Greater DNA Damage Research Articles

Estudio de fotoreactivación en cultivos microbiológicos obtenidos de carga microbiana de la superficie de fresas sometidas a diferentes dosis de luz ultra violeta de onda corta UV-C

Currently the government agency US FDA (Food and Drug Administration) has approved the use of (UV-C) ultraviolet short-wavelength light disinfection of fruit, because exposure to ultraviolet light can reduce load of pathogenic microorganisms that may contain on their surface and thus extend the life of products favoring producers. However, misuse of ultraviolet light may also have risks developing resistance of microorganisms to UV-C through a process known as photoreactivation. Objective: To determine the degree of photoreactivation in microbiological cultures that have undergone different doses of ultra violet light short-wave UV-C. Materials and methods: samples of common strawberries to which different doses of UV-C were applied including conventional were taken. the antibiotic effect of UV-C on the surface of the strawberries was analyzed by pre-culture - post. Results and conclusions: the sample under the distance currently used by industry (30 cm from the UVC source for 7.5 min) proved effective distance for decrease microbial growth. However, at a high dose (20 cm from the source and 7.5 min) cultures of coliform bacteria and yeasts showed an exponential microbial growth compared to the other samples (test performed in duplicate), which proves the antithesis of theory that the higher was the dose of UV-C rays would be greater DNA damage by inhibiting microorganism’s growth giving strength to the theory the higher the dose, the greater stimulus photoreactivation and UV-C resistance.

Environmental Toxins Found Historically in the Polycythemia Vera Cluster Area and their Potential for Inducing DNA Damage.

In 2006, the Agency for Toxic Substances and Disease Registry received a request to determine whether a cluster of polycythemia vera patients existed in a northeast Pennsylvania community. A significant cluster of PV cases was identified at the nexus of three counties near several hazardous waste sites. The current study evaluated the potential for a select number of environmental contaminants previously detected in the cluster area to induce DNA damage using in vitro assays with hematopoietic stem-cell derived progenitor cells.CD34+ cells were isolated from normal cord blood samples and were cultured for 48–72 hours to generate erythroid progenitor cells. Eighteen compounds were chosen for the assay; arsenic trioxide, benzo(a)pyrene, benzene, methylene chloride, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), trichloroethylene, potassium chloride, ethylbenzene, benzo[k]fluoranthene, styrene, cadmium chloride, hydroquinone, 1,1,1-trichloroethane, sodium cyanide, manganese chloride, chromium oxide, lead oxide, and sodium arsenite. Genotoxicity of the compounds was determined using the comet assay, and toxicity determined via the cell viability assay.Using the comet assay, 16 compounds at 10 nM concentration, induced a significant amount of DNA damage compared to the control. When evaluating whether a dose-dependent relationship was present, seventeen of the eighteen compounds led to greater DNA damage with increasing exposure concentrations. 2,3,7,8-TCDD was particularly potent, inducing DNA damage in virtually all cells at 1 μM. In conclusion, most of the toxins evaluated using the comet assay showed potential to induce DNA damage in hematopoietic cells, and the genotoxic effects were dose-dependent.

Pendimethalin induces oxidative stress, DNA damage, and mitochondrial dysfunction to trigger apoptosis in human lymphocytes and rat bone-marrow cells.

Pendimethalin (PM) is a dinitroaniline herbicide extensively applied against the annual grasses and broad-leaved weeds. There is no report available on PM-induced low-dose genotoxicity in human primary cells and in vivo test models. Such data gap has prompted us to evaluate the genotoxic potential of PM in human lymphocytes and rats. PM selectively binds in the minor groove of DNA by forming covalent bonds with G and C nitrogenous bases, as well as with the ribose sugar. PM induces micronucleus formation (MN) in human lymphocytes, indicating its clastogenic potential. Comet assay data showed 35.6-fold greater DNA damage in PM (200μM)-treated human lymphocytes. Rat bone-marrow cells, at the highest dose of 50mg/kg bw/day of PM also exhibited 10.5-fold greater DNA damage. PM at 200μM and 50mg/kg bw/day induces 193.4 and 229% higher reactive oxygen species generation in human lymphocytes and rat bone-marrow cells. PM-treated human lymphocytes and rat bone-marrow cells both showed dysfunction of mitochondrial membrane potential (ΔΨ m). PM exposure results in the appearance of 72.2 and 35.2% sub-G1 apoptotic peaks in human lymphocytes and rat bone-marrow cells when treated with 200μM and 50mg/kg bw/day of PM. Rats exposed to PM also showed imbalance in antioxidant enzymes and histological pathology. Overall, our data demonstrated the genotoxic and apoptotic potentials of PM in human and animal test models.

