n-caproylsphingosine and Ovarian-Neoplasms

Exosomes, which are nanovesicles secreted by almost all the cells, mediate intercellular communication and are involved in various physiological and pathological processes. We aimed to investigate the effects of graphene oxide (GO) on the biogenesis and release of exosomes in human ovarian cancer (SKOV3) cells.. Exosomes were isolated using ultracentrifugation and ExoQuick and characterized by various analytical techniques. The expression levels of exosome markers were analyzed via quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay.. Graphene oxide (10-50 μg/mL), cisplatin (2-10 μg/mL), and C6-ceramide (5-25 μM) inhibited the cell viability, proliferation, and cytotoxicity in a dose-dependent manner. We observed that graphene oxide (GO), cisplatin (CIS), and C6-Ceramide (C6-Cer) stimulated acetylcholine esterase and neutral sphingomyelinase activity, total exosome protein concentration, and exosome counts associated with increased level of apoptosis, oxidative stress and endoplasmic reticulum stress. In contrast, GW4869 treatment inhibits biogenesis and release of exosomes. We observed that the human ovarian cancer cells secreted exosomes with typical cup-shaped morphology and surface protein biomarkers. The expression levels of TSG101, CD9, CD63, and CD81 were significantly higher in GO-treated cells than in control cells. Further, cytokine and chemokine levels were significantly higher in exosomes isolated from GO-treated SKOV3 cells than in those isolated from control cells. SKOV3 cells pre-treated with N-acetylcysteine or GW4869 displayed a significant reduction in GO-induced exosome biogenesis and release. Furthermore, endocytic inhibitors decrease exosome biogenesis and release by impairing endocytic pathways.. This study identifies GO as a potential tool for targeting the exosome pathway and stimulating exosome biogenesis and release. We believe that the knowledge acquired in this study can be potentially extended to other exosome-dominated pathologies and model systems. Furthermore, these nanoparticles can provide a promising means to enhance exosome production in SKOV3 cells.

Topics: Carcinoma, Ovarian Epithelial; Cisplatin; Exosomes; Female; Humans; Ovarian Neoplasms

Ceramide (Cer) is an active cellular sphingolipid that can induce apoptosis or proliferation-arrest of cancer cells. Nanoparticle-based delivery offers an effective approach for overcoming bioavailability and biopharmaceutics issues attributable to the pronounced hydrophobicity of Cer. Missense mutations of the protein p53, which have been detected in approximately 42% of cancer cases, not only lose the tumor suppression activity of wild-type p53, but also gain oncogenic functions promoting tumor progression and drug resistance. Our previous works showed that cellular Cer can eradicate cancer cells that carry a p53 deletion-mutation by modulating alternative pre-mRNA splicing, restoring wild-type p53 protein expression. Here, we report that new ceramide-rubusoside (Cer-RUB) nanomicelles considerably enhance Cer

Topics: Animals; Cell Line, Tumor; Ceramides; Diterpenes, Kaurane; Female; Glucosides; Humans; Mice; Mice, Nude; Mice, Transgenic; Micelles; Mutation, Missense; Nanoparticles; Ovarian Neoplasms; Random Allocation; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Platinum-based therapies are the first line treatments for most types of cancer including ovarian cancer. However, their use is associated with dose-limiting toxicities and resistance. We report initial translational studies of a theranostic nanoemulsion loaded with a cisplatin derivative, myrisplatin and pro-apoptotic agent, C6-ceramide.. The surface of the nanoemulsion is annotated with an endothelial growth factor receptor (EGFR) binding peptide to improve targeting ability and gadolinium to provide diagnostic capability for image-guided therapy of EGFR overexpressing ovarian cancers. A high shear microfludization process was employed to produce the formulation with particle size below 150 nm.. Pharmacokinetic study showed a prolonged blood platinum and gadolinium levels with nanoemulsions in nu/nu mice. The theranostic nanoemulsions also exhibited less toxicity and enhanced the survival time of mice as compared to an equivalent cisplatin treatment.. Magnetic resonance imaging (MRI) studies indicate the theranostic nanoemulsions were effective contrast agents and could be used to track accumulation in a tumor. The MRI study additionally indicate that significantly more EGFR-targeted theranostic nanoemulsion accumulated in a tumor than non-targeted nanoemulsuion providing the feasibility of using a targeted theranostic agent in conjunction with MRI to image disease loci and quantify the disease progression.

