Kinetic Suppression of Microtubule Dynamic Instability by Griseofulvin: Implications for Its Possible Use in the Treatment of Cancer

Kinetic suppression of microtubule dynamic instability by griseofulvin: Implications for its possible use in the treatment of cancer Dulal Panda*†, K. Rathinasamy*, Manas K. Santra*, and Leslie Wilson†‡ *School of Biosciences and Bioengineering, Indian Institute of Technology, Bombay 400076, India; and ‡Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, CA 93106 Edited by John A. Carbon, University of California, Santa Barbara, CA, and approved May 19, 2005 (received for review March 5, 2005) The antifungal drug griseofulvin inhibits mitosis strongly in fungal caused by the impairment of the organization or function of cells and weakly in mammalian cells by affecting mitotic spindle the spindle MTs rather than their depolymerization. Griseo- microtubule (MT) function. Griseofulvin also blocks cell-cycle pro- fulvin is able to bind weakly to mammalian-brain tubulin and gression at G2͞M and induces apoptosis in human tumor cell lines. to inhibit the polymerization of MTs in vitro; however, inhi- Despite extensive study, the mechanism by which the drug inhibits bition of brain tubulin polymerization requires very high mitosis in human cells remains unclear. Here, we analyzed the concentrations of griseofulvin (5, 7). ability of griseofulvin to inhibit cell proliferation and mitosis and MTs exhibit two forms of nonequilibrium dynamics, tread- to affect MT polymerization and organization in HeLa cells to- milling and dynamic instability (3). Most of the earlier work on gether with its ability to affect MT polymerization and dynamic griseofulvin was carried out before the realization that mitotic instability in vitro. Griseofulvin inhibited cell-cycle progression at spindle MTs are highly dynamic, and that the rapid dynamics of prometaphase͞anaphase of mitosis in parallel with its ability to the MTs, not just their presence, are critical for proper spindle function (3, 8–13). Further, most antimitotic MT-targeted drugs inhibit cell proliferation. At its mitotic IC50 of 20 ␮M, spindles in blocked cells displayed nearly normal quantities of MTs and MT that act on animal cells suppress MT dynamics at concentrations organization similar to spindles blocked by more powerful MT- that often are far below the concentrations required to inhibit targeted drugs. Similar to previously published data, we found (e.g., the vinca alkaloids) (9) or increase (e.g., paclitaxel) (10, 11) that very high concentrations of griseofulvin (>100 ␮M) were MT polymerization. Further, several antimitotic drugs that weakly inhibit MT polymerization in cells and in vitro, including required to inhibit MT polymerization in vitro. However, much noscapine (12), estramustine (13), and benomyl (14), suppress lower drug concentrations (1–20 ␮M) strongly suppressed the MT dynamic instability and treadmilling remarkably strongly in dynamic instability behavior of the MTs. We suggest that the the absence of appreciable inhibition of polymerization. primary mechanism by which griseofulvin inhibits mitosis in hu- In pursuing the earlier results of Grisham et al. (4), we man cells is by suppressing spindle MT dynamics in a manner reasoned that, like the aforementioned drugs, griseofulvin might qualitatively similar to that of much more powerful antimitotic inhibit proliferation and mitosis in human cancer cells primarily drugs, including the vinca alkaloids and the taxanes. In view of by suppressing MT dynamics. Thus, we analyzed the ability of griseofulvin’s lack of signiﬁcant toxicity in humans, we further griseofulvin to inhibit the polymerization of brain tubulin into suggest that it could be useful as an adjuvant in combination with MTs and its ability to modulate MT dynamic instability in vitro more powerful drugs for the treatment of cancer. together with its ability to inhibit cell proliferation and mitosis and to affect MT organization in HeLa cells. We found that cancer chemotherapy ͉ mitosis griseofulvin inhibited mitosis in parallel with its ability to inhibit cell proliferation, and that at its IC50 for inhibition of mitosis (20 riseofulvin, an orally active nontoxic antifungal antimitotic ␮M), the organization of the MTs in the blocked spindles Gdrug derived from several species of Penicillium (1), has appeared nearly normal. In addition, griseofulvin bound weakly been used for many years for the treatment of Tinea capitis to tubulin, and inhibition of MT polymerization in vitro required Ͼ ␮ (ringworm) and other dermatophyte infections (2). Its mecha- griseofulvin concentrations 100 M. In contrast, griseofulvin nism of action has been thought to involve the selective inhibi- suppressed the dynamic instability of the MTs remarkably Ͻ ␮ tion of fungal cell mitosis in association with its accumulation in strongly (IC50 for overall dynamicity, 1 M). Our data strongly the keratin layers of the epidermis (1). Early studies demon- support the hypothesis that inhibition of mitosis in human cancer strated that griseofulvin inhibits mitosis in sensitive fungi in a cells is due to the suppression