Lipid-heparin infusion suppresses the IL-10 response to trauma in subcutaneous adipose tissue in humans.

An imbalance between pro- and anti-inflammatory cytokine productions in adipose tissue is thought to contribute to chronic, systemic, low-grade inflammation and consequently to an increased risk of cardiovascular complications in obese and type 2 diabetic patients. Nonesterified fatty acids (NEFA), whose serum levels are elevated in such patients, have been shown to interfere with cytokine production in vitro. In order to evaluate the effects of elevated NEFA levels on cytokine production in adipose tissue in vivo we used an 18-gauge open-flow microperfusion (OFM) catheter to induce local inflammation in the subcutaneous adipose tissue (SAT) of healthy volunteers and to sample interstitial fluid (IF) specifically from the inflamed tissue. In two crossover studies, nine subjects received either an intravenous lipid-heparin infusion to elevate circulating NEFA levels or saline over a period of 28 h. The former increased the circulating levels of triglycerides (TGs), NEFA, glucose, and insulin over the study period. NEFA effects on locally induced inflammation were estimated by measuring the levels of a panel adipokines in the OFM probe effluent. Interleukin-6 (IL-6), IL-8, tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) levels increased during the study period but were not affected by lipid-heparin infusion. In contrast, the level of IL-10, an anti-inflammatory cytokine, was significantly reduced during the final hour of lipid-heparin infusion (saline: 449.2 ± 105.9 vs. lipid-heparin: 65.4 ± 15.4 pg/ml; P = 0.02). These data provide the first in vivo evidence that elevated NEFA can modulate cytokine production by adipose tissue.

Transcriptionally targeted nonviral gene transfer using a beta-catenin/TCF-dependent promoter in a series of different human low passage colon cancer cells.

Nonviral transfections of six low passage human colon cancer cell lines using the artificial beta-catenin/TCF-dependent promoter CTP4 demonstrated a high promoter activity which was 1000- to 70000-fold higher than in HeLa control cells. Luciferase gene expression levels obtained with CTP4 in epithelial-like tumor cell cultures were only slightly lower than with the strong viral CMV promoter/enhancer, whereas in less differentiated tumor cultures CTP4 expression levels exceeded the CMV expression levels up to 28-fold. Three cell lines representing different morphology typical of the original tumors, more differentiated epithelial-like (COGA-5), piled-up (COGA-12), and poorly differentiated rounded-up (COGA-3), were selected for further investigation. Gene transfer was optimized using lipopolyplex formulation of cationic lipid DOSPER and polycation PEI25br. Lipopolyplexes enabled up to 1300-fold or 400-fold higher luciferase expression compared to the corresponding lipoplexes or polyplexes, respectively. Lipopolyfection of an interleukin-2 (IL-2) gene expression construct driven by the CTP4 promoter resulted in very high levels of up to 95 ng of secreted IL-2 per 105 cells and 24 h. The lipopolyplexes were also able to transfect multicellular spheroids that mimic the three-dimensional structure of real tumors.

Structural and mechanistic studies of Escherichia coli nitroreductase with the antibiotic nitrofurazone. Reversed binding orientations in different redox states of the enzyme.

The antibiotics nitrofurazone and nitrofurantoin are used in the treatment of genitourinary infections and as topical antibacterial agents. Their action is dependent upon activation by bacterial nitroreductase flavoproteins, including the Escherichia coli nitroreductase (NTR). Here we show that the products of reduction of these antibiotics by NTR are the hydroxylamine derivatives. We show that the reduction of nitrosoaromatics is enzyme-catalyzed, with a specificity constant approximately 10,000-fold greater than that of the starting nitro compounds. This suggests that the reduction of nitro groups proceeds through two successive, enzyme-mediated reactions and explains why the nitroso intermediates are not observed. The global reaction rate for nitrofurazone determined in this study is over 10-fold higher than that previously reported, suggesting that the enzyme is much more active toward nitroaromatics than previously estimated. Surprisingly, in the crystal structure of the oxidized NTR-nitrofurazone complex, nitrofurazone is oriented with its amide group, rather than the nitro group to be reduced, positioned over the reactive N5 of the FMN cofactor. Free acetate, which acts as a competitive inhibitor with respect to NADH, binds in a similar orientation. We infer that the orientation of bound nitrofurazone depends upon the redox state of the enzyme. We propose that the charge distribution on the FMN rings, which alters upon reduction, is an important determinant of substrate binding and reactivity in flavoproteins with broad substrate specificity.

Antitumor immune responses mediated by adenoviral GDEPT using nitroreductase/CB1954 is enhanced by high-level coexpression of heat shock protein 70.

