IL-4 and IL-17A Cooperatively Promote Hydrogen Peroxide Production, Oxidative DNA Damage, and Upregulation of Dual Oxidase 2 in Human Colon and Pancreatic Cancer Cells.

Dual oxidase 2 (DUOX2) generates H2O2 that plays a critical role in both host defense and chronic inflammation. Previously, we demonstrated that the proinflammatory mediators IFN-γ and LPS enhance expression of DUOX2 and its maturation factor DUOXA2 through STAT1- and NF-κB‒mediated signaling in human pancreatic cancer cells. Using a panel of colon and pancreatic cancer cell lines, we now report the induction of DUOX2/DUOXA2 mRNA and protein expression by the TH2 cytokine IL-4. IL-4 activated STAT6 signaling that, when silenced, significantly decreased induction of DUOX2. Furthermore, the TH17 cytokine IL-17A combined synergistically with IL-4 to increase DUOX2 expression in both colon and pancreatic cancer cells mediated, at least in part, by signaling through NF-κB. The upregulation of DUOX2 was associated with a significant increase in the production of extracellular H2O2 and DNA damage-as indicated by the accumulation of 8-oxo-dG and γH2AX-which was suppressed by the NADPH oxidase inhibitor diphenylene iodonium and a DUOX2-specific small interfering RNA. The clinical relevance of these experiments is suggested by immunohistochemical, microarray, and quantitative RT-PCR studies of human colon and pancreatic tumors demonstrating significantly higher DUOX2, IL-4R, and IL-17RA expression in tumors than in adjacent normal tissues; in pancreatic adenocarcinoma, increased DUOX2 expression is adversely associated with overall patient survival. These data suggest a functional association between DUOX2-mediated H2O2 production and induced DNA damage in gastrointestinal malignancies.

NADPH oxidase 1 supports proliferation of colon cancer cells by modulating reactive oxygen species-dependent signal transduction.

Reactive oxygen species (ROS) play a critical role in cell signaling and proliferation. NADPH oxidase 1 (NOX1), a membrane-bound flavin dehydrogenase that generates O2̇̄, is highly expressed in colon cancer. To investigate the role that NOX1 plays in colon cancer growth, we used shRNA to decrease NOX1 expression stably in HT-29 human colon cancer cells. The 80-90% decrease in NOX1 expression achieved by RNAi produced a significant decline in ROS production and a G1/S block that translated into a 2-3-fold increase in tumor cell doubling time without increased apoptosis. The block at the G1/S checkpoint was associated with a significant decrease in cyclin D1 expression and profound inhibition of mitogen-activated protein kinase (MAPK) signaling. Decreased steady-state MAPK phosphorylation occurred concomitant with a significant increase in protein phosphatase activity for two colon cancer cell lines in which NOX1 expression was knocked down by RNAi. Diminished NOX1 expression also contributed to decreased growth, blood vessel density, and VEGF and hypoxia-inducible factor 1α (HIF-1α) expression in HT-29 xenografts initiated from NOX1 knockdown cells. Microarray analysis, supplemented by real-time PCR and Western blotting, revealed that the expression of critical regulators of cell proliferation and angiogenesis, including c-MYC, c-MYB, and VEGF, were down-regulated in association with a decline in hypoxic HIF-1α protein expression downstream of silenced NOX1 in both colon cancer cell lines and xenografts. These studies suggest a role for NOX1 in maintaining the proliferative phenotype of some colon cancers and the potential of NOX1 as a therapeutic target in this disease.

NADPH oxidase 5 (NOX5)-induced reactive oxygen signaling modulates normoxic HIF-1α and p27Kip1 expression in malignant melanoma and other human tumors.

