## 4 Discussion  
Looking for new therapeutics and combinations of drugs leading to higher cancer selectivity and fewer side effects is still a big challenge. P\(pa\) is a chlorophyll degradation product which has been shown to exert anticancer properties with or without photo-activation; the so-called photodynamic therapy [3]. Our data show that the most active combination against melanoma cells was induced by fraction E from the diatom _Cylin-drotheca closterium_, plus P\(pa\) followed by 1 h photo-activation. In a previous study, we showed the composition of _C. closterium_ fractions A-D [31] and suggested that the anti-inflammatory activity observed for _C. closterium_ fractions C and D was related to the presence of lysophosphatidylkolines and the chlorophyll-derived molecules P\(pa\) and hydroxypheophorbide \(a\)[31]. Compared to fractions A-D, fractions E also contained a larger amount of Pheophytin \(a\), which also is a breakdown product of chlorophyll but it has a long aliphatic tail making it more non-polar (unpublished data). This composition difference in the fractions may explain why fraction E is more active compared to the others. Pheophytin \(a\) isolated from the seagrass _Springodium isoetifolium_ also showed anticancer properties against adenocarcinoma A549 cells [46]. Similarly, Pheophytin \(a\) from the plant _Leucaemia leuccephala_ showed anticancer, anti-migration and anti-invasion activity in human gastric cancer cell line (AGS) [47]. When we studied the mechanism of action in the current study, P\(pa\) triggered the mitochondrial-related apoptotic pathway in melanoma cells as also previously reported in hepatocellular carcinoma [4], uterine carcinomas [5] and breast tumor [7]. Our results suggested the activation of the intrinsic apoptotic pathway (Bcl-2/Bax disregulation) triggered by P\(pa\) and _Cc_ fraction E treatment followed by 1 h photo-activation. Other transcripts have been found to be differentially expressed as well. Up-regulated genes by P\(pa\), fraction E or the combination of both plus photo-activation included TNF, estrogen receptor and defensin beta 1. The role of TNF in melanoma is still controversial [48]. TNF has been shown to have a key role in immunity and immunomodulation and is associated with both the inhibition and promotion of tumor proliferation [48; 49]. The role of the estrogen receptor in melanoma progression and genesis is still under investigation [50]. [PERSON] et al. [7] also showed that P\(pa\) does not require the estrogen receptor for transport into cells. Defensin beta 1 has been found to be constitutively expressed in various epithelia [51; 52]. Moreover, defensin beta 1 was reduced in squamous cell carcinoma and its down-regulation was directly related to malignancy transformation [53]. Defensin beta 1 was also down-regulated in prostate carcinomas [54] and in renal cell carcinomas (RCC) [55]. The down-regulated transcripts by the three treatments also included the amyloid beta precursor, a glucosidase alpha acid and a transmembrane protein 57. The amyloid beta precursor protein has been always linked to Alzheimer disease, but recently, the increased expression of this protein has been found in multiple types of cancers [56]. Hence, its down-regulation is in line with the reduction in cancer cell proliferation. A transcript coding for \"glucose, alpha; acid\", also known as acid alpha-glucosidase (GAA), is a lysosomal enzyme, and its suppression has shown to be associated with improved chemosensitivity and apoptosis in pancreatic cancer cells [57]. [PERSON] et al. [57] found a reduction in tumor growth and prolonged tumor-related survival after GAA silencing in a murine model. In addition, apoptosis activation was also observed. Transmembrane proteins have been found to be differentially expressed in different tumors, but their function is still mostly unknown [58].