HS-173

Synergistic anticancer activity of HS-173, a novel PI3K inhibitor in combination with Sorafenib against pancreatic cancer cells

Abstract

The RAF/MEK/ERK and PI3K/AKT pathways are highly implicated in the development of pancreatic cancer. The principal objective of this study was to assess the synergic effect between Sorafenib (a RAF inhibitor) and HS-173 (a novel PI3K inhibitor) to gain insight into novel therapeutic strategies for treating pancreatic cancer. We first investigated the cytotoxic effect of co-treatment with Sorafenib and HS-173 using the Calcusyn program. Combined treatment of the two drugs synergistically inhibited the viability of Panc-1 cells (combination index < 1). Concomitantly, the co-treatment induced G2/M arrest and increased apoptosis with the loss of mitochondrial membrane potential. Apoptosis resulting from the co-treatment was accompanied by increased levels of cleaved caspase-3 and PARP as well as greater numbers of TUNEL-positive apoptotic cells compared to treatment with either drug alone. Furthermore, combined treatment with these drugs decreased the expression of HIF-1a and VEGF which play an important role in angiogenesis. This anti-angiogenic effect was confirmed by the suppressed tube forma- tion of VEGF-induced human umbilical vein endothelial cells and inhibition of blood vessel formation in a Matrigel plug assay in mice. Taken together, our study demonstrates that combined treatment with Sorafenib and HS-173 has a synergistic anti-cancer effect on pancreatic cancer cells, indicating that simultaneously targeting the RAF/MEK and PI3K/AKT pathways can induce a synergistic inhibitory effect on pancreatic cancers in which both pathways are activated. Based on the observations from our study, we suggest that the combined administration of these two drugs may be considered to be a new thera- peutic regimen for treating pancreatic cancer. 1. Introduction Pancreatic cancer is a highly malignant disease whose incidence has risen steadily. It is now the fourth leading cause of death from cancer in the Western world [1]. Although recent translational re- search has shown successes of overall 5-year survival rate around 5%, the prognosis of pancreatic cancer is extremely poor regardless of stage [2]. Only a small percent of selected patients are able to undergo complete surgical resection resulting in prolonged sur- vival. To date, treatment for advanced pancreatic cancer is mainly focused on palliation and quality of life, and several regimens are widely used including gemcitabine alone or in combination with other drugs [3]. However, these modalities have not yielded satis- factory results and there is no clearly superior treatment option for locally advanced pancreatic cancer. This has led to the development of novel therapeutic approaches, including the targeting of RAF/MEK or PI3K/AKT pathways by newly developed agents. Pancreatic cancer displays the highest frequency (>90%) of so- matic activating mutations in K-RAS genes amongst all human cancers [1,4]. Thus, aberrant K-RAS signaling is a hallmark of the vast majority of pancreatic cancer cases. Consequently, RAS has been shown to mediate activation of the RAF/mitogen- activated protein kinase (MAPK) kinase 1/2(MEK)/extracellular signal-regulated kinase (ERK) signaling cascade. The RAF/MEK/ ERK pathway is frequently deregulated during neoplastic trans- formation [5,6]. Phosphorylation of theses kinases gives rise to proliferation of pancreatic cancer cells and impacts cell survival and metastasis [7,8]. Because of the importance of the RAF/ MEK/ERK pathway in aberrant cancer cells, this pathway has been the subject of numerous investigations to understand its fundamental role in cancer and a potential target for therapeutic intervention [9,10]. In particular, the inhibition of RAF, which is dependent on aberrant RAS/RAF pathway signaling has been concerned as a promising strategy in oncology therapeutics for controlling tumor growth.

Sorafenib is an orally administered multi-kinase inhibitor that has in vitro and in vivo activity against a variety of solid tumors [11,12]. Among the various receptor kinases, Sorafenib targets the RAS/RAF/MAPK pathway at the level of RAF kinase through its inhibitory effects on this mitogenic cascade, resulting in dis- rupted cell proliferation, differentiation and survival [10,12]. Until now, anticancer activity of single agents such as Sorafenib has been observed in not only experimental pancreatic cancer but also hu- man patients with pancreatic cancer as well as other cancers [13,14]. However, the clinical efficacy of Sorafenib is limited due to its transient effect and drug resistance as a single agent; thus, therapeutic strategies involving Sorafenib has moved toward com- bined administration with conventional chemotherapeutic agents. The phosphatidylinositol 3-kinase (PI3K)/AKT pathway is in- volved in cell survival and is frequently disrupted in several human malignancies including pancreatic cancer. It has also been reported that the PI3K/AKT pathway is activated and AKT2 is amplified in 59% and 20% of pancreatic cancer cases, respectively [15,16]. In addition, the PI3K/AKT and RAF/MAPK pathways are well described as mediators of RAS induced transformation and tumorigenesis [17,18]. This has been demonstrated in reports showing the in vivo effect of PI3K on K-RAS mediated tumorigenesis in mice genetically engineered to carry a PI3K mutation resulting in an inability to bind RAS [19]. Recently, cross-talk between RAF/MAPK and PI3K/AKT pathways has been reported; the inhibition of one can result in compensatory responses by the other [20,21]. Indeed, a combination of MAPK inhibitors with PI3K/AKT inhibitors has proved to be effective for combating human melanoma and hepa- tocellular carcinoma [22,23]. For this reason, combined targeting of RAF/MAPK and PI3K/AKT for treating K-RAS-driven tumors has attracted much interest [24,25].

