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Jonathan A Coulter,1 Suneil Jain,2 Karl T Butterworth,2 Laura Taggart,2 Glenn Dickson,2 Stephen J McMahon,3 Wendy Hyland,1 Mark F Muir,3 Coleman Trainor,2 Alan Hounsell,2,4 Joe M O'Sullivan,2,4 Giuseppe Schettino,2 Fred Currell,3 David G Hirst,1 Kevin M Prise21School of Pharmacy, McClay Research Centre, 2Centre for Cancer Research and Cell Biology, 3School of Mathematics and Physics, Queens University Belfast, 4Belfast Health and Social Care Trust, Belfast, IrelandBackground: This follow-up study aims to determine the physical parameters which govern the differential radiosensitization capacity of two tumor cell lines and one immortalized normal cell line to 1.9 nm gold nanoparticles. In addition to comparing the uptake potential, localization, and cytotoxicity of 1.9 nm gold nanoparticles, the current study also draws on comparisons between nanoparticle size and total nanoparticle uptake based on previously published data.Methods: We quantified gold nanoparticle uptake using atomic emission spectroscopy and imaged intracellular localization by transmission electron microscopy. Cell growth delay and clonogenic assays were used to determine cytotoxicity and radiosensitization potential, respectively. Mechanistic data were obtained by Western blot, flow cytometry, and assays for reactive oxygen species.Results: Gold nanoparticle uptake was preferentially observed in tumor cells, resulting in an increased expression of cleaved caspase proteins and an accumulation of cells in sub G1 phase. Despite this, gold nanoparticle cytotoxicity remained low, with immortalized normal cells exhibiting an LD50 concentration approximately 14 times higher than tumor cells. The surviving fraction for gold nanoparticle-treated cells at 3 Gy compared with that of untreated control cells indicated a strong dependence on cell type in respect to radiosensitization potential.Conclusion: Gold nanoparticles were most avidly endocytosed and localized within cytoplasmic vesicles during the first 6 hours of exposure. The lack of significant cytotoxicity in the absence of radiation, and the generation of gold nanoparticle-induced reactive oxygen species provide a potential mechanism for previously reported radiosensitization at megavoltage energies.Keywords: endocytosis, proliferation, reactive oxygen species, transmission electron microscopy

Gold nanoparticle radiosensitization represents a novel technique in enhancement of ionising radiation dose and its effect on biological systems. Variation between theoretical predictions and experimental measurement is significant enough that the mechanism leading to an increase in cell killing and DNA damage is still not clear. We present the first experimental results that take into account both the measured biodistribution of gold nanoparticles at the cellular level and the range of the product electrons responsible for energy deposition. Combining synchrotron-generated monoenergetic X-rays, intracellular gold particle imaging and DNA damage assays, has enabled a DNA damage model to be generated that includes the production of intermediate electrons. We can therefore show for the first time good agreement between the prediction of biological outcomes from both the Local Effect Model and a DNA damage model with experimentally observed cell killing and DNA damage induction via the combination of X-rays and GNPs. However, the requirement of two distinct models as indicated by this mechanistic study, one for short-term DNA damage and another for cell survival, indicates that, at least for nanoparticle enhancement, it is not safe to equate the lethal lesions invoked in the local effect model with DNA damage events.

Therapeutic inhibition of poly(ADP-ribose) polymerase (PARP), as monotherapy or to supplement the potencies of other agents, is a promising strategy in cancer treatment. We previously reported that the first PARP inhibitor to enter clinical trial, rucaparib (AG014699), induced vasodilation in vivo in xenografts, potentiating response to temozolomide. We now report that rucaparib inhibits the activity of the muscle contraction mediator myosin light chain kinase (MLCK) 10-fold more potently than its commercially available inhibitor ML-9. Moreover, rucaparib produces additive relaxation above the maximal degree achievable with ML-9, suggesting that MLCK inhibition is not solely responsible for dilation. Inhibition of nitric oxide synthesis using L-NMMA also failed to impact rucaparib's activity. Rucaparib contains the nicotinamide pharmacophore, suggesting it may inhibit other NAD+-dependent processes. NAD+ exerts P2 purinergic receptor-dependent inhibition of smooth muscle contraction. Indiscriminate blockade of the P2 purinergic receptors with suramin abrogated rucaparib-induced vasodilation in rat arterial tissue without affecting ML-9-evoked dilation, although the specific receptor subtypes responsible have not been unequivocally identified. Furthermore, dorsal window chamber and real time tumor vessel perfusion analyses in PARP-1-/- mice indicate a potential role for PARP in dilation of tumor-recruited vessels. Finally, rucaparib provoked relaxation in 70% of patient-derived tumor-associated vessels. These data provide tantalising evidence of the complexity of the mechanism underlying rucaparib-mediated vasodilation.

