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Cancer Biology & Therapy Dec 2017Glioblastoma is a lethal form of brain tumour usually treated by surgical resection followed by radiotherapy and an alkylating chemotherapeutic agent. Key to the success... (Review)
Review
Glioblastoma is a lethal form of brain tumour usually treated by surgical resection followed by radiotherapy and an alkylating chemotherapeutic agent. Key to the success of this multimodal approach is maintaining apoptotic sensitivity of tumour cells to the alkylating agent. This initial treatment likely establishes conditions contributing to development of drug resistance as alkylating agents form the O-methylguanine adduct. This activates the mismatch repair (MMR) process inducing apoptosis and mutagenesis. This review describes key juxtaposed drivers in the balance between alkylation induced mutagenesis and apoptosis. Mutations in MMR genes are the probable drivers for alkylation based drug resistance. Critical to this interaction are the dose-response and temporal interactions between adduct formation and MMR mutations. The precision in dose interval, dose-responses and temporal relationships dictate a role for alkylating agents in either promoting experimental tumour formation or inducing tumour cell death with chemotherapy. Importantly, this resultant loss of chemotherapeutic selective pressure provides opportunity to explore novel therapeutics and appropriate combinations to minimise alkylation based drug resistance and tumour relapse.
Topics: Antineoplastic Agents, Alkylating; Apoptosis; DNA Adducts; DNA Mismatch Repair; DNA Repair; Drug Resistance, Neoplasm; Glioblastoma; Guanine; Humans; Mutation; Neoplasm Recurrence, Local
PubMed: 29020502
DOI: 10.1080/15384047.2017.1385680 -
Chemical Research in Toxicology Nov 2020The reversible generation and capture of certain electrophilic quinone methide intermediates support dynamic reactions with DNA that allow for migration and transfer of...
The reversible generation and capture of certain electrophilic quinone methide intermediates support dynamic reactions with DNA that allow for migration and transfer of alkylation and cross-linking. This reversibility also expands the possible consequences that can be envisioned when confronted by DNA repair processes and biological machines. To begin testing the response to such an encounter, quinone methide-based modification of DNA has now been challenged with a helicase (T7 bacteriophage gene protein four, T7gp4) that promotes 5' to 3' translocation and unwinding. This model protein was selected based on its widespread application, well characterized mechanism and detailed structural information. Little over one-half of the cross-linking generated by a bisfunctional quinone methide remained stable to T7gp4 and did not suppress its activity. The helicase likely avoids the topological block generated by this fraction of cross-linking by its ability to shift from single- to double-stranded translocation. The remaining fraction of cross-linking was destroyed during T7gp4 catalysis. Thus, this helicase is chemically competent to promote release of the quinone methide from DNA. The ability of T7gp4 to act as a Brownian ratchet for unwinding DNA may block recapture of the QM intermediate by DNA during its transient release from a donor strand. Most surprisingly, T7gp4 releases the quinone methide from both the translocating strand that passes through its central channel and the excluded strand that was typically unaffected by other lesions. The ability of T7gp4 to reverse the cross-link formed by the quinone methide does not extend to that formed irreversibly by the nitrogen mustard mechlorethamine.
Topics: Alkylation; Cross-Linking Reagents; DNA; Indolequinones; Molecular Structure
PubMed: 33147957
DOI: 10.1021/acs.chemrestox.0c00413 -
Analytical Chemistry Aug 2023Temozolomide (TMZ) is considered a first line chemotherapy drug for glioblastoma (GBM). Unfortunately, the GBM without methylation of O-methylguanine-DNA...
Temozolomide (TMZ) is considered a first line chemotherapy drug for glioblastoma (GBM). Unfortunately, the GBM without methylation of O-methylguanine-DNA methyltransferase (MGMT), accounting for about 70% of all GBM, shows an inherent resistance to TMZ treatment. Aberrant accumulation of neutral lipids, primarily triglycerides (TGs) and cholesteryl esters (CEs), in lipid droplets (LDs) has been recognized as metabolic vulnerability for GBM therapy. However, it is not known whether MGMT methylation affects lipid accumulation in GBM. Herein, we employed label-free Raman spectromicroscopy, which integrated stimulated Raman scattering (SRS) microscopy and confocal Raman spectroscopy, to quantitatively analyze both the amount and composition of intracellular LDs in intact GBM tissues obtained from patients who had undergone resection surgery. Our results showed significant reductions in both the LD amount and the CE percentage in MGMT unmethylated GBMs (MGMT methylation < 15%) compared to MGMT methylated ones (MGMT methylation ≥ 15%). Due to a big variation of lipid accumulation in the MGMT methylated GBMs, these patients were further divided into hypermethylated group (MGMT methylation ≥ 50%) and intermediate-methylated group (MGMT methylation 15∼50%), according to the significantly different median survival rates of these two groups. Remarkable differences in LD amount, CE percentage, and also lipid saturation degree were found between the hypermethylated group and the other two groups, but not between the unmethylated and intermediate-methylated groups. To elucidate the possible underlying mechanism, we analyzed the differential expression of lipid metabolism-related genes in GBM with different levels of MGMT methylation using The Cancer Genome Atlas Program (TCGA) dataset. It was shown that the genes related to lipid oxidation and lipid efflux were upregulated, and the genes related to lipid synthesis were downregulated in unmethylated group. These findings unravel the relationship between MGMT methylation and lipid accumulation in GBM, which may offer new opportunities for the diagnosis and treatment of TMZ-resistant GBM.
