Significant Growth Delay Research Articles

P21. Synthesis, structure and biological study of Gallium(III) complexes as model radiopharmaceuticals

Introduction 67Ga radiopharmaceuticals have been associated with the diagnosis of lymphomas and inflammation. This isotope has a half-life of approximately three days and can be utilized for quick and effective basic biological testing. Our interest is therefore the design and synthesis of various gallium complexes which would be evaluated in solid and solution state via nuclear magnetic resonance (NMR), infrared spectroscopy (IR) and X-ray diffraction (XRD). The chemical behaviour in solution state is compared to the absolute configuration of the gallium complexes to improve the understanding of any factors which may influence the biological properties of the metallo-organic complexes. An important aspect in pharmaceutical design is the synthesis of complexes to accommodate aspects such as the ease of synthesis; purity; environmental influences; as well as the synthesis conducted by medical personnel and not synthetic chemists. Material and methods Typical synthetic procedures of chemical entities (pharmaceuticals, ligands, complexes, assemblies, etc.) are classically performed by means of various solution based methods (solvothermal). An (almost) solvent-free method of synthesis (neat, liquid-, seeding-, ion-assisted grinding) has been recognized as potentially faster, environmentally friendly and economically acceptable ways to prepare new and known compounds. We have therefore also re-evaluated the synthetic methods of the carboxamide and Schiff base ligands which could potentially be coordinated to gallium(III). Due to their high charge density, Ga(III) prefer hard donors, such as amine-N and carboxylate-O atoms. Furthermore the nuclear biological potential of the ligands and 67Ga coordinated ligand complexes would be evaluated to determine whether they could potentially be used for PET and SPECT imaging as diagnostic agents. Results Three novel Schiff base ligands (S1, S2 and S3) were prepared by economical and environmentally friendly methods described above and used for the radiolabelling with 67Ga. These ligands were characterized by NMR, IR and XRD. All three radiolabelled ligands utilized by CHO-K1 cells and showed an increase in cellular uptake when compared to the 67Ga chloride control (67 GaCl 3 ). Compared to 67 GaCl 3 , no significant growth delay was observed in the cells regardless of the ligand used for the radiolabeling experiment. An increased amount of radioactivity per cell is required to fully report the conditions needed for effective growth inhibition of cells and the possibility of it being suitable as a potential diagnostic and/or therapeutic pharmaceutical. Conclusion We hereby report the synthesised complexes, their solid and solution state character including the crystal structures of several ligand systems used. In addition to the nuclear biological behaviour compared to gallium(III) chloride control.

Targeting the HER Family with Pan-HER Effectively Overcomes Resistance to Cetuximab.

Cetuximab, an antibody against the EGFR, has shown efficacy in treating head and neck squamous cell carcinoma (HNSCC), metastatic colorectal cancer, and non-small cell lung cancer (NSCLC). Despite the clinical success of cetuximab, many patients do not respond to cetuximab. Furthermore, virtually all patients who do initially respond become refractory, highlighting both intrinsic and acquired resistance to cetuximab as significant clinical problems. To understand mechanistically how cancerous cells acquire resistance, we previously developed models of acquired resistance using the H226 NSCLC and UM-SCC1 HNSCC cell lines. Cetuximab-resistant clones showed a robust upregulation and dependency on the HER family receptors EGFR, HER2, and HER3. Here, we examined pan-HER, a mixture of six antibodies targeting these receptors on cetuximab-resistant clones. In cells exhibiting acquired or intrinsic resistance to cetuximab, pan-HER treatment decreased all three receptors' protein levels and downstream activation of AKT and MAPK. This correlated with decreased cell proliferation in cetuximab-resistant clones. To determine whether pan-HER had a therapeutic benefit in vivo, we established de novo cetuximab-resistant mouse xenografts and treated resistant tumors with pan-HER. This regimen resulted in a superior growth delay of cetuximab-resistant xenografts compared with mice continued on cetuximab. Furthermore, intrinsically cetuximab-resistant HNSCC patient-derived xenograft tumors treated with pan-HER exhibited significant growth delay compared with vehicle/cetuximab controls. These results suggest that targeting multiple HER family receptors simultaneously with pan-HER is a promising treatment strategy for tumors displaying intrinsic or acquired resistance to cetuximab. Mol Cancer Ther; 15(9); 2175-86. ©2016 AACR.

