Human Ovarian Cancer Model Research Articles

Imaging of Treatment Response to the Combination of Carboplatin and Paclitaxel in Human Ovarian Cancer Xenograft Tumors in Mice Using FDG and FLT PET

IntroductionA combination of carboplatin and paclitaxel is often used as first line chemotherapy for treatment of ovarian cancer. Therefore the use of imaging biomarkers early after initiation of treatment to determine treatment sensitivity would be valuable in order to identify responders from non-responders. In this study we describe the non-invasive PET imaging of glucose uptake and cell proliferation using 2-deoxy-2-[18F]fluoro-D-glucose (FDG) and 3’-deoxy-3’-[18F]fluorothymidine (FLT) for early assessment of treatment response in a pre-clinical mouse model of human ovarian cancer treated with carboplatin and paclitaxel.Methods In vivo uptake of FLT and FDG in human ovarian cancer xenografts in mice (A2780) was determined before treatment with carboplatin and paclitaxel (CaP) and repeatedday 1, 4 and 8 after treatment start. Tracer uptake was quantified using small animal PET/CT. Tracer uptake was compared with gene expression of Ki67, TK1, GLUT1, HK1 and HK2.ResultsTumors in the CaP group was significantly smaller than in the control group (p=0.03) on day 8. On day 4 FDG SUVmax ratio was significantly lower in the CaP group compared to the control group (105±4% vs 138±9%; p=0.002) and on day 8 the FDG SUVmax ratio was lower in the CaP compared to the control group (125±13% vs 167±13%; p=0.05). On day 1 the uptake of FLT SUVmax ratio was 89±9% in the CaP group and 109±6% in the control group; however the difference was not statistically significant (p=0.08). ConclusionsOur data suggest that both FDG and FLT PET may be used for the assessment of anti-tumor effects of a combination of carboplatin and paclitaxel in the treatment of ovarian cancer. FLT provides an early and transient signal and FDG a later and more prolonged response. This underscores the importance of optimal timing between treatment and FLT or FDG imaging since treatment response may otherwise be overlooked.

Benefits of Vascular Normalization Are Dose and Time Dependent—Letter

Arjaans and colleagues reported that treatment with the anti-VEGF antibody bevacizumab hampers antibody uptake in an ectopic xenograft model of human ovarian cancer in mice ([1][1]). They found that bevacizumab decreased vessel number and increased pericyte coverage and concluded that treatment-

Evaluation of PNC-27-mediated toxicity in an intraperitoneal mouse model of human ovarian cancer

Objectives: PNC-27 is a novel anti-cancer peptide explicitly cytotoxic against cancer cells via formation of trans-membrane pores while sparing normal cells. We sought to determine the effect of PNC-27 on the established ovarian cancer cell line SKOV3-luc-D3 in vitro. Then, utilizing murine nu/nu mouse models, we sought to determine the maximum tolerated dose (MTD) of daily bolus intraperitoneal (IP) injections in healthy mice and finally to determine the toxicity of PNC-27 around the determined MTD using SKOV3-luc-D3 cells in vivo.

Research Perspective: Potential Role of Nitazoxanide in Ovarian Cancer Treatment. Old Drug, New Purpose?

Among gynecological malignancies epithelial ovarian cancer (EOC) is the leading cause of death. Despite improvements in conventional chemotherapy combinations, the overall cure rate has remained mostly stable over the years, and only 10%–15% of patients maintain a complete response following first-line therapy. To improve the efficacy of ovarian cancer chemotherapy it is essential to develop drugs with new mechanisms of action. Compared to normal tissues, protein disulfide isomerase (PDI) is overexpressed in ovarian tumors. PDI is a cellular enzyme in the lumen of the endoplasmic reticulum (ER) of eukaryotes or the periplasmic region of prokaryotes. This protein catalyzes the formation and breakage of disulphide bonds between cysteine residues in proteins, which affects protein folding. Selective inhibition of PDI activity has been exhibited both in vitro and in vivo anticancer activity in human ovarian cancer models. PDI inhibition caused accumulation of unfolded or misfolded proteins, which led to ER stress and the unfolded protein response (UPR), and in turn resulted in cell death. Nitazoxanide [NTZ: 2-acetyloxy-N-(5-nitro-2-thiazolyl)benzamide] is a thiazolide antiparasitic agent with excellent activity against a wide variety of protozoa and helminths. In this article, we propose that NTZ, acting as PDI inhibitor, may be a new and potent addition to the chemotherapeutic strategy against ovarian cancer.

