ABSTRACT
ObjectiveLymphatic epithelial cells (LECs) are important links involved in lymphatic metastasis in the microenvironment of cholangiocarcinoma. This study aims to detect the modulation of inflammatory factors and chemokines secreted by LECs after stimulation of cholangiocarcinoma cells, and observe the effects of highly expressed factors on lymphangiogenesis.MethodsThe culture medium of cholangiocarcinoma (RBE, HCCC9810), LECs stimulated by cholangiocarcinoma cell culture medium (CCM), and normal LECs were prepared. Inflammatory factors and chemokines in the culture medium were detected using protein chip. The experiments are divided into the following groups, including a blank control group, CCM group, CCM coupled with Anti-ENA-78 group, Anti-ENA-78 group, ENA-78 group, ENA-78 coupled with SB2252002, and SB225002 group. The relationship between the content of factor and time was investigated using ELISA, while the relation between target factors and lymphangiogenesis obtained by cell proliferation and tubule formation assay.ResultsWe found ENA-78, IP-10, GCP-2, MCP-2, MCP-3, MIP-3a, HCC-1, and Lymphotactin expression increased in LECs supernatant after CCM stimulation. However, I-TAC, MIP-1d, IL-10, MIG, PDGF-BB, and CXCL16 factors showed down-regulation. The secretion of ENA-78 in CCM was relatively low. By ELISA, we found that the ENA-78 protein in RBE-LECs and HCCC9810-LECs gradually increased over time, and reached the plateau phase at the point of 48h. The lymphatic tube forming ability of LECs cultured in CCM was significantly increased compared with that of the control group, and this ability could be partially weakened by ENA-78 neutralizing antibodies. In the exogenous ENA-78 protein group, the lymphatic tube formation ability was as well significantly increased compared with that in the control group, and this ability could be effectively blocked by the IL-8B inhibitor.ConclusionThe increased secretion ENA-78 of lymphatic epithelial cells induced by cholangiocarcinoma may play a role in promoting lymphangiogenesis through the IL-8B receptor.
ABSTRACT
ObjectiveLymphatic epithelial cells (LECs) are important links involved in lymphatic metastasis in the microenvironment of cholangiocarcinoma. This study aims to detect the modulation of inflammatory factors and chemokines secreted by LECs after stimulation of cholangiocarcinoma cells, and observe the effects of highly expressed factors on lymphangiogenesis.MethodsThe culture medium of cholangiocarcinoma (RBE, HCCC9810), LECs stimulated by cholangiocarcinoma cell culture medium (CCM), and normal LECs were prepared. Inflammatory factors and chemokines in the culture medium were detected using protein chip. The experiments are divided into the following groups, including a blank control group, CCM group, CCM coupled with Anti-ENA-78 group, Anti-ENA-78 group, ENA-78 group, ENA-78 coupled with SB2252002, and SB225002 group. The relationship between the content of factor and time was investigated using ELISA, while the relation between target factors and lymphangiogenesis obtained by cell proliferation and tubule formation assay.ResultsWe found ENA-78, IP-10, GCP-2, MCP-2, MCP-3, MIP-3a, HCC-1, and Lymphotactin expression increased in LECs supernatant after CCM stimulation. However, I-TAC, MIP-1d, IL-10, MIG, PDGF-BB, and CXCL16 factors showed down-regulation. The secretion of ENA-78 in CCM was relatively low. By ELISA, we found that the ENA-78 protein in RBE-LECs and HCCC9810-LECs gradually increased over time, and reached the plateau phase at the point of 48h. The lymphatic tube forming ability of LECs cultured in CCM was significantly increased compared with that of the control group, and this ability could be partially weakened by ENA-78 neutralizing antibodies. In the exogenous ENA-78 protein group, the lymphatic tube formation ability was as well significantly increased compared with that in the control group, and this ability could be effectively blocked by the IL-8B inhibitor.ConclusionThe increased secretion ENA-78 of lymphatic epithelial cells induced by cholangiocarcinoma may play a role in promoting lymphangiogenesis through the IL-8B receptor.
ABSTRACT
Objective Solamargine (SM), with its anti-inflammatory and anti-tumor effects, inhibits the proliferation and promotes the apoptosis of various tumor cells. This study was to investigate the effects of SM on the proliferation and apoptosis of human esophageal cancer KYSE150 cells and its action mechanism. Methods We treated KYSE150 cells with SM at the concentrations of 0 (the blank control group), 2, 4, 6 and 8 μmol/L for 24 hours. Then, we observed the morphological changes of the cells under the inverted microscope, detected their proliferation and apoptosis by MTT assay and flow cytometry respectively, and determined the expressions of the classical NF-κB signaling pathway-related proteins NF-κB, p-NF-κB, IKKα, IKKβ, IkBα and p-IkBα) and apoptosis-related proteins Bax, caspase-3, cleaved caspase-3 and Bcl-2 in different groups of the cells by Western blot. Results Compared with the blank control, the inhibition rate of the proliferation of the KYSE150 cells in the 2, 4, 6 and 8 μmol/L SM groups was increased significantly in a concentration-dependent manner (0 vs [15.03 ± 0.15]%, [47.94 ± 1.74]%, [68.72 ± 0.47]% and [77.51 ± 1.70]%, P<0.05), and so was the apoptosis rate ([8.17 ± 0.51]% vs [14.50 ± 0.73]%, [18.57 ± 2.08]%, [65.10 ± 10.88]% and [81.55 ± 5.48]%, P<0.05). The expression of the apoptosis-related protein Bax in the SM treated cells was up-regulated, those of Bcl-2, IKKα, IKKβ and p-IkBα down-regulated, and the activity of caspase-3 and cleaved caspase-3 promoted, all in a concentration-dependent manner, with statistically significant differences between the blank control and the 4, 6 and 8 μmol/L SM groups (P<0.05). Statistically significant differences were also found in the expressions of NF-κB, p-NF-κB and IkBα between the blank control and the 6 and 8 μmol/L SM groups (P<0.05). Conclusion Solamargine can significantly inhibit the proliferation and promote the apoptosis of KYSE150 cells, probably by suppressing the classical NF-κB signaling pathway.