In vitro activation of macrophages by a novel proteoglycan isolated from corms of Crocus sativus L

Cancer Letters 144 (1999) 107±114

In vitro activation of macrophages by a novel proteoglycan isolated from corms of Crocus sativus L Julio Escribano a,1, M. Jose M. DõÂaz-Guerra b, 1, Hans H. Riese c, JesuÂs OntanÄoÂn d, DamiaÂn GarcõÂa-Olmo d, Dolores C. GarcõÂa-Olmo d, Angela Rubio a, Jose A. FernaÂndez a,* b

a SeccioÂn de BiotecnologõÂa IDR, Universidad de Castilla-La Mancha, Campus Universitario s/n, E-02071 Albacete, Spain Departamento de BioquõÂmica y BiologõÂa Molecular, Instituto de BioquõÂmica CSIC-UCM, Universidad Complutense, Av. RamoÂn y Cajal s/n, E-28040 Madrid, Spain c Pharmacia Upjohn, Departamento de InmunologõÂa y OncologõÂa, Centro Nacional de BiotecnologõÂa, Campus Universidad AutoÂnoma, E-28049 Madrid, Spain d Unidad de InvestigacioÂn ClõÂnico-Experimental, Hospital General de Albacete, c/ Hermanos Falco s/n, E-02006 Albacete, Spain

Received 10 February 1999; received in revised form 19 May 1999; accepted 24 May 1999

Abstract Saffron corms contain a proteoglycan that is highly cytotoxic on human tumor cells. The present work was undertaken to study the possible immunomodulatory and anti-invasive properties of this compound. Non-cytotoxic concentrations of this glycoconjugate promoted signi®cant macrophage activation, detected by the release of nitric oxide. A rapid activation of protein kinase C and NF-kB was obtained after proteoglycan treatment, which could explain the induction of nitric oxide synthase. Proteoglycan concentrations ranging from 10±1000 ng/ml speci®cally promoted apoptosis of macrophages, probably triggered by their activation. This molecule did not inhibit in vitro migration or invasion of human tumor cells. Altogether these results support a plausible immuno-modulating activity for this saffron Crocus compound. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Crocus sativus; Immuno-stimulating capacity; Macrophage activation; Proteoglycan

1. Introduction In the continued search for new anti-tumor agents, many efforts are directed to the research of natural compounds. The past decade has seen a dramatic resurgence in the research of carbohydrates involved in disease, and their potential use in therapeutics [1]. * Corresponding author. Tel.: 134-967-599-200; fax: 134-967599-233. E-mail address: [email protected] (J.A. FernaÂndez) 1 These authors have contributed equally. Present address: Facultad de Medicina, Universidad de Castilla-La Mancha, E02071 Albacete, Spain.

Several plant polysaccharides have been described with in vitro and in vivo immuno-stimulating activity [2±6]. Their major effect seems to be the activation of macrophages cytotoxicity against tumor cells. Likewise, other branched plant heteropolymers have been reported to enhance cytotoxicity of human natural killer (NK) cells by inducing the production and/or release of cytokines [7±9]. Some polysaccharides have shown potent activity against various tumors when tested in implanted animals [10]. The mechanism proposed has been the blockage of metastasis by covering galactose-speci®c binding sites [11]. These activities may have possible therapeutic impli-

0304-3835/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00211-6

108

J. Escribano et al. / Cancer Letters 144 (1999) 107±114

cations in cancer treatment, from the approach of modulating the immunological functions or by blocking metastasis. Recently, we have isolated a novel cytotoxic proteoglycan, structurally related to arabinogalactanproteins [12], from the corm of the saffron plant (Crocus sativus L.). Doses between 7±22 mg/ml of proteoglycan caused 50% in vitro growth inhibition in tumor cells. To our knowledge, this is the ®rst proteoglycan described to be cytotoxic to human malignant cells. Our aim in the present work has been to initiate investigations about the possible immuno-stimulating and anti-invasive effects of this molecule. We have observed activation of macrophages by the proteoglycan, detected by release of nitric oxide (NO), in non-cytotoxic concentrations.

