Journal: International journal of cancer

Article Title: Inhibition of sonic hedgehog pathway and pluripotency maintaining factors regulate human pancreatic cancer stem cell characteristics.

doi: 10.1002/ijc.26323

Figure Lengend Snippet: Figure 1. Effects of EGCG on tumor spheroids and cell viability of pancreatic CSCs. (a) Pancreatic CSCs were seeded in suspension and treated with EGCG (0–60 lM) for 7 days. Pictures of spheroids formed in suspension were taken by a microscope. (b) Pancreatic CSCs were seeded in suspension and treated with EGCG (0–60 lM) for 7 days. At the end of the incubation period, sheroids were collected and dissociated with Accutase (Innovative Cell Technologies, Inc.). For secondary spheroids, cells were reseeded and treated with EGCG (0–60 lM) for 7 days. Cell viability was measured by trypan blue assay. Data represent mean 6 SD. *, &, @, or # ¼ significantly different from respective controls, p < 0.05. (c) EGCG inhibits colony formation by CSCs. Pancreatic CSCs were seeded in soft agar and treated with various doses of EGCG and incubated at 4C for 21 days. At the end of incubation period, colonies were counted. Data represent mean 6 SD. *, &, or # ¼ significantly different from respective controls, p < 0.05. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: Human pancreatic CSCs (CD133þ/CD44þ/CD24þ/ESAþ) were obtained from Celprogen Inc. (San Pedro, CA).

Techniques: Suspension, Microscopy, Incubation

Journal: International journal of cancer

Article Title: Inhibition of sonic hedgehog pathway and pluripotency maintaining factors regulate human pancreatic cancer stem cell characteristics.

doi: 10.1002/ijc.26323

Figure Lengend Snippet: Figure 2. Regulation of caspase-3/7 activity, apoptosis and apoptosis-related proteins by EGCG on CSCs derived from human primary pancreatic tumors. (a) Regulation of caspase-3/7 activity by EGCG. CSCs were treated with EGCG (0–60 lM) for 24 hr, and caspase-3/7 activity was measured as per manufacturer’s instructions. Data represent mean 6 SD. *, #, or % ¼ significantly different from control, p < 0.05. (b) Regulation of apoptosis by EGCG. CSCs were treated with EGCG (0–60 lM) for 48 hr, and apoptosis was measured by TUNEL assay. Data represent mean 6 SD. *, #, or % ¼ significantly different from control, p < 0.05. (c) Regulation of apoptosis-related proteins. Pancreatic CSCs were treated with EGCG (0–60 lM) for 36 hr. Real time PCR (q-RT-PCR) was performed to examine the expression of Bcl-2, survivin, XIAP, and GAPDH. Data represent mean 6 SD. *, #, or % ¼ significantly different from control, p < 0.05. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: Human pancreatic CSCs (CD133þ/CD44þ/CD24þ/ESAþ) were obtained from Celprogen Inc. (San Pedro, CA).

Techniques: Activity Assay, Derivative Assay, Control, TUNEL Assay, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: International journal of cancer

Article Title: Inhibition of sonic hedgehog pathway and pluripotency maintaining factors regulate human pancreatic cancer stem cell characteristics.

doi: 10.1002/ijc.26323

Figure Lengend Snippet: Figure 3. Regulation of pluripotency maintaining transcription factors by EGCG in pancreatic CSCs. (a) Pancreatic CSCs were treated with EGCG (0–60 lM) for 36 hr. At the end of the incubation period, cells were harvested and the expression of Nanog, Sox-2, c-Myc and Oct-4 was measured by the q-RT-PCR. Data represent mean 6 SD. *, #, or % ¼ significantly different from respective controls, p < 0.05. (b) Nanog shRNA enhances the inhibitory effects of EGCG on CSC’s spheroid viability. Pancreatic CSCs were transduced with either scrambled shRNA or Nanog shRNA expressing lentiviral vector (pLKO.1), and cell lysates were collected and western blot analysis was performed using anti-Nanog antibody (data not shown). CSC/scrambled and CSC/Nanog shRNA were seeded as described above and treated with EGCG (0–60 lM). After 7 days, spheroids were collected and cell suspensions were prepared and viable cells were counted by trypan blue assay. Data represent mean 6 SD. *, &, @, #, ** or % ¼ significantly different from control, p < 0.05. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: Human pancreatic CSCs (CD133þ/CD44þ/CD24þ/ESAþ) were obtained from Celprogen Inc. (San Pedro, CA).

