Involvement of Reactive Oxygen Species in the Hepatorenal Toxicity of Actinomycin V In Vitro and In Vivo.

The high toxicity of actinomycin D (Act D) severely limits its use as a first-line chemotherapeutic agent in the clinic. Actinomycin V (Act V), an analog of Act D, exhibited strong anticancer activity in our previous studies. Here, we provide evidence that Act V has less hepatorenal toxicity than Act D in vitro and in vivo, associated with the reactive oxygen species (ROS) pathway. Compared to Act D, Act V exhibited considerably stronger sensitivity for cancer cells and less toxicity to human normal liver LO-2 and human embryonic kidney 293T cells using the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay. Notably, Act V caused less damage to both the liver and kidney than Act D in vivo, indicated by organ to body weight ratios, as well as alanine aminotransferase (ALT), aspartate aminotransferase (AST), and serum creatinine (Scr) levels. Further experiments showed that the ROS pathway is involved in Act V-induced hepatorenal toxicity. Act V generates ROS and accumulates malondialdehyde (MDA), reducing levels of superoxide dismutase (SOD) and glutathione (GSH) in LO-2 and 293T cells. These findings indicate that Act V induces less hepatorenal toxicity than Act D in vitro and in vivo and merits further development as a potential therapeutic agent for the treatment of cancer.

SLP-2 is required for stress-induced mitochondrial hyperfusion.

Mitochondria are dynamic organelles, the morphology of which results from an equilibrium between two opposing processes, fusion and fission. Mitochondrial fusion relies on dynamin-related GTPases, the mitofusins (MFN1 and 2) in the outer mitochondrial membrane and OPA1 (optic atrophy 1) in the inner mitochondrial membrane. Apart from a role in the maintenance of mitochondrial DNA, little is known about the physiological role of mitochondrial fusion. Here we report that mitochondria hyperfuse and form a highly interconnected network in cells exposed to selective stresses. This process precedes mitochondrial fission when it is triggered by apoptotic stimuli such as UV irradiation or actinomycin D. Stress-induced mitochondrial hyperfusion (SIMH) is independent of MFN2, BAX/BAK, and prohibitins, but requires L-OPA1, MFN1, and the mitochondrial inner membrane protein SLP-2. In the absence of SLP-2, L-OPA1 is lost and SIMH is prevented. SIMH is accompanied by increased mitochondrial ATP production and represents a novel adaptive pro-survival response against stress.

The novel p53 target gene IRF2BP2 participates in cell survival during the p53 stress response.

The tumor suppressor p53 contributes to the cellular fate after genotoxic insults, mainly through the regulation of target genes, thereby allowing e.g. repair mechanisms resulting in cell survival or inducing apoptosis. Unresolved so far is the issue, which exact mechanisms lead to one or the other cellular outcome. Here, we describe the interferon regulatory factor-2-binding protein-2 (IRF2BP2) as a new direct target gene of p53, influencing the p53-mediated cellular decision. We show that upregulation of IRF2BP2 after treatment with actinomycin D (Act.D) is dependent on functional p53 in different cell lines. This occurs in parallel with the down-regulation of the interacting partner of IRF2BP2, the interferon regulatory factor-2 (IRF2), which is known to positively influence cell growth. Analyzing the molecular functions of IRF2BP2, it appears to be able to impede on the p53-mediated transactivation of the p21- and the Bax-gene. We show here that overexpressed IRF2BP2 has an impact on the cellular stress response after Act.D treatment and that it diminishes the induction of apoptosis after doxorubicin treatment. Furthermore, the knockdown of IRF2BP2 leads to an upregulation of p21 and faster induction of apoptosis after doxorubicin as well as Act.D treatment.

Caspase inhibitor z-VAD-FMK increases the survival of hair cells after Actinomycin-D-induced damage in vitro.

