The metastasis of hepatocarcinoma can be inhibited by T lymphocytes reconstitution in nude mice model.

BACKGROUND/AIMS: The metastasis of hepatic carcinoma is correlated with the body's immune status. T lymphocytes play a big part role in tumor immune. The aim of the present study is to investigate the inhibition effects of metastatsis in nude mice bearing hepatic carcinoma after T lymphocytes reconstitution. METHODOLOGY: An immune reconstitution model was established in nude mice. Then, 42 nude mice were distributed into 4 groups for T lymphocytes reconstitution. The lymph nodes of each group were obtained to investigate the tumor metastasis. And the secretion of cytokines and the apoptosis of tumor cells in each group were also detected. RESULTS: The ratio of CD3, CD4, CD8, CD4/CD8 in reconstituted groups were higher than controlled groups. The average time of tumor formation in Balb/c nu/nu mice was 7.7 +/- 0.6 days and in Balb/c mice was 11.5 +/- 1.3 days. After active T lymphocytes reconstitution, the extent lymph nodes metastasis in reconstitution groups was lower than control groups (p < 0.05), but proximal metastasis has no significant difference. The level of serum IL-10 in nude mice after immune reconstitution was significantly lower and VEGF was significantly higher than that of the control group (p < 0.05). Apoptosis of the hepatic carcinoma cells was increased significantly after immune reconstitution (p < 0.05). CONCLUSIONS: The metastasis of hepatic carcinoma can be inhibited by reconstitution of actived T lymphocytes in nude mice, which indicated that tumor metastasis can be affected by the immune status in host body.

[The level of transgelin related to the function of dendritic cells pulsed with liver cancer].

OBJECTIVE: To explore the relationships between the expression of transgelin in dendritic cells (DCs) pulsed with hepatocellular carcinoma lysates and the functions of the DCs. METHODS: DCs derived from healthy human white blood cells were divided into 3 groups: one was pulsed with high metastatic potential hepatocellular carcinoma cell line (MHCC97H) lysates, one with lysates of a low metastatic potential cell line (MHCC97L), and one un-pulsed DCs served as the control. The morphology of the DCs was observed by confocal microscopy and scanning electron microscopy. The phenotypes of the DCs were detected by flowcytometric analysis. The mixed leucocyte reaction (MLR) test and IL-12 secretion of DCs in the supernatants of MLR were employed to determine the functions of the DCs; the expression of transgelin was detected by Western blot. RESULTS: There were no morphological changes in the different DCs, but the levels of HLA-DR, CD80, CD83, CD86, MLR and IL-12 and transgelin were significantly higher in the two pulsed groups than those in the control group (P less than 0.01). In MHCC97H pulsed DCs, their CD80, CD83, CD86, and the expression of transgelin were also higher than those in the control group (P less than 0.05). The expression of transgelin was significantly higher in the MHCC97H pulsed group than in the MHCC97L loaded group, but CD80, CD83, CD86 and the level of IL-12 were all lower in the MHCC97H loaded DC group in comparison with those in the MHCC97 pulsed group (P less than 0.05). CONCLUSION: The expression of transgelin in DCs pulsed with HCC lysates is related to the functions of the DCs.

Down-regulation of beta-centractin might be involved in dendritic cells dysfunction and subsequent hepatocellular carcinoma immune escape: a proteomic study.

AIMS: Proteomic study was used to clarify the mechanism of hepatocellular carcinoma (HCC) immune escape concerning Dendritic cells (DCs') dysfunction and their association with HCC invasion. METHODS: Human peripheral blood mononuclear cells (PBMCs) derived DCs from healthy donors were pulsed with soluble cell lysates prepared from different metastatic potential human HCC cell lines. The total protein of these DCs was analyzed by two-dimensional electrophoresis and Electro-Spray Mass Spectrometry. The allostimulatoy capacity and phenotype of these DCs were also evaluated. The clinical significance of beta-centractin, one of the largest quantitative changed spot, down-regulation in DCs was further evaluated in autologous PBMCs derived DCs pulsed with auto-tumor lysates in 26 HCC patients. RESULTS: The expression of beta-centractin was found to be considerably lower either in DCs pulsed with HCCLM6 (high metastatic potential HCC cell line) lysates, accompanied by down-regulation of CD86 molecule and impaired allostimulatory capacity, than those of DCs pulsed with lysates from HCC cell lines with low or without metastatic potential or in DCs pulsed with lysates from HCC with invasiveness than those without invasiveness. CONCLUSIONS: The down-regulation of beta-centractin in DCs pulsed with high metastatic potential HCC lysates might associate with DCs dysfunction and HCC invasiveness.

