Glycogen synthase kinase‐3β activity plays a key role in the antitumor effect of nafamostat mesilate in pancreatic cancer cells

Abstract Pancreatic cancer is often resistant to chemotherapy. We previously showed the efficacy of combination treatment using gemcitabine and nafamostat mesilate (FUT‐175) for patients with unresectable pancreatic cancer. However, the mechanisms that affect the sensitivity of FUT‐175 are not fully understood. The purpose of the present study was to clarify the mechanism of the sensitivity to FUT‐175, with a focus on the activity of glycogen synthase kinase‐3β (GSK‐3β). In vitro, we assessed sensitivity to FUT‐175 in human pancreatic cancer cell lines (PANC‐1 and MIAPaCa‐2) and difference of signaling in these cells by cell proliferation assay, Western blot analysis and microarray. Next, we assessed cell viability, apoptotic signal and nuclear factor‐kappa B (NF‐κB) activity in response to treatment with FUT‐175 alone and in combination with GSK‐3 inhibitor or protein phosphatase 2A (PP2A) by cell proliferation assay, Western blot analysis and enzyme‐linked immunosorbent assay. Phosphorylated GSK‐3β level was significantly higher in MIAPaCa‐2 (high sensitivity cell) than in PANC‐1 (low sensitivity cell). Cell viability and NF‐κB activity were significantly decreased by addition of GSK‐3 inhibitor to FUT‐175, and levels of cleaved caspase‐8 were increased by inhibition of GSK‐3. PP2A inhibitor increased the levels of phosphorylated GSK‐3β and sensitized both cell lines to FUT‐175 as measured by cell viability and apoptotic signal. The results indicate that GSK‐3β activity plays a key role in the antitumor effect of FUT‐175 in pancreatic cancer cells, and regulation of GSK‐3β by PP2A inhibition could be a novel therapeutic approach for pancreatic cancer.


| INTRODUCTION
Pancreatic cancer is one of the most lethal and aggressive of all malignancies and the fourth leading cause of death in developed countries. 1 Because of rapid tumor growth and high potential for distant metastasis at the time of diagnosis, one-half of patients present with metastatic disease and approximately the other 30% with locally advanced unresectable disease. 2 Multidisciplinary therapy has been applied to advanced pancreatic cancer, but the median overall survival remains unsatisfactory.
Recent studies have shown that nuclear factor-kappa B (NF-jB) plays an important role in regulation of cell apoptosis, inflammation, and oncogenesis, 3,4 and constitutive NF-jB activation contributes to the aggressive behavior of pancreatic cancer. 5 Chemotherapeutic agents and ionizing radiation activates NF-jB, which leads to resistance against chemoradiation therapy. 6,7 Therefore, inhibition of NF-jB activation in cancer cells may be one of the potential options to enhance the antitumor effect of chemoradiation therapy. We previously reported that nafamostat mesilate (FUT-175), a serine protease inhibitor 8 that is widely used in Japan for the treatment of pancreatitis and disseminated intravascular coagulation as well as an anticoagulant for hemodialysis, inhibited NF-jB activation; it also induced caspase-8mediated apoptosis of pancreatic cancer cells in vitro 9 and in vivo. 10 Moreover, FUT-175 inhibited anticancer agents-or radiation therapyinduced NF-jB activation and enhanced antitumor effects in pancreatic, [11][12][13] gallbladder, 14 and gastric cancer. 15 We also reported the usefulness of gemcitabine in combination with intra-arterial FUT-175 administration for unresectable pancreatic cancer patients in phases 1 and 2 clinical studies. 16,17 Other studies investigated new therapeutic options to sensitize pancreatic cancer cells to chemotherapeutic agent 18 and several clinical trials of pancreatic cancer have already targeted molecular factors, either as monotherapy or in combination with gemcitabine. 19 Our treatment strategy has targeted NF-jB activation using FUT-175 in pancreatic cancer cells. However, similar to other chemotherapeutic agents, the sensitivity to FUT-175 was different in each cell line. To improve the therapeutic outcome of pancreatic cancer and to develop a novel treatment strategy, it is important to investigate the mechanisms that affect the sensitivity of FUT-175.
The purpose of the present study was to clarify the mechanism of the sensitivity of FUT-175 in order to improve therapeutic outcome of pancreatic cancer by combination therapy with FUT-175.

