IGROV-1 Cells Research Articles

Nanostructures based on monoolein or diolein and amphiphilic gadolinium complexes as MRI contrast agents.

Highly ordered two or three dimensional mesophases in aqueous solution could be usefully obtained by using monoolein (MO) or diolein (DO) monomers. Nanostructures (also indicated as nanoparticles, NPs) of MO or DO containing different amounts (1%, 5%, 10% and 20%) of the synthetic amphiphilic gadolinium complex (C18)2DTPA(Gd) have been prepared and characterized for their relaxometric and structural behaviors. The nanostructure is found in the 110-200 nm range for all investigated systems, while the presence of the gadolinium containing monomer produces a partial loss of the cubic symmetry, as shown by Cryo-TEM images of NPs doped with 10% w/w of (C18)2DTPA(Gd). Gadolinium containing nanostructures display high relaxivity values (in the 10-15 mM-1 s-1 range at 25° and 20 MHz, with a further increase at 37 °C for DO based NPs), and interesting relaxometric properties for their possible use as MRI contrast agents. NPs containing 10% w/w of (C18)2DTPA(Gd) (MO3-NPs and DO3-NPs) have been also derivatized by introducing 3% wt of (C18)2-Peg3000-FA to obtain targeted aggregates (MO3-NP-FA, DO3-NP-FA). A preferential uptake efficiency of DO3-NP-FA in IGROV-1 cells with respect to DO-NPs without folic acid is observed, especially when cells are incubated with low concentrations of nanostructures or at short incubation times, thus indicating its potential use as a target-selective delivery system for MRI contrast agents on tumor cells overexpressing the folate receptor.

Navitoclax augments the activity of carboplatin and paclitaxel combinations in ovarian cancer cells

ObjectivesTo evaluate the efficacy of combination of navitoclax, carboplatin and paclitaxel in ovarian cancer. Methods8 ovarian cancer cell lines were treated with either doublet or triplet combinations of navitoclax, carboplatin and paclitaxel. Interactions were assessed by determining a combination index or measuring caspase activity. The effect of the combinations was also evaluated by measuring the inhibition of cells grown as spheroids. ResultsNavitoclax exhibited modest (IC50=3–8μM) single agent potency. Antagonism between carboplatin and paclitaxel was evident in Ovcar-4, Ovcar-8 and Skov-3 cells. Drug combinations including navitoclax with carboplatin and/or paclitaxel showed significantly less antagonism, or even synergy, in several cell lines than carboplatin/paclitaxel alone. Navitoclax enhanced the activation of caspase 3/7 induced by carboplatin and/or paclitaxel in Igrov-1 cells. Combinations of navitoclax, carboplatin and paclitaxel showed more than additive activity against Igrov-1 spheroids. ConclusionsNavitoclax improves the activity of combinations of carboplatin and paclitaxel in vitro. Our observations, taken with other published data, provide a rationale for clinical trials of navitoclax in ovarian cancer in combination with chemotherapy.

The effects of DNA methylation and epigenetic factors on the expression of CD133 in ovarian cancers

BackgroundTo identifying the effects of DNA methylation and epigenetic factors on the expression of CD133, a cancer stem cell marker, in gynecologic cancer cell lines.MethodsOvarian cancer cell lines (OVCAR-8 and IGROV-1) and an endometrial cancer cell line (Ishikawa) were treated with 5-aza-2`-deoxycytidine (DAC) or Trichostatin A (TSA). Expression of CD133 was evaluated by quantitative real-time PCR, methylation-specific PCR (MSP), reverse transcription-PCR, western blot, and FACS analysis. All results are representative of three independent experiments.ResultsCD133 mRNA expression varied among the different cell lines; the weakest expression was observed in OVCAR-8 cells, while it was strongly expressed in Ishikawa cells. The degree of methylation of the CD133 P2 promoter was 61% in OVCAR-8 cells, 53% in IGROV-1 cells, and 43% in Ishikawa cells. CD133 expression was increased at both the mRNA and protein level after DAC treatment. On the contrary, CD133 mRNA expression decreased after TSA treatment decreased in all cell lines except OVCAR-8. In addition, MSP of the CD133 P2 promoter revealed that methylation was reduced after treatment with either DAC or TSA.ConclusionsThe expression of the CD133 antigen in primary ovarian and endometrial cancer cell lines is regulated by epigenetics, as indicated by its increased expression following DAC treatment and irregular expression pattern followed by TSA treatment. In addition, the expression of CD133 was negatively correlated with the degree of methylation of the CD133 P2 promoter.

