Glycogen Synthase Kinase-3 Inhibition Sensitizes Pancreatic Cancer Cells to TRAIL-Induced Apoptosis

<div><p>Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) induces apoptosis in a variety of cancer cell lines with little or no effect on normal cells. However, its effect is limited as some cancers including pancreatic cancer show de novo resistance to TRAIL induced apoptosis. In this study we report that GSK-3 inhibition using the pharmacologic agent AR-18, enhanced TRAIL sensitivity in a range of pancreatic and prostate cancer cell lines. This sensitization was found to be caspase-dependent, and both pharmacological and genetic knock-down of GSK-3 isoforms resulted in apoptotic features as shown by cleavage of PARP and caspase-3. Elevated levels of reactive oxygen intermediates and disturbance of mitochondrial membrane potential point to a mitochondrial amplification loop for TRAIL-induced apoptosis after GSK-3 inhibition. Consistent with this, overexpression of anti-apoptotic mitochondrial targets such as Bcl-XL, Mcl-1, and Bcl-2 rescued PANC-1 and PPC-1 cells from TRAIL sensitization. However, overexpression of the caspase-8 inhibitor CrmA also inhibited the sensitizing effects of GSK-3 inhibitor, suggesting an additional role for GSK-3 that inhibits death receptor signaling. Acute treatment of mice bearing PANC-1 xenografts with a combination of AR-18 and TRAIL also resulted in a significant increase in apoptosis, as measured by caspase-3 cleavage. Sensitization to TRAIL occurred despite an increase in β-catenin due to GSK-3 inhibition, suggesting that the approach might be effective even in cancers with dysregulated β-catenin. These results suggest that GSK-3 inhibitors might be effectively combined with TRAIL for the treatment of pancreatic cancer.</p> </div

Genetic knockdown of GSK-3 renders cells sensitive to TRAIL-induced apoptosis.

<p>PANC-1 cells were pre-treated using AR-18 or siRNA against both isoforms of GSK-3, and then exposed to TRAIL. Immunoblotting confirms successful knockdown of GSK3α and β. This resulted in increased cleavage of PARP and caspase-3 following exposure to TRAIL. Scr: Scrambled non-specific siRNA.</p

TRAIL sensitization by AR18 is associated with loss of ΔΨm and increased ROI generation.

<p>PANC-1 and BxPC-3 cells were untreated or incubated with AR-18 (25 µM), TRAIL (10 ng/mL), or their combination for 24 h, and then analyzed by flow cytometry. Dot density plots of propidium iodide (PI) uptake vs ΔΨm show that loss of ΔΨm precedes PI uptake. TRAIL induced loss of ΔΨm in BxPC-3 but not in PANC-1, consistent with their greater sensitivity seen using the SRB assay (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041102#pone-0041102-g001" target="_blank">Figure 1</a>). Combined treatment with TRAIL and AR18 clearly sensitized both cell lines to loss of ΔΨm, and this was accompanied by increased ROI generation and loss of out membrane integrity.</p

GSK-3 inhibition sensitizes TRAIL-resistant pancreatic cancer cells to apoptosis.

<p>PANC-1 (A) and BxPC-3 (B) were treated with TRAIL after 24 h pre-exposure to AR-18 at the indicated concentrations, and cell proliferation measured by SRB assay. Each bar graph signifies mean from three separate experiments with six replicates. error bars  =  ± SEM.</p

GSK-3 inhibition enhances TRAIL sensitization through PARP and caspase cleavage.

<p>(A) BxPC-3 and PANC-1 cells were treated with AR-18, in combination with or without TRAIL for 24 h and analysed by for PARP cleavage. (B) BxPC-3 (left) and PANC-1 (right) cells were treated with AR-18 (25 uM), TRAIL (10 ng/mL), z-VAD-fmk 50 µM, or the combinations indicated and SRB assay after 22 h incubation. Each bar graph signifies mean from three experiments with six replicates. error bars  =  ± SEM. *p<0.005, **p<0.0001.</p