Diazepam induces mitotic defects and cytotoxicity through modulation of tubulin and Eg5

Diazepam (DZP) is a widely prescribed drug for central nervous system disorders. However, it also exhibits a significant inhibitory effect on tubulin and the mitotic kinesin Eg5. These novel mitotic mechanisms explain its cytotoxicity and provide valuable insights for designing safer, more targeted antimitotic agents. Studies in mammalian cancer and noncancerous cell lines, including HeLa, MCF-7, A549, and L929, demonstrated that DZP inhibits cell proliferation in a concentration-dependent manner, with IC₅₀ values ranging from 42 μM to 76 μM. Fluorescence spectroscopy confirmed direct binding of DZP to tubulin and Eg5, with dissociation constants (K_d) of 33.6 μM and 50.5 μM, respectively. This binding disrupted GTPase activity of tubulin and ATPase activity of Eg5, both of which are essential for mitotic progression. Consequently, DZP caused microtubule disorganization, impaired centrosome separation, and induced monopolar spindle formation, collectively leading to mitotic arrest in various cancer cell lines as well as non-cancerous cells. Additionally, DZP induced mitochondrial membrane potential loss and apoptosis, while inhibiting cell migration and colony formation, highlighting its cytotoxic effects. Furthermore, DZP synergistically enhanced the mitotic inhibition induced by the antimitotic agent vinblastine, further suppressing cancer cell proliferation. The findings indicate that DZP exhibits a probable, multi-target antimitotic effect, involving interactions with both tubulin and Eg5, which suggests a need for careful evaluation of its biological safety window. This revised approach addresses concerns regarding the cytotoxic risks and potential antiproliferative effects of DZP in conventional anxiolytic applications.