Effects Of UDCA On SMMC-7721 Human Hepatocellular Carcinoma Cell Line And Xenografts In Nude Mice

Backgrounds and ObjectiveHCC is one of the common malignant tumors, surgery is an effective means of treating patients with HCC early in patients,without underlying liver dysfunction HCC can be surgically removed, but the vast majority of patients with HCC, often loss surgery opportunity, in addition to poor liver function, physical condition and other factors, it can not be surgery. Many current HCC treatment are developing very rapidly, except for surgical excision, the liver transplantation, ablation, interventional radiology, new molecular targeted therapy and palliative symptomatic treatment and offer a choice of treatment methods, but each treatment method has its own indications, contraindications, special effects and adverse reactions, therefore, not suitable for all patients. From the perspective of improving the survival rate of patients, to explore an economical, simple, less targeted therapy to prevent adverse reactions, drug therapy has been one of the direction and goals to explore.Ursodeoxycholic acid is a non-cytotoxic, hydrophilic bile acid, its physiological role is to increase bile acid secretion, and bile composition changes, lower cholesterol and bile cholesterol fat, cholesterol gallstones in favor of gradual dissolution, for the treatment of inoperable cholesterol stones, but also the treatment of primary biliary cirrhosis first-line treatment. Its pharmacological effect is to promote endogenous bile acid secretion, reducing reabsorption; antagonistic cytotoxicity of hydrophobic bile acids protect the liver cell membrane; dissolves cholesterol stones; and immunomodulatory effects.80 years later, due to the gallbladder, cytoprotective, antioxidant and immunomodulatory effects of UDCA, has been widely used in a variety of clinical hepatobiliary disease COPS5 are five subunits COP9 signal body, play a positive role in regulating protein. Studies have shown that, COPS5 expression in HCC was significantly higher than in adjacent liver tissue and adjacent normal liver tissue hemangioma. The expression of increasing COPS5 may play an important role in the occurrence of HCC. COPS5 also induce p53 tumor suppressor factor from the nucleus to cytoplasm translocation, and to promote the degradation of p53. The growth and metabolism of HCC require sustained growth of blood vessels, angiogenesis and tumor invasion, metastasis and prognosis of growth.Recent studies have confirmed that some of the tumor suppressor gene inactivation and mutation can lead to tumor angiogenesis. The inactivation of p53 mutations contribute to tumor formation angiogenic phenotype. p53 is a tumor suppressor gene, can promote infiltration of tumor growth in various ways, and have a significant effect on prognosis.Studies have shown that, UDCA can reduce the incidence of colon cancer, breast cancer cell lines,and also induce apoptosis, inhibit Chemical carcinogen-induced liver cancer in rats. Therefore, it is important to further explore the role of^UDCA on liver tumor cell apoptosis and proliferation. Because of the role of UDCA above, then UDCA may be able to inhibit hepatocellular carcinoma through a common mechanism of action COPS5 and p53. We need to be further explored whether UDCA in vivo effect of anti-hepatocellular carcinoma and possible mechanisms. To test this idea, This topic will be to evaluate UDCA in SMMC-7721 human hepatocellular carcinoma cell line and xenografts in nude mice, and observe its inhibition in vitro and vivo anticancer. To explore its mechanism, by observing its effect on COPS5 and p53 protein expression. The new treatment options may lay a theoretical foundation for the treatment of hepatocellular carcinoma.Part ⅠEffects of UDCA on SMMC-7721 human hepatocellular carcinoma cell lineObjective1、To detect the different concentrations of UDCA on the growth inhibition of SMMC-7721 cells.2、To detect the expression of the COPS5 and p53 on SMMC-7721 cells by different concentrations of UDCA.Methods1、SMMC-7721 cells and L-02 were used by cryopreservation, recovery, cultured and passaged.2、Transwell migration assay was used to observe the growth inhibition of UDCA on SMMC-7721 cells.3、Cell immunofluorescence assay was used to observe the expression of COPS5 and p53 on SMMC-7721 cells cells by the treatment of different concentrations of UDCA.4、Western blot was used to observe the expression of COPS5 and p53 on SMMC-7721 cells cells by the treatment of different concentrations of UDCA.Results1、Transwell migration assay was used to observe the growth inhibition of UDCA on SMMC-7721 cells in vitro.