The effects of high-voltage nanosecond electric pulses (nsEPs) on metastatic melanoma are still unclear. Hence, we applied one, two, three, and four 300 ns 40 kV/cm pulses to murine B16-F10 melanoma cells. Cell attachment ability was determined by comparing the number of floating cells and the percentage of attached cells. Melanoma inhibitory activity (MIA) is a secretory protein that is highly correlated with the malignancy and metastasis of malignant melanomas. We used MIA as our target to evaluate the effect of nsEPs on metastasis. Pulsed (experimental) and unpulsed (control) cells were incubated at 37°C under a 5% CO2 atmosphere. To determine cell attachment ability, the culture medium supernatant and attached cells were collected at 6, 12, 18, and 24 h after a single pulse. The live, dead, and total floating cells in the culture medium supernatant were counted. In addition, the live, dead, and total attached cells were counted after multiple pulses. Total RNA was extracted from the attached cells and reverse transcribed into cDNA. The MIA mRNA expression levels were measured using the cDNA temple via quantitative real-time PCR, with β–actin as the internal control. The experiment was repeated three times (n=3). The results show that a single pulse did not affect the cell attachment ability, cell morphology, and the MIA mRNA expression levels (P=0.8058). Two pulses significantly decreased the cell attachment ability (P=0.014), cell viability (P<0.0001), and changed the cell morphology, but did not change the MIA mRNA expression. The three-pulse and the four-pulse treatments significantly decreased the cell attachment ability (P=0.004, 0.00002, respectively), cell viability (P<0.0001), changed the cell morphology, and increased the MIA mRNA expression levels within the first 12 h (P=0.041, 0.001, respectively). These indices were almost normal at 24 h after pulsing. We speculate that the two-, three-, and four-pulse treatments would be optimal for treating melanoma metastasis, whereas the single pulse treatment was not. Therefore, nsEPs provides a great opportunity for treating metastatic melanomas.
|School:||Old Dominion University|
|School Location:||United States -- Virginia|
|Source:||DAI-B 75/03(E), Dissertation Abstracts International|
|Subjects:||Morphology, Molecular biology, Cellular biology|
|Keywords:||Attachment ability, B16-F10 cells, Melanoma inhibitory activity, Melanoma metastasis, Nanosecond electric pulses|
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