Actinomycin-D (Act-D) is a highly effective chemotherapeutic agent that induces apoptosis in systemic tissues. Act-D combined with other chemotherapeutic agents exhibits ototoxic effects and causes hearing impairment. To investigate the potential toxic effects of Act-D in the inner ear, we treated cochlear organotypic cultures with varying concentrations of Act-D for different durations. For the first time, we found that Act-D specifically induced HC loss and apoptosis in a dose- and time-dependent manner but not neuronal degeneration. Co-treatment with benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-FMK), a pan cysteine protease inhibitor, significantly reduced HC loss and apoptosis induced by Act-D, indicating increased cell survival. Taken together, Act-D exposure has ototoxic effects on the auditory system, while z-VAD-FMK prevents Act-D-induced hair cell damage.

Novel molecular mechanism for actinomycin D activity as an oncogenic promoter G-quadruplex binder.

Actinomycin D (ActD) is a natural antibiotic that inhibits the transcription of genes by interacting with a GC-rich duplex, a single-stranded or hairpin form of DNA, and then interfering with the action of RNA polymerase. In this study, we identified a novel molecular mechanism of anticancer activity of ActD as an oncogenic c-Myc promoter G-quadruplex binder. ActD selectively inhibits the elongation of oligonucleotides containing c-Myc promoter G-quadruplex sequence in PCR-stop assays. UV-vis spectroscopic and circular dichroism studies suggest that ActD interacts with c-Myc promoter G-quadruplex via a surface end stacking interaction, inducing a mixed-type conformation of the G-quadruplex. ActD selectively inhibits the cellular growth and synthesis of c-Myc mRNA in Ramos cells having the NHEIII(1) region in the translocated c-Myc gene. In addition, the results of promoter assays using two kinds of NHEIII(1) region mutants and wild-type constructs strongly support the idea that binding of ActD with G-quadruplex formed in the promoter region results in the reporter gene being turned off. Our study reveals a novel mechanism underlying the anticancer activity of ActD, whereby ActD interacts with oncogenic promoter G-quadruplex DNA to repress gene expression.

Genotoxic stress increases cytoplasmic mitochondrial DNA editing by human APOBEC3 mutator enzymes at a single cell level.

Human cells are stressed by numerous mechanisms that can lead to leakage of mitochondrial DNA (mtDNA) to the cytoplasm and ultimately apoptosis. This agonist DNA constitutes a danger to the cell and is counteracted by cytoplasmic DNases and APOBEC3 cytidine deamination of DNA. To investigate APOBEC3 editing of leaked mtDNA to the cytoplasm, we performed a PCR analysis of APOBEC3 edited cytoplasmic mtDNA (cymtDNA) at the single cell level for primary CD4+ T cells and the established P2 EBV blast cell line. Up to 17% of primary CD4+ T cells showed signs of APOBEC3 edited cymtDNA with ~50% of all mtDNA sequences showing signs of APOBEC3 editing - between 1500-5000 molecules. Although the P2 cell line showed a much lower frequency of stressed cells, the number of edited mtDNA molecules in such cells was of the same order. Addition of the genotoxic molecules, etoposide or actinomycin D increased the number of cells showing APOBEC3 edited cymtDNA to around 40%. These findings reveal a very dynamic image of the mitochondrial network, which changes considerably under stress. APOBEC3 deaminases are involved in the catabolism of mitochondrial DNA to circumvent chronic immune stimulation triggered by released mitochondrial DNA from damaged cells.

A Chemical Screen Identifies Compounds Limiting the Toxicity of C9ORF72 Dipeptide Repeats.

The expansion of GGGGCC repeats within the first intron of C9ORF72 constitutes the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Through repeat-associated non-ATG translation, these expansions are translated into dipeptide repeats (DPRs), some of which accumulate at nucleoli and lead to cell death. We here performed a chemical screen to identify compounds reducing the toxicity of ALS-related poly(PR) peptides. Our screening identified sodium phenylbutyrate, currently in clinical trials, and BET Bromodomain inhibitors as modifiers of poly(PR) toxicity in cell lines and developing zebrafish embryos. Mechanistically, we show that BET Bromodomain inhibitors rescue the nucleolar stress induced by poly(PR) or actinomycin D, alleviating the effects of the DPR in nucleolus-related functions such as mRNA splicing or translation. Our work suggests that BET Bromodomain inhibitors might have beneficial effects in diseases linked to nucleolar stress such as ALS/FTD.

