Beta2-GPI: a novel factor in the development of hepatocellular carcinoma.

PURPOSE: This study investigated the effect and clinical significance of beta2-GPI in hepatocellular carcinoma (HCC). METHODS: Double fluorescent immunostaining analysis was performed in paraffin wax-embedded histological sections of nine HCC parenchyma, seven adjacent non-cancerous tissues and seven control liver tissues from hepatitis B virus (HBV) infected patients using a beta2-GPI polyclonal antibody and a HBV surface antibody. NF-κB activation was assessed by a non-radioactive electrophoretic mobility shift assay (EMSA) and immunofluorescence assay in SMMC-7721 HCC cells exposed to various treatments. The cells were transiently transfected with vectors expressing beta2-GPI (group one), HBsAg (group two), both beta2-GPI and HBsAg (group three), or with a control vector (group four). Untransfected cells (group five) were also used as a control. Alpha fetoprotein (AFP) expressions were also detected by ELISA in all groups. RESULTS: The highest degree of co-localization of beta2-GPI and HBsAg proteins was seen in the endochylema and occurred at the nuclear border in the cancer tissues. Weak beta2-GPI protein staining was present in the endochylema, with a strong signal for HBsAg protein in HBV control samples. Adjacent non-tumorous liver tissue samples also showed HBsAg staining but stronger beta2-GPI signals in the endochylema. In experiments with SMMC-7721 HCC cells, groups one and two had induced activation of NF-κB with the relative NF-κB DNA-binding activities of 55.84 and 51.12, respectively. However, the highest relative NF-κB DNA-binding activity was observed in group three (80.5). The percentages of cells with NF-κB translocated from the cytoplasm to nucleus in groups one, two, three, four and five compared with total cells were 13.5, 8.7, 24.9, 5.7 and 0.95%, respectively. The mean AFP levels were significantly higher in group three (0.0640 ± 0.0059) than in group five (P < 0.001). It appeared higher in group three than in group one (0.0562 ± 0.0060, P < 0.05) and group four (0.0585 ± 0.0040, P < 0.05), while no significant differences were seen between groups three and two, and between groups four and five. CONCLUSIONS: Beta2-GPI may play a role in the development of HBV-related HCC by activating NF-κB via interaction of beta2-GPI and HBsAg.

Silencing of signal transducer and activator of transcription 3 expression by RNA interference suppresses growth of human hepatocellular carcinoma in tumor-bearing nude mice.

AIM: To explore the effect of silencing of signal transducer and activator of transcription 3 (STAT3) expression by RNA interference (RNAi) on growth of human hepatocellular carcinoma (HCC) in tumor-bearing nude mice in vivo. METHODS: To construct the recombinant plasmid of pSilencer 3.0-H1-STAT3-siRNA-GFP (pSH1-siRNA-STAT3) and establish the tumor-bearing nude mouse model of the HCC cell line SMMC7721, we used intratumoral injection together with electroblotting to transfect the recombinant plasmid pSH1-siRNA-STAT3 into the transplanted tumor. The weight of the nude mice and tumor volumes were recorded. STAT3 gene transcription was detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Level of protein expression and location of STAT3 were determined by Western blotting and immunohistochemical staining. STAT3-related genes such as survivin, c-myc, VEGF, p53 and caspase3 mRNA and protein expression were detected in tumor tissues at the same time. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was used to detect apoptosis of tumor cells. RESULTS: The weight of the treated nude mice increased, and the tumor volume decreased markedly compared with those of the mock-treated and negative control groups (P < 0.01). The results of RT-PCR and Western blotting showed that mRNA and protein levels of STAT3 declined markedly in the treated group. The change in STAT3-related gene expression in tumor tissues at the mRNA and protein level also varied, the expression of survivin, VEGF and c-myc were obviously reduced, and expression of p53 and caspase3 increased (P < 0.01). Most of the tumor tissue cells in the treated group developed apoptosis that was detected by TUNEL assay. CONCLUSION: Silencing of STAT3 expression by RNAi significantly inhibits expression of STAT3 mRNA and protein, and suppresses growth of human HCC in tumor-bearing nude mice. The mechanism may be related to down-regulation of survivin, VEGF and c-myc and up-regulation of p53 and caspase3 expression. Accordingly, the STAT3 gene may act as an important and effective target in gene therapy of HCC.

