Sanguinarine inhibits pancreatic cancer stem cell characteristics by inducing oxidative stress and suppressing sonic hedgehog-Gli-Nanog pathway
Abstract
Sonic hedgehog pathway is highly activated in pancreatic cancer stem cells (CSC) which play crucial roles in cancer initiation, progression and metastasis. However, the molecular mechanisms by which sanguinarine regulates pancreatic CSC characteristics is not well understood. The objectives of this study were to examine the molecular mechanisms by which sanguinarine regulates pancreatic CSC characteristics. Sanguinarine inhibited cell proliferation and colony formation and induced apoptosis through oxidative damage. Sanguinarine inhibited self-renewal capacity of pancreatic CSCs isolated from human and KrasG12D mice. Furthermore, sanguinarine suppressed epithelial-mesenchymal transition (EMT) by up-regulating E-cadherin and inhibiting N-cadherin. Significant decrease in expression level of Snail, Slug and Zeb1 corroborated the suppression of EMT in sanguinarine treated pancreatic CSCS. The ability of sanguinarine to inhibit pluripotency maintaining factors and CSC markers suggest that sanguinarine can be an effective agent for inhibiting pancreatic cancer growth and development by targeting CSCs. Furthermore, sanguinarine inhibited Shh-Gli pathway leading to modulation of Gli target genes in pancreatic CSCs. Chromatin immunoprecipitation assay demonstrated that Nanog directly binds to promoters of Cdk2, Cdk6, FGF4, c-Myc and Oct4, and sanguinarine inhibits the binding of Nanog with these genes, suggesting the direct involvement of Nanog in cell cycle, pluripotency and self-renewal. To further investigate the role of Shh-Gli-Nanog pathway, we regulated Shh signaling either by Shh protein or Nanog overexpression. Enforced activation of Shh or overexpression of Nanog counteracted the inhibitory effects of sanguinarine on pancreatic CSC proliferation, suggesting the actions of sanguinarine are mediated, at least in part, through Shh-Gli-Nanog pathway. Our studies suggest that sanguinarine can be used for the treatment and/or prevention of pancreatic cancer by targeting CSCs.
Introduction
Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease and is the fourth leading cause of cancer-related death in the USA (1). The incidence of PDAC is increasing and is expected to be the second leading cause of cancer death in the USA by the year 2030 (2). With an overall 5-year survival rate of only 6% (3,4), pancreatic cancer has one of the poorest prognoses among all cancers (5). It is clinically apparent at late stages and resists all forms of conventional chemotherapy and radiotherapy (6,7). Although in recent years significant progress has been made in the areas of diagnosis and therapeutics, enhanced drug toxic- ity, poor bioavailability and undesirable side effects to normal tissues are major challenges for the effective management of PDAC patients. Therefore, understanding pancreatic carcinogen- esis and developing effective strategies to prevent and/or treat pancreatic neoplasms by nontoxic agents are urgently needed.
Sanguinarine is an isoquinoline alkaloid derived from the root of Sanguinaria canadensis and other poppy fumaria species (8–10). It exerts a broad spectrum of properties such as anti- oxidant, anticancer, antiviral and anti-inflammatory activities (11–14). It induces apoptosis by inducing oxidative stress, dam- aging mitochondria and activating caspases (12,15). However, the molecular mechanisms by which it inhibits pancreatic car- cinogenesis by targeting cancer stem cells (CSCs) have never been examined. Thus, it holds great promise for development as a chemopreventive or therapeutic agent.
