ESR studies on DNA cleavage induced by enediyne C-1027 chromophore

C-1027 belongs to the family of chromoprotein antitumor antibiotics, which contain a carrier apoprotein and a highly unstable enediyne chromophore. The enediyne spontaneously aromatizes to generate p-benzyne biradical, and subsequently abstracts hydrogens from the DNA sugar backbone, resulting in cleavage of the double strand. Using spin-trapping methods, we obtained direct proof of radical intermediates during an DNA cleavage, and found intriguing difference in behavior between the trapping agents 2-methyl-2-nitrosopropane (MNP) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO): MNP added to the sugar radicals of the DNA, whereas DMPO directly trapped a phenyl radical or p-benzyne biradical derived from the C-1027 chromophore.     

DNA damage by the enediyne C-1027 results in the inhibition of DNA replication by loss of replication protein A function and activation of DNA-dependent protein kinase.

Treatment of cells with the enediyne C-1027 is highly efficient at inducing single- and double-strand DNA breaks. This agent is highly cytotoxic when used at picomolar levels over a period of days. For this study, C-1027 has been used at higher levels for a much shorter time period to look at early cellular responses to DNA strand breaks. Extracts from cells treated with C-1027 for as little as 2 h are deficient in SV40 DNA replication activity. Treatment with low levels of C-1027 (1-3 nM) does not result in the presence of a replication inhibitor in cell extracts, but they are deficient in replication protein A (RPA) function. Extracts from cells treated with high levels of C-1027 (10 nM) do show the presence of a trans-acting inhibitor of DNA replication. The deficiency in RPA in extracts from cells treated with low levels of C-1027 can be fully complemented by the addition of exogenous RPA, and may be due to a C-1027-induced decrease in the extractability of RPA. This decrease in the extractability of RPA correlates with the appearance of many extraction-resistant intranuclear RPA foci. The trans-acting inhibitor of DNA replication induced by treatment of cells with high levels of C-1027 (10 nM) is DNA-dependent protein kinase (DNA-PK). DNA-PK is activated by the presence of DNA fragments induced by C-1027 treatment, and can be abrogated by removal of the DNA fragments. Although it is activated by DNA damage and phosphorylates RPA, DNA-PK is not required for either RPA focalization or loss of RPA replication activity.     

The radiomimetic enediyne C-1027 induces unusual DNA damage responses to double-strand breaks

Cells lacking the protein kinase ataxia telangiectasia mutated (ATM) have defective responses to DNA double-strand breaks (DSBs), including an inability to activate damage response proteins such as p53. However, we previously showed that cells lacking ATM robustly activate p53 in response to DNA strand breaks induced by the radiomimetic enediyne C-1027. To gain insight into the nature of C-1027-induced ATM-independent damage responses to DNA DSBs, we further examined the molecular mechanisms underlying the cellular response to this unique radiomimetic agent. Like ionizing radiation (IR) and other radiomimetics, breaks induced by C-1027 efficiently activate ATM by phosphorylation at Ser1981, yet unlike other radiomimetics and IR, DNA breaks induced by C-1027 result in normal phosphorylation of p53 and the cell cycle checkpoint kinases (Chk1 and Chk2) in the absence of ATM. In the presence of ATM, but under ATM and Rad3-related kinase (ATR) deficient conditions, C-1027 treatment resulted in a decrease in the level of Chk1 phosphorylation but not in the level of p53 and Chk2 phosphorylation. Only when cells were deficient in both ATM and ATR was there a reduction in the level of phosphorylation of each of these DNA damage response proteins. This reduction was also accompanied by an increased level of cell death in comparison to that of wild-type cells or cells lacking either ATM or ATR. Our findings demonstrate a unique cellular response to C-1027-induced DNA DSBs in that DNA damage response proteins are unaffected by the absence of ATM, as long as ATR is present.     

C-1027-induced alterations in Epstein-Barr viral DNA replication in latently infected cultured human Raji cells: relationship to DNA damage.

