Human Ovarian Carcinoma SKOV3 Cells Research Articles

Effects of combining Taxol and cyclooxygenase inhibitors on the angiogenesis and apoptosis in human ovarian cancer xenografts

The present study aimed to investigate the combined effects of Taxol and cyclooxygenase (COX) inhibitors on angiogenesis and cell apoptosis of SKOV-3 human ovarian carcinoma cell xenograft-bearing mice. The experiments were continued for 28 days. Animals were treated with 3 mg/kg SC-560 (a COX-1-selective inhibitor) alone, 100 mg/kg celecoxib (a COX-2-selective inhibitor) alone or SC-560/celecoxib by gavage twice a day, 20 mg/kg Taxol alone intraperitoneally once a week or in combination with SC-560 or celecoxib or SC-560/celecoxib/Taxol for three weeks. The mRNA levels of vascular endothelial growth factor (VEGF) was determined by reverse transcription-polymerase chain reaction (RT-PCR). The microvessel density (MVD) of ovarian carcinoma was determined by immunohistochemistry with anti-CD34 as the label. The apoptotic index was detected by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) method. The MVD value and apoptotic index in the SC-560/Taxol group were notably inhibited compared with the Taxol group (P<0.001). Moreover, the VEGF mRNA levels, MVD value and apoptotic index in the SC-560/Taxol group were significantly different from the celecoxib/Taxol group (P<0.05, P<0.05 and P<0.001, respectively). The present study demonstrated that SC-560 enhances the anti-angiogenic and pro-apoptotic effects of Taxol and these effects are better than with celecoxib.

Anti-ovarian cancer potential of two acidic polysaccharides from the rhizoma of Menispermum dauricum

Two acidic polysaccharides (MDP-A1 and MDP-A2) were isolated from the rhizome of Menispermum dauricum and their apparent molecular weight are 9.1×104 and 5.8×104Da, respectively. Both contained glucose, mannose, galactose, arabinose, glucuronic acid and galacturonic acid, but differed in the molar ratio. We also investigated the antitumor activities and mechanisms of MDP-A1 and MDP-A2 in human ovarian carcinoma SKOV3 cells. The MTT assay showed that MDP-A1 and MDP-A2 were able to inhibit cell proliferation of SKOV3 cells in a bell-shaped concentration–response manner, due to a significant increase in the number of apoptotic cells. Furthermore, treatment with MDP-A1 or MDP-A2 caused significant induction of caspase-3 and caspase-8, but slightly affected caspase-9 activity. In addition, a significant reduction in tumor volume was observed in mice treated with MDP-A1 or MDP-A2. Taken together, these results suggested that MDP-A1 and MDP-A2 had a potential application as natural antitumor drugs.

Characterization and antitumor activities of a polysaccharide from the rhizoma of Menispermum dauricum

The rhizome of Menispermum dauricum DC (Menispermaceae) is one of the most commonly used traditional Chinese medicines officially listed in Chinese Pharmacopeia. In present study, we purified a water-soluble polysaccharide (WMDP) from this plant and investigated its physicochemical properties. WMDP was a homogeneous polysaccharide, with an average molecular weight of approximately 3.5×104Da, as determined by high-performance gel-permeation chromatography (HPGPC). Gas chromatography (GC) analysis identified that WMDP was composed of Glc, Gal, Xyl, Rha, Ara and Man in the ratio of 2.45:2.13:1.05:1.29:1.63:1.45. The interreaction between Gongo Red and WMDP in NaOH solutions resulted in the shift of maximum absorption, indicating WMDP had a triple-helix conformation. We also investigated the antitumor activities and mechanisms of WMDP in human ovarian carcinoma SKOV3 cells. The experimental evidence showed that WMDP significantly inhibited cell proliferation and DNA synthesis in SKOV3 cells in a concentration-dependent manner, due to a significant increase in the number of apoptotic cells. Furthermore, treatment with WMDP caused a rapid loss of intracellular glutathione (GSH) content and stimulation of reactive oxygen species (ROS). In addition, nuclear factor-kappa B (NF-κB) in SKOV3 cells received WMDP treatment was inactivated. Taken together, induction of apoptosis on SKOV3 cells by WMDP was mainly associated with ROS production, GSH depletion and NF-κB inactivation.

