Data_Sheet_1_Deficiencies in Natural Killer Cell Numbers, Expansion, and Function at the Pre-Neoplastic Stage of Pancreatic Cancer by KRAS Mutation in the Pancreas of Obese Mice.docx

<p>The combined/synergistic effect of genetic mutation of KRAS in the pancreas and obesity, a life-style factor on suppression of natural killer (NK) cells at the pre-neoplastic stage of pancreatic cancer has not been investigated and is the subject of this report. Obese mice with KRAS (KC) mutation in the pancreas fed with high-fat calorie diet (HFCD) exhibit severe deficiencies in the NK cell expansion and function at the pre-neoplastic stage of pancreatic cancer. Decreased NK cell-mediated cytotoxicity is observed in the peripheral blood, spleen, pancreas, and peri-pancreatic adipose tissue in obese KC mice, whereas in bone marrow an increased NK cell-mediated cytotoxicity is observed when compared to lean WT mice fed with control diet (CD). Obese KC mice on HFCD demonstrated the least ability to expand NK cells or induce NK cell-mediated cytotoxicity when compared to the other groups of mice. Indeed, the following profile WT/CD > WT/HFCD > KC/CD > KC/HFCD was seen for the ability to expand NK cells or mediate cytotoxicity among four groups of mice in spleen, peripheral blood, pancreas, and peri-pancreatic adipose tissue. Sorted NK cells from the splenocytes of four groups of mice also exhibited the same profiles for the cytotoxicity as the unsorted splenocytes, and a decreased IFN-γ secretion could be seen in cultures of NK cells from KC mice fed with either CD or HFCD. Cultures of NK cells with autologous monocytes from obese KC mice fed with HFCD exhibited decreased cytotoxicity and IFN-γ secretion, whereas cultures of allogeneic NK cells from WT mice fed with CD with osteoclasts of obese mice fed with HFCD demonstrated decreased cytotoxicity but augmented IFN-γ secretion. Increased IL-6 along with decreased IFN-γ and cell-mediated cytotoxicity by the NK cells, within NK-adipose tissue of KC/HFCD mice, may provide safe microenvironment for the expansion of pancreatic tumors.</p

Dose-Dependent AMPK-Dependent and Independent Mechanisms of Berberine and Metformin Inhibition of mTORC1, ERK, DNA Synthesis and Proliferation in Pancreatic Cancer Cells

<div><p>Natural products represent a rich reservoir of potential small chemical molecules exhibiting anti-proliferative and chemopreventive properties. Here, we show that treatment of pancreatic ductal adenocarcinoma (PDAC) cells (PANC-1, MiaPaCa-2) with the isoquinoline alkaloid berberine (0.3–6 µM) inhibited DNA synthesis and proliferation of these cells and delay the progression of their cell cycle in G1. Berberine treatment also reduced (by 70%) the growth of MiaPaCa-2 cell growth when implanted into the flanks of nu/nu mice. Mechanistic studies revealed that berberine decreased mitochondrial membrane potential and intracellular ATP levels and induced potent AMPK activation, as shown by phosphorylation of AMPK α subunit at Thr-172 and acetyl-CoA carboxylase (ACC) at Ser⁷⁹. Furthermore, berberine dose-dependently inhibited mTORC1 (phosphorylation of S6K at Thr³⁸⁹ and S6 at Ser^240/244) and ERK activation in PDAC cells stimulated by insulin and neurotensin or fetal bovine serum. Knockdown of α₁ and α₂ catalytic subunit expression of AMPK reversed the inhibitory effect produced by treatment with low concentrations of berberine on mTORC1, ERK and DNA synthesis in PDAC cells. However, at higher concentrations, berberine inhibited mitogenic signaling (mTORC1 and ERK) and DNA synthesis through an AMPK-independent mechanism. Similar results were obtained with metformin used at doses that induced either modest or pronounced reductions in intracellular ATP levels, which were virtually identical to the decreases in ATP levels obtained in response to berberine. We propose that berberine and metformin inhibit mitogenic signaling in PDAC cells through dose-dependent AMPK-dependent and independent pathways.</p></div

Berberine inhibits mTORC1 signaling and ERK activation in PDAC cells.

<p>Cultures of MiaPaCa-2 (Panels <b>A</b> and <b>B</b>) or PANC-1 cells (panels <b>C</b> and <b>D</b>) were incubated in the absence or in the presence of increasing concentrations of berberine. Then, the cells were stimulated for 1 h with 5 nM neurotensin and 10 ng/ml insulin and lysed with 2X SDS-PAGE sample buffer. The samples were analyzed by SDS-PAGE and immunoblotting with antibodies that detect the phosphorylated state of S6K at Thr³⁸⁹, S6 at Ser^240/244, and ERK at Thr²⁰² and Tyr²⁰⁴. Immunoblotting with total S6K, S6 and ERK was used to verify equal gel loading. The quantification of the immune signals was performed using Multi Gauge V3.0. The results are presented in the plots shown in panels <b>B</b> and <b>D.</b> The values represent the mean ± SEM (n = 3) of S6K, S6 and ERK phosphorylation expressed as a percentage of the maximal response obtained in 3 independent experiments.</p

Knockdown of the α subunits of AMPK reverses inhibition of mTORC1, ERK and DNA synthesis induced by low but not high doses of berberine.