Relationship and significance between anti-beta2-glycoprotein I antibodies and platelet activation state in patients with ulcerative colitis.

AIM: To study the relationship between anti-beta2-glycoprotein I (abeta2GPI) antibodies and platelet activation state in patients with ulcerative colitis (UC) and its significance. METHODS: Peripheral blood samples were collected from 56 UC patients (34 males and 22 females, aged 43.5 years, range 21-66 years), including 36 at active stage and 20 at remission stage, and 25 sex-and age-matched controls. The level of abeta2GPI was measured by ELISA. The platelet activation markers, platelet activation complex-I (PAC-I) and P-selectin (CD62P) were detected by flow cytometry. RESULTS: The A value for IgG abeta2GPI in the active UC group was 0.61 +/- 0.13, significantly higher than that in the remittent UC and control groups (0.50 +/- 0.13 and 0.22 +/- 0.14, P < 0.01). There was a significant difference between the two groups (P < 0.01). The A value for IgM abeta2GPI in the active and remittent UC groups was 0.43 +/- 0.13 and 0.38 +/- 0.12, significantly higher than that in the control group (0.20 +/- 0.12, P < 0.01). However, there was no significant difference between the two groups (P > 0.05). The PAC-I positive rate for the active and remittent UC groups was 30.6% +/- 7.6% and 19.6% +/- 7.8% respectively, significantly higher than that for the control group (6.3% +/- 1.7%, P < 0.01). There was a significant difference between the two groups (P < 0.01). The CD62P positive rate for the active and remittent UC groups was 45.0% +/- 8.8% and 31.9% +/- 7.8% respectively, significantly higher than that for the control group (9.2% +/- 2.7%, P < 0.01). There was a significant difference between the two groups (P < 0.01). In the active UC group, the more severe the state of illness was, the higher the A value for IgG abeta2GPI was, and the positive rate for PAC-I and CD62P was positively correlated with the state of illness (Fabeta2GPI = 3.679, P < 0.05; FPAC-I (%) = 5.346, P < 0.01; and FCD62P (%) = 5. 418, P < 0.01). Meanwhile, in the same state of illness, the A value for IgG abeta2GPI was positively correlated to the positive rates for PAC-I and CD62P. CONCLUSION: abeta2GPI level, platelet activation state and their relationship of them are closely correlated with the pathogenesis and development of UC.

[Studies on the association between beta 2-glycoprotein I and hepatotropism of hepatitis B virus].

OBJECTIVE: To further study the binding character of hepatitis B surface antigen (HBsAg) and beta 2-glycoprotein I (beta2GP I) and to explore whether beta2GP I plays an important role in the hepatotropism of hepatitis B virus. METHODS: Using Western blot technique, we observed the binding character of the HBsAg with reduced and non-reduced beta2GP I. RESULTS: rHBsAgs with reduced and non-reduced beta2GP I showed identical binding activity. CONCLUSIONS: The binding activity of HBsAg is dependent on tandem residues, but not on conformational structures of beta2GP I. There is a specific binding between HBV and beta2GP I, which may play an important role in HBV infection and is one of the reasons of hepatotropism of HBV.

[Relations of ulcerative colitis to anti-beta2-glycoprotein antibodies and to platelet activation status].

