TBK1 recruitment to STING mediates autoinflammatory arthritis caused by defective DNA clearance.

Defective DNA clearance in DNase II-/- mice leads to lethal inflammatory diseases that can be rescued by deleting cGAS or STING, but the role of distinct signaling pathways downstream of STING in the disease manifestation is not known. We found that the STING S365A mutation, which abrogates IRF3 binding and type I interferon induction, rescued the embryonic lethality of DNase II-/- mice. However, the STING S365A mutant retains the ability to recruit TBK1 and activate NF-κB, and DNase II-/-STING-S365A mice exhibited severe polyarthritis, which was alleviated by neutralizing antibodies against TNF-α or IL-6 receptor. In contrast, the STING L373A mutation or C-terminal tail truncation, which disrupts TBK1 binding and therefore prevents activation of both IRF3 and NF-κB, completely rescued the phenotypes of DNase II-/- mice. These results demonstrate that TBK1 recruitment to STING mediates autoinflammatory arthritis independently of type I interferons. Inhibiting TBK1 binding to STING may be a therapeutic strategy for certain autoinflammatory diseases instigated by self-DNA.

Anticancer Potential of Damnacanthal and Nordamnacanthal from Morinda elliptica Roots on T-lymphoblastic Leukemia Cells.

BACKGROUND: This study reports on the cytotoxic properties of nordamnacanthal and damnacanthal, isolated from roots of Morinda elliptica on T-lymphoblastic leukaemia (CEM-SS) cell lines. METHODS: MTT assay, DNA fragmentation, ELISA and cell cycle analysis were carried out. RESULTS: Nordamnacanthal and damnacanthal at IC50 values of 1.7 µg/mL and10 µg/mL, respectively. At the molecular level, these compounds caused internucleosomal DNA cleavage producing multiple 180-200 bp fragments that are visible as a "ladder" on the agarose gel. This was due to the activation of the Mg2+/Ca2+-dependent endonuclease. The induction of apoptosis by nordamnacanthal was different from the one induced by damnacanthal, in a way that it occurs independently of ongoing transcription process. Nevertheless, in both cases, the process of dephosphorylation of protein phosphates 1 and 2A, the ongoing protein synthesis and the elevations of the cytosolic Ca2+ concentration were not needed for apoptosis to take place. Nordamnacanthal was found to have a cytotoxic effect by inducing apoptosis, while damnacanthal caused arrest at the G0/G1 phase of the cell cycle. CONCLUSION: Damnacanthal and nordamnacanthal have anticancer properties, and could act as potential treatment for T-lymphoblastic leukemia.

Alpha lipoic acid attenuates cadmium-induced nephrotoxicity via the mitochondrial apoptotic pathways in rat.

Alpha lipoic acid (α-LA), a potent antioxidant, is protective against acute nephrotoxicity. In the present study, the attenuation of cadmium (Cd)-induced kidney injury by α-LA on was investigated in a rat model. Exposure to 50 mg/L Cd for 12 weeks increased kidney index and Cd content, malondialdehyde (MDA) levels, and histological damage to the renal cortex, and decreased the activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH). Treatment with 50 mg/L Cd also damaged renal cell mitochondria and nuclei, and activated the mitochondrial apoptosis pathway, indicated by increased gene and protein expression/activation of caspase-9, caspase-3, poly ADP-ribose polymerase (PARP) and Bcl-2 adenovirus E1a nineteen kilodalton interacting protein 3 (BNIP3), and translocation of cytochrome c (cyt c), apoptosis-inducing factor (AIF), and endonuclease G (Endo G). However, simultaneous supplementation with α-LA (50 mg/kg·bw) protected kidney cells from Cd-induced cytotoxicity by reducing MDA levels and Cd content, restoring endogenous enzyme activities, renewing mitochondrial function, and preventing activation of the mitochondria apoptosis pathway.

Systematic deletion of the ER lectin chaperone genes reveals their roles in vegetative growth and male gametophyte development in Arabidopsis.