of spindle MT dynamics. A recent manner resembling the actions of colchicine and other antimi- report indicates that griseofulvin either alone or in combination with nocodazole inhibits tumor production in athymic mice (15). totic drugs that act in mammalian cells by disrupting spindle Taking these observations together with the data described in microtubule (MT) function (3), although its precise mechanism this work, we suggest that griseofulvin might be useful in of action in sensitive fungi remains unclear. The antiproliferative combination with more powerful MT-targeted drugs or with and antimitotic effects of griseofulvin in mammalian cells are agents acting on novel targets for the treatment of cancer. very weak, with inhibition requiring high micromolar concentrations (4, 5). Materials and Methods The question of whether inhibition of mitosis by griseofulvin Reagents. Griseofulvin, GTP, EGTA, colchicine, piperazine-1,4- involves MT depolymerization or some other action on MTs in bis(2-ethanesulfonic acid) (Pipes), and Mes were purchased human cells has remained unsettled. Specifically, griseofulvin clearly inhibits MT polymerization coincident with inhibition of mitosis in echinoderm eggs (6), and inhibition of mitosis at This paper was submitted directly (Track II) to the PNAS ofﬁce. high griseofulvin concentrations in 3T3 cells occurs together Abbreviations: MT, microtubule; MAP, MT-associated protein; Pipes, piperazine-1,4-bis(2- with depolymerization of the spindle MTs (5). In contrast, ethanesulfonic acid). inhibition of mitosis by griseofulvin in HeLa cells occurred in †To whom correspondence may be addressed. E-mail: [email protected] or wilson@ the absence of significant spindle MT depolymerization (4), lifesci.ucsb.edu. which was interpreted as indicating that mitotic inhibition was © 2005 by The National Academy of Sciences of the USA 9878–9883 ͉ PNAS ͉ July 12, 2005 ͉ vol. 102 ͉ no. 28 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0501821102 Downloaded by guest on October 1, 2021 from Sigma. Phosphocellulose (P11) was obtained from What- Immunofluorescence microscopy was performed as described man. All other chemicals used were of analytical grade. in ref. 14. The cells were seeded on poly(L-lysine)-coated coverslips at a density of 1 ϫ 105 cells per ml contained in 24-well Purification of Goat and Bovine Brain Tubulins. Goat brain MT tissue culture plates. After 40 h of drug treatment, cells were protein, tubulin plus MT-associated proteins (MAPs), was iso- fixed in 3.7% formaldehyde for 30 min at 37°C and then treated lated as described in refs. 14 and 16. Bovine MT protein was with cold 100% methanol (Ϫ20°C) for 20 min. After the obtained as described in ref. 17. Goat and bovine tubulin were nonspecific sites had been blocked with 2% BSA͞PBS, the cells separated from the MAPs by using phosphocellulose chromatog- were incubated with mouse monoclonal anti-␣-tubulin antibody raphy and stored at Ϫ80°C. The tubulin concentration was deter- (Sigma) at 1:300 dilution for 2 h at 37°C. Coverslips were then mined by the Bradford method using BSA as the standard (18). rinsed with BSA͞PBS and incubated with anti-mouse IgG antibody labeled with Alexa Fluor 568 (Molecular Probes) at The Effects of Griseofulvin on Glutamate-Induced MT Polymerization 1:100 dilution for1hat37°C. Coverslips were then rinsed with in Vitro. Goat brain tubulin (11 ␮M) was polymerized for 30 min PBS and incubated with DAPI (1 ␮g͞ml) for 20 s. The MTs and at 37°C in 25 mM Pipes buffer, pH 6.8, containing 1 M sodium DNA were observed with an Eclipse TE-2000 U microscope glutamate, 1 mM GTP, and 5 mM MgCl2 in the absence or (Nikon). The images were analyzed by using IMAGEPRO PLUS presence of griseofulvin. The polymerized MTs were sedimented software (Media Cybernetics, Silver Spring, MD). by centrifugation (52,000 ϫ g for 40 min), and the MT polymer Mitotic indices were determined by Wright–Giemsa staining mass was determined. Samples for transmission electron micros- (14). Briefly, the cells were plated at a density of 5 ϫ 104 cells copy were prepared as described in ref. 14, and MT structures per ml in 24-well plates 24 h before the addition of griseofulvin. were observed with a Tecnai G212 electron microscope (FEI, The cells were then treated with different concentrations of The Netherlands) at ϫ43,000 magnification. griseofulvin and incubated for an additional 20 or 40 h. Both the attached and unattached cells were harvested and combined Effects of Griseofulvin on Seeded MT Assembly. MT nucleating seeds after trypsinization (0.025% trypsin, 10 min), washed with PBS, were constructed by polymerizing goat brain tubulin (3.5 mg͞ml) and then treated with 0.5ϫ PBS for 10 min on ice. The cells were in the presence of 6 M glycerol and 1 mM GTP at 37°C for 45 fixed with methanol͞acetic acid solution (3:1, vol͞vol). Finally, min and shearing the MTs into small fragments by repeated the cell suspensions were spread onto cold slides, air-dried, and passage through a 25-gauge needle. For measurement of poly- stained with a 10% Giemsa solution.

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