The Michael addition of thiols to 13-oxo-octadecadienoate (13-oxo-ODE) with implications for LC-MS analysis of glutathione conjugation.

Unsaturated fatty acid ketones with αß,γδ conjugation are susceptible to Michael addition of thiols, with unresolved issues on the site of adduction and precise structures of the conjugates. Herein we reacted 13-keto-octadecadienoic acid (13-oxo-ODE or 13-KODE) with glutathione (GSH), N-acetyl-cysteine, and ß-mercaptoethanol and identified the adducts. HPLC-UV analyses indicated none of the products exhibit a conjugated enone UV chromophore, a result that conflicts with the literature and is relevant to the mass spectral interpretation of 1,4 versus 1,6 thiol adduction. Aided by the development of an HPLC solvent system that separates the GSH diastereomers and thus avoids overlap of signals in proton NMR experiments, we established the two major conjugates are formed by 1,6 addition of GSH at the 9-carbon of 13-oxo-ODE with the remaining double bond α to the thiol in the 10,11 position. N-acetyl cysteine reacts similarly, while ß-mercaptoethanol gives equal amounts of 1,4 and 1,6 addition products. Equine glutathione transferase catalyzed 1,6 addition of GSH to the two major diastereomers in 44:56 proportions. LC-MS in positive ion mode gives a product ion interpreted before as evidence of 1,4-thiol adduction, whereas here we find this ion using the authentic 1,6 adduct. LC-MS with negative ion APCI gave a fragment selective for 1,4 adduction. These results clarify the structures of thiol conjugates of a prototypical unsaturated keto-fatty acid and have relevance to the application of LC-MS for the structural analysis of keto-fatty acid glutathione conjugation.

Tumor therapy by targeting extracellular hydroxyapatite using novel drugs: A paradigm shift.

BACKGROUND: It has been shown that tumor microenvironment (TME) hydroxyapatite (HAP) is typically associated with many malignancies and plays a role in tumor progression and growth. Additionally, acidosis in the TME has been reported to play a key role in selecting for a more aggressive tumor phenotype, drug resistance and desensitization to immunotherapy for many types of cancers. TME-HAP is an attractive target for tumor detection and treatment development since HAP is generally absent from normal soft tissue. We provide strong evidence that dissolution of hydroxyapatite (HAP) within the tumor microenvironment (TME-HAP) using a novel therapeutic can be used to kill cancer cells both in vitro and in vivo with minimal adverse effects. METHODS: We developed an injectable cation exchange nano particulate sulfonated polystyrene solution (NSPS) that we engineered to dissolve TME-HAP, inducing localized acute alkalosis and inhibition of tumor growth and glucose metabolism. This was evaluated in cell culture using 4T1, MDA-MB-231 triple negative breast cancer cells, MCF10 normal breast cells, and H292 lung cancer cells, and in vivo using orthotopic mouse models of cancer that contained detectable microenvironment HAP including breast (MMTV-Neu, 4T1, and MDA-MB-231), prostate (PC3) and colon (HCA7) cancer using 18 F-NaF for HAP and 18 F-FDG for glucose metabolism with PET imaging. On the other hand, H292 lung tumor cells that lacked detectable microenvironment HAP and MCF10a normal breast cells that do not produce HAP served as negative controls. Tumor microenvironment pH levels following injection of NSPS were evaluated via Chemical Exchange Saturation (CEST) MRI and via ex vivo methods. RESULTS: Within 24 h of adding the small concentration of 1X of NSPS (~7 µM), we observed significant tumor cell death (~ 10%, p < 0.05) in 4T1 and MDA-MB-231 cell cultures that contain HAP but ⟨2% in H292 and MCF10a cells that lack detectable HAP and in controls. Using CEST MRI, we found extracellular pH (pHe) in the 4T1 breast tumors, located in the mammary fat pad, to increase by nearly 10% from baseline before gradually receding back to baseline during the first hour post NSPS administration. in the tumors that contained TME-HAP in mouse models, MMTV-Neu, 4T1, and MDA-MB-231, PC3, and HCA7, there was a significant reduction (p<0.05) in 18 F-Na Fuptake post NSPS treatment as expected; 18 F- uptake in the tumor = 3.8 ± 0.5 %ID/g (percent of the injected dose per gram) at baseline compared to 1.8 ±0.5 %ID/g following one-time treatment with 100 mg/kg NSPS. Of similar importance, is that 18 F-FDG uptake in the tumors was reduced by more than 75% compared to baseline within 24 h of treatment with one-time NSPS which persisted for at least one week. Additionally, tumor growth was significantly slower (p < 0.05) in the mice treated with one-time NSPS. Toxicity showed no evidence of any adverse effects, a finding attributed to the absence of HAP in normal soft tissue and to our therapeutic NSPS having limited penetration to access HAP within skeletal bone. CONCLUSION: Dissolution of TME-HAP using our novel NSPS has the potential to provide a new treatment paradigm to enhance the management of cancer patients with poor prognosis.

