Effects of 5-hydroxytamine and its antagonists on hepatic stellate cells.

BACKGROUND: Hepatic stellate cell (HSC) plays a key role in hepatic fibrosis. This study was undertaken to investigate the expression of 5-hydroxytamine receptors in HSC and the effect of 5-hydroxytamine on biological characteristics of HSC. METHODS: Liver ex vivo perfusion of collagenase and density gradient centrifugation were used to isolate HSCs. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect the expression of 5-hydroxytamine receptor subtypes 1A, 2A, 2B and 3. Western blot hybridization was used to elucidate the effect of 5-hydroxytamine and its 2A receptor antagonist ketanserin and 3 receptor antagonist ondanosetron on the expression of transforming growth factor-beta1 (TGF-beta1) and Smad4 in HSC. RESULTS: HSC expressed 5-hydroxytamine receptor subtypes 1A, 2A and 2B. 5-hydroxytamine significantly increased the expression of TGF-beta1 and Smad4 in HSC (P<0.05). This action can be antagonized by ketanserin, not by ondanosetron. CONCLUSIONS: HSC expresses 5-hydroxytamine receptors. 5-Hydroxytamine could effect the biological characteristics of HSC through its receptor mediation, and may play a role in the pathogenesis of liver cirrhosis and portal hypertension.

Expression of cancer/testis (CT) antigens in Chinese hepatocellular carcinoma and its correlation with clinical parameters.

For investigating the expression of cancer/testis (CT) antigens in patients with hepatocellular carcinoma (HCC) in China, and evaluating the correlations between the expression of these CT antigens and clinical parameters, we collected tumors and adjacent non-cancerous tissues of 43 HCC patients from Beijing and 30 HCC patients from Guangxi province. Expression of the mRNA of 14 CT antigens was evaluated by reverse transcription PCR (RT-PCR). The correlation between CT antigen expression and clinical parameters was statistically analyzed. The mRNA expression frequencies of CT antigens in tumor tissue were: MAGE-A1, 69.9%; MAGE-A3, 47.9%; MAGE-A4, 20.0%; MAGE-A10, 36.7%; SSX-1, 67.4%; SSX-2, 35.6%; SSX-4, 48.8%; SSX-5, 30.2%; NY-ESO-1, 42.5%; MAGE-B1, 52.0%; MAGE-B2, 60.0%; MAGE-C1, 48.0%; MAGE-C2, 68.0%; and SCP-1, 33.3%. However, in adjacent tissues, no CT antigen mRNA expression was detected, except SSX-1 in 9.3% patients. In each HCC tissue, the expression of a minimum of one, two, or three CT antigens was in the range of 80-90, 70-80 or 50-70%, respectively. MAGE-A3 mRNA expression differed between the HCC patients in Beijing and Guangxi (P=0.002). The average age of the HCC patients bearing CT antigen positive tumors was higher than that of the HCC patients bearing CT antigen negative tumors. The expression of MAGE-A3, SSX-1, SSX-2, SSX-4, MAGE-B2, MAGE-C1, and MAGE-C2 correlated significantly with older age (P<0.05). Moreover, the expressions of MAGE-A4 and SCP-1 were related to alpha-fetoprotein abnormality (P<0.05), and the expression of NY-ESO-1 was related to early tumor stage (P<0.05). There was no correlation observed between the expression of CT antigens and the sex, HBV infection or tumor size.

Expression of cancer-testis antigens in hepatocellular carcinoma.