Selective Gene Delivery to Cancer Cells Using an Integrated Cationic Amphiphilic Peptide

Gene therapy provides a number of potential treatments that could be applied in clinic to prevent deaths from cancer. However, the transfer of gene therapy to the clinical application has proven difficult because many problems remain to be solved concerning the transfection efficiency, target specificity, and safety issues. To overcome these barriers, a peptide-based vector, K(12)H(6)V(8)SSQHWSYKLRP (KHV-LHRH) that comprises four functional blocks, is studied in this work for the targeted delivery of a model gene drug to cancer cells. KHV-LHRH peptide, which contains a luteinizing hormone-releasing hormone (LHRH) sequence, can specifically target cancer cells expressing LHRH receptors. The gene expression, cytotoxicity, and cellular uptake mediated by this vector were evaluated against MCF-7 human breast cancer cells (LHRH-receptor-positive) and SKOV-3 human ovarian carcinoma cells (LHRH-receptor-negative) and compared to a peptide vector (K(12)H(6)V(8)) (KHV) without the LHRH ligand and poly(ethylenimine) (PEI). The results showed that KHV-LHRH enhanced the DNA internalization and induced significantly higher gene expression than KHV in LHRH-receptor-positive MCF-7 cells. Also, the peptide-based vectors had low cytotoxicity compared to that of PEI. The high specificity and transfection efficiency of the integrated peptide-based vector make it a very promising material for targeted gene delivery in cancer therapy.

Differential expression of the P2X7 receptor in ovarian surface epithelium during the oestrous cycle in the mouse

Purinergic signalling has been proposed as an intraovarian regulatory mechanism. Of the receptors responsible for purinergic transmission, the P2X7 receptor is an ATP-gated cationic channel that displays a broad spectrum of cellular functions ranging from apoptosis to cell proliferation and tumourigenesis. In the present study, we investigated the functional expression of P2X7 receptors in ovarian surface epithelium (OSE). P2X7 protein was detected in the OSE layer of the mouse, both in situ and in primary cultures. In cultures, 2'(3')-O-(4-Benzoylbenzoyl)adenosine-5'-triphosphate (BzATP) activation of P2X7 receptors increased [Ca(2+)]i and induced apoptosis. The functionality of the P2X7 receptor was investigated in situ by intrabursal injection of BzATP on each day of the oestrous cycle and evaluation of apoptosis 24h using the terminal deoxyribonucleotidyl transferase-mediated dUTP-fluorescein nick end-labelling (TUNEL) assay. Maximum effects of BzATP were observed during pro-oestrus, with the effects being blocked by A438079, a specific P2X7 receptor antagonist. Immunofluorescence staining for P2X7 protein revealed more robust expression during pro-oestrus and in OSE regions behind the antral follicles, strongly supporting the notion that the differences in apoptosis can be explained by increased receptor expression, which is regulated during the oestrous cycle. Finally, P2X7 receptor expression was detected in the OSE layer of human ovaries, with receptor expression maintained in human ovaries diagnosed with cancer, as well as in the human ovarian carcinoma SKOV3 cell line.

Expression of Peroxiredoxin 1, 2, 3, and 6 Genes in Cancer Cells during Drug Resistance Formation

We studied the expression of peroxiredoxin genes (PRDX1, PRDX2, PRDX3, and PRDX6) in human erythroleukemia K652, human breast carcinoma MCF-7, and human ovarian carcinoma SKOV-3 cells during cisplatin resistance development. It was found that drug resistance formation was accompanied by a significant increase in the expression of PRDX1, PRDX2, PRDX3, PRDX6 genes in all cancer cell strains, which confirms the important contribution of redox-dependent mechanisms into the development of cisplatin resistance of cancer cells.

The Effect of Metal-Chelating Polymers (MCPs) for 111In Complexed via the Streptavidin-Biotin System to Trastuzumab Fab Fragments on Tumor and Normal Tissue Distribution in Mice