In recent years, several studies have highlighted the sig- nificance of CSCs/progenitor cells in pancreatic cancer because they appear to be the cause of cancer initiation, progression, metastasis and chemotherapy failure (6). Recent findings of the activated Sonic hedgehog (Shh) signaling pathway in pan- creatic cancer have underlined its role as a target for treatment and prevention of PDAC (16,17). A better understanding of the biological behavior of CSCs may further improve therapeutic approaches and outcomes in patients with this devastating dis- ease. Our studies described here are very novel, and significant because we examine the molecular mechanisms by which san- guinarine inhibits pancreatic CSC characteristics, and assess the preclinical potential of sanguinarine for the treatment and/or prevention of pancreatic cancer.Hedgehog (Hh) pathway is one of the crucial stem cell signaling pathways that regulate embryonic development and adult tissue repair (18). The Hh signaling pathway regulates embryonic growth and patterning in organisms ranging from insects to mammals
(18). Hh signaling also regulates tissue homeostasis by control- ling the populations of stem or progenitor cells (19). Inappropriate activity of the Hh signaling pathway has also been linked to tumor growth, development and metastasis (18). In the absence of Hh ligand binding, the receptor Patched (PTCH1) repress the transmembrane G-protein coupled receptor Smoothened (SMO). Binding of one of the ligands Sonic hedgehog (Shh), Indian hedge- hog (Ihh), or Desert hedgehog (Dhh)] to PTCH1 releases SMO to induce Hh pathway activation via intracellular signaling cascade (20,21). As a result of pathway activation, the transcription factors glioma-associated oncogenes 1–3 (GLI1, GLI2 and GLI3) transmit the cytoplasmic signal and induce transcription of proliferative and antiapoptotic target genes such as GLI1, PTCH1, hedgehog interacting protein (HHIP), Bcl-2 and Cyclin D1 (22).
Pathway activation via Smo thus can occur either by Hh protein stimulation or through loss of Ptch activity. Constitutive pathway activation resulting from mutations of pathway components is frequently detected in some cancers (23,24). Excessive positive feedback or collapsed negative feedback of Hh signaling due to epigenetic or genetic alterations can lead to carcinogenesis. Activation of Hh pathway can induce markers of stem cells (BMI1, LGR5, CD44 and CD133), cell proliferation and survival (XIAP and Bcl2), cell cycle (CyclinD1) and epithelial-to-mesenchymal transition and metas- tasis (Snail, Slug, ZEB1, ZEB2 and TWIST2) (19,25). These studies highlight the importance of Shh-Gli pathway in cancer initiation, progression, metastasis and tumor growth.The main objective of this study is to examine the molec- ular mechanisms by which sanguinarine inhibits pancreatic CSCs characteristics by modulating Shh-Gli-Nanog pathway. The studies described will not only fill the gap in our knowledge but also guide us in formulating strategies for prevention and/ or treatment of human pancreatic cancer by targeting CSCs. Our data indicate that sanguinarine inhibits self-renewal capacity of pancreatic CSCs and cell proliferation of pancreatic cancer cell lines. Sanguinarine is nontoxic to human pancreatic nor- mal ductal epithelial cells (HPNE) and notably inhibits invasion and migration. Further, sanguinarine inhibits the expression of pluripotency promoting factors and stem cell markers in CSCs. Furthermore, our data demonstrate that sanguinarine inhibits Shh-Gli pathway and Nanog transcription and expression. In conclusion, sanguinarine can be used for the treatment and pre- vention of pancreatic cancer by eliminating CSCs.
Antibodies against CD24, CD44, CD133, Nanog, Oct4, Sox2, KLF4, c-Myc, Gli1, Gli2, Patched 1, Patched2, Smoothened, Bcl-2, Cyclin D1, N-Cadherin, E-cadherin, Snail, Slug and ZEB1 were purchased from Cell Signaling Technology (Danvers, MA). Shh protein and anti-β-actin antibody were pur- chased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Sanguinarine chloride hydrate (SCH) (98% pure) was purchased from the LKT (St. Paul, MN). Accutase was purchased from Innovative Cell Technologies, Inc. (San Diego, CA). Matrigel was purchased from BD Bioscience (San Jose, CA). Crystal violet was purchased from Sigma–Aldrich (St. Louis, MO). TRIZOL was purchased from Invitrogen (Grand Island, NY).Pancreatic cancer cell lines (AsPC-1 and PANC-1) and HPNEs were pur- chased from American Type Culture Collection (Manassas, VA). Human pancreatic CSCs were isolated from primary tumors, and they were posi- tive for CD133+/CD44+/CD24+/ESA+, as described previously (26). The isola- tion and characterization of pancreatic CSCs isolated from KrasG12D mice have been described elsewhere (26). Specialized pancreatic CSC growth medium (Celprogen, Inc., Torrance, CA) containing 1% N2 Supplement (Invitrogen), 2% B27 Supplement (Invitrogen), 20 ng/ml human plate- let growth factor (Sigma–Aldrich), 100 ng/ml epidermal growth factor (Invitrogen) and 1% antibiotic-antimycotic (Invitrogen) was used. Cells were cultured at 37°C in a humidified atmosphere of 95% air and 5% CO2.
