Increase In DNA Strand Breaks Research Articles

The RNA polymerase I transcription inhibitor CX-5461 cooperates with topoisomerase 1 inhibition by enhancing the DNA damage response in homologous recombination-proficient high-grade serous ovarian cancer

BackgroundIntrinsic and acquired drug resistance represent fundamental barriers to the cure of high-grade serous ovarian carcinoma (HGSC), the most common histological subtype accounting for the majority of ovarian cancer deaths. Defects in homologous recombination (HR) DNA repair are key determinants of sensitivity to chemotherapy and poly-ADP ribose polymerase inhibitors. Restoration of HR is a common mechanism of acquired resistance that results in patient mortality, highlighting the need to identify new therapies targeting HR-proficient disease. We have shown promise for CX-5461, a cancer therapeutic in early phase clinical trials, in treating HR-deficient HGSC.MethodsHerein, we screen the whole protein-coding genome to identify potential targets whose depletion cooperates with CX-5461 in HR-proficient HGSC.ResultsWe demonstrate robust proliferation inhibition in cells depleted of DNA topoisomerase 1 (TOP1). Combining the clinically used TOP1 inhibitor topotecan with CX-5461 potentiates a G2/M cell cycle checkpoint arrest in multiple HR-proficient HGSC cell lines. The combination enhances a nucleolar DNA damage response and global replication stress without increasing DNA strand breakage, significantly reducing clonogenic survival and tumour growth in vivo.ConclusionsOur findings highlight the possibility of exploiting TOP1 inhibition to be combined with CX-5461 as a non-genotoxic approach in targeting HR-proficient HGSC.

Frontline Selinexor and Chemotherapy Is Highly Active in Older Adults with Acute Myeloid Leukemia (AML)

Background: Acute myeloid leukemia (AML) is an aggressive malignancy of the bone marrow characterized by resistance to treatment and dismal outcomes, especially in the elderly. Novel approaches are desperately needed. Exportin 1 (XPO1) is involved in the selective nuclear export of certain proteins and RNA species. It is overexpressed in a subset of AML, conferring an adverse prognosis. Selinexor is a small-molecule inhibitor of XPO1 with activity in AML. Selinexor sensitizes AML cells to anthracyclines by retaining topoisomerase II in the nucleus resulting in increased DNA strand breaks. Furthermore, selinexor has shown encouraging results when combined with chemotherapy in AML. This abstract reports the ongoing results of a randomized phase II study of induction and consolidation with or without selinexor in newly diagnosed patients with AML, 60 years of age or older and preclinical studies to assess the mechanisms of chemo-sensitization. Methods: Patients 60 years of age or older with newly diagnosed de novo AML were randomized 3:1 between 7+3+selinexor or 7+3. Responding patients could go on to high dose cytarabine consolidation with or without selinexor as per initial randomization. Patients in the selinexor arm who completed all consolidation could then move to maintenance therapy with selinexor alone. Induction consisted of cytarabine 100 mg/m2/d by continuous infusion for 7 days and daunorubicin 60 mg/m2 on days 1-3. Consolidation consisted of cytarabine at 1.5 gm/m2 given Q12 hours days 1-3 with G-CSF given 24 hours following the last dose of cytarabine. Selinexor was dosed at 60 mg PO on days 1, 3, 8, 10, 15 and 17 during induction and consolidation and on days 1 and 8 every 21 days during maintenance. Preclinical studies were conducted with murine AML cell lines. Results: Twenty-seven of a planned twenty-eight patients were enrolled to date. Of the 27 evaluable patients, 20 were randomized to the selinexor arm and 7 to the control arm. Baseline demographics are listed in Table 1. In the standard arm, both 30- and 60-day mortality were 14% (1/7). In the selinexor arm, both 30- and 60-day mortality were 10% (2/20). In the standard arm, 43% (3/7) of patients achieved a complete remission (CR) or complete remission with incomplete count recovery (CRi). Of the 3 responders 1 has gone on to transplant. In the selinexor arm, 85% (17/20) of patients achieved a CR or CRi. Of the 17 responders, 4 have gone on to transplant. Progression free and overall survival both favor the selinexor arm with trends towards significance despite the small size of the trial (Figure 1 and Table 2). No difference in the AE profile was noted between arms and no unexpected side effects were observed. Selinexor induces retention of topoisomerase II in the nucleus increasing sensitivity to anthracyclines. To determine if selinexor sensitized to cytarabine viability assays using murine AML cell lines were done (Figure 2A). Selinexor significantly sensitized both cell lines to cytarabine. AML cells increase mitochondrial oxygen consumption in response to cytarabine leading to resistance. The ability of selinexor to interfere with this response was assessed using Seahorse flux analysis. AML cells treated with cytarabine for 16 hours showed a diminished mitochondrial oxygen response when co-treated with selinexor (Figure 2B). Conclusions: Selinexor in combination with standard induction and consolidation therapy appears highly active in older patients with de novo AML. Selinexor may increase response to cytarabine by interfering with nuclear-mitochondrial communication. Enrollment is ongoing. Disclosures Pardee: Rafael Pharmaceuticals: Consultancy; AbbVie: Consultancy; Genentech, Inc.: Consultancy; BMS: Consultancy, Honoraria, Speakers Bureau; Karyopharm: Research Funding; Rafael: Research Funding; Celgene: Consultancy, Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Pharmacyclics: Speakers Bureau. Ellis:Rafael Pharmaceuticals: Consultancy. Howard:Jazz Pharmaceuticals: Consultancy. Powell:Jazz Pharmaceuticals: Consultancy, Other: Advisor, Research Funding; Genentech: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; Rafael Pharmaceuticals: Consultancy, Other: Advisor, Research Funding. OffLabel Disclosure: Selinexor for the treatment of AML

