1-2-oleoylphosphatidylcholine and Ovarian-Neoplasms

MicroRNAs (miRNAs) are small RNA molecules that affect cellular processes by controlling gene expression. Recent studies have shown that hypoxia downregulates Drosha and Dicer, key enzymes in miRNA biogenesis, causing a decreased pool of miRNAs in cancer and resulting in increased tumor growth and metastasis. Here we demonstrate a previously unrecognized mechanism by which hypoxia downregulates Dicer. We found that miR-630, which is upregulated under hypoxic conditions, targets and downregulates Dicer expression. In an orthotopic mouse model of ovarian cancer, delivery of miR-630 using 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) nanoliposomes resulted in increased tumor growth and metastasis, and decreased Dicer expression. Treatment with the combination of anti-miR-630 and anti-vascular endothelial growth factor antibody in mice resulted in rescue of Dicer expression and significantly decreased tumor growth and metastasis. These results indicate that targeting miR-630 is a promising approach to overcome Dicer deregulation in cancer. As demonstrated in the study, use of DOPC nanoliposomes for anti-miR delivery serves as a better alternative approach to cell line-based overexpression of sense or antisense miRNAs, while avoiding potential in vitro selection effects. Findings from this study provide a new understanding of miRNA biogenesis downregulation observed under hypoxia and suggest therapeutic avenues to target this dysregulation in cancer.

Topics: Animals; Cell Hypoxia; Cell Line, Tumor; DEAD-box RNA Helicases; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Humans; Liposomes; Mice; MicroRNAs; Neoplasms; Ovarian Neoplasms; Phosphatidylcholines; Ribonuclease III; Vascular Endothelial Growth Factor A

A deeper mechanistic understanding of tumour angiogenesis regulation is needed to improve current anti-angiogenic therapies. Here we present evidence from systems-based miRNA analyses of large-scale patient data sets along with in vitro and in vivo experiments that miR-192 is a key regulator of angiogenesis. The potent anti-angiogenic effect of miR-192 stems from its ability to globally downregulate angiogenic pathways in cancer cells through regulation of EGR1 and HOXB9. Low miR-192 expression in human tumours is predictive of poor clinical outcome in several cancer types. Using 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) nanoliposomes, we show that miR-192 delivery leads to inhibition of tumour angiogenesis in multiple ovarian and renal tumour models, resulting in tumour regression and growth inhibition. This anti-angiogenic and anti-tumour effect is more robust than that observed with an anti-VEGF antibody. Collectively, these data identify miR-192 as a central node in tumour angiogenesis and support the use of miR-192 in an anti-angiogenesis therapy.

Topics: Animals; Cell Line, Tumor; Down-Regulation; Early Growth Response Protein 1; Female; Gene Regulatory Networks; Genetic Therapy; Homeodomain Proteins; Humans; Kidney Neoplasms; Mice; MicroRNAs; Neovascularization, Pathologic; Ovarian Neoplasms; Phosphatidylcholines; Tumor Burden

Development of improved RNA interference-based strategies is of utmost clinical importance. Although siRNA-mediated silencing of EphA2, an ovarian cancer oncogene, results in reduction of tumor growth, we present evidence that additional inhibition of EphA2 by a microRNA (miRNA) further "boosts" its antitumor effects. We identified miR-520d-3p as a tumor suppressor upstream of EphA2, whose expression correlated with favorable outcomes in two independent patient cohorts comprising 647 patients. Restoration of miR-520d-3p prominently decreased EphA2 protein levels, and suppressed tumor growth and migration/invasion both in vitro and in vivo. Dual inhibition of EphA2 in vivo using 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) nanoliposomes loaded with miR-520d-3p and EphA2 siRNA showed synergistic antitumor efficiency and greater therapeutic efficacy than either monotherapy alone. This synergy is at least in part due to miR-520d-3p targeting EphB2, another Eph receptor. Our data emphasize the feasibility of combined miRNA-siRNA therapy, and will have broad implications for innovative gene silencing therapies for cancer and other diseases.. This study addresses a new concept of RNA inhibition therapy by combining miRNA and siRNA in nanoliposomal particles to target oncogenic pathways altered in ovarian cancer. Combined targeting of the Eph pathway using EphA2-targeting siRNA and the tumor suppressor miR-520d-3p exhibits remarkable therapeutic synergy and enhanced tumor suppression in vitro and in vivo compared with either monotherapy alone.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cohort Studies; Drug Therapy, Combination; Female; Gene Silencing; Humans; Mice; Mice, Nude; MicroRNAs; Molecular Targeted Therapy; Ovarian Neoplasms; Phosphatidylcholines; Receptor, EphA2; Receptor, EphB2; RNA, Small Interfering; Xenograft Model Antitumor Assays