Topics: Humans; Glioblastoma; Antineoplastic Agents, Alkylating; Dacarbazine; DNA Methylation; Brain Neoplasms; Temozolomide; DNA Modification Methylases; DNA Repair Enzymes; Lipids; Tumor Suppressor Proteins
PubMed: 37417930
DOI: 10.1021/acs.analchem.3c00967 -
Nature Communications Oct 2022Type I restriction-modification systems help establish the prokaryotic DNA methylation landscape and provide protection against invasive DNA. In addition to classical...
Type I restriction-modification systems help establish the prokaryotic DNA methylation landscape and provide protection against invasive DNA. In addition to classical m6A modifications, non-canonical type I enzymes catalyze both m6A and m4C using alternative DNA-modification subunits M1 and M2. Here, we report the crystal structures of the non-canonical PacII_M1M2S methyltransferase bound to target DNA and reaction product S-adenosylhomocysteine in a closed clamp-like conformation. Target DNA binds tightly within the central tunnel of the M1M2S complex and forms extensive contacts with all three protein subunits. Unexpectedly, while the target cytosine properly inserts into M2's pocket, the target adenine (either unmethylated or methylated) is anchored outside M1's pocket. A unique asymmetric catalysis is established where PacII_M1M2S has precisely coordinated the relative conformations of different subunits and evolved specific amino acids within M2/M1. This work provides insights into mechanisms of m6A/m4C catalysis and guidance for designing tools based on type I restriction-modification enzymes.
Topics: DNA Restriction-Modification Enzymes; DNA; Cytosine; DNA Methylation; Methyltransferases
PubMed: 36302770
DOI: 10.1038/s41467-022-34085-z -
Cancer Biology & Medicine Apr 2023Malignant gliomas are known to be one of the most difficult diseases to diagnose and treat because of the infiltrative growth pattern, rapid progression, and poor... (Review)
Review
Malignant gliomas are known to be one of the most difficult diseases to diagnose and treat because of the infiltrative growth pattern, rapid progression, and poor prognosis. Many antitumor drugs are not ideal for the treatment of gliomas due to the blood-brain barrier. Temozolomide (TMZ) is a DNA alkylating agent that can cross the blood-brain barrier. As the only first-line chemotherapeutic drug for malignant gliomas at present, TMZ is widely utilized to provide a survival benefit; however, some patients are inherently insensitive to TMZ. In addition, patients could develop acquired resistance during TMZ treatment, which limits antitumor efficacy. To clarify the mechanism underlying TMZ resistance, numerous studies have provided multilevel solutions, such as improving the effective concentration of TMZ in tumors and developing novel small molecule drugs. This review discusses the in-depth mechanisms underlying TMZ drug resistance, thus aiming to provide possibilities for the establishment of personalized therapeutic strategies against malignant gliomas and the accelerated development and transformation of new targeted drugs.
Topics: Humans; Glioblastoma; Antineoplastic Agents, Alkylating; Expert Testimony; Translational Research, Biomedical; Brain Neoplasms; Temozolomide; Glioma
PubMed: 37092846
DOI: 10.20892/j.issn.2095-3941.2023.0012 -
Experimental Eye Research Nov 2020This review details the current understanding of the mechanism of action and corneal effects of mitomycin C (MMC) for prophylactic prevention of stromal fibrosis after... (Review)
Review
This review details the current understanding of the mechanism of action and corneal effects of mitomycin C (MMC) for prophylactic prevention of stromal fibrosis after photorefractive keratectomy (PRK), and includes discussion of available information on dosage and exposure time recommended for MMC during PRK. MMC is an alkylating agent, with DNA-crosslinking activity, that inhibits DNA replication and cellular proliferation. It acts as a pro-drug and requires reduction in the tissue to be converted to an active agent capable of DNA alkylation. Although MMC augments the early keratocyte apoptosis wave in the anterior corneal stroma, its most important effect responsible for inhibition of fibrosis in surface ablation procedures such as PRK is via the inhibition of mitosis of myofibroblast precursor cells during the first few weeks after PRK. MMC use is especially useful when treating eyes with higher levels of myopia (≥approximately 6 D), which have shown higher risk of developing fibrosis (also clinically termed late haze). Studies have supported the use of MMC at a concentration of 0.02%, rather than lower doses (such as 0.01% or 0.002%), for optimal reduction of fibrosis after PRK. Exposure times for 0.02% MMC longer than 40 s may be beneficial for moderate to high myopia (≥6D), but shorter exposures times appear to be equally effective for lower levels of myopia. Although MMC treatment may also be beneficial in preventing fibrosis after PRK treatments for hyperopia and astigmatism, more studies are needed. Thus, despite the clinical use of MMC after PRK for nearly twenty years-with limited evidence of harmful effects in the cornea-many decades of experience will be needed to exclude late long-term effects that could be noted after MMC treatment.