Cells (1.5 × 104) were treated with various doses of sanguinarine (0–1 µM) for 48 h. Cell viability was determined by trypan blue assay. Apoptosis was measured by terminal deoxynucleotidyl transferase dUTP nick end labe- ling (TUNEL) assay as described elsewhere (27).
Spheroid formation assays were performed as described elsewhere (27). In brief, cells were plated in six-well ultralow attachment plates (Corning, Inc., Corning, NY) at a density of 1000 cells/ml in complete pancreatic CSC medium at 37°C with 95% air and 5% CO2. Spheroids were collected after 7 days and dissociated with Accutase. Cell viability in spheroids was counted using trypan blue assay.Cell motility assay was performed as described elsewhere (27). In brief, a scratch was made through the monolayer, using a standard 200 μl plastic pipette tip, which gave rise to an in vitro wound. Plates were washed twice with PBS and filled with culture medium in the presence or absence of sanguinarine. The width of the scratch gap was viewed under the micro- scope in four separate areas each day until the gap was completely filled in the untreated control wells. For each experimental condition, we have used three replicate wells.For transwell migration assays, 1 × 105 cells were plated in the top cham- ber onto the noncoated membrane (24-well insert; pore size, 8 μm) and allowed to migrate toward serum-containing medium in the lower cham- ber. Cells were fixed after 24 h of incubation with methanol and stained with 0.1% crystal violet (2 mg/ml, Sigma–Aldrich). The number of cells invading through the membrane was counted under a light microscope (three random fields per well).
For invasion assay, 1 × 105 cells were plated in the top chamber con- taining matrigel-coated membrane (24-well insert; pore size, 8 μm). Each well was freshly coated with matrigel (60 μg; BD Bioscience). Cells were plated in medium without serum or growth factors, and medium supple- mented with serum was used as a chemoattractant in the lower cham- ber. The cells were incubated for 48 h. Cells on the lower surface of the membrane were fixed with methanol and stained with crystal violet. The number of cells invading through the membrane was counted under a light microscope.The western blot analysis was performed as we described earlier (28). In brief, cell lysates were subjected to SDS-PAGE, and gels were blotted on nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ). The membranes were blocked with 5% bovine serum albumin in Tris-Tween buffered saline at 37°C for 2 h and then incubated with primary anti- body diluted in Tris-buffered saline (1:1000 dilutions) overnight at 4°C, with gentle shaking. The membranes were then washed three times with Tris-buffered saline-T (TBS-T) and incubated with secondary antibody linked to horseradish peroxidase (1:5000) for 1 h. After incubation with secondary antibody, the membranes were washed again three times with TBS-T. Finally, protein–antibody complexes were detected by the addi- tion of enhanced chemiluminescence substrate (Thermo Fisher Scientific, Rockford, IL).Gli and Nanog reporter activities were measured as we described else- where (29). In brief, lentiviral particles expressing cop-GFP and luciferase genes (pGreen Fire1-4xGli-mCMV-EF1-Neo) were prepared. Cells were stably transduced with lentiviral particles. For transcription assay, cells (5–10 000 cells per well) were seeded in 96-well plates and treated with or without sanguinarine (0–1 µM) for various time points. At the end of incubation period, luciferase reporter activity was measured as per the manufacturer’s instructions (Promega Corp., Madison, WI).