2. Materials and methods 2.1. Materials Corms of `La Mancha' saffron (C. sativus L.) were acquired from farmers in Lezuza, Albacete, Spain. The proteoglycan was puri®ed from corms by ultra®ltration, gel ®ltration-, anion exchange- and reversed phase-chromatography [12]. The samples used were homogeneous both by reversed-phase chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Basement membrane Matrigel was purchased from Collaborative Biomedical Products (Bedford, MA). Human recombinant basic ®broblast growth factor (bFGF) was obtained from Farmitalia Carlo Erba, Italy. Human ®bronectin and gelatin were from Sigma Chemical Co. (St. Louis, MO). BCECF-AM (3 0 -O-Acetyl-2 0 ,7 0 -bis(carboxyethyl)-4 or 5-carboxy¯uorescein, diacetoxymethyl ester) was from Molecular Probes, Inc., Eugene, OR. Anti-mouse p65, p50, p52 and c-Rel NF-kB proteins were obtained from Santa Cruz Biotechnology, Santa Cruz, CA. All other chemicals were reagent grade or of highest available quality. MDA-MB-231 human breast carcinoma and HT1080 ®brosarcoma cell lines were obtained from the American Type Culture Collection (Rockville, MD). Peritoneal resident macrophages were prepared from male rats following a previous protocol [13].

2.2. Cell culture conditions Cell lines were grown at 378C in DMEM (Dulbecco's modi®ed Eagle's medium, Gibco, UK) containing 10% fetal calf serum (FCS) (Labtech Int., France), 2 mM glutamine (ICN, Costa Mesa, CA), 1% vitamins (HT-1080 cells) (Gibco, UK), 100 units/ml penicillin and 0.1 mg/ml streptomycin, in a humidi®ed atmosphere of 5% CO2. Cell lines were routinely checked by the Gen-Probe rapid detection system (Gen-Probe, San Diego, CA) for mycoplasma contamination. Peritoneal resident macrophages were seeded in DMEM containing 10% (v/v) FCS and antibiotics as described above. 2.3. In vitro migration and invasion assays Polyvinyl-pyrrolidone-free polycarbonate ®lter Transwell inserts (6.5 mm diameter) with 8 mm pores (Costar, Cambridge, MA) were incubated overnight with 0.1 mg/ml gelatine in 0.1% (v/v) acetic acid and dried. For invasion assays basement membrane Matrigel (5 mg in 50 ml cold phosphate-buffered saline (PBS)) was further applied to the upper surface of each ®lter and dried at room temperature under a hood. Inserts were set in 24-well culture plates (Falcon, Franklin Lakes, NJ) and human ®bronectin (5 mg in 600 ml culture medium) was added to the lower wells as chemoattractant. HT-1080 and MDAMB-231 tumor cells (150 000 cells/well) were seeded on the Matrigel side of the ®lter in 100 ml culture medium with 2% (v/v) FCS in the presence of 10 mg/ml proteoglycan. After incubation for 24 h at 378C under a 5% (v/v) CO2 atmosphere, cells on the upper side of the ®lter were removed with cotton swabs. Filters were then quantitated with the BCECF-AM ¯uorometric method as described previously [14]. Brie¯y, inserts were transferred to a 24-well culture plate and then incubated for 10 min at 378C, with culture medium containing 6.25 mg/ml BCECF-AM. Excess reagent was discarded and inserts were read in a ¯uorescence multiwell plate reader (Millipore Cyto¯uor 2350), using 485 and 530 nm as excitation and emission wavelengths, respectively. 2.4. Determination of NO Macrophages were treated 24 h with the indicated

