The Role Of DLL1 In B16 Melanoma Development

The Notch signaling pathway is an evolutionarily conserved signaling system that is required for normal embryonic development, the regulation of tissue homeostasis, and the maintenance of stem cells in adults by regulating cell fate decisions such as cell differentiation, proliferation, apoptosis. In the process of embryonic vascular development, Notch signaling pathway plays a key role in maintaining the normal structure and function of embryonic vascular network by regulating the arteriovenous differentiation and endothelial Tip cell and Stalk cell specification. Aberrant regulation of Notch signaling pathway always leads to embryonic death due to defective development of vascular networks. In adult, Notch signaling pathway limits the excessive blood vessels formation to maintain homeostasis during physiological angiogenesis. In tumor development, increased expression of DLL4 was detected in tumor vasculature. Targeted blocking of DLL4 mediated Notch signaling pathway impaired blood perfusion and inhibited tumor growth due to non-productive tumor vessels formation; whereas stimulation Notch signaling with DLL4-overexpressed tumor cells reduced tumor angiogenesis but promoted tumor growth by improving structure and function of tumor blood vessels. Meanwhile, other researches have also demonstrated that Jagged1, another Notch ligand, could significantly enhance the formation of tumor vessels and promote tumor growth.According to the Context-dependent role of Notch signaling pathway and the specific function of Notch molecules, we examined the effects of Notch ligand DLL1 in tumor angiogenesis. DLL1 was reported to be required for arteriovenous differentiation and postnatal vascular remodeling after injury. But its role in tumor blood vessels has not yet reported. In this study, several works have been done to elucidate the relationship between DLL1 and tumor angiogenesis.1. Construction a eukaryotic expression vector which co-express of DLL1 protein and green fluorescent protein (GFP). DLL1 gene was amplified with specific primers by PCR; the products was then cloned into the pIRES2-EGFP plasmid after sequencing, the new pIRES2-EGFP-DLL1 plasmid was confirmed by restriction enzyme digestion and sequencing.2. Over-expression of DLL1 promoted tumor cell proliferation in vitro. B16 melanoma cells were trasfected pIRES2-EGFP-DLL1 plasmid or pIRES2-EGFP plasmid respectively, and stablely trasfected cells were selected. Activation of Notch signaling was detected in B16-DLL1 tumor cells but not in control cells. MTT and Clony forming assay showed that DLL1 over-expression enhaced B16 tumor cell proliferation.3. DLL1 reduced tumor growth by attenuating vasculization in B16 tumors. Tumors growth was reduced in DLL1 over-expression group after subcutaneously inoculation tumor cells into mice. Activation of Notch signaling was detected in B16-DLL1 tumor tissues. Immuno-staining indicated that tumor angiogenesis and tumor blood supply was reduced, which resulted in aggravated hypoxia and necrosis in tumors.Conclusion: In view above data, our study indicated that over-expression of DLL1 promoted tumor cell proliferation and clone formation through activating transcription of Notch downstream gene HES1 and HEY1. However, the growth of B16-DLL1 tumors in vivo was not accelerated but inhibited. The possible reason was that the rapid growth of the tumor needed to adapt to the supply of nutrients,but, on the one hand, expression of DLL1 promoted tumor cell proliferation, on the other hand expression of DLL1 reduced tumor blood vessels, impaired vessel structure, disrupted the tumor's blood supply, which lead to increased hypoxia and necrosis in tumor, Thus inhibited tumor growth.