PDF(Pubmed)
Abstract：
Podophyllotoxin (PPT) is an active pharmaceutical ingredient (API) with established antitumor potential. However, due to its systemic toxicity, its use is restricted to topical treatment of anogenital warts. Less toxic PPT derivatives (e.g., etoposide and teniposide) are used intravenously as anticancer agents. PPT has been exploited as a scaffold of new potential therapeutic agents; however, fewer studies have been conducted on the parent molecule than on its derivatives. We have undertaken a study of ultrastructural changes induced by PPT on HaCaT keratinocytes. We have also tracked the intracellular localization of PPT using its fluorescent derivative (PPT-FL). Moreover, we performed molecular docking of both PPT and PPT-FL to compare their affinity to various binding sites of tubulin. Using the Presto blue viability assay, we established working concentrations of PPT in HaCaT cells. Subsequently, we have used selected concentrations to determine PPT effects at the ultrastructural level. Dynamics of PPT distribution by confocal microscopy was performed using PPT-FL. Molecular docking calculations were conducted using Glide. PPT induces a time-dependent cytotoxic effect on HaCaT cells. Within 24 h, we observed the elongation of cytoplasmic processes, formation of cytoplasmic vacuoles, progressive ER stress, and shortening of the mitochondrial long axis. After 48 h, we noticed disintegration of the cell membrane, progressive vacuolization, apoptotic/necrotic vesicles, and a change in the cell nucleus\'s appearance. PPT-FL was detected within HaCaT cells after ~10 min of incubation and remained within cells in the following measurements. Molecular docking confirmed the formation of a stable complex between tubulin and both PPT and PPT-FL. However, it was formed at different binding sites. PPT is highly toxic to normal human keratinocytes, even at low concentrations. It promptly enters the cells, probably via endocytosis. At lower concentrations, PPT causes disruptions in both ER and mitochondria, while at higher concentrations, it leads to massive vacuolization with subsequent cell death. The novel derivative of PPT, PPT-FL, forms a stable complex with tubulin, and therefore, it is a useful tracker of intracellular PPT binding and trafficking.
摘要：
鬼臼毒素（PPT）是一种具有抗肿瘤潜力的活性药物成分（API）。然而,由于其全身毒性，它的使用仅限于肛门生殖器疣的局部治疗。毒性较小的PPT衍生物(例如，依托泊苷和替尼泊苷）静脉内用作抗癌剂。PPT已被用作新的潜在治疗剂的支架；然而，对母体分子的研究少于对其衍生物的研究。我们已经对PPT诱导的HaCaT角质形成细胞的超微结构变化进行了研究。我们还使用其荧光衍生物(PPT-FL)追踪了PPT的细胞内定位。此外，我们对PPT和PPT-FL进行了分子对接，以比较它们对微管蛋白各种结合位点的亲和力。使用Prestoblue活力测定法，我们在HaCaT细胞中建立了PPT的工作浓度。随后，我们使用选定的浓度来确定PPT在超微结构水平的影响。使用PPT-FL通过共聚焦显微镜进行PPT分布的动力学。使用Glide进行分子对接计算。PPT诱导对HaCaT细胞的时间依赖性细胞毒性作用。24小时内,我们观察到细胞质过程的延伸，细胞质液泡的形成，进行性ER应激，线粒体长轴的缩短。48小时后，我们注意到细胞膜的崩解，渐进的空泡化，凋亡/坏死囊泡，和细胞核外观的变化。在孵育约10分钟后,在HaCaT细胞内检测到PPT-FL,并在以下测量中保留在细胞内。分子对接证实在微管蛋白与PPT和PPT-FL之间形成稳定的复合物。然而,它是在不同的结合位点形成的。PPT对正常人角质形成细胞有很强的毒性,即使在低浓度。它迅速进入细胞，可能是通过胞吞作用.在较低的浓度下，PPT会导致ER和线粒体的破坏，而在较高的浓度下，它导致大量的空泡化和随后的细胞死亡。PPT的新颖衍生物，PPT-FL,与微管蛋白形成稳定的复合物，因此,它是细胞内PPT结合和贩运的有用跟踪器。