USP7 promotes cell proliferation through the stabilization of Ki-67 protein in non-small cell lung cancer cells

The Ki-67 antigen (Ki-67) is the most reliable immunohistochemical marker for evaluation of cell proliferation in non-small cell lung cancer. However, the mechanisms underlying the regulation of protein levels of Ki-67 in non-small cell lung cancer have remained elusive. In this study, we found that Ki-67 and ubiquitin-specific processing protease 7 (USP7) protein were highly expressed in the nucleus of non-small cell lung cancer cells. Furthermore, statistical analysis uncovered the existence of a strong correlation between Ki-67 and USP7 levels. We could also show that the protein levels of Ki-67 in non-small cell lung cancer cells significantly decreased after treatment with P22077, a selective chemical inhibitor of USP7, while the Ki-67 mRNA levels were unperturbed. Similar results were obtained by knocking down USP7 using short hairpin RNA (shRNA) in lung cancer cells. Interestingly, we noticed that ubiquitination levels of Ki-67 increased dramatically in USP7-silenced cells. The tests in vitro and vivo showed a significant delay in tumor cell growth upon knockdown of USP7. Additionally, drug sensitivity tests indicated that USP7-silenced A549 cells had enhanced sensitivity to paclitaxel and docetaxel, while there was no significant change in sensitivity toward carboplatin and cisplatin. Taken together, these data strongly suggest that the overexpression of USP7 might promote cell proliferation by deubiquitinating Ki-67 protein, thereby maintaining its high levels in the non-small cell lung cancer. Our study also hints potential for the development of deubiquitinase-based therapies, especially those targeting USP7 to improve the condition of patients diagnosed with non-small cell lung cancer.

IκB-Kinase-epsilon (IKKε) over-expression promotes the growth of prostate cancer through the C/EBP-β dependent activation of IL-6 gene expression.

The inflammatory cytokine IL-6 has been shown to induce the nuclear translocation of androgen receptors in prostate cancer cells and to activate the androgen receptors in a ligand-independent manner, suggesting it may contribute to the development of a castrate-resistant phenotype. Elevated IL-6 serum levels have also been associated with metastasis-related morbidity in prostate cancer patients. We have previously established that over-expression of I-kappa-B-kinase-epsilon (IKKε also named IKKi or IκBKε) in hormone-sensitive prostate cancer cell lines induces IL-6 secretion. We have also reported that prostate cancer cell lines lacking androgen receptor expression exhibit high constitutive IKKε expression and IL-6 secretion. In the present study, we validated the impact of IKKε depletion on the in vitro proliferation of castrate-resistant prostate cancer cells, and characterized how IKKε depletion affects tumor growth and IL-6 tumor secretion in vivo through a mouse xenograft-based approach. We observed a significant growth delay in IKKε-silenced PC-3 cells injected in SCID mice fed with a doxycycline-supplemented diet in comparison with mice fed with a normal diet. We also found a decrease in IL-6 secretion levels that strongly correlated with tumor growth inhibition. Finally, using constructs with various IL-6-mutated promoters, we demonstrated that IKKε over-expression induces a NF-κB-independent stimulation of the IL-6 gene promoter through the activation and nuclear accumulation of the transcription factor C/EBP-β. Our study demonstrates the pro-proliferative role of the oncogene IKKε in castrate-resistant prostate cancer cell lines, involving the phosphorylation and nuclear translocation of C/EBP-β that initiates IL-6 gene expression.

Abstract 4985: Radiation potentiates systemic anti-tumor immunity unleashed by a novel TLR9 agonist (SD-101)

Abstract Background: SD-101 is a CpG oligonucleotide-based TLR9 agonist that engages its target within early and late endosomes of plasmacytoid dendritic cells (pDCs), resulting in DC activation, robust IFN-α responses, and priming of tumor-specific T-cells. We have previously shown that local radiotherapy (RT) may also act as an immune adjuvant and thereby contribute to immune-mediated tumor rejection (J Natl Cancer Inst, 2013: 105:256-265). We hypothesized that the effects of SD-101 may be potentiated by RT and trigger immune-mediated abscopal responses, outside the irradiated field (Int. J. Radiat. Oncol. Biol. Phys., 58:862-870). Methods: BALB/c mice were inoculated s.c. with syngeneic TSA mammary tumor cells in 2 sites (primary [ipsilateral flank] and secondary [contralateral flank]) to detect abscopal responses. When tumors reached an average of 5 mm in diameter, mice were randomly assigned to 1 out of 4 treatment groups: control (untreated), SD-101, RT, or SD-101+RT. RT was delivered to primary tumors (8 Gy x3 fractions) in 3 consecutive days; secondary tumors were completely shielded from RT. SD-101 was injected into primary tumors (50 μg) three times per week starting on the last day of RT. Mice were followed for tumor growth in both the primary and secondary tumors. In a parallel experiment, mice were euthanized 10 days after initiation of therapy for immune analysis. Results: Significant growth delays (p <0.05) were observed in the primary tumors of mice treated with monotherapy (RT or SD-101), as compared to the primary tumors of the untreated controls. In contrast, no significant growth delays were observed in the secondary tumors of these mice, when compared to the secondary tumors of their untreated counterparts. The combination of SD-101+RT, however, effectively controlled the primary tumors (100% of mice exhibited complete tumor rejection) of treated mice, while significant growth delays were also observed in the secondary tumors (p <0.05), when compared to the secondary tumors of mice from the other groups. SD-101 alone or in combination with RT, induced intratumoral enrichment of CD8α+ DCs (3.02% of CD11c+ cells in SD-101-treated mice vs 1.08% in untreated mice), whereby these CD8α+ DCs preferentially expressed high levels of activation markers (CD40, CD80, and CD86). Yet, mice treated with SD-101+RT, when compared to the other groups, also demonstrated an expansion of tumor-reactive T-cells (CD8+/PD-1+) both locally and systemically (J Clin Investig 2014 124(5):2246-2259). Conclusions: Our studies support the rational use of RT as a powerful therapeutic partner to potentiate the anti-tumor effects of SD-101. Citation Format: Karsten Pilones, Jeffrey Kraynak, Sandra Demaria, Encouse Golden, Silvia Formenti. Radiation potentiates systemic anti-tumor immunity unleashed by a novel TLR9 agonist (SD-101). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4985.

