Electric pulses do not change expression profile of genes involved in development of cancer in malignant melanoma cells. Homo sapiens

Electroporation is a versatile method for in vitro or in vivo delivery of different molecules into cells. However, no study so far has analysed the effects of electric pulses used in electrochemotherapy (ECT pulses) or electric pulses used in electrogene therapy (EGT pulses) on malignant cells. We studied the effect of ECT and EGT pulses in human malignant melanoma cells in vitro in order to understand and predict possible effect of electric pulses on gene expression and their possible effect on cell behaviour. We used microarrays with 2698 different oligonucleotides to obtain expression profile of genes involved in apoptosis and development of cancer in a malignant melanoma cell line (SK-MEL28) exposed to ECT pulses and EGT pulses. Cells exposed to ECT pulses showed 68.8% average survival rate and 31.4% average survival rate in cells exposed to EGT pulses. Only seven common genes were found differentially expressed in cells 16 h after exposure to ECT and EGT pulses. We found that ECT and EGT pulses induce HSP70 stress response mechanism, repress histone protein H4, a major protein involved in chromatin assembly, and down-regulate components involved in protein synthesis. Our results show that electroporation does not significantly change expression profile of major tumour suppressor genes or oncogenes of cell cycle. Moreover, electroporation also does not changes the expression of genes involved in stability of DNA, supporting the current evidence that electroporation is a safe method that does not promote tumorigenesis. However, in spite of being considered an isothermal method it does to some extent induce stress that resulted in expression of environmental stress response mechanism, HSP70. Overall design: The difference in expression of genes involved in development of cancer was obtained by comparison of malignant melanoma cells exposed to EGT or ECT pulses against the same untreated malignant melanoma cells. In our experimental design, microarrays with 2698 different genes were used as a dual colour system in which exposed and non-exposed cells’ mRNA were separately labelled, mixed and hybridised together on each array. Only microarrays expressing at least 50% of genes were used for further analysis. All oligonucleotides on the same array were spotted in quadruplicates and each microarray analysis was performed in duplicates, therefore obtaining eight measurements of the same oligonucleotide. The acquired data were analysed with Acuity 4.0 to select reliable signals. Only genes, present in both duplicated microarrays were considered for further processing.

电穿孔（electroporation）是一种可用于体外（in vitro）或体内（in vivo）向细胞递送各类分子的通用方法。然而迄今为止，尚无研究分析电化学疗法（electrochemotherapy, ECT）所用电脉冲（ECT脉冲）与基因电疗法（electrogene therapy, EGT）所用电脉冲对恶性细胞的影响。本研究以体外培养的人恶性黑色素瘤细胞为模型，探究ECT与EGT脉冲对基因表达的潜在影响及其对细胞行为的作用，以期阐明并预测相关效应。我们采用包含2698种不同寡核苷酸（oligonucleotide）的微阵列（microarray），对经ECT脉冲与EGT脉冲处理的恶性黑色素瘤细胞系SK-MEL28中参与细胞凋亡与癌症发生的基因表达谱进行检测。经ECT脉冲处理的细胞平均存活率为68.8%，经EGT脉冲处理的细胞平均存活率则为31.4%。在暴露于两种脉冲16小时后的细胞中，仅发现7个共有基因存在差异表达。我们发现，ECT与EGT脉冲均可诱导热休克蛋白70（HSP70）应激响应通路，抑制参与染色质组装的核心组蛋白H4的表达，并下调蛋白质合成相关组分的表达。本研究结果显示，电穿孔不会显著改变细胞周期相关主要抑癌基因或癌基因的表达谱。此外，电穿孔亦不会改变参与DNA稳定性维持的基因的表达，这一结果佐证了现有研究结论，即电穿孔是一种安全的方法，不会促进肿瘤发生。不过，尽管电穿孔被认为是一种等温操作方法，但其仍可在一定程度上诱导应激反应，引发环境应激响应通路HSP70的表达。
实验整体设计：
通过对比经EGT或ECT脉冲处理的恶性黑色素瘤细胞与未处理的同源细胞，我们获取了癌症发生相关基因的表达差异数据。在本实验设计中，我们采用包含2698个不同基因的双色微阵列系统，将处理组与对照组细胞的mRNA分别进行标记，随后混合并在同一张微阵列上进行杂交。仅对至少50%的基因均有检测信号的微阵列数据进行后续分析。同一张微阵列上的所有寡核苷酸均设置4次重复点样，且每次微阵列分析均设置2次生物学重复，因此每个寡核苷酸可获得8次检测数据。我们使用Acuity 4.0软件对获取的数据进行分析，以筛选可靠的信号值。仅保留在两次重复微阵列中均检测到的基因用于后续处理。