Meanwhile, we have designed and synthesized a new series of imidazo[1,2-a]pyridine derivatives with a goal of developing a new structural class of potent PI3K inhibitors. Among these com- pounds, HS-173 strongly inhibited the PI3K activity, leading to a potent anticancer effect on various cancer cells [26]. Therefore, the present study was designed to characterize the combined ef- fect and molecular mechanism underlying the interaction of Sorafenib and HS-173 in pancreatic cancer. We hypothesized that inhibition of the PI3K/AKT signaling pathway by HS-173 would promote a potential synergistic effect with Sorafenib in pancreatic cancer. In this study, we showed that the combination of Sorafenib and HS-173 enhanced anti-cancer activity in pancreatic cancer cells possibly by targeting the RAF/MEK and PI3K/AKT pathways. Our findings suggest that the combination of Sorafenib and HS- 173 may be a promising therapeutic strategy for treating pancre- atic cancer.

2. Materials and methods

2.1. Cells and materials

Human pancreatic cancer cells (Panc-1, Miapaca-2, and Aspc-1) were purchased from American Type Culture Collection (Manassas, VA). Panc-1 and Miapaca-2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Aspc-1 cell was cul- tured in Roswell Park Memorial Institute 1640 (RPMI-1640) media supplemented with 10% FBS and 1% penicillin/streptomycin. Human umbilical vein endothelial cells (HUVECs) were grown in a gelatin-coated 75-cm2 flask with M199 medium containing 20 ng/mL basic fibroblast growth factor (bFGF), 100 U/mL heparin, and 20% FBS. Cell cultures were maintained at 37 °C in a CO2 incubator with a controlled humidified atmosphere composed of 95% air and 5% CO2. Sorafenib [N-(3-trifluoro-methyl-4-chlorophenyl)-N0 -(4-(2-methylcarbamoyl pyridin-4-yl) oxyphenyl) urea] was synthesized at Bayer Corporation (West Haven, CT). Sorafenib was dissolved in dimethylsulfoxide (DMSO) at a concentration of 10 mM before use. HS-173 (ethyl 6-(5-(phenylsulfonamido) pyridin-3-yl)imidazo[1,2-a]pyridine-3-carboxylate), a new PI3Ka inhibitor was synthesized according to our previous methods [27]. For all in vitro studies, HS-173 was dissolved in DMSO at a concentration of 10 mM be- fore use. DMSO was added to the cells at 0.1% (V/V) as a solvent control.

2.2. Measurement of cell proliferation

Cell viability was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Briefly, cells were plated at a density of 0.5–1 × 104 cells/well in 96-well plates and incubated for 24 h. The medium was then removed, and the cells were treated with Sorafenib (10 lM) and/or HS-173 (1 lM). After the cells were incubated for 48 h, 100 lL MTT solutions (2 mg/mL) was added to each well and the plate was incubated for another 4 h at 37 °C. The formed formazan crystals were dissolved in DMSO (200 lL/well) with constant shaking for 5 min. Absorbance of the solution was then measured with a microplate reader at 540 nm. This assay was conducted in triplicate. Drug interaction between Sorafenib and HS-173 was assessed at a fixed concentration ratio using combina- tion index (CI), where CI < 1, CI = 1, CI > 1 indicate synergistic, additive and antago- nistic effects, respectively (Calcusyn software, Biosoft, Ferguson, MO).

2.3. Western blotting

Total cellular proteins were extracted with lysis buffer containing 1% Triton X- 100, 1% Nonidet P-40, and the following protease and phosphatase inhibitors: apro- tinin (10 mg/mL), leupeptin (10 mg/mL; ICN Biomedicals, Asse-Relegem, Belgium), phenylmethylsulfonyl fluoride (1.72 mM), NaF (100 mM), NaVO3 (500 mM), and Na4P2O7 (500 mg/mL; Sigma–Aldrich). The proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto nitrocellulose membranes. The blots were immunostained with the appropri- ate primary antibodies followed by secondary antibodies conjugated to horseradish peroxidase. Antibody binding was detected with an enhanced chemiluminescence reagent (Amersham Biosciences). Antibodies against p-MEK (Thr286), MEK, p-ERK (Thr202/Tyr204), ERK, p-mTOR (Ser2448), mTOR, p-AKT (Ser473), AKT, p-P70S6K (Thr389), P70S6K, PARP, cleaved caspase-9, cleaved caspase-3, p-cdc2 (Tyr15), p-cdc25c (Ser216), cyclin B1, HIF-1a, VEGF, and b-actin were purchased from Cell Signaling Technology (Danvers, MA).