Sulfonates-PMMA nanoparticles conjugates: A versatile system for multimodal application

We report herein the viability of a novel nanoparticles (NPs) conjugated system, namely the attachment, based on ionic and hydrophobic interactions, of different sulfonated organic salts to positively charged poly(methylmethacrylate) (PMMA)-based core-shell nanoparticles (EA0) having an high density of ammonium groups on their shells. In this context three different applications of the sulfonates@EA0 systems have been described. In detail, their ability as cytotoxic drugs and pro-drugs carriers was evaluated in vitro on NCI-H460 cell line and in vivo against human ovarian carcinoma IGROV-1 cells. Besides, 8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt (HPTS) was chosen for NPs loading, and its internalization as bioimaging probe was evaluated on Hep G2 cells. Overall, the available data support the interest for these PMMA NPs@sulfonates systems as a promising formulation for theranostic applications. In vivo biological data strongly support the potential value of these core-shell NPs as delivery system for negatively charged drugs or biologically active molecules. Additionally, we have demonstrated the ability of these PMMA core-shell nanoparticles to act as efficient carriers of fluorophores. In principle, thanks to the high PMMA NPs external charge density, sequential and very easy post-loading of different sulfonates is achievable, thus allowing the preparation of nanocarriers either with bi-modal drug delivery behaviour or as theranostic systems.

Synergistic interaction between the novel histone deacetylase inhibitor ST2782 and the proteasome inhibitor bortezomib in platinum-sensitive and resistant ovarian carcinoma cells

The ability of histone deacetylase inhibitors to modulate the expression of genes relevant for growth or apoptotis regulation supports their interest in combination treatments of resistant tumors. We explored the effect of the combination of the histone deacetylase inhibitor ST2782 and the proteasome inhibitor bortezomib in ovarian carcinoma cell lines, including the IGROV-1 cell line and two p53 mutant platinum-resistant sublines (IGROV-1/OHP and IGROV-1/Pt1). We found a synergistic interaction between the two drugs, more evident in the p53-mutant resistant sublines, which was associated with increa sed apoptosis. The treatment with ST2782 resulted in early induction of Bax as well as in cleavage of caspase 3 and poly (ADP-ribose) polymerase only in the resistant cell lines. The inhibition of p53-transcriptional transactivation by pifithrin alpha in IGROV-1 cells enhanced the synergism. Conversely, knockdown of endogenous wild-type p53 in IGROV-1 cells determined synergism reduction. These opposite effects support the relevance of the transactivation-deficient mutant p53 as a synergism determinant. Moreover, in vivo studies indicated that tumor growth inhibition tended to be more evident in mice receiving the drug combination than in those treated with bortezomib alone. Overall, our study supports the potential effectiveness of the combination in platinum drug-resistant ovarian cancer carrying mutant p53.

The peritoneal tumour microenvironment of high-grade serous ovarian cancer.

High-grade serous ovarian cancer (HGSC) disseminates early and extensively throughout the peritoneal space, causing multiple lesions that are a major clinical problem. The aim of this study was to investigate the cellular composition of peritoneal tumour deposits in patient biopsies and their evolution in mouse models using immunohistochemistry, intravital microscopy, confocal microscopy, and 3D modelling. Tumour deposits from the omentum of HGSC patients contained a prominent leukocyte infiltrate of CD3+ T cells and CD68+ macrophages, with occasional neutrophils. Alpha-smooth muscle actin+ (α-SMA+) pericytes and/or fibroblasts surrounded these well-vascularized tumour deposits. Using the murine bowel mesentery as an accessible mouse peritoneal tissue that could be easily imaged, and two different transplantable models, we found multiple microscopic tumour deposits after i.p. injection of malignant cells. Attachment to the peritoneal surface was rapid (6–48 h) with an extensive CD45+ leukocyte infiltrate visible by 48 h. This infiltrate persisted until end point and in the syngeneic murine ID8 model, it primarily consisted of CD3+ T lymphocytes and CD68+ macrophages with α-SMA+ cells also involved from the earliest stages. A majority of tumour deposits developed above existing mesenteric blood vessels, but in avascular spaces new blood vessels tracked towards the tumour deposits by 2–3 weeks in the IGROV-1 xenografts and 6 weeks in the ID8 syngeneic model; a vigorous convoluted blood supply was established by end point. Inhibition of tumour cell cytokine production by stable expression of shRNA to CXCR4 in IGROV-1 cells did not influence the attachment of cells to the mesentery but delayed neovascularization and reduced tumour deposit size. We conclude that the multiple peritoneal tumour deposits found in HGSC patients can be modelled in the mouse. The techniques described here may be useful for assessing treatments that target the disseminated stage of this disease. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Abstract 4653: 5F203-induced reactive oxidative species, DNA damage and apoptosis involves aryl hydrocarbon activation in ovarian cancer cells

