The Effect Of Genistein On Tumor Recurrence And Metastasis After Partial Hepatectomy In Liver Cancer

Primary hepato-cellular carcinoma(HCC) is a common cancer in China and some Asian and African regions. The disease incidence of HCC ranges the fifth in various kinds of cancers. Nowadays, curative resection is generally accepted as the most effective treatment for HCC, but the recurrence rate after radical operation is up to 60-80%. The high recurrence rate becomes the major obstacle of improving prognosis. However, not a effective method has been found so far to prevent recurrence and metastasis of HCC. HCC shows low sensitivity to most anticancer drugs. Traditional drugs, such as 5-FU, cisplatin, ADM and MMC, show low curative effect(10-20%). So, it's meaningful to find a drug, which is effective and hypotoxic, or could decrease chemotherapeutics dosis and increase curative effect when it's combined with chemotherapy drugs. Genistein is a secondary metabolic product in the process of growth of soyabean, and it shows prominent anticarcinogenic effect in recent reseaches. We designed vitro experiment to study the impact of genistein on the proliferation of high-metabasis HCC cell line(HCCLM3), and on adhering, moving and invasive abilities of HCC cell which are closely relative to metastasis, and correlating molecular mechanisms. We built high-metabasis-and-recurrent athymic mouse model simulating post-operation human HCC, to study the impact of genistein on metastasis and recurrence of post-operation HCC and correlating molecular mechanisms. We arranged vivo and vitro experiments to study genistein's synergistic effect with cisplatin in resisting tumor proliferation, recurrence and metastasis, and correlating molecular mechanisms of the congenerous effct of the two drugs.Partâ… The impact of genistein on the proliferative, invasive and metabasis ability of HCCLM3 and the mechanismobjective: To approach the impact of genistein on the proliferation and apoptosis of high-metabasis HCC cell line(HCCLM3), and on adhering, moving and invasive abilities of HCC cell which are closely related to tumor metastasis, and the functional mechanisms of genistein. Methods: Take human high-metabasis HCC cell line(HCCLM3) as the research object. Observe the influence of genistein on the morphology of HCC cells using inverted microscope. Detect the impact of genistein of different concentration on the growth of HCC cells using MTT. Detect cell apoptosis rate and cell cycle variation after treated with genistein with flow cytometry. Observe the variation of cell adhering, moving and invasive abilities after drug action using adhering, moving and invasion experiment. And detect the influence of genistein on the expression of tumor metastasis relative factors VEGF,MMP-2,TIMP₂ and E-Cadherin with immunocytochemical methods. Results: (1) In genistein-treated group, cell survival rate decreased compared to control group. And this effect became more significant with the increase of time and drug concentration. (2) In control group, apoptosis rate was 10.30%, G₀/G₁stage cell occupied 33.48%. In genistein-treated group, apoptosis rate was 13.90%, G₀/G₁stage cell occupied 41.85%. The differences were both statistical significant. (3) Cells in genistein-treated group show adhere inhibition ratio to Matrigel and FN respectively as 55.65% and 45.33% (genistein concerntration 40umol/L) , as 75.16% and 66.67% (80 umol/L). In Transwell booth, relative invasive rate of HCCLM3 cell were 69.73% (40umol/L) and 53.59%(80 umol/L). Relative number of moving cell were 79.70% (40 umol/L) and 70.17% (80 umol/L) .The differences compared with control group were all statistical significant. (4) In genistein-treated group, MMP-2 and VEGF HSCORE are 2.13±0.11 and 2.45±0.16 respectively, while in control group, they are 2.44±0.23 and 3.08±0.16 respectively. The differences were statistical significant. Conclusions: Genistein could inhibit the growth of HCCLM3 cell. The inhibiting effect is time and concentration dependent. Genistein could detain HCCLM3 cells at G₀/G₁ stage and increase the apoptosis rate of HCCLM3. Genistein could also inhibit the adhering, moving and invasive abilities of HCCLM3 cells which are relative to tumor metastasis, and this is because of that genistein could decrease the expression of MMP-2. Besides, genistein may reduce tumor angiogenesis through decreasing the expression of MMP-2 and VEGF. Partâ…¡The impact of genistein on tumor recurrence and metastasis after hepatectomy inathymic mouse hepatoma and the mechanismObjective: To approach the impact of genistein on tumor recurrence and metastasis after hepatectomy in athymic mouse hepatoma and the mechanism. Methods: Build high-metabasis-and-recurrent athymic mouse model simulating post-operation human HCC. For experimental group, administer the mice with 2mg/kg genistein solution through peritoneal injection every other, day, altogether for 4 weeks, and observe general state of health and tumor recurrence and metastasis of the mice. Detect the expression of MMP-2, TIMP-2, E-Cadherin and VEGF in recurrent tumor using immunohistochemical methods. Results: (1) Athymic mice in control group had an average liver recurrent focus volume 1917.00±647.55 mm³. They had intro-liver disseminating focus as 1.67±1.21 pieces per mouse, pulmonary metastasis focus 5.50±1.05 pieces per mouse, metastasis to other organs 2.17±1.17 pieces per mouse. In genistein-treated group, mice had an average liver recurrent focus volume 1011.884±4.1.14 mm³. They had intro-liver disseminating focus as 0.50±0.76 pieces per mouse, pulmonary metastasis focus 3.75±1.58 pieces per mouse, metastasis to other organs 1.00±0.76pieces per mouse. The differences were all statistical