| Background:In recent years, a lot of research was carried out by abroad scholars and had made a progress that how to reduce recurrence and mortality of liver cancer, but the survival rate is very low in a long time, the prognosis of liver cancer is no significant improve with many of treatments. Chinese medicine has become a hot topic in research of anticancer and wildly accepted for the people. Euphorbia helioscopia L as a traditional herbal, widely distributed in China, and its effect on liver cancer, nasopharyngeal cancer, esophageal cancer had documented in the ancient times. Modern pharmacological studies reveal that the water extract of E. helioscopia L play a inhibit role on cancer in vivo and in vitro, the antitumor effect of E. helioscopia L components were confirmed in vitro, such as quercetin, lupeol, gallic acid and hyperoside. Currently, chemical composition and anticancer activity of E. helioscopia L have determined in basically, and found that its ethyl acetate extract significant inhibited hepatocellular carcinoma cell growth and induce apoptosis in vitro. However, to achieve clinical value of E. helioscopia L in anti-tumor, we have to determine the effect on tumor growth in vivo and mechanism. Therefore, this study is to explore the effect of E. helioscopia L ethyl acetate extract on HCC growth in vivo and mechanism, which as a important experiment evidence of E. helioscopia L in anticancer is.Objective:To study the effect on HCC of E. helioscopia L ethyl acetate extract in vivo, and exploring the mechanism of inhibits growth on HCC. Finding some new antitumor targets in E. helioscopia L and preparing for a preclinical trial in anti-hepatoma of E. helioscopia L.Methods:The chemical compositions of E. helioscopia L were isolated and purified by system solvent method, and selected ethyl acetate extract component as experimental reagents. Incubation human hepatoma cell line SMMC-7721, BEL-7402, MHCC97, randomly distributed84nude mice into SMMC-7721group, BEL-7402group and MHCC97group, and then each group was further divided into a control group,5-Fu200μg/mL group, EAE (50μg/mL,100μg/mL,200μg/mL) group, the control group was4mice, and the experiment groups were6mice, raised in SPF environment. HCC cells were transplant into mice subcutaneous at logarithmic growth phase. Xenograft model was treated5-Fu and EAE in different concentrations by intraperitoneal injection, a once a day, for30days, the control group no treatment. Measuring diameter (a) and short axis (b) of xenografts once every5days, following intraperitoneal treatment, and calculate its volume by V=ab2/2. After6weeks, all mice were sacrificed, the xenografts were collected as specimen, and preserved in different way, xenografts fixed in formalin were detected the expression CyclinD1, Bcl-2, Bax Caspase-3, nm23-H1and MMP-9by HE staining and immunohischemical. The xenografts fixed in0.5%glutaraldehyde were observed ultrastructural by transmission electron microscopy, the xenografts preserved in liquid nitrogen, to detect the CyclinDl, Bax, Bcl-2, Caspase-3, nm-23H1and MMP-9protein expression by Western Blotting.Results:The volume of xenograft was significantly decrease by EAE treatment, compared with the controls(P<0.05), and showed that the volume of xenograft decrease with EAE concentration increasing, but this trend only emerged in the same cell line (P<0.05), while no significantly change between the different cell lines (P>0.05). Immunohistochemical detection found that in xenografts the degree and number of cells coloration were significantly decreased of CyclinDl, Bcl-2and MMP-9after EAE treatment, compared to the controls. But Bax, Caspase-3and nm23-H1appears colored deeper and the number of staining cells were significantly increased. Ultrastructure of xenografts was observed by transmission electron microscopy, compared to the controls, after EAE treatment the tumor cells were changed significantly, and the mainly morphology changes occurred in cell interior. such as cell nuclear shrinkage, chromatin condense, mitochondrial and organelle swelling at low concentrations of EAE; With increasing of the EAE concentration, chromatin margination, nucleus gap widened intercellular fibrosis, apoptotic bodies within cells appears in xenografts; high concentration of EAE induced nuclear fragmentation, dissolution, formation large vacuoles. Western blot analysis showed that in xenografts CyclinDl protein expression with in EAE treatment group was significantly decreased (P<0.05), compared to the controls, with concentration gradient increasing, the protein expression present to a reduce trendency; in EAE treatment group the Bcl-2protein expression was significantly lower than the controls (P<0.01), showing a negative correlation with EAE concentration gradient, and in EAE200μg/mL group is less than5-Fu group (P<0.05), the difference was significantly. The expression of Bax and Caspase-3protein was significantly higher in EAE treatment group (P <0.01), showing a positive correlation with EAE concentration gradient, which is in EAE100μg/mL and EAE200μg/mL group higher than5-Fu group (P<0.05), the difference was significantly; the expression of MMP-9protein was significantly lower in the EAE treatment groups than in the control group, and the expression of nm23-H protein was significantly increased, and presented a dose dependent. As above show, the difference of protein expression only in the same cell line’s xenografts by EAE treated, and no significantly change between different cell lines (P>0.05), the difference was no significantly.Conclusion:1.EAE can significantly inhibited growth of HCC in vivo, and the effect were correlated to concentrations gradient, which is showed a significantly dose dependence.2. EAE can downregulated CyclinDl, Bcl-2and MMP-9protein expression within HCC, and upregulated Bax, Caspase-3and nm23-H1protein expression, this regulation is correlation to the EAE concentration.3.The effect of EAE on HCC in vivo, which may be related with inhibit growth, induce apoptosis, weakening invasive and metastasis. |
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