Journal: Cancers

Article Title: TPH1 and 5-HT 7 Receptor Overexpression Leading to Gemcitabine-Resistance Requires Non-Canonical Permissive Action of EZH2 in Pancreatic Ductal Adenocarcinoma

doi: 10.3390/cancers13215305

Figure Lengend Snippet: Expression levels of EZH2, TPH1, and 5-HT 7 correlate with gemcitabine resistance in pancreatic cancer cells. ( A ) Sensitivity of PANC-1, MIA PaCa-2, Capan-1, Capan-2, and H6c7 cells to gemcitabine was determined by measuring cell viability after gemcitabine treatment for 48 h. * p < 0.05 compared to H6c7 cells. ( B ) Expression levels of EZH1, EZH2, TPH1, and 5-HT receptors (5-HT 7, 5-HT 1A, 5-HT 1B ) in PANC-1, MIA PaCa-2, Capan-1, Capan-2, and H6c7. ( C ) Treatment of PANC-1 and MIA PaCa-2 cells with GSK-126 for 48 h inhibited 5-HT 7 and TPH1 expressions. ( D ) Down-regulation of 5-HT 7 , TPH1, EZH1 and EZH2 expression by EZH2 siRNA (100 nM) in PANC-1 and MIA PaCa-2 cells. NT represents non-target. ( E , F ) After transfection of PANC-1 and MIA PaCa-2 cells with HTR7, TPH1, or EZH2 siRNA, expression levels of 5-HT 7 , TPH1, and EZH2 ( E ) and gemcitabine sensitivity ( F ) were measured. * p < 0.05 compared to non-target siRNA (siNT)-transfected group. # p < 0.05 compared to the vehicle-treated control. ( G , H ) H6c7 and Capan-1 cells turned out to be gemcitabine-resistant phenotype by treatment with 5-HT (10 μM) for 96 h ( G ). Time-dependent treatment with 5-HT up-regulated 5-HT 7 , TPH1, and EZH2 expression ( H ). ( I ) Sphere forming ability of Capan-1 cells pretreated with 5-HT (10 μM) for 96 h was measured. Images of spheres were captured after 14 days of culture in the serum-free medium. Scale bar (white colored) represents 200 μm at original magnification of 4×. The number of spheroids over 50 μm in diameter was counted. * p < 0.05, compared to untreated control group. The uncropped Western Blot images can be found in .

Article Snippet: Antibodies against p-PI3K, PI3K, p-Akt, Akt, p-JAK2, JAK2, p-STAT3, STAT3, EZH2, and NF-κB P65 were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA); 5-HT 1A , 5-HT 1B , Nanog, CD44, Sp1, and EZH1 were from Abcam (Cambridge, MA, USA); TPH1 from Invitrogen (Carlsbad, CA, USA); β-Actin and Lamin B from Santa Cruz Biotechnology (Dallas, Texas, USA).

Techniques: Expressing, Transfection, Western Blot

Journal: Cancers

Article Title: TPH1 and 5-HT 7 Receptor Overexpression Leading to Gemcitabine-Resistance Requires Non-Canonical Permissive Action of EZH2 in Pancreatic Ductal Adenocarcinoma

doi: 10.3390/cancers13215305

Figure Lengend Snippet: Effects of silencing 5-HT 7 , TPH1 and EZH2 on pancreatic CSC population. ( A ) PANC-1, MIA PaCa-2, Capan-1, and Capan-2 cells stained with antibodies specific to pancreatic CSC surface markers, CD44-FITC and CD24-APC, were analyzed by FACS. Bar graph indicates relative number of CSCs population (CD24 + CD44 + ) determined from three independent experiments. ( B ) Relative number of CD24 + CD44 + population after KD of HTR7, TPH1, or EZH2 in PANC-1 and MIA PaCa-2 cells. ( C , D ) The relative CSC population expressing CD24, CD44, and 5-HT 7 in PANC-1 ( C ) and MIA PaCa-2 ( D ) cells after KD of HTR7, TPH1, and EZH2. * p < 0.05, compared to siNT-transfected group. # p < 0.05, compared to siHTR7- or siTPH1-transfected group.

Article Snippet: Antibodies against p-PI3K, PI3K, p-Akt, Akt, p-JAK2, JAK2, p-STAT3, STAT3, EZH2, and NF-κB P65 were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA); 5-HT 1A , 5-HT 1B , Nanog, CD44, Sp1, and EZH1 were from Abcam (Cambridge, MA, USA); TPH1 from Invitrogen (Carlsbad, CA, USA); β-Actin and Lamin B from Santa Cruz Biotechnology (Dallas, Texas, USA).

