In Vitro Anticancer Activity and Mechanism of Action of an Aziridinyl Galactopyranoside

We recently screened a series of new aziridines β-D-galactopyranoside derivatives for selective anticancer activity and identified 2-methyl-2,3-[N-(4-methylbenzenesulfonyl)imino]propyl 2,3-di-O-benzyl-4,6-O-(S)-benzylidene-β-D-galactopyranoside (AzGalp) as the most promising compound. In this article, we explore the possible mechanisms involved in the cytotoxicity of this aziridine and evaluate its selective anticancer activity using cancer cells and normal cells from a variety of tissues. Our data show that AzGalp induces DNA damage (comet assay). Cells deficient in the nucleotide excision repair (NER) pathway were hypersensitive to the cytotoxicity of this compound. These results suggest that AzGalp induces bulky DNA adducts, and that cancer cells lacking a functional NER pathway may be particularly vulnerable to the anticancer effects of this aziridine. Several experiments revealed that neither the generation of oxidative stress nor the inhibition of glycolysis played a significant role in the cytotoxicity of AzGalp. Combinations of AzGalp with oxaliplatin or 5-fluorouracil slightly improved the ability of both anticancer drugs to selectively kill cancer cells. AzGalp also showed selective cytotoxicity against a panel of malignant cells versus normal cells; the highest selectivity was observed for two acute promyelocytic leukemia cell lines. Additional preclinical studies are necessary to evaluate the anticancer potential of AzGalp.

Variable inhibition of unwinding rates of DNA catalyzed by the SARS-Cov-2 (COV19) helicase nsp13 by structurally distinct single DNA lesions.

The SARS 2 (Covid 19) helicase nsp13 plays a critically important role in the replication of the Corona virus by unwinding double-stranded RNA (and DNA) with a 5 prime to 3 prime strand polarity. Here we explored the impact of single, structurally defined covalent DNA lesions on the helicase activity of nsp13 in aqueous solutions, The objectives were to derive mechanistic insights into the relationships between the structures of DNA lesions, the DNA distortions that they engender, and the inhibition of helicase activity. The lesions included two bulky stereoisomeric N2-guanine adducts derived from the reactions of benzo[a]pyrene diol epoxide with DNA. The trans-adduct assumes a minor groove conformation, while the cis-product adopts a base-displaced intercalated conformation. The non-bulky DNA lesions included the intra-strand cross-linked thymine dimers, the cis-syn-cyclobutane pyrimidine dimer, and the pyrimidine (6–4) pyrimidone photoproduct. All four lesions strongly inhibit the helicase activity of nsp13, The UV photolesions feature a 2 - 5-fold smaller inhibition of the nsp13 unwinding activity than the bulky DNA adducts, and the kinetics of these two pairs of DNA lesions are also different. The connections between the structural features of these four DNA lesions and their impact on nsp13 unwinding efficiencies are discussed.

Checkpoint Control of DNA Repair in Yeast

Budding yeast has been a model organism for understanding how DNA damage is repaired and how cells minimize genetic instability caused by arresting or delaying the cell cycle at well-defined checkpoints. However, many DNA damage insults are tolerated by mechanisms that can both be error-prone and error-free. The mechanisms that tolerate DNA damage and promote cell division are less well-understood. This review summarizes current information known about the checkpoint response to agents that elicit both the G2/M checkpoint and the intra-S phase checkpoint and how cells adapt to unrepaired DNA damage. Tolerance to particular bulky DNA adducts and radiomimetic agents are discussed, as well as possible mechanisms that may control phosphatases that deactivate phosphorylated proteins.

PrimPol-dependent single-stranded gap formation mediates homologous recombination at bulky DNA adducts

Stalled replication forks can be restarted and repaired by RAD51-mediated homologous recombination (HR), but HR can also perform post-replicative repair after bypass of the obstacle. Bulky DNA adducts are important replication-blocking lesions, but it is unknown whether they activate HR at stalled forks or behind ongoing forks. Using mainly BPDE-DNA adducts as model lesions, we show that HR induced by bulky adducts in mammalian cells predominantly occurs at post-replicative gaps formed by the DNA/RNA primase PrimPol. RAD51 recruitment under these conditions does not result from fork stalling, but rather occurs at gaps formed by PrimPol re-priming and resection by MRE11 and EXO1. In contrast, RAD51 loading at double-strand breaks does not require PrimPol. At bulky adducts, PrimPol promotes sister chromatid exchange and genetic recombination. Our data support that HR at bulky adducts in mammalian cells involves post-replicative gap repair and define a role for PrimPol in HR-mediated DNA damage tolerance.