[Relation between changes of dendritic cell function and down-regulation of beta-centractin in hepatocellular carcinoma].

OBJECTIVE: To investigate the relation between the changes of dendritic cell (DC) function and down-regulation of beta-centractin in hepatocellular carcinoma. METHODS: DC derived from peripheral blood were cultured and then pulsed by lysates from hepatocarcinoma cells (HCC) with high, low, or none metastatic potential of the lines HCCLM6, MHCC97L, and Hep3B, and from normal human liver cell of the line Chang liver. DC not pulsed were used as control group. Three days later scanning electron microscopy and inverted microscopy were used to observe the morphology of the DC. Flow cytometry was used to observe the phenotype. The protein expression of beta-centractin was detected by Western blotting and immunocytochemistry. RESULTS: Scanning electron microscopy and inverted microscopy showed no change in the morphology of the DC pulsed by different antigens. The expression levels of HLA-DR, CD80, CD83, and CD86 of the 4 pulsed groups were all significantly higher than that of the un-pulsed group (all P < 0.05). The expression of CD86 of the DC + LM6 group was significantly lower than those of the DC + Chang, DC + Hep3B, and DC + 97L groups (all P < 0.05). The mixed lymphocyte reaction (MLR) levels of the 4 pulsed groups were all significantly higher than that of the control group (all P < 0.05). The MLR level of the DC + LM6 group was significantly lower than those of the DC + Chang, DC + Hep3B, and DC + 97L groups (all P < 0.05). Western blotting showed that beta-centractin was not expressed in the control DC and was expressed in the 3 pulsed DC groups, and the beta-centractin expression levels of the DC + Chang, DC + Hep3B, and DC + 97L groups were all higher than that of the DC + LM6 group. The results of immunocytochemistry were similar to that of Western blotting. CONCLUSION: The down-regulation of beta-centractin in the DC pulsed with high metastatic potential HVV cell lysates is associated DC dysfunction and may be one of the mechanisms of HCC immune escape.

[A preliminary study of the killing function in vitro by T lymphocytes activated by dendritic cells loaded with exosomes secreted by hepatic cancer cell lines with high or low metastatic potentials].

OBJECTIVE: To study the tumor cell killing function of T lymphocytes stimulated by dendritic cells (DC) and to analyze the differences of protein contents of exosomes in each type of cell. METHODS: The exosomes of hepatic cell lines with high (P group) or low (F group) metastatic potentials were isolated by a process of four-step centrifugation and the collected exosomes were observed under an electron microscope (EM). The tumor cell killing experiment was performed by adding T lymphocytes activated by DC loaded with exosomes from corresponding P and F group cells and was studied using 3H-TdR experiments. The proteomic analysis was performed by surface-enhanced laser desorption/ ionization time of flight mass spectrometry (SELDI-TOF-MS ) on the exosomes of P and F group cells. RESULTS: The density distribution and content of exosomes in the P group were not equal to those in the F group observed by EM. The CD80, CD86, MHC-I and MHC-II in the P group were 64.27+5.00, 44.89+10.11, 84.35+19.89 and 59.03+19.37, and those in the F group were 71.53+4.85, 50.01+9.50, 80.68+29.87 and 58.86+21.11, respectively (P>0.05, compared with the control group). The counts per minute value in the P group was 528.40+179.06 and 78.80+24.44 in the F group after being loaded with exosomes (P<0.01, compared with the control group). There were significant differences between the proteins in the exosomes of hepatic cancer cell lines with high or low metastatic potentials. CONCLUSION: Exosomes have potential values of application in immunotherapy and in biotherapy for recurrences and metastases of hepatic carcinomas.

[The relationship between lymphangiogenesis and lymphatic metastasis in murine hepatic carcinoma of high and low metastatic potentialities].