J. Escribano et al. / Cancer Letters 144 (1999) 107±114

concentrations of proteoglycan (10±50 ng/ml). NO was measured as the accumulation of nitrite and nitrate in the macrophages incubation medium. Nitrate was reduced to nitrite with nitrate reductase (EC 1.6.6.1). Nitrite was determined spectrophotometrically with Griess reagent [13]. The absorbance at 548 nm was compared with a standard of NaNO2. 2.5. Protein kinases and cAMP determinations Macrophages were incubated with 100 ng/ml of proteoglycan for 5 and 30 min. They were then rinsed with cold PBS and rapidly arrested in liquid N2. Protein kinase C (PKC) (EC 2.7.1.37) was detected by its histone kinase activity after partial puri®cation as previously described [15]. Mitogen activated protein (MAP) kinase activity was determined by its capacity to phosphorylate mieline basic protein (MBP) [16]. cAMP was assayed by binding protein using the Amersham Kit (Amersham, Little Chalfont, UK). Cells were incubated in the presence of 0.5 mM isobutyl methyl xanthine (BMX) in order to prevent adenosine 3 0 -5 0 -cyclic monophosphate degradation [17]. 2.6. NF-k B activation NF-kB activation was detected by the electrophoretic mobility shift assay (EMSA). Macrophages were treated for 1 h with different proteoglycan concentrations (10±50 ng/ml) or with bacterial lipopolysaccharides (LPS) (500 ng/ml) as a positive control. The cell layers of macrophages (3 £ 106 ) were washed twice with ice-cold PBS and collected in PBS by centrifugation. The cell pellets were homogenized with 0.2 ml of buffer A (10 mM Hepes (pH 7.9), 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM EGTA, 10 mM KCl, 1 mM 1,4-dithio-dlthreitol (DTT), 0.5 mM phenylmethanesulfonyl ¯uoride, 2 g/ml aprotinin, 10 g/ml leupeptin, 5 mM NaF, 1 mM NaVO4, 10 mM Na2MO4). After 10 min at 48C Nonidet P-40 was added to a concentration of 0.5% (v/v). The tubes were gently vortexed for 15 s and nuclei were sedimented by centrifugation at 8000 £ g for 15 s. The pellet was resuspended in 70 ml of buffer A supplemented with 20% (v/v) glycerol and 0.4 M KCl. Incubation was continued for 30 min at 48C with gentle vortexing. Nuclear proteins were extracted by centrifugation at 13 000 £ g for 15 min

109

and aliquots of the supernatant were stored at 2 808C. Protein concentrations were measured using the BioRad (Richmond, CA) protein reagent following the recommendations of the supplier. Synthetic oligonucleotides were prepared using an oligonucleotide synthesizer (Pharmacia, Uppsala, Sweden). NF-Bp, corresponding to proximal kB motif (nucleotides 292 to 265) of iNOS (nitric oxide synthase) promoter [18]: 5 0 -tcgaCCAACTGGGGACTCTCCCTTTGGGAACA-3 0 and 3 0 -GGTTGACCCCTGAGAGGGAAACCCTTGTagct-5 0 . The oligonucleotides were annealed after incubation for 5 min at 858C in 10 mM Tris±HCl (pH 8.0), 50 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol. Fifty-nanogram aliquots of these annealed oligonucleotides were endlabeled with the Klenow enzyme fragment in the presence of 50 mCi of [a ± 32P]dCTP and the other unlabeled dNTPs in a ®nal volume of 50 ml. 50 000 dpm of the DNA probe were used for each binding assay of nuclear extracts as follows: 5 mg of protein extract were incubated for 30 min at 48C with the DNA and 1 mg/ml poly (dI-dC), 5% (v/v) glycerol, 1 mM EDTA, 100 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol and 10 mM Tris±HCl (pH 7.8), in a ®nal volume of 20 ml. The incubation mixture was applied to a 6% (w/v) polyacrylamide gel, which had been previously electrophoresed for 30 min at 100 V. Gels were run at 0.8 V/cm 2 in 45 mM Tris± borate buffer, followed by transfer to 3MM Whatman paper, drying under vacuum at 808C and exposure at 2808C to X-ray ®lm (Hyper®lm, Amersham) using an intensifying screen. Analysis of competition with unlabeled oligonucleotide was performed using a 50-fold excess of dsDNA in the binding reaction. Supershift assays were carried out after addition of anti-mouse-p65, p50, p52 and c-Rel NF-kB proteins. 2.7. Analysis of DNA fragmentation Programmed cell death (apoptosis) induced by proteoglycan treatment has been studied both by cellular DNA fragmentation analysis and ¯ow cytometry in macrophages. Cells were incubated for 24 h with proteoglycan concentrations ranged from 50± 1000 ng/ml. Internucleosomal DNA fragmentation was analyzed by agarose gel electrophoresis, by detection of mono- and oligonucleosomes in the cytosol [19]. Cells were also analyzed in a FAC-Star

