Effect On CacyBP/SIP Nuclear Translocation By S100A1and S100a6in SW480Colon Cancer Cells

Calcyclin binding protein （CacyBP/SIP）, a30kDa protein originally discovered inEhrlich ascites tumor cells as a S100A6（calcyclin） target by Filipek and Wojda and later onas a Siah-1interacting protein （SIP） by studying P53induced new degradation path ofβ-catenin ubiquitination. Siah-1Interacting Protein （SIP） was confirmed as a humanortholog’s CacyBP by sequence analysis. Hence, CalCyclin Binding Protein was formallynamed as CacyBP/SIP.CacyBP/SIP bound several calcium binding proteins of the S100family such asS100A1、S100A6、S100A12、S100B and S100P. S100proteins were the largest subgroupwithin the EF-hand Ca²⁺-binding protein family. Binding of Ca²⁺induced a conformationalchange in the S100molecule which in consequence increased its overall hydrophobicity andallowed for interaction with target proteins to play their own specific biological effect.CacyBP/SIP was present in the cytoplasm of unstimulated cultured neurons in which restingCa²⁺concentration which was known to be50nM. When Ca²⁺concentration was increasedto above300nM, the CacyBP/SIP was mainly apparent as a ring around the nucleus. Theseresults suggested that Ca²⁺concentration played an important role in CacyBP/SIP nucleartranslocation. whether CacyBP/SIP might enter nuclear to transfer signal as the thirdmessenger after binding of the S100proteins which were activated by binding of Ca²⁺. linvestigated it.Objective1. Effect on the nuclear translocation of CacyBP/SIP by the changes of Ca²⁺concentration in SW480colon cancer cells.2.The binding and the changes of binding capacity of S100A1、S100A6and CacyBP/SIP respectively before and after the nucleartranslocation in SW480colon cancer cells.Effect on the nuclear translocation of CacyBP/SIPby inhibiting the corresponding S100protein expression in SW480colon cancer cells.Methods1.The changes of CacyBP/SIP position and relative levels of the Ca²⁺concentration in SW480colon cancer cells after stimulation by ionomycin（0μmol/L,1μmol/L,2μmol/L,5μmol/L,10μmol/L）were detected by indirect immunofluorescence andlaser copolymerization focal.2.The changes of CacyBP/SIP position and relative levels of theCa²⁺concentration in SW480colon cancer cells after stimulation by ionomycin（5μmol/L）and BAPTA/AM（0μM,5μM,10μM,25μM）were evaluated by indirect immunofluorescenceand laser copolymerization focal.3.To exclude the influence of intracellular Ca²⁺concentration overload in this experiment, viability of cells was detected by MTT assay,meanwhile, Annexin V/PI assay was used to detected the apoptosis in colon cancer cells.4.The binding and the changes of binding capacity of S100A1、S100A6and CacyBP/SIPrespectively were detected by co-immunoprecipitation before and after the nucleartranslocation by increasing Ca²⁺concentration.5.l designed three S100A6siRNA andselected the best group of siRNA silencing effect to transfect the SW480colon cancer cellsby Real-Time PCR and Western blot. It was detected by immunofluorescence that the effecton the nuclear translocation of CacyBP/SIP by inhibiting S100A6expression in SW480coloncancer cells.Results1.CacyBP/SIP was mainly located in the cytoplasm of colon cancer cells in the absenceof stimulation. CacyBP/SIP translocated into nucleus after stimulation by ionomycin, and thenuclear translocation of CacyBP/SIP was the most obvious after stimulation byionomycin（5μmol/L）. CacyBP/SIP redistributed in the cytoplasm and nucleus with the furtherincreasing of ionomycin concentration（10μmol/L）. The intracellular Ca²⁺concentration wasgradually increased after stimulation by ionomycin,and was the highest after stimulation by ionomycin（5μmol/L）. After that,with the further increasing of ionomycin concentration,Ca²⁺concentration tended to smooth（P=0.000）. The results suggested that CacyBP/SIPtranslocated into nucleus with the increasing of Ca²⁺concentration and redistributed in thecytoplasm and nucleus with the further increasing of Ca²⁺concentration.2. With the increasing of BAPTA/AM concentration gradually,CacyBP/SIP translocatedfrom the nucleus to cytoplasm. With the increasing of BAPTA/AM concentration gradually,the intracellular Ca²⁺concentration reduce at the same time（P=0.000）. The results suggestedthat CacyBP/SIP translocated from the nucleus to cytoplasm with the reducing of Ca²⁺concentration.3. MTT assay showed that proportion of cells viability between the treated group anduntreated group was not statistically significant（P=0.985）. Annexin V/PI assay also found noobvious early apoptotic（P=0.168）. The results suggested that the overloading of intracellularCa²⁺concentration had no effect on the experimental results and the experimental resultswere accurate and reliable.4. The changes of binding capacity of S100A1、S100A6and CacyBP/SIP respectivelywas apparent different before and after the nuclear translocation. The binding capacity ofS100A1and CacyBP/SIP was very little and the changes of binding capacity was notstatistically significant before and after the nuclear translocation（P>0.05）.The resultssuggested that S100A1may have no or little effect on the nuclear translocation ofCacyBP/SIP. However, the binding capacity of S100A6and CacyBP/SIP was significantlyincreased（P<0.05）,and was significantly higher than the combined amount of S100A1beforeand after the nuclear translocation（P<0.05）. These results suggested that S100A6may play animportant role in CacyBP/SIP nuclear translocation.5. The Real-Time PCR and Western blot test results suggested that the group ofsiRNA-S100A6-2was the best group of siRNA silencing effect and the inhibition rates were87.9%and85.0%respectively, so the group of siRNA-S100A6-2was used to transfect the SW480colon cancer cells.The nuclear translocation of CacyBP/SIP was significantlyinhibited after stimulation by ionomycin（5μmol/L）, locateing in the cytoplasm and nucleusof colon cancer cells. CacyBP/SIP did not translocate to the nucleus perfectly.These resultsfurther suggested that S100A6played an important role in CacyBP/SIP nucleartranslocation.Conclusions1. CacyBP/SIP translocated into nucleus with the increasing of Ca²⁺concentration, andredistributed in the cytoplasm and nucleus with the further increasing of Ca²⁺concentration inSW480colon cancer cells. With the reducing of Ca²⁺concentration, CacyBP/SIP translocatedfrom the nucleus to cytoplasm. In a word, CacyBP/SIP could translocate into and out of thenucleus with the changing of intracellular Ca²⁺concentration.2. S100A1and S100A6could combine CacyBP/SIP before and after the nucleartranslocation in SW480colon cancer cells. The binding capacity of S100A1and CacyBP/SIPwas very little and the changes of binding capacity was not statistically significant before andafter the nuclear translocation. These results suggested that the influence of S100A1onCa²⁺-mediated nuclear translocation of CacyBP/SIP was may little or inexistence. However,the binding capacity of S100A6and CacyBP/SIP was significantly increased.CacyBP/SIPnuclear translocation was significantly inhibited by reducing S100A6expression at the sametime.These results suggested that the Ca²⁺-mediated combination of S100A6and CacyBP/SIPplayed an important role in CacyBP/SIP nuclear translocation in SW480colon cancer cells.