The Special Inhibition Of Notch Signaling With DOG Fusion Protein In Hypoxic CT26 Cells

Experiments in vitro indicate that Notch signaling mediates hypoxia-induced tumor cell migration and invasion. In order to explore the relationship between Notch signaling of hypoxic tumor cells in colon cancer and tumor growth in vivo, a vector expressing DOG fusion protein was constructed. The DOG fusion protein can inhibit Notch signaling of hypoxic tumor cells. The contents of this study as follows: At first, we constructed three vectors expressing DNMAML1-GFP, ODD-DNMAML1-GFP(ODG) and DNMAML1-ODD-GFP (DOG) fusion proteins. Secondly, the fusion proteins expressed in 293T cells were observed by fluorescence microscopy. The result showed that expression of DOG fusion protein was induced in hypoxia. Furthermore, DOG protein was expressed in CT26 cells by retrovirus. The hypoxia-dependence and the Notch signaling inhibition with DOG fusion protein were tested using fluorescence microscopy, Western blot as well as RFQ-PCR. Finally, the effects of DOG on CT26 cells'anoikis in vitro and tumor formation in vivo were examined.1. Three vectors expressing DNMAML1-GFP, ODG and DOG fusion proteins were successfully constructed.The results of restriction endonuclease analysis and sequence test confirmed that three vectors expressing DNMAML1-GFP, ODG and DOG fusion proteins were successfully constructed.2. The expression of DOG fusion protein was specially up-regulated in hypoxic 273T cells.GFP reflected the relative level of fusion proteins in 293T cells, as DNMAML1, ODD and GFP were assembled together. The expressions of DNMAML1-GFP, DOG and ODG were observed using fluorescence microscopy. The results revealed that expression of DOG fusion protein was only up-regulated in hypoxic 273T cells.3. The DOG fusion protein only existed in hypoxic CT26 cells.CT26 cells were successfully infected with retrovirus (MID-GFP/CT26 and MID-DOG/CT26). The expression of DOG was observed using fluorescence microscopy. The result revealed that expression of DOG fusion protein in CT26 cells was dependent on hypoxia. It was further demonstrated using Western blot that DOG fusion protein only existed in hypoxic CT26 cells.4. Notch signaling in hypoxic CT26 cells was specially inhibited with DOG fusion protein.Hes1 is Notch signaling target gene, whose mRNA level reflects the activation of Notch signaling. The relative level of Hes1 mRNA was examined by RFQ-PCR. No significant difference of Hes1 mRNA between DOG and control in normoxia was observed. Hes1 mRNA of CT26 cells infected with DOG retrovirus was significantly decreased in hypoxia, compared with control (P <0.01).5. The DOG fusion protein potentiated anoikis of hypoxic CT26 cells in vitro.There was no significant difference of anoikis between DOG and control group in normoxia. However, DOG markedly potentiated anoikis of CT26 colon cancer cells in hypoxia (P <0.01).6. Growth of CT26 in BALB/c was inhibited with DOG fusion protein.Tumor formation assay indicated that DOG fusion protein inhibited tumor formation of CT26 cells in vivo compared with GFP control. Compared to MID-GFP/CT26, MID-DOG/ CT26 resulted in the marked decline of tumor weight (P <0.01) in vivo at 21 d post-injection.The results obtained in this work suggest that DOG fusion protein specially inhibits Notch signaling of hypoxic CT26 cells. Notch signaling of hypoxic tumor cells in colon cancer promotes tumor growth in vivo.