神经母细胞瘤细胞对射频电流的响应与信号频率的关系。

BACKGROUND: Capacitive-resistive electric transfer (CRET) is a non-invasive therapeutic strategy that applies radiofrequency electric currents within the 400-600 kHz range to tissue repair and regeneration. Previous studies by our group have shown that 48 h of intermittent exposure to a 570 kHz CRET signal at a subthermal density of 50 μA/mm causes significant changes in the expression and activation of cell cycle control proteins, leading to cycle arrest in human cancer cell cultures. The present study investigates the relevance of the signal frequency in the response of the human neuroblastoma cell line NB69 to subthermal electric treatment with four different signal frequency currents within the 350-650 kHz range. METHODS: Trypan blue assay, flow cytometry, immunofluorescence and immunoblot were used to study the effects of subthermal CRET currents on cell viability, cell cycle progression and the expression of several marker proteins involved in NB69 cell death and proliferation. RESULTS: The results reveal that among the frequencies tested, only a 448 kHz signal elicited both proapoptotic and antiproliferative, statistically significant responses. The apoptotic effect would be due, at least in part, to significant changes induced by the 448 kHz signal in the expression of p53, Bax and caspase-3. The cytostatic response was preceded by alterations in the kinetics of the cell cycle and in the expression of proteins p-ERK1/2, cyclin D1 and p27, which is consistent with a potential involvement of the EGF receptor in electrically induced changes in the ERK1/2 pathway. This receives additional support from results indicating that the proapototic and antiproliferative responses to CRET can be transiently blocked when the electric stimulus is applied in the presence of PD98059, a chemical inhibitor of the ERK1/2 pathway. CONCLUSION: The understanding of the mechanisms underlying the ability of slowing down cancer cell growth through electrically-induced changes in the expression of proteins involved in the control of cell proliferation and apoptosis might afford new insights in the field of oncology.

背景：电容电阻电转移（CRET）是一种非侵入性的治疗策略，它在 400-600kHz 范围内应用射频电流来修复和再生组织。我们小组的先前研究表明，48 小时间歇性暴露于 570kHz 的 CRET 信号下，在亚热密度为 50μA/mm 时，会导致细胞周期控制蛋白的表达和激活发生显著变化，导致人类癌细胞培养物的周期停滞。本研究调查了信号频率在人类神经母细胞瘤细胞系 NB69 对亚热电处理的反应中的相关性，使用四种不同信号频率的电流（350-650kHz）进行了实验。

方法：采用台盼蓝检测法、流式细胞术、免疫荧光和免疫印迹法研究亚热 CRET 电流对 NB69 细胞活力、细胞周期进程和涉及 NB69 细胞死亡和增殖的几种标记蛋白表达的影响。

结果：结果表明，在所测试的频率中，只有 448kHz 的信号引起了明显的促凋亡和抗增殖反应。这种凋亡作用至少部分是由于 448kHz 信号引起的 p53、Bax 和 caspase-3 表达的显著变化所致。细胞抑制反应之前是细胞周期动力学和蛋白 p-ERK1/2、cyclin D1 和 p27 表达的改变，这与 EGF 受体在电诱导的 ERK1/2 途径改变中的潜在作用一致。这一结果得到了以下结果的支持：当在 PD98059（ERK1/2 途径的化学抑制剂）存在下施加电刺激时，可暂时阻断 CRET 对促凋亡和抗增殖的反应。

结论：理解通过电诱导参与细胞增殖和凋亡控制的蛋白表达变化来减缓癌细胞生长的能力的机制，可能为肿瘤学领域提供新的见解。