DNA Damage In Cancer Cells Research Articles

TRAIL causes deletions at the HPRT and TK1 loci of clonogenically competent cells

When chemotherapy and radiotherapy are effective, they function by inducing DNA damage in cancerous cells, which respond by undergoing apoptosis. Some adverse effects can result from collateral destruction of non-cancerous cells, via the same mechanism. Therapy-related cancers, a particularly serious adverse effect of anti-cancer treatments, develop due to oncogenic mutations created in non-cancerous cells by the DNA damaging therapies used to eliminate the original cancer. Physiologically achievable concentrations of direct apoptosis inducing anti-cancer drugs that target Bcl-2 and IAP proteins possess negligible mutagenic activity, however death receptor agonists like TRAIL/Apo2L can provoke mutations in surviving cells, probably via caspase-mediated activation of the nuclease CAD. In this study we compared the types of mutations sustained in the HPRT and TK1 loci of clonogenically competent cells following treatment with TRAIL or the alkylating agent ethyl methanesulfonate (EMS). As expected, the loss-of-function mutations in the HPRT or TK1 loci triggered by exposure to EMS were almost all transitions. In contrast, only a minority of the mutations identified in TRAIL-treated clones lacking HPRT or TK1 activity were substitutions. Almost three quarters of the TRAIL-induced mutations were partial or complete deletions of the HPRT or TK1 genes, consistent with sub-lethal TRAIL treatment provoking double strand breaks, which may be mis-repaired by non-homologous end joining (NHEJ). Mis-repair of double-strand breaks following exposure to chemotherapy drugs has been implicated in the pathogenesis of therapy-related cancers. These data suggest that TRAIL too may provoke oncogenic damage to the genomes of surviving cells.

Self‐Assembled Oleic Acid Nanoparticle Mediated Inhibition of Mitogen‐Activated Protein Kinase Signaling in Combination with DNA Damage in Cancer Cells

AbstractMitogen‐activated protein kinase (MAPK) signaling has been dysregulated in different types of cancers. However, targeting MAPK signaling with small molecules leads to severe toxic side effects to the patients as well as manifestation of drug resistance. To address these, we have developed 120 nm sized self‐assembled, biocompatible, biodegradable oleic acid nanoparticles (OA‐NPs) which can simultaneously contain AZD6244 (MAPK inhibitor) and cisplatin (DNA damaging drug). These OA‐NPs released AZD6244 and cisplatin in increased amount in pH 5.5 compared to pH 7.4 in a slow and sustained manner over 4 days with excellent stability at 4 °C for 2 months in water and in blood circulation mimic for 6 days. Moreover, these OA‐NPs showed much improved in vitro cytotoxicity in cervical cancer (HeLa) and triple negative breast cancer (MDA‐MB‐231) cells at 48 h and in hepatocellular carcinoma (Hep3B) and cisplatin‐resistant hepatocellular carcinoma (Hep3B‐R) cells at 24 h. In HeLa cells, these OA‐NPs induced apoptosis through inhibiting MAPK signaling and damaging DNA after being internalized through macropinocytosis and homed into the acidic lysosomal compartments. These OA‐NPs have the potential to be translated into the clinic for targeting multiple oncogenic signaling pathways and damaging DNA concurrently for augmented efficacy, reduced toxicity, and overcoming drug resistance in next‐generation cancer treatment.

Cancer cell-targeted two-photon fluorescence probe for the real-time ratiometric imaging of DNA damage.

Real-time imaging of DNA damage in cancer cells could provide valuable information on the formation and development of cancer. Herein, a two-photon fluorescence probe was discovered. Through sequential ICT processes, it allows successful in vivo visualization of DNA damage in cancer cells by one/two-photon microscopic imaging or by the unaided eye and a hand-held ultraviolet lamp.