Pancreatic CSCs were fixed with 1% formaldehyde for 15 min (RT), quenched with 125 mM glycine for 5 min (RT), centrifuged and resus- pended in RIPA Buffer containing protease inhibitors and incubated on ice (10 min). Samples were sonicated (Heat Systems-Ultrasonic device) to shear chromatin to an average length of about 1 Kb and transferred to 1.5 ml tubes, microcentrifuged for 10 min (max speed). Supernatants were collected in 1.5 ml tubes containing 1 ml of the dilution buffer (0.01% SDS, 1.1% Triton, 1.2 mM ethylenediaminetetraacetic acid (EDTA), 167 mM NaCl, 17 mM Tris, pH 8). Three micrograms of Nanog antibodies were added to each samples, samples were incubated overnight at 4°C, followed by addi- tion of 5 μl of protein-A and protein-G magnetic beads (Invitrogen) for 2 h. Beads were collected with a magnet (Thermo), washed 4 times with 1 ml of each of four wash buffers (wash buffer 1: 0.1% SDS, 1% Triton, 2 mM EDTA, 150 mM NaCl, 20 mM Tris, pH 8; wash buffer 2: 0.1% SDS, 1% Triton, 2 mM EDTA, 500 mM NaCl, 20 mM Tris, pH 8; wash buffer 3: 0.25 M LiCl, 1% NP-40,1% deoxycholate, 1 mM EDTA, 10 mM Tris, pH 8; wash buffer 4:10 mM Tris, pH 8, 1 mM EDTA). After the last wash, 50 μl of a 10% Chelex-100 (Bio-Rad) resin solution was added to the beads, samples were boiled for 10 min, and microcentrifuged for 1 min at 6000 g. After collecting the supernatant, 50 μl of MQ water was added back to the beads, microcentrifuged again 1 min at 6000 g, and the new supernatant was pooled with the previous one. One to three microliters of elutions were used for PCR reaction.Each experiment was performed at least three times. Differences between groups were analyzed by Student’s t-test or ANOVA using the GraphPad Prism software. A statistically significant difference among groups was defined as P < 0.05. Results We have characterized pancreatic CSCs isolated from human and mice pancreata as described elsewhere (26). These pan- creatic CSCs express high levels of stem cells markers (CD133, CD44, CD24 and ESA), pluripotency maintaining factors (Nanog and Oct4) and drug resistance genes (MDR1 and ABCG2) as compared with normal pancreatic epithelial cells and pancre- atic cancer cells (26). We further showed that the CSCs with CD133+CD44+CD24+ESA+ immunophenotype were highly tumo- rigenic and formed tumors with 50 cells when sc injected into NOD/SCID mice as compared with CD133−CD44−CD24−ESA− cells, where no tumors were formed (26).We first examined the effects of sanguinarine on cell prolif- eration in pancreatic CSCs (CD133+CD44+CD24+ESA+), and cancer cell lines (AsPC-1 and PANC-1). Sanguinarine inhibited cell pro- liferation of pancreatic CSCs and cell lines in a dose-dependent manner (Figure 1A). By comparison, sanguinarine had no effect on cell viability of HPNE. These data suggest that sanguinarine is effective in inhibiting the growth of pancreatic CSCs and cell lines, and exerts no toxicity to normal HPNE cells.Colony formation assay is commonly used to assess the anti- cancer activity of a drug. We, therefore, measured the effects of sanguinarine on colony formation by pancreatic CSCs and can- cer cell lines. Cells were seeded and treated with various doses of sanguinarine for 21 days. Dishes were photographed to visu- alize the colonies, and a number of colonies formed in each dish were also counted. As shown in Figure 1B and C, sanguinarine inhibited colony formation by pancreatic CSCs and cell lines in a dose-dependent manner. We next examined the effects of sanguinarine on apoptosis. Sanguinarine-induced apoptosis in pancreatic CSCs and cell lines in a dose-dependent manner (Figure 2A and C). These data collectively suggest that sanguinarine exerts anticancer activity by inhibiting cell proliferation and colony formation, and induc- ing apoptosis. Since sanguinarine inhibited cell proliferation and induced apoptosis, we next sought to examine the involvement of oxi- dative damage in this process. N-acetyl-L-cysteine (NAC) and glutathione (GSH) act as antioxidants. Pancreatic CSCs were pre- treated with either NAC (5 mM) or GSH (1 mM) for 2 h, followed by treatment with sanguinarine (0.5 μM) for 48 h (Figure 2D and E). At the end of incubation period, viable cells were counted. Sanguinarine inhibited the proliferation of pancreatic CSCs. While NAC or GSH alone was ineffective, pretreatment with NAC or GSH diminished antiproliferative effects of sanguinar- ine. These studies demonstrate the role of oxidative damage in mediating antiproliferative effects of sanguinarine where pre- treatment with the thiol antioxidants NAC and GSH diminished the antiproliferative effects of sanguinarine.We next examined whether GDC-0449/Vismodegib (smooth- ened inhibitor) exerts inhibitory effects on CSC’s proliferation similar to that of sanguinarine. GDC-0449 inhibited prolif- eration of pancreatic CSCs (Figure 2F). As shown above, treat- ment of CSCs with NAC was ineffective on CSC’s proliferation. Pretreatment of pancreatic CSCs with NAC diminished antipro- liferative effects of GDC-0449. These studies suggest that both agents sanguinarine and GDC-0449 inhibit CSC’s proliferation by activating oxidative damage pathway.Sanguinarine inhibits spheroid formation in pancreatic CSCs isolated from human and KrasG12D mice, expression of stem cell markers and pluripotency maintaining factors. The ability of CSCs to form spheroids in suspension is the major characteristics of stemness (26). We next examined the effects of sanguinarine on the growth of pancreatic CSCs by measuring cell viability in spheroids and colony formation. Sanguinarine inhibited cell viability of primary, secondary and tertiary human (Figure 3A) and mouse spheroids (Figure 3B). Sanguinarine treated groups formed smaller and fewer spheroids compared with control group (data not shown).The expression levels of CSC markers may provide an indica- tion of the effectiveness of an anticancer drug. We, therefore, sought to measure the effects of sanguinarine on markers of pancreatic CSCs by western blot analysis and quantitative real time PCR (q-RT-PCR) (Figure 3C). Protein expression was meas- ured at 48 h, and mRNA expression was measured at 12, 24 and 36 h of treatments. Sanguinarine inhibited the expression of CD24, CD44 and CD133 markers at both protein and mRNA levels. These data suggest that sanguinarine can suppress the tumor mass by targeting pancreatic CSCs, as it inhibited the spheroid formation and the expression of CSC’s markers.Since pluripotency maintaining factors are highly expressed in CSCs, we examined the effects of sanguinarine on the expres- sion of Nanog, Oct4, Sox2, KLF4 and c-Myc in pancreatic CSCs by western blot analysis and q-RT-PCR (Figure 3D). Sanguinarine inhibited the expression of Nanog, Oct4, Sox2, KLF4 and c-Myc at both protein and mRNA levels. Based on these findings, it can be suggested that sanguinarine can regulate self-renewal capacity and pluripotency of pancreatic CSCs. Aberrant activation of Shh signaling pathway plays a sig- nificant role in pancreatic cancer growth, development and metastasis (18,30,31). We, therefore, examined the effects of sanguinarine on the components (Gli1, Gli2, Patched 1, Patched 2 and Smoothened) of Shh pathway by western blot analysis and q-RT-PCR. Sanguinarine inhibited the expression of Gli1, Gli2, Patched 1, Patched 2 and smoothened at both protein and mRNA levels (Figure 4A). Since Gli transcription is regulated by Shh pathway, we measured Gli transcription in pancreatic CSCs by luciferase reporter assay. Sanguinarine inhibited Gli reporter activity in a dose-dependent manner (Figure 4B). We next measured the effects of sanguinarine on the expression of Gli targets which control apoptosis and cell cycle. Sanguinarine inhibited the expression of Bcl-2 and cyc- lin D1 at both protein and mRNA levels (Figure 4C). Similar to pancreatic CSCs, sanguinarine inhibited Gli reporter activities in AsPC-1 and