Organomodified nanoclays induce less inflammation, acute phase response, and genotoxicity than pristine nanoclays in mice lungs

Surface modification by different quaternary ammonium compounds (QAC) makes nanoclays more compatible with various polymeric matrices, thereby expanding their potential applications. The growing industrial use of nanoclays could potentially pose a health risk for workers. Here, we assessed how surface modification of nanoclays modulates their pulmonary toxicity. An in vitro screening of the unmodified nanoclay Bentonite (montmorillonite) and four organomodified nanoclays (ONC); coated with various QAC, including benzalkonium chloride (BAC), guided the selection of the materials for the in vivo study. Mice were exposed via a single intratracheal instillation to 18, 54, and 162 µg of unmodified Bentonite or dialkyldimethyl-ammonium-coated ONC (NanofilSE3000), or to 6, 18, and 54 µg of a BAC-coated ONC (Nanofil9), and followed for one, 3, or 28 days. All materials induced dose- and time-dependent responses in the exposed mice. However, all doses of Bentonite induced larger, but reversible, inflammation (BAL neutrophils) and acute phase response (Saa3 gene expression in lung) than the two ONC. Similarly, highest levels of DNA strand breaks were found in BAL cells of mice exposed to Bentonite 1 day post-exposure. A significant increase of DNA strand breaks was detected also for NanofilSE3000, 3 days post-exposure. Only mice exposed to Bentonite showed increased Tgf-β gene expression in lung, biomarker of pro-fibrotic processes and hepatic extravasation, 3 days post-exposure. This study indicates that Bentonite treatment with some QAC changes main physical-chemical properties, including shape and surface area, and may decrease their pulmonary toxicity in exposed mice.

Abstract A092: DNA repair proficiency predicts disparities in triple negative breast cancer outcomes

Abstract Breast cancer remains as one of the most lethal type of gynecological cancers in women. Among various subtypes, triple negative breast cancer (TNBC) is the most aggressive and difficult to treat subtype due to limited therapeutic options. African American women have higher death rate from breast cancer than women of other racial and ethnic groups. Additionally, the incidence of germline BRCA1 mutations is much lower among African American women diagnosed with TNBC, than the women of the other races or ethnicities. Mechanistically, increased DNA repair and cell cycle checkpoint activation remain as the foremost reasons behind TNBC tumor resistance to chemotherapy and recurrence. We used a CHK1 inhibitor prexasertib to inhibit cell cycle checkpoint activation in TNBC cells. Interestingly, we found that prexasertib treatment promotes both proteasome-mediated degradation of BRCA1 and RAD51 proteins and affects their transcript levels. Analysis of DNA repair efficiency in prexasertib treated TNBC cells revealed reduction in homologous recombination efficiency compared to control cells. Based on these results, we hypothesized that prexasertib treatment induced homologous recombination deficiency (HRD) should cause synergistic lethality when combined with PARP inhibitors (PARPi) in TNBC cells. As predicted, combined treatment of TNBC cells with prexasertib and PARPi olaparib resulted in increased DNA strand breaks, gH2AX foci and nuclear disintegration, an indicator of mitotic catastrophe. Similarly, these drug combinations also caused synergistic lethality in multiple TNBC cell lines when compared to single drug-treatments, as indicated by combination index (CI) values. Since RAD51 is the downstream effector repair protein in FA-BRCA pathway-mediated HR, we evaluated TCGA data for RAD51 gene expression using UALCAN portal. Computational analysis revealed that RAD51 overexpression in breast tumors compared to normal breast tissues, in particular, TNBC subtype showed highest RAD51 expression compared to other subtypes of breast cancer. Overall, these data show RAD51 as a poor prognostic marker for breast cancer patients. Interestingly, there was a discrepancy in RAD51 expression levels in distinct racial groups, where African American and Asian breast cancer patients showed high RAD51 expression compared to Caucasian breast cancer patients. Consistent with these observations, African American and Asian TNBC patients show decreased survival probability. Based on these data, RAD51 could be a biomarker for aggressive TNBC and racial disparity in breast cancer therapeutic outcomes. As positive correlation exists between RAD51 and CHEK1 expression in breast cancer, in-vitro preclinical data presented here provides additional mechanistic insights for further evaluation of the rational combination of prexasertib and olaparib for improved prognosis and to reduce racial disparity in TNBC. Citation Format: Chinnadurai Mani, Jonnalagadda Shirisha, Awasthi Sanjay, Manne Upender, Palle Komaraiah. DNA repair proficiency predicts disparities in triple negative breast cancer outcomes [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr A092.

