Clinical and biological significance of tissue transglutaminase in ovarian carcinoma.
ABSTRACT: Tissue type transglutaminase (TG2) is a unique multifunctional protein that plays a role in many steps in the cancer metastatic cascade. Here, we examined the clinical (n = 93 epithelial ovarian cancers) and biological (in vitro adhesion, invasion, and survival and in vivo therapeutic targeting) significance of TG2 in ovarian cancer. The overexpression of TG2 was associated with significantly worse overall patient survival in both univariate and multivariate analyses. Transfection of TG2 into SKOV3ip1 cells promoted attachment and spreading on fibronectin-coated surfaces and increased the in vitro invasive potential of these cells. Conversely, TG2 silencing with small interfering RNA (siRNA) of HeyA8 cells significantly decreased the invasive potential of the cells and also increased docetaxel-induced cell death. In vivo therapy experiments using chemotherapy-sensitive (HeyA8) and chemotherapy-resistant (HeyA8-MDR and RMG2) models showed significant antitumor activity both with TG2 siRNA-1,2-dioleoyl-sn-glycero-3-phosphatidylcholine alone and in combination with docetaxel chemotherapy. This antitumor activity was related to decreased proliferation and angiogenesis and increased tumor cell apoptosis in vivo. Taken together, these findings indicate that TG2 overexpression is an adverse prognostic factor in ovarian carcinoma and TG2 targeting may be an attractive therapeutic approach.
Project description:The purpose of this study was to investigate the antitumor effects of a combination of metronomic doses of a novel delivery vehicle, PLGA-PRINT nanoparticles containing docetaxel, and antiangiogenic mEZH2 siRNA incorporated into chitosan nanoparticles. In vivo dose-finding studies and therapeutic experiments were conducted in well-established orthotopic mouse models of epithelial ovarian cancer. Antitumor effects were determined on the basis of reduction in mean tumor weight and number of metastatic tumor nodules in the animals. The tumor tissues from these in vivo studies were stained to evaluate the proliferation index (Ki67), apoptosis index (cleaved caspase 3), and microvessel density (CD31). The lowest dose of metronomic regimen (0.5 mg/kg) resulted in significant reduction in tumor growth. The combination of PLGA-PRINT-docetaxel and CH-mEZH2 siRNA showed significant antitumor effects in the HeyA8 and SKOV3ip1 tumor models (P < 0.05). Individual as well as combination therapies showed significant antiangiogenic, antiproliferative, and proapoptotic effects, and combination therapy had additive effects. Metronomic delivery of PLGA-PRINT-docetaxel combined with CH-mEZH2 siRNA has significant antitumor activity in preclinical models of ovarian cancer.
Project description:BACKGROUND:Interleukin-8 (IL-8) is a proangiogenic cytokine that is overexpressed in many human cancers. We investigated the clinical and biologic significance of IL-8 in ovarian carcinoma using human samples and orthotopic mouse models. METHODS:Tumor expression of IL-8 was assessed by immunohistochemistry among ovarian cancer patients (n = 102) with available clinical and survival data. We examined the effect of IL-8 gene silencing with small interfering RNAs incorporated into neutral liposomes (siRNA-DOPCs), alone and in combination with docetaxel, on in vivo tumor growth, angiogenesis (microvessel density), and tumor cell proliferation in mice (n = 10 per treatment group) bearing orthotopic taxane-sensitive (HeyA8 and SKOV3ip1) and taxane-resistant (SKOV3ip2.TR) ovarian tumors. All statistical tests were two-sided. RESULTS:Of the 102 cancer specimens, 43 (42%) had high IL-8 expression and 59 (58%) had low or no IL-8 expression; high IL-8 expression was associated with advanced tumor stage (P = .019), high tumor grade (P = .031), and worse survival (median survival for patients with high vs low IL-8 expression: 1.62 vs 3.79 years; P < .001). Compared with empty liposomes, IL-8 siRNA-DOPC reduced the mean tumor weight by 32% (95% confidence interval [CI] = 14% to 50%; P = .03) and 52% (95% CI = 27% to 78%; P = .03) in the HeyA8 and SKOV3ip1 mouse models, respectively. In all three mouse models, treatment with IL-8 siRNA-DOPC plus the taxane docetaxel reduced tumor growth the most compared with empty liposomes (77% to 98% reduction in tumor growth; P < .01 for all). In the HeyA8 and SKOV3ip1 models, tumors from mice treated with IL-8 siRNA-DOPC alone had lower microvessel density than tumors from mice treated with empty liposomes (HeyA8: 34% lower, 95% CI = 32% to 36% lower [P = .002]; SKOV3ip1: 39% lower, 95% CI = 34% to 44% lower [P = .007]). Compared with empty liposomes, IL-8 siRNA-DOPC plus docetaxel reduced tumor cell proliferation by 35% (95% CI = 25% to 44%; P < .001) and 38% (95% CI = 28% to 48%; P < .001) in the HeyA8 and SKOV3ip1 models, respectively. CONCLUSIONS:Increased IL-8 expression is associated with poor clinical outcome in human ovarian carcinoma, and IL-8 gene silencing decreases tumor growth through antiangiogenic mechanisms.
