Metallodrugs against Breast Cancer: Combining the Tamoxifen Vector with Platinum(II) and Palladium(II) Complexes

Abstract

The luminal A-subtype of breast cancer, where the oestrogen receptor α (ERα) is overexpressed, is the most frequent one. The prodrug tamoxifen (1) is the clinically used agent, inhibiting the ERα activity via the formation of several active metabolites, such as 4-hydroxytamoxifen (2) or 4,4'-dihydroxytamoxifen (3). In this study, we present the tamoxifen derivative 4-[1,1-bis(4-methoxyphenyl)but-1-en-2-yl]-2,2'-bipyridine (4), which was combined with platinum or palladium dichloride, the former a well-known scaffold in anticancer treatment, to give [PtCl₂(4-κ²N,N')] (5) or [PdCl₂(4-κ²N,N'] (6). To prevent fast exchange of weakly coordinating chlorido ligands in aqueous solution, a bulky, highly stable and hydrophobic nido-carborate(-2) ([C₂B₉H₁₁]^2-) was incorporated. The resulting complexes [3-(4-κ²N,N')-3,1,2-PtC₂B₉H₁₁] (7) and [3-(4-κ²N,N')-3,1,2-PdC₂B₉H₁₁] (8) exhibit a dramatic change in electronic and biological properties compared to 5 and 6. Thus, 8 is highly selective for triple-negative MDA-MB-231 cells (IC₅₀ = 3.7 μM, MTT test), while 7 is completely inactive against this cell line. The observed cytotoxicity of compounds 4-6 and 8 against this triple-negative cell line suggests off-target mechanisms rather than only ERα inhibition, for which these compounds were originally designed. Spectroscopic properties and electronic structures of the metal complexes were investigated for possible explanations of the biological activities.

Keywords: breast cancer; cytotoxicity; oxidative stress; palladacarboranes; palladium dichloride; platinacarboranes; platinum dichloride; tamoxifen derivative.

Figure 1

Schematic molecular structures of tamoxifen and tamoxifen-based derivatives: literature-known reference compounds tamoxifen (1), 4-hydroxytamoxifen (2), and 4,4′-dihydroxytamoxifen (3), compounds investigated in this study 4-[1,1-bis(4-methoxyphenyl)but-1-en-2-yl]-2,2′-bipyridine (4, L), [PtCl₂(L-κ²N,N′)] (5), [PdCl₂(L-κ²N,N′)] (6), [3-(L-κ²N,N′)-3,1,2-PtC₂B₉H₁₁] (7), [3-(L-κ²N,N′)-3,1,2-PdC₂B₉H₁₁] (8), as well as previously reported compounds 4-[1,1-bis(4-hydroxyphenyl)but-1-en-2-yl]-2,2′-bipyridine (9, L¹), [3-(L-κ²N,N′)-3-(CO)₂-3,1,2-MoC₂B₉H₁₁] (10), and [3-(L¹-κ²N,N′)-3-(CO)₂-3,1,2-MoC₂B₉H₁₁] (11) [22]. Compounds reported here are framed.

Scheme 1

Incorporation of the nido-carborate dianion in 2,2′-bipyridine complexes: (A) initial formation of metallacarboranes (not shown) followed by reaction with ligand 4; (B) initial formation of metal(II) dichloride complexes 5 and 6 followed by reaction with Tl[closo-TlC₂B₉H₁₁] for chloride replacement.

Figure 2

QTAIM analysis: molecular graphs of metal(II) dichlorido complexes (5 and 6, left) and the metallacarboranes (7 and 8, right). The bond paths of the non-covalent interactions are shown as dashed lines. The bond critical points (BCPs) and the ring critical points (RCPs) are indicated as blue or red dots.

Figure 3

The effect of compounds 4−6 on the viability of peritoneal exudate cells. Peritoneal exudate cells were treated with 4−6 and subjected to CV viability assay after 72 h. The data are expressed as percentage of viability of untreated control (set as 100%) from one representative out of three independent experiments and presented as mean ± SD of triplicate cultures (* p < 0.05 in comparison to untreated cells).

Figure 4

Tamoxifen derivatives attenuated MCF-7 cell division and induced programmed cell death type 1 and 2. MCF-7 cells were exposed to IC₅₀ doses of compounds 4, 5 and 6 for 60 or 72 h and analysed by flow cytometry: (A) cell proliferation rate, fluorescence intensity from FL1 channel (exc. 488 nm/em. 536 nm); (B) ApoStat staining for caspase-dependent apoptosis, fluorescence intensity from FL1 channel; (C) apoptosis detection (AnnV-FITC/PI staining) after 60 h (upper panels) or 72 h (lower panels), fluorescence intensity from FL2 channel (y axis, exc. 488 nm/em. 590 nm) vs. FL1 channel (x axis); (D) autophagy detection after 60 h and determination of the role of autophagy by specific inhibitor chloroquine (Chloq) (20 µM), fluorescence intensity from FL3 channel (exc. 488 nm/em. 675 nm). The data are presented as mean ± SD of triplicate cultures (** p < 0.01; *** p < 0.001 refers to compound-treated cultures. Representative dot plots and histograms are shown from at least three independent experiments).

Figure 5

Compounds 4−6 differently affect the production of reactive oxygen and nitrogen species (ROS/RNS) in MCF-7 cells. MCF-7 cells were pre-stained with dihydrorhodamine 123 (DHR 123) and treated with 4, 5 or 6 (IC₅₀ dose) for 60 h. Intracellular accumulation of ROS/RNS were measured by flow cytometry. Representative histograms from three independent experiments are shown.