Inhibitory Effect Of Akebia Fruit Extracts On Adhesion,Migration And Invasion Of Human HepG2 Hepatoma Cells And Related Mechanisms

Objective: To observe the inhibitory effect and mechanism of Traditional Chinese Medicine Akebia Fruit extracts on the adhesion,migration,and invasion of human HepG2 hepatoma cells.Methods:(1)Different concentrations of Traditional Chinese Medicine Akebia Fruit extracts(ATKSE,0.6?3.0 mg/m L)were applied to HepG2 cells,and integrin inhibitor Cilengitide(CIL,0.125?32 ?mol/L)was used as control simultaneously.MTT method was adopted to observe the effect of ATKSE on cell viability,in order to obtain a suitable dose for subsequent experiments.(2)Four groups were set up as follow: the blank control group,ATKSE group(1.0 mg/m L),CIL group(1.0 ?mol/L),and a half-dose combination group(ATKSE 0.5 mg/m L + CIL 0.5 ?mol/L).Cell-matrix adhesion assay,Transwell invasion assay,Transwell Matrigel invasion were used separately to detect effects of ARKSE on the adhesion,migration and invasion of HepG2 cells.(3)Scanning electron microscopy(SEM)was used to observe on the surface ultrastructure of HepG2 hepatoma cells.(4)Observing related moleculars on the Integrin/Focal Adhesion pathway and matrix metalloproteinases by Western Blot,including integrins such as ITGA2,ITGB5,ITGB1 and ITGAV,FAK and its phosphorylated proteins p-FAK(Y397)and p-FAK(Y576+577),other related molecules in the pathway,such as ERK1/2,Rhoa,p JNK and Ras,classical adhesion molecules such as E-cad and N-cad,and matrix metalloproteinases MMP2 and MMP9.(5)Further,using the FAK inhibitor PF-573224,five groups including the blank control group,ATKSE group(1.0 mg / m L),FAK inhibitor group(FAKi 2.0 ?mol / L),a half-dose combination group(ATKSE 0.5 mg /m L+ FAK i 1.0 ?mol/L)and a full-dose combined group(ATKSE 1.0 mg/m L + FAKi 2.0 ?mol/L).Scratch healing assay and Transwell invasion assay were applied to observe the role of FAK inhibition in ATKSE affecting the migration and invasion ability of HepG2 cells.Results:(1)There was almost no change in the morphology and quantity of HepG2 cells under the concentration of 0.6 mg/m L and 0.9 mg/m L of ATKSE;the number of cells decreased relatively from 1.2 mg/m L,the number of cells decreased sharply from 1.8 mg/m L and the state became worse.The overlapping growth characteristics of HepG2 cells disappeared and the cytotoxicity of emerged.(2)MTT results showed that after ATKSE intervension,the IC50 value was about 2.59 mg/m L at 24 h,the IC50 value was about 2.39 mg/m L at 48 h,and the IC50 value at 72 h was about 2.0 mg/m L.The IC50 value was not obtained after 24 h of CIL treatment.The IC50 value was nearly 2.0 ?mol/L at 48 h,and 1.57 ?mol/L at 72 h.The 1.0 mg/m L ATKSE and 1.0 ?mol/L CIL were selected for subsequent experiments.(3)In the cell-matrix adhesion assay,compared with the blank control group,the adhesion rate of each group showed a downward trend(P <0.05),the adhesion rate(%)was 100.00±4.94 in the blank control group,and 83.67±4.04 in ATKSE group,79.69±3.26 in CIL group,and 78.64±2.71 in the semi-dose combination group.(4)In the scratch healing assay,the migration ability of HepG2 cells decresed post different treatment(P < 0.05).The migration rate(%)of the blank control group was 10.32,the ATKSE was 3.10,the CIL group was negative 2.58,and the semi-dose combination group was 0.02.(5)In the invasion experiment,the number of invaded cells declined after administration.Wherein the blank control group was 173 ± 20,ATKSE group was 142 ± 2,CIL group was 130 ± 19,and half dose combination group was 90 ± 13.The difference between the above medication groups and the blank control group was statistically significant(P <0.05);besides,the number of the semi-dose combination group was significantly lower than that of the ATKSE group and the CIL group(P <0.05).(6)The SEM results showed that the overall proliferation of the cells in the blank control group was not active,and the cilia shortened to a sucker-like shape on the cell surface,as a whole the cells morphology is plump.In the ATKSE group,the cilia shortening trend is obvious,the length is different,the thickness is uneven,and the suction cup shape has an expanding trend.The number of cilia connecting cells in the CIL group was significantly reduced,and a small number of cells showed that the cilia atrophy was exhausted;the semi-dose group manifested the feature of the above two groups,and the cilia shortening trend was obvious and the length was different.(7)WB results showed that compared with the blank control group,the Integrin-related protein showed a similar downward trend after ATKSE or CIL treatment,and ITGA2 and ITGB5 showed a dosage reduction and synergistic effect after semi-dose combination(P < 0.05).The expression levels of p-FAK(Y576+577)and p-FAK(Y397)decreased treated by ATKSE or CIL,seperatly(P < 0.05).The expression of E-cad decreased after CIL treatment(P < 0.05);while N-cad increased(P < 0.05)after ATKSE or CIL.The expression of p-ERK1/2 decreased after ATKSE or CIL intervention,and there was a dosage reduction and synergistic effect after semi-dose combination(P < 0.05).After CIL or half-dose combined treatment,the expression of p-JNK decreased(P < 0.05).The expression of MMP2 was down-regulated by ATKSE or CIL(P < 0.05).(8)In the further migration ablility test using FAK inhibitor,the migration rate of other groups decreased compared with the blank control group(P < 0.05).The values of each group(%)was 14.28 in the blank control group,7.02 in the ATKSE group,2.93 in the FAKi group,4.71% in the half-dose comination group,and negative 2.75 in the full-dose combination group.(9)In the further invasion assay,compared with the blank control group 100±10.03(%),the invasive rate of all medication groups decreased(P <0.05),and the degree of combined group was the most.The invasive rate of each group was 56.85±4.35,64.62±6.09,64.62±6.68,and 34.43±4.32,respectively.Conclusions:(1)Chinese Medicine Akebia Fruit,possessing traditional function of promoting qi and activating blood circulationits,extracts from which can inhibit the proliferation,adhesion,migration and invasion of HepG2 hepatoma cells.(2)Akebia Fruit extracts can affect the surface cilia structure of HepG2 liver cancer cells,thus weakening the tight junction between cells.(3)The above effects of the Akebia Fruit extracts may be achieved through the following mechanisms: inhibition of upstream proteins on Integrin/Focal pathway such as ITGA2,ITGB5 and p-FAK,then downstream moleculars such as p-ERK1/2,p-JNK were partially deactivated,further,the MMP2 is inhibited indirectly or directly,that is,by suppressing different links of proliferation,adhesion,migration and invasion,inhibitory effect of migration and invasion are finally achieved.(4)In combination with the integrin inhibitor Cilengitide,there is a dosage reduction and synergistic effect in inhibiting the invasion ability of HepG2 cells.However,the mechanism is not entirely same.