The effect of IGF-1R siRNA on apoptosis of pancreatic cancer cells.

<p>(A) IGF1R inhibition induces apoptosis in PANC1 & HPAC cells. Post transfection cells were stained with Annexin-V-FITC and propidium iodide followed by flow cytometry. The percentage of early apoptotic (bottom right quadrant), apoptotic (top right quadrant), late apoptotic and necrotic cells (top left quadrant), and live healthy cells (bottom left quadrant) are shown. (B) Percentage apoptosis and cell death is summarized for three independent experiments in PANC-1 and HPAC cells. (C) IGF-1R inhibition induces death receptor and mitochondrial mediated apoptosis in PANC1 & HPAC cells. Bax, Bcl-2, caspase 8, caspase 3 and cleaved PARP and β-actin expression was assayed using Western blot in IGF-1R siRNA-transfected PANC-1 and HPAC cells. (D) Representative densitometry analysis shows significant potentiation of apoptosis via intrinsic and extrinsic pathways. PS-PANC-1 Scrambled, PI-PANC-1 IGF-1R silenced, HS-HPAC Scrambled, HI-HPAC IGF-1R silenced.</p

Effect of silencing IGF-1R on proliferation and colony formation in pancreatic cancer cell lines.

<p>(A) Expression levels of IGF-1R in PANC-1, HPAC and MIA PaCa-2 were compared with normal pancreatic cells from rat using western blot. (B) Representative immunohistochemical analysis of IGF-1R by tumor stage in pancreatic adenocarcinoma tissues and normal pancreas tissue. (C) PANC-1 and HPAC cells were transfected with three predesigned IGF-1R siRNAs (A, B and C) at three different concentrations (10, 30 and 50 nM) along with scrambled control siRNA. Silencing efficacy of IGF-1R siRNA was determined using western blot in PANC-1 and HPAC cells. (D) Effect of IGF-1R siRNA on cell viability of PANC-1 and HPAC. Cells were transfected with 30 nM and 50 nM of IGF-1R siRNA in PANC-1 and HPAC cells respectively. Cell viability was assayed at 48 h post transfection using MTS assay kit. Results represented as mean ± standard deviation (n = 3). (E) Inhibition of IGF1R expression blocks colony forming capabilities of pancreatic cancer cells, PANC-1 and HPAC. A soft agar assay was used to study the colony formation ability of PANC-1 and HPAC cells. Forty eight hours after the siRNA transfection, PANC-1 and HPAC cells were allowed to grow in 0.7% agarose in RPMI-1640-supplemented with 10% FBS for 16 and 22 days, respectively. Shown here are representative pictures of colony formation from two independent experiments done in triplicate. (F) Percentage colonies in both PANC-1 and HPAC cells were calculated with scrambled control (SCR) serving as the baseline.</p

IGF-1R silencing inhibits invading ability and epithelial-mesenchymal transition of pancreatic cancer cells.

<p>(A) PANC-1 and HPAC cell invasion was assessed in transwell chambers coated with matrigel. Cells that invaded the matrigel-coated insert were fixed, stained and captured at 20× magnification. (B) Number of invaded cells were counted and expressed as percentage invasion. Experiments were done in triplicate (C) IGF-1R silencing inhibits expression of several epithelial-mesenchymal transition markers. Total protein lysates from scrambled control and IGF-1R silenced PANC-1 and HPAC cells were analyzed for expression of Notch-2, Snail, E-cadherin, N-Cadherin, Zeb, Vimentin, and Slug along with internal control β-actin. (D) Densitometic values of EMT markers are shown as % expression. PS-PANC-1 Scrambled, PI-PANC-1 IGF-1R silenced, HS-HPAC Scrambled, HI-HPAC IGF-1R silenced.</p

Silencing IGF-1R alters ERK and STAT signaling in PANC-1 and HPAC cells.

<p>(A & C): The effect of IGF-1R suppression on ERK and STAT signaling was examined in pancreatic cancer cells. Whole cell lysates were separated by SDS-PAGE and analyzed by Western blot for expression levels of phospho-ERK, ERK, IR-β, phospho-IRS-1, IRS, phospho-STAT3, STAT3, COX-2 and β-actin. (B & D): Representative blots are presented and corresponding densitometric analysis is shown to the right of each image. PS-PANC-1 Scrambled, PI-PANC-1 IGF-1R silenced, HS-HPAC Scrambled, HI-HPAC IGF-1R silenced.</p

IGF1R silencing suppressed cell migration in pancreatic cancer cell lines.

