Melanoma is a malignant tumor that arises from melanocytes present in the basal layer of the epidermis, in hair follicles, more rarely in mucosa eye, inner ear and meninges. Skin is affected in 85% of cases. Human malignant melanoma is an highly aggressive and incurable cancer due to intrinsic resistance to apoptosis, reprogramming proliferation and survival pathways during tumor progression. Several studies have shown that many natural dietary compounds can potently modulate various molecular targets, conducting prevention of cancer initiation, promotion and progression. In particular, dietary fruits and vegetables have been regarded as rich sources of chemopreventive compounds and are widely investigated due to their low toxicity but significant chemopreventive efficacy. Sulforaphane (SFN), an isothiocyanate (ITC) found in cruciferous vegetables, is a common dietary component that has been proven to have a huge cancer chemopreventive potential. It modulates cell death, cell cycle, angiogenesis, susceptibility to carcinogens, invasion and metastasis and possesses antioxidant activities. Pluralities of clinical effects are reported in various experimental diseases as well as human clinical studies. In B16F-10 murine melanoma cell line SFN reduces invasion, inhibits activation of matrix metalloproteinases and, consequently, the developing of lung metastases, and prevents melanin synthesis and tyrosinase expression by affecting the phosphorylation of MAP kinase family. Epidemiological studies have reported association between the consumption of SFN-rich vegetables and reduction in cancer risk at several sites including the bladder, prostate and breast. Moreover, SFN is in phase II trial for prostate cancer. The aim of this project was to evaluate the effects of sulforaphane on human melanoma cell lines, in order to prove its ability to modulate specific cellular and molecular events involved in this type of tumor. A human primary (A375) and metastatic (501Mel) melanoma cell lines has been used in these studies. I demonstrated that SFN inhibits significantly A375 and 501Mel cell proliferation, induces cell cycle arrest and apoptosis. Interestingly, SFN-treated melanoma cells accumulated in the G2/M and sub-G1 phases of the cell cycle. Moreover SFN showed to induce apoptosis through caspase-9/caspase-3 pathway. As control, human epidermal melanocytes cell lines (HEMa) treated with the same concentration of sulforaphane didn’t shown an alteration. Besides its ability to promote tumor cell death, SFN hampers cell migration and invasion. One important process in chemoprotection by SFN involves modulation of the activity of the so-called phase II enzymes, which convert carcinogens to inactive metabolites that are readily excreted from the body, thus preventing their reaction with DNA. Interesting, the results showed that in melanoma cell lines SFN increased significantly the mRNA levels of phase II enzymes such as hemoxygenase-1 (HMOX-1) and NADPH quinone oxidoreductase (NQO1). To better characterize the molecular events induced by SFN on melanoma, I investigated the expression of nerve growth factor (NGF) receptors, known to be involved in melanoma progression. Using RT-PCR and fluorescence-activated cell sorting analysis, it was found that SFN causes up-regulation of neurotrophin receptors expression (p75NTR and TrkA) in A375 cell lines. Moreover, SFN is able to decrease the A375 migration induced by exogenous β-NGF. These observations led to the hypothesis that SFN induces apoptosis through p75NTR and that p75NTR-dependent apoptosis could represent a homeostatic mechanism to eliminate damaged cells –similar to Fas-dependent apoptosis associated with inflammation – and possibly a ‘class effect’ of death receptors. Finally, in this study, it was also established SFN-resistant melanoma cell lines in order to elucidate mechanisms leading to drug resistance. All together, these results indicate that, in vitro, sulforaphane has a strong antitumor effect on human melanoma cell lines and this raises the possibility that SFN might be a promising candidate for molecular-targeting chemotherapy against melanoma.
|Titolo:||The effect of sulforaphane in human melanoma cells|
|Data di pubblicazione:||19-mag-2014|
|Appartiene alla tipologia:||07b Tesi di Dottorato (EX-Padis)|