SMT3IP1, a nucleolar SUMO-specific protease, deconjugates SUMO-2 from nucleolar and cytoplasmic nucleophosmin.

Sumoylation is reversibly regulated by SUMO-specific proteases. We characterized a nucleolar SUMO-specific protease, SMT3IP1, which has a preference for SUMO-2/3. To elucidate SMT3IP1 function, we screened for its interacting proteins that may be its substrates or regulate its activity. By using yeast two-hybrid screening, we identified nucleophosmin (NPM) as an SMT3IP1-binding partner. SMT3IP1 could preferentially remove SUMO-2 from sumoylated NPM. A catalytically inactive SMT3IP1 mutant increased intracellular accumulation of SUMO-2-modified NPM in a dominant-negative manner. Sumoylation of cytoplasmic mutated NPM was markedly elevated in an ARF-dependent manner. Despite the divergence in their localization, ectopic expression of SMT3IP1 could desumoylate a SUMO-2-modified NPM mutant. Additionally, genotoxic drugs caused the dissociation of NPM from nucleolar co-localization with SMT3IP1, but did not affect desumoylation of NPM by SMT3IP1. Our findings suggest that SMT3IP1-mediated desumoylation might control NPM physiological functions at both the nucleolus and other subcellular compartments.

Potent antitumor effects of a novel actinomycin D analog Leu5AMD.

Leu5AMD ([D-Val2, L-MeLeu5]2 AMD) is a novel actinomycin D (AMD) analog, in which both N-methylvalines were replaced by N-methylleucines. In the present study, an attempt has been made to investigate the effects of Leu5AMD on the proliferation of human gastric carcinoma cell line SGC-7901. The results showed that Leu5AMD inhibited the proliferation and induces apoptosis in SGC-7901 cells in a dose-dependent manner. Apoptosis induced by Leu5AMD was further confirmed by annexin V-FITC/PI dual staining assay. After treatment with Leu5AMD, the loss of mitochondrial potential and the decrease of bcl-2 gene expression were observed in apoptotic cells, suggesting that Leu5AMD may be involved in mitochondria and bcl-2 related apoptotic pathway. In addition, the in vivo antitumor effects of Leu5AMD on S-180 bearing mice and the acute toxicity on healthy mice were investigated. Treatment with Leu5AMD markedly suppressed the growth of Sarcoma xenograft. These results suggest that Leu5AMD may be used as a promising chemotherapeutical agent for patients affected by gastric carcinoma and other solid cancer.

Quantitative measure of cytotoxicity of anticancer drugs and other agents.

Many anticancer drugs act on cancer cells to promote apoptosis, which includes impairment of cellular respiration (mitochondrial O(2) consumption). Other agents also inhibit cellular respiration, sometimes irreversibly. To investigate the sensitivity of cancer cells to cytotoxins, including anticancer drugs, we compare the profiles of cellular O(2) consumption in the absence and presence of these agents. Oxygen measurements are made at 37 degrees C, using glucose as a substrate, with [O(2)] obtained from the phosphorescence decay rate of a palladium phosphor. The rate of respiration k is defined as -d[O(2)]/dt in a sealed container. Different toxins produce different profiles of impaired respiration, implying different mechanisms for the drug-induced mitochondrial dysfunction. The decrease in the average value of k over a fixed time period, I, is proposed as a characteristic value to assess mitochondrial injury. The value of I depends on the nature of the toxin, its concentration, and the exposure time as well as on the cell type. Results for several cell types and 10 cytotoxins are presented here.

p53 induction and cell viability modulation by genotoxic individual chemicals and mixtures.

