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.

A series of novel podophyllotoxin derivatives containing benzothiazole scaffolds were synthesized and evaluated for their in vitro cytotoxic activity against five cancer cell lines (MCF-7, SKOV-3, B16F10, LOVO, and HeLa). Two compounds, 7 and 11, which are different only by the absence or presence of the ester group, showed the strongest cytotoxic effect towards all tested cancer cell lines with the IC₅₀ 0.68–2.88 µM. In addition, it was demonstrated that these compounds inhibit cancer cell proliferation by inducing G2/M phase arrest in HeLa cells. The structure–activity relationship was analyzed and it confirmed the importance of the core structural features like a dioxolane ring and free-rotating trimethoxyphenyl group for cytotoxicity. Moreover, the R configuration of the ester group at the C-8′ position proved to be substantial since its epimer was inactive. The molecular docking studies revealed that the most potent compounds have a different binding mode to β-tubulin than podophyllotoxin; however, the benzothiazole fragment docked in a similar location as the trimethoxyphenyl group of podophyllotoxin, exhibiting similar hydrophobic interactions. These findings clearly indicate that podophyllotoxin–benzothiazole derivatives could be addressed for further pharmacological studies in anticancer research.