This study is the first detailing drug-induced changes in EBV DNA replication intermediates (RIs). Both EBV replication inhibition and damage induction were studied in latently infected human Raji cells treated with the enediyne DNA strand-scission agent C-1027. Analysis of RIs on two-dimensional agarose gels revealed a rapid loss in the EBV bubble arc. When elongation of nascent chains was blocked by aphidicolin, this loss was inhibited, suggesting that C-1027-induced disappearance of RIs was related to maturation of preformed replication molecules in the absence of initiation of new RIs. C-1027 damage to EBV DNA was limited at concentrations where loss of the bubble arc was nearly complete, and none was detected within the replicating origin (ori P)-containing fragment, indicating that replication inhibition occurred in trans. By contrast, the non-nuclear mitochondrial genome was insensitive to replication inhibition but highly sensitive to damage induced by C-1027. C-1027-induced trans inhibition of nuclear but not mitochondrial DNA replication is consistent with a cell cycle checkpoint response to a DNA-damaging agent. EBV replication and Raji cell growth were inhibited at equivalent C-1027 doses.     

Characterization of SgcE6, the flavin reductase component supporting FAD-dependent halogenation and hydroxylation in the biosynthesis of the enediyne antitumor antibiotic C-1027

The C-1027 enediyne antitumor antibiotic from Streptomyces globisporus possesses an ( S )-3-chloro-5-hydroxy-β-tyrosine moiety, the chloro- and hydroxy-substituents of which are installed by a flavin-dependent halogenase SgcC3 and monooxygenase SgcC, respectively. Interestingly, a single flavin reductase, SgcE6, can provide reduced flavin to both enzymes. Bioinformatics analysis reveals that, similar to other flavin reductases involved in natural product biosynthesis, SgcE6 belongs to the HpaC-like subfamily of the Class I flavin reductases. The present study describes the steady-state kinetic characterization of SgcE6 as a strictly NADH- and FAD-specific enzyme.     

Solution structures of C-1027 apoprotein and its complex with the aromatized chromophore

C-1027 is one of the most potent antitumor antibiotic chromoproteins, and is a 1:1 complex of an enediyne chromophore having DNA-cleaving ability and a carrier apoprotein. The three-dimensional solution structures of the 110 residue (10.5 kDa) C-1027 apoprotein and its complex with the aromatized chromophore have been determined separately by homonuclear two-dimensional nuclear magnetic resonance methods. The apoprotein is mainly composed of three antiparallel beta-sheets: four-stranded beta-sheet (43-45, 52-54; 30-38; 92-94; 104-106), three-stranded beta-sheet (4-6; 17-22; 61-66), and two-stranded beta-sheet (70-72; 83-85). The overall structure of the apoprotein is very similar to those of other chromoprotein apoproteins, such as neocarzinostatin and kedarcidin. A hydrophobic pocket with approximate dimensions of 14 A x 12 A x 8 A is formed by the four-stranded beta-sheet and the three loops (39-42; 75-79; 97-100). The holoprotein (complex form with the aromatized chromophore) structure reveals that the aromatized chromophore is bound to the hydrophobic pocket found in the apoprotein. The benzodihydropentalene core of the chromophore is located in the center of the pocket and other substituents (beta-tyrosine, benzoxazine, and aminosugar moieties) are arranged around the core. Major binding interactions between the apoprotein and the chromophore are likely the hydrophobic contacts between the core of the chromophore and the hydrophobic side-chains of the pocket-forming residues, which is supplemented by salt bridges and/or hydrogen bonds. Based on the holoprotein structure, we propose possible mechanisms for the stabilization and the release of chromophore by the apoprotein.     

The cellular response to DNA damage induced by the enediynes C-1027 and neocarzinostatin includes hyperphosphorylation and increased nuclear retention of replication protein a (RPA) and trans inhibition of DNA replication

This study examined the cellular response to DNA damage induced by antitumor enediynes C-1027 and neocarzinostatin. Treatment of cells with either agent induced hyperphosphorylation of RPA32, the middle subunit of replication protein A, and increased nuclear retention of RPA. Nearly all of the RPA32 that was not readily extractable from the nucleus was hyperphosphorylated, compared to < or =50% of the soluble RPA. Enediyne concentrations that induced RPA32 hyperphosphorylation also decreased cell-free SV40 DNA replication competence in extracts of treated cells. This decrease did not result from damage to the DNA template, indicating trans-acting inhibition of DNA replication. Enediyne-induced RPA hyperphosphorylation was unaffected by the replication elongation inhibitor aphidicolin, suggesting that the cellular response to enediyne DNA damage was not dependent on elongation of replicating DNA. Neither recovery of replication competence nor reversal of RPA effects occurred when treated cells were further incubated in the absence of drug. C-1027 and neocarzinostatin doses that caused similar levels of DNA damage resulted in equivalent increases in RPA32 hyperphosphorylation and RPA nuclear retention and decreases in replication activity, suggesting a common response to enediyne-induced DNA damage. By contrast, DNA damage induced by C-1027 was at least 5-fold more cytotoxic than that induced by neocarzinostatin.     

