Insensitive Cells Research Articles

Abstract A72: Punicic acid, a fatty acid from pomegranate seed oil, inhibits breast cancer cell proliferation

Abstract Introduction: Pomegranate (Punica granatum) is a well-known fruit used for alternative medicine. Various pomegranate extracts are utilized as supplements with a wide variety of purported health benefits including anticancer actions. These supplements contain a number of different components that may either activate or inhibit estrogen's action. Additionally factors associated with pomegranate fruit or extracts may lower oxidation of LDL cholesterol, reduced arterial plaques, lower oxidative stress, enhanced wound healing, inhibition of angiogenesis and tumor proliferation and increased tumor apoptosis. However, these studies have been conducted with fruit, seeds, pericarp or partially purified pomegranate extracts making it hard to determine what the active component(s) may be. Punicic acid is an omega-5 long chain polyunsaturated fatty acid found in pomegranate seed oil. A number of long chain fatty acids have been reported to have cancer preventive actions. Here we investigated the potential ability of punicic acid to affect growth of both an estrogen insensitive breast cancer cell line (MDA-MB-231) and an estrogen sensitive cell line developed from the MDAMB-231 cells (MDA-ER 7). Methods: The two breast cancer cell lines were cultured in vitro in the presence of various amounts of punicic acid, conjugated linoleic acid, α-linolenic acid and other substances to evaluate cell proliferation, apoptosis, mitochondrial membrane permeability and cell cycle. Results: We found that 40 µM punicic acid inhibited proliferation of MDA-MB-231 and MDA-ER 7 cells 92% and 96% respectively compared to untreated cells. Furthermore, punicic acid induced apoptosis in the MDA-MB-231 and MDA-ER 7 cells by 86% and 91% respectively compared to untreated control cells. Punicic acid also disrupted mitochondrial membrane potential of both cell lines. We next investigated whether lipid oxidation was required for the function of punicic acid by adding 20 µM of the antioxidant tocotrienol to the assays. This resulted in reversal of the effects of punicic acid on proliferation inhibition, apoptosis and disruption of the mitochondrial membrane potential in both the MDA-MB-231 and MDA-ER 7 cells. Finally, we evaluated the role of PKC signaling in the anticancer effects of punicic acid by performing proliferation assays in the presence of the PKC inhibitor bisindolymaleimide I. Proliferation inhibition by punicic acid was partially blocked in both the MDA-MB-231 and MDA-ER 7 cells. Conclusions: Our in vitro assays indicated that punicic acid inhibited both estrogen sensitive and estrogen insensitive cell lines equally. Additionally the results indicate that the antiproliferative effect of punicic acid on human breast cancer cells appears to be at least partially dependent on lipid peroxidation and the PKC pathway. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A72.

Association of Bordetella dermonecrotic toxin with the extracellular matrix

BackgroundBordetella dermonecrotic toxin (DNT) causes the turbinate atrophy in swine atrophic rhinitis, caused by a Bordetella bronchiseptica infection of pigs, by inhibiting osteoblastic differentiation. The toxin is not actively secreted from the bacteria, and is presumed to be present in only small amounts in infected areas. How such small amounts can affect target tissues is unknown.ResultsFluorescence microscopy revealed that DNT associated with a fibrillar structure developed on cultured cells. A cellular component cross-linked with DNT conjugated with a cross-linker was identified as fibronectin by mass spectrometry. Colocalization of the fibronectin network on the cells with DNT was also observed by fluorescence microscope. Several lines of evidence suggested that DNT interacts with fibronectin not directly, but through another cellular component that remains to be identified. The colocalization was observed in not only DNT-sensitive cells but also insensitive cells, indicating that the fibronectin network neither serves as a receptor for the toxin nor is involved in the intoxicating procedures. The fibronectin network-associated toxin was easily liberated when the concentration of toxin in the local environment decreased, and was still active.ConclusionsComponents in the extracellular matrix are known to regulate activities of various growth factors by binding and liberating them in response to alterations in the extracellular environment. Similarly, the fibronectin-based extracellular matrix may function as a temporary storage system for DNT, enabling small amounts of the toxin to efficiently affect target tissues or cells.

Punicic acid is an ω-5 fatty acid capable of inhibiting breast cancer proliferation