OBJECTIVES: To study the relationship between lymphangiogenesis and lymphatic metastasis in mice bearing hepatic carcinoma and analyze the mechanism of the lymphatic metastasis. METHODS: Hepatic carcinoma cell lines of high and low potentialities of lymphatic metastasis were injected into the footpads of Balb/c mice. Their metastases to lymph nodes were examined. The tumor tissues of each group were stained with 5'-nucleotidase-ALP to observe the lymphoangiogenesis. The total RNA of high and low metastatic potential cell lines were extracted for metastasis gene DNA array. The vascular endothelial cell growth factor C (VEGF-C) and VEGF-D of each cell line were detected using semi-quantitative RT-PCR and were further quantatively analyzed using real time PCR. RESULTS: The para-common iliac a. and renal hilar lymph nodes metastases of the high metastatic potential cells were significantly higher than in the controls (P>0.05). The quantity of lymphatic vessels in the high metastasis group was significantly larger than that of the control group (P<0.05). The expressions of CD44, E-cadherin, HER2/neu, H-Ras and VEGF-C in the high metastasis group were higher than those in the low metastasis group shown by the cDNA micro array experiment but the expressions of nm23A, nm23-E4, p16ink4a, CD61 were lower. The VEGF-C expression was higher and the VEGF-D was lower in the high metastasis group compared to those of the low metastasis group shown by semi-quantitative RT-PCR. The secretion of VEGF-D was significantly lower and the ratio of VEGF-C/VEGF-D was significantly higher in the high metastasis group than the low metastasis group (P<0.05). CONCLUSIONS: The lymphatic metastasis of hepatic carcinoma is related to lymphoangiogenesis. The changes of VEGF-C and VEGF-D expressions might be a cause influencing the lymphoangiogenesis. VEGF-C/VEGF-D might be an effective parameter in affecting lymphatic metastases.

[Relationship between DLC-1 expressions and metastasis in hepatocellular carcinoma].

OBJECTIVES: To study the relationship between the expression level of DLC-1 mRNA (located in 8p) and the invasion/metastasis of human hepatocellular carcinoma (HCC). METHODS: Fifty-one surgical specimens of human HCC were divided into high-invasive and low invasive groups according to their clinicopathological features. DLC-1 mRNA expression was studied in the 51 HCC specimens as well as 5 different metastasis potential cell lines using real-time quantitative PCR (RQ-PCR). RESULTS: The expression level of DLC-1 mRNA in HCC specimens with high invasiveness was significantly lower than that with low invasiveness (P < 0.05). The expression levels of DLC-1 mRNA were significantly different between non-metastatic (Hep3B and HepG2) and metastatic (MHCC97-H, MHCC97-L and HCCLM3) cell lines (P < 0.05). From MHCC97-L to HCCLM3, with an increase of invasiveness and metastatic potentials, the expression level of DLC-1 decreased correspondingly, and its expression level in HCCLM3 was significantly lower than that in MHCC97-L (P < 0.01). CONCLUSION: The expression of DLC-1 mRNA may play an important role in inhibiting the invasiveness and metastasis of HCC.

[Influence of reconstruction of immunological functions of T lymphocytes on mouse hepatocarcinoma metastasis].

OBJECTIVE: To investigate the effectiveness of reconstruction of immunological functions of T cells on the degree of metastases of mouse hepatocarcinoma and the mechanisms of their functioning. METHODS: The T cell model of immunological functions in Balb/c nu/nu mice was established and the effectiveness of the model was evaluated. The mice were divided into 4 groups. The immunological functions of T cells in experiment groups of Balb/c nu/nu mice were reconstructed. Metastases of the cancer in lymph nodes in each group were examined histologically. The formation time and growth rate of the tumors were calculated. The expression of MHCI and II of the tumor cell line and the difference of expression of immune associated gene were detected by Th1-Th2-Th3 gene array. RESULTS: The ratio of CD3, CD4, CD8 and CD4/CD8 in the reconstructed group was higher than that in the control group. The average formation time was 7.7+/-0.6 days in Balb/c nu/nu mice and 11.5+/-1.3 days in Balb/c mice. The extent of metastases of the experiment group was lower than that of the control group (P < 0.05). The expression of MHCI of the high metastasis cell line was lower than that of the low metastasis cell line (P < 0.05). The expressions of Th1/Th2 associated genes in lymphocytes of high metastasis mice were lower than those of the low metastasis mice. CONCLUSION: Reconstruction of the immunological function of T cells can influence the metastasis of mouse hepatocarcinoma. The alteration of MHC molecule and low expression of Th1/Th2 correlated genes in lymphocytes may be a factor influencing the metastasis of liver cancer.