Modulation of DNA damage response and induction of apoptosis mediates synergism between doxorubicin and a new imidazopyridine derivative in breast and lung cancer cells

DNA damage response machinery (DDR) is an attractive target of cancer therapy. Modulation of DDR network may alter the response of cancer cells to DNA damaging anticancer drugs such as doxorubicin. The aim of the present study is to investigate the effects of a newly developed imidazopyridine (IAZP) derivative on the DDR after induction of DNA damage in cancer cells by doxorubicin. Cytotoxicity sulphrhodamine-B assay showed a weak anti-proliferative effect of IAZP alone on six cancer cell lines (MCF7, A549, A549DOX11, HepG2, HeLa and M8) and a normal fibroblast strain. Combination of IAZP with doxorubicin resulted in synergism in lung (A549) and breast (MCF7) cancer cells but neither in the other cancer cell lines nor in normal fibroblasts. Molecular studies revealed that synergism is mediated by modulation of DNA damage response and induction of apoptosis. Using constant-field gel electrophoresis and immunofluorescence detection of γ-H2AX foci, IAZP was shown to inhibit the repair of doxorubicin-induced DNA damage in A549 and MCF7 cells. Immunoblot analysis showed that IAZP suppresses the phosphorylation of the ataxia lelangiectasia and Rad3 related (ATR) protein, which is an important player in the response of cancer cells to chemotherapy-induced DNA damage. Moreover, IAZP augmented the doxorubicin-induced degradation of p21, activation of p53, CDK2, caspase 3/7 and phosphorylation of Rb protein. These effects enhanced doxorubicin-induced apoptosis in both cell lines. Our results indicate that IAZP is a promising agent that may enhance the cytotoxic effects of doxorubicin on some cancer cells through targeting the DDR. It is a preliminary step toward the clinical application of IAZP in combination with anticancer drugs and opens the avenue for the development of compounds targeting the DDR pathway that might improve the therapeutic index of anticancer drugs and enhance their cure rate.

Therapeutic gold, silver, and platinum nanoparticles.

There are an abundance of nanoparticle technologies being developed for use as part of therapeutic strategies. This review focuses on a narrow class of metal nanoparticles that have therapeutic potential that is a consequence of elemental composition and size. The most widely known of these are gold nanoshells that have been developed over the last two decades for photothermal ablation in superficial cancers. The therapeutic effect is the outcome of the thickness and diameter of the gold shell that enables fine tuning of the plasmon resonance. When these metal nanoparticles are exposed to the relevant wavelength of light, their temperature rapidly increases. This in turn induces a localized photothermal ablation that kills the surrounding tumor tissue. Similarly, gold nanoparticles have been developed to enhance radiotherapy. The high-Z nature of gold dramatically increases the photoelectric cross-section. Thus, the photoelectric effects are significantly increased. The outcome of these interactions is enhanced tumor killing with lower doses of radiation, all while sparing tissue without gold nanoparticles. Silver nanoparticles have been used for their wound healing properties in addition to enhancing the tumor-killing effects of anticancer drugs. Finally, platinum nanoparticles are thought to serve as a reservoir for platinum ions that can induce DNA damage in cancer cells. The future is bright with the path to clinical trials is largely cleared for some of the less complex therapeutic metal nanoparticle systems.

Abstract 817: Sepantronium is a DNA damaging agent that potentiates PLK1 inhibitor volasertib

Abstract In a search for novel agents that boost the anti-neoplastic effects of polo-like kinase 1(PLK1) inhibitor volasertib, we found that a sepantronium and volasertib combination at the nano mole concentration potently inhibited growth of various non-small cell lung cancer (NSCLC) cell lines than either drug alone in vitro. Combination use of volasertib with sepantronium inhibited adaptation of cells to polo arrest. Addition of sepantronium to volasertib prevented accumulation of survivin and cyclin B protein at a concentration causing no appreciable survivin down regulation. Sepantronium, was originally developed as a survivin inhibitor. However, sepantronium induced cell cycle arrest from G1 or G2/M phase. Further studies demonstrated DNA damage of cancer cells when they are treated with sepantronium, which is evidenced by induction of phospho-gammaH2AX. In line with induction of a DNA damage response in cancer cells, known DNA damage response sensors and transducers ATM, ATR, CHK1, CHK2, p53 are phosphorylated following drug treatment. Meanwhile, expression of CDKN1A, BAX and XRCC5 are induced at the mRNA level as determined by quantitative real time PCR. A single cell electrophoresis assay (Comet assay) of cells treated with sepantronium revealed severe DNA strand breaks. M-phase arrest does not increase the lethality of DNA damage by sepantronium as compared to G1 phase arrest. Knock down of survivin did not cause DNA damage. Hence, sepantronium is a DNA damaging agent that synergizes with volasertib and down- regulation of survivin is likely the consequence of DNA damage induced by sepantronium. Citation Format: Mei Hong, Mingqiang Ren, Jeane Silva, Zhonglin Hao. Sepantronium is a DNA damaging agent that potentiates PLK1 inhibitor volasertib. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 817. doi:10.1158/1538-7445.AM2014-817