OBJECTIVE: To explore the relations of ulcerative colitis (UC) to anti-beta2-glycoproteinI antibodies (abeta(2)GPI) and platelet activation status. METHODS: Peripheral blood samples were collected from 56 UC patients, 34 males and 22 females, aged 43.5 (21 - 66), including 36 in active stage and 20 in remission stage, and 25 sex- and age-matched controls. The level of abeta(2)GPI was detected by ELISA. The platelet activation markers, platelet activation complex-1 (PAC-1) and P-selectin (CD62P) were detected by immunofluorescence staining and flow cytometry. RESULTS: The A value of IgG abeta(2)GPI of the active UC group was 0.61 +/- 0.13, significantly higher than those of the remittent UC group and the control group (0.50 +/- 0.13 and 0.22 +/- 0.14, both P < 0.01), and there was a significant difference between the remittent UC group and the control group (P < 0.01). The A value of IgM abeta(2)GPI of the active and remittent UC groups were 0.43 +/- 0.13 and 0.38 +/- 0.12, both significantly higher than that of the control group (0.20 +/- 0.12, both P < 0.01), however, there was no significant difference between the active UC and remittent UC groups (P > 0.05). The PAC-I positive rate of the active UC and remittent UC groups were 30.6% +/- 7.6% and 19.6% +/- 7.8%, both significantly higher than that of the control group (6.3% +/- 1.7%, both P < 0.01), and there was a significant difference between the active and remittent groups too (P < 0.01). The CD62P positive rates of the active UC and remittent UC groups were 45.0% +/- 8.8% and 31.9% +/- 7.8% respectively, both significantly higher than that of the control group (9.2% +/- 2.7%, both P < 0.01), and there was a significant difference between the active and remittent groups too (P < 0.01). In the active UC group, more severe the state of illness, the higher the A value of IgG abeta(2)GPI, and the positive rates of PAC-I and CD62P, and these values were all positively correlated with the state of illness (F(abeta2GPI) = 3.679, P < 0.05, F(PAC-I(%)) = 5.346, P < 0.01, and F(CD62P(%)) = 5.418, P < 0.01 respectively). CONCLUSION: The abeta(2)GPI level and the platelet activation state are closely correlated with the pathogenesis and development of UC.

Relationship between microvessel density and telomerase activity in hepatocellular carcinoma.

AIM: To study the relationship between microvessel density (MVD), telomerase activity and biological characteristics in hepatocellular carcinoma (HCC). METHODS: S-P immunohistochemical method and telomeric repeat amplification protocol (TRAP) were respectively used to analyze the MVD and telomerase activity in 58 HCC and adjacent normal tissues. RESULTS: The MVD in HCC with metastasis, lower differentiation or without intact capsule was significantly higher than that in HCC with intact capsule, higher differentiation, or without metastasis. While MVD had no relationship with tumor size, hepatic virus infection and other clinical factors. Telomerase activity was related to differentiation degree, but not to tumor size or histological grade. MVD in HCC with telomerase activity was higher than that in HCC without telomerase activity. CONCLUSION: MVD and telomerase activity may serve as diagnostic criteria of HCC in earlier stage. Meanwhile, there may be a cooperative effect between MVD and telomerase on the growth and metastasis of HCC.

Construction of eukaryotic expression plasmids of hepatitis B surface antigen and helper T lymphocyte epitope.

BACKGROUND: DNA immunization provides a promising approach to elicit protective humoral and cellular immune responses against HBV. This study was to construct an eukaryotic expression plasmid containing helper T lymphocyte epitope, which will enhance the immunogenicity of a novel hepatitis B virus (HBV) fusion protein DNA vaccine. METHODS: The target gene containing pan-DR helper T cell epitopes (PADRE) and HBsAg was amplified by polymerase chain reaction (PCR). The PCR products were linked with PMD-18T vector. Plasmid DNA was purified from transformed E.coli competent cell JM109 and digested with Hind III and EcoR I. Then, the target gene was cloned in pcDNA3.1(+) digested by Hind III and EcoR I. Finally, the identity of DNA was verified by digestion and DNA sequencing. RESULTS: The recombinant expression vectors of pcDNA3.1(+)-PADRE/HBs were identified by restriction enzyme digestion and DNA sequencing. The insert DNA fragment was consistent with the expected sequence. CONCLUSIONS: The constructed eukaryotic expression plasmid of pcDNA3.1(+)-PADRE/HBs is convenient for further study of eukaryotic transfection and response for cellular and humoral immunity against HBV.

Relationship between genetic polymorphism of cytochrome P450IIE1 and fatty liver.

AIM: To study the correlation between genetic polymorphism of cytochrome P450IIE1 (CYPIIE1) and fatty liver. METHODS: Peripheral blood mononuclear cells were collected in 56 patients with fatty liver, 26 patients without fatty liver and 20 normal controls. Then PCR-RFLP was used to analyze genetic polymorphism of CYPIIE1 in monocytes on the region of Pst I and Rsa I. RESULTS: The frequency of homozygotic C1 gene in patients with alcoholic fatty liver (28.6%), obese fatty liver (38.5%), or diabetic fatty liver (33.3%) was significantly lower than that of the corresponding patients without fatty liver (100%, 100% and 80% respectively), while the frequency of C2 genes, including C1/C2 and C2/C2, was significantly higher (71.4%/0%, 61.5%/0%, and 66.7%/20%) (P<0.01). The frequency distribution of the above genes of non-fatty liver patients (100%/0, 100%/0, and 80%/20%) was not significantly different from that of the normal controls (85%/15%) (P>0.05). CONCLUSION: The genetic polymorphism of CYPIIE1 on the position of Pst I and Rsa I is related to the susceptibility of fatty liver. Besides, C2 gene may play a key role in the pathogenesis of fatty liver.

