A modified nucleoside O6-methyl-2'-deoxyguanosine-5'-triphosphate exhibits anti-glioblastoma activity in a caspase-independent manner.

Resistance to temozolomide (TMZ), the frontline chemotherapeutic agent for glioblastoma (GBM), has emerged as a formidable obstacle, underscoring the imperative to identify alternative therapeutic strategies to improve patient outcomes. In this study, we comprehensively evaluated a novel agent, O6-methyl-2'-deoxyguanosine-5'-triphosphate (O6-methyl-dGTP) for its anti-GBM activity both in vitro and in vivo. Notably, O6-methyl-dGTP exhibited pronounced cytotoxicity against GBM cells, including those resistant to TMZ and overexpressing O6-methylguanine-DNA methyltransferase (MGMT). Mechanistic investigations revealed that O6-methyl-dGTP could be incorporated into genomic DNA, disrupting nucleotide pools balance, and inducing replication stress, resulting in S-phase arrest and DNA damage. The compound exerted its anti-tumor properties through the activation of AIF-mediated apoptosis and the parthanatos pathway. In vivo studies using U251 and Ln229 cell xenografts supported the robust tumor-inhibitory capacity of O6-methyl-dGTP. In an orthotopic transplantation model with U87MG cells, O6-methyl-dGTP showcased marginally superior tumor-suppressive activity compared to TMZ. In summary, our research, for the first time, underscores the potential of O6-methyl-dGTP as an effective candidate against GBM, laying a robust scientific groundwork for its potential clinical adoption in GBM treatment regimens.

Exogenous 8-Oxo-7,8-dihydro-2'-deoxyguanosine: Biomedical Properties, Mechanisms of Action, and Therapeutic Potential.

8-Oxo-7,8-dihydroguanine (8-oxo-G) is a key biomarker of oxidative damage to DNA in cells, and its genotoxicity is well-studied. In recent years, it has been confirmed experimentally that free 8-oxo-G and molecules containing it are not merely inert products of DNA repair or degradation, but they are actively involved in intracellular signaling. In this review, data are systematized indicating that free 8-oxo-G and oxidized (containing 8-oxo-G) extracellular DNA function in the body as mediators of stress signaling and initiate inflammatory and immune responses to maintain homeostasis under the action of external pathogens, whereas exogenous 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo) exhibits pronounced antiinflammatory and antioxidant properties. This review describes known action mechanisms of oxidized guanine and 8-oxo-G-containing molecules. Prospects for their use as a therapeutic target are considered, as well as a pharmaceutical agent for treatment of a wide range of diseases whose pathogenesis is significantly contributed to by inflammation and oxidative stress.

8-Oxo-2'-deoxyguanosine ameliorates features of metabolic syndrome in obese mice.

Metabolic syndrome describes a group of clinical features that together increase the incidence of coronary artery disease, stroke and type 2 diabetes. Insulin resistance is a major risk factor for developing metabolic syndrome. A chronic state of inflammation accompanies the accumulation of surplus lipids in adipose and liver tissue, frequently involved in insulin resistance. 8-Oxo-2'-deoxyguanosine (8-Oxo-dG) is a potent anti-inflammatory agent that inactivates both Rac1 and Rac2 which are critical to initiating the inflammatory responses in various cell types, including macrophages. In this study, we explored whether 8-Oxo-dG suppressed a series of systemic inflammatory cascades, resulting in the amelioration of typical features of metabolic syndrome in obese mice. The results demonstrate that 8-Oxo-dG effectively improved hyperglycemia, dyslipidemia and fatty liver changes in obese mice. The level of biochemical markers indicative of systemic inflammation were reduced in 8-Oxo-dG treated mice, whereas serum levels of adiponectin, a crucial factor associated with improved metabolic syndrome, were enhanced. Our results demonstrate that 8-Oxo-dG effectively disrupts the pathogenesis of insulin resistance and obesity-associated metabolic syndrome.

8-hydroxy-2'-deoxyguanosine and cardiovascular disease: a systematic review.