Toxicity and Gene Expression Profiling of Copper- and Titanium-Based Nanoparticles Using Air-Liquid Interface Exposure.

To assess the toxicity of nanomaterials, most in vitro studies have been performed under submerged conditions, which do not reflect physiological conditions upon inhalation. An air-liquid interface (ALI) exposure may provide more reliable data on dosimetry and prevent interactions with cell culture media components. Therefore, an ALI exposure was combined with a high-throughput RT-qPCR approach to evaluate the toxicological potential of CuO and TiO2 nanoparticles (NP) in A549 cells. While TiO2 NP did not show any cytotoxicity or other effects compromising genomic stability up to 25.8 μg/cm2, CuO NP revealed a dose-dependent cytotoxicity, starting at 4.9 μg/cm2. Furthermore, CuO NP altered distinct gene expression patterns indicative for disturbed metal homeostasis, stress response, and DNA damage induction. Thus, induction of metal homeostasis associated genes (MT1X, MT2A) at 0.4 μg/cm2 and higher suggested uptake and intracellular dissolution of CuO NP, which was verified by a dose-dependent increase in intracellular copper concentration. Starting at 4.9 μg/cm2, oxidative stress markers (HMOX1, HSPA1A) were induced dose-dependently, supported by elevated ROS levels. Furthermore, a dose-dependent induction of genes associated with DNA damage response (DDIT3, GADD45A) was observed, in concordance with an increase in DNA strand breaks. Finally, transcriptional data suggested the induction of apoptosis at high doses, while flow cytometric analysis revealed increased numbers of either late apoptotic or necrotic cells and clearly necrotic cells at the highest concentrations. Thus, an ALI cell culture system was successfully combined with a comprehensive high-throughput RT-qPCR system, allowing the quantification of NP deposition and their impact on genomic stability. For CuO NP, in principle the data confirm observations made under submerged conditions with respect to intracellular copper ion release, as well as oxidative and genotoxic stress response. However, the results derived from ALI exposure allow the assessment of dose-response-relationships as well as the comparison of relative toxic potencies of different NP.

The lipogenic LXR-SREBF1 signaling pathway controls cancer cell DNA repair and apoptosis and is a vulnerable point of malignant tumors for cancer therapy.

Cancer cells are defective in DNA repair, so they experience increased DNA strand breaks, genome instability, gene mutagenesis, and tumorigenicity; however, multiple classic DNA repair genes and pathways are strongly activated in malignant tumor cells to compensate for the DNA repair deficiency and gain an apoptosis resistance. The mechanisms underlying this phenomenon in cancer are unclear. We speculate that a key DNA repair gene or signaling pathway in cancer has not yet been recognized. Here, we show that the lipogenic liver X receptor (LXR)-sterol response element binding factor-1 (SREBF1) axis controls the transcription of a key DNA repair gene polynucleotide kinase/phosphatase (PNKP), thereby governing cancer cell DNA repair and apoptosis. Notably, the PNKP levels were significantly reduced in 95% of human pancreatic cancer (PC) patients, particularly deep reduction for sixfold in all of the advanced-stage PC cases. PNKP is also deficient in three other types of cancer that we examined. In addition, the expression of LXRs and SREBF1 was significantly reduced in the tumor tissues from human PC patients compared with the adjacent normal tissues. The newly identified LXR-SREBF1-PNKP signaling pathway is deficient in PC, and the defect in the pathway contributes to the DNA repair deficiency in the cancer. Strikingly, further diminution of the vulnerable LXR-SREBF1-PNKP signaling pathway using a small molecule triptonide, a new LXR antagonist identified in this investigation, at a concentration of 8 nM robustly activated tumor-suppressor p53 and readily elevated cancer cell DNA strand breaks over an apoptotic threshold, and selectively induced PC cell apoptosis, resulting in almost complete elimination of tumors in xenograft mice without obvious complications. Our findings provide new insight into DNA repair and apoptosis in cancer, and offer a new platform for developing novel anticancer therapeutics.

Mechanisms and Antitumor Activity of a Binary EGFR/DNA-Targeting Strategy Overcomes Resistance of Glioblastoma Stem Cells to Temozolomide.