To study the effects of backbone composition and charge of biotin-functionalized metal-chelating polymers (Bi-MCPs) for (111)In complexed to streptavidin (SAv)-trastuzumab Fab fragments on tumor and normal tissue localization. Bi-MCPs were synthesized with a polyacrylamide [Bi-PAm(DTPA)(40)], polyaspartamide [Bi-PAsp(DTPA)(33)] or polyglutamide [Bi-PGlu(DTPA)(28)] backbone and harboured diethylenetriaminepentaacetic acid (DTPA) chelators for (111)In. Bi-PAm(DTPA)(40) had a net negative charge; Bi-PAsp(DTPA)(33) and Bi-PGlu(DTPA)(28) were zwitterionic with a net neutral charge. Binding to HER2+ SKOV-3 human ovarian carcinoma cells was determined. Tissue uptake was studied in Balb/c mice by MicroSPECT/CT imaging and biodistribution studies. Tumor and normal tissue uptake of (111)In-labeled Bi-PAsp(DTPA)(33) or Bi-PGlu(DTPA)(28) complexed to SAv-Fab was evaluated 48h post-injection in athymic mice with subcutaneous SKOV-3 xenografts. SAv-Fab complexed to MCPs bound specifically to SKOV-3 cells; but specific binding was decreased 2-fold. Liver uptake was 5-13 fold higher for Bi-PAm(DTPA)(40) than Bi-PAsp(DTPA)(33) and Bi-PGlu(DTPA)(28) but was reduced by decreasing negative charges by saturation with indium. (111)In-Bi-PAsp(DTPA)(33) complexed to SAv-Fab accumulated in SKOV-3 tumors; low tumor uptake was found for (111)In-Bi-PGlu(DTPA)(28)-SAv-Fab. Zwitterionic MCPs composed of polyaspartamide with a net neutral charge are most desirable for constructing radioimmunoconjugates.

Effect of the combination of a cyclooxygenase-1 selective inhibitor and taxol on proliferation, apoptosis and angiogenesis of ovarian cancer in vivo.

This study was designed to investigate the effects of a cyclooxygenase (COX)-1 inhibitor, SC-560, administered in combination with taxol, on the molecular mechanisms of antitumor efficacy in a SKOV-3 human ovarian carcinoma cell xenograft-bearing mouse model. The mice were treated with 6 mg/kg/day SC-560 by gavage twice every other day and 20 mg/kg taxol by intraperitoneal injection once a week for three weeks. Microvessel density (MVD) and vascular endothelial growth factor (VEGF) mRNA levels of ovarian cancer were detected in the tumor tissues using immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR), respectively. The index of proliferating and apoptotic cells in the tumor tissues was determined by staining for Ki-67 and using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method, respectively. On day 7 after the end of administration, the tumor volume of mice in the combination group was reduced by 55.35% compared with that of the control mice, and the difference was statistically significant (P<0.05). In the combination group, the expression of VEGF, MVD and the cell proliferation index were inhibited significantly, while the apoptotic index was notably increased (all P<0.01, compared with the control group). Our results indicate that the molecular mechanisms of the antitumor efficacy of SC-560 combined with taxol therapy may act in part by inhibiting tumor angiogenesis, reducing cell proliferation and inducing cell apoptosis.

Abstract 4904: Acetaminophen enhances the chemotherapeutic effect of cisplatin and paclitaxel on human ovarian carcinoma cells in vitro

Abstract Chemoresistance is commonly found in treating cancer patients. Acetaminophen enhances the cytotoxicity of cisplatin in human liver cancer cells by depleting glutathione and increasing production of toxic metabolites in vitro. The objective of this study was to investigate potential molecular mechanisms for chemoenhancement in non-hepatic cells. Human ovarian carcinoma SKOV3 cells were treated with cisplatin or paclitaxel with or without acetaminophen in vitro. Cellular mitochondrial membrane potential and reactive oxygen species concentration was measured by using JC-1 and DHR123 stain, respectively. Cellular platinum level was analyzed using ICP-MS. Apoptosis and signaling pathways were examined by immunoblotting and immunohistochemical analyses. A 2 h incubation with acetaminophen (10 mM) decreased cellular glutathione levels and increased mitochondrial membrane potential without affecting reactive oxygen species. Overnight acetaminophen treatment increased reactive oxygen species and enhanced both cisplatin- and paclitaxel-induced apoptosis. In contrast, the combination of acetaminophen with cisplatin but not paclitaxel blocked nuclear translocation of nrf2, a transcription factor that has been implicated in regulation of the antioxidant response. Pretreatment with acetaminophen for 2 h did not affect cellular platinum uptake when compared to cisplatin alone. Regardless of different oxidative stress pathways involved between cisplatin and paclitaxel, the addition of acetaminophen enhanced the cytotoxic effects of both chemotherapeutic drugs in human ovarian carcinoma cells. Our results suggest that acetaminophen could be used as a chemoenhancing adjuvant for conventional chemotherapy drugs in treating ovarian and other malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4904. doi:1538-7445.AM2012-4904

