Rictor down-regulation sensitizes chemoresistant OVCA cells to CDDP-induced apoptosis by facilitating Akt proteasomal degradation.

<p>Chemoresistant OVCA cells (C13*) were transfected with rictor siRNA (100 nM siRNA, 48 h) and pretreated with epoxomycin (0-12.5 nM) 30 minutes prior to CDDP treatment (0-10 µM CDDP; 24 h). Rictor, Akt, phospho- Akt (Thr450 and Ser473), and GAPDH content, as well as apoptosis were assessed. Rictor knock-down significantly enhanced CDDP-induced apoptosis in chemoresistant OVCA cells (P<0.01 and P<0.001, respectively) and this effect was rescued by epoxomycin (P<0.05 and p<0.001, respectively) Total-Akt was significantly decreased during rictor silencing with CDDP treatment (P<0.05) and a massive increase in the ratio between phospho-Akt (Ser473) and total-Akt was also observed, which significantly decreased when epoxomycin was added (P<0.001 and P<0.05, respectively) Results are presented as mean ± SEM of three independent experiments. *p<0.05, **p<0.01, ***p<0.001 (vs respective CTL siRNA), # #p<0.01, # # #p<0.05 (vs without epoxomycin). Hoechst 33258 staining was used for assessment of apoptosis as mentioned in experimental procedures.</p

Rictor knockdown sensitizes chemoresistant OVCA cells to CDDP-induced apoptosis.

<p>C13* cells were transfected with rictor siRNA (0-100 nM; 48 h) and cultured with or without CDDP (10 µM; 24 h). Rictor knockdown significantly enhanced CDDP-induced apoptosis in C13* cells in a concentration-dependent manner. Results are expressed as mean ± SEM of three independent experiments. *p<0.05, ***p<0.001 (vs respective CTL siRNA). Hoechst 33258 staining was used for assessment of apoptosis as mentioned in experimental procedures.</p

A hypothetical model illustrating the role of rictor in regulation of CDDP sensitivity in OVCA cells.

<p>In chemosensitive cells, CDDP activates caspase-3 and induces proteasomal degradation for rictor processing, and consequently destabilizes mTORC2, The unstable mTORC2 complex then facilitates Akt phosphorylation at Ser473, an event known to promote Akt proteasomal degradation and the induction of apoptosis. However, in chemoresistant cells, high level of stabilized rictor promotes Akt activation and stabilization, thereby contributing to CDDP resistance.</p

CDDP down-regulates rictor content and induces apoptosis in chemosensitive but not resistant OVCA cells <i>in vitro</i>.

<p>Rictor protein expression is down-regulated in chemosensitive (OV2008 and A2780s) but not chemoresistant OVCA (C13* and A2780cp*) during CDDP treatment (0-10 µM CDDP; 24 h). Decreased rictor content in OV2008 and A2780s following CDDP treatment was associated with increased apoptosis. Rictor protein content in chemoresistant OVCA (OVCAR433, OCC1 and SKOV3) was not affected by CDDP. Rictor content was normalized against GAPDH (loading control). A representative Western blot from three independent experiments is shown. Results are presented as mean ± SEM of three independent experiments. *p<0.05, **p<0.01, ***p<0.001, #p<0.05 (vs CTL in sensitive cells). Hoechst 33258 staining was used for assessment of apoptosis as mentioned in experimental procedures.</p

Apoptotic response of chemoresistant OVCA cells to CDDP following rictor down-regulation is dependent on p53 status.

<p>Chemoresistant OVCA cell lines with varying p53 status (wt-p53, C13* and OVCAR433; p53-mutant, OCC1 and A2780cp; p53-null, SKOV3) were transfected with rictor siRNA (100 nM siRNA, 48 h) with or without adenoviral wt-p53 infection (0-10 MOI; 24 h; p53-deficient cells) and CDDP treatment (0-10 µM CDDP; 24 h). Rictor, p-p53 ser15, p53, PARP and GAPDH content, as well as apoptosis were assessed. Rictor knock-down significantly sensitized chemoresistant wt-p53 cells (<b>A</b>, C13* and OVCAR433; P<0.001) but not p53-difficient cells (<b>A</b>, OCC1; <b>B</b>, A2780cp and SKOV3) to CDDP (10 µM)-induced apoptosis. Reconstitution in A2780cp and SKOV-3 of wt-p53 significantly enhanced CDDP-induced apoptosis (P<0.001). Results are presented as mean ± SEM of three independent experiments. *p<0.05, **p<0.01, ***p<0.001 (vs respective CTL siRNA), # # #p<0.05 (vs controls Adenoviral infection). Hoechst 33258 staining was used for assessment of apoptosis as mentioned in experimental procedures.</p

Proteasome-mediated degradation and caspase-3 activity are responsible for CDDP-induced rictor down-regulation in chemosensitive OVCA.

