Characterising the effects of 1,4,5-Oxathiazinane-4,4-dioxide (OTD) on non-neoplastic cells and primary and metastatic melanoma cells in vitro
Melanoma is an aggressive form of skin cancer, responsible for the largest proportion of skin cancer related deaths in Ireland (69.4%) despite only accounting for 9% of all skin cancers leading to the development of several adjuvant therapies aiming to reduce mortality. Tissue damage inflicted by surgical excision of tumours, can induce an exaggerated sterile proinflammatory immune response followed by immunosuppression, providing an optimal environment for residual tumour cells to differentiate, proliferate and propagate into surrounding and distant tissues resulting in cancer recurrence. Surgical adjuvants have the potential to induce cytotoxicity in residual tumour cells and reduce excessive inflammation, possibly preventing subsequent immunosuppression. The synthesised compound 1,4,5-oxathiazinane-4,4-dioxide (OTD) is a structural analogue of Taurultam derived from anti-inflammatory Taurolidine. OTD demonstrates anti-neoplastic actions against several cancers, however, its effects on inflammation and wound healing remain undetermined. The overall aim of this study was to assess the potential of OTD as an adjuvant treatment following melanoma excision by investigating its effects on cells involved in wound healing (Human Dermal Fibroblast (HDF) cells), tumourigenesis (primary and metastatic melanoma cells) and the adaptive immune system (CD8+ T lymphocytes). The physiological and pathological functions of these cell types were assessed in vitro with five concentrations of OTD (0.25mM, 0.5mM, 0.75mM, 1mM and 1.25mM). Given the critical role of wound healing in the post-operative period, this thesis initially assessed the effects of OTD on the wound healing capacity of HDF cells. At concentrations up to 0.75mM of OTD this study demonstrated that OTD, does not significantly impair HDF migratory function in a wound healing assay (n=3, p>0.05). Additionally, OTD effectively suppressed the release of pro-inflammatory cytokines, IL-6 and IL-8, in IL-1α stimulated HDF cells at concentrations above 0.5mM (n=6, p>0.05). Moreover, OTD preserved HDF cell viability at all tested concentrations compared to untreated controls (n=6, p>0.05). Further investigations aimed to determine the cytotoxic effect of OTD on primary and metastatic melanoma cells. Cytotoxicity was observed in primary (A375/WM3248) and metastatic (SK-MEL-2) cell lines at all OTD concentrations (n=6, p*<0.05). However, metastatic WM164 cells demonstrated resistance to OTD treatment with no observed cytotoxicity (n=6, p>0.05). Furthermore, cell death in primary (WM3248) and metastatic (SK-MEL-2) cell lines occurred via apoptosis (n=3, p*>0.05). Notably, instead of a dose-dependent effect, a phasic response was observed, with prominent cytotoxicity at concentrations of 0.5mM in WM3248 cells (p****<0.0001) and SK-MEL-2 cells (p** <0.01) followed by recovery of cell viability at higher concentrations. This response may be attributed to metabolic rewiring of melanoma cells in response to glycolytic inhibition, promoting tumour survival. Immunosuppression post-operatively results in impairment of adaptive immune responses such as anti-tumoural CD8+ T lymphocyte function. Thus, the final aspect of this thesis explores the effect of OTD on CD8+ T lymphocyte viability and cytotoxic function measured by IFNγ release. Results indicated a negative effect on CD8+ T lymphocyte function with suppression of IFNγ release in cells stimulated with phorbol myristate acetate (PMA)/ionomycin or anti-CD3, at all concentrations of OTD, despite preservation of cell viability (n=3, p <0.05). Considering the infiltration of CD8+ T lymphocytes into the surgical site approximately one week postoperatively, our findings suggest that use of OTD might be best confined to the early postoperative period, although further studies in vivo are required to clarify this. In conclusion, this thesis demonstrates that OTD is a potential surgical adjuvant in melanoma treatment. However, the findings also highlight important considerations for its clinical application including optimal timing and duration of therapy as well as potential combination with alternative metabolic inhibitors to prevent resistance. Further studies are essential to fully characterise the effects of OTD and guide its potential clinical applications.
Melanoma, OTD, Postoperative inflammation, Immunosuppression, Wound healing, Surgical adjuvant
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