RESUMEN

Large-scale phase III clinical trials of Olaparib have revealed benefits for ovarian cancer patients with BRCA gene mutations or homologous recombination deficiency (HRD). However, fewer than 50% of ovarian cancer patients have both BRCA mutations and HRD. Therefore, improving the effect of Olaparib in HR-proficient patients is of great clinical value. Here, a combination strategy comprising Olaparib and CDK12-IN-3 effectively inhibited the growth of HR-proficient ovarian cancer in cell line, patient-derived organoid (PDO), and mouse xenograft models. Furthermore, the combination strategy induced severe DNA double-strand break (DSB) formation, increased NHEJ activity in the G2 phase, and reduced HR activity in cancer cells. Mechanistically, the combination treatment impaired Ku80 poly(ADP-ribosyl)ation (PARylation) and phosphorylation, resulting in PARP1-Ku80 complex dissociation. After dissociation, Ku80 occupancy at DSBs and the resulting Ku80-primed NHEJ activity were increased. Owing to Ku80-mediated DNA end protection, MRE11 and Rad51 foci formation was inhibited after the combination treatment, suggesting that this treatment suppressed HR activity. Intriguingly, the combination strategy expedited cGAS nuclear relocalization, further suppressing HR and, conversely, increasing genomic instability. Moreover, the inhibitory effect on cell survival persisted after drug withdrawal. These findings provide a rationale for the clinical application of CDK12-IN-3 in combination with Olaparib.

Asunto(s)

Inestabilidad Genómica , Neoplasias Ováricas , Ftalazinas , Piperazinas , Ftalazinas/farmacología , Ftalazinas/uso terapéutico , Piperazinas/farmacología , Piperazinas/uso terapéutico , Femenino , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/genética , Humanos , Animales , Línea Celular Tumoral , Ratones , Inestabilidad Genómica/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Quinasas Ciclina-Dependientes/metabolismo , Autoantígeno Ku/metabolismo , Roturas del ADN de Doble Cadena/efectos de los fármacos

RESUMEN

In this study, the protective effects of Panax notoginseng saponins (PNS) against gamma radiation-induced DNA damage and associated physiological alterations in Swiss albino mice were investigated. Exposure to gamma radiation led to a dose-dependent increase in cytokinesis-blocked micronuclei (CBMN) double-strand DNA breaks (DSBs), dicentric aberrations (DC), formation in peripheral blood mononuclear cells. However, pretreatment with PNS at concentrations of 1, 5, and 10 µg/mL significantly attenuated the frequencies of DC and CBMN in a concentration-dependent manner. PNS administration before radiation exposure also reduced radiation-induced DSBs in BL, indicating protection against reactive oxygen species generation and DNA damage. Notably, pretreatment with PNS at 10 µg/mL prevented the overexpression of γ-H2AX, proteins associated with DNA damage response, in irradiated mice. In addition, in vivo studies showed intraperitoneal administration of PNS (25 mg/kg body weight) for 1 h before radiation exposure mitigated lipid peroxidation levels and restored antioxidant status, countering oxidative damage induced by gamma radiation. Furthermore, PNS pretreatment reversed the decrease in hemoglobin (Hb) content, white blood cell count, and red blood cell count in irradiated mice, indicating preservation of hematological parameters. Overall, PNS demonstrated an anticlastogenic effect by modulating radiation-induced DSBs and preventing oxidative damage, thus highlighting its potential as a protective agent against radiation-induced DNA damage and associated physiological alterations. Clinically, PNS will be beneficial for cancer patients undergoing radiotherapy, but their pharmacological properties and toxicity profiles need to be studied.