Ornithine Decarboxylase in Gastric Epithelial Cells Promotes the Immunopathogenesis of Helicobacter pylori Infection.

Colonization by Helicobacter pylori is associated with gastric diseases, ranging from superficial gastritis to more severe pathologies, including intestinal metaplasia and adenocarcinoma. The interplay of the host response and the pathogen affect the outcome of disease. One major component of the mucosal response to H. pylori is the activation of a strong but inefficient immune response that fails to control the infection and frequently causes tissue damage. We have shown that polyamines can regulate H. pylori-induced inflammation. Chemical inhibition of ornithine decarboxylase (ODC), which generates the polyamine putrescine from l-ornithine, reduces gastritis in mice and adenocarcinoma incidence in gerbils infected with H. pylori However, we have also demonstrated that Odc deletion in myeloid cells enhances M1 macrophage activation and gastritis. Here we used a genetic approach to assess the specific role of gastric epithelial ODC during H. pylori infection. Specific deletion of the gene encoding for ODC in gastric epithelial cells reduces gastritis, attenuates epithelial proliferation, alters the metabolome, and downregulates the expression of immune mediators induced by H. pylori Inhibition of ODC activity or ODC knockdown in human gastric epithelial cells dampens H. pylori-induced NF-κB activation, CXCL8 mRNA expression, and IL-8 production. Chronic inflammation is a major risk factor for the progression to more severe pathologies associated with H. pylori infection, and we now show that epithelial ODC plays an important role in mediating this inflammatory response.

Cystathionine γ-lyase exacerbates Helicobacter pylori immunopathogenesis by promoting macrophage metabolic remodeling and activation.

Macrophages play a crucial role in the inflammatory response to the human stomach pathogen Helicobacter pylori, which infects half of the world's population and causes gastric cancer. Recent studies have highlighted the importance of macrophage immunometabolism in their activation state and function. We have demonstrated that the cysteine-producing enzyme cystathionine γ-lyase (CTH) is upregulated in humans and mice with H. pylori infection. Here, we show that induction of CTH in macrophages by H. pylori promoted persistent inflammation. Cth-/- mice had reduced macrophage and T cell activation in H. pylori-infected tissues, an altered metabolome, and decreased enrichment of immune-associated gene networks, culminating in decreased H. pylori-induced gastritis. CTH is downstream of the proposed antiinflammatory molecule, S-adenosylmethionine (SAM). Whereas Cth-/- mice exhibited gastric SAM accumulation, WT mice treated with SAM did not display protection against H. pylori-induced inflammation. Instead, we demonstrated that Cth-deficient macrophages exhibited alterations in the proteome, decreased NF-κB activation, diminished expression of macrophage activation markers, and impaired oxidative phosphorylation and glycolysis. Thus, through altering cellular respiration, CTH is a key enhancer of macrophage activation, contributing to a pathogenic inflammatory response that is the universal precursor for the development of H. pylori-induced gastric disease.

VAP-A and its binding partner CERT drive biogenesis of RNA-containing extracellular vesicles at ER membrane contact sites.

RNA transfer via extracellular vesicles (EVs) influences cell phenotypes; however, lack of information regarding biogenesis of RNA-containing EVs has limited progress in the field. Here, we identify endoplasmic reticulum membrane contact sites (ER MCSs) as platforms for the generation of RNA-containing EVs. We identify a subpopulation of small EVs that is highly enriched in RNA and regulated by the ER MCS linker protein VAP-A. Functionally, VAP-A-regulated EVs are critical for miR-100 transfer between cells and in vivo tumor formation. Lipid analysis of VAP-A-knockdown EVs revealed reductions in the EV biogenesis lipid ceramide. Knockdown of the VAP-A-binding ceramide transfer protein CERT led to similar defects in EV RNA content. Imaging experiments revealed that VAP-A promotes luminal filling of multivesicular bodies (MVBs), CERT localizes to MVBs, and the ceramide-generating enzyme neutral sphingomyelinase 2 colocalizes with VAP-A-positive ER. We propose that ceramide transfer via VAP-A-CERT linkages drives the biogenesis of a select RNA-containing EV population.