Background and Purpose: Gold nanoparticles (GNPs) are novel agents that have been shown to cause radiosensitisation in vitro and in vivo. Tumour hypoxia is associated with radiation resistance and reduced survival in cancer patients. The interaction of GNPs with cells in hypoxia is explored., Materials and Methods: GNP uptake, localization, toxicity and radiosensitisation were assessed in vitro under oxic and hypoxic conditions., Results: GNP cellular uptake was significantly lower under hypoxic than oxic conditions. A significant reduction in cell proliferation in hypoxic MDA-MB-231 breast cancer cells exposed to GNPs was observed. In these cells significant radiosensitisation occurred in normoxia and moderate hypoxia. However, in near anoxia no significant sensitisation occurred., Conclusions: GNP uptake occurred in hypoxic conditions, causing radiosensitisation in moderate, but not extreme hypoxia in a breast cancer cell line. These findings may be important for the development of GNPs for cancer therapy., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

The relationship between the biological activity of NO and its chemistry is complex. The objectives of this study were to investigate the influence of oxygen tension on the cytotoxicity of the NO• donor DETA/NO and to determine the effects of oxygen tension on the key RNS (reactive nitrogen species) responsible for any subsequent toxicity. The findings presented in this study indicate that the DETA/NO-mediated cytotoxic effects were enhanced under hypoxic conditions. Further investigations revealed that neither ONOO⁻ (peroxynitrite) nor nitroxyl was generated. Fluorimetric analysis in the presence of scavengers suggest for the first time that another RNS, dinitrogen trioxide may be responsible for the cytotoxicity with DETA/NO. Results showed destabilization of HIF (hypoxia inducible factor)-1α and depletion of GSH levels following the treatment with DETA/NO under hypoxia, which renders cells more susceptible to DETA/NO cytotoxicity, and could account for another mechanism of DETA/NO cytotoxicity under hypoxia. In addition, there was significant accumulation of nuclear p53, which showed that p53 itself might be a target for S-nitrosylation following the treatment with DETA/NO. Both the intrinsic apoptotic pathway and the Fas extrinsic apoptotic pathway were also activated. Finally, GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is another important S-nitrosylated protein that may possibly play a key role in DETA/NO-mediated apoptosis and cytotoxicity. Therefore this study elucidates further mechanisms of DETA/NO mediated cytotoxicity with respect to S-nitrosylation that is emerging as a key player in the signalling and detection of DETA/NO-modified proteins in the tumour microenvironment.

FK506 binding protein-like (FKBPL) and its peptide derivatives exert potent anti-angiogenic activity in vitro and in vivo and control tumour growth in xenograft models, when administered exogenously. However, the role of endogenous FKBPL in angiogenesis is not well characterised. Here we investigated the molecular effects of the endogenous protein and its peptide derivative, AD-01, leading to their anti-migratory activity. Inhibition of secreted FKBPL using a blocking antibody or siRNA-mediated knockdown of FKBPL accelerated the migration of human microvascular endothelial cells (HMEC-1). Furthermore, MDA-MB-231 tumour cells stably overexpressing FKBPL inhibited tumour vascular development in vivo suggesting that FKBPL secreted from tumour cells could inhibit angiogenesis. Whilst FKBPL and AD-01 target CD44, the nature of this interaction is not known and here we have further interrogated this aspect. We have demonstrated that FKBPL and AD-01 bind to the CD44 receptor and inhibit tumour cell migration in a CD44 dependant manner; CD44 knockdown abrogated AD-01 binding as well as its anti-migratory activity. Interestingly, FKBPL overexpression and knockdown or treatment with AD-01, regulated CD44 expression, suggesting a co-regulatory pathway for these two proteins. Downstream of CD44, alterations in the actin cytoskeleton, indicated by intense cortical actin staining and a lack of cell spreading and communication were observed following treatment with AD-01, explaining the anti-migratory phenotype. Concomitantly, AD-01 inhibited Rac-1 activity, up-regulated RhoA and the actin binding proteins, profilin and vinculin. Thus the anti-angiogenic protein, FKBPL, and AD-01, offer a promising and alternative approach for targeting both CD44 positive tumours and vasculature networks.

Gold nanoparticles are emerging as promising agents for cancer therapy and are being investigated as drug carriers, photothermal agents, contrast agents and radiosensitisers. This review introduces the field of nanotechnology with a focus on recent gold nanoparticle research which has led to early-phase clinical trials. In particular, the pre-clinical evidence for gold nanoparticles as sensitisers with ionising radiation in vitro and in vivo at kilovoltage and megavoltage energies is discussed.

