The Effect of SIN1 and Microtubules on Insulin Induced PKC ζ Activation.

BACKGROUND Protein kinase C zeta (PKC ζ) plays an important role in insulin induced glycometabolism and insulin receptor (IR) associated signaling pathways. The full activation of PKC ζ depends on its translocation from cytosol to membrane and phosphorylation at Thr410. However, the mechanism of PKC ζ activation remains elusive. In this study, the effect of SIN1 and microtubules on insulin-induced PKC ζ activation was investigated. MATERIAL AND METHODS HepG2 cells were stimulated with insulin for co-immunoprecipitation (co-IP) assay. The immunocomplex was captured by using anti-PKC ζ, anti-SIN1 or anti-FLAG antibodies and was subjected to western blotting analysis for detecting PKC ζ, SIN1, and ß-tubulin protein expression level. The cells were intervened by small interfering RNA (siRNA) that targeted exon regions of SIN1. Then the glucose uptake ratio after cells were stimulated by insulin was measured. The PKC ζ insulin receptor levels in the membranes were analyzed. Cells stained with anti-PKC ζ, anti-SIN1 antibodies and probed with molecular probes were observed by immunofluorescence confocal microscopy. RESULTS SIN1 interacted and co-located with PKC ζ by pleckstrin homology (PH) domain. Downregulation of SIN1 severely impaired PKC ζ translocation and phosphorylation induced by insulin. PKC ζ co-immunoprecipitated with ß-tubulin at different intervals upon insulin stimulus, and the activation of PKC ζ was affected by paclitaxel and nocodazole. CONCLUSIONS PKC ζ translocated from cytosol to membrane depending on SIN1, which suggested that PKC ζ may be activated directly by PI3K and the reaction probably carried out on microtubules in HepG2 cells.

Research on optimum resistance factors of paclitaxel against benign biliary scar fibrosis / 中国生化药物杂志

Objective To discuss the best resistance factors of paclitaxel(Taxinol)on benign biliary scar fibrosis,in order to provide an effective basis for clinical prevention and treatment of benign biliary scar fibrosis.Methods Human bile duct epithelial cells were cultured in vitro,the prepared PTX at 0.001 uM,0.005 uM,0.1 uM,0.5 uMand 1 uMconcentration were separately added into cells for 48 h.The half inhibitory rate of BEC (IC50) were determined by MTT and the optimal concentration were confirmed.Human bile duct epithelial cells were cultured in 0 h,24 h,48 h and 72 h,the inhibitory rate of BEC at 100 nM,250 nM,and 500 nM PTX-Chitosan Sustained release membranes and the optimal concentration of PTX were determined by MTT and the optimal concentration of PTX-SRM were obtained.Human bile duct epithelial cells were cultured for 48 h and 72 h,the mRNA and protein expression ofα-SMA,E-cadherin,Vimentin were detected by Western Blot and Real-time PCR methods.Results The optimum resistance concentration of PTX to benign biliary scar was 250 nM.PTX and PTX-SRM could effectively inhibit the proliferation and transformation of BEC,and the best effective treatments to resist benign biliary scar fibrosis were low and middle concentrations of PTX-SRM,the best drug loading were 100 nMand 250 nM.The inhibition duration of PTX-SRMon BEC was longer than PTX alone(P<0.05).Conclusion The inhibition of PTX-SRMon BEC proliferation and transformation is better than the single drug of PTX,which provides a new scientific and feasible method for clinical prevention and treatment of benign biliary scar.

Inhibitation of Paclitaxel-Chitosan Sustain iflm on biliary ifbroblasts cell proliferation / 中国生化药物杂志

Objective To explore the effect of Paclitaxel-Chitosan Sustain film on growth, apoptosis and cell cycle of biliary fibroblasts cells. Methods Human biliary fibroblasts cells were cultured and treated with PTX-CSF and naked PTX,separately, untreated cells as blank control. The experiment was divided into five groups:untreated group, simple PTX-treated group (250nM) and low, medium and high chitosan sustained-release film PTX-treated group (100 nM, 250 nM, 500 nM). The proliferations of cells were determined by MTT assay. The apoptosis and cell cycle of cells were detected by FCM. Results The proliferation of biliary fibroblasts cells was inhibited by PTX-CSF with time-dependent and dose-dependent, and the inhibiting effect was more obvious than naked PTX treatment as the time went on. Meanwhile, PTX-CSF could inhibit the magration of bile duct fibroblasts induced by TGF-β1,and had longer effect than naked PTX. After 72 h, the apoptosis rate of cells treated with PTX-CSF was significantly higher than cells treated with naked PTX or untreated cells(P<0.05), the difference between naked PTX or untreated cells was not significant. Compared with untreated cells, the proportion of G 2/M in cells treated with PTX or PTX-CSF were significantly increased, and the former was sinificantly higher than the latter(P<0.05). Conclusion Compared with naked PTX, PTX-CSF has strong cytotoxic effects and obviously sustained-release effect. The effective concentration can be maintain for a long time by PTX-CSF, and it could be as the novel drug delivery system to continuously inhibit proliferation of bile duct fibroblasts.