Abstract Ovarian cancer is the 2nd cause of death by gynecological malignancies after breast cancer. These tumors have poor prognoses because they are detected at advanced stages of disease and show acquired or intrinsic resistance to therapy.Benzothiazoles, agents with preclinical antitumor activity against ovarian and breast cancer models, belong to a novel mechanistic class which bind to the arylhydrocarbon receptor (AhR). Phortress the lysylamide prodrug of 2-(4-amino-3-methylphenyl)-5- fluorobenzothiazole (5F203) is currently under phase I clinical evaluation. We previously demonstrated that these compounds activate AhR signaling in sensitive breast cancer cells, the goal of this project was to evaluate the role of AhR in 5F203 activity in two human ovarian cell lines: IGROV-1 sensitive to 5F203 and SKOV-3 resistant to this agent. We observed by MTS assay IGROV-1 cell growth inhibition of 31.9±12.2% by 0.01 μM 5F203 and almost total growth inhibition (91.9±3.4%) after treatment of IGROV-1 cells with 1 μM 5F203. Pre-incubation with the AhR inhibitor ΔNF (1 μM), partially blocked the effect of 5F203 (inhibition of 21.6±4.7% and 27.7±5.3% respectively). In contrast, 5F203 failed to inhibit growth of SKOV-3 cells.5F203 (1 μM) induced AhR translocation from cytosolic to nuclear fractions evaluated by Western blot and confocal microscopy in IGROV-1 cells only. Moreover, activation of CYP1A1 related promoter sequences and EROD activity further indicated activation of AhR signaling. 5F203 treatment for 1h increased ROS and induced NFκB nuclear translocation only in IGROV-1 cells, effects which were attenuated by pre-treatment with ΔNF. Detection of γH2AX revealed DNA double strand breaks 2h post 5F203 treatment of IGROV-1 cells only. After 24h exposure, 5F203 resulted in increased IGROV-1 cell apoptosis, evaluated by DAPI staining of apoptotic bodies, increased pP53, caspase 3 and PARP cleavage. We also observed G1 cell cycle arrest, increased P21 expression and decreased cyclin D1 expression induced by 5F 203. We conclude that the antitumor/pro-apoptotic effects of 5F203 observed in IGROV-1 cells is mediated by AhR which may be a new molecular target in the treatment of ovarian tumors and that this agent may represent a potential novel treatment for ovarian cancer. 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 4653. doi:1538-7445.AM2012-4653

Abstract 3915: Insulin-like growth factor 1 promotes cancer cell growth via RAC1 activation in ovarian epithelial cancer cells

Abstract Ovarian cancer is the most lethal of all gynecological malignancies, due in part to the diagnosis at an advanced stage caused by the lack of specific signs and symptoms and the absence of reliable tests for screening and early detection. Furthermore, survival is reduced by recurrence of chemo-resistant tumors. The insulin-like growth factor 1 (IGF1)/IGF1 receptor (IGFR) signaling system exerts a broad anti-apoptotic function and plays a crucial role in human cancer progression, including ovarian cancers. The small GTP-binding protein RAC1 regulates a diverse array of cellular events, including the control of cell growth, cytoskeleton reorganization for motility, and the activation of protein kinases. Although RAC1 is expressed in most tissues, it can be elevated in tumors at various stages of neoplastic progression. This study was conducted to explore the role of RAC1 in IGF1-stimulated ovarian cancer cell growth. While IGFR was expressed in normal human ovarian surface epithelial (HOSE) cells and all OSE cancer cell lines examined, the IGF1R was over expressed in IGROV1 ovarian cancer cells. Exposure of IGROV1 cells to IGF1 rapidly (<0.5 min) induced IGFR phosphorylation which triggered the phosphorylation of AKT (S473) (≥0.5 min), ERK1[[Unsupported Character - ⁄]] 2 (>1.0 min) and p70S6K (T389) (≥1.0 min); indicating that IGF1 actives PI3K and MAPK signaling pathways that are known to be important in cell growth. We observed a maximal effect of IGF1 on IGROV1 cell growth at 50 ng/ml (2.2 fold increase, p<0.001) after 24 h. In contrast, exposure of IGROV1 cells to the PI3K inhibitor LY294002 or the MEK1/2 inhibitor UO126 decreased cell viability. Pull-down assays revealed that IGF1 stimulated GTP loading of RAC1 in less than 1.0 min with maximal increases observed after 5 min (2.6 fold, p<0.05). Treatment with the PI3K inhibitor LY294002 significantly reduced RAC1 activation in response to IGF1. Pre-treatment of IGROV1 cells with the RAC1 inhibitor (NSC23766) for 1 h inhibited IGF1-induced activation of RAC1; and decreased cell survival in a concentration-dependent manner (5- 50 µM NSC23766, p<0.05). The reduction in viability after 48 hr was accompanied by an increase in apoptotic cells, cleaved PARP and elevated cytosolic cytochrome C. In contrast, treatment of HOSE cells with NSC23766 did not alter cell viability. Of interest, following 6 h treatment of IGROV1 cells with 20-50 µM NSC23766 we observed increases in the phosphorylation of Akt (S473), GSK3β (S9), and p70S6K (T389), suggesting the activation of PI3K/AKT pathway was not sufficient to rescue cells from death. Furthermore, treatment with NSC23766 (50 µM) and cisplatin (20 µM) reduced cell viability to an extent greater than achieved by either agent alone (p<0.05). Thus, disruption of RAC1 activation reduces IGF1-stimulated cell growth, induces apoptosis, and enhances cell death by anti-cancer agents in ovarian cancer cells. 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 3915. doi:1538-7445.AM2012-3915