Association of H3K79 monomethylation (an epigenetic signature) with arsenic-induced skin lesions

Arsenic, a non mutagenic carcinogen, poses a profound health risk upon prolonged exposure. The objective of the study was to analyze the post-translational modifications of the major histone H3 and the associated molecular crosstalk to identify the epigenetic signature of arsenic susceptibility. Herein, we identified significant upregulation of H3K79me1, in individuals with arsenic-induced skin lesion (WSL), and H3K79me1 was found to be regulated by the upstream methyltransferase DOT1L. Moreover, the downstream target molecule 53BP1, a tumor suppressor protein that has a docking preference for H3K79me1 at a site of a double-strand break (DSB), was downregulated, indicating greater DNA damage in the WSL group. Western blot data confirmed higher levels of γH2AX, a known marker of DSBs, in group WSL. In vitro dose–response analysis also confirmed the association of the H3K79me1 signature with arsenic toxicity. Taken together, our findings revealed that H3K79me1 and DOT1L could be a novel epigenetic signature of the arsenic-exposed WSL group.

Abstract 264: The cell cycle checkpoint kinase 1/2 inhibitor, LY2606368 with PARP inhibition results in synergistic cytotoxicity in high-grade serous ovarian cancer (HGSOC) at lower than physiologically administered concentrations

Abstract Background: Chk1/2 are major cell cycle regulators in p53-deficient tumors, such as HGSOC. Chk1 plays a critical role in DNA repair, facilitating the BRCA2-RAD51 interaction by phosphorylating the BRCA2 C-terminal domain and Thr309-RAD51. Clinical activity with the Chk1/2 inhibitor, LY2606368 mesylate monohydrate (LY), has been observed in solid tumors. We hypothesize that Chk1 inhibition would sensitize HGSOC to PARP inhibition (PARPi) by preventing nuclear RAD51 foci formation, thus impairing DNA repair. We investigated potential synergy of the combination of LY and the PARPi, olaparib (O), in HGSOC cell lines, testing lower concentrations than clinically attained. Materials and Methods: We examined cytotoxicity, DNA damage, and nuclear RAD51 foci formation by LY and/or O using XTT assay, comet assay and immunofluorescence (IF), in 3 HGSOC cell lines: two BRCA1/2 wild type (CAOV3, OV90), and one with BRCA2 mutation (PEO1). We examined a dose range based on clinically achievable concentrations of LY (0.53μM–1.34μM) and O (7.8μM–11.0μM) and calculated IC50 concentrations for cytotoxicity. The combination index (CI) was calculated to evaluate synergism. DNA damage and nuclear RAD51 foci formation were examined. DMSO was used as vehicle control. All experiments were performed in at least three replicates in all cell lines. Results are presented as mean ± SEM. Results: LY alone yielded cytotoxicity in CAOV3, OV90, and PEO1 with IC50 6.34nM, 35.2nM, 12.6nM, respectively. O 5μM monotherapy resulted in 47%, 13%, and 69% cytotoxicity in CAOV3, OV90, and PEO1. LY/O combination showed 51%, 58% and 82% cell injury in CAOV3, OV90 and PEO1. CI values indicated cytotoxicity synergism with the combination. LY/O (5nM/5μM) treatment showed greater DNA damage than O alone in OV90 and PEO1 (p<0.05), and a similar trend was observed in CAOV3 (20.2% ±3.10 vs. 6.71% ±1.08, p = 0.05). The mean percentage of cells with ≥ 5 nuclear RAD51 foci decreased with LY/O (5nM/5μM) combination therapy compared to O alone in CAOV3 (9.03% ±1.85 vs. 36.3% ±7.45, p<0.01) and OV90 (3.66% ±1.84 vs. 22.9% ±3.08, p<0.01), but not in PEO1 (1.26% ± 1.26 vs. 3.73% ±3.73). Ongoing work includes cell cycle analysis and immunoblotting to examine biochemical changes in DNA repair and cell cycle checkpoint pathways. Conclusions: Our preliminary results suggest synergistic activity of the combination of LY and PARPi in HGSOC cell lines using concentrations that are lower than clinically attainable in patients. Citation Format: Yeong-ran Ahn, Takuhei Yokoyama, Minshu Yu, Nicolas Gordon, Elise C. Kohn, Jung-min Lee. The cell cycle checkpoint kinase 1/2 inhibitor, LY2606368 with PARP inhibition results in synergistic cytotoxicity in high-grade serous ovarian cancer (HGSOC) at lower than physiologically administered concentrations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 264.

