| Tumor angiogenesis plays an important role in the tumor growth, invasion and metastasis. It supplies nutrients to tumors for their growth and removes metabolic wastes from tumors. In addition, new blood vessels provide passageways to tumor cells for their entrance. Tumor angiogenesis is a complex process, tumor cells and endothelial cells secrete angiogenic growth factors, which can bind activated endothelial cells to endothelial receptors, then cells secrete protease which can degrade vessel basal membranes. The activated endothelial cells proliferate, migrate and form capillary blood vessels and induce angiogenesis finally.Abundant studies have demonstrated that many flavonoids which exist in plants have extensive effects including antioxidant, anti-inflammatory, immunomodulatory and selective estrogen-like properties and they have good prevention on tumor and angiocardiopathy. In addition, they possess antiangiogenesis effects and have received considerable attention in cancer research. Liquirigenin(LQ) is a flavanone existed in Radix glycyrrhizae glycyrrhizae, with the polyphenolic structure. Our former research indicates that LQ can suppress cell proliferation of human hepatocarcinoma cells (SMMC-7721) and human cervical carcinoma cells (HeLa) and induce cells apoptosis via the mitochondrial pathway. LQ has a significant inhibitory effect in H22 tumor-bearing mice in vivo. However, the effects and mechanisms of LQ on antiangiogenesis are not clear. This study was aimed to elucidate the angiogenic effects of LQ on human umbilical vein endothelial cells (HUVECs) and human cervical carcinoma (HeLa) cells. To further clarify the molecular mechanisms of its anti-angiogenesis effects, we detected the angiogenic growth factors such as VEGF and HIF-1αand explored the mediate role of PI3K/AKT and ERK1/2 signaling pathways in anti-angiogenesis to supply evidence for antitumor of LQ. Objective: To study the effects of LQ on cell viability, migration and capillary-like tube formation in Human Umbilical Vein Endothelial Cells (HUVECs) and investigate the role of PI3K/AKT/p70S6K and ERK1/2 pathways in mediating angiogenic growth factors VEGF and HIF-1αexpression.Methods: MTT assay was used to detect the cell viability of LQ on HUVECs. The effect of LQ on HUVECs migration was measured by wound healing method.The effect of LQ on tubular structure of HUVECs was measured by capillary-like tube formation assay. The protein levels of VEGF, HIF-1α, PI3K/AKT/p70S6K and ERK1/2 pathways were detected by Western Blot.Results:(1) LQ (25, 50, 75, 100, 200 and 300μM) significant decreased cell viability of HUVECs for 12, 24 and 48h in a dose-and time-dependent manner. The viability rates were 90.76%, 88.54% and 86.3% with 25μM LQ treatment for 12 h, 24 h and 48 h, respectively, and were 68.16%, 63.68% and 57.19% at the same time with 300μM LQ treatment. (2) HUVECs were treated with LQ (25~100μM) for 12h. LQ inhibited the migration and formation of capillary-like structures of HUVECs in a dose-dependent manner. (3) LQ inhibited the expression of VEGF and HIF-1αby PI3K/AKT/p70S6K pathway in HUVECs. To further confirm the result, HUVECs pretreated with PI3K inhibitor LY294002, p70S6K inhibitor Rapamycin, and ERK1/2 inhibitor U0126, respectively, and the expression of VEGF and HIF-1αwhich mediated by PI3K/AKT/p70S6K and ERK1/2 pathways were also decreased. The results were consistent with the previous study.Conclusion: LQ can inhibit cell viability, migration and capillary-like tube formation of HUVECs and inhibit VEGF and HIF-1αexpression through PI3K/AKT/p70S6K and ERK1/2 pathways. Objective: To investigate the anti-angiogenesis effects of LQ on angiogenic growth factors VEGF and HIF-1αin human cervical carcinoma (HeLa) cells and study the mechanism of the effects on the gene expression and PI3K/AKT/mTOR-p70S6K and ERK1/2 pathways.Methods: RT- PCR was used to detect mRNA levels of VEGF and HIF-1α. The protein levels of VEGF, HIF-1α, PI3K/AKT/mTOR-p70S6K and ERK1/2 pathways were detected by western blot, and the degradation and synthesis of HIF-1αprotein were detected.Results: (1) HeLa cells were treated with LQ (25,50,75 and 100μM) for 12h, the expression of VEGF at the protein and mRNA levels were both decreased, and the HIF-1αprotein accumulation was inhibited, but no obvious effects on HIF-1αmRNA were observed. (2) HeLa cells were treated with cycloheximide (CHX) or proteasome inhibitor MG132, LQ reduced HIF-1αprotein accumulation by reducing its stability and decreasing its synthesis. (3) LQ inhibited the PI3K/AKT/mTOR-p70S6K pathway in HeLa cells. To further confirm the result, HeLa cells pretreated with PI3K inhibitor LY294002 and p70S6K inhibitor Rapamycin, respectively, the expression of VEGF and HIF-1αwhich mediated by PI3K/AKT/mTOR-p70S6K pathway were also decreased. The results were consistent with the previous study, but there was no obvious inhibitory effect on ERK1/2.Conclusion: LQ inhibited angiogenic growth factors VEGF and HIF-1αexpression in HeLa cells and the molecular mechanism was related to PI3K/AKT/mTOR-p70S6K signaling pathway. |
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