NADPH oxidase 5 (NOX5) generated reactive oxygen species (ROS) have been implicated in signaling cascades that regulate cancer cell proliferation. To evaluate and validate NOX5 expression in human tumors, we screened a broad range of tissue microarrays (TMAs), and report substantial overexpression of NOX5 in malignant melanoma and cancers of the prostate, breast, and ovary. In human UACC-257 melanoma cells that possesses high levels of functional endogenous NOX5, overexpression of NOX5 resulted in enhanced cell growth, increased numbers of BrdU positive cells, and increased γ-H2AX levels. Additionally, NOX5-overexpressing (stable and inducible) UACC-257 cells demonstrated increased normoxic HIF-1α expression and decreased p27Kip1 expression. Similarly, increased normoxic HIF-1α expression and decreased p27Kip1 expression were observed in stable NOX5-overexpressing clones of KARPAS 299 human lymphoma cells and in the human prostate cancer cell line, PC-3. Conversely, knockdown of endogenous NOX5 in UACC-257 cells resulted in decreased cell growth, decreased HIF-1α expression, and increased p27Kip1 expression. Likewise, in an additional human melanoma cell line, WM852, and in PC-3 cells, transient knockdown of endogenous NOX5 resulted in increased p27Kip1 and decreased HIF-1α expression. Knockdown of endogenous NOX5 in UACC-257 cells resulted in decreased Akt and GSK3ß phosphorylation, signaling pathways known to modulate p27Kip1 levels. In summary, our findings suggest that NOX5 expression in human UACC-257 melanoma cells could contribute to cell proliferation due, in part, to the generation of high local concentrations of extracellular ROS that modulate multiple pathways that regulate HIF-1α and networks that signal through Akt/GSK3ß/p27Kip1 .

NADPH oxidases and cancer.

The mechanism by which reactive oxygen species (ROS) are produced by tumour cells remained incompletely understood until the discovery over the last 15 years of the family of NADPH oxidases (NOXs 1-5 and dual oxidases DUOX1/2) which are structural homologues of gp91phox, the major membrane-bound component of the respiratory burst oxidase of leucocytes. Knowledge of the roles of the NOX isoforms in cancer is rapidly expanding. Recent evidence suggests that both NOX1 and DUOX2 species produce ROS in the gastrointestinal tract as a result of chronic inflammatory stress; cytokine induction (by interferon-γ, tumour necrosis factor α, and interleukins IL-4 and IL-13) of NOX1 and DUOX2 may contribute to the development of colorectal and pancreatic carcinomas in patients with inflammatory bowel disease and chronic pancreatitis, respectively. NOX4 expression is increased in pre-malignant fibrotic states which may lead to carcinomas of the lung and liver. NOX5 is highly expressed in malignant melanomas, prostate cancer and Barrett's oesophagus-associated adenocarcinomas, and in the last it is related to chronic gastro-oesophageal reflux and inflammation. Over-expression of functional NOX proteins in many tissues helps to explain tissue injury and DNA damage from ROS that accompany pre-malignant conditions, as well as elucidating the potential mechanisms of NOX-related damage that contribute to both the initiation and the progression of a wide range of solid and haematopoietic malignancies.

Activation of TLR4 is required for the synergistic induction of dual oxidase 2 and dual oxidase A2 by IFN-γ and lipopolysaccharide in human pancreatic cancer cell lines.

Pancreatitis is associated with release of proinflammatory cytokines and reactive oxygen species and plays an important role in the development of pancreatic cancer. We recently demonstrated that dual oxidase (Duox)2, an NADPH oxidase essential for reactive oxygen species-related, gastrointestinal host defense, is regulated by IFN-γ-mediated Stat1 binding to the Duox2 promoter in pancreatic tumor lines. Because LPS enhances the development and invasiveness of pancreatic cancer in vivo following TLR4-related activation of NF-κB, we examined whether LPS, alone or combined with IFN-γ, regulated Duox2. We found that upregulation of TLR4 by IFN-γ in BxPC-3 and CFPAC-1 pancreatic cancer cells was augmented by LPS, resulting in activation of NF-κB, accumulation of NF-κB (p65) in the nucleus, and increased binding of p65 to the Duox2 promoter. TLR4 silencing with small interfering RNAs, as well as two independent NF-κB inhibitors, attenuated LPS- and IFN-γ-mediated Duox2 upregulation in BxPC-3 cells. Induction of Duox2 expression by IFN-γ and LPS may result from IFN-γ-related activation of Stat1 acting in concert with NF-κB-related upregulation of Duox2. Sustained extracellular accumulation of H(2)O(2) generated by exposure to both LPS and IFN-γ was responsible for an ∼50% decrease in BxPC-3 cell proliferation associated with a G(1) cell cycle block, apoptosis, and DNA damage. We also demonstrated upregulation of Duox expression in vivo in pancreatic cancer xenografts and in patients with chronic pancreatitis. These results suggest that inflammatory cytokines can interact to produce a Duox-dependent pro-oxidant milieu that could increase the pathologic potential of pancreatic inflammation and pancreatic cancer cells.