AIM: To investigate the expression of cancer-testis (CT) antigens MAGE-1, SSX-1,CTp11 and HCA587 genes in hepatocellular carcinoma (HCC) and the possibility of applying these antigens as targets for specific immunotherapy for HCC. METHODS: Expression levels of MAGE-1, SSX-1, CTp11 and HCA587 mRNA were detected with reverse transcription polymerase chain reaction (RT-PCR) in HCC tissues and corresponding adjacent non-cancerous tissues from 105 HCC patients, 40 samples of cirrhosis and normal liver tissues. Genes of five samples with positive PCR results were sequenced. RESULTS: Of 105 HCC tissues, MAGE1, SSX-1,CTp11 and HCA587 mRNA expressions were detectable in 75.2%(79/105), 72.4%(76/105), 62.9%(66/105) and 56.2%(59/105) of HCC samples, respectively. About 93.3%(98/105), 72.4%(76/105), 48.6%(51/105) and 37.1%(39/105) of HCC tissues positively expressed at least one, two, three, and four members of CT antigens, respectively. Conversely, only SSX-1 could be detectable in 2.9%(3/105) of the corresponding adjacent non-HCC tissues in which no metastatic lesion was found. Of the latter 3 patients, biopsy samples far from tumor were obtained in 2 patients and RT-PCR indicated no expression of SSX-1 mRNA in these two samples. In addition, none of 40 samples of cirrhotic and normal liver tissues expressed CT antigen gene mRNA. DNA sequences confirmed that the RT-PCR products were true target cDNA. No relationship was found between expression of CT antigens and clinico pathological indicators such as age, gender, tumor size, degree of tumor differentiation, serum alpha-fetoprotein level and infection of hepatitis B virus or hepatitis C virus (P>0.05). CONCLUSION: CT antigens genes (MAGE-1, SSX-1, CTp11 and HCA587) are expressed with high percentage and specificity in HCC and their products are promising targets for antigen-specific immunotherapy of HCC. High frequent co-expression of multiple members of CT antigens in HCC provides possibility of polyvalent vaccinations for HCC.

[Expression of MAGE-B genes in hepatocellular carcinoma].

OBJECTIVE: To investigate the expression of MAGE-B genes in hepatocellular carcinoma (HCC) in order to find new targets for immunotherapy. METHODS: The expression of MAGE-B1, B2, A1 and A3 mRNA was detected using RT-PCR in HCC tissues and the corresponding adjacent non-HCC tissues from 47 HCC patients, 30 samples of cirrhosis and normal liver tissues. Four samples selected randomly from MAGE-B1 or B2 with positive RT-PCR results were sequenced to confirm the results of RT-PCR. The relationship between the expression of MAGE-B and some clinicopathological parameters was analyzed. RESULTS: MAGE-B1 mRNA and MAGE-B2 mRNA were detected in 44.7% (21/47) and 61.7% (29/47) of HCC samples, respectively, while neither MAGE-B1 nor MAGE-B2 could be detected in the corresponding adjacent non-HCC liver tissues. In addition, none of 30 samples of cirrhosis and normal liver tissues was shown to express both MAGE-B genes. The DNA sequence confirmed that the RT-PCR products were truly target cDNA. The frequency of the expression of MAGE-A1 and A3 was 74.5% (35/47) and 44.7% (21/47), respectively. There was significant correlation between the expression of MAGE-B and MAGE-A (P < 0.05). However, the positive expression of MAGE-B was observed in 5 out of 12 HCC tissues without expression of MAGE-A1 and/or A3. When all four MAGE genes were examined, the positive rate of expression of one, two, three and four genes was 83.0% (39/47), 55.3% (26/47), 48.9% (23/47), and 38.3% (18/47) of 47 HCC tissues, respectively. No correlation was found between the expression of MAGE-B and clinical parameters such as age, sex, tumor size, degree of tumor differentiation, serum alpha-fetoprotein level and hepatitis B virus or hepatitis C virus infection (P > 0.05). CONCLUSION: MAGE-B genes are expressed with relatively high frequency and specificity in HCC. Most HCC patients with positive expression of at least one member of MAGE-B or MAGE-A gene family are adequate candidates to receive specific immunotherapy. Frequent co-expression of multiple members of MAGE-B and MAGE-A subfamilies provides the possibility of using polyvalent vaccines to achieve more effective immunotherapeutic results.