2.4. Immunofluorescence

Panc-1 cells were plated on 18-mm cover glasses in DMEM medium and incu- bated for 24 h so that approximately 70% confluence was reached. The cells were then treated with Sorafenib (10 lM) and/or HS-173 (1 lM) for 6 h, washed twice with PBS, and fixed in an acetone: methanol solution (1:1) for 10 min at —20 °C. Cells were blocked in 1.5% horse serum in PBS for 30 min at room temperature, and then incubated overnight at 4 °C with primary antibodies including rabbit anti-p-MEK, p-AKT, p-P70S6K, cyclin B1 and p-cdc2 (Cell Signaling Technology, Danvers, MA) in a humidified chamber. After washing twice with PBS, the cells were incubated with rabbit TRITC secondary antibody (1:100, Dianova, Germany) for 1 h at room temperature. The cells were also stained with 4,6-diamidino-2-phenylin- dole (DAPI) to visualize the nuclei. The slides were then washed twice with PBS and covered with DABCO (Sigma–Aldrich) before viewing with a confocal laser scanning microscope (Olympus, Tokyo, Japan).

2.5. Measurement of mitochondrial membrane potential

Mitochondrial membrane potential (wm) was assessed with a Mitochondria staining kit (MitoPT, Immunohistochemistry Technologies, Bloomington, MN) using 5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethylbenzimidazol-carbocyanine iodide (JC-1), which exhibits potential-dependent accumulation in mitochondria. Panc-1 cells were plated on 18-mm cover glasses in DMEM medium and incubated for 24 h.
When cell confluency reached approximately 70%, the cells were then treated with Sorafenib (10 lM) and/or HS-173 (1 lM) for 4 h, and then 100 lL JC-1 solutions at a final concentration of 12.5 lg/mL was added to each well and the plate was incu- bated for another 30 min at 37 °C. After washing twice with PBS, the cells were also stained with DAPI to visualize the nuclei. The slides were then washed twice with PBS and covered with fluorescent mounting medium (Dako North America Inc., Car- pinteria, CA) before viewing with a confocal laser scanning microscope (Olympus, Tokyo, Japan). For measurement of JC-1 fluorescence level, Panc-1 cells were plated into 96-well plates with black well (Corning Incorporated, Corning, NY, USA) at a density of 1 × 104 cells/well. After 24 h, cells were treated with 10 lM Sorafenib
and/or 1 lM HS-173 for 4 h. 5 lL JC-1 (12.5 lg/mL) was added and incubated for 30 min at 37 °C in the dark. After the cells were seeded to the plate bottom using centrifugation at 400g for 5 min, JC-1 and culture medium were aspirated. Cells were washed three times with Assay buffer and 100 lL Assay buffer was added to each well. The plates were immediately read by using a LS 55 Luminescence
Spectrometer (Waltham, MA). The results (green/red fluorescence ratio, G/R ratio) were expressed as the percentage of the control.

2.6. Analysis of cytochrome c localization

Panc-1 cells were plated on 18-mm cover glasses in DMEM medium and incu- bated for 24 h so that approximately 70% confluence was reached. The cells were then treated with Sorafenib (10 lM) and/or HS-173 (1 lM) for 6 h and washed twice with PBS, and fixed in an acetone: methanol solution (1:1) for 10 min at —20 °C. Cells were blocked in 1.5% horse serum in PBS for 30 min at room temper- ature, and then incubated overnight at 4 °C with cytochrome c antibody (1:50; San- ta Cruz Biotechnologies, Santa Cruz, CA). After washing twice with PBS, the cells were incubated with mouse fluorescein-labeled secondary antibody (1:100, Diano- va, Germany) containing 100 nM of a mitochondrion-specific dye (Mitotracker green FM: Molecular Probes Inc., Eugene, OR) for 40 min at 37 °C. The cells were also stained with DAPI to visualize the nuclei. The slides were then washed twice with PBS and covered with fluorescent mounting medium (Dako North America Inc.) before viewing with a confocal laser scanning microscope (Olympus, Tokyo, Japan).

2.7. DAPI staining and TUNEL staining

Panc-1 cells were plated in an 18-mm cover glass with DMEM at ~70% conflu- ence and incubated for 24 h at 37 °C. The cells were then treated with combination of 10 lM Sorafenib and 1 lM HS-173 for 24 h. The cells were fixed in ice-cold 2% para-formaldehyde (PFA), washed with PBS, and then stained with 2 lg/mL of DAPI (Sigma–Aldrich) for 20 min at 37 °C. The stained cells were examined under a fluo- rescence microscope for evidence of nuclear fragmentation. Terminal deoxynucleo- tidyl transferase-mediated nick end labeling (TUNEL) was subsequently performed using the TUNEL kit (Chemicon, Temecula, CA) following manufacturer’s instructions.

2.8. TUNEL assay

DNA fragmentation was detected using an APO-BrdU™ TUNEL assay kit (Invit- rogen) according to the manufacturer’s instructions. Briefly, 24 h after incubating with 10 lM of Sorafenib and/or 1 lM of HS-173, supernatants and trypsinized cells were collected. Each pellet of cells was fixed with 1% PFA in PBS (w/v) for 1 h at 4 °C. Apoptotic cells were then detected with the APO-BrdU TUNEL assay kit. Analysis of the flow cytometric data was conducted using FlowJo software (Tree Star, Inc., San Carlos, CA).