PARP-1 is a critical enzyme in the repair of DNA strand breaks. Inhibition of PARP-1 increases the effectiveness of radiation in killing tumor cells. However, although the mechanism(s) are well understood for these radiosensitizing effects in vitro, the underlying mechanism(s) in vivo are less clear. Nicotinamide, a drug structurally related to the first generation PARP-1 inhibitor, 3-aminobenzamide, reduces tumor hypoxia by preventing transient cessations in tumor blood flow, thus improving tumor oxygenation and sensitivity to radiotherapy. Here, we investigate whether olaparib, a potent PARP-1 inhibitor, enhances radiotherapy, not only by inhibiting DNA repair but also by changing tumor vascular hemodynamics in non-small cell lung carcinoma (NSCLC). In irradiated Calu-6 and A549 cells, olaparib enhanced the cytotoxic effects of radiation (sensitizer enhancement ratio at 10% survival = 1.5 and 1.3) and DNA double-strand breaks persisted for at least 24 hours after treatment. Combination treatment of Calu-6 xenografts with olaparib and fractionated radiotherapy caused significant tumor regression (P = 0.007) relative to radiotherapy alone. To determine whether this radiosensitization was solely due to effects on DNA repair, we used a dorsal window chamber model to establish the drug/radiation effects on vessel dynamics. Olaparib alone, when given as single or multiple daily doses, or in combination with fractionated radiotherapy, increased the perfusion of tumor blood vessels. Furthermore, an ex vivo assay in phenylephrine preconstricted arteries confirmed olaparib to have higher vasodilatory properties than nicotinamide. This study suggests that olaparib warrants consideration for further development in combination with radiotherapy in clinical oncology settings such as NSCLC.

The role of endogenous nitric oxide (NO) in the growth and vascularization of a rat carcinosarcoma (P22) has been investigated. Tumor-bearing animals were treated with (i) nitric oxide synthase (NOS) inhibitors, administered via the drinking water, including N(G)-nitro-l-arginine methyl ester (L-NAME), a nonisoform-selective inhibitor, and 2 others that target the inducible (NOS II) enzyme preferentially, namely 1-amino-2-hydroxyguanidine or N-[3-(aminomethyl)benzyl]acetamidine hydrochloride; or (ii) daily injections (intraperitoneally) of 2 Ru(III) polyaminocarboxylates, AMD6221 and AMD6245, both of which are effective NO scavengers. L-NAME, AMD6221, and AMD6245 reduced tumor growth by approximately 60% to 75% of control rates. Tumor sections stained with abs to CD-31/platelet endothelial cell adhesion molecule-1 or NOS III showed that this was associated with a marked reduction (60%-77%) of tumor microvascular densitiy (MVD). Tumors resumed growing promptly when treatment was discontinued, accompanied by partial or complete restoration of MVDs. In contrast, NOS-II selective inhibitors had no effect on tumor growth or vascularization, indicating that both responses require complete blockade of NO production. The results corroborate the view that endogenous NO facilitates tumor development. We suggest that NO deprivation causes tumor feeder vessels to constrict, reducing tumor blood flow. The delivery of oxygen and essential nutrients to the developing tumor is impaired as a consequence, hampering further growth. Normalizing NO levels by withholding treatment causes tumor feeder vessels to dilate, increasing tumor perfusion and reestablishing conditions that allow tumors to begin growing again.

Background and Purpose: The addition of gold nanoparticles (GNPs) to tumours leads to an increase in dose due to their high density and energy absorption coefficient, making it a potential radiosensitiser. However, experiments have observed radiosensitisations significantly larger than the increase in dose alone, including at megavoltage energies where gold's relative energy absorption is lowest. This work investigates whether GNPs create dose inhomogeneities on a sub-cellular scale which combine with non-linear dose dependence of cell survival to be the source of radiosensitisation at megavoltage energies., Materials and Methods: Monte Carlo simulations were carried out to calculate dose in the vicinity of a single GNP on the nanoscale. The effect of this nanoscale dose distribution was then modelled for MDA-MB-231 cells exposed to 2 nm GNPs, and compared to experimental results., Results: Dramatic dose inhomogeneities occur around GNPs exposed to megavoltage radiation. When analysed using the Local Effect Model, these inhomogeneities lead to significant radiosensitisation, in agreement with experimental results., Conclusions: This work suggests that GNP radiosensitisation is driven by inhomogeneities in dose on the nanoscale, rather than changes in dose over the entire cell, which may contribute to the similar radiosensitisation observed in megavoltage and kilovoltage experiments. The short range of these inhomogeneities and the variation in enhancement in different cells suggests sub-cellular localisation is important in determining GNP radiosensitisation., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Bone marrow mesenchymal stem cells (BMSCs) represent an important source of cells for tissue repair. The tropism of these cells to the sites of injury and tumors has been well established. Their tumor-homing properties make BMSCs good candidates as antitumor agent delivery vehicles. In this study, we showed that BMSCs have the ability to migrate toward various cancer cells, including prostate cancer cells in vitro and in vivo and incorporating into the tumor mass. When infected with herpes simplex virus thymidine kinase (TK) gene by lentiviral transduction, TK-BMSCs maintained their tumor tropism capabilities and significantly inhibited the growth of subcutaneous PC3 prostate cancer xenografts in nude mice, in the presence of prodrug ganciclovir (GCV). Xenogenic TK-BMSCs also survived and exerted a significant antitumor effect in an animal model bearing metastastic RIF-1 (fibrosarcoma) tumor in the presence of prodrug GCV. The present study demonstrated that overexpression of TK in BMSCs did not affect their multidifferentiation potentials and their specific homing capacities toward the tumor mass, and the TK-BMSCs alone did not cause any harmful side effects in vivo. The use of TK-BMSCs as tumor-specific delivery vehicles together with controlled prodrug treatment may be a safe and novel anticancer therapy approach.