Pomegranate extracts have been used as anticancer agents and they contain a large number of potentially bioactive substances. Punicic acid is an omega-5 long chain polyunsaturated fatty acid found in Punica granatum (pomegranate) seed oil. A number of long chain fatty acids have been reported to have cancer preventive actions. Here we investigated the potential ability of punicic acid to affect growth of both an estrogen insensitive breast cancer cell line (MDA-MB-231) and an estrogen sensitive cell line developed from the MDA-MB-231 cells (MDA-ERalpha7). Proliferation was inhibited 92 and 96% for MDA-MB-231 and MDA-ERalpha7 cells, respectively compared to untreated cells by 40 microM punicic acid. Furthermore, punicic acid induced apoptosis in the MDA-MB-231 and MDA-ERalpha7 cells by 86 and 91%, respectively compared to untreated control cells and disrupted cellular mitochondrial membrane potential. We also investigated whether lipid oxidation was required for the function of punicic acid by adding 20 microM of the antioxidant tocotrienol to the assays. This resulted in reversal of the effects of punicic acid on proliferation inhibition, apoptosis and disruption of the mitochondrial membrane potential. Finally, we evaluated the role of PKC signaling in the anti-cancer effects of punicic acid by performing proliferation assays in the presence of the PKC inhibitor bisindolymaleimide I. Proliferation inhibition by punicic acid was partially blocked in both the MDA-MB-231 and MDA-ERalpha7 cells. These results suggest that punicic acid has breast cancer inhibitor properties that are dependent on lipid peroxidation and the PKC pathway.

Abstract B29: SMAC-mimetic induced apoptosis of SKOV3 cells requires NIK and activation of canonical NFκB signaling

Abstract By stimulating the autoubiquitination and proteosome mediated degradation of CIAP1, Smac mimetics hypersensitize tumor cells to TNFα mediated apoptosis. Indeed, Smac mimetics induce the production of TNFα in sensitive but not insensitive tumor cell lines. The precise mechanism of NFκB activation and subsequent TNFα induction is not well understood, and identifying the mechanism by which LBW242 induces TNFα may provide potential biomarkers for patient stratification. Paradoxically, these agents have been reported to both activate and inhibit the canonical NFκB pathway and to activate the non-canonical pathway. To elucidate this mechanism, we knocked down nodes of the canonical and non-canonical NFκB pathways and assessed activation of an NFκB/p65-reporter, induction of TNFα expression and cell death after treatment with the Smac mimetic compound LBW242. In the canonical pathway, knockdown of IKKB and p65 ablated TNF induction and rescued SKOV cells from LBW242-mediated cell death, suggesting that activation of the canonical pathway is required for LBW242-mediated cell death. In the noncanonical pathway, knockdown of NIK, but surprisingly not RelB, rescued SKOV cells from LBW242-mediated cell death. This data suggests a role for NIK in LBW242-mediated activation of the canonical but not the non-canonical NFκB pathway. Consistent with this model, we find overexpression of NIK is sufficient to activate the p65 reporter. Moreover, activation of the non-canonical NFκB pathway was robustly observed in both sensitive and insensitive tumor cell lines, further invalidating the contribution of the non-canonical arm to Smac mimetic induced TNF production. We propose NIK-mediated activation of the canonical NFκB signaling is responsible for Smac mimetic induced TNFα production. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B29.

Abstract A204: Investigating a novel antitumor molecular target using a chemical proteomic strategy

Abstract Three novel, synthetic biarylheterocycles bearing imidazole terminal groups were discovered with extreme potency (IC50 16–640 nM) against the drug-resistant (Mitomycin C and EO9) colon carcinoma cell line BE, and also the human ovarian carcinoma A2780 and human non-small cell lung cancer H460 cell lines. Notably, this biological activity was independent of duplex DNA binding affinity. The compounds were tested in the NCI 60-cell line panel and COMPARE analysis suggests they are targeting the product of a ‘gene-like sequence’ of unidentified function. The identity of the target proteins was explored using an affinity chromatography proteomic strategy. Bespoke affinity matrices were prepared in which test compounds were attached to a solid support through a biotin tag. A synthetic route to compounds containing a biotin moiety in place of one of the imidazole sidechains was developed. Chemosensitivity studies confirmed that the biotinylated compounds retain their activity (IC50 6.25 µM in a susceptible cell line, compared with > 100 µM for an insensitive cell line). The biotinylated ligands were exposed to protein extracts from the susceptible cell lines and ligand-protein complexes collected using a streptavidin-activated affinity column. Bound proteins were eluted from the column and separated using SDS-PAGE. Proteins were characterised by MALDI MS/MS and identified using database searches. In parallel, the mechanism of action of the compound series has been investigated using NCI 3D MIND data mining. This suggests the mechanisms of action to be associated with phosphatase- and kinase-mediated cell cycle regulation. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A204.

Heterogeneity in Simvastatin-Induced Cytotoxicity in AML Is Related to Differential Ras-Isoprenylation, Rather Than to Blockade of Cholesterol Synthesis.

