Transcription factors: targets for inhibiting carcinogenesis
Tersigni, Mariaroberta (2010) Transcription factors: targets for inhibiting carcinogenesis. [Tesi di dottorato] (Inedito)
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The perceived hypothetical role of inflammation in carcinogenesis has been bolstered by epidemiological observations linking infections and chronic inflammatory conditions to cancer. Given their place as master regulators at the center of inflammation, NF-kappaB transcription factors were natural suspects in providing a mechanistic link between inflammation and carcinogenesis. Of major importance is the recognition that various effects of NF-kappaB on cancer initiation, promotion, and progression are cell-type, tissue and context specific, ascertainment of which was only possible due to recent advances in genetically dissectable mouse models of inflammation-linked cancer [Naugler WE and Karin M, 2008]. Therefore, an increasing number of compounds able to block NF-kappaB have been tested and have shown to suppress the growth of those cancer cells whose tumorigenicity depends on NF-kappaB activity [Karin M et al, 2002]. Malignant melanoma is an aggressive, therapy-resistant malignancy of melanocytes that exhibits constitutive activation of NF-kappaB which confers tumor survival capacity and escape from apoptosis. The major aim of this work of thesis was to investigate the anti-carcinogenic effect on human melanoma cell lines of Yellow Extract (YE), the methanolic extract of the yellow fruits of Opuntia ficus-indica, indicaxanthin and Lauroside B (LB) a megastigmane glycoside isolated from Laurus nobilis L. leaves, and to clarify the mechanisms involved in their anti-cancer properties, particularly in relation to the transcription factor NF-kappaB. Both the Yellow Extract and indicaxanthin extracted from the Opuntia Ficus-indica inhibited A375 human melanoma, A549 adenocarcinomic human alveolar basal epithelial and Caco-2 human colorectal adenocarcinoma cell growth, as evaluated by the MTT assay. The treatment with YE and indicaxanthin inhibited the three cell lines growth in a time- and concentration-dependent manner. To better understand the mechanisms of YE and indicaxanthin effects on human melanoma, we investigated the involvement of the transcription factor NF-kappaB. Treatment of A375 human melanoma cells with YE inhibited the high constitutive NF-kappaB activation as well as treatment with indicaxanthin in a time-dependent fashion. Moreover, A375 cells were treated with YE and indicaxanthin and analized with flow cytometry. Treatment of A375 cells with YE and indicaxanthin determined a time-dependent rise of the Annexin V positive cells, demonstrating that the antiproliferative effect of YE and indicaxanthin on A375 was due to induction of apoptosis and not just to a mere cytotoxic effect. As for Lauroside B, a megastigmane glycoside isolated from Laurus nobilis L. leaves, we showed that it induces apoptosis in human melanoma cell lines. In fact, LB inhibited cell proliferation of three different human melanoma cell lines: A375, WM115 and SK-Mel-28, indicating that this compound has a broad spectrum of activity against human melanoma cell lines. Treatment of A375 cell line with LB induced cell-surface Annexin V binding thus demonstrating the ability of LB to induce apoptosis of A375 human melanoma cell line. We also investigated whether caspase-3, which is the main “effector” caspase in the apoptotic pathway, was activated following treatment of A375 cells with LB. We clearly observed a time-dependent degradation of both pro-caspase-3 and PARP (its substrate) following treatment of A375 cells with LB, thus supporting the data obtained by FACS analysis. To further investigate the mechanisms underlying the LB-induced apoptosis of human melanoma cells we analyzed, by western blot, the expression of IkappaB-alfa in the cytosolic fractions of A375 cells and, by EMSA, the NF-kappaB DNA-binding activity on A375 whole extracts. Our results clearly demonstrate that LB was able to inhibit, in a time-dependent fashion, both IkappaB-alfa degradation and constitutive NF-kappaB-DNA binding activity, suggesting that the apoptotic effect of LB may be partly mediated via reduction of NF-kappaB signaling. Another important result we obtained is the demonstration that exposure of human melanoma cells to LB negatively affected the expression of two anti-apoptotic genes, XIAP and c-FLIP, whose expression is regulated by NF-kappaB [Baud V and Karin M, 2009]. The present work of thesis demonstrates, for the first time, the pro-apoptotic activity of the Yellow Extract, indicaxanthin and Lauroside B against human melanoma cell lines and that this effect is associated to inhibition of constitutive NF-kappaB activation. These natural compounds may also represent potential chemotherapeutic agents against other malignancies that exhibit constitutive NF-kappaB activation and resistance to regular chemotherapy. Future studies will be needed to further elucidate the molecular mechanisms of action of these compounds as well as to address their effects in animal models, in order to promote their development as potential chemotherapeutic agents to treat human melanoma.
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