Effects of oil-related environmental pollutants on gonads of the freshwater leech Limnatis nilotica (Annelida, Hirudinea)

The main objective of this study was to determine the association between exposure to a mixture of benzene, toluene, ethylbenzene, and xylene (BTEX) and reproductive toxicity in the freshwater leech Limnatis nilotica. It was observed that exposure to 1.4 and 2.8 mg L−1 BTEX had a significant effect on previtellogenic and vitellogenic oocytes, but no effect on oogonia or early oocytes. In the 1.4 mg L−1 treatment group, previtellogenic and vitellogenic oocytes were characterized by clumping and shrinkage of the cytoplasm. In the 2.8 mg L−1 treatment group, the effect was more severe and the number of degenerating oocytes increased. The oocytes were easily identified by the vacuolization of their cytoplasm and nuclei. Exposure to 1.4 mg L−1 BTEX affected spermatogenesis by damaging spermatogenic cysts. 2.8 mg L−1 BTEX treatment caused severe damage to spermatogenesis manifested by the complete loss of normal cysts. The histopathological effects were confirmed by the comet assay because DNA damage was detected in both ovarian and testicular cells and was BTEX dose-dependent. The greatest DNA damage was recorded at 2.8 mg L−1 of BTEX in both ovarian and testicular cells compared to the control.

SGN-CD33A in Combination with Hypomethylating Agents Is Highly Efficacious in Preclinical Models of AML

Acute myeloid leukemia (AML) remains a therapeutic challenge. Long-term survival rates, in particular for older AML patients, remain poor, highlighting the need for improved and well-tolerated treatment options. AML patients who are unfit for high dose chemotherapy in the US are often prescribed hypomethylating agents (azacitidine or decitabine) although the efficacy of these agents in this population was modest (Estey, Leukemia 2013).SGN-CD33A is a CD33-directed antibody drug conjugate (ADC) that is comprised of a cysteine-engineered anti-CD33 antibody, a cleavable dipeptide linker that is highly stable in circulation, and a PBD dimer that binds DNA with high intrinsic affinity. The ADC is active as a single agent in preclinical models of AML that are characteristically resistant to chemotherapy (multi-drug-resistant, MDR-positive) (Sutherland et al. Blood 2013). In the present study, we tested the activity of SGN-CD33A in combination with hypomethylating agents (HMA), azacitidine or decitabine. We hypothesized that the combination of SGN-CD33A with an HMA will have greater impact on the DNA repair pathway in leukemic cells, furthering the processes of apoptosis and cell death. MDR-positive AML cells were treated for 96 hours with SGN-CD33A and each of the HMAs alone and in combination, evaluating both simultaneous as well as sequential administration. Enhanced tumor cell killing was observed when AML cells were treated concomitantly with the combination of SGN-CD33A and an HMA or pre-treated with HMAs prior to the addition of SGN-CD33A.Superior anti-leukemic activity of subtherapeutic doses of SGN-CD33A in combination with HMAs was observed in mouse xenograft models generated with MDR-positive AML cell lines. In the difficult to treat HEL9217 model, decitabine or a single dose of 200 mcg/kg SGN-CD33A delayed tumor growth, while significant reductions in tumor growth were observed for the combination treatment (p=0.0001). Improved antitumor activity in this model was also observed for SGN-CD33A in combination with azacitidine. SGN-CD33A in combination with decitabine significantly reduced tumor burden compared to either agent alone in the TF1-α AML model (p=0.0002). Similarly, sequenced dosing of azacitidine followed by a subtherapeutic dose of 30 mcg/kg SGN-CD33A in mice bearing KG-1 xenografts delivered greater antitumor activity compared to the individual agents (p=0.0001).To investigate the mechanism underlying the enhancement in antileukemic activity, we looked at the impact of HMAs alone and in combination with SGN-CD33A on myeloid marker expression, PBD drug release, and impact on the DNA damage and apoptotic pathways. In AML cell lines that did not show improved cytotoxic activity with the combination of SGN-CD33A and HMA, HMA treatment appeared to have no positive effect on the cell surface levels of CD33. However in responsive cell lines such as TF1-α, HMA treatment resulted in time- and dose-dependent increases in the levels of CD33. Significant increases in expression were observed between 2 and 4 days with decitabine and after 4 days with azacitidine. In addition, more PBD dimer drug from SGN-CD33A was incorporated into DNA in HMA treated cells. Concomitant, the treatment of AML cells with the combination of SGN-CD33A and HMA resulted in greater DNA damage and apoptosis as shown by the increased levels of the early DNA damage marker, γH2AX, and the formation of cleaved PARP, an apoptosis marker, compared to either agent alone. Studies are in progress to investigate the effects on other components of the DNA repair and cell cycle pathways.These findings demonstrate that SGN-CD33A can be successfully combined with hypomethylating agents to deliver marked antitumor activity in preclinical drug-resistant models of AML. DisclosuresSutherland:Seattle Genetics, Inc.: Employment. Yu:Seattle Genetics, Inc.: Employment. O'Day:Seattle Genetics,Inc: Employment. Alley:Seattle Genetics,Inc.: Employment. Anderson:Seattle Genetics, Inc.: Employment. Emmerton:Seattle Genetics, Inc.: Employment. Zeng:Seattle Genetics, Inc.: Employment. O'Meara:Seattle Genetics, Inc: Employment, Equity Ownership. Feldman:Seattle Genetics,Inc: Employment. Kennedy:Seattle Genetics,Inc: Employment, Equity Ownership, Honoraria, Speakers Bureau. Ryan:Seattle Genetics, Inc.: Employment. Benjamin:Seattle Genetics, Inc: Employment.