Techniques: Incubation, Expressing, Reverse Transcription Polymerase Chain Reaction, shRNA, Transduction, Plasmid Preparation, Western Blot, Control

Journal: International journal of cancer

Article Title: Inhibition of sonic hedgehog pathway and pluripotency maintaining factors regulate human pancreatic cancer stem cell characteristics.

doi: 10.1002/ijc.26323

Figure Lengend Snippet: Figure 4. Inhibition of components of SHh pathway, Gli transcription and nuclear translocation by EGCG. (a) Inhibition of components of SHh pathway and Gli transcription. Pancreatic CSCs were treated with EGCG (0–60 lM) for 36 hr. The expression of SMO, PTCH-1 and PTCH-2 was measured by q-RT-PCR. Data represent mean 6 SD. *, #, or % ¼ significantly different from respective controls, p < 0.05. (b) Inhibition of Gli1 and Gli2 expression and Gli transcription. Pancreatic CSCs were treated with EGCG (0–60 lM) for 36 hr. The expression of Gli1 and Gli2 was measured by q-RT-PCR. Gli reporter activity. CSCs were transduced with Gli-responsive GFP/firefly luciferase viral particles (pGreen Fire1-Gli with EF1, System Biosciences). After transduction, culture medium was replaced and CSCs were treated with EGCG (0–60 lM) for 24 hr. Gli- responsive reporter activity was measured by luciferase assay (Promega Corporation). Data represent mean 6 SD. *, #, %, or & ¼ significantly different from respective controls, p < 0.05. (c) EGCG inhibits nuclear translocation of Gli1 and Gli2. Pancreatic CSCs were treated with or without EGCG (40 or 60 lM) for 24 hr. At the end of incubation period, CSCs were fixed with paraformaldehyde, permeabilized with titron 100, and blocked with 5% normal goat serum. Cells were then treated with either anti-Gli1 or anti-Gli2 antibody, followed by secondary antibody plus DAPI. Stained cells were mounted and visualized under a fluorescence microscope. Blue fluorescence of DAPI was changed to red color for a better contrast. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: Human pancreatic CSCs (CD133þ/CD44þ/CD24þ/ESAþ) were obtained from Celprogen Inc. (San Pedro, CA).

Techniques: Inhibition, Translocation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Transduction, Luciferase, Incubation, Staining, Microscopy

Journal: International journal of cancer

Article Title: Inhibition of sonic hedgehog pathway and pluripotency maintaining factors regulate human pancreatic cancer stem cell characteristics.

doi: 10.1002/ijc.26323

Figure Lengend Snippet: Figure 5. Regulation of EMT factors, migration, invasion and TCF/LEF activity by EGCG in pancreatic CSCs. (a) Pancreatic CSCs were treated with EGCG (0–60 lM) for 48 hr. At the end of incubation period, the expression of Snail, ZEB1 and Slug was measured by q-RT-PCR. Data represent mean 6 SD. * ¼ significantly different from respective controls, p < 0.05. (b) Transwell migration assay. Pancreatic CSCs were plated in the top chamber of the transwell and treated with EGCG (0–60 lM) for 24 hr. Cells migrated to the lower chambered were fixed with methanol, stained with crystal violet and counted. Data represent mean 6 SD. *, #, or % ¼ significantly different from respective controls, p < 0.05. (c) Matrigel invasion assay. CSCs were plated onto the Matrigel-coated membrane in the top chamber of the transwell and treated with EGCG (0–60 lM) for 48 hr. Cells invaded to the lower chambered were fixed with methanol, stained with crystal violet, and counted. Data represent mean 6 SD. *, #, or % ¼ significantly different from respective controls, p < 0.05. (d) Effects of EGCG on TCF- 1/LEF activity. Pancreatic CSCs were transduced with TCF/LEF responsive GFP/firefly luciferase viral particles (pGreen Fire-TCF/LEF with EF1). Transduced CSCs were treated with EGCG (0–60 lM) for 48 hr and the GFP fluorescence was measured. Data represent mean 6 SD. *, #, or ** ¼ significantly different from control, p < 0.05. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: Human pancreatic CSCs (CD133þ/CD44þ/CD24þ/ESAþ) were obtained from Celprogen Inc. (San Pedro, CA).