Calnexin (CNX) and calreticulin (CRT) are homologous lectin chaperones in the endoplasmic reticulum (ER) that facilitate glycoprotein folding and retain folding intermediates to prevent their transit via the secretary pathway. The Arabidopsis genome has two CNX (CNX1 and CNX2) and three CRT (CRT1, CRT2 and CRT3) homologs. Despite growing evidence of the biological roles of CNXs and CRTs, little is understood about their function in Arabidopsis growth and development under normal conditions. Here, we report that the deletion of CNX1, but not of CNX2, in the crt1 crt2 crt3 triple mutation background had an adverse effect on pollen viability and pollen tube growth, leading to a significant reduction in fertility. The cnx1 crt1 crt2 crt3 quadruple mutation also conferred severe defects in growth and development, including a shortened primary root, increased root hair length and density, and reduced plant height. Disruption of all five members of the CNX/CRT family was revealed to be lethal. Finally, the abnormal phenotype of the cnx1 crt1 crt2 crt3 quadruple mutants was completely rescued by either the CNX1 or CNX2 cDNA under the control of the CNX1 promoter, suggesting functional redundancy between CNX1 and CNX2. Taken together, these results provide genetic evidence that CNX and CRT play essential and overlapping roles during vegetative growth and male gametophyte development in Arabidopsis.

Protective effects of phillyrin on H2O 2-induced oxidative stress and apoptosis in PC12 cells.

Oxidative stress is a major component of harmful cascades activated in neurodegenerative disorders. We sought to elucidate possible effects of phillyrin, an active constituent isolated from the Chinese medicinal herb Forsythia suspense, on hydrogen peroxide (H2O2)-induced cell death and determine the underlying molecular mechanisms in neuron-like PC12 cells. By MTT assay and lactate dehydrogenase (LDH) leakage assay, we found that phillyrin treatment effectively protected PC12 cells against H2O2-induced cell damage. H2O2 exposure induced oxidative stress in PC12 cells, as revealed by enhanced oxidative stress and decreased activities of antioxidative enzymes, which were inhibited by phillyrin pretreatment. ROS activated mitochondria-dependent apoptosis. The anti-apoptotic effects of phillyrin were also confirmed by acridine orange/ethidium bromide (AO/EB) staining. Mitochondrial membrane potential decrease, cytochrome c release, caspases activation, activation of AIF and Endo G were observed in H2O2-treated cells by rhodamine 123 or western blot. Interestingly, phillyrin effectively suppressed these changes. Moreover, phillyrin could inhibit H2O2-induced up-regulation of Bax/Bcl-2 ratio. In conclusion, phillyrin effectively inhibited H2O2-induced oxidative stress and apoptosis in PC12 cells.

Curcumin-enhanced chemosensitivity of FDA-approved platinum (II)-based anti-cancer drugs involves downregulation of nuclear endonuclease G and NF-κB as well as induction of apoptosis and G2/M arrest.

Curcumin, an active natural compound in turmeric and curry, has been reported to exhibit anti-cancer effect. Cisplatin, carboplatin and oxaliplatin are used to treat various types of cancers. However, acquired resistance and toxicities are observed. Here, the addition of curcumin significantly increased cytotoxicity of the anti-cancer drugs on human colorectal cancer HT-29 cells, producing synergistic (cisplatin and carboplatin) and additivity (oxaliplatin) effects. Treatments in combination with curcumin resulted in a significantly increased induction of apoptosis and occurrence of G2/M arrest. Nuclear apoptosis-inducing factor (AIF), EndoG and NF-κB were elevated by anti-cancer drugs, suggesting the involvement of AIF and EndoG. The addition of curcumin suppressed nuclear AIF and EndoG and reversed anti-cancer drugs-induced NF-κB expression, suggesting the association of EndoG and NF-κB in curcumin-enhanced chemosensitivity. Therefore, the intake of foods rich in curcumin or curcumin-containing supplements should be taken into consideration for patients receiving chemotherapy to optimize the outcome of treatments.

Nuclease-mediated genome editing: At the front-line of functional genomics technology.