Abstract C32: Characterization of molecular targets of the novel platinum agent PT-112 in human colon cancer cells

Abstract Background: The purpose of this study was to delineate differences in the mechanism of action (MOA) and to identify unique molecular targets responsible for the antineoplastic effects of PT-112 when compared to oxaliplatin. PT-112 is a novel platinum-based chemotherapeutic agent currently under clinical development that has demonstrated superior efficacy in resistant cell lines and xenograft models. In this study we focus on the differential mechanistic effects of PT-112 and oxaliplatin treatment of HCT-116 colon cancer cells and report data derived from multiple cancer signaling pathways. Results: Treatment of HCT-116 with the IC50 dose of PT-112 and oxaliplatin for 24 and 48h resulted in growth inhibition and apoptosis induction as measured by MTT assay, pro-caspase 8 and PARP cleavage. At equipotent doses, oxaliplatin treatment resulted in greater DNA damage as measured by H2A.X serine phosphorylation when compared to PT-112, whereas PT-112 induced markedly greater degrees of expression of several proteins in multiple pathways, including p53, p16, and FasL. PT-112's activity may derive more directly from p53 expression, when compared to oxaliplatin, as revealed by growth inhibition assays using and a p53-null HCT-116 cell line. Additionally, PT-112 triggered the cleavage of executioner pro-caspases 3, 6 and 7 to a greater extent vs oxaliplatin. When compared to oxaliplatin, PT-112 treatment resulted in the significant inhibition of gp130 and JAK/STAT signaling and transcriptional activation mediated by IL-6. A decrease, relative to oxaliplatin, in TNF-mediated NF-κB signaling and expression of cell-cycle factors E2F3, CDK4 and CDK1, as well as cell survival protein c-FLIPshort were also observed. PT-112 was more potent than oxaliplatin in inducing the release of High-mobility group protein B1, a marker for immunogenic cell death (ICD) processes. Additionally, the inhibition of the malignant phenotype, as assessed by anchorage independent growth, was significantly greater in cells incubated with PT-112 when compared to oxaliplatin. When treating non-malignant 1459 colon cells or biopsied benign normal colon tissue grown in culture, PT-112 did not result in apoptosis at concentrations much higher than those that were used in the HCT-116 experiments. In contrast, oxaliplatin remained equivalently potent in the non-malignant cell lines. Conclusions: PT-112's ability to affect numerous intracellular proteins, and the evidence of extracellular initiation of anticancer signaling, along with the apparent reduced dependence on DNA-damage, makes it an attractive and versatile compound, particularly as it relates to potential drug resistance mechanisms. PT-112's inhibition of STAT3 activation and induction of ICD also offers the intriguing proposition of downstream immune-potentiating therapeutic effects. These results underline a unique rationale for further clinical evaluation of PT-112. In conclusion, our study has demonstrated that PT-112 treatment simultaneously regulates multiple cellular targets including apoptosis, cell survival, tumor suppressor and cell cycle proteins and pathways in a manner that is clearly differentiated from oxaliplatin. Citation Format: Justin Q. Wang, Tyler Ames, Emily Arciero, Marie-Therese Hehenberger, Devasis Chatterjee. Characterization of molecular targets of the novel platinum agent PT-112 in human colon cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C32.

CSIG-03INTEGRATING COMPUTATIONAL AND EXPERIMENTAL APPROACHES TO UNDERSTAND THE SMG1 KINASE NETWORK IN GLIOBLASTOMA STEM CELLS

The SMG1 kinase was identified and validated from an RNAi screen as a gene that can preferentially inhibit the growth of selected glioblastoma stem cell lines under hypoxia (1% oxygen). In addition, we have found that SMG1 knockdown can substantially enhance the inhibitory effects of the DNA damaging drug, temozolomide (TMZ), both in vitro and in mouse xenografts. In order to evaluate the relative contributions of nonsense mediated decay, the DNA damage response (DDR), and autophagy in the cellular response to SMG1 inhibition, we have used the Cell Collective software platform (BMC Systems Biology, 6, 96) to model the SMG1 signaling network. To do this, we integrating our data with results in the literature in order to simulate the cell growth and survival of GBM stem cells under hypoxia and TMZ induced DNA damage. Our model initially predicted that the preferential growth inhibition of SMG1 inhibition in GS7-2 cells under hypoxia specifically was likely due to the role of NMD pathway in regulating autophagy. We tested the model-generated hypothesis and found induction of the autophagy marker, LC3-II, when SMG1 is knocked down under hypoxia. However, either inhibition of autophagy-related protein, ATG7, or inhibition of NMD pathway does not fully account for the preferential growth inhibition by loss of SMG1 under hypoxia. We have refined the model based on these results and are currently testing the prediction that a defect in the DDR pathway accounts for growth inhibition when SMG1 is knocked down under hypoxia. Our preliminary results indicate greater DNA damage, possibly leading to cell cycle block, when SMG1 is knocked down under hypoxia. The long-term goal of this study will be to develop therapeutic predictions for targeting SMG1 and other kinase targets in a given tumor and in order to develop personalized therapies for GBM.

