Adiponectin receptor agonists inhibit leptin induced pSTAT3 and in vivo pancreatic tumor growth

Figures

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  Figure 1. Adiponectin receptor levels are reduced in pancreatic cancer Human and murine normal and pancreatic cancer samples were immunostained for ADIPOR1 and ADIPOR2. (A) strong staining is present in the acinar tissue of human normal samples for both receptors but is decreased in the human pancreatic tumor samples. (B) murine pancreatic cancer tissue samples from PKT mice demonstrate a high level of staining in the adjacent acinar tissue and a reduced staining in the dysplastic pancreatic tumor. Graphs represent quantitative analysis of mean fluorescence from single channel images. Scale bar is 100μm. (C-D) quantitative real time PCR analysis was used to determine the relative expression of AdipoR1 (white bars) and AdipoR2 (black bars) in human and murine pancreatic tumor samples (PDAC) as well as in pancreatic cancer cell lines, relative to the level expressed in the normal pancreas (NP) tissue. (E) quantitative real time PCR analysis for adiponectin expression in human and mouse pancreatic cancer cell lines and mouse tissue, relative to human adipose tissue (AT), 3T3L1 cells differentiated to adipocytes (3T3L1) and mouse AT, respectively. Statistical analysis was performed separately…

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  Figure 2. Adiponectin receptor agonists inhibit pancreatic cancer cell proliferation (A) recombinant globular adiponectin (gADN) at 1μg/mL or full length adiponectin (fADN) at 10μg/mL were incubated with human (Panc1 and MiaPaca2) and mouse (Panc02) pancreatic cancer cell lines, labeled with EdU and measured for active proliferation. DMSO was used as a control. Statistical analysis was performed using unpaired t-test (***P ≤ 0.001, ****P ≤ 0.0001). (B) human (Panc1 and MiaPaca2) and mouse PDAC cells (Panc02, P-4313 and K-8484) were treated with increasing concentration of AdipoRon for 48h before measuring proliferation via EdU, which showed dose-dependent inhibition for each PDAC cell line. Statistical significance was determined using ordinary one-way Anova (*P ≤ 0.05, ****P ≤ 0.0001).

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  Figure 3. Adiponectin receptor agonist AdipoRon promotes pancreatic cancer cell apoptosis Annexin V staining was used to evaluate the change in percentage of apoptotic cells by comparing 50μM AdipoRon treatment (+) versus control (-) in multiple PDAC cell lines. Statistical significance was determined using ordinary one-way Anova test (**P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001).

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  Figure 4. AdipoRon inhibits colony formation of pancreatic cancer cells PDAC cell lines were treated with an increasing concentration of AdipoRon for 2 weeks. (A) representative images show a drastic decrease in colony number and size after AdipoRon treatment compared to control. (B) crystal violet staining was performed and quantification of stain was determined by reading absorbance at 590nm. Colony formation was decreased for both murine and human pancreatic cancer cell lines. Statistical analysis was performed using ordinary one-way Anova test (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001).

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  Figure 5. AdipoRon inhibits anchorage independent growth PDAC cell lines were seeded in soft agar and treated with an increasing concentration of AdipoRon for 2 weeks. (A) anchorage independent growth was evaluated in the presence or absence of 50μM AdipoRon. (B) quantitative assessment of spheroid area as well as spheroid diameter were both decreased with AdipoRon treatment compared to control. Statistical analysis was performed using ordinary one-way Anova (***P ≤ 0.001, ****P ≤ 0.0001).

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  Figure 6. AdipoRon inhibits cytokine-induced pSTAT3 (A) qualitative representation of mouse K-8484 and human Panc1 PDAC cells starved overnight and then treated with increasing concentrations of AdipoRon for 12h. At the endpoint cells were lysed and probed with either pAMPK, tAMPK, pSTAT3, or tSTAT3 antibody. (B) correspondent relative quantitation of triplicate experiments. (C) AdipoRon inhibits Leptin-induced STAT3 activation. K-8484 and Panc1 PDAC cell lines were starved overnight and then treated with Leptin at lean concentration of 5ng/ml or an obese concentration of 70ng/ml alone or in combination with 50μM AdipoRon for 12h. Western analysis demonstrated suppression of leptin-induced pSTAT3 in the presence of AdipoRon. (D) correspondent relative quantitation of triplicate experiments. β-actin levels are shown as a total loading control. Statistical analysis was performed using ordinary one-way Anova (*P ≤ 0.05, **P ≤ 0.01***P ≤ 0.001, ****P ≤ 0.0001).

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  Figure 7. AdipoRon inhibits orthotopic pancreatic tumor growth and tumor proliferation in vivo Murine P-4313 and K-8484 cancer cells were orthotopically injected into syngeneic wildtype mice and allowed to grow for two weeks, after which mice were treated with vehicle or AdipoRon 5mg/kg/day for 2 weeks. (A) at endpoint, AdipoRon treated mice had a significantly reduced tumor size. (B) tumor proliferation measured by Ki67 positive tumor area per tumor section was significantly reduced after AdipoRon treatment. (C) no significant difference was detected in relative expression of AdipoR1 (white bars) and AdipoR2 (black bars) by quantitative real time PCR analysis between control and AdipoRon treated tumors. (D) ELISA analysis showed no significant difference in circulating serum adiponectin levels between the two groups of mice. (P-4313 filled symbols and K-8484 open symbols). Statistical analysis was performed using unpaired t-test (*P ≤ 0.05, **P ≤ 0.01).