As such, zebrafish transplanted with MDA MB 231 or M4 cells, treated with and without SB 431542 or LY 294002 were subjected to im munohistochemistry to determine if pSmad2 in tumour cells had been prevented sellectchem and or inhibited upon inhibitor treatment. Zebrafish that displayed invasion, regardless of treatment, were positive for pSmad2. This posed a conundrum, as it may be expected that invasive or metastatic cells that have overcome the inhibition of TGF B would continue to phosphorylate Smad2, but cells that were sensitive to the treatment would not invade, and thus are not able to be examined for pSmad2. Therefore, zebra fish transplanted with MDA MB 231 or M4 cells were kept without treatment for 5 days, and then treated with SB 431542, LY 294002 or a vehicle control for up to 24 h.

This system allowed the cells to invade and metastasise, giving us the best option to visualise the impact small molecular inhibitors of the TGF B pathway have on the zebrafish Inhibitors,Modulators,Libraries xenograft model. As seen Inhibitors,Modulators,Libraries in Figure 3B, phosphorylation of Smad2 can be effectively switched off in breast cancer cells, MDA MB 231, when treated with a TKRI, for as little as 1 h. Thus, pharmacological inhibition of TKRI activity in hibits TGF B Smad2 signalling of transplanted breast tumour cells in zebrafish and inhibits their invasiveness. Smad4 knockdown in breast cancer cells inhibits their invasion and metastasis in the zebrafish model Next we set out to examine the effect on breast cancer in vasion and metastasis by antagonizing TGF B Smad signal ling in breast cancer cells in a Inhibitors,Modulators,Libraries cell autonomous manner.

MDA MB 231 cells with stable transfection of shRNA tar geting Inhibitors,Modulators,Libraries Smad4 have been previously shown to inhibit the frequency of bone metastasis in nude mice by 75% and sig nificantly increased metastasis free survival in intracardiac mouse models. Using the zebrafish embryo xenograft model, MDA MB 231 cells stably transfected with shRNA targeting Inhibitors,Modulators,Libraries Smad4, or the empty vector, were examined for invasion. Similar to the mouse model, at 6 dpi, Smad4 knockdown provided a significant reduction of invasion. Furthermore, given the relatively short time required to visualise invasion and metastasis, transient transfection of siRNA may be a useful tool. To examine the effect of siRNAs targeting Smad4 on invasion, MDA MB 231, M2, and M4 cells were transiently trans fected with Smad4 specific siRNA, or a scrambled control. Significant best inhibition of invasion was seen in each cell line. MMP inhibition MMPs are known to have tumour promoting and tumour inhibitory effects but unfortunately, several clinical trials of broad spectrum MMP inhibitors have failed to show promising effects. The specific MMP2 9 Inhibitor II, has been shown to mitigate TGF B induced invasion of breast cancer cells.