In honour of N. Yngve Öhrn: surveying proton cancer therapy reactions with Öhrn's electron nuclear dynamics method. Aqueous clusters radiolysis and DNA-base damage by proton collisions

Proton cancer therapy (PCT) utilises high-energy H+ projectiles to cure cancer. PCT healing arises from its DNA damage in cancerous cells, which is mostly inflicted by the products from PCT water radiolysis reactions. While clinically established, a complete microscopic understanding of PCT remains elusive. To help in the microscopic elucidation of PCT, Professor Öhrn's simplest-level electron nuclear dynamics (SLEND) method is herein applied to H+ + (H2O)3–4 and H+ + DNA-bases at ELab = 1.0 keV. These are two types of computationally feasible prototypes to study water radiolysis reactions and H+-induced DNA damage, respectively. SLEND is a time-dependent, variational, non-adiabatic and direct-dynamics method that adopts a nuclear classical-mechanics description and an electronic single-determinantal wavefunction. Additionally, our SLEND + effective-core-potential method is herein employed to simulate some computationally demanding PCT reactions. Due to these attributes, SLEND proves appropriate for the simulation of various types of PCT reactions accurately and feasibly. H+ + (H2O)3–4 simulations reveal two main processes: H+ projectile scattering and the simultaneous formation of H and OH fragments; the latter process is quantified through total integrals cross sections. H+ + DNA-base simulations reveal atoms and groups displacements, ring openings and base-to-proton electron transfers as predominant damage processes.

Parmelia sulcata Taylor and Usnea filipendula Stirt induce apoptosis-like cell death and DNA damage in cancer cells.

Successful cancer treatments still require more compounds to be isolated from natural sources. Thus, we have investigated anti-proliferative/apoptotic effects of methanolic extracts of lichen species Parmelia sulcata Taylor and Usnea filipendula Stirt on human lung cancer (A549, PC3), liver cancer (Hep3B) and rat glioma (C6) cells. Anti-proliferative effects were monitored by MTT and adenosine triphosphate viability assays, while genotoxic activity was studied using the comet assay. Additionally, cell death mode and apoptosis assays (fluorescence staining, caspase-cleaved cytokeratin 18, caspase-3 activity and PARP cleavage) were performed. Extracts produced anti-population growth effects in a dose-dependent manner (1.56-100 μg/ml) by inducing apoptosis-like cell death. This resulted in the lines having the presence of pyknotic cell nuclei. In addition, significant increase in genetic damage in the cell lines was seen, indicating that DNA damage may have been responsible for apoptotic cell death. In this study, methanolic extracts of Parmelia sulcata and Usnea filipendula induced apoptosis-like cell death by causing DNA damage, to cancer cells.

Genotoxic effect of doxorubicin–transferrin conjugate on human leukemia cells

Doxorubicin (DOX) is an effective anthracycline antibiotic against a wide spectrum of tumors and hematological malignancies. It mainly interacts with DNA, but can also generate reactive oxygen species (ROS), which damage cell components. Unfortunately, numerous side effects, such as severe cardiotoxicity and bone-marrow suppression, limit its use. To reduce this obstacle and improve its pharmacokinetics, we conjugated DOX to transferrin (TRF), a human plasma protein. In our study, we compared the effect of DOX and the doxorubicin–transferrin conjugate (DOX-TRF) on human leukemic lymphoblasts (CCRF-CEM), and on normal peripheral blood mononuclear cells (PBMC). In parallel, experiments were carried out on two human chronic myeloid leukemia (CML) cell lines derived from K562 cells, of which one was sensitive and the other resistant to doxorubicin (K562/DOX). By use of the alkaline comet assay, the effect of the agents on the induction of DNA damage in normal human cells and human leukemia cells was determined. Oxidative and alkylating DNA damage were assayed by a slightly modified comet assay that included the use of the DNA-repair enzymes endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg). To investigate whether DNA breaks are the result of apoptosis, we examined the induction of DNA fragmentation visualized as oligosomal ladders after simple agarose electrophoresis under neutral conditions. Modifications of the genome induced by the different drugs were analyzed following assessment of the cell-cycle phase.The DOX-TRF conjugate caused more DNA damage than the free drug, the degree of DNA fragmentation being dependent on the duration of treatment and the cell type analyzed. With neutral agarose electrophoresis we showed that the test compounds caused the formation of a characteristic DNA-ladder pattern. Furthermore, the DOX-TRF conjugate generated a higher percentage of apoptotic cells in the subG1 fraction and blocked more cells in the G2/M phase of the cell cycle than did free DOX. In summary, both agents induced DNA damage in cancer cells, but the DOX-TRF conjugate generated more genotoxic effects and apoptosis than the unconjugated drug.

