Exploration of irreversible electroporation induced apoptotic pathways and mechanisms in pancreatic adenocarcinoma cells

Abstract

Background: Most patients with pancreatic cancer have unresectable disease upon diagnosis. Locally advanced pancreatic cancer has limited therapeutic options, but in vitro and in vivo studies have demonstrated that irreversible electroporation (IRE) is safe and efficacious. Using electrical pulses of variable duration, strength, frequency, and number, IRE can cause cell death via apoptosis induction. Incomplete IRE treatment effect leads to aggressive recurrence, and reversible electroporation with electrochemotherapy or electrogene therapy may improve efficacy. This pilot study aims to demonstrate the underlying apoptotic mechanisms of electroporation, and tests the hypothesis that IRE induces early apoptosis. Methods: In-vitro Panc1 cells were exposed to electroporation while warmed to 36–38 °C. Cells underwent 10, 30, or 50 pulses with 0.1 milliseconds duration, 0.5 seconds pulse interval, and variable electric field strengths from 0 to 2500 Volt/centimeter. Trypan blue viability assay and flow cytometry with Annexin V-FITC and propidium iodide were performed at 1 or 24 hours after exposure. The mean viability of triplicate tests were reported. Results: At 30 pulses there was an increase in late apoptosis/necrosis that plateaued as field strength increased at both time points (Fig. 1). Percent viability using 30 pulses was lower at 24 hours compared to 1 hour. Preliminary 1 hour data at 10 pulses reveals increasing early apoptosis with increasing field strength. Conclusion: Preliminary data suggests that field strenth >1000 V/cm reproducibly induces cell death. Thirty pulses induces late apoptosis or necrosis. There is also less viability at 24 hours for equal field strength. Finally, as few as 10 pulses may induce early apoptosis without creating necrosis. Future studies will gather more data at 10 pulses and conduct assays of apoptosis mediators.