Abstract 4722: Selective inhibition of DNA methyltransferases and efficacy of novel DNMT inhibitors in leukemia xenografts

Abstract Background. DNA methyltransferase (DNMT) 1 plays an important role in tumorigenesis and is responsible for maintenance and propagation of DNA methylation patterns, and DNMT3A and DNMT3B are involved in de novo DNA methylation. 4’-Thio-5-aza-2’-deoxycytidine (5-aza-T-dCyd) and 4’-thio-2’-deoxycytidine (T-dCyd) are novel DNMT inhibitors in development. In this investigation, we test the hypothesis that DNMTs are differentially modulated by DNMT inhibitors that may contribute to treatment efficacy. Methods. Animals (5 per group) bearing HL-60 leukemia cell xenografts were treated with saline or aza-T-dCyd at 2 mg/kg with and without 100 mg/kg tetrahydrouridine (ThU) for 5 days weekly for 2 weeks and cycle repeat after 9-day rest. Two other groups were dosed with T-dCyd at 4 mg/kg and decitabine at 0.75 mg/kg, respectively. Tumor growth was assessed by measuring tumor size. Expression of DNMT1, DNMT3A and DNMT3B was examined by immunohistochemistry in paraffin-embedded tumors resected at day 11. Results. Expression of DNMT1 was high, DNMT3B intermediate, and DNMT3A at low levels in saline control group. Significant growth delay was achieved in the aza-T-dCyd (86%) and aza-T-dCyd plus ThU cohorts (45%), compared with the controls. In the aza-T-dCyd treated xenografts, histological examination confirmed a significant reduction (77%) in tumor area (P = 0.002). Expression of DNMT1 and DNMT3B (n = 2 in aza-TdCyd group) was reduced in aza-T-dCyd cohort (P = 0.01 and P = 0.04) as well as aza-T-dCyd in combination with ThU group (P = 0.01 and P = 0.03). In contrast, no growth inhibition but decrease in DNMT3A and DNMT1 were observed by dosing with decitabine. There were neither delays for tumor growth nor changes of all three DNMTs by T-dCyd treatment with the dose administered. Conclusions. Aza-T-dCyd and aza-T-dCyd plus ThU treatments result in significant tumor growth inhibition in HL-60 leukemia xenografts. The anti-tumor activity is associated with simultaneous inhibition of the two drug targets — DNMT1 and DNMT3B. The preclinical results provide compelling evidence to advance aza-T-dCyd for clinical development and perhaps in the context of companion biomarker approach. Citation Format: Dat Nguyen, Melinda Hollingshead, Larry Rubinstein, James Doroshow, Sherry X. Yang. Selective inhibition of DNA methyltransferases and efficacy of novel DNMT inhibitors in leukemia xenografts. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4722.

Abstract 4884: Intratumoral expression of IL-12 from a dendritic cell-targeting chimeric lentiviral vector from the ZVex platform cures established tumors in multiple models and induces systemic anti-tumor responses