A novel role of nucleostemin in maintaining the genome integrity of dividing hepatocytes during mouse liver development and regeneration

During liver development and regeneration, hepatocytes undergo rapid cell division and face an increased risk of DNA damage associated with active DNA replication. The mechanism that protects proliferating hepatocytes from replication-induced DNA damage remains unclear. Nucleostemin (NS) is known to be up-regulated during liver regeneration, and loss of NS is associated with increased DNA damage in cancer cells. To determine whether NS is involved in protecting the genome integrity of proliferating hepatocytes, we created an albumin promoter-driven NS conditional-null (albNS(cko) ) mouse model. Livers of albNS(cko) mice begin to show loss of NS in developing hepatocytes from the first postnatal week and increased DNA damage and hepatocellular injury at 1-2 weeks of age. At 3-4 weeks, albNS(cko) livers develop bile duct hyperplasia and show increased apoptotic cells, necrosis, regenerative nodules, and evidence suggestive of hepatic stem/progenitor cell activation. CCl4 treatment enhances degeneration and DNA damage in NS-deleted hepatocytes and increases biliary hyperplasia and A6(+) cells in albNS(cko) livers. After 70% partial hepatectomy, albNS(cko) livers show increased DNA damage in parallel with a blunted and prolonged regenerative response. The DNA damage in NS-depleted hepatocytes is explained by the impaired recruitment of a core DNA repair enzyme, RAD51, to replication-induced DNA damage foci. This work reveals a novel genome-protective role of NS in developing and regenerating hepatocytes.

MiR-23a-mediated inhibition of topoisomerase 1 expression potentiates cell response to etoposide in human hepatocellular carcinoma

BackgroundmicroRNAs have been shown to regulate the chemosensitivity of cancer cells. The aim of this study is to investigate the role and mechanism of mir-23a in enhancing the anti-tumor effect of topoisomerase 2A (TOP2A) poison etoposide in human hepatocellular carcinoma (HCC).MethodsThe anti-tumor effect of chemotherapeutic agents in HCC cells were examined in vitro and in vivo xenograft model. Expression of mRNA and miRNAs were determined by quantitative real-time PCR. Protein expression was analyzed by immunoblotting.ResultsOverexpression of mir-23a could significantly potentiate the in vitro and in vivo anti-tumor effect of etoposide; however, ectopic expression of miR-23a fails to sensitize HCC cells to 5-fluorouracil treatment, indicating the miR-23a-induced cancer cell hypersensitivity in chemotherapy is TOP2A-specific though miR-23a overexpression could not directly up-regulate TOP2A expression. Topoisomerase 1(TOP1) is down-regulated in miR-23a-overexpressed HCC cells. MiR-23a could directly bind to 3′untranslated region of TOP1 mRNA, and suppress the corresponding protein expression and inhibition of miR-23a further arguments the expression of TOP1. MiR-23a was up-regulated during DNA damage in cancer cells in line with the p53 expression. Up-regulation of p53 induces mir-23a expression, while suppression of p53 inhibits miR-23a in HCC cells.ConclusionOur study sheds light on the role of miR-23a as a potential target in regulating chemosensitivity of HCC cells.

Newcastle Disease Virus (NDV) Iraqi Strain AD2141 Induces DNA Damage and FasL in Cancer Cell Lines