Genome editing with engineered endonucleases is rapidly becoming a staple method in developmental biology studies. Engineered nucleases permit random or designed genomic modification at precise loci through the stimulation of endogenous double-strand break repair. Homology-directed repair following targeted DNA damage is mediated by co-introduction of a custom repair template, allowing the derivation of knock-out and knock-in alleles in animal models previously refractory to classic gene targeting procedures. Currently there are three main types of customizable site-specific nucleases delineated by the source mechanism of DNA binding that guides nuclease activity to a genomic target: zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR). Among these genome engineering tools, characteristics such as the ease of design and construction, mechanism of inducing DNA damage, and DNA sequence specificity all differ, making their application complementary. By understanding the advantages and disadvantages of each method, one may make the best choice for their particular purpose.

Base excision repair AP endonucleases and mismatch repair act together to induce checkpoint-mediated autophagy.

Cellular responses to DNA damage involve distinct DNA repair pathways, such as mismatch repair (MMR) and base excision repair (BER). Using Caenorhabditis elegans as a model system, we present genetic and molecular evidence of a mechanistic link between processing of DNA damage and activation of autophagy. Here we show that the BER AP endonucleases APN-1 and EXO-3 function in the same pathway as MMR, to elicit DNA-directed toxicity in response to 5-fluorouracil, a mainstay of systemic adjuvant treatment of solid cancers. Immunohistochemical analyses suggest that EXO-3 generates the DNA nicks required for MMR activation. Processing of DNA damage via this pathway, in which both BER and MMR enzymes are required, leads to induction of autophagy in C. elegans and human cells. Hence, our data show that MMR- and AP endonuclease-dependent processing of 5-fluorouracil-induced DNA damage leads to checkpoint activation and induction of autophagy, whose hyperactivation contributes to cell death.

Zinc finger nucleases: looking toward translation.

Genetic engineering has emerged as a powerful mechanism for understanding biological systems and a potential approach for redressing congenital disease. Alongside, the emergence of these technologies in recent decades has risen the complementary analysis of the ethical implications of genetic engineering techniques and applications. Although viral-mediated approaches have dominated initial efforts in gene transfer (GT) methods, an emerging technology involving engineered restriction enzymes known as zinc finger nucleases (ZFNs) has become a powerful new methodology for gene editing. Given the advantages provided by ZFNs for more specific and diverse approaches in gene editing for basic science and clinical applications, we discuss how ZFN research can address some of the ethical and scientific questions that have been posed for other GT techniques. This is of particular importance, given the momentum currently behind ZFNs in moving into phase I clinical trials. This study provides a historical account of the origins of ZFN technology, an analysis of current techniques and applications, and an examination of the ethical issues applicable to translational ZFN genetic engineering in early phase clinical trials.

Oleifolioside A mediates caspase-independent human cervical carcinoma HeLa cell apoptosis involving nuclear relocation of mitochondrial apoptogenic factors AIF and EndoG.

Apoptosis, the main type of programmed cell death, plays an essential role in a variety of biological events. Whereas "classical" apoptosis is dependent on caspase activation, caspase-independent death is increasingly recognized as an alternative pathway. To develop new anticancer agents, oleifolioside A was isolated from Dendropanax morbifera Leveille and the biochemical mechanisms of oleifolioside A-induced apoptosis in HeLa cells were investigated. Exposure to oleifolioside A resulted in caspase activation and typical features of apoptosis, although cell death was not prevented by caspase inhibition. Oleifolioside A treatment induced up-regulation of Bad, loss of mitochondrial membrane potential, nuclear relocation of mitochondrial factors, apoptosis-inducing factor (AIF), endonuclease G (EndoG), and apoptosis induction. This is the first report of anticancer activity of oleifolioside A, and nuclear translocation of AIF and EndoG in oleifolioside A-treated HeLa cells might represent an alternative death signaling pathway in the absence of caspase activity.

Copper-induced immunotoxicity involves cell cycle arrest and cell death in the spleen and thymus.