A Specific A/T Polymorphism in Western Tyrosine Phosphorylation B-Motifs Regulates Helicobacter pylori CagA Epithelial Cell Interactions

Helicobacter pylori persistently colonizes the human stomach, with mixed roles in human health. The CagA protein, a key host-interaction factor, is translocated by a type IV secretion system into host epithelial cells, where its EPIYA tyrosine phosphorylation motifs (TPMs) are recognized by host cell kinases, leading to multiple host cell signaling cascades. The CagA TPMs have been described as type A, B, C or D, each with a specific conserved amino acid sequence surrounding EPIYA. Database searching revealed strong non-random distribution of the B-motifs (including EPIYA and EPIYT) in Western H. pylori isolates. In silico analysis of Western H. pylori CagA sequences provided evidence that the EPIYT B-TPMs are significantly less associated with gastric cancer than the EPIYA B-TPMs. By generating and using a phosphorylated CagA B-TPM-specific antibody, we demonstrated the phosphorylated state of the CagA B-TPM EPIYT during H. pylori co-culture with host cells. We also showed that within host cells, CagA interaction with phosphoinositol 3-kinase (PI3-kinase) was B-TPM tyrosine-phosphorylation-dependent, and the recombinant CagA with EPIYT B-TPM had higher affinity to PI3-kinase and enhanced induction of AKT than the isogenic CagA with EPIYA B-TPM. Structural modeling of the CagA B-TPM motif bound to PI3-kinase indicated that the threonine residue at the pY+1 position forms a side-chain hydrogen bond to N-417 of PI3-kinase, which cannot be formed by alanine. During co-culture with AGS cells, an H. pylori strain with a CagA EPIYT B-TPM had significantly attenuated induction of interleukin-8 and hummingbird phenotype, compared to the isogenic strain with B-TPM EPIYA. These results suggest that the A/T polymorphisms could regulate CagA activity through interfering with host signaling pathways related to carcinogenesis, thus influencing cancer risk.     

Substrate specificity of the adenylation enzyme SgcC1 involved in the biosynthesis of the enediyne antitumor antibiotic C-1027

C-1027 is an enediyne antitumor antibiotic composed of a chromophore with four distinct chemical moieties, including an (S)-3-chloro-4,5-dihydroxy-beta-phenylalanine moiety that is derived from l-alpha-tyrosine. SgcC4, a novel aminomutase requiring no added co-factor that catalyzes the formation of the first intermediate (S)-beta-tyrosine and subsequently SgcC1 homologous to adenylation domains of nonribosomal peptide synthetases, was identified as specific for the SgcC4 product and did not recognize any alpha-amino acids. To definitively establish the substrate for SgcC1, a full kinetic characterization of the enzyme was performed using amino acid-dependent ATP-[(32)P]PP(i) exchange assay to monitor amino acid activation and electrospray ionization-Fourier transform mass spectroscopy to follow the loading of the activated beta-amino acid substrate to the peptidyl carrier protein SgcC2. The data establish (S)-beta-tyrosine as the preferred substrate, although SgcC1 shows promiscuous activity toward aromatic beta-amino acids such as beta-phenylalanine, 3-chloro-beta-tyrosine, and 3-hydroxy-beta-tyrosine, but all were <50-fold efficient. A putative active site mutant P571A adjacent to the invariant aspartic acid residue of all alpha-amino acid-specific adenylation domains known to date was prepared as a preliminary attempt to probe the substrate specificity of SgcC1; however the mutation resulted in a loss of activity with all substrates except (S)-beta-tyrosine, which was 142-fold less efficient relative to the wild-type enzyme. In total, SgcC1 is now confirmed to catalyze the second step in the biosynthesis of the (S)-3-chloro-4,5-dihydroxy-beta-phenylalanine moiety of C-1027, presenting downstream enzymes with an (S)-beta-tyrosyl-S-SgcC2 thioester substrate, and represents the first beta-amino acid-specific adenylation enzyme characterized biochemically.     

