DCPIP (2,6-dichlorophenolindophenol) as a genotype-directed redox chemotherapeutic targeting NQO1*2 breast carcinoma

Accumulative experimental evidence suggests feasibility of chemotherapeutic intervention targeting human cancer cells by pharmacological modulation of cellular oxidative stress. Current efforts aim at personalization of redox chemotherapy through identification of predictive tumour genotypes and redox biomarkers. Based on earlier research demonstrating that anti-melanoma activity of the pro-oxidant 2,6-dichlorophenolindophenol (DCPIP) is antagonized by cellular NAD(P)H:quinone oxidoreductase (NQO1) expression, this study tested DCPIP as a genotype-directed redox chemotherapeutic targeting homozygous NQO1*2 breast carcinoma, a common missense genotype [rs1800566 polymorphism; NP_000894.1:p.Pro187Ser] encoding a functionally impaired NQO1 protein. In a panel of cultured breast carcinoma cell lines and NQO1-transfectants with differential NQO1 expression levels, homozygous NQO1*2 MDA-MB231 cells were hypersensitive to DCPIP-induced caspase-independent cell death that occurred after early onset of oxidative stress with glutathione depletion and loss of genomic integrity. Array analysis revealed upregulated expression of oxidative (GSTM3, HMOX1, EGR1), heat shock (HSPA6, HSPA1A, CRYAB) and genotoxic stress response (GADD45A, CDKN1A) genes confirmed by immunoblot detection of HO-1, Hsp70, Hsp70B', p21 and phospho-p53 (Ser15). In a murine xenograft model of human homozygous NQO1*2-breast carcinoma, systemic administration of DCPIP displayed significant anti-tumour activity, suggesting feasibility of redox chemotherapeutic intervention targeting the NQO1*2 genotype.     

DCPIP (2,6-dichlorophenolindophenol) as a genotype-directed redox chemotherapeutic targeting NQO1*2 breast carcinoma

Abstract

Accumulative experimental evidence suggests feasibility of chemotherapeutic intervention targeting human cancer cells by pharmacological modulation of cellular oxidative stress. Current efforts aim at personalization of redox chemotherapy through identification of predictive tumour genotypes and redox biomarkers. Based on earlier research demonstrating that anti-melanoma activity of the pro-oxidant 2,6-dichlorophenolindophenol (DCPIP) is antagonized by cellular NAD(P)H:quinone oxidoreductase (NQO1) expression, this study tested DCPIP as a genotype-directed redox chemotherapeutic targeting homozygous NQO1*2 breast carcinoma, a common missense genotype [rs1800566 polymorphism; NP_000894.1:p.Pro187Ser] encoding a functionally impaired NQO1 protein. In a panel of cultured breast carcinoma cell lines and NQO1-transfectants with differential NQO1 expression levels, homozygous NQO1*2 MDA-MB231 cells were hypersensitive to DCPIP-induced caspase-independent cell death that occurred after early onset of oxidative stress with glutathione depletion and loss of genomic integrity. Array analysis revealed upregulated expression of oxidative (GSTM3, HMOX1, EGR1), heat shock (HSPA6, HSPA1A, CRYAB) and genotoxic stress response (GADD45A, CDKN1A) genes confirmed by immunoblot detection of HO-1, Hsp70, Hsp70B’, p21 and phospho-p53 (Ser15). In a murine xenograft model of human homozygous NQO1*2-breast carcinoma, systemic administration of DCPIP displayed significant anti-tumour activity, suggesting feasibility of redox chemotherapeutic intervention targeting the NQO1*2 genotype.     

A direct interaction between JNK1 and CrkII is critical for Rac1-induced JNK activation.

