EGCG inhibits the degredation of CTR1 induced by cDDP.

<p>(A, B) The effect of cDDP on the expression of CTR1 in OVCAR3 and SKOV3 cells. The cells were treated with 10 μM cDDP for the indicated time and CTR1 protein expression were detected by western blot analysis. (C)The effect of MG132 on the degradation of CTR1. After OVCAR3 cells were pretreated with 5 μM MG132 for 10h, the cells were incubation with 10 μM cDDP for 14 h. Then followed by western blot analysis. (D, E) The effect of EGCG on cDDP-trigged decrease of CTR1. The OVCAR3 and SKOV3 cells were treated with/without 10μM EGCG in the presence/absence of 10μMcDDP for 24 h. CTR1 protein expression was detected. The bands were quantified with Image J software. (*<i>P</i><0.05, **<i>P</i><0.01)</p

Effect of knock-down of CTR1 on the sensitivity of cells to cDDP.

<p>(A) Effect of knock-down of CTR1 on the sensitivity of ovary cancer cells to cDDP. OVCAR3 cells were transfected with three company sythetic si-RNAs or siRNA control and the western blost analysis showed si-RNA 3 exhibiting the best effect. After transfected with si-RNA3 or siRNA control, OVCAR3 and SKOV3 cells were treated with cDDP at various doses for 48 h and the cell survival fraction was detected by MTT assay. (B) Embryonic kidney HEK-293 cells were tranfected with human CTR1 si-RNA3 or siRNA control. Then the cells were exposed by indicated doses of cDDP for 48 h and the cell survival fraction was detected by MTT assay. (*<i>P</i><0.05).</p

Effect of EGCG on the sensitivity of the ovarian cancer to cDDP.

<p>(A) Effect of EGCG on ovary cancer cells survival fraction. OVCAR3 and SKOV3 cells were treated with the indicated concentrations of EGCG for 24 h, then followed by MTT assay to detect cell the survival fraction. (B) Effect of the combination of EGCG and cDDP on cell survival fraction. The cells were treated with indicated concentration of cDDP alone, or in combination with the EGCG (10 μM, shown as E10), and followed by MTT assay. (C) EGCG in combination with cDDP repressed colony formation. OVCAR3 cells were treated with 10 μM of EGCG alone or in combination of 10 μM of cDDP for 48 h. When the colonies formed two weeks later, colony formation assay was carried out. (D) The combination of EGCG and cDDP on cells apoptosis, Hoechst 33258 staining was used to detect apoptosis caused by the indicated treatments. (*<i>P</i><0.05, **<i>P</i><0.01)</p

Effects of EGCG on Pt and DNA-Pt adducts accumulation in the cells.

<p>OVCAR3 cells were treated with/without 10μM EGCG and 30 μMcDDP for 4 h, and then followed by ICP-MC assay. (A) Whole-cell Pt accumulation. (B) DNA-Pt adducts accumulation. (**<i>P</i><0.01)</p

EGCG enhances the efficacy of cDDP on tumor responsiveness and attenuates the nephrotoxicity induced by cDDP in vivo.

<p>Four groups (control, EGCG, cDDP and EGCG+cDDP) were set up. Except there were 6 mice in control group, there were 8 mice for each of the other groups. The body weight (A) and the tumor size (A) were measured twice a week. (B) The mRNA expression of the CTR1 in tumor tissues was measured by RT-PCR and real qPCR. (C) The expression of CTR1 in tumor tissue was assessed by western blotting. (D) The expression of CTR1 in kidney tissue was measured by western blotting. The bands were quantified by Image J software. (*P<0.05, **P<0.01)</p

A New Dimension to Ras Function: A Novel Role for Nucleotide-Free Ras in Class II Phosphatidylinositol 3-Kinase Beta (PI3KC2 beta) Regulation

The intersectin 1 (ITSN1) scaffold stimulates Ras activation on endocytic vesicles without activating classic Ras effectors. The identification of Class II phosphatidylinositol 3-kinase beta, PI3KC2 beta, as an ITSN1 target on vesicles and the presence of a Ras binding domain (RBD) in PI3KC2 beta suggests a role for Ras in PI3KC2 beta activation. Here, we demonstrate that nucleotide-free Ras negatively regulates PI3KC2 beta activity. PI3KC2 beta preferentially interacts in vivo with dominant-negative (DN) Ras, which possesses a low affinity for nucleotides. PI3KC2 beta interaction with DN Ras is disrupted by switch 1 domain mutations in Ras as well as RBD mutations in PI3KC2 beta. Using purified proteins, we demonstrate that the PI3KC2 beta-RBD directly binds nucleotide-free Ras in vitro and that this interaction is not disrupted by nucleotide addition. Finally, nucleotide-free Ras but not GTP-loaded Ras inhibits PI3KC2 beta lipid kinase activity in vitro. Our findings indicate that PI3KC2 beta interacts with and is regulated by nucleotide-free Ras. These data suggest a novel role for nucleotide-free Ras in cell signaling in which PI3KC2 beta stabilizes nucleotide-free Ras and that interaction of Ras and PI3KC2 beta mutually inhibit one another

Reduced cardiac muscle damage in α-MHC-tTA hearts subjected to I/R injury.

<p>Lack of cardiac muscle cell damage was apparent in α-MHC-tTA compared to control where abundant creatine kinase levels were observed within 15 min of start of reperfusion. (N = 4, p<0.05).</p

α-MHC-tTA hearts were protected against I/R injury <i>in vitro</i> using the Langendorff-perfused heart.

<p>(A–B) LVDP was similar between α-MHC-tTA and control hearts at baseline; however, after 30 min of ischemia, control hearts recovered to 35% of baseline LVDP values whereas α-MHC-tTA had 90% recovery. (C–D) LV systolic and diastolic functions assessed by dP/dt_max and dP/dt_min respectively were significantly higher in α-MHC-tTA compared to control after 30 min of ischemia. (N = 3 for control and N = 4 for αMHC-tTA, p<0.05).</p

Effect of PI3K inhibition on the protection against I/R injur in α-MHC-tTA hearts.

<p>Administration of LY294002 did not abolish the protective effect seen in α-MHC-tTA hearts however it did abolish protection imparted by IPC. (N = 5 for α-MHC-tTA and N = 3 for α-MHC-tTA+8 µM LY294002, p<0.05).</p

Mutations in the effector region of Ras disrupt PI3KC2β binding.

<p>(A) Point mutations in the effector region of Ras12V disrupt interactions with specific Ras targets. (B) VC-tagged PI3KC2β was co-transfected with either VN-tagged Ras17N, 17N/69N, or one the effector mutants in the background of Ras17N/69N. BiFC signal is pseudo-colored green. Effector mutations that disrupt Class I PI3K binding to Ras12V disrupt Class II PI3K binding to Ras17N/69N. CFP (red) was used as a transfection control. (C) Graph represents the average fluorescence intensity per cell ± S.E.M. from at least three independent experiments. (*p = 0.02). (D) Western blot analysis demonstrates equal expression of all constructs. (E) Mutation of Thr392 to Asp or Lys379 to Ala in full-length PI3KC2β disrupts interaction with Ras17N. The ΔRBD mutant was also included as a negative control. Graph represents the average of three independent experiments (*p<0.05).</p