<p><b>A</b>, PANC-1 cells were transfected with either non-targeting negative control (Non Target.) or 10 nM AMPKα1 and 10 nM AMPKα2 siRNA (AMPKα1, α2 siRNA) in DMEM containing 5 mM glucose and 10% FBS. After 3 days the cells were incubated either in the absence or presence of berberine for 17 h in serum free DMEM containing 5 mM glucose. Then, the cells were stimulated for 1 h with 5 nM neurotensin and 10 ng/ml insulin and lysed with 2X SDS-PAGE sample buffer. The samples were analyzed by SDS-PAGE and immunoblotting with the following phospho antibodies: S6K at Thr³⁸⁹, S6 at Ser^240/244, and ERK at Thr²⁰² and Tyr²⁰⁴, ACC at Ser⁷⁹ and Raptor at Ser⁷⁹². Shown here is a representative autoluminogram; similar results were obtained in 4 independent experiments. <b>B,</b> Quantification was performed using Multi Gauge V3.0. Results are expressed as the percentage of the maximum (mean ±SEM: n = 4). <b>C,</b> PANC-1 cells were transfected with either non-targeting negative control (open bars) or 10 nM AMPKα1 and 10 nM AMPKα2 siRNA (black bars) in DMEM containing 5 mM glucose and 10% FBS. After 3 days the cells were incubated for 6 h in serum-free medium containing 5 mM glucose. Then, 5 nM neurotensin and 10 ng/ml insulin and the indicated concentration of berberine were added for 17 h at 37°C prior to the addition of [³H]-thymidine for 6 h. The radioactivity incorporated into acid-insoluble pools was measured in a scintillation counter, as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114573#s2" target="_blank">Materials and Methods</a>”. Results are expressed as the percentage of maximum mean ± SEM obtained in 4 independent experiments (3 replicate cultures per point in each experiment).</p

Berberine inhibits the growth of MiaPaCa-2 tumor xenografts as effectively as metformin.

<p>Xenografts of MiaPaca-2 were generated by implantation of 2×10⁶ cells into the right flanks of male <i>nu/nu</i> mice. When the tumors reached a mean diameter of 2 mm the animals were randomized into control and treated groups (10 mice per group). Berberine was given once daily intraperitoneally at 5 mg/kg for the duration of the experiment. For comparison, metformin was given intraperitoneally to another group of mice at 250 mg/kg. The 1^st day of treatment was designated as day 0. Control animals received an equivalent volume of saline. <b>A</b>, Tumor volumes were measured every 4 days as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114573#s2" target="_blank">Materials and Methods</a>”. At the end of the experiment (day 29, the tumors were removed, weighted and measured and tumor volumes estimated as <i>V</i> = 0.52 (length × width × depth). The results are shown in panel <b>B</b> (mean ± SEM). Treatment of mice with berberine significantly reduced the volume and weight of the tumors as compared with the tumors from the control group (p<0.001), as indicated. A similar inhibition of tumor growth was obtained by administration of metformin (p<0.001). The curves corresponding to the growth of MiaPaca-2 xenografts in mice treated with berberine or metformin were superimposable during the first 24 days. At day 29, metformin was slightly more effective than berberine as assessed by tumor volume (p<0.05) but the difference of the effects between these drugs did not reach statistical significance (p>0.07) when scored by tumor weight (<b>Fig. 2, B</b>). At the concentrations used, berberine and metformin were well tolerated with no apparent toxicity based on body weight changes.</p

Berberine inhibits DNA synthesis, cell cycle progression and proliferation in PANC-1 and Mia PaCa-2 cells.

<p><b>A,</b> Mia PaCa-2 or PANC-1 cells were incubated without (<i>open bars</i>) or with 5 nM neurotensin and 10 ng/ml insulin (<i>closed bars</i>) in the presence of increasing concentration of berberine for 17 h at 37°C prior to the addition of [³H]-thymidine for 6 h. The radioactivity incorporated into acid-insoluble pools was measured in a scintillation counter, as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114573#s2" target="_blank">Materials and Methods</a>. The values shown are the mean ± SEM obtained in 3 independent experiments; <b>B,</b> PANC-1 cells were treated without (control) or with berberine at 1.5 µM or 3 µM in medium containing 2.5% FBS for 3 days (indicated by cont., FBS, FBS +1.5 Ber and FBS +3 Ber). Cell cycle was analyzed by PI-staining and flow cytometry. Similar results were obtained in 3 independent experiments. <b>C,</b> Single-cell suspension of Mia PaCa-2 or PANC-1 cells were plated on tissue culture dishes at a density of 2×10⁴ cells per dish. After 24 h of incubation the medium was removed and the cultures shifted to medium without or with 3% FBS in the absence (open bars) or presence (closed bars) of 3 µM berberine. The cultures were incubated for 4 days as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114573#s2" target="_blank">Materials and Methods</a>”. Cell count was determined from 4 to 6 replicate plates per condition using a Coulter Counter. Results are presented as mean ± SEM of 3 biological replicates.</p

