- Email: [email protected]
Anticancer activity of HS-527, a novel inhibitor targeting PI3-kinase in human pancreatic cancer cells
Cancer Letters 353 (2014) 68–77
Contents lists available at ScienceDirect
Cancer Letters journal homepage: www.elsevier.com/locate/canlet
Anticancer activity of HS-527, a novel inhibitor targeting PI3-kinase in human pancreatic cancer cells Ye-Lim Ryu a,1, Kyung Hee Jung a,1, Mi Kwon Son a, Hong Hua Yan a, Soo Jung Kim a, Sanghye Shin b, Sungwoo Hong b,⇑, Soon-Sun Hong a,⇑ a b
Department of Drug Development, College of Medicine, Inha University, 3-ga, Sinheung-dong, Jung-gu, Incheon 400-712, Republic of Korea Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Republic of Korea
a r t i c l e
i n f o
Article history: Received 16 April 2014 Received in revised form 2 July 2014 Accepted 3 July 2014
Keywords: PI3K HS-527 Pancreatic cancer Apoptosis Angiogenesis
a b s t r a c t Pancreatic cancer is known to have low 5-year survival rate and poor response to treatment. In this study, we synthesized HS-527, a new PI3-kinase inhibitor, and investigated not only its anticancer activity, but also its mechanism of action in pancreatic cancer cells. HS-527 had higher specificity for PI3K than other kinases and inhibited PI3K/Akt signaling pathway by down-regulating Akt and P70S6K. And HS-527 inhibited the cell growth and proliferation of the pancreatic cancer in a time- and dose-dependent manner, with greater activity than gemcitabine. Even HS-527 showed lower cytotoxicity than gemcitabine in normal cells. When treated with HS-527, the cancer cells appeared apoptotic, increasing the expression of cleaved PARP, cleaved caspase-3, and Bax. Furthermore, HS-527 showed an anti-angiogenic activity by decreasing the expression of HIF-1a and VEGF, and inhibited the migration of endothelial cells, and the formation of new blood vessel in mouse Matrigel plug assay. In this study, we found that HS-527 showed anti-cancer activity through an inhibition of the PI3K/Akt pathway in pancreatic cancer cells, suggesting that HS-527 could be used as a promising therapeutic agent for pancreatic cancer. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
Introduction Pancreatic cancer is ranked fourth among the causes of cancer that is related to deaths [1]. Over 40,000 people were diagnosed with pancreatic cancer, and 36,800 died as a result of the disease in the United States during the year 2013 [2,3]. Even if it accounts for 5% of all cancer-related deaths, the overall 5-year survival rate is 3–5% due to advanced staging at the time of diagnosis, along with poor response to current treatment [4]. In advanced pancreatic cancer, gemcitabine has been used as the standard drug for the past several decades. However, the benefit of a single agent in advanced pancreatic cancer is minimal [5]. Further, a number of combinations with gemcitabine have been studied to gain more effective results; however, these studies were not satisfactory. Therefore, the development of potential agent for pancreatic cancer is urgently needed. To overcome these limitations of cancer therapy, many recent studies are focusing on therapeutic targets. Among them, one of the most potential targets is phosphotidylinositol-3-kinase ⇑ Corresponding authors. Tel.: +82 42 350 2811; fax: +82 42 350 2810 (S. Hong). Tel.: +82 32 890 3683; fax: +82 32 890 2462 (S.S. Hong). E-mail addresses: [email protected] (S. Hong), [email protected] (S.-S. Hong). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.canlet.2014.07.001 0304-3835/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
(PI3K). PI3-kinases are composed of three classes: class I, class II and class III PI3Ks, according to the structure and function [6]. Class IA PI3K plays the most important role in human cancers because it is activated directly by the cell surface receptors [7]. It consists of p85 regulatory subunit and p110 catalytic subunit that is focused more on curing human cancers [8]. Recently, not only mutations in the catalytic subunit of PI3Ka, but also upregulation of the PI3K/Akt signaling pathway, occur commonly in most cancers [7,9]. It is widely known that the activated PI3K phosphorylates Akt and P70S6K, and is involved in numerous biological functions, such as cell proliferation, angiogenesis, differentiation, and survival [10]. Also, it is greatly associated with the treatment limitation of pancreatic cancer. Even though some studies have reported that there are no mutations in PIK3CA or AKT1, activated Akt is regarded as an important molecule for the survival of pancreatic cancer [11,12]. Based on a number of previous reports, we synthesized a potent novel PI3K inhibitor HS-527, 2-amino-N,N-dimethyl-5-(3(2-methylpyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)pyridine-3sulfonamide). HS-527 is especially targeted to a p110 catalytic subunit of PI3Ka. In this study, we studied the effect of HS-527 on anti-cancer activity through the PI3K/Akt signaling pathway in terms of cell growth and proliferation, apoptosis, and angiogenesis in pancreatic cancer.
Y.-L. Ryu et al. / Cancer Letters 353 (2014) 68–77 Material and methods Cells and materials Human pancreatic cancer cells PANC-1, MIA PaCa-2 and AsPC-1 were purchased from the American Type Culture Collection (AATC, Manassas, VA), and normal human fibroblast cells Hs677 were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea). PANC-1 and MIA PaCa-2 were cultured in Dulbecco’s modified Eagle’s medium (DMEM), and AsPC-1 and Hs677 were cultured in Roswell Park Memorial Institute Media 1640 (RPMI-1640). These cells were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. FBS and all other agents used in cell culture studies were purchased from Invitrogen (Carlsbad, CA). Human umbilical vein endothelial cells (HUVECs) were purchased from PromoCell (Heidelberg, GM). HUVECs were cultured in a 2% gelatin coated 75 cm2 flask in Endothelial Cell Growth Medium 2 with supplement mixture. The cultures were maintained at 37 °C in a CO2 incubator with a humidified atmosphere composed of 5% CO2 and 95% air. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and proteinase K were purchased from Sigma–Aldrich (St. Louis, MO). HS527 was dissolved in dimethyl sulfoxide (DMSO) as a stock at concentration of 100 mM, and was stored at 20 °C. Synthesis and binding assay of HS-527 A 5-bromo-7-azaindole in acetone was added N-Iodosuccinimide, and the mixture was stirred at room temperature for 3 h under N2 condition. The resulting precipitate was filtrated and washed with cold acetone to give 5-bromo-3-iodo1H-pyrrole[2,3-b]pyridine. An obtained intermediates in CH2Cl2 were added benzenesulfonyl chloride, Bu4NHSO4, and 50% aqueous NaOH. After stirring the mixture at room temperature for 6 h, aqueous work-up was carried out using CH2Cl2. The organic extracts were dried over MgSO4, which were then filtered and concentrated. It was washed with cold MeOH and the precipitate was collected by filtration to afford the N-protected intermediate as a white solid. The N-protected intermediates in 1,4-dioxane:H2O (3:1) were added 3-methyl-4-pyridinylboronic acid, Cs2CO3, and PdCl2(dppf). It was heated to 80 °C for 3 h. After cooling down the resulting mixture to room temperature, it was treated with 4 N KOH for deprotection of the benzene sulfonyl group. The mixture was heated to 80 °C for 1 h again, which was then cooled to room temperature. The resulting solution was neutralized with 4 N HCl, extracted with CH2Cl2, and washed with brine. The organic layer was dried over MgSO4 and concentrated in vacuo. The residue was purified with flash column chromatography to give a deprotected intermediate. The deprotected intermediate, boronic ester, K2CO3 and PdCl2(dppf) in 1,4-dioxane:H2O (3:1) were heated to 100 °C for 100 min using microwave. After cooling down the resulting mixture to room temperature, it was concentrated in vacuo. The residue was diluted with CH2Cl2 and MeOH, and then filtered through a short plug of silica and Celite. The filtrate was concentrated and purified with flash column chromatography to obtain HS-527 as a white solid (Fig. 1A). And, in the binding assay, POC and Kd of HS-527 were determined at the Ambit Bioscience Corp. (San Diego, CA, USA). HS-527 was profiled at 1 lM against a panel of 20 representative kinases in a high-throughput binding assay (Ambit Bioscience). Measurement of cell proliferation Cell viability was measured by the MTT assay. Pancreatic cancer cells PANC-1, MIA PaCa-2, and AsPC-1 were plated at a density of 3–10 103 cells/well in 96-well plates, and then incubated for 24 h. Then, the media was removed and the cells were treated with either DMSO, as a negative control, or various concentrations of HS-527 and gemcitabine. The final concentration of DMSO in the media was 0.1% (v/v). After incubating the cells for 24, 48, and 72 h, a 10% of MTT solution (2 mg/mL) was added to each well, and the cells were incubated for another 4 h at 37 °C. After incubation for 4 h, 100 lL DMSO was added to each well to dissolve the formed formazan crystals with constant shaking for 5 min. The plate was read at 540 nm using a microplate reader. Each analysis was measured from three replicate wells.
69
substrate was added, and the plate was incubated for 10 min. Next, 1 mM H2SO4 was added to terminate the reaction. Each well was measured using a microplate reader at 450 nm. Four replicate wells were used for each analysis.
Immunodetection of incorporation of 50 -bromo-20 -deoxyuridine (BrdU) PANC-1 and MIA PaCa-2 were plated onto 18-mm cover glass in DMEM and grown to approximately 70% confluence for 24 h. The cells were then treated with 5 lM of HS-527 for 3 h, and then labeled for 4 h with 10 lM 50 -bromo-20 -deoxyuridine (BrdU). After labeling, the cells were fixed in an acetic acid: ethanol solution (3:1) for 10 min at 20 °C and washed with 1% Triton X-100 in PBS (pH 7.4). Then, the cells were incubated in HCl (4 N) for 15 min at 37 °C incubator to disrupt the DNA structure in the BrdU-labeled cells. After denaturation, borate buffer (0.1 M) was added for neutralization. The cells were blocked with 5% goat and horse serums for 30 min at room temperature. And then the cells were incubated with anti-BrdU antibody (Abcam, Cambridge, MA) for overnight in a humidified chamber. After washing with PBS, FITC-labeled anti-mouse secondary antibody was diluted with PBS at room temperature in the dark for 1 h. The nuclei were stained with 4,6diamidino-2-phenylindole (DAPI) in the dark for 30 min at room temperature. The slides were washed with PBS, and then covered with DABCO (Sigma–Aldrich). Confocal laser scanning microscopy was performed.
Western blot PANC-1 and MIA PaCa-2 cells were washed with ice-cold PBS before lysis using a buffer containing 1% Nonidet P-40, 1% Triton X-100, and the following protease and phosphatase inhibitors: aprotinin (10 mg/mL), leupeptin (10 mg/mL) (ICN Biomedicals, Asse-Relegem, Belgium), phenylmethylsulfonyl fluoride (1.72 mM), NaVO3 (500 mM), NaF (100 mM), and Na4P2O7 (500 mg/mL) (Sigma–Aldrich). Equal amounts of protein were separated using 8–15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). In addition, the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes. The protein transfer was checked by Ponceau S solution staining (Sigma–Aldrich). Immunostaining of the blots was performed by the primary antibodies. After washing three times, the blots were incubated with the secondary antibody conjugated to horseradish peroxidase (HRP) and detection by enhanced chemiluminescence reagent (Amersham Biosciences, Piscataway, NJ). Primary antibodies were purchased as follows: Bax, HIF1a (Santa Cruz Biotechnology, Santa Cruz, CA), b-actin (Abcam, Cambridge, UK), Akt, p-Akt (Thr308), P70S6K, p-P70S6K (Thr389), cleaved caspase-3, Mcl-1, survivin, and cleaved PARP (Cell Signaling Technology, Beverly, MA).
Terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay A TUNEL assay was performed using a commercially available kit (Chemicon, Temecula, CA), following the manufacturer’s protocol. The cells were plated on an 18-mm cover glass in DMEM, at a density of 70% confluence, and incubated for 24 h. The cells were then treated with 5 lM HS-527 for 24 h before being fixed in an ice-cold mixture of acetic acid and ethanol. After the cells were washed with PBS, they were stained for TUNEL. The stained cells were examined under a light microscope for nuclear fragmentation.
Measurement of mitochondrial membrane potential (JC-1) Pancreatic cancer cells were plated onto 18-mm cover glass in the media and incubated for 24 h in order to reach approximately 70% confluence. The cells were incubated in the presence or absence of 5 lM HS-527 treatment for 6 h and then incubated with 5 lM JC-1 fluorescence dye (JC-1 Mitochondrial Membrane Potential Assay Kit Cayman Chemical Company, Ann Arbor, MI) for 20 min in a CO2 incubator. The slides were washed with PBS and covered with DABCO (Sigma–Aldrich) before being viewed with a confocal laser scanning microscope (Olympus, Tokyo, Japan).
Measurement of 50 -bromo-20 -deoxyuridine (BrdU) cell proliferation assay Enzyme-linked immunosorbent assay (ELISA) PANC-1 cells were plated at a density of 8 103 cells/well in 96-well plates, and then incubated for 24 h. Further, MIA PaCa-2 cells were plated at a density of 6 103 cells/well in 96-well plates, which were then incubated for 24 h. The pancreatic cancer cells were then treated with various concentrations of HS-527 at 37 °C for 2 h. After incubation, DNA content synthesis was assessed by 50 -bromo20 -deoxyuridine (BrdU) cell proliferation assay kit according to the manufacturer’s instructions (Merck, Darmstadt, Germany). Briefly, cells in each well were labeled by the addition of BrdU (5 lM) for 2 h at 37 °C. After removing the labeling media, 100 lL per well fixing/denaturing solution was added to each well and incubated for 30 min at room temperature. After removing fixing/denaturing solution, 100 lL per well of detection antibody solution was added, and the microplate was incubated for 1 h at room temperature. After washing, horseradish peroxidase (HRP)-conjugate solution was added, and the plate was incubated for 30 min. TMB
The amount of VEGF secreted into the media was measured by a sandwich ELISA. The plates were coated with 100 lL of 2 lg/mL anti-VEGF antibody (R&D Systems, Minneapolis, MN) diluted in PBS for 24 h at room temperature. The plates were washed three times with PBS containing 0.1% Tween-20 and incubated with 100 lL/well of 1% bovine serum albumin (BSA, Sigma–Aldrich) in PBS for 1 h at room temperature. The conditioned medium and various concentrations of recombinant human VEGF were incubated overnight at 4 °C with 100 lL of 50 ng/mL biotinylated anti-VEGF antibody. The plates were then washed and incubated for 30 min with 100 lL of HRP-conjugated streptavidin (Vector Laboratories, Burlingame, CA). After washing, the reaction was stopped by adding 50 lL of 2 N H2SO4. The absorbance was measured at 450 nm using a microplate reader. Three replicate wells were used for each analysis.
70
Y.-L. Ryu et al. / Cancer Letters 353 (2014) 68–77
Fig. 1. Characteristics of HS-527. (A) 2-amino-N,N-dimethyl-5-(3-(2-methylpyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)pyridine-3-sulfonamide. (B) Binding mode of HS527 in the ATP-binding site of PI3Ka. (C) Binding affinity of HS-527 was measured by KINOMEscan. A panel of 20 kinases was tested at 1 lM of HS-527. Lower percent of control (POC) values represent stronger hits. Values shown are the mean of duplicate measurements.
Immunofluorescence microscopy PANC-1 and MIA PaCa-2 cells were plated on 18-mm cover glasses in DMEM at a density of 70% confluence and incubated for 24 h. Next, the cells were incubated in the presence or the absence of 5 lM HS-527 for 4 h. The cells were washed with PBS and fixed in an acetic acid: ethanol (2:1) solution for 5 min at 20 °C. Nonspecific binding was blocked with 5% goat and horse serum/PBS for 1 h at room temperature, and the cells were then incubated with primary antibodies against p-Akt (Thr308) (Abcam, Cambridge, UK), p-Stat3 (Tyr705), and p-Mek (Ser221) (Cell Signaling Technology, Beverly, MA) in a humidified chamber. After washing twice in PBS, the cells were incubated with fluorescein-labeled secondary antibody (1:50; Dianova) in an antibody dilution solution for 1 h at room temperature in the dark. The nuclei were stained with DAPI in the dark for 30 min at room temperature. The slides were washed twice with PBS, covered with DABCO (Sigma–Aldrich), and examined with confocal laser scanning microscopy (Olympus, Tokyo, Japan).
HS-527 (0.1–10 lM), and/or VEGF (100 ng/mL). HUVECs were cultured for 20 h to migrate in a CO2 incubator. The cells were washed with a serum-free medium and photographed under a phase-contrast microscope at 200 magnification.
Matrigel plug assay BALB/c mice were obtained from Orient-Bio Laboratory Animal Research Center Co., Ltd. (Gyeonggi-do, Kapyung, Korea). The animals were 6 weeks old and kept in a temperature and humidity controlled environment, with 12 h light–dark cycles. They were provided with standard rat chow and free access to tap water. After stabilization, the mice were subcutaneously injected with 800 lL of Matrigel mixed with VEGF (100 ng/mL) and either PBS or HS-527 (5 lM). After 7 days, the mice were sacrificed and the Matrigel plugs were isolated and fixed in 10% buffered formaldehyde, embedded in paraffin, and sectioned.
Histopathological staining Wound migration assay HUVECs were seeded on 60 mm2 culture dishes and cultured until 90% confluence. HUVECs were wounded with a razor blade. This step made the injury line that HUVECs were marked. After wounding, HUVECs were washed with PBS and further incubated in Endothelial Cell Growth Medium 2 with 2% FBS, 20 unit/mL heparin (Sigma–Aldrich), 1 mM thymidine (Sigma–Aldrich), and various concentrations of
The Matrigel plug samples were fixed in 10% buffered formaldehyde, embedded in paraffin, and then sectioned. The 8 lm-thick sections were stained with hematoxylin and eosin (H&E) for histology observation. On the other slides, ten micrometer thick frozen sections were incubated overnight with anti-CD34 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 4 °C after the hydration process. After washing three times with PBS, the sections were performed with anti rabbit Texas red
Y.-L. Ryu et al. / Cancer Letters 353 (2014) 68–77 labeled secondary antibodies. Then, DAPI was used to stain the nucleus. The slides were washed twice with PBS and covered with DABCO (Sigma–Aldrich), and examined with confocal laser scanning microscopy.
Statistical analysis Data were expressed as the mean ± SD. Statistical analysis was performed using ANOVA. A P-value of 0.05 or less was considered statistically significant. Statistical calculations were performed using SPSS software for Windows operating system (Version 10.0; SPSS, Chicago, IL).
Results HS-527 binds to ATP binding site of PI3Ka with strong binding affinity We synthesized HS-527, a novel PI3K inhibitor, which targeted the ATP binding site against PI3K (Fig. 1A). The binding mode in the ATP-binding site of PI3Ka was investigated in a comparative fashion in order to obtain some structural insight into the inhibitory mechanisms of the identified HS-527. Fig. 1B shows the lowestenergy conformation of HS-527 in the PI3Ka ATP binding site, calculated using the modified AutoDock program. On the basis of the docking model results, hydrogen bonding group on the 7-azaindole moiety appeared to point toward the hinge region (Val851), while the pyridyl sulfonamide group is lined by Tyr836 and Asp810. 7azaindole contains both H-donor and H-acceptor for interaction with the hinge region of the kinase. The NH group of 7-azaindole donates a hydrogen bond to the backbone oxygen of Val851. In addition, the nitrogen at the 7-position of the azaindole scaffold appeared to form a hydrogen bond with the backbone nitrogen
71
of Val851. Furthermore, HS-527 could be stabilized further in the PI3Ka ATP binding site via additional hydrogen bonds of the 5position pyridyl sulfonamide in azaindole. The pyridyl group was anchored by a hydrogen bond to Tyr836. The anilic group of C5 aryl ring donated hydrogen bonds to the backbone amino carbonyl oxygen of Asp810. In addition, the sulfonamide binds to the catalytic lysine (Lys802). These multiple hydrogen bonds appeared to be the most significant binding force stabilizing HS-527 in the active site of PI3Ka. To obtain more detailed characteristics of HS-527, we performed a high-throughput binding with a panel of 20 cancer-related protein kinases by KINOMEscan. As shown in Fig. 1C, HS-527 showed the strongest binding affinity to PI3KCA at a concentration of 1 lM, with modest binding affinity to other cancer-related protein kinases. Therefore, the result of a KINOMEscan shows that HS-527 is specific enough to PI3KCA.
HS-527 inhibits PI3K/Akt pathway In order to investigate which pathway was especially regulated by HS-527, we performed the immunofluorescence and Western blot assay in MIA PaCa-2 cells. In Fig. 2A, HS-527 significantly inhibited the p-Akt expression, which is the downstream signal of the PI3K pathway. On the other hand, both p-Stat3 and p-Mek were not inhibited by HS-527. To identify the anti-cancer effects of HS-527 through the PI3K pathway, we determined the phosphorylation of Akt and P70S6K, downstream of PI3K pathway, in pancreatic cancer cells using a Western blot. In Fig. 2B, HS-527
Fig. 2. Effect of HS-527 on the PI3K/Akt signaling pathway in pancreatic cancer cells. (A) The phosphorylation levels of Akt (Thr308), Stat3 (Tyr705), and Mek (Ser221) were detected by confocal microscopy and Western blot. MIA PaCa-2 cells were treated with 5 lM HS-527 for staining or for Western blot 1 or 5 lM HS-527 for 4 h. (B) PANC-1 and MIA PaCa-2 cells were treated with HS-527 (0–10 lM) for 24 h. The expression of p-Akt (Thr308), Akt, p-P70S6K (Thr389) and P70S6K were performed by Western blot.
72
Y.-L. Ryu et al. / Cancer Letters 353 (2014) 68–77
inhibited both the expression of p-Akt and p-P70S6K in a dosedependent manner.
HS-527 inhibits cell growth of human pancreatic cancer cells with low toxicity in normal fibroblast cells To examine the effect of HS-527 on the viability of pancreatic cancer cells, the cells were treated with various concentrations (0–20 lM) of HS-527 for 24, 48, and 72 h. As shown in Fig. 3A, the growth of pancreatic cancer cells treated with HS-527 was inhibited in a dose- and time-dependent manner. Furthermore, to compare the effect of HS-527 with gemcitabine, a conventional drug for pancreatic cancer, we treated various concentrations of the drugs to the cells. HS-527 was more effective than gemcitabine in the MIA PaCa-2 cells given the 24 h treatment (Fig. 3B). Next, to observe the cytotoxicity of HS-527 in the normal cells, we performed MTT assay in a human normal fibroblast cell line, Hs677. The cells were exposed to various concentrations of HS-527 and
gemcitabine for 24 h. HS-527 represented a cell viability of approximately 90% compared to the control. On the other hand, the gemcitabine revealed 70% cell viability compared to the control (Fig. 3C). Therefore, the cytotoxicity of HS-527 seemed to be less toxic to the normal cells compared with gemcitabine in all tested concentrations.