Topics: Alkylating Agents; Corneal Opacity; Corneal Stroma; Fibrosis; Humans; Lasers, Excimer; Mitomycin; Myopia; Photorefractive Keratectomy; Postoperative Complications; Visual Acuity
PubMed: 32905844
DOI: 10.1016/j.exer.2020.108218 -
Proceedings of the National Academy of... May 2024DNA base damage is a major source of oncogenic mutations and disruption to gene expression. The stalling of RNA polymerase II (RNAP) at sites of DNA damage and the...
DNA base damage is a major source of oncogenic mutations and disruption to gene expression. The stalling of RNA polymerase II (RNAP) at sites of DNA damage and the subsequent triggering of repair processes have major roles in shaping the genome-wide distribution of mutations, clearing barriers to transcription, and minimizing the production of miscoded gene products. Despite its importance for genetic integrity, key mechanistic features of this transcription-coupled repair (TCR) process are controversial or unknown. Here, we exploited a well-powered in vivo mammalian model system to explore the mechanistic properties and parameters of TCR for alkylation damage at fine spatial resolution and with discrimination of the damaged DNA strand. For rigorous interpretation, a generalizable mathematical model of DNA damage and TCR was developed. Fitting experimental data to the model and simulation revealed that RNA polymerases frequently bypass lesions without triggering repair, indicating that small alkylation adducts are unlikely to be an efficient barrier to gene expression. Following a burst of damage, the efficiency of transcription-coupled repair gradually decays through gene bodies with implications for the occurrence and accurate inference of driver mutations in cancer. The reinitation of transcription from the repair site is not a general feature of transcription-coupled repair, and the observed data is consistent with reinitiation never taking place. Collectively, these results reveal how the directional but stochastic activity of TCR shapes the distribution of mutations following DNA damage.
Topics: DNA Repair; DNA Damage; Transcription, Genetic; RNA Polymerase II; Animals; Stochastic Processes; Mice; DNA; Humans; Alkylation; Mutation; Excision Repair
PubMed: 38717857
DOI: 10.1073/pnas.2403871121 -
Analytical Chemistry Nov 2022Apurinic/apyrimidinic (AP) sites, that is, abasic sites, are among the most frequently induced DNA lesions. Spontaneous or DNA glycosylase-mediated β-elimination of the...
Apurinic/apyrimidinic (AP) sites, that is, abasic sites, are among the most frequently induced DNA lesions. Spontaneous or DNA glycosylase-mediated β-elimination of the 3'-phosphoryl group can lead to strand cleavages at AP sites to yield a highly reactive, electrophilic 3'-phospho-α,β-unsaturated aldehyde (3'-PUA) remnant. The latter can react with amine or thiol groups of biological small molecules, DNA, and proteins to yield various damaged 3'-end products. Considering its high intracellular concentration, glutathione (GSH) may conjugate with 3'-PUA to yield 3-glutathionyl-2,3-dideoxyribose (GS-ddR), which may constitute a significant, yet previously unrecognized endogenous lesion. Here, we developed a liquid chromatography tandem mass spectroscopy method, in combination with the use of a stable isotope-labeled internal standard, to quantify GS-ddR in genomic DNA of cultured human cells. Our results revealed the presence of GS-ddR in the DNA of untreated cells, and its level was augmented in cells upon exposure to an alkylating agent, -methyl--nitrosourea (MNU). In addition, inhibition of AP endonuclease (APE1) led to an elevated level of GS-ddR in the DNA of MNU-treated cells. Together, we reported here, for the first time, the presence of appreciable levels of GS-ddR in cellular DNA, the induction of GS-ddR by a DNA alkylating agent, and the role of APE1 in modulating its level in human cells.