Iron deficiency linked to altered bile acid metabolism promotes Helicobacter pylori-induced inflammation-driven gastric carcinogenesis.

Gastric carcinogenesis is mediated by complex interactions among Helicobacter pylori, host, and environmental factors. Here, we demonstrate that H. pylori augmented gastric injury in INS-GAS mice under iron-deficient conditions. Mechanistically, these phenotypes were not driven by alterations in the gastric microbiota; however, discovery-based and targeted metabolomics revealed that bile acids were significantly altered in H. pylori-infected mice with iron deficiency, with significant upregulation of deoxycholic acid (DCA), a carcinogenic bile acid. The severity of gastric injury was further augmented when H. pylori-infected mice were treated with DCA, and, in vitro, DCA increased translocation of the H. pylori oncoprotein CagA into host cells. Conversely, bile acid sequestration attenuated H. pylori-induced injury under conditions of iron deficiency. To translate these findings to human populations, we evaluated the association between bile acid sequestrant use and gastric cancer risk in a large human cohort. Among 416,885 individuals, a significant dose-dependent reduction in risk was associated with cumulative bile acid sequestrant use. Further, expression of the bile acid receptor transmembrane G protein-coupled bile acid receptor 5 (TGR5) paralleled the severity of carcinogenic lesions in humans. These data demonstrate that increased H. pylori-induced injury within the context of iron deficiency is tightly linked to altered bile acid metabolism, which may promote gastric carcinogenesis.

Intravitreal HDAC Inhibitor Belinostat Effectively Eradicates Vitreous Seeds Without Retinal Toxicity In Vivo in a Rabbit Retinoblastoma Model.

Purpose: Current melphalan-based regimens for intravitreal chemotherapy for retinoblastoma vitreous seeds are effective but toxic to the retina. Thus, alternative agents are needed. Based on the known biology of histone deacetylases (HDACs) in the retinoblastoma pathway, we systematically studied whether the HDAC inhibitor belinostat is a viable, molecularly targeted alternative agent for intravitreal delivery that might provide comparable efficacy, without toxicity. Methods: In vivo pharmacokinetic experiments in rabbits and in vitro cytotoxicity experiments were performed to determine the 90% inhibitory concentration (IC90). Functional toxicity by electroretinography and structural toxicity by optical coherence tomography (OCT), OCT angiography, and histopathology were evaluated in rabbits following three injections of belinostat 350 µg (2× IC90) or 700 µg (4× IC90), compared with melphalan 12.5 µg (rabbit equivalent of the human dose). The relative efficacy of intravitreal belinostat versus melphalan to treat WERI-Rb1 human cell xenografts in rabbit eyes was directly quantified. RNA sequencing was used to assess belinostat-induced changes in RB cell gene expression. Results: The maximum nontoxic dose of belinostat was 350 µg, which caused no reductions in electroretinography parameters, retinal microvascular loss on OCT angiography, or retinal degeneration. Melphalan caused severe retinal structural and functional toxicity. Belinostat 350 µg (equivalent to 700 µg in the larger human eye) was equally effective at eradicating vitreous seeds in the rabbit xenograft model compared with melphalan (95.5% reduction for belinostat, P < 0.001; 89.4% reduction for melphalan, P < 0.001; belinostat vs. melphalan, P = 0.10). Even 700 µg belinostat (equivalent to 1400 µg in humans) caused only minimal toxicity. Widespread changes in gene expression resulted. Conclusions: Molecularly targeted inhibition of HDACs with intravitreal belinostat was equally effective as standard-of-care melphalan but without retinal toxicity. Belinostat may therefore be an attractive agent to pursue clinically for intravitreal treatment of retinoblastoma.

Hypusination Orchestrates the Antimicrobial Response of Macrophages.