Asunto(s)

Rayos gamma , Panax notoginseng , Saponinas , Animales , Rayos gamma/efectos adversos , Saponinas/farmacología , Ratones , Panax notoginseng/química , Humanos , Masculino , Daño del ADN/efectos de los fármacos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/efectos de la radiación , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/efectos de la radiación , Protectores contra Radiación/farmacología , Roturas del ADN de Doble Cadena/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de la radiación , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Monocitos/efectos de la radiación , Especies Reactivas de Oxígeno/metabolismo , Antioxidantes/farmacología

RESUMEN

In clinics, chemotherapy is often combined with surgery and radiation to increase the chances of curing cancers. In the case of glioblastoma (GBM), patients are treated with a combination of radiotherapy and TMZ over several weeks. Despite its common use, the mechanism of action of the alkylating agent TMZ has not been well understood when it comes to its cytotoxic effects in tumor cells that are mostly non-dividing. The cellular response to alkylating DNA damage is operated by an intricate protein network involving multiple DNA repair pathways and numerous checkpoint proteins that are dependent on the type of DNA lesion, the cell type, and the cellular proliferation state. Among the various alkylating damages, researchers have placed a special on O6-methylguanine (O6-mG). Indeed, this lesion is efficiently removed via direct reversal by O6-methylguanine-DNA methyltransferase (MGMT). As the level of MGMT expression was found to be directly correlated with TMZ efficiency, O6-mG was identified as the critical lesion for TMZ mode of action. Initially, the mode of action of TMZ was proposed as follows: when left on the genome, O6-mG lesions form O6-mG: T mispairs during replication as T is preferentially mis-inserted across O6-mG. These O6-mG: T mispairs are recognized and tentatively repaired by a post-replicative mismatched DNA correction system (i.e., the MMR system). There are two models (futile cycle and direct signaling models) to account for the cytotoxic effects of the O6-mG lesions, both depending upon the functional MMR system in replicating cells. Alternatively, to explain the cytotoxic effects of alkylating agents in non-replicating cells, we have proposed a "repair accident model" whose molecular mechanism is dependent upon crosstalk between the MMR and the base excision repair (BER) systems. The accidental encounter between these two repair systems will cause the formation of cytotoxic DNA double-strand breaks (DSBs). In this review, we summarize these non-exclusive models to explain the cytotoxic effects of alkylating agents and discuss potential strategies to improve the clinical use of alkylating agents.

Asunto(s)

Roturas del ADN de Doble Cadena , Reparación del ADN , Humanos , Reparación del ADN/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de los fármacos , Alquilación , Temozolomida/farmacología , ADN/metabolismo , Antineoplásicos Alquilantes/farmacología , Animales , Glioblastoma/metabolismo , Glioblastoma/patología , Glioblastoma/genética , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , O(6)-Metilguanina-ADN Metiltransferasa/genética

RESUMEN

Rhabdomyosarcoma (RMS) represents one of the most lethal soft-tissue sarcomas in children. The toxic trace element arsenic has been reported to function as a radiosensitizer in sarcomas. To investigate the role of arsenic sulfide (As4S4) in enhancing radiation sensitization in RMS, this study was conducted to elucidate its underlying mechanism in radiotherapy. The combination of As4S4 and radiotherapy showed significant inhibition in RMS cells, as demonstrated by the cell counting kit-8 (CCK-8) assay and flow cytometry. Subsequently, we demonstrated for the first time that As4S4, as well as the knockdown of NFATc3 led to double-strand break (DSB) through increased expression of RAG1. In vivo experiment confirmed that co-treatment efficiently inhibited RMS growth. Furthermore, survival analysis of a clinical cohort consisting of 59 patients revealed a correlation between NFATc3 and RAG1 expression and overall survival (OS). Cox regression analysis also confirmed the independent prognostic significance of NFATc3 and RAG1.Taken together, As4S4 enhances radiosensitivity in RMS via activating NFATc3-RAG1 mediated DSB. NFATc3 and RAG1 are potential therapeutic targets. As4S4 will hopefully serve as a prospective radio-sensitizing agent for RMS.

Asunto(s)

Arsenicales , Roturas del ADN de Doble Cadena , Factores de Transcripción NFATC , Tolerancia a Radiación , Rabdomiosarcoma , Sulfuros , Humanos , Roturas del ADN de Doble Cadena/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de la radiación , Sulfuros/farmacología , Sulfuros/uso terapéutico , Rabdomiosarcoma/metabolismo , Rabdomiosarcoma/tratamiento farmacológico , Rabdomiosarcoma/radioterapia , Rabdomiosarcoma/patología , Rabdomiosarcoma/genética , Línea Celular Tumoral , Masculino , Femenino , Arsenicales/farmacología , Arsenicales/uso terapéutico , Animales , Tolerancia a Radiación/efectos de los fármacos , Factores de Transcripción NFATC/metabolismo , Ratones , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/genética , Ratones Desnudos , Niño , Fármacos Sensibilizantes a Radiaciones/farmacología , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Ratones Endogámicos BALB C