<p><b>A</b>. OV2008 cells were pretreated (30 min) with the proteasomal inhibitors [epoxomycin (10 nM) and lacytasystin (4 µM)] and subjected to CDDP challenge (0-10 µM; 24 h). Rictor content and apoptosis were analyzed by Western blotting and nuclear morphology assessment. Both epoxomicin and lactacystin effectively blocked CDDP-induced rictor degradation (P<0.001) but only partially attenuated apoptosis. <b>B</b>. OV2008 cells were cultured in the same conditions as in A, but pretreated with proteasome inhibitor and/or Z-DEVD (50 µM). No synergistic effect between the two inhibitors was observed. <b>C</b>. Incubation of OV2008 whole cell lysate (30 min, 30°C) with recombinant active caspase-3 (5-20 µg/ml) resulted in Rictor cleavage as evidenced by decreased intact rictor content (200 kDa) and increased in the cleaved forms (130 kDa and 160 kDa). Results are presented as mean ± SEM of three independent experiments. *p<0.05, **p<0.01, ***p<0.001 (vs respective controls). Hoechst 33258 staining was used for assessment of apoptosis as mentioned in experimental procedures.</p

CDDP-induced rictor downregulation in CDDP-sensitive cells involves caspase-3-mediated cleavage.

<p>OV2008 and A2780s were pretreated with Z-VAD FMK (10 µM) and Z-DEVD FMK (50 µM) for 30 minutes before and during CDDP challenge (0-10 µM; 24 h) and rictor content and apoptosis were assessed. OV2008 cells treated with CDDP alone exhibited an intact rictor (200 kDa) and two cleaved products (160 kDa and 130 kDa). CDDP decreased intact rictor content and 160 kDa protein but markedly increased levels of the 130 kDa band. Treatment of A2780s with CDDP resulted in down-regulation of intact rictor but increased the level of the 160 kDa protein and had no effect on the 130 kDa protein. Pre-treatment of the cells with the pan-caspase inhibitor (Z-VAD) or the specific caspase-3 inhibitor (Z-DEVD) significantly attenuated the CDDP-induced changes in intact and cleaved rictor contents in both sensitive cells, and CDDP-induced apoptosis was significantly but not completely attenuated by the presence of the inhibitors in both chemosensitive cell lines. Rictor content was normalized against GAPDH (loading control). Results are presented as mean ± SEM (n=3 and n=5 in OV2008 and in A2780s, respectively). *p<0.05, **p<0.01, ***p<0.001 (vs respective controls). Hoechst 33258 staining was used for assessment of apoptosis as mentioned in experimental procedures.</p

Immunohistochemistry was used to demonstrate expression of SERPINB3 protein in cancerous ovaries, but not in normal ovaries from women.

<p>(a and a′) Weak, (b and b′) moderate and (c and c′) strong expression of SERPINB3 protein was detected in women with epithelial ovarian cancer. There was either no expression of very weak expression of SERPINB3 in normal ovaries, whereas SERPINB3 protine was easily detectable in cancerous ovaries from women. The negative control used was mouse IgG instead of primary antibody. <i>Scale bar</i> represents 200 μm (the first horizontal panels and IgG) or 50 μm (the second horizontal panels).</p

Expression of <i>SERPINB3</i> mRNA and protein is unique to glandular epithelium of cancerous ovaries from laying hens.

<p>[A] <i>In situ</i> hybridization analyses of <i>SERPINB3</i> mRNA. Cross-sections of normal and cancerous ovaries from laying hens were hybridized with sense or anti-sense chicken <i>SERPINB3</i> cRNA probes. [B] Immunohistochemical expression of SERPINB3 protein: For negative control, the primary antibody was substituted with purified non-immune mouse IgG. F, follicle; GE, glandular epithelium; S, stroma; <i>Scale bar</i> represents 200 μm (the first columnar panels and sense) or 50 μm (the second columnar panels).</p

Multivariate Cox’s proportional hazard analysis for poor prognostic factors affecting progression-free survival.

<p>Abbreviation: FIGO, International Federation of Gynecology and Obstetrics; HR, hazard ratio; CI, confidence interval.</p