Glioblastoma (GBM) is a fatal primary malignant brain tumor. GBM stem cells (GSC) contribute to resistance to the DNA-damaging chemotherapy, temozolomide. The epidermal growth factor receptor (EGFR) displays genomic alterations enabling DNA repair mechanisms in half of GBMs. We aimed to investigate EGFR/DNA combi-targeting in GBM. ZR2002 is a "combi-molecule" designed to inflict DNA damage through its chlorethyl moiety and induce irreversible EGFR tyrosine kinase inhibition. We assessed its in vitro efficacy in temozolomide-resistant patient-derived GSCs, mesenchymal temozolomide-sensitive and resistant in vivo-derived GSC sublines, and U87/EGFR isogenic cell lines stably expressing EGFR/wild-type or variant III (EGFRvIII). We evaluated its antitumor activity in mice harboring orthotopic EGFRvIII or mesenchymal TMZ-resistant GSC tumors. ZR2002 induced submicromolar antiproliferative effects and inhibited neurosphere formation of all GSCs with marginal effects on normal human astrocytes. ZR2002 inhibited EGF-induced autophosphorylation of EGFR, downstream Erk1/2 phosphorylation, increased DNA strand breaks, and induced activation of wild-type p53; the latter was required for its cytotoxicity through p53-dependent mechanism. ZR2002 induced similar effects on U87/EGFR cell lines and its oral administration significantly increased survival in an orthotopic EGFRvIII mouse model. ZR2002 improved survival of mice harboring intracranial mesenchymal temozolomide-resistant GSC line, decreased EGFR, Erk1/2, and AKT phosphorylation and was detected in tumor brain tissue by MALDI imaging mass spectrometry. These findings provide the molecular basis of binary EGFR/DNA targeting and uncover the oral bioavailability, blood-brain barrier permeability, and antitumor activity of ZR2002 supporting potential evaluation of this first-in-class drug in recurrent GBM.

Trophic transfer affects cytogenetic and antioxidant responses of the mussel Mytilus galloprovincialis to copper and benzo(α)pyrene

The impacts of environmental pollutants on marine organisms can be determined by the routes of exposure. Various routes of exposure, including dietary exposure and waterborne exposure with or without feeding, were applied to study the cytogenetic responses in marine mussels Mytilus galloprovincials to typical pollutants, BaP (53.74 ± 19.79 μg/L) and Cu (47.38 ± 3.10 μg/L). The increased DNA strand breaks and micronucleus formation were found in haemocytes of mussels via the dietary exposure, indicating the vital role of trophic transfer in toxicity induction. The deeper exploration to relate BaP induced cytogenetic alterations with key antioxidant defense factors, SOD and GST, was performed under different exposure routes. The results revealed the significantly inhibited SOD activity via the trophic transfer, suggesting more direct or prompt role of SOD in antioxidant defense. On contrary, gene expressions of both sod and gst were up-regulated upon all routes of exposures, and showed negative correlation with enzyme activities. The results suggested the asynchronous regulation of studied antioxidant factors at transcriptional and enzyme functional level in mussels upon the change of exposure routes. The study brings out the first observation of trophic transfer influenced cytogenetic and antioxidant responses to pollutants and their alterative risk to marine organisms.

Selinexor in Combination with Induction and Consolidation Therapy in Older Adults with AML Is Highly Active

