G-One Ahn

Inhibition of Mac-1 (CD11b/CD18) enhances tumor response to radiation by reducing myeloid cell recruitment

Despite recent advances in radiotherapy, loco-regional failures are still the leading cause of death in many cancer patients. We have previously reported that bone marrow-derived CD11b+ myeloid cells are recruited to tumors grown in irradiated tissues, thereby restoring the vasculature and tumor growth. In this study, we examined whether neutralizing CD11b monoclonal antibodies could inhibit the recruitment of myeloid cells into irradiated tumors and inhibit their regrowth. We observed a significant enhancement of antitumor response to radiation in squamous cell carcinoma xenografts in mice when CD11b antibodies are administered systemically. Histological examination of tumors revealed that CD11b antibodies reduced infiltration of myeloid cells expressing S100A8 and matrix metalloproteinase-9. CD11b antibodies further inhibited bone marrow-derived cell adhesion and transmigration to C166 endothelial cell monolayers and chemotactic stimuli, respectively, to levels comparable to those from CD11b knockout or CD18 hypomorphic mice. Given the clinical availability of humanized CD18 antibodies, we tested two murine tumor models in CD18 hypomorphic or CD11b knockout mice and found that tumors were more sensitive to irradiation when grown in CD18 hypomorphic mice but not in CD11b knockout mice. When CD18 hypomorphism was partially rescued by reconstitution with the wild-type bone marrow, the resistance of the tumors to irradiation was restored. Our study thus supports the rationale of using clinically available Mac-1 (CD11b/CD18) antibodies as an adjuvant therapy to radiotherapy.

Loss of CDKN2B Promotes p53-Dependent Smooth Muscle Cell Apoptosis and Aneurysm Formation

Objective—Genomewide association studies have implicated allelic variation at 9p21.3 in multiple forms of vascular disease, including atherosclerotic coronary heart disease and abdominal aortic aneurysm. As for other genes at 9p21.3, human expression quantitative trait locus studies have associated expression of the tumor suppressor gene CDKN2B with the risk haplotype, but its potential role in vascular pathobiology remains unclear. Methods and Results—Here we used vascular injury models and found that Cdkn2b knockout mice displayed the expected increase in proliferation after injury, but developed reduced neointimal lesions and larger aortic aneurysms. In situ and in vitro studies suggested that these effects were attributable to increased smooth muscle cell apoptosis. Adoptive bone marrow transplant studies confirmed that the observed effects of Cdkn2b were mediated through intrinsic vascular cells and were not dependent on bone marrow–derived inflammatory cells. Mechanistic studies suggested that the observed increase in apoptosis was attributable to a reduction in MDM2 and an increase in p53 signaling, possibly due in part to compensation by other genes at the 9p21.3 locus. Dual inhibition of both Cdkn2b and p53 led to a reversal of the vascular phenotype in each model. Conclusion—These results suggest that reduced CDKN2B expression and increased smooth muscle cell apoptosis may be one mechanism underlying the 9p21.3 association with aneurysmal disease.

Optimized Clostridium-Directed Enzyme Prodrug Therapy Improves the Antitumor Activity of the Novel DNA Cross-Linking Agent PR-104

We have previously shown that spores of the nonpathogenic clostridial strain C. sporogenes genetically engineered to express the E. coli-derived cytosine deaminase gene are effective in converting systemically injected nontoxic 5-fluorocytosine into the toxic anticancer drug 5-fluorouracil, thereby producing tumor-specific antitumor activity. To improve the expression of E. coli-derived genes with this system, we first replaced the original fdP promoter in the vector with one of two powerful endogenous clostridial promoters: that of the thiolase gene (thlP) and that for the clostridial transcription factor abrB310 (abrBP). These substitutions improved protein expression levels of the prodrug-activating genes by 2- to 3-fold in comparison with fdP-driven expression. However, despite these strong promoters, we found much higher expression of the nitroreductase (NTR) protein in the E. coli host compared with the clostridial host, which we hypothesized could be the result of different codon use between the two organisms. To test this, we constructed new expression vectors with an artificially synthesized NTR gene using optimized clostridial codons (sNTR). Results from both enzymatic assays and Western blots of cell extracts from clostridial transformants harboring plasmid constructs of thlP-sNTR and abrBP-sNTR showed that the expression and activity of the NTR gene product was increased by approximately 20-fold compared with the original construct. In vivo studies with i.v. administered sNTR-expressing C. sporogenes spores in SiHa tumor-bearing mice showed significantly improved antitumor efficacy when combined with either 5-aziridinyl-2,4-dinitrobenzamide (CB1954) or the novel dinitrobenzamide mustard prodrug, PR-104.

Transcriptional activation of hypoxia-inducible factor-1 (HIF-1) in myeloid cells promotes angiogenesis through VEGF and S100A8.