Helicobacter pylori Promotes Epithelial–Mesenchymal Transition in Gastric Cancer by Downregulating Programmed Cell Death Protein 4 (PDCD4)

Helicobacter pylori, a Gram-negative, microaerophilic bacterium found in the stomach, is assumed to be associated with carcinogenesis, invasion and metastasis in digestive diseases. Cytotoxin-associated gene A (CagA) is an oncogenic protein of H. pylori that is encoded by a Cag pathogenicity island related to the development of gastric cancer. The epithelial–mesenchymal transition (EMT) is the main biological event in invasion or metastasis of epithelial cells. H. pylori may promote EMT in human gastric cancer cell lines, but the specific mechanisms are still obscure. We explored the underlying molecular mechanism of EMT induced by H. pylori CagA in gastric cancer. In our article, we detected gastric cancer specimens and adjacent non-cancerous specimens by immunohistochemistry and found increased expression of the EMT-related regulatory protein TWIST1 and the mesenchymal marker vimentin in cancer tissues, while programmed cell death factor 4 (PDCD4) and the epithelial marker E-cadherin expression decreased in cancer specimens. These changes were associated with degree of tissue malignancy. In addition, PDCD4 and TWIST1 levels were related. In gastric cancer cells cocultured with CagA expression plasmid, CagA activated TWIST1 and vimentin expression, and inhibited E-cadherin expression by downregulating PDCD4. CagA also promoted mobility of gastric cancer cells by regulating PDCD4. Thus, H. pylori CagA induced EMT in gastric cancer cells, which reveals a new signaling pathway of EMT in gastric cancer cell lines.     

A Unified Mechanism for Aminopeptidase N-based Tumor Cell Motility and Tumor-homing Therapy

Tumor cell surface aminopeptidase N (APN or CD13) has two puzzling functions unrelated to its enzymatic activity: mediating tumor cell motility and serving as a receptor for tumor-homing peptides (peptides that bring anti-cancer drugs to tumor cells). To investigate APN-based tumor-homing therapy, we determined the crystal structure of APN complexed with a tumor-homing peptide containing a representative Asn-Gly-Arg (NGR) motif. The tumor-homing peptide binds to the APN enzymatic active site, but it resists APN degradation due to a distorted scissile peptide bond. To explore APN-based tumor cell motility, we examined the interactions between APN and extracellular matrix (ECM) proteins. APN binds to, but does not degrade, NGR motifs in ECM proteins that share similar conformations with the NGR motif in the APN-bound tumor-homing peptide. Therefore, APN-based tumor cell motility and tumor-homing therapy rely on a unified mechanism in which both functions are driven by the specific and stable interactions between APN and the NGR motifs in ECM proteins and tumor-homing peptides. This study further implicates APN as an integrin-like molecule that functions broadly in cell motility and adhesion by interacting with its signature NGR motifs in the extracellular environment.     

Conformational Analysis of Isolated Domains of Helicobacter pylori CagA

The CagA protein of Helicobacter pylori is associated with increased virulence and gastric cancer risk. CagA is translocated into the host cell by a H. pylori type IV secretion system via mechanisms that are poorly understood. Translocated CagA interacts with numerous host factors, altering a variety of host signalling pathways. The recently determined crystal structure of C-terminally-truncated CagA indicated the presence of two domains: the smaller, flexible N-terminal domain and the larger, middle domain. In this study, we have investigated the conformation, oligomeric state and stability of the N-terminal, middle and glutamate-proline-isoleucine-tyrosine-alanine (EPIYA)-repeats domains. All three domains are monomeric, suggesting that the multimerisation of CagA observed in infected cells is likely to be mediated not by CagA itself but by its interacting partners. The middle and the C-terminal domains, but not the N-terminal domain, are capable of refolding spontaneously upon heat denaturation, lending support to the hypothesis that unfolded CagA is threaded C-terminus first through the type IV secretion channel with its N-terminal domain, which likely requires interactions with other domains to refold, being threaded last. Our findings also revealed that the C-terminal EPIYA-repeats domain of CagA exists in an intrinsically disordered premolten globule state with regions in PPII conformation - a feature that is shared by many scaffold proteins that bind multiple protein components of signalling pathways. Taken together, these results provide a deeper understanding of the physicochemical properties of CagA that underpin its complex cellular and oncogenic functions.     

