Resveratrol inhibits pancreatic cancer stem cell characteristics in human and KrasG12D transgenic mice by inhibiting pluripotency maintaining factors and epithelialmesenchymal transition. PLoS One 6: e16530

Background: Cancer stem cells (CSCs) can proliferate and self-renew extensively due to their ability to express anti-apoptotic and drug resistant proteins, thus sustaining tumor growth. Therefore, the strategy to eradicate CSCs might have significant clinical implications. The objectives of this study were to examine the molecular mechanisms by which resveratrol inhibits stem cell characteristics of pancreatic CSCs derived from human primary tumors and Kras G12D transgenic mice. Methodology/Principal Findings: Human pancreatic CSCs (CD133 + CD44 + CD24 + ESA +) are highly tumorigenic and form subcutaneous tumors in NOD/SCID mice. Human pancreatic CSCs expressing high levels of CD133, CD24, CD44, ESA, and aldehyde dehydrogenase also express significantly more Nanog, Oct-4, Notch1, MDR1 and ABCG2 than normal pancreatic tissues and primary pancreatic cancer cells. Similarly, CSCs from Kras G12D mice express significantly higher levels of Nanog and Oct-4 than pancreatic tissues from Pdx-Cre mice. Resveratrol inhibits the growth (size and weight) and development (PanIN lesions) of pancreatic cancer in Kras G12D mice. Resveratrol inhibits the self-renewal capacity of pancreatic CSCs derived from human primary tumors and Kras G12D mice. Resveratrol induces apoptosis by activating capase-3/7 and inhibiting the expression of Bcl-2 and XIAP in human CSCs. Resveratrol inhibits pluripotency maintaining factors (Nanog, Sox-2, c-Myc and Oct-4) and drug resistance gene ABCG2 in CSCs. Inhibition of Nanog by shRNA enhances the inhibitory effects of resveratrol on self-renewal capacity of CSCs. Finally, resveratrol inhibits CSC’s migration and invasion and marker

Nanog shRNA enhances the inhibitory effects of resveratrol on self-renewal capacity of pancreatic cancer stem cells.

<p>(A), Expression of Nanog. Pancreatic CSCs were transduced with scrambled or Nanog shRNA, and Western blot analyses were performed to examine the expression of Nanog and GAPDH. (B), Transduced cells were treated with resveratrol (0–30 µM) and spheres in suspension were grown for one week. Spheres were harvested, resuspended and cell viability was determined by trypan blue assay.</p

Resveratrol inhibits the pancreatic cancer stem cell characteristics Kras^G12D mice.

<p>(A), Pancreatic CSCs were isolated from Kras^G12D mice (about 12 months old) using CD133, CD44, CD24, and ESA antibodies. The expression of these markers and Nanog and Oct-4 was confirmed by qRT-PCR. Data represent mean ± SD. *  =  significantly different from the same gene of Pdx-Cre mice at P<0.05. (B), CSCs were grown in suspension and treated with resveratrol (0–30 µM) for 7 days to obtain primary spheroids. At the end of incubation period, spheroids were collected, resuspended and treated with resveratrol for another week to obtain secondary spheroids. Cell viability in spheroids was measured by trypan blue assay at the end of 7 and 14 days. Data represent mean ± SD. *, %, &, ** and ##  =  significantly different from respective controls, P<0.05.</p

Resveratrol activates caspase-3/-7, induces apoptosis, and inhibits the expression of XIAP, Bcl-2 and cyclin D1 in pancreatic CSCs.

<p>(A and B), Activation of caspase-3/-7 and induction of apoptosis. Pancreatic CSCs were treated with resveratrol (0–30 µM) for 48 h, and caspase-3/-7 activity and apoptosis were measured by colorometric and TUNEL assay, respectively. Data represent mean ± SD. *, & and #  =  significantly different from control, P<0.05. (C and D), Pancreatic CSCs were treated with resveratrol (0–20 µM) for 48 h, and western blot analysis was performed to measure the expression of XIAP, Bcl-2, caspase-3 and cyclin D1. GAPDH was used as a loading control.</p

Regulation of EMT markers, invasion and migration by resveratrol in pancreatic CSCs.