Oxidative stress due to an excess of reactive oxygen species (ROS) may play a role in the development and progression of cardiovascular disease (CVD). 8-hydroxy-2'-deoxyguanosine (8-OHdG) is a marker of oxidative DNA damage caused by ROS. This review aimed to assess the association between 8-OHdG and CVD by reviewing the literature. Studies in human subjects using either plasma or urine to determine 8-OHdG concentrations were surveyed. Eighteen relevant studies were found, of which 13 were case-control studies and five had a prospective design. Without exception, the case-control studies showed significant positive associations between 8-OHdG and CVD. In agreement, two prospective studies showed a significant association of 8-OHdG and heart failure. Furthermore, two prospective studies found a significant association between 8-OHdG and stroke, and finally, one prospective study showed a borderline significant (p = 0.08) association between coronary artery disease (CAD) patients developing a cardiac event and 8-OHdG concentrations. In conclusion, high levels of 8-OHdG in blood and urine are associated with atherosclerosis and heart failure, but further large prospective studies are needed to investigate 8-OHdG as a predictor for cardiovascular diseases.

The Effects of Black Raspberry as a Whole Food-Based Approach on Biomarkers of Oxidative Stress in Buccal Cells and Urine of Smokers.

Cigarette smoke is a rich source of free radicals that can promote oxidative stress and carcinogenesis, including head and neck squamous cell carcinoma (HNSCC) development; importantly, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-iso-prostaglandin F2α (8-isoprostane) are biomarkers of oxidative stress. Several mechanisms, including the antioxidant properties of black raspberry (BRB), account for their chemopreventive effects. In the present clinical trial, we tested the hypothesis that BRB administration reduces biomarkers levels of oxidative stress in buccal cells and urine of smokers. One week after enrolling 21 smokers, baseline buccal cells and urine samples were collected before the administration of BRB lozenges for 8 weeks (5/day, 1 gm BRB/lozenge). Buccal cells and urine samples were collected at the middle and the end of BRB administration. The last samples were collected after the BRB cessation (washout period). We analyzed levels of 8-oxodG and 8-isoprostane (LC/MS-MS), urinary cotinine (ELISA), and creatinine (spectrophotometry). BRB significantly reduced the levels of 8-oxodG by 17.08% (P = 0.00079) in buccal cells and 12.44% (P = 0.034) in urine at the middle of BRB administration as compared with baseline; the corresponding values at the end of BRB administration were 16.46% (P = 0.026) in buccal cells and 25.72% (P = 0.202) in urine. BRB had no significant effect on the levels of urinary 8-isoprostane. BRB's capacity to inhibit 8-oxodG formation of smokers' buccal cells and urine is clearly evident and the reduction in 8-oxodG suggests that antioxidant abilities are central to BRB's HNSCC chemopreventive properties. PREVENTION RELEVANCE: Cigarette smoke contains highly active components namely free radicals that can promote oxidative stress and oral cancer. We found that black raspberry (BRB) inhibited the formation of oxidative stress markers in the oral cavity and urine of smokers suggesting the antioxidant abilities of BRB in preventing oral cancer.

Hydrogen exerts neuroprotective effects by inhibiting oxidative stress in experimental diabetic peripheral neuropathy rats.

Diabetic peripheral neuropathy (DPN) is a complex disorder caused by long-standing diabetes. Oxidative stress was considered the critical creed in this DPN pathophysiology. Hydrogen has antioxidative effects on diabetes mellitus and related complications. However, there is still no concern on the beneficial effects of hydrogen in DPN. This paper aimed to evaluate the effects of exogenous hydrogen to reduce the severity of DPN in streptozotocin-induced diabetic rats. Compared with hydrogen-rich saline treatment, hydrogen inhalation significantly reduced blood glucose levels in diabetic rats in the 4th and 8th weeks. With regard to nerve function, hydrogen administration significantly attenuated the decrease in the velocity of motor nerve conduction in diabetic animals. In addition, hydrogen significantly attenuated oxidative stress by reducing the level of malondialdehyde, reactive oxygen species, and 8-hydroxy-2-deoxyguanosine and meaningfully enhanced the antioxidant capability by partially restoring the activities of superoxide dismutase. Further studies showed that hydrogen significantly upregulated the expression of nuclear factor erythroid-2-related factor 2 and downstream proteins such as catalase and hemeoxygenase-1 in the nerves of diabetic animals. Our paper showed that hydrogen exerts significant protective effects in DPN by downregulating oxidative stress via the pathway of nuclear factor erythroid-2-related factor 2, which suggests its potential value in clinical applications.