Ovarian cancer remains a major threat to women's health, partly due to difficulty in early diagnosis and development of metastases. A critical need exists to identify novel targets that curb the progression and metastasis of ovarian cancer. In this study, we examined whether the nuclear receptor coregulator PELP1 (proline-, glutamic acid-, leucine-rich protein-1) contributes to progression and metastatic potential of ovarian cancer cells and determined whether blocking of the PELP1 signaling axis had a therapeutic effect.. Ovarian cancer cells stably expressing PELP1-shRNA (short hairpin RNA) were established. Fluorescent microscopy, Boyden chamber, invasion assays, wound healing, and zymography assays were performed to examine the role of PELP1 in metastasis. Expression analysis of the model cells was conducted using tumor metastasis microarray to identify PELP1 Target genes. Therapeutic potential of PELP1-siRNA in vivo was determined using a nanoliposomal formulation of PELP1-siRNA-DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine) administered systemically in a xenograft model.. PELP1 knockdown caused cytoskeletal defects and significantly affected the migratory potential of ovarian cancer cells. Microarray analysis revealed that PELP1 affected the expression of selective genes involved in metastasis including Myc, MTA1, MMP2, and MMP9. Zymography analysis confirmed that PELP1 knockdown caused a decrease in the activation of matrix metalloproteases (MMP) 2 and MMP9. Compared with control siRNA-DOPC-treated mice, animals injected with PELP1-siRNA-DOPC had 54% fewer metastatic tumor nodules, exhibited a 51% reduction in tumor growth and an 84% reduction in ascites volume.. The results suggest that PELP1 signaling axis is a potential druggable target and liposomal PELP1-siRNA-DOPC could be used as a novel drug to prevent or treat ovarian metastasis.

Topics: Animals; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Co-Repressor Proteins; Cytoskeleton; Female; Gene Expression Profiling; Humans; Liposomes; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Nude; Neoplasm Metastasis; Oligonucleotide Array Sequence Analysis; Ovarian Neoplasms; Phosphatidylcholines; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; Transcription Factors; Tumor Burden; Xenograft Model Antitumor Assays

We investigated the clinical and biological significance of p130cas, an important cell signaling molecule, in ovarian carcinoma.. 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.. 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.. 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.

Topics: Adult; Aged; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Carcinoma; Cell Survival; Crk-Associated Substrate Protein; Disease-Free Survival; Docetaxel; Down-Regulation; Electrophoresis, Polyacrylamide Gel; Female; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Gene Silencing; Green Fluorescent Proteins; Humans; Immunoblotting; Immunohistochemistry; Kaplan-Meier Estimate; Membrane Proteins; Mice; Microscopy, Electron, Transmission; Microtubule-Associated Proteins; Middle Aged; Multivariate Analysis; Ovarian Neoplasms; Phosphatidylcholines; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; RNA, Small Interfering; Signal Transduction; Taxoids; Transfection; Transplantation, Heterologous; Up-Regulation

RNA interference (RNAi) is a powerful approach for silencing genes associated with a variety of pathologic conditions; however, in vivo RNAi delivery has remained a major challenge due to lack of safe, efficient, and sustained systemic delivery. Here, we report on a novel approach to overcome these limitations using a multistage vector composed of mesoporous silicon particles (stage 1 microparticles, S1MP) loaded with neutral nanoliposomes (dioleoyl phosphatidylcholine, DOPC) containing small interfering RNA (siRNA) targeted against the EphA2 oncoprotein, which is overexpressed in most cancers, including ovarian. Our delivery methods resulted in sustained EphA2 gene silencing for at least 3 weeks in two independent orthotopic mouse models of ovarian cancer following a single i.v. administration of S1MP loaded with EphA2-siRNA-DOPC. Furthermore, a single administration of S1MP loaded with-EphA2-siRNA-DOPC substantially reduced tumor burden, angiogenesis, and cell proliferation compared with a noncoding control siRNA alone (SKOV3ip1, 54%; HeyA8, 57%), with no significant changes in serum chemistries or in proinflammatory cytokines. In summary, we have provided the first in vivo therapeutic validation of a novel, multistage siRNA delivery system for sustained gene silencing with broad applicability to pathologies beyond ovarian neoplasms.