CrkII, a cellular homolog of v-crk, belongs to a family of adaptor proteins that play a central role in signal transduction cascades. We demonstrate that CrkII interacts directly with c-Jun N-terminal kinase 1 (JNK1). A proline-rich sequence of JNK1 is critical for the interaction of the kinase with the N-terminal Src homology 3 (SH3) domain of CrkII. JNK1 is localized with CrkII in membrane ruffles of Crk-overexpressing cells in a Rac1-dependent manner. A JNK1 mutant (K340A) that fails to interact with CrkII is defective in Rac/epidermal growth factor-induced activation, but remains responsive to UVC irradiation. Furthermore, CrkII recruits JNK1 to a p130Cas multiprotein complex where it may be activated through a hematopoietic progenitor kinase 1- and mitogen-activated protein kinase kinase 4-dependent pathway. Together, the results presented here argue for a new mechanism of regulation of the JNK pathway through the CrkII-p130Cas adaptor complex.     

An association between a NQO1 genetic polymorphism and risk of lung cancer

NAD(P)H:quinone oxidoreductase (NQO1) is a detoxification enzyme that protects against the regeneration of reactive oxygen species chemically induced by oxidative stress, cytotoxicity, mutagenicity, and carcinogenicity. The protection conferred by NQO1 protein reduces certain environmental carcinogens, such as nitroaromatic compounds, heterocyclic amines, and possible cigarette smoke condensate. The gene coding for NQO1 has a genetic polymorphism (C-->T) at nucleotide position 609 (i.e. amino acid codon 187) of the NQO1 cDNA. This polymorphism was shown to reduce NQO1 enzyme activity, thereby diminishing the protection provided by NQO1. Therefore, we hypothesized that individuals with the variant NQO1 genotype are at higher risk for lung cancer. Using a case-control study, we genotyped the NQO1 variants successfully by PCR-RFLP in 826 lung cancer patients and 826 healthy control subjects matched for age, sex, ethnicity, and smoking status. The frequency of the NQO1 T-allele was statistically significantly different among three ethnic groups (p<0.001). In further analysis of Caucasians, the variant NQO1 genotypes (CT and TT) were associated with a marginally increased lung cancer risk (OR=1.19; 95% CI: 0.95-1.50). The elevated lung cancer risk was only evident in younger individuals (age <62 years old) (OR=1.46; 95% CI: 1.04-2.05), women (OR=1.89; 95% CI: 1.33-2.68), and never smokers (OR=1.80; 95% CI: 1.03-3.13). Furthermore, we found a statistically significant trend in the development of lung cancer at an early age in women with increasing copies of the variant allele (p=0.03). These results suggest that the NQO1 variant genotype may modulate lung cancer risk, especially in younger individuals (age<62), women, and never smokers.     

Lack of direct interaction between enalaprilat and the kinin B1 receptors

It has been recently proposed that the second extracellular loop of the human bradykinin (BK) B1 receptor (B1R) contains a conserved HExxH motif also present in peptidases possessing a Zn2+ prosthetic group, such as angiotensin converting enzyme (ACE), and that ACE inhibitors directly activate B1R signaling in endothelial cells. However, the binding of ACE inhibitors to the B1Rs has never been directly evaluated. Information about binding of a radiolabeled inhibitor to natural or recombinant ACE in intact cells (physiologic ionic composition) was also collected. We used the tritiated form of an ACE inhibitor previously proposed to activate the B1R, enalaprilat, to address these questions using recombinant human B1Rs and naturally expressed or recombinant ACE. [3H]Lys-des-Arg9-BK bound to the human recombinant B1Rs with high affinity (KD 0.35 nM) in HEK 293a cells. [3H]Enalaprilat (0.25-10 nM) did not bind to cells expressing recombinant human B1R, but bound with a subnanomolar affinity to recombinant ACE or to naturally expressed ACE in human umbilical vein endothelial cells. The radioligand was further validated using a binding competition assay that involved unlabeled ACE inhibitors or their prodrug forms in endothelial cells. Membranes of HEK 293a cells that expressed B1Rs did not hydrolyze hippuryl-glycylglycine (an ACE substrate). Enalaprilat did not stimulate calcium signaling in HEK 293a cells that expressed B1Rs. A typical ACE inhibitor did not bind to nor stimulate the human B1Rs; nevertheless, several other indirect mechanisms could connect ACE inhibition to B1R stimulation in vivo.     