Techniques: Migration, Activity Assay, Incubation, Expressing, Reverse Transcription Polymerase Chain Reaction, Transwell Migration Assay, Staining, Invasion Assay, Membrane, Transduction, Luciferase, Control

Journal: International journal of cancer

Article Title: Inhibition of sonic hedgehog pathway and pluripotency maintaining factors regulate human pancreatic cancer stem cell characteristics.

doi: 10.1002/ijc.26323

Figure Lengend Snippet: Figure 6. Quercetin synergizes with EGCG to inhibit self-renewal capacity, invasion, migration, and TCF/LEF and Gli transcriptional activities in pancreatic CSCs. (a) Effects of EGCG and quercetin on spheroid and colony formation. Upper panel: Quercetin synergizes with EGCG to inhibit spheroid’s cell viability. CSCs were seeded in suspension and treated with EGCG (0–60 lM) with or without quercetin (20 lM) for 7 days. At the end of incubation period, all the spheroids were collected and resuspended. Cell viability was measured by trypan blue assay. Data represent mean 6 SD. *, #, %, **, ##, or %% ¼ significantly different from control, p < 0.05. Lower panel: Quercetin synergizes with EGCG to inhibit colony formation. Pancreatic CSCs were seeded in soft agar and treated with various doses of EGCG (0–60 lM) with or without quercetin (20 lM) and incubated at 4C for 21 days. At the end of incubation period, colonies were counted. Data represent mean 6 SD. *, #, %, **, ##, or %% ¼ significantly different from control, p < 0.05. (b) Effects of EGCG and quercetin on invasion and migration. Upper panel: Matrigel invasion assay. CSCs were plated onto the Matrigel-coated membrane in the top chamber of the transwell and treated with EGCG (0–60 lM) with or without quercetin (20 lM) for 48 hr. Cells invaded to the lower chambered were fixed with methanol, stained with crystal violet and counted. Data represent mean 6 SD. *, #, %, **, ##, or %% ¼ significantly different from control, p < 0.05. Lower panel: Transwell migration assay. Pancreatic CSCs were plated in the top chamber of the transwell and treated with EGCG (0–60 lM) with or without quercetin (20 lM) for 48 hr. Cells migrated to the lower chambered were fixed with methanol, stained with crystal violet and counted. Data represent mean 6 SD. *, #, %, **, ##, or %% ¼ significantly different from respective controls, p < 0.05. (c) Quercetin synergizes with EGCG to induce apoptosis. CSCs were seeded in suspension and treated with EGCG (0–60 lM) with or without quercetin (20 lM) for 7 days. At the end of incubation period, all the spheroids were collected. Apoptosis was measured by TUNEL assay. Data represent mean 6 SD. *, #, %, **, ##, or %% ¼ significantly different from control, p < 0.05. (d) Effects of EGCG and quercetin on TCF/LEF and Gli transcriptional activities. Pancreatic CSCs were transduced with either lentivirus encoding TCF/LEF responsive GFP and luciferase genes or Gli-responsive GFP and luciferase genes. Transduced CSCs were treated with EGCG (40 lM) with or without quercetin (20 lM) for 48 hr and the luciferase activity was measured. Data represent mean 6 SD. *, #, or % ¼ significantly different from respective control, p < 0.05. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: Human pancreatic CSCs (CD133þ/CD44þ/CD24þ/ESAþ) were obtained from Celprogen Inc. (San Pedro, CA).

