Verification of Apoptosis in MDA-MB-231 Triple Negative Breast Cancer Cells Post NBA Photodynamic Therapy Using DNA Fragmentation Assay and Cell Death Dyes

Cell death is a biological mechanism of a cell ceasing to carry out its functions and is considered as a crucial process as it promotes the removal of unwanted and or damaged cells and maintains a balance between the growth, development, proliferation, and death to maintain tissue homeostasis. Cell death is generally characterized into 2 basic types: Programmed cell death and un-programmed cell death, each type further divided into a set of sub-types. Programmed cell death is the mechanism that is usually carried out by the body in order to eliminate unwanted or damaged cells. A crucial characteristic of programmed cell death is that it retains the integrity of the cell membrane and cells are engulfed by macrophages or neighboring cells. Un-programmed cell death is a mechanism followed by the cells under conditions of tissue damage, stress or injury to the cell. Un-programmed cell death, as opposed to programmed cell death, is characterized by non-specific swelling of cells and loss of membrane integrity. In contrast to programmed cell death, it also results in the generation of an inflammatory response that is essential for recognition of the cell for host-defense and tissue repair. A type of programmed cell death is apoptosis, which is characterized by cell shrinkage and formation of extensive, unretracted plasma membrane blebs. A characteristic of apoptotic cell death is flipping of phosphatidylserine from the inner to the outer plasma membrane. In addition, cleavage of nuclear DNA into oligonucleosomal fragments represented by multiples of 180-200 base pairs is also an important event of apoptosis. Our lab is currently investigating a light activated compound, o-nitrobenzaldehyde (NBA), as a photodynamic therapy to causes decreases in intracellular pH followed by cell death. We have observed that this photodynamic therapy causes cell death in MDA-MB-231 triple negative breast cancer cells; however, the mechanism of cell death induced by this therapy is unknown. We hypothesize that our photodynamic therapy causes these triple negative breast cancer cells to undergo apoptosis. We test our hypothesis, by using DNA gel electrophoresis assay to characterize the type of cell death followed by MDA-MB-231 cells. Additionally, we also used the apoptosis marker death dye Annexin-V, following photoactivation of NBA in-vitro to further test this hypothesis. Our results lead us to the conclusion that NBA photodynamic therapy does not cause MDA-MB-231 triple negative breast cancer cells to undergo apoptosis to the extent of causing fragmentation of oligonucleosomal DNA but induce apoptosis that enough to cause a positive expression of the apoptosis death dye, Annexin V.