Abstract Interleukin-12 (IL-12), produced by antigen-presenting cells, plays a pivotal role in the interplay between innate and adaptive arms of the immune system. IL-12 treatment has been shown to augment cytotoxic T lymphocyte (CTL) and T-helper 1 responses and anti-tumor effects. However, its use as a systemic therapeutic agent is limited due to toxicity. Intratumoral administration of IL-12 is thus being explored as a local alternative route of administration. Such strategies involve plasmid electroporation or other methods that randomly direct cells in the tumor to express IL-12. Here, we evaluated whether targeting expression of IL-12 to intratumoral dendritic cells (DC), thus mimicking the cytokine's physiological biosynthesis and localization, would result in local and systemic immune responses and tumor control in preclinical models. Six murine tumor models were used, including melanoma (B16F10), colon carcinoma (CT26), breast cancer (4T1), lymphoma (A20) and mastocytoma (P815). Tumors were implanted unilaterally, and for some models also bilaterally, to study systemic (abscopal) effects of therapy. A chimeric third-generation lentiviral vector from the ZVex platform, pseudotyped with the DC-tropic envelope glycoprotein of Sindbis virus, was engineered to express murine IL-12 (p35-p40) (ZVex/IL-12). ZVex/IL-12 was administered as a single intratumoral injection into palpable tumors, alone or in combination with the synthetic TLR4-agonist, glucopyranosyl lipid A (GLA). In some models, systemic anti-CTLA4 treatment was added to enhance clinical efficacy. Animals were monitored 2-3 times per week for tumor size and survival. Greatest curative efficacy of ZVex/IL-12 was observed in the 100% lethal CT26 flank model, where all treated animals cleared their tumors and survived until end-of-study at 70 days post-challenge. In the B16 footpad and flank models, curative efficacy varied from 40% to 90%, with abscopal effects being observed after addition of anti-CTLA4. In the A20 and P815 models, efficacy varied from 30% to 60%. In the aggressive orthotopic 4T1 breast cancer model, co-administration of ZVex/IL-12 with GLA-AF resulted in significantly delayed tumor growth and increased survival time compared to either ZVex/IL-12 or GLA-AF used alone. A single intratumoral injection of ZVex/IL-12 resulted in complete tumor regression or significant growth delay in all mouse tumor models investigated and was accompanied by significant survival benefit. The therapeutic effect could be enhanced by ad-mixing with GLA. Abscopal effects were observed in some models after treatment with ZVex/IL12 only and in others after addition of anti-CTLA4. These results point to the powerful modification of the tumor/tumor microenvironment by intratumorally expressed IL-12 by a ZVex vector and the induction of local and systemic immunity. Citation Format: Tina C. Albershardt, Anshika Bajaj, Jacob F. Archer, Rebecca S. Reeves, Andrea J. Parsons, Lisa Y. Ngo, Jan ter Meulen, Peter Berglund. Intratumoral expression of IL-12 from a dendritic cell-targeting chimeric lentiviral vector from the ZVex platform cures established tumors in multiple models and induces systemic anti-tumor responses. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4884.

Abstract 2054: Improved delivery of paclitaxel to prostate tumors: a membrane-type matrix metalloproteinase (MT-MMP)-targeted approach

Abstract Introduction : Membrane-type matrix metalloproteinases (MT-MMPs) are highly active in prostate tumours, but absent or inactive in normal tissues. MT-MMPs are also known to be elevated in the majority of solid human tumours and to be central to tumor invasion and angiogenesis. Our objective has been to design inactive prodrugs of paclitaxel that are converted to the active drug by selected MMPs within the prostate tumor microenvironment. Methods and Results : We report the synthesis and biological evaluation of a new series of peptide-based conjugates of paclitaxel designed to be selectively cleaved by MT-MMPs in the tumour microenvironment. Paclitaxel is conjugated to the peptide C-terminus via a self-immolative linker, while the N-terminus is protected from non-specific exopeptidase cleavage through the use of a masking group. The relative importance of individual amino acids within the MT-MMP peptide recognition sequence has been investigated following extensive ex vivo metabolic studies. These studies employed tissue homogenates to assess activation of prodrug conjugates in tumour (PC-3) tissues and stability in normal tissues (liver kidney lung). ICTTax5 emerged as our lead agent, demonstrating in vitro stability in normal tissue with differential release of free paclitaxel in tumour tissue. Further in vivo murine pharmacokinetic studies monitoring paclitaxel release in liver, lung, kidney heart and plasma revealed a substantial increase in tumour exposure to paclitaxel following ICTTax5 prodrug administration (20mg/kg) compared to the molar equivalent dose of paclitaxel alone (7mg/kg). AUC (uM.h) ratios of paclitaxel released from ICTTax5 compared to paclitaxel administered alone were 16.2 for tumour and in the range of 0.05-0.99 for normal tissues. Similar highly significant changes in Cmax were demonstrated with a 10-fold (9.77) increase in tumour concentrations and a substantially decreased Cmax ratio (0.02 - 0.19) in other tissues when administered as ICTTax5. Anti-tumour efficacy studies (PC3 xenografts) resulted in a highly significant growth delay following single dose administration of ICTTax5 [20mg/kg] with paclitaxel alone (7mg/kg; the molar equivalent dose to the paclitaxel released from ICTTax5) having no significant anti-tumour effect. Conclusion : A series of peptide-based prodrug conjugates of paclitaxel were synthesized. ICTTax5 was identified as the lead molecule, enabling selective delivery of active paclitaxel to PC3 prostate tumours resulting in superior pharmacokinetics and efficacy when compared to delivery of paclitaxel alone. Citation Format: Paul M. Loadman, Javier Giménez-Warren, Andrew Mitchell, Amanda D. Race, Jade A. Spencer, Steve D. Shnyder, Jason H. Gill, Robert A. Falconer. Improved delivery of paclitaxel to prostate tumors: a membrane-type matrix metalloproteinase (MT-MMP)-targeted approach. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2054.

Identification and characterization of a library of surfactins and fengycins from a marine endophytic Bacillus sp.