Immune Cells in the Normal Ovary and Spontaneous Ovarian Tumors in the Laying Hen (Gallus domesticus) Model of Human Ovarian Cancer

BackgroundSpontaneous ovarian cancer in chickens resembles human tumors both histologically and biochemically. The goal was to determine if there are differences in lymphocyte content between normal ovaries and ovarian tumors in chickens as a basis for further studies to understand the role of immunity in human ovarian cancer progression.MethodsHens were selected using grey scale and color Doppler ultrasound to determine if they had normal or tumor morphology. Cells were isolated from ovaries (n = 6 hens) and lymphocyte numbers were determined by flow cytometry using antibodies to avian CD4 and CD8 T and B (Bu1a) cells. Ovarian sections from another set of hens (n = 26) were assessed to verify tumor type and stage and to count CD4, CD8 and Bu1a immunostained cells by morphometric analysis.ResultsT and B cells were more numerous in ovarian tumors than in normal ovaries by flow cytometry and immunohistochemistry. There were less CD4+ cells than CD8+ and Bu1a+ cells in normal ovaries or ovarian tumors. CD8+ cells were the dominant T cell sub-type in both ovarian stroma and in ovarian follicles compared to CD4+ cells. Bu1a+ cells were consistently found in the stroma of normal ovaries and ovarian tumors but were not associated with follicles. The number of immune cells was highest in late stage serous tumors compared to endometrioid and mucinous tumors.ConclusionsThe results suggest that similar to human ovarian cancer there are comparatively more immune cells in chicken ovarian tumors than in normal ovaries, and the highest immune cell content occurs in serous tumors. Thus, this study establishes a foundation for further study of tumor immune responses in a spontaneous model of ovarian cancer which will facilitate studies of the role of immunity in early ovarian cancer progression and use of the hen in pre-clinical vaccine trials.

Effect of Plant Polyphenol Genistein on Growth of Human Ovarian Carcinoma Transplanted Subcutaneously in Nude Mice

In this study we investigated the effect of plant polyphenol PTK inhibitor genistein on subcutaneous human transplant ovarian cancer tumor of nude mice. All 30 cases of nude mice are used to establish subcutaneous xenograft models of human ovarian cancer, and divided into 4 groups: Control group (containing 0.04% DMSO of saline); Genistein group (containing 0.2 mg / kg and 0.4 mg/kg two concentrations, subcutaneous injection); Cisplatin group (4 mg/kg, i.v.); Genistein and Cisplatin combination group. The transplanted tumor growth and weight changes of nude mice in different groups on 7, 14, 21 and 28 days were observed and histopathological examined. The results showed that the growth of SKOV3 xenograft tumor was significantly inhibited in 0.4 mg/kg Genistein group. Compared with control group, the tumor weights were decreased, the tumor volumes were reduced, and there was a significant increase in the area of necrosis, but no significant effects were showed on the weights of nude mice. 0.4 mg/kg Genistein (s.c.) in combination with 4 mg/kg Cisplatin (i.v.) enhanced the inhibitory effect. The results provide evidence for the potential usefulness of Genistein in the prevention and treatment of human ovarian carcinoma.

Highlights of This Issue

Therapeutics targeting Her2 have shown impressive clinical activity, although there are resistance mechanisms leading to treatment failure. Granzyme B (GrB) is an intensely cytotoxic protein used by NK and CTL to kill target cells. Cao and colleagues fused GrB to the anti-Her2 scFv 4D5. This construct showed specific apoptosis induction and cytotoxicity to Her2 cells in vitro through a defined, multimodal mechanism and was active against 1apatanib, Herceptin-resistant, and MDR-expressing cells. Tumor-targeted GrB localized in tumor xenografts and resulted in tumor suppression. These studies suggest that tumor-directed GrB constructs have excellent potential as novel therapeutic agents.Drug resistance is a major cause of treatment failure in ovarian cancer, emphasizing the need for drugs with novel mechanism of action. Xu and colleagues identified gp130 as an important drug target in ovarian cancer and have developed a first-in-class small-molecule gp130 inhibitor. SC144 can be used as a new tool to interrogate the gp130-implicated signaling pathways affecting several malignancies. More significantly, SC144 shows efficacy in in vivo models of human ovarian cancer and displays no significant off-target toxicity in normal tissues. Our study offers a new approach to treat diseases with altered gp130 signaling, including multiple types of cancers.ATM signals DNA double strand breaks (DSB) to cell cycle checkpoints and for repair. Defects in ATM confer radiosensitivity and chemosensitivity, making ATM an attractive target for anticancer chemotherapy. Batey and colleagues have identified a potent ATM inhibitor, KU59403, that sensitizes human tumor cells to radiation and topoisomerase poisons in a p53-independent manner. Importantly, KU59403 is the first ATM inhibitor to demonstrate good tissue distribution and chemosensitization of human tumor xenografts without major toxicity, providing the first advanced pre-clinical proof-of-principle data to justify developing ATM inhibitors for clinical use.Alternate angiogenic pathways and/or hypoxia-induced eptithelial-to-mesenchymal transition (EMT) may facilitate resistance to anti-VEGF therapy. Kutluk Cenik and colleagues investigated the efficacy and biological effects of BIBF 1120 (nintedanib), a balanced inhibitor of VEGF, PDGF, and FGF pathways in xenografts of lung and pancreatic cancer. BIBF 1120 potently reduced tumor vascular density and function and inhibited primary tumor growth and metastasis. Despite induction of hypoxia, chronic BIBF 1120 did not enhance EMT or tumor invasiveness, suggesting a balanced multitarget strategy may combat the development of resistance. Clinical studies of BIBF 1120 are underway.

