Identification through high-throughput screening of 4'-methoxyflavone and 3',4'-dimethoxyflavone as novel neuroprotective inhibitors of parthanatos.

BACKGROUND AND PURPOSE: The current lack of disease-modifying therapeutics to manage neurological and neurodegenerative conditions justifies the development of more efficacious agents. One distinct pathway leading to neuronal death in these conditions and which represents a very promising and attractive therapeutic target is parthanatos, involving overactivation of PARP-1. We therefore sought to identify small molecules that could be neuroprotective by targeting the pathway. EXPERIMENTAL APPROACH: Using HeLa cells, we developed and optimized an assay for high-throughput screening of about 5120 small molecules. Structure-activity relationship (SAR) study was carried out in HeLa and SH-SY5Y cells for molecules related to the initial active compound. The neuroprotective ability of each active compound was tested in cortical neuronal cultures. KEY RESULTS: 4'-Methoxyflavone (4MF) showed activity by preventing the decrease in cell viability of HeLa and SH-SY5Y cells caused by the DNA-alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), which induces parthanatos. A similar compound from the SAR study, 3',4'-dimethoxyflavone (DMF), also showed significant activity. Both compounds reduced the synthesis and accumulation of poly (ADP-ribose) polymer and protected cortical neurones against cell death induced by NMDA. CONCLUSIONS AND IMPLICATIONS: Our data reveal additional neuroprotective members of the flavone class of flavonoids and show that methoxylation of the parent flavone structure at position 4' confers parthanatos-inhibiting activity while additional methoxylation at position 3', reported by others to improve metabolic stability, does not destroy the activity. These molecules may therefore serve as leads for the development of novel neurotherapeutics for the management of neurological and neurodegenerative conditions.

Role of recombinant human erythropoietin in mitomycin C-induced genotoxicity: analysis of DNA fragmentation, chromosome aberrations and micronuclei in rat bone-marrow cells.

Mitomycin C (MMC) is one of the most effective chemotherapeutic agents. However, during clinical use several side effects may occur. Recombinant human erythropoietin (rhEPO), a glycoprotein that regulates haematopoiesis, has been shown to exert an important cyto-protective effect in many tissues. The aim of this study was to explore whether rhEPO protects against MMC-induced genotoxicity in rat bone-marrow cells. Adult male Wistar rats were divided into six groups of 18 animals each: a control group, a 'rhEPO alone' group, an 'MMC alone' group and three 'rhEPO+MMC' groups (pre-, co- and post-treatment conditions). Our results show that MMC induced a noticeable genotoxic effect in rat bone-marrow cells. rhEPO reduced the effects of MMC significantly in every type of experiment conducted, such as the frequency of micronuclei, the percentage of chromosome aberrations and the level of DNA damage measured with the comet assay. The protective effect of rhEPO was more efficient when it was given 24h prior to MMC treatment.

Protection by Salvia extracts against oxidative and alkylation damage to DNA in human HCT15 and CO115 cells.

DNA damage induced by oxidative and alkylating agents contributes to carcinogenesis, leading to possible mutations if replication proceeds without proper repair. However, some alkylating agents are used in cancer therapy due to their ability to induce DNA damage and subsequently apoptosis of tumor cells. In this study, the genotoxic effects of oxidative hydrogen peroxide (H2O2) and alkylating agents N-methyl-N-nitrosourea (MNU) and 1,3-bis-(2-chloroethyl)-1-nitosourea (BCNU) agents were examined in two colon cell lines (HCT15 and CO115). DNA damage was assessed by the comet assay with and without lesion-specific repair enzymes. Genotoxic agents were used for induction of DNA damage in both cell lines. Protective effects of extracts of three Salvia species, Salvia officinalis (SO), Salvia fruticosa (SF), and Salvia lavandulifolia (SL), against DNA damage induced by oxidative and alkylating agents were also determined. SO and SF protected against oxidative DNA damage in HCT15 cells. SO and SL decreased DNA damage induced by MNU in CO115 cells. In addition to chemopreventive effects of sage plant extracts, it was also important to know whether these plant extracts may interfere with alkylating agents such as BCNU used in cancer therapy, decreasing their efficacy. Our results showed that sage extracts tested and rosmarinic acid (RA), the main constituent, protected CO115 cells from DNA damage induced by BCNU. In HCT15 cells, only SF induced a reduction in BCNU-induced DNA damage. Sage water extracts and RA did not markedly change DNA repair protein expression in either cell line. Data showed that sage tea protected colon cells against oxidative and alkylating DNA damage and may also interfere with efficacy of alkylating agents used in cancer therapy.

