The Mechanism Of Extract Of Fungi Of Huaier In The Interventional Therapy Of Hepatocarcinoma: An Experimental Study

ObjectiveTo explore the mechanism that extract of fungi of huaier inhibits the growth of thehuman hepatocellular carcinoma cells (Hep-G2) in vitro.To investigate the therapeutic effect of transcatheter arterial chemoembolizationcombined with extract of fungi of huaier on the expression of VEGF,MVD and hepaticfunction, the growth and metastasis of hepatic tumor of rabbit VX2.To investigate the therapeutic effect of extract of fungi of huaier combined withtranscatheter arterial chemoembolization on the apoptosis and the expression of associatedprotein P53,Bax and Bcl-2 of rabbit VX2 hepatocarcinoma.To provide the theory of treatment Hepatocellular carcinoma by extract of fungi ofhuaier combined with transcatheter arterial chemoembolization (TACE).Materials and MethodsHuman liver cancer cell line Hep-G2 was cultured in DMEM medium containing 10%fetal bovine serum. The human hepatocellular carcinoma cells lines (Hep-G2) in log phasewere cultured in DMEM medium containing 10% fetal bovine serum and extract of fungiof huaier of different concentrations (1mg/ml, 2mg/ml, 4mg/ml, 8mg/ml) in DMEMmedium containing 10% fetal bovine serum for 24h, 48h and 72h respectively. The growthinhibitory effect of extract of fungi of huaier on Hep-G2 was observed by MTT assay andthe apoptosis rates of cells were also observed by flow cytometry.VX2 tumor pieces were surgically implanted into the left liver lobe of New Zealandwhite rabbits, two weeks later, 36 rabbits with experimental hepatic tumors diagnosed byMRI were randomly divided into 3 groups, 12 rabbits in each group. The rabbits in Group A (control group) were infused with 0.2ml/kg physiological saline via the hepatic artery;infused with lipiodol 0.2ml/kg+MMC 0.5mg/kg via the hepatic artery in Group B (TACEgroup); given by oral administration with extract of fungi of huaier 500mg/kg.d afterTACE in Group C (TACE+administration with extract of fungi of huaier).The commonconditions of the rabbits were observed daily after operation. The level of AST and ALT inserum was respectively tested at 1 day before operation, 3 days, 7 days and 14 days afteroperation. Two weeks after treatment, all experimental rabbits were sacrificed and thetumors were taken out. The volume and necrotic area of the implanted tumor weremeasured. The metastases in the liver, lungs and celiac lymph node were recorded.Immunohistochemical staining was performed to detect the expression and localization offactorⅧ,VEGF,P53,Bax and Bcl-2 proteins. The MVD was calculated by counting thefactorⅧpositive endothelial cells. The apoptosis index of tumor cells was assessed withthe method of TUNEL.ResultsThe growth inhibitory rates of liver cancer Hep-G2 cells were observed by MTT assay:The growth of Hep-G2 cells were significantly inhibited by extract of fungi huaier. Uponthe extract of fungi of huaier administration in the concentration of 1-8mg/ml and culturedfor 24h, 48h and 72h respectively, the growth inhibitory rates was an obvious difference ascompared to the control group (p＜0.05). The growth inhibitory rates raised with increasingthe concentration of extract of fungi huaier and there is evident dose-dependent manner.The apoptosis rates of liver cancer Hep-G2 cells were observed by flow cytometry:Extract of fungi of huaier significantly induced apoptosis of liver cancer Hep-G2 cells.Upon the extract of fungi of huaier administration in the concentration of 1-8mg/ml andcultured for 24h, 48h and 72h respectively, there was an obvious difference as compared tothe control group (p＜0.05). With the increased concentration and the prolonged time ofextract of fungi of huaier with Hep-G2 cells, the apoptosis rates induced by extract of fungiof huaier gradually elevated. There were no significant difference in rabbits' weight and tumors' volume among 3groups at 1 day before operation (P＞0.05). The average rabbits' weight in group B wassignificant lower than those in group A and Group C at 14 days after operation (P＜0.05),but there was no significant difference between group A and group C (P＞0.05).There was no significant difference of rabbits' AST and ALT in serum among 3 groups at1 day before operation; There was significant difference of rabbits' AST and ALT in serumamong 3 groups at 3 and 7 days after operation (F=28.411, 55.537 P＜0.05; F=8.565,6.401 P＜0.05), and there was significant difference between group B and group C(P＜0.05).Two weeks after treatment, the average volume, the necrotic rate and growth rate of theimplanted tumor was significantly different between group B, group C versus group A(P＜0.01); Meanwhile there was statistical different between group B and group C(P＜0.05).The metastases rate in the livers and lungs was not significantly different between groupA and group B (P＞0.05), while was significantly different in group A and group C(P＜0.05), group B and group C(P＜0.05). The incidence of metastases in celiac lymphnode was of no statistical difference among group A, B and C(P＞0.05).The MVD and the positive express of VEGF is the lowest in group C, the highest ingroup B, there was significant difference among 3 groups (F=5.22, P=0.011;χ~2=7.247,P=0.027 P＜0.05), and there was significant difference between group B and group C(P＜0.05).The apoptosis index of tumor cells was the minimum in Group A(7.31±2.85%) and themaximum in Group C(15.73±2.89%); there was significant difference of the apoptosisindex of tumor cells among 3 groups (P＜0.05), and there was significant differencebetween group B(12.83±3.51%) and group C (P＜0.05).The positive expression rate of Bax proteins was the minimum in Group A(16.67%) andthe maximum in Group C(83.33%); the positive expression rate of P53 and Bcl-2 proteins was the minimum in Group C(25.00%, 16.67%) and the maximum in Group A (91.67%,83.33%); there was significant difference of the positive expression rate of P53,Bax andBcl-2 proteins among 3 groups (P＜0.05), and there was significant difference betweengroup B and group C(P＜0.05).ConclusionThe extract of fungi of huaier can induce apoptosis and inhibit the proliferation ofHep-G2 cells.The extract of fungi of huaier can improve the rabbits' quality of life after TACE. It cannot only inhibit the growth and promote the necrosis of tumor, but also prevent the tumorfrom developing metastasis to the liver and lungs.The extract of fungi of huaier can improve the hepatic function of rabbit VX2hepatocarcinoma after TACE; it can treat hepatocarcinoma through diminishing theexpression of VEGF and inhibiting the angiogenesis of tumor.The extract of fungi of huaier may increase the expression of Bax proteins and decreasethe expression of P53 and Bcl-2 proteins; it may treat hepatocarcinoma through increasingthe apoptosis of hepatoma carcinoma cell.The extract of fungi of huaier is an ideal drug for clinical treatment of hepatic cancer.