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FoxM1 axis
Journal of Functional Foods 56 (2019) 102–109
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Anti-pancreatic cancer activity of Z-ajoene from garlic: An inhibitor of the Hedgehog/Gli/FoxM1 axis Hwa Jin Leea,1, Ji Hye Jeongb,1, Jae-Ha Ryub, a b
T
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School of Industrial Bio-Pharmaceutical Science, Semyung University, Jecheon, Chungbuk 27136, Republic of Korea College of Pharmacy and Research Center for Cell Fate Control, Sookmyung Women’s University, Seoul 04310, Republic of Korea
A R T I C LE I N FO
A B S T R A C T
Keywords: Ajoene Garlic Pancreatic cancer Hedgehog pathway Gli FoxM1
Overexpression of glioma-associated oncogene (Gli), a transcription factor at the final step of Hedgehog (Hh) pathway, is involved in pancreatic cancer development. Therefore, inhibition of Gli can be a therapeutic strategy to treat pancreatic cancer. The objective of this study was to determine effects of Z-ajoene from garlic, on Glimediated transcription and proliferation of pancreatic cancer cells. We found that Z-ajoene could inhibit Gli transcriptional activity in Sonic Hedgehog (Shh) stimulated C3H10T1/2 mesenchymal stem cells. Z-Ajoene suppressed Gli transcriptional activity and Gli-target protein expressions in PANC-1 human pancreatic cancer cells. Z-Ajoene also reduced expression of FoxM1, one of Gli-target proteins, and subsequently down-regulated expressions of cell cycle-related proteins. Moreover, Z-ajoene reduced cell proliferation and increased G2/M phase of PANC-1 cells. These results suggest that Z-ajoene can repress pancreatic cancer cell proliferation by inhibiting Gli signaling. Thus, Z-ajoene might be a lead compound for the development of anti-pancreatic cancer agent.
1. Introduction The Hedgehog (Hh) signaling pathway regulates embryonic organogenesis, tissue patterning, and cell differentiation (Armas-Lopez, Zuniga, Arrieta, & Avila-Moreno, 2017; Ingham & McMahon, 2013). Aberrant activation of Hh signaling pathway contributes to tumor formation and development of various cancers including pancreatic cancer (Jiang & Hui, 2008; Pasca di Magliano & Hebrok, 2003). In the presence of Sonic hedgehog (Shh) ligand, the 12-pass transmembrane receptor (Patched, Ptch) can release repressed Smoothened (Smo). This induces nuclear translocation of Gli activator and activation of target gene expressions (Lum & Beachy, 2004; McMahon, Ingham, & Tabin, 2003). In the absence of Hh ligands, Ptch restrains Smo, resulting in nuclear translocation of Gli repressor to switch off Hh target gene expression (Pan, Bai, Joyner, & Wang, 2006). There are three members of Gli transcription factors: Gli1, Gli2, and Gli3 (Hu et al., 2006). Gli1 and Gli2 can activate the expression of target genes related to cell growth and survival (Aberger & Ruiz, 2014; Hui, Slusarski, Platt, Holmgren, & Joyner, 1994; Persson et al., 2002). Gli2 may be an effective
transcription factor in the absence of Gli1 (Bai & Joyner, 2001). However, Gli3 is a transcriptional repressor of Hh signaling (Hu et al., 2006). Direct target genes by Gli-mediated transcription are Hh pathway regulators such as Gli1, Gli2, Ptch (Agren, Kogerman, Kleman, Wessling, & Toftgard, 2004; Hegde et al., 2008; Regl et al., 2002), and cell cycle regulator such as FoxM1 (Teh et al., 2002). Recently, many studies have demonstrated that Hh signaling pathway is constitutively activated in pancreatic cancer. High level of Gli1 as the last stage in Hh signaling pathway is a significant indicator of tumor growth and aggressiveness in pancreatic cancer (Lauth & Toftgard, 2011; Liu et al., 2015; Onishi & Katano, 2014; Thayer et al., 2003). Therefore, interference of Gli might be a good therapeutic strategy to treat cancers accompanying highly activated Hh signaling pathway. Z-Ajoene, an organosulfur compound from garlic (Allium sativum), has antioxidant, antimicrobial, antithrombotic, and anti-inflammatory activities (Choi et al., 2018; Kay et al., 2010; Lee et al., 2012; Srivastava & Tyagi, 1993). Z-Ajoene has been reported to possess anti-proliferative and apoptotic effects on several cancer cells by activating endoplasmic reticulum stress, caspase-3, and ERK/p38 (Hassan, 2004; Jung et al.,
Abbreviations: Hh, Hedgehog; Shh, Sonic Hedgehog; Ptch, 12-pass transmembrane spanning receptor Patched; Smo, 7-pass transmembrane receptor Smoothened; Gli, glioma-associated oncogene; Shh CM, Sonic Hedgehog conditioned medium; FoxM1, Forkhead box protein M1 ⁎ Corresponding author at: College of Pharmacy and Research Center for Cell Fate Control, Sookmyung Women’s University, 100 Chungparo-47-Gil, Yongsan-Gu, Seoul 04310, Republic of Korea. E-mail address: [email protected] (J.-H. Ryu). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.jff.2019.03.010 Received 14 November 2018; Received in revised form 7 March 2019; Accepted 8 March 2019 1756-4646/ © 2019 Published by Elsevier Ltd.
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Fig. 1. Structure of Z-ajoene and effects of Z-ajoene on Sonic Hedgehog signaling pathway in C3H10T1/2-Gli1-luc cells. (A) Structure of Z-ajoene from garlic (Allium sativum). (B) C3H10T1/2-Gli1-luc cells were treated with Sonic Hedgehog conditioned medium (Shh CM) or recombinant human Sonic Hedgehog ligand (rh-Shh, 500 ng/mL) for 30 h. Relative luciferase activity was expressed as relative Gli1-mediated transcriptional activity compared to vehicle CM treated cells. *p < 0.05 indicates a significant difference from vehicle CM. (C) C3H10T1/2-Gli1-luc cells were treated with Shh CM and various concentrations of Z-ajoene or cyclopamine (5 μM) for 30 h. The relative luciferase activity was expressed as the relative Gli1-mediated transcriptional activity compared to vehicle treated cells. *p < 0.05 indicates a significant difference from Shh CM alone. (D) C3H10T1/2-Gli1-luc cells were stimulated with SAG (500 nM) in the presence of Z-ajoene for 30 h. The relative luciferase activity was expressed as the relative Gli1-mediated transcriptional activity compared to vehicle treated cells. *p < 0.05 indicates a significant difference from SAG alone.
construct gifted from Prof. Gyu-Un Bae (Sookmyung Women’s University, Korea) and Prof. Jong-Sun Kang (Sungkyunkwan University, Korea) (Zhang, Kang, Cole, Yi, & Krauss, 2006) was transiently transfected into HEK293 cells. Shh-producing HEK293 cells were then grown to 80% confluency in DMEM containing 10% FBS. The medium was replaced with DMEM containing 2% FBS and cells were grown for 5 days. Conditioned medium (CM) was harvested, filtered through a 0.22 μm membrane, and stored at 4 °C until use.
2014; Kaschula et al., 2016; Kaschula, Hunter, & Parker, 2010). Herein, we report effects of Z-ajoene on Gli-mediated transcription at Hh pathway and proliferation of pancreatic cancer cells. 2. Materials and methods 2.1. Plant materials Z-Ajoene (Fig. 1A) was prepared as previously described (Lee et al., 2012).