2.9. Cell cycle analysis

Panc-1 cells were plated in 100-mm culture dishes with DMEM at ~70% conflu- ence and incubated for 24 h at 37 °C. The next day, the cells were treated with Sorafenib (10 lM) and/or HS-173 (1 lM). Floating and adherent cells were collected and fixed overnight in cold 70% ethanol at 4 °C. After washing with PBS, the cells were subsequently stained with 50 lg/mL propidium iodide (PI) and 100 lg/mL RNase A for 1 h at room temperature in the dark, and subjected to flow cytometry to determine the percentage of cells in specific phases of the cell cycle (sub-G1, G0/ G1, S, and G2/M). Flow cytometric analysis was performed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA). Analysis of the flow cytometric data was conducted using FlowJo software (Tree Star, Inc.). All the experiments were performed in triplicate.

2.10. Capillary tube formation assay

Matrigel (10 mg/mL, 200 lL; BD Biosciences) was polymerized for 30 min at 37 °C. HUVECs were suspended in M199 medium supplemented with 2% FBS at a density of 2.5 × 105 cells/mL. Aliquots (0.2 mL) of the cell suspension were added to each well coated with Matrigel with or without 10 lM of Sorafenib and/or 1 lM of HS-173 or VEGF (50 ng/mL) and incubated for 6 h at 37 °C. Morphological changes of the cells and tube formation were observed with a phase-contrast microscope. The cells were photographed at 200 × magnification.

2.11. Matrigel plug assay

Animal care and experimental procedures were conducted in accordance with the Guide for Animal Experiments by the Korean Academy of Medical Sciences. Male BALB/c 6-week-old mice were obtained from Orient-Bio Laboratory Animal Research Center Co., Ltd. (Gyeonggi-do, Kapyung, South Korea). The animals were fed with standard rat chow with free access to tap water in a temperature- and humidity controlled animal house alternating 12 h light–dark cycles. The mice were subcutaneously injected with 500 lL of Matrigel containing concentrated VEGF (100 ng/mL), and 10 lM of Sorafenib and/or 1 lM of HS-173 or PBS (10 lL). Matri- gel plugs were surgically removed on day 7 and photographed. The plugs were fixed in 10% buffered formaldehyde, embedded in paraffin, and sectioned. The 8 lm-thick sections were stained with hematoxylin and eosin (H&E) or anti-CD31 antibody (Chemicon).

2.12. Statistical analysis

Data are expressed as the mean ± standard deviation (S.D.). Statistical analysis was performed using an ANOVA and unpaired Student0 s t-test. p-values of 0.05 or less were considered statistically significant. Statistical calculations were per- formed using SPSS software for the MS Windows operating system (version 10.0; SPSS, Chicago, IL).

3. Results

3.1. The combination of Sorafenib and HS-173 synergistically inhibited cell proliferation in pancreatic cancer cell lines

