Mechanisms of tumour cell death induced by novel titanium-based agents.

摘要

Titanocene dichloride (TDC) and its water soluble derivatives are possible alternatives to heavy metal based anticancer drugs due to their reduced toxicity and increased activity in preclinical cisplatin-resistant model systems. The aim of this study was to determine how TDC and its derivative TDC-11 induce tumour cell death. Immunoblotting experiments and fluorescence-based cleavage assays indicated that TDC and TDC-11 do not activate the executioner caspase-3 in human non-small cell lung cancer A549 cells, suggesting that these agents do not induce caspase-dependent apoptosis, at least in this cell type. To test whether TDC and TDC-11 induce other cell death pathways in A549 cells, parameters of apoptosis-like program mediated cell death (PCD), necrosis-like PCD and necrosis were measured. Mitochondrial membrane permeabilization (MMP) (JC-1 staining) and DNA fragmentation (propidium iodide (PI) staining) were significantly increased in TDC and TDC-11-treated A549 cells, suggesting cell death is mediated via apoptosis-like and/or necrosis-like PCD pathway(s). To determine which form(s) of PCD is induced by TDC and TDC-11 in A549 cells, phosphatidylserine externalization (annexin V (AV) staining and PI co-staining) was examined by flow cytometry. Apoptosis (AV positive), necrosis (PI positive) and late stage apoptosis/necrosis (AV and PI positive) were all observed in TDC and TDC-11-treated A549 cells, indicating the induction of both apoptosis-like and necrosis-like PCD. In addition, NAD+ levels (a measure of poly(ADP-ribose) polymerase-1 activation) were substantially decreased in TDC and TDC-11-treated A549 cells. This decrease in NAD+ occurred before and in parallel with increases in DNA fragmentation and MMP. It also accompanied the increase in number of cells undergoing apoptosis and necrosis. These observations suggest that TDC and TDC-11 induce apoptosis-like and necrosis-like PCD via PARP-1-mediated pathways in A549 cells. Finally, the toxicity of 10 newly synthesized TDC derivatives was tested against A549 cells. The two most potent of these compounds, GP 3.2 and GP 4.2, are TDC derivatives disubstituted with 3-picolylium and 4-picolylium groups on the cyclopentadienyl rings respectively. The IC50s were 24.20 +/- 0.34 muM & 23.40 +/- 1.52 muM for GP 3.2, and 21.73 +/- 1.00 muM and 25.31 +/- 2.69 muM for GP 4.2. These values compare favorably with IC50s of 196.39 +/- 3.74 muM and 128.92 +/- 2.33 muM for TDC.