2.4. Gli1/Gli-dependent luciferase reporter assay
2.2. Cell lines, chemicals and biochemical
C3H10T1/2 cells were transiently transfected with Gli1, Gli-dependent firefly luciferase, and β-galactosidase expression constructs (C3H10T1/2-Gli1-Luc cells) to assess Gli1-mediated transcriptional activity. Gli1 plasmid and Gli reporter plasmid were gifts from Prof. Gyu-Un Bae (Sookmyung Women’s University, Korea) and Prof. JongSun Kang (Sungkyunkwan University, Korea) (Zhang et al., 2006). Transfected cells were treated with Z-ajoene in the presence of either Shh CM or control medium. C3H10T1/2-Gli1-Luc cells were also induced by SAG, a Smo inhibitor, in the presence of Z-ajoene. After incubation at 37 °C for 30 h, Gli1-dependent firefly luciferase and β-galactosidase activities were measured using a microplate luminometer (Perkin Elmer, Waltham, MA, USA). For assessment of Gli-mediated transcriptional activity in pancreatic cancer cells, plasmids of Gli-dependent firefly luciferase reporter construct and β-galactosidase expression construct were transiently transfected into PANC-1 cells (PANC-1-Gli-Luc cells). These cells were
C3H10T1/2 (mouse mesenchymal stem cell line), PANC-1 and AsPC-1 (human pancreatic cancer cell lines) (ATCC, Manassas, VA, USA) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin (Life Technologies, Carlsbad, CA, USA). SAG (purity: ≥98%), a Smo agonist, was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Cyclopamine (purity: ≥98%, Sigma-Aldrich), a Smo inhibitor in the Hh pathway, was used as a positive control to inhibit the Hh pathway. 2.3. Preparation of sonic hedgehog conditioned media For induction of Hh signaling, we prepared Sonic Hedgehog conditioned medium (Shh CM) as described previously (Chen, Taipale, Young, Maiti, & Beachy, 2002; Lee et al., 2016). Briefly, Shh expression 103
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3. Results
treated with various concentrations of Z-ajoene at 37 °C for 20 h. Cellular firefly luciferase and β-galactosidase activities were then measured using a microplate luminometer. Luciferase activity was normalized against β-galactosidase reporter activity as a control. All luciferase reporter activities were presented as relative luciferase activities (fold).
3.1. Effects of Z-ajoene on Hh/Gli1-mediated transcriptional activity in C3H10T1/2 mouse mesenchymal stem cells To investigate the effect of Z-ajoene on Hh pathway, Shh ligand induced Gli1- transcriptional activity was measured as luciferase activity in C3H10T1/2-Gli1-Luc cells transiently transfected with Gli1, Gli-dependent firefly, and β-galactosidase reporters. C3H10T1/2-Gli1Luc cells were activated with Shh conditioned medium (Shh CM). The potency of Shh CM-induced activation was similar to that of recombinant Shh ligand (500 ng/mL, PeproTech Inc., Rocky Hill, NJ, USA) (Fig. 1B). Luciferase activity was normalized against β-galactosidase reporter activity as a control. As shown in Fig. 1C, Z-ajoene inhibited Shh CM-induced luciferase activity in a concentration-dependent manner. Cyclopamine (5 μM), a Smo inhibitor, was used as a positive control (Lee et al., 2014). We also examined the inhibitory effect of Z-ajoene on Hh pathway by Smo activation. C3H10T1/2-Gli1Luc cells were treated with SAG (500 nM), a synthetic Smo agonist (Chen et al., 2002), to activate Gli1 transcriptional activity. Z-Ajoene suppressed SAG-activated Gli1 activity in a dose-dependent manner (Fig. 1D). From the fact that Z-ajoene modulated the Hh/Gli signaling pathway by suppressing Shh CM and SAG-induced Gli1 transcriptional activity, we postulated that Z-ajoene could inhibit the downstream events of Smo to regulate the Hh pathway. These results led us to investigate the suppressive effect of Z-ajoene on activity of Gli1 as downstream transcription factor of Smo in Hh pathway.
2.5. Reverse transcription-polymerase chain reaction (RT-PCR) analysis PANC-1 cells (2.5 × 105 cells/60 mm dish) were treated with Zajoene for 20 h. Total RNA was isolated from the cell pellet using Trizol reagent (Life Technologies, Paisley, UK). RNA was then reverse transcribed into cDNA using reverse transcriptase and random hexamer. Sense and antisense primers for Gli1 and GAPDH were: 5′-GCCGTGTA AAGCTCCAGTGAACACA-3′ and 5′-TCCCACTTTGAGAGGCCCATAGC AAG-3′; 5′-TGTGATGGGTGTGAACCACGAG-3′ and 5′-TGCTGTTGAAG TCGCAGGAGAC-3′, respectively. Amplified DNA was separated on 1.5% agarose gels and stained with ethidium bromide.
2.6. Western blot analysis Cells were treated with various concentrations of Z-ajoene for 20 h, harvested, and lysed gently with cell lysis buffer (Cell Signaling Technology Inc., Danvers, MA, USA). Cell lysates were centrifuged at 14,000g at 4 °C for 20 min and supernatants were collected. Protein lysates (20 μg) were then subjected to SDS-PAGE and transferred onto PVDF membranes. After blocking with 5% non-fat milk, membranes were incubated with the primary antibodies overnight at 4 °C. Antibodies to Gli1, Gli2, phospho-Akt (p-Akt), total Akt, c-Myc, survivin, cyclin B1 (Cell Signaling Technology), Ptch1 (Abcam, Cambridge, UK), and FoxM1 (Bethyl Laboratories, Inc., Montgomery, TX, USA) were used for immunoblot analysis. After incubation with the secondary antibody for 1 h at room temperature, proteins were detected by VersaDoc 3000 (Bio-Rad, Hercules, CA, USA) with ECL reagents (GE Health Care Life Sciences, Marlborough, MA, USA).
3.2. Effects of Z-ajoene on Gli transcriptional activity and Gli related protein levels in PANC-1 human pancreatic cancer cells
PANC-1 cells were treated with increasing concentrations (5, 10, and 20 μM) of Z-ajoene for 24 h, harvested, and fixed with 70% ethanol at 4 °C overnight. Fixed cells were resuspended in PBS and incubated with RNase A solution for 15 min at 37 °C. Propidium iodide (PI) was added to cells and incubated 4 °C for 20 min. Then 10,000 events were captured on a flow-cytometer (BD Biosciences, San Jose, CA, USA). Fractions of cells in different phases of cell cycle were quantified using BD CellQues™ Pro software.
To elucidate the effect of Z-ajoene on Gli activity, we measured Gli transcriptional activity in PANC-1 human pancreatic cancer cells. PANC-1 cells known to have aberrant activation of Gli1 (Guo et al., 2013) were transiently transfected with Gli-dependent firefly and βgalactosidase reporters (PANC-1-Gli-Luc cells). Luciferase activity was normalized against β-galactosidase reporter activity as a control. Glidependent luciferase activities were presented as relative luciferase activities (fold). Z-Ajoene concentration-dependently suppressed luciferase activity whereas the vehicle showed high level of luciferase activity (Fig. 2A). This suggested that Gli transcriptional activity was reduced by Z-ajoene in human pancreatic cancer cells. Next, we investigated mRNA level of Gli1 by Z-ajoene to investigate whether reduced Gli transcriptional activity could affect the transcription of Glitarget gene in PANC-1 cells. Z-Ajoene reduced mRNA level of Gli1 in PANC-1 cells (Fig. 2B). We then examined the effect of Z-ajoene on expression levels of Glimediated protein such as Gli1, Gli2, Ptch (Wickstrom et al., 2013), and FoxM1 (Teh et al., 2002). As shown in Fig. 2C, Z-ajoene reduced protein expression levels of Gli1, Gli2, Ptch, and FoxM1 in PANC-1 cells in a concentration-dependent manner compared to vehicle control. Phosphatidylinositol 3-kinase (PI3K)/Akt has been reported to be involved in activation of Hh signaling by controlling Gli inactivation (Riobo, Lu, Ai, Haines, & Emerson, 2006). Z-Ajoene showed no effect on p-Akt/Akt levels in PANC-1 cells (Fig. 2C). These results indicate that Z-ajoene can down-regulate protein levels of Gli1, Gli2, Ptch, and FoxM1 by repressing Gli-mediated transcription without disruption PI3K/Akt signaling.
2.9. Statistical analysis
3.3. Effects of Z-ajoene on FoxM1-Cell cycle related protein levels in PANC1 pancreatic cancer cells
2.7. Cell proliferation assay Cells were plated in a 96 well plate at a density of 3 × 103 cells/ well. Cells were incubated with various concentrations of Z-ajoene for 72 h, treated with MTT (5 mg/mL) solution for 4 h, and lysed with DMSO. Absorbance at 540 nm was measured using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).
2.8. Cell cycle analysis
Data are presented as mean ± standard deviation. P-values were determined using unpaired Student’s t-test. A p-value < 0.05 was considered significant.