NQO1 and NQO2 regulation of humoral immunity and autoimmunity

NAD(P)H:quinone oxidoreductase 1 (NQO1) and NRH:quinone oxidoreductase 2 (NQO2) are cytosolic enzymes that catalyze metabolic reduction of quinones and derivatives. NQO1-null and NQO2-null mice were generated that showed decreased lymphocytes in peripheral blood, myeloid hyperplasia, and increased sensitivity to skin carcinogenesis. In this report, we investigated the in vivo role of NQO1 and NQO2 in immune response and autoimmunity. Both NQO1-null and NQO2-null mice showed decreased B-cells in blood, lower germinal center response, altered B cell homing, and impaired primary and secondary immune responses. NQO1-null and NQO2-null mice also showed susceptibility to autoimmune disease as revealed by decreased apoptosis in thymocytes and pre-disposition to collagen-induced arthritis. Further experiments showed accumulation of NADH and NRH, cofactors for NQO1 and NQO2, indicating altered intracellular redox status. The studies also demonstrated decreased expression and lack of activation of immune-related factor NF-kappaB. Microarray analysis showed altered chemokines and chemokine receptors. These results suggest that the loss of NQO1 and NQO2 leads to altered intracellular redox status, decreased expression and activation of NF-kappaB, and altered chemokines. The results led to the conclusion that NQO1 and NQO2 are endogenous factors in the regulation of immune response and autoimmunity.     

Evidence for direct interaction between Sprouty and Cbl

Sprouty (SPRY) was first identified in a genetic screen in Drosophila as an antagonist of fibroblast and epidermal growth factor receptors and Sevenless signaling, seemingly by inhibiting the receptor tyrosine kinase (RTK)/Ras/MAPK pathway. To date, four mammalian Sprouty genes have been identified; the primary sequences of the gene products share a well conserved cysteine-rich C-terminal domain with their Drosophila counterpart. The N-terminal regions do not, however, exhibit a large degree of homology. This study was aimed at identifying proteins with which human SPRY2 (hSPRY2) interacts in an attempt to understand the mechanism by which Sprouty proteins exert their down-regulatory effects. Here, we demonstrate that hSPRY2 associates directly with c-Cbl, a known down-regulator of RTK signaling. A short sequence in the N terminus of hSPRY2 was found to bind directly to the Ring finger domain of c-Cbl. Parallel binding was apparent between the Drosophila homologs of Sprouty and Cbl, with cross-species associations occurring at least in vitro. Coexpression of hSPRY2 abrogated an increase in the rate of epidermal growth factor receptor internalization induced by c-Cbl, whereas a mutant hSPRY2 protein unable to bind c-Cbl showed no such effect. Our results suggest that one function of hSPRY2 in signaling processes downstream of RTKs may be to modulate c-Cbl physiological function such as that seen with receptor-mediated endocytosis.     

Structural basis for a direct interaction between FGFR1 and NCAM and evidence for a regulatory role of ATP

The neural cell adhesion molecule (NCAM) promotes axonal outgrowth, presumably through an interaction with the fibroblast growth factor receptor (FGFR). NCAM also has a little-understood ATPase activity. We here demonstrate for the first time a direct interaction between NCAM (fibronectin type III [F3] modules 1 and 2) and FGFR1 (Ig modules 2 and 3) by surface plasmon resonance (SPR) analysis. The structure of the NCAM F3 module 2 was determined by NMR and the module was shown by NMR to interact with the FGFR1 Ig module 3 and ATP. The NCAM sites binding to FGFR and ATP were found to overlap and ATP was shown by SPR to inhibit the NCAM-FGFR binding, indicating that ATP probably regulates the NCAM-FGFR interaction. Furthermore, we demonstrate that the NCAM module was able to induce activation (phosphorylation) of FGFR and to stimulate neurite outgrowth. In contrast, ATP inhibited neurite outgrowth induced by the module.     

A direct interaction between Photosystem I and the chloroplast coupling factor.