An endophytic bacterial strain from a marine green alga, Ulva lactuca, was isolated and identified by 16S rRNA gene sequencing method. The bacterial isolate was found to secrete two major families of cyclic depsilipopeptides, surfactins, and fengycins. Sequencing of the isolated lipopeptides was carried out using the MSn data obtained from an electrospray ionization (ESI) ion trap mass spectrometer coupled to an HPLC system. The assigned sequences were confirmed by a chemical derivatization approach involving esterification followed by mass spectrometric analysis. Distinction of leucine residues from isoleucine was established through a combined electron transfer dissociation-collision-induced dissociation (ETD-CID) method. The fengycins described in this study were found to cause significant delay of growth of two plants, Vigna radiata (mung bean) and Oryza sativa (rice). To the best of our knowledge, this is the first study describing identification and characterization of cyclic peptides from an endophytic Bacillus sp. isolated from marine algae.

Life without complex I: proteome analyses of an Arabidopsis mutant lacking the mitochondrial NADH dehydrogenase complex

The mitochondrial NADH dehydrogenase complex (complex I) is of particular importance for the respiratory chain in mitochondria. It is the major electron entry site for the mitochondrial electron transport chain (mETC) and therefore of great significance for mitochondrial ATP generation. We recently described an Arabidopsis thaliana double-mutant lacking the genes encoding the carbonic anhydrases CA1 and CA2, which both form part of a plant-specific 'carbonic anhydrase domain' of mitochondrial complex I. The mutant lacks complex I completely. Here we report extended analyses for systematically characterizing the proteome of the ca1ca2 mutant. Using various proteomic tools, we show that lack of complex I causes reorganization of the cellular respiration system. Reduced electron entry into the respiratory chain at the first segment of the mETC leads to induction of complexes II and IV as well as alternative oxidase. Increased electron entry at later segments of the mETC requires an increase in oxidation of organic substrates. This is reflected by higher abundance of proteins involved in glycolysis, the tricarboxylic acid cycle and branched-chain amino acid catabolism. Proteins involved in the light reaction of photosynthesis, the Calvin cycle, tetrapyrrole biosynthesis, and photorespiration are clearly reduced, contributing to the significant delay in growth and development of the double-mutant. Finally, enzymes involved in defense against reactive oxygen species and stress symptoms are much induced. These together with previously reported insights into the function of plant complex I, which were obtained by analysing other complex I mutants, are integrated in order to comprehensively describe 'life without complex I'.

Investigating the role of tumour cell derived iNOS on tumour growth and vasculature in vivo using a tetracycline regulated expression system.

Nitric oxide (NO) is a free radical signalling molecule involved in various physiological and pathological processes, including cancer. Both tumouricidal and tumour promoting effects have been attributed to NO, making its role in cancer biology controversial and unclear. To investigate the specific role of tumour-derived NO in vascular development, C6 glioma cells were genetically modified to include a doxycycline regulated gene expression system that controls the expression of an antisense RNA to inducible nitric oxide synthase (iNOS) to manipulate endogenous iNOS expression. Xenografts of these cells were propagated in the presence or absence of doxycycline. Susceptibility magnetic resonance imaging (MRI), initially with a carbogen (95% O2/5% CO2) breathing challenge and subsequently an intravascular blood pool contrast agent, was used to assess haemodynamic vasculature (ΔR2*) and fractional blood volume (fBV), and correlated with histopathological assessment of tumour vascular density, maturation and function. Inhibition of NO production in C6 gliomas led to significant growth delay and inhibition of vessel maturation. Parametric fBV maps were used to identify vascularised regions from which the carbogen-induced ΔR2* was measured and found to be positively correlated with vessel maturation, quantified ex vivo using fluorescence microscopy for endothelial and perivascular cell staining. These data suggest that tumour-derived iNOS is an important mediator of tumour growth and vessel maturation, hence a promising target for anti-vascular cancer therapies. The combination of ΔR2* response to carbogen and fBV MRI can provide a marker of tumour vessel maturation that could be applied to non-invasively monitor treatment response to iNOS inhibitors.

Application of non-starter lactic acid bacteria as biopreservative agents to control fungal spoilage of fresh cheese

The effects of six non-starter lactic acid bacteria (NSLAB) as biopreservative agents against mycotoxigenic moulds in fresh cheeses were investigated. The potential of NSLAB to inhibit the growth of contaminating fungi was evaluated using end point (antifungal overlay assay and mycelium inhibition growth) and descriptive (antifungal assay on cell-free cheese extract) methods. The application of all NSLAB except Lactobacillus plantarum KU13 as the sole starter culture of fresh cheese resulted in significant delay of mycelial growth of Aspergillus flavus and Aspergillus parasiticus on the cheese surface. Compared with other NSLAB strains, Lb. plantarum PIN exhibited the most antifungal activity and delayed the mycelia growth of A. flavus and A. parasiticus on the cheese surface by 19 and 22 days, respectively (at 4 °C). The present study indicated that NSLAB could be used as a natural biopreservatives, for controlling the problems caused by mould growth in cheese.