Abstract 1718Poster Board I-744 IntroductionCholesterol synthesis is enhanced in blast cells of acute myeloid leukemia (AML) patients, as indicated by high mRNA levels of 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoAR) and LDL receptor (LDLR) and increased LDL uptake. In addition, AML cells exposed to chemotherapeutic drugs acutely increase their cholesterol content, which renders these cells less susceptible to chemotherapy. Statins inhibit HMG-CoAR, the rate-limiting enzyme of the mevalonate pathway. As a result, both the cholesterol synthesis route and the isoprenylation route are blocked. The latter route leads to the production of the isoprenoids farnesyl (FPP) and geranylgeranyl pyrophosphate (GGPP). Small GTP-ases Ras and Rho require farnesyl or geranylgeranyl isoprenylation for their proper function, a process which is catalyzed by farnesyltransferase (FTase) or geranylgeranyltransferase (GGTase). Simvastatin has shown to be cytotoxic to primary (CD34+) AML cells in vitro, however, these effects are heterogeneous. The aim of the present study was to elucidate the mechanism of simvastatin-induced cytotoxicity and the heterogeneity therein. This could be extensively studied by making use of cell lines that display a strong heterogeneity in responsiveness to simvastatin. MethodsIn 9 myeloid leukemic cell lines we mimicked or rescued the cytotoxic effects of simvastatin with compounds acting on either the cholesterol synthesis route or the isoprenylation route. ResultsIn the cell lines, we identified 3 subgroups: sensitive (EC50 3-15 μM: NB4, OCI-M3, HL60), medium sensitive (EC50 16-50 μM: THP-1, U937, K562), and insensitive (EC50 60-100 μM: UT7-GM, TF-1, KG1a) to simvastatin, as determined by assessing cytotoxicity using a chemoluminescence assay. In all cell lines, simvastatin-induced cytotoxicity could completely be rescued by mevalonate, a compound directly downstream of HMG-CoAR. This indicates that the mevalonate pathway is indeed the key pathway involved in statin-induced cytotoxicity. To assess the role of the cholesterol synthesis route, mRNA levels of HMG-CoAR and LDLR were determined following simvastatin-treatment. In all cell lines, HMG-CoAR (1.5-7.8 fold) and LDLR (1.5-3.2 fold) mRNA expression were increased. No relationship existed between the basal or induced mRNA expression levels of both genes and simvastatin-induced cytotoxicity. In addition, squalene, a cholesterol precursor, did not rescue simvastatin-induced cell death. The role of the isoprenylation route, which is linked to the GTP-ases Ras and Rho, was assessed by rescue studies: simvastatin-induced cytotoxicity could especially be rescued by GGPP (68%), but also by FPP (42%), in the sensitive cell lines. Rescue was less pronounced in the insensitive cell lines (12% and 26% by GGPP and FPP, respectively), indicating a difference between the cell lines at the level of isoprenylation. This observation was confirmed by the finding that GGTI-298, a GGTase inhibitor, and to a lesser extent tipifarnib, an FTase inhibitor, were more effective in the sensitive cell lines compared to the insensitive cell lines (60% versus 96% viability at 0.2 μM, p=0.005; and 55% versus 78% viability at 5 μM, p=0.001, respectively). Also the ERK inhibitor U0126, acting downstream of Ras, was more effective in the sensitive cell lines when looking at cell viability (48% versus 70%, p=0.01) and pERK inhibition, as determined by Western blot. This is in line with the observed relationship between simvastatin-induced cytotoxicity and inhibition of pERK. Y27632, an inhibitor of ROCK (downstream target of Rho), and LY294,002, an Akt inhibitor (downstream of Ras), were equally cytotoxic in all cell lines. Currently, experiments are ongoing to obtain more detailed information on the mechanism involved by assessing the activity of Ras, GGTase and FTase, and we will confirm our findings in primary AML cells. ConclusionOur data indicate that the Ras-isoprenylation route, rather than the cholesterol synthesis route of the mevalonate pathway is responsible for simvastatin-induced cytotoxicity and the heterogeneous response between AML cells.Supported by a grant of the Dutch Cancer Society DisclosuresNo relevant conflicts of interest to declare.

Displacement of Bim From Anti-Apoptotic Proteins Is the Primary Factor for Determining ABT-737 Activity in Multiple Myeloma Cell Lines.