HS-527 inhibits the proliferation of pancreatic cancer cells To investigate the effect of HS-527 on pancreatic cancer cell proliferation, we performed the BrdU proliferation assay and BrdU staining. In the BrdU proliferation assay, pancreatic cancer cells were treated with various concentrations (0–50 lM) of HS-527 for 2 h. As shown in Fig. 3D, the BrdU incorporation was inhibited by HS-527 in a dose-dependent manner. Also, the result showed that HS-527 significantly decreased the number of BrdU positive cells in a dose-dependent manner (Fig. 3E). These results revealed that HS-527 inhibited the proliferation of pancreatic cancer cells.
Fig. 3. Effect of HS-527 on the viability and proliferation of pancreatic cancer cells. (A) The growth inhibition by HS-527 in pancreatic cancer cells (PANC-1, MIA PaCa-2 and AsPc-1) for 24 h, 48 h and 72 h. Viability of (B) MIA PaCa-2 cells or (C) Hs677 cells treated with various concentrations (0–50 lM) of HS-527 or gemcitabine was measured at 24 h. (D) The effect of HS-527 (0.01–50 lM) on the proliferation of pancreatic cancer cells was performed by a BrdU proliferation assay for 2 h. (E) PANC-1 and MIA PaCa-2 cells were treated with 0.1–5 lM of HS-527 for 3 h. After BrdU staining, the cells were detected by confocal microscopy. The representative confocal image showed that HS527 (5 lM) effectively inhibited proliferation of pancreatic cancer cells. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to the control. Data represent as mean ± SD from the (A– C) three or (D) four replicate wells.
Y.-L. Ryu et al. / Cancer Letters 353 (2014) 68–77
73
Fig. 3 (continued)
HS-527 induces apoptotic cell death in pancreatic cancer cells In order to examine the apoptotic effect of HS-527, we performed TUNEL assay, JC-1 assay, and Western blot. When treated with 5 lM HS-527, the cells showed morphological features, such as DNA fragmentation and loss of mitochondrial transmembrane potential (wm), which are characteristics of apoptotic cells. In Fig. 4A, the treatment of HS-527 increased the number of TUNEL positive cells in a dose dependent manner. We also investigated the change of mitochondria potential using a JC-1 assay kit. As shown in Fig. 4B, the control cells showed both the red and green fluorescence in the cytoplasm. Treatment with 5 lM of HS-527 decreased the red fluorescence intensity, whereas it increased the green fluorescence intensity. To confirm these results, we examined the protein expression related to apoptosis by a Western blot in the pancreatic cancer cells. HS-527 increased the pro-apoptotic protein expression of cleaved PARP, cleaved caspase-3, and Bax. In contrast, HS-527 decreased the anti-apoptotic protein expression, such as Mcl-1 and survivin (Fig. 4C). These results implied that HS-527 promoted apoptosis in pancreatic cancer cells. HS-527 inhibits the angiogenesis in pancreatic cancer and HUVECs Next, to investigate the effect of HS-527 on the expression of HIF-1a and VEGF under hypoxic condition, MIA PaCa-2 cells were treated with 100 lM CoCl2 and HS-527 (0–10 lM) for 24 h. First, we measured the expression of HIF-1a by a Western blot, as well as VEGF by ELISA. As shown in Fig. 5A and B, the expression of both HIF-1a and VEGF was increased in hypoxic condition by a CoCl2 treatment. However, the expression of HIF-1a and VEGF was
decreased by a treatment of HS-527, in a dose-dependent manner. To confirm these data, we performed immunofluorescence analyses with the red fluorescence intensity for HIF-1a expression and the green for VEGF expression. In Fig. 5C, HIF-1a signal appeared in the nucleus, whereas VEGF was stained in the cytoplasm. In contrast to the control, the cells treated with HS-527 significantly showed a low expression of HIF-1a and VEGF. HS-527 decreased the expression of HIF-1a and VEGF in the MIA PaCa-2 cells in a hypoxic condition. Migration of endothelial cells is one of the essential steps during angiogenesis [13]. To investigate the movement of endothelial cells, HUVECs were wounded by raze blade and incubated with various concentrations of HS-527 (0–10 lM) for 20 h. The movements of HUVECs were significantly blocked by HS-527 treatment in a dose-dependent manner (Fig. 5D). HS-527 was considered to have an anti-angiogenic activity in both the pancreatic cancer cells and endothelial cells. HS-527 inhibits the formation of blood vessel in vivo As shown in Fig. 6, we confirmed that HS-527 significantly inhibited the angiogenesis in vitro. Further, we speculated that HS-527 has the ability of anti-angiogenesis in vivo, as well. We subcutaneously injected Matrigel mixed with VEGF (100 ng/mL) or HS-527 (5 lM) into the BALB/c mice. After 7 days, we removed the Matrigel plug from the BALB/c mice. The plug injected with VEGF alone became red because the blood vessels and red blood cells were induced by VEGF. On the contrary, when the Matrigel was mixed with VEGF and HS-527, the plug slightly changed to red compared to the treatment with VEGF alone. In the H&E
74
Y.-L. Ryu et al. / Cancer Letters 353 (2014) 68–77
Fig. 4. Effect of HS-527 on apoptosis of pancreatic cancer cells. (A) The induction of apoptosis by HS-527 treatment was evaluated by TUNEL staining. Pancreatic cancer cells were treated with 1 and 5 lM HS-527 for 24 h. Characteristics of apoptosis was shown with dark brown staining. (B) JC-1 staining was performed to detect mitochondrial transmembrane potential during apoptosis. HS-527 (5 lM) was treated to pancreatic cancer cells for 6 h. (C) The expression of PARP, cleaved caspase-3, Bax, survivin and Mcl1 were assayed by Western blot analysis after PANC-1 and MIA PaCa-2 cells were treated with HS-527 at the indicated doses (0–10 lM) for 24 h. **p < 0.01 and ***p < 0.001 compared to the control.
staining, a number of red blood cells were recruited in the section of the VEGF plug, while HS-527 efficiently inhibited the recruit of red blood cells into the plug (Fig. 6A). In addition, we stained CD34, an angiogenesis marker, in the section of the plug using immunofluorescence. Like the previous results, HS-527 significantly decreased the expression of CD34, which stained to a red color.
Discussion To date, it has been reported that the PI3K pathway plays important roles in numerous human cancers, including pancreatic cancer [14–16]. As the overexpression of PI3K/p110a plays an important role in pancreatic cancer, targeting the PI3K/Akt pathway was considered as the therapeutic strategy [17]. Recently, Li
Y.-L. Ryu et al. / Cancer Letters 353 (2014) 68–77
75
Fig. 5. Effect of HS-527 on angiogenesis of pancreatic cancer cells. (A) MIA PaCa-2 cells were treated with 100 lM CoCl2 and various concentrations (0, 0.1, 1, 5, 10 lM) of HS527 for 24 h. Expression of HIF-1a in hypoxia mimic condition was detected by a Western blot. (B) Production of VEGF in MIA PaCa-2 cells treated with CoCl2 and/or HS-527 was examined using ELISA. Data represented as the mean ± SD from the triplicate wells. (C) The expression of HIF-1a and VEGF by immunofluorescence microscope in MIA PaCa-2 cells treated with HS-527 in hypoxia mimic condition (CoCl2, 100 lM). (D) HUVECs were plated at 60 mm2 dish until 90% confluence. And then the HUVECs were scratched with a razor blade and treated with VEGF (100 ng/mL) and HS-527 as indicated concentrations (0.1–10 lM) for 20 h. **p < 0.01 and ***p < 0.001 compared to the control.
et al. have reported that the inhibition of the PI3K/Akt pathway led to gemcitabine resistance to be overcome in pancreatic cancer [18]. This circumstance prompted us to investigate further into the novel PI3K inhibitors in pancreatic cancer. Considering these facts, we identified a novel PI3K inhibitor, HS-527, and evaluated its anticancer activities on pancreatic cancer. The present study revealed that our novel molecule, HS-527, had a prominent inhibitory effect on the PI3K/Akt signaling pathway in pancreatic cancer cells, which may lead to the inhibition of cell growth and angiogenesis as well as induction of apoptosis. Over-activation of PI3K pathways have been observed in many cancer types. Especially, the PI3K pathway is one of major pathways that can be directly activated by a mutated KRAS of pancreatic cancer, thereby leading to cell proliferation and tumor growth [19]. Also, the PI3K/Akt pathway is a potent pathway to block the apoptotic effects of chemotherapy drugs and radiation therapy in a variety of cancer types including pancreatic cancer [20]. In addition, Schlieman et al. have reported that 59% of pancreatic cancer showed overexpression of Akt, a main mediator of PI3K/
Akt pathway [21]. Furthermore, most of the cancers acquire drug resistance due to the activation of PI3K/Akt pathway [22]. Indeed, drug resistance of paclitaxel and doxorubicin in breast cancer and that of gemcitabine in pancreatic cancer were mediated by hyperactivation of PI3K/Akt pathway [23–25]. For this reason, a lot of researchers have concerned with PI3K/Akt pathway as an attractive molecular target. First, we confirmed the chemical characteristics of HS-527. This compound not only had the highest binding affinity to PI3K (POC = 1.1), but it also bound to the ATP binding site of PI3K with a stable force. Since HS-527 had the highest binding affinity to the ATP binding site of PI3K, we investigated whether HS-527 inhibited the expression of p-Akt, p-Mek, and pStat3 which are key downstream molecules of the PI3K, MAPK, and JAK/Stat3 signaling pathways related to pancreatic cancer therapy [11]. HS-527 dramatically inhibited the p-Akt expression in the pancreatic cancer cells, whereas the expression of p-MEK and p-STAT3 were scarcely altered. To understand the mechanisms of anticancer activity of HS-527, we evaluated the change of the downstream signals of PI3K/Akt pathway. HS-527 blocked the
76
Y.-L. Ryu et al. / Cancer Letters 353 (2014) 68–77
Fig. 6. Effect of HS-527 on the formation of blood vessel in vivo. (A) Matrigel plugs mixed with VEGF (100 ng/mL) or HS-527 (5 lM) were injected into Balb/c mice, and sacrificed after 7 days. Photographs were taken of the plugs and a section of the plug was stained with H&E. (B) For staining the recruited new vessels, CD34 were stained by red color at 200 magnification.