SLC43A3 Is a Biomarker of Sensitivity to the Telomeric DNA Damage Mediator 6-Thio-2'-Deoxyguanosine.

Cell membrane transporters facilitate the passage of nucleobases and nucleosides for nucleotide synthesis and metabolism, and are important for the delivery of nucleoside analogues used in anticancer drug therapy. Here, we investigated if cell membrane transporters are involved in the cellular uptake of the nucleoside analogue DNA damage mediator 6-thio-2'-deoxyguanosine (6-thio-dG). A large panel of non-small cell lung cancer (NSCLC) cell lines (73 of 77) were sensitive to 6-thio-dG; only four NSCLC lines were resistant to 6-thio-dG. When analyzed by microarray and RNA sequencing, the resistant NSCLC cell lines clustered together, providing a molecular signature for patients that may not respond to 6-thio-dG. Significant downregulation of solute carrier family 43 A3 (SLC43A3), an equilibrative nucleobase transporter, was identified as a candidate in this molecular resistance signature. High levels of SLC43A3 mRNA predicted sensitivity to 6-thio-dG and therefore SLC43A3 could serve as a promising biomarker for 6-thio-dG sensitivity in patients with NSCLC. SIGNIFICANCE: These findings identify a biomarker of resistance to the telomeric DNA damage mediator 6-thio-2'-deoxyguanosine.

Telomere Stress Potentiates STING-Dependent Anti-tumor Immunity.

Telomerase is an attractive target for anti-tumor therapy as it is almost universally expressed in cancer cells. Here, we show that treatment with a telomere-targeting drug, 6-thio-2'-deoxyguanosine (6-thio-dG), leads to tumor regression through innate and adaptive immune-dependent responses in syngeneic and humanized mouse models of telomerase-expressing cancers. 6-thio-dG treatment causes telomere-associated DNA damages that are sensed by dendritic cells (DCs) and activates the host cytosolic DNA sensing STING/interferon I pathway, resulting in enhanced cross-priming capacity of DCs and tumor-specific CD8+ T cell activation. Moreover, 6-thio-dG overcomes resistance to checkpoint blockade in advanced cancer models. Our results unveil how telomere stress increases innate sensing and adaptive anti-tumor immunity and provide strong rationales for combining telomere-targeting therapy with immunotherapy.

Therapeutic Effects of Topical 8-Oxo-2'-deoxyguanosine on Ethanol-Induced Ocular Chemical Injury Models.

PURPOSE: To evaluate the therapeutic effects of topical 8-oxo-2'-deoxyguanosine (8-oxo-dG) on experimental ocular chemical injury models. METHODS: We created ocular chemical injury models with 8-week-old BALB/c mice (n = 70) by applying 100% ethanol; the mice were then treated with 8-oxo-dG eye drops 10 and 5 mg/mL and phosphate-buffered saline (PBS) twice daily. After 7 days, clinical findings such as corneal integrity, clarity, and neovascularization were assessed. Histology, immunohistochemistry findings, and inflammatory cytokine levels using real-time polymerase chain reactions in the corneas of the mice were also analyzed. RESULTS: Topical application of 8-oxo-dG eye drops resulted in a significant improvement of epithelial defects and clarity, dose dependently (each P < 0.001). Inflammatory cell infiltration and corneal stromal edema were also decreased in the 8-oxo-dG-treated mice compared with PBS-treated controls, based on hematoxylin and eosin staining. The expressions of F4/80 and neutrophil elastase-positive inflammatory cells and IL-1 and TNF-α cytokine levels were significantly reduced in the 8-oxo-dG group compared with the PBS group (each P < 0.01). CONCLUSIONS: Topical 8-oxo-dG application showed an excellent therapeutic effect in ocular chemical injury models by suppressing inflammation.

