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DOI: 10.1055/s-2002-36342
Microtubule-Interacting Activity and Cytotoxicity of the Prenylated Coumarin Ferulenol
Claudia Bocca
Department of Experimental Medicine and Oncology, University of Torino
Corso Raffaello 30
10125 Torino
Italy
Phone: +39-0116707751
Fax: +39-0116707753
Email: claudia.bocca@unito.it
Publication History
Received: April 9, 2002
Accepted: July 13, 2002
Publication Date:
20 December 2002 (online)
Abstract
In this study we investigated biological activity of ferulenol, a prenylated 4-hydroxycoumarin from Ferula communis. Ferulenol stimulates tubulin polymerization in vitro, and inhibits the binding of radiolabeled colchicine to tubulin. It rearranges cellular microtubule network into short fibres, and alters nuclear morphology. Remarkably, ferulenol exerts a dose-dependent cytotoxic activity against various human tumor cell lines.
#Abbreviations
DAPI:4,6-diamidino-2-phenylindole
DMEM:Dulbecco's modified Eagle's medium
EGTA:ethylenebis(oxyethylenenitrilo)tetraacetic acid
FBS:fetal bovine serum
FITC:fluoresceine isothiocyanate
GTP:guanosine 5′-triphosphate
MES:2-[N-morpholino]ethane-sulfonic acid
MTP:microtubule protein
SDS:sodium dodecyl sulfate
The C-3 prenylated 4-hydroxycoumarin ferulenol (Fig. [1]) represents the toxic principle of Ferula communis [1] (Umbelliferae), widespread in the Mediterranean area. Ferulenol has been reported to cause ferulosis [2] and produces toxicity and anticoagulant activity [3].
Initial reports indicated that some coumarins exhibit antitumor activity in a range of advanced malignancies, including prostate cancer [4]. More recently, a series of natural and synthetic coumarin derivatives have been reported to exert cytotoxic effects in different melanoma cell lines [5] and have been proposed for a chemotherapeutic role. In addition, we previously showed that some coumarin compounds inhibit the growth of various tumor cell lines, probably by an interaction with the microtubular system [6], [7].
Our goal was to identify whether ferulenol may represent a new product from plant sources that targets microtubules. We examined the tubulin-interacting activity of ferulenol, both in vitro and at a cellular level, and cytotoxic effects exerted against various human tumor cell lines.
Ferulenol did not influence GTP-induced tubulin assembly (not shown), but stimulated tubulin polymerization in the absence of GTP, with a less extensive polymerization profile at 100 pM concentration (Fig. [2]). The polymerizing mode of action of ferulenol is apparently similar to that of paclitaxel, a well known antitumor natural diterpene compound that promotes tubulin polymerization into stable microtubules [8], [9]. Calcium inhibited ferulenol-induced tubulin polymerization, and ferulenol-induced tubulin polymers were disassembled by calcium addition as well (not shown). These effects did not account for stabilizing activity, unlike paclitaxel and other related taxanes [10]. Microtubule-interacting agents can be classified into three main families: microtubule stabilizing compounds, Vinca alkaloid site interacting agents, colchicine site binders. Ferulenol decreased radiolabeled colchicine bound by tubulin, in a dose-dependent manner and significantly with 1 μM concentration (Fig. [3]), thus it could be included into the third one.
Microtubules of ferulenol-treated MCF-7 breast cancer cells were arrayed into thin and short fibres (Fig. [4] B), less numerous than microtubules of untreated cells (Fig. [4] A). Ferulenol thus appeared to reorganize the microtubule network rather than produce bundled microtubules in living cells, another characteristic of paclitaxel [9]. In addition, normal nuclear morphology of MCF-7 cells (Fig. [5] A), was altered by ferulenol, inducing swallen nuclei containing vesicle-like areas (Fig. [5] B).
Cytotoxic effects of ferulenol were assessed in human breast (MCF-7), colon (Caco-2), ovarian (SK-OV-3) and leukemic (HL-60) cancer cells, respectively. Ferulenol induced a dose-dependent reduction of cell viability, following 24 h of treatment (Fig. [6]). The inhibitory effect was significant at the concentrations of 100 nM and 1 μM, compared with untreated control cells. The good cytotoxic potency exhibited at the same concentrations inducing tubulin assembly in vitro likely indicates a reasonable accord with biochemical effects of ferulenol.