Beetroot juice protects against N-nitrosodiethylamine-induced liver injury in rats.

Red beetroot, a common ingredient of diet, is a rich source of a specific class of antioxidants, betalains. Our previous studies have shown the protective role of beetroot juice against carcinogen induced oxidative stress in rats. The aim of this study was to examine the effect of long term feeding (28 days) with beetroot juice on phase I and phase II enzymes, DNA damage and liver injury induced by hepatocarcinogenic N-nitrosodiethylamine (NDEA). Long term feeding with beetroot juice decreased the activities of enzymatic markers of cytochrome P450, CYP1A1/1A2 and CYP2E1. NDEA treatment also reduced the activities of these enzymes, but increased the activity of CYP2B. Moreover, combined treatment with beetroot juice and NDEA enhanced significantly CYP2B only. Modulation of P450 enzyme activities was accompanied by changes in the relevant proteins levels. Increased level and activity of NQO1 was the most significant change among phase II enzymes. Beetroot juice reduced the DNA damage increased as the result of NDEA treatment, as well as the biomarkers of liver injury. Collectively, these results confirm the protective effect of beetroot juice against oxidative damage shown in our previous studies and indicate that metabolic alterations induced by beetroot feeding may protect against liver damage.

Phenylbutyrate inhibits homologous recombination induced by camptothecin and methyl methanesulfonate.

Homologous recombination is accompanied by extensive changes to chromatin organization at the site of DNA damage. Some of these changes are mediated through acetylation/deacetylation of histones. Here, we show that recombinational repair of DNA damage induced by the anti-cancer drug camptothecin (CPT) and the alkylating agent methyl methanesulfonate (MMS) is blocked by sodium phenylbutyrate (PBA) in the budding yeast Saccharomyces cerevisiae. In particular, PBA suppresses CPT- and MMS-induced genetic recombination as well as DNA double-strand break repair during mating-type interconversion. Treatment with PBA is accompanied by a dramatic reduction in histone H4 lysine 8 acetylation. Live cell imaging of homologous recombination proteins indicates that repair of CPT-induced DNA damage is redirected to a non-recombinogenic pathway in the presence of PBA without loss in cell viability. In contrast, the suppression of MMS-induced recombination by PBA is accompanied by a dramatic loss in cell viability. Taken together, our results demonstrate that PBA inhibits DNA damage-induced homologous recombination likely by mediating changes in chromatin acetylation. Moreover, the combination of PBA with genotoxic agents can lead to different cell fates depending on the type of DNA damage inflicted.

Vesicular flavonoid in combating diethylnitrosamine induced hepatocarcinoma in rat model.

Reactive oxygen species (O2(*-), OH(-), H2O2) are known to play an important role in tumor initiation in hepatocarcinoma. Hepatocarcinoma was developed in the Swiss Albino rats by administration three doses of diethylnitrosamine (DEN) (200 mg/kg b. wt.) (i.p.) at 15 days interval. Quercetin (QC), herbal polyphenolic compound, is a potent anticancer drug. Clinical trials are difficult for its hydrophobic nature. To overcome this problem, our study was aimed to formulate soluble liver specific, galactosylated liposomal QC and to investigate its efficacy against hepatocarcinoma in rat model. Galactosylated liposomal QC was formulated and the suspension was introduced intravenously to rats (8.98 microM/kg) once in a week for 16 weeks. Hepatocarcinoma in rat model and its pathological improvement were evaluated histopathologically, histochemically and electron microscopically. Severe oxidative damage was noticed in the whole liver and its microsomal fraction of DEN treated rats. Huge numbers of hyperplastic nodules (HNs) with pre-neoplastic lesions appeared in rat liver by DEN administration. Galactosylated liposomal QC injections prevented DEN mediated development of hepatocarcinoma and oxidative damage in rat liver. Quercetin in liver specific galactosylated liposomal drug delivery system may be recommended as a potent therapeutic formulation against DEN-induced hepatocarcinoma.

Hepatoprotective effect of vitamin B12 on dimethylnitrosamine-induced liver injury.