A possible receptor for beta 2 glycoprotein I on the membrane of hepatoma cell line smmc7721.

OBJECTIVES: To study the interaction of beta-2-glycoprotein I (beta 2GPI) with the membrane of hepatocytes and determine whether beta 2GPI participates in HBV infection. METHODS: Ligand blotting, fluorescence microscopy, and fluorescence activated cell sorter (FACS) analysis were used to detect the specific interaction of beta 2GPI with the hepatoma cell line smmc7721, the gastric carcinoma cell line SGC7901, and the lymphoma cell line HL-60. RESULTS: A specific 40 kDa beta 2GPI band was observed by ligand blotting in the case of smmc7721 cells. No such band was observed in SGC7901 or HL-60 cells. Fluorescence microscopy also revealed specific binding of FITC-beta 2GPI to smmc7721 cells, but neither to SGC7901 nor HL-60 cells. FACS analysis demonstrated that the binding rate of FITC-beta 2GPI to smmc7721 cells was significantly higher than these in SGC7901 and HL-60 cells (P < 0.01). The binding rate to smmc7721 cells did not increase with increasing amounts of FITC-beta 2GPI. CONCLUSIONS: There is a specific beta 2GPI-binding protein on the membrane of hepatoma cells in cell line smmc7721 which as the beta 2GPI receptor may participate in HBV infection of hepatocytes.

Studies on specific interaction of beta-2-glycoprotein I with HBsAg.

AIM: To observe the binding activity of beta-2-glycoprotein I(beta(2)GPI) to hepatitis B surface antigen (HBsAg) and the possible roles of beta(2)GPI in hepatitis B virus (HBV) infection. METHODS: The rationale of ELISA methods and ELISA-based research method and ligand-blotting technique were used to detect the specific interaction of beta(2)GPI with HBsAg. RESULTS: With the increase of rHBsAg, the binding of beta(2)GPI to rHBsAg elevated, and these changes had statistic significance. When we added non- biotinlyated beta(2)GPI, the OD value significantly decreased though they still were positively relevant to rHBsAg, suggesting non- biotinlyated beta(2)GPI competed with biotinlyated beta(2)GPI to saturate the binding sites on rHBsAg. Meanwhile BSA was used as negative control to substitute for rHBsAg coating the plates. The results indicated no interaction between beta(2)GPI and BSA, suggesting the affinity of beta(2)GPI to rHBsAg was specific. The ligand blotling indicated that beta(2)GPI might bind to rHBsAg no matter whether it was under reduced condition or not. CONCLUSION: The binding of beta(2)GPI to HBsAg suggests that beta(2)GPI may be a carrier of HBV and that beta(2)GPI may play important roles in HBV infection.

Inhibitory effect of all-trans retinoic acid on human hepatocellular carcinoma cell proliferation.

AIM: To study the inhibitory effect of all-trans retinoic acid on human hepatocellular carcinoma cell line SMMC-7721 and to explore the mechanism of its effect. METHODS: SMMC-7721 cells were divided into two groups, one treated with all-trans retinoic acid (ATRA) for 5 days and the other as a control group. Light microscope and electron microscope were used to observe the morphological changes. Telomerase activity was analyzed with silver-stained telomere repeated assay protocal (TRAP). Expression of Caspase-3 was demonstrated with western blot. RESULTS: ATRA-treated cells showed differentiation features including small and pyknotic nuclei, densely stained chromatin and fewer microvilli. Besides, ATRA could inhibit the activity of telomerase, promote the expression of Caspase-3 and its activation. CONCLUSION: Telomerase activity and Caspase-3 expression are changed in human hepatocellular carcinoma cell line SMMC-7721 treated with all-trans retinioc acid. The inhibition of telomerase activity and the activation of Caspase-3 may be the key steps through which ATRA inhibits the proliferation of SMMC-7721 cell line.