Purpose: Antiangiogenic therapies can be an important adjunct to the management of many malignancies. Here we investigated a novel protein, FKBPL, and peptide derivative for their antiangiogenic activity and mechanism of action., Experimental Design: Recombinant FKBPL (rFKBPL) and its peptide derivative were assessed in a range of human microvascular endothelial cell (HMEC-1) assays in vitro. Their ability to inhibit proliferation, migration, and Matrigel-dependent tubule formation was determined. They were further evaluated in an ex vivo rat model of neovascularization and in two in vivo mouse models of angiogenesis, that is, the sponge implantation and the intravital microscopy models. Antitumor efficacy was determined in two human tumor xenograft models grown in severe compromised immunodeficient (SCID) mice. Finally, the dependence of peptide on CD44 was determined using a CD44-targeted siRNA approach or in cell lines of differing CD44 status., Results: rFKBPL inhibited endothelial cell migration, tubule formation, and microvessel formation in vitro and in vivo. The region responsible for FKBPL's antiangiogenic activity was identified, and a 24-amino acid peptide (AD-01) spanning this sequence was synthesized. It was potently antiangiogenic and inhibited growth in two human tumor xenograft models (DU145 and MDA-231) when administered systemically, either on its own or in combination with docetaxel. The antiangiogenic activity of FKBPL and AD-01 was dependent on the cell-surface receptor CD44, and signaling downstream of this receptor promoted an antimigratory phenotype., Conclusion: FKBPL and its peptide derivative AD-01 have potent antiangiogenic activity. Thus, these agents offer the potential of an attractive new approach to antiangiogenic therapy., (©2011 AACR.)

