Design of novel small molecule inhibitors of tubulin polymerization with high apoptosis-inclucing activity

A262 Attacking the microtubule system is a successful strategy to inhibit cancer cell proliferation and to induce selectively cell death of tumor cells in clinical settings. This feature makes them one of the most prominent targets for the development of anticancer agents and for the treatment of solid tumors. To improve anticancer therapy we designed novel tubulin interacting agents and evaluated the drugs for apoptosis inducing activity.
 New synthesized tubulin-binding 9-benzylidene-naphtho[2,3-b]thiophen-4-one and benzylidene-9(10H)-anthracenone derivatives were evaluated for their ability to induce cell death. As determined by flow cytometry, cancer cells are predominantely arrested in metaphase with 4N DNA before cell death occurs. By using indirect immunofluorescence technique we visualized microtubule depolymerization recognizable by short microtubule fragments scattered around the nucleus. Exposure of breast cancer cells to novel agents caused fragmentation of cells, chromatin condensation and cell shrinkage, which are among others, typical features of apoptotic cell death. Furthermore, time- and dose-dependent induction of apoptosis in SH-SY5Y cells was detected via cleavage of Ac-DEVD-AMC, a fluorigenic substrate for caspase-3. We observed a lower apoptotic activity in neuroblastoma cells overexpressing Bcl-xL, suggesting activation of the mitochondrial apoptosis pathway. Western blot analysis demonstrated that caspase-3 and its active subunits were upregulated in a time-dependent manner after exposure of SH-SY5Y cells to drugs. The agents investigated in the present study display strong apoptosis inducing activity and therefore show promise for the development of novel chemotherapeutics.