RESUMEN

The efficiency and outcome of CRISPR/Cas9 editing depends on the chromatin state at the cut site. It has been shown that changing the chromatin state can influence both the efficiency and repair outcome, and epigenetic drugs have been used to improve Cas9 editing. However, because the target proteins of these drugs are not homogeneously distributed across the genome, the efficacy of these drugs may be expected to vary from locus to locus. Here, we systematically analyzed this chromatin context-dependency for 160 epigenetic drugs. We used a human cell line with 19 stably integrated reporters to induce a double-stranded break in different chromatin environments. We then measured Cas9 editing efficiency and repair pathway usage by sequencing the mutational signatures. We identified 58 drugs that modulate Cas9 editing efficiency and/or repair outcome dependent on the local chromatin environment. For example, we find a subset of histone deacetylase inhibitors that improve Cas9 editing efficiency throughout all types of heterochromatin (e.g. PCI-24781), while others were only effective in euchromatin and H3K27me3-marked regions (e.g. apicidin). In summary, this study reveals that most epigenetic drugs alter CRISPR editing in a chromatin-dependent manner, and provides a resource to improve Cas9 editing more selectively at the desired location.

Asunto(s)

Sistemas CRISPR-Cas , Cromatina , Epigénesis Genética , Edición Génica , Inhibidores de Histona Desacetilasas , Humanos , Edición Génica/métodos , Epigénesis Genética/efectos de los fármacos , Cromatina/metabolismo , Cromatina/genética , Inhibidores de Histona Desacetilasas/farmacología , Reparación del ADN , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genética , Heterocromatina/metabolismo , Heterocromatina/genética , Línea Celular , Histonas/metabolismo , Eucromatina/genética , Roturas del ADN de Doble Cadena/efectos de los fármacos

RESUMEN

In recent years, various poly(ADP-ribose) polymerase (PARP) inhibitors (PARPis) have been approved for the treatment of several cancers to target the vulnerability of homologous recombination (HR) deficiency (e.g., due to BRCA1/2 dysfunction). In this review we analyze the ongoing debates and recent breakthroughs in the use of PARPis for BRCA1/2-deficient cancers, juxtaposing the 'double-strand break (DSB)' and 'single-stranded DNA (ssDNA) gap' models of synthetic lethality induced by PARPis. We spotlight the complexity of this interaction, highlighting emerging research on the role of DNA polymerase theta (POLθ) and ssDNA gaps in shaping therapy responses. We scrutinize the clinical ramifications of these findings, especially concerning PARPi efficacy and resistance mechanisms, underscoring the heterogeneity of BRCA-mutated tumors and the urgent need for advanced research to bridge the gap between laboratory models and patient outcomes.

Asunto(s)

Proteína BRCA1 , Proteína BRCA2 , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Humanos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Proteína BRCA2/genética , Proteína BRCA1/genética , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , ADN Polimerasa theta , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/genética , ADN de Cadena Simple , Reparación del ADN/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Animales , Recombinación Homóloga/efectos de los fármacos , Mutaciones Letales Sintéticas

RESUMEN

BACKGROUND/AIM: Despite the established antitumor effectiveness and synergistic interactions of melatonin with photon irradiation, its role in carbon-ion radiotherapy remains uncertain. This study aimed to elucidate the mechanisms and potential clinical advantages of combining exogenous melatonin therapy with carbon-ion radiotherapy. MATERIALS AND METHODS: The investigation assessed the impact of combining exogenous melatonin with photon or carbon-ion irradiation on cell-cycle modulation and DNA-repair capability using the melanoma cell line B16F10. RNA sequencing and bioinformatics analysis were conducted to explore mechanisms and evaluate potential clinical benefits, with validation performed on the osteosarcoma cell line LM8. RESULTS: Pre-treatment with melatonin reduced the survival fraction of B16F10 and LM8 cells upon exposure to photon and carbon-ion radiation. Mechanistically, melatonin was found to inhibit G2/M arrest, preserve DNA damage, and suppress key genes involved in DNA double-strand break repair after 8 Gy carbon-ion radiation. Furthermore, RNA sequencing and bioinformatics analysis revealed favorable changes in genes associated with survival and metastasis, highlighting potential clinical significance. LM8 cells treated with melatonin exhibited increased radiosensitivity and suppression of DNA-repair proteins. CONCLUSION: The combination of exogenous melatonin not only heightened radiosensitivity and modulated hallmark tumor gene sets in vitro but also markedly suppressed the efficiency of DNA double-strand break-repair pathway, thus enhancing the cytotoxicity of carbon-ion radiotherapy.

