Induction of apoptosis and inhibition of cell migration and tube-like formation by dihydroartemisinin in murine lymphatic endothelial cells.
ABSTRACT Dihydroartemisinin (DHA) is a semisynthesized agent from the artemisinin first extracted from the Chinese plant Artemisia annua. Previous studies have shown that artemisinin derivates, apart from their antimalarial activity, possess antitumor, antiangiogenic, and anti-inflammatory effects. In the present investigation, DHA was found to have a potent ability in influencing lymphatic endothelial cells (LECs) behavior. Murine LECs were isolated from benign lymphangiomas induced by intraperitoneal injection of incomplete Freund's adjuvant and identified by indirect immunofluorescence assay and fluorescence-activated cell sorting analysis to examine the expression of the specific marker VEGFR-3/Flt-4. When LECs were treated with DHA at 10 microg/ml, the growth of LECs was inhibited, and LECs showed typical apoptotic morphological features, with a higher apoptotic rate as compared with the controls. DHA also exerted a significant inhibitory effect on migration and tube-like formation of LECs in a dose-dependent manner. Quantitative RT-PCR further showed that DHA remarkably downregulated the expression of antiapoptotic bcl-2 mRNA, but upregulated that of the proapoptotic gene bax mRNA. In addition, DHA could strongly attenuate the mRNA and protein levels of VEGFR-3/Flt-4. In summary, these findings indicate that DHA may be useful as a potential lymphangiogenesis inhibitor under induction of cell apoptosis, inhibition of the migration, and formation of tube-like structures in LECs.
- SourceAvailable from: James E Talmadge[show abstract] [hide abstract]
ABSTRACT: Tumor production of vascular endothelial cell growth factor (VEGF)-C is associated with tumor lymphangiogenesis and lymph node metastasis. In this study, we examined the effects of small interfering RNA (siRNA)-mediated inhibition of VEGF-C on murine mammary tumor growth, metastasis, and survival. The mRNA and protein expression of VEGF-C in murine mammary tumor cells stably transfected with a VEGF-C siRNA vector were significantly lower compared with VEGF-C-control vector-transfected cells. Cl66-siVEGFC tumors had lower levels of lymphangiogenesis and lymph node and spontaneous lung metastasis than Cl66-control tumors. However, we did not observe significant differences in primary tumor growth and experimental lung metastasis between mice injected with Cl66-siVEGFC and Cl66-control cells. In addition, mice bearing Cl66-siVEGFC tumors lived significantly longer than mice bearing Cl66-control tumors. Furthermore, our data suggest that inhibition of VEGF-C modulates immune cell infiltration and their function, which might be critical in tumor immunity. In summary, our data show that inhibition of VEGF-C expression using siRNA-mediated gene silencing vectors reduces lymphangiogenesis and lymph node and spontaneous lung metastasis, and enhances survival.Cancer Research 11/2005; 65(19):9004-11. · 8.65 Impact Factor
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ABSTRACT: Based on a previous report on the effect of a matrix metalloproteinase (MMP) inhibitory compound, MMI270, in regulating tumor-induced angiogenesis, as well as recent findings concerning functional correlations among tumor metastasis, angiogenesis and lymphangiogenesis, we investigated the anti-metastatic efficacy of MMI270 in a murine model of lymph node metastasis of lung cancer, and analyzed whether this inhibitor could also regulate lymphangiogenesis-related properties of murine lymphatic endothelial cells (LECs) and invasive properties of Lewis lung cancer (LLC) cells. The observation that MMI270 led to a significant decrease in the weight of tumor-metastasized lymph nodes of mice led us to test its anti-lymphangiogenic and anti-invasive effects in vitro. Murine LECs were characterized by an in vitro tube formation assay, by semi-quantitative RT-PCR assay to examine the expression of mRNAs for flt-4, Flk-1, Tie-1, Tie-2, CD54/ICAM1, vWF, MMPs and uPA, and by western blotting to confirm the protein expression of flt-4 and CD31/PECAM. This is the first report on the expression of MMP-2, MMP-9 and MT1-MMP in murine LECs, as well as on the inhibition of their enzymatic activity, and of the invasive ability and tube-forming property of LECs by an MMP inhibitor. Furthermore, MMI270 was shown to strongly inhibit the activity of MMP-2 and -9 produced by LLC cells and the invasion of these cells through Matrigel. In summary, the present results indicate that MMI270, apart from its anti-tumor angiogenic application, might be useful as an anti-metastatic drug, on the basis of its downregulatation of both the lymphangiogenesis-related properties of LECs and the invasive properties of LLC cells in vitro.Cancer Science 02/2004; 95(1):25-31. · 3.48 Impact Factor
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ABSTRACT: Vascular endothelial growth factor receptor-3 (VEGFR-3/Flt4) binds two known members of the VEGF ligand family, VEGF-C and VEGF-D, and has a critical function in the remodelling of the primary capillary vasculature of midgestation embryos. Later during development, VEGFR-3 regulates the growth and maintenance of the lymphatic vessels. In the present study, we have isolated and cultured stable lineages of blood vascular and lymphatic endothelial cells from human primary microvascular endothelium by using antibodies against the extracellular domain of VEGFR-3. We show that VEGFR-3 stimulation alone protects the lymphatic endothelial cells from serum deprivation-induced apoptosis and induces their growth and migration. At least some of these signals are transduced via a protein kinase C-dependent activation of the p42/p44 MAPK signalling cascade and via a wortmannin-sensitive induction of Akt phosphorylation. These results define the critical role of VEGF-C/VEGFR-3 signalling in the growth and survival of lymphatic endothelial cells. The culture of isolated lymphatic endothelial cells should now allow further studies of the molecular properties of these cells.The EMBO Journal 09/2001; 20(17):4762-4773. · 9.82 Impact Factor