| BackgroundNew research indicates that primary liver cancer with high incidence and high mortality having become internationally famous (ranks fifth in male and seventh in female about malignant tumor incidence, ranks second in male and sixth in female about mortality). Even several treatment technologies are increasingly perfect, such as operation excision, TACE, Gama ray, radiofrequency ablation and liver transplantation, etc., when HCC patients are diagnosed with advanced disease, they generally have a poor prognosis with median survival times of about 6-8 months. Therefore, how to prevent metastasis or recurrence of primary liver cancer so as to raise survival rate of the patients with liver cancer, which is the main direction of current research. Our research group for the first time on the basis of previous research results put forward the point of view:the main reason for the high rate of recurrence and metastasis of primary liver cancer is the hypothesis of "liver cancer having residual toxin be not yet clear". We believe that on one hand, the risk factors of primary liver cancer including hepatitis virus, aflatoxin, chlorinated organic compounds and toxin has not been effectively controlled; on the other hand, appropriate postoperative recurrence and metastasis of primary liver cancer tumor microenvironment persist. Therefore, how to continue to remove "liver cancer having residual toxin be not yet clear" may become the main path to prevent recurrence and metastasis of liver cancer. So venenum bufonis preparation, which for the above type of disease, comes into being. The venenum bufonis preparation has the effectiveness of clearing damp, dispersing blood stasis, clearing away heat and toxic materials. The recent researches about bufalin, which is the extractive of venenum bufonis preparation, are comfirmed having suppress functions for hepatoma cell lines of proliferation, invasion and migration. The mechanism for AKT targets in vitro experiment effects the corresponding factors in AKT/GSK3β/β-catenin/E-cadherin signal path. Bufalin also combining with the perspective of the tumor microenvironment has been comfirmed to suppress functions for several hepatoma cell lines of invasion and migration, gradually become the focused research at home and abroad. Primary liver cancer has organ-site tropism. So seting up the monoclonal human liver cell lines and lung targeting nude mice transplantation model, clarifying the previous extracorporal molecular mechanism with bufalin treatment and further determining the effective treatment method are imperative.ObjectiveThis study investigated the effect of bufalin on growth of orthotopic liver transplantation tumor mice bearing hepatocellular carcinoma cell line, orthotopic transplanted tumor tissues undergo necrosis and apoptosis, pulmonary metastases formation in vivo experiment. And with AKT as the target, it is further to explored the change of corresponding factors in AKT/GSK3 β/β-catenin/E-cadherin signal path and the mechanism being related to these phenomena.MethodsBased on the HCCLM3-R and corresponding nude mice transplantation model, we divided these animals which meet a criterion into four groups randomly and used physiological saline to dilute the bufalin and LY294002. Bufalin was intraperitoneally injected into two groups of mice at doses of 1 mg/kg and 1.5 mg/kg from day 8 to 38, and LY294002 was intraperitoneally into one group of mice at a dose of 100 mg/kg thrice weekly as a positive control. The control group was injected with an equal volume of saline (0.15 ml) from day 8 to 38. All mice were sacrificed and weighed on day 39. By measuring the orthotopic transplantation tumor weights, tumor volumes, the tumor inhibition rate to evaluate the effect of bufalin on tumor growth in nude mice in a dose-dependent manner. H&E staining revealed that the degree of necrosis and positive TUNEL staining was located in the nuclei to show the number of apoptosis, which can indicate orthotopic transplanted tumor tissues undergo necrosis and apoptosis induced by bufalin treatment. Bioluminescence showed the pixel numbers of pulmonary metastatic foci and H&E staining measure the cell number and area of pulmonary metastatic nodules. Then taken them together, analysis of serial lung sections indicated the influence of pulmonary metastases in bufalin-treated mice. For further detection of IHC and western blot analysis, bufalin was evaluated about the change of corresponding factors in AKT/GSK3 β/β-catenin/E-cadherin signal path and variation tendency about them.Result1.Bufalin effects on tumor growth of orthotopic transplanted tumor tissuesThe tumor weights were 1.93±0.45 g in NS control group,1.65±0.60 g in the low-dose bufalin-treated group,1.10±0.23 g in the high-dose bufalin-treated group and 0.76±0.23 g in LY294002-treated group. The tumor volumes were 1379.75± 741.48 mm3 in the control group,1174.69±692.42 mm3 in the low-dose bufalin-treated group,712.88±212.12 mm3 in the high-dose bufalin-treated group and 438.35±303.37mm3 in LY294002-treated group. Among them, the inhibition rates of tumors were 13.51±1.35% in low-dose bufalin-treated mice,46.27±2.06% in high-dose bufalin-treated mice and 66.84±1.40% in LY294002-treated group. Compared