Validation of a hypoxia-inducible factor-1 alpha specimen collection procedure and quantitative enzyme-linked immunosorbent assay in solid tumor tissues.

Hypoxia-inducible factor-1 alpha (HIF-1α) is an important marker of hypoxia in human tumors and has been implicated in tumor progression. Drugs targeting HIF-1α are being developed, but the ability to measure drug-induced changes in HIF-1α is limited by the lability of the protein in normoxia. Our goal was to devise methods for specimen collection and processing that preserve HIF-1α in solid tumor tissues and to develop and validate a two-site chemiluminescent quantitative enzyme-linked immunosorbent assay (ELISA) for HIF-1α. We tested various strategies for HIF-1α stabilization in solid tumors, including nitrogen gas-purged lysis buffer, the addition of proteasome inhibitors or the prolyl hydroxylase inhibitor 2-hydroxyglutarate, and bead homogenization. Degassing and the addition of 2-hydroxyglutarate to the collection buffer significantly increased HIF-1α recovery, whereas bead homogenization in sealed tubes improved HIF-1α recovery and reduced sample variability. Validation of the ELISA demonstrated intra- and inter-assay variability of less than 15% and accuracy of 99.8±8.3% as assessed by spike recovery. Inter-laboratory reproducibility was also demonstrated (R(2)=0.999). Careful sample handling techniques allow us to quantitatively detect HIF-1α in samples as small as 2.5µg of total protein extract, and this method is currently being applied to analyze tumor biopsy specimens in early-phase clinical trials.

Optimal function of the DNA repair enzyme TDP1 requires its phosphorylation by ATM and/or DNA-PK.

Human tyrosyl-DNA phosphodiesterase (TDP1) hydrolyzes the phosphodiester bond at a DNA 3' end linked to a tyrosyl moiety. This type of linkage is found at stalled topoisomerase I (Top1)-DNA covalent complexes, and TDP1 has been implicated in the repair of such complexes. Here we show that Top1-associated DNA double-stranded breaks (DSBs) induce the phosphorylation of TDP1 at S81. This phosphorylation is mediated by the protein kinases: ataxia-telangiectasia-mutated (ATM) and DNA-dependent protein kinase (DNA-PK). Phosphorylated TDP1 forms nuclear foci that co-localize with those of phosphorylated histone H2AX (gammaH2AX). Both Top1-induced replication- and transcription-mediated DNA damages induce TDP1 phosphorylation. Furthermore, we show that S81 phosphorylation stabilizes TDP1, induces the formation of XRCC1 (X-ray cross-complementing group 1)-TDP1 complexes and enhances the mobilization of TDP1 to DNA damage sites. Finally, we provide evidence that TDP1-S81 phosphorylation promotes cell survival and DNA repair in response to CPT-induced DSBs. Together; our findings provide a new mechanism for TDP1 post-translational regulation by ATM and DNA-PK.

Exogenous DNA enhances DUOX2 expression and function in human pancreatic cancer cells by activating the cGAS-STING signaling pathway.

