Recent evidences indicate that activation of nuclear factor-kappa B (NF-kB) could play a role in melanoma progression. NF-kB is able to regulate the expression and the function of a wide spectrum of genes involved in cell cycle control, apoptosis, cell growth, tissue invasiveness and inflammation. Inactivation of NF-kB may contribute to the loss of their tumorigenic potential due to an increased susceptibility to apoptosis. Moreover, NF-kB blocked activity could inhibit the metastatic potential or reduce the tumor size. The identification of the NEMO (NF-kB essential modulator) binding domain (NBD) peptide that can block the activation of the IkB kinase (IKK) complex, have provided the possibility to selectively modulate the activation of NF-kB by targeting the NBD-NEMO interaction. Methods: Protein extracts were separated by 10% SDS PAGE, and transferred to polyvinylidine difluoride membrane (Millipore). Primary antibodies used included rabbit anti-caspase-3 and rabbit anti-caspase-8 antibodies, and goat anti-rabbit secondary antibodies. Horseradish Peroxidase conjugated anti-Rabbit secondary antibodies were used for signal detection. Western blot was performed by electro-transferring proteins from 12% acrylamide gel to a PVDF membrane using a Bio-Rad apparatus, followed by immunochemical analysis. Densytometric determinations of immunopositive bands were carried out with a Quantity one Program in a Densytometric Analysis. Annexin V FITC (Becton Dickinson) staining was used in conjunction with propidium iodide. Viable cells were both FITC Annexin V and PI negative while cells that were in early apoptosis are FITC Annexin V positive and PI negative or cells that were in late apoptosis or already dead are both FITC Annexin V and PI positive. Results: NBD peptide is able to induce apoptosis in A375 melanoma cell line through a caspase mediated pathway. This confirms a possible role of NF-kB in melanoma progression. Conclusions: The administration of NBD peptide could modulate NF-kB activity which could be critical for melanoma cell survival in A375 cell line and could offer new possible therapeutic strategies for melanoma.
The role of NEMO (NF-kB essential modulator) binding domain (NDB) peptide in inducing apoptosis in A375 melanoma cell line
G Palmieri
2008
Abstract
Recent evidences indicate that activation of nuclear factor-kappa B (NF-kB) could play a role in melanoma progression. NF-kB is able to regulate the expression and the function of a wide spectrum of genes involved in cell cycle control, apoptosis, cell growth, tissue invasiveness and inflammation. Inactivation of NF-kB may contribute to the loss of their tumorigenic potential due to an increased susceptibility to apoptosis. Moreover, NF-kB blocked activity could inhibit the metastatic potential or reduce the tumor size. The identification of the NEMO (NF-kB essential modulator) binding domain (NBD) peptide that can block the activation of the IkB kinase (IKK) complex, have provided the possibility to selectively modulate the activation of NF-kB by targeting the NBD-NEMO interaction. Methods: Protein extracts were separated by 10% SDS PAGE, and transferred to polyvinylidine difluoride membrane (Millipore). Primary antibodies used included rabbit anti-caspase-3 and rabbit anti-caspase-8 antibodies, and goat anti-rabbit secondary antibodies. Horseradish Peroxidase conjugated anti-Rabbit secondary antibodies were used for signal detection. Western blot was performed by electro-transferring proteins from 12% acrylamide gel to a PVDF membrane using a Bio-Rad apparatus, followed by immunochemical analysis. Densytometric determinations of immunopositive bands were carried out with a Quantity one Program in a Densytometric Analysis. Annexin V FITC (Becton Dickinson) staining was used in conjunction with propidium iodide. Viable cells were both FITC Annexin V and PI negative while cells that were in early apoptosis are FITC Annexin V positive and PI negative or cells that were in late apoptosis or already dead are both FITC Annexin V and PI positive. Results: NBD peptide is able to induce apoptosis in A375 melanoma cell line through a caspase mediated pathway. This confirms a possible role of NF-kB in melanoma progression. Conclusions: The administration of NBD peptide could modulate NF-kB activity which could be critical for melanoma cell survival in A375 cell line and could offer new possible therapeutic strategies for melanoma.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
