| Objectives:Holotrichia diomphalia Bates(Qi Cao)is a species of Melolonthidae that is indigenous to eastern Asian areas.Qi Cao was recorded in Shennong’s Herbal as traditional Chinese medicine.The previous activity screening of Qi Cao polysaccharides showed that HDPS-2Ⅱcould significantly inhibit tumor growth,but the pharmacological activity and mechanism of HDPS-2Ⅱagainst tumor angiogenesis were not clear.Therefore,this article aims to comprehensively evaluate the anti-tumor and anti-angiogenesis activities of HDPS-2Ⅱand make a preliminary exploration of HDPS-2Ⅱon its anti-tumor angiogenic molecular mechanism,so as to provide more data for promoting the research and development of a new anti-tumor drug of traditional Chinese medicine.Methods:1.The human umbilical vein endothelial cells(HUVEC)were induced by the conditioned medium from human hepatocarcinoma SMMC-7721 cells(SMMC-7721 CM)to produce the tumor-derived endothelial cells(H-TDEC)that were used as a pathological cell model in vitro.The MTT,scratch and Transwell assays were used to evaluate the inhibitory activities of HDPS-2Ⅱon the proliferation,migration and invasion of H-TDEC.The xenograft tumor model was created to evaluate the anti-tumor and anti-angiogenesis activities of HDPS-2Ⅱ.The tumor tissues of nude mice were observed using H&E staining.The immunohistochemical assay was used to detect the tumor microvascular density.2. The MTT,scratch and Transwell assays were used to evaluate the inhibitory activities of HDPS-2Ⅱon the proliferation and migration of HUVEC in vitro.The vascular fluorescent transgenic zebrafish were used to evaluate the inhibitory activity of HDPS-2Ⅱon physiological angiogenesis in vivo.3. The direct interactions between HDPS-2Ⅱand both Gal-1 and Gal-3 were determined by microscale therophoresis(MST).Western blot was used to determine the effects of HDPS-2Ⅱon the expressions of Gal-1,Vimentin and VE-cadherin in HUVEC and H-TDEC.4.Using the mouse brain microvascular endothelial cells(b End.3)as the tool cells,the inhibitory activities of HDPS-2Ⅱon their proliferation,migration,invasion and microtubule formation in vitro were investigated by MTT,scratch,Transwell and microtubule formation assays,respectively.Meanwhile,the expressions of Gal-1,vimentin and VE-cadherin in b End.3 and b-TDEC,which were induced by SMMC-7721CM,were determined by western blot.So that we could evaluate the feasibility of b End.3 replacing HUVEC to construct Gal-1 overexpressed endothelial cells for subsquent signal pathway research.5.The Gal-1 overexpressed b End.3 cells OE-Gal-1were constructed by lentivirus transfection.OE-NEG-CON cells were negtive control.Western blot was used to detect the effects of HDPS-2Ⅱon the expressions of Gal-1,p-VEGFR2,VEGFR2,vimentin and VE-cadherin in Gal-1 stably overexpressed b End.3 cells.Results:1.Compared with HUVEC,0.03～300 mg/L HDPS-2Ⅱhad a stronger inhibitory effect on H-TDEC proliferation at 24,48 and 72 h.The H-TDEC migration and invasion were significantly inhibited by 10 mg/L and 30 mg/L HDPS-2Ⅱ.The HDPS-2Ⅱcould suppress the growth of transplanted hepatocellular carcinoma in a dose-dependent manner in nude mice,and the tumor inhibition rate of 40 mg/kg HDPS-2Ⅱwas 65.58%(93.47%in sorafenib group).The tumor tissue in 40 mg/kg HDPS-2Ⅱgroup had a large area of necrosis,and the inhibition rate of tumor microvascular formation in vivo was 74.36%(87.18%in sorafenib group).2.Compared with control,0.03～300 mg/L HDPS-2Ⅱhad no significant inhibitory effect on HUVEC proliferation at 24,48 and 72h,while 30 mg/L HDPS-2Ⅱhad inhibitory effect on HUVEC migration.HDPS-2Ⅱhad no significant inhibition on the physiological angiogenesis of zebrafish internode.3. The MST results showed that HDPS-2Ⅱhad a stronger affinity with Gal-1(the affinity constant K_d=17.2±7.3μM)compared with Gal-3(K_d=527.18±0.3μM).Western blot results showed that compared with HUVEC,the expressions of Gal-1 and vimentin were up-regulated and the expression of VE-cadherin was down-regulated in H-TDEC after induction by SMMC-7721CM.After 24 hours of treatment,HDPS-2Ⅱcould down-regulate the expressions of Gal-1,vimentin and at the same time up-regulate the expression of VE-cadherin in concentration-dependent manners.Among them,the same concentration of HDPS-2Ⅱhad more significant effects on the expressions of Gal-1,vimentin and VE-cadherin in H-TDEC.4. The effects of HDPS-2Ⅱon the proliferation,migration,microtubule formation and the expressions of related proteins(Gal-1,vimentin and VE-cadherin)in b End.3and b-TDEC cells induced by SMMC-7721CM were similar to those of HDPS-2Ⅱon HUVEC and H-TDEC,so that the HUVEC could be replaced by b End.3 cells to construct the Gal-1 overexpressed endothelial cells for subsequent signaling pathway study.5. The western blot results showed that compared with OE-NEG-CON,the expression of Gal-1 was significantly increased in OE-Gal-1 cells,indicating that the Gal-1 overexpressed b End.3 cells were successfully constructed and screened.After 24hours of treatment,HDPS-2Ⅱcould down-regulate the expressions of Gal-1,p-VEGFR2,vimentin and up-regulate the expressions of VEGFR2 and VE-cadherin in concentration-dependent manners.Among them,the same concentration of HDPS-2Ⅱhad more significant effects on the expressions of Gal-1,p-VEGFR2,VEGFR2,vimentin and VE-cadherin in OE-Gal-1-TDEC cells induced by SMMC-7721CM.Conclusions:1.HDPS-2Ⅱcan inhibite the migration,invasion and microtubule formation of TDEC in vitro and tumor growth and tumor microvascular formation in vivo.HDPS-2Ⅱis an active anti-tumor polysaccharide of traditional Chinese medicine with low toxic and side effects on physiological angiogenesis but obvious inhibition on tumor angiogenesis.2.HDPS-2Ⅱmay inhibit the migration,invasion and microtubule formation of TDEC by blocking the Gal-1-VEGFR2 signaling pathway,thus producing the inhibitory effect on tumor angiogenesis. |
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