Pro-inflammatory cytokines upregulate the expression of the H2O2-producing NADPH oxidase dual oxidase 2 (DUOX2)2 which, when elevated, adversely affects survival from pancreatic ductal adenocarcinoma (PDAC). Because the cGAS-STING pathway is known to initiate pro-inflammatory cytokine expression following uptake of exogenous DNA, we examined whether activation of cGAS-STING could play a role in the generation of reactive oxygen species by PDAC cells. Here, we found that a variety of exogenous DNA species markedly increased the production of cGAMP, the phosphorylation of TBK1 and IRF3, and the translocation of phosphorylated IRF3 into the nucleus, leading to a significant, IRF3-dependent enhancement of DUOX2 expression, and a significant flux of H2O2 in PDAC cells. However, unlike the canonical cGAS-STING pathway, DNA-related DUOX2 upregulation was not mediated by NF-κB. Although exogenous IFN-ß significantly increased Stat1/2-associated DUOX2 expression, intracellular IFN-ß signaling that followed cGAMP or DNA exposure did not itself increase DUOX2 levels. Finally, DUOX2 upregulation subsequent to cGAS-STING activation was accompanied by the enhanced, normoxic expression of HIF-1α and VEGF-A as well as DNA double strand cleavage, suggesting that cGAS-STING signaling may support the development of an oxidative, pro-angiogenic microenvironment that could contribute to the inflammation-related genetic instability of pancreatic cancer.

Up-regulation and sustained activation of Stat1 are essential for interferon-gamma (IFN-gamma)-induced dual oxidase 2 (Duox2) and dual oxidase A2 (DuoxA2) expression in human pancreatic cancer cell lines.

Dual oxidase 2 is a member of the NADPH oxidase (Nox) gene family that plays a critical role in the biosynthesis of thyroid hormone as well as in the inflammatory response of the upper airway mucosa and in wound healing, presumably through its ability to generate reactive oxygen species, including H2O2. The recently discovered overexpression of Duox2 in gastrointestinal malignancies, as well as our limited understanding of the regulation of Duox2 expression, led us to examine the effect of cytokines and growth factors on Duox2 in human tumor cells. We found that exposure of human pancreatic cancer cells to IFN-γ (but not other agents) produced a profound up-regulation of the expression of Duox2, and its cognate maturation factor DuoxA2, but not other members of the Nox family. Furthermore, increased Duox2/DuoxA2 expression was closely associated with a significant increase in the production of both intracellular reactive oxygen species and extracellular H2O2. Examination of IFN-γ-mediated signaling events demonstrated that in addition to the canonical Jak-Stat1 pathway, IFN-γ activated the p38-MAPK pathway in pancreatic cancer cells, and both played an important role in the induction of Duox2 by IFN-γ. Duox2 up-regulation following IFN-γ exposure is also directly associated with the binding of Stat1 to elements of the Duox2 promoter. Our findings suggest that the pro-inflammatory cytokine IFN-γ initiates a Duox2-mediated reactive oxygen cascade in human pancreatic cancer cells; reactive oxygen species production in this setting could contribute to the pathophysiologic characteristics of these tumors.

Inhibitors of human tyrosyl-DNA phospodiesterase (hTdp1) developed by virtual screening using ligand-based pharmacophores.

Human tyrosyl-DNA phosphodiesterase (hTdp1) inhibitors have become a major area of drug research and structure-based design since they have been shown to work synergistically with camptothecin (CPT) and selectively in cancer cells. The pharmacophore features of 14 hTdp1 inhibitors were used as a filter to screen the ChemNavigator iResearch Library of about 27 million purchasable samples. Docking of the inhibitors and hits obtained from virtual screening was performed into a structural model of hTdp1 based on a high resolution X-ray crystal structure of human Tdp1 in complex with vanadate, DNA and a human topoisomerase I (TopI)-derived peptide (PDB code: 1NOP). A total of 46 compounds matching the three-dimensional arrangement of the pharmacophoric features were assayed. Using a high-throughput screening assay, we have identified an 1H-indol-3-yl-acetic acid derivative as a potent Tdp1 inhibitor with an IC(50) value of 7.94 microM. The obtained novel chemotype may provide a new scaffold for developing inhibitors of Tdp1.

Virtual screening using ligand-based pharmacophores for inhibitors of human tyrosyl-DNA phospodiesterase (hTdp1).

Human tyrosyl-DNA phosphodiesterase (hTdp1) inhibitors have become a major area of drug research and structure-based design since they have been shown to work synergistically with camptothecin (CPT) and selectively in cancer cells. The pharmacophore features of 14hTdp1 inhibitors were used as a filter to screen the ChemNavigator iResearch Library of about 27 million purchasable samples. Docking of the inhibitors and hits obtained from virtual screening was performed into a structural model of hTdp1 based on a high resolution X-ray crystal structure of human Tdp1 in complex with vanadate, DNA and a human topoisomerase I (Top1)-derived peptide (PDB code: 1NOP). We present and discuss in some detail 46 compounds matching the three-dimensional arrangement of the pharmacophoric features. The presented novel chemotypes may provide new scaffolds for developing inhibitors of Tdp1.