Significance Here, we are reporting our findings that hypoxia-inducible factor 1 (HIF-1) activation in monocytes promotes neovascularization in matrigel and improves blood flow in hindlimb ischemia through production of vascular endothelial growth factor and S100A8. We found that HIF-1 regulates S100A8 expression specifically in monocytes isolated from our unique strain of transgenic mice targeting HIF pathways. Emerging evidence indicates that myeloid cells are essential for promoting new blood vessel formation by secreting various angiogenic factors. Given that hypoxia-inducible factor (HIF) is a critical regulator for angiogenesis, we questioned whether HIF in myeloid cells also plays a role in promoting angiogenesis. To address this question, we generated a unique strain of myeloid-specific knockout mice targeting HIF pathways using human S100A8 as a myeloid-specific promoter. We observed that mutant mice where HIF-1 is transcriptionally activated in myeloid cells (by deletion of the von Hippel–Lindau gene) resulted in erythema, enhanced neovascularization in matrigel plugs, and increased production of vascular endothelial growth factor (VEGF) in the bone marrow, all of which were completely abrogated by either genetic or pharmacological inactivation of HIF-1. We further found that monocytes were the major effector producing VEGF and S100A8 proteins driving neovascularization in matrigel. Moreover, by using a mouse model of hindlimb ischemia we observed significantly improved blood flow in mice intramuscularly injected with HIF-1–activated monocytes. This study therefore demonstrates that HIF-1 activation in myeloid cells promotes angiogenesis through VEGF and S100A8 and that this may become an attractive therapeutic strategy to treat diseases with vascular defects.

Synthesis and Evaluation of Stable Bidentate Transition Metal Complexes of 1-(Chloromethyl)-5-hydroxy-3-(5,6,7-trimethoxyindol-2-ylcarbonyl)-2,3-dihydro-1H-pyrrolo[3,2-f]quinoline (seco-6-azaCBI-TMI) as Hypoxia Selective Cytotoxins

A series of metal complexes were prepared as potential prodrugs of the extremely toxic DNA minor groove alkylator 1-(chloromethyl)-5-hydroxy-3-[(5,6,7-trimethoxyindol-2-yl)carbonyl]-2,3-dihydro-1H-pyrrolo[3,2-f]quinoline (seco-6-azaCBI-TMI) and close analogues. The pyrrolo[3,2-f]quinoline cytotoxins were prepared from 2-methoxy-4-nitroaniline in a nine-step synthesis involving a Skraup construction of a quinoline intermediate, its appropriate functionalization, and a final radical cyclization. The metal complexes were prepared from these and the labile metal complex synthons [Co(cyclen)(OTf)(2)](+), [Cr(acac)(2)(H(2)O)(2)](+), and [Co(2)(Me(2)dtc)(5)](+). The cobalt complexes were considerably more stable than the free effectors and showed significant attenuation of the cytotoxicity of the latter, with IC(50) ratios (complex/effector) of 50- to 150-fold, and substantial hypoxic cell selectivity, with IC(50) ratios (oxic/hypoxic cells) of 20- to 40-fold. The cobalt complexes were also efficiently activated by ionizing radiation, with G values for loss of the compound close to the theoretical value for one-electron reduction of 0.68 micromol/J. This work extends earlier observations that cobalt cyclen complexes are suitable for both the bioreductive and radiolytic release of potent pyrrolo[3,2-f]quinoline effectors.

Quantum Dots in an Amphiphilic Polyethyleneimine Derivative Platform for Cellular Labeling, Targeting, Gene Delivery, and Ratiometric Oxygen Sensing.

Amphiphilic polyethyleneimine derivatives (amPEIs) were synthesized and used to encapsulate dozens of quantum dots (QDs). The QD-amPEI composite was ∼100 nm in hydrodynamic diameter and had the slightly positive outer surface that suited well for cellular internalization. The QD-amPEI showed very efficient QD cellular labeling with the labeled cell fluorescence intensity more than 10 times higher than conventional techniques such as Lipofectamine-assisted QD delivery. QD-amPEI was optimal for maximal intracellular QD delivery by the large QD payload and the rapid endocytosis kinetics. QD-amPEI platform technology was demonstrated for gene delivery, cell-specific labeling, and ratiometric oxygen sensing. Our QD-amPEI platform has two partitions: positive outer surface and hydrophobic inside pocket. The outer positive surface was further exploited for gene delivery and targeting. Co-delivery of QDs and GFP silencing RNAs was successfully demonstrated by assembling siRNAs to the outer surfaces, which showed the transfection efficiency an order of magnitude higher than conventional gene transfections. Hyaluronic acids were tethered onto the QD-amPEI for cell-specific targeted labeling which showed the specific-to-nonspecific signal ratio over 100. The inside hydrophobic compartment was further applied for cohosting oxygen sensing phosphorescence Ru dyes along with QDs. The QD-Ru-amPEI oxygen probe showed accurate and reversible oxygen sensing capability by the ratiometric photoluminescence signals, which was successfully applied to cellular and spheroid models.