Background: Acute myeloid leukemia (AML) is an aggressive myeloid cancer of the hematopoietic system that primarily affects older adults and is characterized by therapy resistance and dismal outcomes. Novel approaches to treat AML are desperately needed. Selinexor is a small-molecule inhibitor of the nuclear export protein XPO1. Treatment of AML cells with selinexor was shown to sensitize them to chemotherapy in part by blocking the nuclear export of topoisomerase II, resulting in increased DNA strand breaks when an anthracycline is present. Furthermore, selinexor has shown encouraging results when combined with chemotherapy in the relapsed setting. This abstract reports on the initial results of a randomized phase II study of induction and consolidation with or without selinexor in newly diagnosed patients with AML 60 years of age or older. Methods: Patients 60 years of age or older with newly diagnosed de novo AML were randomized between 7+3 or 7+3+selinexor induction. Responding patients could then go on to high dose cytarabine consolidation with or without selinexor as per their initial treatment assignment. Patients in the selinexor arm who completed all planned consolidation could then move to maintenance therapy with selinexor alone. During induction cytarabine was dosed at 100 mg/m2 by continuous infusion for 7 days and daunorubicin was dosed at 60 mg/m2 on days 1-3. In consolidation cytarabine was dosed at 1.5 gm/m2 given Q12 hours on days 1, 3 and 5. Selinexor was dosed at 60 mg PO on days 1, 3, 8, 10, 15 and 17 during induction and consolidation and on days 1 and 8 every 21 days in maintenance. Optional blood samples were collected just prior to and at several time points after selinexor during induction. Mononuclear cells were isolated from these samples, lysed and lysates evaluated for protein pathway drug target activation mapping by reverse phase phosphoprotein array (RPPA). Results: Thirteen patients have been enrolled to date with 12 available for evaluation. Of the 12 evaluable patients 6 were randomized to each arm. Baseline demographics are listed in Table 1. Overall the combination was well tolerated with no patients discontinuing selinexor secondary to treatment related adverse events. In the standard arm 3 of 6 patients achieved a complete remission. Of the 3 responders 1 has relapsed, 1 has gone on to transplant and one has completed consolidation chemotherapy. In the selinexor arm 5 of 6 patients achieved a complete remission and 1 patient achieved a morphologic leukemia free state. All patients given selinexor achieved a response. Of the 5 responders 3 have gone on to transplant, 1 is on maintenance therapy (completed 14 cycles) and one is completing consolidation therapy. In the standard arm no patients died during induction and 3 of 6 patients have died from progressive disease. In the selinexor arm 1 patient died during induction and none of the remaining patients have progressed (Figure 1 and Table 2). No difference in the AE profile was noted between arms and no unexpected side effects were observed. The patient on long term maintenance selinexor is tolerating it without significant AEs to date. Blood samples were available from 3 patients from the selinexor arm. RPPA based protein pathway activation mapping analysis of PBMCs revealed a significant increase in phosphorylation of AMPK, BAD and LC3B at one-hour post treatment that returned to baseline by 2 hours suggesting a transient increase in these pathways that was subsequently suppressed (Figure 2). Conclusions: Selinexor in combination with standard induction and consolidation therapy appears highly active in older de novo AML patients. Selinexor may increase response by modulation of AMPK, autophagy and BAD phosphorylation. Enrollment is ongoing. Disclosures Pardee: Spherix Intellectual Property: Research Funding; CBM Bipharma: Membership on an entity's Board of Directors or advisory committees; Amgen: Speakers Bureau; Celgene: Speakers Bureau; Pharmacyclics/Janssen: Speakers Bureau; Karyopharm: Research Funding; Rafael Pharmaceuticals: Consultancy, Research Funding. Manuel:Novartis: Speakers Bureau; Jazz Pharmaceuticals: Speakers Bureau. Powell:Pfizer: Consultancy, Research Funding; Rafael Pharmaceuticals: Consultancy, Research Funding; Novartis: Consultancy, Speakers Bureau; Jazz Pharmaceuticals: Consultancy, Research Funding, Speakers Bureau; Janssen: Research Funding. OffLabel Disclosure: Selinexor is not approved for the treatment of AML

Caffeic Acid Genotoxicity: Correlation of the Pig-a Assay with Regulatory Genetic Toxicology In Vivo Endpoints.

Caffeic acid is found in variety of fruits and vegetables. It is considered as possible human carcinogen (Group 2B). It is negative in Ames and mouse micronucleus (MN), but positive in mouse lymphoma and chromosomal aberration assays. The objective of this study was to evaluate the in vivo genotoxicity of caffeic acid using three different endpoints: in vivo MN, Pig-a, and comet assay. Two sets of six rats per group were administered vehicle (0.5% hydroxypropyl methylcellulose), 500, 1,000, or 2,000 mg/kg/day of caffeic acid for three consecutive days via oral gavage. One set of animals was used for the Pig-a and MN assay and the other set was used for the comet assay. N-Ethyl N-Nitrosourea was used as positive control for the Pig-a and MN assay, and ethyl methanesulfonate for the comet assay. From one set of animals, peripheral blood was collected on Days -1, 14, and 30 for the Pig-a assay and on Day 4 for the MN assay. The other set of animals was euthanized 3 hr after the last dose; liver and blood were collected for the comet assay. A statistically significant increase in the MN frequency was observed at 2,000 mg/kg/day. No increase in the red blood cells (RBCCD59- ) or reticulocytes (RETCD59- ) Pig-a mutant frequencies was observed on Days 14 or 30. No increase in DNA strand breaks was observed in the peripheral blood or liver in the comet assay. Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc.

The cyanobacterial oligopeptides microginins induce DNA damage in the human hepatocellular carcinoma (HepG2) cell line

Microginins (MGs) are bioactive metabolites mainly produced by Microcystis spp., (Cyanobacteria) commonly found in eutrophic environments. In this study, the cytotoxic and genotoxic activities of four MG congeners (MG FR3, MG GH787, cyanostatin B, MGL 402) and a well characterized cyanobacterial extract B-14-01 containing these metabolites were evaluated in the human hepatocellular carcinoma (HepG2) cell line. The cytotoxicity was measured with the MTT assay, while genotoxicity was studied with the comet, γH2AX and cytokinesis block (CBMN) micronucleus assays. The viability of cells after 24 h was significantly affected only by the extract, whereas after 72 h a concentration dependent decrease in cell proliferation was observed for the extract and tested microginins, with MGL 402 being the most potent and MG FR3 the least potent congener. The extract and all tested congeners induced DNA strand breaks after 4 and 24 h exposure. The most potent was the extract, which induced concentration and time dependent increase in DNA damage at concentrations ≥0.01 μg mL−1. Among microginins the most potent was MGL 402 (increase in DNA strand breaks at ≥ 0.01 μg mL−1) and MG FR3 was the least potent (increase in DNA strand breaks at ≥ 1 μg mL−1). However, no induction of DNA double strand breaks was observed after 24 and 72-h exposure to the cyanobacterial extract or MGs. Induction of genomic instability was observed in cells exposed to MG GH787, cyanostatin B and the extract B-14-01. This study is the first to provide the evidence that microginins exert genotoxic activity.