Cytotoxicity of PAMAM, PPI and maltose modified PPIdendrimers in Chinese hamster ovary (CHO) and human ovarian carcinoma (SKOV3) cells

Characterization of dendrimers as potential therapeutics or drug carriers is complete only when toxicity is assessed. There are numerous studies on the influence of surface modification of PAMAM and PPI dendrimers on their cytotoxic properties but without proposing a mechanism for their toxic effect. In this study cytotoxicity profiles of acid-terminated PAMAM G3.5 and amino-terminated PAMAM G4 in comparison to unmodified amino-terminated PPI-G4 and maltotriose modified PPI-G4 dendrimers were checked. Also the mechanism of cell death in Chinese hamster ovary (CHO) and human ovarian carcinoma (SKOV3) cell lines was investigated. The anionic PAMAM G3.5 dendrimers seem to be the most suitable dendrimers for therapeutic applications, because of their high biocompatibility and low cytotoxicity. Cationic PPI-G4 and PAMAM G4 were the most harmful for both CHO and SKOV3 cell lines, especially in high doses. Maltotriose modification has significantly reduced toxicity within the series of PPI-G4 dendrimers. The moderately doxorubicin and cisplatin resistant human ovarian carcinoma SKOV3 cell line was more vulnerable to modified PPI dendrimers than Chinese hamster ovary CHO cell line which does not show resistance to majority of anticancer agents. This unique property makes these dendrimers potentially interesting for an anticancer therapy.

Abstract C47: Acetaminophen enhances the chemotherapeutic effect of cisplatin and paclitaxel on human ovarian carcinoma cells in vitro

Abstract Chemoresistance is commonly found in treating cancer patients. Acetaminophen enhances the cytotoxicity of cisplatin in human liver cancer cells by depleting glutathione and increasing production of toxic metabolites in vitro. The objective of this study was to investigate potential molecular mechanisms for chemoenhancement in nonhepatic cells. Human ovarian carcinoma SKOV3 cells were treated with cisplatin or paclitaxel with or without acetaminophen in vitro. Cellular mitochondrial membrane potential and reactive oxygen species concentration was measured by using JC-1 and DHR123 stain, respectively. Cellular platinum level was analyzed using ICP-MS. Apoptosis and signaling pathways were examined by immunoblotting and immunohistochemical analyses. A 2-h incubation with acetaminophen (10 mM) decreased cellular glutathione levels and increased mitochondrial membrane potential without affecting reactive oxygen species. Overnight acetaminophen treatment increased reactive oxygen species and enhanced both cisplatin- and paclitaxel-induced apoptosis. In contrast, the combination of acetaminophen with cisplatin but not paclitaxel blocked nuclear translocation of nrf2, a transcription factor that has been implicated in regulation of the antioxidant response. Pretreatment with acetaminophen for 2 h did not affect cellular platinum uptake when compared to cisplatin alone. Regardless of different oxidative stress pathways involved between cisplatin and paclitaxel, the addition of acetaminophen enhanced the cytotoxic effects of both chemotherapeutic drugs in human ovarian carcinoma cells. Our results suggested that acetaminophen could be used as a chemoenhancing adjuvant for conventional chemotherapy drugs in treating ovarian and other malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C47.

Blocking effects of siRNA on VEGF expression in human triple negetive breast l cancer cell line

Prodrug activation gene therapy for cancer involves expressing prodrug-activating enzymes in tumour cells, so they can be selectively killed by systemically administered prodrug. For example, Escherichia coli nfsB nitroreductase (E.C. 1.6.99.7)(NTR), sensitises cells to the prodrug CB1954 (5-[aziridin-1-yl]-2,4-dinitrobenzamide), which it converts to a potent DNA-crosslinking agent. However, low catalytic efficiency with this non-natural substrate appears to limit the efficacy of this enzyme prodrug combination for eliminating the target cancer cells. To improve this, we aim to engineer NTR for improved prodrug activation. Previously, a number of single amino acid substitutions at six positions around the active site of the enzyme were found to increase activity, resulting in up to ∼5-fold enhanced cell sensitisation to CB1954. In this study we have made pairwise combinations among some of the best mutants at each of these 6 sites. A total of 53 double mutants were initially screened in E. coli, then the 7 most promising were inserted into an adenovirus vector and compared in SKOV3 human ovarian carcinoma cells for sensitisation to CB1954 and two alternative prodrugs. The most effective mutants, T41L/N71S and T41L/F70A, were 14–17-fold more potent than WT NTR at sensitising the cancer cells to CB1954. The best mutant for activation of the dinitrobenzamide mustard prodrug SN23862 was T41L/F70A (4.8-fold improvement); and S40A/F124M showed 1.7-fold improvement over WT with the nitrobenzylphosphoramide mustard prodrug LH7. In two tumour xenograft models using SKOV3 or human prostate carcinoma PC3, T41L/N71S NTR demonstrated greater CB1954-dependent anti-tumour activity than WT NTR.