with the control group, the high-dose bufalin-treated group has statistical significance on above three respects (P<0.01, P<0.05, P<0.01, respectively), which is similar to LY294002-treated positive control group. The low-dose bufalin-treated group has statistical significance only on the inhibition rate.2.Orthotopic transplanted tumor tissues undergo necrosis and apoptosis effected by bufalin treatmentOrthotopic transplanted tumor tissues of control group, low-dose bufalin-treated group, high-dose bufalin-treated group and LY294002-treated positive group were made by H&E staining, which was one of the routine pathological examinations. There are focal necrosis in tumor tissues, nucleus dissolving and cytoplasm being eosinophilic in these four groups. Necrosis are mainly concentrated in the central part but cancer cells on the edge of tumor tissues are still active. The degrees of these four groups are mild, moderate, severe and severe necrosis, respectively.Positive TUNEL staining was located in the nuclei. The numbers of apoptotic cells in control, low-dose bufalin-treated, high-dose bufalin-treated and LY294002-treated positive groups were 63.20±10.33,107.80±14.20,146.80± 14.20 and 106.20±7.12 per 400x magnification field, respectively. The bufalin-treated groups were obviously different NS control group statistically (P<0.01), which is similar to LY294002-treated positive control group. 3.Bufalin effects on pulmonary metastasesFluorescence microscope with 580 nm bioluminescence showed that the pixel numbers of pulmonary metastatic foci in control, high-dose and low-dose bufalin-treated and LY294002-treated positive group mice were 17937.33±3997.06 pixels,2217.00±475.07 pixels,512.00±95.83 pixels and 356.50±83.29 pixels per lung. Further routine pathologic examination for lung serial section by H&E staining, each alveolar interstitials have tumor cells, which assemble, clustering, pleomorphism and pale nucleus dying. But they have different number of metastatic cells, being 112.17 ±19.05,68.17±9.47,39.67±3.72 and 36.33±5.99 per lung, respectively. Fluorescence quantitative was similar with HE staining results, which showed the experimental groups being obviously statistical different the control group (P<0.01). Also the high-dose bufalin-treated group is similar to LY294002-treated positive control group.4.Bufalin was evaluated about the change of corresponding factors in AKT/GSK3 β/ β-catenin/E-cadherin signal path and variation tendency by IHC.IHC staining was also scored combined with percentage of positive cells in cell nucleus or cytoplasm and degree of staining about orthotopic transplanted tumor tissues of control group, low-dose bufalin-treated group, high-dose bufalin-treated group and LY294002-treated. At the same time record the average optical density as immunohistochemical semi-quantitative evaluation:p-AKT, p-GSK3β,β-catenin, MMP-2 and MMP9 protein levels expressed positive and trendency of decline;total GSK3β expression of tumors was IHC 0 in the control, and IHC in the other groups showed positive; IHC exhibited weak staining of E-cadherin in the control group; AKT protein levels in four groups were negative. Observation expressions of several proteins about AKT/GSK33β/β-catenin/E-cadherin signaling pathways and do statistical research, it is concluded that above factors’change trendency are related with bufalin and its dose, and also it is similar to LY294002-treated positive control group.5.Bufalin was evaluated about the change of corresponding factors in AKT/GSK3β/ β-catenin/E-cadherin signal path and variation tendency by western blotThe control, low-dose bufalin-treated and high-dose bufalin-treated hepatomas were subjected to western blot analysis. Bufalin inhibited the expression of pAKT, pGSK3β,β-catenin, MMP-2 and MMP-9 in HCCLM3-R in a dose-dependent manner. On the other hand, the expression of GSK3β, GSK3βand E-cadherin raised gradually in a dose-dependent manner. Furthermore, the expression of AKT essentially unchanged. Observation expressions of several proteins about AKT/GSK3 β/β-catenin/E-cadherin signaling pathways and do statistical research, it is concluded that above factors’change trendency are related with bufalin and its dose, and also it is similar to LY294002-treated positive control group.Conclusion1.Bufalin inhibited tumor growth in nude mice in a dose-dependent manner, including reducing orthotopic transplantation tumor liver weight and volumes, and increasing the inhibition rates of tumors.2.Orthotopic transplanted tumor tissues undergo necrosis and apoptosis induced by bufalin treatment. Bufalin-treated groups’ necrosis were observed severely and numbers of apoptotic cells were increasing in a dose-dependent manner.3.Analysis of serial lung sections indicated significant inhibition of pulmonary metastases in bufalin-treated mice by decreasing area and numbers.4.Changes in the expression of numerous important regulatory proteins in the AKT/GSK3 β/β-catenin/E-cadherin signaling pathway occurred in a dose-dependent manner by treatment with bufalin, And the mechanism was related to above phenomena about inhibiting tumor growth in nude mice, inducing necrosis and apoptosis, inhibiting pulmonary metastases by bufalin-treated. |
PDF Full Text Request