| Pancreatic cancer is one of the most lethal human cancers. It is usually discovered and diagnosed at its advanced stage. The only chance of cure is represented by surgical resection, but it is feasible in less than one half of the patients. And the overall survival rate of all patients is less than 1% at 5year, with most patients died within 1 year after diagnosed as pancreatic cancer. It is critical need to discover a specific early detection marker and molecular targets to fight against pancreatic cancers.The CacyBP/SIP protein (S100A6 binding protein and Siah-1 interacting protein) was initially identified, purified, and characterized in Ehrlich ascites tumor cells as a S100A6 (calcyclin) target and later on as a Siah-1 interacting protein. CacyBP/SIP can also bind several target proteins such as some calcium binding proteins of the S100 family (S100A1, S100A12, S100B and S100P), Skp1, tubulin and ERK1/2[1]. Reports showed that CacyBP/SIP took part in several celluar processes such as ubiquitination, proliferation, differentiation, tumorigenesis, cytoskeletal rearrangement or regulation of transcription through interaction with these target proteins. In our previous studies, CacyBP/SIP was identified as an up-regulated gene in a multidrug-resistant gastric cancer cell line, SGC7901/ADR, compared to its parental cells SGC7901, and Up-regulation of CacyBP/SIP is associated with multiple drug-resistant in gastric cancer cells. By using monoclonal antibodies against CacyBP/SIP firstly produced in our laboratory, we found that CacyBP/SIP was detected almost in all kinds of tumor tissues and was highly expressed in pancreatic cancer, nasopharyngeal carcinoma, and osteogenic sarcoma. These data suggested that CacyBP/SIP might be involved in malignant behaviours of pancreatic cancer and have a role in the carcinogenesis of pancreatic cancer. But the exact role that CacyBP/SIP played in the carcinogenesis of pancreatic cancer is still unknown.【Objectives】1、To investigate the expression and clinical significance of CacyBP/SIP in pancreatic cancer. 2、To explore the possible role of CacyBP/SIP in pancreatic cancer and the molecular mechanism underlying it.【Methods】1、The expression of CacyBP/SIP in human pancreatic cancer tissues and the normal pancreatic tissues was examined by immunohistochemistry and the relationship between the expression of CacyBP/SIP and the clinical characteristics was statistically analyzed by using SPSS version 10.0 software.2、The protein and mRNA level of CacyBP/SIP in fresh pancreatic cancer tissues and pancreatic cancer cell lines were examined by Western-blot and RT-PCR, respectively. 3、The small interference RNA of CacyBP/SIP were constructed and further indentified by sequence test. 4、The eukaryotic expression vector of CacyBP/SIP and the corresponding empty vector were transfected into the pancreatic cancer cell PC-2. And the stable clones were further identified using RT-PCR and Western-blot. 5、The effect of CacyBP/SIP on the cell growth ability was detected by MTT assay. And the ability of clony formation of pancreatic cancer cells was studied by clony formation assay. 6、The effect of CacyBP/SIP on the pancreatic cancer cell’s tumorigenesis ability was examined using in vivo tumorigenesis assay in nude mice. 7、The cell cycle distribution of cells transfected with different transfectants were detected using Flow cytometry analysis. 8、The possible downstream effectors were explored using Western-blot and RT-PCR.【Results】1、The expression level of CacyBP/SIP in pancreatic cancer tissues is significant higher than in normal pancreatic tissues. Expression and subcellular location of CacyBP/SIP protein were studied by immunohistochemistry of 68 pancreatic cancers and 37 normal pancreatic specimens. It was revealed that CacyBP/SIP was mainly located in the cytoplasm of the pancreatic cancerous cells, and only occasionally in the nuclei. CacyBP/SIP staining was found positive (grade III and IV) in 28 (41.2%) cases of pancreatic cancer specimens, but no positive(grade III and IV) expression of CacyBP/SIP was observed in normal