EGCG and gemcitabine inhibit cell viability and STAT3 target genes.

<p>(A), AsPC-1 and PANC-1 cells were treated with EGCG (0, 20, 40, 60 µM) with or without gemcitabine (0.5 µM) for 72 h. Cell viability was measured by XTT assay. Data represent mean ± SD. * or ** = significantly different from respective controls, P<0.05. (B), AsPC-1 and PANC-1 cells were treated with EGCG (0, 20, 40, 60 µM) with or without gemcitabine (0.5 µM) for 72 h. Apoptosis was measured by TUNEL assay. Data represent mean ± SD. * or ** = significantly different from respective controls, P<0.05. (C), Inhibition of STAT3 target genes by EGCG and gemcitabine. AsPC-1 and PANC-1 cells were treated with EGCG (20 µM) or gemcitabine (0.5 µM) for 48 h. The expression of VEGF, c-Myc, survivin and cyclin D1was was measured by qRT-PCR. Data represent mean ± SD. * = significantly different from respective controls, P<0.05.</p

GDC-0449 differentially regulates genes involved in the balance between cell death and cell survival.

<p>Pancreatic CSCs were treated with GDC-0449 (0, 1, 5 and 10 µM) for 48 h. The expression of Fas, DR4/TRAIL-R1, DR5/TRAIL-R2, PARP cleavage, Bcl-2, and Caspase-3 by the Western blot analysis. β-Actin was used as a loading control.</p

Schematic representation of the inhibition of SHH signaling and genes involved in the balance between cellular proliferation and cell death.

<p>Activated Gli1 and Gli2, downstream of SHH-Patched-Smoothened, regulate targets of SHH signaling including Bcl-2, PDGFRα, Fas, and DRs. GDC-0449 (targeting Smoothened) blocks the indirect functions of Gli activators, resulting in cell death.</p

Impact of SHH signaling pathway on the regulation of cell survival and antiproliferative effects of GDC-0449.

<p>(A), Knockout of Gli1 shRNA and Gli2 shRNA in human pancreatic CSCs. Pancreatic CSCs were transduced with lentiviral particles expressing Scrambled, Gli1 shRNA, Gli2 shRNA or Gli1 plus Gli2 shRNA (KO). (B), Pancreatic CSCs were treated with GDC-0449 (0, 1, 5 and 10 µM) for 72 h, and cell viability and apoptosis was measured in scrambled and Gli1 plus Gli2 shRNA CSCs. Data represent mean ± SD, n = 4. @ or # significantly different from respective control (P<0.05). (C), Scrambled and Gli1 plus Gli2 shRNA pancreatic CSCs were treated with GDC-0449 (0 and 10 µM) for 48 h, and lysates were extracted to determine the expression of DR4, DR5, PDGFRα, Fas and Bcl-2 by Western blot analysis. β-Actin was used as the loading control. (D), Inhibition of primary and secondary spheroids by GDC-0449. Pancreatic CSCs (scrambled, and Gli1 + Gli2 shRNA) were seeded in suspension and treated with GDC-0449 (10 µM) for 7 days. At the end of incubation period, spheroids were collected, and dissociated with Accutase (Innovative Cell Technologies, Inc.). For secondary spheroids, cells were reseeded and treated with GDC-0449 (10 µM) for additional 7 days. Cell viability was measured by trypan blue assay. Data represent mean ± SD. @ or # significantly different from respective controls, P<0.05.</p

Inhibition of STAT3 enhances the inhibitory effects of EGCG on motility and cell viability of pancreatic cancer cells.

<p>(A), AsPC-1 and PANC-1 were transfected with STAT3 shRNA. The expression of STAT3 was performed by Western blotting. (B), AsPC-1 scratch assay. AsPC-1 scrambled and STAT3 shRNA cells were cultured in 6 well dishes. The scratch was marked when the dishes were 50% confluent. Pictures were taken after the cells were treated with EGCG and incubated for 0, 24 and 48 h. (C), Cell viability assay. AsPC-1 and PANC-1 (scrambled and STAT3 shRNA) cells were seeded and treated with EGCG (0, 20, 40, 60 µM). After 72 h of treatment, cell viability was performed by XTT assay. Data represent mean ± SD. * or ** = significantly different from respective controls, P<0.05.</p

EGCG inhibits JAK3/STAT3 pathway in pancreatic cancer.