Abstract 2851Poster Board II-827ABT-737 and its orally active analog ABT-263 are Bcl-2-family inhibitors that are currently in clinical trials for a variety of cancers including hematological malignancies such as multiple myeloma. Previously, we reported that the sensitivity of multiple myeloma cell lines to ABT-737 correlates with the interactions, but not the expression, of Bcl-2 proteins. Analysis of 6 multiple myeloma cell lines revealed that expression of Bcl-2 proteins did not correlate with sensitivity, however the sensitive cells (8226/S, MM.1S and KMS-11) have a substantial amount of their pro-apoptotic Bcl-2 protein, Bak, bound to Bcl-xL. On the other hand, in the insensitive cell lines (U266, KMS-11 and OPM2), Bak was found to be associated with Mcl-1, a family member that does not bind ABT-737 and thereby confers resistance to this drug. Furthermore, we also showed that release of the BH3-only protein Bim by ABT-737 from Bcl-xL and Bcl-2 also contributes to cell death in 8226/S and MM.1S. The purpose of the current study is to further investigate the role of Bim in ABT-737-induced cell death in the multiple myeloma lines. Similar to Bak, a substantial amount of Bim is bound to Bcl-xL and Bcl-2 in the ABT-737-sensitive cell lines, MM.1S and KMS-18, while in the insensitive cell lines, it is highly bound to Mcl-1. Surprisingly, in the ABT-737-sensitive 8226/S cells, Bim appears to bind to Mcl-1. However in these cells, ABT-737 treatment resulted in upregulation of Noxa, which is a BH3-only protein that binds Mcl-1 and can release Bim. Taken together these data suggest that although binding of Bim to Mcl-1 may confer resistance to ABT-737, in certain cell types this treatment could also induce Noxa expression that antagonizes Mcl-1-mediated resistance. Consistent with this hypothesis, Mcl-1 overexpression as well as knockdown of Noxa expression significantly protected 8226/S cells from ABT-737-induced cell death while they had no effect in MM.1S cells. To further demonstrate the role of Bim in ABT-737-induced cell death, ABT-resistant 8226/S, KMS-11, KMS-18 and U266 cell lines were generated. In the resistant cell lines of 8226/S and KMS-18, Bim is exclusively bound to Mcl-1, which was overexpressed as compared to the parental cells. Bak binding was not affected by acquisition of ABT-737 resistance. This result is in agreement with the findings that interaction of Bim and Mcl-1 confers resistance to ABT-737. On the other hand, in ABT-resistant U266 and KMS-11 cell lines, Bim expression was down-regulated while Mcl-1 levels were not changed. Thus, it appears that in cells where Bim is already bound to Mcl-1, further resistance is achieved by down-regulating the expression of this BH3-only protein. Overall, these results suggest that the complex interactions between Bcl-2 proteins need to be investigated in order to understand how multiple myeloma cells may respond to ABT-737 treatment. Disclosures:Boise:University of Chicago: Patents & Royalties.

Extracellular Metabolite Biomarkers of Bortezomib Resistance in Multiple Myeloma Indicate Involvement of Unexpected Metabolic Pathways.

Abstract 1839Poster Board I-865 Introduction and Objectives:Bortezomib (BZ) is a chemotherapeutic agent approved for the treatment of multiple myeloma (MM). BZ acts through proteasome inhibition, inducing significant ER stress and ideally resulting in cell death. Unfortunately, nearly 20% of MM patients are primarily resistant to BZ treatment and responses to BZ are difficult to predict based on the currently available clinical, cytogenetic and genomic biomarkers. Our function hypothesis is that extracellular metabolites have a greater potential to be found in circulating biofluids as biomarkers. As a result we used a metabolite ‘footprinting' approach in cell growth media to examine the metabolic consequences of BZ treatment using eight human MM cell lines, three of which have been determined to be less sensitive to BZ treatment than the others with a 10 fold difference in their IC50 at 24 hours (5 nM vs 50 nM). Our aims were 1) to establish whether analysis of growth media was suitable for monitoring metabolic changes and 2) to determine specific biopatterns of BZ resistance. Methods:Eight MM cell lines (MM1S, MM1R, INA6, U266, RPMI8266, OPM2, KMS11 and PCL1) were cultured under standard conditions without (control group, 10% FBS) or with bortezomib added (10nM). Media samples were taken for metabolic analysis at 6 and 24 hours for a total of 32 media profiles. Metabolite profiling was accomplished using gas chromatography mass spectrometry (GC-MS) and nuclear magnetic resonance spectroscopy (NMR). GC-MS data was analysed using AMDIS (NIST), and NMR data using Chenomx NMR Suite. Significant metabolites were identified using multivariate regression analysis by supervised projection methods (two-way orthogonal partial least squares discriminant analysis, O2PLS-DA) using SIMCA-P (Umetrics). Results:An average of 756 chemical or metabolite components per sample were profiled, which was reduced to a subset of 116 unique features that were shared in at least 75% of samples. An initial O2PLS-DA model was successfully built from the GC-MS feature set using both growth time (p=0.03) and BZ status (p=6.9e-13) in the Y-matrix. Figure 1 shows a scores plot, where each point represents a single sample, and the position is calculated as a combination of the underlying metabolite concentrations. Changes in cell growth were consistent with the known uptake of carbohydrate substrates and elimination of various amino acids and waste products such as lactate. The remarkable metabolic difference between BZ-treated and untreated cells resulted from reduced energy-related metabolites such as citric acid cycle intermediates and sugars, with a concomitant increase in selected amino acids. Intriguingly, the BZ-insensitive cell cultures exhibit overall metabolic phenotypes much more similar to the BZ-sensitive cultures than to the untreated group, with the exception of a single sample after 6 hours (denoted with an asterisk in Figure 1) which showed an averaged profile. To further probe the phenomenon of BZ resistance, the treated group was analyzed independently, with the 37 most influential components providing discriminating ability between the BZ-insensitive and BZ-sensitive cells (p= 0.04) in an OPLS-DA model. Conclusions:We conclude that metabolite footprinting is a reliable and robust method for monitoring metabolic events for both cell growth and BZ treatment. Furthermore, BZ-insensitivity is accompanied by a notable shift from carbohydrate metabolism to fatty acid metabolism, while the overall metabolic phenotype remains very similar in both BZ-sensitive and insensitive strains in the presence of the drug. This result suggests that BZ function remains largely intact in both sensitive and insensitive cell lines, and resistance is conferred through alternate mechanisms with measureable metabolic endpoints. Success in measuring extracellular metabolites also supports the notion of serum-accessible biomarkers or biopatterns of BZ resistance. The unique genetic instability underlying each cell line may provide a further avenue for characterizing resistance mechanisms along with analysis of various intracellular components. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Increased anti-tumour effects of doxorubicin and zoledronic acid in prostate cancer cells in vitro: supporting the benefits of combination therapy