110

J. Escribano et al. / Cancer Letters 144 (1999) 107±114

Fig. 1. The saffron glycoconjugate induces NO release in macrophages. Macrophages were treated for 24 h with the indicated concentrations of glycoconjugate, and NO release was measured in the medium. Results show the mean^SE of three experiments.

Plus ¯ow cytometer (Becton Dickinson) equipped with a 5 W argon laser. The forward scatter was plotted against the propidium iodide ¯uorescence [20]. 2.8. Data analysis. The number of experiments analyzed is indicated in the legend of each ®gure. Statistical differences (P , 0:05) between mean values were determined by one-way ANOVA followed by Student's test. 3. Results 3.1. The proteoglycan activates NO production and NF-k B in macrophages We have analyzed macrophage triggering by the saffron compound following NO production. Macrophages were incubated with proteoglycan concentrations below toxic limits. Treatment with 50 ng/ml of proteoglycan doubled the release of nitrate and nitrite, the degradative products of NO (Fig. 1). Higher concentrations (up to 500 ng/ml) resulted in a decreased NO production in parallel with a marked fall in cell viability (data not shown). Maximal NO accumulation represents a 10% of that observed with optimal doses of bacterial LPS, a standard macrophage activator (data not shown).

In macrophages, NO is synthesized by an inducible nitric oxide synthase (iNOS). Discrete interactions of proteoglycan with the plasma membrane could lead to different modulation of signaling transduction pathways, some involved in iNOS transcription. We have studied two characterized pathways implicated in iNOS induction, one dependent on cAMP generation or the other on protein kinase C activation. Basal cAMP levels in macrophages were 2:0^0:2 pmol/10 ±6 cells (n ˆ 4). A proteoglycan concentration of 100 ng/ ml did not stimulate cAMP generation in macrophages, at least in the ®rst 30 min. As we have shown previously, an increase in intracellular calcium was observed after proteoglycan treatment (unpublished data), accordingly, we studied protein kinase C activity, as it depends on calcium and membrane phospholipids for activation. An increase of cytosolic and particulate PKC activity was observed rapidly after treatment with proteoglycan (100 ng/ml), followed by a decrease after 30 min (Table 1). A decrease in PKC levels is observed usually after PKC activation as result of its proteolysis [15]. We have not detected activation of other kinases such as MAP kinases (data not shown). PKC activation could be the result of the increase in calcium permeability of plasma membrane, as observed after ionophores treatment. Induction of iNOS expression in macrophages depends on activation of different transcription factors [16]. NF-kB participates in the regulation of the expression of multiple genes involved in the immune response, including iNOS [17]. Triggering macrophages with proteoglycan concentrations that induce NO production, increased the binding of NF-kB Table 1 Effect of saffron proteoglycan on protein kinase C activity a Protein kinase C activity

Control Proteoglycan 5 min Proteoglycan 30 min

Cytosol

Membrane

100 230 ^ 20 50 ^ 18

100 150 ^ 12 80 ^ 10

a Macrophages were treated with 100 ng/ml of proteoglycan for 5 and 30 min. Cells were arrested in liquid nitrogen and kinase activity was determined as described. The results are expressed as percentage of control cells and show the mean^SE of three experiments.