Asunto(s)

Reparación del ADN , Radioterapia de Iones Pesados , Melatonina , Tolerancia a Radiación , Fármacos Sensibilizantes a Radiaciones , Melatonina/farmacología , Línea Celular Tumoral , Tolerancia a Radiación/efectos de los fármacos , Ratones , Animales , Humanos , Reparación del ADN/efectos de los fármacos , Reparación del ADN/efectos de la radiación , Fármacos Sensibilizantes a Radiaciones/farmacología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Melanoma Experimental/radioterapia , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/patología , Roturas del ADN de Doble Cadena/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de la radiación

RESUMEN

Hexavalent chromium [Cr(VI)] is an established human lung carcinogen, but the carcinogenesis mechanism is poorly understood. Chromosome instability, a hallmark of lung cancer, is considered a major driver of Cr(VI)-induced lung cancer. Unrepaired DNA double-strand breaks are the underlying cause, and homologous recombination repair is the primary mechanism preventing Cr(VI)-induced DNA breaks from causing chromosome instability. Cell culture studies show acute Cr(VI) exposure causes DNA double-strand breaks and increases homologous recombination repair activity. However, the ability of Cr(VI)-induced DNA breaks and repair impact has only been reported in cell culture studies. Therefore, we investigated whether acute Cr(VI) exposure could induce breaks and homologous recombination repair in rat lungs. Male and female Wistar rats were acutely exposed to either zinc chromate particles in a saline solution or saline alone by oropharyngeal aspiration. This exposure route resulted in increased Cr levels in each lobe of the lung. We found Cr(VI) induced DNA double-strand breaks in a concentration-dependent manner, with females being more susceptible than males, and induced homologous recombination repair at similar levels in both sexes. Thus, these data show this driving mechanism discovered in cell culture indeed translates to lung tissue in vivo.

Asunto(s)

Cromatos , Cromo , Roturas del ADN de Doble Cadena , Pulmón , Ratas Wistar , Reparación del ADN por Recombinación , Animales , Femenino , Roturas del ADN de Doble Cadena/efectos de los fármacos , Masculino , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Cromo/toxicidad , Reparación del ADN por Recombinación/efectos de los fármacos , Ratas , Cromatos/toxicidad , Compuestos de Zinc/toxicidad

RESUMEN

BACKGROUND: Poly (ADP-ribose) polymerase inhibitors (PARPis) can effectively treat ovarian cancer patients with defective homologous recombination (HR). Loss or dysfunction of PTEN, a typical tumour suppressor, impairs double-strand break (DSB) repair. Hence, we explored the possibility of inhibiting PTEN to induce HR deficiency (HRD) for PARPi application. METHODS: Functional studies using PTEN inhibitor VO-OHpic and PARPi olaparib were performed to explore the molecular mechanisms in vitro and in vivo. RESULTS: In this study, the combination of VO-OHpic with olaparib exhibited synergistic inhibitory effects on ovarian cancer cells was demonstrated. Furthermore, VO-OHpic was shown to enhance DSBs by reducing nuclear expression of PTEN and inhibiting HR repair through the modulation of MRE11-RAD50-NBN (MRN) complex, critical for DSB repair. TCGA and GTEx analysis revealed a strong correlation between PTEN and MRN in ovarian cancer. Mechanistic studies indicated that VO-OHpic reduced expression of MRN, likely by decreasing PTEN/E2F1-mediated transcription. Moreover, PTEN-knockdown inhibited expression of MRN, increased sensitivities to olaparib, and induced DSBs. In vivo experiments showed that the combination of VO-OHpic with olaparib exhibited enhanced inhibitory effects on tumour growth. CONCLUSIONS: Collectively, this study highlights the potential of PTEN inhibitors in combination therapy with PARPis to create HRD for HRD-negative ovarian cancers.

