upon 5AzaCdR treatment in 4C11+ melanoma cell (Figure 2A, Tables S1–S4). Finally, the resulting genes were plotted in heatmaps (Pearson correlation metric distance; average linkage algorithm) organized according to the sequential stages of melan-a malignant transformation (Figure 3B–3D). In total, 11 genes showed responsiveness to 5AzaCdR treatment at stage I (Figure 3B), 9 genes at stage II (Figure 3C), and 136 genes at stage III (Figure 3D) of melanoma progression. Three genes were selected for further validation of gene expression (Xist, Hspb1 and Serpine1) (Figure 3B–3D). These genes, except for Hspb1 [18], were not previously reported to be modulated by 5AzaCdR treatment in melanoma cells. Despite the fact that the genes Xist and Hspb1 were found deregulated earlier in the process of malignant transformation of melan-a melanocytes, studies have shown they are likely to have distinct functional roles in the tumor progression [20–22]. Candidate Markers for melan-a Malignant Transformation In recent years, it became evident that HDAC inhibitors such as Trichostatin A (TSA) in combination to DNA demethylation agents such as 5AzaCdR are attractive epigenetic agents to synergistically increase the expression of tumor silenced-genes in cancer [13,23–25]. Along this line of evidence, the differential expression of selected genes were validated using quantitative real- time approach (RT-qPCR) in melan-a, 4C, 4C112 and 4C11+ cell lines previously exposed to three protocols based on epigenetic drug therapy: 40 nM TSA for 16 hours, 10 mM 5AzaCdR for 48 hours, and 10 mM 5AzaCdR for 48 hours plus 40 nM TSA for 16 hours (Figure 4). The rationale for the establishment of these treatment conditions for TSA and 5AzaCdR combined with TSA was based on the same explanation as those for 5AzaCdR alone, but also exploring, directly or indirectly, the acetylated state of genes. Cytotoxicity and cell viability were monitored by MTT assays, as previously described [11]. RT-qPCR revealed that the expression of Xist was negative in all melan-a-derived cell lines compared to melan-a melanocytes. The treatment of the 4C pre- malignant melanocyte as well as 4C112 non-metastatic melano- ma cell line with 5AzaCdR plus TSA resulted in the highest up- regulation of Xist expression. On the other hand, we observed a more robust up-regulation of Xist gene expression when 5AzaCdR alone was given to 4C11+ metastatic melanoma cells. Further- more, a slight decrease in the relative intensity of Xist gene expression was noted when TSA alone was given to melan-a melanocytes as well as 4C11+ metastatic melanoma cell line (Figure 4A). The dynamic of Hspb1 gene expression was characterized by a significant enrichment found at pre-malignant and non-metastatic stages (4C melanocyte lineage and 4C112 melanoma cell line, respectively) in relation to melan-a melano- cytes, followed by a progressive loss in Hspb1 expression in 4C11+ metastatic melanoma cell line when compared with 4C pre- malignant melanocyte and 4C112 non-metastatic melanoma cell. The treatment of the 4C112 non-metastatic melanoma cell line with TSA resulted in no significant change of Hspb1 mRNA expression. Despite this fact, the highest up-regulation of Hspb1 expression was induced by the combination of 5AzaCdR and TSA (. 12 times increase). At metastatic stage (4C11+ melanoma cell line), treatment with either TSA or 5AzaCdR was able to enhance Hspb1 expression, but when TSA and 5AzaCdR were combined, a slight increase in the relative intensity of Hspb1 gene expression was noted above individual drugs. As a whole, there was no significant change in Hspb1 expression at pre-malignant stage (4C melanocyte lineage), except a slight increase when 5AzaCdR treatment was done. At immortalized, but non-tumorigenic stage (melan-a melanocytes), we observed a more robust up-regulation Novel Candidate Biomarkers in Melanomas of Hspb1 gene expression by 5AzaCdR and TSA combination than by 5AzaCdR