Discovery of a Novel Orally Active Small-Molecule gp130 Inhibitor for the Treatment of Ovarian Cancer

Interleukin (IL)-6 and Stat3 play key roles in ovarian cancer progression. However, the role of glycoprotein 130 (gp130), the signal transducer of this signaling axis, is not well-established. Currently, there are no small-molecule inhibitors of gp130 under clinical development. In this study, we show that gp130 is an attractive drug target in ovarian cancer due to its role in promoting cancer progression via the activation of its downstream Stat3 signaling. We also present preclinical studies of SC144, the first-in-class orally active small-molecule gp130 inhibitor. SC144 shows greater potency in human ovarian cancer cell lines than in normal epithelial cells. SC144 binds gp130, induces gp130 phosphorylation (S782) and deglycosylation, abrogates Stat3 phosphorylation and nuclear translocation, and further inhibits the expression of downstream target genes. In addition, SC144 shows potent inhibition of gp130 ligand-triggered signaling. Oral administration of SC144 delays tumor growth in a mouse xenograft model of human ovarian cancer without significant toxicity to normal tissues.

Induction of Apoptosis and Inhibition of Angiogenesis by PEGylated Liposomal Quercetin in Both Cisplatin-Sensitive and Cisplatin-Resistant Ovarian Cancers

The clinical efficiency of cisplatin against ovarian cancer is often limited by the development of drug resistance. In this work, we investigated PEGylated liposomal quercetin (Lipo-Que) on cisplatin-sensitive (A2780s) and cisplatin-resistant (A2780cp) human ovarian cancer models in vitro and in vivo to reveal whether a cisplatin-resistant ovarian cancer has susceptibility to quercetin (Que) and the mechanism of its antitumor activity. Lipo-Que was prepared using a solid dispersion method, and the obtained Lipo-Que is monodisperse with a mean diameter of 163 +/-10 nm. Besides, in vitro drug release assay showed a sustained release behavior of Lipo-Que. In vitro experiments suggested that Lipo-Que inhibited cell proliferation, induced apoptosis, and induced cell cycle arrest in both A2780s and A2780cp cells. Furthermore, antitumor activity of Lipo-Que was investigated in both cisplatin-sensitive and cisplatin-resistant human ovarian tumor xenograft models in nude mice. Lipo-Que significantly suppressed tumor growth in both models in comparison with free Que, blank liposomes (Lipo), or normal saline (NS). Furthermore, immunohistochemistry and immunofluorescence tests revealed that Lipo-Que induced apoptosis, decreased microvessel density, and inhibited proliferation of tumors in both A2780s and A2780cp tumor models. Therefore, our results suggest that Lipo-Que is an effective agent to inhibit tumor growth in both cisplatin-sensitive and cisplatin-resistant human ovarian cancers.

Bevacizumab-Induced Normalization of Blood Vessels in Tumors Hampers Antibody Uptake

In solid tumors, angiogenesis occurs in the setting of a defective vasculature and impaired lymphatic drainage that is associated with increased vascular permeability and enhanced tumor permeability. These universal aspects of the tumor microenvironment can have a marked influence on intratumoral drug delivery that may often be underappreciated. In this study, we investigated the effect of blood vessel normalization in tumors by the antiangiogenic drug bevacizumab on antibody uptake by tumors. In mouse xenograft models of human ovarian and esophageal cancer (SKOV-3 and OE19), we evaluated antibody uptake in tumors by positron emission tomographic imaging 24 and 144 hours after injection of (89)Zr-trastuzumab (SKOV-3 and OE19), (89)Zr-bevacizumab (SKOV-3), or (89)Zr-IgG (SKOV-3) before or after treatment with bevacizumab. Intratumor distribution was assessed by fluorescence microscopy along with mean vessel density (MVD) and vessel normalization. Notably, bevacizumab treatment decreased tumor uptake and intratumoral accumulation compared with baseline in the tumor models relative to controls. Bevacizumab treatment also reduced MVD in tumors and increased vessel pericyte coverage. These findings are clinically important, suggesting caution in designing combinatorial trials with therapeutic antibodies due to a possible reduction in tumoral accumulation that may be caused by bevacizumab cotreatment.

