A New Inhibitor of Tubulin Polymerization Kills Multiple Cancer Cell Types and Reveals p21-Mediated Mechanism Determining Cell Death after Mitotic Catastrophe

Abstract

Induction of mitotic catastrophe through the disruption of microtubules is an established target in cancer therapy. However, the molecular mechanisms determining the mitotic catastrophe and the following apoptotic or non-apoptotic cell death remain poorly understood. Moreover, many existing drugs targeting tubulin, such as vincristine, have reduced efficacy, resulting from poor solubility in physiological conditions. Here, we introduce a novel small molecule 2-aminoimidazoline derivative—OAT-449, a synthetic water-soluble tubulin inhibitor. OAT-449 in a concentration range from 6 to 30 nM causes cell death of eight different cancer cell lines in vitro, and significantly inhibits tumor development in such xenograft models as HT-29 (colorectal adenocarcinoma) and SK-N-MC (neuroepithelioma) in vivo. Mechanistic studies showed that OAT-449, like vincristine, inhibited tubulin polymerization and induced profound multi-nucleation and mitotic catastrophe in cancer cells. HeLa and HT-29 cells within 24 h of treatment arrested in G2/M cell cycle phase, presenting mitotic catastrophe features, and 24 h later died by non-apoptotic cell death. In HT-29 cells, both agents altered phosphorylation status of Cdk1 and of spindle assembly checkpoint proteins NuMa and Aurora B, while G2/M arrest and apoptosis blocking was consistent with p53-independent accumulation in the nucleus and largely in the cytoplasm of p21/waf1/cip1, a key determinant of cell fate programs. This is the first common mechanism for the two microtubule-dissociating agents, vincristine and OAT-449, determining the cell death pathway following mitotic catastrophe demonstrated in HT-29 cells.

New Multi-Targeted Antiproliferative Agents: Design and Synthesis of IC261-Based Oxindoles as Potential Tubulin, CK1 and EGFR Inhibitors

“…The ability of microtubule poisons to restrict tumor cell growth at low doses renders them efficient tools in cancer therapy. However, it is important such drugs should have limited side effects and enhanced physicochemical parameters [53].…”

“…The ability of compound 4b to inhibit tubulin polymerization was detected using a fluorescent kit (cytoskeleton, Catalog number BK011P) and Tecan-spark reader according to prescribed manufacturer protocol (Danvers, MA, USA) [53]. A solution of compound 4b in DMSO (10%, 5 µL) was added (in triplicate) to the wells of a 96-well plate.…”

Cisplatin resistant ovarian cancer cells reveal a polyploid phenotype with remarkable activation of nuclear processes

“…As well, the top central genes of the constructed network typically attributed to nuclear process. According to our previous studies indicating the importance of central genes in promotion of cellular phenotype [45,46], we think that the central nodes as well as genes in the densely-connected modules which belonged to chromatid segregation, microtubule polymerization and membrane budding, indicate the key role of nuclear processes and polyploidy in cancer resistance [47][48][49][50][51][52][53][54].…”

Novel Dicarboximide BK124.1 Breaks Multidrug Resistance and Shows Anticancer Efficacy in Chronic Myeloid Leukemia Preclinical Models and Patients’ CD34+/CD38− Leukemia Stem Cells

“…After 24 h pre-incubation, BK124.1 in DMSO was added and incubated for the specified time. Next, cytosolic and nuclear cell fractions were separated as described [ 37 ]. Samples for immunoblotting contained the same amount of protein (at least 15 µg) in the same final volume.…”