Laboratory of Biomedical Ultrasonics/Gynecological Oncology Laboratory, West China Second University Hospital, Sichuan University, Chengdu, P.R. China.
Eur Rev Med Pharmacol Sci. 2013 Jan;17(2):152-60.
Intense nanosecond pulsed electric fields (nsPEFs) have been known to promote apoptosis without physically changing membrane structure or damaging morphology of tumor cells. To determine the contribution of centrosome to the progression of apoptosis by nsPEFs, HeLa cells were exposed to high intensity (6 kV/cm) nsPEFs (8-32 ns) in normal culture condition and cell biology and molecular parameters of cells were investigated.
Apoptotic cell death was identified by TUNEL assay after being exposed to the nsPEFs with various pulse durations, while immunofluorescent staining was performed to detect the number and distribution of centrosomes. To clarify whether nsPEFs-induced centrosome over-duplication is the consequence of DNA damage, we used comet assay to detect simultaneous DNA damage. And additionally Western Blot was used to detect PLK1 protein level to explore the correlation between apoptotic cell death and nsPEFs-induced centrosome over-duplication. Correlation between nsPEFs and molecular parameters was statistically analyzed.
NsPEFs induced a clear apoptosis reaching a maximum at 24ns, 24h after exposure (p < 0.05), where DNA fragmentation and over-duplicated centrosomes were observed. This apoptosis may be promoted in a time- and pulse duration-dependent manner. Polo-like kinase (PLK1) protein levels were significantly decreased by such nsPEFs (p < 0.05). Control treatment without the nsPEFs did not cause any damage to the cultured HeLa cells.
Intense nsPEFs promote cell apoptosis through a centrosome-mediated pathway involving a reduction in the level of PLK1, which may provide new therapeutic targets for human cancer treatment.
高强度纳秒脉冲电场（nsPEFs）已被证实可以促进细胞凋亡，而不会改变细胞膜的物理结构或破坏肿瘤细胞的形态。为了确定中心体在 nsPEFs 诱导的细胞凋亡过程中的作用，我们在正常培养条件下用高强度（6 kV/cm）nsPEFs（8-32 ns）处理 HeLa 细胞，并研究了细胞的生物学和分子参数。
用不同脉宽的 nsPEFs 处理细胞后，通过 TUNEL 实验鉴定凋亡细胞死亡，同时通过免疫荧光染色检测中心体的数量和分布。为了阐明 nsPEFs 诱导的中心体过度复制是否是 DNA 损伤的结果，我们使用彗星实验检测同时发生的 DNA 损伤。此外，我们还使用 Western blot 检测 PLK1 蛋白水平，以探索凋亡细胞死亡与 nsPEFs 诱导的中心体过度复制之间的关系。统计分析了 nsPEFs 与分子参数之间的相关性。
nsPEFs 在暴露后 24 小时（p < 0.05）诱导了明显的凋亡，达到最大值为 24ns，此时观察到 DNA 片段化和中心体过度复制。这种凋亡可能是时间和脉宽依赖性的。这种 nsPEFs 还显著降低了 Polo-like 激酶（PLK1）蛋白水平（p < 0.05）。没有 nsPEFs 的对照处理对培养的 HeLa 细胞没有造成任何损伤。
强烈的 nsPEFs 通过一种涉及 PLK1 水平降低的中心体介导的途径促进细胞凋亡，这可能为人类癌症治疗提供新的治疗靶点。
