Disruption of tubulin polymerization and cell proliferation by 1-naphthylarsonic acid Roya Mahinpour*, Gholamhossein Riazi 1{ , Mohammad A. Shokrgozar 1{ , Mohammad N. Sarbolouki { , Shahin Ahmadian { , Masoumeh Douraghi " , Hamid Hadi Alijanvand { , Kayhan Azadmanesh 1 , Maryam Heidari { , Zahra Naghdi Gheshlaghi { and Ali A. Moosavi-Movahedi { * Department of Organic Chemistry, Faculty of Chemistry, University of Kashan, Kashan, Iran { Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran { National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran 1 Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, Iran " Division of Microbiology, Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran Abstract Arsenical compounds exhibit a differential toxicity to cancer cells. Microtubules are a primary target of a number of anticancer drugs, such as arsenical compounds. The interaction of 1-NAA (1-naphthylarsonic acid) has been investigated on microtubule polymerization under in vitro and cellular conditions. Microtubules were extracted from sheep brain. Transmission electron microscopy was used to show microtubule structure in the presence of 1-NAA. Computational docking method was applied for the discovery of ligand-binding sites on the microtubular proteins. Proliferation of HeLa cells and HF2 (human foreskin fibroblasts) was measured by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H- tetrazolium bromide] assay method following their incubation with 1-NAA. Fluorescence microscopic labelling was done with the help of a-tubulin monoclonal antibody and Tunel kit was used to investigate the apoptotic effects of 1-NAA on the HeLa cells. 1-NAA inhibits the tubulin polymerization by the formation of abnormal polymers having high affinity to the inner cell wall. Keywords: anti-mitotic agent; HeLa cell; human foreskin fibroblasts (HF2); microtubule; 1-naphthylarsonic acid (1-NAA) 1. Introduction Tubulin, the major building block of microtubules is present in almost all eukaryotic cells. It is a heterodimer consisting of a- and b- tubulin. Both subunits have an approximate molecular mass of 55 kDa and share considerable homology. The a-/b-tubulins bind head to tail during the construction of protofilaments, forming a cylindrical polymer. Microtubules can switch between assem- bling/disassembling both in vivo and in vitro. This unequilibrated behaviour, known as dynamic instability, is based on the hydrolysis of GTP by tubulin subunits (McNally, 1996; Desai and Mitchison, 1997; Hirokawa et al., 1998; Nogales, 2000). Microtubule dynamics are regulated by MAPs (microtubule-associated proteins). Several classes of MAPs act by stabilizing microtubules (Sloboda et al., 1976; Cleveland et al., 1977; Parysek et al., 1984; Wiche et al., 1991). Tau protein is a neuronal MAP that forms cross-bridges between microtubules. This protein promotes microtubule growth and nucleation, in vitro and in vivo, and is regulated by phosphorylation and dephosphorylation. Tau’s C- and N-termini differentially affect the microtubule growth and nucleation (Hirokawa et al., 1988; Aizawa et al., 1990; West et al., 1991; Brandt and Lee, 1993; Chau et al., 1998; Makrides et al., 2004). Since microtubules are the target of anti-mitotic compounds, they play crucial role in cell division and cancer therapy (Jordan and Wilson, 2004; Andre ´ and Meille, 2006; Attard et al., 2006; Pasquier and Kavallaris, 2008). While arsenic and relative compounds are poisonous, there are abundant elements in nature and have been used therapeutically for .2400 years in Oriental medicine (Forkner and Scott, 1931). As 2 O 3 (arsenic trioxide) exhibits differential toxicity to cancer cells, while it is an effective drug for the treatment of APL (acute promyelocytic leukaemia) (Jackson and Grainge, 1975; Cuzick et al., 1982; Buchner, 1997; Chan and Huff, 1997; Huff et al., 1999; Li and Broome, 1999; Waxman and Anderson, 2001; Miller Jr et al., 2002; Rojewski et al., 2004). Ling et al. (2002) showed that As 2 O 3 induces M-phase arrest due to interaction with tubulin, resulting in their polymerization. They have also found that tubulin poly- merization by As 2 O 3 did not affect GTP binding to b-tubulin (Ling et al., 2002). However, these results are not in agreement with those of Li and Broome (1999), who have reported that As 2 O 3 caused blockade of GTP binding to b-tubulin and thus inhibited tubulin polymerization. Organo-arsenical compounds have lower toxicity than As 2 O 3 as an inorganic molecule (Kaise et al., 1988); for example, DMA (dimethylarsinic acid), an anticancer agent, disrupts mitotic spindle and induces mitotic arrest in cultured mammalian cells (Kawata et al., 2001). Moreover, 1-NAA (1-naphthylarsonic acid) is an organo–arsenic compound, which contains greater hydrophobic moieties than that of DMA. 1 Correspondence may be addressed to either of these authors (email ghriazi@ibb.ut.ac.ir or mashokrgozar@pasteur.ac.ir). Abbreviations: As 2 O 3 , arsenic trioxide; DAPI, 49,6-diamidino-2-phenylindole; DMA, dimethylarsinic acid; FBS, fetal bovine serum; HF2, human foreskin fibroblasts; MAP, microtubule-associated protein; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; 1-NAA, 1-naphthylarsonic acid; PI, propidium iodide. Cell Biol. Int. (2012) 36, 403–408 (Printed in Great Britain) Research Article E The Author(s) Journal compilation E 2012 Portland Press Limited Volume 36 (4) N pages 403–408 N doi:10.1042/CBI20100603 N www.cellbiolint.org 403