A regulation of the ATP-synthesizing complex by electron-transport rate has been found. The site of regulation could be localized within the Photosystem I region. The regulatory effect probably is produced by direct interactions between neighbouring charged protein complexes. The primary result is an increase in the percentage of those binding sites adopting a low-affinity state. This seems to lead to an enhanced leakage of protons out of the thylakoids, especially under those experimental conditions employing low nucleotide concentrations. Changes in the P/2e ratio can be observed, especially if the total ADP + ATP concentration used in the experiment is below 200 microM.     

Functional and direct interaction between the RNA binding protein HuD and active Akt1

The RNA binding protein HuD plays essential roles in neuronal development and plasticity. We have previously shown that HuD stimulates translation. Key for this enhancer function is the linker region and the poly(A) binding domain of HuD that are also critical for its function in neurite outgrowth. Here, we further explored the underlying molecular interactions and found that HuD but not the ubiquitously expressed HuR interacts directly with active Akt1. We identify that the linker region of HuD is required for this interaction. We also show by using chimeric mutants of HuD and HuR, which contain the reciprocal linker between RNA-binding domain 2 (RBD2) and RBD3, respectively, and by overexpressing a dominant negative mutant of Akt1 that the HuD-Akt1 interaction is functionally important, as it is required for the induction of neurite outgrowth in PC12 cells. These results suggest the model whereby RNA-bound HuD functions as an adapter to recruit Akt1 to trigger neurite outgrowth. These data might also help to explain how HuD enhances translation of mRNAs that encode proteins involved in neuronal development.     

Evidence of direct interaction between actin and membrane lipids

Actin is a protein component of the cystoskeleton and is involved in cell motility. It is believed generally that actin filaments are attached to the cell membrane through an interaction with membranous actin-binding proteins. By using an in vitro system composed of liposomes and actin, we have shown that actin may also interact directly with the phospholipids of the membrane. Actin deposited at the surface of the liposome is organized in two regular patterns: a paracrystalline sheet of parallel filaments in register, or a netlike organization. These interactions of actin with membrane lipids occur only in the presence of millimolar concentrations of Mg2+. These results suggest that the interaction of the cytoskeleton with the membrane involves, at least in part, a direct association of actin with phospholipids.     

A mutually inhibitory feedback loop between the 20S proteasome and its regulator, NQO1

NAD(P)H:quinone-oxidoreductase-1 (NQO1) is a cytosolic enzyme that catalyzes the reduction of various quinones using flavin adenine dinucleotide (FAD) as a cofactor. NQO1 has been also shown to rescue proteins containing intrinsically unstructured domains, such as p53 and p73, from degradation by the 20S proteasome through an unknown mechanism. Here, we studied the nature of interaction between NQO1 and the 20S proteasome. Our study revealed a double negative feedback loop between NQO1 and the 20S proteasome, whereby NQO1 prevents the proteolytic activity of the 20S proteasome and the 20S proteasome degrades the apo form of NQO1. Furthermore, we demonstrate, both in vivo and in vitro, that NQO1 levels are highly dependent on FAD concentration. These observations suggest a link between 20S proteolysis and the metabolic cellular state. More generally, the results may represent a regulatory mechanism by which associated cofactors dictate the stability of proteins, thus coordinating protein levels with the metabolic status.     

A direct interaction between cytoplasmic dynein and kinesin I may coordinate motor activity

Cytoplasmic dynein and kinesin I are both unidirectional intracellular motors. Dynein moves cargo toward the cell center, and kinesin moves cargo toward the cell periphery. There is growing evidence that bi-directional motility is regulated in the cell, potentially through direct interactions between oppositely oriented motors. We have identified a direct interaction between cytoplasmic dynein and kinesin I. Using the yeast two-hybrid assay and affinity chromatography, we demonstrate that the intermediate chain of dynein binds to kinesin light chains 1 and 2. The interaction is both direct and specific. Co-immunoprecipitation experiments demonstrate an interaction between endogenous proteins in rat brain cytosol. Double-label immunocytochemistry reveals a partial co-localization of vesicle-associated motor proteins. Together these observations suggest that soluble motors can interact, potentially allowing kinesin I to actively localize dynein to cellular sites of function. There is also a vesicle population with both dynein and kinesin I bound that may be capable of bi-directional motility along cellular microtubules.     