Inhibiting the Activity of NADPH Oxidase in Cancer.

Significance: The primary function of NADPH oxidases (NOX1-5 and dual oxidases DUOX1/2) is to produce reactive oxygen species (ROS). If inadequately regulated, NOX-associated ROS can promote oxidative stress, aberrant signaling, and genomic instability. Correspondingly, NOX isoforms are known to be overexpressed in multiple malignancies, thus constituting potential therapeutic targets in cancer. Recent Advances: Multiple genetic studies aimed at suppressing the expression of NOX proteins in cellular and animal models of cancer have provided support for the notion that NOXs play a pro-tumorigenic role. Further, large drug screens and rational design efforts have yielded inhibitor compounds, such as the diphenylene iodonium (DPI) analog series developed by our group, with increased selectivity and potency over "first generation" NOX inhibitors such as apocynin and DPI. Critical Issues: The precise role of NOX enzymes in tumor biology remains poorly defined. The tumorigenic properties of NOXs vary with cancer type, and precise tools, such as selective inhibitors, are needed to deconvolute NOX contribution to cancer development. Most NOX inhibitors developed to date are unspecific, and/or their mechanistic and pharmacological characteristics are not well defined. A lack of high-resolution crystal structures for NOX functional domains has hindered the development of potent and selective inhibitors. Future Directions: In-depth studies of NOX interactions with the tumor microenvironment (e.g., cytokines, cell-surface antigens) will help identify new approaches for NOX inhibition in cancer.

NADPH oxidase 1 is highly expressed in human large and small bowel cancers.

To facilitate functional investigation of the role of NADPH oxidase 1 (NOX1) and associated reactive oxygen species in cancer cell signaling, we report herein the development and characterization of a novel mouse monoclonal antibody that specifically recognizes the C-terminal region of the NOX1 protein. The antibody was validated in stable NOX1 overexpression and knockout systems, and demonstrates wide applicability for Western blot analysis, confocal microscopy, flow cytometry, and immunohistochemistry. We employed our NOX1 antibody to characterize NOX1 expression in a panel of 30 human colorectal cancer cell lines, and correlated protein expression with NOX1 mRNA expression and superoxide production in a subset of these cells. Although a significant correlation between oncogenic RAS status and NOX1 mRNA levels could not be demonstrated in colon cancer cell lines, RAS mutational status did correlate with NOX1 expression in human colon cancer surgical specimens. Immunohistochemical analysis of a comprehensive set of tissue microarrays comprising over 1,200 formalin-fixed, paraffin-embedded tissue cores from human epithelial tumors and inflammatory disease confirmed that NOX1 is overexpressed in human colon and small intestinal adenocarcinomas, as well as adenomatous polyps, compared to adjacent, uninvolved intestinal mucosae. In contradistinction to prior studies, we did not find evidence of NOX1 overexpression at the protein level in tumors versus histologically normal tissues in prostate, lung, ovarian, or breast carcinomas. This study constitutes the most comprehensive histopathological characterization of NOX1 to date in cellular models of colon cancer and in normal and malignant human tissues using a thoroughly evaluated monoclonal antibody. It also further establishes NOX1 as a clinically relevant therapeutic target in colorectal and small intestinal cancer.

Von Hippel-Lindau-coupled and transcription-coupled nucleotide excision repair-dependent degradation of RNA polymerase II in response to trabectedin.