Topics: Animals; Cell Line, Tumor; Female; Gene Silencing; Genetic Therapy; Humans; Liposomes; Mice; Mice, Nude; Nanoparticles; Ovarian Neoplasms; Phosphatidylcholines; Receptor, EphA2; RNA, Small Interfering; Silicon; Xenograft Model Antitumor Assays

Discovery of RNA interference (RNAi) has been one of the most important findings in the last ten years. In recent years, small interfering RNA (siRNA)-mediated gene silencing is beginning to show substantial promise as a new treatment modality in preclinical studies because of its robust gene selective silencing. However, until recently, delivery of siRNA in vivo was a major impediment to its use as a therapeutic modality. We have used a neutral liposome, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), for highly efficient in vivo siRNA delivery. Using siRNA tagged with Alexa-555, incorporated in DOPC liposomes, we have demonstrated efficient intra-tumoral delivery following either intraperitoneal or intravenous injection. Furthermore, EphA2-targeted siRNA in DOPC liposomes showed significant target modulation and anti-tumor efficacy.

Topics: Animals; Carcinoma; Female; Fluorescent Dyes; Gene Silencing; Genetic Therapy; Humans; Liposomes; Mice; Mice, Nude; Ovarian Neoplasms; Phosphatidylcholines; Receptor, EphA2; RNA, Small Interfering

Focal adhesion kinase (FAK) plays a critical role in ovarian cancer cell survival and in various steps in the metastatic cascade. Based on encouraging in vitro results with FAK silencing, we examined the in vivo therapeutic potential of this approach using short interfering RNA (siRNA) in the neutral liposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC).. Therapy experiments of FAK siRNA with or without docetaxel were done using human ovarian cancer cell lines SKOV3ip1, HeyA8, and HeyA8MDR in nude mice. Additional experiments with a cisplatin-resistant cell line (A2780-CP20) were also done. Assessments of angiogenesis (CD31), cell proliferation (proliferating cell nuclear antigen), and apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) were done using immunohistochemical analysis.. A single dose of FAK siRNA-DOPC was highly effective in reducing in vivo FAK expression for up to 4 days as assayed by Western blot and immunohistochemical analysis. Therapy experiments were started 1 week after injection of the ovarian cancer cells. Treatment with FAK siRNA-DOPC (150 mug/kg twice weekly) reduced mean tumor weight by 44% to 72% in the three cell lines compared with the control group (Ps < 0.05 for HeyA8, A2780-CP20, and SKOV3ip1). When FAK siRNA-DOPC was combined with docetaxel, there was even greater reduction in mean tumor weight in all models (all Ps < 0.05). Similar results were observed in combination with cisplatin. Treatment with FAK siRNA-DOPC plus docetaxel resulted in decreased microvessel density, decreased expression of vascular endothelial growth factor and matrix metalloproteinase-9, and increased apoptosis of tumor-associated endothelial cells and tumor cells.. Taken together, these findings suggest that FAK siRNA-DOPC plus docetaxel or platinum might be a novel therapeutic approach against ovarian cancer.

Topics: Animals; Apoptosis; Cell Proliferation; Down-Regulation; Female; Focal Adhesion Protein-Tyrosine Kinases; Humans; Liposomes; Mice; Mice, Nude; Neovascularization, Pathologic; Ovarian Neoplasms; Phosphatidylcholines; RNA, Small Interfering; Xenograft Model Antitumor Assays

Intravenous (IV) delivery of siRNA incorporated into neutral liposomes allows efficient delivery to tumor tissue, and has therapeutic efficacy in preclinical proof-of-concept studies using EphA2-targeting siRNA. We sought to determine whether intraperitoneal (IP) delivery of these siRNA complexes was as effective at delivery and therapy as IV delivery.. SiRNA was incorporated into the neutral liposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). Alexa555-siRNA-DOPC was injected IP into nude mice bearing established ovarian tumors, and organs were collected for microscopic fluorescent examination. Subsequently, therapeutic efficacy of the IP versus IV routes was directly compared.. Alexa555-siRNA in DOPC liposomes injected IP was diffusely distributed into intraperitoneal ovarian tumors. Delivery was also seen deeply into the liver and kidney parenchyma, suggesting that the predominant means of distribution was through the vasculature, rather than direct diffusion from the peritoneal cavity. In mice with orthotopic ovarian tumors, treatment with combined paclitaxel and IP EphA2-targeting siRNA-DOPC reduced tumor growth by 48-81% compared to paclitaxel/control siRNA-DOPC IP (HeyA8: 0.34 g v 0.66 g; SKOV3ip1: 0.04 v 0.21, p<0.01). This reduction was comparable to concurrently-treated mice with paclitaxel and EphA2 siRNA-DOPC injected IV, which showed a reduction in growth by 45-69% compared to paclitaxel/control siRNA-DOPC injected IV (HeyA8: 0.23g v. 0.42g; SKOV3ip1: 0.04 v. 0.13 g).. IP injection of siRNA incorporated in DOPC allows intra-tumoral delivery and has therapeutic efficacy in orthotopic ovarian tumors. These findings may have therapeutic implications for siRNA-based strategies.