Tetramethylpyrazine inhibits migration of SKOV3 human ovarian carcinoma cells and decreases the expression of interleukin-8 via the ERK1/2, p38 and AP-1 signaling pathways

Interleukin-8 (IL-8) expression by melanoma cells may influence their metastatic capabilities. Tetramethylpyrazine (TMP) from Ligusticum wallichil Franch. possesses anti-inflammatory and antitumor activities. It has recently been suggested that autocrine IL-8 may play a role in tumor cell survival, invasion and migration. The role of TMP in association with IL-8 in the tumor cell migratory process remains unclear. The purpose of the present study was to determine whether TMP influences the migratory ability of a human ovarian carcinoma cell line (SKOV3) via regulation of IL-8 expression invitro. Cell counts showed that treatment of SKOV3 with TMP (25-100 µg/ml) for 24 h did not decrease cell numbers, while an effect of TMP on the down-regulation of the expression of IL-8 was observed. In addition, migration of SKOV3 cells was suppressed after treatment with TMP (25-100 µg/ml) for 24h. Therefore, expression of IL-8 by SKOV3 cells correlates with their metastatic potential. Western blot analysis revealed that ERK1/2 and p38 phosphorylation was blocked by TMP. Furthermore, IL-8 mRNA expression was inhibited significantly after co-incubation with PD98059 (ERK inhibitor) and SB203580 (p38 inhibitor), respectively. Notably, these changes were the results of activator protein-1 (AP-1) activity suppression rather than that of NF-κB. Our data suggest that TMP may inhibit tumor cell invasion and migration, at least in part, through its down-regulation of IL-8 expression. Our results provide evidence that anti-inflammation plays an important role in integrative cancer therapies.

Influence of clusterin gene silencing on proliferation and invasion of human ovarian carcinoma SKOV3 cell line

Objective To investigate the influence of secretory clusterin( sCLU) gene silencing by RNA interference on theproliferation,apoptosis and invasion of human ovarian carcinoma SKOV3 cell line. Methods The human ovarian carcinoma SKOV3cells were transfected with sCLU-targeting siRNA by Lipofectamine 2000. The study included four groups: the experimental group( sCLU-siRNA + Lipofectamine 2000) ,negative groupⅠ ( negative control siRNA + Lipofectamine 2000) ,negative groupⅡ( sCLU-siRNA) and blank control group. The expressions of mRNA and protein were examined by RT-PCR and Western blotting analysis. Theproliferation,apoptosis and invasion ability of SKOV3 cells were evaluated by MTT assay,flow cytometry and Transwell assay. ResultsThe sequence-specific siRNA of sCLU effectively and specifically down-regulated the expression of sCLU at both mRNA and proteinlevels. The MTT results showed that the proliferation of cells in the experimental group was significantly inhibited compared with thosein the other groups( P 0. 05) . Flow cytometry demonstrated that the apoptotic rate of cells in the experimental group( [15. 84 ± 1.53]% ) increased by 9% compared with those in the rest groups( P 0. 01) . We also found that the matrigel penetrating capacity ofcells in the experimental group ( [26. 52 ± 6. 22]cells/field) was significantly inhibited compared with those in the rest groups( P 0.01) . Conclusion Silencing of sCLU can effectively inhibit the proliferation and invasion of human ovarian carcinoma SKOV3 cells,and it can also greatly promote their apoptosis,making sCLU gene a potential new target for ovarian carcinoma treatment.