Inhibition of JNK Sensitizes Hypoxic Colon Cancer Cells to DNA-Damaging Agents.

We showed previously that in HT29 colon cancer cells, modulation of hypoxia-induced stress signaling affects oxaliplatin cytotoxicity. To further study the significance of hypoxia-induced signaling through JNK, we set out to investigate how modulation of kinase activities influences cellular responses of hypoxic colon cancer cells to cytotoxic drugs. In a panel of cell lines, we investigated effects of pharmacologic and molecular inhibition of JNK on sensitivity to oxaliplatin, SN-38, and 5-FU. Combination studies for the drugs and JNK inhibitor CC-401 were carried out in vitro and in vivo. Hypoxia-induced JNK activation was associated with resistance to oxaliplatin. CC-401 in combination with chemotherapy demonstrates synergism in colon cancer cell lines, although synergy is not always hypoxia specific. A more detailed analysis focused on HT29 and SW620 (responsive), and HCT116 (nonresponsive) lines. In HT29 and SW620 cells, CC-401 treatment results in greater DNA damage in the sensitive cells. In vivo, potentiation of bevacizumab, oxaliplatin, and the combination by JNK inhibition was confirmed in HT29-derived mouse xenografts, in which tumor growth delay was greater in the presence of CC-401. Finally, stable introduction of a dominant negative JNK1, but not JNK2, construct into HT29 cells rendered them more sensitive to oxaliplatin under hypoxia, suggesting differing input of JNK isoforms in cellular responses to chemotherapy. These findings demonstrate that signaling through JNK is a determinant of response to therapy in colon cancer models, and support the testing of JNK inhibition to sensitize colon tumors in the clinic.

A combination of high dose rate (10X FFF/2400 MU/min/10 MV X-rays) and total low dose (0.5 Gy) induces a higher rate of apoptosis in melanoma cells in vitro and superior preservation of normal melanocytes.

The aim of this study was to determine the apoptotic effects, toxicity, and radiosensitization of total low dose irradiation delivered at a high dose rate in vitro to melanoma cells, normal human epidermal melanocytes (HEM), or normal human dermal fibroblasts (HDF) and to study the effect of mitochondrial inhibition in combination with radiation to enhance apoptosis in melanoma cells. Cells irradiated using 10X flattening filter-free (FFF) 10 MV X-rays at a dose rate of 400 or 2400 MU/min and a total dose of 0.25–8 Gy were analyzed by cell/colony counting, MitoTracker, MTT, and DNA-damage assays, as well as by quantitative real-time reverse transcriptase PCR in the presence or absence of mitochondrial respiration inhibitors. A dose rate of 2400 MU/min killed on average five-fold more melanoma cells than a dose rate 400 MU/min at a total dose of 0.5 Gy and preserved 80% survival of HEM and 90% survival of HDF. Increased apoptosis at the 2400 MU/min dose rate is mediated by greater DNA damage, reduced cell proliferation, upregulation of apoptotic genes, and downregulation of cell cycle genes. HEM and HDF were relatively unharmed at 2400 MU/min. Radiation induced upregulation of mitochondrial respiration in both normal and cancer cells, and blocking the respiration with inhibitors enhanced apoptosis only in melanoma cells. A high dose rate with a low total dose (2400 MU/min, 0.5 Gy/10X FFF 10 MV X-rays) enhances radiosensitivity of melanoma cells while reducing radiotoxicity toward HEM and HDF. Selective cytotoxicity of melanoma cells is increased by blocking mitochondrial respiration.

