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Pdcd4 (programmed cell death 4) is a new tumour suppressor. Pdcd4 levels are reduced in various tumours and during malignant transformation. Furthermore, high amounts of Pdcd4 showed the potential to repress malignant transformation. So far, the effects of Pdcd4 seemed to be cell type specific. The growth factor Ang2 (Angiopoietin2), which is involved in angiogenesis, could be a cell type independent target gene of Pdcd4. Selective down-regulation of Pdcd4 by siRNA (siPdcd4) showed enhanced Ang2 levels in Western Blots of tumour cell-lines of the mammary, liver, pancreas, gastrointestinal tract and in endothelial cells. Additionally, semiquantitative RT- and RTD-PCRs indicated a transcriptional regulation. Therefore Ang2 represents a target gene of Pdcd4 in various tumour cell-lines.
After proving the elevated Ang2 expression by Pdcd4 suppression in Western Blots, investigations of supernatants of stably (shPdcd4) and transiently (siPdcd4) transfected cell-lines revealed higher Ang2 amounts in comparison to the control, indicating an augmented secretion of Ang2 protein. Tube Formation- and Boyden Chamber Assays were done to show the relevance of high Ang2 levels. We used the immortalized microvascular endothelial cell-line HMEC-1 for these experiments. The higher Ang2 levels in the conditioned medium of stably shPdcd4 transfected BON-1- und HCT116- cells caused an increased angiogenic activity in Tube Formation Assays in comparison to the mock control. Length and junctions of the tubes were software based evaluated. Tube Formation Assays with recombinant Ang2 confirmed these results. According to the existing literature Ang2 acted in two ways. On the one hand Ang2 increased the angiogenic activity in medium with 10% FBS, on the other hand Ang2 repressed angiogenesis in medium with 1% FBS. The predictable reasons are the different VEGF amounts in Medium with 1% and 10% FBS. Next to angiogenesis Ang2 had migrational potential on HMEC-1 cells in Boyden Chamber Assays. The higher levels of Ang2 in the conditioned medium of stably shPdcd4 transfected BON-1- and HCT116 cells with 1% and 10% FBS caused an increased migration on HMEC-1 cells in comparison to the mock control.
In summary, this study shows that Ang2 is a target gene of Pdcd4 regulated in various tumour cell-lines. The suppression of Pdcd4 by siRNA led to a higher transcription, translation and secretion of Ang2. The functional importance of increased Ang2 amounts was seen in Tube Formation and Boyden Chamber Assays. Pdcd4 controls angiogenesis via regulation of Ang2 and is thus involved in the development of malignant neoplasms. All in all, a putative restoring of Pdcd4 in tumours and with it an active role as a tumour suppressor could be a promising therapeutic target.