The present study determines whether the novel designer biomimetic vector (DBV) can condense and deliver the cytotoxic iNOS gene to breast cancer cells to achieve a therapeutic effect. We have previously shown the benefits of iNOS for cancer gene therapy but the stumbling block to future development has been the delivery system. The DBV was expressed, purified and complexed with the iNOS gene. The particle size and charge were determined via dynamic light scattering techniques. The toxicity of the DBV/iNOS nanoparticles was quantified using the cell toxicity and clonogenic assays. Over expression of iNOS was confirmed via Western blotting and Griess test. The DBV delivery system fully condensed the iNOS gene with nanoparticles less than 100nm. Transfection with the DBV/iNOS nanoparticles resulted in a maximum of 62% cell killing and less than 20% clonogenicity. INOS overexpression was confirmed and total nitrite levels were in the range of 18μM. We report for the first time that the DBV can successfully deliver iNOS and achieve a therapeutic effect. There is significant cytotoxicity coupled with evidence of a bystander effect. We conclude that the success of the DBV fusion protein in the delivery of iNOS in vitro is worthy of future in vivo experiments., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Purpose: Gold nanoparticles (GNPs) have been shown to cause sensitization with kilovoltage (kV) radiation. Differences in the absorption coefficient between gold and soft tissue, as a function of photon energy, predict that maximum enhancement should occur in the kilovoltage (kV) range, with almost no enhancement at megavoltage (MV) energies. Recent studies have shown that GNPs are not biologically inert, causing oxidative stress and even cell death, suggesting a possible biological mechanism for sensitization. The purpose of this study was to assess GNP radiosensitization at clinically relevant MV X-ray energies., Methods and Materials: Cellular uptake, intracellular localization, and cytotoxicity of GNPs were assessed in normal L132, prostate cancer DU145, and breast cancer MDA-MB-231 cells. Radiosensitization was measured by clonogenic survival at kV and MV photon energies and MV electron energies. Intracellular DNA double-strand break (DSB) induction and DNA repair were determined and GNP chemosensitization was assessed using the radiomimetic agent bleomycin., Results: GNP uptake occurred in all cell lines and was greatest in MDA-MB-231 cells with nanoparticles accumulating in cytoplasmic lysosomes. In MDA-MB-231 cells, radiation sensitizer enhancement ratios (SERs) of 1.41, 1.29, and 1.16 were achieved using 160 kVp, 6 MV, and 15 MV X-ray energies, respectively. No significant effect was observed in L132 or DU145 cells at kV or MV energies (SER 0.97-1.08). GNP exposure did not increase radiation-induced DSB formation or inhibit DNA repair; however, GNP chemosensitization was observed in MDA-MB-231 cells treated with bleomycin (SER 1.38)., Conclusions: We have demonstrated radiosensitization in MDA-MB-231 cells at MV X-ray energies. The sensitization was cell-specific with comparable effects at kV and MV energies, no increase in DSB formation, and GNP chemopotentiation with bleomycin, suggesting a possible biological mechanism of radiosensitization., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Gold nanoparticles (GNPs) are being proposed as contrast agents to enhance X-ray imaging and radiotherapy, seeking to take advantage of the increased X-ray absorption of gold compared to soft tissue. However, there is a great discrepancy between physically predicted increases in X-ray energy deposition and experimentally observed increases in cell killing. In this work, we present the first calculations which take into account the structure of energy deposition in the nanoscale vicinity of GNPs and relate this to biological outcomes, and show for the first time good agreement with experimentally observed cell killing by the combination of X-rays and GNPs. These results are not only relevant to radiotherapy, but also have implications for applications of heavy atom nanoparticles in biological settings or where human exposure is possible because the localised energy deposition high-lighted by these results may cause complex DNA damage, leading to mutation and carcinogenesis.

Nitric oxide (NO) is just one member of a new class of gaseous signalling molecules with fundamental actions in biology. In higher vertebrates it has key roles in maintaining haemostasis and in smooth muscle (especially vascular smooth muscle), neurons and the gastrointestinal tract. It is intimately involved in regulating all aspects of our lives from waking, digestion, sexual function, perception of pain and pleasure, memory recall and sleeping. Finally, the way it continues to function in our bodies will influence how we degenerate with age. It will likely play a role in our deaths through cardiovascular disease, stroke, diabetes and cancer. Our ability to control NO signalling and to use NO effectively in therapy must therefore have a major bearing on the future quality and duration of human life.

Background: Using the tumour type specific human osteocalcin (hOC) promoter, we have previously reported strong promoter activation in hormone independent prostate cancer cells in vitro. In the present study, we present a comparative study of the tissue specific promoter prostate specific membrane antigen (PSMA), and the tumour-type specific hOC promoter driving the inducible nitric oxide synthase (iNOS) transgene using both in vitro and in vivo models., Methods: In vitro cytotoxicity was assessed by clonogenic assay. Quantification of nitric oxide expression was determined by the Griess test. In vivo anti-tumour efficacy was determined by tumour growth delay following direct intra-tumoural injection of the constructs into PC3 xenografts. In addition, tumours were dissected post mortem and examined for morphological differences as well as changes in apoptotic protein expression., Results: PSMA/iNOS produced cytotoxicity in both androgen dependant and independent cell lines. Nitric oxide quantification confirmed that increased cytotoxicity was directly associated with nitric oxide production. Tumour growth delays were observed in all groups treated with the iNOS-expressing constructs ranging from 10.7 days for the hOC/iNOS single dose treatment group to a maximum of 52.2 days for the hOC/iNOS multiple dose group. Intra-tumoural assessment of iNOS and cleaved poly (ADP-ribose) polymerase protein expression demonstrated a significant up-regulation of both proteins, indicating cytotoxicity mediated through the intrinsic apoptotic pathway., Conclusions: Highly significant tumour growth delay coupled with no detrimental side-effects were observed following treatment with the PSMA/iNOS and hOC/iNOS constructs. We consider that these findings provide a basis for the development of systemically delivered PSMA/iNOS or hOC/iNOS targeting early stage and advanced prostate cancer.