The effect of Sorafenib and HS-173 in combination, on Panc-1, Miapaca-2 and Aspc-1 pancreatic cancer cells was determined by MTT assay. Cells were treated with a fixed concentration of Sorafe- nib (10 lM) and/or various concentrations of HS-173 (0, 0.1, 1, 5, or 10 lM) for 48 h. Combination of Sorafenib with HS-173 significantly inhibited growth of the three cell lines compared to treat- ment with a single agent. To identify synergistic effect between Sorafenib and HS-173, we evaluated the combination index (CI) values using CalcuSyn software. Of those, the greatest synergistic effect was observed in the Panc-1 and Miapaca-2 cells (Fig. 1C). In- deed, the CI values were <1 in combination of 10 lM Sorafenib and 1 lM HS-173 in Panc-1 cells, and combination of 10 lM Sorafenib and 5 lM HS-173 in Miapaca-2 cells. (CI = 0.11 for Panc-1 cells and 0.66 for Miapaca-2 cells) 3.2. The combination of Sorafenib and HS-173 inhibited key enzymes in both RAF/MAPK and PI3K/AKT signaling pathways RAF kinases are key mediators of the MEK/ERK cascade, and up- regulated signaling through this pathway has an important role in pancreatic cancer cell growth [14]. Additionally, the PI3K/AKT pathway is highly activated in pancreatic cancer, consequently provoking cell proliferation, survival, and tumorigenesis [16]. Since Sorafenib is a RAF/MEK inhibitor and HS-173 is a PI3K inhibitor, we examined the phosphorylation level of key proteins of these path- ways in Panc-1 and Miapaca-2 cells treated with Sorafenib and HS- 173 by Western blotting (Fig. 2A). Sorafenib obviously inhibited the expression of p-MEK and p-ERK, main mediators of the RAF/ MAPK pathways, while HS-173 inhibited the expression of p- AKT, p-mTOR, and p-P70S6K to a greater degree than p-MEK and p-ERK in both Panc-1 and Miapaca-2 cells. However, combined treatment with both drugs decreased all the expression of p- MEK, p-ERK, p-AKT, p-mTOR and p-P70S6K compared to single agent treatment, indicating the synergistic suppression of both RAF/MAPK and PI3K/AKT pathways (Fig. 2A). These results were confirmed by confocal fluorescent microscopy (Fig. 2B). 3.3. The combination of Sorafenib with HS-173 synergistically induced apoptosis Since combined treatment with Sorafenib and HS-173 caused a significant reduction in cell proliferation, the underlying mecha- nisms were further investigated. First, we identified the apoptotic effect of Sorafenib and HS-173 in Panc-1 cells by DAPI staining and a TUNEL assay. As shown in Fig. 3A, the cells simultaneously treated with the two drugs presented more prominent morpholog- ical features characteristic of apoptotic cells such as bright nuclear condensation and perinuclear apoptotic bodies by DAPI staining. Similarly, increase of apoptosis by combined treatment with the two drugs was observed by TUNEL staining. At this time, combined treatment of Sorafenib and HS-173 also represented significant in- creases on the percentage of TUNEL-positive cells compared to agent alone (p < 0.01). To confirm the induction of apoptosis by this combination, Panc-1 cells were treated with the agents individu- ally or in combination and examined by TUNEL staining using flow cytometry (Fig. 3B). Consistent with above results, flow cytometric analysis of TUNEL staining revealed that the population of TUNEL- positive cells was obviously increased by combined treatment of Sorafenib and HS-173. In addition, to identify involvement of combined treatment of Sorafenib and HS-173 on changes of mitochondrial membrane potential, correlating with intrinsic apoptosis, we performed JC-1 staining. As shown in Fig. 3C, control cells showed heterogeneous staining of the cytoplasm with both red and green fluorescence coexisting in the same cells. Consistent with mitochondrial localization, red fluorescence (corresponding to high mitochondrial membrane potential) was mostly found in granular structures distributed throughout the cytoplasm. Treat- ment of Panc-1 cells with Sorafenib and HS-173 decreased the red fluorescence and frequent clusters of mitochondria were ob- served. Combined treatment of Sorafenib and HS-173 induced marked changes in mitochondrial membrane potential (wm) as evi- dent from the disappearance of red fluorescence or the increase of green fluorescence in most cells. These results were supported by JC-1 fluorescence microplate reader (Fig. 3D). Mitochondrial mem- brane potential induces the release of mitochondrial cytochrome c into the cytosol, a hallmark of intrinsic pathway-mediated apopto- sis. As shown in Fig. 3E, we observed that combined treatment of two agents synergistically increased cytochrome c release along with a concomitant decrease in the-co-localization of cytochrome c and mitochondria. In addition, combination treatment of Sorafe- nib and HS-173 significantly increased the expression levels of cleaved caspase-3 and caspase-9 as well as PARP expression in Panc-1 and Miapaca-2 cells (Fig. 3F). Collectively, these results indicate that the combination of Sorafenib with HS-173 synergisti- cally induced apoptosis of pancreatic cancer cells. Fig. 1. Synergistic cytotoxic effect of Sorafenib and HS-173 on Panc-1, Miapaca-2, and Aspc-1 pancreatic cancer cells. (A) Chemical structures of Sorafenib and HS-173. Sorafenib; 4-[4-[[4-chloro-3-trifluoromethyl)phenyl]carbamoylamino]phenoxy]-N-methyl-pyridine-2-carboxamide. HS-173; ethyl 6-(5-(phenylsulfonamido)pyridin-3- yl)imidazo[1,2-a]pyridine-3-carboxylate. (B) Panc-1, Miapaca-2 and Aspc-1 cells were treated with Sorafenib (10 lM) and/or HS-173 (0, 0.1, 1, 5, or 10 lM) for 48 h. MTT assay was performed to determine the cytotoxic effect of Sorafenib and/or HS-173. The combination index (CI) values for Sorafenib and HS-173 in Panc-1, Miapaca-2, and Aspc-1 cells were determined by Chou–Talalay method and CalcuSyn software (Biosoft). A CI of <1 was considered as synergistic