The oncolytic viruses are promising form of cancer therapy which is based on the selectively killing of the cancer cells. This study was aimed to investigate the role of Newcastle disease virus (NDV) Iraqi strain AD2141 in apoptosis. Firstly, the virulence of AD2141 was detected in embryonated chicken eggs after 48hrs of infection. It was observed a hemorrhage in the skin of infected embryos that led to death. Then, the ability of this strain for regression cancer cell lines was examined. By using cytotoxicity test, it was found 128 HAU/ml of AD2141 had a potent inhibition against growth of RD and AMN3 after 72hrs of exposure time; the inhibition rate was 86.8% and 86.98% respectively. Moreover, the a poptotic activity of AD2141 was examined by comet assay. A significant induction of DNA damage in cancer cells was determined after 48hrs of exposure time; the average DNA tail length in RD, AMN3, and AMGM was 55.46, 79.1, and 84.4 respectively, and the percentage of apoptosis in treated RD, AMN-3, and AMGM was 78%, 92%, and 94% respectively. Furthermore, the concentration of FasL in treated and untreated cells was measured by immunofluoresent technique. FasL was elevated particularly in treated human cancer cell lines, RD and AMGM; it was 62.5pg/ml and 31.25pg/ml respectively. From these results, it can be concluded that NDV Iraqi strain AD2141 is a velogenic type; it caused death in infected chicken embryos. In addition it is an oncolytic virus due to induced apoptosis by damaging DNA and elevating FasL concentration in human cancer cells.

Abstract B11: YM155 synergizes with Plk1 inhibitor volasertib in killing of non-small cell lung cancer

Abstract Non-small cell lung cancer (NSCLC) remains the leading cause of cancer death in the US. There is a pressing need for more regimens that are both effective and well tolerated by the typical metastatic lung cancer population. The synthetic lethal approach is emerging as an attractive strategy in mining such regimens. Volasertib is a highly selective and potent small molecule inhibitor of polo-like kinase 1(Plk1) that is essential for cell cycle progression. Its toxicity is mainly hematological and reversible. Single agent performance is less than optimal. In a search for novel agents that boost the anti-neoplastic effects of Plk1 inhibitor volasertib, we found that a YM155 and volasertib combination at the nano mole concentration potently inhibited growth of various NSCLC cell lines than either drug alone could achieve in vitro. Flow cytometry analysis showed that the combination regimen is much more potent in inducing apoptosis than either alone. Volasertib normally arrests cells at G2/M. Cells treated with both drugs initially arrest in G2 and then distribute equally in both G1/S and G2/M suggesting that fewer cells escaped to G1 from the polo arrest compared to volasertib treatment alone in which the majority of them were able to transit to G1 from G2 phase. Western blot analysis demonstrated that addition of YM155 to volasertib prevented accumulation of survivin and cyclin B, which is consistent with a cell cycle arrest before the end of mitosis and cytokinesis when compared to volasertib alone. All these data suggest that YM155 induces cell cycle arrest. However, YM155, originally developed as a survivin inhibitor, only caused modest suppression of survivin expression and could not explain the degree of cell growth inhibition it caused. Further studies demonstrated DNA damage of cancer cells when they are treated with YM155, which is evidenced by induction of phospho-γH2AX, a marker for DNA double strand break (DSB). In line with induction of a DNA damage response in cancer cells, known DNA damage response transducers ATM, ATR, CHK1, CHK2, p53 and BRCA1 are phosphorylated following drug treatment. Meanwhile, expression of CIP1, BAX and KARP1 are induced at the mRNA level as determined by quantitative real time PCR. Therefore, YM155 suppresses cancer cell growth by causing DNA damage and illicits a DNA damage response, cell cycle arrest and programmed cell death. Furthermore, cells with wild type p53 (H661) seem to be more sensitive to YM155 induced growth arrest than cells with p53 deletion (H1299, H520), which is consistent with a role of p53 in mediating programmed cell death after DNA damage. Polo-like kinase is known to play an essential role in recovery from checkpoint-triggered cell cycle arrest. We speculate that volasertib synergizes with YM155 by preventing recovery from DNA damage checkpoint triggered cell cycle arrest. On the other hand, since YM155 causes DNA damage, fewer cells arrested in G2 by volasertib may be able to escape from the polo arrest when their DNAs are severely damaged. Currently, we are testing the two hypotheses. Combination of the two inhibitors deserves close scrutiny in the clinical arena if future studies in an animal model support this prediction. Citation Format: Mei Hong, Mingqiang Ren, John Cowell, Zhonglin Hao. YM155 synergizes with Plk1 inhibitor volasertib in killing of non-small cell lung cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B11.

