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Liu, Xiangrui (2012) Evaluation of transcriptional cyclin dependent kinase inhibitors as potential cancer therapeutics. PhD thesis, University of Nottingham.

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Abstract

Cancer cells depend heavily on sustained expression of anti-apoptotic proteins. Targeting transcription to suppress these anti-apoptotic proteins seems a promising strategy for anti-cancer therapy. Cyclin-dependant kinase 9 (CDK9) regulates transcription elongation by phosphorylating Ser2 on the C-terminal domain of RNA polymerase II, while CDK7 phosphorylates Ser5 during transcription initiation. A screening cascade comprised of an MTT assay, a caspase-3 activation assay, a p53 stabilization assay and a mitotic index assay was developed to classify compounds and identify lead transcriptional CDK inhibitors from a novel class of 2,4,5-trisubstituted pyrimidines. Compounds S3-41 and CDKI-71 are the most potent CDK9 inhibitors identified by the screening cascade. They showed potent anti-proliferative activity in the MTT assay and induce both caspase-3 activity and p53 protein level at the GI50 concentration. In addition, no significant effect on mitotic index was observed.

The detailed mechanism of action of CDKI-71 was further investigated and compared with the clinical compound, flavopiridol. Like flavopiridol, CDKI-71 displayed potent cytotoxicity and caspase-dependent apoptosis that were closely associated with the inhibition of RNAPII phosphorylation at Ser2. This indicated effective targeting of cyclinT-CDK9 and the downstream inhibition of anti-apoptotic proteins such as Mcl-l in cells. Similar to flavopiridol, CDKI-71 down-regulated a large range of genes, including Mcl-l and Bcl-2. No correlation between apoptosis and inhibition of cell cycle CDKs 1 and 2 was observed. Non-transformed lung fibroblast cell lines showed resistance to CDKI-71 treatment. In contrast, flavopiridol showed little selectivity between cancer and normal cells. Flavopiridol also induced genotoxic stress through the induction of DNA double-strand breakage. These results suggest that CDKI-71 has a great potential to be developed as an anti-cancer agent.

Another study focused on in vitro anti-tumour mechanism of CDKI-83, a dual inhibitor of CDK9 and CDKI, was performed in A2780 ovarian cancer cells. CDKI-71 presented potent anti-proliferation activity and induced apoptosis in A2780 cells. By inhibiting cellular CDK1 and CDK9 activities, CDKI-83 arrested cells in G2 phase and reduced anti-apoptotic proteins at both mRNA and protein levels, respectively. This study suggests that the combination of CDK9 and CDK1 inhibition results in effective induction of apoptosis in cancer cells.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Dekker, L. Wang, S.
Keywords:	cancer cells, anti-apoptotic proteins, cyclin-dependent kinases
Subjects:	Q Science > QP Physiology > QP501 Animal biochemistry R Medicine > RC Internal medicine > RC 254 Neoplasms. Tumors. Oncology (including Cancer)
Faculties/Schools:	UK Campuses > Faculty of Science > School of Pharmacy
Item ID:	29653
Depositing User:	Hatton, Mrs Kirsty
Date Deposited:	25 Aug 2015 08:12
Last Modified:	20 Oct 2017 00:53
URI:	https://eprints.nottingham.ac.uk/id/eprint/29653

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