Thus, we performed the prolifera tion experiments using 10% FCS as stimulant. The results selleck screening library mirrored the situation Inhibitors,Modulators,Libraries previously observed in melan a Hm cells. Proliferation was blocked by the MMP inhibitor mix, and the only inhibitor responsible for this effect was MMP 9 13. The progression of starved A375 cells into S phase, which is seen 20 and 24 h after FCS stimulation, was prevented in presence of MMP9 13. MMP13 mediates cell proliferation in melanocytes and melanoma cells Ilomastat efficiently inactivates MMP1, MMP2, MMP3, MMP8, and MMP9, while the only described targets of the MMP9 13 inhibitor are MMP9 and MMP13. There fore we concluded that the effect of the MMP9 13 inhi bitor is MMP13 specific.

Supportingly, the application of another inhibitor, targeting MMP1, 2, 3, 9, and 13, as well as an independent MMP13 specific inhibitor showed the same effect on the Hm and A375 cells. To validate this, we transfected melan a Hm cells with a retroviral plasmid expressing Mmp13 specific shRNA, which resulted in a reduction of Mmp13 expression Inhibitors,Modulators,Libraries on RNA Inhibitors,Modulators,Libraries and protein level. Melan a Hm shMMP13 cells proliferated much slower than cells expressing a control plasmid. Interestingly, we also observed that Mmp13 down regulation went along with a strong increase in pigmen tation, as visible by a 100% increase in melanin content. This was accompa nied by enhanced levels of tyrosinase RNA. A similar approach was done with the human mela noma cell line A375. As several tested shRNA con structs did not efficiently knock down the gene, we used commercial siRNA for this cell line, which reduced MMP13 transcript levels to approx.

33%. Western blot analysis also confirmed a reduction in the pro and active forms of the protein, with 60 and 48 kDa, respectively. Instead of the previously conducted Inhibitors,Modulators,Libraries long term proliferation assays, we performed a BrdU incorporation assay as Inhibitors,Modulators,Libraries a measure of DNA replication 72 h after transfection of the respective siRNA. Knockdown of MMP13 decreased BrdU incorporation to 60%. We also observed an increased fraction of siMMP13 transfected cells in the G0 G1 phase of the cell cycle when compared to control cells. However, the effect was weaker than the effect seen in presence of the MMP 9 13 inhibitor displayed in figures 3C and 5C. Possibly, this is due to the incomplete MMP13 knock down.

It is also likely that the arrest is more enhanced in starved cells that are confronted with growth stimulus and MMP inhibitor at the same time. If MMP13 is knocked down in the normal growing cell culture, it may block cell cycle progression in general, irrespective of the selleck chemicals cell cycle phase. This kind of behaviour is remi niscent of the effect of growth factor withdrawal, which can block the cell cycle in G1 and G2, and might point to the possibility that MMP13 releases an unidentified growth factor.