Anti-inflammatory effects of 8-hydroxydeoxyguanosine in LPS-induced microglia activation: suppression of STAT3-mediated intercellular adhesion molecule-1 expression.

To elucidate the roles of 8-hydroxydeoxyguanosine (oh(8)dG), the nucleoside of 8-hydroxyguanine (oh(8)Gua), we examined the effects of oh(8)dG upon LPS-induced intercellular adhesion molecule-1 (ICAM-1) expression and the underlying mechanisms in brain microglial cells. We found that oh(8)dG reduces LPS-induced reactive oxygen species (ROS) production, STAT3 activation, and ICAM-1 expression. oh(8)dG also suppresses pro-inflammatory cytokines, such as TNF-alpha, IL-6 and IFN-gamma. Overexpression of dominant negative STAT3 completely diminshed STAT3-mediated ICAM-1 transcriptional activity. Chromatin immunoprecipitation studies revealed that oh(8)dG inhibited recruitment of STAT3 to the ICAM-1 promoter, followed by a decrease in ICAM-1 expression. Using mice lacking a functional Toll-like receptor 4 (TLR4), we demonstrated that, while TLR4+/+ microglia were activated by LPS, TLR4-/- microglia exhibited inactivated STAT3 in response to LPS. Evidently, LPS modulates STAT3-dependent ICAM-1 induction through TLR4-mdiated cellular responses. Oh(8)dG apparently plays a role in anti-inflammatory actions via suppression of ICAM-1 gene expression by blockade of the TLR4-STAT3 signal cascade in inflammation-enhanced brain microglia. Therefore, oh(8)dG in the cytosol probably functions as an anti-inflammatory molecule and should be considered as a candidate for development of anti-inflammatory agents.

2,6-Diaminopurinedeoxyriboside as a prodrug of deoxyguanosine in L1210 cells.

The mode of action of the antiproliferative nucleoside analogue 2,6-diaminopurinedeoxyriboside (DAPdR) has been characterized in cultured L1210 cells. A marked concentration-dependent decrease in DNA synthesis and ribonucleotide reductase activity occurred in L1210 cells exposed to 0.05 to 1.0 mM DAPdR. Concomitantly, dGTP levels increased as much as 1100-fold as compared to untreated controls. Adenosine deaminase efficiently catalyzed DAPdR conversion to deoxyguanosine in vitro. In a comparative study, DAPdR and deoxyguanosine gave similar results. A 50% inhibition of cell growth during a 72-h incubation was achieved with 0.14 mM DAPdR or 0.26 mM deoxyguanosine. Deoxycytidine rescued the L1210 cells from DAPdR and deoxyguanosine toxicity to the same extent. DAPdR and deoxyguanosine counteracted the toxic effects of mycophenolic acid with the same efficiency. While DAPdR was not metabolized to its 5'-triphosphate, 2,6-diaminopurine was converted to 2,6-diaminopurineriboside 5'-triphosphate in L1210 cells; accordingly 50% inhibition of cell growth occurred at 0.015 mM 2,6-diaminopurine. Combinations of DAPdR with erythro-9-(2-hydroxy-3-nonyl)adenine or deoxycoformycin resulted in antagonism instead of an expected synergism. These data suggest that DAPdR exerts its toxicity on L1210 cells as a prodrug of deoxyguanosine.

Thresholds of O6-alkylguanine-DNA alkyltransferase which confer significant resistance of human glial tumor xenografts to treatment with 1,3-bis(2-chloroethyl)-1-nitrosourea or temozolomide.

