Another evidence of pro apoptotic signals in D6 treated cells expression pro file is the over expression of the BCL10 gene, encoding for a pro apoptotic member of the Bcl2 family proteins. Bcl10 protein contains a caspase recruitment domain motif and promotes the activation of caspase 9. The p53 full read signalling pathway has resulted to be signifi cantly affected also in fibroblasts, be ing CDKN1A and GADD45 A/B partially up regulated. Again, this molecular response in fibroblasts is weaker than that in melanoma cells, without causing in normal cells block of proliferation or cell death. Our analyses pointed out a down modulation of cell cycle regulators cyclin B1, cdc25B, and CDK4, which certainly contributes to the inhibition of cell prolif eration exerted by D6 on melanoma cells.

Block of cell cycle in G2/M phase perfectly matches with a decrease in expression of both cyclin B and cdc25, whereas the decrease in CDK4 expression Inhibitors,Modulators,Libraries indicates that cells lack entering the cell cycle while are driven to age and die, as demonstrated by the G1 cell population decrease after D6 treatment. Interestingly, a lower or ab sent down modulation of these mitosis promoters has been evidenced in fibroblasts, suggesting that D6 treatment specifically inhibits cell proliferation pathways in melanoma cells. Another gene down modulated by D6 in melanoma cells is the CCNF gene, codifying for cyclin F, the founding member of the F box protein family. In addition to an F box do main, cyclin F contains a cyclin box domain, but, in con trast to typical cyclins, it does not bind or activate any cyclin dependent kinases.

However, like other cyclins, cyclin F protein levels vary during the cell division cycle, peaking in G2. During G2, cyclin F is involved in ubiquitination and degradation of proteins as well as in spindle formation and it is required for the fidelity of mi tosis and Inhibitors,Modulators,Libraries genome. In our system, down modulation of such a protein is in agreement with the block of cell cycle in G2/M Inhibitors,Modulators,Libraries phase demonstrated by cytofluorimetry. A further contribution to D6 anticancer activity on mel anoma cells is given by the down Inhibitors,Modulators,Libraries modulation of the c KIT Inhibitors,Modulators,Libraries proto oncogene. The c kit protein be longs to class III receptor tyrosine kinases. its extracellular domain binds the SCF to stimulate sev eral processes, including melanogenesis, gametogenesis, and haematopoiesis.

The c KIT up regulation is often associated with increased Ruxolitinib cell proliferation. its down regulation in D6 treated melanoma cells was confirmed by western blot analysis. One could also hypothesize that a big contribution to the anticancer activity of D6 is given by down regulation of both phosphatidylinositol 3 kinase and NF kB signalling pathways. There is growing evidence that activa tion of the PI3K/Akt pathway plays a significant role in melanoma.