FACS analysis of oxidative stress induced on tumour cells by SERMs

doi:10.1016/j.ica.2004.11.027

Inorganica Chimica Acta

Volume 358, Issue 6, 30 March 2005, Pages 1993-1998

https://doi.org/10.1016/j.ica.2004.11.027 Get rights and content

Abstract

This study confirms previous reports of the antiproliferative effect of OH–ferrocifen on both hormone-dependent MCF-7 and hormone-independent MDA-MB-231 breast cancer cell models. Contrastive analysis of the proliferative effects of classical agonists (including estradiol and ethynylestradiol) and selective estrogen receptor modulators (SERMs) such as tamoxifen and its active metabolite, 4-hydroxy-tamoxifen, was also performed. Previous studies have attributed the effects of OH–ferrocifen on hormone-independent cells to an underlying mechanism based on intracellular oxidation, production of hydroxyl radicals and oxidative damage of DNA. Here we report that the chemically generated OH–ferrocifen cation (as well as the decamethylferrocenium cation) is less cytotoxic to both cell lines than the neutral parent complex. Moreover, fluorescence activated cell sorting (FACS) analysis of 8-oxo-guanine production indicates that, even at relatively high concentrations, OH–ferrocifen produces negligible oxidative DNA damage as compared to other SERMs, namely tamoxifen and 4-hydroxy-tamoxifen. Alternative pathways to explain the remarkable activity of OH–ferrocifen must therefore be sought.

Graphical abstract

This study confirms previous reports on the anti-proliferative effect of OH–ferrocifen on both ER(+) and ER(−) cell lines. The growth inhibition observed cannot be attribute to oxidative stress.

Introduction

Selective estrogen-receptor modulators (SERMs)offer great potential for applications in clinical practice. Unlike estrogens and their pure antagonists, SERMs exert selective activity on various estrogen target tissues [1], [2], [3]. For example, the SERM tamoxifen 1, which is converted in vivo into its more active metabolite, 4-hydroxy-tamoxifen 2 (Scheme 1), exhibits estrogen receptor (ER) antagonist activity in breast tissue, but agonist activity in uterus and bone tissue [4].

SERMs are a set of chemically diverse compounds whose structure lacks the steroid skeleton of estrogens but is still capable of interacting with the estrogen receptor-binding site. Like estrogens, SERMs operate by diffusing into the cell and binding to the receptor. This causes conformational changes and dimerization, leading to direct interaction of the adduct with specific DNA promoter portions (estrogen response elements, ERE) present on target genes [5]. However, while it has been very successfully employed in the treatment of breast cancer, compound 1 has also been associated to thromboembolism, vasomotor symptoms, and an increased risk for developing endometrial cancer and cataracts [6]. Further complexity derives from the fact that two subtypes of estradiol receptor (ER), namely ERα and ERβ, can coexist in breast cancer cells [7]. A clearer understanding of the molecular mechanisms underlying the effects of SERMs is thus necessary if we are to take full advantage of their therapeutic potential.

Transition-metal (especially platinum) complexes have been widely employed as anti-cancer agents [8]. However, due to their high general toxicity and narrow spectrum of activity, effective “drug targeting and delivery” strategies are being explored. A typical example using OH–ferrocifen 3 [9], [10], [11], [12], is shown in Scheme 1. The significant ER binding affinity of compound 3, coupled with its lipophilicity, led to suppose that the organometallic moiety could be directed towards specific intracellular targets.

This paper presents results from a study on the putative relationship between the antiproliferative activity of compound 3 and oxidative damage to DNA. We used compounds 1 and 2 as reference molecules to estimate the basic DNA oxidative stress level caused by the hormone carrier. Oxidative damage was assessed by measuring 8-oxo-G, a marker of nucleobase oxidative damage [13], [14].

In addition, we investigated the biological effects of ferrocenium cation derivative 3⁺, the postulated first active species produced by oxidation of compound 3 in the cell. The biological effects of decamethylferrocenium tetrafluoroborate (4⁺), a stable and cytotoxic ferrocenium derivative lacking SERM activity, was used as the experimental control [15].

Section snippets

Chemicals

All chemicals and solvents were purchased from Sigma, unless otherwise noted. Compound 3 was synthesized as described in previous reports [9], [10], [11], [12]. Compound 4⁺ was synthesized according to standard procedures [15], [16].