Inhibition of P-glycoprotein functionality by vandetanib may reverse cancer cell resistance to doxorubicin

P-glycoprotein belongs to the ATP binding cassette transporters, responsible for the multidrug resistance of cancer cells. These transporters efflux hydrophobic drugs outside cells and decrease their therapeutic efficacy. The aim of this study was to investigate the effect of vandetanib, an oral tyrosine kinase inhibitor of EGFR, VEGFR 2 and RET kinases, on the functionality of P-gp after a 24h-treatment at therapeutic concentration (2μM), and its ability to increase the cytotoxicity of chemotherapeutic agents in multidrug resistance cancer cells. In this study we found that IGROV1-DXR and IGROV1-CDDP cells were resistant to doxorubicin and cisplatin respectively, compare to parental cell line IGROV1. The parental sensitive and the two resistant cell lines similarly expressed MRP1 and did not express BCRP. Moreover, in contrast to the IGROV1 and IGROV1-CDDP cells, IGROV1-DXR cell line overexpressed P-gp. Functional activity studies demonstrated that MRP1 was not functional and the MDR phenotype in IGROV1-DXR cells was linked to P-gp functionality. Results also showed that vandetanib reversed resistance to doxorubicin in IGROV1-DXR cells, but not to cisplatin in IGROV1-CDDP cells. After 24h of treatment, vandetanib increased the accumulation of rhodamine 123 and calcein AM, demonstrating a functional inhibition of the transporter. In IGROV1-DXR cell line, vandetanib reverse resistance to doxorubicin by inhibiting the functionality of P-gp. In conclusion, vandetanib should be an option for drug combination in patients already developing a P-gp mediated multidrug resistance.

Abstract C34: Synergistic interaction between a novel histone deacetylase inhibitor and the proteasome inhibitor bortezomib in platinum-resistant ovarian carcinoma cells.

Abstract Based on the ability of histone deacetylase inhibitors to modulate the expression of genes relevant for growth regulatory or apoptotic pathways, the purpose of the present study was to explore the interest of novel combination treatments for drug-resistant tumors. Thus, using cellular and biochemical pharmacology approaches, we examined the effect of the combination between the novel histone deacetylase inhibitor ST2782 and the proteasome inhibitor bortezomib in ovarian carcinoma cell lines, including the parental IGROV-1 cell line and two platinum-resistant sublines. The IGROV-1/OHP and IGROV-1/Pt1 cells, selected for resistance to oxaliplatin and cisplatin respectively, are characterized by a mutant p53 gene. Using the combination index method and cell sensitivity assays, we found a synergistic interaction between ST2782 and bortezomib, more evident in the p53-mutant resistant sublines. Such an interaction was associated with increased apoptotic cell death, as shown by TUNEL assays. The treatment with ST2782 resulted in early induction of the pro-apoptotic protein Bax as well as in cleavage of caspase 3 and PARP only in the resistant cell lines, as supported by Western blot analyses. The inhibition of p53-transcriptional transactivation by pifithrin alpha in IGROV-1 cells enhanced the synergism between the two drugs. Conversely, knockdown of endogenous wild-type p53 in IGROV-1 cells following transfection of small interfering RNAs determined a considerable synergism reduction. These opposite effects support the relevance of the transactivation-deficient mutant p53 as a determinant of the synergism. Moreover, in vivo studies in IGROV-1/Pt1 xenografts indicated that tumor growth inhibition tended to be more marked in mice receiving the drug combination with respect to those treated with bortezomib alone. In conclusion, the present study supports the potential effectiveness of this combination in p53-mutant ovarian cancer models resistant to platinum compounds. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C34.

Abstract A230: Discovery and characterization of ARQ 092, an ATP-independent, potent and selective inhibitor of AKT kinases.