In vivo studies of the PARP inhibitor, AZD-2281, in combination with fractionated radiotherapy: An exploration of the therapeutic ratio

Background and purposePre-clinical data have shown that PARP inhibitors (PARPi) may increase the efficacy of radiotherapy in prostate cancer. However, it is uncertain as to whether PARPi lead to clonogenic kill when combined with radiotherapy (RT). Material and methodsWe tested the PARP inhibitor AZD-2281 as a radiosensitizing agent under oxic and hypoxic conditions for clonogenic survival in vitro and in vivo using the human prostate cancer cell line, 22Rv1. In addition, the effects of PARPi+RT on normal tissue were investigated using a crypt clonogenic assay. ResultsAZD-2281 inhibited cellular PARP activity under both oxic and hypoxic conditions. The addition of AZD-2281 radiosensitized 22Rv1 cells under oxia, acute hypoxia and chronic hypoxia in vitro. The combination of AZD-2281 with fractionated radiotherapy resulted in a significant growth delay and clonogenic kill in vivo. No increased gut toxicity was observed using this combined PARPi+radiotherapy regimen. ConclusionsThis is the first preclinical study to demonstrate direct clonogenic kill in vivo by the addition of AZD-2281 to radiotherapy. As we did not observe gut toxicity, the use of PARPi in the context of prostate cancer radiotherapy warrants further investigation in clinical trials.

Abstract 2484: IR700-conjugated anti-CD44 monoclonal antibody for phototherapy theranostics of triple-negative breast cancer

Abstract CD44 transmembrane glycoproteins are cell adhesion molecules that play a role in cancer cell proliferation, migration, and invasion.1 CD44 expression is associated with stem-like breast cancer cells1 and is upregulated under hypoxia2 making it a significant target. Monoclonal antibody (mAb)-linked to photosensitizers have achieved targeted phototherapy.3 We developed a CD44 mAb-targeted photosensitizer complex for combined fluorescent detection and phototherapy (PT) of CD44 expressing triple negative breast cancer cells. The mAb conjugate is activated by near-infrared (NIR) light, and demonstrates phototoxic effects selectively in CD44 expressing breast cancer cells following cell membrane binding. CD44 mAb was conjugated with a NIR phthalocyanine dye, IR700, to form CD44-IR700. MB-MDA-231 (CD44 positive), MCF-7 and BT-474 (CD44 negative) were selected for the cell studies. SDS-PAGE, UV spectroscopy and confocal fluorescence imaging were performed to determine purity, composition and cellular localization of the conjugate. Microscopic observation and cell viability tests were performed to study CD44-specific cell death. Tumor models were established by inoculating 2×106 MB-MDA-231 or BT-474 cells in the upper-right mammary fat pad of athymic female mice. In vivo fluorescence imaging was performed on a Li-Cor Pearl® Impulse scanner. Tumor bearing mice (n = 5 per group) injected with PBS, IR700, anti-CD44 mAb, IgG-IR700 and CD44-IR700 were monitored over a 3 week-period and sacrificed to harvest tumors. NIR light at a dose of 30 J/cm2 was applied on day 1 after injection. CD44-IR700 demonstrated preferential binding to the cell membrane of MB-MDA-231 but not MCF-7 or BT-474 cells. CD44-specific cell death was initiated by CD44-IR700-mediated PT (>90% of MB-MDA-231 cells were killed immediately, while 88% of MCF-7 cells and 90% of BT-474 cells were alive). Irradiation itself was harmless to cells and there was no cytotoxicity associated with IR700 and CD44 antibody or with CD44-IR700 in the absence of irradiation. In vivo fluorescence imaging verified the 3-fold increased retention of CD44-IR700 in MB-MDA-231 tumors compared to BT-474 tumors. CD44-IR700 injection together with a therapeutic dose of NIR light exposure resulted in significant growth delay in MB-MDA-231 tumors, but not BT-474 tumors. We did not observe tumor growth delay in separate groups of mice injected with PBS, IR700, anti-CD44 mAb, or IgG-IR700, irrespective of NIR light application. Histological studies confirmed a significant increase of necrosis in the treated MDA-MB-231 tumors. This novel PT theranostic strategy provides a promising opportunity for treating CD44 expressing primary and metastatic triple negative breast cancer.

Abstract 2568: S49076, a MET, AXL, FGFR inhibitor, potentiates radiation therapy in subcutaneous and orthotopic models of lung cancer

