CIL-102 binds to tubulin at colchicine binding site and triggers apoptosis in MCF-7 cells by inducing monopolar and multinucleated cells

• Published in: Biochemical pharmacology 2012-09, Vol.84 (5), p.633-645
• Main Authors: Gireesh, K.K., Rashid, Aijaz, Chakraborti, Soumyananda, Panda, Dulal, Manna, Tapas
• Format: Article
• Language: English
• Subjects: anticarcinogenic activity; Apoptosis; Binding Sites; Breast Neoplasms - metabolism; Breast Neoplasms - pathology; Calorimetry; Cell Line, Tumor; Cell Proliferation; CIL-102; colchicine; dissociation; Enthalpy; Entropy; heat; Humans; Kinetics; microtubules; p21; p53; Spectrometry, Fluorescence; Tetrazolium Salts - metabolism; Thermodynamics; Thiazoles - metabolism; titration; Tubulin; Tubulin - metabolism
• ISSN: 0006-2952; 1873-2968
• DOI: 10.1016/j.bcp.2012.06.008

A plant dictamine analog, 1-[4-(furo[2,3-b]quinolin-4-ylamino)phenyl]ethanone (CIL-102) has been shown to exert potent anti-tumor activity. In this study, we examined the mode of interaction of CIL-102 with tubulin and unraveled the cellular mechanism responsible for its anti-tumor activity. CIL-102 bound to tubulin at a single site with a dissociation constant ∼0.4μM. Isothermal titration calorimetry revealed that CIL-102–tubulin interaction is highly enthalpy driven and that the binding affords a large negative heat capacity change (ΔCp=−790calmol−1K−1) with an enthalpy–entropy compensation. An analysis of the modified Dixon plot suggested that CIL-102 competitively inhibited the binding of podophyllotoxin, a colchicine-binding site agent, to tubulin. Computational modeling indicated that CIL-102 binds exclusively at the β-subunit of tubulin and that CIL-102 and colchicine partially share their binding sites on tubulin. It bound to tubulin reversibly and the binding was estimated to be ∼1000 times faster than that of colchicine. CIL-102 potently inhibited the proliferation of MCF-7 cells, induced monopolar spindle formation and multi-nucleation. At half-maximal inhibitory concentration, the spindle microtubules were visibly depolymerized and disorganized. CIL-102 reduced the inter-polar distances of bipolar mitotic cells indicating that it impaired microtubule–kinetochore attachments. CIL-102-treatment induced apoptosis in MCF-7 cells in association with increased nuclear accumulation of p53 and p21 suggesting that apoptosis is triggered through a p53–p21 dependent pathway. The results indicated that CIL-102 exerted anti-proliferative activity by disrupting microtubule functions through tubulin binding and provided important insights into the differential mode of tubulin binding by CIL-102 and colchicine.