Techniques: Staining, Expressing, Transfection

Journal: Cancers

Article Title: TPH1 and 5-HT 7 Receptor Overexpression Leading to Gemcitabine-Resistance Requires Non-Canonical Permissive Action of EZH2 in Pancreatic Ductal Adenocarcinoma

doi: 10.3390/cancers13215305

Figure Lengend Snippet: Effects of EZH2-TPH1-5-HT 7 axis inhibition on the activation of PI3K, Akt, JAK2, and STAT3 in PANC-1 and MIA PaCa-2 cells. ( A ) Cells were treated with siRNA of HTR7, TPH1, and EZH2. * p < 0.05, compared to siNT-transfected group. ( B ) Cells were treated with GSK-126 for 48 h. * p < 0.05, compared to vehicle-treated control group. The uncropped Western Blot images can be found in .

Article Snippet: Antibodies against p-PI3K, PI3K, p-Akt, Akt, p-JAK2, JAK2, p-STAT3, STAT3, EZH2, and NF-κB P65 were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA); 5-HT 1A , 5-HT 1B , Nanog, CD44, Sp1, and EZH1 were from Abcam (Cambridge, MA, USA); TPH1 from Invitrogen (Carlsbad, CA, USA); β-Actin and Lamin B from Santa Cruz Biotechnology (Dallas, Texas, USA).

Techniques: Inhibition, Activation Assay, Transfection, Western Blot

Journal: Cancers

Article Title: TPH1 and 5-HT 7 Receptor Overexpression Leading to Gemcitabine-Resistance Requires Non-Canonical Permissive Action of EZH2 in Pancreatic Ductal Adenocarcinoma

doi: 10.3390/cancers13215305

Figure Lengend Snippet: Effects of signaling inhibitors and EZH2-TPH1-5-HT 7 KD on sphere formation and expression of DAB2IP and PTEN in PANC-1 and MIA PaCa-2 cells. ( A – C ) PANC-1 and MIA PaCa-2 cells were treated with 1 or 3 μM of gemcitabine, wortmannin, SC66, fedratinib, stattic, SB-269970, telotristat, and GSK-126 for 11 days, and measured sphere formation. Images of spheres captured after 11 days of the inhibitor treatment ( A ). Scale bar (white colored) represents 200 μm at original magnification of 4×. The number of spheroids over 50 μm in diameter were counted ( B ). * p < 0.05 compared to the vehicle-treated control group. Immunoblots of CD44, Nanog, EZH2, TPH1, and 5-HT 7 from the 1 μM of each inhibitor-treated spheres ( C ). * p < 0.05 compared to the vehicle. ( D ) DAB2IP and PTEN expression in the cells treated with siRNA of EZH2, TPH1, or 5-HT 7 gene. * p < 0.05, compared to siNT-transfected group. The uncropped Western Blot images can be found in .

Article Snippet: Antibodies against p-PI3K, PI3K, p-Akt, Akt, p-JAK2, JAK2, p-STAT3, STAT3, EZH2, and NF-κB P65 were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA); 5-HT 1A , 5-HT 1B , Nanog, CD44, Sp1, and EZH1 were from Abcam (Cambridge, MA, USA); TPH1 from Invitrogen (Carlsbad, CA, USA); β-Actin and Lamin B from Santa Cruz Biotechnology (Dallas, Texas, USA).

Techniques: Expressing, Western Blot, Transfection

Journal: Cancers

Article Title: TPH1 and 5-HT 7 Receptor Overexpression Leading to Gemcitabine-Resistance Requires Non-Canonical Permissive Action of EZH2 in Pancreatic Ductal Adenocarcinoma

doi: 10.3390/cancers13215305

Figure Lengend Snippet: Transcription factors, NF-κB, SP-1, and p-STAT3, are associated with PI3K/Akt and JAK2/STAT3 signaling, leading to 5-HT 7 , TPH1 and EZH2 expression. ( A ) Immunoblots of 5-HT 7 , TPH1 and EZH2 in PANC-1 and MIA PaCa-2 cells treated with mithramycin A (Mith A, 0.1 μM), SR11302 (10 μM), KG-501 (10 μM), PDTC (10 μM), or stattic (10 μM) for 24 h. ( B ) Nuclear levels of SP-1, p-STAT3, and NF-κB (p65) in PANC-1 and MIA PaCa-2 cells treated with gallein (10 μM), wortmannin (3 μM), SC66 (3 μM), fedratinib (3 μM), stattic (3 μM), SB-269970 (3 μM), telotristat (3 μM), or SB203580 (10 μM) for 48 h. Immunoblots are the representative of three independent experiments, and results are the mean ± SEM in the bar diagram. * p < 0.05 compared to the vehicle-treated control group. ( C , D ) Immunoblots of 5-HT 7 , TPH1 and EZH2 expression in Capan-1 cells. Cells were pretreated with 5-HT (10 μM) for 96 h prior to the treatment with signaling molecule inhibitors for 48 h ( C ) and with transcription factor inhibitors for 24 h ( D ). The bar graphs represent the means ± SEM from three independent experiments. * p < 0.05 compared to the vehicle-treated control. # p < 0.05 compared to the 5-HT-treated group. The uncropped Western Blot images can be found in .

