Functionalised symmetrical flexicates 2.1 Introduction
2.5.4 Effects on the cell cycle
The cell division cycle is the process by which a cell prepares for and carries out self-replication. In cancerous cells this is often disrupted where mutations have occurred which switch off important regulatory processes.40 Arresting cancerous
cells at a certain stage of this division cycle is an important indicator in the mode of action of an anticancer agent and can be used to give direction to further study of the effect of the compound on certain key biomolecules. The effect flexicates have on the cell cycle of HCT116 p53+/+ cells was assessed.
Figure 2.20 | % population of HCT116 p53+/+ cells in each of the phases of the cell cycle after treatment with flexicates (10 µM) for 24 h compared to a control of untreated cells.
HCT116 p53+/+ cells (5 × 105 cells in 1 ml RPMI-1640 medium) were incubated
with each flexicate (10 µM) for 24 h and treated with the fluorescent dye propidium iodide (PI) which stains DNA quantitatively. The proportion of cells in the various phases of the cell cycle were determined by fluorescence via FACS analysis as a result of the differing amounts of DNA in the cells.41 [Fe2LS3]Cl4 flexicates show a
dramatic increase in the proportion of cells in G2/M phase compared to the control
(from ca 20 to 40 % of cells); such arresting of cell growth at this phase is likely to be a significant factor in the mechanism of action. Interestingly [Fe2L13]Cl4
flexicates did not show a significant increase in the G2/M phase, indicating a
different mechanism. There is a very pronounced increase in population of sub G1
cells, from 4 % in the control to between 13 and 30 %, the latter result being, again, for [Fe2LS3]Cl4. Cells in the sub G1 phase are considered apoptotic42, 43 and this
Rebecca A. Kaner | Chapter 2 suggests that these compounds may well be inducing programmed cell death at a much higher rate than in an untreated cell over 24 h.
2.5.5 Induction of apoptosis
In the early stages of apoptosis cell membrane changes occur, without the loss of membrane integrity.44-46 These changes are recognised by Annexin-V-FLUOS (a
fluorescent-conjugated calcium(II) dependant phospholipid binding protein).47 As
these cell membrane changes are also present in necrotic cells Annexin-V-FLOUS cannot differentiate between the two cell types. Propidium iodide (PI, a DNA stain) is thus introduced to distinguish between apoptotic and necrotic cells. PI will stain necrotic cells exclusively as the loss of membrane integrity membranes permits access to the intracellular environment. The induction of apoptosis and necrosis by flexicates was investigated by Dr Simon Allison, Bradford Institute of Cancer therapeutics.
HCT116 p53+/+ cells were seeded (5 × 105 cells per flask) and incubated for
24 h. Upon addition of fresh media containing no drug (control) or freshly dissolved drug (20 µM) the cells were incubated for a further 72 h. Cells were then harvested by trypsinisation with non-adhered cells also collected, washed with PBS and stained with propidium iodide (100 µl) and Annexin-V-FLUOS labelling solution (100 µl). The proportion of live, early apoptotic and late apoptotic/necrotic cells were then quantitated by flow cytometry.48
Figure 2.21 | Amount of early apoptotic (dark grey) and late apoptotic / necrotic (light grey) HCT116
p53+/+ cells after treatment with flexicates (20 µM) for 72 h, along with an untreated control.
Compared to the untreated control both class Ia and class Ib flexicates increased the number of both early apoptotic and necrotic cells significantly, with ΛFe-[Fe2LS3]Cl4
in particular having an extreme effect. This, with the cell cycle data, was a strong positive indication that these compounds are triggering early cell death as part of their mode of action.
Rebecca A. Kaner | Chapter 2
2.6
Summary
At the beginning of this work we set out to make a library of functionalised flexicates by including different moieties in the structure of the ligand. This we have been able to achieve successfully and can now access the widest library of water- soluble complexes of this size and function available. Each complex of this library exists as a single diastereomerically pure species and is fully characterised by NMR spectroscopy, mass spectrometry, thermogravimetry, microanalysis and circular dichroism.
These novel compounds have also been found to be active and selective anticancer agents, particularly potent towards the human colon carcinoma cell lines, giving IC50 values in the nM range – substantially more toxic than cisplatin. For
some flexicates the range of sensitivity in between human tumour and healthy cells is up to three orders of magnitude; very promising selectively. Enantiomers of class Ia [Fe2LS3]Cl4 have been shown previously to be selective DNA binders,1, 2 however
they, and the non-DNA binding class Ib flexicates, do not induce DNA damage as demonstrated by single gel electrophoresis and γ-H2AX expression analysis; the chemosensitivity is not caused by DNA damage. These compounds do, however, induce dramatic changes in cell-cycle population and a high level of apoptosis, which indicates that it is this promotion of programmed cell death that is crucial in their mode of action. Moving forwards in this project, further study into the mode of action is required. Whether an extrinsic or intrinsic apoptotic pathway is being triggered as well as interactions with key biomolecules, such as p53, are currently being investigated with our collaborators at the Institute of Cancer Therapeutics.
2.7
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