Multidrug resistance is a major limitation for microtubule-binding agents in cancer treatment. Here we report a novel microtubule inhibitor (2-morpholin-4-yl-5-nitro-benzoic acid 4-methylsulfanyl-benzyl ester, IMB5046), its cytotoxicity against multidrug-resistant cell lines and its antitumor efficacy in animal models. IMB5046 disrupted microtubule structures in cells and inhibited purified tubulin polymerization in vitro. It bound to the colchicine pocket of tubulin. IMB5046 displayed potent cytotoxicity against multiple tumor cell lines with an IC₅₀ range of 0.037–0.426 μM. Notably, several multidrug-resistant cell lines which were resistant to colchicine, vincristine and paclitaxel remained sensitive to IMB5046. IMB5046 was not a P-glycoprotein substrate. IMB5046 blocked cell cycle at G2/M phase and induced cell apoptosis. Microarray assay indicated that the differentially expressed genes after IMB5046 treatment were highly related to immune system, cell death and cancer. In a mouse xenograft model IMB5046 inhibited the growth of human lung tumor xenograft by 83% at a well-tolerated dose. It is concluded that IMB5046 is a tubulin polymerization inhibitor with novel chemical structure and can overcome multidrug resistance. It is a promising lead compound for cancer chemotherapy, especially for treatment of multidrug-resistant tumors.

Gemcitabine (Gem) is currently used as the first-line therapy for liver and pancreatic cancer but has limited efficacy in most cases. Dexamethasone (Dex) have been applied as a chemoprotectant and chemosensitizer in cancer chemotherapy. This study further explored the potential of combination of Gem and Dex and tested the hypothesis that glucocorticoid receptor signaling is essential for the synergistic antitumor activity. In the HepG2 and AsPC-1 xenograft models, the combination treatment showed a significantly synergistic antitumor activity. Immunohistochemistry of post-treatment tumors showed a significant decrease in proliferation and angiogenesis as compared to either of the treatments alone. Dex alone and the combination with Gem inhibited the expression of glucocorticoid receptor. The combination of Dex and Gem showed synergistic cytotoxicity in cell lines in vitro. The antiproliferative synergism is prevented by used glucocorticoid receptor (GR) small interfering RNA, demonstrating that the glucocorticoid receptor is required for the antiproliferative synergism of Gem and Dex. The inhibition of glucocorticoid receptor signaling pathway and induction of apoptosis via activation of caspases 3, 8 and 9, PARP, contributed to the synergistic effect of this combination therapy. These results demonstrate that Dex could potentiate the antitumor efficacy of Gem. The synergistic antitumor activity of the combination of Dex and Gem was through glucocorticoid receptor signaling. Taken together, a combination of Dex and Gem shows a significant synergistic antitumor activity and lesser toxicity both in vitro and in vivo and could be a combination chemotherapy for the treatment of highly expression of glucocorticoid receptor patients.

Microtubule‐depolymerizing agents can selectively disrupt tumor vessels via inducing endothelial membrane blebbing. However, the mechanism regulating blebbing is largely unknown. IMB5046 is a newly discovered microtubule‐depolymerizing agent. Here, the functions of focal adhesion kinase (FAK) during IMB5046‐induced blebbing and the relevant mechanism are studied. We found that IMB5046 induced membrane blebbing and reassembly of focal adhesions in human vascular endothelial cells. Both FAK inhibitor and knock‐down expression of FAK inhibited IMB5046‐induced blebbing. Mechanism study revealed that IMB5046 induced the activation of FAK via GEF‐H1/ Rho/ ROCK/ MLC2 pathway. cRGD peptide, a ligand of integrin, also blocked IMB5046‐induced blebbing. After activation, FAK further promoted the phosphorylation of MLC2. This positive feedback loop caused more intensive actomyosin contraction and continuous membrane blebbing. FAK inhibitor blocked membrane blebbing via inhibiting actomyosin contraction, and stimulated stress fibre formation via promoting the phosphorylation of HSP27. Conclusively, these results demonstrate that FAK is a molecular switch controlling endothelial blebbing and stress fibre formation. Our study provides a new molecular mechanism for microtubule‐depolymerizing agents to be used as vascular disrupting agents.