Topics: Humans; Animals; DNA-(Apurinic or Apyrimidinic Site) Lyase; DNA Repair; Methylnitrosourea; DNA Damage; DNA; Alkylating Agents; Mammals
PubMed: 36332130
DOI: 10.1021/acs.analchem.2c02003 -
Journal of Experimental & Clinical... Oct 2023Temozolomide (TMZ) treatment efficacy in glioblastoma (GBM) patients has been limited by resistance in the clinic. Currently, there are no clinically proven therapeutic...
BACKGROUND
Temozolomide (TMZ) treatment efficacy in glioblastoma (GBM) patients has been limited by resistance in the clinic. Currently, there are no clinically proven therapeutic options available to restore TMZ treatment sensitivity. Here, we investigated the potential of albumin-bound paclitaxel (ABX), a novel microtubule targeting agent, in sensitizing GBM cells to TMZ and elucidated its underlying molecular mechanism.
METHODS
A series of in vivo and in vitro experiments based on two GBM cell lines and two primary GBM cells were designed to evaluate the efficacy of ABX in sensitizing GBM cells to TMZ. Further proteomic analysis and validation experiments were performed to explore the underlying molecular mechanism. Finally, the efficacy and mechanism were validated in GBM patients derived organoids (PDOs) models.
RESULTS
ABX exhibited a synergistic inhibitory effect on GBM cells when combined with TMZ in vitro. Combination treatment of TMZ and ABX was highly effective in suppressing GBM progression and significantly prolonged the survival oforthotopic xenograft nude mice, with negligible side effects. Further proteomic analysis and experimental validation demonstrated that the combined treatment of ABX and TMZ can induce sustained DNA damage by disrupting XPC and ERCC1 expression and nuclear localization. Additionally, the combination treatment can enhance ferroptosis through regulating HOXM1 and GPX4 expression. Preclinical drug-sensitivity testing based on GBM PDOs models confirmed that combination therapy was significantly more effective than conventional TMZ monotherapy.
CONCLUSION
Our findings suggest that ABX has the potential to enhance TMZ treatment sensitivity in GBM, which provides a promising therapeutic strategy for GBM patients.
Topics: Animals; Mice; Humans; Temozolomide; Glioblastoma; Albumin-Bound Paclitaxel; Antineoplastic Agents, Alkylating; Ferroptosis; Mice, Nude; Proteomics; Drug Resistance, Neoplasm; DNA Damage; Cell Line, Tumor; Brain Neoplasms; Xenograft Model Antitumor Assays
PubMed: 37891669
DOI: 10.1186/s13046-023-02843-6 -
Cancer Chemotherapy and Pharmacology Oct 2022The DNA alkylating agent temozolomide (TMZ), is the first-line therapeutic for the treatment of glioblastoma (GBM). However, its use is confounded by the occurrence of...
PURPOSE
The DNA alkylating agent temozolomide (TMZ), is the first-line therapeutic for the treatment of glioblastoma (GBM). However, its use is confounded by the occurrence of drug resistance and debilitating adverse effects. Previously, we observed that letrozole (LTZ), an aromatase inhibitor, has potent activity against GBM in pre-clinical models. Here, we evaluated the effect of LTZ on TMZ activity against patient-derived GBM cells.
METHODS
Employing patient-derived G76 (TMZ-sensitive), BT142 (TMZ-intermediately sensitive) and G43 and G75 (TMZ-resistant) GBM lines we assessed the influence of LTZ and TMZ on cell viability and neurosphere growth. Combination Index (CI) analysis was performed to gain quantitative insights of this interaction. We then assessed DNA damaging effects by conducting flow-cytometric analysis of ˠH2A.X formation and induction of apoptotic signaling pathways (caspase3/7 activity). The effects of adding estradiol on LTZ-induced cytotoxicity and DNA damage were also evaluated.
RESULTS
Co-treatment with LTZ at a non-cytotoxic concentration (40 nM) reduced TMZ IC by 8, 37, 240 and 640 folds in G76, BT-142, G43 and G75 cells, respectively. The interaction was deemed to be synergistic based on CI analysis. LTZ co-treatment also significantly increased DNA damaging effects of TMZ. Addition of estradiol abrogated these LTZ effects.
CONCLUSIONS
LTZ increases DNA damage and synergistically enhances TMZ activity in TMZ sensitive and TMZ-resistant GBM lines. These effects are abrogated by the addition of exogenous estradiol underscoring that the observed effects of LTZ may be mediated by estrogen deprivation. Our study provides a strong rationale for investigating the clinical potential of combining LTZ and TMZ for GBM therapy.
Topics: Antineoplastic Agents, Alkylating; Aromatase Inhibitors; Brain Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; Estradiol; Glioblastoma; Humans; Letrozole; Temozolomide
PubMed: 36050497
DOI: 10.1007/s00280-022-04469-5