Topics: Animals; Antineoplastic Agents; Blood Platelets; Ceramides; Drug Delivery Systems; ErbB Receptors; Female; Gadolinium; Mice; Microfluidics; Organoplatinum Compounds; Ovarian Neoplasms; Particle Size; Survival Analysis; Theranostic Nanomedicine; Tissue Distribution

Platinum-based chemotherapy is the treatment of choice for malignant epithelial ovarian cancers, but generalized toxicity and platinum resistance limits its use. Theranostic nanoemulsion with a novel platinum prodrug, myrisplatin, and the pro-apoptotic agent, C6-ceramide, were designed to overcome these limitations.. The nanoemulsions, including ones with an EGFR binding peptide and gadolinium, were made using generally regarded as safe grade excipients and a high shear microfluidization process. Efficacy was evaluated in ovarian cancer cells, SKOV3, A2780 and A2780CP.. The nanoemulsion with particle size <150 nm were stable in plasma and parenteral fluids for 24 h. Ovarian cancer cells in vitro efficiently took up the non-targeted and EGFR-targeted nanoemulsions; improved cytotoxicity was observed for the these nanoemulsions with the latter showing a 50-fold drop in the IC50 in SKOV3 cells as compared to cisplatin alone. The addition of gadolinium did not affect cell viability in vitro, but showed relaxation times comparable to Magnevist(®).. The myrisplatin/C6-ceramide nanoemulsion synergistically enhanced in vitro cytotoxicity. An EGFR binding peptide addition further increased in vitro cytotoxicity in EGFR positive cancer cells. The diagnostic version showed MR imaging similar to the clinically relevant Magnevist® and may be suitable as a theranostic for ovarian cancer.

Topics: Amino Acid Sequence; Antineoplastic Agents; Cell Line, Tumor; Cell Survival; Ceramides; Drug Carriers; Drug Delivery Systems; Emulsions; Female; Gadolinium; Green Fluorescent Proteins; Humans; Magnetic Resonance Imaging; Molecular Sequence Data; Organoplatinum Compounds; Ovarian Neoplasms; Ovary; Peptides

Ovarian cancer is a debilitating disease, which needs multi-pronged approach of targeted drug delivery and enhanced efficacy with the use of combination therapeutics. In this study, we have examined the anticancer activity of PIK75 incorporated in surface functionalized nanoemulsions for targeted delivery to SKOV-3 cells. A pro-apoptotic molecule C(6)-ceramide was also co-delivered to augment therapeutic efficacy.. EGFR and FR functionalized nanoemulsions incorporating PIK75 and C(6)-ceramide were characterized for particle size, surface charge, entrapment efficiency and morphology. Fluorescence and quantitative uptake studies were conducted in SKOV-3 cells to determine intracellular distribution. Cell viability was assessed using MTT assay while mechanism of cytotoxicity was evaluated using capsase-3/7, TUNEL and hROS assay.. Cytotoxicity assay showed 57% decrease in IC(50) value of PIK75 following treatment with EGFR targeted nanoemulsion and 40% decrease following treatment with FR targeted nanoemulsion. Combination therapy with PIK75 and ceramide enhanced the cytotoxicity of PIK75 compared to therapy with individual formulations. The increase in cytotoxicity was attributed to increase in cellular apoptosis and hROS activity.. The results of this study showed that the targeted system improved cytotoxicity of PIK75 compared to the non-targeted system. Combination therapy with ceramide augmented PIK75's therapeutic activity.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Caspase 3; Caspase 7; Cell Line, Tumor; Cell Survival; Ceramides; Chemistry, Pharmaceutical; Drug Delivery Systems; Emulsions; ErbB Receptors; Female; Humans; Nanoparticles; Ovarian Neoplasms; Particle Size; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Reactive Oxygen Species

P-glycoprotein (P-gp) antagonists inhibit ceramide metabolism at the juncture of glycosylation. The purpose of this study was to test whether targeting P-gp would be a viable alternative to targeting glucosylceramide synthase (GCS) for enhancing ceramide cytotoxicity. A2780 wild-type, and multidrug-resistant 2780AD and NCI/ADR-RES human ovarian cancer cell lines and the cell-permeable ceramide analog, C6-ceramide (C6-cer), were employed. Compared to P-gp-poor A2780 cells, P-gp-rich 2780AD cells converted 3.7-fold more C6-cer to nontoxic C6-glucosylceramide (C6-GC), whereas cell-free GCS activities were equal. 2780AD cells displayed resistance to C6-cer (10 μM) that was reversed by inclusion of the P-gp antagonist tamoxifen (5 μM) but not by inclusion of a GCS inhibitor. Co-administration of C6-cer and P-gp antagonists was also effective in NCI/ADR-RES cells. For example, C6-cer, VX-710 (Biricodar), and cyclosporin A (cyc A) exposure resulted in viabilities of ~90% of control; however, C6-cer/VX-710 and C6-cer/cyc A additions were synergistic and resulted in viabilities of 22% and 17%, respectively. Further, whereas C6-ceramide and cyc A imparted 1.5- and 0-fold increases in caspase 3/7 activity, the combination produced a 3.5-fold increase. Although the upstream elements of cell death have not been elucidated, the novel C6-ceramide/P-gp antagonist combination merits further study and assessment of clinical translational potential.

Topics: Antineoplastic Agents, Hormonal; Antineoplastic Combined Chemotherapy Protocols; ATP Binding Cassette Transporter, Subfamily B, Member 1; Blotting, Western; Ceramides; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Drug Synergism; Female; Glucosyltransferases; Humans; Ovarian Neoplasms; Tamoxifen; Tumor Cells, Cultured