| Reagents
FUT-175 was kindly given to us by Torii Pharmaceutical Co., Ltd (Tokyo, Japan), which was dissolved in sterile distilled water (5 mg/ mL) and stored at À20°C until use. Glycogen synthase kinase (GSK)-3 Inhibitor IX, and cantharidic acid as protein phosphatase 2A (PP2A) inhibitor was purchased from Merck Millipore (Darmstadt, Germany), which were dissolved in dimethyl sulfoxide (10 mmol/L) and stored at 4°C and room temperature until use. Protease inhibitor cocktail and phosphatase inhibitor cocktail tablets were purchased from Roche Diagnostics (Indianapolis, IN, USA).

| Antibodies
Antibodies specific to phosphorylated AKT, phosphorylated GSK-3b and cleaved caspase-8 were obtained from Cell Signaling Technology  Cy3-labeled cRNA was prepared with 500 ng RNA using a Quick Amp labeling Kit, one-color (Agilent, Santa Clara, CA, USA) according to the manufacturer's protocol. Expressed genes were screened using SurePrint G3 Human GE 8960 K v2 Microarray (Agilent) according to the manufacturer's instructions. Gene expression was compared using a log ratio, which was defined as log 2 (PANC-1/MIAPaCa-2).

| Quantitative analysis of NF-jB activity
Concentrations of NF-jB p65 in the nuclear extracts were measured.
Nuclear extracts from in vitro experiments were prepared using a nuclear extract kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer's protocol. The nuclear extracts were assayed using an ELISA kit (TransAM TM NF-jB; Active Motif) to detect and quantify the NF-jB activity according to the manufacturer's instructions.
Briefly, 20 lg of the nuclear extract protein was incubated for 1 hour at 25°C in microwells coated with an oligonucleotide containing an NF-jB p65 or p50-binding consensus sequence. Next, the wells were incubated with rabbit anti-NF-jB p65 antibodies (1:1000 dilution) for 1 hour at 25°C, followed by incubation with peroxidaseconjugated goat anti-rabbit IgG (1:1000 dilution) for 1 hour at 25°C.
Peroxidase activity was visualized by the tetramethylbenzidine reaction, and the optical density was measured at 450 nm.

| Western blot analysis
Lysate protein was extracted from whole cells (5910 6 cells for each) after each treatment for 24 hours. This protocol for Western blot analysis was described in a previous study. 22 After incubating the blots in each primary antibody overnight, the membranes were incubated with the peroxidase-labeled secondary antibody (Histofine; Nichirei, Tokyo, Japan) for 2 hours and detected by using the ImmunoStar LD â chemiluminescence reagent (WAKO Chemical, Tokyo, Japan). Protein bands were detected using a Chemi Doc XRS+ system (Bio-Rad, Hercules, CA, USA).

| Reverse transcription polymerase chain reaction
Total RNA was extracted using the SV Total RNA Isolation System (Promega), and reverse transcription (RT) was carried out using a Transcriptor First Strand cDNA synthesis Kit (Roche Applied Science, Mannheim, Germany) according to the manufacturer's instructions.
Primer sequences for the genes were as follows:

| Statistical analysis
Data were expressed as meanAESD. Non-paired t-test (two-tailed) was used for statistical studies. All P-values were considered statistically significant when the associated probability was <.05.

| Assessment of expression changes of genes by FUT-175
To investigate the change in mRNA expression of PANC-1 and MIA-PaCa-2, we carried out microarray analyses. As to GSK signaling, AKT2 and DVL-1 were greater in PANC-1 than in MIAPaCa-2.
PPP2R2A was lower in PANC-1, whereas the expressions of PPP2R2B and PPP2R2C were greater in PANC-1 than in MIAPaCa-2 (Table 1) Figure 3A). In Western blot analysis, the levels of cleaved caspase-8 of both pancreatic cancer cell lines were increased by the addition of GSK-3 inhibitor to FUT-175, which were greater than those of FUT-175 alone ( Figure 3B). Furthermore, we evaluated whether GSK-3 inhibition affected NF-jB activity. NF-jB is typically a heterodimer that consists of p65 and p50 proteins. To assess NF-jB activity, we measured concentrations of p65 in the nuclear extracts by ELISA. Concentrations of NF-jB p65 in the nuclear extracts of PANC-1 and MIAPaCa-2 cells were significantly decreased by the addition of the GSK-3 inhibitor to FUT-175, which were lower than those of FUT-175 alone (PANC-1, P=.0002; MIAPaCa-2, P=.0049, Figure 3C).

| PP2A regulated GSK-3 activity and inhibition of PP2A improved sensitivity to FUT-175
To investigate the detailed molecular mechanism for the difference of GSK-3 activity between PANC-1 and MIAPaCa-2, we examined