Antiproliferative effect of ferrocifen drug candidates on malignant pleural mesothelioma cell lines

Inorganica Chimica Acta 362 (2009) 4037–4042

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Antiproliferative effect of ferrocifen drug candidates on malignant pleural mesothelioma cell lines q Ilaria Zanellato a, Jan-Martin Heldt b,1, Anne Vessières b, Gérard Jaouen b, Domenico Osella a,* a b

Dipartimento di Scienze dell’Ambiente e della Vita, Università del Piemonte Orientale ‘‘Amedeo Avogadro”, Via T. Michel 11, 15100 Alessandria, Italy Laboratoire de Chimie et Biochimie des Complexes Moléculaires, UMR CNRS 7576, Ecole Nationale Supérieure de Chimie de Paris, 11, rue Pierre et Marie Curie, 75231 Paris 05, France

a r t i c l e

i n f o

Article history: Received 15 April 2009 Accepted 23 May 2009 Available online 31 May 2009 Keywords: Malignant mesothelioma Antiproliferative activity Ferrocifens Bio-orgometallic chemistry

a b s t r a c t The purpose of this study was to investigate the antiproliferative potential of two novel bio-organometallic drug candidates, based on hydroxyl-phenyl-but-1-ene skeleton and containing the ferrocenyl (Fc) moiety, namely ferrociphenol (Fc-diOH) and ferrocifen (Fc-OH-TAM), on two cell lines, named BR95 (epithelial-like) and MM98 (sarcomatous-like), obtained from pleural effusions of previously untreated malignant pleural mesothelioma (MPM) patients. In vitro chemosensitivity of MPM cells towards the title compounds was evaluated by cell viability assay, alkaline Single Cell Gel Electrophoresis (Comet test) and western blotting evaluation of p53 induction. The two bio-organometallic derivatives were found to be more potent in inhibiting cell proliferation than the reference metallo-drug cisplatin (CDDP). This antiproliferative effect cannot be attributed to estrogenic/antiestrogenic activity, since both cell lines resulted to be estrogen insensitive (ER ). Fc-diOH and CDDP were able to upregulate wild type p53 present in MM98 cell line, while Fc-OH-TAM was not. Similarly, Fc-diOH and CDDP induced early DNA damage, while Fc-OH-TAM did not. This indicates that, albeit the similar structures, the two ferrocifens exhert different mechanisms of cytotoxicity on MPM cells. Ó 2009 Elsevier B.V. All rights reserved.

1. Introduction Malignant mesothelioma (MM) is an aggressive tumor that arises from the mesothelial surfaces of the pleura and the peritoneum cavities, and, less commonly, from the pericardium and the tunica vaginalis [1]. The most important causal factor for the development of malignant pleural mesothelioma (MPM) is occupational exposure to asbestos [2,3]. MPM was once rare, but its incidence has been increasing in several countries as a result of widespread exposure to asbestos in the past, and patients generally die within a year after initial diagnosis [4]. In any given case of MM, microscopic features may range from the pure epithelioid (most comAbbreviations: cisplatin or CDDP, cis-diamminedichloroplatinum (II); Comet assay, single cell gel electrophoresis; CT, continuous treatment; E2, estrogen; ER , estrogen insensitive; ER, estrogen receptor; ER+, estrogen sensitive; Fc-diOH, (1,1bis(4-hydroxy-phenyl)-2-ferrocenyl-but-1-ene: ferrociphenol; Fc-OH-TAM, 1-[4-(O(CH2)3NMe2)phenyl]-1-(4-hydroxy-phenyl)-2-ferrocenyl-but-1-ene): ferrocifen; HRP, horse radish peroxidase; MM, malignant mesothelioma; MPM, malignant pleural mesothelioma; MTS, (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium); PBS, phosphate buffered saline; R, recovery; TAM, tamoxifen. q Based on the presentation given at the 8th workshop on pharmaco bio-metallics, 24–26 October 2008, Ravenna, Italy. * Corresponding author. Tel.: +39 0131 360 366; fax: +39 0131 360 250. E-mail address: [email protected] (D. Osella). 1 Present address: Forschungszentrum Dresden-Rossendorf e.V., Bautzner Landstraße 128, 01328 Dresden, Germany.