Combination treatment using the chemotherapy drug doxorubicin and the anti-resorptive agent zoledronic acid has shown to be very effective in inducing apoptosis in breast cancer cells, and also to eradicate breast tumour growth in vivo. Here, we investigated whether apoptotic cell death is increased when zoledronic acid and doxorubicin are given in sequence or in combination in prostate cancer cells in vitro. PC3, DU145 and LNCaP prostate cancer cells were treated with zoledronic acid or doxorubicin alone, in sequence or in combination, and apoptosis was measured by evaluation of nuclear morphology following staining with Hoechst and PI. The involvement of the mevalonate pathway in the induction of apoptosis was assessed through the addition of the mevalonate pathway intermediate geranylgeraniol. Both agents induced PC3 cell death, with 5 microM zoledronic acid inducing 1.73% apoptosis and 50 nM doxorubicin 3.60% apoptosis following 24 h of exposure. In contrast, sequential exposure (doxorubicin followed by zoledronic acid) caused 8.87% apoptosis. Doxorubicin followed by zoledronic acid induced 4.77% apoptosis in LNCaP cells, compared to 1.53% caused by zol alone, 2.23% by dox alone and 2.5% following the reverse sequence (P < 0.001 in all cases). In DU145 cells doxorubicin followed by zoledronic acid induced 5.73% apoptosis, compared to 1.8% following zol alone, 2.93% by dox alone, and 3.20% following the reverse sequence (P < 0.001 in all cases). This is the first detailed study to show that an increased anti-tumour effect is generated when doxorubicin and zoledronic acid are given in sequence in both hormone-sensitive and insensitive prostate cancer cells in vitro. Our results suggest that combined treatment with these agents is superior to single agent therapy, and should be explored in a tumour model of prostate cancer.

Split Hopkinson pressure bar multiple reloading and modeling of a 316 L stainless steel metallic hollow sphere structure

The high strain rate (600 s −1) compression deformation of a 316 L metallic hollow sphere (MHS) structure (density: 500 kg m −3; average outer hollow sphere diameter: 2 mm and wall thickness: 45 μm) was determined both numerically and experimentally. The experimental compressive stress–strain behavior at high strain rates until about large strains was obtained with multiple reloading tests using a large-diameter compression type aluminum Split Hopkinson Pressure Bar (SHPB) test apparatus. The multiple reloading of MHS samples in SHPB was analyzed with a 3D finite element model using the commercial explicit finite element code LS-DYNA. The tested MHS samples showed increased crushing stress values, when the strain rate increased from quasi-static (0.8 × 10 −4 s −1) to high strain rate (600 s −1). Experimentally and numerically deformed sections of MHS samples tested showed very similar crushing characteristics; plastic hinge formation, the indentation of the spheres at the contact regions and sphere wall buckling at intermediate strains. The extent of micro-inertial effects was further predicted with the strain rate insensitive cell wall material model and with the strain rate sensitive behavior of MHS structure similar to that of the cell wall material. Based on the predictions, the strain rate sensitivity of the studied 316 L MHS sample was attributed to the strain rate sensitivity of the cell wall material and the micro-inertia.

Endonucleases induced TRAIL-insensitive apoptosis in ovarian carcinoma cells

TRAIL induced apoptosis of tumor cells is currently entering phase II clinical settings, despite the fact that not all tumor types are sensitive to TRAIL. TRAIL resistance in ovarian carcinomas can be caused by a blockade upstream of the caspase 3 signaling cascade. We explored the ability of restriction endonucleases to directly digest DNA in vivo, thereby circumventing the caspase cascade. For this purpose, we delivered enzymatically active endonucleases via the cationic amphiphilic lipid SAINT-18 ®:DOPE to both TRAIL-sensitive and insensitive ovarian carcinoma cells (OVCAR and SKOV-3, respectively). Functional nuclear localization after delivery of various endonucleases (BfiI, PvuII and NucA) was indicated by confocal microscopy and genomic cleavage analysis. For PvuII, analysis of mitochondrial damage demonstrated extensive apoptosis both in SKOV-3 and OVCAR. This study clearly demonstrates that cellular delivery of restriction endonucleases holds promise to serve as a novel therapeutic tool for the treatment of resistant ovarian carcinomas.