Project description:<h4>Background</h4>We investigated the clinical and biological significance of p130cas, an important cell signaling molecule, in ovarian carcinoma.<h4>Methods</h4>Expression of p130cas in ovarian tumors, as assessed by immunohistochemistry, was associated with tumor characteristics and patient survival. The effects of p130cas gene silencing with small interfering RNAs incorporated into neutral nanoliposomes (siRNA-DOPC), alone and in combination with docetaxel, on in vivo tumor growth and on tumor cell proliferation (proliferating cell nuclear antigen) and apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling) were examined in mice bearing orthotopic taxane-sensitive (HeyA8 and SKOV3ip1) or taxane-resistant (HeyA8-MDR) ovarian tumors (n = 10 per group). To determine the specific mechanisms by which p130cas gene silencing abrogates tumor growth, we measured cell viability (MTT assay), apoptosis (fluorescence-activated cell sorting), autophagy (immunoblotting, fluorescence, and transmission electron microscopy), and cell signaling (immunoblotting) in vitro. All statistical tests were two-sided.<h4>Results</h4>Of 91 ovarian cancer specimens, 70 (76%) had high p130cas expression; and 21 (24%) had low p130cas expression. High p130cas expression was associated with advanced tumor stage (P < .001) and higher residual disease (>1 cm) following primary cytoreduction surgery (P = .007) and inversely associated with overall survival and progression-free survival (median overall survival: high p130cas expression vs low expression, 2.14 vs 9.1 years, difference = 6.96 years, 95% confidence interval = 1.69 to 9.48 years, P < .001; median progression-free survival: high p130cas expression vs low expression, 1.04 vs 2.13 years, difference = 1.09 years, 95% confidence interval = 0.47 to 2.60 years, P = .01). In mice bearing orthotopically implanted HeyA8 or SKOV3ip1 ovarian tumors, treatment with p130cas siRNA-DOPC in combination with docetaxel chemotherapy resulted in the greatest reduction in tumor growth compared with control siRNA therapy (92%-95% reduction in tumor growth; P < .001 for all). Compared with control siRNA therapy, p130cas siRNA-DOPC reduced SKOV3ip1 cell proliferation (31% reduction, P < .001) and increased apoptosis (143% increase, P < .001) in vivo. Increased tumor cell apoptosis may have persisted despite pan-caspase inhibition by the induction of autophagy and related signaling pathways.<h4>Conclusions</h4>Increased p130cas expression is associated with poor clinical outcome in human ovarian carcinoma, and p130cas gene silencing decreases tumor growth through stimulation of apoptotic and autophagic cell death.