specimens. The frequency of higher grade expression (grade III and IV) of CacyBP/SIP in pancreatic cancer tissues was significantly (P<0.01) higher than that in nonneoplastic pancreatic tissues. The results also revealed a positive association of CacyBP/SIP staining intensities with the degree of tumor differentiation. With respect to the TNM stage, the frequency of CacyBP/SIP high grade expression was much higher in patients of III+IV than those of I+II (P<0.01) stage. There was a statistically significant difference (P<0.01) in the frequency of CacyBP/SIP high grade expression between tumors with metastasis and those without. The mRNA and protein level of CacyBP/SIP detected by RT-PCR and Western-blot also confirmed that the expression of CacyBP/SIP in the pancreatic cancer tissues was significantly up-regulated compared with that in the matched adjacent normal tissues.2、CacyBP/SIP promotes proliferation of pancreatic cancer in vitro and in vivo.We expressed siRNA from two different CacyBP/SIP sequences (siRNA1 or siRNA2) in the poorly differentiated pancreatic cancer cell line PC-2. Both of the siRNAs greatly reduced the expression of CacyBP/SIP in PC-2 cell. Western blot and RT-PCR showed that both of protein and mRNA level were substantially decreased, and its mRNA level were reduced by about 90% and 75% in siRNA1 and siRNA2 expressing cells, respectively. CacyBP/SIP knockdown decreased proliferation of CacyBP/SIP siRNA1 and CacyBP/SIP siRNA2 cells in a medium containing 10% fetal calf serum, compared with the parental cell line and control pSilencer vector cells (p<0.05). Our data also showed that depletion of CacyBP/SIP dramatically decreased the ability of colony formation in these cells in plate and in soft agar (P <0.05). We next examined the in vivo effect of CacyBP/SIP in tumorigenicty in nude mice. The tumor sizes of mice injected with PC-2 cells expressing siRNA1 and siRNA2 were smaller and slower than those injected with PC-2 cells expressing control pSilencer vector (P < .05). Immunoblotting analysis of tumor sections derived from mice receiving different treatments also confirmed that CacyBP/SIP was down-regulated in siRNA1 and siRNA2 expressing groups compared to control group. 3、Inhibition of CacyBP/SIP expression induces the Cell Cycle Arrest of Pancreatic Cancer CellsTo further probe the mechanism by which CacyBP/SIP promotes pancreatic cancer cell growth, we studied the effects of down-regulation of CacyBP/SIP expression on the cell cycle by Flowcytometry. The results indicated that at 24 hours after the release of synchronized cultures, 67.2% of PC-2 cells expressing siRNA1 and 61.4% of PC-2 cells expressing siRNA2 were in G1-phase, respectively, whereas 46.3% of PC-2 cells expressing pSilencer were in G1-phase (P < .05). To further investigate the mechanism by which CacyBP/SIP induced cell cycle progress in pancreatic cancer cells, we next analyze cell cycle effectors expressions by Western blot analysis. Our data showed that down-regulation of CacyBP/SIP protein was associated with a reducing in cyclin A, cyclin E, cdk2 and p-Rb proteins, but with an increase in p27 and Rb proteins, but it has no effect on the level of cyclinB, cyclin D1 and P53 proteins. ERK1/2, Skp1, S100A6 and beta-catenin can interact with CacyBP/SIP, which was confirmed by other researchers, were also examined. The expression levels of these proteins have not been changed in the PC-2 cells after CacyBP/SIP knockdown.【Conclusion】1、The expression of CacyBP/SIP protein in pancreatic cancer tissues was significant higher than in normal pancreatic tissues. There are positive association between the expression level of CacyBP/SIP and the degree of tissue differentiation、TNM stage and metastasis. 2、Down-regulation of CacyBP/SIP by small interference RNA severely suppresses the proliferation and tumorigenesis in pancreatic cancer. 3、G1/S transition arrest induced by inhibition of CacyBP/SIP is at least partly mediated by down-regulation of Cyclin A、Cyclin E、CDK2 and pRb as well as up-regulation of p27 and Rb. |
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