Ahmed, Ola Ahmed, Auhood Nassar, Mai

Lotfy, Abeer Bahnassy Background/Aims: Hypoxia is deprivation of an adequate oxygen supply and induces hypoxic apoptosis. Hypoxia inducible factor-1α (HIF-1α) and interleukin (IL-8) activate tumor survival in different pathways. We evaluated whether adenovirus-mediated small hairpin RNAs for HIF-1α (shHIF-1α) and IL-8 (shIL-8) induced apoptosis in hepatocellular carcinoma (HCC) and endothelial cell lines. see more Methods: The HCC cell line was infected with adenovirus expressing shRNA for HIF-1α and IL-8 and maintained under hypoxic conditions (1% O2, 24 h). The expression levels of HIF-1α and apoptotic and growth factors were examined by real-time quantitative polymerase chain reaction and Western blotting. We also investigated apoptosis by terminal deoxynucleotidyl ICG-001 in vivo transferase dUTP nick-end-labeling assay (flow assisted cytometry and immunofluorescence) and measured cytochrome c levels. Results: Inhibiting HIF-1α and IL-8 up-regulated the expression of apoptotic factors

while simultaneously down-regulating anti-apoptotic factors. Knockdown of HIF-1α and IL-8 increased cytochrome c concentration and enhanced DNA fragmentation in the HCC cell line and human umbilical vein endothelial cells (HUVECs). Moreover, the culture supernatant collected from knockdown of HIF-1α and IL-8 in the HCC cell line induced apoptosis in HUVECs under hypoxia. Conclusions: These data suggest that adenovirus-mediated knockdown of HIF-1α and IL-8 induced apoptosis

in HCC and triggered apoptosis in vascular endothelial cells. MCE公司 Disclosures: The following people have nothing to disclose: Sung Hoon Choi, Seung Up Kim, Do Young Kim, Weon Sang Ro, Sang Hoon Ahn, Seungtaek Kim, Chae Ok Yun, Kwang-Hyub Han, Jun Yong Park [Backgrounds] Transforming growth factor (TGF)-β induces epithelial-mesenchymal transition (EMT) which is a crucial step for invasion and metastasis in various types of cancer. Reduced expression of E-cadherin, a hallmark of EMT, is reported in hepatocellular carcinoma (HCC), however, involvement of microRNAs (miRNAs) in this process is poorly understood. CDH1, which encodes E-cadherin, has CpG islands in the promoter region. DNA methylation of CpG islands are regulated by DNA methyltransferases (DNMTs) which are the targets of miR-29a. We investigated the involvement and role of miR-29a in epigenetic regulation of E-cadherin expression during the process of EMT induced by TGF-β. [Methods] Human HCC cell lines, PLC/PRF/5 and HepG2 were treated with 1-10 ng/ml of TGF- β for ∼72 hours to induce EMT. Expression of E-cadherin was examined by using real-time qPCR and immunoblotting. Methylation specific PCR (MSP) was performed to determine the methylation level of CpG islands in the E-cadherin promoter that contains E-boxes. To force the expression of miR-29a, cells were electroporated with synthetic precursor miR-29a.