Bis-2-chloroethylnitrosourea (BCNU) or temozolomide (TMZ) were tested alone or in combination with the AGT inhibitors O6-benzyl-2'-deoxyguanosine (dBG) or O6-benzylguanine (BG) against human glial tumor xenografts growing s.c. in athymic mice. Four glioblastoma (SWB77, SWB40, SWB39, and D-54) and one anaplastic oligodendroglioma (SWB61) xenografts having O6-alkylguanine-DNA alkyltransferase (AGT) activities of 75, 45, 10, < 10, and 16 fmol/mg protein, respectively, were used. BCNU at 35 mg/m2 was ineffective against these tumors, although 70 mg/m2 (LD10, 75 mg/m2) produced a marked tumor growth delay (T-C) in D54 but had no effect against SWB40 or SWB77. Coadministration of BG or dBG and BCNU necessitated reduction of the BCNU dose to a maximum of 30 and 35 mg/m2, respectively, because of increased toxicity. Optimized treatment with dBG (250 mg/m2) and BCNU (35 mg/m2) resulted in T-Cs of 30, 29, 11, 16, and 14 days for SWB77, SWB40, SWB39, D-54 and SWB61, respectively. These delays were more pronounced than those induced with optimized, isotoxic treatments with BG (180 mg/m2) and BCNU (30 mg/m2). In comparison to BCNU, TMZ was less toxic, with an LD10 of 400 mg/m2. TMZ (300 mg/m2) was more effective than BCNU against SWB77, SWB40, and SWB61, inducing T-Cs of 23, 53, and 56 days, respectively. BG and dBG enhanced the toxicity of TMZ in athymic mice by decreasing the LD10 from 400 to 200 mg/m2. TMZ (180 mg/m2) with either BG (180 mg/m2) or dBG (250 mg/m2) resulted in T-Cs of 31 and 49 days in SWB77, respectively, as compared with 16 days for TMZ (180 mg/m2) alone. In SWB40, the combination of TMZ with dBG, but not with BG, was significantly more effective than the maximum tolerated dose of TMZ (300 mg/m2) alone. The combination of TMZ with AGT inactivators had no benefit, as compared with TMZ alone, against xenografts with marginal AGT activity. In conclusion, at equimolar doses dBG was less toxic than BG in athymic mice when combined with either BCNU or TMZ. In this regard, BCNU or TMZ can be used at higher doses in combination with dBG than with BG. This study further demonstrates that there is a significant benefit of depleting AGT with nonspecific AGT inhibitors prior to treatment with either BCNU or TMZ in tumors having AGT activity >45 fmol/mg protein.

Eradication of human medulloblastoma tumor xenografts with a combination of O6-benzyl-2'-deoxyguanosine and 1,3-bis(2-chloroethyl)1-nitrosourea.

O6-Benzyl-2'-deoxyguanosine (dBG), a water-soluble inhibitor of O6-methylguanine-DNA methyltransferase (MGMT), potentiates the efficacy of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) against MGMT-positive, BCNU-resistant Daoy human medulloblastoma tumor xenografts in athymic mice (S. C. Schold et al., Cancer Res., 56: 2076-2081, 1996). Such potentiation was comparable to that observed for O6-benzylguanine, the prototype MGMT inhibitor that is currently undergoing clinical trials. In this study, we optimized the therapeutic effect of the dBG and BCNU combination against brain tumor xenografts without inducing substantial toxicity in the host by adjusting the doses of both compounds. dBG was escalated from 133 mg/m2 to 200 and 300 mg/m2, whereas corresponding doses of BCNU were reduced from 25 mg/m2 to 17 and 11 mg/m2, respectively. The growth delays of 30.2, 38.4, and 22.3 days, respectively, observed for the above regimens suggest that the optimal drug combination is not achieved with maximum doses of dBG. In fact, the highest doses of dBG (300 mg/m2) contributed to more frequent BCNU-related toxicities, despite the reduced BCNU dosage, and a reduction of the therapeutic effect. Toxicity was related to the depletion of MGMT activity in the gut of host mice and was manifested by edema, inflammation, and hemorrhage in the bowel wall by subsequent BCNU administration. With additional dosage adjustments, we found that tumor suppression of >90 days without toxicity was observed at 200 mg/m2 dBG and 23 mg/m2 BCNU. At these doses, tumors were eradicated (regressed to an undetectable size for >90 days) in 8 of 12 animals. Thus, dBG is the first of the MGMT inhibitors to show a curative effect in combination with BCNU against a human central nervous system tumor xenograft in athymic mice.