Project description:<h4>Purpose</h4>Src is an attractive target because it is overexpressed in a number of malignancies, including ovarian cancer. However, the effect of Src silencing on other Src family kinases (SFKs) is not known. We hypothesized that other SFK members could compensate for the lack of Src activity.<h4>Experimental design</h4>Cell viability after either Src or Fgr silencing was examined in ovarian cancer cell lines by MTT assay. Expression of SFKs after Src silencing in ovarian cancer cells was examined by real-time reverse transcriptase (RT)-PCR. Therapeutic effect of in vivo Src and/or Fgr silencing was examined using siRNA incorporated into chitosan nanoparticles (siRNA/CH-NP). Microvessel density, cell proliferation, and apoptosis markers were determined by immunohistochemical staining in ovarian tumor tissues.<h4>Results</h4>Src silencing enhanced cytotoxicity of docetaxel in both SKOV3ip1 and HeyA8 cells. In addition, Src silencing using siRNA/CH-NP in combination with docetaxel resulted in significant inhibition of tumor growth compared with control siRNA/CH-NP (81.8% reduction in SKOV3ip1, P = 0.017; 84.3% reduction in HeyA8, P < 0.005). These effects were mediated by decreased tumor cell proliferation and angiogenesis, and increased tumor cell apoptosis. Next, we assessed the effects of Src silencing on other SFK members in ovarian cancer cell lines. Src silencing resulted in significantly increased Fgr levels. Dual Src and Fgr silencing in vitro resulted in increased apoptosis that was mediated by increased caspase and AKT activity. In addition, dual silencing of Src and Fgr in vivo using siRNA/CH-NP resulted in the greatest reduction in tumor growth compared with silencing of either Src or Fgr alone in the HeyA8 model (68.8%, P < 0.05).<h4>Conclusions</h4>This study demonstrates that, in addition to Src, Fgr plays a biologically significant role in ovarian cancer growth and might represent an important target.
Project description:EphA2 gene silencing has been shown to result in antitumor efficacy. Here we considered whether silencing additional targets downstream of EphA2 would further enhance the therapeutic effect. EphA2 targeted siRNA was tested in combination with either FAK or Src targeted siRNA using DOPC nanoliposomes in orthotopic models of ovarian carcinoma. The effects of therapy were determined by changes in tumor weight, proliferation (Ki-67), and microvessel density (CD31). In our initial in vivo study, EphA2 plus FAK silencing resulted in the greatest reduction in tumor growth (by 73%, p < 0.005) as compared to control siRNA alone. In the SKOV3ip1 and HeyA8 ovarian cancer models, EphA2 siRNA-DOPC treatment resulted in a 50-67% decrease in tumor growth (p < 0.02, for both), and FAK siRNA-DOPC resulted in a 61-62% decrease in tumor growth (p < 0.009, p < 0.05, respectively). EphA2 plus FAK siRNA-DOPC treatment resulted in a significant reduction (SKOV3ip1: 76%, p < 0.007, HeyA8: 90%, p < 0.003) in tumor growth compared to control siRNA-DOPC. Combination treatment with EphA2 + FAK siRNA-DOPC resulted in significant decreases in tumor cell proliferation (p < 0.001) and microvessel density compared to control siRNA-DOPC (80%; p < 0.001), or the monotherapy groups (p values <0.001). These data suggest that the antitumor efficacy of in vivo EphA2 targeting is enhanced in combination with FAK silencing. Dual targeting of EphA2 and FAK may have therapeutic implications for ovarian cancer management.
Project description:The Aurora kinase family plays pivotal roles in mitotic integrity and cell cycle. We sought to determine the effects of inhibiting Aurora kinase on ovarian cancer growth in an orthotopic mouse model using a small molecule pan-Aurora kinase inhibitor, MK-0457.We examined cell cycle regulatory effects and ascertained the therapeutic efficacy of Aurora kinase inhibition both alone and combined with docetaxel using both in vitro and in vivo ovarian cancer models.In vitro cytotoxicity assays with HeyA8 and SKOV3ip1 cells revealed >10-fold greater docetaxel cytotoxicity in combination with MK-0457. After in vivo dose kinetics were determined using phospho-histone H3 status, therapy experiments with the chemosensitive HeyA8 and SKOV3ip1 as well as the chemoresistant HeyA8-MDR and A2780-CP20 models showed that Aurora kinase inhibition alone significantly reduced tumor burden compared with controls (P values<0.01). Combination treatment with docetaxel resulted in significantly improved reduction in tumor growth beyond that afforded by docetaxel alone (P <or= 0.03). Proliferating cell nuclear antigen immunohistochemistry revealed that MK-0457 alone and in combination with docetaxel significantly reduced cellular proliferation (P values<0.001). Compared with controls, treatment with MK-0457 alone and in combination with docetaxel also significantly increased tumor cell apoptosis by approximately 3-fold (P<0.01). Remarkably, compared with docetaxel monotherapy, MK-0457 combined with docetaxel resulted in significantly increased tumor cell apoptosis.Aurora kinase inhibition significantly reduces tumor burden and cell proliferation and increases tumor cell apoptosis in this preclinical orthotopic model of ovarian cancer. The role of Aurora kinase inhibition in ovarian cancer merits further investigation in clinical trials.