(1) SMMC-7721 were co-cultured with UDCA 48 hours, cells were counted, the low-dose group was 7.42 ±0.14 × 105/ml, and the high-dose group was 5.16 ± 0.10 × 105 /ml, and cells counted in the control group was 11.89 × 0.24 × 105/ml, UDCA show inhibitory effect on the SMMC-7721 significantly, the difference was statistically significant (compared with the control group, P<0.001), while no significant difference (L-02 cells,P> 0.05).(2) As a reference to the control group, the experimental group to calculate the number of cells in the liver cell line with the control group decreased inhibition ratio, SMMC-7721 cells in the low dose was 37.59 ± 0.78%, the high-dose group was 56.60 ± 1.35,The inhibition of the high-dose group was higher than the low-dose group, the high-dose group and low dose group was significantly different (P<0.01); L-02 cells in each dose group had no significant change (P> 0.05).2、Cell immunofluorescence assay was used to observe the expression of COPS5 and p53 on SMMC-7721 cells cells by the treatment of different concentrations of UDCA.(1) Cells by immunofluorescence experiments show, COPS5 on SMMC-7721 cells mainly localized in the nucleus, cytoplasm also visible part of the expression, after UDCA treatment, the decrease in the fluorescence intensity of the nucleus, cytoplasm is more dispersed. p53 is mainly localized in the nucleus, with the UDCA treatment, the fluorescence intensity was significantly enhanced compared with the control group.(2) Calculate the relative fluorescence intensity of the two proteins, the results show that compared with the control group, the relative fluorescence intensity of COPS5 on SMMC-7721 cells between low-dose group and high dose group was 0.42 ± 0.008,0.22 ± 0.005, were lower group (P<0.001), the difference was statistically significant, with concentration of UDCA increases, the relative fluorescence intensity of COPS5 showed decreasing trends. The relative fluorescence intensity of p53 between low-dose group and high dose group on SMMC-7721 cells, was 1.26 ± 0.023,1.34 ± 0.031,were higher (P 0.001), the difference was statistically significant, with concentration of UDCA increases, the relative fluorescence intensity of p53 tended to increase.3% Western blot was used to observe the expression of COPS5 and p53 on SMMC-7721 cells cells by the treatment of different concentrations of UDCA.(1) With the UDCA concentration increased, COPS5 expression gradually weakened, in order to further determine COPS5 and p53 expression by computer grayscale scanning and comparative analysis with the internal reference,0.4 mmol/L group relative value compared with control group(P<0.001), there was a significant difference, 1.0mmol/L group relative to the control group (P ＜0.001), the difference was significant. With UDCA concentration,the COPS5 expression showed a weakening trend.(2) With the UDCA concentration increased, p53 expression gradually increased, the computer grayscale scanning and comparative analysis with the internal reference,0.4 mmol/L group relative value compared with control group (P<0.001), the difference was significant,0.4 mmol / L group relative value compared with control group (P <0.001), the difference was statistically significant; the results suggest that, SMMC-7721 cells, treatment with UDCA concentration, p53 expression gradually increased.Conclusion1、UDCA can selectively inhibit the growth of SMMC-7721 human hepatocellular carcinoma cell line.2、The mechanism of UDCA on SMMC-7721 human hepatocellular carcinoma cell line may regulat through p53-related COPS5,and can up-regulate the expression of p53, down COPS5 expression, blocking cell proliferation.