Incidence of pancreatic cancer is dramatically increased by a high fat, high calorie diet in KrasG12D mice

<div><p>Epidemiologic data has linked obesity to a higher risk of pancreatic cancer, but the underlying mechanisms are poorly understood. To allow for detailed mechanistic studies in a relevant model mimicking diet-induced obesity and pancreatic cancer, a high-fat, high-calorie diet (HFCD) was given to <i>P48</i>^<i>+/Cre</i><i>;LSL-KRAS</i>^<i>G12D</i> (KC) mice carrying a pancreas-specific oncogenic Kras mutation. The mice were randomly allocated to a HFCD or control diet (CD). Cohorts were sacrificed at 3, 6, and 9 months and tissues were harvested for further analysis. Compared to CD-fed mice, HFCD-fed animals gained significantly more weight. Importantly, the cancer incidence was remarkably increased in HFCD-fed KC mice, particularly in male KC mice. In addition, KC mice fed the HFCD showed more extensive inflammation and fibrosis, and more advanced PanIN lesions in the pancreas, compared to age-matched CD-fed animals. Interestingly, we found that the HFCD reduced autophagic flux in PanIN lesions in KC mice. Further, exome sequencing of isolated murine PanIN lesions identified numerous genetic variants unique to the HFCD. These data underscore the role of sustained inflammation and dysregulated autophagy in diet-induced pancreatic cancer development and suggest that diet-induced genetic alterations may contribute to this process. Our findings provide a better understanding of the mechanisms underlying the obesity-cancer link in males and females, and will facilitate the development of interventions targeting obesity-associated pancreatic cancer.</p></div

HFCD leads to greater weight gain in male and female KC mice.

<p><b>(A)</b> A schematic view of the study design. <b>(B)</b> Weight gain of male (left panel) and female (right panel) KC mice fed the CD or HFCD. The values are means ± SD. *<i>P</i><0.05, Student’s <i>t</i>-tests. For the male mice collected at 3, 6, and 9 months, n = 12 (5 on CD and 7 on HFCD), 12 (6 on CD and 6 on HFCD), and 11 (5 on CD and 6 on HFCD), respectively. For the female mice collected at 3, 6, and 9 months, n = 11 (6 on CD and 5 on HFCD), 12 (5 on CD and 7 on HFCD), and 11 (5 on CD and 6 on HFCD), respectively. <b>(C)</b> Plasma levels of insulin, leptin, cholesterol, glucose, and triglycerides in 9-month-old KC mice fed the CD or HFCD. Data are depicted as means ± SD. *<i>P</i><0.05 vs. control, Student’s <i>t</i>-tests.</p

HFCD markedly accelerates stroma formation, extracellular matrix deposition and exocrine atrophy in KC mice.

<p><b>(A)</b> The extent of pancreatic collagen deposition was evaluated by Sirius red staining. Graph shows percentage of Sirius red-stained area in pancreas tissue sections at the indicated ages. Data represent mean ± SEM; 8–10 random pancreatic sections were evaluated per mouse; 3–4 mice per group. *<i>P</i><0.05 vs. CD. <b>(B)</b> Pictures illustrate Sirius red staining in pancreatic tissue sections of KC mice fed the CD or HFCD for 6 months, the time-point displaying the highest differences in collagen deposition between CD-fed and HFCD-fed mice. <b>(C)</b> Pancreatic levels of fibrosis-related proteins were analyzed by Western blotting in pancreas lysates from KC mice fed the CD or HFCD for 9 months. Picture shows representative immunoblots of fibronectin; prolyl-4-hydroxylase (P4HA2), a key collagen processing enzyme; cadherin 11, a mesenchymal marker expressed by activated myofibroblasts; α-SMA, a myofibroblast marker; and p-STAT3 (Y705)/ total STAT3. Picture also shows protein levels of pancreatic amylase, a digestive enzyme produced by acinar cells and GAPDH used as loading control. Each lane represents an individual mouse; three mice per group are shown. <b>(D)</b> Graphs show optical density of immunoblots depicted in panel D. Data in graphs represent mean ± SEM, n = 3. *<i>P</i><0.05 vs. CD.</p

HFCD promotes inflammation in the pancreas of KC mice.

<p>Inflammatory parameters in the histological sections of pancreas were evaluated quantitatively. Specifically, acinar loss scores, inflammatory scores, fibrosis scores, and pancreatitis indices were determined for male and female KC mice fed the CD or HFCD for different time periods. The values are means ± SD. *<i>P</i><0.05, Student’s <i>t</i>-tests.</p