0020-1693/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ica.2009.05.047

mon) to the pure mesenchymal-sarcomatous, with any combination of these phenotypes [5]. MPM cell lines have been established from tumor biopsies followed by enzymatic digestion [6] or by plating overnight of pleural effusions [7], thereby providing useful in vitro models to investigate the molecular biology of the tumor and to test novel therapeutic strategies. Diagnosis and staging of MPM are often inaccurate [8], so that therapy usually addresses late-stage mesotheliomas. Chemotherapy is a promising approach for the clinical management of MPM, because it induces reasonable responses while improving the patients’ quality of life. Among the chemotherapic drugs have evaluated in patients with MPM [9], cisplatin (CDDP) was identified as the most active agent in polychemotherapeutic regimens [10]. However, no effective standard chemotherapeutic treatment has emerged so far, and the identification of more active agents for MPM remains a great challenge. Ferrocifen (Fc-OH-TAM), a ferrocenyl derivative of 4-OHtamoxifen (4-OH-TAM), the active metabolite of tamoxifen, which is widely prescribed for the treatment of hormone-dependent breast cancer, and ferrociphenol (Fc-diOH) (Fig. 1) have been recently reported to possess a strong antiproliferative effect at micromolar level in vitro on both hormone-dependent, e.g. MCF7 (ER+), and independent, e.g. MDA-MB-231 (ER ) breast cancer cell lines [11–14].

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Fig. 1. Structures of compounds: ferrociphenol (Fc-diOH), ferrocifen (Fc-OH-TAM), 4-OH-tamoxifen (4-OH-TAM) and estradiol (E2).

The antiproliferative effect observed on ER+ cells with Fc-OHTAM results from both antiestrogenic and cytotoxic activities, while the effect observed on ER cells with Fc-OH-TAM and on both cell lines with Fc-diOH can only be linked to cytotoxicity. Recently, another report showed the specific antiproliferative effect of Fc-OH-TAM on melanoma cells with respect to normal melanocytes [15]. The precise mechanism underlying the cytotoxic effect of FcdiOH and Fc-OH-TAM has not been fully understood. The cellular oxidative stress caused by reactive oxygen species (ROS) produced by preformed ferricenium cation, through the initiation of Fentontype cycle, has been demonstrated to be responsible for the cytotoxicity of such derivatives in vitro [16–19]. In a similar vein, water soluble ferrocene derivatives could inflict oxidative damage as consequence of in vivo redox equilibrium between ferrocene/ferrocinium couple [20]. The preliminary oxidation step, albeit the reducing milieu of the tumor (hypoxic) cells, could occur in lysosomal compartiments, where ferrocenes are known to accumulate [21]. Recently, a more complex pathway has been proposed: it corresponds to intracellular oxidation of ferrocenyl moiety, followed by removal of a phenolic proton, leading to the formation of a quinone methide, an electrophilic species able to react with macromolecules, especially DNA [22]. Bioactivation of tamoxifen to quinone methide is a drawback of the long-term treatment with this popular selective estrogen-receptor modulator (SERM) and may contribute to its genotoxic effect of tamoxifen [23].

were prepared in DMSO and stored at months.

