Cryptophycin: a New Antimicrotubule Agent Active Against Drug-Resistant Cells'
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[CANCERRESEARCH54,3779—3784,July15, 1994] Cryptophycin: A New Antimicrotubule Agent Active against Drug-resistant Cells' Charles D. Smith,2 Xinqun Zhang, Susan L. Mooberry, Gregory M. L. Patterson, and Richard E. Moore Cancer Research Center ofHawai4 University ofHawaii at Manoa, Honolulu, Hawaii 96813 ABSTRACT MATERIALS AND METHODS Cryptophycin is a cytotoxic dioxadiazacyclohexadecenetetrone isolated Materials. Vinblastine, cytochalasin B, TRITC3-phalloidin, sulforho from cyanobacteria of the genus Nostoc. Incubation of L1210 leukemia damine B, and antibodies against f3-tubulin and vimentin were obtained ceils with cryptophycin resulted In dose-dependent inhibition of cell pro from the Sigma Chemical Company. Cryptophycin (Fig. 1) was purified liferatlon in parallel with increases in the percentage of cells in mitosis from extracts of Nostoc sp. GSV 224 as described elsewhere (15). Briefly, (half-maximal effects at <10 pM). Indirect immunofluorescence studies lyophilized bacteria were extracted with methanol and the resulting material demonstrated that treatment of A-10 vascular smooth muscle cells with was concentratedby evaporationunder a vacuum.The extract was partitioned cryptophycin results in marked depletion of cellular microtubules and between water and methylene chloride, and the lipophilic portion was reorganization ofvlmentin intermediate filaments, similar to the effects of chromatographed on octadecylsilane-coated silica beads. Cryptophycin vinblastine. Cytochalasm B caused the depolymerization of microffla eluted with 75% methanol and was purified by high performance liquid ments in these cells, while neither vinblastine nor cryptophycin affected chromatography on Econosil C18 eluted with 17% methanol. The corn this cytoskeletal component. Pretreatment of cells with taxol prevented pound was dissolved in ethanol and stored at —20°C.BMEwas from microtubule depolymerization in response to either vinblastine or cryp Gibco BRL (Grand Island, NY) and FBS was purchased from Hyclone tophycin. While microtubule depolymerization in response to vinblastine Laboratories, Inc. (Logan, UT). was rapidly reversed by removal of the drug, cells treated with crypto Cell Lines. L1210 munne leukemia, MCF-7 breast carinoma, and MCF phycin remained microtubule depleted for at least 24 h after removal of 7/ADR, a MDR subline, was obtained from the Division of Cancer Treatment the compound. Combinational treatments with vinbiastine and crypto of the National Cancer Institute and was cultured in RPM] 1640 containing 10% FBS and 50 @.tg/mlgentamicinsulfate. A-lO rat aortic smooth muscle phycin resulted in additive cytotoxicity. Ovarian carcinoma and breast cells were obtained from the American Type Culture Collection (CRL 1476) carcinoma cells which are multiply drug resistant due to overexpression of and were grown in BME containing 10% FBS and 50 @tWmlgentamicin P-glycoprotein are markedly less resistant to cryptophycin than they are sulfate. Human ovarian carcinoma cells (SKOV3) and a subline which to vinbiastine, colchicine, and taxol. Therefore, cryptophycin is a new has been selected for resistance to vinblastine (SKVLB1) were a generous gift antimicrotubule compound which appears to be a poorer substrate for from Dr. Victor Ling of the Ontario Cancer Institute (16). Both cell lines P-glycoprotein than are the Vinca alkaloids. This property may confer an were maintained in BME containing 10% FBS and 50 ,.@g/mlgentamicin advantage to cryptophycin in the chemotherapy ofdrug-resistant tumors. sulfate. Vinblastine was added to a final concentration of 1 j@g/ml to SKVLB1 cells 24 h after passage to maintain selection pressure for P-glycoprotein-overexpressing cells. INTRODUCTION Cell Proliferation and Cycle Arrest Assays. For adherent lines, cells were plated into 96-well tissue culture plates to a density of approximately 10% of The cytoskeleton of eukaryotic cells is involved in the regulation of confluency. After 24 h, the indicated concentrations of drugs were added, and a number of cellular processes, including motility, secretion, and the cells were incubated for an additional 48 h. Subsequently, the medium was proliferation. Because of their involvement in mitotic spindle func removed from each well and cells were washed with PBS and then fixed with tion, microtubules are the molecular targets of several currently uti 10% trichloroacetic acid. The fixed cells were stained with 0.4% SRB in 1% lized anticancer drugs (1—3).Microtubule depolymerization is induced acetic acid for 30 mm as described by Skehan et aL (17). Unbound SRB was by Vinca alkaloids such as vinblastine and vincristine (3, 4), colchi removed by