J. Escribano et al. / Cancer Letters 144 (1999) 107±114

111

Fig. 2. NF-kB activation in macrophages treated with the glycoconjugate. Cells were treated for 1 h with different glycoconjugate concentrations or with bacterial LPS (500 ng/ml), and nuclear protein extracts were prepared. The protein binding to the kB sequence corresponding to the iNOS promoter was measured by EMSA. Controls to ensure the speci®city of the interaction were performed using a 50-fold excess of the unlabeled probe. Speci®c complexes are indicated by arrows (A). To analyze the proteins that bind to the iNOS NF-kB sequence, supershift assays were carried out with 0.5 mg of the indicated anti-mouse antibodies (B). The results show one representative experiment out of three.

proteins to the kB motif of the iNOS gene promoter (Fig. 2). This NF-kB activation, as observed with NO production, is limited when compared with that induced by bacterial lipopolysaccharides that are intense immune activators. However, this effect is similar to that obtained with other macrophage-stimulating factors, such as cecropin and mellitin-derived peptides [18]. The nature of the NF-kB binding proteins was characterized using a supershift assay. As ®gure 2B shows, the presence of p65, p50 and cRel proteins were detected in NF-kB-complexes after macrophage activation with 50 ng/ml of proteoglycan. 3.2. Proteoglycan induces apoptosis in macrophages The possible apoptotic effect of the proteoglycan in macrophages was studied. Although untreated macrophages showed a certain level of apoptosis, probably

due to cell culture and/or handling, incubation of macrophages with non-cytotoxic concentrations of proteoglycan (50±1000 ng/ml) resulted in an increase of the DNA laddering characteristic of apoptotic cells (Fig. 3A). A dose dependent increase of apoptotic cells was also detected by ¯ow cytometry, con®rming this effect (Fig. 3B-C). This behavior was speci®c for macrophages as no apoptosis was observed in HeLa cells treated with similar proteoglycan concentrations (data not shown). These results suggest that apoptosis could be a consequence of macrophage activation, as has been suggested previously [20]. 3.3. Migration and invasion assays At non-cytotoxic concentrations the saffron corm proteoglycan did not inhibit in vitro migration and

112

J. Escribano et al. / Cancer Letters 144 (1999) 107±114

Fig. 3. Low concentrations of proteoglycan induce apoptosis in macrophages. Macrophages were incubated for 24 h with the indicated concentrations of the glycoconjugate. Apoptosis was analyzed by the appearance of a DNA ladder in agarose gel electrophoresis (A) and by ¯ow cytometry (B). A representative ¯ow cytometry plot of untreated (control) or proteoglycan stimulated cells is shown in panel C. Results of panel B show the mean ^ SE of three experiments.

invasion of MDA-MB-231 or HT-1080 cells (data not shown). 4. Discussion We have recently reported the cytotoxicity of this proteoglycan extracted from saffron corms on tumor cells [12]. The present work was designed to extend the investigation of possible immuno-modulator and anti-tumor effects of this compound. Macrophages are important in the body's defense against tumors. Selective stimulation of this cell population could be important to the development of therapeutic applications, therefore this approach has been carried out with a variety of agents [21]. Some plant polysaccharides, including mannans and arabinogalactans have shown modulating activity in the immune system, increasing bactericidal and tumoricidal activities [2,22]. Macrophages are implicated in this response by the synthesis of free radicals and oxidative species such as nitric oxide (NO). A wide

array of extracellular signals induces macrophage activation and nitric oxide synthase expression increasing the release of NO to the extracellular medium [16]. NO is a reactive molecule involved in multiple responses including inhibition of tumor cell proliferation. Some membrane-interacting glycoproteins and peptides have shown speci®c effects in the immune system as consequence of signal transduction pathways activation. Acemannan [22], cecropin A and mellitin-derived peptides [18] induce macrophage activation followed by an increase in the free radical, NO and peroxides production. The saffron corm proteoglycan is induces NO liberation after macrophage treatment with concentrations below toxic limits. Indeed, after treatment with proteoglycan, macrophages increase its apoptotic pattern as occurs after activation with bacterial lipopeptides and lipopolysaccharides [13,20]. Nevertheless, high concentrations of proteoglycan induce membrane damage of macrophages as occurs in tumor cells. The presence of apoptotic or necrotic effects depending on the dose of proteoglycan have been observed for other