FoxM1, one of target genes of Gli, can regulate expression of genes related to cell proliferation and anti-apoptosis such as c-Myc (Wierstra & Alves, 2007), cyclin B1 (Wang, Banerjee, Kong, Li, & Sarkar, 2007), and survivin (Radhakrishnan et al., 2006). In the present study, we 104
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Fig. 2. Effects of Z-ajoene on Gli transcriptional activity and expression of Gli-target proteins in PANC-1 human pancreatic cancer cells. (A) PANC-1-Gli-luc cells were treated with various concentrations of Z-ajoene for 20 h and then luciferase activity was measured using a microplate luminometer. The relative luciferase activity was expressed as the relative Gli-mediated transcriptional activity compared to vehicle treatment. *p < 0.05 indicates a significant difference from the vehicle treatment. (B) Effects of Z-ajoene on Gli1 mRNA level in PANC-1 human pancreatic cancer cells. mRNA levels of Gli1 and GAPDH were determined by RT-PCR. (C) PANC-1 human pancreatic cancer cells were treated with various concentrations of Z-ajoene for 20 h. Cell lysates were prepared. Gli1, Gli2, Ptch, and FoxM1 protein levels were determined by Western blotting. Images are representatives of three independent experiments with similar results.
3.4. Effects of Z-ajoene on proliferation and cell cycle distribution of PANC1 pancreatic cancer cells Next, we investigated the effect of Z-ajoene on proliferation of PANC-1 pancreatic cells. To evaluate whether Z-ajoene selectively suppressed the proliferation of pancreatic cancer cells accompanying the activation of Gli, we used C3H10T1/2 cells as control. C3H10T1/2 cells are Hh signaling responsive without depending on Hh signaling for survival (Hosoya, Arai, Koyano, Kowithayakorn, & Ishibashi, 2008). As seen in Fig. 4A, Z-ajoene concentration-dependently suppressed viability of PANC-1 pancreatic cancer cells. By treatment with 20 μM Zajoene for 72 h, the cell viability of PANC-1 cells was 19.1% compared to vehicle treatment whereas that of C3H10T1/2 cells was 70.5% (Fig. 4A). The inhibitory effect of Z-ajoene on the proliferation of PANC-1 cells was much higher than that on the proliferation of C3H10T1/2 cells. Gli positive PANC-1 pancreatic cancer cells were much more susceptible to Z-ajoene than Gli independent C3H10T1/2 cells. These results indicate that the anti-proliferative effect of Z-ajoene is mainly mediated by down-regulating Gli-FoxM1 related protein levels in pancreatic cancer. To further examine whether Z-ajoene could affect cell cycle distribution for the anti-proliferative activity of PANC-1 cells, we conducted cell cycle analysis by propidium iodide staining using flow cytometry. As shown in Fig. 4B, Z-ajoene induced a significant accumulation of cells in G2/M phase and a decrease of cell population in S phase compared to control cells. These results disclosed that Zajoene caused cell cycle arrest in G2/M phase of PANC-1 cells. This coincides with the finding that ajoene can induce G2/M cell cycle arrest and apoptosis in oesophageal cancer cells and leukemia cells (Dirsch, Antlsperger, Hentze, & Vollmar, 2002; Kaschula et al., 2012; Li et al.,
Fig. 3. Effects of Z-ajoene on levels of FoxM1-target proteins in PANC-1 pancreatic cancer cells. Cells were treated with Z-ajoene at indicated concentrations for 20 h. Cell lysates were prepared and protein levels of c-Myc, cyclin B1, and survivin were determined by Western blotting. Images are representatives of three independent experiments with similar results.
investigated whether Z-ajoene could affect expression levels of c-Myc, cyclin B1, and survivin because Z-ajoene down-regulated FoxM1 level by suppressing Gli-mediated transcription (Fig. 2C). Immunoblot analysis revealed that Z-ajoene concentration-dependently reduced expression levels of c-Myc, cylcin B1, and survivin in PANC-1 cells (Fig. 3). These results led us to hypothesize that Z-ajoene could suppress the proliferation of pancreatic cancer cells by down-regulating levels of Gli-FoxM1-Cell cycle related proteins.
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Fig. 4. Effects of Z-ajoene on proliferation of PANC-1 and C3H10T1/2 cells and cell cycle distribution of PANC-1 human pancreatic cancer cells. (A) PANC-1 cells and C3H10T1/2 cells were treated with Z-ajoene at indicated concentrations for 72 h. Cell viability was determined by MTT assay. Values are presented as mean ± S.D. *, p < 0.05, indicating significant difference from vehicle treatment. (B) PANC-1 cells were treated with 0, 5, 10, or 20 μM of Z-ajoene for 24 h and cell cycle distribution was analyzed by flow cytometry.
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Gli at the downstream of Smo in Hh pathway. Here, we found that Z-ajoene could inhibit Gli transcriptional activity and mRNA level of Gli1 in PANC-1 human pancreatic cancer cells known to have high level of Gli1 with resistance to Smo inhibitor (Guo et al., 2013; Thayer et al., 2003) (Fig. 2A and B). These results suggest that regulation of Gli-mediated transcription might contribute to the inhibitory potential of Z-ajoene on Hh signaling pathway. Our colleagues demonstrated that an ajoene analog suppressed NFκB transcriptional activity through modulating the interaction between NF-κB and PPARγ. The analog did not affect nuclear localization and DNA binding affinity of the p65 subunit, but enhanced its physical interaction with PPARγ, which resulted in suppression of NF-κB transcriptional activity (Hwang et al., 2016). Here, we found that Z-ajoene also did not alter Gli1 protein level in nuclear fraction in PANC-1 cells (Supplementary material Fig. S2). The mode of action for inhibition of Gli transcriptional activity by Z-ajoene in PANC-1 cells might be derived from altered interaction between Gli and regulatory cofactors. The exact mechanism needs to be elucidated in a further study. Gli1, one of target genes of the Gli transcription factor, can regulate the transcription of Hh responsive genes by itself. Gli1 is upregulated in pancreatic cancer tissues. Its expression level is positively correlated with Hh signaling (Liu et al., 2015). FoxM1, another Gli target gene, acts as a cell cycle regulator and plays an important role in carcinogenesis (Wang et al., 2017; Wierstra, 2013). Z-Ajoene down-regulated Gli target proteins such as Gli1, Gli2, Ptch, and FoxM1 protein levels whereas basal levels of Gli1 and FoxM1 were elevated in PANC-1 cells (Fig. 2C). The level of FoxM1 was more susceptible than Gli1 level by treatment with 10 μM Z-ajoene. This means that Z-ajoene might have another mechanism for modulating FoxM1 protein level besides the Gli pathway. Further study is needed to determine the molecular target of Z-ajoene so that we could explain the detailed mechanism. Moreover, Zajoene did not alter levels of phosphorylated Akt in PANC-1 cells (Fig. 2C), suggesting that Z-ajoene could specifically regulate the Hh/ Gli signaling pathway without affecting the PI3K/Akt signaling pathway. FoxM1, a downstream target of Gli, has been mainly related to cell cycle regulation and cellular proliferation in tumor (Alvarez-Fernandez & Medema, 2013; Halasi & Gartel, 2013; Teh et al., 2002). Especially, FoxM1 is a regulator of G1/S and G2/M transitions and M-phase progression (Wonsey & Follettie, 2005). It controls cell cycle-related molecules such as c-Myc (Wierstra & Alves, 2006), cyclin B1 (Leung et al., 2001) and survivin (Nestal de Moraes et al., 2015). c-Myc, an oncoprotein, is responsible for cellular proliferation and cell cycle regulation of cancer cells. c-Myc expression is elevated and deregulated in neoplastic cells (Albihn, Johnsen, & Henriksson, 2010; DePinho, SchreiberAgus, & Alt, 1991). Cyclin B1, another target of FoxM1, is usually expressed at very low levels. It accumulates sharply only at G2/M cell cycle transition under normal conditions. In cancer cells, cyclin B1 is overexpressed throughout cell cycle progression (Ye, Wang, Wu, Li, & Chai, 2017). Survivin, an anti-apoptotic protein, plays a role in the regulation of cell division and spindle formation. It is essential for cell cycle progression (Mita, Mita, Nawrocki, & Giles, 2008). Overexpression of survivin is frequently associated with poor prognosis and drug resistance in neoplastic mammary cells (Hinnis, Luckett, & Walker, 2007). As shown in Fig. 3, Z-ajoene suppressed target gene expression of FoxM1, c-Myc, cyclin B1, and survivin, together with depressed proliferation of PANC-1 pancreatic cancer cells (Fig. 4A). These results are in agreement with down-regulation of FoxM1 by Z-ajoene through inhibition of Gli activation. Furthermore, cell cycle analysis demonstrated that Z-ajoene significantly elevated the percentage of G2/M phase cells (Fig. 4B). These results suggest that Z-ajoene can reduce the proliferation of PANC-1 pancreatic cancer cells through inducing cell cycle arrest in G2/M phase. Taken together, Z-ajoene exhibits antiproliferative activity in pancreatic cancer cells by down-regulating Gli in aberrantly activated Hh signaling pathway. Therefore, inhibition of Gli is a well-organized approach to regulate the Hh signaling pathway