Prexasertib treatment induces homologous recombination deficiency and synergizes with olaparib in triple-negative breast cancer cells

BackgroundBreast cancer remains as one of the most lethal types of cancer in women. Among various subtypes, triple-negative breast cancer (TNBC) is the most aggressive and hard to treat type of breast cancer. Mechanistically, increased DNA repair and cell cycle checkpoint activation remain as the foremost reasons behind TNBC tumor resistance to chemotherapy and disease recurrence.MethodsWe evaluated the mechanism of prexasertib-induced regulation of homologous recombination (HR) proteins using 20S proteasome inhibitors and RT-PCR. HR efficiency and DNA damages were evaluated using Dr-GFP and comet assays. DNA morphology and DNA repair focus studies were analyzed using immunofluorescence. UALCAN portal was used to evaluate the expression of RAD51 and survival probability based on tumor stage, subtype, and race in breast cancer patients.ResultsOur results show that prexasertib treatment promotes both post-translational and transcriptional mediated regulation of BRCA1 and RAD51 proteins. Additionally, prexasertib-treated TNBC cells revealed over 55% reduction in HR efficiency compared to control cells. Based on these results, we hypothesized that prexasertib treatment induced homologous recombination deficiency (HRD) and thus should synergize with PARP inhibitors (PARPi) in TNBC cells. As predicted, combined treatment of prexasertib and PARPi olaparib increased DNA strand breaks, γH2AX foci, and nuclear disintegration relative to single-agent treatment. Further, the prexasertib and olaparib combination was synergistic in multiple TNBC cell lines, as indicated by combination index (CI) values. Analysis of TCGA data revealed elevated RAD51 expression in breast tumors compared to normal breast tissues, especially in TNBC subtype. Interestingly, there was a discrepancy in RAD51 expression in racial groups, with African-American and Asian breast cancer patients showing elevated RAD51 expression compared to Caucasian breast cancer patients. Consistent with these observations, African-American and Asian TNBC patients show decreased survival.ConclusionsBased on these data, RAD51 could be a biomarker for aggressive TNBC and for racial disparity in breast cancer. As positive correlation exists between RAD51 and CHEK1 expression in breast cancer, the in vitro preclinical data presented here provides additional mechanistic insights for further evaluation of the rational combination of prexasertib and olaparib for improved outcomes and reduced racial disparity in TNBC.

DNA damage in human whole blood caused by radiopharmaceuticals evaluated by the comet assay.

Radiopharmaceuticals used for diagnosis or therapy induce DNA strand breaks, which may be detectable by single-cell gel electrophoresis (called comet assay). Blood was taken from patients before and at different time points after treatment with radiopharmaceuticals; blood cells were investigated by the comet assay using the percentage of DNA in the tail as the critical parameter. Whereas [225Ac]Ac-prostate-specific membrane antigen (PSMA)-617 alpha therapy showed no difference relative to the blood sample taken before treatment, beta therapy with [177Lu]Lu-PSMA-617 3 h post-injection revealed a small but significant increase in DNA strand breaks. In blood of patients who underwent positron emission tomography (PET) with either [18F]2-fluor-2-deoxy-D-glucose (FDG) or [68Ga]Ga-PSMA-11, an increase of DNA migration determined by the comet assay was not found when analysed at different time points (2–70 min) after intravenous tracer injection. Human whole blood was incubated with the targeted clinically relevant therapeutic radiopharmaceuticals [225Ac]Ac-PSMA-617, [177Lu]Lu-PSMA-617 and [90Y]Y-DOTA(0)-Phe(1)-Tyr(3)-octreotide (DOTA-TOC) at different activity concentrations (kBq/ml) for 5 days and then analysed by the comet assay. DNA damage increased with higher concentrations of all radiolabeled compounds tested. [177Lu]Lu-PSMA-617 caused higher blood cell radiotoxicity than equal activity concentrations of [90Y]Y-DOTA-TOC. Likewise, whole human blood was exposed to the positron emitters [18F]FDG and [68Ga]Ga-PSMA-11 in vitro for 24 h with activity concentrations ranging between 5 and 40 MBq/ml. The same activity concentration dependent elevated DNA migration was observed for both compounds although decay energies are different. This study demonstrated that the amount of DNA damage detected by the comet assay in whole human blood is similar among different positron emitters and divergent by a factor of 200 between alpha particles and beta radiation.