The binding of the p53 tumor suppression protein to DNA response elements after genotoxic stress can be quantified by cell-based reporter gene assays as a DNA damage endpoint. Currently, bioassay evaluation of environmental samples requires further knowledge on p53 induction by chemical mixtures and on cytotoxicity interference with p53 induction analysis for proper interpretation of results. We investigated the effects of genotoxic pharmaceuticals (actinomycin D, cyclophosphamide) and nitroaromatic compounds (4-nitroquinoline 1-oxide, 3-nitrobenzanthrone) on p53 induction and cell viability using a reporter gene and a colorimetric assay, respectively. Individual exposures were conducted in the absence or presence of metabolic activation system, while binary and tertiary mixtures were tested in its absence only. Cell viability reduction tended to present direct correlation with p53 induction, and induction peaks occurred mainly at chemical concentrations causing cell viability below 80%. Mixtures presented in general good agreement between predicted and measured p53 induction factors at lower concentrations, while higher chemical concentrations gave lower values than expected. Cytotoxicity evaluation supported the selection of concentration ranges for the p53 assay and the interpretation of its results. The often used 80% viability threshold as a basis to select the maximum test concentration for cell-based assays was not adequate for p53 induction assessment. Instead, concentrations causing up to 50% cell viability reduction should be evaluated in order to identify the lowest observed effect concentration and peak values following meaningful p53 induction.

Renal scattered tubular-like cells confer protective effects in the stenotic murine kidney mediated by release of extracellular vesicles.

To test the hypothesis that intrinsic renal scattered tubular cells (STC-like cells) contribute to repairing injured tubular epithelial cells (TEC) by releasing extracellular vesicle (EV). EV released from primary cultured pig STC-like cells were confirmed by electron microscopy. Antimycin-A (AMA)-induced injured proximal TEC (PK1 cells) were co-cultured with STC-like cells, STC-like cells-derived EV, or EV-free conditioned-medium for 3 days. Cellular injury, oxidative stress and mitochondrial function were assessed. Transfer of mitochondria from STC-like cells to TEC was assessed using Mito-trackers, and their viability by mitochondrial membrane potential assays. STC-like cells-derived EV were intra-arterially injected into mice 2 weeks after induction of unilateral renal artery stenosis. Two weeks later, renal hemodynamics were studied using magnetic-resonance-imaging, and renal fibrosis assessed ex-vivo. Cultured STC-like cells released EV that were uptaken by TEC. A protective effect conferred by STC-like cells in AMA-induced TEC injury was partly mimicked by their EV. Furthermore, STC-like cells-EV carried and transferred mitochondrial material to injured TEC, which partly restored mitochondrial function. In vivo, STC-like cells-derived EV engrafted in the stenotic kidney, and improved its perfusion and oxygenation. STC-like cells-EV exert protective effects on injured tubular cells in vitro and in vivo, partly by transferring STC-like cells mitochondria, which remain at least partly functional in recipient TEC.

Impaired elimination of DNA double-strand break-containing lymphocytes in ataxia telangiectasia and Nijmegen breakage syndrome.

The repair of DNA double-strand breaks is critical for genome integrity and tumor suppression. Here we show that following treatment with the DNA-intercalating agent actinomycin D (ActD), normal quiescent T cells accumulate double-strand breaks and die, whereas T cells from ataxia telangiectasia (AT) and Nijmegen breakage syndrome (NBS) patients are resistant to this death pathway despite a comparable amount of DNA damage. We demonstrate that the ActD-induced death pathway in quiescent T lymphocytes follows DNA damage and H2AX phosphorylation, is ATM- and NBS1-dependent and due to p53-mediated cellular apoptosis. In response to genotoxic 2-Gy gamma-irradiation, on the other hand, quiescent T cells from normal donors survive following complete resolution of the damage thus induced. T cells from AT and NBS patients also survive, but retain foci of phosphorylated H2AX due to a subtle double-strand break (DSB) repair defect. A common consequence of these two genetic defects in the DSB response is the apparent tolerance of cells containing DNA breaks. We suggest that this tolerance makes a major contribution to the oncogenic risk of patients with chromosome instability syndromes.

The anti-cancer drug-induced pica in rats is related to their clinical emetogenic potential.