Optimization of the Auxiliary Ligand Shell of Cobalt(III)(8-hydroxyquinoline) Complexes as Model Hypoxia-Selective Radiation-Activated Prodrugs

Abstract Ahn, G-O., Ware, D. C., Denny, W. A. and Wilson, W. R. Optimization of the Auxiliary Ligand Shell of Cobalt(III)(8-hydroxyquinoline) Complexes as Model Hypoxia-Selective Radiation-Activated Prodrugs. Radiat. Res. 162, 315–325 (2004). A potential approach for activating prodrugs in hypoxic regions of tumors is to use ionizing radiation, rather than bioreductive enzymes, to effect reduction. This study investigates radiolytic release of 8-hydroxyquinoline (8-HQ), as a model for hydroxyaza-chloromethylbenzindoline DNA minor groove alkylators, from Co(III) complexes under hypoxia. 8-HQ release, measured by HPLC, showed higher efficiency (one-electron stoichiometry) when the auxiliary ligand was a tetraazamacrocycle [e.g. 1,4,7,10-tetraazacyclododecane (cyclen)] rather than a triazamacrocycle [1,4,7-triazacyclononane (TACN)]. These complexes differ from the bioreductive cobalt complex SN 24771 in that their reduction provides stable cobalt-containing products rather than free (aquated) Co2+. Radiolytic release of 8-HQ from Co(cyclen)(8-HQ) and Co(TACN)(CN)(8-HQ) was also demonstrated in deoxygenated human plasma, selectively in the absence of oxygen, again with higher efficiency for the cyclen system. The cobalt complexes were >1000-fold less potent than free 8-HQ as inhibitors of cell proliferation and were metabolically stable in aerobic and hypoxic cell cultures. Investigation of cell uptake of total cobalt, by inductively coupled plasma mass spectrometry, showed that these complexes enter cells but do not accumulate to the high concentrations seen with SN 24771. The results demonstrate the feasibility of masking the cytotoxicity of hydroxyquinoline-based cytotoxins as Co(III) complexes and demonstrate the utility of cyclen-based auxiliary ligands for optimizing radiolytic activation of these novel prodrugs under hypoxia.

Influence of bone marrow-derived hematopoietic cells on the tumor response to radiotherapy: Experimental models and clinical perspectives

In this review, we highlight some of recent studies underscoring the importance of the tumor microenvironment, especially the role of bone marrow-derived myeloid cells, in restoring tumor growth after irradiation. Myeloid cells are hematopoietic cells that give rise to monocytes and macrophages in the peripheral blood and tissues. These cells have been shown to be proangiogenic in tumors promoting tumor growth. We also discuss our previously unpublished results on the effect of irradiation on the tumor vasculature including pericyte and basement membrane coverage to the endothelium of tumor blood vessels. We summarize the clinical significance of these studies including the use of MMP-9 inhibitors, administering white blood cell boosters, or planning safety margin of tumor volumes, in order to improve overall clinical benefits in cancer patients treated with radiotherapy.

Light-responsible DNA hydrogel–gold nanoparticle assembly for synergistic cancer therapy

Assembled AuNPs in a DNA hydrogel (Dgel) showed strongly coupled plasmon modes, and the Dgel vehicle can co-load anticancer drugs such as doxorubicin (Dox) as a light-controlled releasing cargo by DNA intercalations. Upon laser excitation, local heat shock generation was accompanied by the release of Dox. A highly synergistic combination of thermo- and chemotherapy was demonstrated in cellular and animal models. Our Dgel vehicle can be fragmented after the excitation-induced heat generations, which subsequently causes the dispersion of the AuNPs. Our system may be less toxic because it uses small sizes of AuNPs, and the inherently biocompatible scaffold may reduce the long-term toxicity by rapid clearance.

Tumor hypoxia and reoxygenation: the yin and yang for radiotherapy

Tumor hypoxia, a common feature occurring in nearly all human solid tumors is a major contributing factor for failures of anticancer therapies. Because ionizing radiation depends heavily on the presence of molecular oxygen to produce cytotoxic effect, the negative impact of tumor hypoxia had long been recognized. In this review, we will highlight some of the past attempts to overcome tumor hypoxia including hypoxic radiosensitizers and hypoxia-selective cytotoxin. Although they were (still are) a very clever idea, they lacked clinical efficacy largely because of ‘reoxygenation’ phenomenon occurring in the conventional low dose hyperfractionation radiotherapy prevented proper activation of these compounds. Recent meta-analysis and imaging studies do however indicate that there may be a significant clinical benefit in lowering the locoregional failures by using these compounds. Latest technological advancement in radiotherapy has allowed to deliver high doses of radiation conformally to the tumor volume. Although this technology has brought superb clinical responses for many types of cancer, recent modeling studies have predicted that tumor hypoxia is even more serious because ‘reoxygenation’ is low thereby leaving a large portion of hypoxic tumor cells behind. Wouldn’t it be then reasonable to combine hypoxic radiosensitizers and/or hypoxia-selective cytotoxin with the latest radiotherapy? We will provide some preclinical and clinical evidence to support this idea hoping to revamp an enthusiasm for hypoxic radiosensitizers or hypoxia-selective cytotoxins as an adjunct therapy for radiotherapy.