20 °C in the dark for several

2.3. Cell lines Two primary cell lines, having epithelioid-like and sarcomatous-like phenotype, derived from pleural effusion of previously untreated patients suffering from MPM, called BR95 [24,25] and MM98 [26], respectively, were used. Cells were grown in DMEM (Gibco, Invitrogen Life Science, S. Giuliano Milanese) supplemented with L-glutamine 2 mM, penicillin 100 IU/ml, streptomycin (100 g) and 10% fetal bovine serum (FBS, Euroclone, Pero, Italy) at 37 °C in a 5% CO2 humified chamber. The number of passages was between 30th and 40th. To test the compounds, 1  104 cells/well were seeded in 96-well flat bottom plates 1 day before the treatment. Population doubling takes about 28 h for BR95 and 20 h for MM98. 2.4. Estrogen sensitivity test On day 1, 1  104 cells/well were seeded in 96-well plates in Ham’s F10 medium (phenol red free) supplemented as reported above. On day 2, 1 nM 17b-estradiol (E2) or 1 lM 4-OH-TAM or fresh complete medium were added and cell viability was assessed after 3 days using the MTS assay (CellTiter Aqueous Solution, PromegaCorp., Madison, WI, USA). The final data have been calculated from three replicates of the same experiment.

2. Experimental 2.5. Cell treatment with organometallic putative drugs 2.1. Reagents Unless specified, all reagents were from Sigma–Aldrich (St. Louis, MO, USA). 2.2. Bio-organometallic complexes The ferrocenyl derivatives Fc-OH-TAM and Fc-diOH were synthesized as previously described [12,13]. Stock solutions (1 mM)

Compounds were diluted in complete medium and controls (having the same concentration of DMSO, less than 1% in every experiment) were used as reference. CDDP was dissolved in 0.9% NaCl, acidified at pH 3 with HCl, and stored at 80 °C. Three treatment protocols were chosen: 24 h continuous treatment (CT), 24 h CT followed by PBS washing and 48 h of recovery (R) in fresh medium, and 72 h CT. Cell viability measurement followed three PBS washings. The MTS test was assessed according

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to manufacturer’s instruction. In each experiment, the MPM cells were challenged with the two drug candidates at different concentrations and the final data were calculated from at least three replicates of the same experiment carried out in triplicate. Absorbances were recorded at 490/620 nM by a spectrophotometric plate reader (Sirio S, SEAC, Florence, Italy); the absorbance of an MTS solution (without cells) was used as blank.

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2.6. Comet assay Cells were treated for 2 h at 37 °C with sublethal, equitoxic doses of the compounds under investigation, namely, 0.5 lM of Fc-diOH or Fc-OH-TAM for MM98, and 8 lM of Fc-diOH or 2.5 lM of Fc-OH-TAM for BR95 cells. CDDP (at a concentration of 10 lM) was chosen as positive control since it is known to cause

Fig. 2. Cell viability normalized with respect to untreated cells (NT) after 72 h CT with estradiol (E2 1 nM) or 4-hydroxy-tamoxifen (4-OH-TAM 1 lM). Untreated cells (NT) were used as controls.

Fig. 3. Cell viability of BR95 (left column) and MM98 (right column) following CDDP, Fc-diOH or Fc-OH-TAM at 24 h CT (24 CT), 24 h CT followed by 48 h of R (24 h CT + 48 h R), or 72 h CT (72 h). Results are means of 3–8 replications of the same experiment, p < 0.05.

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DNA strand-breaks [27–29]. Fresh medium was used as negative control (non-treated, NT). About 3  104 cells were suspended in 0.5% low melting point (LPM) agar and then deposited on NMP (Normal Melting Point) 1% agarose pre-coated microscope slides and overheld by another layer of 0.5% LMP agar. Each experiment was replicated twice. Lysis was carried out at 4 °C overnight in 100 mM EDTA, 2.5 M sodium chloride, 0.5% N-lauroylsarcosine, 10 mM Tris base, 1% TritonTM X-100, 10% DMSO. Alkali DNA unwinding (20 min) and electrophoresis (20 min, 300 mA, 25 V) were performed in 0.5 M disodium EDTA, 300 mM NaOH, pH > 14. Slides were neutralized by three washes in Tris–acetate 0.5 M pH 7.5 and DNA was stained by DAPI 0.2 lg/ml in PBS. Digital images of the nuclei were recorded using a camera mounted on a fluorescence microscope (Axiocam and Axiovert, Carl Zeiss Inc., Thornwood, NY). 100 and 200 Magnification ratios were used.

