**3.7 Cardiac glycosides, such as digoxin, may be able to suppress LC population**

Cardiac glycosidases (CGs) are a family of drugs used for the treatment of congestive heart failure and cardiac arrhythmia by regulating cardiac muscle contraction through the inhibition of the NA<sup>+</sup> -K+ -ATPase pump [136]. The first anti-proliferative effects of CGs were reported more than five decades ago in HeLa cells [137] and since then, multiple studies have highlighted the anti-neoplastic effects of CGs by inducing cancer cell apoptosis [138], activating autophagic cell death through the Ras-dependent extracellular signal-regulated kinase (ERK1/2) pathway [139], inhibiting hypoxia-inducible factor-1 alpha (HIF-1α) protein synthesis [140] and inhibiting FA/BRCA pathway activation [141]. CGs have been shown to have a higher cytotoxicity effect when combined with chemotherapy in prostate, breast, non-small cell lung, colorectal, and pancreatic cell lines as well as advanced stage melanoma patients compared to single agents [141–144]. However, so far epidemiological studies have yielded inconsistent results. For example, while digoxin was found to inhibit tumour growth *in vitro* and was associated with a 25% lower prostate cancer risk [145], systematic review and meta-analyses indicated an increased prostate cancer risk in digoxin users [146]. Nevertheless, the number of clinical trials specifically designed for cancer patients being treated with CGs is very limited and most of these conflicting results come from re-analysing data present in the medical databases with limited numbers of patients. So far, there are no clinical

trials designed to investigate the relationship between CGs and OC. Despite this, there is a recent study retrospectively analysing the Surveillance, Epidemiology, and End Results (SEER) program, the national cancer institute (NCI), and Medicare healthcare claim record data to assess whether digoxin use enhances chemotherapeutic responses in OC treatment [147]. The study suggested that digoxin use during chemotherapy did not have any survival benefits in patients with EOC, however, the research was limited by small sample size. Furthermore, 46% of the patients had a prior history of heart disease complicating the interpretation of subject fatality rates. More importantly, only 7% of the studied population were treated with digoxin during chemotherapy which may describe the opposing results with other cancer types. Since cardiac glycosidases regulate ion transport via the NA<sup>+</sup> /K+ - ATPase, they interact with a wide variety of the intracellular signalling pathways, including those driving cellular proliferation and apoptosis [148], therefore, future clinical trials specifically designed for OC patients is highly expected. Our laboratory drug screening pipeline used to identify therapies against LCs has identified digoxin as a potent LC inhibitor, demonstrating synergistic effects when sublethal concentrations of digoxin were combined with platinum-based chemotherapies (result not published).