Innate responses of myeloid cells defend against pathogenic bacteria via inducible effectors. Deoxyhypusine synthase (DHPS) catalyzes the transfer of the N-moiety of spermidine to the lysine-50 residue of eukaryotic translation initiation factor 5A (EIF5A) to form the amino acid hypusine. Hypusinated EIF5A (EIF5AHyp) transports specific mRNAs to ribosomes for translation. We show that DHPS is induced in macrophages by two gastrointestinal pathogens, Helicobacter pylori and Citrobacter rodentium, resulting in enhanced hypusination of EIF5A. EIF5AHyp was also increased in gastric macrophages from patients with H. pylori gastritis. Furthermore, we identify the bacteria-induced immune effectors regulated by hypusination. This set of proteins includes essential constituents of antimicrobial response and autophagy. Mice with myeloid cell-specific deletion of Dhps exhibit reduced EIF5AHyp in macrophages and increased bacterial burden and inflammation. Thus, regulation of translation through hypusination is a critical hallmark of the defense of eukaryotic hosts against pathogenic bacteria.

Pharmacokinetics, Tissue Localization, Toxicity, and Treatment Efficacy in the First Small Animal (Rabbit) Model of Intra-Arterial Chemotherapy for Retinoblastoma.

Purpose: Current intra-arterial chemotherapy (IAC) drug regimens for retinoblastoma have ocular and vascular toxicities. No small-animal model of IAC exists to test drug efficacy and toxicity in vivo for IAC drug discovery. The purpose of this study was to develop a small-animal model of IAC and to analyze the ocular tissue penetration, distribution, pharmacokinetics, and treatment efficacy. Methods: Following selective ophthalmic artery (OA) catheterization, melphalan (0.4 to 1.2 mg/kg) was injected. For pharmacokinetic studies, rabbits were euthanized at 0.5, 1, 2, 4, or 6 hours following intra-OA infusion. Drug levels were determined in vitreous, retina, and blood by liquid chromatography tandem mass spectrometry. To assess toxicity, angiograms, photography, fluorescein angiography, and histopathology were performed. For in situ tissue drug distribution, matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) was performed. The tumor model was created by combined subretinal/intravitreal injection of human WERI-Rb1 retinoblastoma cells; the tumor was treated in vivo with intra-arterial melphalan or saline; and induction of tumor death was measured by cleaved caspase-3 activity. Results: OA was selectively catheterized for 79 of 79 (100%) eyes in 47 of 47 (100%) rabbits, and melphalan was delivered successfully in 31 of 31 (100%) eyes, without evidence of vascular occlusion or retinal damage. For treated eyes, maximum concentration (Cmax) in the retina was 4.95 µM and area under the curve (AUC0→∞) was 5.26 µM·h. Treated eye vitreous Cmax was 2.24 µM and AUC0→∞ was 4.19 µM·h. Vitreous Cmax for the treated eye was >100-fold higher than for the untreated eye (P = 0.01), and AUC0→∞ was ∼50-fold higher (P = 0.01). Histology-directed MALDI-IMS revealed highest drug localization within the retina. Peripheral blood Cmax was 1.04 µM and AUC0→∞ was 2.07 µM·h. Combined subretinal/intravitreal injection of human retinoblastoma cells led to intra-retinal tumors and subretinal/vitreous seeds, which could be effectively killed in vivo with intra-arterial melphalan. Conclusions: This first small-animal model of IAC has excellent vitreous and retinal tissue drug penetration, achieving levels sufficient to kill human retinoblastoma cells, facilitating future IAC drug discovery.

Glutathionylated γG and γA subunits of hemoglobin F: a novel post-translational modification found in extremely premature infants by LC-MS and nanoLC-MS/MS.

Oxidative stress plays an important role in the development of various disease processes and is a putative mechanism in the development of bronchopulmonary dysplasia, the most common complication of extreme preterm birth. Glutathione, a major endogenous antioxidant and redox buffer, also mediates cellular functions through protein thiolation. We sought to determine if post-translational thiol modification of hemoglobin F occurs in neonates by examining erythrocyte samples obtained during the first month of life from premature infants, born at 23 0/7 - 28 6/7 weeks gestational age, who were enrolled at our center in the Prematurity and Respiratory Outcomes Program (PROP). Using liquid chromatography-mass spectrometry (LC-MS), we report the novel finding of in vivo and in vitro glutathionylation of γG and γA subunits of Hgb F. Through tandem mass spectrometry (nanoLC-MS/MS), we confirmed the adduction site as the Cys-γ94 residue and through high-resolution mass spectrometry determined that the modification occurs in both γ subunits. We also identified glutathionylation of the ß subunit of Hgb A in our patient samples; we did not find modified α subunits of Hgb A or F. In conclusion, we are the first to report that glutathionylation of γG and γA of Hgb F occurs in premature infants. Additional studies of this post-translational modification are needed to determine its physiologic impact on Hgb F function and if sG-Hgb is a biomarker for clinical morbidities associated with oxidative stress in premature infants.