phosphorylation of Akt and P70S6K, the downstream of the PI3K/ Akt pathway, in a dose-dependent manner. In order to understand the anti-cancer effects of HS-527, we investigated the cell viability and proliferation in three pancreatic cancer cell lines. In our results, HS-527 strongly suppressed the cell growth and DNA synthesis of the three pancreatic cancer cells in a dose-, time-dependent manner. Also, we observed that HS-527 strongly inhibited PI3K activity with an IC50 of <10 lM in MIA PaCa-2 cells and was much more potent than gemcitabine. In addition, HS-527 has a lower toxicity compared to gemcitabine in the normal cells. Based on the above results, we propose that HS-527 inhibited the cell growth/proliferation by the highest binding affinity to PI3K in the pancreatic cancer cells, while having low toxicity to normal cells. Along with the inhibition of cell proliferation, induction of apoptosis is a major point in cancer therapy. Especially, mitochondrion 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 [26,27]. Change in the mitochondrial membrane potential induces to create pores of mitochondria, which leads to the release of cytochrome c into the cytoplasm, resulting in expression of various pro-apoptotic proteins in cytoplasm [28]. In this process, Bax, as pro-apoptotic molecule, actively induce
cytochrome c release from mitochondria within cells. Thus, Bax overexpression induces apoptosis induction in response to various apoptosis stimuli [29]. Also, XIAP, Mcl-1, and survivin, as antiapoptotic protein family, have been reported to inhibit apoptosis signaling by binding to caspases [30]. In addition, Tsuruta et al. have reported that PI3K/Akt pathway suppressed Bax translocation to mitochondria, which promoted cell survival and inhibited apoptosis [31]. Thus, to identify involvement of the HS-527 in mitochondrial membrane potential, we conducted JC-1 staining. As a result, HS-527 induced marked changes in mitochondrial membrane potential (wm). Also, HS-527 decreased the expression of Mcl-1 and survivin, whereas increased the expression of Bax. These changes led to increase the expression of cleaved caspase-3 and cleavage of PARP. Overall, our study showed that HS-527 strongly induced mitochondria-mediated apoptosis in the pancreatic cancer cells. Angiogenesis is a necessary process for the growth of solid tumors, including pancreatic cancer. As the tumor grows, hypoxia condition emerges inside the tumor, and imbalance of key regulators begins between HIF-1a and VEGF, which are key mediators in angiogenesis [32–34]. In angiogenesis of cancer, PI3K/Akt pathway is activated by various stimuli including VEGF in endothelial cells and plays important role for regulating HIF-1a and VEGF expressions [35]. In addition, angiogenesis occurs in multi-steps,
Y.-L. Ryu et al. / Cancer Letters 353 (2014) 68–77
including migration and proliferation of the endothelial cells, as well as the formation of a new blood vessel [36]. In this study, we observed that HS-527 efficiently suppressed the expression of HIF-1a and VEGF in a hypoxia condition of the pancreatic cancer cells. Also, HS-527 strongly inhibited VEGF-induced migration of HUVECs in a dose-dependent manner. Much like these in vitro results, HS-527 prominently inhibited the formation of new blood vessel and the recruitment of endothelial cells into the plug by VEGF induction, using the in vivo Matrigel plug assay. Moreover, it decreased the expression of CD34 well. These results indicated that HS-527 significantly suppresses VEGF induced-angiogenesis both in vitro and in vivo. In conclusion, the present study demonstrates that a novel PI3K inhibitor, HS-527, shows remarkable potent anti-cancer activity by inhibiting cell growth/proliferation and angiogenesis, along with inducing apoptosis. The anticancer effect of HS-527 appears to be associated with the inhibition of the PI3K/Akt pathway. Therefore, we suggest that HS-527 can be considered as a potential therapeutic agent targeting the PI3K/Akt pathway in pancreatic cancer. Conflict of Interest None. Role of the Funding Source None declared. Acknowledgments This research was supported by the National Research Foundation (NRF) Grant (2012R1A2A2A010) and Medical Research Center (2014009392) funded by MSIP, Korea. References [1] A. Jemal, T. Murray, E. Ward, A. Samuels, R.C. Tiwari, A. Ghafoor, E.J. Feuer, M.J. Thun, Cancer statistics, CA Cancer J. Clin. 55 (2005) 10–30. [2] R. Siegel, D. Naishadham, A. Jemal, Cancer statistics, CA Cancer J. Clin. 63 (2013) 11–30. [3] H. Zhong, C. Sanchez, D. Spitrzer, S. Plambeck-Suess, J. Gibbs, W.G. Hawkins, D. Denardo, F. Gao, R.A. Pufahl, A.C. Lockhart, et al., Synergistic effects of concurrent blockade of PI3K and MEK pathways in pancreatic cancer preclinical models, PLoS ONE 8 (2013) e77243. [4] Z. Wang, Y. Li, A. Ahmad, S. Banerjee, A.S. Azmi, D. Kong, F.H. Sarkar, Pancreatic cancer: understanding and overcoming chemoresistance, Nat. Rev. Gastroen. Hepatol. 8 (2011) 27–33. [5] H.A. Burris 3rd, M.J. Moore, J. Andersen, M.R. Green, M.L. Rothenberg, M.R. Modiano, M.C. Cripps, R.K. Portenoy, A.M. Storniolo, P. Tarassoff, et al., Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial, J. Clin. Oncol. 15 (1997) 2403–2413. [6] A. Akinleye, P. Avvaru, M. Furqan, Y. Song, D. Liu, Phosphatidylinositol 3-kinase (PI3K) inhibitors as cancer therapeutics, J. Hematol. Oncol. 6 (2013) 88. [7] T.L. Yuan, L.C. Cantley, PI3K pathway alterations in cancer: variations on a theme, Oncogene 27 (2008) 5497–5510. [8] B. Geering, P.R. Cutillas, B. Vanhaesebroeck, Regulation of class IA PI3Ks: is there a role for monomeric PI3K subunits?, Biochem Soc. Trans. 35 (2007) 199– 203. [9] J.J. Wallin, K.A. Edgar, J. Guan, M. Berry, W.W. Prior, L. Lee, J.D. Lesnick, C. Lewis, J. Nonomiya, J. Pang, et al., GDC-0980 is a novel class I PI3K/mTOR kinase inhibitor with robust activity in cancer models driven by the PI3K pathway, Mol. Cancer Ther. 10 (2011) 2426–2436.
77
[10] I. Vivanco, C.L. Sawyers, The phosphatidylinositol 3-Kinase AKT pathway in human cancer, Nat. Rev. Cancer 2 (2002) 489–501. [11] D. Matthaios, P. Zarogoulidis, I. Balgouranidou, E. Chatzaki, S. Kakolyris, Molecular pathogenesis of pancreatic cancer and clinical perspectives, Oncology 81 (2011) 259–272. [12] A. Maitra, R.H. Hruban, Pancreatic cancer, Annu. Rev. Pathol. 3 (2008) 157– 188. [13] I. Zachary, VEGF signalling: integration and multi-tasking in endothelial cell biology, Biochem. Soc. Trans. 31 (2003) 1171–1177. [14] V.M. Bondar, B. Sweeney-Gotsch, M. Andreeff, G.B. Mills, D.J. McConkey, Inhibition of the phosphatidylinositol 30 -kinase-AKT pathway induces apoptosis in pancreatic carcinoma cells in vitro and in vivo, Mol. Cancer Ther. 1 (2002) 989–997. [15] A. Hollestelle, F. Elstrodt, J.H. Nagel, W.W. Kallemeijn, M. Schutte, Phosphatidylinositol-3-OH kinase or RAS pathway mutations in human breast cancer cell lines, Mol. Cancer Res. 5 (2007) 195–201. [16] H. Lee, S.J. Kim, K.H. Jung, M.K. Son, H.H. Yan, S. Hong, S.S. Hong, A novel imidazopyridine PI3K inhibitor with anticancer activity in non-small cell lung cancer cells, Oncol. Rep. 30 (2013) 863–869. [17] C.E. Edling, F. Selvaggi, R. Buus, T. Maffucci, P. Di Sebastiano, H. Friess, P. Innocenti, H.M. Kocher, M. Falasca, Key role of phosphoinositide 3-kinase class IB in pancreatic cancer, Clin. Cancer Res. 16 (2010) 4928–4937. [18] J. Li, X. Liang, X. Yang, Ursolic acid inhibits growth and induces apoptosis in gemcitabine-resistant human pancreatic cancer via the JNK and PI3K/Akt/NFkappaB pathways, Oncol. Rep. 28 (2012) 501–510. [19] R.A. Perugini, T.P. McDade, F.J. Vittimberga Jr., M.P. Callery, Pancreatic cancer cell proliferation is phosphatidylinositol 3-kinase dependent, J. Surg. Res. 90 (2000) 39–44. [20] B.N. Fahy, M. Schlieman, S. Virudachalam, R.J. Bold, AKT inhibition is associated with chemosensitisation in the pancreatic cancer cell line MIAPaCa-2, Br. J. Cancer 89 (2003) 391–397. [21] M.G. Schlieman, B.N. Fahy, R. Ramsamooj, L. Beckett, R.J. Bold, Incidence, mechanism and prognostic value of activated AKT in pancreas cancer, Br. J. Cancer 89 (2003) 2110–2115. [22] H.G. Wendel, E. De Stanchina, J.S. Fridman, A. Malina, S. Ray, S. Kogan, C. Cordon-Cardo, J. Pelletier, S.W. Lowe, Survival signalling by Akt and eIF4E in oncogenesis and cancer therapy, Nature 428 (2004) 332–337. [23] M. Gehrmann, M. Schmidt, J.C. Brase, P. Roos, J.G. Hengstler, Prediction of paclitaxel resistance in breast cancer: is CYP1B1⁄3 a new factor of influence?, Pharmacogenomics 9 (2008) 969–974 [24] L. Smith, M.B. Watson, S.L. O’Kane, P.J. Drew, M.J. Lind, L. Cawkwell, The analysis of doxorubicin resistance in human breast cancer cells using antibody microarrays, Mol. Cancer Ther. 5 (2006) 2115–2120. [25] S. Mori-Iwamoto, Y. Kuramitsu, S. Ryozawa, K. Taba, M. Fujimoto, K. Okita, K. Nakamura, I. Sakaida, A proteomic profiling of gemcitabine resistance in pancreatic cancer cell lines, Mol. Med. Rep. 1 (2008) 429–434. [26] I.M. Ghobrial, T.E. Witzig, A.A. Adjei, Targeting apoptosis pathways in cancer therapy, CA Cancer J. Clin. 55 (2005) 178–194. [27] K. Horikawa, H. Nakakuma, T. Kawaguchi, N. Iwamoto, S. Nagakura, T. Kagimoto, K. Takatsuki, Apoptosis resistance of blood cells from patients with paroxysmal nocturnal hemoglobinuria, aplastic anemia, and myelodysplastic syndrome, Blood 90 (1997) 2716–2722. [28] V. Petronilli, D. Penzo, L. Scorrano, P. Bernardi, F. Di Lisa, The mitochondrial permeability transition, release of cytochrome c and cell death. Correlation with the duration of pore openings in situ, J. Biol. Chem. 276 (2001) 12030– 12034. [29] S.H. Kaufmann, M.O. Hengartner, Programmed cell death: alive and well in the new millennium, Trends Cell Biol. 11 (2001) 526–534. [30] D.C. Altieri, Survivin and IAP proteins in cell-death mechanisms, Biochem. J. 430 (2010) 199–205. [31] F. Tsuruta, N. Masuyama, Y. Gotoh, The phosphatidylinositol 3-kinase (PI3K)Akt pathway suppresses Bax translocation to mitochondria, J. Biol. Chem. 277 (2002) 14040–14047. [32] J. Folkman, Angiogenesis, Annu. Rev. Med. 57 (2006) 1–18. [33] S.M. Weis, D.A. Cheresh, Tumor angiogenesis: molecular pathways and therapeutic targets, Nat. Med. 17 (2011) 1359–1370. [34] G.L. Semenza, Targeting HIF-1 for cancer therapy, Nat. Rev. Cancer 3 (2003) 721–732. [35] A.M. Arsham, D.R. Plas, C.B. Thompson, M.C. Simon, Akt and hypoxia-inducible factor-1 independently enhance tumor growth and angiogenesis, Cancer Res. 64 (2004) 3500–3507. [36] B.H. Jiang, L.Z. Liu, PI3K/PTEN signaling in tumorigenesis and angiogenesis, Biochim. Biophys. Acta 1784 (2008) 150–158.