PURPOSE: Ecteinascidin 743 (Et743; trabectedin, Yondelis) has recently been approved in Europe for the treatment of soft tissue sarcomas and is undergoing clinical trials for other solid tumors. Et743 selectively targets cells proficient for TC-NER, which sets it apart from other DNA alkylating agents. In the present study, we examined the effects of Et743 on RNA Pol II. EXPERIMENTAL DESIGN AND RESULTS: We report that Et743 induces the rapid and massive degradation of transcribing Pol II in various cancer cell lines and normal fibroblasts. Pol II degradation was abrogated by the proteasome inhibitor MG132 and was dependent on TC-NER. Cockayne syndrome (CS) cells and xeroderma pigmentosum (XP) cells (XPD, XPA, XPG, and XPF) were defective in Pol II degradation, whereas XPC cells whose defect is limited to global genome NER in nontranscribing regions were proficient for Pol II degradation. Complementation of the CSB and XPD cells restored Pol II degradation. We also show that cells defective for the VHL complex were defective in Pol II degradation and that complementation of those cells restores Pol II degradation. Moreover, VHL deficiency rendered cells resistant to Et743-induced cell death, a similar effect to that of TC-NER deficiency. CONCLUSION: These results suggest that both TC-NER-induced and VHL-mediated Pol II degradation play a role in cell killing by Et743.

Novel high-throughput electrochemiluminescent assay for identification of human tyrosyl-DNA phosphodiesterase (Tdp1) inhibitors and characterization of furamidine (NSC 305831) as an inhibitor of Tdp1.

By enzymatically hydrolyzing the terminal phosphodiester bond at the 3'-ends of DNA breaks, tyrosyl-DNA phosphodiesterase (Tdp1) repairs topoisomerase-DNA covalent complexes and processes the DNA ends for DNA repair. To identify novel Tdp1 inhibitors, we developed a high-throughput assay that uses electrochemiluminescent (ECL) substrates. Subsequent to screening of 1981 compounds from the 'diversity set' of the NCI-Developmental Therapeutics Program, here we report that furamidine inhibits Tdp1 at low micromolar concentrations. Inhibition of Tdp1 by furamidine is effective both with single- and double-stranded substrates but is slightly stronger with the duplex DNA. Surface plasmon resonance studies show that furamidine binds both single- and double-stranded DNA, though more weakly with the single-stranded substrate DNA. Thus, the inhibition of Tdp1 activity could in part be due to the binding of furamidine to DNA. However, the inhibition of Tdp1 by furamidine is independent of the substrate DNA sequence. The kinetics of Tdp1 inhibition by furamidine was influenced by the drug to enzyme ratio and duration of the reaction. Comparison with related dications shows that furamidine inhibits Tdp1 more effectively than berenil, while pentamidine was inactive. Thus, furamidine represents the most potent Tdp1 inhibitor reported to date.

Predicting cisplatin and trabectedin drug sensitivity in ovarian and colon cancers.

Molecular profiling of markers involved in the activity of chemotherapeutic agents can shed light on the successes and failures of treatment in patients and can also provide a basis for individualization of therapy. Toward those ends, we have used reverse-phase protein lysate microarrays to evaluate expression of protein components of the nucleotide excision repair (NER) pathways. Those pathways strongly influence the anticancer activities of numerous drugs, including those that are the focus here, cisplatin and ecteinascidin 743 (Et-743; Yondelis, Trabectedin). Cisplatin is generally more active in cell types deficient in NER, whereas Et-743 tends to be less active in those cells. We measured protein expression and sensitivity to those drugs in 17 human ovarian and colon cancer cell lines (13 of them from the NCI-60 panel) and five xeroderma pigmentosum (XP) patient cell types, each containing a different NER defect. Of the NER proteins giving reliable signals, XPF and XPG showed the highest correlations of protein expression with drug activity across all three tissue-of-origin groups. When we compared protein expression data with mRNA expression data from Affymetrix U133A chips, we found no consistent correlation between the two across the cell lines studied, which reinforces the conclusion that protein measurements can give more interpretable mechanistic information than can transcript measurements. The work reported here provides motivation for larger proteomic studies with more cell types focused on potential biomarkers in additional pharmacologically pertinent pathways.

Covalent binding of the natural antimicrobial peptide indolicidin to DNA abasic sites.