Effect of plasmid-mediated stable interferon-γ expression on proliferation and cell death in the SKOV-3 human ovarian cancer cell line

Introduction: High interferon-γ (IFN-γ) expression in tumors has been reported to be a favorable prognostic marker. Continuous exposure of ovarian cancer cells to IFN-γ was previously shown to result in significant growth inhibition and apoptosis. Our goal in this study was to evaluate the effect of plasmid-mediated stable IFN-γ expression on the SKOV-3 human ovarian carcinoma cell line.Methods: SKOV-3 cells were stably transfected with the pEGFP-IFN-γ plasmid. IFN-γ mRNA was detected by RT-PCR and IFN-γ protein expression was measured by ELISA. Proliferation and cell death in transfected SKOV-3 cells were measured by methyl-thiazolyl tetrazolium (MTT) assay and Hoechst 33258 staining, respectively and compared with untransfected and empty vector-transfected cells.Results: pEGFP-IFN-γ SKOV-3 cells efficiently expressed and secreted IFN-γ. They exhibited significantly decreased cellular proliferation when compared with control untransfected or empty vector-transfected cells (P < 0.05). The mode of cell death was primarily apoptosis.Conclusions: Stable expression of IFN-γ significantly inhibits the proliferation of ovarian carcinoma cells and has the potential to be used in clinical applications to treat ovarian carcinoma in the future.

N-Glycosylation of total cellular glycoproteins from the human ovarian carcinoma SKOV3 cell line and of recombinantly expressed human erythropoietin

Ovarian carcinoma is the leading cause of death from gynecological cancers in many Western countries. Aberrant glycosylation is an important aspect in malignant transformation and consequently in ovarian cancer. In this study, a detailed structure analysis of the N-linked glycans from total glycoproteins from the SKOV3 ovarian carcinoma cell line and from a recombinantly expressed secretory glycoprotein, erythropoietin (EPO), produced from the same cells has been performed using high-performance anion exchange chromatography with pulsed amperometric detection and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Total cellular N-glycans contained high-mannose type and proximally fucosylated complex type partially agalactosylated structures. On the other hand, the recombinant human EPO secreted from SKOV3 cells contained predominantly core-fucosylated tetraantennary structures, which were partially lacking one or two galactose residues, and partially contained the LacdiNAc motif. Only minor amounts of di- and triantennary complex-type glycans were found, and high-mannose-type glycans were not present in the secreted EPO protein. A large amount of N-acetylneuraminic acid in α2,3-linkage was detected as well. Endogenous glycoproteins were also found to contain the LacdiNAc motif in N-linked glycans. This work contributes to the knowledge of the glycosylation of a human ovarian cancer cell line. It also establishes the basis to further explore high-mannose-type glycans, and the LacdiNAc motif as possible markers of ovarian carcinoma.

Exosomes from human ovarian carcinoma SKOV3 cells are enriched in glycosylated proteins

Exosomes are small vesicles released by various cell types, including tumor cells, as a result of fusion of the multivesicular bodies with the plasma membrane. Exosomes can interact with target cells and mediate different processes, such as, tumor progression and intercellular transfer of proteins and RNA. Although heavily glycosylated forms of some proteins were found in exosomes, the role of glycosylation on protein sorting to exosomes and on the recognition and uptake of these vesicles by cells is still not known. The analysis of the glycosylation profiles of cellular extracts and exosomes from ovarian carcinoma SKOV3 cells, using lectins with different specificities, showed higher levels of protein glycosylation, in particular, sialylation in exosomes. SKOV3 cells were found to internalize SKOV3 derived exosomes, labeled with carboxyfluoresceine diacetate succinimidyl-ester (Exos-CFSE), through an energy-dependent process that was almost completely inhibited at 4 oC. Digestion of Exos-CFSE and SKOV3 cells with proteinase K resulted in considerably lower levels of uptake indicating that proteins were required, at least in part, for internalization. Exos-CFSE uptake was slightly increased after exosomes desialylation with neuraminidase and blocking of cell surface glycan-receptors with different monosaccharides had no effect when compared with the control D-glucose. In conclusion, the increase in protein glycosylation was not essential for exosomes recognition and uptake by target cells. The presence of some heavily glycosylated proteins in exosomes might be a consequence of their enrichment in specific domains of the plasma membrane from where exosomes emerge.

