[[(Ethoxycarbonyl)difluoromethyl]thio]phthalimide: A Shelf-Stable, Electrophilic Reagent with a Convertible Group for the Synthesis of Diversified Fluoroalkylthiolated Compounds

A shelf-stable and easily convertible reagent for the preparation of diversified fluoroalkylthiolated compounds, [[(ethoxycarbonyl)­difluoromethyl]­thio]­phthalimide, was developed. [[(Ethoxycarbonyl)­difluoromethyl]­thio]­phthalimide is an efficient electrophilic fluoroallylthiolating reagent that reacted with electron-rich heteroarenes/arenes, β-ketoesters, oxindoles, benzofuranones, and thiols. More importantly, the ethoxycarbonyl group of the resulting fluoroalkylthiolated compounds could be easily converted into various other functional groups such as chloride, alkynyl, hydrocarbonyl, carbomoyl, hydromethyl, or heteroaryl groups

Roles of GSK-3 in EGF-promoted glioma cell invasion.

<p>(A) EGF promoted U87 cell invasion in the transwell assay. ** <i>P</i><0.01 versus DMSO .(B) GSK-3 knock-down and uniform up-regulation of GSK-3β activities inhibited U87 cell invasion. * <i>P</i><0.05, ** <i>P</i><0.01 versus pcDNA3 (<i>ctl</i>) or non-sense control (<i>si-ctl</i>). (C) Up-regulation of p-Ser-GSK-3 levels during EGF-induced wound recovery of U251 cells. <i>Left</i>: representative western blots of U251 cell lysates collected at indicated time points after scratching. <i>Right</i>: statistics of pSer-GSK-3 levels normalized to GSK-3 levels and then compared with the value at 0hr. *<i>P</i><0.05, ** <i>P</i><0.01 versus the value of pGSK-3/GSK-3 at 0hr. (D) Location of increased pGSK-3 staining in EGF-induced wound recovery of U251 cells 3 hrs after scratching. <i>Green</i>: anti-pSer21-GSK-3αor anti-pSer9-GSK-3β; <i>Red</i>: rhodamine phalloidin. <i>Original magnification, 400×</i>. si-3α, siRNA against GSK-3α; si-3β, siRNA against GSK-3β; WT, wild-type form of GSK-3β; S9A, constitutively active form of GSK-3β. Data were mean ± SD of three independent experiments. EGF, 40ng/ml.</p

Localized inactivation of GSK-3β was associated with the formation of cell polarity.

<p>(A) Up-regulation of pSer-GSK-3 during the wound recovery of U251 cells. <i>Left</i>: representative western blot results of U251 cell lysates collected at indicated time points after scratching. <i>Right</i>: statistics of p-Ser-GSK-3 levels normalized to GSK-3 levels and compared with the value at 0hr. *<i>P</i><0.05, ** <i>P</i><0.01 versus the value of pGSK-3/GSK-3 at 0hr. (B) Location of increased pGSK-3 staining in the wound recovery of U251 cells at 0 hr or 3 hrs after scratching. Green: anti-pSer21-GSK-3α or anti-pSer9-GSK-3β; Red: rhodamine phalloidin. <i>Original magnification, 400×</i>.(C) MTOC mis-orientation in U251 caused by GSK-3 inhibitors. <i>Left</i>: representative images of MTOC in different treatments. Green: anti-α-tubulin; Red: anti-pericentrin; Blue: Hoechst 33258. <i>Original magnification, 400×</i>. <i>Right</i>: quantification of centrosome polarization as described in <i>Materials and Methods</i> in U251 with or without GSK-3β inhibitors. ** <i>P</i><0.01 versus DMSO. LiCl 20mM, SB216763, 20μM, NaCl, 20 mM. Data were mean ± SD of three independent experiments.</p

Inhibition of GSK-3 influenced directional persistence and locomotion speed of human glioma cell migration.

<p>(A) Scheme to define the speed and relative step angles as described in <i>Materials and Method</i>. (B) Morphological changes of the U87 cells treated with LiCl. <i>Red</i>: rhodamine phalloidin. <i>Original magnification, 400×</i>. (C) Decreased locomotion speed of the U87 cells upon LiCl treatment. Data were mean ± SD of ten cells in each group ** <i>P</i><0.01 versus DMSO. (D) The representative migration traces of the cells in the presence or absence of LiCl, with the starting point of migration superimposed at the origin of the X-Y plot. (E) The relative step angle of representative four migrating cells in the presence or absence of LiCl. Relative step angle and direction change were defined as described in <i>Materials and Methods</i>. Negative or positive values in <i>y-axis</i> indicated angles in clockwise or counter-clockwise turn, respectively. Red line at 60°or -60° outlined time points when a direction change occurred (beyond the field of -60°<⊿α<60°). LiCl, 20mM.</p

Atypical PKC and MAPK were involved in GSK-3 inactivation in EGF-promoted glioma invasion.

<p>(A) EGF-induced increase in pSer-GSK-3 levels was eliminated by inhibitors of aPKC and MAPK. <i>Left</i>: representative western blots of U251 lysates collected 3 hrs after wound recovery in the presence of EGF and indicated agents. <i>Right</i>: quantification of changes in pSer-GSK-3levels normalized to GSK-3 levels and then compared with the group without EGF treatment. (B) BothGSK-3β and PKCζ were present in the same complexes. Western blot analysis of the complexes precipitated by the indicated antibodies (U251 lysates collected at 2 hrs after EGF stimulation). (C) Inhibition of aPKC and MAPK suppressed U87 cell invasion in the transwell assay. #<i>P</i><0.05 versus DMSO; * <i>P</i><0.05, ** <i>P</i><0.01 versus DMSO+EGF. Wortmannin, 1μM; LY294002, 5μM; Gö6976, 0.1μM; Gö6983, 10μM; U0126, 10μM, EGF, 40ng/ml. Data were mean ± SD of three independent experiments in triplicate.</p

Inhibiting GSK-3 blocked migration and invasion of human glioma cells.

<p>(A) Wound recovery of U251 and U87 cells in the presence of indicated agents. ** <i>P</i><0.01 versus DMSO. (B and C) Blockade of cell migration and invasion in the presence of GSK-3 inhibitors. (B) Representative images of U251 cells in the transwell invasion assay in different treatments. <i>Original magnification, 100×</i>. (C) Quantifications of transwell migration in invasion assays of U251 and U87 cells. ** <i>P</i><0.01 versus DMSO. (D) Western blot analysis of U87 cell lysates transfected with indicated constructs. The values indicated the amount of indicated proteins normalized to that of GAPDH and compared with non-sense (<i>si-ctl</i>) or pcDNA3 (<i>ctl</i>) control group. (E) GSK-3 down-regulation by small interfering RNA inhibited U87 cell migration and invasion in the transwell assay. * <i>P</i><0.05 versus non-sense control group (<i>si-ctl</i>). (F) Uniform up- or down-regulation of GSK-3β activities inhibited U87 cell migration and invasion in the transwell assay. ** <i>P</i><0.01 versus pcDNA3 (<i>ctl</i>). si-3α, siRNA against GSK-3α; si-3β, siRNA against GSK-3β; WT, wild-type form of GSK-3β; S9A, constitutively active form of GSK-3β; GID, inhibitory peptide of GSK-3β; LP, mutant of GID without inhibitory effect. LiCl, 20mM, SB216763, 20μM, NaCl, 20mM. Data were mean ± SD of three independent experiments in triplicate. </p

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

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

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