Responses to genotoxicity in mouse testicular germ cells and epididymal spermatozoa are affected by increased age

The increased number of cell divisions undergone by spermatogonia of older fathers cannot fully account for the observed increase in germline genetic damage. Studies have shown that the mechanisms induced in germ cells in response to oxidative damage varies with age, that DNA repair efficiency declines, and both sperm DNA damage and spontaneous mutations increase. However, it is not known whether the altered response with age is a cause, or consequence, of an age-associated change in cell susceptibility to genetic damage.Following a single 150 mg/kg dose of cyclophosphamide (CP), young (8-weeks old) and aged (17-month old) male mice were examined 24 h later for induced genetic damage in epididymal spermatozoa using the alkaline comet and sperm chromatin stability assays. Apoptosis among testicular cells was examined on tissue cross-sections using the TUNEL assay.Sperm showed no significant increase in DNA strand breaks with age (detected by the comet assay) and no change in sperm chromatin stability (detected by the SCSA assay). Following CP treatment, there was no effect on DNA-strand breakage but sperm chromatin instability was significantly higher. Furthermore, it was also significantly elevated in old treated, compared with young treated, animals suggesting that increased age affects the sensitivity of epididymal sperm to chromatin damage.There was no difference in apoptosis in testicular germ cells from either young or old control animals, while CP administration resulted in a significant increase in apoptosis among young animals but not old animals. Following genotoxin exposure, an increase in chromatin instability in the spermatozoa of old animals and a decrease in the ability of their testicular germ cells undergo apoptosis suggests an age-related decrease in genome protection mechanisms. Since those germ cells are only transiently present in the testis, it is likely that this age-related deterioration originates in the spermatogonial stem cells. The findings are also evidence that the safety evaluation of reproductive genotoxins should consider young and old individuals separately.

The genotoxicity of organic extracts from particulate truck emissions produced at various engine operating modes using diesel or biodiesel (B100) fuel: A pilot study

An analysis of the toxic effects of emissions should reflect real traffic conditions. The exhaust emissions of particulate matter from diesel engines strongly depend on their operating conditions, with low-speed, low-load "urban creep" conditions, common for truck traffic in heavily congested urban areas, being one of the worst. We aimed to detect the genotoxicity of organic extracts from particulate matter in the exhaust of the diesel engine Zetor 1505 running on diesel and biodiesel (B100) fuels at characteristic modes of extended "urban creep", typical for transit truck traffic in Prague, comparing the first 5 min of idling with extended (20–80 min) idling, full load after idle, "stabilized" full load, and 30% load. The diluted exhaust was sampled with high volume samplers on glass fiber fluorocarbon coated filters. The filters were extracted with dichloromethane and DNA damage was analyzed in A549 cells using comet assay, with the inclusion of formamidopyrimidine DNA glycosylase (FPG) and endonuclease III (ENDOIII) to recognize oxidized DNA bases. The cells were exposed to extractable organic matter (EOM) for 4 and 24 h at non-cytotoxic dose corresponding to 0.001 m3 of undiluted exhaust gas per ml cell media. At the 4 h exposure interval, all samples from B100 and diesel emissions induced DNA damage. EOM from the extended idle engine mode exerted the strongest genotoxic effect for both fuels. Twenty hours later, the cells exposed to diesel EOM exhibited a further increase of DNA strand breaks compared to the preceding interval. In contrast, DNA damage seemed to be fully repaired in cells treated with EOM derived from biodiesel B100. The preliminary results suggest that (i) diesel emissions are more genotoxic than the emissions from B100, (ii) biodiesel induced DNA lesions are repaired within 24 h.

In vitro evaluation of cytotoxic and genotoxic effects of Di(2-ethylhexyl)-phthalate (DEHP) on European sea bass (Dicentrarchus labrax) embryonic cell line

Marine litter is extensively distributed in the marine environment, and plastic debris, of which litter is mostly composed, can be a major source of pollutants. Among them, Di(2-ethylhexyl)-phthalate (DEHP) is the most abundantly used plastic additive, and it has been reported to affect biochemical processes both in humans and wildlife; however, studies on its toxicological effects on marine organisms are still scarce. In this survey, we studied the cytotoxic, genotoxic, and mutagenic effects of DEHP in European sea bass embryonic cell line (DLEC) by applying specific in vitro tests. Results showed a significant decrease in cell viability starting at 0.01 mM of DEHP after 24 h together with a significant increase in apoptosis and necrosis, morphological changes and cell detachment. Consistently, we detected a moderate increase in DNA strand breaks from 0.02 mM, and a dose-dependent increase in of micronucleus frequency from 0.01 mM, accompanied by a significant inhibition of cell proliferation, which suggested a possible aneugenic effect of this phthalate. Our results demonstrate that in vitro exposure to DEHP had a dose-dependent cytotoxic and genotoxic effects in DLEC cell line, encouraging further investigation into its effects in in vivo and/or ex vivo cell systems of marine organisms.