Reduction of vitreous prostaglandin E2 levels after topical administration of ketorolac 0.45%.

IMPORTANCE: Inhibition of proinflammatory prostaglandins in the retina may have therapeutic effects for retinal disease. OBJECTIVE: To determine vitreous levels of ketorolac and prostaglandin E2 (PGE2) in eyes treated with topical ketorolac tromethamine 0.45% (Acuvail). DESIGN, SETTING, AND PARTICIPANTS: A prospective comparative interventional study, performed in a university academic hospital, included 24 eyes in 22 consecutive patients undergoing pars plana vitrectomy. INTERVENTION: Application of topical ketorolac 0.45%, 4 times daily, for 3 days before pars plana vitrectomy in the first 12 consecutive eyes. The next 12 eyes were untreated and served as controls. Undiluted vitreous samples were obtained at the time of surgery and immediately frozen at -80 °C. MAIN OUTCOMES AND MEASURES: Vitreous ketorolac and PGE2 levels. RESULTS: Seven of the 12 eyes (58%) had ketorolac levels above the lower limit of quantitation. All 7 were in pseudophakic eyes, and 4 of the 5 below this limit were phakic (P = .01). The mean ketorolac level in the 7 eyes was 7.55 ng/mL (range, 5.0-14.9 ng/mL). The mean (SD) PGE2 levels were 13.8 (3.8) pg/mL in control eyes and 11.7 (4.4) pg/mL in ketorolac-treated eyes (P = .04). Treatment with ketorolac resulted in a 15% reduction in PGE2 levels. When only pseudophakic eyes were analyzed, mean (SD) PGE2 levels were 14.1 (4.1) pg/mL in control eyes and 11.6 (4.5) pg/mL in ketorolac-treated eyes (P < .05). CONCLUSIONS AND RELEVANCE: Topical ketorolac 0.45% can obtain a vitreous level that exceeds its median inhibitory concentration and can significantly decrease vitreous PGE2 levels. Vitreous levels of ketorolac were significantly higher in pseudophakic eyes than in phakic eyes. The results of this study suggest that topically administered ketorolac 0.45% may allow meaningful inhibition of prostaglandins in the retina. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01609881.

Identification of Hedgehog pathway responsive glioblastomas by isocitrate dehydrogenase mutation.

The Hedgehog (Hh) pathway regulates the growth of a subset of adult gliomas and better definition of Hh-responsive subtypes could enhance the clinical utility of monitoring and targeting this pathway in patients. Somatic mutations of the isocitrate dehydrogenase (IDH) genes occur frequently in WHO grades II and III gliomas and WHO grade IV secondary glioblastomas. Hh pathway activation in WHO grades II and III gliomas suggests that it might also be operational in glioblastomas that developed from lower-grade lesions. To evaluate this possibility and to better define the molecular and histopathological glioma subtypes that are Hh-responsive, IDH genes were sequenced in adult glioma specimens assayed for an operant Hh pathway. The proportions of grades II-IV specimens with IDH mutations correlated with the proportions that expressed elevated levels of the Hh gene target PTCH1. Indices of an operational Hh pathway were measured in all primary cultures and xenografts derived from IDH-mutant glioma specimens, including IDH-mutant glioblastomas. In contrast, the Hh pathway was not operational in glioblastomas that lacked IDH mutation or history of antecedent lower-grade disease. IDH mutation is not required for an operant pathway however, as significant Hh pathway modulation was also measured in grade III gliomas with wild-type IDH sequences. These results indicate that the Hh pathway is operational in grades II and III gliomas and glioblastomas with molecular or histopathological evidence for evolvement from lower-grade gliomas. Lastly, these findings suggest that gliomas sharing this molecularly defined route of progression arise in Hh-responsive cell types.

Oxidation of methyl and ethyl nitrosamines by cytochrome P450 2E1 and 2B1.