<p>Pancreatic CSCs were treated with resveratrol (0–0 µM) for 24 h. The expression of Zeb-1 (A), Snail (B) and Slug (c) was measured by the qRT-PCR. Data represent the mean ± S.D. * or #  =  significantly different from respective controls, P<0.05. (D), Invasion assay. Pancreatic CSCs were plated onto the Matrigel-coated membrane in the top chamber of the transwell and treated with resveratrol (0–30 µM) for 48 h. Cells invaded to the lower chambered were fixed with methanol, stained with crystal violet and counted. Data represent mean ± SD. *, & or #  =  significantly different from control, P<0.05. (E) Migration assay. Pancreatic CSCs were plated in the top chamber of the transwell and treated with resveratrol (0–30 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 control, P<0.05.</p

Resveratrol inhibits cell viability in spheroids and colony formation by pancreatic CSCs.

<p>(A), Human pancreatic CSCs were grown in six-well ultralow attachment plates (Corning Inc., Corning, NY) at a density of 1,000 cells/ml in Celprogen pancreatic CSC medium at 37°C in a humidified atmosphere of 95% air and 5% CO₂ and treated with resveratrol (0–30 µM) for 7 days to obtain primary spheroids. At the end of incubation period, spheroids were collected, reseeded and treated with resveratrol for another week to obtain secondary spheroids. (B) Cell viability in spheroids was measured by trypan blue assay at the end of 7 and 14 days from above experiment. Data represent mean ± SD. *, &, #, % and @  =  significantly different from controls, P<0.05. (C), Pancreatic CSCs were seeded in soft agar and treated with resveratrol (0–30 µM) for 21 days. At the end of incubation period, numbers of colonies were counted. Data represent mean ± SD. *, % and &  =  significantly different from control, P<0.05.</p

Isolation and characterization of human pancreatic CSCs from human primary tumors.

<p>(A–H), Pancreatic cancer cells were isolated from primary tumors and analyzed by flow cytometry using antibody against CD44, CD24, ESA, CD133, Oct4, and ALDH. (I), Expression of stem cell markers. RNA was isolated from normal pancreatic tissues, primary pancreatic cancer and pancreatic CSCs and the expression of CD24, CD133, CD44, ESA, Nanog, Notch1, MDR1 and ABCG2 was measured by q-RT-PCR. Data represent mean ± SD. *, ** @  =  significantly different from controls, P<0.05.</p

Inhibition of Nanog, Sox-2, cMyc, Oct-4 and ABCG2 in human pancreatic CSCs by resveratrol.

<p>(A), Pancreatic CSCs were treated with resveratrol (0–20 µM) for 24 h. The expression of Nanog, Sox-2 and cMyc was measured by qRT-PCR. Data represent mean ± SD. * and @  =  significantly different from control, P<0.05. (B), Pancreatic CSCs were treated with resveratrol (0–30 µM) for 48 h. The expression of Nanog, Oct-4 and Sox-2 was measured by the Western blot analysis. GAPDH was used as a loading control. (C), Inhibition of Nanog transcription by resveratrol. Pancreatic CSCs were transduced with Nanog reporter construct. Transduced cells were treated with resveratrol to examine the Nanog transcriptional activity. Data represent mean ± SD. *, #, @, & and **  =  significantly different from control, P<0.05. (D), Pancreatic CSCs were treated with resveratrol (0–30 µM) for 36 h. The expression of ABCG2 was measured by qRT-PCR. Data represent mean ± SD. * and #  =  significantly different from control, P<0.05.</p

Tumorigenic potential of pancreatic CSCs in NOD/SCID mice.

<p>(A), NOD/SCID mice were sc injected with 50 CD133⁻CD44⁻CD24⁻ESA⁻ cells on right flank and CD133⁺CD44⁺CD24⁺ESA⁺ CSCs on left flanks and tumor growth was observed for 20 days. (B), Pancreatic CSCs are similar to human primary tumors. Tumor sections from human primary pancreatic tumors, and pancreatic CSCs grown in NOD/SCID mice were stained with H&E, Statifin and S100P.</p

Resveratrol inhibits pancreatic cancer growth and development in Kras^G12D mice, and pancreatic CSC characteristics.

<p>(A), Inhibition of pancreatic cancer growth by resveratrol. Kras^G12D transgenic mice were treated with resveratrol (40 mg/kg) by gavage once everyday (Monday – Friday) for about 10 months (mice age 12 months). Pancreas weights from control and resveratrol treated mice were taken. Weight of pancreas from Pdx-cre mice was also taken. (B) Picture of pancreas obtained from control and resveratrol treated Kras^G12D mice. (C), Quantification of PanIN lesions in the pancreas of control and resveratrol treated Kras^G12D mice. Data represent the mean ± S.D. *, @, %, #  =  significantly different from respective controls, P<0.05.</p