Copper is an essential trace element for human physiological processes. To evaluate the potential adverse health impact/immunotoxicological effects of this metal in situ due to over exposure, Swiss albino mice were treated (via intraperitoneal injections) with copper (II) chloride (copper chloride) at doses of 0, 5, or 7.5 mg copper chloride/kg body weight (b.w.) twice a week for 4 wk; these values were derived from LD50 studies using copper chloride doses that ranged from 0 to 40 mg/kg BW (2×/wk, for 4 wk). Copper treated mice evidenced immunotoxicity as indicated by dose-related decreases and increases, respectively, in thymic and splenic weights. Histomorphological changes evidenced in these organs were thymic atrophy, white pulp shrinkage in the spleen, and apoptosis of splenocytes and thymocytes; these observations were confirmed by microscopic analyses. Cell count analyses indicated that the proliferative functions of the splenocytes and thymocytes were also altered because of the copper exposures. Among both cell types from the copper treated hosts, flow cytometric analyses revealed a dose related increase in the percentages of cells in the Sub-G0/G1 state, indicative of apoptosis which was further confirmed by Annexin V binding assay. In addition, the copper treatments altered the expression of selected cell death related genes such as EndoG and Bax in a dose related manner. Immunohistochemical analyses revealed that there was also increased ubiquitin expression in both the cell types. In conclusion, these studies show that sublethal exposure to copper (as copper chloride) induces toxicity in the thymus and spleen, and increased Sub G0/G1 population among splenocytes and thymocytes that is mediated, in part, by the EndoG-Bax-ubiquitin pathway. This latter damage to these cells that reside in critical immune system organs are likely to be important contributing factors underlying the immunosuppression that has been documented by other investigators following acute high dose/chronic low-medium dose exposures to copper agents.

The roles of AIF and Endo G in the apoptotic effects of benzyl isothiocyanate on DU 145 human prostate cancer cells via the mitochondrial signaling pathway.

It is well known that the response of cancer cells to chemotherapeutic drugs involves the activation of apoptotic pathways. Benzyl isothiocyanate (BITC) is an important compound found in plant food and has been shown to have anti-cancer effects on human cancer cells, but its effect on prostate cancer cells in vitro remains unknown. The aim of the present study was to investigate the effects of BITC on DU 145 human prostate cancer cells in order to clarify whether a time/concentration range for optimal BITC-induced apoptosis exists and to find the associated signaling pathway. Cell morphological changes, percentage of cell viability, DNA damage and apoptosis in DU 145 cells were examined by phase-contrast microscopy, flow cytometric assay, 4',6-diamidine-20-phenylindole dihydrochloride staining, comet assay and Western blotting analysis. The results indicate that BITC induces cell morphological changes, decreases the percentage of viable cells (induction of cell cytotoxicity), and induces DNA damage and apoptosis in DU 145 cells in a time- and dose-dependent manner. Flow cytometric assays indicated that BITC promoted reactive oxygen species and Ca2+ productions and decreased the levels of mitochondrial membrane potential (ΤYm), while the pre-treatment with N-acetylcysteine caused an increase in the percentage of viable cells. BITC also promoted caspase-3, -8 and -9 activities. Furthermore, when cells were pre-treated with the caspase-3 inhibitor and then treated with BITC, this led to an increase in the percentage of viable cells. Confocal laser microscopy examination indicated that BITC promoted the expression of AIF and Endo G, which were released from the mitochondria in DU 145 cells. In conclusion, BITC induces apoptosis in DU 145 cells through the release of AIF and Endo G from the mitochondria and also promotes caspase-3 activation.

Identification of a chemical that inhibits the mycobacterial UvrABC complex in nucleotide excision repair.