Cloning, expression and characterization of gene sgcD involved in the biosynthesis of novel antitumor lidamycin

Lidamycin with high antitumor activity is a novel enediyne antitumor antibiotic produced by Streptomyces globisporus C1027. The 75 kb biosynthesis gene cluster of lidamycin containing 33 open reading frames has been cloned from S. globisporus C1027. In this paper, the function of sgcD (ORF24) is investigated. Gene disruption experiment proved that sgcD is involved in lidamycin biosynthesis. With homologous comparing analysis, we deduce that sgcD codes aminomutase catalyzing a-tyrosine to β-tyrosine which is one motif for lidamycin. To identify the function of enzyme coded by sgcD, sgcD is cloned into vector pET30a for inducing expression and the activity of expression product is analyzed. The result showed that the expression product of sgcD has the activity of aminomutase. Aminomutase coded by sgcD is the first characterized enzyme involved in the biosynthesis of enediyne antitumor antibiotics. Our research will be helpful to clarifying the biosynthesis mechanism of such kind of antibiotic and to producing new antitumor compounds.     

Amplification of C1027-induced DNA cleavage and apoptosis by a quinacrine-netropsin hybrid molecule in tumor cell lines

We examined the effect of a newly synthesized DNA-binding ligand, quinacrine-netropsin hybrid molecule (QN), on cytotoxicity, apoptosis, and DNA strand breaks induced by an enediyne antitumor antibiotic, C1027. QN significantly enhanced C1027-induced cellular DNA strand breaks, caspase-3 activation, and DNA ladder formation, characteristic of apoptosis, in human HL-60 cells. Flow cytometry revealed that C1027-induced intracellular H(2)O(2) generation was enhanced by QN, suggesting that QN enhances C1027-induced cytotoxic effect through H(2)O(2)-mediated apoptosis. QN also significantly enhanced C1027-induced apoptosis in BJAB cells, and the inhibition of apoptosis was observed in BJAB cells transfected with Bcl-2 gene. The experiment using (32)P-labeled DNA fragments showed that the addition of QN enhanced C1027-induced double-stranded DNA cleavage at the 5'-AGG-3'/3'-TCC-5' sequence (cutting sites are underlined). These results suggest that QN enhances C1027-induced antitumor effect via DNA cleavage and apoptosis. The present study shows a novel approach to the potentially effective anticancer therapy.     

Helicobacter pylori Counteracts the Apoptotic Action of Its VacA Toxin by Injecting the CagA Protein into Gastric Epithelial Cells

Infection with Helicobacter pylori is responsible for gastritis and gastroduodenal ulcers but is also a high risk factor for the development of gastric adenocarcinoma and lymphoma. The most pathogenic H. pylori strains (i.e., the so-called type I strains) associate the CagA virulence protein with an active VacA cytotoxin but the rationale for this association is unknown. CagA, directly injected by the bacterium into colonized epithelium via a type IV secretion system, leads to cellular morphological, anti-apoptotic and proinflammatory effects responsible in the long-term (years or decades) for ulcer and cancer. VacA, via pinocytosis and intracellular trafficking, induces epithelial cell apoptosis and vacuolation. Using human gastric epithelial cells in culture transfected with cDNA encoding for either the wild-type 38 kDa C-terminal signaling domain of CagA or its non-tyrosine-phosphorylatable mutant form, we found that, depending on tyrosine-phosphorylation by host kinases, CagA inhibited VacA-induced apoptosis by two complementary mechanisms. Tyrosine-phosphorylated CagA prevented pinocytosed VacA to reach its target intracellular compartments. Unphosphorylated CagA triggered an anti-apoptotic activity blocking VacA-induced apoptosis at the mitochondrial level without affecting the intracellular trafficking of the toxin. Assaying the level of apoptosis of gastric epithelial cells infected with wild-type CagA⁺/VacA⁺ H. pylori or isogenic mutants lacking of either CagA or VacA, we confirmed the results obtained in cells transfected with the CagA C-ter constructions showing that CagA antagonizes VacA-induced apoptosis. VacA toxin plays a role during H. pylori stomach colonization. However, once bacteria have colonized the gastric niche, the apoptotic action of VacA might be detrimental for the survival of H. pylori adherent to the mucosa. CagA association with VacA is thus a novel, highly ingenious microbial strategy to locally protect its ecological niche against a bacterial virulence factor, with however detrimental consequences for the human host.     