Abstract CT326: Pharmacokinetic/pharmacodynamic study of sequence specificity of the PARP inhibitor, olaparib and carboplatin in recurrent women's cancers

Abstract Background: Olaparib/carboplatin are active in gBRCA1/2m+ or BRCA-like breast and ovarian cancer (Br/OvCa). Our in vitro data suggest that pretreatment with olaparib before carboplatin attenuates C-induced DNA double stranded breaks (DSBs) and cytotoxicity. We hypothesize olaparib/carboplatin sequence may affect DNA DSBs and toxicity in pts. Methods: Eligible pts had recurrent women's cancers, normal end-organ function, and evaluable disease. Pts were randomized to arm A or B for intra-pt and inter-cohort analysis of PK/PD endpoints. 21 pts were required per arm for 80% power to determine one SD difference between arms. PBMCs were collected prior to and 24hrs after olaparib or carboplatin on cyc 1&2 for comet DNA damage assay and PAR incorporation ELISA. Toxicity was evaluated q3 wks, and response q2 cyc by RECISTv1.1. Results: 59 women (age 59 [25-74]; 47 OvCa (26 gBRCAm+)/10 triple negative BrCa [TNBC; 4 gBRCAm+]/1 uterine carcinosarcoma/1 endometrial Ca) were treated. All had prior therapy (median 5[2-14]). Intra-pt comparisons of PD endpoints indicated olaparib/carboplatin yields greater DNA DSBs than olaparib or carboplatin alone (median fold change compared to baseline; 1.21+/- 0.30 SD v. 1.13 or 0.97 [arm A], 1.33 +/- 0.67 SD v. 1.02 or 1.04 [arm B], both p<0.05). Intra-pt and inter-cohort comparisons show no significant differences in DNA DSBs, PAR incorporation and frequencies of Gr3/4 AEs as a function of the order of the schedule. Gr3/4 AEs included neutropenia (22%), anemia (12%), thrombocytopenia (10%), and carboplatin hypersensitivity (3%). Responses (54pts) included 1 CR (2%, 23mo; TNBC) and 23 PR (43%, 9[5-15]mo; 20 OvCa/3 TNBC). gBRCAm+ pts had a higher response rate (RR; 1CR/19 PR) compared to BRCAwt/unknown (4 PR; 65% v. 17%, p<0.001). Conclusions: Combination O/C induced greater DNA damage than single agents, consistent with the higher than expected RR. However, the O/C sequence did not impact DNA damage, PAR incorporation or toxicity. Olaparib tablets 200mg bid x 7d with carboplatin AUC 4 q 21d is active and tolerable in recurrent women's cancers, especially for gBRCAm+ pts. (NCT01237067) Treatment schedule armsCycle (cyc) 1Cyc 2Cyc 3-8Cyc 9+Arm AO tablet 200mg bid d1-7⇒C AUC4 d8C AUC4 d1⇒O tablet 200mg bid d2-8O tablet 200mg bid d1-7, C AUC4 d1or2maintenance O tablet 300mg bid Citation Format: Victoria L. Chiou, Christina Annunziata, Stanley Lipkowitz, Lori Minasian, Nicolas Gordon, Minshu Yu, Seth Steinberg, Nicole Houston, Elise Kohn, Jung-min Lee. Pharmacokinetic/pharmacodynamic study of sequence specificity of the PARP inhibitor, olaparib and carboplatin in recurrent women's cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT326. doi:10.1158/1538-7445.AM2015-CT326

DNA damage caused by organic extracts of contaminated sediment, crude, and weathered oil and their fractions recovered up to 5 years after the 2007 Hebei Spirit oil spill off Korea

We examined the degree of DNA damage caused by three fractions (F1, aliphatic hydrocarbons; F2, aromatic hydrocarbons; and F3, polar compounds) of the organic extract of sediments taken from Taean, Korea, following the Hebei Spirit oil spill. DNA damage was measured using the comet assay with blood cells of the striped beakfish (Oplegnathus fasciatus). DNA damage was also examined for fractions of crude oil (Iranian Heavy Crude Oil, IHC), weathered oil and six subfractions (F2.1–F2.6). The greatest DNA damage was found from the Sinduri dune region and DNA damage decreased to 40% weathered oil in F2 fraction compared with crude oil. The DNA damage of the sum of fractions was found higher than the organic extracts of sediments, suggesting antagonistic interactions between the genotoxic compounds. This study confirmed the persistence of potential genotoxicity in sediments of the severely affected regions as long as 5 years after the oil spill.

Impairment of DNA in a freshwater gastropod (Lymnea luteola L.) after exposure to titanium dioxide nanoparticles.

The apoptotic and genotoxic potential of titanium dioxide nanoparticles (TiO2NPs) were evaluated in hemocyte cells of freshwater snail Lymnea luteola L. Before evaluation of the toxic potential, mean size of the TiO2NPs was determined using a transmission electron microscopy and dynamic light scattering. In this study, L. luteola were exposed to different concentrations of TiO2NPs (28, 56, and 84μg/ml) over 96 h. Induction of oxidative stress in hemolymph was observed by a decrease in reduced glutathione and glutathione-S-transferase levels at different concentration of TiO2NPs and, in contrast, an increase in malondialdehyde and reactive oxygen species levels. Catalase activity was decreased at lower concentrations but increased at greater concentration of TiO2NPs. The extent of DNA fragmentation occurring in L. luteola due to ecotoxic impact TiO2NPs was further substantiated by alkaline single-cell gel electrophoresis assay and expressed in terms of % tail DNA and olive tail moment. The alkaline single-cell gel electrophoresis assay for L. luteola clearly shown relatively greater DNA damage at the highest concentration of TiO2NPs.The results indicate that the interaction of TiO2NPs with snail influences toxicity, which is mediated by oxidative stress according dose and in a time-dependent manner. The results of this study showed the importance of a multibiomarker approach for assessing the injurious effects of TiO2NPs to freshwater snail L. luteola, which may be vulnerable due to the continuous discharge of TiO2NPs into the aquatic ecosystems. The measurement of DNA integrity in L. luteola thus provides an early warning signal of contamination of the aquatic ecosystem by TiO2NPs.

