(177e) Identification of Various Stages of Induced Apoptosis and Treatment Sensitivity in Non-Small Cell Lung Carcinoma Using Dielectrophoresis

It is important to understand various biomolecular events that contribute towards classification of programmed cell death (apoptosis) and how tumors evade apoptotic death. Small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) are two major categories of lung cancer that differ in their sensitivity to undergo apoptosis. With new drugs being formulated to control abnormal cell proliferation (cancer) caused by mutations, effective drug-induced apoptosis will yield the relationship between cancer genetics and treatment sensitivity. The objective of this study was to build non-invasive techniques (without altering inherent cell properties) to isolate cells that are undergoing varying stages of apoptosis for a high-throughput screening application. We have used dielectrophoresis to determine and isolate various stages of apoptotic cells (early, mid and late) for two NSCLCs adenocarcinoma cell lines (HCC1833 and H1755). Our studies have shown significant differences in apoptotic cells by chromatin condensation, formation of apoptotic bodies and exposure of phosphatidylserine (PS) on the extracellular surface when the cells where exposed to a potent Bcl-2 family inhibitor drug (ABT-263). Time lapse dielectrophoretic studies were performed over a period of 48 hours upon exposure of varying concentrations of ABT-263 ranging between 50 nM to 500 nM. As a result of physical and biochemical changes, inherent dielectric properties of cells undergoing varying stages of apoptosis showed amplified changes in their cytoplasmic and membrane capacitance. In addition, zeta potential of these fixed isolated cells were measured to obtain direct correlation to biomoelcular events. Western blot was used to examine the changes in the expression levels of Bax, Bcl-2, caspase-3, Hsp-90. As a control, the obtained results were compared against standard apoptotic assays for respective stages; JC-1, a mitochondrial membrane potential dye for marking early stage apoptosis, Annexin-V for the detection of PS as a mid-stage marker and defragmentation of DNA, verified by immunofluorescence staining using fluorescein-deoxyuridnie triphosphate (FITC-dUTP) and flow cytometry. The use of non-invasive dielectrophoresis is to provide a sophisticated method to characterize and isolate cells for potential downstream analysis. Effective separation of carcinoma cells at different apoptotic stages should enable a more rational approach to anticancer drug design and therapy.