title: Indicine N-oxide binds to tubulin at a distinct site and inhibits the assembly of microtubules: a mechanism for its cytotoxic activity.
abstract: Indicine N-oxide, a pyrrolizidine alkaloid present in the plant Heliotropium indicum had shown promising cytotoxic activity in various tumor models. The compound exhibited severe toxicity to hepatocytes and bone marrow cells. The present work was aimed to evaluate the molecular mechanism of the toxicity of indicine N-oxide. We found that indicine N-oxide inhibited the proliferation of various cancer cell lines in a concentration dependent manner with IC50 ranging from 46 to 100 μM. At the half maximal inhibitory concentration it blocked the cell cycle progression at mitosis without significantly altering the organization of the spindle and interphase microtubules. The toxicities of the compound at higher concentrations are attributed to its severe depolymerizing effect on both the interphase and spindle microtubules. Binding studies using purified goat brain tubulin indicated that indicine N-oxide binds to tubulin at a distinct site not shared by colchicine or taxol. It decreased the polymer mass of both purified tubulin and MAP-rich tubulin. It was found to induce cleavage of DNA using pUC18 plasmid. The interactions of indicine N-oxide on DNA were also confirmed by computational analysis; which predicted its binding site at the minor groove of DNA. These studies bring to light that the toxicities of indicine N-oxide were due to its DNA damaging effects and depolymerization of microtubules.
authors.affiliation: School of Biotechnology, National Institute of Technology Calicut, Calicut, Kerala, India.
authors.forename: Prakash
authors.initials: P
authors.lastname: Appadurai
authors.affiliation: School of Biotechnology, National Institute of Technology Calicut, Calicut, Kerala, India. Electronic address: rathin@nitc.ac.in.
authors.forename: Krishnan
authors.initials: K
authors.lastname: Rathinasamy
mesh_terms: D000970:Antineoplastic Agents / Q000378:metabolism / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001665:Binding Sites; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D006367:HeLa Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D061986:MCF-7 Cells; D008870:Microtubules / Q000187:drug effects* / Q000378:metabolism; D062105:Molecular Docking Simulation; D009690:Nucleic Acid Conformation; D011763:Pyrrolizidine Alkaloids / Q000378:metabolism / Q000494:pharmacology*; D014404:Tubulin / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D050257:Tubulin Modulators / Q000378:metabolism / Q000494:pharmacology*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D011763:Pyrrolizidine Alkaloids; C495901:TP53 protein, human; D014404:Tubulin; D050257:Tubulin Modulators; D016159:Tumor Suppressor Protein p53; C013095:indicine-N-oxide; D004247:DNA
keywords: Antiproliferative; Cytotoxic; Docking; Indicine N-oxide; Microtubules; Mitosis