Indolicidin is a host defense tridecapeptide that inhibits the catalytic activity of HIV-1 integrase in vitro. Here we have elucidated its mechanism of integrase inhibition. Using crosslinking and mass spectrometric footprinting approaches, we found that indolicidin interferes with formation of the catalytic integrase-DNA complex by directly binding DNA. Further characterization revealed that the peptide forms covalent links with abasic sites. Indolicidin crosslinks single- or double-stranded DNAs and various positions of the viral cDNA with comparable efficiency. Using truncated and chemically modified peptides, we show that abasic site crosslinking is independent of the PWWP motif but involves the indolicidin unique lysine residue and the N- and C- terminal NH2 groups. Because indolicidin can also inhibit topoisomerase I, we believe that multiple actions at the level of DNA might be a common property of antimicrobial peptides.

4-nitroquinoline-1-oxide induces the formation of cellular topoisomerase I-DNA cleavage complexes.

RecQ helicase BLM-deficient cells are characteristically hypersensitive to 4-nitroquinoline-1-oxide (4NQO). We recently reported that isogenic BLM-deficient cells (PNSG13) are more sensitive than BLM-complemented cells (PNSF5) to camptothecin, which specifically traps topoisomerase I cleavage complexes (Top1cc). We now report that PNSG13 are also 3.5-fold more sensitive to 4NQO compared with PNSF5 and that 4NQO induces higher levels of Top1cc and reduced histone gamma-H2AX in PSNG13 than in PNSF5. Similarly, 4NQO induces more Top1cc in primary fibroblasts from a patient with Bloom syndrome than in normal human fibroblasts. 4NQO also induces Top1cc in colon cancer HCT116 and HT29 cells in a time- and concentration-dependent fashion. Of note, distinct from camptothecin, the Top1cc produced by 4NQO accumulate progressively after 4NQO addition and persist following 4NQO removal. The Top1cc induced by 4NQO are detectable by alkaline elution. To examine the functional relevance of the Top1cc induced by 4NQO, we used two stable topoisomerase I small interfering RNA (siRNA) cell lines derived from HCT116 and MCF7 cells. Both topoisomerase I siRNA cell lines are resistant to 4NQO, indicating that Top1cc contribute to the cellular activity of 4NQO. Collectively, these data show that 4NQO is an effective inducer of cellular Top1cc. Because 4NQO does not directly trap Top1cc in biochemical assays, we propose that active metabolites of 4NQO trap Top1cc by forming DNA adducts. Induction of Top1cc and histone gamma-H2AX by 4NQO may contribute to the cellular effects of 4NQO, including its selective activity toward RecQ helicase BLM-deficient cells.

Optimization of the indenone ring of indenoisoquinoline topoisomerase I inhibitors.

Two series of indenoisoquinoline topoisomerase I inhibitors have been prepared to investigate optimal substituents on the indenone ring at the 9-position. The more exhaustive series was prepared using a nitrated isoquinoline ring that has been previously demonstrated to enhance biological activity. After preliminary biological evaluation, a more focused series of inhibitors was prepared utilizing a 2,3-dimethoxy-substituted isoquinoline ring. The results of the two series indicate the existence of superior functional groups such as methoxy, fluorine, and cyano for the indenoisoquinoline 9-position. Interestingly, these functional groups coincide with established structure-activity relationships for the 11-position of camptothecin.

Nitrated indenoisoquinolines as topoisomerase I inhibitors: a systematic study and optimization.

The biological activity of indenoisoquinoline topoisomerase I (Top1) inhibitors can be greatly enhanced depending on the choice of substituents on the aromatic rings and lactam side chain. Previously, it was discovered that a 3-nitro group and a 9-methoxy group afforded enhanced biological activity. In the present investigation, indenoisoquinoline analogues were systematically prepared using combinations of nitro groups, methoxy groups, and hydrogen atoms in an effort to understand the contribution of each group toward cytotoxicity and Top1 inhibition. Analysis of the biological results suggests that the nitro group is important for Top1 inhibition and the methoxy group improves cytotoxicity. In addition, previously identified structure-activity relationships were utilized to select favorable lactam side chain functionalities for incorporation on the aromatic skeleton of analogues in this study. As a result, this investigation has provided optimal Top1 inhibitors equipotent to camptothecin that demonstrate low nanomolar cytotoxicities toward cancer cells.