Abstract S49076 is an oral ATP-competitive inhibitor of the receptor tyrosine kinases MET, AXL, and FGFR1-3 currently in phase I/II clinical development in patients with advanced solid tumors. Here, we investigated the effect of S49076 on both MET- and non-MET- dependent cell lines alone and in association with ionizing radiation (IR) both in vitro and in vivo. We performed proliferation and clonogenic survival assays using the low MET-expressing H460 and A549 non-small-cell lung cancer (NSCLC) cell lines, the high MET-expressing H441 NSCLC cell line and the MET-dependent GTL16 gastric cancer cell line. Activity against the MET receptor and Aurora B kinase was assessed by analyzing the phosphorylation status of MET Tyr1234/1235 and Histone H3 Ser10 residues. Pharmacologic and RNA interference approaches against Aurora B and MET were used to determine the molecular mechanism of the cell response to S49076. S49076 inhibited MET activity in the nanomolar range (1 - 100 nM). At higher, but still clinically-relevant concentrations (0.5 - 1 μM), S49076 was also found to inhibit Aurora B kinase activity in all cell lines tested. In non MET-dependent cell lines, the antiproliferative and anti-clonogenic effects of the drug correlated with its activity against Aurora B, whereas in the MET-dependent cell line, these effects could be attributed to direct inhibition of the MET receptor. When treatment with S49076 was combined with IR, an enhancement of IR effects was observed in all cell lines tested in vitro. In vivo, a significant growth delay of subcutaneous tumors was observed at 50 mg/kg bid oral S49076 in non-MET dependent models and at 3 mg/kg bid oral S49076 in the GTL16 MET-dependent model. For non MET-dependent models, immunohistochemistry revealed that treatment with S49076 reduced Histone H3 phosphorylation on Ser10. This is consistent with the finding that S49076 concentrations above 1 μM were attained in the tumors, as determined by LC-MS/MS analysis. In all tumors tested, S49076 treatment significantly delayed tumor growth when combined to a fractionated IR (4 fractions of 2.5 Gy) compared to IR alone. An orthotopic model of lung cancer using luciferase-expressing H460 NSCLC cells was also used to evaluate the combination of S49076 (50mg/kg bid) and locally fractionated thoracic IR (4*2.5 Gy). In this model, the median survival after cell implantation was 22 days for control mice, 29.5 and 29 days for S49076 treated and irradiated mice respectively and 41 days for mice receiving the combined treatment (p<0.002 for combined treatment versus the irradiated arm or the S49076 treated arm). Our findings indicate that S49076 improves the efficacy of radiotherapy in both MET- and non-MET- dependent cell, supporting the use of combined treatment with S49076 and radiation in clinical trials without patient selection. Citation Format: Céline Clémenson, Cyrus Chargari, Michele Mondini, Charles Ferté, Winchygn Liu, Mike Burbridge, Valérie Cattan, Anne Jacquet-Bescond, Eric Deutsch. S49076, a MET, AXL, FGFR inhibitor, potentiates radiation therapy in subcutaneous and orthotopic models of lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2568. doi:10.1158/1538-7445.AM2015-2568

B7-H1/PD-1 signals in carcinoma and lymphoma immunopathogenesis and immunotherapy (VAC12P.1118)

Abstract αB7-H1 (αPD-L1) reportedly treats cancer by blocking PD-1 signals, but few formal data are reported. We showed B16 melanoma is CD80-PD-1-B7-H1+. αB7-H1 slowed subcutaneous (SQ) B16 in wild type (WT), B7-H1 KO and CD80 KO, with little difference between groups. αB7-H1 did not slow B16/B7-H1lo tumors in B7-H1 KO mice, formally showing tumor B7-H1 mediates αB7-H1 effects. αPD-1 slowed B16 in WT or B7-H1 KO, but not B16/B7-H1lo in B7-H1 KO, formally showing B7-H1/PD-1 effects in αB7-H1. Tumor B7-H1 variably predicts αB7-H1 efficacy, possibly due to variable tumor B7-H1 expression. To model variable B7-H1, we gave B16 or B16/B7-H1lo on both flanks, or each on opposite flanks of the same WT mice. αB7-H1 slowed B16, but not B16/B7-H1lo, unless B16 was on the other flank, suggesting spread of anti-tumor immunity, helping explain variable αB7-H1 effects by tumor B7-H1 status. We found EL4 T cell lymphoma is CD80loB7-H1loPD-1hi. αB7-H1 and αPD-1 did not slow SQ EL4. Unexpectedly, EL4/B7-H1hi exhibited significant growth delay after SQ or intravenous challenge in WT. EL4/B7-H1hi grew well in vitro, but in clusters as opposed to EL4. αPD-1 or αB7-H1 in vitro blocked clusters but such pre-treated cells still grew slowly in vivo suggesting negative inter-cell B7-H1/PD-1 signals. Our data formally show B7-H1/PD-1 signals in αB7-H1 treatment effects, help explain variable αB7-H1 effects by tumor B7-H1 status, and suggest novel and specific effects in T cell lymphomas requiring more study.

Virus-induced dormancy in the archaeon Sulfolobus islandicus.

ABSTRACTWe investigated the interaction between Sulfolobus spindle-shaped virus (SSV9) and its native archaeal host Sulfolobus islandicus. We show that upon exposure to SSV9, S. islandicus strain RJW002 has a significant growth delay where the majority of cells are dormant (viable but not growing) for 24 to 48 hours postinfection (hpi) compared to the growth of controls without virus. We demonstrate that in this system, dormancy (i) is induced by both active and inactive virus particles at a low multiplicity of infection (MOI), (ii) is reversible in strains with active CRISPR-Cas immunity that prevents the establishment of productive infections, and (iii) results in dramatic and rapid host death if virus persists in the culture even at low levels. Our results add a new dimension to evolutionary models of virus-host interactions, showing that the mere presence of a virus induces host cell stasis and death independent of infection. This novel, highly sensitive, and risky bet-hedging antiviral response must be integrated into models of virus-host interactions in this system so that the true ecological impact of viruses can be predicted and understood.