Helicobacter pylori promotes epithelial-to-mesenchymal transition by downregulating CK2β in gastric cancer cells

Strains of Helicobacter pylori that are positive for the oncoprotein CagA (cytotoxin-associated gene A) are associated with gastric cancer and might be related to the epithelial-to-mesenchymal transition (EMT). Casein kinase 2 (CK2) is a serine/threonine protein kinase that plays a major role in tumorigenesis through signaling pathways related to the EMT. However, the role played by the interaction between CagA and CK2 in gastric carcinogenesis is poorly understood. Although CK2α protein expression remained unchanged during H. pylori infection, we found that CK2α kinase activity was increased in gastric epithelial cells. We also found that the CK2β protein level decreased in H. pylori-infected gastric cancer cells in CagA-dependent manner and demonstrated that CagA induced CK2β degradation via HDM2 (human double minute 2; its murine equivalent is MDM2). We observed that CagA induced HDM2 protein phosphorylation and that p53 levels were decreased in H. pylori-infected gastric cancer cells. In addition, downregulation of CK2β induced AKT Ser473 phosphorylation and decreased the AKT Ser129 phosphorylation level in gastric cancer cells. We also found that the downregulation of CK2β triggered the upregulation of Snail levels in gastric cancer cells. Furthermore, our in vivo experiments and functional assays of migration and colony formation suggest that CK2β downregulation is a major factor responsible for the EMT in gastric cancer. Therefore, CK2 could be a key mediator of the EMT in H. pylori-infected gastric cancer and could serve as a molecular target for gastric cancer treatment.     

Cloning, expression and characterization of gene sgcD involved in the biosynthesis of novel antitumor lidamycin

Lidamycin, an antitumor antibiotic composed of a macro-molecule peptide and enediyne chromophore[1] and originally named C1027, is produced by Streptomyces globisporus C1027 isolated from the soil in Qianjiang County, Hubei Province, China. It has extremely high antitu- mor activity, which has been proved to be the highest among antitumor compounds[2], being 1000- fold higher than that of adriamycin commonly used in clinic. The structure of lidamycin consists of an acid apoprotein and a chr…     

Stability of Helicobacter pylori CagA oncoprotein in human gastric epithelial cells

Upon delivery into gastric epithelial cells, Helicobacter pylori cytotoxin-associated gene A (CagA) binds and deregulates cellular proteins such as Src homology 2 domain-containing protein tyrosine phosphatase 2 and partitioning-defective 1 (PAR1), thereby acting as an epigenetic oncoprotein that promotes early phases of gastric cancer development. To elucidate the spatial and temporal contribution of CagA to carcinogenesis, it is crucial to know the stability of CagA in host cells. Here we show that the biological half-life of CagA is about 200 min in gastric epithelial cells. Furthermore, deletion of the PAR1-binding sequence accelerates CagA degradation. Thus, CagA is a relatively short half-life protein whose stability may be modulated through complex formation with PAR1.     

Design,synthesis and biological evaluation of phenol-linked uncialamycin antibody-drug conjugates

Uncialamycin is one of the structurally simpler and newer members of enediyne family of natural products. It exhibits highly potent activity against several types of bacteria and cancer cells. Described herein is a strategy for the targeted delivery of this cytotoxic agent to tumors using an antibody-drug conjugate (ADC) approach. Central to the design of ADC were the generation of potent and chemically stable uncialamycin analogues and attachment of protease cleavable linkers to newly realized phenolic handles to prepare linker-payloads. Conjugation of the linker-payloads to tumor targeting antibody, in vitro activity and in vivo evaluation are presented.