Regeneration of isthmic tissue is the result of a specific and direct interaction between rhombomere 1 and midbrain.

The midbrain-hindbrain boundary, or isthmus, is the source of signals that are responsible for regional specification of both the midbrain and anterior hindbrain. Fibroblast growth factor 8 (Fgf8) is expressed specifically at the isthmus and there is now good evidence that it forms at least part of the patterning signal. In this study, we use Fgf8 as a marker for isthmic cells to examine how interactions between midbrain and hindbrain can regenerate isthmic tissue and, thereby, gain insight into the normal formation and/or maintenance of the isthmus. We show that Fgf8-expressing tissue with properties of the isthmic organiser is generated when midbrain and rhombomere 1 tissue are juxtaposed but not when midbrain contacts any other rhombomere. The use of chick/quail chimeras shows that the isthmic tissue is largely derived from rhombomere 1. In a few cases a small proportion of the Fgf8-positive cells were of midbrain origin but this appears to be the result of a local respecification to a hindbrain phenotype, a process mimicked by ectopic FGF8. Studies in vitro show that the induction of Fgf8 is the result of a direct planar interaction between the two tissues and involves a diffusible signal.     

Synergism through direct covalent bonding between agents: a strategy for rational design of chemotherapeutic combinations

Self-assembling chemotherapeutic agents are mixtures of relatively nontoxic precursors that can combine chemically under physiological conditions to form products with greater cytotoxic and/or antimicrobial activity than either of the precursors. Combinations that form products more rapidly in or near the target (tumor, pathogen, virally infected cell) than in normal tissues will exhibit target-selective synergism, thus exhibiting an antitarget selectivity that is greater than the selectivities of the product (e.g., a hydrazone) and of either precursor (e.g., a hydrazine derivative or ketone) used singly. This paper describes the target-selective cytotoxic synergism of a cationic aldehyde (A) and a cationic acylhydrazine (B) containing a triarylalkylphosphonium moiety against Ehrlich ascites carcinoma cells (ELA) in culture, in addition to reviewing previous work on self-assembling cytotoxins. The synergism between A and B is carcinoma selective when the ELA cells (the target) are compared to CV-1, an untransformed African green monkey kidney epithelial line. Like tetraphenylphosphonium and rhodamine 123, which are selectively concentrated in ELA cells relative to CV-1, A, B and the hydrazone C resulting from their reaction are lipophilic delocalized cations that selectively inhibit ELA growth relative to CV-1 growth. The hydrazone C is more growth inhibitory than either A or B for both cell lines. A combination of A with an unreactive analogue of B and a combination of B with an unreactive analogue of A did not synergistically inhibit ELA proliferation. The degree of synergism is greater against the ELA cells than against the CV-1 cells. These data, together with hydrazone formation kinetics, suggest that A and B are both concentrated together selectively inside the ELA due to the transmembrane potentials, reacting inside the ELA cells at a higher velocity than inside the CV-1 cells to form the more growth-inhibitory hydrazone C.     

Parkin stabilizes PINK1 through direct interaction

Parkinson disease (PD) is the most common movement disorder and is characterized by dopaminergic dysfunction. The majority of PD cases are sporadic; however, the discovery of genes linked to rare familial forms of the disease has provided crucial insight into the molecular mechanisms of disease pathogenesis. Multiple genes mediating familial forms of Parkinson’s disease (PD) have been identified, such as parkin (PARK2) and phosphatase and tensin homologue deleted on chromosome ten (PTEN)-induced putative kinase 1: PINK1 (PARK6). Here, we showed that Parkin directly interacts with PINK1, but did not bind to pathogenic PINK1 mutants. Parkin, but not its pathogenic mutants, stabilizes PINK1 by interfering with its degradation via the ubiquitin-mediated proteasomal pathway. In addition, the interaction between Parkin and PINK1 resulted in reciprocal reduction of their solubility. Our results indicate that Parkin regulates PINK1 stabilization via direct interaction with PINK1, and operates through a common pathway with PINK1 in the pathogenesis of early-onset PD.     