[Expression of 5-hydroxytamine receptors in hepatic stellate cell and action of 5-hydroxytamine on biological characteristics of hepatic stellate cell].

OBJECTIVE: To investigate the expression of 5-hydroxytamine receptors in hepatic stellate cells HSCs and action of 5-hydroxytamine on biological characteristics of HSC. METHODS: Liver ex vivo perfusion of collagenase and density gradient centrifugation were used to isolate hepatic stellate cell. RT-PCR was used to detect the expression of 5-hydroxytamine receptor subtypes 1A, 2A, 2B and 3. Western blot hybridization was used to elucidate the effect of 5-hydroxytamine and its 2A receptor antagonist ketanserin and 3 receptor antagonist ondanosetron on expression of transforming growth factor-beta1 (TGF-beta1) and Smad4 in HSC. HSCs were cultured on silicone membrane. The effect of 5-hydroxytamine, ketanserin and ondanosetron on cell contraction were studied. RESULTS: HSC expressed 5-hydroxytamine receptors subtypes 1A, 2A and 2B. 5-hydroxytamine significantly increased the expression of TGF-beta1 and Smad4 in HSC (P < 0.05). This was antagonized by ketanserin, not by ondanosetron. 5-hydroxytamine induced cell contraction in a dose-dependant manner. Ketanserin antagonized this action, but ondanosetron did not. CONCLUSIONS: HSCs express 5-hydroxytamine receptors. 5-hydroxytamine could affect the biological characteristics of HSC through its receptor mediation, and may play a role in the pathogenesis of liver cirrhosis and portal hypertension.

Diagnostic value of cancer-testis antigen mRNA in peripheral blood from hepatocellular carcinoma patients.

AIM: To evaluate the diagnostic value of cancer-testis antigen (CTA) mRNA in peripheral blood samples from hepatocellular carcinoma (HCC) patients. METHODS: Peripheral blood samples were taken from 90 patients with HCC before operation. Expression of melanoma antigen-1 (MAGE-1), synovial sarcoma X breakpoint-1 (SSX-1), and cancer-testis-associated protein of 11 kDa (CTp11) mRNA in peripheral blood mononuclear cells (PBMC) was tested by nested reverse transcripts-polymerase chain reaction (RT-PCR). Serum alpha-fetoprotein (AFP) in these patients was also determined. RESULTS: The positive rate of MAGE-1, SSX-1 and CTp11 transcripts was 37.7%, 34.4%, 31.1% in PBMC samples, and 74.4%, 73.3%, 62.2% in their resected tumor samples, respectively. The positive rate for at least one of the transcripts of three CTA genes was 66.7% in PBMC samples and 91.1% in their resected tumor samples. MAGE-1, SSX-1 and/or CTp11 mRNA were not detected in the PBMC of those patients from whom the resected tumor samples were MAGE-1, SSX-1 and/or CTp11 mRNA negative, nor in the PBMC samples from 20 healthy donors and 10 cirrhotic patients. Among the 90 patients, the serum AFP in 44 patients met the general diagnostic standard (AFP > 400 microg/L) for HCC, and was negative (AFP < or = 20 microg/L) or positive with a low concentration (20 microg/L < AFP < or = 400 microg/L) in the other patients. The positive rate for at least one of the transcripts of three CTA genes in PBMC samples from the AFP negative or positive patients with a low concentration was 69.2% and 45.0%, respectively. Of the 90 patients, 71 (78.9%) were diagnosed as HCC by nested RT-PCR and serum AFP. Although the positive rate for at least one of the transcripts of three CTA genes in PBMC samples from 53 patients at TNM stage III or IV was obviously higher than that in PBMC samples from 37 patients at stage I or II (77.9% vs 51.4%, P = 0.010), the CTA mRNA was detected in 41.7% and 56.0% of PBMC samples from HCC patients at stages I and II, respectively. CONCLUSION: Detecting MAGE-1, SSX-1 and CTp11 mRNA in PBMC improves the total diagnostic rate of HCC.