Effects of signal regulatory proteinα1 on proliferation of hepatocellular carcinoma: a preliminary study

BACKGROUND: Signal regulatory protein alpha1 (Sirpα1) is a negative regulatory factor, and inhibits receptor tyro- sine kinase-dependent cell proliferating signal. This study was undertaken to observe the effect of signal regulatory proteinα1 ( Sirpα1) on gankyrin, cyclin D1, CDK4 and Fas expression in Sk-hep1 mouse hepatoma carcinoma cell line. METHODS: BOSC 23 packed cells were respectively trans- fected by means of recombinated retrovirus including pLX- SN, pLXSN-Sirpα1 and pLXSN-Sirpα1Δ4Y2 with lipofec- tin, and various plasmid virus media (viral titer 2.1 × 106 CFU/ml) were collected and infected respectively in 80% confluent Sk-hepl cells. Transfected Sk-hep1 cells were se- lectively screened with G418 (1200 μg/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: Sirpα1 transfection remarkably downregulated gankyrin and cyclin D1 expression. Sirpα1Δ4Y2 downregu- lation of gankyrin expression was greater than that of Sirpα1(P <0.05), but no significant effect of Sirpα1 and Sirpα1Δ4Y2 on CDK4 and Fas protein expression was ob- served in transfected Sk-hep1 lines (P >0.05). The per- centage of S phase cells significantly decreased in Sk-hep1 cells transfected with Sirpα1 and Sirpα1Δ4Y2 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 Sirpα1 was the lowest ( vs pLXSN and Sirpα1Δ4Y2 Sk- hepl, P<0.05). The percentage of S phase cells in trans- fected pLSXN Sk-hep1 cells was the largest (vs Sirpα1 and Sirpα1Δ4Y2 Sk-hepl, P <0. 05). There was no significant difference between the transfected Sirpα1 Sk-hepl cells and Sirpα1Δ4Y2 Sk-hep1 cells (P>0.05). CONCLUSIONS: Sirpα1 decreases gankyrin and cyclin D1 expression, and inhibits proliferation of liver carcinoma cells. It may be one of the forms for an Sirpα1 negative regulation of carcinogenesis and development of hepatocel- lular carcinoma.

Effect of Sirpα1 on the expression of nuclear factor-kappa B in hepatocellular carcinoma

BACKGROUND:Signal regulatory protein alpha1(Sirpα1) 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 Sirpα1 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 Sirpα1 gene and preliminarily clarified the mechanisms by which Sirpα1 negatively regulates the carcinogenesis and development of HCC. METHODS:Liver cancer Sk-Hep1 cell was respectively transfected with plasmids of pLXSN,pLXSN-Sirpα1 and pLXSN-Sirpα1Δ4Y 2 ,screened with the drug of G418(1200 μg/ml),and various transfected Sk-Hep1 cell lines were obtained.The protein expressions of P65,P50,IκBα,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:Sirpα1 could significantly upregulate the protein expression of IκBα(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 mutantSirpα1Δ4Y 2 ,but in nucleus of the Sk-Hep1 cell with wild Sirpα1.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 Sirpα1 and mutant Sirpα1Δ4Y 2 plasmid. CONCLUSIONS:Sirpα1 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 Sirpα1 negatively regulates the carcinogenesis and development of HCC.

Negatively regulating mechanism of Sirpal 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 nonhema-topoietic cells. Sirp alphal ( Sirpα1) is a member of Sirp families. Sirpal 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 Sirpal in liver cancer. METHODS: pLXSN, Sirpα1 and Sirpα1Δ4Y2 plasmids were respectively transfected into Sk-Hepl liver cancer cell line, and various stable Sk-Hepl cell lines were obtained with screening agent of G418 (1200 μg/ml). The expressing levels of cyclin D1, CDK4, Fas, β-catenin and gankyrin in various cell lines were determined with Western blotting. Cell cycles were determined at 0, 12 and 24 hours with flow cytotnetry after various synchronous cell lines were cultured without serum for 72. Cell apoptosis induced with agent of TNF-α (50 ng/ml) was determined with flow cytotnetry at 0,0.5,1,3,6 and 12 hours. RESULTS: Sirpα1 could significantly decrease the expression of cyclin D1, β-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 Sirpal plasmid was the lowest [(31.92 ± 0.22)% vs. other cell lines, P <0.05], and the cell line was highly sensitive to TNF-α agent for 1 hour. (59.31 ±0.59)% of apoptotic cells occurred (vs. the other time points, P < 0.05). CONCLUSIONS: Sirpal might block the cell cycle of liver cancer, inhibit cell proliferation, promote cell apoptosis by decreasing the expression of cyclin D1, β-catenin and gankyrin. It is one of the important mechanisms inhibiting the occurrence and development of hepatocellular carcinoma.

Engineering SIRPα cellular membrane-based nanovesicles for combination immunotherapy

Immune checkpoint inhibitors(ICIs)have revolutionized cancer treatment for their unprecedented clinical efficacy.Signal regulatory proteinα(SIRPα)is a phagocytic checkpoint expressed on macrophages,dendritic cells,other myeloid cells.Cancer cells inhibit macrophage phagocytosis through the interaction of the CD47-SIRPαaxis.Disrupting the CD47-SIRPαaxis has therefore been a promising strategy in restoring the immune attack against cancer.Herein,we engineered cellular membrane nanovesicles(NVs)presenting SIRPαreceptors for phagocytosis checkpoint blockade to augment the antitumor immune response.Furthermore,zebularine(Zeb),an inhibitor of DNA methyltransferase,was encapsulated into SIRPαNVs to reprogram the immunosuppressive tumor microenvironment together with blockade of phagocytosis checkpoint.It is demonstrated that SIRPα@Zeb can improve tumor immunogenicity,the polarization of tumor-associated macrophages to the M1 phenotype,increase the infiltration of CD8^(+)T lymphocytes in tumors.The robust antitumor immune response induced by SIRPα@Zeb significantly suppressed tumor growth and extended mice-bearing melanoma xenograft survival.

Two-component system GacS/GacA,a global response regulator of bacterial physiological behaviors

The signal transduction system of microorganisms helps them adapt to changes in their complex living environment.Two-component system(TCS)is a representative signal transduction system that plays a crucial role in regulating cellular communication and secondary metabolism.In Gram-negative bacteria,an unorthodox TCS consist-ing of histidine kinase protein GacS(initially called LemA)and response regulatory protein GacA is widespread.It mainly regulates various physiological activities and behaviors of bacteria,such as quorum sensing,secondary metabolism,biofilm formation and motility,through the Gac/Rsm(Regulator of secondary metabolism)signaling cascade pathway.The global regulatory ability of GacS/GacA in cell physiological activities makes it a potential research entry point for developing natural products and addressing antibiotic resistance.In this review,we summarize the progress of research on GacS/GacA from various perspectives,including the reaction mechanism,related regulatory pathways,main functions and GacS/GacA-mediated applications.Hopefully,this review will facilitate further research on GacS/GacA and promote its application in regulating secondary metabolism and as a therapeutic target.