High atomic number (Z) materials such as gold preferentially absorb kilovoltage x-rays compared to soft tissue and may be used to achieve local dose enhancement in tumours during treatment with ionizing radiation. Gold nanoparticles have been demonstrated as radiation dose enhancing agents in vivo and in vitro. In the present study, we used multiple endpoints to characterize the cellular cytotoxic response of a range of cell lines to 1.9 nm gold particles and measured dose modifying effects following transient exposure at low concentrations. Gold nanoparticles caused significant levels of cell type specific cytotoxicity, apoptosis and increased oxidative stress. When used as dose modifying agents, dose enhancement factors varied between the cell lines investigated with the highest enhancement being 1.9 in AGO-1522B cells at a nanoparticle concentration of 100 microg ml(-1). This study shows exposure to 1.9 nm gold particles to induce a range of cell line specific responses including decreased clonogenic survival, increased apoptosis and induction of DNA damage which may be mediated through the production of reactive oxygen species. This is the first study involving 1.9 nm nanometre sized particles to report multiple cellular responses which impact on the radiation dose modifying effect. The findings highlight the need for extensive characterization of responses to gold nanoparticles when assessing dose enhancing potential in cancer therapy.

The HSP90 chaperone and immunophilin FKBPL is an estrogen-responsive gene that interacts with estogen receptor alpha (ERalpha) and regulates its levels. In this study, we explored the effects of FKBPL on breast cancer proliferation. Breast cancer cells stably overexpressing FKBPL became dependent on estrogen for their growth and were dramatically more sensitive to the antiestrogens tamoxifen and fulvestrant, whereas FKBPL knockdown reverses this phenotype. FKBPL knockdown also decreased the levels of the cell cycle inhibitor p21WAF1 and increased ERalpha phosphorylation on Ser(118) in response to 17beta-estradiol and tamoxifen. In support of the likelihood that these effects explained FKBPL-mediated cell growth inhibition and sensitivity to endocrine therapies, FKBPL expression was correlated with increased overall survival and distant metastasis-free survival in breast cancer patients. Our findings suggest that FKBPL may have prognostic value based on its impact on tumor proliferative capacity and sensitivity to endocrine therapies, which improve outcome.

Nitric oxide (NO) is now recognised as one of the most important molecules influencing the development, progression and treatment of cancer. A key component of its action is as a negative and positive regulator of apoptosis. Broadly, constitutive levels of NO(nM), are capable of inhibiting numerous signalling pathways in both normal and cancer cells. These include soluble guanylate cyclase, leading to reduced Ca++ signalling, inhibition of caspases and scavenging of reactive oxygen species, all of which promote survival signalling. High concentrations (M-mM) on the other hand, generally promote apoptosis. Pathways involving cGMP, cytochrome c release, mitogen activated kinases, ceramide and poly(ADP)ribose polymerase have all been implicated. The role of p53 in NO-induced cell death has been widely studied. In many cell types p53-dependent signalling is involved, while in others, apoptosis occurs in the absence of functional p53. There is also evidence that the tumour microenvironment, where low oxygen and glucose levels prevail, enhances cell death signalling by NO and peroxynitrite, thus tumours may be more sensitive to high levels of NO than their normal tissue counterpart. The cytotoxicity of NO has been studied directly in many tumour models, both in vitro and in vivo. In all cases, high concentrations of NO, generated by donor drugs or by iNOS gene transfer caused extensive tumour cell death, which was enhanced by the ability of NO to diffuse readily from its source of generation to most cells within tumours. NO was also a very effective enhancer of conventional chemo- and radiotherapy. Thus, NO therapy has great potential to improve the treatment of cancer.

Purpose: Poly(ADP-ribose) polymerase (PARP) plays an important role in DNA repair, and PARP inhibitors can enhance the activity of DNA-damaging agents in vitro and in vivo. AG014699 is a potent PARP inhibitor in phase II clinical development. However, the range of therapeutics with which AG014699 could interact via a DNA-repair based mechanism is limited. We aimed to investigate a novel, vascular-based activity of AG014699, underlying in vivo chemosensitization, which could widen its clinical application., Experimental Design: Temozolomide response was analyzed in vitro and in vivo. Vessel dynamics were monitored using "mismatch" following the administration of perfusion markers and real-time analysis of fluorescently labeled albumin uptake in to tumors established in dorsal window chambers. Further mechanistic investigations used ex vivo assays of vascular smooth muscle relaxation, gut motility, and myosin light chain kinase (MLCK) inhibition., Results: AG014699 failed to sensitize SW620 cells to temozolomide in vitro but induced pronounced enhancement in vivo. AG014699 (1 mg/kg) improved tumor perfusion comparably with the control agents nicotinamide (1 g/kg) and AG14361 (forerunner to AG014699; 10 mg/kg). AG014699 and AG14361 relaxed preconstricted vascular smooth muscle more potently than the standard agent, hydralazine, with no impact on gut motility. AG014699 inhibited MLCK at concentrations that relaxed isolated arteries, whereas AG14361 had no effect., Conclusion: Increased vessel perfusion elicited by AG014699 could increase tumor drug accumulation and therapeutic response. Vasoactive concentrations of AG014699 do not cause detrimental side effects to gut motility and may increase the range of therapeutics with which AG014699 could be combined with for clinical benefit.