2002). Taken together, Z-ajoene could reduce proliferation of PANC-1 pancreatic cancer cell by inducing G2/M cell cycle arrest through down-regulation of Gli-FoxM1 signaling pathway. In response to Shh ligand binding, Ptch abrogates the suppression of Smo, resulting in nuclear accumulation of Gli and initiation of Glimediated transcription. Ectopic expression of Shh has been reported to be sufficient to induce multiple carcinomas including pancreatic cancer whose Hh signaling pathway is dysregulated by Shh dependent-Gli activation (Fendrich et al., 2011a; Rimkus, Carpenter, Qasem, Chan, & Lo, 2016). However, inhibition of Gli could be a worthy approach to control several cancer types through upregulation of Hh signaling pathway by overexpression of Shh ligands (Supplementary material Fig. S1). Thus, Z-ajoene might be useful for targeting Gli on pancreatic cancers that are driven by ectopic activation of Hh signaling pathway. 4. Discussion Garlic has been consumed as a spice and a folk medicine since ancient times for cardio-protective and health benefits as well as for treatment of cancers (Adaki, Adaki, Shah, & Karagir, 2014; Block et al., 1984; Khatua, Adela, & Banerjee, 2013). Z-Ajoene, an organosulfur compound derived from allicin, has been isolated from garlic (Allium sativum). Biological activities of Z-ajoene include inhibition of platelet activation, enhancement of host defense against mycobacteria, and suppression of cancer cell proliferation (Choi et al., 2018; Jung et al., 2014; Teranishi, Apitz-Castro, Robson, Romano, & Cooper, 2003). Several plant-derived molecules such as resveratrol, curcumin, zerumbone, and physalin B have been reported as modulators of Hh/Gli signaling pathway (Hosoya et al., 2008; Mohapatra et al., 2015). Our previous studies have demonstrated that a sesquiterpene lactone and diarylheptanoids from Siegesbeckia glabrescens, Alpinia officinarum and Alnus japonica can inhibit the Hh/Gli signaling pathway (Dong et al., 2017; Lee et al., 2016). Herein, we report that Z-ajoene (Fig. 1A) from garlic can also affect the Hh/Gli signaling pathway. While tightly regulated Hh pathway is responsible for coordinating cellular growth and embryo development, dysregulation of Hh pathway is involved in the formation, development, and aggressiveness of tumors, especially pancreatic cancer (Lauth & Toftgard, 2011; Xie, Bartels, Barton, & Gu, 2013). Hh pathway is activated with a series of signal cascade such as Shh ligand binding to Ptch, Smo activation and initiation of Gli-mediated transcription. Recent evidence suggests that constitutive activation of Gli is associated with tumor formation and growth (Kasper, Regl, Frischauf, & Aberger, 2006; Pietrobono et al., 2018). Although Smo antagonists have been reported as therapuetic candidates for various cancers (Fendrich et al., 2011b; Ibuki et al., 2013), their therapeutic efficacies in preclinical and clinical cancer models are not promising (Rimkus et al., 2016). Recent studies have shown that acquired resistance to Smo inhibition is linked to mutations in Smo and aberrant activation of Gli or upregulation of synergic signals such as PI3K signaling (Benvenuto et al., 2016; Lauth & Toftgard, 2007). Resistance against Smo antagonist (vismodegib) has already been reported in patients with advanced or metastatic solid tumors (Atwood, Chang, & Oro, 2012; Gonnissen, Isebaert, & Haustermans, 2015; LoRusso et al., 2011). Another study has reported that GANT 61 (a Gli inhibitor) shows therapeutic effects on colon cancer cells that are resistance to Smo inhibitors (cyclopamine or GDC-0449) (Agyeman, Mazumdar, & Houghton, 2012). Therefore, the inhibition of Gli has been recognized as an efficient strategy to regulate the Hh signaling pathway for cancer therapy. In our reporter assay system, C3H10T1/2-Gli1-Luc cells were incubated with Shh CM to induce Gli1-mediated luciferase activity (Fig. 1B). Z-Ajoene inhibited Shh CM-induced luciferase activity in a concentration-dependent manner as seen in Fig. 1C. Z-Ajoene also inhibited SAG (a Smo activator)-induced luciferase activity (Fig. 1D). These findings suggest that Z-ajoene could modulate the activation of 107
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for cancer therapy.
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5. Conclusion Modulation of aberrant overexpression of Gli level in the Hh pathway is a promising strategy to develop therapeutic agent for cancer. Z-Ajoene from garlic can modulate the Hh/Gli signaling pathway by reducing Gli transcriptional activity in Shh activated C3H10T1/2 cells as well as in PANC-1 pancreatic cancer cells. Z-Ajoene down-regulates expressions of Gli-target proteins and FoxM1-target proteins in PANC-1 cancer cells. This compound exhibits anti-proliferative activity against PANC-1 pancreatic cancer cells by inducing G2/M cell cycle arrest through down-regulation of Gli/FoxM1. Thus, ZAjoene from garlic might be a valuable candidate for developing new therapeutic agents against pancreatic cancer accompanying aberrant activation of Hh/Gli signaling pathway. Ethics statements file This research does not include any human subjects and animal experiments. Conflict of interest The authors declare no conflict of interest. Acknowledgements This study was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MSIP) (Nos. 2011-0030074 and 2018R1D1A3B07050361) and Sookmyung Women’s University Specialization Program Funding. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jff.2019.03.010. References Aberger, F., & Ruiz, I. A. A. (2014). Context-dependent signal integration by the GLI code: The oncogenic load, pathways, modifiers and implications for cancer therapy. Seminars in Cell & Developmental Biology, 33, 93–104. https://doi.org/10.1016/j. semcdb.2014.05.003. Adaki, S., Adaki, R., Shah, K., & Karagir, A. (2014). Garlic: Review of literature. Indian Journal of Cancer, 51, 577–581. https://doi.org/10.4103/0019-509X.175383. Agren, M., Kogerman, P., Kleman, M. I., Wessling, M., & Toftgard, R. (2004). Expression of the PTCH1 tumor suppressor gene is regulated by alternative promoters and a single functional Gli-binding site. Gene, 330, 101–114. https://doi.org/10.1016/j. gene.2004.01.010. Agyeman, A., Mazumdar, T., & Houghton, J. A. (2012). Regulation of DNA damage following termination of Hedgehog (HH) survival signaling at the level of the GLI genes in human colon cancer. Oncotarget, 3, 854–868. https://doi.org/10.18632/ oncotarget.586. Albihn, A., Johnsen, J. I., & Henriksson, M. A. (2010). MYC in oncogenesis and as a target for cancer therapies. Advances in Cancer Research, 107, 163–224. https://doi.org/10. 1016/S0065-230X(10)07006-5. Alvarez-Fernandez, M., & Medema, R. H. (2013). Novel functions of FoxM1: From molecular mechanisms to cancer therapy. Frontiers in Oncology, 3, 30. https://doi.org/ 10.3389/fonc.2013.00030. Armas-Lopez, L., Zuniga, J., Arrieta, O., & Avila-Moreno, F. (2017). The Hedgehog-GLI pathway in embryonic development and cancer: Implications for pulmonary oncology therapy. Oncotarget, 8, 60684–60703. https://doi.org/10.18632/oncotarget. 19527. Atwood, S. X., Chang, A. L., & Oro, A. E. (2012). Hedgehog pathway inhibition and the race against tumor evolution. Journal of Cell Biology, 199, 193–197. https://doi.org/ 10.1083/jcb.201207140. Bai, C. B., & Joyner, A. L. (2001). Gli1 can rescue the in vivo function of Gli2. Development, 128, 5161–5172. http://dev.biologists.org/content/128/24/5161. Benvenuto, M., Masuelli, L., De Smaele, E., Fantini, M., Mattera, R., Cucchi, D., et al. (2016). In vitro and in vivo inhibition of breast cancer cell growth by targeting the Hedgehog/GLI pathway with SMO (GDC-0449) or GLI (GANT-61) inhibitors. Oncotarget, 7, 9250–9270. https://doi.org/10.18632/oncotarget.7062.