Metronomic chemotherapy following the maximum tolerated dose as a multitarget antitumor therapy affecting angiogenesis, tumor dissemination, and cancer stem cells.

e22057 Background: Metronomic chemotherapy has been suggested as a maintenance administration strategy after the Maximum Tolerated Dose (MTD) treatment in a multi-targeted chemo-switch schedule (C-S). We report the effectiveness of this chemo-switch schedule in mice models of human pancreatic and ovarian cancer. Methods: In pancreas cancer models, Gemcitabine (G) was administered on four different schedules: metronomic (METG), Maximum Tolerated Dose (MTDG), chemo-switch schedule (C-SG, MTDG dosing followed by the METG) and anti-angiogenic (MTDG followed by the administration of monoclonal anti-VEGF receptor antibody DC101). In ovarian cancer model, animals were treated following a Cyclophosphamide chemo-switch schedule (C-S CTX). Results: In all these models C-S schedule had the most favorable effect, reaching at least 80% of tumor growth inhibition in absence of increased toxicity. Moreover, in the pancreas cancer model while peritoneal metastases were observed in control and MTD groups, no dissemination was observed in the MET and C-S groups. C-S treatment caused a decrease in angiogenesis and its effect on tumor growth was similar to obtained by the MTD followed by anti-angiogenic DC101 treatment. At the molecular level, C-S treatment combined an increase in thrombospondin-1 expression with a decrease in number of CD133+ cancer cells and triple positive CD133+/CD44+/CD24+ cancer stem cells. Conclusions: These findings provide confirmation that the chemo-switch schedule is a challenging clinical strategy with demonstrated inhibitory effects on tumor dissemination, angiogenesis and cancer stem cells.

Development of PIK-75 nanosuspension formulation with enhanced delivery efficiency and cytotoxicity for targeted anti-cancer therapy

PIK-75 is a phosphatidylinositol 3-kinase (PI3K) inhibitor that shows selectivity toward p110-α over the other PI3K class Ia isoforms p110-β and p110-δ, but it lacks solubility, stability and other kinase selectivity. The purpose of this study was to develop folate-targeted PIK-75 nanosuspension for tumor targeted delivery and to improve therapeutic efficacy in human ovarian cancer model. High pressure homogenization was used to prepare the non-targeted and targeted PIK-75 nanosuspensions which were characterized for size, zeta potential, entrapment efficiency, morphology, saturation solubility and dissolution velocity. In vitro analysis of drug uptake, cell viability and cell survival was conducted in SKOV-3 cells. Drug pharmacokinetics and pAkt expression were determined in SKOV-3 tumor bearing mice. PIK-75 nanosuspensions showed an improvement in dissolution velocity and an 11-fold increase in saturation solubility over pre-milled PIK-75. In vitro studies in SKOV-3 cells indicated a 2-fold improvement in drug uptake and 0.4-fold decrease in IC50 value of PIK-75 following treatment with targeted nanosuspension compared to non-targeted nanosuspension. The improvement in cytotoxicity was attributed to an increase in caspase 3/7 and hROS activity. In vivo studies indicated a 5-10-fold increased PIK-75 accumulation in the tumor with both the nanosuspension formulations compared to PIK-75 suspension. The targeted nanosuspension showed an enhanced downregulation of pAkt compared to non-targeted formulation system. These results illustrate the opportunity to formulate PIK-75 as a targeted nanosuspension to enhance uptake and cytotoxicity of the drug in tumor.

Abstract 901: Overexpression of inhibitors of apoptosis (IAP) family members in human breast and ovarian cancer models of non-MDR1 taxane resistance.