HSPH1 inhibition downregulates Bcl-6 and c-Myc and hampers the growth of human aggressive B-cell non-Hodgkin lymphoma

AbstractWe have shown that human B-cell non-Hodgkin lymphomas (B-NHLs) express heat shock protein (HSP)H1/105 in function of their aggressiveness. Here, we now clarify its role as a functional B-NHL target by testing the hypothesis that it promotes the stabilization of key lymphoma oncoproteins. HSPH1 silencing in 4 models of aggressive B-NHLs was paralleled by Bcl-6 and c-Myc downregulation. In vitro and in vivo analysis of HSPH1-silenced Namalwa cells showed that this effect was associated with a significant growth delay and the loss of tumorigenicity when 104 cells were injected into mice. Interestingly, we found that HSPH1 physically interacts with c-Myc and Bcl-6 in both Namalwa cells and primary aggressive B-NHLs. Accordingly, expression of HSPH1 and either c-Myc or Bcl-6 positively correlated in these diseases. Our study indicates that HSPH1 concurrently favors the expression of 2 key lymphoma oncoproteins, thus confirming its candidacy as a valuable therapeutic target of aggressive B-NHLs.

Evaluation of Nutritional Status in a Teaching Hospital Neonatal Intensive Care Unit

Background:Extrauterine growth restriction remains a common and serious problem in newborns especially who are small, immature, and critically ill. Very low birth weight infants (VLBW) had 97% and 40% growth failure at 36 weeks and 18-22 months post-conceptual age respectively. The postnatal development of premature infants is critically dependent on an adequate nutritional intake that mimics a similar gestational stage. Deficient protein or amino acid administration over an extended period may cause significant growth delay or morbidity in VLBW infants. The purpose of this study was to evaluate current nutritional status in the neonatal intensive care unit in a teaching hospital.Methods:During this prospective observational study, the nutritional status of 100 consecutive critically ill neonates were evaluated by anthropometric and biochemical parameters in a tertiary neonatal intensive care unit. Their demographic characteristics (weight, height and head circumference), energy source (dextrose and lipid) and protein were recorded in the first, 5th, 10th, 15th and 20th days of admission and blood samples were obtained to measure serum albumin and prealbumin. The amount of calorie and protein were calculated for all of preterm and term neonates and compared to standard means separately. Results: The calorie and amino acids did not meet in the majority of the preterm and term neonates and mean daily parenteral calorie intake was 30% or lower than daily requirements based on neonates’ weight. Mortality rate was significantly higher in neonates with lower serum albumin and severity of malnutrition but not with serum prealbumin concentration. Conclusion: Infants were studied did not receive their whole of daily calorie and protein requirements and it is recommended early and enough administration of calorie source (dextrose, lipids) and amino acids. Prealbumin was a more benefit biochemical parameter than albumin to evaluate short term nutrition especially in critically ill patients.

Superior Antibacterial Activity of Photochemical Synthesized Ag-CNT Composites and their Synergistic Effects in Combination with other Antimicrobial Agents

Silver nanoparticle-modified multiwalled carbon nanotubes (MWCNTs) nanocomposites (AgCNTs) were synthesized by photochemical reduction method, their antimicrobial effect on both Gram negative and Gram positive bacteria, using E. coli and Bacillus anthracis as model bacteria, respectively, were investigated. The results indicated that AgCNTs exhibited more potent antibacterial effects against both Gram negative and Gram positive bacterium compared to Ag nanoparticles (Ag NPs). The minimal inhibitory concentrations (MICs) of AgCNTs against E. coli cells (0.5 μg/mL) and B. anthracis cells (0.8 μg/mL) was 1/20 times and 1/17.5 of the MICs of Ag NPs against E. coli and B. anthracis, respectively. Further study on the antibacterial effect of combination treatment of AgCNTs with oxidizing antimicrobial agents (NaOCl or H2O2) to E. coli cells indicated a partial synergistic or a synergistic effect using the fractional inhibitory concentration (FIC) index test or isololograms. The combination treatment of AgCNTs with a natural peptide, nisin, also exhibited enhanced inhibitory effect on E. coli growth, as significant delays in growth of E. coli cells treated by the combination of 0.2 μg/mL AgCNTs and 4 or 8 μg/mL nisin was observed compared to AgCNTs alone nisin alone treatment. The synergistic or enhanced effect of the combination of AgCNTs with other antimicrobial relied on the combination of different action mechanisms in which AgCNTs played a role to damage cell membrane which allowed easier access for other small antimicrobial molecules to penetrate into cells. Such combination strategy could be broadly applicable to the improvement of existing antimicrobial methods or design/discover new effective antimicrobial agents/methods.