J. Escribano et al. / Cancer Letters 144 (1999) 107±114

membrane perturbing agents such as cecropin A and melittin-derived peptides [18]. These data suggest that the saffron glycoconjugate may be useful in activating macrophages to defend against tumors. It is possible to hypothesize that non-cytotoxic immuno-stimulating concentrations could be reached in certain tissues when administrated in vivo. The molecular mechanisms that originate macrophage activation by the saffron proteoglycan are still poorly understood. Nevertheless, we have detected speci®c stimulation of PKC activity, probably triggered by the increase of calcium levels caused by treatment with this glycoconjugate. Recently, we have found that this compound alters plasma membrane permeability on HeLa cells, causing calcium in¯ux (unpublished data). The rise of intracellular calcium levels and activation of PKC induce NF-kB DNA-binding, a process necessary to activate iNOS transcription and NO production in macrophages, as has been described for some membrane-interacting peptides as melittin [13,17,18]. In vivo experiments, currently taking place in our laboratory, are required to determine the appropriate amounts of glycoconjugate to be applied, as well as to evaluate the anti-tumoral roles of cytotoxicity and macrophage activation in animal cancer models. Plant polysaccharides of the arabinogalactan group have also been reported to enhance phagocyte activity, to activate cytotoxicity of macrophages against tumor cells and to induce macrophages to produce lymphokines in vivo as well as in vitro [1,2,4,5]. In liver tumors induced in mice, treatment with an arabinogalactan polysaccharide has been described to reduce the amount of liver metastasis and prolonged the survival times of the animals [11]. It was proposed an effect of arabinogalactan blockade of potential liver receptors by covering galactose speci®c binding sites. We investigated a similar effect of this related molecule. The saffron proteoglycan did not show any anti-invasive effect as checked on MDA-MB-231 or HT-1080 cells. Therefore, cytostatic effects based on migration and invasion could not be demonstrated with these techniques. Our data indicate that low doses of proteoglycan activate macrophages in vitro, as revealed by the release of NO in treated cells. Thus, this compound may be useful in activating macrophages to defend against tumors.

113

Acknowledgements This study was supported in part by a grant (SAF97-0149) from the `Plan Nacional de I 1 D' (Spain). Jesus OntanÄoÂn and Angela Rubio are recipients of fellowships from the `Cultural Albacete' Public Consortium, and Junta de Comunidades de Castilla-La Mancha, respectively.