Abstract Four breast cancer cell lines (MCF-7, BT-549, MDA-MB-231 and T-47D) and four ovarian cancer cell lines (1A9/A2780, ES-2, MES-OV and OVCAR-3) were selected for taxane resistance by exposing cells to either docetaxel or paclitaxel in the presence of the P-glycoprotein inhibitor, PSC-833 (valspodar, 2 μM). All of these co-selected variants are MDR1/ABCB1(-), and the resistance to taxanes is not transporter-mediated. We have previously reported elevated class III β-tubulin (TUBB3), reduced BRCA1, elevated CDKN1A (p21), and altered epithelial-mesenchymal transition (EMT) genes in the majority of the non-MDR1 taxane variants. Inhibitors of apoptosis (IAP) proteins directly bind and inhibit several caspases, and may play a critical role in determining cell fate after exposure to chemotherapeutic agents. In this study, we profiled the expression of IAP proteins and found elevated content in this panel of taxane-resistant human breast and ovarian cancer cell lines. In both the paclitaxel- (TP) and docetaxel-selected (TxTP) ovarian cancer cell lines we observed significantly overexpressed cIAP1 (BIRC2), cIAP2 (BIRC3), XIAP (BIRC4), and Livin (BIRC7) relative to parental cell lines. Expression of cIAP2 was undetectable in OVCAR-3 and its taxane resistant cell lines, OVCAR-3/TP20 and OVCAR-3/TxTP5. Elevated cIAP1 and XIAP levels were detected in the human breast cancer variant BT-549/TxTP50, and Livin was overexpressed in all taxane-resistant breast cancer cell models. Survivin (BIRC5) is highly expressed in cancers and has been associated with taxane resistance via its effects on apoptosis and the cell cycle. We observed reduced Survivin content in the majority of the variants established in our laboratory except for the ovarian ES-2/TP80 cell line where we found slightly elevated expression resulting from paclitaxel selection relative to its parental control. In addition to these alterations in IAP content, we observed elevated Bcl-2 in ovarian (MES-OV/TP40 and MES-OV/TxTP50) and breast (MCF-7/TxTP50, BT-549/TxTP50, and MDA231/TxTP50) cancer cell lines at the transcript level by rt-PCR and confirmed by immunoblotting. Specific gene silencing by RNAi and treatment with small molecule inhibitors will test the functional significance of IAP alterations in these models of taxane resistance. Citation Format: George E. Duran, Anamaria Brozovic, Francisco J. Martinez, E Brian Francisco, Yan C. Wang, Branimir I. Sikic. Overexpression of inhibitors of apoptosis (IAP) family members in human breast and ovarian cancer models of non-MDR1 taxane resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 901. doi:10.1158/1538-7445.AM2013-901

Abstract 948: In vivo evaluation of IGF2 RNA interference in a mouse xenograft model of paclitaxel-resistant human ovarian cancer.

Abstract Introduction. We previously showed that paclitaxel-resistance of human ovarian carcinoma cell lines was reverted by knockdown of IGF2 with RNA interference. The aim of this study was to determine if our paclitaxel-resistant cell line HEY-T30 was resistant to paclitaxel treatment in vivo and if this resistance could be reverted by IGF2 knockdown through short hairpin RNA. Methods. Paclitaxel-resistant HEY-T30 cells, derived in our laboratory from HEY ovarian carcinoma cells, were transfected with a plasmid containing shRNA to target IGF2 (shIGF2-p) or a nontargeting shRNA (shScrambled). Stably transfected clones were evaluated by reverse transcription real-time PCR for IGF2 mRNA expression and by sulforhodamine cytotoxicity assay for paclitaxel sensitivity. For in vivo experiments, HEY, HEY-T30, shIGF2-p or shScrambled cells were suspended in OptiMem and one million cells/animal injected subcutaneously into 4-week-old female nude mice. Tumors were allowed to grow to a mean volume of 125 mm3, prior to treatment initiation with paclitaxel (20 mg/kg administered intraperitoneally every 3 days x 5 doses) or vehicle (5% dextrose in water; D5W). Animals were weighed and tumors were measured with digital calipers three times per week. Effect of treatment was calculated as effect=1-(T/C) at the indicated time point, where T is the mean tumor volume of the paclitaxel treatment group and C is the mean tumor volume of the vehicle control group. Significance was calculated with a 2-way repeated measures ANOVA with a Bonferroni multiple comparisons post-test. Results. IGF2 mRNA levels of shIGF2-p cells were comparable to HEY, whereas shScrambled was similar to HEY-T30. The IC50s (concentration of 50% inhibition of proliferation) for paclitaxel in vitro were HEY: 2.3 nM; HEY-T30: 164.0 nM; shIGF2-p: 13.89 nM; shScrambled: 140.0 nM. HEY xenografts responded well to paclitaxel treatment, showing a significant growth retardation compared to the D5W group beginning at 9 days after treatment initiation (p=0.0005), with an effect of treatment of 83.2% at day 19. In contrast, HEY-T30 xenografts showed no significant difference in tumor volume between the paclitaxel-treated and the D5W group at any time point, confirming its resistance to paclitaxel (the effect of treatment was 5.5%). shScrambled xenografts were similarly resistant to paclitaxel as HEY-T30 xenografts. shIGF2-p xenografts responded well to paclitaxel treatment, showing a difference in tumor volume from day 15 onward (day 15, p=0.0457; day 19, p=0.0006) with an effect of treatment of 64.1% after 22 days. Discussion. HEY-T30 xenografts are resistant to paclitaxel treatment, whereas HEY xenografts respond well. IGF2 knockdown by shRNA restores paclitaxel sensitivity to the HEY-T30 xenografts. These in vivo results confirm our prior observations in cell lines and suggest a novel target for the treatment of paclitaxel-resistant disease. Citation Format: Jurriaan Brouwer-Visser, Jiyeon Lee, María J. Cossio, Gloria S. Huang. In vivo evaluation of IGF2 RNA interference in a mouse xenograft model of paclitaxel-resistant human ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 948. doi:10.1158/1538-7445.AM2013-948

