Investigation of the effects of phytochemical induced changes in TG2 activity on three cell line models of cancer chemotherapy

The use of Phytochemicals as chemotherapeutic agents is a growing area of research, with biologicaly active agents such as the flavonoids, curcumin, resveratrol and berberine all having proven to be both anti-tumourgenic and well tolerated compounds. As such agents are easily purified from plant sources, require no NICE/FDA approval for use and generally exhibit favourable pharmakokinetic behavior, continued research in this area is suggested. Transglutaminase 2 (TG2) is an enzyme that is abnormally expressed in a variety of cancers with retinoic acid (a TG2 activity modulator) having been shown to increase suceptability of a variety of cancer models to chemotherapeutics. Some research has been conducted into the use of Phytochemicals to modulate TG2 activity/levels/localization in an effort to sensitise cancers to other therapies, but there are at the time of writing large gaps in the current body of work. This work compared and contrasted how the phytochemicals resveratrol and berberine affect cancer cell model systems and the activity of the enzyme TG2. The aim was to use established cancer cell lines to test the effectiveness of the phytochemicals in isolation and in combination as chemotherapeutics and to determine how they influence cell viability, cellular adhesion/migration and the cell cycle and if this relates to TG2 activity. The model systems used in this study involved 3 cancer cell lines; these were HepG2 (a human hepatocarcinoma model system), Caki-2 (human renal carcinoma model system) and 1321N1 (a human astrocytoma model system). The results of this study would determine whether resveratrol and berberine have the potential to be used at clinically-achievable doses to treat cancer and whether the mechanism by which they induce any chemotherapeutic effects involves the modulation of TG2. This research is important as existing cancer therapies show limited effectiveness and are in general not well tolerated, finding and evaluating new therapeutics is therefore highly desirable. Analyses of cell viability was performed using CCK-8 and flow cytometry. Cell cycle and TG2 enzymatic activity were analysed using flow cytometry. Analyses of cellular adhesion and metastasis were performed using scratch test assays and a series of microscopic observations. mRNA levels were measured using qPCR, and inhibition of TG2 employed the TG2 competitive inhibitor cystamine. A model combined drug therapy was developed that modulated TG2 activity. The phytochemical berberine increased TG2 enzymatic activity in both Caki-2 and HepG2 cells and induced cell death in the HepG2 model. Resveratrol treatment inhibited both the cell cycle and cellular migration and also increased the strength of cellular adhesion in both cell lines. Potential binding sites for resveratrol on the TG2 enzyme were determined using the SwissDock software. The well-tolerated phytochemicals berberine and resveratrol in combination proved effective at inducing cell death, inhibiting the cell cycle and preventing metastasis at clinically-achievable doses in the HepG2 model system. Resveratrol alone proved affective at inhibiting the cell cycle and inhibiting migration at clinically-achievable doses in the Caki-2 model system. These results show the potential for development of a combined Resveratrol/Berberine therapy to treat human Hepatocarcinoma.