CacyBP/SIP nuclear translocation induced by gastrin promotes gastric cancer cell proliferation

AIM:To investigate the role of nuclear translocation of calcyclin binding protein,also called Siah-1 interacting protein(CacyBP/SIP),in gastric carcinogenesis.METHODS:The expression of CacyBP/SIP protein in gastric cancer cell lines was detected by Western blot.Immunofluorescence experiments were performed on gastric cancer cell lines that had been either unstimulated or stimulated with gastrin.To confirm the immunofluorescence findings,the relative abundance of CacyBP/SIP in nuclear and cytoplasmic compartments was assessed by Western blot.The effect of nuclear translocation of CacyBP/SIP on cell proliferation was examined using MTT assay.The colony formation assay was used to measure clonogenic cell survival.The effect of CacyBP/SIP nuclear translocation on cell cycle progression was investigated.Two CacyBP/SIPspecific siRNA vectors were designed and constructed to inhibit CacyBP/SIP expression in order to reduce the nuclear translocation of CacyBP/SIP,and the expression of CacyBP/SIP in stably transfected cells was determined by Western blot.The effect of inhibiting CacyBP/SIP nuclear translocation on cell proliferation was then assessed.RESULTS:CacyBP/SIP protein was present in most of gastric cancer cell lines.In unstimulated cells,CacyBP/SIP was distributed throughout the cytoplasm;while in stimulated cells,CacyBP/SIP was found mainly in the perinuclear region.CacyBP/SIP nuclear translocation generated a growth-stimulatory effect on cells.The number of colonies in the CacyBP/SIP nuclear translocation group was significantly higher than that in the control group.The percentage of stimulated cells in G1phase was significantly lower than that of control cells(69.70%±0.46%and 65.80%±0.60%,control cells and gastrin-treated SGC7901 cells,P=0.008;72.99%±0.46%and 69.36%±0.51%,control cells and gastrin-treated MKN45 cells,P=0.022).CacyBP/SIPsi1effectively down-regulated the expression of CacyBP/SIP,and cells stably transfected by CacyBP/SIPsi1 were then chosen for further cellular assays.In CacyBP/SIPsi1 stably transfected cells,CacyBP/SIP was shown to be distributed throughout the cytoplasm,irregardless of whether they were stimulated or not.After CacyBP/SIP nuclear translocation was reduced,there had no major effect on cell proliferation,as shown by MTT assay.There had no enhanced anchoragedependent growth upon stimulation,as indicated by colony formation in flat plates.No changes appeared in the percentage of cells in G0-G1 phase in either cell line(71.09%±0.16%and 70.86%±0.25%,control cells and gastrin-treated SGC7901-CacyBP/SIPsi1 cells,P=0.101;74.17%±1.04%and 73.07%±1.00%,control cells and gastrin-treated MKN45-CacyBP/SIPsi1cells,P=0.225).CONCLUSION:CacyBP/SIP nuclear translocation promotes the proliferation and cell cycle progression of gastric cancer cells.

World Journal of GastroenterologyDevelopment and validation of a risk prediction score for the severity of acute hypertriglyceridemic pancreatitis in Chinese patients

BACKGROUND The frequency of acute hypertriglyceridemic pancreatitis(AHTGP) is increasing worldwide. AHTGP may be associated with a more severe clinical course and greater mortality than pancreatitis caused by other causes. Early identification of patients with severe inclination is essential for clinical decision-making and improving prognosis. Therefore, we first developed and validated a risk prediction score for the severity of AHTGP in Chinese patients.AIM To develop and validate a risk prediction score for the severity of AHTGP in Chinese patients.METHODS We performed a retrospective study including 243 patients with AHTGP. Patients were randomly divided into a development cohort(n = 170) and a validation cohort(n = 73). Least absolute shrinkage and selection operator and logistic regression were used to screen 42 potential predictive variables to construct a risk score for the severity of AHTGP. We evaluated the performance of the nomogram and compared it with existing scoring systems. Last, we used the best cutoff value(88.16) for severe acute pancreatitis(SAP) to determine the risk stratification classification.RESULTS Age, the reduction in apolipoprotein A1 and the presence of pleural effusion were independent risk factors for SAP and were used to construct the nomogram(risk prediction score referred to as AAP). The concordance index of the nomogram in the development and validation groups was 0.930 and 0.928, respectively. Calibration plots demonstrate excellent agreement between the predicted and actual probabilities in SAP patients. The area under the curve of the nomogram(0.929) was better than those of the Bedside Index of Severity in AP(BISAP), Ranson, Acute Physiology and Chronic Health Evaluation(APACHE II), modified computed tomography severity index(MCTSI), and early achievable severity index scores(0.852, 0.825, 0.807, 0.831 and 0.807, respectively). In comparison with these scores, the integrated discrimination improvement and decision curve analysis showed improved accuracy in predicting SAP and better net benefits for clinical decisions. Receiver operating characteristic curve analysis was used to determine risk stratification classification for AHTGP by dividing patients into high-risk and low-risk groups according to the best cutoff value(88.16). The high-risk group(> 88.16) was closely related to the appearance of local and systemic complications, Ranson score ≥ 3, BISAP score ≥ 3, MCTSI score ≥4, APACHE II score ≥ 8, C-reactive protein level ≥ 190, and length of hospital stay.CONCLUSION The nomogram could help identify AHTGP patients who are likely to develop SAP at an early stage, which is of great value in guiding clinical decisions.

CacyBP/SIP nuclear translocation regulates p27Kip1 stability in gastric cancer cells

AIM: To investigate the mechanism of calcyclin binding protein/Siah-1 interacting protein(Cacy BP/SIP) nuclear translocation in promoting the proliferation of gastric cancer(GC) cells. METHODS: The effect of Cacy BP/SIP nuclear translocation on cell cycle was investigated by cell cycle analysis. Western blot analysis was used to assess the change in expression of cell cycle regulatory proteins and proteasome-mediated degradation of p27Kip1. Coimmunoprecipitation(co-IP) analysis was performed to examine the binding of Cacy BP/SIP with Skp1. A Cacy BP/SIP truncation mutant which lacked the Skp1 binding site was constructed and fused to a fluorescent protein. Subsequently, the effect on Skp1 binding with the fusion protein was examined by co-IP, while localization of fluorescent fusion protein observed by confocal laser microscopy, and change in p27Kip1protein expression assessed by Western blot analysis.RESULTS: Cacy BP/SIP nuclear translocation induced by gastrin promoted progression of GC cells from G1 phase. However, while Cacy BP/SIP nuclear translocation was inhibited using si RNA to suppress Cacy BP/SIP expression, cell cycle was clearly inhibited. Cacy BP/SIP nuclear translocation significantly decreased the level of cell cycle inhibitor p27Kip1, increased Cyclin E protein expression whereas the levels of Skp1, Skp2, and CDK2 were not affected. Upon inhibition of Cacy BP/SIP nuclear translocation, there were no changes in protein levels of p27Kip1 and Cyclin E, while p27Kip1 decrease could be prevented by the proteasome inhibitor MG132. Moreover, Cacy BP/SIP was found to bind to Skp1 by immunoprecipitation, an event that was abolished by mutant Cacy BP/SIP, which also failed to stimulate p27Kip1 degradation, even though the mutant could still translocate into the nucleus.CONCLUSION: Cacy BP/SIP nuclear translocation contributes to the proliferation of GC cells, and Cacy BP/SIP exerts this effect, at least in part, by stimulating ubiquitin-mediated degradation of p27Kip1.