Mistretta, Carmela (2011) Apoptosis induction in different melanoma cell lines by NEMO Binding Domain (NBD) peptide treatment. [Tesi di dottorato] (Unpublished)


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Item Type: Tesi di dottorato
Resource language: English
Title: Apoptosis induction in different melanoma cell lines by NEMO Binding Domain (NBD) peptide treatment
Mistretta, Carmelacarmela.mistretta@libero.it
Date: 30 November 2011
Number of Pages: 69
Institution: Università degli Studi di Napoli Federico II
Department: Biologia strutturale e funzionale
Scuola di dottorato: Scienze biologiche
Dottorato: Biochimica e biologia cellulare e molecolare
Ciclo di dottorato: 24
Coordinatore del Corso di dottorato:
Arcari, Paoloarcari@unina.it
Faraone Mennella, Maria Rosariafaraone@unina.it
De Maio, Annaandemaio@unina.it
Date: 30 November 2011
Number of Pages: 69
Keywords: Melanoma NBD-peptide Poly(ADP-ribosyl)ation
Settori scientifico-disciplinari del MIUR: Area 05 - Scienze biologiche > BIO/10 - Biochimica
Date Deposited: 06 Dec 2011 15:52
Last Modified: 17 Jun 2014 06:03
URI: http://www.fedoa.unina.it/id/eprint/8924

Collection description

Melanoma is the most aggressive form of skin cancer that originates from neoplastic trasformation of melanocyte cells. Important predisposing factors are considered UV exposure and genetic susceptibility. Due to the complex nature of the disease, metastatic melanomas have proven to be typically resistant to different therapy and chemotherapy treatments. On this basis, recently, key signalling pathways that are important in promoting melanoma tumourigenesis, have been identified thus providing dynamic targets for therapy. The NF-kB is a well known transcriptional nuclear factor involved in melanoma progression. It is constitutively activated in several melanoma cell lines and regulates the expression and function of several genes involved in immediate early pathogen response, in inflammation, cell proliferation and survival and in apoptosis. Based on this knowledge, it was hypothesized that its inhibition could represent a new way to induce programmed death in tumoral cells. Therefore, A375 melanoma cells were incubated with different concentrations of NBD-peptide at different times. The anti-inflammatory activity of this peptide was already known. In this study, PARP-1 was used as marker of apoptotic process. In fact, it is a substrate of caspase-3, responsible for its cleavage in two stable and inactive fragments (89kDa and 24kDa). The results showed that the NBD peptide treatment induces a concentration-dependent inhibition of melanoma cell proliferation. FACS analysis (flow cytometry) using the monoclonal antibody (mAb) PE-conjugated anti-human-active caspase-3, together with PARP-1 expression and activity analyses, confirmed that the treated cells die by apoptosis. FACS analysis showed that in treated cells, a concentration-dependent activation of caspase-3 occurs. Partial fragmentation and inactivation of PARP-1, is also observed by western blotting and enzimatic assay, respectively. EMSA assay (electrophoretic mobility shift assay) carried out to study both NF-kB and IKK kinase complex activities, confirmed the hypothesis that the peptide induces apoptosis by modulating activity of this transcriptional nuclear factor. Such modulation is due to the block of IKK complex activity, responsible of NF-kB activation. In fact, it’ s known that the latter, inactive, cannot translocate into nucleus, where it modulates transcription of different genes among which the antiapoptotic genes. Preliminary data also suggest that partial PARP-1 inactivation could allow the maintenance of apoptotic process. PARP-1, in fact, is one of the main consumers of intracellular energy. Since it was demonstrated that poly-ADP-ribose (PAR) levels play an important role both in cellular survival and death, in the second part of the present research it was studied whether NBD peptide treatment influences poly-ADP-ribose turnover. To this aim, beside poly-ADP-ribose synthesis, its degradation, by poly(ADPR)glycohydrolase (PARG) was studied. In both NBD treated and untreated A375 melanoma cells, the same PARG isoform is expressed (about 75kDa), localized into the nucleus. Its activity is more high in untreated melanoma cells, in which an active PAR turnover is guaranteed and tumoral cell survival occurs. In treated cells, the lowest activity of PARG could depend on both length and intranuclear concentration of PAR. The partial PARP-1 inactivation suggests that PAR levels in treated cells are lower than those produced in melanoma cells. Moreover, a comparison of PAR isolated from treated cells and that from melanoma cells, indicates that the former is unbranched and shorter. In conclusion, it is possible to hypothesize that in treated A375 cells, where the apoptotic process occurs, the low PARP activity produces low and no toxic PAR levels that combined with a low PARG activity, might allow the cells to maintain the apoptotic state rather than undergo necrosis. In the last part of research the efficience of NBD peptide was also tested on other seven melanoma cell lines with different gene mutations. So, this peptide might represent a new and efficient drug in melanoma care. In addition, respect to common drugs used to inhibit NF-kB activity, NBD-peptide doesn’t block nuclear factor activity completely, allowing its physiological roles in immunity, inflammation and cellular homeostasis.


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