Synthesis of OH–ferrocifen cation (3⁺)

The oxidant AgBF₄ (38.16 mg, 0.196 mmol) was added under stirring conditions to 10 ml of acetone solution of compound 3 (100 mg, 0.196 mmol). A dark grey precipitate appeared immediately. The reaction mixture was filtered to remove metallic silver. The solvent was

Results and discussion

In the present work we have built on previous studies on the antiproliferative activity of compound 3 in MCF-7 (ERα positive) and MDA-MB-231 (ERα negative, ERß positive) breast cancer cell lines [9], [10], [11], [12]. The effects of ER agonists (estradiol (E₂) and ethynylestradiol (EE)) and ER antagonists (compounds 1 and 2) on cell proliferation were similar to those reported by other authors [18], [19], [20]. The results given in Fig. 1 represent the reference for 1 μM concentration.

The

Acknowledgements

This work received the financial support of MIUR (Roma) within the auspices of the COFIN2001 project. Research was carried out within the framework of the European Cooperation COST D20 action (Metal compounds in the treatment of cancer and viral diseases) and COST B16 action (Multidrug resistance reversal). G.C. gratefully acknowledges the EU for a Marie Curie training fellowship (HMPT-CT-2000-00186). Thanks are due to Dr. L. McLean for her assistance in writing this manuscript.

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In the present work, a new para-ferrocenylbenzoate copper(I) complex (a4) has been synthesized and characterized by numerous spectroscopic techniques such as: FT-IR, AAS, and CHNS analysis. Moreover, the single-crystal X-ray structure of the compound a4 was also explored. The DNA binding potency was determined by cyclic voltammetry, UV-visible spectroscopy, and viscometry. The redox behavior of the complex studied by cyclic voltammetry revealed a single quasi-reversible voltammogram, attributed to the dominant redox-active ferrocenyl moiety. The electrochemical study reveals that the complex binds to DNA non-covalently, the mode of interaction in particular, was found to be an electrostatic. The DNA binding constant was found to be 4.3 × 10⁴ M⁻¹, which is very significant and impressive for non-covalent interactions, and is in close agreement with the UV-visible spectroscopic results. DFT/B3LYP method was used to ascertain the energies of frontier molecular orbitals (HOMO/LUMO) and the charge distribution on the molecular structure. The ferrocene-based copper(I) complex (a4) demonstrated to be a decent candidate in terms of cytotoxicity, although less active than that of the standard chemotherapeutic drug (cisplatin).
Synthesis, structural and electrochemical characterization of novel 1,3-ketoureas bearing a ferrocenyl group
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Twenty-two novel 1,3-ketourea derivatives bearing ferrocene moiety were synthesized in good-to-excellent yields (up to 99%) via simple and efficient protocol. This solvent- and catalyst-free synthesis was achieved by additions of different ferrocene-containing Mannich bases – 3-(arylamino)-1-ferrocenylpropan-1-ones to phenyl isocyanate promoted only by ultrasound irradiations at ambient temperature. All synthesized 1-aryl-3-phenyl-1-(3-ferrocenyl-3-oxopropyl)ureas were characterized by standard spectral data (¹H NMR, ¹³C NMR and IR), and their electrochemical behavior were investigated by cyclic voltammetry. Detailed single-crystal X-ray diffraction analysis of three representative ferrocene-containing 1,3-ketoureas, among which one crystallized with two independent molecules in an asymmetric unit, were done.
Ferrocene-steroid conjugates: Synthesis, structure and biological activity
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One of the important point on this finding is the presence of ferrocene enhances the anti-proliferative activity. Although we do not have experimental data on these complexes, their enhancement in anti-proliferative activities must likely could be related to the ROS production capacity, as previously reported for ferrocenium and ferrocene complexes [37–40]. However, we cannot separate quantitatively which portion of the anti-proliferative activity belongs to receptor recognition and ROS contribution.
Four ferrocene-steroid conjugates (steroid = 3α-hydroxy androstan-17-one (androsterone, 3β-hydroxy androstan-17-one (trans-androsterone), 3β-hydroxy pregn-5-en-18-one (pregnenolone) and 3β-hydroxy dehydroandrostan-17-one (dehydroepiandrosterone, DHEA)) were synthesized and structurally characterized by analytical methods. The molecular structures of 16-ferrocenylidene-3α-hydroxy androstan-17-one, 1, 16-ferrocenylidene-3β-hydroxy androstan-17-one, 2, and 21-ferrocenylidene-3β-hydroxy pregn-5-en-20-one, 3, were determined by single crystal X-ray diffraction techniques. The crystallographic data confirmed the aldol condensation was achieved in C-16 for 1 and 2, and C-21 in 3. 1 and 2 showed ferrocene is positioned in the beta face of the steroid while for 3 is positioned between alpha and beta faces. The anti-proliferative activity of these conjugates was determined on colon cancer HT-29 and breast cancer MCF-7 cell lines. The conjugates displayed moderate to high anti-proliferative activity on both cell lines, 16-ferrocenylidene-3α-hydroxy androstan-17-one being the most active on HT-29.