Methylation of SUV39H1 by SET7/9 results in heterochromatin relaxation and genome instability

Suppressor of variegation 3-9 homolog 1 (SUV39H1), a histone methyltransferase, catalyzes histone 3 lysine 9 trimethylation and is involved in heterochromatin organization and genome stability. However, the mechanism for regulation of the enzymatic activity of SUV39H1 in cancer cells is not yet well known. In this study, we identified SET domain-containing protein 7 (SET7/9), a protein methyltransferase, as a unique regulator of SUV39H1 activity. In response to treatment with adriamycin, a DNA damage inducer, SET7/9 interacted with SUV39H1 in vivo, and a GST pull-down assay confirmed that the chromodomain-containing region of SUV39H1 bound to SET7/9. Western blot using antibodies specific for antimethylated SUV39H1 and mass spectrometry demonstrated that SUV39H1 was specifically methylated at lysines 105 and 123 by SET7/9. Although the half-life and localization of methylated SUV39H1 were not noticeably changed, the methyltransferase activity of SUV39H1 was dramatically down-regulated when SUV39H1 was methylated by SET7/9. Consequently, H3K9 trimethylation in the heterochromatin decreased significantly, which, in turn, led to a significant increase in the expression of satellite 2 (Sat2) and α-satellite (α-Sat), indicators of heterochromatin relaxation. Furthermore, a micrococcal nuclease sensitivity assay and an immunofluorescence assay demonstrated that methylation of SUV39H1 facilitated genome instability and ultimately inhibited cell proliferation. Together, our data reveal a unique interplay between SET7/9 and SUV39H1--two histone methyltransferases--that results in heterochromatin relaxation and genome instability in response to DNA damage in cancer cells.

Ellagic Acid: Cytodifferentiating and Antiproliferative Effects In Human Prostatic Cancer Cell Lines

Recently, increasing attention has been given to neuroendocrine differentiation (NED) of Prostate Cancer and its diagnostic, prognostic and therapeutic potential. During multistep carcinogenesis, cytodifferentiation of malignant/premalignant cells into more mature or normal-like cells, has become an attractive modality of treatment and promises to be a less toxic and a more specific targeting strategy than conventional chemotherapy. In this study we investigated the capacity of a polyphenol, ellagic acid (EA), to induce differentiation of two prostate cancer cell lines: LnCap and DU145. NED markers, Chromogranin A (CgA) and p75NGFR levels were evaluated by immunocytochemistry. DNA methyltransferase- 1 (DNMT-1) and phospho-Rb (p-Rb) expression were evaluated by western blotting. Akt activation was evaluated by ELISA. Finally the ability of EA to induce DNA damage in cancer cells was examined using the COMET assay. Treatment with EA significantly reduced CgA levels and increased p75NGFR expression. Moreover p-Rb, DNMT-1 levels and Akt activation/phosphorylation were decreased. EA treatment induced, in a dose-dependent manner, a marked increase in DNA damage, both in LnCap and DU145 cell lines. The results of this study demonstrate that EA treatment represents a new approach and highly effective strategy in reducing carcinogenesis. Therefore, EA may be considered in a promising new class of cancer therapeutic agent, with both antiproliferative and pro-differentiation properties.

The MAPK Pathway Signals Telomerase Modulation in Response to Isothiocyanate-Induced DNA Damage of Human Liver Cancer Cells

4-methylthiobutyl isothiocyanate (MTBITC), an aliphatic, sulphuric compound from Brassica vegetables, possesses in vitro and in vivo antitumor activity. Recently we demonstrated the potent growth inhibitory potential of the DNA damaging agent MTBITC in human liver cancer cells. Here we now show that MTBITC down regulates telomerase which sensitizes cells to apoptosis induction. This is mediated by MAPK activation but independent from production of reactive oxygen species (ROS). Within one hour, MTBITC induced DNA damage in cancer cells correlating to a transient increase in hTERT mRNA expression which then turned into telomerase suppression, evident at mRNA as well as enzyme activity level. To clarify the role of MAPK for telomerase regulation, liver cancer cells were pre-treated with MAPK-specific inhibitors prior to MTBITC exposure. This clearly showed that transient elevation of hTERT mRNA expression was predominantly mediated by the MAPK family member JNK. In contrast, activated ERK1/2 and P38, but not JNK, signalled to telomerase abrogation and consequent apoptosis induction. DNA damage by MTBITC was also strongly abolished by MAPK inhibition. Oxidative stress, as analysed by DCF fluorescence assay, electron spin resonance spectroscopy and formation of 4-hydroxynonenal was found as not relevant for this process. Furthermore, N-acetylcysteine pre-treatment did not impact MTBITC-induced telomerase suppression or depolarization of the mitochondrial membrane potential as marker for apoptosis. Our data therefore imply that upon DNA damage by MTBITC, MAPK are essential for telomerase regulation and consequent growth impairment in liver tumor cells and this detail probably plays an important role in understanding the potential chemotherapeutic efficacy of ITC.