Cytochrome P450 (P450) 2E1 is the major enzyme that oxidizes N-nitrosodimethylamine [N,N-dimethylnitrosamine (DMN)], a carcinogen and also a representative of some nitrosamines formed endogenously. Oxidation of DMN by rat or human P450 2E1 to HCHO showed a high apparent intrinsic kinetic deuterium isotope effect (KIE), ≥8. The KIE was not attenuated in noncompetitive intermolecular experiments with rat liver microsomes {(D)V = 12.5; (D)(V/K) = 10.9 [nomenclature of Northrop, D. B. (1982) Methods Enzymol. 87, 607-625]} but was with purified human P450 2E1 [(D)V = 3.3; (D)(V/K) = 3.7], indicating that C-H bond breaking is partially rate-limiting with human P450 2E1. With N-nitrosodiethylamine [N,N-diethylnitrosamine (DEN)], the intrinsic KIE was slightly lower and was not expressed [e.g., (D)(V/K) = 1.2] in noncompetitive intermolecular experiments. The same general pattern of KIEs was also seen in the (D)(V/K) results with DMN and DEN for the minor products resulting from the denitrosation reactions (CH(3)NH(2), CH(3)CH(2)NH(2), and NO(2)(-)). Experiments with deuterated N-nitroso-N-methyl-N-ethylamine demonstrated that the lower KIEs associated with ethyl versus methyl oxidation could be distinguished within a single molecule. P450 2E1 oxidized DMN and DEN to aldehydes and then to the carboxylic acids. No kinetic lags were observed in acid formation; pulse-chase experiments with carrier aldehydes showed only limited equilibration with P450 2E1-bound aldehydes, indicative of processive reactions, as reported for P450 2A6 [Chowdhury, G., et al. (2010) J. Biol. Chem. 285, 8031-8044]. These same features (no lag phase for HCO(2)H formation and a lack of equilibration in pulse-chase assays) were also seen with (rat) P450 2B1, which has a lower catalytic efficiency for DMN oxidation and a larger active site. Thus, the processivity of dialkyl nitrosamine oxidation appears to be shared by a number of P450s.

Menaquinone biosynthesis potentiates haem toxicity in Staphylococcus aureus.

Staphylococcus aureus is a pathogen that infects multiple anatomical sites leading to a diverse array of diseases. Although vertebrates can restrict the growth of invading pathogens by sequestering iron within haem, S. aureus surmounts this challenge by employing high-affinity haem uptake systems. However, the presence of excess haem is highly toxic, necessitating tight regulation of haem levels. To overcome haem stress, S. aureus expresses the detoxification system HrtAB. In this work, a transposon screen was performed in the background of a haem-susceptible, HrtAB-deficient S. aureus strain to identify the substrate transported by this putative pump and the source of haem toxicity. While a recent report indicates that HrtAB exports haem itself, the haem-resistant mutants uncovered by the transposon selection enabled us to elucidate the cellular factors contributing to haem toxicity. All mutants identified in this screen inactivated the menaquinone (MK) biosynthesis pathway. Deletion of the final steps of this pathway revealed that quinone molecules localizing to the cell membrane potentiate haem-associated superoxide production and subsequent oxidative damage. These data suggest a model in which membrane-associated haem and quinone molecules form a redox cycle that continuously generates semiquinones and reduced haem, both of which react with atmospheric oxygen to produce superoxide.

Berberine induces caspase-independent cell death in colon tumor cells through activation of apoptosis-inducing factor.

Berberine, an isoquinoline alkaloid derived from plants, is a traditional medicine for treating bacterial diarrhea and intestinal parasite infections. Although berberine has recently been shown to suppress growth of several tumor cell lines, information regarding the effect of berberine on colon tumor growth is limited. Here, we investigated the mechanisms underlying the effects of berberine on regulating the fate of colon tumor cells, specifically the mouse immorto-Min colonic epithelial (IMCE) cells carrying the Apc(min) mutation, and of normal colon epithelial cells, namely young adult mouse colonic epithelium (YAMC) cells. Berberine decreased colon tumor colony formation in agar, and induced cell death and LDH release in a time- and concentration-dependent manner in IMCE cells. In contrast, YAMC cells were not sensitive to berberine-induced cell death. Berberine did not stimulate caspase activation, and PARP cleavage and berberine-induced cell death were not affected by a caspase inhibitor in IMCE cells. Rather, berberine stimulated a caspase-independent cell death mediator, apoptosis-inducing factor (AIF) release from mitochondria and nuclear translocation in a ROS production-dependent manner. Amelioration of berberine-stimulated ROS production or suppression of AIF expression blocked berberine-induced cell death and LDH release in IMCE cells. Furthermore, two targets of ROS production in cells, cathepsin B release from lysosomes and PARP activation were induced by berberine. Blockage of either of these pathways decreased berberine-induced AIF activation and cell death in IMCE cells. Thus, berberine-stimulated ROS production leads to cathepsin B release and PARP activation-dependent AIF activation, resulting in caspase-independent cell death in colon tumor cells. Notably, normal colon epithelial cells are less susceptible to berberine-induced cell death, which suggests the specific inhibitory effects of berberine on colon tumor cell growth.