Topics: Animals; Cell Line, Tumor; Female; Genetic Therapy; Humans; Liposomes; Mice; Mice, Nude; Ovarian Neoplasms; Phosphatidylcholines; RNA, Small Interfering; Transplantation, Heterologous

Inducing destruction of specific mRNA using small interfering RNA (siRNA) is a powerful tool in analysis of protein function, but its use as a therapeutic modality has been limited by inefficient or impractical delivery systems. We have used siRNA incorporated into the neutral liposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) for efficient in vivo siRNA delivery. In nude mice bearing i.p. ovarian tumors, nonsilencing siRNA tagged with the fluorochrome Alexa 555 was encapsulated into DOPC liposomes and shown to be taken up by the tumor as well as many major organs. Furthermore, DOPC-encapsulated siRNA targeting the oncoprotein EphA2 was highly effective in reducing in vivo EphA2 expression 48 hours after a single dose as measured by both Western blot and immunohistochemistry. Therapy experiments in an orthotopic mouse model of ovarian cancer were initiated 1 week after injection of either HeyA8 or SKOV3ip1 cell lines. Three weeks of treatment with EphA2-targeting siRNA-DOPC (150 microg/kg twice weekly) reduced tumor growth when compared with a nonsilencing siRNA (SKOV3ip1: 0.35 versus 0.70 g; P = 0.020; HeyA8: 0.98 versus 1.51 g; P = 0.16). When EphA2-targeting siRNA-DOPC was combined with paclitaxel, tumor growth was dramatically reduced compared with treatment with paclitaxel and a nonsilencing siRNA (SKOV3ip1: 0.04 versus 0.22 g; P < 0.001; HeyA8: 0.21 versus 0.84 g; P = 0.0027). These studies show the feasibility of siRNA as a clinically applicable therapeutic modality.

Topics: Animals; Cell Line, Tumor; Down-Regulation; Female; Genetic Therapy; Humans; Liposomes; Mice; Mice, Nude; Neovascularization, Pathologic; Ovarian Neoplasms; Phosphatidylcholines; Receptor, EphA2; RNA, Small Interfering; Xenograft Model Antitumor Assays

A unique method for formulation of plasmid DNA with phospholipids has been devised for the purpose of producing vehicles that can mediate gene delivery and transfection of living cells. The polycation, spermine, was used to condense plasmid DNA within a water-in-chloroform emulsion stabilized by phospholipids. After organic solvent removal, the particles formed could be extruded to a number average size of about 200 nm and retained DNA that was protected from nuclease digestion. This resulted in a relatively high protected DNA-to-lipid ratio of approximately 1 microg DNA/micromol lipid. The size distribution of the preparation was relatively homogeneous as judged by light microscopy and quasi-elastic light scattering. Electron microscopic studies showed structural heterogeneity, but suggested that at least some of the plasmid DNA in this preparation was in the form of the previously observed spermine-condensed bent rods and toroids and was encapsulated within liposomal membranes. Preparations with the fusogenic phospholipid composition, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-dodecanoyl/ 1, 2-dioleoyl-sn-glycero-3-phosphocholine, showed transfection activity for several cells lines, particularly OVCAR-3 cells. The transfection activity sedimented with the lipid during centrifugation, confirming the association of active plasmid DNA with phospholipids. Transfection efficiency in culture was found to be of the same order of magnitude as cationic lipoplexes but much less toxic to the cells. Significant transfection of OVCAR-3 cells in tissue culture could also be observed, even in the presence of the intraperitoneal fluid from a mouse with an OVCAR-3 ascites tumor. These data indicate a new type of liposomal gene delivery system devoid of cationic lipids, phosphatidylethanolamine, cationic polymers and viral components.

Topics: Animals; Artificial Gene Fusion; Female; Freeze Fracturing; Genetic Therapy; Genetic Vectors; Humans; Liposomes; Mice; Microscopy, Electron; Microscopy, Fluorescence; Ovarian Neoplasms; Phosphatidylcholines; Phosphatidylethanolamines; Spermine; Transfection; Tumor Cells, Cultured