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  • br Discussion br Angiogenesis is considered a fundamental st

    2020-07-29


    4. Discussion
    Angiogenesis is considered a fundamental step in tumor develop-ment at all stages. Cancer POM 1 produce and secrete numerous growth factors to induce tumor neovascularization and improve the blood supply and nutrient deficiency of the vascular system in response to environmental stimuli. Upregulated expression of proangiogenic factor can activate dormant microvascular endothelial cells, facilitating them to lose the their cell–cell connections, migrate into the surrounding space of blood vessels, decrease endothelial cell apoptosis, proliferate extensively, and form vascular structures.
    To the best of our knowledge, the present study provides important 
    data concerning the role of ANGPLT6 in AFPGC development. First, ANGPTL6 was identified as a major driver gene for angiogenesis among eight members of the ANGPTL family in AFPGC. Second, recombinant ANGPTL6 or cell supernatants from AFPGC promoted the endothelial cell migration, invasion, and tubule formation, and decreased apop-tosis, but not cell proliferation. Furthermore, ANGPTL6 prevented cancer cells from undergoing apoptosis and enhanced cancer cell mi-gration and invasion. Importantly, anti-angiogenesis drugs, such as bevacizumab may be a useful drug for AFPGC.
    ANGPTL6 is a versatile member of the ANGPTL family. Most ANGPTLs are acknowledged to have angiogenic effects [23] and to modulate glucose, lipid, and energy metabolism [24–28]. Although some of them are not fully characterized and, ANGPTL6 in particular is a less prominent member of the family. The protein is secreted as 52 kDa monomers. Originally, ANGPTL6 was discovered in human liver [29]. As a hepatocyte-derived factor, it appears to be involved in the regulation of metabolic homeostasis and epidermal regeneration [29]. Several studies have reported that some ANGPTLs exert angiogenesis in several types of human tumors, including breast cancer, nonsmall lung cancer, colorectal cancer, prostate cancer, pancreatic cancer, ovarian cancer and glioblastoma. ANGPTL3 induces endothelial cell adhesion, and migration and promoted blood vessel formation [30]. ANGPTL7 was revealed as a pro-angiogenic factor and led to the formation of blood vessels in a Matrigel sponge experiment [15]. In contrast, it was
    Fig. 4. ANGPTL6 loss promoted the apoptosis, migration, and invasion of AFPGC. (A and B) RT-PCR and western blotting revealed downregulation of ANGPTL6 in GCIY cells. (C) Tube formation assays and quantification of the numbers are shown. Magnification, 200 × . Migration assays (D) and invasive experiments (E) were performed after the knockdown of ANGPTL6 in GCIY cells. Magnification, 200 × . The data are expressed as the mean ± SD of three independent experiments, each performed in triplicate. *P < 0.05; **P < 0.01 compared with the control.
    hypothesized that certain ANGPTLs may play an inhibitory role in tumor development. ANGPTL1 inhibits epithelial-mesenchymal transi-tion and suppresses lung cancer by abrogating SLUG [31]. ANGPTL1 also decreases angiogenesis by inhibiting the phosphorylation of ERK1/ 2 and AKT and interacts with integrin α1β1 to suppress the JAK2/ STAT3 signaling pathway [32]. Until now, the role of ANGPTL6 in gastric cancer has not been clearly defined. The current study is the first to show that ANGPTL6 is preferentially expressed in AFPGC compared to common gastric cell lines, while the other seven members of ANGPTLs were nearly undetectable in eight gastric cancer cell lines. 
    Moreover, our results revealed that the expression of ANGPTL6 is po-sitively correlated with the expression of micro-vessel density in AFPGC patients. The above results indicated that the overexpression of ANGPTL6 is involved in AFPGC progression.
    ANGPTL6, signals through ERK1/2. However, the role of this se-cretory protein in the tumor microenvironment remains largely un-known. Indeed, we consistently found that ANGPTL6 enhances the migration and capillary network formation ability of HUVECs. Notably, we also consistently observed that ANGPTL6 is able to support the survival and motility of endothelial cells, as indicated by activation of
    Fig. 5. ANGPTL6 knockdown inhibits tumor growth in vivo. (A) Tumors are shown, along tumor volumes (B) and tumor weights (C). All tumors derived from the indicated cells were shown and tumor volumes and weights were measured. The results are shown as the mean ± SEM of tumor volumes (n = 6) (**P < 0.01, Mann–Whitney test). (D) Tumor samples were formalin-fixed, paraffin-embedded, and sliced for IHC assay. Representative images of the indicated ANGPTL6, Ki-67, and CD34 staining are shown. Magnification, 200 × . *P < 0.05; **P < 0.01 compared with POM 1 the control.
    the pro-survival kinase AKT and by ERK activation, as well as by mi-gration and invasion assays. In sum, ANGPTL6 is likely to exert a wide and complete biofunction in endothelial cells, thereby favoring in cancer angiogenesis. This finding represents an attractive therapeutic target for anti-tumor therapy. In vitro studies revealed that the bio-function of ANGPTL6 in gastric cancer progression not only increased endothelial cell migration and formation tube structures, but also de-creased cancer cell apoptosis. In previous studies, ANGPTL6 was con-firmed to have a powerful effect on the migration of endothelial cells, and we demonstrated that ANGPTL6 leads to endothelial cell pro-liferation and tube formation. Together, these observations underlie the notion that neovascularization of tumors is mediated by ANGPTL6 in AFPGC. Further investigation confirmed that the activation of FAK-, ERK1/2 and signaling pathways in endothelial cells may exert a key role in these ANGPLT6-mediated effects.