| Purpose:To investigate the inhibitory effect on the metastasis of B16-F10 melanoma and related mechanisms in vitro and in vivo by Ginkgo biloba exocarp extracts(GBEE).Methods:The C57BL/6J mice with B16-F10 melanoma lung metastasis model was established.The C57BL/6J mice were randomly divided into normal control group,model control group,positive control group,GBEE treatment group,and each group contained ten mice.Normal control group and model control group were given normal saline(NS)at a volume of 0.1 mL/10 g by intraperitoneal injection(ig),once a day for 17 d;the positive control group was given cis-Dichlorodiamineplatinum(?)(DDP)at a dose of 5 mg/kg by intraperitoneal injection(ip),once every other day for 7 d;GBEE treatment groups were given GBEE at a dose of 50,100,200 mg/kg by ig,once a day for 17 d.On the 2nd day after the end of administration,the weight of the transplanted tumor was measured by removing the tumor tissue and then the tumor inhibition rate was calculated.After lung tissue was removed,the lung metastasis foci was observed under dissecting microscope,and the lung metastatic rate and anti-metastatic rate were calculated.Sections of lung tissue and tumor tissue were prepared.The pathological changes of lung tissue were observed by hematoxylin-eosin(HE)staining.The expression of CD34 in the transplanted tumor was detected by immunohistochemistry and then the microvessel density(MVD)was calculated.At the same time,the expression of HIF-la,VEGF and MMP-9 were detected with immunohistochemistry.B16-F10 cells and human umbilical vein endothelial cells(HUVEC)were cultured in vitro.MTT assay was used to detect the effect of GBEE on the proliferation of B16-F10 cells in vitro.The effect of GBEE on the heterogeneous adhesion of B16-F10 cells to HUVEC was observed by cell adhesion assay.The effect of GBEE on the migration of B16-F10 cells was detected by cell wound healing assay.qRT-PCR assay and Western Blot assay were used to detect the effect of GBEE on the level of HIF-1?,VEGF,VEGFR2,NF-?B,MMP-9,p-PI3K/PI3K,p-Akt/Akt in vitro and in vivo.Results:The results of the experiment in vivo found that GBEE(50,100,200 mg/kg)inhibited the growth and lung metastasis of B16-F10 melanoma transplanted tumor.And GBEE made the expression of MMP-9 decreased.Moreover,GBEE inhibited the expression of CD34 and made MVD reduced.All of these effects were dose-dependent.Meanwhile,GBEE inhibited the mRNA and protein expression of HIF-la,VEGF and VEGFR2 in B16-F10 transplanted tumor tissue,and the inhibitory effect showed a dose-effect relationship.GBEE made the protein expression of p-PI3K,p-Akt in B16-F10 transplanted tumor tissue decreased in a dose-dependent manner,but had no obvious effect on the protein level of PI3K and Akt.The results of the experiment in vitro showed that GBEE significantly inhibited the proliferation of B16-F10 cells in the concentration range of 5-320 ?g/mL.And with the increase of drug concentration,the inhibitory rate increased.GBEE(10,20,40 ?g/mL)inhibited the migration of B16-F10 cells and the heterogeneous adhesion of B16-F10 cells to HUVEC in a concentration-dependent manner.At the level of mRNA and protein,GBEE suppressed the expression of NF-?B,MMP-9,p-PI3K,and p-Akt in B16-F10 cells in a concentration-dependent manner.GBEE had no significant effect on the level of PI3K and Akt.Conclusion:The mechanism of GBEE inhibiting the metastasis of B16-F10 melanoma may be involved inhibiting tumor cell proliferation,migration,heterogeneous adhesion,and ECM and BM degradation via regulation of PI3K/Akt/NF-?B/MMP-9 signaling pathway,inhibiting angiogenesis by interfering PI3K/Akt/HIF-1?/VEGF signaling pathway. |
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