significant. (2) In control group, MMP-2 and VEGF HSCORE of liver recurrent tumor are 2.75±0.04 and 2.58±0.05 respectively, while in genistein-treated group, they are 2.56±0.19 and 2.43±0.04 respectively. The differences were statistical significant. Conclusions: Genistein could restrain the growth of recurrent focus and intro-liver metastasis in athymic mice after hepatectomy, and it could also restrain metastasis to lung and other organs. The mechanism may be that genistein could decrease the expression of MMP-2 and VEGF in recurrent tumor.Partâ…¢The impact of genistein combined with cisplatin on tumor recurrence and metastasis after hepatectomy and the mechanismObjective: To approach the synergistic inhibiting effect of genistein and cisplatin on tumor recurrence and metastasis after hepatectomy and the mechanism. Methods: Use MTT to detect the effect of cisplatin combined with genistein of different concentration on the proliferation of HCCLM3 cells, use median-effect principle and formula to calculate median-effect dose and combination index. Use flow cytometry to detect cell apoptosis rate and cell cycle variation after treated with genistein combined with cisplatin. Observe the variation of cell adhering, moving and invasive abilities after drug action (ginistein combined with cisplatin)using adhering, moving and invasion experiment. Detect the influence of the drugs(genistein combined with cisplatin) on the expression of VEGF, MMP-2 and NF-Kb of HCCLM3 cells with immunocytochemical methods. Build high-metabasis-and-recurrent athymic mouse model simulating post-operation human HCC, and observe the effect of the drugs(genistein combined with cisplatin) on tumor recurrence and metastasis of the athymic mice after hepatectomy, then use Jin Zhengjun formula to evaluate whether the two drugs perform coordinate repression on liver recurrent focus and pulmonary metastasis. Use immunohistochemical methods to detect the effect of genistein combined with cisplatin on the expression of NF-κB, MMP-2 and VEGF in liver recurrent tumor. Use real time fluorescent quantitation PCR to detect the effect of genistein combined with cisplatin on the expression of MMP-2 mRNA of liver recurrent tumors. Results: (1) In genistein combining with cisplatin group, cell survival rate decreased more than single-drug group. When the drugs are used Single, the median-effect dose of genistein and cisplatin were 152.44umol/L and 52.75umol/L respectively, but when they are used combined, the median-effect dose of genistein and cisplatin were 59.30umol/L and 14.83umol/L respectively. Genistein and cisplatin performed antagonism at low concentration, but when inhibition ratio exceeded 41.1%, I.e. genistein concentration exceeded 41.50umol/L, cisplatin concentration esceeded 10.38umol/L, they performed synergism when used together. (2) In genistein combining with cisplatin group, apoptosis rate was 67.30%, G₀/G₁ stage cells occupied 93.88%. In single genistein group, apoptosis rate was 13.90%, G₀/G₁ stage cells occupied 41.85%. In single cisplatin group, apoptosis rate was 48.54%, G₀/G₁ stage cells occupied 63.95%. The difference between combined drugs and single drug are statistical significant. (3) Between genistein combined with cisplatin group and single genistein group, the difference of cell adhere inhibition ratio to Matrigel and FN was statistical insignificant. The difference of relative invasive rate and Relative number of moving cell between the two groups were statistical insignificant either. (4) In single cisplatin group, MMP-2 and NF-κB HSCORE of HCCLM3 cells were 2.78±0.19 and 2.95±0.08 respectively, while in genistein and cisplatin combined group they are 2.12±0.13 and 2.46±0.07, in control group they are 2.44±0.23 and 2.79±0.06. The difference among three groups are statistical significant. (5) Compared with single drug group, mice in genistein combined with cisplatin group had smaller liver recurrent focus volumn, lesser pulmonary metastasis. Using Jin Zhengjun formula to calculate q value of hepatoma recurrent focus volumn suppression and pulmonary metastasis suppression, they were 1.09 and 1.55 respectively. (6) In single cisplatin group, NF-κB and MMP-2 HSCORE of mice recurrent tumor were 2.98±0.04 and 3.00±0.04 respectively, while in genistein and cisplatin combined group they are 2.38±0.03 and 2.47±0.10, in control group they are 2.51±0.04 and 2.75±0.04. The difference among three groups are statistical significant. (7) Using real time fluorescent quantitation PCR to detect the expression of MMP-2 mRNA, single cisplatin group was 2.06 times that of control group, while genistein combined with cisplatin group was 0.44 times that of control group. Conclusions: In vitro, when genistein was combined with cisplatin, accompanying the increase of concentration, the two drugs perform synergistic repression on the proliferation of HCCLM3 cells. When genistein was combined with cisplatin, the effect of inducing cell apoptosis is more significant than the two drugs are single used, and the major mechanism is blocking cells at G₀/G₁ stage. In vitro, when genistein was combined with cisplatin, the depressing effect on adhering, invasing and moving abilities of HCCLM3 cells was not significantly greater than single drug group. In vivo, genistein and cisplatin add their depressing effect on tumor recurrent focus after hepatectomy, and they perform synergistic repression on pulmonary metastasis after hepatectomy. The mechanism of their synergistic repression may be that genistein could inhibit the heightening of NF-κB which is induced by cisplatin, thus increase apoptosis of tumor cells, inhibit proliferation. Meanwhile, genistein could inhibit the increase of MMP-2 which is due to the heightening of NF-κB.