Evidence for a direct,nucleotide-sensitive interaction between actin and liver cell membranes

We have investigated the association of actin with membranes isolated from rat liver. A plasma membrane-enriched fraction prepared by homogenization in a low salt/CaCl2 buffer was found to contain a substantial amount of residual actin which could be removed by treatment with 1 M Na2CO3/NaHCO3, pH 10.5. Using a sedimentation binding assay that uses gelsolin to shorten actin filaments and render membrane binding saturable (Schwartz, M. A., and E. J. Luna. 1986. J. Cell Biol. 102:2067-2075), we found that membranes stripped of endogenous actin bound 125I-actin in a specific and saturable manner. Scatchard plots of binding data were linear, indicating a single class of binding sites with a Kd of 1.6 microns; 66 micrograms actin bound/mg membrane protein at saturation. Binding of actin to liver cell membranes was negligible with unstripped membranes, was competed by excess unlabeled actin, and was greatly reduced by preheating or proteolytic digestion of the membranes. Kinetic measurements showed that binding had an initial lag phase and was strongly temperature dependent. The binding of actin to liver cell membranes was also found to be competitively inhibited by ATP and other nucleotides, including the nonhydrolyzable analogue AMP-PNP. We conclude that we have reconstituted an interaction between actin and integral membrane proteins from the rat liver. This interaction exhibits a number of distinctive features which have not been observed in other actin- membrane systems.     

Evidence for NQO1 and NQO2 catalyzed reduction of ortho- and para-quinone methides

Abstract

NAD(P)H:quinone oxidoreductase (NQO1) and NRH:quinone oxidoreductase 2 (NQO2) catalyze the two-electron reduction of quinones and thereby prevent generation of toxic radicals. Quinone methides (QMs) covalently react with cellular macromolecules to form DNA adducts and/or protein conjugates resulting in toxicity and carcinogenesis. Based on similar structural features of quinones and QMs, it is logical to assume that NQO1 and/or NQO2 could also catalyze the two-electron reduction of QMs. However, hitherto the reduction of QMs, as both endogenous and/or exogenous biological substrates, by either NQO1/NQO2 has never been demonstrated. Here we show for the first time that both NQO1 and NQO2 can catalyze the reduction of electrophilic ortho-/para-QMs. The involvement of the enzyme in the reduction of p-cresol quinone methide (PCQM) and o-cresol quinone methide (OCQM) was demonstrated by reappearance of NQO1/NQO2-FAD peak at 450 nm after addition of the QMs to the assay mixture. Further reduction of methides by NQO1/NQO2 was confirmed by analyzing the assay mixture by tandem mass spectrometry. Preliminary kinetic studies show that NQO2 is faster in reducing QMs than its homolog NQO1, and moreover, ortho-QMs are reduced faster than para-QMs. Enzyme-substrate docking studies showed results consistent with enzyme catalysis. Thus, NQO1/NQO2 can play a significant role in deactivation of QMs.     

A functional interaction between sprouty proteins and caveolin-1

Growth factor-mediated signal transduction cascades can be regulated spatio-temporally by signaling modulators, such as Sprouty proteins. The four mammalian Sprouty family members are palmitoylated phosphoproteins that can attenuate or potentiate numerous growth factor-induced signaling pathways. Previously, we have shown that Sprouty-1 and Sprouty-2 associate with Caveolin-1, the major structural protein of caveolae. Like Sprouty, Caveolin-1 inhibits growth factor-induced mitogen-activated protein kinase activation. Here, we demonstrate that all four mammalian Sprouty family members physically interact with Caveolin-1. The C terminus of Caveolin-1 is the major Sprouty-binding site, whereas Sprouty binds Caveolin-1 via its conserved C-terminal domain. A single point mutation in this domain results in loss of Caveolin-1 interaction. Moreover, we demonstrate that the various Sprouty isoforms differ dramatically in their cooperation with Caveolin-1-mediated inhibition of mitogen-activated protein kinase activation and that such cooperation is also highly dependent on the type of growth factor investigated and on cell density. Together, the data suggest that the Sprouty/Caveolin-1 interaction modulates signaling in a growth factor- and Sprouty isoform-specific manner.