effect. (C) Cell viability by 10 lM Sorafenib and 1 lM HS-173 in Panc-1 cells. A CI value of 0.11 indicated strong synergism. Cell viability by 10 lM Sorafenib and 5 lM HS-173 in Miapaca-2 cells. A CI value of 0.66 indicated synergism. Data are expressed as the mean ± S.D. from the triplicate wells ωp < 0.05, ωωp < 0.01, and ωωωp < 0.001 vs Sorafenib or HS-173 treated groups for Panc-1 and Miapaca-2 cells. Fig. 2. The combination of Sorafenib with HS-173 inhibits key enzymes in the RAF/MAPK and PI3K/AKT signaling pathways. (A) Effects of Sorafenib and HS-173 on the expression levels of AKT, mTOR, P70S6K, ERK, and MEK and phosphorylation were determined by Western blot analysis. Panc-1 cells were treated with Sorafenib (10 lM) and/or HS-173 (1 lM) for 6 h. Miapaca-2 cells were treated with Sorafenib (10 lM) and/or HS-173 (5 lM) for 6 h. (B) After treatment with 10 lM Sorafenib and 1 lM HS-173 for 6 h in Panc-1 cells, p-MEK, p-AKT, p-P70S6K, and p-mTOR were detected by immunofluorescence. 3.4. Combined treatment of Sorafenib with HS-173 resulted in synergistic G2/M phase arrest of cell cycle progression To better understand the anticancer mechanism responsible for the synergistic anti-proliferative activity of Sorafenib and HS-173 in Panc-1 cells, the cell cycle distribution was evaluated using flow cytometric analysis. The cell cycle data showed that combined treatment of the two drugs synergistically induced the accumula- tion of cells in the G2/M phase (44%) with an accompanying de- crease of cells in the G0/G1 phase (Fig. 4A). Furthermore, we investigated the expression of cyclin B1, p-cdc2 and p-cdc25c, which typically cause cell arrest in the G2/M phase of the cell cycle. As expected, the combined treatment not only decreased the expression of cyclin B1 but also increased that of p-cdc2 and p-cdc25c in Panc-1 cells (Fig. 4B). These results indicated the syn- ergistic effect of the two drugs on the G2/M phase arrest, which was confirmed by results of confocal fluorescent microscopy (Fig. 4C). Fig. 3. Combined treatment with Sorafenib and HS-173 induces synergistic apoptosis. (A) Induction of apoptosis by the combination of Sorafenib and HS-173 was determined in Panc-1 cells by DAPI and TUNEL staining. Data are expressed as the mean ± S.D. from the three experiments. ωωp < 0.01 vs HS-173 treated group or Sorafenib treated groups. (B) After treatment with 10 lM Sorafenib and 1 lM HS-173 for 24 h, DNA fragmentation in Panc-1 cells was evaluated by flow cytometric analysis. (C) After treatment with 10 lM Sorafenib and 1 lM HS-173 for 4 h, the Panc-1 cells were stained with a JC-1 probe and analyzed with an Olympus confocal laser scanning microscope. (D) Panc-1 cells were treated with Sorafenib (10 lM) and/or HS-173 (1 lM) for 24 h, and the mitochondrial membrane potential was determined by JC-1 staining and analyzed by LS 55 Luminescence Spectrometer. The results (green/red ratio) are expressed as the percentage of cells treated with Sorafenib and HS-173. Data are expressed as the mean ± S.D. of triplicate wells. ωp < 0.05 vs HS-173 treated group and ωωp < 0.01 vs Sorafenib treated group. (E) After treatment with 10 lM Sorafenib and 1 lM HS-173 for 6 h, Panc-1 cells were stained with anti-cytochrome c antibody, Mitotracker and DAPI. The immunostained cells were analyzed under an Olympus confocal laser scanning microscope. (F) Panc-1 cells were treated with Sorafenib (10 lM) and/or HS-173 (1 lM) for 24 h. Miapaca-2 cells were treated with Sorafenib (10 lM) and/or HS-173 (5 lM) for 24 h. Cell lysates were prepared and subjected to Western blotting for PARP, cleaved caspase-9 and cleaved caspase-3. 3.5. The combination of Sorafenib with HS-173 synergistically inhibited the expression of HIF-1a and VEGF in Panc-1 cells Considering the importance of HIF-1a in hypoxia, we attempted to examine the effect of the combination of two drugs on the expression pattern of HIF-1a and VEGF in Panc-1 cells. Cells were simultaneously treated with two drugs under hypoxia conditions induced by 100 lM CoCl2 for 24 h. As shown in Fig. 5A, HIF-1a expression was increased under the hypoxic condition. However, the combined treatment of Sorafenib with HS-173 synergistically inhibited the hypoxia-induced HIF-1a expression. To further examine the effect of the combination of two drugs on hypoxia-in- duced expression of VEGF, which is an immediate downstream tar- get gene of HIF-1a, the VEGF levels in Panc-1 cells were determined by Western blotting and an ELISA. A notable increase of VEGF was observed after exposure to hypoxia, and combined treatment of the two drugs synergistically suppressed hypoxia-in- duced VEGF expression (Fig. 5A and B). 3.6. The combination of Sorafenib with HS-173 synergistically suppressed VEGF-induced tube formation of HUVECs To examine the effect of the combined treatment of two drugs on the angiogenesis, we examined its inhibitory effect in a capillary tube formation assay using HUVECs, which are a well-known in vitro angiogenesis model. When the HUVECs were seeded on Matrigel, robust tubular-like structures were formed in the pres- ence of VEGF. However, the combined treatment of Sorafenib with HS-173 synergistically inhibited the VEGF-induced formation of vessel-like structures, consisting of the elongation and alignment of the cells compared to treatment with either drug alone (Fig. 5C). Considering that endothelial tube formation is property highly relevant to the process of angiogenesis, our results shows that combined treatment of Sorafenib with HS-173 has the ability to synergistically block VEGF-induced in vitro angiogenesis. 