Contents lists available at ScienceDirect
Cancer Letters journal homepage: www.elsevier.com/locate/canlet
Anticancer activity of HS-527, a novel inhibitor targeting PI3-kinase in human pancreatic cancer cells Ye-Lim Ryu a,1, Kyung Hee Jung a,1, Mi Kwon Son a, Hong Hua Yan a, Soo Jung Kim a, Sanghye Shin b, Sungwoo Hong b,⇑, Soon-Sun Hong a,⇑ a b
Department of Drug Development, College of Medicine, Inha University, 3-ga, Sinheung-dong, Jung-gu, Incheon 400-712, Republic of Korea Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Republic of Korea
a r t i c l e
i n f o
Article history: Received 16 April 2014 Received in revised form 2 July 2014 Accepted 3 July 2014
Keywords: PI3K HS-527 Pancreatic cancer Apoptosis Angiogenesis
a b s t r a c t Pancreatic cancer is known to have low 5-year survival rate and poor response to treatment. In this study, we synthesized HS-527, a new PI3-kinase inhibitor, and investigated not only its anticancer activity, but also its mechanism of action in pancreatic cancer cells. HS-527 had higher specificity for PI3K than other kinases and inhibited PI3K/Akt signaling pathway by down-regulating Akt and P70S6K. And HS-527 inhibited the cell growth and proliferation of the pancreatic cancer in a time- and dose-dependent manner, with greater activity than gemcitabine. Even HS-527 showed lower cytotoxicity than gemcitabine in normal cells. When treated with HS-527, the cancer cells appeared apoptotic, increasing the expression of cleaved PARP, cleaved caspase-3, and Bax. Furthermore, HS-527 showed an anti-angiogenic activity by decreasing the expression of HIF-1a and VEGF, and inhibited the migration of endothelial cells, and the formation of new blood vessel in mouse Matrigel plug assay. In this study, we found that HS-527 showed anti-cancer activity through an inhibition of the PI3K/Akt pathway in pancreatic cancer cells, suggesting that HS-527 could be used as a promising therapeutic agent for pancreatic cancer. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
Introduction Pancreatic cancer is ranked fourth among the causes of cancer that is related to deaths [1]. Over 40,000 people were diagnosed with pancreatic cancer, and 36,800 died as a result of the disease in the United States during the year 2013 [2,3]. Even if it accounts for 5% of all cancer-related deaths, the overall 5-year survival rate is 3–5% due to advanced staging at the time of diagnosis, along with poor response to current treatment [4]. In advanced pancreatic cancer, gemcitabine has been used as the standard drug for the past several decades. However, the benefit of a single agent in advanced pancreatic cancer is minimal [5]. Further, a number of combinations with gemcitabine have been studied to gain more effective results; however, these studies were not satisfactory. Therefore, the development of potential agent for pancreatic cancer is urgently needed. To overcome these limitations of cancer therapy, many recent studies are focusing on therapeutic targets. Among them, one of the most potential targets is phosphotidylinositol-3-kinase ⇑ Corresponding authors. Tel.: +82 42 350 2811; fax: +82 42 350 2810 (S. Hong). Tel.: +82 32 890 3683; fax: +82 32 890 2462 (S.S. Hong). E-mail addresses: [email protected] (S. Hong), [email protected] (S.-S. Hong). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.canlet.2014.07.001 0304-3835/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
(PI3K). PI3-kinases are composed of three classes: class I, class II and class III PI3Ks, according to the structure and function [6]. Class IA PI3K plays the most important role in human cancers because it is activated directly by the cell surface receptors [7]. It consists of p85 regulatory subunit and p110 catalytic subunit that is focused more on curing human cancers [8]. Recently, not only mutations in the catalytic subunit of PI3Ka, but also upregulation of the PI3K/Akt signaling pathway, occur commonly in most cancers [7,9]. It is widely known that the activated PI3K phosphorylates Akt and P70S6K, and is involved in numerous biological functions, such as cell proliferation, angiogenesis, differentiation, and survival [10]. Also, it is greatly associated with the treatment limitation of pancreatic cancer. Even though some studies have reported that there are no mutations in PIK3CA or AKT1, activated Akt is regarded as an important molecule for the survival of pancreatic cancer [11,12]. Based on a number of previous reports, we synthesized a potent novel PI3K inhibitor HS-527, 2-amino-N,N-dimethyl-5-(3(2-methylpyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)pyridine-3sulfonamide). HS-527 is especially targeted to a p110 catalytic subunit of PI3Ka. In this study, we studied the effect of HS-527 on anti-cancer activity through the PI3K/Akt signaling pathway in terms of cell growth and proliferation, apoptosis, and angiogenesis in pancreatic cancer.
Y.-L. Ryu et al. / Cancer Letters 353 (2014) 68–77 Material and methods Cells and materials Human pancreatic cancer cells PANC-1, MIA PaCa-2 and AsPC-1 were purchased from the American Type Culture Collection (AATC, Manassas, VA), and normal human fibroblast cells Hs677 were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea). PANC-1 and MIA PaCa-2 were cultured in Dulbecco’s modified Eagle’s medium (DMEM), and AsPC-1 and Hs677 were cultured in Roswell Park Memorial Institute Media 1640 (RPMI-1640). These cells were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. FBS and all other agents used in cell culture studies were purchased from Invitrogen (Carlsbad, CA). Human umbilical vein endothelial cells (HUVECs) were purchased from PromoCell (Heidelberg, GM). HUVECs were cultured in a 2% gelatin coated 75 cm2 flask in Endothelial Cell Growth Medium 2 with supplement mixture. The cultures were maintained at 37 °C in a CO2 incubator with a humidified atmosphere composed of 5% CO2 and 95% air. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and proteinase K were purchased from Sigma–Aldrich (St. Louis, MO). HS527 was dissolved in dimethyl sulfoxide (DMSO) as a stock at concentration of 100 mM, and was stored at 20 °C. Synthesis and binding assay of HS-527 A 5-bromo-7-azaindole in acetone was added N-Iodosuccinimide, and the mixture was stirred at room temperature for 3 h under N2 condition. The resulting precipitate was filtrated and washed with cold acetone to give 5-bromo-3-iodo1H-pyrrole[2,3-b]pyridine. An obtained intermediates in CH2Cl2 were added benzenesulfonyl chloride, Bu4NHSO4, and 50% aqueous NaOH. After stirring the mixture at room temperature for 6 h, aqueous work-up was carried out using CH2Cl2. The organic extracts were dried over MgSO4, which were then filtered and concentrated. It was washed with cold MeOH and the precipitate was collected by filtration to afford the N-protected intermediate as a white solid. The N-protected intermediates in 1,4-dioxane:H2O (3:1) were added 3-methyl-4-pyridinylboronic acid, Cs2CO3, and PdCl2(dppf). It was heated to 80 °C for 3 h. After cooling down the resulting mixture to room temperature, it was treated with 4 N KOH for deprotection of the benzene sulfonyl group. The mixture was heated to 80 °C for 1 h again, which was then cooled to room temperature. The resulting solution was neutralized with 4 N HCl, extracted with CH2Cl2, and washed with brine. The organic layer was dried over MgSO4 and concentrated in vacuo. The residue was purified with flash column chromatography to give a deprotected intermediate. The deprotected intermediate, boronic ester, K2CO3 and PdCl2(dppf) in 1,4-dioxane:H2O (3:1) were heated to 100 °C for 100 min using microwave. After cooling down the resulting mixture to room temperature, it was concentrated in vacuo. The residue was diluted with CH2Cl2 and MeOH, and then filtered through a short plug of silica and Celite. The filtrate was concentrated and purified with flash column chromatography to obtain HS-527 as a white solid (Fig. 1A). And, in the binding assay, POC and Kd of HS-527 were determined at the Ambit Bioscience Corp. (San Diego, CA, USA). HS-527 was profiled at 1 lM against a panel of 20 representative kinases in a high-throughput binding assay (Ambit Bioscience). Measurement of cell proliferation Cell viability was measured by the MTT assay. Pancreatic cancer cells PANC-1, MIA PaCa-2, and AsPC-1 were plated at a density of 3–10 103 cells/well in 96-well plates, and then incubated for 24 h. Then, the media was removed and the cells were treated with either DMSO, as a negative control, or various concentrations of HS-527 and gemcitabine. The final concentration of DMSO in the media was 0.1% (v/v). After incubating the cells for 24, 48, and 72 h, a 10% of MTT solution (2 mg/mL) was added to each well, and the cells were incubated for another 4 h at 37 °C. After incubation for 4 h, 100 lL DMSO was added to each well to dissolve the formed formazan crystals with constant shaking for 5 min. The plate was read at 540 nm using a microplate reader. Each analysis was measured from three replicate wells.
69
substrate was added, and the plate was incubated for 10 min. Next, 1 mM H2SO4 was added to terminate the reaction. Each well was measured using a microplate reader at 450 nm. Four replicate wells were used for each analysis.
Immunodetection of incorporation of 50 -bromo-20 -deoxyuridine (BrdU) PANC-1 and MIA PaCa-2 were plated onto 18-mm cover glass in DMEM and grown to approximately 70% confluence for 24 h. The cells were then treated with 5 lM of HS-527 for 3 h, and then labeled for 4 h with 10 lM 50 -bromo-20 -deoxyuridine (BrdU). After labeling, the cells were fixed in an acetic acid: ethanol solution (3:1) for 10 min at 20 °C and washed with 1% Triton X-100 in PBS (pH 7.4). Then, the cells were incubated in HCl (4 N) for 15 min at 37 °C incubator to disrupt the DNA structure in the BrdU-labeled cells. After denaturation, borate buffer (0.1 M) was added for neutralization. The cells were blocked with 5% goat and horse serums for 30 min at room temperature. And then the cells were incubated with anti-BrdU antibody (Abcam, Cambridge, MA) for overnight in a humidified chamber. After washing with PBS, FITC-labeled anti-mouse secondary antibody was diluted with PBS at room temperature in the dark for 1 h. The nuclei were stained with 4,6diamidino-2-phenylindole (DAPI) in the dark for 30 min at room temperature. The slides were washed with PBS, and then covered with DABCO (Sigma–Aldrich). Confocal laser scanning microscopy was performed.
Western blot PANC-1 and MIA PaCa-2 cells were washed with ice-cold PBS before lysis using a buffer containing 1% Nonidet P-40, 1% Triton X-100, and the following protease and phosphatase inhibitors: aprotinin (10 mg/mL), leupeptin (10 mg/mL) (ICN Biomedicals, Asse-Relegem, Belgium), phenylmethylsulfonyl fluoride (1.72 mM), NaVO3 (500 mM), NaF (100 mM), and Na4P2O7 (500 mg/mL) (Sigma–Aldrich). Equal amounts of protein were separated using 8–15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). In addition, the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes. The protein transfer was checked by Ponceau S solution staining (Sigma–Aldrich). Immunostaining of the blots was performed by the primary antibodies. After washing three times, the blots were incubated with the secondary antibody conjugated to horseradish peroxidase (HRP) and detection by enhanced chemiluminescence reagent (Amersham Biosciences, Piscataway, NJ). Primary antibodies were purchased as follows: Bax, HIF1a (Santa Cruz Biotechnology, Santa Cruz, CA), b-actin (Abcam, Cambridge, UK), Akt, p-Akt (Thr308), P70S6K, p-P70S6K (Thr389), cleaved caspase-3, Mcl-1, survivin, and cleaved PARP (Cell Signaling Technology, Beverly, MA).
Terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay A TUNEL assay was performed using a commercially available kit (Chemicon, Temecula, CA), following the manufacturer’s protocol. The cells were plated on an 18-mm cover glass in DMEM, at a density of 70% confluence, and incubated for 24 h. The cells were then treated with 5 lM HS-527 for 24 h before being fixed in an ice-cold mixture of acetic acid and ethanol. After the cells were washed with PBS, they were stained for TUNEL. The stained cells were examined under a light microscope for nuclear fragmentation.
Measurement of mitochondrial membrane potential (JC-1) Pancreatic cancer cells were plated onto 18-mm cover glass in the media and incubated for 24 h in order to reach approximately 70% confluence. The cells were incubated in the presence or absence of 5 lM HS-527 treatment for 6 h and then incubated with 5 lM JC-1 fluorescence dye (JC-1 Mitochondrial Membrane Potential Assay Kit Cayman Chemical Company, Ann Arbor, MI) for 20 min in a CO2 incubator. The slides were washed with PBS and covered with DABCO (Sigma–Aldrich) before being viewed with a confocal laser scanning microscope (Olympus, Tokyo, Japan).
Measurement of 50 -bromo-20 -deoxyuridine (BrdU) cell proliferation assay Enzyme-linked immunosorbent assay (ELISA) PANC-1 cells were plated at a density of 8 103 cells/well in 96-well plates, and then incubated for 24 h. Further, MIA PaCa-2 cells were plated at a density of 6 103 cells/well in 96-well plates, which were then incubated for 24 h. The pancreatic cancer cells were then treated with various concentrations of HS-527 at 37 °C for 2 h. After incubation, DNA content synthesis was assessed by 50 -bromo20 -deoxyuridine (BrdU) cell proliferation assay kit according to the manufacturer’s instructions (Merck, Darmstadt, Germany). Briefly, cells in each well were labeled by the addition of BrdU (5 lM) for 2 h at 37 °C. After removing the labeling media, 100 lL per well fixing/denaturing solution was added to each well and incubated for 30 min at room temperature. After removing fixing/denaturing solution, 100 lL per well of detection antibody solution was added, and the microplate was incubated for 1 h at room temperature. After washing, horseradish peroxidase (HRP)-conjugate solution was added, and the plate was incubated for 30 min. TMB
The amount of VEGF secreted into the media was measured by a sandwich ELISA. The plates were coated with 100 lL of 2 lg/mL anti-VEGF antibody (R&D Systems, Minneapolis, MN) diluted in PBS for 24 h at room temperature. The plates were washed three times with PBS containing 0.1% Tween-20 and incubated with 100 lL/well of 1% bovine serum albumin (BSA, Sigma–Aldrich) in PBS for 1 h at room temperature. The conditioned medium and various concentrations of recombinant human VEGF were incubated overnight at 4 °C with 100 lL of 50 ng/mL biotinylated anti-VEGF antibody. The plates were then washed and incubated for 30 min with 100 lL of HRP-conjugated streptavidin (Vector Laboratories, Burlingame, CA). After washing, the reaction was stopped by adding 50 lL of 2 N H2SO4. The absorbance was measured at 450 nm using a microplate reader. Three replicate wells were used for each analysis.
70
Y.-L. Ryu et al. / Cancer Letters 353 (2014) 68–77
Fig. 1. Characteristics of HS-527. (A) 2-amino-N,N-dimethyl-5-(3-(2-methylpyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)pyridine-3-sulfonamide. (B) Binding mode of HS527 in the ATP-binding site of PI3Ka. (C) Binding affinity of HS-527 was measured by KINOMEscan. A panel of 20 kinases was tested at 1 lM of HS-527. Lower percent of control (POC) values represent stronger hits. Values shown are the mean of duplicate measurements.
Immunofluorescence microscopy PANC-1 and MIA PaCa-2 cells were plated on 18-mm cover glasses in DMEM at a density of 70% confluence and incubated for 24 h. Next, the cells were incubated in the presence or the absence of 5 lM HS-527 for 4 h. The cells were washed with PBS and fixed in an acetic acid: ethanol (2:1) solution for 5 min at 20 °C. Nonspecific binding was blocked with 5% goat and horse serum/PBS for 1 h at room temperature, and the cells were then incubated with primary antibodies against p-Akt (Thr308) (Abcam, Cambridge, UK), p-Stat3 (Tyr705), and p-Mek (Ser221) (Cell Signaling Technology, Beverly, MA) in a humidified chamber. After washing twice in PBS, the cells were incubated with fluorescein-labeled secondary antibody (1:50; Dianova) in an antibody dilution solution for 1 h at room temperature in the dark. The nuclei were stained with DAPI in the dark for 30 min at room temperature. The slides were washed twice with PBS, covered with DABCO (Sigma–Aldrich), and examined with confocal laser scanning microscopy (Olympus, Tokyo, Japan).
HS-527 (0.1–10 lM), and/or VEGF (100 ng/mL). HUVECs were cultured for 20 h to migrate in a CO2 incubator. The cells were washed with a serum-free medium and photographed under a phase-contrast microscope at 200 magnification.
Matrigel plug assay BALB/c mice were obtained from Orient-Bio Laboratory Animal Research Center Co., Ltd. (Gyeonggi-do, Kapyung, Korea). The animals were 6 weeks old and kept in a temperature and humidity controlled environment, with 12 h light–dark cycles. They were provided with standard rat chow and free access to tap water. After stabilization, the mice were subcutaneously injected with 800 lL of Matrigel mixed with VEGF (100 ng/mL) and either PBS or HS-527 (5 lM). After 7 days, the mice were sacrificed and the Matrigel plugs were isolated and fixed in 10% buffered formaldehyde, embedded in paraffin, and sectioned.
Histopathological staining Wound migration assay HUVECs were seeded on 60 mm2 culture dishes and cultured until 90% confluence. HUVECs were wounded with a razor blade. This step made the injury line that HUVECs were marked. After wounding, HUVECs were washed with PBS and further incubated in Endothelial Cell Growth Medium 2 with 2% FBS, 20 unit/mL heparin (Sigma–Aldrich), 1 mM thymidine (Sigma–Aldrich), and various concentrations of
The Matrigel plug samples were fixed in 10% buffered formaldehyde, embedded in paraffin, and then sectioned. The 8 lm-thick sections were stained with hematoxylin and eosin (H&E) for histology observation. On the other slides, ten micrometer thick frozen sections were incubated overnight with anti-CD34 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 4 °C after the hydration process. After washing three times with PBS, the sections were performed with anti rabbit Texas red
Y.-L. Ryu et al. / Cancer Letters 353 (2014) 68–77 labeled secondary antibodies. Then, DAPI was used to stain the nucleus. The slides were washed twice with PBS and covered with DABCO (Sigma–Aldrich), and examined with confocal laser scanning microscopy.
Statistical analysis Data were expressed as the mean ± SD. Statistical analysis was performed using ANOVA. A P-value of 0.05 or less was considered statistically significant. Statistical calculations were performed using SPSS software for Windows operating system (Version 10.0; SPSS, Chicago, IL).
Results HS-527 binds to ATP binding site of PI3Ka with strong binding affinity We synthesized HS-527, a novel PI3K inhibitor, which targeted the ATP binding site against PI3K (Fig. 1A). The binding mode in the ATP-binding site of PI3Ka was investigated in a comparative fashion in order to obtain some structural insight into the inhibitory mechanisms of the identified HS-527. Fig. 1B shows the lowestenergy conformation of HS-527 in the PI3Ka ATP binding site, calculated using the modified AutoDock program. On the basis of the docking model results, hydrogen bonding group on the 7-azaindole moiety appeared to point toward the hinge region (Val851), while the pyridyl sulfonamide group is lined by Tyr836 and Asp810. 7azaindole contains both H-donor and H-acceptor for interaction with the hinge region of the kinase. The NH group of 7-azaindole donates a hydrogen bond to the backbone oxygen of Val851. In addition, the nitrogen at the 7-position of the azaindole scaffold appeared to form a hydrogen bond with the backbone nitrogen
71
of Val851. Furthermore, HS-527 could be stabilized further in the PI3Ka ATP binding site via additional hydrogen bonds of the 5position pyridyl sulfonamide in azaindole. The pyridyl group was anchored by a hydrogen bond to Tyr836. The anilic group of C5 aryl ring donated hydrogen bonds to the backbone amino carbonyl oxygen of Asp810. In addition, the sulfonamide binds to the catalytic lysine (Lys802). These multiple hydrogen bonds appeared to be the most significant binding force stabilizing HS-527 in the active site of PI3Ka. To obtain more detailed characteristics of HS-527, we performed a high-throughput binding with a panel of 20 cancer-related protein kinases by KINOMEscan. As shown in Fig. 1C, HS-527 showed the strongest binding affinity to PI3KCA at a concentration of 1 lM, with modest binding affinity to other cancer-related protein kinases. Therefore, the result of a KINOMEscan shows that HS-527 is specific enough to PI3KCA.
HS-527 inhibits PI3K/Akt pathway In order to investigate which pathway was especially regulated by HS-527, we performed the immunofluorescence and Western blot assay in MIA PaCa-2 cells. In Fig. 2A, HS-527 significantly inhibited the p-Akt expression, which is the downstream signal of the PI3K pathway. On the other hand, both p-Stat3 and p-Mek were not inhibited by HS-527. To identify the anti-cancer effects of HS-527 through the PI3K pathway, we determined the phosphorylation of Akt and P70S6K, downstream of PI3K pathway, in pancreatic cancer cells using a Western blot. In Fig. 2B, HS-527
Fig. 2. Effect of HS-527 on the PI3K/Akt signaling pathway in pancreatic cancer cells. (A) The phosphorylation levels of Akt (Thr308), Stat3 (Tyr705), and Mek (Ser221) were detected by confocal microscopy and Western blot. MIA PaCa-2 cells were treated with 5 lM HS-527 for staining or for Western blot 1 or 5 lM HS-527 for 4 h. (B) PANC-1 and MIA PaCa-2 cells were treated with HS-527 (0–10 lM) for 24 h. The expression of p-Akt (Thr308), Akt, p-P70S6K (Thr389) and P70S6K were performed by Western blot.
72
Y.-L. Ryu et al. / Cancer Letters 353 (2014) 68–77
inhibited both the expression of p-Akt and p-P70S6K in a dosedependent manner.
HS-527 inhibits cell growth of human pancreatic cancer cells with low toxicity in normal fibroblast cells To examine the effect of HS-527 on the viability of pancreatic cancer cells, the cells were treated with various concentrations (0–20 lM) of HS-527 for 24, 48, and 72 h. As shown in Fig. 3A, the growth of pancreatic cancer cells treated with HS-527 was inhibited in a dose- and time-dependent manner. Furthermore, to compare the effect of HS-527 with gemcitabine, a conventional drug for pancreatic cancer, we treated various concentrations of the drugs to the cells. HS-527 was more effective than gemcitabine in the MIA PaCa-2 cells given the 24 h treatment (Fig. 3B). Next, to observe the cytotoxicity of HS-527 in the normal cells, we performed MTT assay in a human normal fibroblast cell line, Hs677. The cells were exposed to various concentrations of HS-527 and
gemcitabine for 24 h. HS-527 represented a cell viability of approximately 90% compared to the control. On the other hand, the gemcitabine revealed 70% cell viability compared to the control (Fig. 3C). Therefore, the cytotoxicity of HS-527 seemed to be less toxic to the normal cells compared with gemcitabine in all tested concentrations.