Bacterial DNA can be damaged by reactive nitrogen and oxygen intermediates (RNI and ROI) generated by host immunity, as well as by antibiotics that trigger bacterial production of ROI. Thus a pathogen's ability to repair its DNA may be important for persistent infection. A prominent role for nucleotide excision repair (NER) in disease caused by Mycobacterium tuberculosis (Mtb) was suggested by attenuation of uvrB-deficient Mtb in mice. However, it was unknown if Mtb's Uvr proteins could execute NER. Here we report that recombinant UvrA, UvrB, and UvrC from Mtb collectively bound and cleaved plasmid DNA exposed to ultraviolet (UV) irradiation or peroxynitrite. We used the DNA incision assay to test the mechanism of action of compounds identified in a high-throughput screen for their ability to delay recovery of M. smegmatis from UV irradiation. 2-(5-Amino-1,3,4-thiadiazol-2-ylbenzo[f]chromen-3-one) (ATBC) but not several closely related compounds inhibited cleavage of damaged DNA by UvrA, UvrB, and UvrC without intercalating in DNA and impaired recovery of M. smegmatis from UV irradiation. ATBC did not affect bacterial growth in the absence of UV exposure, nor did it exacerbate the growth defect of UV-irradiated mycobacteria that lacked uvrB. Thus, ATBC appears to be a cell-penetrant, selective inhibitor of mycobacterial NER. Chemical inhibitors of NER may facilitate studies of the role of NER in prokaryotic pathobiology.

An experimental study on the antileukemia effects of gypenosides in vitro and in vivo.

PURPOSE: Gypenosides (Gyp), found in Gynostemma pentaphyllum Makino, have been used as folk medicine for centuries and have exhibited diverse pharmacological effects, including antileukemia effects in vitro and in vivo. In the present study, Gyp were used to examine effects on cell viability, cell cycle, and induction of apoptosis in vitro. They were administered in the diet to mice injected with WEHI-3 cells in vivo. EXPERIMENTAL DESIGN: Effects of Gyp on WEHI-3 cells were determined by flow cytometric assay and Western blotting. RESULTS: Gyp inhibited the growth of WEHI-3 cells. These effects were associated with the induction of G0/G1 arrest, morphological changes, DNA fragmentation, and increased sub-G1 phase. Gyp promoted the production of reactive oxygen species, increased Ca(2+) levels, and induced the depolarization of the mitochondrial membrane potential. The effects of Gyp were dose and time dependent. Moreover, Gyp increased levels of the proapoptotic protein Bax, reduced levels of the antiapoptotic proteins Bcl-2, and stimulated release of cytochrome c, AIF (apoptosis-inducing factor), and Endo G (endonuclease G) from mitochondria. The levels of GADD153, GRP78, ATF6-α, and ATF4-α were increased by Gyp, resulting in ER (endoplasmic reticular) stress in WEHI-3 cells. Oral consumption of Gyp increased the survival rate of mice injected with WEHI-3 cells used as a mouse model of leukemia. CONCLUSIONS: Results of these experiments provide new information on understanding mechanisms of Gyp-induced effects on cell cycle arrest and apoptosis in vitro and in an in vivo animal model.

Evodiamine induces caspase-dependent apoptosis and S phase arrest in human colon lovo cells.

Evodiamine, one of the major bioactive components derived from Wu-Chu-Yu, a long-standing Chinese herb, was reported to possess anticancer activity. In this study, we investigated the in-vitro and in-vivo anticancer effects of evodiamine on human colon lovo cells and their potential mechanisms. The 3-(4, 5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay showed that the in-vitro proliferation of lovo cells was inhibited by evodiamine of various concentrations. Flow cytometry showed a time-dependent increase in the percentage of apoptotic cells and cells arrested in the S phase after treatment with 60 micromol/l evodiamine. Western blot indicated that evodiamine treatment decreased the expression of procaspase-8, procaspase-9, and procaspase-3 in lovo cells, accompanied by the activation of caspase-8, caspase-9, and caspase-3. However, the translocation of apoptosis-inducing factor and endonuclease G was not affected by evodiamine. Moreover, western blot assay also suggested that evodiamine-induced S phase arrest in lovo cells was associated with a marked decrease in the protein expression of cyclinA, cyclinA-dependent kinase 2, and cdc25c. In-vivo antineoplastic characteristics of evodiamine were examined in a human colon carcinoma lovo xenograft model and results showed that evodiamine increased the number of TUNEL-positive cells accompanied by the downregulated expression of procaspase-8, procaspase-9, and procaspase-3. In conclusion, these findings indicated that evodiamine could inhibit the in-vitro and in-vivo proliferation of human colon lovo cells by inducing caspase-dependent apoptosis and S phase arrest.