The Indian Medicinal Plant Giloe (Tinospora cordifolia) induces Cytotoxic Effects by Damaging Cellular DNA in HeLa Cells: A Comet Assay Study

Environmental science is moving through a dreaded and drastic crisis. Tinospora cordifolia or giloe is used in Ayurvedic system of medicine for the treatment of many diseases. Our earlier studies have shown that giloe exerts cytotoxic effect in vitro and in vivo, which stimulated us to understand the mechanism of cell death is HeLa cells receving various concentrations of giloe. Therefore, induction of molecular DNA damage by different concentrations of dichloromethane extract of giloe, Tinospora cordifolia (TCE) was studied in HeLa cells by alkaline comet assay and the DNA damage induced by TCE has been expressed as olive tail moment (OTM). Incubation of HeLa cells with TCE for 4 h caused greater DNA damage as indicated by increased OTM when compared with 2 h treatment and therefore 4 h exposure time was considered as an optimum duration for TCE treatment. Exposure of HeLa cells to 0, 1, 2, 4, 6 or 8 μg/ml TCE resulted in a concentration dependent elevation in the DNA damage and the lowest concentration of 1 μg/ ml TCE increased the baseline DNA damage approximately by 10 folds, whereas 8 μg/ml TCE, the highest concentration tested caused an approximate 68 folds elevation in the DNA damage, when compared with the untreated control.

Anticancer Property of Iron Oxide Nanoparticle-Drug Complexes: An In Vitro Study.

Tumor-specific targeting of chemotherapeutic drugs can increase the therapeutic efficacy of most anticancer drugs. On surface-modified iron oxide nanoparticles (NPs), we complexed a hypoxic cell-targeting agent, sanazole (SAN), and a cytotoxic isoquinoline alkaloid, berberine (BBN). The major objective of this study was to elucidate the molecular mechanism of cytotoxicity in murine tumor cells (DLA) induced by NP-BBN-SAN complexes. The cytotoxicity of these complexes was determined using the dye exclusion method. The induction of apoptosis and cellular DNA damage in these cells was analyzed using dual staining and comet assay, respectively. The expression of genes in the treated cells elucidated the molecular mechanism underlying cytotoxicity. Cells treated with NP-BBN-SAN complexes showed significant increases in cytotoxicity and apoptosis, as well as extensive damage to cellular DNA compared to control cells. The cells treated with NP-BBN-SAN complexes showed greater DNA damage compared with other treatments. The increase in the expression of a pro-apoptotic gene suggested that apoptosis was the mechanism underlying cytotoxicity induced by NP-BBN-SAN complexes. Complexing with SAN increased the cytotoxic potential of NP-BBN complexes. Further in vivo studies are needed to evaluate the potential application of this method in controlling tumors.

Methyl pyropheophorbide-a-mediated photodynamic therapy induces apoptosis in human ovarian cancer cells

Objective To investigate the mechanism by which methyl pyropheophorbide-a-mediated photodynamic therapy(Mppa-PDT)inhibit cell viability and induce apoptosis in human ovarian cancer cell line SKOV3.Methods Human ovarian cancer cells SKOV3 at the logarithmic growth phase were divided into Mppa-PDT treated group(both Mppa and PDT treated group)and control groups(the blank group,the only Mppa treated group and only PDT treated group).After Mppa-PDT treatment,the cell viability was examined with CCK-8assay;cell apoptosis was detected by flow cytometry with Annexin Ⅴ-FITC/PI;and nuclear morphological changes during cell apoptosis was detected by DAPI staning.Moreover,the celluar reactive oxygen species(ROS) were detected by DCFH-DA staining;DNA damage was observed by single cell gel electrophoresis;and the protein expression of p53,Caspase-3,Bax,and Bcl-2 were assessed by Western blotting analysis.Results(1)Mppa-PDT could greatly suppress the cell viability of human ovarian cancer cells SKOV3 in a dose-dependent manner.(2)The cell apoptosis rate of Mppa-PDT treated group was significanlty higher than those of three control groups(blank group,Mppagroup and PDT group)(P0.05),and there was no difference among the three control groups(P0.05).(3)After treating with Mppa-PDT,DAPI staining showed strongly stained nuclei of the apoptotic cells;DCFH-DA staining displayed higher level of ROS than those of the three control groups;single cell gel electrophoresis showed greater DNA damage than those of the three control groups;and Western blotting analysis showed that the expression of p53,Caspase-3and Bax protein was increased and Bcl-2protein was decreased(P0.05).Conclusion Mppa-PDT can significantly suppress cell viability and induce apoptosis in human ovarian cancer cell SKOV3,accompanied by DNA damage and the activation of mitochondrial apoptosis pathway.