Abstract The AKT family of serine-threonine kinases is a critical signal transduction node serving a variety of cellular functions including survival, proliferation, protein synthesis, and glucose metabolism. Small molecule inhibitors of AKT are emerging as potential targeted agents to treat cancers that exhibit aberrant AKT pathway signaling. A screening strategy was employed to identify inhibitors which utilize the intrinsic negative regulatory function of hydrophobic clusters in the ATP-binding cleft to inhibit the kinase activity of AKT1. ARQ 092 was identified through this screening paradigm following in vitro and in vivo optimization and was selected for further development. Crystallographic studies provided evidence that this mechanism of inhibition of AKT had been achieved. ARQ 092 binds to inactive, unphosphorylated AKT1 with sub-nanomolar affinity (Kd = 0.28 nM measured by surface plasma resonance) and biochemically inhibits all three isoforms (IC50 values of 3 nM, 4.5 nM, and 5.5 nM respectively for AKT1, AKT2, and AKT3). ARQ 092 displayed inhibition kinetics against AKT1 which were not affected by ATP concentrations up to 1000 M. When screened against a panel of over 300 kinases, ARQ 092 inhibited only three kinases within 100-fold of its IC50 against AKT1. ARQ 092 potently inhibited AKT phosphorylation (Ser473 & Thr308) in AN3CA human endometrial carcinoma cells (EC50 values of 39 nM and 61 nM, respectively for p-Ser473 & p-Thr308) and demonstrated concentration-dependent inhibition of phosphorylation of the downstream AKT substrate PRAS40. ARQ 092 inhibited growth of AN3CA cells (GI50 = 555 nM), A2780 cells (GI50 = 400 nM), and IGROV-1 cells (GI50 = 66 nM), in addition to LNCaP, ZR-75–1, and BT-474 human cancer cell lines (GI50 values ranging from 1 to 4 μM). Following a single oral dose of ARQ 092, inhibition of AKT and PRAS40 phosphorylation was documented in vivo in both AN3CA human endometrial tumor and BT474 human breast tumor xenograft models. The growth of AN3CA tumor xenografts was markedly suppressed after daily oral administration of ARQ 092 for 10 days. Finally, ARQ 092 was shown to have good pharmaceutical properties and was advanced into preclinical development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A230.

5.58 Advances in the Use of Targeted Nanoparticles for the Treatment of B-CLL

Objective: To demonstrate the therapeutic effect of chlorambucil and hydroxichloroquine (CLB/HCQ) as well as Vorinostat and PEITC (V/PEITC) loaded biological nanoparticles (BNPs) coated with the anti-CD20 monoclonal antibody Rituximab targeted to B-CLL cells. Materials and methods: We produced four types of BNPs: BNP0, empty PLGA nanoparticles without a drug or monoclonal antibody; BNP1, Rituximab-coated but without drugs; BNP2, CLB/HCQ-loaded and Rituximab coated; and BNP3, V/PEITC-loaded and Rituximab coated. To evaluate the cytotoxic effect of BNPs, B-CLL cells obtained from 10 B-CLL patients were incubated with defined amounts of all types of BNPs, measuring residual viable cells after 48 hours. The cytotoxic effect of BNPs was also tested on CD20-negative CHO, HUVEC, MEL-28 and IGROV1 cells incubated with BNPs (2 l at 0.9mg/ml of polymer). Results: BNP2 was able to induce cell cytotoxicity in in a dosedependent manner, while BNP0 and BNP1 were almost ineffective. Ninety five percent of tumor cells from all patients were killed using only 2 l of BNP2 at 0.9mg/ml of polymer and containing 5.4 g of CLB/HCQ. The same amount of free cytotoxic agents induced an approx 80% kill rate. No specific cell cytotoxicity was measured using BNP0 and 1 in non-CD20 cells. BNP2 was effective in CHO and melanoma treated cells, but values never exceeded 20% of killing. B-CLL cells with the lowest expression levels of CD20 were killed by BNP2 at about 82%. V/PEITC BNP3 had similar effects to BNP2, but when tested with the lowest CD20 expressing cells the killing effect was around 95%. Discussion: BNPs seem to be a novel and effective strategy to treat B-CLL. CD20 expressed on cell surface seems to be the limiting factor for the binding of a sufficient amount of Rituximab for the activation of all the effector systems. This antibody limitation seems not to be the case for our BNPs. Both CLB/ HCQ and V/PEITC BNPs had a significant killing effect on B-CLL cells independent of the concentration of CD20 on cell surfaces. This was especially noticeable with V/PEITC nanoparticles. This last combination of drugs is a very interesting for use in B-CLL. Its delivery inside biodegradable nanoparticles could provide a new therapeutic option in the near future.

