Greciano, Olga; (2008) Studies of the effects on cancer cells of cisplatin in combination with the G-quadruplex stabilising agent BRACO19. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Cisplatin is one of the most widely used drugs in the clinic in a variety of cancers such as ovarian, lung, head and neck, and is especially active in testicular cancer. Nevertheless, its side effects, principally nephrotoxicity, limit its use. Therefore it is important to combine this agent with drugs that can potentiate its effect, so that doses can be reduced and side-effects minimised. A series of 3,6,9-trisubstituted acridines has been developed which act by binding to the 3'-single strand telomeric DNA overhang, stabilising G-quadruplex structures and producing inhibition of telomerase activity and subsequent arrest of cell growth. It has been previously shown that the lead compound from this series, BRACO19, is able to produce telomerase inhibition, which leads to telomere attrition and senescence phenotype in vitro in different human cancer cell lines, and in vivo in human tumour xenografts. Cisplatin reacts preferentially with purine residues, especially with the N7 atom of guanines, producing DNA interstrand and intrastrand adducts, making telomeric DNA potentially a target of cisplatin. It is known that inhibition of telomerase activity enhances the effect of cisplatin in different cell lines. Therefore the combination of cisplatin with a G-quadruplex stabilising agent could be used as a new improved strategy of therapy. The aim of this study was to establish the mechanism of action of this combination therapy at the molecular and cellular levels in different human cancer cell lines representing phenotypically varied cancers. Initially, determination of the grade of synergism using short-term cytotoxicity combination studies was carried out with both agents in the following cell lines: A2780, A2780cis, A431 and MCF7, using the Calcusyn program. All the cell lines followed the same pattern at the ratios used in this study, when the concentration of BRACO19 compared to cisplatin was greater the combination was better and synergistic for all of them. Following this, long-term viability studies were performed to investigate the effect after longer period of exposures. The results of these studies were definitely showing synergism for A431 and MCF-7 cells whereas for the ovarian cell lines the effects were additive at the concentrations used in this study. Subsequently, detailed molecular and cellular studies were carried out to determine the type of cell death using specific markers for apoptosis such as caspase 3 and cell cycle. Induction of senescence was studied assessing cells for β-galactosidase activity. Gene and protein expression studies of important cell cycle regulators such as p53 and p21 were assessed. Also, an early DNA damage response was shown by the detection of γH2AX after treatment with both compounds in combination and alone. Inhibition of telomerase activity was observed for both agents and down-regulation of h-TERT in the case of BRACO19 and in the combination. However the compounds did not have an effect on telomere length. This study has revealed that G-quadruplex stabilising ligands such as BRACO19 can be effectively used in combination with cisplatin to produce antitumour activity in various cancer cell lines. This can be of significant therapeutic utility in cancers that do not respond well to cisplatin.

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