An investigation into the role of transglutaminase 2 in the uptake of cisplatin in parental and chemoresistant cancer cells

A major problem in the successful treatment of cancer is a loss in the effectiveness of chemotherapeutic drugs, such as cisplatin, due to development of drug resistance by tumour cells. Such resistance necessitates prescription of higher and potentially toxic concentrations of drugs. The side-effects of such treatments therefore both reduces the quality of patients’ life, and also contributes to the failure of effective chemotherapy. At present, the mechanisms by which such drug resistance operates are not fully understood. However, candidate markers for drug resistance are emerging, one of which is transglutaminase 2 (TG2). TG2 is a ubiquitous, multifunctional enzyme that is implicated in several pathological and physiological processes, including the regulation of cell division and cell death. Two TG2 isoforms, TG2-L and TG2-S, have been characterised. The overexpression of TG2 has been observed in various aspects of cancer, including the development of drug-resistance and cell survival. However, the amount to which TG2 contributes to the cellular environment of normal and drug-resistant cancer cells is poorly understood and is still controversial. In order to establish whether TG2 is directly involved in drug resistance mechanisms, TG2 gene expression at the mRNA level and protein levels was modulated in the hepatocarcinoma (HepG2) cell line, and the effects were measured, using RT-PCR, Western blotting, flow cytometry, confocal microscopy, and a range of assays for determination of TG2-specific enzymatic activity. This study provides evidence that TG2 expression and its associated transamination activity are inhibited during the initiation of apoptosis by cisplatin, an observation that was reversed by increasing the expression of TG2 following the treatment of cells with retinoic acid. Single-dose treatment of HepG2 cells with IC80 concentrations of cisplatin generated drug-resistant progeny cells that exhibited reduced TG2 expression but increased enzyme activity, and this induced chemoresistance was accompanied by a reduced ability of cells to take up Alexa fluor 546/488-labelled cisplatin. Interestingly, treatment of cells with a classic TG2 inhibitor (cystamine), or treatment by TG2 mRNA silencing (siRNA), both reversed this effect and increased the level of uptake of fluorescent cisplatin in both parental and chemoresistant cancer cells. In conclusion, these results indicate that the presence of elevated levels of TG2 in hepatocellular carcinoma prevents and/or limits the entry of anti-cancer drugs. Thus, TG2 may represent a novel biomarker and possibly a therapeutic target for overcoming the chemotherapeutic resistance in hepatocarcinoma.