Effect Of Tri-Ortho-cresyl Phosphate On Activity And Translocation Of Protein Kinase C In SH-SY5Y Human Neuroblastoma Cells

Objective:To study the effect of tri-ortho-cresyl phosphate (TOCP), with orwithout PKC activator phorbol12-myristate13-acetate（PMA）and PKCinhibitors staurosporine（SP）on activity and translocation of proteinkinase C in SH-SY5Y human neuroblastoma cellsMethods:1.The cell line of human neuroblastoma SH-SY5Y were treated withfresh medium containing0.25mmol/L,0.5mmol/L, and1.0mM TOCP. Atthe indicated time, PKC activity was measured after exposure to TOCPfor12h,24h, and48h by PKC activity kit. Furthermore, cells werepretreated with PKC activator PMA (25nmol/L and100nmol/L) and PKCinhibitor SP (100nmol/L) for15min, prior to their challenge with1.0mmol/L TOCP.2. The distribution of PKC was localized by indirectimmunoflorescence assay through fluorescence microscopy.3. The level of PKCα、PKCβI、PKCβⅡ、total PKC and phosphorylatedPKC were evaluated by western blotting.Results:1. When compared with the control, the ratio of p-PKC/PKC increasedsignificantly after TOCP exposure (0.5mmol/L,1.0mmol/L) for24h and1.0mmol/L TOCP exposure for12h (P<0.05). It showed a time course dependent increasing. The activity of PKC increased in1.0mM TOCP-treatedSH-SY5Y cell for24h. Subsequently, cells were preincubated with100nmol/L PMA or100nmol/L SP for15min, followed by stimulation with1.0mmol/L TOCP for12h,24h and48h. The content of phosphorylated PKCincreased gradually with a time-effect realationship. The change inp-PKC/PKC densitometry ratio of cells treated with0.50mmol/L TOCP for24h（P<0.05）and1.0mmol/L TOCP for12h and24h（P<0.05）showed significantdifferrences compared to control. There exist time and concentrationdependent manner.2. Under the fluorescent inverted microscopy SH-SY5Y cells appearedto be fibroblast-like cells, irregular and small cell boby, theimmunostaining intensity in cells were weak. The results showed that1.0mmol/L TOCP treatment to SH-SY5Y for24h may characterized bymodification in the shape of the cells from fusiform and polygons shapeinto round shape and a green fluorescent from cytoplasm to cell membrane.3. SH-SY5Y cells were treated with TOCP with or without PMA and SP for24h. Our study found that PKC protein expression of PKC activator phorbol12-myristate13-acetate（PMA）and PKC inhibitors staurosporine（SP）were enhanced. PKC protein expression of PKC inhibitors staurosporine（SP）and tri-ortho-cresyl phosphate (TOCP) were weakened.Conclsions:1. Treatment with1.0mmol/L TOCP for24hours may activate PKC andinduce PKCα、PKCβI and PKCβⅡ membrane translocation in SH-SY5Y cells..2. PKC activator phorbol12-myristate13-acetate （PMA） and PKCinhibitors staurosporine（SP）can enhance and inhibit PKC activationinduced by TOCP and effect membrane translocation respectively.