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Anti-pancreatic cancer activity of Z-ajoene from garlic: An inhibitor of the Hedgehog/Gli/FoxM1 axis Hwa Jin Leea,1, Ji Hye Jeongb,1, Jae-Ha Ryub, a b
T
⁎
School of Industrial Bio-Pharmaceutical Science, Semyung University, Jecheon, Chungbuk 27136, Republic of Korea College of Pharmacy and Research Center for Cell Fate Control, Sookmyung Women’s University, Seoul 04310, Republic of Korea
A R T I C LE I N FO
A B S T R A C T
Keywords: Ajoene Garlic Pancreatic cancer Hedgehog pathway Gli FoxM1
Overexpression of glioma-associated oncogene (Gli), a transcription factor at the final step of Hedgehog (Hh) pathway, is involved in pancreatic cancer development. Therefore, inhibition of Gli can be a therapeutic strategy to treat pancreatic cancer. The objective of this study was to determine effects of Z-ajoene from garlic, on Glimediated transcription and proliferation of pancreatic cancer cells. We found that Z-ajoene could inhibit Gli transcriptional activity in Sonic Hedgehog (Shh) stimulated C3H10T1/2 mesenchymal stem cells. Z-Ajoene suppressed Gli transcriptional activity and Gli-target protein expressions in PANC-1 human pancreatic cancer cells. Z-Ajoene also reduced expression of FoxM1, one of Gli-target proteins, and subsequently down-regulated expressions of cell cycle-related proteins. Moreover, Z-ajoene reduced cell proliferation and increased G2/M phase of PANC-1 cells. These results suggest that Z-ajoene can repress pancreatic cancer cell proliferation by inhibiting Gli signaling. Thus, Z-ajoene might be a lead compound for the development of anti-pancreatic cancer agent.
1. Introduction The Hedgehog (Hh) signaling pathway regulates embryonic organogenesis, tissue patterning, and cell differentiation (Armas-Lopez, Zuniga, Arrieta, & Avila-Moreno, 2017; Ingham & McMahon, 2013). Aberrant activation of Hh signaling pathway contributes to tumor formation and development of various cancers including pancreatic cancer (Jiang & Hui, 2008; Pasca di Magliano & Hebrok, 2003). In the presence of Sonic hedgehog (Shh) ligand, the 12-pass transmembrane receptor (Patched, Ptch) can release repressed Smoothened (Smo). This induces nuclear translocation of Gli activator and activation of target gene expressions (Lum & Beachy, 2004; McMahon, Ingham, & Tabin, 2003). In the absence of Hh ligands, Ptch restrains Smo, resulting in nuclear translocation of Gli repressor to switch off Hh target gene expression (Pan, Bai, Joyner, & Wang, 2006). There are three members of Gli transcription factors: Gli1, Gli2, and Gli3 (Hu et al., 2006). Gli1 and Gli2 can activate the expression of target genes related to cell growth and survival (Aberger & Ruiz, 2014; Hui, Slusarski, Platt, Holmgren, & Joyner, 1994; Persson et al., 2002). Gli2 may be an effective
transcription factor in the absence of Gli1 (Bai & Joyner, 2001). However, Gli3 is a transcriptional repressor of Hh signaling (Hu et al., 2006). Direct target genes by Gli-mediated transcription are Hh pathway regulators such as Gli1, Gli2, Ptch (Agren, Kogerman, Kleman, Wessling, & Toftgard, 2004; Hegde et al., 2008; Regl et al., 2002), and cell cycle regulator such as FoxM1 (Teh et al., 2002). Recently, many studies have demonstrated that Hh signaling pathway is constitutively activated in pancreatic cancer. High level of Gli1 as the last stage in Hh signaling pathway is a significant indicator of tumor growth and aggressiveness in pancreatic cancer (Lauth & Toftgard, 2011; Liu et al., 2015; Onishi & Katano, 2014; Thayer et al., 2003). Therefore, interference of Gli might be a good therapeutic strategy to treat cancers accompanying highly activated Hh signaling pathway. Z-Ajoene, an organosulfur compound from garlic (Allium sativum), has antioxidant, antimicrobial, antithrombotic, and anti-inflammatory activities (Choi et al., 2018; Kay et al., 2010; Lee et al., 2012; Srivastava & Tyagi, 1993). Z-Ajoene has been reported to possess anti-proliferative and apoptotic effects on several cancer cells by activating endoplasmic reticulum stress, caspase-3, and ERK/p38 (Hassan, 2004; Jung et al.,
Abbreviations: Hh, Hedgehog; Shh, Sonic Hedgehog; Ptch, 12-pass transmembrane spanning receptor Patched; Smo, 7-pass transmembrane receptor Smoothened; Gli, glioma-associated oncogene; Shh CM, Sonic Hedgehog conditioned medium; FoxM1, Forkhead box protein M1 ⁎ Corresponding author at: College of Pharmacy and Research Center for Cell Fate Control, Sookmyung Women’s University, 100 Chungparo-47-Gil, Yongsan-Gu, Seoul 04310, Republic of Korea. E-mail address: [email protected] (J.-H. Ryu). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.jff.2019.03.010 Received 14 November 2018; Received in revised form 7 March 2019; Accepted 8 March 2019 1756-4646/ © 2019 Published by Elsevier Ltd.
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Fig. 1. Structure of Z-ajoene and effects of Z-ajoene on Sonic Hedgehog signaling pathway in C3H10T1/2-Gli1-luc cells. (A) Structure of Z-ajoene from garlic (Allium sativum). (B) C3H10T1/2-Gli1-luc cells were treated with Sonic Hedgehog conditioned medium (Shh CM) or recombinant human Sonic Hedgehog ligand (rh-Shh, 500 ng/mL) for 30 h. Relative luciferase activity was expressed as relative Gli1-mediated transcriptional activity compared to vehicle CM treated cells. *p < 0.05 indicates a significant difference from vehicle CM. (C) C3H10T1/2-Gli1-luc cells were treated with Shh CM and various concentrations of Z-ajoene or cyclopamine (5 μM) for 30 h. The relative luciferase activity was expressed as the relative Gli1-mediated transcriptional activity compared to vehicle treated cells. *p < 0.05 indicates a significant difference from Shh CM alone. (D) C3H10T1/2-Gli1-luc cells were stimulated with SAG (500 nM) in the presence of Z-ajoene for 30 h. The relative luciferase activity was expressed as the relative Gli1-mediated transcriptional activity compared to vehicle treated cells. *p < 0.05 indicates a significant difference from SAG alone.
construct gifted from Prof. Gyu-Un Bae (Sookmyung Women’s University, Korea) and Prof. Jong-Sun Kang (Sungkyunkwan University, Korea) (Zhang, Kang, Cole, Yi, & Krauss, 2006) was transiently transfected into HEK293 cells. Shh-producing HEK293 cells were then grown to 80% confluency in DMEM containing 10% FBS. The medium was replaced with DMEM containing 2% FBS and cells were grown for 5 days. Conditioned medium (CM) was harvested, filtered through a 0.22 μm membrane, and stored at 4 °C until use.
2014; Kaschula et al., 2016; Kaschula, Hunter, & Parker, 2010). Herein, we report effects of Z-ajoene on Gli-mediated transcription at Hh pathway and proliferation of pancreatic cancer cells. 2. Materials and methods 2.1. Plant materials Z-Ajoene (Fig. 1A) was prepared as previously described (Lee et al., 2012).