The precise mode of action of coumarins remains to be defined, although cell cycle arrest and decrease in DNA, RNA and protein synthesis have been shown as key cellular events in coumarins toxicity. Unexpectedly, ferulenol did not arrest cell cycle progression (not shown), denoting lack of apoptotic pathways. This report, together with unusual nuclear modifications observed, corroborates the differences with paclitaxel, which induces apoptosis via peculiar G2/M cell cycle arrest [11].
Hydroxycoumarins are cytotoxic and cytostatic across a variety of neoplastic cell types [12]. Geiparvarin and some derivatives inhibit cell proliferation by interacting with tubulin [6], [7], and this could also represent a mode of action for ferulenol, despite any structural similarities with all known microtubule-interacting agents.
Fig. 1 Chemical structure of ferulenol.
Fig. 2 Stimulation of tubulin assembly by ferulenol. Different concentrations of ferulenol and paclitaxel were added to the reaction mixtures containing 1 mg/ml tubulin; the polymerization reaction was initiated by raising the temperature to 37 °C and turbidity was followed at 350 nm every minute. Each point represents the mean of three different experiments, standard deviations are < 10 %.
Fig. 3 Effect of ferulenol on colchicine binding activity. Reaction mixtures containing purified tubulin (2 mg/ml) and different concentrations of ferulenol were preincubated for 20 min and further incubated for 60 min with [3 H]-colchicine. Data are percentages of controls (100 %) and represent the means of three different experiments (bar, SD). *P < 0.01 as compared with control.
Fig. 4 Images of microtubule perturbation by ferulenol. MCF-7 cells were untreated (A) or treated for 24 h with 100 nM ferulenol (B) and processed for immunofluorescence. Bar = 10 μm.
Fig. 5 Effects of ferulenol on nuclear structure. MCF-7 cells were untreated (A) or treated for 24 h with 100 nM ferulenol (B), and DNA was visualized by DAPI staining. Bar = 10 μm.
Fig. 6 Cytotoxic effects of ferulenol. Four human tumor cell lines were untreated or treated for 24 h with the indicated concentrations of ferulenol. Viability data are shown as means of percentages (bar, SD), calculated by standardizing untreated cells to 100 %, of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, compared with untreated cells.
Materials and Methods
Ferulenol was extracted according to Valle et al. [13] and was available from the library of Prof. G. Appendino, DiSCAFF, Università del Piemonte Orientale, Novara (Italy). For all assays, the compound was dissolved in DMSO, and the control mixtures contained solvent equivalent to the drug-treated mixtures. MCF-7 cells and Caco-2 cells were a gift from Prof. A. Corsini, University of Milano, Italy; HL-60 and SK-OV-3 cells were a gift from Prof. G. Barrera and Prof. Di Renzo, respectively, University of Torino, Italy. Murine monoclonal anti-β-tubulin antibody, FITC-conjugated sheep-anti-mouse antibody were purchased from Sigma Chemical Co. (St. Louis, MO). Culture media and FBS were obtained from Gibco-BRL/Life Technologies (Renfrewshire, UK). Colchicine was from Amersham International. Electrophoretically homogeneous MTP was prepared from bovine brain and stored in liquid nitrogen until use.
Polymerization reaction was followed turbidimetrically at 350 nm in a spectrophotometer equipped with a temperature controller. Drug or equivalent amount of DMSO (1 % final concentration) was added to the reaction mixtures containing a final MTP of 1 - 2 mg/ml in 0.1 M MES, 1 mM EGTA, 0.5 mM MgCl2.
Colchicine binding activity was determined by the DEAE-cellulose filter assay of Borisy [14]. After a pre-incubation with ferulenol, tubulin solutions were incubated with 0.1 mM colchicine containing trace amounts of [3 H]-colchicine (specific activity 5 μCi/ml) and repeatedly washed and filtrated through DEAE-cellulose filter. The radioactivity was measured in a liquid scintillation counter.
MCF-7 cells and Caco-2 cells were grown as monolayers in DMEM containing 10 % and 20 % FBS, respectively. SK-OV-3 cells were grown as monolayers and HL-60 cells were grown as suspension cultures, in RPMI medium containing 10 % FBS. All cell lines were maintained at 37 °C in a humidified 5 % CO2 atmosphere. Viable cells were scored microscopically by counting trypan blue positive or negative cells.