Techniques: Migration, Suspension, Incubation, Control, Invasion Assay, Membrane, Staining, Transwell Migration Assay, TUNEL Assay, Transduction, Luciferase, Activity Assay

Journal: Colloids and surfaces. B, Biointerfaces

Article Title: Peptide-targeted, stimuli-responsive polymersomes for delivering a cancer stemness inhibitor to cancer stem cell microtumors.

doi: 10.1016/j.colsurfb.2017.12.036

Figure Lengend Snippet: Fig. 5. Cellular uptake studies in monolayer and microtumor cultures of prostate and pancreatic cancer stem cells. Panels (A–F): prostate cancer stem cells (scale bar: 25 m), and

Article Snippet: Prostate cancer stem cells were maintained in human prostate cancer stem cell complete growth media with serum and antibiotics and pancreatic cancer stem cell were maintained in human pancreatic cancer stem cell complete growth media with serum and antibiotics from Celprogen.

Techniques:

Journal: Colloids and surfaces. B, Biointerfaces

Article Title: Peptide-targeted, stimuli-responsive polymersomes for delivering a cancer stemness inhibitor to cancer stem cell microtumors.

doi: 10.1016/j.colsurfb.2017.12.036

Figure Lengend Snippet: Fig. 6. Expression of neuropilin-1 in prostate and pancreatic cancer stem cells as determined

Article Snippet: Prostate cancer stem cells were maintained in human prostate cancer stem cell complete growth media with serum and antibiotics and pancreatic cancer stem cell were maintained in human pancreatic cancer stem cell complete growth media with serum and antibiotics from Celprogen.

Techniques: Expressing

Journal: Colloids and surfaces. B, Biointerfaces

Article Title: Peptide-targeted, stimuli-responsive polymersomes for delivering a cancer stemness inhibitor to cancer stem cell microtumors.

doi: 10.1016/j.colsurfb.2017.12.036

Figure Lengend Snippet: Fig. 7. The viability of prostate and pancreatic cancer stem cells in monolayer and spheroid cultures. (A) Monolayer cultures of prostate cancer stem cells, (B) monolayer cultures

Article Snippet: Prostate cancer stem cells were maintained in human prostate cancer stem cell complete growth media with serum and antibiotics and pancreatic cancer stem cell were maintained in human pancreatic cancer stem cell complete growth media with serum and antibiotics from Celprogen.

Techniques:

Journal: Colloids and surfaces. B, Biointerfaces

Article Title: Peptide-targeted, stimuli-responsive polymersomes for delivering a cancer stemness inhibitor to cancer stem cell microtumors.

doi: 10.1016/j.colsurfb.2017.12.036

Figure Lengend Snippet: Fig. 9. Flow cytometry analysis of the effect of napabucasin on prostate (A) and pancreatic cancer stem cells (B) with Annexin V and PI staining.

Article Snippet: Prostate cancer stem cells were maintained in human prostate cancer stem cell complete growth media with serum and antibiotics and pancreatic cancer stem cell were maintained in human pancreatic cancer stem cell complete growth media with serum and antibiotics from Celprogen.

Techniques: Flow Cytometry, Staining

Journal: Journal of materials chemistry. B

Article Title: Design and evaluation of nanoscale materials with programmed responsivity towards epigenetic enzymes.

doi: 10.1039/d4tb00514g

Figure Lengend Snippet: Fig. 6 Interaction of the block copolymers and HDAC8-responsive nanoparticles with different types of PDAC cells and non-cancerous HPNE cells in vitro. (A) PEG-block-poly(acetylated L-lysine) block copolymers studied in this work do not trigger cytotoxicity in different cell lines. (B) Chemical structure of the STAT3 inhibitor, Napabucasin (NAPA), which has been used as the model hydrophobic drug to demonstrate the encapsulation and HDAC-mediated release activity of the nanoparticles in the context of drug delivery. (C) HDAC 8 (1 mM) triggers the release of NAPA, and over 90% of the encapsulated drug is released from these nanoparticles after incubation with the enzyme for 3 h. The standard deviation of mean is taken for N = 3 replicates. Without the enzyme, the drug release rate and extent were significantly decreased. (D) NAPA-loaded nanoparticles showed a concentration- dependent effect on different types of cancer cells, with a more prominent effect on cancer stem cells (CSCs).