Relationship between 31P metabolites and oncolytic viral therapy sensitivity in human colorectal cancer xenografts

Studies using phosphorus magnetic resonance spectroscopy (MRS) have pointed to the significance of phospholipid metabolite alterations as biochemical markers for tumour progression or therapy response. Spectroscopic imaging was performed in colorectal flank tumours in nude mice. In vivo tumour doubling times for each cell line were measured. In vivo sensitivity of each tumour line to treatment with G207 and NV1020 oncolytic viruses was assessed. Correlations between viral sensitivity and tumour doubling time and phosphorus MRS were estimated. For G207 virus, in vitro cytotoxicity tests showed cell viability at multiplicities of infection (ratio of viral particles per tumour cell) of 0.1 on day 6 as follows: C85, less than 1 per cent; HCT8, 1 per cent; LS174T, 9 per cent; HT29, 18 per cent; and C18, 92 per cent. Respective values for NV1020 were 1, 18, 4, 18 and 86 per cent. The phosphoethanolamine to phosphocholine ratio was significantly lower in virus-sensitive than -insensitive cells, and was dependent on tumour doubling time. Alterations in membrane phospholipid metabolites that relate to proliferation of cancer cells affect the efficacy of oncolytic viral therapy. MRS proved a highly sensitive non-invasive tool for predicting the efficacy of viruses.

Force Characterization and Rigidity Analysis of a Monolithic Cochlear Prosthesis Actuator

Cochlear implants continue to be used in the treatment of profound deafness. Because of the tonotopic nature of the cochlea, more controlled insertion is perhaps the most important factor affecting device performance. The implant stiffness, and therefore the scala tympani (ST) wall contact force, contributes to insertion difficulties. Attempts to correlate the implant carrier structural properties and the intracochlear contact forces during insertion are limited. Researchers in the Michigan Center for Wireless Integrated Microsystems are developing perimodiolar-shaped silicon and parylene-based thin film cochlear electrode arrays and backing devices for a more controllable implantation. We report a method developed for measuring the thin film actuated electrode array rigidity to quantify the ST and modiolus wall contact forces during and after insertion. The method used a pneumatically actuated polyethylene terephthalate (PET) monolithic electrode actuator using pressurized air (0–200 kPa) for actuation. The prototype actuators consisted of PET tubes with an ID of 365 μm and a wall thickness of 58 μm. Force calculations using cantilever beam bending theory were performed to estimate the tube bending forces as a function of internal pressure and therefore variable structural stiffness. Based on estimations, a method was developed to measure such small forces avoiding the use of commercially available, relatively insensitive load cells. A fixture was fabricated incorporating two brass microcantilevers (reference and deflection arms) sensitive to sub-mN forces applied by the actuator on the deflection arm of the cantilevers. Microcantilver deflection data, captured by an interferometric microscope, was used to calculate the actuator force and eventually the reaction force acting on the actuator. The implant actuation forces ranged from 0–0.76 mN over an actuation pressure range of 0–140 kPa, from nearly straight to the relaxed perimodiolar post-implantation shape. For estimating the implant rigidity (EI), the actuator stiffness and the actuation pressure was correlated. The actuator stiffness at different actuation pressures was obtained both theoretically (using beam bending theory and PET tube structural properties) and experimentally (using the derived unconstrained actuator deflections at measured actuator forces). The theoretical and experimental stiffness values ranged from 3.6E-08 to 5.34E-07 N/μm and 2.5E-08 to 7.8E-06 N/μm respectively over the working pressure range. The calculated rigidity constant (EI) of the best prototype insertion tool from the experimental stiffness measurement was 6.71E06 Nμm2. The insertion tool-ST wall contact forces were calculated, using the estimated rigidity, in a hypothetical insertion situation. Force calculations assumed that the implant is equipped with actuator deflection feedback sensors and the actuator's stiffness remains constant over its entire length for a given operating pressure. A contact force of 1.19 mN was found acting on the cochlear ST wall when the insertion tool hits the wall and deflects by 200 μm at an actuation pressure of 140 kPa.

Effects of sorafenib on proliferation of hormone-sensitive and hormone-insensitive prostate cancer cells

e16092 Background: Sorafenib is a multi-targeted kinase inhibitor approved for treatment of renal and hepatocellular cancer. In patients with castration therapy-resistant prostate cancer sorafenib treatment is associated with discordant prostate-specific antigen (PSA) and clinical responses, possibly due to an effect on PSA expression. The mechanisms of its action in advanced prostate tumors are not investigated so far. The aim of the present study was to evaluate the effects of sorafenib in androgen-sensitive (LNCaP) and -insensitive (PC 3) prostate cancer cell lines. Methods: Proliferation was evaluated by measurement of cell number and protein. Expression of cell cycle and apoptosis regulatory proteins of the Bcl-2 family was determined by Western blot. Results: Treatment of those cells by sorafenib for 48 hours resulted in a concentration-dependent inhibition of proliferation. Maximal inhibition was achieved with 25 μM of the compound. Cell number was reduced by 50% in both cell lines. We observed inhibition of expression of cyclin-dependent kinase 2 in LNCaP and PC3 cells. In addition, Mcl-1 protein, that is frequently overexpressed in prostate cancer, was down-regulated by sorafenib in both cellular models. Conclusions: Sorafenib caused inhibition of growth of prostate cancer cells regardless of their androgen sensitivity. Its inhibitory effects on cyclin- dependent kinase 2 and Mcl-1 imply that sorafenib regulates cell cycle progression and apoptosis in prostate cancer. On the basis of these results experiments with chemotherapy-resistant prostate cells are being performed. [Table: see text]

