Cellular Distribution and Ultrastructural Changes in HaCaT Cells, Induced by Podophyllotoxin and Its Novel Fluorescent Derivative, Supported by the Molecular Docking Studies.

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.

New gold (III) cyanide complex TGS 121 induces ER stress, proteasome inhibition and death of Ras-hyperactivated cells.

Metal-based agents in cancer therapy, like cisplatin and its derivates, have established clinical applications but also can induce serious side effects. Thus, metallotherapeutic alternatives for platinum derivatives are developed and intensively studied. Platinum is replaced by several transition metals including gold. Especially gold (III) complexes can have the same square-planar structure and are isoelectric with platinum (II). Hence, they are developed as potential anti-cancer drugs. Thus, our group projected and developed a group of novel cyanide-based gold (III) complexes. Within this work, we aimed to characterize the safety and effectivity of one of them, TGS 121. TGS 121 in our preliminary work was selective for Ras-hyperactivated cells. Here we studied the effects of the novel complex in cancerous Ras-3 T3 and non-cancerous NIH-3 T3 cells. The complex TGS 121 turned out to be non-toxic for NIH-3 T3 cells and to induce death and alternations in Ras-hyperactivated cells. We found induction of ER stress, mitochondria swelling, proteasome inhibition, and cell cycle block. Moreover, TGS 121 inhibited cell migration and induced the accumulation of perinuclear organelles that was secondary to proteasome inhibition. Results presented in this report suggest that stable gold-cyanide TGS 121 complex is non-toxic, with a targeted mechanism of action and it is promising in anticancer drug discovery.

Novel podophyllotoxin and benzothiazole derivative induces transitional morphological and functional changes in HaCaT cells.

Podophyllotoxin (PPT) is an antimitotic drug used topically in the treatment of anogenital warts. Due to its toxicity it cannot be administered systemically as an anticancer agent. However, modified PPT derivatives such as etoposide and teniposide are used clinically as systemic agents. Thus, we invented novel PPT derivative KL3 that was synthesized by photocyclization. Earlier we have shown that KL3 has an anticancer effect in various cell lines. Here we compared the toxicity of KL3 vs PPT on non-cancerous normal human keratinocytes (HaCaT) and peripheral blood mononuclear cells (PBMC) showing that KL3 is less toxic than PPT to non-cancerous cells. At concentrations that neither induced cell death, nor affected cell cycle, KL3 in HaCaT cells evoked transient ultrastructural features of ER stress, swelling of mitochondria and elongation of cytoplasmic processes. Those changes partially reversed with prolonged incubation while features of autophagy were induced. PPT in equivalent concentrations induced HaCaT cell death by cell cycle arrest, intrinsic apoptosis and finally disintegration of cell membranes followed by secondary necrosis. In conclusion, we show that the KL3 derivative of PPT in contrast to PPT allows repair of normal keratinocytes and triggers mechanisms that restore non-tumor cell homeostasis.