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Molecules (Basel, Switzerland) Jan 2024Two novel sizable multicharged cationic complexes, of the formulae [(--[12]CPP)[Ru(--Cp)]]Χ and [(--[11]CPP)[Ru(--Cp)]]Χ, CPP = cycloparaphenylene, Cp =...
Two novel sizable multicharged cationic complexes, of the formulae [(--[12]CPP)[Ru(--Cp)]]Χ and [(--[11]CPP)[Ru(--Cp)]]Χ, CPP = cycloparaphenylene, Cp = cyclopentadienyl, X = [PF], (), () and [Cl], (), (), were synthesized and characterized using NMR techniques, high-resolution mass spectrometry, and elemental analyses. Complexes () and () were stable in acetone and acetonitrile solutions over 48 h. In contrast, the water-soluble () and () begin to decompose in aqueous media after 1 h, due to the [Cl] tendency for nucleophilic attack on ruthenium of the {Ru(--Cp)} units. Fluorescence quenching experiments conducted during the stability window of () with the d(5'-CGCGAATTCGCG-3')-EtBr adducts revealed remarkably high values for K = 1.185 × 10 ± 0.025 M and K = 3.162 × 10 ± 0.001 M. Furthermore, the cytotoxic activity of () against A2780, A2780res, and MCF-7 cancer cell lines shows that it is highly cytotoxic with IC values in the range of 4.76 ± 1.85 to 16 ± 0.81 μΜ.
Topics: Humans; Female; Cell Line, Tumor; Ruthenium; Ovarian Neoplasms; Antineoplastic Agents; Cations; DNA; Coordination Complexes
PubMed: 38276592
DOI: 10.3390/molecules29020514 -
Cell Death & Disease Jan 2024DNA topoisomerase II (TOP2) is an enzyme that performs a critical function in manipulating DNA topology during replication, transcription, and chromosomal compaction by...
DNA topoisomerase II (TOP2) is an enzyme that performs a critical function in manipulating DNA topology during replication, transcription, and chromosomal compaction by forming a vital intermediate known as the TOP2-DNA cleavage complex (TOP2cc). Although the TOP2cc is often transient, stabilization can be achieved by TOP2 poisons, a family of anti-cancer chemotherapeutic agents targeting TOP2, such as etoposide (VP-16), and then induce double-strand breaks (DSBs) in cellular DNA. TOP2cc first needs to be proteolyzed before it can be processed by TDP2 for the removal of these protein adducts and to produce clean DNA ends necessary for proper repair. However, the mechanism by which TOP2βcc is proteolyzed has not been thoroughly studied. In this study, we report that after exposure to VP-16, MDM2, a RING-type E3 ubiquitin ligase, attaches to TOP2β and initiates polyubiquitination and proteasomal degradation. Mechanistically, during exposure to VP-16, TOP2β binds to DNA to form TOP2βcc, which promotes MDM2 binding and subsequent TOP2β ubiquitination and degradation, and results in a decrease in TOP2βcc levels. Biologically, MDM2 inactivation abrogates TOP2β degradation, stabilizes TOP2βcc, and subsequently increases the number of TOP2β-concealed DSBs, resulting in the rapid death of cancer cells via the apoptotic process. Furthermore, we demonstrate the combination activity of VP-16 and RG7112, an MDM2 inhibitor, in the xenograft tumor model and in situ lung cancer mouse model. Taken together, the results of our research reveal an underlying mechanism by which MDM2 promotes cancer cell survival in the presence of TOP2 poisons by activating proteolysis of TOP2βcc in a p53-independent manner, and provides a rationale for the combination of MDM2 inhibitors with TOP2 poisons for cancer therapy.
Topics: Animals; Humans; Mice; Disease Models, Animal; DNA; DNA Topoisomerases, Type II; DNA-Binding Proteins; Etoposide; Phosphoric Diester Hydrolases; Proteolysis; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53
PubMed: 38263255
DOI: 10.1038/s41419-024-06474-3 -
BioRxiv : the Preprint Server For... Jan 2024Acetaldehyde is the primary metabolite of alcohol and is present in many environmental sources including tobacco smoke. Acetaldehyde is genotoxic, whereby it can form...