Quantum dot-based quantification revealed differences in subcellular localizationof EGFR and E-cadherin between EGFR-TKI sensitive and insensitive cancercells

Nanoparticle quantum dots (QDs) provide sharper and more photostable fluorescent signalsthan organic dyes, allowing quantification of multiple biomarkers simultaneously.In this study, we quantified the expression of epidermal growth factor receptor(EGFR) and E-cadherin (E-cad) in the same cells simultaneously by using secondaryantibody-conjugated QDs with two different emission wavelengths (QD605 and QD565)and compared the cellular distribution of EGFR and E-cad between EGFR-tyrosine kinaseinhibitor (TKI)-insensitive and -sensitive lung and head and neck cancer cell lines.Relocalization of EGFR and E-cad upon treatment with the EGFR-TKI erlotinib in thepresence of EGF was visualized and analyzed quantitatively. Our results showed thatQD-immunocytochemistry (ICC)-based technology can not only quantify basal levels ofmultiple biomarkers but also track the localization of the biomarkers upon biostimulation.With this new technology we found that in EGFR-TKI-insensitive cells, EGFRand E-cad were located mainly in the cytoplasm; while in sensitive cells, theywere found mainly on the cell membrane. After induction with EGF, both EGFRand E-cad internalized to the cytoplasm, but the internalization capability insensitive cells was greater than that in insensitive cells. Quantification also showedthat inhibition of EGF-induced EGFR and E-cad internalization by erlotinib inthe sensitive cells was stronger than that in the insensitive cells. These studiesdemonstrate substantial differences between EGFR-TKI-insensitive and -sensitivecancer cells in EGFR and E-cad expression and localization both at the basallevel and in response to EGF and erlotinib. QD-based analysis facilitates theunderstanding of the features of EGFR-TKI-insensitive versus -sensitive cancercells and may be used in the prediction of patient response to EGFR-targetedtherapy.

GnRH receptor expression in human prostate cancer cells is affected by hormones and growth factors

GnRH receptors (GnRH-R) have been found in various malignancies, including prostate cancer (PCa). They mediate the direct antitumor effects of GnRH analogs. Nevertheless, few reports concern drug-induced modulation of GnRH-R levels. In this study, we investigated GnRH-R expression in androgen-sensitive (LNCaP) and -insensitive (PC-3) PCa cells treated for 4 and 6 days with a GnRH agonist (Leuprorelin acetate, LA, 10(-11) or 10(-6) M), Dihydrotestosterone (DHT, 10(-9) M), Cyproterone acetate (CA, 10(-7) M), and Epidermal growth factor (EGF, 10 ng/ml), either alone or combined. The RT-PCR analysis showed no variation in GnRH-R mRNA levels of both treated LNCaP and PC-3 cells. On the contrary, immunoblotting indicated that in LNCaP and PC-3 cells, LA upregulated membrane GnRH-R expression (up to 92%). In androgen-sensitive cells, DHT induced a GnRH-R increase (up to 119%) always comparable to that occurring in the presence of CA. GnRH-R upregulation by LA/DHT or CA/DHT association was similar to that promoted by the single agents. In PC-3 cells, EGF upregulated GnRH-R (up to 110%). A prolonged treatment (for 12 days) determined a greater EGF-induced increase in GnRH-R levels (142%). Lower (or no) receptor enhancement occurred when LA and EGF were associated. Our findings indicate that LA post-transcriptionally upregulates its own membrane receptor in androgen-sensitive and -insensitive PCa cells, counteracting the receptor enhancement produced by DHT and EGF. The effects, obtained with a relatively long and continuous treatment, may have implications in the choice of therapy modality with GnRH analogs and may render the receptor a novel therapeutic target, particularly in hormone-refractory PCa.

Toxicity caused by Chelidonium majus in primary human, canine, monkey, and rat hepatocytes

Herbal extracts of Chelidonium majus have been widely used in European countries and in Chinese herbal medicines for different applications, based on their anti‐viral, anti‐inflammatory and anti‐microbial properties and their use against various liver disorders including cancer. Recently, several cases of liver disease have been related to C. majus intake. Therefore, we have used primary hepatocyte cultures of human, rat, canine and monkey to elucidate the potential hepatotoxicity of C. majus.Water‐soluble extracts were prepared and incubated with primary hepatocytes. Cellular viability and functions were analyzed by means of lactate dehydrogenase (LDH) and urea release and via the MTT assay. The amounts of different alkaloids present in C. majus extracts were determined by HPLC analysis.The liquid extract showed a significant concentration‐dependent toxicity in human hepatocytes and at concentrations of 7.5 mg/ml for animal hepatocytes. In addition, C. majus lead to a decrease in urea release. The decline in urea production varied between the different species with regard to cellular sensitivity and to its maximal extent.The data suggest, that human hepatocytes seemed to be more sensitive to C. majus extracts than canine, rat and monkey hepatocytes, with rat hepatocytes being the most insensitive cells. In addition, these results demonstrate the necessity to use human hepatocytes in order to evaluate possible toxicological effects of complex plant extracts in humans.

