Novel therapies for cancer treatment : designing high affinity selective ligands against SIRT1 enzyme

Al-Sudani, BT 2017, Novel therapies for cancer treatment : designing high affinity selective ligands against SIRT1 enzyme , PhD thesis, University of Salford.

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Abstract

The word ‘Sirtuin’ or Sir2 proteins are a class of proteins that possess either mono-ADP-ribosyltransferase, or deacylase activity, including deacetylase. SIRT1 is the most studied mammalian Sirtuins and predominantly localised in the nucleus and cytoplasm. Many Sirtuins targets are involved in cancer and in many types of cancers, SIRT1 is found to be overexpressed. Recent observations support SIRT1 being both an oncogene and a tumour suppressor, depending on the cancer etiology and type of tissue. To answer the question “How can SIRT1 behave as a tumour suppressor?”, highly selective ligands (aptamers) were developed against SIRT1 enzyme as the first step towards the development of an alternative chemotherapy for cancer diseases. The objectives of current study are to: (i) produce by in vitro SELEX procedures, SIRT1 binding single-stranded DNA (ssDNA) aptamers; (ii) characterise the interactions between selected aptamers and SIRT1 in vitro and determine their equilibrium dissociation constant (KD) values and; (iii) investigate the effects of selected aptamers on cancer cell lines. To achieve these objectives, ssDNA aptamers capable of binding SIRT1 enzyme were generated in vitro using a sequential approach known as SELEX. A total of eight novel SIRT1 aptamers (circular and linear), four circular aptamers from 8 rounds of circularisation-SELEX procedure, and the other four linear aptamers from 12 rounds of a basic-SELEX procedure were generated. The initial screening using the Fluor de Lys-SIRT1 assay for SIRT1 enzymatic activity in vitro indicated that an activator SIRT1 enzyme (circular3, circular4, linear3 and linear4) were obtained, these aptamers showed acceptable values of Km and Vmax to SIRT1 enzyme in kinetic characterisation studies. After equilibrium binding characterisation study of both linear and circular aptamers by SPR, it was show that circular3 and linear3 aptamers are good binder to SIRT1 enzyme, with the low KD constant (27.07± 0.959 nM and 48.3± 0.986 nM) respectively with highly exhibited stability for circular3 in human plasma.

To investigate the effects of aptamers in cancer cell lines, it has been found that the lung cancer epithelial model A549, the colorectal adenocarcinoma model Caco-2, the liver hepatic model HepG2 were very sensitive with an IC50 (0.32, 0.67, 0.2μM) respectively. Both breast cancer models (MCF-7 and MDA-MB-468) were highly sensitive with an IC50 (0.14 and 0.13 μM), the very difficult to treat MCF-7 and the extremely challenging oestrogen negative MDA-MB-468 proved to be substantially sensitive with the longer exposure. A special mention should be the osteosarcoma model U2OS, this cancer is very prevalent as bone cancer in children and adults over 60 years of age, with prognosis being related to the cancer stage and current treatment leaving extremely non-desirable side effects, the aptamer was very effective on this cell line with IC50 = 0.06 μM. Notably, pre-treatment of adult human keratinocyte HaCaT cells with aptamers resulted in markedly decreased cell viability and the IC50 = 0.123 μM. The most interesting point who that the aptamer was very safe on normal cell line Beas-2b, which indicated that it is safe to non-cancerous tissue.

In conclusion, a pharmacological activation of SIRT1 enhanced cell death suggesting a tumour suppressive function of SIRT1 and the high-affinity SIRT1- aptamers identified in this study may be used in the future for cancer treatment.

Item Type: Thesis (PhD)
Schools: Schools > School of Environment and Life Sciences > Biomedical Research Centre
Funders: Iraqi Ministry of Higher Education and Scientific Research
Depositing User: Basma Talib Al-Sudani
Date Deposited: 19 Jan 2018 12:57
Last Modified: 19 Jan 2018 12:57
URI: http://usir.salford.ac.uk/id/eprint/42748

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