PANC-1 cell lines in a dose-dependent manner (Figure 4D).Since sanguinarine inhibited self-renewal capacity of pan- creatic CSCs by suppressing Gli expression and transcription, we next examined whether hyperactivation of Shh pathway by Shh protein counteracts the inhibitory effects of sanguinarine on cell proliferation. Incubation of CSCs with Shh protein slightly enhanced cell proliferation (Figure 4E). Furthermore, Shh protein diminished the inhibitory effects of sanguinarine on cell prolif- eration. These data suggest that sanguinarine can inhibit self- renewal capacity of pancreatic CSCs by targeting Shh pathway. Sanguinarine inhibits binding of Nanog to its target genes (Cdk2, Cdk6, FGF4, c-Myc and Oct4) and Nanog transcription, and overexpression of Nanog counteracts the inhibitory effects of α-sanguinarine on cell proliferation.Nanog is a transcription factor which regulates self-renewal and pluripotency (6,32). We have shown that Nanog is highly expressed in pancreatic CSCs and cell lines. Chromatin immu- noprecipitation assays are performed to examine the binding of a transcription factor to its targets. We, therefore, employed chromatin immunoprecipitation assay to examine the binding of Nanog to the promoters of Cdk2, Cdk6, FGF4, c-Myc and Oct4, and also assess whether Sanguinarine inhibits the binding of Nanog to its target genes. As shown in Figure 5A, Nanog can bind to Cdk2, Cdk6, FGF4, c-Myc and Oct4 target genes. In addi- tion, sanguinarine inhibited the binding of Nanog to Cdk2, Cdk6, FGF4, c-Myc and Oct4 genes. These data suggest that sangui- narine can regulate pluripotency-, cell survival- and cell cycle- related genes through Nanog.Nanog is a direct transcriptional target of Gli (33). We next examined the effects of sanguinarine on Nanog transcription by measuring luciferase reporter activity. Sanguinarine inhibited Nanog reporter activity in pancreatic CSCs, AsPC-1 and PANC-1 cell lines (Figure 5B–D). Overall, these data suggest that Nanog can regulate cell cycle, self-renewal and pluripotency of pan- creatic CSCs by modulating the expression of Cdk2, Cdk6, FGF4, c-Myc and Oct4. Since one of the direct targets of Gli is a transcription factor Nanog. We next examined whether overexpression of Nanog can counteract the inhibitory effects of sanguinarine on pancreatic CSC proliferation. Pancreatic CSCs were transduced with lenti- viral particles expressing either empty vector or Nanog cDNA. Transduced pancreatic CSCs were treated with sanguinarine to measure cell proliferation. Sanguinarine inhibited cell pro- liferation of pancreatic CSC/Empty vector group (Figure 5E). Overexpression of Nanog counteracted the inhibitory effects of sanguinarine on cell proliferation in pancreatic CSC/Nanog cDNA CSCs. Overall, these data suggest that sanguinarine exerts its biological effects through inhibition of Shh-Nanog pathway which regulates pluripotency and self-renewal of pancreatic CSCs. Sanguinarine inhibits cell motility, migration and invasion and markers of epithelial-mesenchymal transition .Epithelial-mesenchymal transition (EMT) is a biological process by which cells undergo genetic changes that allow them to leave the primary site and migrate to a distant location (secondary site) to reestablish and proliferate (34). Since CSCs have been shown to be the cause of metastasis, we next measured the effects of sanguinarine on cell motility, migration, invasion and on the expression of EMT markers. Sanguinarine inhibited cell motility, invasion and migration of pancreatic CSCs (Figure 6B and C). Similarly, sanguinarine inhibited cell migration and invasion in pancreatic cancer AsPC-1 and PANC-1 cell lines (data not shown).Since sanguinarine showed inhibitory effects on cell motil- ity, migration and invasion, we next examined its effects on the expression of EMT regulators by western blot analysis and q-RT-PCR. Transcription factors Snail, Slug and Zeb1 regulate the expression of cadherins. As