Glycogen Synthase Kinase-3β Is Associated with the Prognosis of Hepatocellular Carcinoma and May Mediate the Influence of Type 2 Diabetes Mellitus on Hepatocellular Carcinoma

<div><p>Background</p><p>Although many studies have shown glycogen synthase kinase-3β(GSK-3β) was associated with type 2 diabetes mellitus(T2DM) and implicated with a wide range of cancers, the role of GSK-3β in hepatocellular carcinoma(HCC) and the correlation among GSK-3β, T2DM and HCC remains unclear. Our objectives were to identify the effect of p-Ser9-GSK-3β on the prognosis of patients with HCC and to learn more about the interaction among T2DM, GSK-3β and the prognosis of HCC.</p><p>Methods</p><p>Firstly we used reverse transcriptase-PCR(RT-PCR) and western blotting to determine the expression levels of GSK-3β and p-Ser9-GSK-3β in human HCC samples. We then used immunohistochemical staining to evaluate the expression pattern of p-Ser9-GSK-3β in 178 patients with HCC after curative partial hepatectomy. Finally we statistically analyzed the association of p-Ser9-GSK-3β and T2DM with the prognosis of patients with HCC.</p><p>Results</p><p>P-Ser9-GSK-3β was over-expressed in tumor tissues compared with their normal counterparts. Correlation and regression analysis indicated that the over-expression of p-Ser9-GSK-3β was significantly associated with T2DM, and the correlation coefficient was 0.259 (P = 0.001). Multivariate analysis showed that the over-expression of p-Ser9-GSK-3β(P<0.001) and T2DM(P = 0.008) were independently associated with poor prognosis of HCC, respectively. Further analysis demonstrated that these two variables are closely related with each other.</p><p>Conclusion</p><p>The over-expression of p-Ser9-GSK-3β and T2DM are strongly correlated with worse surgical outcome of HCC. P-Ser9-GSK-3β may play a significant role in mediating the influence of T2DM on the prognosis of HCC.</p></div

Immunohistochemical analysis of p-Ser9-GSK-3β expressions in HCCs and the adjacent nontumorous liver tissues.

<p>(A-D) Immunohistochemical staining of a paired HCC and nontumoral tissue. HCC cells were strongly positive for p-Ser9-GSK-3β expression in the cytoplasm (A,C). Normal hepatocytes in the nontumoral tissue showed no detectable p-Ser9-GSK-3β expression(B,D). (E-H) Immunohistochemical staining of another paired HCC and nontumoral tissue. HCC cells were strongly positive for p-Ser9-GSK-3β expression in the cytoplasm(E,G). Normal hepatocytes in the nontumoral tissue showed no detectable p-Ser9-GSK-3β expression(F,H). Original magnifications: magnification*50 (A,B,E,F); magnification*100 (C,D,G,H).</p

Correlation between p-Ser9-GSK-3β expression and clinicopathologic features.

<p>Abbreviations: AFP, alpha-fetoprotein; HBsAg, hepatitis B surface antigen; HBeAg, Hepatitis E antigen; MVI, microvascular invasion; TBIL total bilirubin; ALB, albumin; ALT, alanine; WBC, white blood cell; RBC, red blood cell; PLT, platelet count; INR, international normalized ratio; CR, creatinine; MELD, model for end-stage liver disease; T2DM, type 2 diabetes mellitus.</p><p>Correlation between p-Ser9-GSK-3β expression and clinicopathologic features.</p

Kaplan-Meier analysis of TTR and OS of patients with HCC.

<p>(A,B) Kaplan-Meier curves of the correlation between p-Ser9-GSK-3β expression level and (A) recurrence or (B) overall survival of HCC patients; (C,D) Kaplan-Meier curves of the correlation between T2DM and (A) recurrence or (B) overall survival of HCC patients.</p

Kaplan-Meier analysis of TTR and OS stratified by tumor diameter of HCC patients.

<p>(A,B) Kaplan-Meier curves of the correlation between p-Ser9-GSK-3β expression level, tumor diameter divided by 5cm and (A) recurrence or (B) overall survival of HCC patients; (C,D) Kaplan-Meier curves of the correlation between T2DM, tumor diameter divided by 5cm and (C) recurrence or (D) overall survival of HCC patients.</p

Expression of GSK-3β and p-Ser9-GSK-3β in HCC.

<p>(A) Relative expression level of GSK-3β mRNA in 60 tumor tissues(TT) and paired non-tumorous tissues(NTT) by RT-PCR (P<0.001). (B) RT-PCR analysis of GSK-3β mRNA expression. (C) Expressions of p-Ser9-GSK-3β were analyzed by Western Blotting in HCC tumor tissues(TT) and paired non-tumorous tissues(NTT).</p

Multivariate analysis for overall survival.

<p>Abbreviations: pG, p-Ser9-GSK-3β; T2DM, type 2 diabetes mellitus.</p><p>Multivariate analysis for overall survival.</p

Surfactant-Free Synthesis of PNIPAM-Based Smart Microgels for Drug Delivery Using a High-Gravity Rotating Packed Bed

Poly(N-isopropylacrylamide) (PNIPAM) microgels have a number of potential biomedical applications, especially being considered as next-generation drug delivery systems. So far, nm-sized PNIPAM microgels have been commonly prepared in lab-scale experiments. In this work, nm-size controllable and monodisperse PNIPAM-based microgels were synthesized for the first time by surfactant-free precipitation polymerization using a high-gravity rotating packed bed reactor (RPB). The microgel size could be synergistically controlled by adjusting the amount of cross-linker and initiator in an RPB. Compared with the conventional stirred tank reactor, the RPB synthesis procedure could obtain a higher yield within 2 h of the reaction time. The higee level exhibited significant influence on the microgel size. Depending on the variation of the higee level, the particle size was tailored from 129 to 325 nm, and the hydrodynamic diameter was tailored from 217.7 to 805.4 nm without the usage of surfactants. In addition, different comonomers were introduced to regulate the lower critical solution temperature (LCST), achieve multiresponsiveness, and control microgel size. The in vitro DOX-loaded release demonstrated that PNIPAM-based microgels dramatically contributed to the sustained release of drugs

Activation of NMDA receptors with D-serine obviously limited the decrease of p-CREB in the hippocampus of the surviving LPS-treated mice.

<p>(A) Images of the p-CREB staining, Bar = 50μm. (B) The relative mean optical densities for p-CREB staining in the CA1 and dentate gyrus. Data are expressed as the mean ±S.D.*<i>P</i><0.05 LPS+NS vs. LPS+D-serine for corresponding time points after LPS i.p. (n = 5 per time point per group).</p

Activation of NMDA receptors with D-serine obviously inhibited the increase of ROS in the hippocampus of the surviving LPS-treated mice.

<p>(A) Representative images of ROS fluorescence in the CA1, Bar = 100μm. (B) Representative relative mean Et fluorescence in the CA1 and dentate gyrus, *<i>P</i><0.05 LPS+NS vs. LPS+D-serine for corresponding time points. Data are expressed as the mean ±S.D. (n = 5 per time point per group).</p