Abstract 4642: GRP78 in association with VEGFR-2 detects early stage ovarian cancer.

Abstract Background: The lack of an effective early detection test makes ovarian cancer (OVCA) a fatal malignancy of women. Changes in cellular metabolic processes during ovarian malignant transformation lead to the endoplasmic reticular (ER) and mitochondrial stress. Glucose-regulated protein of 78kDa (GRP78) is a marker of ER stress. Tumor-associated ER stress causes relocalization of GRP78 from ER to cell surface with an increase in its serum levels. Secreted GRP78 also stimulates tumor associated neoangiogenesis (TAN). Vascular endothelial growth factor receptor-2 (VEGFR-2) is a marker of ovarian TAN. Thus GRP78 represents a marker of ovarian malignant transformation and in association with VEGFR-2 may constitute an early detection test for OVCA. However, information on OVCA related changes in GRP78 expression and its association with ovarian TAN is unknown. Objectives: The goals of this study were to examine (i) whether GRP78 expression increases during malignant changes in the ovary and (ii) whether the frequency of TAN vessels is associated with ovarian tumor-associated GRP78 expression at early stage of OVCA in the laying hen, a spontaneous model of human OVCA. Materials and Methods: 3-4 years old White Leghorn laying hens with normal (n=25) or ovarian tumors (n=30) were selected by contrast enhanced transvaginal ultrasound scanning. Serum samples were collected, hens were euthanized, and ovarian tissues were processed for routine histology or immunohistochemistry (IHC) and protein extraction. Ovarian tumors were confirmed by gross morphology and microscopy. Expression of GRP78 by ovarian malignant cells and VEGFR-2 by TAN vessels was determined by IHC. IHC observations were confirmed by immunoproteomic studies. Results: As compared with normal ovaries (n=25), the intensity of GRP78 expression was significantly (p<0.001) higher in hens with early stage OVCA (n=12) and increased further in hens with late stage OVCA (n=18) (p<0.05). GRP78 expression was higher in hens with serous OVCA followed by endometrioid OVCA and was least in hens with mucinous and clear cell OVCA irrespective of their stages of OVCA. A band of approximately 80kDa was observed for GRP78 in all ovaries examined and the patterns of expression were similar to that of IHC. The frequency of VEGFR-2 expressing TAN vessels was significantly (p<0.001) higher in OVCA hens than in normal hens. Increase in the frequency of VEGFR-2 expressing TAN vessels was positively correlated with the intensity of GRP78 expression. Conclusion: The results of this study suggest that increase in the GRP78 expression is associated with ovarian malignant transformation, and this enhanced expression may stimulate ovarian tumor-associated neoangiogenesis. Thus GRP78 in combination with VEGFR-2 may constitute an early detection test for OVCA. These results will constitute a foundation for a clinical study to establish an early detection test for OVCA. Support: DOD Award # W81XWH-11-1-0510. Citation Format: Doungkamol Alongkronrusmee, Pincas Bitterman, Jacques S. Abramowicz, Janice M. Bahr, Sanjib Basu, Salvatore Grasso, Sameer Sharma, Jacob Rotmensch, Animesh Barua. GRP78 in association with VEGFR-2 detects early stage ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4642. doi:10.1158/1538-7445.AM2013-4642

Characterization of Ascites-Derived Ovarian Tumor Cells from Spontaneously Occurring Ovarian Tumors of the Chicken: Evidence for E-Cadherin Upregulation