2.4. Gli1/Gli-dependent luciferase reporter assay
2.2. Cell lines, chemicals and biochemical
C3H10T1/2 cells were transiently transfected with Gli1, Gli-dependent firefly luciferase, and β-galactosidase expression constructs (C3H10T1/2-Gli1-Luc cells) to assess Gli1-mediated transcriptional activity. Gli1 plasmid and Gli reporter plasmid were gifts from Prof. Gyu-Un Bae (Sookmyung Women’s University, Korea) and Prof. JongSun Kang (Sungkyunkwan University, Korea) (Zhang et al., 2006). Transfected cells were treated with Z-ajoene in the presence of either Shh CM or control medium. C3H10T1/2-Gli1-Luc cells were also induced by SAG, a Smo inhibitor, in the presence of Z-ajoene. After incubation at 37 °C for 30 h, Gli1-dependent firefly luciferase and β-galactosidase activities were measured using a microplate luminometer (Perkin Elmer, Waltham, MA, USA). For assessment of Gli-mediated transcriptional activity in pancreatic cancer cells, plasmids of Gli-dependent firefly luciferase reporter construct and β-galactosidase expression construct were transiently transfected into PANC-1 cells (PANC-1-Gli-Luc cells). These cells were
C3H10T1/2 (mouse mesenchymal stem cell line), PANC-1 and AsPC-1 (human pancreatic cancer cell lines) (ATCC, Manassas, VA, USA) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin (Life Technologies, Carlsbad, CA, USA). SAG (purity: ≥98%), a Smo agonist, was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Cyclopamine (purity: ≥98%, Sigma-Aldrich), a Smo inhibitor in the Hh pathway, was used as a positive control to inhibit the Hh pathway. 2.3. Preparation of sonic hedgehog conditioned media For induction of Hh signaling, we prepared Sonic Hedgehog conditioned medium (Shh CM) as described previously (Chen, Taipale, Young, Maiti, & Beachy, 2002; Lee et al., 2016). Briefly, Shh expression 103
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3. Results
treated with various concentrations of Z-ajoene at 37 °C for 20 h. Cellular firefly luciferase and β-galactosidase activities were then measured using a microplate luminometer. Luciferase activity was normalized against β-galactosidase reporter activity as a control. All luciferase reporter activities were presented as relative luciferase activities (fold).
3.1. Effects of Z-ajoene on Hh/Gli1-mediated transcriptional activity in C3H10T1/2 mouse mesenchymal stem cells To investigate the effect of Z-ajoene on Hh pathway, Shh ligand induced Gli1- transcriptional activity was measured as luciferase activity in C3H10T1/2-Gli1-Luc cells transiently transfected with Gli1, Gli-dependent firefly, and β-galactosidase reporters. C3H10T1/2-Gli1Luc cells were activated with Shh conditioned medium (Shh CM). The potency of Shh CM-induced activation was similar to that of recombinant Shh ligand (500 ng/mL, PeproTech Inc., Rocky Hill, NJ, USA) (Fig. 1B). Luciferase activity was normalized against β-galactosidase reporter activity as a control. As shown in Fig. 1C, Z-ajoene inhibited Shh CM-induced luciferase activity in a concentration-dependent manner. Cyclopamine (5 μM), a Smo inhibitor, was used as a positive control (Lee et al., 2014). We also examined the inhibitory effect of Z-ajoene on Hh pathway by Smo activation. C3H10T1/2-Gli1Luc cells were treated with SAG (500 nM), a synthetic Smo agonist (Chen et al., 2002), to activate Gli1 transcriptional activity. Z-Ajoene suppressed SAG-activated Gli1 activity in a dose-dependent manner (Fig. 1D). From the fact that Z-ajoene modulated the Hh/Gli signaling pathway by suppressing Shh CM and SAG-induced Gli1 transcriptional activity, we postulated that Z-ajoene could inhibit the downstream events of Smo to regulate the Hh pathway. These results led us to investigate the suppressive effect of Z-ajoene on activity of Gli1 as downstream transcription factor of Smo in Hh pathway.
2.5. Reverse transcription-polymerase chain reaction (RT-PCR) analysis PANC-1 cells (2.5 × 105 cells/60 mm dish) were treated with Zajoene for 20 h. Total RNA was isolated from the cell pellet using Trizol reagent (Life Technologies, Paisley, UK). RNA was then reverse transcribed into cDNA using reverse transcriptase and random hexamer. Sense and antisense primers for Gli1 and GAPDH were: 5′-GCCGTGTA AAGCTCCAGTGAACACA-3′ and 5′-TCCCACTTTGAGAGGCCCATAGC AAG-3′; 5′-TGTGATGGGTGTGAACCACGAG-3′ and 5′-TGCTGTTGAAG TCGCAGGAGAC-3′, respectively. Amplified DNA was separated on 1.5% agarose gels and stained with ethidium bromide.
2.6. Western blot analysis Cells were treated with various concentrations of Z-ajoene for 20 h, harvested, and lysed gently with cell lysis buffer (Cell Signaling Technology Inc., Danvers, MA, USA). Cell lysates were centrifuged at 14,000g at 4 °C for 20 min and supernatants were collected. Protein lysates (20 μg) were then subjected to SDS-PAGE and transferred onto PVDF membranes. After blocking with 5% non-fat milk, membranes were incubated with the primary antibodies overnight at 4 °C. Antibodies to Gli1, Gli2, phospho-Akt (p-Akt), total Akt, c-Myc, survivin, cyclin B1 (Cell Signaling Technology), Ptch1 (Abcam, Cambridge, UK), and FoxM1 (Bethyl Laboratories, Inc., Montgomery, TX, USA) were used for immunoblot analysis. After incubation with the secondary antibody for 1 h at room temperature, proteins were detected by VersaDoc 3000 (Bio-Rad, Hercules, CA, USA) with ECL reagents (GE Health Care Life Sciences, Marlborough, MA, USA).
3.2. Effects of Z-ajoene on Gli transcriptional activity and Gli related protein levels in PANC-1 human pancreatic cancer cells
PANC-1 cells were treated with increasing concentrations (5, 10, and 20 μM) of Z-ajoene for 24 h, harvested, and fixed with 70% ethanol at 4 °C overnight. Fixed cells were resuspended in PBS and incubated with RNase A solution for 15 min at 37 °C. Propidium iodide (PI) was added to cells and incubated 4 °C for 20 min. Then 10,000 events were captured on a flow-cytometer (BD Biosciences, San Jose, CA, USA). Fractions of cells in different phases of cell cycle were quantified using BD CellQues™ Pro software.
To elucidate the effect of Z-ajoene on Gli activity, we measured Gli transcriptional activity in PANC-1 human pancreatic cancer cells. PANC-1 cells known to have aberrant activation of Gli1 (Guo et al., 2013) were transiently transfected with Gli-dependent firefly and βgalactosidase reporters (PANC-1-Gli-Luc cells). Luciferase activity was normalized against β-galactosidase reporter activity as a control. Glidependent luciferase activities were presented as relative luciferase activities (fold). Z-Ajoene concentration-dependently suppressed luciferase activity whereas the vehicle showed high level of luciferase activity (Fig. 2A). This suggested that Gli transcriptional activity was reduced by Z-ajoene in human pancreatic cancer cells. Next, we investigated mRNA level of Gli1 by Z-ajoene to investigate whether reduced Gli transcriptional activity could affect the transcription of Glitarget gene in PANC-1 cells. Z-Ajoene reduced mRNA level of Gli1 in PANC-1 cells (Fig. 2B). We then examined the effect of Z-ajoene on expression levels of Glimediated protein such as Gli1, Gli2, Ptch (Wickstrom et al., 2013), and FoxM1 (Teh et al., 2002). As shown in Fig. 2C, Z-ajoene reduced protein expression levels of Gli1, Gli2, Ptch, and FoxM1 in PANC-1 cells in a concentration-dependent manner compared to vehicle control. Phosphatidylinositol 3-kinase (PI3K)/Akt has been reported to be involved in activation of Hh signaling by controlling Gli inactivation (Riobo, Lu, Ai, Haines, & Emerson, 2006). Z-Ajoene showed no effect on p-Akt/Akt levels in PANC-1 cells (Fig. 2C). These results indicate that Z-ajoene can down-regulate protein levels of Gli1, Gli2, Ptch, and FoxM1 by repressing Gli-mediated transcription without disruption PI3K/Akt signaling.
2.9. Statistical analysis
3.3. Effects of Z-ajoene on FoxM1-Cell cycle related protein levels in PANC1 pancreatic cancer cells
2.7. Cell proliferation assay Cells were plated in a 96 well plate at a density of 3 × 103 cells/ well. Cells were incubated with various concentrations of Z-ajoene for 72 h, treated with MTT (5 mg/mL) solution for 4 h, and lysed with DMSO. Absorbance at 540 nm was measured using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).
2.8. Cell cycle analysis
Data are presented as mean ± standard deviation. P-values were determined using unpaired Student’s t-test. A p-value < 0.05 was considered significant.