Vitamin B(12) contains a cobalt complex and accumulates at high levels in the liver. Vitamin B(12) was examined for its hepatoprotective effect on dimethylnitrosamine-induced liver injury in mice. Vitamin B(12) decreased the blood levels of aspartate aminotransferase and alanine aminotransferase, and clearly inhibited the overaccumulation of collagen fibrils. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of the liver showed that the gene expression of alpha-smooth muscle actin and heat-shock protein 47, which are markers of fibrosis, were suppressed by vitamin B(12) administration. Our findings indicate that vitamin B(12) could be an effective hepatoprotective agent.

Protective effects of Cuscutae semen against dimethylnitrosamine-induced acute liver injury in Sprague-Dawley rats.

We investigated the protective effect of Cuscutae semen (CS) on acute liver injury induced by dimethylnitrosamine (DMN) in Sprague-Dawley rats. CS is an important traditional herbal medicine widely used as a tonic and aphrodisiac to nourish the liver and kidney and to treat impotence and seminal emission. Rats were given a single intraperitoneal injection of DMN (40 mg/kg), and were then treated with CS daily by oral gavage for 4 d. Immunohistochemical studies for alpha-smooth muscle actin (alpha-SMA) and proliferating cell nuclear antigen (PCNA) were performed, along with hydroxyproline and biological assay. Liver injury caused by DMN-injection was significantly inhibited in the CS-treated group compared to the silymarin-treated group. The results of blood biological assay were significantly protected by CS in serum total protein (T-protein), T-bilirubin (T-bili), D-bilirubin (D-bili), GOT, GPT, and ALP. The hydroxyproline content and amount of active alpha-SMA and PCNA were significantly decreased in the CS-treated group than in the silymarin-treated group. CS exhibited an in vivo hepatoprotective effect and anti-fibrogenic effects against DMN-induced acute liver injury and inhibited the formation of hydroxyproline, which suggests that CS may be useful in preventing fibrogenesis after liver injury.

Impediment of diethylnitrosamine induced hepatotoxicity in male Balb/c mice by pretreatment with aqueous Azadirachta indica leaf extract.

Considering the hepatoprotective properties of Azadirachta indica, the present study was designed to evaluate its preventive effects against diethylnitrosamine (NDEA) induced hepatotoxicity in male Balb/c mice. Exposure of NDEA caused a significant increase in micronucleated cell score, lipid peroxidation levels (LPO) and activity of lactate dehydrogenase (LDH). A significant decrease in reduced glutathione (GSH) contents and activity of glutathione-S-transferase (GST) was also observed upon NDEA treatment, whereas their activities of cytochrome P450 and cytochrome b5 showed non-significant alterations. Aqueous A. indica leaf extract (AAILE) pretreatment showed protective effects against NDEA induced toxicity by decreasing the frequency of micronucleated cell, levels of LPO and LDH activity. Also, a decreased activity of GST, cytochrome P450 and an increased activity of cytochrome b5, GSH contents was observed when AAILE pretreated mice were injected with NDEA. Only AAILE treatment caused a noticeable decrease in the frequency of micronuclei, activity of cytochrome P450 and cytochrome b5, but a significant increase in the activity of GST and GSH contents, whereas, non significant alterations were observed in the activity of LDH and levels of LPO. Significance of these observations with respect to hepatoprotective efficacy of A. indica has been discussed in the present manuscript.

Prevention of 1,2-dimethylhydrazine-induced circulatory oxidative stress by bis-1,7-(2-hydroxyphenyl)-hepta-1,6-diene-3,5-dione during colon carcinogenesis.

We have performed this study to investigate the modulatory effect of bis-1,7-(2-hydroxyphenyl)-hepta-1,6-diene-3,5-dione, a bisdemethoxy curcumin analog (BDMCA) on circulatory lipid peroxidation (LPO) and antioxidant status during 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis in male Wistar rats. The effects were compared with that of the reference drug, curcumin. Increased tumor incidence as well as enhanced LPO in the circulation of tumor bearing rats was accompanied by a significant decrease in the level of reduced glutathione and activities of glutathione peroxidase (GPx), glutathione S-transferase (GST), superoxide dismutase (SOD) and catalase (CAT). Intragastric administration of BDMCA or curcumin to DMH-treated rats significantly decreased colon tumor incidence and the circulatory LPO, with simultaneous enhancement of GSH content and GPx, GST, SOD and CAT activities. We report that BDMCA exert its chemopreventive effect by decreasing the colon tumor incidence as well as by modulating circulatory oxidative stress in DMH-treated rats through its influence on LPO and antioxidant status. The effects of BDMCA were comparable with that of the reference compound curcumin, a well known anticarcinogen and antioxidant. Thus, it would be suggested that the methoxy group is not responsible for the beneficial effects, however, the terminal phenolic moieties or the central 7-carbon chain may play a role.