3. Results and discussion Actually, only two cases of estrogen receptor-positive peritoneal mesotheliomas have been reported [31], and tamoxifen has been successfully used in one case of rare benign cystic mesothelioma [32,33].

2.7. Upregulation of p53 Following 24 h CT with CDDP, Fc-diOH or Fc-OH-TAM, 2  106 cells were washed and lysed for 30 min on ice in RIPA buffer (50 mM Tris–HCl, 150 mM NaCl, 1% Igepal CA-630, 0.5% sodium deoycholate, 0.1% SDS, pH 8) supplemented with protease inhibitor cocktail. Cell debris were removed by centrifugation (30 min, 13 200 rpm, 4 °C). Protein concentrations were determined in the supernatants by the bicinchoninic acid method (BCA assay [30], Pierce Biotechnology, Celbio SpA, Milan, Italy); 75 lg of proteins were run on a 10% SDS–PAGE, blotted onto nitrocellulose (Immobilon-P, Millipore, MA, USA), blocked with 5% w/V non-fat milk and detected by anti-p53 primary antibody (1C12, Cell Signaling Technology, Celbio S.p.A., Milan, Italy). HRP-conjugated anti-mouse (Bethyl Laboratories Inc., Tema Ricerca, Bologna, Italy) chemiluminescence was determined by ECL (Immobilon Western, Millipore, MA, USA) using a ChemiDoc apparatus (Bio-Rad Laboratories, Hercules, CA). 2.8. Statistical data analysis Absorbance data were normalized to 100% cell viability for nontreated cells at the same percentage of DMSO co-solvent and fitted to a dose-response sigmoid using Origin 7 (Microcal) and Prism 3 (Graphpad). The statistical significance of observed effects was assessed by Student’s t-test or one-way ANOVA (p < 0.05). For the Comet assay, bitmap-saved 200 magnification images were analyzed using CometScore (TriTeK CometScore TM Freeware v1.5) for determining tail length and head diameter of nuclei.

Table 1 IC50 values as means of at least two repetitions of the same experiment (p < 0.05). Cell line

Compound

Treatment

IC50 (lM) ± SD

BR95

CDDP

24 h 24 h 72 h 24 h 24 h 72 h 24 h 24 h 72 h

CT CT + 48 h R CT CT CT + 48 h R CT CT CT + 48 h R CT

31.8 ± 8.7 9.9 ± 2.8 3.1 ± 1.4 23.1 ± 4.2 1.6 ± 0.3 1.3 ± 0.1 7.5 ± 0,3 1.8 ± 0.2 4.0 ± 0.4

24 h 24 h 72 h 24 h 24 h 72 h 24 h 24 h 72 h

CT CT + 48 h R CT CT CT + 48 h R CT CT CT + 48 h R CT

45.7 ± 0.4 11.7 ± 1.8 6.4 ± 0.6 8.8 ± 0.5 1.3 ± 0.05 0.6 ± 0.03 9.9 ± 0.07 1.4 ± 0.3 1.7 ± 0.09

Fc-diOH

Fc-OH-TAM

MM98

CDDP

Fc-diOH

Fc-OH-TAM

Fig. 4. Comet assay: (a) BR95 comet head diameter, (b) BR95 comet tail length, (c) MM98 comet head diameter, and (d) MM98 comet tail length. Data are expressed in pixels (px). Untreated cells (NT) were used as controls for the unpaired t-test (*p < 0.05, **p < 0.01, and ***p < 0.001).