FoxM1, one of target genes of Gli, can regulate expression of genes related to cell proliferation and anti-apoptosis such as c-Myc (Wierstra & Alves, 2007), cyclin B1 (Wang, Banerjee, Kong, Li, & Sarkar, 2007), and survivin (Radhakrishnan et al., 2006). In the present study, we 104
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Fig. 2. Effects of Z-ajoene on Gli transcriptional activity and expression of Gli-target proteins in PANC-1 human pancreatic cancer cells. (A) PANC-1-Gli-luc cells were treated with various concentrations of Z-ajoene for 20 h and then luciferase activity was measured using a microplate luminometer. The relative luciferase activity was expressed as the relative Gli-mediated transcriptional activity compared to vehicle treatment. *p < 0.05 indicates a significant difference from the vehicle treatment. (B) Effects of Z-ajoene on Gli1 mRNA level in PANC-1 human pancreatic cancer cells. mRNA levels of Gli1 and GAPDH were determined by RT-PCR. (C) PANC-1 human pancreatic cancer cells were treated with various concentrations of Z-ajoene for 20 h. Cell lysates were prepared. Gli1, Gli2, Ptch, and FoxM1 protein levels were determined by Western blotting. Images are representatives of three independent experiments with similar results.
3.4. Effects of Z-ajoene on proliferation and cell cycle distribution of PANC1 pancreatic cancer cells Next, we investigated the effect of Z-ajoene on proliferation of PANC-1 pancreatic cells. To evaluate whether Z-ajoene selectively suppressed the proliferation of pancreatic cancer cells accompanying the activation of Gli, we used C3H10T1/2 cells as control. C3H10T1/2 cells are Hh signaling responsive without depending on Hh signaling for survival (Hosoya, Arai, Koyano, Kowithayakorn, & Ishibashi, 2008). As seen in Fig. 4A, Z-ajoene concentration-dependently suppressed viability of PANC-1 pancreatic cancer cells. By treatment with 20 μM Zajoene for 72 h, the cell viability of PANC-1 cells was 19.1% compared to vehicle treatment whereas that of C3H10T1/2 cells was 70.5% (Fig. 4A). The inhibitory effect of Z-ajoene on the proliferation of PANC-1 cells was much higher than that on the proliferation of C3H10T1/2 cells. Gli positive PANC-1 pancreatic cancer cells were much more susceptible to Z-ajoene than Gli independent C3H10T1/2 cells. These results indicate that the anti-proliferative effect of Z-ajoene is mainly mediated by down-regulating Gli-FoxM1 related protein levels in pancreatic cancer. To further examine whether Z-ajoene could affect cell cycle distribution for the anti-proliferative activity of PANC-1 cells, we conducted cell cycle analysis by propidium iodide staining using flow cytometry. As shown in Fig. 4B, Z-ajoene induced a significant accumulation of cells in G2/M phase and a decrease of cell population in S phase compared to control cells. These results disclosed that Zajoene caused cell cycle arrest in G2/M phase of PANC-1 cells. This coincides with the finding that ajoene can induce G2/M cell cycle arrest and apoptosis in oesophageal cancer cells and leukemia cells (Dirsch, Antlsperger, Hentze, & Vollmar, 2002; Kaschula et al., 2012; Li et al.,
Fig. 3. Effects of Z-ajoene on levels of FoxM1-target proteins in PANC-1 pancreatic cancer cells. Cells were treated with Z-ajoene at indicated concentrations for 20 h. Cell lysates were prepared and protein levels of c-Myc, cyclin B1, and survivin were determined by Western blotting. Images are representatives of three independent experiments with similar results.
investigated whether Z-ajoene could affect expression levels of c-Myc, cyclin B1, and survivin because Z-ajoene down-regulated FoxM1 level by suppressing Gli-mediated transcription (Fig. 2C). Immunoblot analysis revealed that Z-ajoene concentration-dependently reduced expression levels of c-Myc, cylcin B1, and survivin in PANC-1 cells (Fig. 3). These results led us to hypothesize that Z-ajoene could suppress the proliferation of pancreatic cancer cells by down-regulating levels of Gli-FoxM1-Cell cycle related proteins.
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Fig. 4. Effects of Z-ajoene on proliferation of PANC-1 and C3H10T1/2 cells and cell cycle distribution of PANC-1 human pancreatic cancer cells. (A) PANC-1 cells and C3H10T1/2 cells were treated with Z-ajoene at indicated concentrations for 72 h. Cell viability was determined by MTT assay. Values are presented as mean ± S.D. *, p < 0.05, indicating significant difference from vehicle treatment. (B) PANC-1 cells were treated with 0, 5, 10, or 20 μM of Z-ajoene for 24 h and cell cycle distribution was analyzed by flow cytometry.
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Gli at the downstream of Smo in Hh pathway. Here, we found that Z-ajoene could inhibit Gli transcriptional activity and mRNA level of Gli1 in PANC-1 human pancreatic cancer cells known to have high level of Gli1 with resistance to Smo inhibitor (Guo et al., 2013; Thayer et al., 2003) (Fig. 2A and B). These results suggest that regulation of Gli-mediated transcription might contribute to the inhibitory potential of Z-ajoene on Hh signaling pathway. Our colleagues demonstrated that an ajoene analog suppressed NFκB transcriptional activity through modulating the interaction between NF-κB and PPARγ. The analog did not affect nuclear localization and DNA binding affinity of the p65 subunit, but enhanced its physical interaction with PPARγ, which resulted in suppression of NF-κB transcriptional activity (Hwang et al., 2016). Here, we found that Z-ajoene also did not alter Gli1 protein level in nuclear fraction in PANC-1 cells (Supplementary material Fig. S2). The mode of action for inhibition of Gli transcriptional activity by Z-ajoene in PANC-1 cells might be derived from altered interaction between Gli and regulatory cofactors. The exact mechanism needs to be elucidated in a further study. Gli1, one of target genes of the Gli transcription factor, can regulate the transcription of Hh responsive genes by itself. Gli1 is upregulated in pancreatic cancer tissues. Its expression level is positively correlated with Hh signaling (Liu et al., 2015). FoxM1, another Gli target gene, acts as a cell cycle regulator and plays an important role in carcinogenesis (Wang et al., 2017; Wierstra, 2013). Z-Ajoene down-regulated Gli target proteins such as Gli1, Gli2, Ptch, and FoxM1 protein levels whereas basal levels of Gli1 and FoxM1 were elevated in PANC-1 cells (Fig. 2C). The level of FoxM1 was more susceptible than Gli1 level by treatment with 10 μM Z-ajoene. This means that Z-ajoene might have another mechanism for modulating FoxM1 protein level besides the Gli pathway. Further study is needed to determine the molecular target of Z-ajoene so that we could explain the detailed mechanism. Moreover, Zajoene did not alter levels of phosphorylated Akt in PANC-1 cells (Fig. 2C), suggesting that Z-ajoene could specifically regulate the Hh/ Gli signaling pathway without affecting the PI3K/Akt signaling pathway. FoxM1, a downstream target of Gli, has been mainly related to cell cycle regulation and cellular proliferation in tumor (Alvarez-Fernandez & Medema, 2013; Halasi & Gartel, 2013; Teh et al., 2002). Especially, FoxM1 is a regulator of G1/S and G2/M transitions and M-phase progression (Wonsey & Follettie, 2005). It controls cell cycle-related molecules such as c-Myc (Wierstra & Alves, 2006), cyclin B1 (Leung et al., 2001) and survivin (Nestal de Moraes et al., 2015). c-Myc, an oncoprotein, is responsible for cellular proliferation and cell cycle regulation of cancer cells. c-Myc expression is elevated and deregulated in neoplastic cells (Albihn, Johnsen, & Henriksson, 2010; DePinho, SchreiberAgus, & Alt, 1991). Cyclin B1, another target of FoxM1, is usually expressed at very low levels. It accumulates sharply only at G2/M cell cycle transition under normal conditions. In cancer cells, cyclin B1 is overexpressed throughout cell cycle progression (Ye, Wang, Wu, Li, & Chai, 2017). Survivin, an anti-apoptotic protein, plays a role in the regulation of cell division and spindle formation. It is essential for cell cycle progression (Mita, Mita, Nawrocki, & Giles, 2008). Overexpression of survivin is frequently associated with poor prognosis and drug resistance in neoplastic mammary cells (Hinnis, Luckett, & Walker, 2007). As shown in Fig. 3, Z-ajoene suppressed target gene expression of FoxM1, c-Myc, cyclin B1, and survivin, together with depressed proliferation of PANC-1 pancreatic cancer cells (Fig. 4A). These results are in agreement with down-regulation of FoxM1 by Z-ajoene through inhibition of Gli activation. Furthermore, cell cycle analysis demonstrated that Z-ajoene significantly elevated the percentage of G2/M phase cells (Fig. 4B). These results suggest that Z-ajoene can reduce the proliferation of PANC-1 pancreatic cancer cells through inducing cell cycle arrest in G2/M phase. Taken together, Z-ajoene exhibits antiproliferative activity in pancreatic cancer cells by down-regulating Gli in aberrantly activated Hh signaling pathway. Therefore, inhibition of Gli is a well-organized approach to regulate the Hh signaling pathway