CDNA microarray analysis of pigment epithelium‐derived factor‐regulated gene expression profile in prostate carcinoma cells

To clarify molecular mechanisms involved in the action of pigment epithelium-derived factor (PEDF) in hormone insensitive prostate cancer cells. Total ribonucleic acid from untreated and PEDF-treated cells was subjected to microarray analysis using BioStar 8464 microarray. Real-time polymerase chain reaction analysis was conducted to confirm the microarray data. Twenty-seven out of 8464 genes were found altered in both cell lines. Common gene responses altered by PEDF were identified and included genes known to alter cell signaling as well as genes involved in catalytic activity, cell proliferation, angiogenesis and apoptosis. Real-time reverse transcription polymerase chain reaction, in accordance with the microarray analysis, indicated that PEDF treatment caused an upregulation in the mRNA expression level of stanniocalcin 2, brain-specific angiogenesis inhibitor 2 and growth arrest, DNA-damage-inducible, alpha, and downregulation in the messenger ribonucleic acid level of fibroblast growth factor 3, teratocarcinoma-derived growth factor, neuropilin1, and endothelial Per/ARNT/Sim domain protein1, respectively. These findings demonstrate that PEDF administration causes significant changes in the gene expression of the prostate, providing insights into the potential role of PEDF in the treatment of prostate cancer.

MTOR Inhibitor RAD001 (Everolimus) Has Antiangiogenic/Vascular Properties Distinct from a VEGFR Tyrosine Kinase Inhibitor

Comparison of the antiangiogenic/vascular properties of the oral mammalian target of rapamycin (mTOR) inhibitor RAD001 (everolimus) and the vascular endothelial growth factor receptor (VEGFR) inhibitor vatalanib (PTK/ZK). Antiproliferative activity against various tumor histotypes and downstream effects on the mTOR pathway were measured in vitro. In vivo, antitumor activity, plasma, and tumor RAD001 levels were measured. Activity in several different angiogenic/vascular assays in vitro and in vivo was assessed and compared with PTK/ZK. RAD001 inhibited proliferation in vitro (IC50 values<1 nmol/L to >1 micromol/L), and in sensitive and insensitive tumor cells, pS6 kinase and 4E-BP1 were inhibited. Activity in vitro did not correlate with activity in vivo and significant responses were seen in tumors with IC50 values>10-fold higher than tumor RAD001 concentrations. In vitro, RAD001 inhibited the proliferation of VEGF-stimulated and fibroblast growth factor-stimulated human endothelial cells but not dermal fibroblasts and impaired VEGF release from both sensitive and insensitive tumor cells but did not inhibit migration of human endothelial cells. In vivo, in tumor models derived from either sensitive or insensitive cells, RAD001 reduced Tie-2 levels, the amount of mature and immature vessels, total plasma, and tumor VEGF. RAD001 did not affect blood vessel leakiness in normal vasculature acutely exposed to VEGF nor did it affect tumor vascular permeability (Ktrans) as measured by dynamic contrast-enhanced magnetic resonance imaging. However, the pan-VEGFR inhibitor PTK/ZK inhibited endothelial cell migration and vascular permeability but had less effect on mature vessels compared with RAD001. VEGFR and mTOR inhibitors show similar but also distinct effects on tumor vascular biology, which has implications for their clinical activity alone or in combination.

AKT inhibitor, GSK690693, induces growth inhibition and apoptosis in acute lymphoblastic leukemia cell lines

AbstractThe PI3K/AKT signaling is activated in various hematologic malignancies. We evaluated the effect of a novel, pan-AKT kinase inhibitor, GSK690693, on the proliferation of 112 cell lines representing different hematologic neoplasia. Fifty-five percent of all cell lines tested were sensitive to AKT inhibitor (EC50 < 1 μM), with acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma, and Burkitt lymphoma showing 89%, 73%, and 67% sensitivity to GSK690693, respectively. The antiproliferative effect was selective for the malignant cells, as GSK690693 did not inhibit the proliferation of normal human CD4+ peripheral T lymphocytes as well as mouse thymocytes. Phosphorylation of downstream substrates of AKT was reduced in both sensitive and insensitive cell lines on treatment with GSK690693, suggesting that the cause of resistance was not related to the lack of AKT kinase inhibition. Consistent with the role of AKT in cell survival, GSK690693 also induced apoptosis in sensitive ALL cell lines. Overall, our data provide direct evidence for the role of AKT signaling in various hematologic malignancies, especially ALL and some lymphomas.