NVX-412, a New Oncology Drug Candidate, Induces S-Phase Arrest and DNA Damage in Cancer Cells in a p53-Independent Manner

The new molecular entity quinoxalinhydrazide derivative NVX-412 was identified as a promising drug candidate for the treatment of various cancer types due to its strong cytotoxic activity and relative specificity. Here, we provide first data about the mechanisms of action of NVX-412. We show that NVX-412 exerts its anti-neoplastic activity in a p53-independent manner and induces S-phase arrest and DNA damage as assessed by γH2AX staining. We suggest a bi-modal (dose-dependent) mode of action of NVX-412, being primarily cytostatic at lower and predominantly cytotoxic at higher concentrations. Based on the broad and consistent anti-neoplastic activity observed, NVX-412 holds promise as an effective drug candidate for the treatment of various cancer types, especially for hematological malignancies with highly unmet medical need.

Biotic and Abiotic Factors Affecting the Content of the Chosen Antioxidant Compounds in Vegetables

Biotic and Abiotic Factors Affecting the Content of the Chosen Antioxidant Compounds in Vegetables Vegetables are a rich source of biologically active substances, which support the body's defense mechanisms. A large group of these substances are compounds with antioxidant properties. Apart from vitamins (A, C and E), tocopherols, carotenoids, glutathiones and thiocyanates, polyphenols are also classified as the compounds of antioxidant properties being found in plants. They include: phenolic acids, flavonoids and hydroxycinnamic acid and among them a large group of anthocyanins. These compounds inhibit DNA damage in cancer cells, induce the production of insulin in the pancreas and protect the human brain from aging. They have also high antioxidant activity, which determines the defense mechanisms of plants under stress, such as temperature varia-tions, UV radiation, attacks by pests and mechanical damage. Their content may vary among individual plants of the same species, which is associated with a number of internal and external conditions, such as genetic factors, environmental and agronomic. The contents of anthocyanins determines plant species, botanical variety and breeding and biological processes associated with ontogenesis. Also climatic and soil factors, the factors influencing the content of antioxidants and agronomic factors such as method, place and date of planting, fertilizing, mulching, salinity may contribute to the formation of stress conditions during plant growth and increase the content of antioxidants in plants. This review focused on the content, composition, and antioxidant capacity of vegetables.

CS07-5. Loss of p53 increases unphosphorylated STAT1, which induces resistance to DNA damage in cancer cells

CS07-5. Loss of p53 increases unphosphorylated STAT1, which induces resistance to DNA damage in cancer cells

Zyxin Is a Critical Regulator of the Apoptotic HIPK2-p53 Signaling Axis

HIPK2 activates the apoptotic arm of the DNA damage response by phosphorylating tumor suppressor p53 at serine 46. Unstressed cells keep HIPK2 levels low through targeted polyubiquitination and subsequent proteasomal degradation. Here we identify the LIM domain protein Zyxin as a novel regulator of the HIPK2-p53 signaling axis in response to DNA damage. Remarkably, depletion of endogenous Zyxin, which colocalizes with HIPK2 at the cytoskeleton and in the cell nucleus, stimulates proteasome-dependent HIPK2 degradation. In contrast, ectopic expression of Zyxin stabilizes HIPK2, even upon enforced expression of its ubiquitin ligase Siah-1. Consistently, Zyxin physically interacts with Siah-1, and knock-down of Siah-1 rescues HIPK2 expression in Zyxin-depleted cancer cells. Mechanistically, our data suggest that Zyxin regulates Siah-1 activity through interference with Siah-1 dimerization. Furthermore, we show that endogenous Zyxin coaccumulates with HIPK2 in response to DNA damage in cancer cells, and that depletion of endogenous Zyxin results in reduced HIPK2 protein levels and compromises DNA damage-induced p53 Ser46 phosphorylation and caspase activation. These findings suggest an unforeseen role for Zyxin in DNA damage-induced cell fate control through modulating the HIPK2-p53 signaling axis.