| MEKKl is a 196-kDa serine-threonine kinase activated in response to a variety of stimuli, including EGF, lysophosphatidic acid, osmotic stress, UV light, and microtubule toxins. In this study, a subline of B16 mouse melanoma cells harboring MEKKl genes (M1B16) was established, and the expression of MEKKl could decrease the colony formation and tumor formation abilities of mouse melanoma B16 cells. The adherence ability and the Calpain-1 activity were also decreased in M1B16 cells referring to control cell lines. The motility and invasive ability were enhanced by the expression of MEKKl in vitro and in vivo. The morphology of B16 melanoma cells harboring MEKKl showed the dendrite outgrowth. All of these data indicate that M1B16 may be a suitable cell model to screen MEKK1 inhibitor for cancer metastasis therapy. MEKK1 is an important kinase in MAPK signaling pathway. Upon activating, MEKK1 participates in the regulation of the JNK and ERK pathways and is involved in the activation of NF-κB. In addition, MEKK1 is activated in response to changes in cell shape and the microtubule cytoskeleton. MEKK1 senses microtubule integrity, protects cells from committing to apoptosis, and contributes to the migration of fibroblasts and epithelial cells. In this study, a direct competitive ELISA to quantify total MEKK1 in human cell lines was developed. The procedure showed a high sensitivity (detection limit: 0.17 ng/mL), good precision (coefficient of variation≤10.12) and acceptable linearity with a large range of MEKK1 concentrations (0.1-10000 ng/mL). In a pilot study, this assay was used to quantify MEKK1 in different cell lines. In cancer cells, the range of MEKK1 is 0.02-85 ng/mg protein and its concentration was higher in pancreas cancer and umbilical vein cells than that in others. And this high expression of MEKK1 in these two cell lines indicates that MEKK1 may be a potential target in interfering with pancreas cancer and angiogenesis. Phenanthroindolizidine alkaloids are found primarily in the genera Cynanchum, Pergularia, and Tylophora of the Asclepiadaceae, but have also been reported from Hypoestes verticillaris (Acanthaceae), Cryptocarya phyllostemmon (Lauraceae), Ficus hispida and F. septica (Moraceae). It is a small group of alkaloids known for its profound cytotoxic activity. It has been shown that the toxicity of phenanthroindolizidine alkaloids is due to inhibition of protein and nucleic acid synthesis. In this paper, the anti-tumor activities of a new phenanthroindolizidine alkaloid (CAT) against cancer cells in vitro were investigated to provide the molecular mechanism for CAT in the treatment of hepatoma.Effects of CAT on the growth of different cancer cell lines were assayed by MTT and SRB assay. MTT test showed that the IC50 of CAT to cancer cells were in the range of 0.044～0.286μmol/L. Human hepatoma cell (Bel7402) was the most sensitive to CAT with the IC50 of 0.044μmol/L. GI50 evaluated by SRB assay was 0.023～0.103μmol/L. TGI evaluated by SRB assay was 0.079～0.390μmol/L. The inhibition of CAT on growth of Bel7402 and HCT-8 cells was in a dose-dependent manner, and the colony formation ability of Bel7402 and HCT-8 cells was inhibited by CAT (P＜0.05) significantly, too. To investigate the nature of growth inhibition caused by CAT, flow cytometric analysis was performed. The cells treated with increasing concentrations of CAT showed progressive accumulations in the G1 phase and S phase of the cell cycle. Topoisomerase I activity was measured by DNA relaxation assay. CAT inhibited Topoisomerase I activity in a dose-dependent manner. Western Blotting analysis was performed to observe the expression of P53, P21, P16, and CyclinA proteins. P53, P21, P16 and CyclinA protein expression were increased in a dose-dependent manner.After treated with CAT for 2h, the adhesive ability of Bl6-BL6 cells with basement membarane components (Matrigel) was markedly reduced in a dose-dependent manner. 0.02, 0.04 and 0.08μmol/L CAT inhibited significantly the migration of Bl6-BL6 cells with the inhibition rate of 57％, 75％and 86％(P＜0.001), respectively. Secretion and activation of MMP-2 in serum-free culture medium of HT-1080 cells treated by CAT for 24h were suppressed by a dose-dependent mode. These results implied that CAT decreased degradation of basement membrane and invasive potentials by inhibiting the adhesion, migration and invasion ability of tumor cells and suppressing the secretion and activation of MMP-2.The anti-proliferation effect of CAT on human umbilical vein endothelial cell (ECV304) was demonstrated by SRB assay with GI50 of 0.10μmol/L and with TGI of 0.22μmol/L. MTT test showed that the IC50 of CAT to ECV304 was 0.088μmol/L. The chemotaxis mobility of ECV304 cells induced by fibronectin (FN) was significantly restrained by CAT at the different concentrations, the inhibitory rate of 0.02, 0.04 and 0.08μmol/L CAT on migration potential was 55％, 65％and 82％, respectively. Three-dimension in vitro angiogenesis system showed that CAT inhibited tube formation of ECV304 cells seeded on Matrigel, the multicellular capillary-like structure formation was suppressed to 81.8％, 54.6％and 16.1％of untreated control by 0.02, 0.04 and 0.08μmol/L CAT, respectively. With RT-PCR analysis, KDR and MMP-9 gene expression of ECV304 cells were inhibited by CAT treatment. CAT inhibited angiogenesis by suppressing endothelial cells proliferation, migration and KDR gene expression.In summary, it was found that CAT inhibited growth of various cancer cells in vitro, the metastasis of Bl6-BL6 cells in vitro and the angiogenesis process. CAT could induce progressive accumulations of Bel7402 cells at the Gl phase and S phase. This blockade was related to the Topoisomerase I inhibition by CAT. The changes of P53, P21, P16, and CyclinA protein expression levels may play a major role in the cell cycle arrest triggered by CAT. Inhibition of CAT on MMP secretion may play a key role in suppression both on tumor growth and on metastasis. The effects of CAT on angiogenesis were associated with inhibition of expression of KDR genes. The results reported herein may suggest potential clinical application of CAT against cancer, especially for those refractory to current chemotherapy.
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