Phase I pharmacokinetic and pharmacodynamic study of SJG-136, a novel DNA sequence selective minor groove cross-linking agent, in advanced solid tumors.

PURPOSE: This phase I study assessed the maximum tolerated dose (MTD), safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of SJG-136, a sequence-specific DNA cross-linking agent, in patients with advanced cancer. EXPERIMENTAL DESIGN: In schedule A, seven patients received escalating doses of SJG-136 (6, 12, 24, and 48 µg/m(2)) daily for 5 of 21 days. Blood samples were collected for PK analysis on days 1 and 5 of cycle 1. In schedule B, SJG-136 was given daily for 3 of 21 days (N = 17; doses 20, 25, 30, and 35 µg/m(2)). Blood samples were collected on days 1 and 3 of cycles 1 and 2 for PK and PD analysis. Patients in schedule B received dexamethasone and early diuretic care. RESULTS: Schedule A-dose-limiting toxicities included grade 3 edema, dyspnea, fatigue, and delayed liver toxicity (grade 3-4). PK analysis revealed dose-dependent increases in AUC and C(max). Substantial changes in volume of distribution at steady-state occurred after repeated dosing in some patients prior to the onset of edema. Schedule B-the same toxicities were manageable with steroid premedication and diuretic support. No significant myelosuppression occurred on either schedule. DNA interstrand cross-links correlated with systemic exposure of SJG-136 following the second dose in cycle 1 and were still detectable immediately before cycle 2. CONCLUSIONS: The MTD of SJG-136 in this study was 30 µg/m(2) administered on a daily 3× basis with no myelosuppression effects. Coupled with supportive management, SJG-136 is now advancing to a phase II trial in ovarian cancer.

Oxidation of N-Nitrosoalkylamines by human cytochrome P450 2A6: sequential oxidation to aldehydes and carboxylic acids and analysis of reaction steps.

Cytochrome P450 (P450) 2A6 activates nitrosamines, including N,N-dimethylnitrosamine (DMN) and N,N-diethylnitrosamine (DEN), to alkyl diazohydroxides (which are DNA-alkylating agents) and also aldehydes (HCHO from DMN and CH(3)CHO from DEN). The N-dealkylation of DMN had a high intrinsic kinetic deuterium isotope effect ((D)k(app) approximately 10), which was highly expressed in a variety of competitive and non-competitive experiments. The (D)k(app) for DEN was approximately 3 and not expressed in non-competitive experiments. DMN and DEN were also oxidized to HCO(2)H and CH(3)CO(2)H, respectively. In neither case was a lag observed, which was unexpected considering the k(cat) and K(m) parameters measured for oxidation of DMN and DEN to the aldehydes and for oxidation of the aldehydes to the carboxylic acids. Spectral analysis did not indicate strong affinity of the aldehydes for P450 2A6, but pulse-chase experiments showed only limited exchange with added (unlabeled) aldehydes in the oxidations of DMN and DEN to carboxylic acids. Substoichiometric kinetic bursts were observed in the pre-steady-state oxidations of DMN and DEN to aldehydes. A minimal kinetic model was developed that was consistent with all of the observed phenomena and involves a conformational change of P450 2A6 following substrate binding, equilibrium of the P450-substrate complex with a non-productive form, and oxidation of the aldehydes to carboxylic acids in a process that avoids relaxation of the conformation following the first oxidation (i.e. of DMN or DEN to an aldehyde).

A phase I trial of SJG-136 (NSC#694501) in advanced solid tumors.

