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

Abstract

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.

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 common) 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-OH-tamoxifen (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. MCF-7 (ER+), and independent, e.g. MDA-MB-231 (ER−) breast cancer cell lines [11], [12], [13], [14].

The antiproliferative effect observed on ER+ cells with Fc-OH-TAM 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 Fc-diOH 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 Fenton-type cycle, has been demonstrated to be responsible for the cytotoxicity of such derivatives in vitro [16], [17], [18], [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].