Acetaldehyde is the primary metabolite of alcohol and is present in many environmental sources including tobacco smoke. Acetaldehyde is genotoxic, whereby it can form DNA adducts and lead to mutagenesis. Individuals with defects in acetaldehyde clearance pathways have increased susceptibility to alcohol-associated cancers. Moreover, a mutation signature specific to acetaldehyde exposure is widespread in alcohol and smoking-associated cancers. However, the pathways that repair acetaldehyde-induced DNA damage and thus prevent mutagenesis are vaguely understood. Here, we used to systematically delete genes in each of the major DNA repair pathways to identify those that alter acetaldehyde-induced mutagenesis. We found that deletion of the nucleotide excision repair (NER) genes, or , led to an increase in mutagenesis upon acetaldehyde exposure. Acetaldehyde-induced mutations were dependent on translesion synthesis as well as DNA inter-strand crosslink (ICL) repair in strains. Moreover, whole genome sequencing of the mutated isolates demonstrated an increase in C→A changes coupled with an enrichment of gCn→A changes in the acetaldehyde-treated isolates. The gCn→A mutation signature has been shown to be diagnostic of acetaldehyde exposure in yeast and in human cancers. We also demonstrated that the deletion of the two DNA-protein crosslink (DPC) repair proteases, and , also led to increased acetaldehyde-induced mutagenesis. Defects in base excision repair (BER) led to a mild increase in mutagenesis, while defects in mismatch repair (MMR), homologous recombination repair (HR) and post replicative repair pathways did not impact mutagenesis upon acetaldehyde exposure. Our results in yeast were further corroborated upon analysis of whole exome sequenced liver cancers, wherein, tumors with defects in ERCC1 and ERCC4 (NER), FANCD2 (ICL repair) or SPRTN (DPC repair) carried a higher gCn→A mutation load than tumors with no deleterious mutations in these genes. Our findings demonstrate that multiple DNA repair pathways protect against acetaldehyde-induced mutagenesis.
PubMed: 38260495
DOI: 10.1101/2024.01.07.574575 -
Cancers Jan 2024O-methylguanine-DNA methyltransferase (MGMT or AGT) is a DNA repair protein with the capability to remove alkyl groups from O-AlkylG adducts. Moreover, MGMT plays a... (Review)
Review
O-methylguanine-DNA methyltransferase (MGMT or AGT) is a DNA repair protein with the capability to remove alkyl groups from O-AlkylG adducts. Moreover, MGMT plays a crucial role in repairing DNA damage induced by methylating agents like temozolomide and chloroethylating agents such as carmustine, and thereby contributes to chemotherapeutic resistance when these agents are used. This review delves into the structural roles and repair mechanisms of MGMT, with emphasis on the potential structural and functional roles of the N-terminal domain of MGMT. It also explores the development of cancer therapeutic strategies that target MGMT. Finally, it discusses the intriguing crosstalk between MGMT and other DNA repair pathways.
PubMed: 38254819
DOI: 10.3390/cancers16020331 -
Biomolecules Dec 2023Occupational exposure to aromatic amines (AAs) is an important risk factor for urinary bladder cancer. This study aimed to evaluate the toxicity of AAs and analyze the...
Evaluation of the Mechanisms Involved in the Development of Bladder Toxicity following Exposure to Occupational Bladder Cancer Causative Chemicals Using DNA Adductome Analysis.
Occupational exposure to aromatic amines (AAs) is an important risk factor for urinary bladder cancer. This study aimed to evaluate the toxicity of AAs and analyze the carcinogenic mechanisms in rat bladder by comprehensive analysis of DNA adducts (DNA adductome). DNA was extracted from the bladder epithelia of rats treated with AAs, including acetoacet--toluidine (AAOT) and -toluidine (OTD), and adductome analysis was performed. Principal component analysis-discriminant analysis revealed that OTD and AAOT observed in urinary bladder hyperplasia could be clearly separated from the controls and other AAs. After confirming the intensity of each adduct, four adducts were screened as having characteristics of the OTD/AAOT treatment. Comparing with the in-house DNA adduct database, three of four candidates were identified as oxidative DNA adducts, including 8-OH-dG, based on mass fragmentation together with high-resolution accurate mass (HRAM) spectrometry data. Therefore, findings suggested that oxidative stress may be involved in the toxicity of rat bladder epithelium exposed to AAs. Consequently, the administration of apocynin, an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase, in six-week-old rats fed with 0.6% OTD in their diet resulted in simple hyperplastic lesions in the bladder that were suppressed by apocynin. The labeling indices of Ki67, γ-H2AX, and 8-OHdG were significantly decreased in an apocynin concentration-dependent manner. These findings indicate that oxidative stress may have contributed to the development of urinary cancer induced by OTD.