3.7. The combination of Sorafenib with HS-173 synergistically suppressed angiogenesis in an in vivo Matrigel plug assay We further explored the anti-angiogenic activity of combined treatment of Sorafenib with HS-173 using in vivo Matrigel plug assay. Matrigel containing VEGF or Sorafenib and/or HS-173 was subcutaneously injected into male BALB/c mice and removed from the mice at 7 days after the implantation. As shown in Fig. 6A, blood vessels were rarely observed in Matrigel plugs without VEGF. The Matrigel plugs containing VEGF alone appeared red in color due to the presence of RBCs, indicating that new blood vessels had formed inside the Matrigel via angiogenesis initiated by VEGF. However, combined treatment of Sorafenib with HS-173 consider- ably inhibited vascular formation compared to treatment with a single agent alone (Fig. 6A). For histological analysis, each section of the Matrigel plug was stained with H&E and CD31, an endothe- lial marker. The stained sections showed that the plug with Sorafe- nib or HS-173 treatment had fewer vessels within the gels than plugs containing only VEGF (Fig. 6B). In particular, the combination of Sorafenib and HS-173 suppressed VEGF-induced formation of blood vessels to a greater degree than treatment with a single agent alone. CD31 expression was also decreased more with the combined treatment than single treatment in the Matrigel plugs containing VEGF. These results suggest that the combination of Sorafenib with HS-173 possessed more potent anti-angiogenic activity in vivo than single agent alone. Fig. 4. Effect of the combination of Sorafenib with HS-173 on cell cycle distribution. (A) Panc-1 cells were treated with 10 lM Sorafenib and 1 lM HS-173 for 24 h. The cell cycle distribution was then assessed by flow cytometry. (B) Effect of Sorafenib and HS-173 on the levels of cyclin B1, p-cdc2, and p-cdc25c in Panc-1 cells was determined by Western blotting analysis. (C) After treatment with 10 lM Sorafenib and 1 lM HS-173 for 6 h, the expression of cyclin B1, p-cdc2, and p-cdc25c was detected by immunofluorescence. Fig. 5. Effect of the combination of Sorafenib with HS-173 on angiogenesis. (A) The expression of HIF-1a and VEGF was synergistically inhibited by the combination of Sorafenib and HS-173 in hypoxic Panc-1 cells. (B) Production of VEGF in hypoxia-induced Panc-1 cells treated with the combination of Sorafenib and HS-173 for 24 h. Data were expressed as the mean ± S.D. of triplicate wells. ωωp < 0.01 vs the Sorafenib-treated group, ωωωp < 0.001 vs the HS-173-treated group, and ###p < 0.001 vs the hypoxia control. (C) Effects of the combination of Sorafenib and HS-173 on VEGF-induced tube formation in vitro. HUVECs were plated on Matrigel (200 ll/well) and simultaneously treated with 10 lM Sorafenib and/or 1 lM HS-173. Capillary tube formation was assessed after 6 h. Morphological changes of the cells and tube formation were observed under a phase-contrast microscope and photographed at 200 × magnification. 4. Discussion Approximately 80% of pancreatic patients are diagnosed with locally advanced or metastatic stage that responds poorly to con- ventional cytotoxic chemotherapy. Due to this problem, the major- ity of the pancreatic cancer patients have limited treatment options. Molecular targeted approaches are thus particularly attractive for the treatment of pancreatic cancer. Numerous molec- ular targeting agents have been developed as cancer therapies and are under clinical evaluation. However, they have often shown suboptimal clinical activity as single agents owing to an inability to sufficiently inhibit the targeted signaling pathway as well as the development of compensatory mechanisms [28]. Therefore,combinations of molecular targeted agents that are mechanisti- cally complementary to overcome monotherapy may represent a more effective strategy and has become a common therapeutic approach. K-RAS is mutated in more than 90% of pancreatic cancers; the high frequency of this genetic aberration is unique to pancreatic cancer [29]. Nevertheless, no specific inhibitors targeting K-RAS have been developed to date. Consequently, many researchers have turned around the downstream targeting of K-RAS pathway, which seems a promising alternative at present [30]. The emer- gence of RAF/MEK inhibitors such as Sorafenib has enabled effec- tive signaling suppression sufficient to produce significant therapeutic activity in various human cancers including pancreatic cancer [31]. The effective use of RAF/MAPK inhibitors to treat pan- creatic cancer will also need to address the issue of the PI3K pathway activation, which indicates the aggressiveness of this disease. Indeed, activated AKT and PI3K/p110a overexpression is important for the progression and survival of pancreatic cancer [32,33]. Addi- tionally, extensive interactions between the PI3K/AKT and RAS/ RAF/MAPK pathways have been reported [34]. These findings lead to strong stimulation to design treatment regimens that suppress signaling through both the RAF/MAPK and PI3K/AKT pathways. For these reasons, we selected Sorafenib, an inhibitor of RAS/RAF/ MAPK pathway, and HS-173, a novel PI3K inhibitor, in the present study. Here, we report for the first time that combined Sorafenib and HS-173 treatment had synergistically enhanced anti-cancer activity by inhibiting the RAF/MAPK and PI3K/AKT pathways, which may lead to the inhibition of cell growth and angiogenesis together with the induction of apoptosis in pancreatic cancer. Fig. 6. In vivo effects of combined treatment with Sorafenib and HS-173 evaluated with a Matrigel plug assay. (A) Matrigel plugs were implanted into mice with VEGF (50 ng/ mL) and/or 10 lM Sorafenib and/or 1 lM HS-173 were implanted in mice. After 7 days, the plugs were removed, sectioned, stained with H&E, and photographed to evaluate the extent of vascularization. (B) The plugs were sectioned and immunostained for CD31. Stained plug sections were viewed with an Olympus confocal laser scanning microscope. Sorafenib with HS-173 significantly inhibited the growth of three types of pancreatic cancer cells compared to treatment with either