To detect microtubules by immunofluorescence, MCF-7 cells were fixed in ice-cold methanol, permeabilized in ice-cold acetone and incubated with primary antibody and successively with secondary antibody. To detect nuclei, MCF-7 cells were treated as described above and stained with DAPI. Fluorescent patterns of microtubules or chromatin were photographed using a fluorescence microscope with optics for fluorescein and DAPI, respectively.
The significance of the differences was analysed using the one-way ANOVA test with the Bonferroni post test.
#Acknowledgements
This work was supported by grants from Ministero della Ricerca Scientifica e Tecnologica of Italy.
#References
- 1 Carboni S, Malaguzzi V, Marsili A. Ferulenol, a new coumarin derivative from Ferula communis. Tetrahedron Lett 1963: 2783-6
- 2 Tligui N, Ruth G R, Felice L J. Plasma ferulenol concentration and activity of clotting factors in sheep with Ferula communis var. brevifolia intoxication. Am J Vet Res. 1994; 55 1564-9
- 3 Fraigui O, Lamnaouer D, Faouzi M Y. Acute toxicity of ferulenol, a 4-hydroxycoumarin isolated from Ferula communis L. Vet Hum Toxicol. 2002; 44 5-7
- 4 Maucher A, Kager M, von Angerer E. Evaluation of the antitumour activity of coumarin in prostate cancer models. J Cancer Res Clin Oncol. 1993; 3 150-4
- 5 Finn G J, Creaven B, Egan D A. Study of the in vitro cytotoxic potential of natural and synthetic coumarin derivatives using human normal and neoplastic skin cell lines. Melanoma Res. 2001; 11 461-7
- 6 Miglietta A, Bocca C, Gadoni E, Gabriel L, Rampa A, Bisi A, Valenti P, Da Re P. Interaction of geiparvarin and related compounds with purified microtubular protein. Anticancer Drug Des. 1996; 11 35-48
- 7 Miglietta A, Bocca C, Rampa A, Bisi A, Gabriel L. Geiparvarin and derivatives in combination with taxol: effect on microtubular organization in 3T3 fibroblasts. Anticancer Drug Des. 1997; 12 607-20
- 8 Schiff P B, Fant J, Horwitz S B. Promotion of microtubule assembly in vitro by taxol. Nature. 1979; 22 665-7
- 9 Schiff P B, Horwitz S B. Taxol stabilizes microtubules in mouse fibroblast cells. Proc Natl Acad Sci USA. 1980; 77 1561-5
- 10 Miglietta A, Bocca C, Gabriel L. Comparative studies on biological activity of certain microtubule-interacting taxanes. Chem Biol Interact. 2002; 139 83-99
- 11 Bhalla K, Ibrado A M, Tourkina E, Tang C, Mahoney M E, Huang Y. Taxol induces internucleosomal DNA fragmentation associated with programmed cell death in human myeloid leukemia cells. Leukemia (Baltimore). 1993; 6 563-8
- 12 Egan D, James P, Cooke D, O’Kennedy R. Studies on the cytostatic and cytotoxic effects and mode of action of 8-nitro-7-hydroxycoumarin. Cancer Lett. 1997; 118 201-11
- 13 Valle M G, Appendino G, Nano G M, Picci V. Prenylated coumarins and sesquiterpenoids from Ferula communis . Phytochemistry. 1987; 26 253-6
- 14 Borisy G G. A rapid method for quantitative determination of microtubule protein using DEAE-cellulose filters. Anal Biochem. 1972; 50 73-85
Claudia Bocca
Department of Experimental Medicine and Oncology, University of Torino
Corso Raffaello 30
10125 Torino
Italy
Phone: +39-0116707751
Fax: +39-0116707753
Email: claudia.bocca@unito.it
References
- 1 Carboni S, Malaguzzi V, Marsili A. Ferulenol, a new coumarin derivative from Ferula communis. Tetrahedron Lett 1963: 2783-6
- 2 Tligui N, Ruth G R, Felice L J. Plasma ferulenol concentration and activity of clotting factors in sheep with Ferula communis var. brevifolia intoxication. Am J Vet Res. 1994; 55 1564-9
- 3 Fraigui O, Lamnaouer D, Faouzi M Y. Acute toxicity of ferulenol, a 4-hydroxycoumarin isolated from Ferula communis L. Vet Hum Toxicol. 2002; 44 5-7