Poly ADP-ribose polymerase (PARP) inhibitors transiently protect leukemia cells from alkylating agent induced cell death by three different effects.

Polyadenosylation of nuclear enzymes is well known to regulate the cellular repair capacity after DNA damage. PARP mediates the transfer of poly-ADP-ribose moieties on itself and other nuclear proteins by the breakdown of NAD+. The present study investigated how modulation of PARP activity interferes with cell death induced by two different alkylating agents used in cancer chemotherapy. 1-methyl-3-nitro-1-nitrosoguanidinium (MNNG) decreased cellular reduction capacity (WST-1 assay) in HL60 and CCRF-CEM cells, accompanied by increased activity of PARP and depletion of intracellular NAD+ and ATP. Pretreatment with the PARP inhibitors 3-AB or 4-AN resulted in transient cell protection, which was associated with a switch from necrosis to apoptosis in CCRF-CEM cells and enhanced apoptosis in HL60 cells. Both PARP inhibitors delayed the drop in WST-1 reduction and retained NAD+ and ATP levels required for apoptosis. Furthermore, 3-AB or 4-AN prevented progressive DNA degradation in MNNG-treated CCRF-CEM cells. In contrast to MNNG, we did not observe early activation of PARP, decrease in WST-1 reduction, or wasteful consumption of NAD+ and ATP after treatment with melphalan. However, preincubation with 3-AB or 4-AN resulted in decreased HL60 cell membrane blebbing and reduced formation of apoptotic bodies. In conclusion, the cell death preventing effects of PARP inhibitors are mediated by their ability to maintain cellular energy metabolism, to inhibit the activation of endonucleolytic DNA degradation and to prevent cell blebbing. Surprisingly, these protective effects of PARP inhibitors on different cell functions seem to be independent of each other and are rather determined by the respective cytotoxic mechanisms implicated by different drugs. Our results support the hypothesis, that PARP activation and/or cleavage plays a regulatory role in the induction of apoptosis.

Nicotinamide prevents N-methyl-N-nitrosourea-induced photoreceptor cell apoptosis in Sprague-Dawley rats and C57BL mice.

In previous studies, it was found that a single systemic administration of N-methyl-N-nitrosourea (MNU) to rats and mice resulted in the retinal degeneration in all treated animals over a 7 day period. Retinal degeneration was due to photoreceptor cell apoptosis that was identical to the apoptosis seen in human retinitis pigmentosa (RP). In the present study, nicotinamide (NAM), a water-soluble B-group vitamin (vitamin B(3)), suppressed photoreceptor cell loss in a dose-dependent manner when administered immediately after MNU treatment. In rats, a dose of NAM >or=25 mg kg(-1) completely suppressed photoreceptor cell loss, and 10 mg kg(-1) partially suppressed photoreceptor cell loss. In mice, doses of 1000 and >or=100 mg kg(-1) were needed for complete and partial suppression, respectively. Thus, rats were more responsive to NAM than mice. The retinoprotective effect of 1000 mg kg(-1) NAM lasted throughout the long-term (35 days) observation period, with no apparent toxicity. Also, in rats, 1000 mg kg(-1) NAM completely suppressed photoreceptor cell loss when administered up to 4 hr after MNU treatment, and partially suppressed photoreceptor cell loss when administered 6 hr after MNU treatment. In mice, administration of NAM 2-6 hr after MNU resulted in partial suppression. NAM did not reduce levels of 7-methyldeoxyguanosine DNA adduct, but did reduce photoreceptor cell apoptosis. Although the mechanism of action underlying this retinoprotection remains to be clarified, NAM may be a potential therapeutic agent for the treatment of retinal degeneration.

Anti-genotoxicity of galangin as a cancer chemopreventive agent candidate.

