Melanoma Morphology Change & Apoptosis Induced by Multiple Nanosecond Pulsed Electric Fields

Abstract

Our initial in vitro (HL-60 cells) and in vivo (B16-F10 murine) studies showed nanosecond pulsed electric fields (nsPEFs) caused intracellular changes and melanoma involution, respectively. We wanted to describe the morphologic changes in cell ultrastructure and investigate the mechanism for change due to nsPEFs in B16-F10 melanoma tumors in SKH-1 mice. We injected B16-F10 cells into 120 female SKH-1 mice to derive melanoma tumors. After multiple nsPEF treatments (40 kV/cm field strength; 5 ns rise time; 300 ns duration), morphologic changes of melanoma size, shape, tumor nesting pattern, blood vessel structure and cell ultrastructure were observed. Nuclear changes were recorded with light-and transmission electron-microscopy (TEM). Apoptosis in situ was detected by several immunohistochemistry (IHC) methods: (1) terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL); (2) caspase 3, 6 and 7; and (3) histone H2AX. After multiple nsPEF treatments the melanoma blood supply was disrupted and melanomas shrank an average of 90% within two weeks. TEM showed increased rough endoplasmic reticulum and condensed dark-staining nucleoli; TUNEL presented fluorescent increase. Together these suggest post-treatment apoptotic activity, which was concurrently allied with increased caspase 3, 6 and 7, and histone H2AX fluorescence (DNA damage) after pulsing. Our study shows that blood flow and apoptotic changes play a central role in the biological effects caused by nsPEFs. Therefore nsPEFs may have application potential in cancer therapy, gene regulation and biophysical research by non-invasively disrupting intracellular components and inducing apoptosis in malignant tumors.