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Opium Alkaloid Noscapine Is an Antitumor Agent That Arrests Metaphase and Induces Apoptosis in Dividing Cells

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Opium Alkaloid Noscapine Is an Antitumor Agent That Arrests Metaphase and Induces Apoptosis in Dividing Cells Proc. Natl. Acad. Sci. USA Vol. 95, pp. 1601–1606, February 1998 Cell Biology Opium alkaloid noscapine is an antitumor agent that arrests metaphase and induces apoptosis in dividing cells KEQIANG YE*†,YONG KE‡,NAGALAKSHMI KESHAVA§,JOHN SHANKS†,JUDITH A. KAPP‡,RAJESHWAR R. TEKMAL§, i JOHN PETROS¶, AND HARISH C. JOSHI*† *Graduate Program in Biochemistry and Molecular Biology, Departments of †Anatomy and Cell Biology, ‡Pathology, §Gynecology–Obstetrics, and ¶Urology, Emory University School of Medicine, Atlanta, GA 30322 Edited by Thomas D. Pollard, Salk Institute for Biological Studies, La Jolla, CA, and approved December 17, 1997 (received for review July 17, 1997) ABSTRACT An alkaloid from opium, noscapine, is used (2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one], TCB as an antitussive drug and has low toxicity in humans and (2,3,4-trimethoxy-49-carbomethoxy-1,19-biphenyl), and TKB mice. We show that noscapine binds stoichiometrically to (2,3,4-trimethoxy-49-acetyl-1,19-biphenyl), and vinca alkaloids. tubulin, alters its conformation, affects microtubule assembly, Taxol and its analogs represent the compounds that promote and arrests mammalian cells in mitosis. Furthermore, the assembly of microtubules. It is now clear that although all noscapine causes apoptosis in many cell types and has potent of these microtubule drugs prevent cell division, only a select antitumor activity against solid murine lymphoid tumors few have been useful clinically. In addition, there are differ- (even when the drug was administered orally) and against ences regarding the toxicity and the efficacy of these drugs for human breast and bladder tumors implanted in nude mice. distinct classes of tumors (6). Because noscapine is water-soluble and absorbed after oral Noscapine (Fig. 1), a phthalideisoquinoline alkaloid consti- administration, its chemotherapeutic potential in human can- tuting 1–10% of the alkaloid content of opium, has been used cer merits thorough evaluation. as a cough suppressant in humans and in experimental animals (7–9). Mechanisms for its antitussive action are unknown, Two important events in the cell division cycle are the dupli- although animal studies have suggested central nervous system cation of the chromosomal DNA and the separation of the as a site of action. Karlsson et al. (10) and Mourey et al. (11) duplicated chromosomes. These events occur in two discrete described high-affinity, saturable, and stereo-specific binding phases: the synthetic phase (S phase) and the mitotic phase (M sites for noscapine in Guinea pig brain homogenates. In addition, noscapine has been shown to have low toxicity (7–9), phase), which are separated from each other by G1 and G2 phases. The proper coordination of these events is achieved by although there are some reports of aneuploidy produced in cell checkpoint pathways that delay the progression of the cell cycle cultures (12, 13). Additionally, noscapine has been used in when proper completion of one phase is disrupted by physical humans orally, and the metabolic fate, bioavailability, and damage or some other means (1, 2). Under normal circum- pharmacokinetics of noscapine by oral administration are stances, if the extent of damage is irreparable, most cells relatively well studied (9, 14, 15). In oral administration of 50 mg, noscapine was rapidly absorbed and gave a maximum initiate a sequence of biochemical events leading to pro- y grammed cell death or apoptosis (for a recent review, see ref. plasma concentration of 182 ng ml after 1 h. From then on, the 3). Deregulation in any one or more of these checkpoint noscapine levels declined with a half-life of 124 min. The mechanisms might lead to genetic instability, which is a absolute oral bioavailability was 30% (8). The main metabo- primary step for a tumor to evolve into invasive malignant lites of noscapine include cotarnine, hydrocotarnine, and state. The chemotherapeutic management of various cancers is meconine, which result from the cleavage of the C–C bond between isoquinoline and phthalide groups (14). achieved by drugs that block S phase, M phase, or regulatory In this article, we show that the opium alkaloid noscapine or metabolic pathways impinging on the cell cycle machinery. shares chemical similarity with colchicine and podophyllo- For example, some drugs affect the functions or structures of toxin, arrests cells at mitosis, and induces apoptosis. Further- DNA or RNA; others interfere with enzymes involved in more, noscapine has also been shown to shrink murine thy- folate, purine, or pyrimidine metabolism or the function of moma and human breast and bladder tumors in vivo.In mitotic spindles (4, 5). Antimitotic drugs such as vinca alka- addition, we show that noscapine binds