Camptothecin and Thiocamptothecin: the Role of Sulfur in Shifting the Hydrolysis Equilibrium towards the Closed Lactone Form

Adverse effects have limited the clinical potential of 20-(S)-camptothecin (CPT) and led to a growing interest in the development of CPT analogues that exhibit less severe drawbacks, while maintaining their therapeutic activity. Recently, a thiopyridone isostere of CPT, 20-(S)-thiocamptothecin (TCPT), was developed that resulted more potent than the parent compound in H460, HT29 and IGROV-1 cell lines. The improved activity of TCPT over CPT might be due to the greater stability of the lactone ring. Here, reversible hydrolysis to the ring-open carboxylate forms of CPT and TCPT was studied by HPLC, both in the presence and absence of human serum albumin (HSA). The amount of TCPT that exists in the lactone form at equilibrium in buffer solution (24 h) was found to be significantly higher than CPT, and the same trend was observed in the presence of HSA. Specifically, HSA caused a shift in the hydrolysis equilibrium of TCPT towards the carboxylate form, but the proportion of lactone form remained higher than that observed for CPT under the same conditions, and also in the presence of a higher excess of the protein. The role of the sulfur atom in the stability of the open and closed lactone derivatives was investigated by theoretical calculations using stabilization energies and comparison between experimental and calculated absorption spectra. Our results suggest that, in aqueous solution, more ionic species (anionic and enolic forms) are present for TCPT. This study provides further insights into the effects of oxygen/sulfur replacement in the CPT pyridone ring.

Systemy ekspresyjne białek cytochromu P450 w badaniach in vitro metabolizmu leków

Cytochrome P450 proteins are the most important enzymes involved in metabolic activation or detoxification of various drugs used in clinical practice. However, some drug metabolism pathways may be responsible for their increased toxicity. New expression systems of cytochrome P450 proteins in mammalian cells, including human, are designed to explore the influence of metabolism on the cellular and molecular mechanisms of action of potential drugs and those used therapeutically. They can also be used to study the effect of tested compounds on activity and expression of metabolizing enzymes. Human tumor cell lines with overexpression of cytochrome P450 isoenzymes are of particular importance, especially in studies of potential chemotherapeutics. The HepG2 cell line, derived from human liver cancer, is the most commonly used in studies on drug metabolism and toxicity. However, due to the low level of metabolizing enzymes in these cells, the Hep3A4 cell line with overexpression of CYP3A4 isoenzyme was developed. The stable overexpression of cytochrome P450 isoenzymes was also obtained in other human cancer cell lines, including hepatoma HepaRG cells, ovarian cancer IGROV-1 cells, colon cancer Caco-2, and LS180 cells. This review describes currently developed bacterial, yeast, insect and mammalian (including human) cytochrome P450 protein expression systems, in terms of their advantages and disadvantages in the context of their suitability for basic research and use on a commercial scale.

Abstract 2544: Preclinical pharmacodynamics (PD) of ONX 0801, a folate receptor-α (FRα) and tumor-targeted thymidylate synthase (TS) inhibitor

Abstract ONX 0801, a first-in-class tumor-targeted antifolate, selectively targets TS in tumor cells due to transport by FRα, thus differentiating it from other antifolates. FRα expression is restricted to the apical membrane of specialised tissues generally inaccessible to drugs in circulation; however, it is highly expressed in several cancers including ovarian. FRα is a high affinity, low capacity transporter and the rate of drug uptake and cell line sensitivity to ONX 0801 is related to the number of FRαs tethered to the cell surface (72h IC50 range: 0.003-0.3µM). Non-FRα mediated uptake becomes relevant at relatively high concentrations (>1µM). The purpose of the study was to investigate the relationship between drug exposure and FRα- or non-FRα mediated TS inhibition in both tumors and normal proliferating tissues. PD endpoints of TS inhibition included an intact TS assay for cultured cells, and tumor and plasma dUrd in mice bearing FRα positive IGROV-1 ovarian tumors. In IGROV-1 cells, the 72h IC50 was 0.003µM; however, blocking FRα-mediated uptake with excess folic acid increased it to 2µM. In FRα negative A431 cells the IC50 was 7µM. Drug washout experiments demonstrated that exposure to high concentrations for < 24h gave a duration of TS inhibition insufficient to produce cytotoxicity as ONX 0801 is not polyglutamated and therefore effluxed from non-FR expressing cells. The IC50 for ONX 0801 in A431 cells was >30µM for 4h exposure (72h endpoint) but was reduced to 8µM and 5µM for a 24h and 48h exposure, respectively. These data suggest that this level of exposure in vivo could produce effects in normal proliferating tissues (NPT). We have tested this in mice bearing IGROV-1 xenografts by injecting ONX 0801, as either a bolus to achieve temporary high exposure, or as a 3 day continuous infusion (CI) with osmotic minipumps. During the 4hr following a bolus of 100-200mg/kg of ONX 0801 the plasma drug levels were high (∼500-1µM) and plasma dUrd was elevated consistent with non FR-mediated TS inhibition in NPT. However, plasma dUrd had normalised by 24h. Consistent with tumor-targeting, tumor dUrd was still elevated 24h after 15mg/kg ONX 0801. CI of ONX 0801 of 5mg/kg/24h produced a steady-state plasma drug level of 0.05µM and increased tumour dUrd for 72h. No increases in plasma dUrd were observed up to a dose of 300mg/kg/24h where a small increase was seen (plasma ONX 0801 ∼4µM). Taken together, these data are consistent with cell line data and predict that increasing exposure in patients to >5µM for >24h is unnecessary and could be associated with toxicity. In conclusion, ONX 0801 has a high therapeutic index in model systems using PD markers of TS inhibition. Further, we have defined guiding pharmacokinetic and PD parameters for the ongoing Phase 1 clinical trial to aid the determination of the biologically effective dose. Supported by grants from Onyx Pharmaceuticals, BTG PLC and Cancer Research UK Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2544. doi:10.1158/1538-7445.AM2011-2544

