title: Metformin induces an intracellular reductive state that protects oesophageal squamous cell carcinoma cells against cisplatin but not copper-bis(thiosemicarbazones).
abstract: BACKGROUND
  Oesophageal squamous cell carcinoma (OSCC) is a highly aggressive carcinoma with a poor survival rate. One of the most commonly used chemotherapeutic drugs, cisplatin, displays varied and often poor efficacy in vivo. Therefore, alternative, cost-effective and more efficacious treatments are required. Metformin has been previously shown to reduce proliferative rates in various carcinoma cell lines. We report for the first time, the effect of metformin on OSCC cell proliferation and show that it antagonises cisplatin-induced but not copper-bis(thiosemicarbazone)-induced cytotoxicity in OSCC cells.
  
  
  METHODS
  Cell proliferation and stage of the cell cycle were quantified by trypan blue counts and flow cytometry, respectively. All cytotoxicity measurements were made using the tetrazolium based MTT assay. Metabolic alterations to cells were determined as follows: glycolysis via a lactate dehydrogenase assay, reducing equivalents by MTT reduction and reduced intracellular thiols by monobromobimane-thiol fluorescence, and glutathione depletion using buthionine sulfoximine. Inductively coupled plasma mass spectrometry was used to quantify cisplatin-DNA adduct formation.
  
  
  RESULTS
  Metformin was found to reduce cell proliferation significantly in all OSCC cell lines, with an accumulation of cells in G0/G1 phase of the cell cycle. However, metformin significantly protected OSCC cells against cisplatin toxicity. Our results indicate that a major mechanism of metformin-induced cisplatin resistance results from a significant increase in glycolysis, intracellular NAD(P)H levels with a concomitant increase in reduced intracellular thiols, leading to decreased cisplatin-DNA adduct formation. The glutathione synthesis inhibitor buthionine sulfoximine significantly ablated the protective effect of metformin. We subsequently show that the copper-bis(thiosemicarbazones), Cu-ATSM and Cu-GTSM, which are trapped in cells under reducing conditions, cause significant OSCC cytotoxicity, both alone and in combination with metformin.
  
  
  CONCLUSIONS
  This is the first study showing that metformin can be used to decrease cell proliferation in OSCC cells. However, metformin protects against cisplatin cytotoxicity by inducing a reducing intracellular environment leading to lower cisplatin-DNA adduct formation. As such, we advise that caution be used when administering cisplatin to diabetic patients treated with metformin. Furthermore, we propose a novel combination therapy approach for OSCC that utilises metformin with metformin-compatible cytotoxic agents, such as the copper-bis(thiosemicarbazones), Cu-ATSM and Cu-GTSM.
authors.forename: Leonard Howard
authors.initials: LH
authors.lastname: Damelin
authors.forename: Rupal
authors.initials: R
authors.lastname: Jivan
authors.forename: Robin Bruce
authors.initials: RB
authors.lastname: Veale
authors.forename: Amanda Louise
authors.initials: AL
authors.lastname: Rousseau
authors.affiliation: School of Molecular and Cell Biology, University of the Witwatersrand, Private Bag X3, Johannesburg 2050, South Africa. Demetra.Mavri-Damelin@wits.ac.za.
authors.forename: Demetra
authors.initials: D
authors.lastname: Mavri-Damelin
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D002294:Carcinoma, Squamous Cell / Q000378:metabolism / Q000473:pathology*; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D002945:Cisplatin / Q000494:pharmacology*; D056831:Coordination Complexes / Q000494:pharmacology*; D018736:DNA Adducts / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004938:Esophageal Neoplasms / Q000378:metabolism / Q000473:pathology*; D005978:Glutathione / Q000378:metabolism; D006019:Glycolysis / Q000187:drug effects; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008687:Metformin / Q000494:pharmacology*; D009942:Organometallic Compounds / Q000494:pharmacology*; D010084:Oxidation-Reduction; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013882:Thiosemicarbazones / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; D056831:Coordination Complexes; D018736:DNA Adducts; D009942:Organometallic Compounds; D013438:Sulfhydryl Compounds; D013882:Thiosemicarbazones; C089548:copper (II) diacetyl-di(N(4)-methylthiosemicarbazone); C000595457:glyoxal-bis(4-methylthiosemicarbazonato)copper(II); D008687:Metformin; D007770:L-Lactate Dehydrogenase; D005978:Glutathione; D002945:Cisplatin