References [1] A. Persidis, The carbohydrate-based drug industry, Nature Biotech 15 (1997) 479±480. [2] B. Luetting, C. SteinmuÈller, G.E. Gifford, H. Wagner, M.L. Lohmann-Matthes, Macrophage activation by the polysaccharide arabinogalactan isolated from plant cell cultures of Echinacea purpurea, J. Natl. Cancer Inst 81 (1989) 669±675. [3] J. Varljen, A. Liptak, H. Wagner, Structural analysis of a rhamnoarabinogalactan and arabinogalactans with immunostimulating activity from Calendula of®cinalis, Phytochemistry 28 (1989) 2379±2383. [4] N. Shimizu, M. Tomoda, R. Gonda, M. Nakari, N. Takanashi, N. Takahashi, The major pectic arabinogalactan having activity on the reticuloendothelial system from the roots and rhizomes of Saposhnikovia divaricata, Chem. Pharm. Bull 37 (1989) 1329±1332. [5] R. Gonda, M. Tomoda, N. Ohara, K. Takada, Arabinogalactan core structure and immunological activities of ukonan C, and acidic polysaccharide from the rhizome of Curcuma longa, Biol. Pharm. Bull 16 (1993) 235±238. [6] M. Tomoda, H. Miyamoto, N. Shimizu, R. Gonda, N. Ohara, Two acidic polysaccharides having reticuloendothelial system-potentiating activity from the raw root of Rehmannia glutinosa, Biol. Pharm Bull 17 (1994) 1456±1459. [7] E.A. Mueller, F.A. Anderer, Chemical speci®city of effector cell/tumor cell bringing by a Viscum album rhamnogalacturonan enhancing cytotoxicity of human NK cells, Immunopharmacology 19 (1990) 69±77. [8] E.A. Mueller, F.A. Anderer, Synergistic action of a plant rhamnogalacturonan enhancing antitumor cytotoxicity of human natural killer cells: chemical speci®city of target cell recognition, Cancer Res 50 (1990) 3646±3651. [9] J. Hauer, F.A. Anderer, Mechanism of stimulation of human natural killer cytotoxicity by arabinogalactan from Larix occidentalis, Cancer Immunol. Immunother 36 (1993) 224±237. [10] H. Yamada, K. Komiyama, H. Kiyohara, J.C. Cyong, Y. Hirakawa, Y. Otsuka, Structural characterization and antitumor activity of a pectic polysaccharide from the roots of Angelica acutiloba, Planta Med 56 (1990) 182±186. [11] B. Hagmar, W. Ryd, H. Skomedal, Arabinogalactan blockade of experimental metastases to liver by murine hepatoma, Invas. Metast. 11 (1991) 348±355. [12] J. Escribano, I. RõÂos, J.A. FernaÂndez, Isolation and cytotoxic properties of a novel glycoconjugate from corms of saffron

114

[13]

[14]

[15] [16] [17]

J. Escribano et al. / Cancer Letters 144 (1999) 107±114 plant (Crocus sativus L.), Biochim. Biophys. Acta 1426 (1999) 217±222. F. Terenz, M.J.M. DõÂaz-Guerra, M. Casado, S. Hortelano, S. Leoni, L. BoscaÂ, Bacterial lipopeptides induce nitric oxide synthase and promote apoptosis through nitric oxide-independent pathways, J. Biol. Chem 270 (1995) 6017±6021. T. Garrido, H.H. Riese, A.R. Quesada, M.M. Barbacid, M. Aracil, Quantitative assay for cell invasion using the ¯uorogenic substrate 2 0 ,7 0 -Bis(2-carboxyethyl)-5 (and -6)-carboxy¯uorescein acetoxymethylester, Anal. Biochem 235 (1996) 234±236. Y. Nishizuka, Protein kinase C and lipid signalling for sustained cellular responses, FASEB J 9 (1995) 484±496. C. Nathan, Q.W. Xie, Nitric oxide synthases: roles, tolls and controls, Cell 78 (1994) 915. D. Thanos, T. Maniatis, NF-kB: a lesson in family values, Cell 80 (1995) 529±532.

[18] M. Velasco, M.J.M. DõÂaz-Guerra, P. DõÂaz-Achirica, D. Andreu, L. Rivas, L. Bosch, Macrophage triggering with cecropin A and melittin-derived peptides induces type II nitric oxide synthase expression, J. Inmunol 158 (1997) 4437±4443. [19] H.U. Bergmeyer, M. Grabl, H.-E. Walter, in: H.U. Bergemeyer, (Ed.), Methods of Enzymatic Analysis, Weinsheim Verlag Chemie,1999, pp. 232±233. [20] E.A. Albina, S. Cui, R.B. Mateo, J.S. Reichner, Nitric oxidemediated apoptosis in murine peritoneal macrophages, J. Immunol. 150 (1993) 5080±5085. [21] C.F. Nathan, Z.A. Cohn, Role of oxygen-dependent mechanisms in antibody-induced lysis of tumor cells by activated macrophages, J. Exp. Med 152 (1980) 198±202. [22] L. Ramammorthy, M.C. Kemp, I.R. Tizard, Suramin, a novel antineoplastic agent with multiple potential mechanisms of action, Mol. Pharmacol 50 (1996) 878±884.