HCC EPIGENETICS · Epigenetic Deregulation of Apoptotic Gene Expression in Hepatocellular Carcinoma (HCC)

Periodic Report Summary - HCC EPIGENETICS (Epigenetic deregulation of apoptotic gene expression in hepatocellular carcinoma (HCC))

The main aim in this period was to determine the role of novel essential oil compounds from Satureja thymbra and Satureja parnassica in inducing cell death in human hepatocellular carcinoma cell lines. For this reason, the following objectives were set: (1) To determine the antioxidant capacity of Satureja thymbra and parnassica essential oils as well as that of their major constituents (carvacrol and thymol). In these experiments, the antioxidant diphenylpicrylhydrazyl (DPPH) assay was performed in three independent experiments for the purpose of statistical analyses. EC50 values were determined for the S. thymbra and S. parnassica essential oil fractions as well as for their monoterpene phenol fractions namely carvacrol and thymol. Results showed that all fractions, under study, possessed significant antioxidant capacity by inhibiting DPPH formation (see attached document). (2) To determine the anti-carcinogenic capacity of Satureja thymbra and parnassica essential oils as well as that of their major constituents (carvacrol and thymol). In these experiments, the cell viability SRB (sulforhodamine B) assay was performed in three independent experiments for the purpose of statistical analyses. EC50 values were determined only for the monoterpene phenol fractions (e.g. namely carvacrol and thymol) whereas such determinations were not possible in the case of the S. thymbra and S. parnassica essential oil fractions. We believe the reason might be due to the storage conditions of these freshly-isolated fractions prior to the implementation of the project. Nevertheless, results showed that the monoterpene phenol fractions possessed significant anti-carcinogenic capacity by promoting cellular viability (see attached document). (3) To determine activation of cell death mechanisms as a means of potential anti-proliferative activity of Satureja thymbra and parnassica essential oils as well as that of their major constituents (carvacrol and thymol). In these experiments, the activation of the cell death (apoptosis) pathway was examined as a means of quantitating an anti-proliferative response in these fractions. Given the previous findings of not being able to document an anti-carcinogenic response in the S. thymbra and parnassica essential oil fractions, we only examined the carvacrol and thymol ones. We observed that both phenolic fractions were capable of inducing the cell death pathway and thus 'killing' cancer cells instead of allowing them to proliferate. In general, we obtained optimal concentrations and exposure times for each fraction after detailed time course and dose response experiments respectively. Cell death was determined by utilising caspase 3 activation, poly(ADP-ribose) polymerase (PARP) cleavage and deoxyribonucleic acid (DNA) fragmentation assays for each fraction tested. The results showed: (i) increased caspase 3 activation (determined as decreased levels of inactive pro-caspase 3) levels; (ii) increased levels of PARP cleavage (a target molecule which is cleaved by active caspase 3); and (iii) increased levels of fragmented DNA under different concentrations of carvacrol and thymol fractions (see attached document). (4) To determine if the anti-carcinogenic capacity is subject to other human cancer cell lines representing other types of malignancies namely of breast and lung origin. In these experiments, we determined the anti-carcinogenic capacity as already described above (second objective). Briefly, we determined that the anti-carcinogenic potential of carvacrol and thymol was not only specific to human hepatocellular cell lines (HepG2 and Hep3B) but also to human mammary (MCF7) and lung (A549) adenocarcinoma cell lines. In fact, carvacrol was a more efficient anti-carcinogen than thymol whereas they showed a higher anti-carcinogenic potential in mammary followed by the lung adenocarcinoma followed by hepatocellular carcinoma cell lines (see attached document). In summary, our results support the following conclusions: (i) The essential oils from the Mediterranean aromatic plants Satureja thymbra and Satureja parnassica as well as their major constituents carvacrol and thymol exhibit significant antioxidant capacity in vitro. (ii) The essential oil extracts and their two major constituents, carvacrol and thymol, demonstrated significant anti-proliferative activity in a panel of cancer cell lines. (iii) Such anti-proliferative activity is associated with the induction of the cell death (apoptotic) pathway in cancer cells. Finally, our findings provide a basis for nutritional intervention in the development of hepatocellular carcinoma. The socio-economic aspect of such outcome is of paramount importance and high relevance to human health because it can open up the way for more efficient cancer intervention strategies where human nutrition is of utmost importance.

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