<p>(A), AsPC-1 and PANC-1 cells were treated with EGCG (0–60 µM) for 48 h, and the expression of STAT3 was measured by q-RT-PCR. Data represent mean ± SD. * or ** = significantly different from respective controls, P<0.05. (B), AsPC-1 and PANC-1 cells were treated with EGCG (0–60 µM) for 48 h. The expression of STAT3, p-STAT3, JAK3 and p-JAK3 was measured by Western blot analysis. β-actin was used as a loading control. (C), Expression of STAT3 in AsPC-1 and PANC-1 cells. Cells were treated with EGCG (0–60 µM) for 48 h. After incubation, the expression of STAT3 was measured by immunoflurescence. DAPI was used to stain nuclei. For better visuality, the color of DAPI was changed from blue to red. The green color represents the expression of STAT3. Red color = nuclei.</p

Effects of STAT3 shRNA on the regulation of cyclin D1, Bcl-X_L and c-Myc by EGCG.

<p>(A), AsPC-1/scrambled and AsPC-1/STAT3 shRNA cells were treated with or without EGCG (60 µM) for 48 h. The expression of cyclin D1 was measured by q-RT-PCR. Data represent mean ± SD. * = significantly different from respective controls, P<0.05. (B), PANC-1/scrambled and PANC-1/STAT3 shRNA cells were treated with or without EGCG (60 µM) for 48 h. The expression of cyclin D1 was measured by q-RT-PCR. Data represent mean ± SD. * = significantly different from respective controls, P<0.05. (C), PANC-1/scrambled and PANC 1/STAT3 shRNA cells were treated with or without EGCG (60 µM) for 48 h. The expression of Bcl-X_L was measured by qRT-PCR. Data represent mean ± SD. * = significantly different from respective controls, P<0.05. (D), PANC-1/scrambled and PANC-1/STAT3 shRNA cells were treated with or without EGCG (60 µM) for 48 h. The expression of c-Myc was measured by q-RT-PCR. Data represent mean ± SD. * = significantly different from respective controls, P<0.05.</p

Inhibition of SHH signaling suppressed cell viability in human pancreatic cancer cell lines and pancreatic CSCs by GDC-0449.

<p>Cells were treated with GDC-0449 (0, 1, 5 and 10 µM) for 48 and 72 h. At the end of incubation period, cell viability was measured by XTT in (A) AsPC-1, (B) MIA PaCa-2, (C) PANC-1, and (D) Pancreatic CSCs. Data represent mean ± SD. @ or # significantly different from respective control (P<0.05).</p

Effects of EGCG on pancreatic cancer cells.

<p>(A), Transwell migration assay. AsPC-1 and PANC-1 cells were plated in the top chamber of the transwell and treated with EGCG (0–60 µM) for 24 h. Cells migrated to the lower chambered were fixed with methanol, stained with crystal violet and counted. Data represent mean ± SD. * or ** = significantly different from respective controls, P<0.05. (B) Matrigel invasion assay. AsPC-1 and PANC-1 cells were plated onto the Matrigel-coated membrane in the top chamber of the transwell and treated with EGCG (0–60 µM) for 48 h. Cells invaded to the lower chamber were fixed with methanol, stained with crystal violet and counted. Data represent mean ± SD. * or ** = significantly different from respective controls, P<0.05. (C), Caspase-3 activity. AsPC-1 and PANC-1 cells were treated with EGCG (0–40 µM) for 48 h, and the caspase-3 activity was measured as per manufacturer's instructions (Invitrogen). Data represent mean ± SD. * = significantly different from respective controls, P<0.05. (D), AsPC-1 and PANC-1 cells were treated with EGCG (0–60 µM) with or without gemcitabine (0.5 µM) for 48 h. Cells were harvested and the Western blot analysis was performed to examine the expression of PARP and caspase-3. β-actin was used as a loading control. PARP antibody recognizes cleaved PARP, and caspase-3 antibody recognizes cleaved/active caspase-3.</p

Expressing components of Sonic Hedgehog (SHH) signaling pathway in human pancreatic cancer cell lines and pancreatic cancer stem cells (CSCs).

<p>Pancreatic cancer cells (AsPC-1, PANC-1 and MIA PaCa-2) and pancreatic CSCs were grown for 48 h. Total RNA was isolated and expression of Shh, Patched-1, Patched-2, Smoothened, Gli-1 and Gli-2 was measured by qRT-PCR. HK-GAPD was used as endogenous normalization control. All assays were performed in triplicate and were calculated on the basis of ΔΔ<i>C</i>t method. The n-fold change in mRNAs expression was determined according to the method of 2^-ΔΔCT with GAPDH employed as the endogenous control.</p