Effect of Sirpalpha1 on the expression of nuclear factor-kappa B in hepatocellular carcinoma.

BACKGROUND: Signal regulatory protein alpha1 (Sirpalpha1) is a member of Sirps families containing four immunoreceptor tyrosine-based inhibitory motifs (ITIMs) domains in the cytoplasm of and an activated substrate of receptor tyrosine kinase (RTK), that negatively regulates the RTK-dependent cell proliferating signal transduction pathway. Previously we found that Sirpalpha1 was closely associated with the occurrence and development of hepatocellular carcinoma (HCC) as well as liver regeneration. Since it is unclear about the regulatory mechanisms, we established the cell line transfected Sirpalpha1 gene and preliminarily clarified the mechanisms by which Sirpalpha1 negatively regulates the carcinogenesis and development of HCC. METHODS: Liver cancer Sk-Hep1 cell was respectively transfected with plasmids of pLXSN, pLXSN-Sirpalpha1 and pLXSN-Sirpalpha1delta4Y2, screened with the drug of G418 (1200 microg/ml), and various transfected Sk-Hep1 cell lines were obtained. The protein expressions of P65, P50, IkappaBalpha, cyclin D1 and Fas in various Sk-Hep1 cell lines were determined by Western blotting, and P65 and P50 were localized by the immunofluorescence technique. RESULTS: Sirpalpha1 could significantly upregulate the protein expression of IkappaBalpha (vs. other cell lines, P<0.05) in the Sk-Hep1 cell, and downregulate the protein expressions of P65, P50 and cyclin D1 (vs. other cell lines, P<0.05) in the Sk-Hep1 cell. P65 protein expression was mainly localized in the cytoplasm in the pLXSN Sk-Hep1 cell, and in the nucleus of the Sk-Hep1 cell with mutant Sirpalpha1delta4Y2, but in nucleus of the Sk-Hep1 cell with wild Sirpalpha1. P50 protein expression was localized in the cytoplasm and nucleus of the pLXSN Sk-Hep1 cell, but in the nucleus of the Sk-Hep1 cell with wild Sirpalpha1 and mutant Sirpalpha1delta4Y2 plasmid. CONCLUSIONS: Sirpalpha1 might negatively regulate and control the abnormal proliferation of liver cancer cells by influencing the protein content and localization of nuclear factor-kappa B, then influence the expression of cyclins such as cyclin D1 in the signal transduction pathway. It may be one of the important mechanisms by which Sirpalpha1 negatively regulates the carcinogenesis and development of HCC.

Negatively regulating mechanism of Sirpalpha1 in hepatocellular carcinoma: an experimental study.

BACKGROUND: Signal regulatory protein (Sirp) is a recently isolated, cloned and identified inhibitor receptor distributed in the membrane of hematopoietic and nonhematopoietic cells. Sirp alpha1 (Sirpalpha1) is a member of Sirp families. Sirpalpha1 can bind SHP-2 in the form of tyrosine phosphorylation by SH2 effect and negatively regulate growth factor, oncogene, or insulin-induced responses as its substrate. This study aimed to preliminarily clarify the negatively regulating proliferation mechanism of Sirpalpha1 in liver cancer. METHODS: pLXSN, Sirpalpha1 and Sirpalpha1P4Y2 plasmids were respectively transfected into Sk-Hep1 liver cancer cell line, and various stable Sk-Hep1 cell lines were obtained with screening agent of G418 (1200 microg/ml). The expressing levels of cyclin D1, CDK4, Fas, beta-catenin and gankyrin in various cell lines were determined with Western blotting. Cell cycles were determined at 0, 12 and 24 hours with flow cytometry after various synchronous cell lines were cultured without serum for 72. Cell apoptosis induced with agent of TNF-alpha (50 ng/ml) was determined with flow cytometry at 0, 0.5, 1, 3, 6 and 12 hours. RESULTS: Sirpalpha1 could significantly decrease the expression of cyclin D1, beta-catenin and gankyrin, but it couldn't affect the expression level of CDK4 and Fas. When synchronous cells were cultured for 12 hours, S phase Sk-Hep1 cell transfected with Sirpalpha1 plasmid was the lowest [(31.92+/-0.22)% vs. other cell lines, P<0.05], and the cell line was highly sensitive to TNF-alpha agent for 1 hour. (59.31+/-0.59)% of apoptotic cells occurred (vs. the other time points, P<0.05). CONCLUSIONS: Sirpalpha1 might block the cell cycle of liver cancer, inhibit cell proliferation, promote cell apoptosis by decreasing the expression of cyclin D1, beta-catenin and gankyrin. It is one of the important mechanisms inhibiting the occurrence and development of hepatocellular carcinoma.

Effects of signal regulatory proteinalpha1 on proliferation of hepatocellular carcinoma: a preliminary study.

