N-Heterocyclic Carbene Iron Complexes as Anticancer Agents: In Vitro and In Vivo Biological Studies

Abstract

Cisplatin and its derivatives are commonly used in chemotherapeutic treatments of cancer, even though they suffer from many toxic side effects. The problems that emerge from the use of these metal compounds led to the search for new complexes capable to overcome the toxic side effects. Here, we report the evaluation of the antiproliferative activity of Fe(II) cyclopentadienyl complexes bearing n-heterocyclic carbene ligands in tumour cells and their in vivo toxicological profile. The in vitro antiproliferative assays demonstrated that complex Fe1 displays the highest cytotoxic activity both in human colorectal carcinoma cells (HCT116) and ovarian carcinoma cells (A2780) with IC₅₀ values in the low micromolar range. The antiproliferative effect of Fe1 was even higher than cisplatin. Interestingly, Fe1 showed low in vivo toxicity, and in vivo analyses of Fe1 and Fe2 compounds using colorectal HCT116 zebrafish xenograft showed that both reduce the proliferation of human HCT116 colorectal cancer cells in vivo.

Keywords: N-heterocyclic carbene; anticancer activity; iron(II)–NHC complexes; zebrafish.

Figure 1

Molecular structure of the Fe–NHC complexes tested in this work.

Figure 2

Time evolution of the UV–vis absorption spectrum of Fe1 (A) and Fe2 (C) in PBS buffer at pH 7 at room temperature under aerobic conditions. Variation of the absorbance, measured at 360 nm of Fe1 (B) and Fe2 (D). Blue colour indicates measurements made at t < 24 h, green colour indicates measurements at 24 h < t < 48 h, and black colour indicates measurements at t 48 h.

Figure 3

Cell viability (%) of A2780 (A) and HCT116 (B) tumour cell lines after 48 h of exposure to Fe1 and Fe2 complexes. Cell viability was determined using the MTS assay. Data expressed as mean ± SEM. * p < 0.05.

Figure 4

Cell viability (%) after 48 h of exposure of fibroblasts to the Fe1 and Fe2 complexes. Cell viability was determined using the MTS assay. Data expressed as mean ± SEM. * p < 0.05.

Figure 5

Cytotoxicity of cisplatin in A2780 (A) and HCT116 (B) cell lines after 48 h of incubation (1.9 ± 0.2 µM and 15.6 ± 5.3 µM for A2780 and HCT116 cell lines, respectively). Cell viability was determined using the MTS assay. Data normalised against the control (0.1% (v/v) DMSO) and expressed as the mean ± SEM of three independent assays. * p < 0.05; **** p < 0.005.

Figure 6

Accumulative mortality over (72 h) for complex Fe1 (A) and complex Fe2 (B) at the different stages evaluated (in hpf).

Figure 7

Mortality–response curve for Fe1 (A) and Fe2 (B).

Figure 8

In vivo effectivity assays of Fe1 and Fe2 against human HCT116 colorectal cancer cell line: (A) Representative images of the caudal hematopoietic tissue (CHT) in the tail region of the zebrafish embryos where cells metastasize and proliferate at 3 dpi. Main images are a superposition of a fluorescence image and a bright field image of the same embryo. Fluorescence images show only the labelled cells of the main image. Scale = 250 µm; (B) fold change comparing 3 dpi against 1 dpi and normalised to the control condition at 3 dpi. The red line state the cell maintenance, above the bar, proliferation occurs, and below the bar, the cells decrease, compared to the control condition (n = 6 embryos/condition).