Multi-log cytotoxicity of carbocyclic 2'-deoxyguanosine in HSV-TK-expressing human tumor cells.

Ganciclovir (GCV) is widely used as a prodrug for selective activation in tumor cells expressing herpes simplex virus thymidine kinase (HSV-TK) because of its ability to induce multi-log cytotoxicity to HSV-TK-expressing as well as nonexpressing bystander cells. We now report that another substrate for HSV-TK, D-carbocyclic 2'-deoxyguanosine (CdG), induces multi-log cytotoxicity in HSV-TK-expressing and bystander cells at concentrations

Synthesis of hypoxanthine, guanine, and 6-thiopurine nucleosides of 6-deoxy-D-allofuranose.

Hypoxanthine, guanine, and 6-thiopurine nucleosides of 6-deoxy-D-allofuranose have been prepared as potential antitumor agents. Thus, reaction of 6-deoxy-beta-D-allofuranosyl bromide (1) with the trimethylsilyl derivatives of hypoxanthine and guanine afforded mixtures of the 9- and the 7-substituted bases, which were separated and deblocked with ammonia to give 9-(6'-deoxy-beta-D-allofuranosyl)hypoxanthine (6), 7-(6'-deoxy-beta-D-allofuranosyl)hypoxanthine (7), 9-(6'-deoxy-beta-D-allofuranosyl)guanine (8), and 7-(6'-deoxy-beta-D-allofuranosyl)guanine (9). The two nucleosides with the purine joined at the N-9 position, namely, 6 and 8, are easily distinguished from the other two nucleosides (7 and 9), having N-7 junctions, by their NMR spectra. Reaction of 1 with the trimethylsilyl derivative of 6-chloropurine afforded 10, which upon treatment with thiourea and deblocking gave 9-(6'-deoxy-beta-D-allofuranosyl)-6-thiopurine (12). The hypoxanthine and guanine nucleosides showed no inhibition of mouse leukemia L1210 when tested in vivo, but the thiopurine nucleoside 12 showed strong inhibition of growth of L1210 both in vivo and in vitro. Compound 7 strongly inhibited purine nucleoside phosphorylase (KI = 8.8 X 10(-5) M), while compounds 8, 9, 6, and 12 were inactive.

Fluorocarbocyclic nucleosides: synthesis and antiviral activity of 2'- and 6'-fluorocarbocyclic 2'-deoxy guanosines.

A series of four isomeric 2'- and 6'-fluorocarbocyclic guanosine analogues have been prepared and evaluated as potential anti-herpes agents. The racemic 2' beta-fluoro isomer 2-amino-1,9-dihydro- 9-[(1 alpha, 2 alpha, 3 beta, 4 alpha)-2-fluoro-3-hydroxy-4- (hydroxymethyl)cyclopentyl]-6H-purin-6-one (11a, C-AFG) and its 2' alpha-fluoro epimer 11b plus the chiral 6' beta-fluoro isomer 2-amino-1,9-dihydro-9-[[1S-(1 alpha, 2 alpha, 3 alpha, 4 beta)]- 2-fluoro-4-hydroxy-3-(hydroxymethyl)cyclopentyl]-6H-purine-6-one (11c) and its 6' alpha-fluoro epimer 11d were prepared from their respective fluoro amino diol hydrochlorides (6a,d). For comparison, the furanosyl compound 9-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)guanine (17, AFG) was prepared by coupling 2-amino-6-chloropurine with 2-deoxy-2-fluoro-3,5-di-O-benzoyl-alpha- D-arabinofuranosyl bromide followed by base hydrolysis. The 6' alpha-fluoro derivative 11d exhibited comparable activity to that of acyclovir (ACV) against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in vitro but was greater than 30-fold more active than ACV against HSV-1 and HSV-2 in vivo in the mouse systemic model. The 2' beta-fluoro derivative (11a, C-AFG) was extremely potent in vitro against HSV-1 and HSV-2 (ID50 0.006 and 0.05 micrograms/mL) and in vivo it was greater than 2 orders of magnitude more potent than ACV against HSV-1 and 70-fold more potent against HSV-2. The 2' alpha-fluoro 11b and 6' beta-fluoro 11c isomers were much less active.