Article Snippet: Antibodies against p-PI3K, PI3K, p-Akt, Akt, p-JAK2, JAK2, p-STAT3, STAT3, EZH2, and NF-κB P65 were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA); 5-HT 1A , 5-HT 1B , Nanog, CD44, Sp1, and EZH1 were from Abcam (Cambridge, MA, USA); TPH1 from Invitrogen (Carlsbad, CA, USA); β-Actin and Lamin B from Santa Cruz Biotechnology (Dallas, Texas, USA).

Techniques: Expressing, Western Blot

Journal: Cancers

Article Title: TPH1 and 5-HT 7 Receptor Overexpression Leading to Gemcitabine-Resistance Requires Non-Canonical Permissive Action of EZH2 in Pancreatic Ductal Adenocarcinoma

doi: 10.3390/cancers13215305

Figure Lengend Snippet: Anti-tumor activities of inhibitors of EZH2-TPH1-5-HT 7 axis in mouse tumor xenograft models with PANC-1 cells. ( A – D ) BALB/c nude mice xenografted with PANC-1 cells were administered i.p. with gemcitabine (50 mg/kg), telotristat (1 and 10 mg/kg), SB-269970 (1 and 10 mg/kg) for 42 days (6 days per week) ( n = 5). Tumor volume (mm 3 ) was measured twice a week ( A ). Representative images showing gross appearance of tumor in mice ( B ) and weight of excised tumor at the time of sacrifice ( C ). Mouse body weight during the drug administration period ( D ). * p < 0.05 compared to the vehicle-treated control. ( E – H ) In a separate set of mouse tumor models, mice were administered i.p. with drug alone (50 mg/kg gemcitabine or 10 mg/kg GSK-126) or gemcitabine plus drug (10 mg/kg of GSK-126, SB-269970, or telotristat) for 38 days ( n = 6). Tumor volume ( E ), gross appearance of tumor in mice ( F ), and tumor weight ( G ) at the time of sacrifice, and mouse body weight during the treatment period ( H ). * p < 0.05 compared to the vehicle-treated control. # p < 0.05 compared to GSK-126-treated group.

Article Snippet: Antibodies against p-PI3K, PI3K, p-Akt, Akt, p-JAK2, JAK2, p-STAT3, STAT3, EZH2, and NF-κB P65 were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA); 5-HT 1A , 5-HT 1B , Nanog, CD44, Sp1, and EZH1 were from Abcam (Cambridge, MA, USA); TPH1 from Invitrogen (Carlsbad, CA, USA); β-Actin and Lamin B from Santa Cruz Biotechnology (Dallas, Texas, USA).

Techniques:

Journal: Cancers

Article Title: TPH1 and 5-HT 7 Receptor Overexpression Leading to Gemcitabine-Resistance Requires Non-Canonical Permissive Action of EZH2 in Pancreatic Ductal Adenocarcinoma

doi: 10.3390/cancers13215305

Figure Lengend Snippet: Schematic summary of noncanonical permissive action of EZH2 on TPH1-5-HT 7 expression in PDAC. 5-HT activates PI3K/Akt and JAK2/STAT3 through Gβγ linked to 5-HT 7 . Together with Ras-activated ERK signaling, these signaling pathways enhance the nuclear level of Sp1, NF-κB, and p-STAT3, leading to EZH2 upregulation. Under the permissive action of EZH2, p-STAT3 is required for upregulation of TPH1 and 5-HT 7 , whereas Sp1 and NF-κB contribute to transcriptional repression of DAB2IP and PTEN. Through its noncanonical action, EZH2 activates intracellular signaling pathways through two ways, (1) activation of PI3K/Akt and JAK2/STAT3 through transactivation of TPH1-5-HT 7 expressions, and (2) maintenance of the PI3K/Akt activity by inhibition of its inhibitory signals through downregulation of DAB2IP and PTEN.