shown in Figure 6D and E, sangui- narine induced the expression of E-cadherin and inhibited the expression of N-Cadherin, and transcription factors (Snail, Slug and ZEB1) at both protein and mRNA levels. These data suggest that sanguinarine has potential to inhibit EMT by inducing ‘cad- herin switch’ and inhibiting EMT-related transcription factors. Discussion To the best of our knowledge, it is a first study demonstrating the anticancer activity of sanguinarine against pancreatic cancer by targeting Shh-Gli-Nanog pathway. Sanguinarine inhibited Shh pathway which controls cell cycle, EMT/metastasis, prolif- eration and stemness. Our data demonstrate that sanguinarine can inhibit cell proliferation and colony formation and induce apoptosis in pancreatic CSCs and cell lines. By comparison, san- guinarine had no effect on HPNE, suggesting the specificity of sanguinarine towards cancer cells and CSCs. Furthermore, san- guinarine inhibited pancreatic CSC characteristics by modulat- ing the expression of pluripotency maintaining factors, stem cell markers and EMT. Our study suggests that sanguinarine can be developed as a useful agent for the treatment and prevention of pancreatic cancer.CSCs and stem cells share similar phenotypic markers. We have characterized pancreatic CSCs isolated from human and KrasG12D and KPC mice (26). These pancreatic CSCs express high levels of pluripotency maintaining factors (Nanog, Sox2, Klf4, c-Myc and Oct4), stem cells markers (CD133, CD44, CD24 and ESA) and drug resistance genes (MDR1 and ABCG2) as compared with normal pancreatic epithelial cells and pancreatic cancer cells (26). We also demonstrated that CD133+CD44+CD24+ESA+ CSCs were highly tumorigenic and formed tumors when sc injected into NOD/SCID mice as com- pared with CD133−CD44−CD24−ESA− cells, where no tumors were formed (26). These data suggest we should design our strategies to target pancreatic CSCs which are responsible for drug resistance and cancer relapse.Our studies have demonstrated the role of oxidative dam- age in mediating antiproliferative effects of sanguinarine where pretreatment with the thiol antioxidants NAC and GSH abro- gated the antiproliferative activity of sanguinarine. Similarly, others have demonstrated the role of oxidative stress in sangui- narine-induced cell death (15). Sanguinarine inhibits vascular endothelial growth factor release by the generation of reac- tive oxygen species in human breast cancer (35). Sanguinarine induces apoptosis in human pancreatic cancer, gastric carci- noma, prostate cancer, skin cancer and neuroblastoma (36–41). Furthermore, sanguinarine demonstrated protective effects in ultraviolet B-mediated skin cancer (42). Sanguinarine sensitizes human gastric adenocarcinoma cells to TRAIL-mediated apop- tosis via down-regulation of AKT and activation of caspase-3 (38). These studies suggest that sanguinarine can induce cell death and also can be a protective agent against radiation- induced damage. We detected the expression of components of Hh pathway Gli1, Gli2, Patched 1, Patched 2 and smoothened in pancre- atic CSCs. The expression of all these proteins was inhibited by sanguinarine. It has been shown that Shh signaling could protect neurons against oxidative stress by increasing super- oxide dismutase 1 (SOD1) activity. Cyclopamine, the classical inhibitor of Shh signaling, aggravated brain damage associated with the down-regulation of Gli1, Ptch1 and SOD1 expression in acute ischemic stroke (43). Target genes responding to Hh pathway stimulation have been shown to be dependent on context and cell type. We observed the inhibition of Gli targets, Bcl-2 and Cyclin D1 expression, by sanguinarine, suggesting that sanguinarine can regulate cell survival, apoptosis and cell cycle in pancreatic CSCs. Furthermore, in the present study, we discovered a new mechanism by which sanguinarine inhibited pluripotency and self-renewal capacity of pancreatic CSCs iso- lated from