Salim, Handi Yuwono; (2024) Does pyroptotic cell death contribute to anthracycline-induced cardiotoxicity? Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Background: Anthracyclines are widely used as effective chemotherapeutic agents. However, research has found that anthracycline-induced cardiotoxicity (AIC) causes significant morbidity and mortality. While the precise mechanism of AIC remains elusive, apoptosis has long been suspected as a key player. The precise mechanism of AIC remains elusive, although apoptosis has long been suspected as a key player. In recent studies, pyroptosis, a programmed lytic cell death pathway, was also found to contribute to AIC potentially. Most studies have focused on evaluating AIC in the myocardium, but emerging evidence suggests that AIC may also affect vascular cells. Furthermore, the relative toxicity of doxorubicin (DOX) or its supposed active metabolite, doxorubicinol (DOXOL), has not been widely investigated in cardiac vascular cells. / Purpose: This thesis examines the hypothesis that pyroptosis plays a role in AIC. Specifically, this thesis investigates DOX and DOXOL cytotoxicity in H9c2 immortalised rat cardiac myoblasts, two types of human cardiac endothelial cells, a human breast cancer cell line MCF-7, and a human monocytic acute myeloid leukaemia cell line THP-1. Subsequently, this thesis evaluates the expression of a pore-forming protein Gasdermin D (GSDMD) and its N-Terminal domain (GSDMD NT), caspase 3 and its cleaved product as the key executioner of pyroptosis and apoptosis, respectively; this being investigated in the above cells treated with DOX and DOXOL. / Methods: In vitro experiments were conducted to ascertain dose-dependent death response in H9c2, and cardiac endothelial cells following an 18-hour 0-10 μM DOX or DOXOL treatment. Morphological changes were observed with Annexin V and Propidium Iodide (PI) following DOX or DOXOL treatment compared to known inducers of pyroptosis (LPS & Nigericin).Western blot experiments were conducted to study the expression of full-length GSDMD, GSDMD-NT, caspase 3 and cleaved caspase 3, following DOX or DOXOL. / Results: My data suggest that dose-dependent cell death following DOX treatment was greater compared to DOXOL in H9c2 and both cardiac endothelial cells. When treated with DOX, these three cell types underwent apoptotic changes (intracellular granulations, cell shrinkage and blebbing) with more severe effects at higher concentrations. In contrast, DOXOL caused milder effects, with these cells undergoing early apoptosis. Despite detecting of GSDMD and caspase 3 protein bands in the Western blot experiment with DOX or DOXOL, the respective cleaved proteins, GSDMD-NT or cleaved caspase 3 protein, were not detected. In MCF-7 cells, DOX-induced apoptotic changes were observed in the absence of caspase 3, but no changes were observed following DOXOL treatment. Interestingly, in THP-1 cells, DOX exerted predominantly apoptotic cell death, whereas DOXOL predominantly exerted pyroptotic cell death (nuclei and membrane swelling, membrane rupture). This was accompanied by the detection of full-length caspase 3 and GSDMD with its cleaved proteins in DOX and DOXOL, respectively. / Conclusion: My data show that DOX and DOXOL primarily induce apoptotic cell death in H9c2 cardiomyocytes and cardiac endothelial cells. In MCF-7 cells, DOX induced apoptotic changes. Interestingly, THP-1 cells can be driven towards apoptotic or pyroptotic cell death induced by DOX or DOXOL, respectively. These findings suggest that pyroptosis is not likely to play a major role in anthracycline-induced cytotoxicity. Instead, anthracycline-induced cytotoxicity is likely to be due to apoptotic injury.

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