Topics: Animals; Rats; Urinary Bladder; DNA Adducts; Urinary Bladder Neoplasms; 8-Hydroxy-2'-Deoxyguanosine; Amines; Databases, Nucleic Acid; Acetophenones; Toluidines
PubMed: 38254636
DOI: 10.3390/biom14010036 -
Toxins Dec 2023In the context of the mysterious Balkan endemic nephropathy of the 1900s, and the discovery in the 1960s of the potent mycotoxin ochratoxin A, experimental research... (Review)
Review
In the context of the mysterious Balkan endemic nephropathy of the 1900s, and the discovery in the 1960s of the potent mycotoxin ochratoxin A, experimental research projects sought to explore any inter-relationship. Experimental lifetime administration of the toxin to male rats had revealed renal DNA adducts with the toxin, correlated with renal tumours, confirmation of which required molecular evidence. Consequently, production of C-ochratoxin A of a high specific radioactivity was required, practical biosynthetic detail of which had not previously been published. A fermentation study of was carried out during 2002 for a European project, to select for the production of high-quality C-ochratoxin A, necessarily exploring for the maximum diversion of C-sodium acetate into the pentaketide portion of mycotoxin. Experimentation necessarily had to optimise the competitive context of fungal growth dynamics and addition of the biosynthetic precursor in the early days of shaken-flask fermentation before adding the radiolabelled precursor. From optimal fermentation, 50 mg of the C ochratoxin A was supplied within a European project for DNA adduct experimentation, but that proved negative as subsequently published. Experimental description of the radiolabelled ochratoxin A production was later made in a doctoral thesis, but is first publicised here. Further review of the literature reveals an explanation for the published failure to confirm rat DNA/ochratoxin A adduct formation, for which further experimentation is now recommended.
Topics: Male; Animals; Rats; Aspergillus ochraceus; Fermentation; Balkan Nephropathy; Mycotoxins; Ochratoxins
PubMed: 38251225
DOI: 10.3390/toxins16010008 -
Antioxidants (Basel, Switzerland) Jan 2024Diabetic kidney disease (DKD) is a leading cause of death in patients with diabetes. An early precursor to DKD is endothelial cell dysfunction (ECD), which often...
Diabetic kidney disease (DKD) is a leading cause of death in patients with diabetes. An early precursor to DKD is endothelial cell dysfunction (ECD), which often precedes and exacerbates vascular disease progression. We previously discovered that covalent adducts formed on DNA, RNA, and proteins by the reactive metabolic by-product methylglyoxal (MG) predict DKD risk in patients with type 1 diabetes up to 16 years pre-diagnosis. However, the mechanisms by which MG adducts contribute to vascular disease onset and progression remain unclear. Here, we report that the most predominant MG-induced nucleoside adducts, -(1-carboxyethyl)-deoxyguanosine (CEdG) and -(1-carboxyethyl)-guanosine (CEG), drive endothelial dysfunction. Following CEdG or CEG exposure, primary human umbilical vein endothelial cells (HUVECs) undergo endothelial dysfunction, resulting in enhanced monocyte adhesion, increased reactive oxygen species production, endothelial permeability, impaired endothelial homeostasis, and exhibit a dysfunctional transcriptomic signature. These effects were discovered to be mediated through the receptor for advanced glycation end products (RAGE), as an inhibitor for intracellular RAGE signaling diminished these dysfunctional phenotypes. Therefore, we found that not only are MG adducts biomarkers for DKD, but that they may also have a role as potential drivers of vascular disease onset and progression and a new therapeutic modality.
PubMed: 38247509
DOI: 10.3390/antiox13010085 -
STAR Protocols Mar 2024UV radiation induces the formation of adducts in genomic DNA within cells that are later found to be present in cell-free fractions associated with extracellular...
UV radiation induces the formation of adducts in genomic DNA within cells that are later found to be present in cell-free fractions associated with extracellular vesicles (EVs) outside of cells. Here, we present a protocol for isolating UV photoproducts in extracellular DNA released from UVB-irradiated cells via differential centrifugation. We then detail steps for monitoring the DNA adducts using DNA immunoblotting. This protocol can be applied for detection of DNA adducts in EVs from cell culture and skin explant models. For complete details on the use and execution of this protocol, please refer to Carpenter et al..
Topics: DNA Damage; DNA Adducts; DNA; Ultraviolet Rays; Skin
PubMed: 38244199
DOI: 10.1016/j.xpro.2024.102838 -
Chemical Research in Toxicology Feb 2024Human tissue three-dimensional (3D) organoid cultures have the potential to reproduce the physiological properties and cellular architecture of the organs from which...
