Studying the effect of differentiation therapy on osteosarcoma

Dimitriadi Evgenidi, Chara (2024) Studying the effect of differentiation therapy on osteosarcoma. PhD thesis, University of Glasgow.

Abstract

Lack of terminal differentiation is widely observed in bone cancer patients, yielding fast-proliferating, immature cancer cells. A potential strategy for cancer treatment is differentiation therapy, where the aim is to “reprogramme” cancer cell behaviour, by inducing differentiation. Around 150 people are diagnosed with osteosarcoma (OS) in England and Wales each year, which is a malignant primary bone cancer, with highest incidence in young people between the ages of 10-24. Identifying small molecules that can restore differentiation potential in OS cells was considered a promising strategy, so known osteogenic conditions for MSCs, including nanokicking, and a known osteogenic medium, containing ascorbic acid, dexamethasone and β-glycerophosphate, were applied on osteosarcoma cell lines MG-63 and SAOS-2, and MSCs. Mechanical and chemical stimulation appeared to drive differentiation in OS cells, as evident by upregulation of RUNX2, OSX and/or ONN in different conditions. After confirming differentiation, a metabolomics analysis was performed, to observe what groups of metabolites and pathways are involved in differentiation, and to identify metabolites of interest.

Mechanical and chemical stimulation were shown to drive metabolic reprogramming in OS cells, by altering bioenergetics, and employing metabolic pathways that are reported to be impaired in OS, including TCA metabolism. Cholesterol sulfate (CS) and taurine (TAU) levels were found to be significantly altered differentiation, so they were tested on OS cells, along with mineralocorticoid fludrocortisone acetate (FA). The small molecules were tested on SAOS-2 and MG-63 cells, and differentiation appeared to occur in a time, and dose dependent manner, with a concentration of 10 μM showing enhanced differentiation after 7 days.

A cancer targeting delivery approach was attempted for glucocorticoid, differentiation agent dexamethasone, where the small molecule was tethered to glucose, via a hydrazone cleavable linker. A hydrazone bond was formed at the C3 position of dexamethasone, and extensive purification was carried out to obtain the hydrazone, in a mixture of cis-trans isomers. The dexamethasone hydrazone was then coupled with the carboxylic acid of the glucose linker, which generated a mixture of cis and trans isomers, which were successfully separated via RP-HPLC. The novel small molecules were individually characterised, and the cis isomer was deprotected under strongly basic conditions, with the aim of obtaining the de-acetylated species. This led to degradation on the C20 sidechain of dexamethasone’s D-ring, but the small molecule was isolated, and characterised, and was found to exhibit biological activity, through initial testing. The small molecule induced a small decrease in viability of MG-63 and SAOS-2 cells, without inducing extensive cytotoxicity. The conjugate presented enhanced differentiation, compared to dexamethasone, in a dose dependent-manner, in MG-63 cells, while more limited response was observed in SAOS-2 cells. Overall, conditions were identified that could drive differentiation in OS cells, while a novel small molecule was successfully synthesised, characterised, and found to present biological response in OS cells.

• Item Type: Thesis (PhD)
• Subjects: Q Science > QH Natural history > QH345 Biochemistry; R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology (including Cancer)
• Colleges/Schools: College of Science and Engineering > School of Chemistry
• Funder's Name: Engineering and Physical Sciences Research Council (EPSRC)
• Supervisor's Name: France, Dr. David, Dalby, Professor Matthew and Tsimbouri, Dr. Monica
• Date of Award: 2024
• Depositing User: Theses Team
• Unique ID: glathesis:2024-84708
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Nov 2024 11:21
• Last Modified: 22 Nov 2024 12:11
• Thesis DOI: 10.5525/gla.thesis.84708
• URI: https://theses.gla.ac.uk/id/eprint/84708