Abstract 4576: IMGN853, an anti-Folate Receptor I antibody-maytansinoid conjugate for targeted cancer therapy

Abstract Previously we reported (O. Ab; EORTC, 2010) that an antibody-maytansinoid conjugate (AMC) composed of an anti-FOLR1 antibody conjugated to the cytotoxic maytansinoid, DM4, via the disulfide-containing linker, SPDB, was potent in killing FOLR1-expressing cancer cells in vitro and in vivo. In light of the favorable results noted, we assessed the optimal antibody, linker, and maytansinoid agent for an AMC targeting FOLR1, as reported here. Antibody selection. Anti-FOLR1 antibodies were generated by immunizing mice with human FOLR1-expressing cells, and a panel of FOLR1-specific antibodies was identified by flow cytometry binding assay. Several FOLR1-antibodies with high binding affinity to both human and monkey FOLR1 were chosen for further evaluation and were humanized using ImmunoGen's resurfacing technology. Antibodies were conjugated to DM1 via the non-cleavable SMCC linker and the conjugates tested for activity against FOLR1-positive KB cells in vitro and in vivo. All conjugates had comparable cytotoxic potencies in vitro. However, the in vivo anti-tumor activity of one conjugate, M9346A-SMCC-DM1, was significantly better than that of SMCC-DM1 conjugates of other FOLR1 antibodies. Based on this finding, the M9346A antibody was chosen for further development. Linker/maytansinoid selection. The M9346A antibody was linked to DM1 or DM4 via the disulfide-containing cleavable linkers SPP, SPDB or sulfo-SPDB, or via the non-cleavable SMCC linker. We compared the in vitro cytotoxic activities of these conjugates on KB, Igrov-1 and Jeg-3 cell lines. The conjugates with cleavable linkers displayed markedly greater in vitro activities than the SMCC conjugate. We then examined the in vivo activities of the conjugates in FOLR1-positive KB- and Ovcar 3-tumor models. Again, we found that the conjugates with cleavable linkers were more active in vivo than the noncleavable conjugate. Among the conjugates with cleavable linkers, the sulfo-SPDB-DM4 conjugate was the most active conjugate against the Ovcar-3 model, it had activity comparable to that of the SPDB-DM4 conjugate against KB tumors, and both were more active than the SPP-DM1 conjugate in the two xenograft models. Taking into consideration that sulfo-SPDB-DM4 was the most efficacious design in vivo and the potential of the hydrophilic sulfo-SPDB-linker to enable better activity against PgP-expressing cells (previously reported data), M9346A-sulfo-SPDB-DM4 was selected to be the candidate for development and designated IMGN853. IMGN853 is a promising candidate for the treatment of FOLR1-expressing tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4576. doi:10.1158/1538-7445.AM2011-4576

Abstract 2591: Folate receptor alpha: A promoter of cancer cell proliferation that is independent of folate

Abstract Folate receptor alpha (FRα) is over-expressed in malignant tumors of epithelial origin, particularly in ovarian cancer, but largely absent in normal tissue. High levels of protein expression in tumor cells are associated with increased proliferative capacity and enhanced chemo-resistance to Cisplatinum (CP). However, the underlying mechanism remains unclear. We sought to evaluate the biological function of FRα in CP-resistant cancer and hypothesized that alterations of FRα expression levels in drug CP-resistant cancers would result in the re-sensitization of tumor cells to CP. We first developed a human ovarian cancer cell line, IGROV-1, with increased CP resistance and found that these cells also showed an increase in FRα expression over parental IGROV-1 cells. Then, we used FOLR1 specific siRNA to completely knock-down FRα expression. Surprisingly, ablation of FRα expression did not restore sensitivity to CP but had a substantial negative impact on proliferation in both parental and CP-resistant cells. Similar phenomena were also observed when FRα was knocked-down in 786-0 cells, a human renal carcinoma line that expresses robust levels of FRα. In contrast, when we applied FRα knock-down to the normal human kidney cells HK-2 (FRα+), we observed no impairment on proliferation. One explanation of these results is that removal of FRα in cancer cells impairs the ability of these cells to uptake reduced folates, a requirement for nucleic acid synthesis and other methylation reactions. However, even pharmacological concentrations of 5-methyltetrahydrofolate, provided only partial restoration of proliferation in these cells. Taken together, these results suggest that FRα plays an essential role in the proliferation of tumor cells, but not in normal cells via a yet undefined pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2591. doi:10.1158/1538-7445.AM2011-2591

