CpG oligodeoxynucleotides inhibit tumor growth and reverse the immunosuppression caused by the therapy with 5-fluorouracil in murine hepatoma.

AIM: To investigate the effect of CpG-containing oligodeoxynucleotides (CpG ODN) alone or in combination with the chemotherapeutic agent 5-fluorouracil (5-FU) on tumor growth and whether CpG ODN can reverse the immunosuppression caused by the chemotherapy with 5-FU in murine hepatoma model. METHODS: Hepatoma model was established by subcutaneous inoculation with hepatoma-22 (H(22)) cells into the right flank of BALB/c mice. Mice with tumor were treated by peritumoral injection of CpG ODN alone or in combination with subcutaneous injection of 5-FU. Tumor size was quantified regularly. Serum levels of IL-12 and IFN-gamma in mice were assayed by enzyme-linked immunosorbent assay (ELISA). The lytic capacity of splenic NK cells was tested by lactate dehydrogenase release assay. RESULTS: Peritumoral injection of CpG ODN alone or in combination with subcutaneous injection of 5-FU, and the treatment with 5-FU alone all led to significant inhibition of hepatoma growth. The mean tumor volumes fell by 46.66% in mice injected with CpG ODN, 68.34% in the 5-FU treated mice, and 70.23% in mice treated with the combination of CpG ODN and 5-FU than in controls. There was no significant difference in tumor size between 5-FU-treated mice and mice treated with the combination of 5-FU and CpG ODN (P>0.05). The serum levels of IL-12 and IFN-gamma of mice treated with CpG ODN alone (IL-12: 464.50+/-24.37 pg/mL; IFN-gamma: 134.20+/-25.76 pg/mL) or with the co-administration of CpG ODN and 5-FU (IL-12: 335.83+/-28.74 pg/mL; IFN-gamma: 111.00+/-5.33 pg/mL) were significantly higher than that of controls (IL-12: 237.50+/-45.31 pg/mL; IFN-gamma: 56.75+/-8.22 pg/mL). The production of IL-12 and IFN-gamma was suppressed moderately in 5-FU-treated mice (IL-12: 166.67+/-53.22 pg/mL; 53.33+/-16.98 pg/mL) compared to control mice (P>0.05), whereas the combination of CpG ODN and 5-FU significantly increased the serum levels of IL-12 and IFN-gamma compared to 5-FU alone (P<0.05). The NK cell killing activity in CpG ODN-treated mice (44.04+/-1.38%) or the mice treated with CpG ODN combined with 5-FU (30.67+/-1.28%) was significantly potentiated compared to controls (19.22+/-0.95%, P<0.05). The co-administration of CpG ODN and 5-FU also significantly enhanced the lytic activity of NK cells when compared with the treatment with 5-FU alone (12.03+/-1.42%, P<0.05). CONCLUSION: The present data suggests that CpG ODN used as single therapeutic agent triggers anti-tumor immune response to inhibit the growth of implanted hepatoma and reverses the immunosuppression caused by the chemotherapy with 5-FU.

[Analysis of proteomic components of sera from patients with hepatocellular carcinomas by two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time of flying mass spectrometry].

OBJECTIVE: To analyze the proteomic components of the sera from the patients with hepatocellular carcinomas (HCC), in search of the diagnostic markers of HCC. METHODS: Immobilized pH gradient two-dimensional polyacrylamide gel electrophoresis (2DE), silver staining, PDQuest 2DE analysis software, peptide mass fingerprint based on matrix-assisted laser desorption/ionization time of flying mass spectrometry (MALDI-TOF-MS) and SWISS-PROT database searching, were used to separate and identify the proteome of the sera from the patients with HCC. RESULTS: The 2DE images were analysed by PDQuest 2DE software. The average matching rate was 70.2%. In IEF direction, the average deviation was (1.02 +/- 0.22) mm, in SDS-PAGE direction, the deviation was (0.97 +/- 0.14) mm. The twenty-three different protein spots were incised from silver staining gel and digested in-gel by TPCK trypsin. 15 peptide mass fingerprints (PMF) maps were obtained by MOLDI-TOF-MS. The typical peptide masses were searched in the SWISS-PROT database using PeptIdent software. CONCLUSIONS: Good reproducibility could be obtained by applying immobilized pH gradient two-dimensional polyacrylamide gel electrophoresis (2DE) to separate and identify the proteome in serum. There is still the problem of efficiently removing a higher level of proteins and lipids from the serum. Identification by MOLDI-TOF-MS peptide mass fingerprint provides useful information for screening diagnostic markers of human HCC.

Expression of IGF-II in early experimental hepatocellular carcinomas and its significance in early diagnosis.

AIM: To investigate the serum level and expression of insulin growth factor II (IGF-II) in liver tissues of rats with early experimental hepatocellular carcinomas (HCC) and its significance in early diagnosis. METHODS: Early experimental hepatocellular carcinomas were induced by diethylnitrosamine (DENA) in 180 male SD rats. Another 20 male SD rats served as control. The IGF-II serum level was measured by ELISA. Immunohistochemistry and electron microscopic immunohistochemistry were used to observe the expression of IGF-II in normal and tumor liver tissues and its ultrastructural location in malignant hepatocytes. The expressions of IGF-II in human hepatoma cell lines HepG2, SMMC7721 and human embryonic liver cell line L-02 were measured by immunocytochemistry. IGF-II mRNA level was studied by in situ hybridization. RESULTS: IGF-II was expressed in the cytoplasm of both sinusoidal cells in paracancerous cirrhotic liver tissue and malignant hepatocytes in early experimental HCC tissues. Gold particles were seen on the rough endoplasmic reticulum and the mitochondrion in malignant hepatocytes. IGF-II was expressed in the human hepatoma cell lines. The mRNA level of IGF-II was higher in rat liver tumor tissue than in normal rat liver tissue. The serum IGF-II level of the early experimental HCC group was 34.67+/-10.53 ng.ml(-1) and that of the control group was 11.75+/-5.84 ng.ml(-1). The rank sum test was used for statistical analysis. There was a significant difference between the two groups (P<0.01). CONCLUSION: During the induction of early experimental HCC by DENA, IGF-II may promote hepatocytic proliferation via a paracrine mechanism in the pre-cancerous stage. When hepatocytes are transformed into malignant cells, they may secrete IGF-II and promote malignant cell proliferation by an autocrine mechanism. IGF-II may be a possible biological marker in the early diagnosis of HCC.