Targeting an Ultrasound Contrast Agent to Folate Receptors on Ovarian Cancer Cells

The purpose of the study was to synthesize and characterize folate-targeted microbubbles (MB(F)) as an ultrasound contrast agent and to evaluate their affinity to the folate receptor (FR) in vitro. Folate-targeted microbubbles were prepared by incorporating 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]-folate into the lipid membrane of microbubbles. The diameter and concentration of the MB(F) were determined by a cell counter and sizer. The MB(F), control microbubbles (MB(C)), and MB(F) with a free folic acid block-ade were tested for binding specificity to human ovarian carcinoma SKOV3 cells, which overexpress the FR, by microscopy and confocal imaging, respectively. The basic physical characteristics of MB(F) were similar to those of MB(C). In the cell binding test, the adherence efficiency of MB(F) to the SKOV3 cells (mean +/- SD, 16 +/- 5 microbubbles per cell; P < .01) was significantly higher than that of MB(C) (0.7 +/- 0.4 microbubbles per cell) or MB(F) with the free folic acid blockade (0.7 +/- 0.6 microbubbles per cell). Folate-targeted microbubbles showed high affinity to SKOV3 cells with FR overexpression. They are potentially useful for ultrasonic molecular imaging and treatment of FR-positive tumors and warrant further investigation.

Potent Preclinical Impact of Metronomic Low-Dose Oral Topotecan Combined with the Antiangiogenic Drug Pazopanib for the Treatment of Ovarian Cancer

Low-dose metronomic chemotherapy has shown promising activity in many preclinical and some phase II clinical studies involving various tumor types. To evaluate further the potential therapeutic impact of metronomic chemotherapy for ovarian cancer, we developed a preclinical model of advanced human ovarian cancer and tested various low-dose metronomic chemotherapy regimens alone or in concurrent combination with an antiangiogenic drug, pazopanib. Clones of the SKOV-3 human ovarian carcinoma cell line expressing a secretable beta-subunit of human choriogonadotropic (beta-hCG) protein and firefly luciferase were generated and evaluated for growth after orthotopic (i.p.) injection into severe combined immunodeficient mice; a highly aggressive clone, SKOV-3-13, was selected for further study. Mice were treated beginning 10 to 14 days after injection of cells when evidence of carcinomatosis-like disease in the peritoneum was established as assessed by imaging analysis. Chemotherapy drugs tested for initial experiments included oral cyclophosphamide, injected irinotecan or paclitaxel alone or in doublet combinations with cyclophosphamide; the results indicated that metronomic cyclophosphamide had no antitumor activity whereas metronomic irinotecan had potent activity. We therefore tested an oral topoisomerase-1 inhibitor, oral topotecan, at optimal biological dose of 1 mg/kg/d. Metronomic oral topotecan showed excellent antitumor activity, the extent of which was significantly enhanced by concurrent pazopanib, which itself had only modest activity, with 100% survival values of the drug combination after six months of continuous therapy. In conclusion, oral topotecan may be an ideal agent to consider for clinical trial assessment of metronomic chemotherapy for ovarian cancer, especially when combined with an antiangiogenic drug targeting the vascular endothelial growth factor pathway, such as pazopanib. Mol Cancer Ther; 9(4); 996-1006. (c)2010 AACR.

LYP, a novel bestatin derivative, inhibits cell growth and suppresses APN/CD13 activity in human ovarian carcinoma cells more potently than bestatin

LYP is a bestatin dimethylaminoethyl ester which inhibits aminopeptidase N (APN/CD13). Our goal in this study was to evaluate LYP as a candidate compound for cancer treatment, beginning by studying its inhibitory effects on tumors and then comparing it to bestatin. Experiments were performed on human ovarian carcinoma (OVCA) ES-2 and SKOV-3 cell lines, which have high and low levels of APN/CD13 respectively. LYP effectively inhibited ES-2 cell growth as estimated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and the trypan blue dye-exclusion test. LYP significantly suppressed APN/CD13 activity on the surface of ES-2 cells as measured by quantifying the enzymatic cleavage of the substrate L-leucine-p-nitroanilide. The inhibitory effects of LYP were greater than those of bestatin at the same concentrations. In contrast, LYP was a weak inhibitor of SKOV-3 cell growth, suggesting that LYP may inhibit ES-2 cell growth via suppression of APN/CD13. Inhibition of APN/CD13 expression was also demonstrated with immunofluorescent flow cytometry and Western blot analysis. Inhibitory effects of LYP were confirmed by using a mouse model in which LYP delayed the growth of ES-2 xenografts in mice after 2 weeks of LYP injections. Inhibition of APN/CD13 expression was demonstrated in the ES-2 xenografts using Western blot analysis. The inhibitory effects of LYP on the ES-2 xenografts were stronger than those of bestatin. These results suggest that LYP has a powerful inhibitory effect on the growth of OVCA cells and that the mechanism may be via a decrease in the expression of APN/CD13.