Ovarian cancer, a highly metastatic disease, is the fifth leading cause of cancer-related deaths in women. Chickens are widely used as a model for human ovarian cancer as they spontaneously develop epithelial ovarian tumors similar to humans. The cellular and molecular biology of chicken ovarian cancer (COVCAR) cells, however, have not been studied. Our objectives were to culture COVCAR cells and to characterize their invasiveness and expression of genes and proteins associated with ovarian cancer. COVCAR cell lines (n = 13) were successfully maintained in culture for up to19 passages, cryopreserved and found to be viable upon thawing and replating. E-cadherin, cytokeratin and α-smooth muscle actin were localized in COVCAR cells by immunostaining. COVCAR cells were found to be invasive in extracellular matrix and exhibited anchorage-independent growth forming colonies, acini and tube-like structures in soft agar. Using RT-PCR, COVCAR cells were found to express E-cadherin, N-cadherin, cytokeratin, vimentin, mesothelin, EpCAM, steroidogenic enzymes/proteins, inhibin subunits-α, βA, βB, anti-müllerian hormone, estrogen receptor [ER]-α, ER-β, progesterone receptor, androgen receptor, and activin receptors. Quantitative PCR analysis revealed greater N-cadherin, vimentin, and VEGF mRNA levels and lesser cytokeratin mRNA levels in COVCAR cells as compared with normal ovarian surface epithelial (NOSE) cells, which was suggestive of epithelial-mesenchymal transformation. Western blotting analyses revealed significantly greater E-cadherin levels in COVCAR cell lines compared with NOSE cells. Furthermore, cancerous ovaries and COVCAR cell lines expressed higher levels of an E-cadherin cleavage product when compared to normal ovaries and NOSE cells, respectively. Cancerous ovaries were found to express significantly higher ovalbumin levels whereas COVCAR cell lines did not express ovalbumin thus suggesting that the latter did not originate from oviduct. Taken together, COVCAR cell lines are likely to improve our understanding of the cellular and molecular biology of ovarian tumors and its metastasis.

Establishment of a new representative model of human ovarian cancer in mice

BackgroundIntraperitoneal (i.p.) models that accurately mimic the feature behavior of human ovarian cancer are required to investigate the pathology and therapeutics of the disease. However, established i.p. models which are well-characterized and reliable are few. The purposes of this study are to establish a representative mice i.p. model of the disease and to analyze the consequent pathology.MethodsFresh tumor cells fiom the ascites of patient were injected into female NOD/SCID mice intraperitoneally. Histology, Cytogenetic, immunohistochemistry,tumor markers of CA125,AFP, CA-199 and CEA were used to analyze the model.ResultsThe mice developed marked abdominal distention within 6 months after inoculated with tumor cells from a patient with epithelial ovarian carcinoma. The mice developed clinically evident intraperitoneal tumors and massive ascites containing numerous tumor cells in clumps. CA125 level in our model was high in both serum and ascites supernatants, while levels of other tumor markers, such as AFP, CA-199 and CEA, were normal. Cytogenetic analysis and immunohistochemical staining confirmed its characteristics resembling human epithelial ovarian tumor.ConclusionsThe model described in this paper accurately mimics the features of ovarian tumor, which may be useful for evaluation of new therapeutics.

Abstract B70: Decreasing ovarian cancer metastases and progression via restoration of microRNAs that target mediators of anoikis- and chemoresistance.

Abstract Background: Epithelial ovarian cancer (EOC) accounts for the majority of deaths from gynecological malignancies, with a 5-year survival rate for women with advanced stage disease of only 46%. In EOC metastasis primarily occurs by a mechanism termed direct seeding, which involves shedding of tumor cells from the primary site into the peritoneal cavity. To allow survival in suspension before attachment at metastatic sites, ovarian cancer cells must acquire resistance to anoikis, a form of apoptosis initiated by detachment from native extracellular matrix. A targeted therapy that could simultaneously affect multiple factors critical for ovarian cancer progression such as anoikis resistance, chemoresistance and attachment to metastatic sites in the peritoneal cavity, would be a revolutionary breakthrough for this aggressive malignancy. Due to their ability to inhibit multiple targets involved in tumor survival, microRNAs (miRNAs) are attractive therapeutic candidates. We previously demonstrated that miR-200c is downregulated in ovarian cancer cell lines and restoration of miR-200c increased sensitivity to taxanes in vitro, by targeting TUBB3, a known mediator of chemoresistance. We tested the effect of miR-200c restoration in an intraperitoneal xenograft model of human ovarian cancer and observed dramatically decreased tumor formation. Further, even in established tumors, inducible restoration of miR-200c alone and in combination with paclitaxel, resulted in significantly reduced tumor burden. Hypothesis: Since restoration of miR-200c to multiple anoikis resistant EOC cell lines increased anoikis sensitivity and reduced adherence to biological substrates in vitro, we hypothesized that the decreased intraperitoneal tumor burden observed upon miR-200c restoration resulted from increased anoikis sensitivity. Methods and results: To elucidate mechanisms of anoikis resistance in ovarian cancer and identify targets involved in the ability of miR-200c to reverse anoikis resistance, we performed gene and miRNA expression profiling of attached Hey ovarian cancer cells as compared to the same cells grown under conditions of forced suspension for 24h. We identified miR-193 as downregulated in anoikis resistant cells in suspension. Among numerous genes predicted as miR-200c and/or miR-193 targets, SCLC7A11 (xCT), which encodes the light chain of the cystine/glutamate antiporter system xc, was increased by 10.7 fold in suspended versus attached cells. The xc- system is involved in glutathione (GSH) synthesis, mediating intracellular defense against oxidative stress. Reactive oxygen species (ROS) increased in anoikis-sensitive (OvCA 433) cells in suspension, but not in anoikis-resistant cells (Hey), suggesting that xCT might protect these cells from ROS-induced cell death. Consistent with this hypothesis, xCT mRNA and protein increased in anoikis resistant EOC cells in suspension, and transient xCT knockdown rendered cells anoikis sensitive. Interestingly, the xCT 3'UTR contains three predicted binding sites for miR-200c and two sites for miR-193b, and restoration of either miRNA blocked the suspension induced-increase in cXT protein, suggesting that both miRNAs target this gene. Conclusions: Our results indicate that the ability of miR-200c to reduce tumor burden results, at least in part, from its ability to restore anoikis sensitivity by inhibiting a suspension-induced increase in xCT that protects tumor cells from ROS. Thus, miR-200c could be a powerful candidate to block progression of ovarian cancer by enhancing anoikis sensitivity and restoring chemo-sensitivity to already attached tumor. Citation Format: Diana M. Cittelly, Dawn R. Cochrane, Erin N. Howe, Irina Dimitrova, Miriam D. Post, Broaddus R. Russell, Monique A. Spillman, Jennifer K. Richer. Decreasing ovarian cancer metastases and progression via restoration of microRNAs that target mediators of anoikis- and chemoresistance.. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B70.