agent alone. When we calculated CI values to further charac- terize the synergistic effect between Sorafenib and HS-173, the combination of 10 lM Sorafenib with 1 lM HS-173 was the most effective on Panc-1, Miapaca-2, and Aspc-1 cells. Among these dif- ferent cell lines, the combined treatment had the greatest synergis- tic effect on Panc-1 cells and Miapaca-2 cells showed the following good synergic effect. More importantly, Sorafenib highly inhibited the expression of p-MEK and p-ERK, main mediators of RAF/MAPK pathway, whereas HS-173 more inhibited the expression of p-AKT, p-mTOR, and p-P70S6K than that of p-MEK and p-ERK. However, combined treatment with two agents decreased all the expression of p-MEK, p-ERK, p-AKT, p-mTOR and p-P70S6K more than single agent alone, indicating synergistic suppression of both RAF/MAPK and PI3K/AKT pathways. Considering these results, this synergic ef- fect by both agents is likely to be mediated by the ability of Sorafe- nib and HS-173 to attenuate feedback induction of RAF/MAPK and PI3K/AKT signaling, respectively. Although previous studies of combinations of various agents for treating pancreatic cancer have been reported, the results were disappointing. Indeed, the combination of gemcitabine and 5-fluo- rouracil (5-FU) [35], as well as gemcitabine with cetuximab [36] or bevacizumab [37] failed to substantially improve survival rates. Recently, Hofmann et al. provided a rationale for testing combina- tions of RAF/MEK inhibitor and PI3K inhibitors in clinical trials comprising a patient population with pancreatic cancer harboring K-RAS mutation [38]. From this point of view, our combination experiment of Sorafenib and HS-173 is a meaningful trial and at the same time, it is noteworthy that combination treatment of both agents exerted potent synergic effects on pancreatic cancer cells. The combination of Sorafenib and HS-173 induced apoptosis of pancreatic cancer cells, which was observed as increased nuclear fragmentation and condensation by TUNEL and DAPI staining. In addition, Annexin-V assay revealed DNA damage, leading to induce apoptosis after combination treatment of two agents. Mitochon- dria, which provide energy in the form of ATP, plays an important role in apoptosis by releasing pro-apoptotic proteins normally sequestered in the intermembrane space into the cytosol where they activate downstream apoptotic signaling pathways [39]. Change in the mitochondrial membrane potential induces to create pores of mitochondria, which dissipates the transmembrane po- tential and leads to the release of cytochrome c into the cytoplasm [40,41]. Thus, to identify involvement of the combination treat- ment of Sorafenib and HS-173 on mitochondrial membrane poten- tial, we conducted JC-1 and cytochrome c immunofluorescence staining. The combination of Sorafenib and HS-173 induced marked changes in mitochondrial membrane potential (wm) and synergistically increased cytochrome c release along with a con- comitant increase in co-localization of cytochrome c with mito- chondria. In addition, the combination treatment significantly increased the expression level of cleaved caspase-3 and caspase- 9 along with PARP expression in Panc-1 and Miapaca-2 cells. Like- wise, the synergistic effect on apoptosis by the two agents was similar to those of previous studies showing that a combination of MEK and PI3K inhibitors enhances the induction of apoptosis in various cancers with K-RAS mutations [24,42,43]. Overall, our study showed that the combination of Sorafenib with HS-173 syn- ergistically induced mitochondria-mediated apoptosis in pancre- atic cancer cells, indicating that simultaneous targeting of the RAF/MAPK and PI3K/AKT pathways can provide a synergistic effect on apoptosis of pancreatic cancer. Angiogenesis is a process that plays an important role in the growth and metastasis of solid tumors [44]. In particular, HIF-1a plays a central role as a main regulator of the hypoxic transcription response during angiogenesis. In addition, since VEGF is a down- stream effector of HIF-1a and a principal mediator of tumor angi- ogenesis, the VEGF/HIF-1a system is considered to be an important target for anti-angiogenic therapies to treat cancer [45]. In this study, we showed that the combination of Sorafenib with HS-173 obviously inhibited the expression of HIF-1a and VEGF in Panc-1 pancreatic cancer cells grown under hypoxic conditions induced by CoCl2. Angiogenesis is also a multistep process linking with endothelial cell migration, proliferation, and capillary tube formation. These steps are also associated with the production of angiogenic factors such as VEGF [46]. We therefore measured the anti-angiogenic effects of Sorafenib with HS-173 on VEGF-induced migration and tube formation of HUVECs together with a Matrigel plug assay. The combination of Sorafenib with HS-173 synergisti- cally inhibited the VEGF-induced tube formation and migration of HUVECs compared to treatment with single agent alone. The anti-angiogenic effect of Sorafenib with HS-173 was also shown by decreased expression of CD31 in the VEGF-mediated Matrigel plugs. Our results demonstrated that the combination of Sorafenib with HS-173 blocks angiogenesis and may suppress cell growth. In conclusion, our data showed that combined treatment with Sorafenib and HS-173 significantly inhibited the growth of pancre- atic cancer cells. This drug combination also had synergistically en- hanced anticancer activity by inhibiting angiogenesis and inducing apoptosis in pancreatic cancer cells. Taken together, these results indicated that targeting the RAF/MAPK and PI3K/AKT pathways can elicit a synergistic inhibitory effect on pancreatic cancers in which both pathways are activated. Our findings suggest that the combination of Sorafenib and HS-173 may be a promising thera- peutic strategy for treating pancreatic cancer.