Anti-angiogenesis Effect Of Cardamonin And Its Mechanism Based On The Target Of MTOR

ObjectiveAngiogenesis plays an important role in the tumor progression; anti-angiogenesisis one of the main methods for current tumor therapy. Mammalian target of rapamycin（mTOR） is a key factor of tumor growth, which involved in the regulation of cellmetabolism and proliferation. In addition, mTOR contribute to tumor angiogenesis.Our previous study had demonstrated that cardamonin （CAR） could inhibit tumorgrowth by targeting mTOR, but whether it could affect tumor angiogenesis is unclear.This study was to investigate whether CAR could inhibit tumor angiogenesis via themTOR target; and the possible mechanism was further explored. This study willprovide the experimental evidence for the development of mTOR inhibitor.Methods1Experimental materialIn vitro: Human ovarian cancer cell line SKOV3; in vivo: Chicken embryoallantois membrane （CAM） model.2ProtocolSKOV3: Normoxia group is divided into solvent control group （0.1%DimethylSulfoxide, DMSO）, RAP group (10^-7mol·L^-1), low-dose CAR group (3×10-6mol·L^-1)and high-dose CAR group (3×10^-5mol·L^-1), each group plus CoCl₂(1.5×10^-4mol·L^-1)to become hypoxia comparison group.CAM: Divided into the PBS blank group, conditioned medium （CM） model group,RAP group (10^-5mol·L^-1), low-dose CAR group (3×10^-4mol·L^-1), medium-dose CARgroup (10^-3mol·L^-1) and high-dose CAR group (3×10^-3mol·L^-1).3Measurements and methodology3.1The morphology of cell was observed by microscope;3.2Cell proliferation was measured by MTT method; 3.3The mRNA expression of hypoxia-inducible factor1α （HIF1α）, hypoxia-induciblefactor2α （HIF2α） and vascular endothelial growth factor （VEGF） were measuredby semi-quantify RT-PCR;3.4The protein expression of HIF1α, HIF2α, VEGF, mTOR, p-mTOR, S6K1, p-S6K1were performed by Western blotting;3.5CAM: Counting the number of vessels converging toward the grafts.Results1SKOV3cell proliferation was significantly inhibited by mimetic hypoxia （P <0.01）.CAR could inhibit the proliferation of SKOV3in a dose-dependent manner;2The phosphorylation of mTOR and S6K1was significantly decreased by CAR （P <0.01）, and high-dose CAR had a stronger inhibitory effect. Nevertheless, CAR hadno influence on the total protein expression of mTOR and S6K1;3CAR decreased the protein expression of HIF1α and HIF2α induced by CoCl₂（P <0.01）; however, the mRNA expression was not affected. High-dose CAR had astronger inhibitory effect on the protein expression of HIF1α （P <0.01）;4CAR could decrease the protein and mRNA expression of VEGF in both normoxiaand mimetic hypoxia SKOV3cells （P <0.01）. There was no significant differencebetween high-dose and low-dose of CAR on the mRNA expression. However, theinhibitory effect of high-dose CAR on protein expression of VEGF was strongerthan that of low-dose CAR;5CAR could inhibit the number of CAM vessels in a dose-dependent manner.Conclusions1CAR decreases the expression of HIF1α, HIF2α and VEGF through themTOR-dependent manner;2CAR may decrease the expression of HIF1α, HIF2α and VEGF through the mTOR-independent manner;3CAR could inhibit angiogenesis stimulated by growth factor.