Date of Award

Summer 2008

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Committee Director

Stephen Beebe

Committee Member

Peter Blackmore

Committee Member

Andrei Pakhomov

Committee Member

Karl Schoenbach

Committee Member

R. James Swanson

Abstract

Nanosecond pulsed electric fields (nsPEFs) are ultra-short pulses that induce direct electric field and biological effects that initiate apoptosis. Here the application of ten 300ns pulses ranging in electric fields from 12kV/cm-60kV/cm was administered to determine the effects on B16F10 melanoma cells evaluated by in vitro studies. Initial application of nsPEFs demonstrated apoptosis induction in an electric field- and pulse number-dependent manner measured by caspase activation that correlated with decrease in cell viability 24hr post pulse. In addition caspase activity was shown to be independent of calcium mobilization though ions may play a part in other aspects of apoptosis. The use of additional apoptotic markers Annexin V and propidium iodide revealed no phosphatidylserine externalization and no direct electric field effects on membrane permeability, respectively. However, characteristic apoptotic morphological features of plasma membrane blebbing and F-actin cytoskeleton disruption were observed. In order to define apoptotic pathway, evaluation of mitochondrial function illustrated no intracellular release of mitochondrial proteins, cytochrome c, second mitochondria-derived activator of caspase (Smac), direct IAP-binding protein (DIABLO) and apoptosis inducing factor (AIF). Further examination of caspases with isozymes specific initiators indicated electric field-dependent increases in caspases -8 and interestingly -9 as well as executioner caspases -3, -6 and -7.

NsPEFs activate signal transduction mechanisms that induce apoptosis through a mitochondria independent pathway that mimics an apparent membrane-dependent extrinsic type I apoptotic mechanism(s). Uniquely, nsPEF activate caspase -9 either via direct cleavage or an unknown mechanism without apoptosome formation in the mitochondria-dependent apoptotic pathway. Therefore nsPEFs induce apoptosis rapidly by mechanisms that are distinct and independent from those that are commonly mutant in the mitochondria pathway in melanomas and those same mechanisms frequently used by chemotherapeutic agents that result in resistant tumors. The short nsPEF treatment time (3 milliseconds) and rapid apoptosis induction (tens of minutes) is less likely to result in cancers that are resistant to nsPEF treatment. The therapeutic application of nsPEFs can provide a new and novel method to the arsenal for cancer treatment strategies.

DOI

10.25777/f464-te67

ISBN

9780549753629

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