The results of an investigation into the damage caused to dry plasmid DNA after irradiation by fast (keV) hydrogen atoms are presented. Agarose gel electrophoresis was used to assess single and double strand break yields as a function of dose in dry DNA samples deposited on a mica substrate. Damage levels were observed to increase with beam energy. Strand break yields demonstrated a considerable dependence on sample structure and the method of sample preparation. Additionally, the effect of high-Z nanoparticles on damage levels was investigated by irradiating DNA samples containing controlled amounts of gold nanoparticles. In contrast to previous (photonic) studies, no enhancement of strand break yields was observed with the particles showing a slight radioprotective effect. A model of DNA damage as a function of dose has been constructed in terms of the probability for the creation of single and double strand breaks, per unit ion flux. This model provides quantitative conclusions about the effects of both gold nanoparticles and the different buffers used in performing the assays and, in addition, infers the proportion of multiply damaged fragments.

Background: Nitric oxide (NO.) derived from donor drugs has been shown to be an effective chemosensitizer in vitro. We investigated the combination of inducible nitric oxide synthase (iNOS) gene transfer, driven by a strong constitutive promoter (cytomegalovirus; CMV) with the DNA cross-linking agent cisplatin in mouse and human tumour cell lines., Methods: Proof of principal experiments were performed in the radiation-induced fibrosarcoma-1 (RIF-1) murine cell line. Cells were transfected with constitutively expressed CMV/iNOS plasmid DNA using a cationic lipid vector, before exposure to cisplatin. In vivo efficacy was determined in an intradermal RIF-1 tumour model, with intraperitoneal administration of cisplatin. Additionally, treatment potential was investigated in various human tumour cell lines including human prostate (DU145 and PC3) and human colon (HT29 and HCT116) cancer cell lines. Experimental endpoints were established using western blot, Greiss test, clonogenic assay and tumour growth delay., Results: Transfection of RIF-1 tumour cells in vitro with the CMV/iNOS significantly enhanced the cytotoxicity of cisplatin (0.2-1.0 microM). In vivo transfer of CMV/iNOS by direct injection into established RIF-1 tumours caused a significant (p = 0.0027) delay in tumour growth. CMV/iNOS gene transfer in vitro resulted in the strong expression of iNOS DNA in all cell lines, and significantly increased levels of NO. in all cell lines except HCT116., Conclusions: Significant chemosensitization of cisplatin cytotoxicity was observed in the presence of NO. derived from the overexpression iNOS. We conclude that p53 status of the various cell lines was unlikely to be responsible for cisplatin-induced apoptosis., (Copyright (c) 2008 John Wiley & Sons, Ltd.)

Cytochrome P450 1B1 (CYP1B1) mRNA is constitutively expressed in most normal extra-hepatic tissues; however the protein is not detectable in these tissues but is expressed in a wide variety of tumors. CYP1B1 is responsible for the activation of a number of carcinogens present in tobacco smoke and food. A surgical model of rat esophageal tumorigenesis, promoted by gastric or duodenal reflux was used to determine CYP1B1 expression in premalignant esophageal tissue. Immunohistochemistry was performed using a modified amplified fluorescein tyramide protocol. CYP1B1 was not observed in normal esophageal mucosa, submucosa, or muscularis mucosa. Animals exposed to gastric reflux developed mild hyperplasia. Varying degrees of hyperplasia were observed in the duodenal reflux group. All regions of hyperplasia showed moderate or strong CYP1B1 immunoreactivity. Duodenal reflux induced a small number of premalignant changes: immunoreactivity was absent from the epithelium of squamous dysplasia (0/10), Barrett's esophagus (0/7), and majority of dysplastic Barrett's esophagus (1/4). Moderate or strong immunoreactivity was observed in the majority (7/8) of squamous cell carcinomas (SCCs) in situ. Immunoreactivity was also observed in the lamina propria and submucosa in association with inflammation, regardless of the severity of inflammation. The expression of CYP1B1 in hyperplasia, SCCs in situ, or in association with inflammation may increase the production of carcinogenic metabolites, which may promote esophageal tumorigenesis., (Copyright 2008 Wiley-Liss, Inc.)