Human tissue three-dimensional (3D) organoid cultures have the potential to reproduce the physiological properties and cellular architecture of the organs from which they are derived. The ability of organoid cultures derived from human stomach, liver, kidney, and colon to metabolically activate three dietary carcinogens, aflatoxin B (AFB), aristolochic acid I (AAI), and 2-amino-1-methyl-6-phenylimidazo[4,5-]pyridine (PhIP), was investigated. In each case, the response of a target tissue (liver for AFB; kidney for AAI; colon for PhIP) was compared with that of a nontarget tissue (gastric). After treatment cell viabilities were measured, DNA damage response (DDR) was determined by Western blotting for p-p53, p21, p-CHK2, and γ-H2AX, and DNA adduct formation was quantified by mass spectrometry. Induction of the key xenobiotic-metabolizing enzymes (XMEs) CYP1A1, CYP1A2, CYP3A4, and NQO1 was assessed by qRT-PCR. We found that organoids from different tissues can activate AAI, AFB, and PhIP. In some cases, this metabolic potential varied between tissues and between different cultures of the same tissue. Similarly, variations in the levels of expression of XMEs were observed. At comparable levels of cytotoxicity, organoids derived from tissues that are considered targets for these carcinogens had higher levels of adduct formation than a nontarget tissue.
Topics: Humans; DNA Adducts; Carcinogens; Neoplasms; Liver; Organoids
PubMed: 38232180
DOI: 10.1021/acs.chemrestox.3c00255 -
BMC Cancer Jan 2024KRAS is the undisputed champion of oncogenes, and despite its prominent role in oncogenesis as mutated gene, KRAS mutation appears infrequent in gliomas. Nevertheless,...
BACKGROUND
KRAS is the undisputed champion of oncogenes, and despite its prominent role in oncogenesis as mutated gene, KRAS mutation appears infrequent in gliomas. Nevertheless, gliomas are considered KRAS-driven cancers due to its essential role in mouse malignant gliomagenesis. Glioblastoma is the most lethal primary brain tumor, often associated with disturbed RAS signaling. For newly diagnosed GBM, the current standard therapy is alkylating agent chemotherapy combined with radiotherapy. Cisplatin is one of the most effective anticancer drugs and is used as a first-line treatment for a wide spectrum of solid tumors (including medulloblastoma and neuroblastoma) and many studies are currently focused on new delivery modalities of effective cisplatin in glioblastoma. Its mechanism of action is mainly based on DNA damage, inducing the formation of DNA adducts, triggering a series of signal-transduction pathways, leading to cell-cycle arrest, DNA repair and apoptosis.
METHODS
Long-term cultures of human glioblastoma, U87MG and U251MG, were either treated with cis-diamminedichloroplatinum (cisplatin, CDDP) and/or MEK-inhibitor PD98059. Cytotoxic responses were assessed by cell viability (MTT), protein expression (Western Blot), cell cycle (PI staining) and apoptosis (TUNEL) assays. Further, gain-of-function experiments were performed with cells over-expressing mutated hypervariable region (HVR) KRAS plasmids.
RESULTS
Here, we studied platinum-based chemosensitivity of long-term cultures of human glioblastoma from the perspective of KRAS expression, by using CDDP and MEK-inhibitor. Endogenous high KRAS expression was assessed at transcriptional (qPCR) and translational levels (WB) in a panel of primary and long-term glioblastoma cultures. Firstly, we measured immediate cellular adjustment through direct regulation of protein concentration of K-Ras4B in response to cisplatin treatment. We found increased endogenous protein abundance and involvement of the effector pathway RAF/MEK/ERK mitogen-activated protein kinase (MAPK) cascade. Moreover, as many MEK inhibitors are currently being clinically evaluated for the treatment of high-grade glioma, so we concomitantly tested the effect of the potent and selective non-ATP-competitive MEK1/2 inhibitor (PD98059) on cisplatin-induced chemosensitivity in these cells. Cell-cycle phase distribution was examined using flow cytometry showing a significant cell-cycle arrest in both cultures at different percentage, which is modulated by MEK inhibition. Cisplatin-induced cytotoxicity increased sub-G1 percentage and modulates G2/M checkpoint regulators cyclins D1 and A. Moreover, ectopic expression of a constitutively active KRAS rescued CDDP-induced apoptosis and different HVR point mutations (particularly Ala 185) reverted this phenotype.
CONCLUSION
These findings warrant further studies of clinical applications of MEK1/2 inhibitors and KRAS as 'actionable target' of cisplatin-based chemotherapy for glioblastoma.
Topics: Humans; Antineoplastic Agents; Cell Line, Tumor; Cisplatin; Glioblastoma; Mitogen-Activated Protein Kinase Kinases; Platinum; Protein Kinase Inhibitors; Proto-Oncogene Proteins p21(ras)
PubMed: 38225605
DOI: 10.1186/s12885-023-11758-6