Project description:There is growing recognition of the important role of metronomic chemotherapy in cancer treatment. On the basis of their unique antiangiogenic effects, we tested the efficacy of nab-paclitaxel, which stimulates thrombospondin-1, and topotecan, which inhibits hypoxia-inducible factor 1-?, at metronomic dosing for the treatment of ovarian carcinoma. In vitro and in vivo SKOV3ip1, HeyA8, and HeyA8-MDR (taxane-resistant) orthotopic models were used to examine the effects of metronomic nab-paclitaxel and metronomic topotecan. We examined cell proliferation (Ki-67), apoptosis (cleaved caspase-3), and angiogenesis (microvessel density, MVD) in tumors obtained at necropsy. In vivo therapy experiments demonstrated treatment with metronomic nab-paclitaxel alone and in combination with metronomic topotecan resulted in significant reductions in tumor weight (62% in the SKOV3ip1 model, P < 0.01 and 96% in the HeyA8 model, P < 0.03) compared with vehicle (P < 0.01). In the HeyA8-MDR model, metronomic monotherapy with either cytotoxic agent had modest effects on tumor growth, but combination therapy decreased tumor burden by 61% compared with vehicle (P < 0.03). The greatest reduction in MVD (P < 0.05) and proliferation was seen in combination metronomic therapy groups. Combination metronomic therapy resulted in prolonged overall survival in vivo compared with other groups (P < 0.001). Tube formation was significantly inhibited in RF-24 endothelial cells exposed to media conditioned with metronomic nab-paclitaxel alone and media conditioned with combination metronomic nab-paclitaxel and metronomic topotecan. The combination of metronomic nab-paclitaxel and metronomic topotecan offers a novel, highly effective therapeutic approach for ovarian carcinoma that merits further clinical development.
Project description:EphA2 is overexpressed in many types of human cancer but is absent or expressed at low levels in normal epithelial tissues. We investigated whether a novel immunoconjugate containing an anti-EphA2 monoclonal antibody (1C1) linked to a chemotherapeutic agent (monomethyl auristatin phenylalanine [MMAF]) through a noncleavable linker maleimidocaproyl (mc) had antitumor activity against ovarian cancer cell lines and tumor models.Specificity of 1C1-mcMMAF was examined in EphA2-positive HeyA8 and EphA2-negative SKMel28 ovarian cancer cells by antibody binding and internalization assays. Controls were phosphate-buffered saline (PBS), 1C1, or control IgG-mcMMAF. Viability and apoptosis were investigated in ovarian cancer cell lines and tumor models (10 mice per group). Antitumor activities were tested in the HeyA8-luc and SKOV3ip1 orthotopic mouse models of ovarian cancer. Endothelial cells were identified by use of immunohistochemistry and anti-CD31 antibodies. All statistical tests were two-sided.The 1C1-mcMMAF immunoconjugate specifically bound to EphA2-positive HeyA8 cells but not to EphA2-negative cells and was internalized by HeyA8 cells. Treatment with 1C1-mcMMAF decreased the viability of HeyA8-luc cells in an EphA2-specific manner. In orthotopic mouse models, treatment with 1C1-mcMMAF inhibited tumor growth by 85%-98% compared with that in control mice (eg, for weight of HeyA8 tumors, 1C1-mcMMAF = 0.05 g and control = 1.03 g; difference = 0.98 g, 95% confidence interval [CI] = 0.40 to 1.58 g; P = .001). Even in bulkier disease models with HeyA8-luc cells, 1C1-mcMMAF treatment, compared with control treatment, caused regression of established tumors and increased survival of the mice (eg, 1C1-mcMMAF vs control, mean = 60.6 days vs 29.4 days; difference = 31.2 days, 95% CI = 27.6 to 31.2 days; P = .001). The antitumor effects of 1C1-mcMMAF therapy, in SKOV3ip1 tumors, for example, were statistically significantly related to decreased proliferation (eg, 1C1-mcMMAF vs control, mean = 44.1% vs 55.8% proliferating cells; difference = 11.7%, 95% CI = 2.45% to 20.9%; P = .01) and increased apoptosis of tumor cells (eg, 1C1-mcMMAF vs control, mean = 8.6% vs 0.9% apoptotic cells; difference = 7.7%, 95% CI = 3.8% to 11.7%; P < .001) and of mouse endothelial cells (eg, 1C1-mcMMAF vs control, mean 2.8% vs 0.4% apoptotic endothelial cells; difference = 2.4%, 95% CI = 1.4% to 4.6%; P = .034).The 1C1-mcMMAF immunoconjugate had antitumor activity in preclinical models of ovarian carcinoma.