Background Aims: Cell-based gene therapy is an alternative to viral and non-viral gene therapy. Emerging evidence suggests that mesenchymal stem cells (MSC) are able to migrate to sites of tissue injury and have immunosuppressive properties that may be useful in targeted gene therapy for sustained specific tissue engraftment., Methods: In this study, we injected intravenously (i.v.) 1x10(6) MSC, isolated from green fluorescent protein (GFP) transgenic rats, into Rif-1 fibrosarcoma-bearing C3H/HeN mice. The MSC had been infected using a lentiviral vector to express stably the luciferase reporter gene (MSC-GFP-luci). An in vivo imaging system (IVIS 200) and Western blotting techniques were used to detect the distribution of MSC-GFP-luci in tumor-bearing animals., Results: We observed that xenogenic MSC selectively migrated to the tumor site, proliferated and expressed the exogenous gene in subcutaneous fibrosarcoma transplants. No MSC distribution was detected in other organs, such as the liver, spleen, colon and kidney. We further showed that the FGF2/FGFR pathways may play a role in the directional movement of MSC to the Rif-1 fibrosarcoma. We performed in vitro co-culture and in vivo tumor growth analysis, showing that MSC did not affect the proliferation of Rif-1 cells and fibrosarcoma growth compared with an untreated control group. Finally, we demonstrated that the xenogenic MSC stably expressing inducible nitric oxide synthase (iNOS) protein transferred by a lentivirus-based system had a significant inhibitory effect on the growth of Rif-1 tumors compared with MSC alone and the non-treatment control group., Conclusions: iNOS delivered by genetically modified iNOS-MSC showed a significant anti-tumor effect both in vitro and in vivo. MSC may be used as a target gene delivery vehicle for the treatment of fibrosarcoma and other tumors.

FKBP-like (FKBPL) protein is a novel immunophilin-like protein that plays a role in the cellular stress response. Its three tetratricopeptide repeat motifs are homologous to the heat shock protein 90 interaction sites of other immunophilins that have roles in steroid hormone receptor signaling. In this study, using biomolecular complementation and coimmunoprecipitation techniques, we show that FKBPL also colocalizes and interacts with the components of the heat shock protein 90-glucocorticoid receptor (GR) complex and demonstrate that the PPIase domain of FKBPL is important for the interaction between this complex and the dynein motor protein, dynamitin. Treatment of DU145 cells with the GR ligand, dexamethasone, induced a rapid and coordinated translocation of both GR and FKBPL to the nucleus; this response was perturbed when FKBPL was knocked down with a targeted small interfering RNA. Furthermore, overexpression of FKBPL increased GR protein levels and transactivation of a luciferase reporter gene in response to dexamethasone in DU145 cells. However, these responses were cell line dependent. In summary, these data suggest that FKBPL can be classed as a new member of the FKBP protein family with a role in steroid receptor complexes and signaling.

Background: Radiation therapy is a treatment modality routinely used in cancer management so it is not unexpected that radiation-inducible promoters have emerged as an attractive tool for controlled gene therapy. The human tissue plasminogen activator gene promoter (t-PA) has been proposed as a candidate for radiogenic gene therapy, but has not been exploited to date. The purpose of this study was to evaluate the potential of this promoter to drive the expression of a reporter gene, the green fluorescent protein (GFP), in response to radiation exposure., Methods: To investigate whether the promoter could be used for prostate cancer gene therapy, we initially transfected normal and malignant prostate cells. We then transfected HMEC-1 endothelial cells and ex vivo rat tail artery and monitored GFP levels using Western blotting following the delivery of single doses of ionizing radiation (2, 4, 6 Gy) to test whether the promoter could be used for vascular targeted gene therapy., Results: The t-PA promoter induced GFP expression up to 6-fold in all cell types tested in response to radiation doses within the clinical range., Conclusions: These results suggest that the t-PA promoter may be incorporated into gene therapy strategies driving therapeutic transgenes in conjunction with radiation therapy., ((c) 2008 John Wiley & Sons, Ltd.)

Much research over the past two decades has focussed on understanding the complex interactions of nitric oxide (NO(.)) in both physiological and pathological processes. As with many other aspects of NO(.) biology, its precise role in tumour pathophysiology has been the cause of intense debate and we now know that it participates in numerous signalling pathways that are crucial to the malignant character of cancer. The available experimental evidence highlights contrasting pro- and anti-tumour effects of NO(.) expression, which appear to be reconciled by consideration of the concentrations involved. This review addresses the complexities of the role of NO(.) in cancer, whilst evaluating various experimental approaches to NO(.)-based cancer therapies, including both inhibition of nitric oxide synthases, and overexpression of NO(.) using donor drugs or nitric oxide synthase gene transfer. The evidence provided strongly supports a role for manipulation of tumour NO(.) either as a stand-alone therapy or in combination with conventional treatments to achieve a significant therapeutic gain.