## 4 Discussion
Finally, treatment with P\(pa\) plus fraction E plus photo-activation also induced the down-regulation of tumor necrosis factor receptor superfamily (member 1A), cathepsin B and S. Tumor necrosis receptor 1 levels were found to be increased in patients with hepatocellular carcinoma [49], but to our knowledge its dysregulation has never been reported to be associated with melanoma. Cathepsin B inhibition has been found to reduce cell invasiveness of melanoma cells [59]. Cathepsin S has been associated with different cancer types. It is a mediator of tumor progression and has been associated with angiogenesis and metastasis formation, hence, representing a promising target for cancer therapy [60]. The microtubule subunit \(\beta\)-tubulin, initially selected as a possible reference gene for Western blotting, was also found to be differentially expressed (i.e., reduced expression induced by the treatment) in our study. In fact, levels of \(\beta\)-tubulin were affected by Pp\(a\) treatment. The intrinsic apoptotic pathway has been found to also be activated by chemotherapeutic agents targeting tubulin proteins, such as paclitaxel ([PERSON](r)) and docetaxel ([PERSON], 2017). However, in addition to the intrinsic apoptotic pathway mediators, we also found that GPX4 and NCOA4 protein levels were reduced after Pp\(a\) treatment and photo-activation suggesting that the treatment may induce lipid peroxidation. This study shows that the known compound Pp\(a\), a specific microalgal fraction plus photo-activation, can have an additive anti-proliferative role on melanoma cells. As reported by [PERSON] et al. (2017), the US Food and Drug Administration (FDA) has granted the GRAS status to various microalgae, including _Auxenochlorella protothecodes_ (formerly _Chlorella protothecodes_), _Spirulina_, _Dunaliella salina_, _Haematococcus pluvialis_, _Prototheca zopfii_, _Ulkenia_ sp., _Crypthecodimium chonii_ and _Schizochytrium_ sp. For some of these, the GRAS status refers to the entire biomass, while for others only a part of the extract or a fraction. _Spirulina_ and _Chlorella_ are most commonly used in Europe and, recently, whole alga (_Odontella aurita_ and _Tetraselmis chlui_) or only specific components (_Ullkenia_ sp. oil, _Schizochytrium_ sp. oil, _Haematococcus pluvialis_ oleoresin) have been approved under the Commission Implementing Regulation (EU) 2017/2470 ([PERSON], 2017). This \"safe\" status has been expanded to a greater number of species in recent years and this trend will positively influence the addition of microalgal extracts, fractions and components in nutraceuticalal and pharmaceutical products. These may have properties that are useful for various human pathologies, such as antioxidant, anti-inflammatory and anticancer properties ([PERSON], 2017; [PERSON], 2017). Future studies on the combinatorial effects of microalgal extracts with known drugs, such as those used for chemotherapy, with or without photo-activation, can shed light on new activities against cancer cells and on possible photodynamic therapies. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app13042590/s1](https://www.mdpi.com/article/10.3390/app13042590/s1)]([https://www.mdpi.com/article/10.3390/app13042590/s1](https://www.mdpi.com/article/10.3390/app13042590/s1)), Figure S1: Antiproliferative activity of the diatom _Cylindricalo discretion_, Pheophorbide \(a\), and their combination on normal human cells HACAT. Results are expressed as percent survival after 24 h exposure followed by 1 h photo-activation (* \(p<0.05\); ** \(p<0.01\); *** \(p<0.001\)); Figure S2: The intensity of the bands of immunodetected Bcl-2 (a) and Bax (b) were normalized with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (* \(p<0.05\); ** \(p<0.01\)); Table S1: Antiproliferative activity of various microalgae on cancer human cells. Conceptualization, A.S., A.I. and C.L. ; formal analysis, A.S., G.R. and C.L. ; writing--original draft preparation, A.S., G.R. and C.L. ; writing--review and editing, A.S., G.R., A.I.