BACKGROUND: Signal regulatory protein alpha1 (Sirpalpha1) is a negative regulatory factor, and inhibits receptor tyrosine kinase-dependent cell proliferating signal. This study was undertaken to observe the effect of signal regulatory proteinalpha1 (Sirpalpha1)on gankyrin,cyclin D1,CDK4 and Fas expression in Sk-hep1 mouse hepatoma carcinoma cell line. METHODS: BOSC 23 packed cells were respectively transfected by means of recombinated retrovirus including pLXSN, pLXSN- Sirpalpha1 and pLXSN- Sirpalpha1P4Y2 with lipofectin, and various plasmid virus media(viral titer 2.1X10(6) CFU/ml) were collected and infected respectively in 80% confluent Sk-hep1 cells. Transfected Sk-hep1 cells were selectively screened with G418 (1200 mug/ml), and Sk-hep1 cell lines transfected with various plasmids were obtained. The protein expressions of gankyrin, cyclin D1, CDK4 and Fas in various Sk-hep1 lines were determined by Western blotting. Various Sk-hep1 lines were recovered to culture with 10% fetal bovine serum at 12 hours and 24 hours after starving culture with free serum for 72 hours, and cells were collected to determine the percentage of S phase cells of proliferating cycle by flow cytometry. RESULTS: Sirpalpha1 transfection remarkably downregulated gankyrin and cyclin D1 expression. Sirpalpha1P4Y2 downregulation of gankyrin expression was greater than that of Sirpalpha1 (P<0.05), but no significant effect of Sirpalpha1 and Sirpalpha1P4Y2 on CDK4 and Fas protein expression was observed in transfected Sk-hep1 lines (P>0.05). The percentage of S phase cells significantly decreased in Sk-hep1 cells transfected with Sirpalpha1 and Sirpalpha1P4Y2 plasmids (vs pLXSN Sk-hep1, P<0.05). The percentage of S phase cells in various Sk-hep1 cells increased when recovering to culture with 10% fetal bovine serum at 12 hours, but the percentage of S phase cells in Sk-hep1 cells transfected with Sirpalpha1 was the lowest (vs pLXSN and Sirpalpha1P4Y2 Sk-hep1, P<0.05). The percentage of S phase cells in transfected pLSXN Sk-hep1 cells was the largest (vs Sirpalpha1 and Sirpalpha1P4Y2 Sk-hep1, P<0.05). There was no significant difference between the transfected Sirpalpha1 Sk-hep1 cells and Sirpalpha1P4Y2 Sk-hep1 cells (P>0.05). CONCLUSIONS: Sirpalpha1 decreases gankyrin and cyclin D1 expression, and inhibits proliferation of liver carcinoma cells. It may be one of the forms for an Sirpalpha1 negative regulation of carcinogenesis and development of hepatocellular carcinoma.

[PTEN expression and its significance in human primary hepatocellular carcinoma].

OBJECTIVES: To explore the significance of PTEN (phosphatase and tensin homolog deleted on chromosome 10) in the development of human primary hepatocellular carcinoma (HCC). METHODS: PTEN protein expression in cancerous liver tissues and paired para-carcinoma liver tissues from 60 HCC patients was detected by immunohistochemistry and PTEN mRNA expression was analyzed by northern blot. The significance of PTEN in the development of HCC was analyzed by investigating the relationship between the expression levels of PTEN protein and mRNA, and the clinicopathological parameters of HCC patients. RESULTS: PTEN protein was immunohistochemically stained in the cytoplasmic region of para-carcinoma liver tissues in all the 60 patients, while only 48.3% (29/60) of the patients were positive for PTEN protein in cancerous liver tissues. The positive rate of PTEN protein in HCC tissues were relative to the histological gading and the presence of tumor thrombus. In grade I - II, III, and IV, the positive rates were 84.0%, 23.8%, and 21.4% respectively, and in the group with tumor thrombus was 26.7%, while in the group without tumor thrombus was 55.6%. Northern blot showed that there existed four PTEN mRNA transcripts with the length of 5.5kb, 4.4kb, 2.4kb, and 1.8kb respectively. The level of PTEN mRNA expression in HCC tissues was much lower than that in the paired para-carcinoma liver tissues. The low expression level of the 5.5kb and 4.4kb transcripts was significantly associated with serum AFP value, presence of tumor thrombus, state of satellite lesion and histological grading. The low expression level of the 2.4kb transcript in HCC was significantly associated with the presence of tumor thrombus and satellite lesions in HCC patients. However, no evident relationship between the lowered expression level of the 1.8kb transcript and the clinicopathological parameters of HCC was observed in these 60 patients. CONCLUSIONS: Down-regulation of PTEN expression may play an important role in the development of HCC and the expression level of PTEN may be a potential adjuvant parameter in forecasting the progress and prognosis of HCC.