HS-527 inhibits the proliferation of pancreatic cancer cells To investigate the effect of HS-527 on pancreatic cancer cell proliferation, we performed the BrdU proliferation assay and BrdU staining. In the BrdU proliferation assay, pancreatic cancer cells were treated with various concentrations (0–50 lM) of HS-527 for 2 h. As shown in Fig. 3D, the BrdU incorporation was inhibited by HS-527 in a dose-dependent manner. Also, the result showed that HS-527 significantly decreased the number of BrdU positive cells in a dose-dependent manner (Fig. 3E). These results revealed that HS-527 inhibited the proliferation of pancreatic cancer cells.
Fig. 3. Effect of HS-527 on the viability and proliferation of pancreatic cancer cells. (A) The growth inhibition by HS-527 in pancreatic cancer cells (PANC-1, MIA PaCa-2 and AsPc-1) for 24 h, 48 h and 72 h. Viability of (B) MIA PaCa-2 cells or (C) Hs677 cells treated with various concentrations (0–50 lM) of HS-527 or gemcitabine was measured at 24 h. (D) The effect of HS-527 (0.01–50 lM) on the proliferation of pancreatic cancer cells was performed by a BrdU proliferation assay for 2 h. (E) PANC-1 and MIA PaCa-2 cells were treated with 0.1–5 lM of HS-527 for 3 h. After BrdU staining, the cells were detected by confocal microscopy. The representative confocal image showed that HS527 (5 lM) effectively inhibited proliferation of pancreatic cancer cells. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to the control. Data represent as mean ± SD from the (A– C) three or (D) four replicate wells.
Y.-L. Ryu et al. / Cancer Letters 353 (2014) 68–77
73
Fig. 3 (continued)
HS-527 induces apoptotic cell death in pancreatic cancer cells In order to examine the apoptotic effect of HS-527, we performed TUNEL assay, JC-1 assay, and Western blot. When treated with 5 lM HS-527, the cells showed morphological features, such as DNA fragmentation and loss of mitochondrial transmembrane potential (wm), which are characteristics of apoptotic cells. In Fig. 4A, the treatment of HS-527 increased the number of TUNEL positive cells in a dose dependent manner. We also investigated the change of mitochondria potential using a JC-1 assay kit. As shown in Fig. 4B, the control cells showed both the red and green fluorescence in the cytoplasm. Treatment with 5 lM of HS-527 decreased the red fluorescence intensity, whereas it increased the green fluorescence intensity. To confirm these results, we examined the protein expression related to apoptosis by a Western blot in the pancreatic cancer cells. HS-527 increased the pro-apoptotic protein expression of cleaved PARP, cleaved caspase-3, and Bax. In contrast, HS-527 decreased the anti-apoptotic protein expression, such as Mcl-1 and survivin (Fig. 4C). These results implied that HS-527 promoted apoptosis in pancreatic cancer cells. HS-527 inhibits the angiogenesis in pancreatic cancer and HUVECs Next, to investigate the effect of HS-527 on the expression of HIF-1a and VEGF under hypoxic condition, MIA PaCa-2 cells were treated with 100 lM CoCl2 and HS-527 (0–10 lM) for 24 h. First, we measured the expression of HIF-1a by a Western blot, as well as VEGF by ELISA. As shown in Fig. 5A and B, the expression of both HIF-1a and VEGF was increased in hypoxic condition by a CoCl2 treatment. However, the expression of HIF-1a and VEGF was
decreased by a treatment of HS-527, in a dose-dependent manner. To confirm these data, we performed immunofluorescence analyses with the red fluorescence intensity for HIF-1a expression and the green for VEGF expression. In Fig. 5C, HIF-1a signal appeared in the nucleus, whereas VEGF was stained in the cytoplasm. In contrast to the control, the cells treated with HS-527 significantly showed a low expression of HIF-1a and VEGF. HS-527 decreased the expression of HIF-1a and VEGF in the MIA PaCa-2 cells in a hypoxic condition. Migration of endothelial cells is one of the essential steps during angiogenesis [13]. To investigate the movement of endothelial cells, HUVECs were wounded by raze blade and incubated with various concentrations of HS-527 (0–10 lM) for 20 h. The movements of HUVECs were significantly blocked by HS-527 treatment in a dose-dependent manner (Fig. 5D). HS-527 was considered to have an anti-angiogenic activity in both the pancreatic cancer cells and endothelial cells. HS-527 inhibits the formation of blood vessel in vivo As shown in Fig. 6, we confirmed that HS-527 significantly inhibited the angiogenesis in vitro. Further, we speculated that HS-527 has the ability of anti-angiogenesis in vivo, as well. We subcutaneously injected Matrigel mixed with VEGF (100 ng/mL) or HS-527 (5 lM) into the BALB/c mice. After 7 days, we removed the Matrigel plug from the BALB/c mice. The plug injected with VEGF alone became red because the blood vessels and red blood cells were induced by VEGF. On the contrary, when the Matrigel was mixed with VEGF and HS-527, the plug slightly changed to red compared to the treatment with VEGF alone. In the H&E
74
Y.-L. Ryu et al. / Cancer Letters 353 (2014) 68–77
Fig. 4. Effect of HS-527 on apoptosis of pancreatic cancer cells. (A) The induction of apoptosis by HS-527 treatment was evaluated by TUNEL staining. Pancreatic cancer cells were treated with 1 and 5 lM HS-527 for 24 h. Characteristics of apoptosis was shown with dark brown staining. (B) JC-1 staining was performed to detect mitochondrial transmembrane potential during apoptosis. HS-527 (5 lM) was treated to pancreatic cancer cells for 6 h. (C) The expression of PARP, cleaved caspase-3, Bax, survivin and Mcl1 were assayed by Western blot analysis after PANC-1 and MIA PaCa-2 cells were treated with HS-527 at the indicated doses (0–10 lM) for 24 h. **p < 0.01 and ***p < 0.001 compared to the control.
staining, a number of red blood cells were recruited in the section of the VEGF plug, while HS-527 efficiently inhibited the recruit of red blood cells into the plug (Fig. 6A). In addition, we stained CD34, an angiogenesis marker, in the section of the plug using immunofluorescence. Like the previous results, HS-527 significantly decreased the expression of CD34, which stained to a red color.
Discussion To date, it has been reported that the PI3K pathway plays important roles in numerous human cancers, including pancreatic cancer [14–16]. As the overexpression of PI3K/p110a plays an important role in pancreatic cancer, targeting the PI3K/Akt pathway was considered as the therapeutic strategy [17]. Recently, Li
Y.-L. Ryu et al. / Cancer Letters 353 (2014) 68–77
75
Fig. 5. Effect of HS-527 on angiogenesis of pancreatic cancer cells. (A) MIA PaCa-2 cells were treated with 100 lM CoCl2 and various concentrations (0, 0.1, 1, 5, 10 lM) of HS527 for 24 h. Expression of HIF-1a in hypoxia mimic condition was detected by a Western blot. (B) Production of VEGF in MIA PaCa-2 cells treated with CoCl2 and/or HS-527 was examined using ELISA. Data represented as the mean ± SD from the triplicate wells. (C) The expression of HIF-1a and VEGF by immunofluorescence microscope in MIA PaCa-2 cells treated with HS-527 in hypoxia mimic condition (CoCl2, 100 lM). (D) HUVECs were plated at 60 mm2 dish until 90% confluence. And then the HUVECs were scratched with a razor blade and treated with VEGF (100 ng/mL) and HS-527 as indicated concentrations (0.1–10 lM) for 20 h. **p < 0.01 and ***p < 0.001 compared to the control.
et al. have reported that the inhibition of the PI3K/Akt pathway led to gemcitabine resistance to be overcome in pancreatic cancer [18]. This circumstance prompted us to investigate further into the novel PI3K inhibitors in pancreatic cancer. Considering these facts, we identified a novel PI3K inhibitor, HS-527, and evaluated its anticancer activities on pancreatic cancer. The present study revealed that our novel molecule, HS-527, had a prominent inhibitory effect on the PI3K/Akt signaling pathway in pancreatic cancer cells, which may lead to the inhibition of cell growth and angiogenesis as well as induction of apoptosis. Over-activation of PI3K pathways have been observed in many cancer types. Especially, the PI3K pathway is one of major pathways that can be directly activated by a mutated KRAS of pancreatic cancer, thereby leading to cell proliferation and tumor growth [19]. Also, the PI3K/Akt pathway is a potent pathway to block the apoptotic effects of chemotherapy drugs and radiation therapy in a variety of cancer types including pancreatic cancer [20]. In addition, Schlieman et al. have reported that 59% of pancreatic cancer showed overexpression of Akt, a main mediator of PI3K/
Akt pathway [21]. Furthermore, most of the cancers acquire drug resistance due to the activation of PI3K/Akt pathway [22]. Indeed, drug resistance of paclitaxel and doxorubicin in breast cancer and that of gemcitabine in pancreatic cancer were mediated by hyperactivation of PI3K/Akt pathway [23–25]. For this reason, a lot of researchers have concerned with PI3K/Akt pathway as an attractive molecular target. First, we confirmed the chemical characteristics of HS-527. This compound not only had the highest binding affinity to PI3K (POC = 1.1), but it also bound to the ATP binding site of PI3K with a stable force. Since HS-527 had the highest binding affinity to the ATP binding site of PI3K, we investigated whether HS-527 inhibited the expression of p-Akt, p-Mek, and pStat3 which are key downstream molecules of the PI3K, MAPK, and JAK/Stat3 signaling pathways related to pancreatic cancer therapy [11]. HS-527 dramatically inhibited the p-Akt expression in the pancreatic cancer cells, whereas the expression of p-MEK and p-STAT3 were scarcely altered. To understand the mechanisms of anticancer activity of HS-527, we evaluated the change of the downstream signals of PI3K/Akt pathway. HS-527 blocked the
76
Y.-L. Ryu et al. / Cancer Letters 353 (2014) 68–77
Fig. 6. Effect of HS-527 on the formation of blood vessel in vivo. (A) Matrigel plugs mixed with VEGF (100 ng/mL) or HS-527 (5 lM) were injected into Balb/c mice, and sacrificed after 7 days. Photographs were taken of the plugs and a section of the plug was stained with H&E. (B) For staining the recruited new vessels, CD34 were stained by red color at 200 magnification.
