Highly Cytotoxic Molybdenocenes with Strong Metabolic Effects Inhibit Tumour Growth in Mice

Abstract

A series of six highly lipophilic Cp-substituted molybdenocenes bearing different bioactive chelating ligands was synthesized and characterized by NMR spectroscopy, mass spectrometry and X-ray crystallography. In vitro experiments showed a greatly increased cytotoxic potency when compared to the non-Cp-substituted counterparts. In vivo experiments performed with the dichlorido precursor, (Ph₂ C-Cp)₂ MoCl₂ and the in vitro most active complex, containing the thioflavone ligand, showed an inhibition of tumour growth. Proteomic studies on the same two compounds demonstrated a significant regulation of tubulin-associated and mitochondrial inner membrane proteins for both compounds and a strong metabolic effect of the thioflavone containing complex.

Keywords: anticancer; biological tests (in vitro and in vivo); bioorganometallic chemistry; molybdenocenes; proteomics.

Figure 1

Structures of titanocene dichloride, Titanocene Y and molybdenocene dichloride (left to right).

Scheme 1

General synthetic route for compounds 1–6.

Figure 2

Molecular structures of compounds 1 (left) and 5 (right) with 50 % displacement ellipsoids. Solvent molecules and counter ion are omitted for clarity.

Figure 3

Cellular accumulation levels of molybdenum complexes in fg [Mo]/cell, measured by ICP‐MS. SW480 cells were treated for 2 h at 37 °C with 50 μM of all test compounds (black columns) and with 5 μM of the more cytotoxic compounds 2, 5 and 6 (grey columns). Values are means±SDs from at least three independent experiments performed in triplicates.

Figure 4

Fractions of viable (AV−/PI−), early apoptotic (AV+/PI−), late apoptotic (AV+/PI+) and necrotic (AV−/PI+) cells, measured by the flow‐cytometric annexin V‐FITC/PI assay upon treatment of SW480 cells with 100 μM of compound 1 or 5, 10, 15, 20 and 25 μM of compounds 2, 5 and 6.

Figure 5

Reactive oxygen species production detected by the DCFH‐DA assay in SW480 cells treated with 0.2, 2 and 20 μM of 1, 2, 5 and 6 for 2 h, measured every 10 min during incubation. Data are means±SDs.

Figure 6

Proteomic analysis of the cellular response of SW480 cancer cells to treatment with A) compound 1 and B) 5. The volcano plots illustrate the significantly regulated proteins as orange squares. Notable protein groups were additionally coloured. The x‐axis depicts Log2 fold‐changes of LFQ protein abundance between control and treatment and the y‐axis depicts the significance of the regulation in Log10‐scale.

Figure 7

Regulation of functional protein groups in SW480 cancer cells upon treatment with compound 1 or 5. A) Similar protein regulations upon treatment with 1 (left) or 5 (right) are associated with mitochondrial inner membrane proteins, microtubules, membrane raft, stress fiber and myosin complex. The bars represent the summed fold‐change of the proteins in each group. Only proteins from the NE fraction were considered. B) The global cellular response to the treatment with 5 is depicted. The ten most significantly regulated functional protein groups are shown. Each bubble represents a labelled functional protein group and its position is determined by the average protein fold‐change in the NE (x‐axis) and CYT (y‐axis) fractions. The size of the bubble represents the number of proteins in each group. OxPhos=oxidative phosphorylation of the cellular respiration.

Figure 8

In vivo anticancer activity of compounds 1 and 5; A and C) depict average tumor volumes over time, B and D) show overall survival of mice.