Sensitive CometChip assay for screening potentially carcinogenic DNA adducts by trapping DNA repair intermediates

Genotoxicity testing is critical for predicting adverse effects of pharmaceutical, industrial, and environmental chemicals. The alkaline comet assay is an established method for detecting DNA strand breaks, however, the assay does not detect potentially carcinogenic bulky adducts that can arise when metabolic enzymes convert pro-carcinogens into a highly DNA reactive products. To overcome this, we use DNA synthesis inhibitors (hydroxyurea and 1-β-d-arabinofuranosyl cytosine) to trap single strand breaks that are formed during nucleotide excision repair, which primarily removes bulky lesions. In this way, comet-undetectable bulky lesions are converted into comet-detectable single strand breaks. Moreover, we use HepaRG™ cells to recapitulate in vivo metabolic capacity, and leverage the CometChip platform (a higher throughput more sensitive comet assay) to create the ‘HepaCometChip’, enabling the detection of bulky genotoxic lesions that are missed by current genotoxicity screens. The HepaCometChip thus provides a broadly effective approach for detection of bulky DNA adducts.

Modulation of smoke-induced DNA and microRNA alterations in mouse lung by licofelone, a triple COX-1, COX-2 and 5-LOX inhibitor

Chronic inflammation plays a crucial role in the carcinogenesis process and, in particular, in smoking-related carcinogenesis. Therefore, anti-inflammatory agents provide an interesting perspective in the prevention of smoking-associated cancers. Among nonsteroidal anti-inflammatory drugs (NSAIDs), licofelone is a triple inhibitor of both cyclooxygenases (COX-1 and COX-2) and of 5-lipooxygenase (5-LOX) that has shown some encouraging results in cancer prevention models. We previously showed that the dietary administration of licofelone, starting after weanling, to Swiss H mice exposed for 4 months to mainstream cigarette smoke since birth attenuated preneoplastic lesions of inflammatory nature in both lung and urinary tract, and had some effects on the yield of lung tumors at 7.5 months of age. The present study aimed at evaluating the early modulation by licofelone of pulmonary DNA and RNA alterations either in smoke-free or smoke-exposed H mice after 10 weeks of exposure. Licofelone protected the mice from the smoke-induced loss of body weight and significantly attenuated smoke-induced nucleotide alterations by decreasing the levels of bulky DNA adducts and 8-hydroxy-2′-deoxyguanosine in mouse lung. Moreover, the drug counteracted dysregulation by smoke of several pulmonary microRNAs involved in stress response, inflammation, apoptosis, and oncogene suppression. However, even in smoke-free mice administration of the drug had significant effects on a broad panel of microRNAs and, as assessed in a subset of mice used in a parallel cancer chemoprevention study, licofelone even enhanced the smoke-induced systemic genotoxic damage after 4 months of exposure. Therefore, caution should be paid when administering licofelone to smokers for long periods.

PrimPol-dependent single-stranded gap formation mediates homologous recombination at bulky DNA adducts

Obstacles on the DNA template can lead to DNA replication fork stalling and genomic rearrangements. RAD51-mediated homologous recombination (HR) can promote restart and repair of stalled forks, but also post-replicative repair once the obstacle has been bypassed. Bulky DNA adducts are important replication-blocking lesions induced by environmental carcinogens, but it is not known whether they activate HR directly at stalled forks, or at gaps left behind ongoing forks. Here we show that in mammalian cells, bulky adducts predominantly induce HR at post-replicative gaps formed by the DNA/RNA primase PrimPol. Using BPDE and other bulky model lesions, we report that RAD51 is not recruited to stalled or collapsed forks, but instead to long gaps formed by PrimPol re-priming activity and resection by MRE11 and EXO1. In contrast, RAD51 loading at DSBs does not require PrimPol. At bulky adducts, PrimPol is required for the induction of sister chromatid exchanges and genetic recombination. Our data support that HR at bulky adducts in mammalian cells involves post-replicative gap repair and define a role for PrimPol in DNA damage tolerance by homologous recombination.

Bulky DNA adducts, microRNA profiles, and lipid biomarkers in Norwegian tunnel finishing workers occupationally exposed to diesel exhaust

ObjectivesThis study aimed to assess the biological impact of occupational exposure to diesel exhaust (DE) including DE particles (DEP) from heavy-duty diesel-powered equipment in Norwegian tunnel finishing workers (TFW).MethodsTFW (n=69) and referents (n=69) were investigated for bulky DNA adducts (by 32P-postlabelling) and expression of microRNAs (miRNAs) (by small RNA sequencing) in peripheral blood mononuclear cells (PBMC), as well as circulating free arachidonic acid (AA) and eicosanoid profiles in plasma (by liquid chromatography–tandem mass spectrometry).ResultsPBMC from TFW showed significantly higher levels of DNA adducts compared with referents. Levels of DNA adducts were also related to smoking habits. Seventeen miRNAs were significantly deregulated in TFW. Several of these miRNAs are related to carcinogenesis, apoptosis and antioxidant effects. Analysis of putative miRNA-gene targets revealed deregulation of pathways associated with cancer, alterations in lipid molecules, steroid biosynthesis and cell cycle. Plasma profiles showed higher levels of free AA and 15-hydroxyeicosatetraenoic acid, and lower levels of prostaglandin D2 and 9-hydroxyoctadecadienoic acid in TFW compared with referents.ConclusionOccupational exposure to DE/DEP is associated with biological alterations in TFW potentially affecting lung homoeostasis, carcinogenesis, inflammation status and the cardiovascular system. Of particular importance is the finding that tunnel finishing work is associated with an increased level of DNA adducts formation in PBMC.