Ferrocenes as potential chemotherapeutic drugs: Synthesis, cytotoxic activity, reactive oxygen species production and micronucleus assay
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Estrogens genotoxic mechanism may involve DNA adduct formation by estrogen metabolites, free radical by redox cycling of estrogens through microsomal, mitochondrial or nuclear processes.35–38 For ferrocene derivatives, DNA damage may be caused by oxygen free radical species, inducing oxidative damage.12–14,27,28,39 Those oxidative species cause DNA fragmentation resulting in micronucleated cells.
Three new ferrocene complexes were synthesized with 4-(1H-pyrrol-1-yl)phenol group appended to one of the Cp ring. These are: 1,1′-4-(1H-pyrrol-1-yl)phenyl ferrocenedicarboxylate, (‘Fc-(CO₂-Ph-4-Py)₂’), 1,4-(1H-pyrrol-1-yl)phenyl, 1′-carboxyl ferrocenecarboxylate (‘Fc-(CO₂-Ph-4-Py)CO₂H’) and 4-(1H-pyrrol-1-yl)phenyl ferroceneacetylate (‘Fc-CH₂CO₂-Ph-4-Py’). The new species were characterized by standard analytical methods. Cyclic voltammetry experiments showed that Fc-CH₂CO₂-Ph-4-Py has redox potential very similar to the Fc/Fc⁺ redox couple whereas Fc-(CO₂-Ph-4-Py)₂ and Fc-(CO₂-Ph-4-Py)CO₂H have redox potentials of over 400 mV higher than Fc/Fc⁺ redox couple. The in vitro studies on Fc-(CO₂-Ph-4-Py)₂ and Fc-(CO₂-Ph-4-Py)CO₂H revealed that these two compounds have moderate anti-proliferative activity on MCF-7 breast cancer cell line. In contrast Fc-CH₂CO₂-Ph-4-Py which displayed low anti-proliferative activity. In the HT-29 colon cancer cell line, the new species showed low anti-proliferative activity. Cytokinesis-block micronucleus assay (CBMN) was performed on these ferrocenes and it was determined they induce micronucleus formation on binucleated cells and moderate genotoxic effects on the MCF-7 breast cancer cell line. There is a correlation between the IC₅₀ values of the ferrocenes and the amount of micronucleus formation activity on binucleated cells and the reactive oxygen species (ROS) production on MCF-7 cell line.
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Triple-negative breast cancers (TNBC) represent the most aggressive form of breast cancers and their treatment are challenging due to the tumor heterogeneity. The high death rate and the limited systemic treatment options for TNBC necessitate the search for alternative chemotherapeutics. We previously found that FcOHTAM, an organometallic derivative of hydroxytamoxifen, showed in vitro a strong antiproliferative effect on various breast cancer cell lines, including MDA-MB-231 cells, the archetype of TNBC. In this study, we developed stealth FcOHTAM loaded lipid nanocapsules (LNCs) to further evaluate this novel drug on a TNBC xenografted model. Cell cycle analysis of MDA-MB-231 cells confirmed the preservation of the drug activity through LNCs causing a cycle arrest in phase S after 48 h exposure at the IC₅₀ concentration (2 μm). Two intraperitoneal injections of FcOHTAM loaded LNCs (20 mg/kg) administered to luciferase-transfected MDA-MB-231 tumors bearing mice led to a marked delay in tumor growth. As a consequence, a significantly lower tumor volume was obtained at the end of the experiment with a difference of 36% at day 38 compared to the untreated group. These results represent the first evidence of an in vivo effect of FcOHTAM and ferrocenyl derivatives in general on xenografted breast tumors.

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¹: Present address: Children’s Hospital Oakland Research Institute (CHORI), Martin Luther King Jr Way, Oakland, CA 94609, USA.

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FACS analysis of oxidative stress induced on tumour cells by SERMs

Abstract

Graphical abstract

Introduction

Section snippets

Chemicals

Synthesis of OH–ferrocifen cation (3+)

Results and discussion

Acknowledgements

J. Steroid Biochem. Mol. Biol.

J. Biol. Chem.

Crit. Rev. Oncol. Hematol.

C. R. Acad. Sci. Paris, Ser. IIc, Chim/Chem.

J. Organomet. Chem.

Inorg. Chim. Acta

J. Immunol. Meth.

J. Biol. Chem.

Cancer Lett.

J. Biol. Chem.

J. Endocrinol. Invest.

Oncologist

J. Natl. Cancer Inst.

Biochem. Pharmacol.

Chem. Commun.

Chem. Eur. J.

Synthesis of OH–ferrocifen cation (3⁺)