The influence of RAPTA moieties on the antiproliferative activity of peripheral-functionalised poly(salicylaldiminato) metallodendrimers

Cationic N,O-chelating dendrimers functionalised on the periphery with RAPTA-like (ruthenium(II)-arene-1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane) moieties have been synthesised and characterised using NMR and IR spectroscopy, elemental analysis and MALDI-TOF/HR-ESI mass spectrometry. Metallodendrimers from the first to the fourth-generation containing up to 32 peripheral ruthenium-arene-PTA moieties were obtained. Model mononuclear analogues, [{Ru(η(6)-p-cymene)((C(7)H(5)NO)-κ(2)-N,O)(PTA)}((CH(2))(3))][PF(6)] and [{Ru(η(6)hexamethylbenzene)((C(7)H(5)NO)-κ(2)-N,O)(PTA)}((CH(2))(3))][PF(6)], have been prepared and their structures were determined by single crystal X-ray diffraction analysis. The cytotoxicities of the metallodendrimers and their mononuclear analogues were established on A2780 and A2780cisR human ovarian carcinoma cancer cells and model human embryonic kidney (HEK) cells.

An In Vitro and In Vivo Investigation of the Antimetastatic Effects of a Chinese Medicinal Decoction, Erxian Decoction, on Human Ovarian Cancer Models

Erxian Decoction (EXD) is a well-documented Chinese medicinal formulation, which has been clinically applied for years for relieving menopausal syndromes by modulating hormonal levels indicating that EXD might also be effective in treating hormone-related tumors. This study aimed to differentially investigate the efficacy of EXD and its antimetastatic property on human ovarian cancer cells, OVCA429. The efficacy and cell cycle progression of EXD on OVCA429 cells was determined by MTT assay and flow cytometry, respectively. The modulated expression of metastatic markers by EXD in OVCA429 cells and xenografts was evaluated at transcriptional and translational levels by Western blotting and real-time polymerase chain reaction, respectively. The migrating and invasive ability of the cancer cells were determined by wound healing and invasive assays. The IC50 value of EXD on OVCA429 cells was determined after 24 hours incubation with EXD at 1 mg/mL. EXD (1.5 mg/mL) mediated S-phase cell cycle arrest and apoptotic cell death at 24 hours posttreatment. EXD repressed the expression of several metastatic mediators, including EGFR, ErbB2, MMP2, MMP7, MMP9, and VEGF in OVCA429 cells and xenografts at transcriptional and/or translational levels. Furthermore, EXD functionally demonstrated significant inhibition of migrating and invasive ability of OVCA429 cells. EXD suppressed tumor size in xenografts without any adverse effects on body weight. This is the first study that illustrates the antimetastatic property of EXD on human ovarian cancer models. This decoction merits serious consideration for further delineation of its multiple pharmacological effects, especially on hormone-related cancers, and these would be valuable for future clinical applications of EXD as an alternative regime for cancers.