Cancer chemotherapy is frequently accompanied by severe emesis. The anti-cancer drugs are classified according to their clinical emetogenic potential. We have already found that kaolin ingestion behavior "pica" is analogous to emesis in rats. The aim of this study was to examine the effects of the clinical emetogenic potential of anti-cancer drugs on the induction of the pica in rats. Rats were housed in individual cages with free access to food and kaolin pellets and the daily food and kaolin intakes were measured for 3 days after the intraperitoneal administration of anti-cancer drugs (cisplatin, cyclophosphamide, actinomycin D, 5-fluorouracil and vincristine). The drugs with high potential for inducing emesis, such as cisplatin and cyclophosphamide, induced pica in all animals on the day of administration and the behavior lasted during the observation period. The drugs with moderate emetogenic potential, i.e. actinomycin D and 5-fluorouracil, also induced pica on the first and second day after the drug administration but the kaolin intake was less than that of the drugs with high potential. Vincristine, a drug with low emetogenic potential, slightly increased the kaolin intake in rats on the only first day of the administration. Cyclophosphamide, actinomycin D and vincristine induced anorexia and decreased their body weight during the observation period. These results suggested that the both amounts of kaolin intake and duration of behavior in the anti-cancer drug-induced pica are related to the clinical emetogenic potential of the drugs and the incidence of the anorexia is not related to their emetogenic potential.

Co-amplification of double minute chromosomes, multiple drug resistance, and cell surface P-glycoprotein in DNA-mediated transformants of mouse cells.

A genetic system comprised of mammalian cell mutants which demonstrate concomitant resistance to a number of unrelated drugs has been described previously. The resistance is due to reduced cell membrane permeability and is correlated with the presence of large amounts of a plasma membrane glycoprotein termed P-glycoprotein. This system could represent a model for multiple drug resistance which develops in cancer patients treated with chemotherapeutic drugs. We demonstrate here that the multiple drug resistance phenotype can be transferred to mouse cells with DNA from a drug-resistant mutant and then amplified quantitatively by culture in media containing increasing concentrations of drug. The amount of P-glycoprotein was correlated directly with the degree of drug resistance in the transformants and amplified transformants. In addition, the drug resistance and expression of P-glycoprotein of the transformants were unstable and associated quantitatively with the number of double minute chromosomes. We suggest that the gene for multiple drug resistance and P-glycoprotein is contained in these extrachromosomal particles and is amplified by increases in double minute chromosome number. The potential use of this system for manipulation of mammalian genes in general is discussed.

Alterations of transcription and translation in HeLa cells exposed to amino acid analogs.

Amino acid analogs, like other effectors of the stress response, induce in mammalian cells the same gene products that are induced upon heat shock; incorporation of the analog into protein is required for induction. We show here that induction by analogs involves controls operating at the levels of both transcription and translation. The electrophoretic patterns of newly made mRNAs simplify with time such that the putative stress protein mRNAs are the only species transported from the nucleus. Concomitantly, the patterns of protein synthesis simplify such that the stress proteins become nearly exclusive polypeptide products. Although the normal mRNAs are either not used or used with greatly reduced efficiency, they are not degraded and retain translatability when transferred to cell-free systems. Soon after the stress response has been induced, there follows a defect in the initiation of polypeptide chains, as evidenced by examination of polysome profiles. Upon prolonged exposure, polysomes are recovered, and although they give rise to stress proteins almost exclusively, the normal mRNAs are still present in these structures. Thus, in addition to the initiation defect, a lesion in elongation may also be involved. The extreme sensitivity of protein synthesis to the inhibition of RNA synthesis, together with the parallel simplifications in the patterns of newly made mRNAs and polypeptides, may imply that only newly made mRNAs are efficiently translated in analog-treated cells.

Comparative study of human neuronal and glial cell sensitivity for in vitro neurogenotoxicity testing.

Cell cultures from neuronal and glial origin have proven to be powerful tools for elucidating cellular and molecular mechanisms of nervous system development and physiology, and as neurotoxicity models to evaluate in vitro the possible effects of chemicals. But cellular heterogeneity of nervous system is considerable and these cells have been shown to respond diversely to neurotoxic insults, leading to disparate results from different studies. To shed more light on suitability of cellular models of nervous origin for neurotoxicity screening, the objective of this study was to compare the sensitivity to genetic damage induction of two nervous cell lines. To this aim, neurons (SH-SY5Y) and glial (A172) cells were treated with differently-acting genotoxic agents (bleomycin, actinomycin-D, methyl methanesulfonate, mitomycin C, and griseofulvin). After discarding cytotoxicity, genotoxicity was evaluated by a battery of assays encompassing detection of different genetic lesions. Results obtained showed that glial cells are generally more resistant to genotoxic damage induced by clastogenic agents, but more sensitive to aneugenic effects. These results highlight the need of proper design of in vitro neurotoxicology studies, especially for neurogenotoxicity screening, emphasizing the importance of employing more than one nervous cell type for testing the potential toxicity of a particular exposure.