Therapeutic potential of a novel synthetic selectin blocker, OJ-R9188, in allergic dermatitis.

1. We investigated the ability of a newly synthesized sugar derivative, OJ-R9188, [N-(2-tetradecylhexadecanoyl)-O-(L-alpha-fucofuranosyl)-D-seryl]-L-glutamic acid 1-methylamide 5-L-arginine salt, to block binding of selectins to their ligands in vitro and inhibit the infiltration of leukocytes in vivo. 2. OJ-R9188 prevented the binding of human E-, P- and L-selectin-IgG fusion proteins to immobilized sialyl Lewis(x) (sLe(x))-pentasaccharide glycolipid, with IC(50) values of 4.3, 1.3, and 1.2 microM, respectively. 3. In a mouse model of thioglycollate-induced peritonitis, OJ-R9188 at 10 mg kg(-1), i.v. inhibited neutrophil accumulation in the peritoneal cavity. In the IgE-mediated skin reaction, OJ-R9188 at 3 and 10 mg kg(-1), i.v. significantly inhibited extravasation of neutrophils and eosinophils into the inflammatory sites and at 10 mg kg(-1), i.v. also inhibited infiltration caused by picryl chloride-induced delayed-type hypersensitivity in mice. These results suggest that OJ-R9188 may be a useful selectin blocker, with activity against human and mouse E-, P- and L-selectins in vitro and in vivo, and that blocking selectin-sLe(x) binding is a promising strategy for the treatment of allergic skin diseases.

Phosphorylation of anticancer nucleoside analogs by human mitochondrial deoxyguanosine kinase.

The kinetic properties of recombinant human mitochondrial deoxyguanosine kinase (dGK, EC 2.7.1.113) for 2'-deoxyguanosine and the clinically important nucleoside analogs 2-chloro-2'-deoxyadenosine (CdA), 9-beta-D-arabinofuranosylguanine (araG) and 2',2',-difluorodeoxyguanosine (dFdG) were determined. The Michaelis-Menten kinetic parameters, comparing ATP and UTP as phosphate donors, demonstrated a marked increase in phosphorylation efficiency (VmaxKm) with UTP in comparison with ATP for both CdA and araG. The difluoro analog dFdG was an efficient substrate for recombinant dGK with an apparent Km of 16 microM with ATP as phosphate donor. We compared the kinetic properties of dGK with those of the related enzyme deoxycytidine kinase (dCK, EC 2.7.1.74). Although the purines 2'-deoxyguanosine (dGuo) and 2'-deoxyadenosine are substrates for both dGK and dCK, only CdA among the purine nucleoside analogs tested was an efficient substrate for both dCK and dGK. In competition with dGuo, the most efficient analog for phosphorylation by dGK was araG, as indicated by a lower Ki value than for CdA and dFdG. Of the purine analogs tested as substrates for dCK, only CdA could compete with 2'-deoxycytidine (dCyd). No inhibition of dCK-mediated dCyd phosphorylation was found by either araG or dFdG. In crude cell extract of HeLa and Capan 2 cells, the major CdA phosphorylation was contributed by dCK, while most araG phosphorylation was a result of dGK activity. Our study with pure recombinant enzymes confirms that dGK is mainly responsible for araG and dFdG phosphorylation, whereas dCK is the most important enzyme for activation of CdA and 2',2'-difluorodeoxycytidine (dFdC).

Immunopharmacological and antitumor effects of second-generation immunomodulatory oligonucleotides containing synthetic CpR motifs.