humans and KrasG12D mice. Sanguinarine not only inhibited Shh-Gli pathway, similar to GDC-0449 (smoothened inhibitor) by inducing oxidative stress, but also Nanog tran- scription and expression. Nanog is also a direct target of Gli (33). Sanguinarine inhibited the binding of Nanog to the pro- moters of Cdk2, Cdk6, FGF4, c-Myc and Oct4. Nanog along with c-Myc, Klf4, Sox2 and Oct4 regulate stemness. The ability of sanguinarine to inhibit the expression of stem cell mark- ers CD24, CD44 and CD133 suggest that sanguinarine can be effective in regressing tumor by reducing the population of pancreatic CSCs. The positive association between inappropriate Hh path- way activation and cancer suggest that inhibition of the path- way may be a viable therapeutic strategy. Since Shh pathway is highly activated in pancreatic cancer cells but not in normal pancreatic tissues, it is an attractive target for studying and reg- ulating molecular oncogenesis. The modulation of Shh pathway, pluripotency promoting factors and EMT markers in CSCs by sanguinarine offer new hope for the treatment and/or preven- tion of pancreatic cancer by targeting CSCs. Shh is abnormally expressed in pancreatic intraepithelial neoplasia (PanIN) and pancreatic adenocarcinoma. Pancreata of Pdx-Shh mice have been shown to develop abnormal tubular structures, which are similar to human PanIN-1 and -2. Furthermore, these PanIN- like lesions also demonstrated mutations in K-ras and overex- press HER-2/neu, which have been commonly observed early in human pancreatic cancer progression. We have demonstrated that small molecule inhibitors of smoothened or Gli inhibited cell proliferation and induced apoptosis in pancreatic cancer (44–46). Similarly, several cancer preventive agents inhibited pancreatic cancer cell growth in vitro and in vivo by modulat- ing Shh pathway (47–49). Overall, our data underline the scope of Shh pathway in the pancreatic cancer initiation, progression and metastasis and inhibition of Shh signaling by sanguinarine can be useful for the treatment and prevention of pancreatic cancer. Metastasis is a multistage process that requires cancer cells to undergo EMT, escape from the primary tumor, survive in the circulation, seed at distant sites, revert to mesenchymal epithe- lial transition (MET) and grow at the new location/secondary sites (33,34). EMT is generally characterized by class switch from E-cadherin to N-cadherin. Accumulating evidence suggests that EMT plays a crucial role during malignant tumor progression where CSCs gain EMT characteristics. Thus, induction of EMT can regulate metastasis and cancer progression. The present study demonstrates that Shh-Gli-Nanog pathway promotes pancreatic CSC proliferation, stem cell self-renewal and meta- static behavior. Sanguinarine inhibits EMT as demonstrated by inhibition in cell motility, invasion and migration. A significant decrease in expression level of Snail, Slug and Zeb1 corroborated the suppression of EMT in sanguinarine treated pancreatic CSCs. The inhibition of EMT by sanguinarine was associated with cadherin switch (upregulation of E-Cadherin and down-regula- tion of N-Cadherin) in pancreatic CSCs, suggesting a potential inhibitory role of sanguinarine in early metastasis. Since Shh pathway is constitutively active in pancreatic cancer, targeting Gli or Nanog can decrease tumor bulk and eradicate CSCs and metastases. In conclusion, our study has demonstrated for the first time that sanguinarine can inhibit the growth of pancreatic CSCs and cancer cell lines, and inhibit the self-renewal capacity of human and mice CSCs. Sanguinarine not only inhibits Shh pathway, but also Nanog, a direct target of Gli. Nanog plays a role in pluri- potency and self-renewal of CSCs. These data suggest that sanguinarine can inhibit pancreatic carcinogenesis by inhibiting Shh-Gli-Nanog axis, and thus offers a great potential for the treatment and prevention of Sanguinarine pancreatic cancer.