Coordinated and sequential activation of neutral and acidic DNases during interdigital cell death in the embryonic limb.

Interdigital tissue regression during embryonic development is one of the most representative model systems of morphogenetic cell death, but the degenerative cascade accounting for this process awaits clarification. Although the canonical apoptotic caspase pathway appears to be activated in the interdigital mesenchyme committed to die, neither genetic nor chemical blockage of caspases or their downstream effectors, is sufficient to prevent cell death. Hence, alternative and/or complementary dying pathways must also be responsible for this degenerative process. In this work we have chosen to study the endonucleases during the regression of the interdigital tissue of avian embryos to gain insights into the molecular mechanisms accounting for programmed cell death in this system. We show that caspase activated DNase, which is a neutral DNase associated with the caspase apoptotic pathway, appears to be the main endonuclease only at an initial phase of interdigit regression. However at peak stages of the degenerative process, the acidic DNases L-DNase II and lysosomal DNase IIB become predominant in the system and markers for cell autophagy become moderately up-regulated. Consistent with the activation of acidic endonucleases we observed that microenvironmental pH value in the interdigits decreased to levels only appropriate for acidic enzymes. Furthermore, we found that overexpression of lysosomal DNase IIB in embryonic limb mesoderm promoted cell death, which was also accompanied by up-regulation and activation of L-DNase II. Up-regulation of acidic DNases was maintained in interdigits explanted to culture dishes, where the participation of exogenous professional phagocytes of hematopoietic origin is avoided. Finally, and consistent with all our findings, up-regulation of acidic DNases was much reduced in the webbed interdigits of duck embryos, characterized by a rudimentary interdigital degenerative process. We conclude that the regression of the interdigital tissue involves a coordinated and sequential activation of the caspase and lysosomal degenerative molecular cascades.

Autoschizis of T-cells is induced by the nutritional supplement, Cr(III)picolinate.

In recent times, Cr(III)(picolinate)(3) [Cr(III)(pic)(3)] a nutritional supplement, is gaining attention because of its clastogenic and mutagenic properties. Earlier studies of ours indicated that Cr(III)(pic)(3) is cytotoxic to lymphocytes with ROS and mitochondrial events playing a role in bringing about apoptosis. Now, we report that, autoschizis is induced in lymphocytes in a concentration and time dependent manner which is confirmed through TEM and SEM. Lymphocytes treated with concentrations of 100microM of Cr(III)(pic)(3) exhibit features such as cytoplasmic bleb, self excision of cytoplasm, cytoplasmic leakage and membrane bound bodies formed from the excised pieces apart from apoptosis and necrosis. Though autoschizis has been described in tumor cell lines treated with menadione and ascorbate, occurrence of this cell death in normal T-lymphocytes is reported here. The cellular events that accompany autoschizis are found to be increase in intracellular Reactive Oxygen Species (ROS) and cytoplasmic lactate dehydrogenase, loss of mitochondrial membrane potential (MMP) and depletion of ATP. Further, autoschizis is effected through increases in DNase I and DNase II activity with a concomitant decrease in caspase-3 activity which leads to a random cleavage of the DNA as demonstrated by a smear like pattern after electrophoresis on agarose gel.

HSP70 and EndoG modulate cell death by heat in human skin keratinocytes in vitro.