Project description:<h4>Purpose</h4>This study aimed to develop an Arg-Gly-Asp (RGD) peptide-labeled chitosan nanoparticle (RGD-CH-NP) as a novel tumor targeted delivery system for short interfering RNA (siRNA).<h4>Experimental design</h4>RGD peptide conjugated with chitosan by thiolation reaction was confirmed by proton-NMR (H-NMR). Binding of RGD-CH-NP with alphanubeta3 integrin was examined by flow cytometry and fluorescence microscopy. Antitumor efficacy was examined in orthotopic mouse models of ovarian carcinoma.<h4>Results</h4>We show that RGD-CH-NP loaded with siRNA significantly increased selective intratumoral delivery in orthotopic animal models of ovarian cancer. In addition, we show targeted silencing of multiple growth-promoting genes (POSTN, FAK, and PLXDC1) along with therapeutic efficacy in the SKOV3ip1, HeyA8, and A2780 models using siRNA incorporated into RGD-CH-NP (siRNA/RGD-CH-NP). Furthermore, we show in vivo tumor vascular targeting using RGD-CH-NP by delivering PLXDC1-targeted siRNA into the alphanubeta3 integrin-positive tumor endothelial cells in the A2780 tumor-bearing mice. This approach resulted in significant inhibition of tumor growth compared with controls.<h4>Conclusions</h4>This study shows that RGD-CH-NP is a novel and highly selective delivery system for siRNA with the potential for broad applications in human disease.
Project description:Platelet-derived growth factor receptor alpha (PDGFR?) is believed to be associated with cell survival. We examined (i) whether PDGFR? blockade enhances the antitumor activity of taxanes in ovarian carcinoma and (ii) potential biomarkers of response to anti-PDGFR? therapy.PDGFR? expression in 176 ovarian carcinomas was evaluated with tissue microarray and correlated to survival outcome. Human-specific monoclonal antibody to PDGFR? (IMC-3G3) was used for in vitro and in vivo experiments with or without docetaxel. Gene microarrays and reverse-phase protein arrays with pathway analyses were performed to identify potential predictive biomarkers.When compared to low or no PDGFR? expression, increased PDGFR? expression was associated with significantly poorer overall survival of patients with ovarian cancer (P=0.014). Although treatment with IMC-3G3 alone did not affect cell viability or increase apoptosis, concurrent use of IMC-3G3 with docetaxel significantly enhanced sensitization to docetaxel and apoptosis. In an orthotopic mouse model, IMC-3G3 monotherapy had no significant antitumor effects in SKOV3-ip1 (low PDGFR? expression), but showed significant antitumor effects in HeyA8-MDR (high PDGFR? expression). Concurrent use of IMC-3G3 with docetaxel, compared with use of docetaxel alone, significantly reduced tumor weight in all tested cell lines. In protein ontology, the EGFR and AKT pathways were downregulated by IMC-3G3 therapy. MAPK and CCNB1 were downregulated only in the HeyA8-MDR model.These data identify IMC-3G3 as an attractive therapeutic strategy and identify potential predictive markers for further development.