Article Snippet: Antibodies against p-PI3K, PI3K, p-Akt, Akt, p-JAK2, JAK2, p-STAT3, STAT3, EZH2, and NF-κB P65 were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA); 5-HT 1A , 5-HT 1B , Nanog, CD44, Sp1, and EZH1 were from Abcam (Cambridge, MA, USA); TPH1 from Invitrogen (Carlsbad, CA, USA); β-Actin and Lamin B from Santa Cruz Biotechnology (Dallas, Texas, USA).

Techniques: Expressing, Activation Assay, Activity Assay, Inhibition

Journal: Frontiers in Physiology

Article Title: Oral Administration of Penicillin or Streptomycin May Alter Serum Serotonin Level and Intestinal Motility via Different Mechanisms

doi: 10.3389/fphys.2020.605982

Figure Lengend Snippet: Co-administration of penicilin-streptomycin (P-S) caused significant changes in serum 5-HT, cecum size and weight and HCN2 expression in cecal epithelium. (A) Photos of the typical cecum of control and P-S-treated mice. (B,C) Comparisons of the wet (with cecum content) and dry (net) cecum weight (normalized to the body weight) between P-S-treated and control mice. (D) Comparisons of the serum 5-HT content between P-S-treated and control mice. Data were normalized to serum 5-HT content in control mice. (E) Western blot detection of HCN2, TPH1, and β-actin in cecal mucosa of the control mice and the mice treated with P-S. (F) Quantification of relative HCN2 and TPH1 protein in the cecal mucosa. ∗ P < 0.05 and ∗∗∗ P < 0.001 vs. control. n = 6 mice for each group.

Article Snippet: After blockade with 5% fat-free milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 for 2 h at room temperature, the membrane was incubated overnight at 4°C with rabbit anti-HCN2 (1:400, Alomone, Israel) and rabbit anti-TPH1 (1:500, Millipore, United States).

Techniques: Expressing, Western Blot

Journal: Frontiers in Physiology

Article Title: Oral Administration of Penicillin or Streptomycin May Alter Serum Serotonin Level and Intestinal Motility via Different Mechanisms

doi: 10.3389/fphys.2020.605982

Figure Lengend Snippet: Effects of penicillin treatment on serum 5-HT level, cecum size and weight, intestinal motility and expression of HCN2 and TPH1 in cecal epithelium. (A–C) Comparison of cecum size and weight between the penicillin-treated and control mice. (D) Comparison of the serum 5-HT content between the penicillin-treated and the control mice. Data were normalized to serum 5-HT content in control mice. (E) Western blot detection of HCN2, TPH1, and β-actin in the cecal mucosa of the control mice and the mice treated with penicillin. (F) Quantification of relative HCN2 and TPH1 protein in the cecal mucosa. (G) Intestinal transit time of the mice detected by carmine dye test. (H) Fecal water content of the mice. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. n = 6 mice for each group.

Article Snippet: After blockade with 5% fat-free milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 for 2 h at room temperature, the membrane was incubated overnight at 4°C with rabbit anti-HCN2 (1:400, Alomone, Israel) and rabbit anti-TPH1 (1:500, Millipore, United States).

Techniques: Expressing, Western Blot

Journal: Frontiers in Physiology

Article Title: Oral Administration of Penicillin or Streptomycin May Alter Serum Serotonin Level and Intestinal Motility via Different Mechanisms

doi: 10.3389/fphys.2020.605982

Figure Lengend Snippet: Effects of streptomycin treatment on serum 5-HT level, cecum size and weight, intestinal motility and expression of HCN2 and TPH1 in the cecal epithelium. (A–C) Comparison of cecum size and weight between the streptomycin-treated and control mice. (D) Comparison of the serum 5-HT content between the streptomycin-treated and the control mice. Data were normalized to serum 5-HT content in control mice. (E) Western blot detection of HCN2, TPH1, and β-actin in the cecal epithelium of the control mice and the mice treated with streptomycin. (F) Quantification of relative HCN2 and TPH1 protein in the cecal mucosa. (G) Intestinal transit time of the mice detected by carmine dye test. (H) Fecal water content of the mice. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. n = 6 mice for each group.

Article Snippet: After blockade with 5% fat-free milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 for 2 h at room temperature, the membrane was incubated overnight at 4°C with rabbit anti-HCN2 (1:400, Alomone, Israel) and rabbit anti-TPH1 (1:500, Millipore, United States).

Techniques: Expressing, Western Blot