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In order to test the presence of estrogen receptor in BR95 and MM98, cells have been treated with 1 nM of 17b-estradiol (E2) (Fig. 2), the natural ligand of estrogen receptor and with 1 lM of 4-OH-TAM, the archetypal selective estrogen-receptor modulator (SERM) drug. For this test, Ham’s F10 was preferred to DMEM medium since this medium does not contain phenol red, that seems to have some hormonal activity [34,35] and, thus, is best suited for study on estrogenic/antiestrogenic behavior. The results shown in Fig. 2 clearly demonstrate that the two compounds did not significantly alter the proliferation with respect to untreated cells at p < 0.05 (unpaired t-test). Thus, one could conclude that these two cell lines are ER , therefore Fc-diOH and Fc-OH-TAM behave on them as purely cytostatic/cytotoxic agents. As previously stated in the introduction, Fc-diOH and Fc-OHTAM showed specific antiproliferative activity on several tumor cell lines. So we have tested these organometallic drug candidates against the very aggressive tumor MPM. Both Fc-diOH and Fc-OH-TAM are more potent than CDDP (assumed as the reference antitumor drug) in inhibiting cell proliferation. The lowest IC50 values (highest activities) are achieved after 72 h CT for CDDP and both ferrocifens, showing a slow cytotoxicity-inducing kinetics (Fig. 3). Similarly, in a previous report [15], ferrocifens were shown to be highly toxic on melanoma cells and melanocytes only after long incubation periods (viz 96 h).

Fig. 5. Representative DAPI-stained nuclei after 2 h treatment at sublethal concentrations, followed by comet assay.

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Based on the comparison of IC50 values of the three compounds measured on the two MPM cell lines (Table 1), there is no clearlycut evidence of a different chemosensitivity related to a given cell phenotype (i.e. epithelial BR95 versus sarcomatous MM98). The alkaline version of the Single Cell Gel Electrophoresis or Comet assay has become a very popular method for the analysis of DNA damage caused by various chemical and physical agents [29]. The DNA damage evidenced by Comet test consists of DNA strand-breaks, alkali-labile sites and incomplete nuclear excision repair sites [36]. We treated BR95 and MM98 cells with equitoxic (sublethal) doses of CDDP, Fc-diOH and Fc-OH-TAM for a short period of time (2 h to exclude DNA fragmentation following apoptosis [37] or necrosis [38]). Fc-diOH clearly induced DNA fragmentation as testified by long comet tails and big head diameters (Figs. 4 and 5). A similar response was obtained with CDDP, the well-known DNAdamaging antiblastic drug. On the contrary, Fc-OH-TAM did not damage DNA within the experimental timeframe, as indicated by the lack of comet tails; however, the increase in nuclear staining could be interpreted as a sign of initial apoptosis [39]. In order to assess whether Fc-diOH activates any cellular responses to DNA damage, we investigated its effect on the protein levels of p53 the so called ‘‘guardian of the genome”. DNA-damaging chemotherapeutics trigger p53 stabilization and transcriptional activation [40,41]. Steady state levels of p53 and transcriptional activity are considerably increased in response to DNA damage or upon diverse forms of cellular stress. The tumor suppressor protein p53 induces cell-cycle arrest, DNA repair or premature senescence and apoptosis [42,43]. The results of western blots are reported in Fig. 6 where the human squamous carcinoma cell line A431 lysate is shown as p53 positive control [44]. In BR95 extracts, immunoblotting with an antip53 antibody evidenced an immunoreactive band at a molecular weight lower than 53 kDa, the intensity of which was unaffected by DNA-damaging treatments. In MM98 extracts, no immunoreactive band corresponding to p53 was detected under baseline conditions, but a band (having the same migration pattern of the positive control) appeared following CDDP treatment and was assigned to wild type p53. Fc-diOH also induced p53 upregulation, while Fc-OH-TAM did not affect p53 levels, thus confirming the Comet assay results. In conclusion, this brief report demonstrates that ferrocifens exhibit higher cytotoxicity on MPM cell line with respect to the reference metallo-drug cisplatin. In particular, Fc-diOH provocates DNA damage and its cytotoxic action is consistent with the intracellular generation of the alkylating quinone methide metabolite (Fig. 7) [22]. Since MPM is a quite aggressive tumor, the elevate activity of ferrocifens on both epithelioid and sarcomatous MPM primary cell lines corroborates the previous promising features for these drug candidates.

Fig. 6. Western blot for phospho-p53. Cells were treated with their respective IC50’s and lysed after 24 h.

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Fig. 7. Proposed intracellular activation of Fc-diOH giving rise to DNA-damaging, alkylating quinine methide.

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