Oesophageal carcinoma, of which there are two subtypes, adenocarcinoma and squamous cell carcinoma, is the sixth most common cause of cancer-related death . Oesophageal squamous cell carcinoma (OSCC) is a highly aggressive carcinoma with a very poor survival rate that occurs with particularly high frequency in developing countries including Iran, China, South Africa, and Brazil, where mortality rates can exceed 100 per 100,000 population. In developed countries, incidence rarely exceeds 10 per 100,000 population [2, 3], with the exceptions of certain regions in North-West France and Northern Italy where incidence may reach 30 and 2 per 100,000 in males and females, respectively . The causes of OSCC are multiple and varied, probably reflecting repeated exposure to dietary components, such as N-nitroso compounds, excessive smoking and alcohol consumption, chronic inflammation and possibly, genetic predisposition . Current commonly used therapies for OSCC include 5-fluorouracil and cisplatin, which show poor efficacy and often display both chemotoxicity and chemoresistance . Cisplatin has multiple mechanisms of cytotoxicity including the formation of DNA and protein adducts, as well as via oxidative stress. Many resistance mechanisms for cisplatin have been identified, including, pertinent to this study, sequestering of cisplatin by glutathione (a major species of intracellular thiols). This results in export of cisplatin-glutathione adducts leading to a reduction in cisplatin-mediated DNA damage [7–9]. Investigations into more effective targeted treatment options for OSCC using monoclonal antibody therapies are very promising  however, access to such therapies in developing countries is extremely limited, primarily due to cost. Therefore, there is a continued and urgent requirement for alternative, effective and economical treatment options.
Recently, the well characterized and tolerated anti-diabetic drug, metformin has been the subject of intense investigations in cancer research. Population studies have shown that this biguanide, conventionally used to decrease peripheral glucose levels and increase insulin sensitivity in diabetic and pre-diabetic patients [11, 12], reduced breast cancer occurrence in female patients with type 2 diabetes . Since then, metformin has been observed to reduce the proliferation of many types of carcinoma cell lines and diabetic patients taking metformin have been found to have better recovery rates from breast cancer [14–17]. Furthermore, metformin has been shown to target cancer stem cells . However, whilst metformin reduces cell proliferation in most cancer types, it rarely causes apoptosis, and is therefore being combined with conventional chemotherapeutic drugs, including cisplatin. This treatment combination has mixed results, with some studies showing that metformin can enhance the effectiveness of chemotherapeutic drugs whilst others have shown increased chemoresistance in the presence of metformin [19, 20]. With regards to cisplatin, metformin has been shown to reduce cisplatin sensitivity through the AMPK-independent upregulation of the Akt survival pathway . A search on clinicaltrials.gov found over 40 clinical trials investigating metformin and a variety of chemotherapeutic drugs, for breast, ovarian and prostate cancer amongst a number of others.
In this study, we investigated the effect of metformin on OSCC cell proliferation and on the cytotoxicity of cisplatin for OSCC cells. We show that whilst metformin markedly reduces OSCC cell proliferation and causes cells to accumulate in the G0/G1 phase of the cell cycle, it also significantly protects against cisplatin cytotoxicity. The protective effect is not solely due to reduced cell-proliferation, as the biguanide minimally to partially protects against the DNA-crosslinker, mitomycin C, but is dependent on a metformin-induced increase in glycolysis and intracellular NAD(P)H levels with a concomitant increase in reduced intracellular thiols, which coincides with decreased cisplatin-DNA adduct formation. The glutathione synthesis inhibitor buthionine sulfoximine (BSO) significantly reverses this protective effect, confirming the role of reduced glutathione in cisplatin detoxification by metformin-treated cells. In light of these findings, we investigated the copper-bis(thiosemicarbazones), copper diacetyl-bis(4-methylthiosemicarbazonato)copper(II) (Cu-ATSM) and copper glyoxal-bis(4-methylthiosemicarbazonato)copper(II) (Cu-GTSM). Copper-bis(thiosemicarbazones) induce cytotoxicity through a number of mechanisms, including inhibition of DNA synthesis . Importantly, as these compounds are known to be trapped in cells under reducing conditions, they are therefore compatible with a reducing intracellular state . We show that both Cu-ATSM and Cu-GTSM display significant levels of cytotoxicity at LD50 values comparable to or lower than cisplatin, both alone or in combination with metformin, highlighting the use of metformin and reduction-compatible cytotoxic drugs as a novel combination therapy strategy for the treatment of OSCC.