Bacterial and synthetic DNAs containing CpG dinucleotides in specific sequence contexts (CpG DNA) activate the vertebrate immune system and produce potent Th1 immune responses. Recently, we reported immunomodulatory oligonucleotides (IMOs) containing 3'-3'-attached novel structures (immunomers) and synthetic immunostimulatory CpR (R=2'-deoxy-7-deazguanosine) dinucleotides show potent stimulatory activity with distinct cytokine secretion profiles. In the present study, we evaluated in vivo immunopharmacological and antitumor properties of second-generation immunomodulatory oligonucleotides (IMOs) either alone or in combination with chemotherapeutic agents. Repeated peritumoral administration of IMOs at 1 mg/kg to mice bearing established subcutaneous CT26 colon tumor or B16.F0 melanoma resulted in complete regression or strong inhibition of tumor growth. Direct peritoneal injection of IMOs at 2.5 mg/kg to mice bearing peritoneally implanted ascites CT26 or B16.F0 tumors completely eradicated or inhibited tumor growth. Treatment of mice bearing beta-gal expressing CT26.CL25 tumor with IMOs resulted in a significant tumor-specific CTL responses compared with treatment with a control non-CpG DNA or PBS. These responses correlated with IFN-gamma, but not IL-4 secreted in IMO, treated mice. A 5-fold increase in beta-gal specific IgG2a antibodies was found in mice, significantly increasing the IgG2a/IgG1 ratio. IMOs showed similar antitumor activity in both wt and IL-6 knockout (ko) C57BL/6 mice but failed to elicit activity in IL-12 ko C57BL/6 mice. Tumor-free mice from the IMO treatment group rejected the same tumor cell rechallenge, suggesting an adaptive immune response against these cells. Moreover, naïve mice quickly developed specific antitumor response without IMO treatment following adoptive transfer of splenocytes obtained from tumor free mice from the IMO treated group. Additionally, the co-administration of IMOs with chemotherapeutic agents, docetaxel and doxorubicin, resulted in synergistic antitumor effects in both B16.F0 melanoma and 4T1 breast carcinoma models. These results demonstrate potent antitumor activity of second-generation IMO compounds containing a synthetic CpR stimulatory motif in a broad spectrum of tumor models through induction of strong Th1 immune responses. IMO treatment resulted in the development of tumor-specific memory immune responses. No treatment-related toxicity was observed in mice at the doses and treatment schedules studied.

8-Hydroxydeoxyguanosine: not mere biomarker for oxidative stress, but remedy for oxidative stress-implicated gastrointestinal diseases.

Reactive oxygen species (ROS) attack guanine bases in DNA easily and form 8-hydroxydeoxyguanosine (8-OHdG), which can bind to thymidine rather than cytosine, based on which, the level of 8-OHdG is generally regarded as a biomarker of mutagenesis consequent to oxidative stress. For example, higher levels of 8-OHdG are noted in Helicobacter pylori-associated chronic atrophic gastritis as well as gastric cancer. However, we have found that exogenous 8-OHdG can paradoxically reduce ROS production, attenuate the nuclear factor-κB signaling pathway, and ameliorate the expression of proinflammatory mediators such as interleukin (IL)-1, IL-6, cyclo-oxygenase-2, and inducible nitric oxide synthase in addition to expression of nicotinamide adenine dinucleotide phosphate oxidase (NOX)-1, NOX organizer-1 and NOX activator-1 in various conditions of inflammation-based gastrointestinal (GI) diseases including gastritis, inflammatory bowel disease, pancreatitis, and even colitis-associated carcinogenesis. Our recent finding that exogenous 8-OHdG was very effective in either inflammation-based or oxidative-stress-associated diseases of stress-related mucosal damage has inspired the hope that synthetic 8-OHdG can be a potential candidate for the treatment of inflammation-based GI diseases, as well as the prevention of inflammation-associated GI cancer. In this editorial review, the novel fact that exogenous 8-OHdG can be a functional molecule regulating oxidative-stress-induced gastritis through either antagonizing Rac-guanosine triphosphate binding or blocking the signals responsible for gastric inflammatory cascade is introduced.