Substituted titanocenes induce caspase-dependent apoptosis in human epidermoid carcinoma cells in vitro and exhibit antitumour activity in vivo

Abstract

Titanocene compounds are a novel series of agents that exhibit cytotoxic effects in a variety of human cancer cells in vitro and in vivo. In this study, the antiproliferative activity of two titanocenes (Titanocenes X and Y) was evaluated in human epidermoid cancer cells in vitro. Titanocenes X and Y induce apoptotic cell death in epidermoid cancer cells, with IC50 values that are comparable to cisplatin. Characterisation of the cell death pathway induced by titanocene compounds in A431 cells revealed that apoptosis is preceded by cell cycle arrest and the inhibition of cell proliferation. The induction of apoptosis is dependent on the activation of caspase-3 and -7 but not caspase-8. Furthermore, the antitumour activity of Titanocene Y was tested in an A431 xenograft model of epidermoid cancer. Results indicate that Titanocene Y significantly reduced the growth of A431 xenografts with an antitumour effect similar to cisplatin. These results suggest that titanocenes represent a novel series of promising antitumour agents.

Figure 1

Molecular structures of Titanocenes X and Y.

Figure 2

Antiproliferative effect of Titanocenes X and Y, Cp₂TiCl₂ and cisplatin on HeLa and A431 cells. HeLa (A and B) and A431 (C and D) cells were treated with vehicle (DMSO (0.5% v/v)) or 0.5, 5, 50 and 250 μM of the indicated compound for 24 h (A and C) and 48 h (B and D). Cell viability was determined using an MTT assay and absorbance was monitored spectrophotometrically at 540 nm. Results represent the mean±s.e.m. of three separate experiments. (E) IC50 values±s.e. from Cp₂TiCl₂, cisplatin, Titanocenes X and Y treatment of HeLa and A431 cells for 48 h, calculated using the PRISM statistical analysis software package.

Figure 3

Effect of Titanocene Y and cisplatin on growth of A431 xenograft mice in vivo. A431 cells (1 × 10⁷) were injected subcutaneously (s.c.) at day 0 into female NMRI:nu/nu mice (eight per group) allowed to reach 5–6 mm in diameter. Mice were treated i.p. with cisplatin (3 mg kg day⁻¹) or Titanocene Y (40 mg kg day⁻¹) for 5 consecutive days starting on day 8. For each treatment group, tumour volume was measured using calipers on days 10, 14, 17 and 21 (A) and mean body weight was calculated (B).

Figure 4

A431 cells undergo apoptosis when treated with Titanocene Y. Cells (0.8 × 10⁶) were treated with (A) vehicle (DMSO (0.5% v/v)) or (B) Titanocene Y (50 μM) or (C) cisplatin (50 μM) for 48 h. Cells were harvested by trypsination and centrifugation and stained with Annexin V/FITC antibody for 15 min according to the manufacturer's instructions. Cells were washed in binding buffer and stained with propidium iodide. Annexin V and propidium iodide fluorescence was measured by flow cytometry.

Figure 5

Effect of Cp₂TiCl₂, cisplatin, Titanocene X and Titanocene Y on A431 and HeLa cell cycle. A431 (A and B) and HeLa (C and D) cells were treated with vehicle (DMSO) (0.5% v/v) or 50 μM Cp₂TiCl₂, cisplatin, Titanocene X or Titanocene Y for (A and C) 24 h and (B and C) 48 h. DNA was stained with propidium iodide, and DNA content was analysed by flow cytometry. Results represent the mean±s.e.m. of three separate experiments.

Figure 6

Western blot analysis of caspase-3, -7 and -8 following Titanocene Y treatment of A431 cells. Cells were treated with either vehicle (0.5% DMSO (v/v)) or Titanocene Y (50 μM) for (A) 24 or 48 h or (B) a range of Titanocene Y concentrations (0–50 μM) for 48 h. Whole cell extracts were prepared and protein (60 μg) and resolved by SDS–PAGE followed by Western blotting using antibodies to detect the active cleavage product of caspase-3, -7 and -8. As a positive control (+VE) for caspase 8 activity, PWR-1 prostate cells were treated with etoposide (10 μM) for 48 h. Blots were stripped and re-probed with β-actin or GAPDH as loading controls. Results are representative of three independent experiments. (C) A431 cells (1 × 10⁵ cells) were treated with vehicle (DMSO (0.5% v/v)), 50 μM Titanocene Y or 50 μM cisplatin for 48 h, or a pretreatment of z-VAD-fmk (150 μM) for 1 h prior to DMSO (0.5% v/v) or 50 μM Titanocene Y or cisplatin for a further 48 h. Cell viability was determined by flow cytometry following propidium iodide staining. The appearance of a pre-G₁ peak represents non-viable cells. ^*P<0.005 with respect to cisplatin treatment, Student's t-test. Results represent the mean±s.e.m. of three separate experiments.