PURPOSE: SJG-136 is a pyrrolobenzodiazepine dimer that forms DNA crosslinks and has demonstrated broad antitumor activity. We undertook this trial to determine the maximum-tolerated dose (MTD), toxicities and pharmacokinetic (PK) profile of SJG-136 in patients with an advanced solid tumor. PATIENTS AND METHODS: In this phase I study, patients were treated with SJG-136 on days 1, 8 and 15 of a 28-day cycle. Dose levels studied were 10, 20, 40 and 60 microg/m2. PK parameters of SJG-136 were assessed following the intravenous administration of SJG-136 on days 1 and 15 of cycle 1. RESULTS: Twenty-one patients with advanced solid tumors were treated. Patients had a median of two prior chemotherapy regimens. Fatigue was dose-limiting with SJG-136 60 microg/m2/day administered on days 1, 8 and 15 of a 28-day cycle. Grade 3 thrombocytopenia and delayed onset liver toxicity were seen in one patient each. PK parameters of SJG-136 indicated dose-proportional increases in systemic exposure with increasing doses. No objective responses were seen. CONCLUSION: For patients with advanced solid tumors, the MTD of SJG-136 is 40 microg/m2/day administered on days 1, 8 and 15 of a 28-day cycle. The major dose limiting toxicity was fatigue. Alternative dosing strategies are now being evaluated.

Kinetic analysis of oxidation of coumarins by human cytochrome P450 2A6.

Human cytochrome P450 (P450) 2A6 catalyzes 7-hydroxylation of coumarin, and the reaction rate is enhanced by cytochrome b5 (b5). 7-Alkoxycoumarins were O-dealkylated and also hydroxylated at the 3-position. Binding of coumarin and 7-hydroxycoumarin to ferric and ferrous P450 2A6 are fast reactions (k(on) approximately 10(6) m(-1) s(-1)), and the k(off) rates range from 5.7 to 36 s(-1) (at 23 degrees C). Reduction of ferric P450 2A6 is rapid (7.5 s(-1)) but only in the presence of coumarin. The reaction of the ferrous P450 2A6 substrate complex with O2 is rapid (k > or = 10(6) m(-1) s(-1)), and the putative Fe2+.O2 complex decayed at a rate of approximately 0.3 s(-1) at 23 degrees C. Some 7-hydroxycoumarin was formed during the oxidation of the ferrous enzyme under these conditions, and the yield was enhanced by b5. Kinetic analyses showed that approximately 1/3 of the reduced b5 was rapidly oxidized in the presence of the Fe2+.O2 complex, implying some electron transfer. High intrinsic and competitive and non-competitive intermolecular kinetic deuterium isotope effects (values 6-10) were measured for O-dealkylation of 7-alkoxycoumarins, indicating the effect of C-H bond strength on rates of product formation. These results support a scheme with many rapid reaction steps, including electron transfers, substrate binding and release at multiple stages, and rapid product release even though the substrate is tightly bound in a small active site. The inherent difficulty of chemistry of substrate oxidation and the lack of proclivity toward a linear pathway leading to product formation explain the inefficiency of the enzyme relative to highly efficient bacterial P450s.

Influence of the a-ring on the redox and nuclease properties of the prodigiosins: importance of the bipyrrole moiety in oxidative DNA cleavage.

Prodigiosin (Prod, 1) is the parent member of a class of polypyrrole natural products that exhibit promising immunosuppressive and cytotoxic activity. They can facilitate copper-promoted oxidative double-strand (ds) DNA cleavage through reductive activation of Cu(II). This is triggered by oxidation of the electron-rich Prod molecule and may provide a basis for the cytotoxicity of the prodigiosins. To gain an understanding of this activity, we prepared several Prod analogues with various A-ring systems to examine their electrochemical properties in acetonitrile (MeCN) as a means to establish a basis for structure-reactivity relationships in copper-promoted nuclease activity. The intact bipyrrole (BP) chromophore is critical for the copper-mediated nuclease properties of the Prods. In fact, simple BP systems are shown to facilitate oxidative single-strand (ss) DNA cleavage. Replacement of the Prod A-pyrrole ring with alternative arenes (phenyl, furan-2-yl, or thiophen-2-yl) inhibits DNA strand scission and raises the half-peak oxidation potential (E(p/2)) of the Prod free base [E(p/2) = 0.44 V vs saturated calomel electrode (SCE) in MeCN] by ca. 200 mV. The same effect was achieved through attachment of an electron-withdrawing group (acetyl) at the 5'-position of the A-pyrrole ring. The structural modifications that inhibit DNA cleavage correlate with known structure-reactivity relationships of Prods against leukemia and melanoma cancer cells. The implications of our findings with regard to the cytotoxicity of the Prods are discussed.