Development of an accommodating intra-ocular lens--in vitro prevention of re-growth of pig and rabbit lens capsule epithelial cells.

Cataract surgery is routinely performed to replace the clouded lens by a rigid polymeric intra-ocular lens unable to accommodate. By implanting a silicone gel into an intact capsular bag the accommodating properties of the natural lens can be maintained or enhanced. The implantation success of accommodating lenses is hampered by the occurrence of capsular opacification (PCO) due to lens epithelial cell (LEC) growth. In order to prevent LEC proliferation, a treatment regime using actinomycin D, cycloheximide and water was developed. The effectiveness of treatment was analyzed using an in vitro, MTT-based cell culture system and an ex vivo pig eye model in which the implanted lens-in-the-bag is cultured as a whole. LEC were exposed to treatment solutions for 5 min, then the cells were allowed to recover and to re-colonize the substratum. MTT conversion by cells was transiently inhibited by cycloheximide dissolved in water and by water alone. Exposure to actinomycin D resulted in a lasting inhibition of MTT conversion and consequently cell proliferation. These in vitro data could not be fully reproduced in the ex vivo pig eye model due to essential differences between both models. Treatment with actinomycin D containing solutions, however, resulted in a nearly complete absence of cells on the capsular wall. The pig eye model is a promising approach to further evaluate the effects of peri-surgical treatment during the accommodating intra-ocular lens implantation.

Muramyl peptides augment cytotoxic effect of tumor necrosis factor-alpha in combination with cytotoxic drugs on tumor cells.

We have demonstrated that biologically active muramyl peptides, in particular, glucosaminylmuramyl dipeptide (GMDP), augmented in vitro cytotoxic activity of tumor necrosis factor-alpha (TNF-alpha) against murine fibrosarcoma L929 cells. The introduction of GMDP resulted in cytotoxic effect characteristic for substantially higher dose of cytokine. Even more potent was the combination of GMDP, TNF-alpha and Actinomycin D (ActD). According to clonogenic and MTT assays 100% L929 cells could be killed in culture with low doses of TNF-alpha and ActD if GMDP was present. When cisplatin was substituted for ActD similar results were obtained. GMDP also enhanced cytotoxicity of TNF-alpha and cisplatin against human breast carcinoma MCF7 and histiocytic lymphoma U937 cells. Normal cells, namely human peripheral blood leucocytes and murine peritoneal macrophages, were resistant to selected doses of TNF-alpha/cisplatin/GMDP.

Anti-apoptotic effect of hepatocyte growth factor from actinomycin D in hepatocyte-derived HL7702 cells is associated with activation of PI3K/Akt signaling.

Actinomycin D (ActD) is a well-known cytotoxic chemotherapeutic reagent and the prevention of ActD-induced apoptotic cell death has been an attractive issue for biomedical investigators. Since phosphatidylinositol-3 kinase (PI3K)/Akt pathway is essential for cell survival, the present study has examined whether the preventive effect of hepatocyte growth factor (HGF) on ActD-induced apoptotic cell death in a human hepatocyte-derived cell line (HL7702) is associated with PI3K/Akt activation. Apoptotic cell death was measured by several methods including Hoechst 33342 staining, DNA fragmentation, and flow cytometry. We found that ActD caused a significant increase in apoptotic cell death, an effect significantly prevented by pre-addition of HGF in the cultures. HGF was found to significantly activate Akt phosphorylation while pre-treatment with PI3K specific inhibitor wortmannin further enhanced ActD-induced apoptotic effect, and also significantly prevented HGF's protection against ActD-induced apoptosis. These results suggest that HGF's prevention of ActD-induced apoptotic cell death in HL7702 cells is associated with the activation of PI3K/Akt signaling.