The mechanism of anti-tumour activity of the DNA binding agent SN 28049

by Drummond, Catherine

Abstract (Summary)
SN 28049 is a novel DNA intercalating anti-cancer drug developed at the Auckland Cancer Society Research Centre as part of ongoing research into topoisomerase II poisons with high activity against solid tumours. SN 28049 was curative against murine Colon 38 tumours, a model of colorectal adenocarcinoma which is generally unresponsive to topoisomerase II poisons (1), while two clinically used topoisomerase II poisons, doxorubicin and etoposide, were respectively moderately active and inactive against this tumour. The aim of this thesis was to use human tumour cell lines in order to develop an understanding of the mechanism of anti-tumour activity of SN 28049. Topoisomerase II poisons induce DNA double strand breaks which signal through multiple pathways, and two of these, the ?-phosphorylation of histone H2AX (?-H2AX) and the induction of p53 protein were investigated in HCT 116 colorectal carcinoma cells and in NZM3, NZM6 and NZM52 melanoma cells. SN 28049 induced only a small increase in ?- H2AX phosphorylation, comparable to that mediated by doxorubicin and etoposide. However, a low concentration of SN 28049 (25 nM) stimulated p53 protein expression in HCT 116 cells to levels in excess of those observed in response to doxorubicin or etoposide. The activation of p53 by SN 28049 cells was investigated using the HCT 116 line and an isogenic line lacking p53 expression. As assessed by expression of the representative p53 transcriptional targets FAS, p21WAF1 and survivin, SN 28049 was considerably more active than doxorubicin or etoposide in stimulating the p53 pathway. However, there was little evidence of SN 28049 inducing either G1 arrest or apoptosis in these cells. SN 28049 instead induced p53-dependent G1 tetraploid arrest following mitotic failure in the absence of cell division. In response to a five-hour drug exposure and further growth in the absence of drug, HCT 116 p53 +/+ cells arrested with a ā€˜4Nā€™ DNA content, expressing high levels of G1-phase cyclin E. At later times, senescence associated ?-galactosidase (SA-?-Gal) activity increased, indicative of senescence. Under similar conditions, HCT 116 p53 -/- cells continued cycling with a DNA content of >4N. In contrast, doxorubicin added under similar conditions to HCT 116 p53 +/+ cells caused cycle arrest with an ā€˜8Nā€™ DNA content and signs of senescence, while etoposide induced only signs of senescence. The activity of SN 28049 and a series of its analogues was also compared to that of doxorubicin and etoposide in both the HCT 116 p53 +/+ and p53 -/- lines using growth inhibition assays. When cells were cultured at a density that allows exponential growth, SN 28049 selectively inhibited HCT 116 cells expressing wild-type p53, and the selectivity was related to potency. However, responses to SN28049 were not distinguished qualitatively from doxorubicin and etoposide. In contrast, under conditions of high cell density, SN28049 and several active analogues maintained selectivity for HCT 116 p53 +/+ over p53 -/- cells while doxorubicin and etoposide lost their selectivity. The ability of SN 28049 to activate p53 was retained at high cell density. It is concluded that SN28049 has two cellular actions. The first, shared with doxorubicin and etoposide, is mediated by topoisomerase II-induced DNA damage, is cell cycle non-specific and leads to cell cycle arrest. The second, which is unique to SN28049 and is most easily observed in a cell line (HCT 116) growing at high cell density to ensure minimal expression of active topoisomerase II, involves bypass of mitosis and entry into a G1 tetraploid phase and is characterised by high expression of p53 and its downstream products. It is hypothesised that this second cellular action could explain the high in vivo activity of SN 28049.
Bibliographical Information:

Advisor:Professor Bruce Baguley; Dr Graeme Finlay

School:The University of Auckland / Te Whare Wananga o Tamaki Makaurau

School Location:New Zealand

Source Type:Master's Thesis



Date of Publication:01/01/2008

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