- 4 Maucher A, Kager M, von Angerer E. Evaluation of the antitumour activity of coumarin in prostate cancer models. J Cancer Res Clin Oncol. 1993; 3 150-4
- 5 Finn G J, Creaven B, Egan D A. Study of the in vitro cytotoxic potential of natural and synthetic coumarin derivatives using human normal and neoplastic skin cell lines. Melanoma Res. 2001; 11 461-7
- 6 Miglietta A, Bocca C, Gadoni E, Gabriel L, Rampa A, Bisi A, Valenti P, Da Re P. Interaction of geiparvarin and related compounds with purified microtubular protein. Anticancer Drug Des. 1996; 11 35-48
- 7 Miglietta A, Bocca C, Rampa A, Bisi A, Gabriel L. Geiparvarin and derivatives in combination with taxol: effect on microtubular organization in 3T3 fibroblasts. Anticancer Drug Des. 1997; 12 607-20
- 8 Schiff P B, Fant J, Horwitz S B. Promotion of microtubule assembly in vitro by taxol. Nature. 1979; 22 665-7
- 9 Schiff P B, Horwitz S B. Taxol stabilizes microtubules in mouse fibroblast cells. Proc Natl Acad Sci USA. 1980; 77 1561-5
- 10 Miglietta A, Bocca C, Gabriel L. Comparative studies on biological activity of certain microtubule-interacting taxanes. Chem Biol Interact. 2002; 139 83-99
- 11 Bhalla K, Ibrado A M, Tourkina E, Tang C, Mahoney M E, Huang Y. Taxol induces internucleosomal DNA fragmentation associated with programmed cell death in human myeloid leukemia cells. Leukemia (Baltimore). 1993; 6 563-8
- 12 Egan D, James P, Cooke D, O’Kennedy R. Studies on the cytostatic and cytotoxic effects and mode of action of 8-nitro-7-hydroxycoumarin. Cancer Lett. 1997; 118 201-11
- 13 Valle M G, Appendino G, Nano G M, Picci V. Prenylated coumarins and sesquiterpenoids from Ferula communis . Phytochemistry. 1987; 26 253-6
- 14 Borisy G G. A rapid method for quantitative determination of microtubule protein using DEAE-cellulose filters. Anal Biochem. 1972; 50 73-85
Claudia Bocca
Department of Experimental Medicine and Oncology, University of Torino
Corso Raffaello 30
10125 Torino
Italy
Phone: +39-0116707751
Fax: +39-0116707753
Email: claudia.bocca@unito.it
Fig. 1 Chemical structure of ferulenol.
Fig. 2 Stimulation of tubulin assembly by ferulenol. Different concentrations of ferulenol and paclitaxel were added to the reaction mixtures containing 1 mg/ml tubulin; the polymerization reaction was initiated by raising the temperature to 37 °C and turbidity was followed at 350 nm every minute. Each point represents the mean of three different experiments, standard deviations are < 10 %.
Fig. 3 Effect of ferulenol on colchicine binding activity. Reaction mixtures containing purified tubulin (2 mg/ml) and different concentrations of ferulenol were preincubated for 20 min and further incubated for 60 min with [3 H]-colchicine. Data are percentages of controls (100 %) and represent the means of three different experiments (bar, SD). *P < 0.01 as compared with control.
Fig. 4 Images of microtubule perturbation by ferulenol. MCF-7 cells were untreated (A) or treated for 24 h with 100 nM ferulenol (B) and processed for immunofluorescence. Bar = 10 μm.
Fig. 5 Effects of ferulenol on nuclear structure. MCF-7 cells were untreated (A) or treated for 24 h with 100 nM ferulenol (B), and DNA was visualized by DAPI staining. Bar = 10 μm.
Fig. 6 Cytotoxic effects of ferulenol. Four human tumor cell lines were untreated or treated for 24 h with the indicated concentrations of ferulenol. Viability data are shown as means of percentages (bar, SD), calculated by standardizing untreated cells to 100 %, of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, compared with untreated cells.