Nanosecond pulsed electric fields induce apoptosis in p53-wildtype and p53-null HCT116 colon carcinoma cells

Abstract

Non-ionizing radiation produced by nanosecond pulsed electric fields (nsPEFs) is an alternative to ionizing radiation for cancer treatment. NsPEFs are high power, low energy (non-thermal) pulses that, unlike plasma membrane electroporation, modulate intracellular structures and functions. To determine functions for p53 in nsPEF-induced apoptosis, HCT116p53(+/+) and HCT116p53(-/-) colon carcinoma cells were exposed to multiple pulses of 60 kV/cm with either 60 ns or 300 ns durations and analyzed for apoptotic markers. Several apoptosis markers were observed including cell shrinkage and increased percentages of cells positive for cytochrome c, active caspases, fragmented DNA, and Bax, but not Bcl-2. Unlike nsPEF-induced apoptosis in Jurkat cells (Beebe et al. 2003a) active caspases were observed before increases in cytochrome c, which occurred in the presence and absence of Bax. Cell shrinkage occurred only in cells with increased levels of Bax or cytochrome c. NsPEFs induced apoptosis equally in HCT116p53(+/+) and HCT116p53(-/-) cells. These results demonstrate that non-ionizing radiation produced by nsPEFs can act as a non-ligand agonist with therapeutic potential to induce apoptosis utilizing mitochondrial-independent mechanisms in HCT116 cells that lead to caspase activation and cell death in the presence or absence of p-53 and Bax.

p53 positively regulates the expression of cancer stem cell marker CD133 in HCT116 colon cancer cells

“…The human colon cancer cell lines HCT116 p53 +/+ and HCT116 p53 -/-were well-established cell lines (11), kindly provided by Professor Xingzhi Xu (College of Life Sciences, Capital Normal University, Beijing, China) and maintained in our laboratory. All HCT116 cells were cultured in McCoy's 5A medium (HyClone; GE Healthcare Life Sciences, Logan, UT, USA).…”

Pulse Power Ablation of Melanoma with Nanosecond Pulsed Electric Fields

“…However, as indicated in Figure 3, we did detect small increases in fluorescence with antibodies to Smac/Diablo and apoptosis initiating factor (AIF), albeit in small populations of cells (10-15%). Given that cytochrome c release was detected in Jurkat cells coincident with activation of caspases (Beebe et al, 2003a] as well as in HCT 116 cells [Hall et al, 2007a] and E4 squamous carcinoma cells [Ren and Beebe, 2011] after caspase activation, this suggests that nsPEFs have cell type-specific effects on mitochondriamediated events associated with apoptosis-like mechanisms.…”

“…In contrast, the cytoskeletal structure of S phase cells exposed to the same pulses was not significantly perturbed. Five hours after treatment with these conditions, control and treated cell were indistinguishable with 90-95% survival [Hall et al, 2007a]. When these cells were pulsed with three 300ns pulses at 60kV/cm, which killed 90% of cells, S-phase cells and unsynchronized cells exhibited rearranged actin cytoskeletons.…”

“…Given that nsPEFs have effects on plasma membranes and intracellular structures and the cytoskeleton is an extension of the plasma membrane, it was likely that nanosecond pulsed electric fields would affect the cytoskeleton. Effects of pulse power ablation on the actin cytoskeleton have been demonstrated [Hall et al, 2007a]. Human HCT 116 colon carcinoma cells were synchronized to S phase (95%) by thymidine block or analyzed unsynchronized (50% S phase).…”

Electropermeabilization

“…When longer pulse trains or higher voltage nsEP are applied, cells undergo necrotic cell death, as a result of damaged plasma membrane [19,22,38,39]. When shorter pulse trains and/or lower voltage nsEP are applied, however, cells undergo apoptotic cell death [4-6, 22, 50].…”