four rapid washes with 1% acetic acid, and the bound SRB was cine (5), and synthetic estrogens (6, 7). Additionally, a number of solubilized in 0.2 ml of 10 mM unbuffered Tris base. The amount of bound SRB was determined by its absorbance at 560 nm. The inhibition of cell natural products from plants, fungi, and marine animals have been growth by the cytotoxin was calculated as the percentage of decrease in SRB demonstrated to induce microtubule depolymerization in cells and in binding compared with untreated cultures. For L1210 cells, cultures were vitro (8—12).The molecular mechanisms and the potential therapeutic treated with drugs as described above and total cell numbers were determined usefulness of most of these later compounds have yet to be defined. by counting the cells in a hemacytometer. The percentage of cells in mitosis Cryptophycin is a cytotoxic dioxadiazacyclohexadecenetetrone was determined by staining with 0.4% Giemsa in PBS followed by three rapid isolated from cyanobacteria of the genus Nostoc (13). Cryptophycin washes with PBS. At least 400 cells/treatment were scored for the presence of was patented in 1990 as an antifungal agent with good activity toward mitotic figures and the mitotic index was calculated as the ratio of cells with filamentous fungi and certain species of yeast (14); however, the mitotic figures to the total number of cells counted. Immunofluorescence Assays. A-b cells were grown to near-confluency mechanism of action was unknown. In the present study, we demon on glass coverslips in BME/10% FBS. Compounds in PBS were added to the strate that this compound potently disrupts microtubule structure in indicated final concentrations and cells were incubated for an additional 24 h. cultured cells. A novel and potentially very important property of For the staining of microtubules and intermediate filaments, the cells were this compound is that, in contrast with the Vinca alkaloids, crypto fixed with cold methanol and incubated with PBS containing 10% calf serum phycin appears to be a poor substrate for the drug-efflux pump to block nonspecific binding sites. Cells were then incubated at 37°Cfor 60 P-glycoprotein. mmwitheithermonoclonalanti-f3-tubulin(SigmaT-4026)ormonoclonal anti-vimentin (Sigma V-5255) at dilutions recommended by the manufacturer. Bound primary antibodies were subsequently visualized by a 30-mm incuba Received 2/4/94; accepted 5/13/94. The costs of publication of this article were defrayed in part by the payment of page tion with fluorescein-conjugated rabbit anti-mouse IgG. The coverslips were charges. This article must therefore be hereby marked advertisement in accordance with mounted on microscope slides and the fluorescence patterns were examined 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by the State of Hawal, Office of Technology Transfer and Economic Development (C. D. S.) and NIH Grant CA 12623 (R. E. M.). 3 The abbreviations used are: TRITC, tetramethylrhodamine isothiocyanate; BME, 2 To whom requests for reprints should be addressed, at Department of Pharmacology, basal medium containing Earle's salts; PBS, phosphate-buffered saline; FBS, fetal bovine Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111. serum; SRB, sulforhodamine B; IC5,@,concentrationof drug which inhibited cell prolif eration by 50%; MDR, multidrug resistant. 3779 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1994 American Association for Cancer Research. ANTIMICROTUBULEEFFECFS OF CRYPTOPHYCIN H vinblastine, or 0.2 nM cryptophycin. These doses of vinblastine and 0 cryptophycin are approximately 100 times the IC5@@sforthese cells, while the dose of cytochalasin has been used previously to selectively disrupt microfilament bundles (18). After 24 h, microtubules and vimentin intermediate filaments were visualized by indirect immuno fluorescence and microfilaments were stained using TRITC-phal loidin. The morphological effects of each drug were examined and imaged as indicated above. Untreated cells displayed bundles of Cryptophycin microfilaments concentrated along the major axis ofthe cell (Fig. 3A). These cells had extensive microtubule networks complete with Fig. 1. Structure of cryptophycin. perinuclear microtubule organizing centers (Fig. 3B), while vimentin intermediate filaments were also evenly distributed throughout the and imaged using a Nikon Optiphot-2 microscope with a Bio-Rad MRC 600 cytoplasm (Fig. 3C). Cytochalasin B caused complete loss of micro Confocal Laser Scanning system. The images were reconstructed using Bio filament bundles along with the accumulation of paracrystalline rem Rad Comos and VoxelView Ultra software and were printed on a KOdak nants (Fig. 3G). This compound did not affect the distribution of Model XL 7700 digital continuous tone printer. For staining of