Assessment of DNA damage in Polish children environmentally exposed to pesticides

Exposure to pesticides leads to complex, long-lasting adverse effects on human health, and poses a substantial risk to those living in areas devoted to agriculture. Children are particularly vulnerable to the pesticide exposure, due to the developmental, dietary and physiological factors. Small body mass and typical exploratory behavior result in increased risk of intoxication. Thus, even exposure to low concentrations of pesticides, if of sufficient duration, may lead to permanent health disorders and limit their harmonious development.In this study 108 children, living in areas of an intense pesticide use and a control group (n = 92) of children from an agrotouristic area were investigated, whether DNA damage increased due to prolonged pesticide exposure. A presence of DNA breaks and oxidative damage to DNA bases, characterized as Fpg-sensitive sites, were detected by comet assay. Micronuclei (MN) formation was evaluated by cytokinesis-block MN assay. The exposure of children to pesticides resulted in increased number of MN in peripheral blood lymphocytes (P = 0.016), increased DNA strand breaks level (P = 0.002) and oxidative damage to DNA (P < 0.001). Negative correlation was demonstrated between the level of DNA strand breaks and acetylcholinesterase (AChE) activity in exposed group. In conclusion, despite just environmental pesticide exposure in the test group of children, significant biological effects were detected.

In vitro and in vivo genotoxicity assessment of gold nanoparticles of different sizes by comet and SMART assays

Due to the increasing use of gold nanoparticles (AuNPs) in different areas such as medicine, biotechnology or food sector, human exposure to them has grown significantly and its toxicity evaluation has become essential. Therefore, the purpose of this study was to compare the potential genotoxic effects of 30, 50 and 90 nm AuNPs, using in vitro comet assay with the in vivo mutagenic and recombinogenic activity (SMART Test) in Drosophila. The results indicated that in both cell lines, 30, 50 and 90 nm (1–10 μg ml−1) AuNPs increased DNA strand breaks following 24 h treatment. This damage was not dose and size-dependent. Moreover, a modified comet assay using endonuclease III and formamidopyrimidine-DNA glycosylase restriction enzymes showed that in both cell lines, pyrimidines and purines were oxidatively damaged by all AuNPs, being 90 nm AuNPs slightly more genotoxic. However, the data obtained with SMART showed that 30 nm AuNPs did not modify the spontaneous frequencies of spots indicating lack of mutagenic and recombinogenic activity. Therefore, further experiments must be carried out to gain a better understanding of the mechanism of action of AuNPs to ensure their safe use.

O 022 – The short-term effects of selective dorsal rhizotomy on gait in cerebral palsy

The increased number of cell divisions undergone by spermatogonia of older fathers cannot fully account for the observed increase in germline genetic damage. Studies have shown that the mechanisms induced in germ cells in response to oxidative damage varies with age, that DNA repair efficiency declines, and both sperm DNA damage and spontaneous mutations increase. However, it is not known whether the altered response with age is a cause, or consequence, of an age-associated change in cell susceptibility to genetic damage.Following a single 150 mg/kg dose of cyclophosphamide (CP), young (8-weeks old) and aged (17-month old) male mice were examined 24 h later for induced genetic damage in epididymal spermatozoa using the alkaline comet and sperm chromatin stability assays. Apoptosis among testicular cells was examined on tissue cross-sections using the TUNEL assay.Sperm showed no significant increase in DNA strand breaks with age (detected by the comet assay) and no change in sperm chromatin stability (detected by the SCSA assay). Following CP treatment, there was no effect on DNA-strand breakage but sperm chromatin instability was significantly higher. Furthermore, it was also significantly elevated in old treated, compared with young treated, animals suggesting that increased age affects the sensitivity of epididymal sperm to chromatin damage.There was no difference in apoptosis in testicular germ cells from either young or old control animals, while CP administration resulted in a significant increase in apoptosis among young animals but not old animals. Following genotoxin exposure, an increase in chromatin instability in the spermatozoa of old animals and a decrease in the ability of their testicular germ cells undergo apoptosis suggests an age-related decrease in genome protection mechanisms. Since those germ cells are only transiently present in the testis, it is likely that this age-related deterioration originates in the spermatogonial stem cells. The findings are also evidence that the safety evaluation of reproductive genotoxins should consider young and old individuals separately.

Modulation of immune genes mRNA levels in mucosal tissues and DNA damage in red blood cells of Sparus aurata by gold nanoparticles

Gold nanoparticles (AuNP) effects on Sparus aurata were evaluated on skin, gills and intestine by assessing the expression of immune genes and in peripheral blood evaluating genetic damage. Fish were exposed to 0.5 and 50 μg/L AuNP for 96 h. Results showed that exposure to 50 μg/L AuNP induced an upregulation in the expression of innate immune genes in gills (c3, lys, il1β, tnfα, il6, il10 and tgfβ) and intestine (il1β, tnfα and il6). Furthermore, mRNA levels of hsp70 and hsp90 were increased in gills after exposure to 0.5 μg/L AuNP, when compared to 50 μg/L. Present data demonstrated the sensitivity of gills and intestines to AuNP exposure supporting their use in the study of fish responses to other nanoparticles. Genotoxic potential of AuNP was demonstrated by increased DNA strand breaks in red blood cells of fish exposed to AuNP, suggesting that AuNP represent a potential hazard to fish.