2002). Taken together, Z-ajoene could reduce proliferation of PANC-1 pancreatic cancer cell by inducing G2/M cell cycle arrest through down-regulation of Gli-FoxM1 signaling pathway. In response to Shh ligand binding, Ptch abrogates the suppression of Smo, resulting in nuclear accumulation of Gli and initiation of Glimediated transcription. Ectopic expression of Shh has been reported to be sufficient to induce multiple carcinomas including pancreatic cancer whose Hh signaling pathway is dysregulated by Shh dependent-Gli activation (Fendrich et al., 2011a; Rimkus, Carpenter, Qasem, Chan, & Lo, 2016). However, inhibition of Gli could be a worthy approach to control several cancer types through upregulation of Hh signaling pathway by overexpression of Shh ligands (Supplementary material Fig. S1). Thus, Z-ajoene might be useful for targeting Gli on pancreatic cancers that are driven by ectopic activation of Hh signaling pathway. 4. Discussion Garlic has been consumed as a spice and a folk medicine since ancient times for cardio-protective and health benefits as well as for treatment of cancers (Adaki, Adaki, Shah, & Karagir, 2014; Block et al., 1984; Khatua, Adela, & Banerjee, 2013). Z-Ajoene, an organosulfur compound derived from allicin, has been isolated from garlic (Allium sativum). Biological activities of Z-ajoene include inhibition of platelet activation, enhancement of host defense against mycobacteria, and suppression of cancer cell proliferation (Choi et al., 2018; Jung et al., 2014; Teranishi, Apitz-Castro, Robson, Romano, & Cooper, 2003). Several plant-derived molecules such as resveratrol, curcumin, zerumbone, and physalin B have been reported as modulators of Hh/Gli signaling pathway (Hosoya et al., 2008; Mohapatra et al., 2015). Our previous studies have demonstrated that a sesquiterpene lactone and diarylheptanoids from Siegesbeckia glabrescens, Alpinia officinarum and Alnus japonica can inhibit the Hh/Gli signaling pathway (Dong et al., 2017; Lee et al., 2016). Herein, we report that Z-ajoene (Fig. 1A) from garlic can also affect the Hh/Gli signaling pathway. While tightly regulated Hh pathway is responsible for coordinating cellular growth and embryo development, dysregulation of Hh pathway is involved in the formation, development, and aggressiveness of tumors, especially pancreatic cancer (Lauth & Toftgard, 2011; Xie, Bartels, Barton, & Gu, 2013). Hh pathway is activated with a series of signal cascade such as Shh ligand binding to Ptch, Smo activation and initiation of Gli-mediated transcription. Recent evidence suggests that constitutive activation of Gli is associated with tumor formation and growth (Kasper, Regl, Frischauf, & Aberger, 2006; Pietrobono et al., 2018). Although Smo antagonists have been reported as therapuetic candidates for various cancers (Fendrich et al., 2011b; Ibuki et al., 2013), their therapeutic efficacies in preclinical and clinical cancer models are not promising (Rimkus et al., 2016). Recent studies have shown that acquired resistance to Smo inhibition is linked to mutations in Smo and aberrant activation of Gli or upregulation of synergic signals such as PI3K signaling (Benvenuto et al., 2016; Lauth & Toftgard, 2007). Resistance against Smo antagonist (vismodegib) has already been reported in patients with advanced or metastatic solid tumors (Atwood, Chang, & Oro, 2012; Gonnissen, Isebaert, & Haustermans, 2015; LoRusso et al., 2011). Another study has reported that GANT 61 (a Gli inhibitor) shows therapeutic effects on colon cancer cells that are resistance to Smo inhibitors (cyclopamine or GDC-0449) (Agyeman, Mazumdar, & Houghton, 2012). Therefore, the inhibition of Gli has been recognized as an efficient strategy to regulate the Hh signaling pathway for cancer therapy. In our reporter assay system, C3H10T1/2-Gli1-Luc cells were incubated with Shh CM to induce Gli1-mediated luciferase activity (Fig. 1B). Z-Ajoene inhibited Shh CM-induced luciferase activity in a concentration-dependent manner as seen in Fig. 1C. Z-Ajoene also inhibited SAG (a Smo activator)-induced luciferase activity (Fig. 1D). These findings suggest that Z-ajoene could modulate the activation of 107
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for cancer therapy.
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5. Conclusion Modulation of aberrant overexpression of Gli level in the Hh pathway is a promising strategy to develop therapeutic agent for cancer. Z-Ajoene from garlic can modulate the Hh/Gli signaling pathway by reducing Gli transcriptional activity in Shh activated C3H10T1/2 cells as well as in PANC-1 pancreatic cancer cells. Z-Ajoene down-regulates expressions of Gli-target proteins and FoxM1-target proteins in PANC-1 cancer cells. This compound exhibits anti-proliferative activity against PANC-1 pancreatic cancer cells by inducing G2/M cell cycle arrest through down-regulation of Gli/FoxM1. Thus, ZAjoene from garlic might be a valuable candidate for developing new therapeutic agents against pancreatic cancer accompanying aberrant activation of Hh/Gli signaling pathway. Ethics statements file This research does not include any human subjects and animal experiments. Conflict of interest The authors declare no conflict of interest. Acknowledgements This study was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MSIP) (Nos. 2011-0030074 and 2018R1D1A3B07050361) and Sookmyung Women’s University Specialization Program Funding. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jff.2019.03.010. References Aberger, F., & Ruiz, I. A. A. (2014). Context-dependent signal integration by the GLI code: The oncogenic load, pathways, modifiers and implications for cancer therapy. Seminars in Cell & Developmental Biology, 33, 93–104. https://doi.org/10.1016/j. semcdb.2014.05.003. Adaki, S., Adaki, R., Shah, K., & Karagir, A. (2014). Garlic: Review of literature. Indian Journal of Cancer, 51, 577–581. https://doi.org/10.4103/0019-509X.175383. Agren, M., Kogerman, P., Kleman, M. I., Wessling, M., & Toftgard, R. (2004). Expression of the PTCH1 tumor suppressor gene is regulated by alternative promoters and a single functional Gli-binding site. Gene, 330, 101–114. https://doi.org/10.1016/j. gene.2004.01.010. Agyeman, A., Mazumdar, T., & Houghton, J. A. (2012). Regulation of DNA damage following termination of Hedgehog (HH) survival signaling at the level of the GLI genes in human colon cancer. Oncotarget, 3, 854–868. https://doi.org/10.18632/ oncotarget.586. Albihn, A., Johnsen, J. I., & Henriksson, M. A. (2010). MYC in oncogenesis and as a target for cancer therapies. Advances in Cancer Research, 107, 163–224. https://doi.org/10. 1016/S0065-230X(10)07006-5. Alvarez-Fernandez, M., & Medema, R. H. (2013). Novel functions of FoxM1: From molecular mechanisms to cancer therapy. Frontiers in Oncology, 3, 30. https://doi.org/ 10.3389/fonc.2013.00030. Armas-Lopez, L., Zuniga, J., Arrieta, O., & Avila-Moreno, F. (2017). The Hedgehog-GLI pathway in embryonic development and cancer: Implications for pulmonary oncology therapy. Oncotarget, 8, 60684–60703. https://doi.org/10.18632/oncotarget. 19527. Atwood, S. X., Chang, A. L., & Oro, A. E. (2012). Hedgehog pathway inhibition and the race against tumor evolution. Journal of Cell Biology, 199, 193–197. https://doi.org/ 10.1083/jcb.201207140. Bai, C. B., & Joyner, A. L. (2001). Gli1 can rescue the in vivo function of Gli2. Development, 128, 5161–5172. http://dev.biologists.org/content/128/24/5161. Benvenuto, M., Masuelli, L., De Smaele, E., Fantini, M., Mattera, R., Cucchi, D., et al. (2016). In vitro and in vivo inhibition of breast cancer cell growth by targeting the Hedgehog/GLI pathway with SMO (GDC-0449) or GLI (GANT-61) inhibitors. Oncotarget, 7, 9250–9270. https://doi.org/10.18632/oncotarget.7062.
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