(2) With the UDCA concentration increased, p53 expression gradually increased, the computer grayscale scanning and comparative analysis with the internal reference,0.4 mmol/L group relative value compared with control group (P<0.001), the difference was significant,0.4 mmol / L group relative value compared with control group (P <0.001), the difference was statistically significant; the results suggest that, SMMC-7721 cells, treatment with UDCA concentration, p53 expression gradually increased.Conclusion1、UDCA can selectively inhibit the growth of SMMC-7721 human hepatocellular carcinoma cell line.2、The mechanism of UDCA on SMMC-7721 human hepatocellular carcinoma cell line may regulat through p53-related COPS5,and can up-regulate the expression of p53, down COPS5 expression, blocking cell proliferation.PartⅡEffects of UDCA on xenografts in nude miceObjective1、Preparation of nude mice.2、To detect the different concentrations of UDCA on the growth inhibition in nude mice.3、To analye the different concentrations of UDCA effect on xenograft tumor tissue.4、To detect the expression of the COPS5 and p53 on xenograft tumor in nude mice by different concentrations of UDCA.Methods1、SMMC-7721 cells was used by cryopreservation, recovery, cultured and passaged.2、Preparation of the second-generation xenograft tumor model in nude mice. 3、The effects of xenograft tumor growth curve by different concentrations of UDCA in nude mice were investigated.4、The effect of different concentrations of UDCA necrosis of tissue in nude mice were observed.5、The Immunohistochemical detection of the COPS5 and p53 on xenograft tumor in nude mice by different concentrations of UDCA were evaluated.6、The Western blot detection of the COPS5 and p53 on xenograft tumor in nude mice by different concentrations of UDCA were evaluated.Result1、The effects of xenograft tumor growth curve by different concentrations of UDCA in nude mice were investigated.UDCA can inhibit tumor growth in liver transplantation, the tumor growth curve in the treatment group is non-exponential growth, slow growth, expressed as the drug concentration increased,it show the trend of inhibiting the growth.2、The effect of different concentrations of UDCA necrosis of tissue in nude mice were observed.Xenografts histopathological observation showed that in the control group, mild necrosis of tumor tissue after treatment, tumor necrosis increased significantly, UDCA high-dose group showed large areas of necrotic tissue, rank test X2= 11.68, P<0.05 suggesting that UDCA dose and necrotic tissue correlated.all nude mice xenografts had no distant metastasis.3、The Immunohistochemical detection of the COPS5 and p53 on xenograft tumor in nude mice by different concentrations of UDCA were evaluated.Light microscopy vision of COPS5 can be seen, there was brown-stained nuclei of tumor cells, irregular in the control group, after UDCA treatment of tumor tissue, the nuclei stained cells reduce the number of brown, pale blue nucleus and cytoplasm of cells were in a larger number,and most cell morphology were regular. And p53 positive cells increased with the concentration of UDCA increased to.4、The Western blot detection of the COPS5 and p53 on xenograft tumor in nude mice by different concentrations of UDCA were evaluated.After UDCA treatment, the expression of COPS5 and p53 in xenograft tumor tissue were follows: With the UDCA concentration increased, COPS5 expression gradually weakened, while p53 expression gradually increased. To further determine the expression of COPS5 and p53 by computer grayscale scanning and comparative analysis with the internal reference, the relative value of the 90mg/kg group compared with control group (P<0.05), the difference was significant, the 270mg/kg relative value group and the control group Compared(P<0.05), the difference was significant. The results suggest that in transplanted tumor tissue with therapeutic concentration COPS5 weakened, and gradually increased p53 expression.Conclusion1、UDCA selectively inhibit tumor growth in nude mice.2、UDCA can promote tumor xenograft tumor tissue necrosis.3、UDCA inhibited tumor growth in nude mice, and the mechanism was relate to regulation COPS5 and p53, it can increase the expression of p53, downregulate COPS5 expression, block the inhibitory cell growth.4、UDCA may prevent hepatocellular carcinoma in nude mice experimentally.