Anestopoulos Ioannis, Paraskevaidis Ioannis, Kyriakou Sotiris, Potamiti Louiza, Trafalis Dimitrios T, Botaitis Sotiris, Franco Rodrigo, Pappa Aglaia, Panayiotidis Mihalis I
Department of Cancer Genetics, Therapeutics & Ultrastructural Pathology, The Cyprus Institute of Neurology & Genetics, 2371 Nicosia, Cyprus.
Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Epigenomes. 2025 Feb 25;9(1):7. doi: 10.3390/epigenomes9010007.
In the present study, we aimed to characterize the cytotoxic efficacy of Zebularine either as a single agent or in combination with various isothiocyanates in an in vitro model consisting of human melanoma (A375, Colo-679) as well as non-tumorigenic immortalized keratinocyte (HaCaT) cells. In this model, we have evaluated the anti-melanoma effect of Zebularine (in single and combinatorial protocols) in terms of cell viability, apoptotic induction and alterations in ultrastructural chromatin configuration, protein expression levels of DNA methyltransferases (DNMTs) and associated histone epigenetic marks capable of mediating gene expression. Exposure to Zebularine resulted in dose- and time-dependent cytotoxicity through apoptotic induction in malignant melanoma cells, while neighboring non-tumorigenic keratinocytes remained unaffected. A more profound response was observed in combinational protocols, as evidenced by a further decline in cell viability leading to an even more robust apoptotic induction followed by a differential response (i.e., activation/de-activation) of various apoptotic genes. Furthermore, combined exposure protocols caused a significant decrease of DNMT1, DNMT3A and DNMT3B protein expression levels together with alterations in ultrastructural chromatin configuration and protein expression levels of specific histone modification marks capable of modulating gene expression. Overall, we have developed a novel experimental approach capable of potentiating the cytotoxic efficacy of Zebularine against human malignant melanoma cells while at the same time maintaining a non-cytotoxic profile against neighboring non-tumorigenic keratinocyte (HaCaT) cells.
在本研究中，我们旨在表征zebularine作为单一药物或与各种异硫氰酸盐联合使用时，在由人黑色素瘤（A375、Colo - 679）以及非致瘤性永生化角质形成细胞（HaCaT）组成的体外模型中的细胞毒性作用。在该模型中，我们从细胞活力、凋亡诱导以及超微结构染色质构型的改变、DNA甲基转移酶（DNMTs）的蛋白质表达水平和能够介导基因表达的相关组蛋白表观遗传标记等方面，评估了zebularine（单一及联合方案）的抗黑色素瘤作用。暴露于zebularine会通过诱导恶性黑色素瘤细胞凋亡导致剂量和时间依赖性细胞毒性，而相邻的非致瘤性角质形成细胞则不受影响。在联合方案中观察到了更显著的反应，细胞活力进一步下降，导致更强有力的凋亡诱导，随后各种凋亡基因出现差异反应（即激活/失活），这证明了这一点。此外，联合暴露方案导致DNMT1、DNMT3A和DNMT3B蛋白质表达水平显著降低，同时超微结构染色质构型以及能够调节基因表达的特定组蛋白修饰标记的蛋白质表达水平也发生改变。总体而言，我们开发了一种新的实验方法，能够增强zebularine对人恶性黑色素瘤细胞的细胞毒性作用，同时对相邻的非致瘤性角质形成细胞（HaCaT）保持无细胞毒性的特性。
