGF-β1 significantly suppressed the expression of E-cadherin and elevated the expression of N-cadherin and vimentin in Panc-1 and BxPC-3 cells (Fig. 5A and B). The wound healing assays showed that treatment with recombinant TGF-β1 promoted the migration of cancer cells (Fig. 5C and D). Accordingly, the Matrigel invasion assays showed that the invasive capability of cancer cells was also increased by recombinant TGF-β1 treatment (Fig. 5E and F).

Next, we set out to observe the effect of TGF-β1 deprivation on the invasion and migration of pancreatic cancer. TGF-β1 neutralizing antibody was used to block the function of TGF-β1. As expected, treatment with TGF-β1 neutralizing antibody elevated the expression of E-cadherin and decreased the levels of N-cadherin and vimentin compared to vehicle-treated cancer cells (Fig. 5A and B). The wound healing and Matrigel invasion assays also showed that TGF-β1 neutralizing antibody inhibited the invasion and migration of Panc-1 and BxPC-3 cells (Fig. 5C-F). We revealed that TGF-β is a potent factor that mediates EMT, invasion and migration in pancreatic cancer cells.

Given the results that ITZ inhibited TGF-β/SMAD2/3 signaling as well as the invasion and migration of pancreatic cancer cells, we wondered whether TGF-β/SMAD2/3 signaling is required for ITZ to exert its anticancer effects on pancreatic cancer. To verify our hypothesis, Panc-1 and BxPC-3 cells were pretreated with ITZ (5 µM) for 24 h, and then, the cells were treated with recombinant TGF-β1 (5 ng/ml). Western blot assays showed that recombinant TGF-β1 recovered the expression of N-cadherin and vimentin, both of which were suppressed by ITZ treatment. Accordingly, recombinant TGF-β1 treatment inhibited the ITZ-induced expression of E-cadherin (Fig. 6A and B). In addition, the invasion and migration capabilities of Panc-1 and BxPC-3 cells were also recovered (Fig. B-E). These results showed that recombinant TGF-β1 reversed the effect of ITZ on pancreatic cancer. We revealed that TGF-β/SMAD2/3 signaling mediates the effects of ITZ on pancreatic cancer.

To further investigate the therapeutic effect of ITZ in vivo, we used KPC transgenic mice harboring spontaneous pancreatic cancer. Starting at 8 weeks of age, when KPC mice have developed widespread advanced pancreatic neoplasia, we treated the KPC mice with vehicle or ITZ. KPC mice were sacrificed when end-staged disease developed. We found that ITZ treatment significantly decreased the tumor weight (Fig. 7A and B). Next, immunohistochemistry was performed to detect the proliferation index, Ki67. We found that treatment with ITZ decreased the number of Ki67-positive cells (Fig. 7C and D). These results suggest the therapeutic effect of ITZ in vivo.