alone (Figure 4B). Serpine1 gene expression was significantly higher in the initial phases of melanoma genesis (4C pre-malignant melanocyte and 4C112 non-metastatic melanoma cell line) than immortalized melanocyte lineage (melan-a). On the other side, a progressive loss of Serpine1 expression was found in 4C11+ metastatic melanoma cell line when compared with 4C pre-malignant melanocyte lineage and 4C112 non-metastatic melanoma cells. Although Serpine1 expression was significantly higher in melan-a melanocyte lineage treated with 5AzaCdR and TSA, a meticulous dynamic in its expression was observed across the therapeutic spectrum. The 4C pre-malignant melanocyte lineage showed a progressive enhancement of Serpine1 gene expression as follows: TSA alone (. 5), 5AzaCdR alone (. 9), and 5AzaCdR plus TSA (. 14 times increase) treatment. These results showed a synergistic effect of epigenetic agents to increase the expression of Serpine1 gene at pre-malignant stage. There was no significant difference in Serpine1 mRNA levels in previously untreated 4C112 non-metastatic melanoma cell line relative to its 5AzaCdR+TSA-treated counterpart cell. When 4C112 melano- ma cell was treated with TSA alone, the increase in Serpine1 gene expression was higher than 5AzaCdR alone (at least 22 times increase). Besides that, 4C11+ metastatic melanoma cell line showed a striking enhancement of Serpine1 gene expression (. 93 fold enrichment) when TSA alone was given, indicating TSA as the most effective epigenetic compound for Serpine1 up-regulation at metastatic stage. Noteworthy, such enhancement of Serpine1 at mRNA level returned close to basal level observed in previously untreated 4C11+ metastatic melanoma cell line when 5AzaCdR alone or in combination to TSA was given (. 10 and . 17 fold enrichment, respectively) (Figure 4C). We also found a set of differentially up-regulated genes in 4C11+ metastatic melanoma cell line in relation to its 4C112 non-metastatic counterpart cell (Figure 2A). Curiously, some of them displayed significantly up- regulation upon 5AzaCdR treatment in 4C11+ melanoma cell such as Fblim1 (Table S4), a cytoskeleton regulator that can control integrin activation and cancer progression [26]. Along this line, an additional transcript was validated by RT-qPCR as a candidate oncogene aberrantly over-expressed in 4C11+ meta- static melanoma cell line. As a result, we found a significant enrichment in Fblim1 gene expression in 4C112 and 4C11+ melanoma cell lines when compared with melan-a melanocyte lineage. The 4C112 non-metastatic melanoma cell line showed no significant change in Fblim1 expression when treated with TSA alone. On the other hand, approximately five times increase in Fblim1 expression was observed after 5AzaCdR treatment, and . 7.5 times increase when 5AzaCdR combined with TSA was given. Besides that, the 4C11+ metastatic melanoma cell line showed a progressive enhancement of Fblim1 gene expression as follows: TSA alone (. 3.5), 5AzaCdR alone (. 5), and 5AzaCdR plus TSA (. 6.5 times increase) treatment. The highest up- regulation of Fblim1 expression was induced by the combination of 5AzaCdR and TSA in melan-a melanocytes. At pre-malignant stage (4C melanocyte lineage) we observed no significant change in Fblim1 expression when TSA treatment was done. On the other side, when 5AzaCdR treatment was given alone or in combination to TSA, a significant up-regulation of Fblim1 expression was found. Therefore, the treatment with 5AzaCdR alone or in combination to TSA showed a great potential to up-regulated Fblim1 gene in cell lines with the lowest Fblim1 expression (melan-a and 4C melanocytes) (Figure 4D). Despite the fact that RT-qPCR approach was more sensitive than microarray-based gene expres- sion studies, there was a good correlation between mRNA profiles detected by microarrays and RT-qPCR techniques for the novel PLOS ONE | www.plosone.org 6 September 2012 | Volume 7 | Issue 9 | e44800