<p>(A) Wound healing assay was performed to evaluate the migration of PANC-1 and HPAC cells after silencing IGF-1R. Forty eight hours after siRNA transfection, wound healing capacity of cells were monitored with automated Nikon Biostation CT at 2 h intervals up to 96 h. (B & C): Cell migration was determined by the rate of cells moving towards the scratched area. The percentage migration was calculated by the NIS-Element AR software. Similar results were obtained in three independent experiments. (D) Silencing IGF-1R expression inhibits migration of PANC-1 and HPAC cells. Cell migratory abilities were determined using uncoated transwell Boyden chambers. Post transfection PANC-1 and HPAC cells were allowed to migrate through pores to the bottom surface of transwell. Migrated cells were fixed and stained with 0.2% crystal violet in 5% formalin. Data are representative of five random microscopic field images taken at 20X magnification (E) Percentage migration for transwell assays is shown for IGF-1R silenced PANC-1 and HPAC cells from the results of three independent experiments.</p

Suppression of IGF-1R alters key signaling molecules in PANC-1 and HPAC cells.

<p>(A, C & E): The effect of IGF-1R suppression on AKT/PI3K signaling was examined in pancreatic cancer cells. PANC-1 and HPAC cells were treated with IGF-1R siRNA for 48 h. The cells were harvested and the expression of phospho-AKT, AKT, phospho-PI3K, PI3K, phospho-PTEN, phospho-mTOR, mTOR, phospho-p70s6kinase, p70s6kinase and the internal control β-actin was measured by Western blotting. (B, D & F): Densitometric analysis is also shown to the right of each representative image.</p

Cell proliferation under hyperglycemia.

<p>Cells were cultured in three concentrations of glucose including 5(normal) and 10 and 25 mM (high glucose). Changes in cell proliferation were assessed via MTS assay at 24, 48, and 72 hr in estrogen-receptor positive breast cancer cells (A), triple negative breast cancer cells (B), and non-tumorigenic breast epithelial cells (C). In each case, results were first normalized to baseline growth at 24 hr for each condition and expressed as fold change in cell proliferation above the 24 hr baseline. Results shown here are representative of at least five separate experiments. Statistical significance was calculated using unpaired Student's t-test. Results are expressed as mean±SEM. ns, not statistically significant *, p-value≤0.05 **, p-value≤0.01 ***, p-value≤0.001.</p

Leptin receptor expression.

<p>Cells were cultured in normal (N) and high glucose (HG) for 24 and 72 hr. Changes in leptin receptor expression were assessed via Western blot for estrogen-receptor positive breast cancer cells (A), triple-negative breast cancer cells (B), and non-tumorigenic breast epithelial cells (C). A total of 10 µg of protein was loaded for each sample. Results are representative of at least three separate experiments. ‘OB-Rs’ is the short isoform of leptin receptor, ‘OB-Rl’ is the long isoform of leptin receptor.</p

Proposed model of hyperglycemia's protumorigenic effects.

<p>Under normal glucose levels or normoglycemia, glucose uptake and leptin/IGF1R signaling remain at relatively low basal levels. Under hyperglycemia, cells respond by increasing glucose uptake through GLUT receptors and this in turn activates metabolic pathways that not only respond to increased glucose levels but also have mitogenic effects on breast epithelial cells. Two key metabolic pathways that can have this effect include the leptin and IGF1R signaling pathways. Breast epithelial cells respond to increased glucose levels by increasing production of leptin and IGF1, which in turn increase the expression and activation of leptin and IGF1 receptors at the cell surface. Together, leptin and IGF1 act synergistically to enhance AKT/mTOR signaling leading to increased cell proliferation, in part, through modulation of PKC proteins and cell cycle-associated proteins.</p

Cell cycle proteins increase with hyperglycemia.

<p>Cells were cultured 24 and 72(N) and high glucose (HG). Levels of CDK2 and Cyclin D1, which enhance progression through the G1 and S phases of the cell cycle, were assessed by Western blot. Levels of these proteins were assessed in estrogen-receptor positive breast cancer cells (A), triple-negative breast cancer cells (B), and non-tumorigenic breast epithelial cells (C). Ten micrograms of protein was loaded in each lane. Results shown are representative of two separate experiments.</p