Gene-directed enzyme prodrug therapy (GDEPT) is a promising approach to local management of cancer through targeted chemotherapy. Killing localized tumors by GDEPT in a manner that induces strong antitumor cellular immune responses might improve local management and allow benefit in disseminated cancer. Here we evaluated the combination of nitroreductase (NTR)/CB1954 GDEPT with high-level expression of heat shock protein 70 (HSP70, a stress protein that can shuttle cytosolic peptides into antigen-presenting cells) for induction of antitumor immunity using adenovirus gene delivery in an aggressive and nonimmunogenic BALB/c syngeneic 4T1 breast cancer model. The mechanism of cell death and spectrum of stress proteins induced are likely to be important determinants of the resulting immune responses. We showed that NTR/CB1954 treatment of 4T1 cells gave both apoptotic and nonapoptotic killing. In vivo killing of 4T1 cells expressing NTR gave weak antitumor immunity and very limited induction of stress proteins including HSP70. High-level coexpression of HSP70 during NTR/CB1954-mediated killing of 4T1 cells in vivo gave much greater protection from tumor challenge (67% long-term survivors compared to 17%) and induced 4T1-specific cytotoxic T-cell responses. The enhancement of antitumor responses resulting from HSP70 coexpression was similar to that conferred by coexpression of GM-CSF.

Optimization of a synthetic beta-catenin-dependent promoter for tumor-specific cancer gene therapy.

We recently published the construction and evaluation of a beta-catenin-dependent, highly active promoter, CTP1, and its possible application for the treatment of colorectal cancer using gene-directed enzyme prodrug therapy with adenoviral (Ad) vectors. Alternative Ad-based approaches such as tumor-specific, replication-competent vectors and/or exploiting therapeutic gene products with intrinsic toxic activity, such as gibbon ape leukemia virus fusogenic membrane glycoprotein, diphtheria toxin A (DTA), and ricin, would demand a very tightly regulated promoter to avoid breakthrough replication and toxicity in nontumor tissue and Ad producer cell lines. In this study we optimized the activity/specificity profile of the synthetic beta-catenin-dependent promoter by varying its basal promoter, the number of Tcf binding sites, and the distance between these and the basal promoter. The optimal promoter, CTP4, showed virtually undetectable expression in cells with normal beta-catenin regulation but high level expression in cells deregulated for beta-catenin. Using CTP4 we were able to generate, for the first time to our knowledge, an Ad vector expressing fully active wild-type DTA without the need for time-consuming and cumbersome production systems. CTP4 should be the promoter of choice for Ad-based gene therapies of tumors deregulated for beta-catenin. We provide preliminary evidence that these may include prostate and ovarian as well as colorectal cancer.

An oncolytic adenovirus vector combining enhanced cell-to-cell spreading, mediated by the ADP cytolytic protein, with selective replication in cancer cells with deregulated wnt signaling.

We have constructed a novel oncolytic adenovirus (Ad) vector named VRX-009 that combines enhanced cell spread with tumor-specific replication. Enhanced spread, which could significantly increase antitumor efficacy, is mediated by overexpression of the Ad cytolytic protein named ADP (also known as E3-11.6K). Replication of VRX-009 is restricted to cells with a deregulated wnt signal transduction pathway by replacement of the wild-type Ad E4 promoter with a synthetic promoter consisting of five consensus binding sites for the T-cell factor transcription factor. Tumor-selective replication is indicated by several lines of evidence. VRX-009 expresses E4ORF3, a representative Ad E4 protein, only in colon cancer cell lines. Furthermore, VRX-009 replicates preferentially in colon cancer cell lines as evidenced by virus productivity 2 orders of magnitude higher in SW480 colon cancer cells than in A549 lung cancer cells. Replication in primary human bronchial epithelial cells and human umbilical vein endothelial cells was also significantly lower than in SW480 cells. When tested in human tumor xenografts in nude mice, VRX-009 effectively suppressed the growth of SW480 colon tumors but not of A549 lung tumors. VRX-009 may provide greater level of antitumor efficacy than standard oncolytic Ad vectors in tumors in which a defect in wnt signaling increases the level of nuclear beta-catenin.

Generation of Escherichia coli nitroreductase mutants conferring improved cell sensitization to the prodrug CB1954.

Escherichia coli nitroreductase (NTR) activates the prodrug CB1954 to a cytotoxic derivative, allowing selective sensitization of NTR-expressing cells or tumors to the prodrug. This is one of several enzyme-prodrug combinations that are under development for cancer gene therapy, and the system has now entered clinical trials. Enhancing the catalytic efficiency of NTR for CB1954 could improve its therapeutic potential. From the crystal structure of an enzyme-ligand complex, we identified nine amino acid residues within the active site that could directly influence prodrug binding and catalysis. Mutant libraries were generated for each of these residues and clones screened for their ability to sensitize E. coli to CB1954. Amino acid substitutions at six positions conferred markedly greater sensitivity to CB1954 than did the WT enzyme; the best mutants, at residue F124, resulted in approximately 5-fold improvement. Using an adenovirus vector, we introduced the F124K NTR mutant into human SK-OV-3 ovarian carcinoma cells and showed it to be approximately 5-fold more potent in sensitizing the cells to CB1954 at the clinically relevant prodrug concentration of 1 micro M than was the WT enzyme. Enhanced mutant NTRs such as F124K should improve the efficacy of the NTR/CB1954 combination in cancer gene therapy.