Article Snippet: The fourth cell variant is patient-derived xenograft pancreatic cancer stem cells (CSCs) obtained from Celprogen.

Techniques: Blocking Assay, In Vitro, Encapsulation, Activity Assay, Incubation, Standard Deviation, Concentration Assay

Journal: Oncology Letters

Article Title: Anticancer effect of arsenite on cell migration, cell cycle and apoptosis in human pancreatic cancer cells

doi: 10.3892/ol.2016.4564

Figure Lengend Snippet: Effects of PDGF-BB on the migration of AsPC-1 and BxPC-3 pancreatic cancer cells, compared with normal PE cells. (A) AsPC-1, (B) BxPC-3 and (C) PE cells (5×104 cells/well) were treated with the indicated doses of arsenite for 24 h, prior to be seeded in the upper Boyden chamber. Following incubation for 16 h, cells were exposed to 30 ng/ml PDGF-BB for 36 h at 37°C. Cells were then fixed, stained and visualized under a microscope. The average number of migrated cells from five randomly selected fields on the lower surface of the membrane was counted. Data were obtained from ≥3 independent experiments. *P<0.05 vs. controls. Right panels show representative images of the migrated cells stained with clonogenic reagent. PDGF, platelet-derived growth factor; PE, pancreatic epithelial.

Article Snippet: Primary normal human pancreatic epithelial (PE) cells were purchased from DS Pharma Biomedical Co., Ltd. (Osaka, Japan) and maintained in CSC medium (Cell Systems Corporation, Kirkland, WA, USA).

Techniques: Migration, Incubation, Staining, Microscopy, Membrane, Derivative Assay

Journal: Oncology Letters

Article Title: Anticancer effect of arsenite on cell migration, cell cycle and apoptosis in human pancreatic cancer cells

doi: 10.3892/ol.2016.4564

Figure Lengend Snippet: Effects of arsenite on PARP cleavage in AsPC-1, BxPC-3 and PE cells. (A) AsPC-1, (B) BxPC-3 and (C) PE cells were exposed to arsenite (30 µM) for the indicated time periods. Protein extracts were then harvested and examined by western blotting using anti-PARP and anti-glyceraldehyde-3-phosphate dehydrogenase antibodies. Background-subtracted signal intensity of each protein band was normalized to GAPDH. Data are presented as the mean ± standard deviation of triplicate assay. *P<0.05. PARP, poly(adenosine diphosphate-ribose) polymerase; PE, pancreatic epithelial; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Primary normal human pancreatic epithelial (PE) cells were purchased from DS Pharma Biomedical Co., Ltd. (Osaka, Japan) and maintained in CSC medium (Cell Systems Corporation, Kirkland, WA, USA).

Techniques: Western Blot, Standard Deviation

Journal: Oncology Letters

Article Title: Anticancer effect of arsenite on cell migration, cell cycle and apoptosis in human pancreatic cancer cells

doi: 10.3892/ol.2016.4564

Figure Lengend Snippet: Effects of arsenite on the phosphorylation of p44/p42 MAPK and Akt in AsPC-1, BxPC-3 and PE cells. (A) AsPC-1, (B) BxPC-3 and (C) PE cells were exposed to arsenite (30 µM) and incubated with 30 ng/ml platelet-derived growth factor-BB for the indicated time periods. Protein extracts were then harvested and examined by western blotting using specific antibodies against phospho-p44/p42 MAPK, p44/p42 MAPK, phospho-Akt and Akt. Background-subtracted signal intensity of each protein band was normalized to Akt. Data are presented as the mean ± standard deviation of triplicate assay. *P<0.05 vs. cells without arsenite exposure. PE, pancreatic epithelial; PDGF, platelet-derived growth factor; phospho, phosphorylated; MAPK, mitogen-activated protein kinase; M, marker.

Article Snippet: Primary normal human pancreatic epithelial (PE) cells were purchased from DS Pharma Biomedical Co., Ltd. (Osaka, Japan) and maintained in CSC medium (Cell Systems Corporation, Kirkland, WA, USA).

Techniques: Phospho-proteomics, Incubation, Derivative Assay, Western Blot, Standard Deviation, Marker