phosphorylation of Akt and P70S6K, the downstream of the PI3K/ Akt pathway, in a dose-dependent manner. In order to understand the anti-cancer effects of HS-527, we investigated the cell viability and proliferation in three pancreatic cancer cell lines. In our results, HS-527 strongly suppressed the cell growth and DNA synthesis of the three pancreatic cancer cells in a dose-, time-dependent manner. Also, we observed that HS-527 strongly inhibited PI3K activity with an IC50 of <10 lM in MIA PaCa-2 cells and was much more potent than gemcitabine. In addition, HS-527 has a lower toxicity compared to gemcitabine in the normal cells. Based on the above results, we propose that HS-527 inhibited the cell growth/proliferation by the highest binding affinity to PI3K in the pancreatic cancer cells, while having low toxicity to normal cells. Along with the inhibition of cell proliferation, induction of apoptosis is a major point in cancer therapy. Especially, mitochondrion 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 [26,27]. Change in the mitochondrial membrane potential induces to create pores of mitochondria, which leads to the release of cytochrome c into the cytoplasm, resulting in expression of various pro-apoptotic proteins in cytoplasm [28]. In this process, Bax, as pro-apoptotic molecule, actively induce
cytochrome c release from mitochondria within cells. Thus, Bax overexpression induces apoptosis induction in response to various apoptosis stimuli [29]. Also, XIAP, Mcl-1, and survivin, as antiapoptotic protein family, have been reported to inhibit apoptosis signaling by binding to caspases [30]. In addition, Tsuruta et al. have reported that PI3K/Akt pathway suppressed Bax translocation to mitochondria, which promoted cell survival and inhibited apoptosis [31]. Thus, to identify involvement of the HS-527 in mitochondrial membrane potential, we conducted JC-1 staining. As a result, HS-527 induced marked changes in mitochondrial membrane potential (wm). Also, HS-527 decreased the expression of Mcl-1 and survivin, whereas increased the expression of Bax. These changes led to increase the expression of cleaved caspase-3 and cleavage of PARP. Overall, our study showed that HS-527 strongly induced mitochondria-mediated apoptosis in the pancreatic cancer cells. Angiogenesis is a necessary process for the growth of solid tumors, including pancreatic cancer. As the tumor grows, hypoxia condition emerges inside the tumor, and imbalance of key regulators begins between HIF-1a and VEGF, which are key mediators in angiogenesis [32–34]. In angiogenesis of cancer, PI3K/Akt pathway is activated by various stimuli including VEGF in endothelial cells and plays important role for regulating HIF-1a and VEGF expressions [35]. In addition, angiogenesis occurs in multi-steps,
Y.-L. Ryu et al. / Cancer Letters 353 (2014) 68–77
including migration and proliferation of the endothelial cells, as well as the formation of a new blood vessel [36]. In this study, we observed that HS-527 efficiently suppressed the expression of HIF-1a and VEGF in a hypoxia condition of the pancreatic cancer cells. Also, HS-527 strongly inhibited VEGF-induced migration of HUVECs in a dose-dependent manner. Much like these in vitro results, HS-527 prominently inhibited the formation of new blood vessel and the recruitment of endothelial cells into the plug by VEGF induction, using the in vivo Matrigel plug assay. Moreover, it decreased the expression of CD34 well. These results indicated that HS-527 significantly suppresses VEGF induced-angiogenesis both in vitro and in vivo. In conclusion, the present study demonstrates that a novel PI3K inhibitor, HS-527, shows remarkable potent anti-cancer activity by inhibiting cell growth/proliferation and angiogenesis, along with inducing apoptosis. The anticancer effect of HS-527 appears to be associated with the inhibition of the PI3K/Akt pathway. Therefore, we suggest that HS-527 can be considered as a potential therapeutic agent targeting the PI3K/Akt pathway in pancreatic cancer. Conflict of Interest None. Role of the Funding Source None declared. Acknowledgments This research was supported by the National Research Foundation (NRF) Grant (2012R1A2A2A010) and Medical Research Center (2014009392) funded by MSIP, Korea. References [1] A. Jemal, T. Murray, E. Ward, A. Samuels, R.C. Tiwari, A. Ghafoor, E.J. Feuer, M.J. Thun, Cancer statistics, CA Cancer J. Clin. 55 (2005) 10–30. [2] R. Siegel, D. Naishadham, A. Jemal, Cancer statistics, CA Cancer J. Clin. 63 (2013) 11–30. [3] H. Zhong, C. Sanchez, D. Spitrzer, S. Plambeck-Suess, J. Gibbs, W.G. Hawkins, D. Denardo, F. Gao, R.A. Pufahl, A.C. Lockhart, et al., Synergistic effects of concurrent blockade of PI3K and MEK pathways in pancreatic cancer preclinical models, PLoS ONE 8 (2013) e77243. [4] Z. Wang, Y. Li, A. Ahmad, S. Banerjee, A.S. Azmi, D. Kong, F.H. Sarkar, Pancreatic cancer: understanding and overcoming chemoresistance, Nat. Rev. Gastroen. Hepatol. 8 (2011) 27–33. [5] H.A. Burris 3rd, M.J. Moore, J. Andersen, M.R. Green, M.L. Rothenberg, M.R. Modiano, M.C. Cripps, R.K. Portenoy, A.M. Storniolo, P. Tarassoff, et al., Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial, J. Clin. Oncol. 15 (1997) 2403–2413. [6] A. Akinleye, P. Avvaru, M. Furqan, Y. Song, D. Liu, Phosphatidylinositol 3-kinase (PI3K) inhibitors as cancer therapeutics, J. Hematol. Oncol. 6 (2013) 88. [7] T.L. Yuan, L.C. Cantley, PI3K pathway alterations in cancer: variations on a theme, Oncogene 27 (2008) 5497–5510. [8] B. Geering, P.R. Cutillas, B. Vanhaesebroeck, Regulation of class IA PI3Ks: is there a role for monomeric PI3K subunits?, Biochem Soc. Trans. 35 (2007) 199– 203. [9] J.J. Wallin, K.A. Edgar, J. Guan, M. Berry, W.W. Prior, L. Lee, J.D. Lesnick, C. Lewis, J. Nonomiya, J. Pang, et al., GDC-0980 is a novel class I PI3K/mTOR kinase inhibitor with robust activity in cancer models driven by the PI3K pathway, Mol. Cancer Ther. 10 (2011) 2426–2436.
77
[10] I. Vivanco, C.L. Sawyers, The phosphatidylinositol 3-Kinase AKT pathway in human cancer, Nat. Rev. Cancer 2 (2002) 489–501. [11] D. Matthaios, P. Zarogoulidis, I. Balgouranidou, E. Chatzaki, S. Kakolyris, Molecular pathogenesis of pancreatic cancer and clinical perspectives, Oncology 81 (2011) 259–272. [12] A. Maitra, R.H. Hruban, Pancreatic cancer, Annu. Rev. Pathol. 3 (2008) 157– 188. [13] I. Zachary, VEGF signalling: integration and multi-tasking in endothelial cell biology, Biochem. Soc. Trans. 31 (2003) 1171–1177. [14] V.M. Bondar, B. Sweeney-Gotsch, M. Andreeff, G.B. Mills, D.J. McConkey, Inhibition of the phosphatidylinositol 30 -kinase-AKT pathway induces apoptosis in pancreatic carcinoma cells in vitro and in vivo, Mol. Cancer Ther. 1 (2002) 989–997. [15] A. Hollestelle, F. Elstrodt, J.H. Nagel, W.W. Kallemeijn, M. Schutte, Phosphatidylinositol-3-OH kinase or RAS pathway mutations in human breast cancer cell lines, Mol. Cancer Res. 5 (2007) 195–201. [16] H. Lee, S.J. Kim, K.H. Jung, M.K. Son, H.H. Yan, S. Hong, S.S. Hong, A novel imidazopyridine PI3K inhibitor with anticancer activity in non-small cell lung cancer cells, Oncol. Rep. 30 (2013) 863–869. [17] C.E. Edling, F. Selvaggi, R. Buus, T. Maffucci, P. Di Sebastiano, H. Friess, P. Innocenti, H.M. Kocher, M. Falasca, Key role of phosphoinositide 3-kinase class IB in pancreatic cancer, Clin. Cancer Res. 16 (2010) 4928–4937. [18] J. Li, X. Liang, X. Yang, Ursolic acid inhibits growth and induces apoptosis in gemcitabine-resistant human pancreatic cancer via the JNK and PI3K/Akt/NFkappaB pathways, Oncol. Rep. 28 (2012) 501–510. [19] R.A. Perugini, T.P. McDade, F.J. Vittimberga Jr., M.P. Callery, Pancreatic cancer cell proliferation is phosphatidylinositol 3-kinase dependent, J. Surg. Res. 90 (2000) 39–44. [20] B.N. Fahy, M. Schlieman, S. Virudachalam, R.J. Bold, AKT inhibition is associated with chemosensitisation in the pancreatic cancer cell line MIAPaCa-2, Br. J. Cancer 89 (2003) 391–397. [21] M.G. Schlieman, B.N. Fahy, R. Ramsamooj, L. Beckett, R.J. Bold, Incidence, mechanism and prognostic value of activated AKT in pancreas cancer, Br. J. Cancer 89 (2003) 2110–2115. [22] H.G. Wendel, E. De Stanchina, J.S. Fridman, A. Malina, S. Ray, S. Kogan, C. Cordon-Cardo, J. Pelletier, S.W. Lowe, Survival signalling by Akt and eIF4E in oncogenesis and cancer therapy, Nature 428 (2004) 332–337. [23] M. Gehrmann, M. Schmidt, J.C. Brase, P. Roos, J.G. Hengstler, Prediction of paclitaxel resistance in breast cancer: is CYP1B1⁄3 a new factor of influence?, Pharmacogenomics 9 (2008) 969–974 [24] L. Smith, M.B. Watson, S.L. O’Kane, P.J. Drew, M.J. Lind, L. Cawkwell, The analysis of doxorubicin resistance in human breast cancer cells using antibody microarrays, Mol. Cancer Ther. 5 (2006) 2115–2120. [25] S. Mori-Iwamoto, Y. Kuramitsu, S. Ryozawa, K. Taba, M. Fujimoto, K. Okita, K. Nakamura, I. Sakaida, A proteomic profiling of gemcitabine resistance in pancreatic cancer cell lines, Mol. Med. Rep. 1 (2008) 429–434. [26] I.M. Ghobrial, T.E. Witzig, A.A. Adjei, Targeting apoptosis pathways in cancer therapy, CA Cancer J. Clin. 55 (2005) 178–194. [27] K. Horikawa, H. Nakakuma, T. Kawaguchi, N. Iwamoto, S. Nagakura, T. Kagimoto, K. Takatsuki, Apoptosis resistance of blood cells from patients with paroxysmal nocturnal hemoglobinuria, aplastic anemia, and myelodysplastic syndrome, Blood 90 (1997) 2716–2722. [28] V. Petronilli, D. Penzo, L. Scorrano, P. Bernardi, F. Di Lisa, The mitochondrial permeability transition, release of cytochrome c and cell death. Correlation with the duration of pore openings in situ, J. Biol. Chem. 276 (2001) 12030– 12034. [29] S.H. Kaufmann, M.O. Hengartner, Programmed cell death: alive and well in the new millennium, Trends Cell Biol. 11 (2001) 526–534. [30] D.C. Altieri, Survivin and IAP proteins in cell-death mechanisms, Biochem. J. 430 (2010) 199–205. [31] F. Tsuruta, N. Masuyama, Y. Gotoh, The phosphatidylinositol 3-kinase (PI3K)Akt pathway suppresses Bax translocation to mitochondria, J. Biol. Chem. 277 (2002) 14040–14047. [32] J. Folkman, Angiogenesis, Annu. Rev. Med. 57 (2006) 1–18. [33] S.M. Weis, D.A. Cheresh, Tumor angiogenesis: molecular pathways and therapeutic targets, Nat. Med. 17 (2011) 1359–1370. [34] G.L. Semenza, Targeting HIF-1 for cancer therapy, Nat. Rev. Cancer 3 (2003) 721–732. [35] A.M. Arsham, D.R. Plas, C.B. Thompson, M.C. Simon, Akt and hypoxia-inducible factor-1 independently enhance tumor growth and angiogenesis, Cancer Res. 64 (2004) 3500–3507. [36] B.H. Jiang, L.Z. Liu, PI3K/PTEN signaling in tumorigenesis and angiogenesis, Biochim. Biophys. Acta 1784 (2008) 150–158.