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Nanosecond pulsed electric fields (nsPEFs) eliminates B16f10 melanoma in mice, but cell death mechanisms and kinetics of molecular events of cell death are not fully characterized. Treatment of B16f10 cells in vitro resulted in coordinate increases in active caspases with YO-PRO-1 uptake, calcium mobilization, decreases in mitochondria membrane potential with decreases in forward light scatter (cell size), increases in ADP/ATP ratio, degradation of actin cytoskeleton and membrane blebbing. However, there was no mitochondrial release of cytochrome c, AIF or Smac/DIABLO or generation of reactive oxygen species. Phosphatidylserine externalization was absent and propidium iodide uptake was delayed in small populations of cells. The results indicate that nsPEFs rapidly recruit apoptosis-like mechanisms through the plasma membrane, mimicking the extrinsic apoptosis pathway without mitochondrial amplification yet include activation of initiator and executioner caspases. nsPEFs provide a new cancer therapy that can bypass cancer-associated deregulation of mitochondria-mediated apoptosis in B16f10 melanoma.

The hamster is a useful model of human reproductive biology because its oocytes are similar to those in humans in terms of size and structural stability. In the present study we evaluated fecundity rate, ovarian follicular numbers, ova production, mitochondrial number, structure and function, and cytoplasmic lamellae (CL) in young (2–4 months) and old (12–18 months) Syrian hamsters (Mesocricetus auratus). Young hamsters had higher fertilisation rates and larger litters than old hamsters (100 vs 50% and 9.3 ± 0.6 vs 5.5 ± 0.6, respectively). Ovarian tissue from superovulated animals showed a 46% decrease in preantral follicles in old versus young hamsters. There was a 39% reduction in MII oocyte number in old versus young hamsters. Young ova had no collapsed CL, whereas old ova were replete with areas of collapsed, non-luminal CL. Eighty-nine per cent of young ova were expanded against the zona pellucida with a clear indentation at the polar body, compared with 58.64% for old ova; the remaining old ova had increased perivitelline space with no polar body indentation. Higher reactive oxygen species levels and lower mitochondrial membrane potentials were seen in ova from old versus young hamsters. A significant decrease in mitochondrial number (36%) and lower frequency of clear mitochondria (31%) were observed in MII oocytes from old versus young hamster. In conclusion, the results of the present study support the theory of oocyte depletion during mammalian aging, and suggest that morphological changes of mitochondria and CL in oocytes may be contributing factors in the age-related decline in fertility rates.

Background In human oocytes, as in other mammalian ova, there is a significant variation in the pregnancy potential, with approximately 20% of oocyte-sperm meetings resulting in pregnancies. This frequency of successful fertilization decreases as the oocytes age. This low proportion of fruitful couplings appears to be influenced by changes in mitochondrial structure and function. In this study, we have examined mitochondrial biogenesis in both hamster (Mesocricetus auratus) and mouse (Mus musculus) ova as models for understanding the effects of aging on mitochondrial structure and energy production within the mammalian oocyte. Methodology/Principal Findings Individual metaphase II oocytes from a total of 25 young and old mice and hamsters were collected from ovarian follicles after hormone stimulation and prepared for biochemical or structural analysis. Adenosine triphosphate levels and mitochondrial DNA number were determined within individual oocytes from young and old animals. In aged hamsters, oocyte adenosine triphosphate levels and mitochondrial DNA molecules were reduced 35.4% and 51.8%, respectively. Reductions of 38.4% and 44% in adenosine triphosphate and mitochondrial genomes, respectively, were also seen in aged mouse oocytes. Transmission electron microscopic (TEM) analysis showed that aged rodent oocytes had significant alterations in mitochondrial and cytoplasmic lamellae structure. Conclusions/Significance In both mice and hamsters, decreased adenosine triphosphate in aged oocytes is correlated with a similar decrease in mtDNA molecules and number of mitochondria. Mitochondria in mice and hamsters undergo significant morphological change with aging including mitochondrial vacuolization, cristae alterations, and changes in cytoplasmic lamellae.

Melanocytes are cells that originate in the neural crest. In the dermis, their well characterized role is to produce melanin and through interactions with keratinocytes transfer this pigment to determine skin color and protect the largest organ in the body, the skin, from ultraviolet light. As an effective free radical scavenger, melanin protects against reactive oxygen species that would otherwise damage DNA [Rozanowska et al., 1999]. Melanocytes may also have other roles such as immune, neuroendocrine, and signaling functions through interactions with cells other than keratinocytes, such as lymphocytes, mast cells and endothelial cells [see Tsatmali et al., 2002 for review]. However, keratinocytes regulate melanocyte number, differentiation and melanin production in response to UV radiation. It may be that the resilience of melanocytes to protect the skin, their extraordinary regenerative capacity and their origin as neural crest migratory cells makes them one of the most deadly forms of metastatic skin cancers when they undergo tumorogenesis. It is known that there is a great deal of common cellular and genetic events among embryonic development, tissue regeneration and cancer. Further, typical self-renewal and migration capacity of cancer are shared with embryonic and regenerative cells [White and Zon, 2008]. The recapitulation of embryonic genetic programs is facilitated by overexposure to extreme sunlight (UVA and UVB) or tanning bed light (mostly UVA). The American Cancer Society estimated that in the US in 2009, 68,720 new cases of melanoma (188 new cases /day) will be diagnosed and 8,650 people will die from the disease (24 melanoma deaths /day). The continued increase in melanoma is a significant cause of morbidity and mortality in the Western world. Thus, metastatic melanoma remains a persistent therapeutic challenge. There are limited successes in preventing this often fatal disease and there are even fewer successes in developing a cure.

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.

Nanosecond pulsed electric fields (nsPEF) have been shown to penetrate into living cells to permeabilize intracellular organelles and release Ca2+ from the endoplasmic reticulum. They provide a new approach for physically targeting intracellular organelles with many applications, including initiation of apoptosis, enhancement of gene transfection efficiency and inhibiting tumor growth. We have been working with the murine melanoma model system and here we show that 40 kV/cm electric field pulses 300 nanoseconds in duration can rapidly stimulate pyknosis, reduce blood flow and fragment DNA in murine melanoma tumors in vivo with a total field exposure time of 1.8 microseconds. Three treatments of 100 pulses each results in a mean tumor size regression of 90% within two weeks. Another round of treatments at this time can completely eliminate the melanoma. This new therapy is the first to simultaneously trigger pyknosis and reduce tumor blood flow.

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