Flavonoids are polyphenolic compounds that are present in plants. They have been shown to possess a variety of biological activities at non-toxic concentrations in organisms. Galangin, a member of the flavonol class of flavonoid, is present in high concentrations in medicinal plants (e.g. Alpinia officinarum) and propolis, a natural beehive product. Results from in vitro and in vivo studies indicate that galangin with anti-oxidative and free radical scavenging activities is capable of modulating enzyme activities and suppressing the genotoxicity of chemicals. These activities will be discussed in this review. Based on our review, galangin may be a promising candidate for cancer chemoprevention.

Stobadine inhibits lysosomal enzyme release in vivo and in vitro.

This study investigated the ability of stobadine, an effective cardioprotective drug with antiarrhythmic, antihypoxic and oxygen free radical scavenging properties, to protect cells against cyclophosphamide-induced toxic and cytotoxic damage in vivo and in vitro. Cyclophosphamide-induced toxic damage in female ICR mice was accompanied by marked increase in the activity of lysosomal enzymes in the spleen and kidney. Administration of stobadine prior to cyclophosphamide inhibited these biochemical changes. The in vivo protective effect of stobadine was comparable with its in vitro effect established in HeLa cells.

Distinct endoplasmic reticulum signaling pathways regulate apoptotic and necrotic cell death following iodoacetamide treatment.

Environmental stress induces the synthesis of glucose-regulated proteins (Grps) in the endoplasmic reticulum (ER) and heat shock proteins (Hsps) in the cytoplasm. Iodoacetamide (IDAM), a prototypical alkyating agent, induces both Grp and Hsp synthesis in renal epithelial cells and causes necrosis which is prevented by prior activation of the ER stress response (pre-ER stress) [Liu, H., et al. (1997) J. Biol. Chem. 272, 21751-21759]. In this study, we examined the biochemical pathways leading to IDAM-induced apoptosis and investigated the role of the ER stress response in apoptotic cell death. The antioxidant N,N'-diphenyl-p-phenylenediamine (DPPD) prevented necrosis after IDAM treatment, but the cells went on to die with hallmarks of apoptosis, i.e., cell detachment, caspase-3 activation, cleavage of poly(ADP-ribose)polymerase (PARP), and DNA-ladder formation, all of which were blocked by the general caspase inhibitor zVAD. As with IDAM-induced necrosis, dithiothreitol protected against apoptosis, but cell permeable calcium chelators did not, suggesting that distinct biochemical pathways mediate these two forms of cell death. Pre-ER stress, but not heat shock, prevented IDAM-induced apoptosis. pkASgrp78 cells are deficient in Grp78 induction due to expression of a grp78 antisense RNA and are more sensitive to necrosis. However, these cells were resistant to IDAM-induced apoptosis and had increased basal levels of Grp94 and a KDEL-containing protein of about 50 kDa. Thus, the expression of grp78 antisense perturbs ER functions and activates expression of other ER stress genes accounting for the resistance to apoptosis. Taken together, the data describe functionally distinct signaling pathways through which the ER regulates apoptosis and necrosis caused by chemical toxicants.

Absence of cross-resistance between two alkylating agents: BCNU vs bifunctional galactitol.

Dianhydrogalactitol (DAG) increased the life span of both BCNU-sensitive and -resistant L1210 tumor-bearing mice. However, the BCNU-resistant strain showed slightly lower sensitivity against DAG, which could be overcome by an increase in drug dose of ca. 20%. The somewhat lower sensitivity was proportional to a slightly reduced DNA cross-linking formation induced by DAG in BCNU-resistant cells. The amount of DNA cross-links was determined by measurement of the 1,6-di(guaninyl)-galactitol content of DNA. The slight reduction in cross-links is not attributable to DNA repair but rather to other factors that seem to prevent the formation of DNA-drug adducts. The absence of cross-resistance is explained by different kinds of DNA damage caused by the two alkylating agents and the presumably different defense mechanisms developed by cells against these lesions.

Anticlastogenic effect of beta-carotene in Chinese hamsters. Time and dose response studies with different mutagens.

beta-Carotene exhibited dose-dependent anticlastogenic effects on aberrations induced by the direct-acting mutagens thio-TEPA, methyl methanesulfonate and busulfan in the in vivo chromosome aberration test (bone marrow cells, Chinese hamsters). No effect was seen when retinol was used. Apparent differences in the action of beta-carotene on aberrations induced by the three applied mutagens may be due to differences in the solubility of the compounds and to the different routes of administration.