We examined how young and old keratinocytes died from heat stress in vitro. We found that keratinocyte cell death was not due to oxidative stress as neither Mn-SOD nor Cu-Zn-SOD was produced in either young or old heated keratinocytes. Instead, analysis of the anti-apoptotic factors, Bcl2 and HSP70, and the pro-apoptotic factors, caspase 3, caspase 8, Apaf-1, cytochrome c, AIF, and EndoG, indicated that keratinocyte cell death occurred via the caspase-independent EndoG apoptotic pathway. We found that both young and old keratinocytes died via the same pathway, and that we could specifically reduce both young and old keratinocyte death by addition of the EndoG inhibitor NEM. Further analysis suggested that the difference between young and old keratinocyte death was due to the synthesis of HSP70 protein, with the increase in response to heat more pronounced in young keratinocytes than in old keratinocytes. When we inhibited HSP70 by adding quercetin, death was increased in both young and old keratinocytes, but more so in old keratinocytes. These data suggest that old keratinocytes may die more readily than young keratinocytes when heated because they synthesize HSP70 at a lower efficiency. Such findings suggest that HSP70 production may be age-dependent.

Tomato-oleoresin supplement prevents doxorubicin-induced cardiac myocyte oxidative DNA damage in rats.

Doxorubicin (DOX) is an efficient chemotherapeutic agent used against several types of tumors; however, its use is limited due to severe cardiotoxicity. Since it is accepted that reactive oxygen species are involved in DOX-induced cardiotoxicity, antioxidant agents have been used to attenuate its side effects. To determine tomato-oleoresin protection against cardiac oxidative DNA damage induced by DOX, we distributed Wistar male rats in control (C), lycopene (L), DOX (D) and DOX+lycopene (DL) groups. They received corn oil (C, D) or tomato-oleoresin (5mg/kg body wt. day) (L, DL) by gavage for a 7-week period. They also received saline (C, L) or DOX (4mg/kg body wt.) (D, DL) intraperitoneally at the 3rd, 4th, 5th, and at 6th week. Lycopene absorption was checked by HPLC. Cardiac oxidative DNA damage was evaluated by the alkaline Comet assay using formamidopyrimidine-DNA glycosylase (FPG) and endonuclease III (endo III). Cardiomyocyte levels of SBs, SBs FPG and SBs Endo III were higher in rats from D when compared to other groups. DNA damage levels in cardiomyocytes from DL were not different when compared to C and L groups. The viability of cardiomyocytes from D or DL was lower than C or L groups (p<0.01). Lycopene levels (mean+/-S.D.nmol/kg) in saponified hearts were similar between L (47.43+/-11.78) and DL (49.85+/-16.24) groups. Our results showed: (1) lycopene absorption was confirmed by its cardiac levels; (2) DOX-induced oxidative DNA damage in cardiomyocyte; (3) tomato-oleoresin supplementation protected against cardiomyocyte oxidative DNA damage.

Caspase-independent death of human osteosarcoma cells by flavonoids is driven by p53-mediated mitochondrial stress and nuclear translocation of AIF and endonuclease G.

Flavonoids have antioxidant and antitumor promoting effects. Rhus verniciflua Stokes (RVS) is a flavonoid-rich herbal medicine that has long been used in Korea as both a food additive and antitumor agent. It was previous reported that a purified flavonoid fraction prepared from RVS, herein named RCMF (the RVS chloroform-methanol fraction), inhibited the proliferation and induced apoptosis in human osteosarcoma (HOS) cells. This study examined the mechanisms involved in the RCMF-mediated apoptosis in HOS cells. RCMF was shown to be capable of inducing apoptosis in HOS cells by inducing p53 in the cells resulting in the decrease in Bcl-2 level, activation of Bax, and cytoplasmic release of cytochrome c, which led to the translocation of apoptosis-inducing factor (AIF) and endonuclease G (EndoG) into the nucleus. However, the RCMF-induced apoptosis was suppressed by transfecting the cells with antisense p53 oligonucleotides but not by treating them with a MAPK or caspase inhibitor. This suppression occurred through the regulation of Bcl-2 members as well as by preventing the nuclear translocation of the mitochondrial apoptogenic factors. Overall, it appears that p53-mediated mitochondrial stress and the nuclear translocation of AIF and EndoG are mainly required for the apoptosis induced by RCMF.