A prevalent cancer susceptibility APOBEC3A hybrid allele bearing APOBEC3B 3'UTR enhances chromosomal DNA damage.

Human APOBEC3A (A3A) cytidine deaminase is a host enzyme that can introduce mutations into chromosomal DNA. As APOBEC3B (A3B) encodes a C-terminal catalytic domain ~91% identical to A3A, we examined its genotoxic potential as well as that of a highly prevalent chimaeric A3A-A3B deletion allele (ΔA3B), which is linked to a higher odds ratio of developing breast, ovarian and liver cancer. Interestingly, breast cancer genomes from ΔA3B(-/-) patients show a higher overall mutation burden. Here it is shown that germline A3B can hypermutate nuclear DNA, albeit less efficiently than A3A. Chimaeric A3A mRNA resulting from ΔA3B was more stable, resulting in higher intracellular A3A levels and greater DNA damage. The cancer burden implied by the higher A3A levels could be considerable given the high penetration of the ΔA3B allele in South East Asia.

Poly (ADP-ribose) polymerase inhibition enhances trastuzumab antitumour activity in HER2 overexpressing breast cancer

AimPoly (ADP-ribose) polymerase (PARP) inhibitors have shown promising results in Breast Cancer (BRCA) deficient breast cancer, but not in molecularly unselected patient populations. Two lines of research in this field are needed: the identification of novel subsets of patients that could potentially benefit from PARP inhibitors and the discovery of suitable targeted therapies for combination strategies. MethodsWe tested PARP inhibition, alone or combined with the anti-HER2 antibody trastuzumab on HER2+ breast cancer. We used two PARP inhibitors in clinical development, olaparib and rucaparib, as well as genetic downmodulation of PARP-1 for in vitro studies. DNA damage was studied by the formation of γH2AX foci and comet assay. Finally, the in vivo anti-tumour effect of olaparib and trastuzumab was examined in nude mice subcutaneously implanted with BT474 cells. ResultsIn a panel of four HER2 overexpressing breast cancer cell lines, both olaparib and rucaparib significantly decreased cell growth and enhanced anti-tumour effects of trastuzumab. Cells exposed to olaparib and trastuzumab had greater DNA damage than cells exposed to each agent alone. Mechanistic exploratory assays showed that trastuzumab downmodulated the homologous recombination protein proliferating cell nuclear antigen (PCNA). Combination treatment in the BT474 xenograft model resulted in enhanced growth inhibition, reduced tumour cell proliferation, and increased DNA damage and apoptosis. ConclusionTaken together, our results show that PARP inhibition has antitumour effects and increases trastuzumab activity in HER2 overexpressing breast cancer. These findings make this novel combination a promising strategy for clinical development.

Combination of YM155, a Survivin Suppressant, with Bendamustine and Rituximab: A New Combination Therapy to Treat Relapsed/Refractory Diffuse Large B-cell Lymphoma

There remains an unmet therapeutic need for patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL). The purpose of this study was to evaluate the therapeutic potential of sepantronium bromide (YM155), a survivin suppressant, in combination with either bendamustine or both bendamustine and rituximab using DLBCL models. Human DLBCL cell lines, DB, SU-DHL-8, and WSU-DLCL2, were treated with YM155 in combination with bendamustine. Cell viability, apoptosis induction, protein expression, and cell-cycle distribution were evaluated. Furthermore, antitumor activities of YM155, in combination with bendamustine or both bendamustine and rituximab, were evaluated in mice bearing human DLBCL xenografts. The combination of YM155 with bendamustine showed greater cell growth inhibition and sub-G1 population than either agent alone. YM155 inhibited bendamustine-induced activation of the ATM pathway and accumulation of survivin at G2-M phase, with greater DNA damage and apoptosis than either single agent alone. In a DLBCL DB murine xenograft model, YM155 enhanced the antitumor activity of bendamustine, resulting in complete tumor regression without affecting body weight. Furthermore, YM155 combined with bendamustine and rituximab, decreased FLT-PET signals in lymph nodes and prolonged overall survival of mice bearing disseminated SU-DHL-8, an activated B-cell-like (ABC)-DLBCL xenografts when compared with the combination of either rituximab and bendamustine or YM155 with rituximab. These results support a clinical trial of the combination of YM155 with bendamustine and rituximab in relapsed/refractory DLBCL.