Dasatinib (BMS-35482) has synergistic activity with paclitaxel and carboplatin in ovarian cancer cells

PurposeTo explore the activity of dasatinib alone and in combination with paclitaxel and carboplatin in ovarian cancer cells and to determine if dasatinib activity can be predicted based on evaluation of the SRC pathway. Experimental designMicroarray analysis was performed for IGROV1, OVCAR3, A2780 and SKOV3 ovarian cancer cells and the status of the genomic SRC signature pathway was determined. Cells were treated with carboplatin, paclitaxel and dasatinib individually and in combination. Pre- and post-treatment phospho-SRC (pSRC) and SRC protein expression was determined. Dose–response curves were constructed, and drug interaction was assessed by the Combination Index (CI) method. ResultsSRC protein expression levels reflected the SRC pathway genomic signature in the cell lines with the lowest (SKOV3) and highest (IGROV1) pathway expression, but not in those with intermediate expression (OVCAR3, A2780). Dasatinib treatment caused loss of pSRC in all cell lines, with 50% growth inhibition for IGROV1 at 70nM, OVCAR3 at 34nM, A2780 at 4.1μM and SKOV3 at 530nM. Dasatinib combined with cytotoxics yielded a synergistic effect (CI=0.46 to 0.79) in all cell lines except SKOV3. ConclusionDasatinib in combination with standard chemotherapeutic agents appears to interact in a synergistic manner in some ovarian cancer cell lines. Further research is needed to evaluate tumor cell characteristics which predict response to dasatinib.

Proteomic Analysis of Cellular Response to Novel Proapoptotic Agents Related to Atypical Retinoids in Human IGROV-1 Ovarian Carcinoma Cells

Novel agents characterized by the scaffold of the atypical retinoid ST1926, but containing different chemical functions (carboxylic or hydroxamic acid), exhibit potent proapoptotic activity. In the present paper, we show that the treatment of the IGROV-1 ovarian cancer cell line with compounds sharing structural features with ST1926 (ST1898, ST3595, ST3056) determines a strong inhibition of proliferation mainly due to apoptotic cell death. In an effort to understand the mechanism of action of these compounds, we performed a proteomics analysis of IGROV-1 total lysates and nuclear extracts. Using this approach, we found that deregulation of calcium homeostasis, oxidative stress, cytoskeleton reorganization, and deregulation of proteasome function may represent important pathways involved in response of IGROV-1 cells to the studied compounds. The most prominent effect was down-regulation of factors involved in protein degradation, an event more marked in cells treated with ST3595. In addition, we identified proteins specifically modulated by each treatment, including prohibitin and cochaperone P23 (ST1898), pre-mRNA splicing factor SF2p32 and clathrin light chain (ST3595), as well as Far upstream element (FUSE) binding protein 1 and DNA-binding protein B (ST3056). By identifying proteins modulated by novel proapoptotic agents, this study provides insights into critical aspects of their mechanism of action.

Inhibition of the Inflammatory Cytokine TNF-α Increases Adenovirus Activity in Ovarian Cancer via Modulation of cIAP1/2 Expression

Oncolytic adenoviruses show promise as a cancer treatment. However, they generate acute inflammatory responses with production of cytokines, including tumor necrosis factor-α (TNF-α). We investigated whether inhibition of TNF-α augments efficacy of the E1A CR2-deleted adenovirus dl922-947 in ovarian cancer. dl922-947 induced transcription of TNF-α and its downstream signaling targets interleukin-6 and -8 (IL-6 and IL-8) in ovarian cancer cells. In vitro, RNAi-mediated knockdown of TNF-α reduced production of multiple inflammatory cytokines after infection and increased ovarian cancer cell sensitivity to virus cytotoxicity, as did treatment with the anti-TNF-α antibody infliximab. In vivo, stable knockdown of TNF-α in IGROV-1 xenografts increased the anticancer activity of dl922-947. In addition, inhibition of TNF-α using monoclonal antibodies also improved dl922-947 efficacy. This increased efficacy resulted from suppression of cellular inhibitor of apoptosis-1 and -2 (cIAP1 and cIAP2) transcription in malignant cells and a consequent increase in caspase-mediated apoptosis. These findings suggest that TNF-α acts as a survival factor in adenovirus-infected cells. Combining TNF-α inhibition with oncolytic adenoviruses could improve antitumor activity in clinical trials.