tubulin and alters loids and taxoids can arrest cells in M phase by interacting with microtubule assembly reactions in vitro. the microtubules of the mitotic spindle. It is now well accepted that dynamic assembly and disas- sembly of microtubules are required for the morphogenesis of MATERIALS AND METHODS mitotic spindle. This has lead to a tremendous interest in small Noscapine. Noscapine (97% purity) was from Aldrich. The organic molecules that modulate the dynamics of microtubules noscapine stock solution was prepared at 0.1 M in dimethyl primarily because some of the microtubule interacting agents sulfoxide (DMSO) and kept at 220°C. are useful for chemotherapeutic management of certain kinds Cells. HeLa cells were grown in DMEM (GIBCOyBRL) of tumors. There are two classes of these antimicrotubule supplemented with 10% fetal calf serum, 1 mM L-glutamine, agents: those that prevent the assembly of tubulin and those and 1% penicillinystreptomycin. The tumor cell line E.G7- that promote the assembly of tubulin. A prototypic example of OVA (H-2b) (16) was grown in RPMI 1640 medium (GIBCOy a potent assembly inhibitor is colchicine. Others are analogs of BRL) with 10% fetal calf serum, 1 mM sodium pyruvate, 1 mM colchicine, such as podophyllotoxin, MTC [2-methoxy-5- L-glutamine, 0.1% gentamycin, and 50 mM 2-mercaptoethanol. y Cells were grown at 37°C in a 5% CO2 95% air atmosphere. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in This paper was submitted directly (Track II) to the Proceedings office. accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: DMSO, dimethyl sulfoxide; TUNEL, terminal de- © 1998 by The National Academy of Sciences 0027-8424y98y951601-6$2.00y0 oxynucleotidyltransferase-mediated dUTP nick-end labeling. i PNAS is available online at http:yywww.pnas.org. To whom reprint requests should be addressed. 1601 Downloaded by guest on October 1, 2021 1602 Cell Biology: Ye et al. Proc. Natl. Acad. Sci. USA 95 (1998) (0.04 M Tris-acetatey0.001 M EDTA, pH 8.0). DNA bands were visualized under UV light. A 123-bp DNA ladder (GIBCOyBRL) was used as molecular size marker. Cytochemical Staining of Apoptotic Cells. Morphological changes in the nuclear chromatin of cells undergoing apoptosis were detected by staining with 49,6-diamidino-2-phenylindole (Boehringer Mannheim) as described (4). In brief, 0.5 3 106 to 3 3 106 cells were fixed with 4% glutaraldehydey0.2% Triton X-100 in PBS, incubated at room temperature for 10 min, then centrifuged at 1,000 3 g for 10 min, and resuspended in 20 ml of 0.1% 4,6-diamidino-2-phenylindole in ethanol. After a 15-min incubation at room temperature, 10 ml was placed on FIG. 1. Chemical structures of noscapine. a glass slide, and 400 cells per slide were scored for the incidence of apoptotic chromatin changes with a Zeiss Axio- Cell viability was assessed by trypan blue exclusion analysis. vert model 135 fluorescence microscope. Cell numbers were determined by using a hemacytometer Terminal Deoxynucleotidyltransferase-Mediated dUTP (VWR Scientific). Nick-End Labeling (TUNEL) Assay for Apoptosis. The Mice. Female C57BLy6 (H-2b) mice, 8 to 12 weeks of age, TUNEL assay was used as described (19). In brief, 2 3 106 cells were obtained from Harian–Sprague–Dawley. Mice were in 10 ml of medium were incubated with 2 mlof20mM maintained on standard laboratory chow and water ad libitum noscapine (2 ml of a 0.1 M solution in DMSO) for 24 h. Cells in a temperature and light controlled environment. were pelleted and washed with ice-cold PBS twice, and lym- Immunofluorescence of Microtubules. Cells in 10 ml of phocytes were fixed in 4% paraformaldehyde in PBS and medium were incubated with 2 ml of DMSO or 20 mM air-dried. The slides were rinsed with PBS and incubated with m noscapine (2 l of a 0.1 M solution in DMSO), respectively. blocking solution (0.3% H2O2 in methanol) for 30 min at room After 24 h, cells were fixed with cold (220°C) methanol for 5 temperature. The slides were rinsed with PBS again and min and then rehydrated by PBS for 1 min. Nonspecific sites incubated in permeability solution (0.1% Triton X-100y0.1% were blocked by incubating with 200 ml of 1% BSA in PBS at sodium citrate) on ice for 2 min. The slides were then washed 37°C for 15 min. A mouse monoclonal antibody against twice with PBS, and 50 ml of TUNEL reaction mixture was a-tubulin (Amersham) was diluted 1:200 in PBS containing added to samples on the slides and the slides were incubated 1% BSA and incubated (200 ml) with the coverslips at 37°C for in a humidified chamber for 60 min at 37°C. After the slides 1 h. Cells were then washed with 1% BSAyPBS for 10 min at were rinsed with PBS, 50 ml of converter-POD solution room temperature before incubating with a 1:200 dilution of a (anti-fluorescein-POD, Fab fragment in 60 mM Tris-Hepes rhodamine-labeled goat anti-mouse IgG antibody at room buffery0.4% bovine immunoglobuliny0.2% Germall) was temperature for 45 min, and then the coverslips were rinsed added on samples and incubated for 30 min at 37°C.
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