Asunto(s)

Ácido Anhídrido Hidrolasas , Proteína Homóloga de MRE11 , Neoplasias Ováricas , Fosfohidrolasa PTEN , Ftalazinas , Piperazinas , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Femenino , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Humanos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Ftalazinas/farmacología , Animales , Ratones , Línea Celular Tumoral , Piperazinas/farmacología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Sinergismo Farmacológico , Roturas del ADN de Doble Cadena/efectos de los fármacos , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/antagonistas & inhibidores

RESUMEN

BACKGROUND: Due to its high incidence and elevated mortality, hepatocellular carcinoma (HCC) has emerged as a formidable global healthcare challenge. The intricate interplay between gender-specific disparities in both incidence and clinical outcomes has prompted a progressive recognition of the substantial influence exerted by estrogen and its corresponding receptors (ERs) upon HCC pathogenesis. Estrogen replacement therapy (ERT) emerged for the treatment of HCC by administering exogenous estrogen. However, the powerful side effects of estrogen, including the promotion of breast cancer and infertility, hinder the further application of ERT. Identifying effective therapeutic targets for estrogen and screening bioactive ingredients without E2-like side effects is of great significance for optimizing HCC ERT. METHODS: In this study, we employed an integrative approach, harnessing data from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, clinical paraffin sections, adenoviral constructs as well as in vivo studies, to unveil the association between estrogen, estrogen receptor α (ESR1) and HCC. Leveraging methodologies encompassing molecular dynamics simulation and cellular thermal shift assay (CETSA) were used to confirm whether ESR1 is a molecular target of DHT. Multiple in vitro and in vivo experiments were used to identify whether i) ESR1 is a crucial gene that promotes DNA double-strand breaks (DSBs) and proliferation inhibition in HCC, ii) Dihydrotanshinone I (DHT), a quinonoid monomeric constituent derived from Salvia miltiorrhiza (Dan shen) exerts anti-HCC effects by regulating ESR1 and subsequent DSBs, iii) DHT has the potential to replace E2. RESULTS: DHT could target ESR1 and upregulate its expression in a concentration-dependent manner. This, in turn, leads to the downregulation of breast cancer type 1 susceptibility protein (BRCA1), a pivotal protein involved in the homologous recombination repair (HRR) process. The consequence of this downregulation is manifested through the induction of DSBs in HCC, subsequently precipitating a cascade of downstream events, including apoptosis and cell cycle arrest. Of particular significance is the comparative assessment of DHT and isodose estradiol treatments, which underscores DHT's excellent HCC-suppressive efficacy without concomitant perturbation of endogenous sex hormone homeostasis. CONCLUSION: Our findings not only confirm ESR1 as a therapeutic target in HCC management but also underscores DHT's role in upregulating ESR1 expression, thereby impeding the proliferation and invasive tendencies of HCC. In addition, we preliminarily identified DHT has the potential to emerge as an agent in optimizing HCC ERT through the substitution of E2.

Asunto(s)

Carcinoma Hepatocelular , Proliferación Celular , Roturas del ADN de Doble Cadena , Receptor alfa de Estrógeno , Neoplasias Hepáticas , Fenantrenos , Carcinoma Hepatocelular/tratamiento farmacológico , Receptor alfa de Estrógeno/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Humanos , Proliferación Celular/efectos de los fármacos , Fenantrenos/farmacología , Animales , Roturas del ADN de Doble Cadena/efectos de los fármacos , Línea Celular Tumoral , Ratones Desnudos , Masculino , Apoptosis/efectos de los fármacos , Ratones , Células Hep G2 , Furanos , Quinonas

RESUMEN

Determining the balance between DNA double strand break repair (DSBR) pathways is essential for understanding treatment response in cancer. We report a method for simultaneously measuring non-homologous end joining (NHEJ), homologous recombination (HR), and microhomology-mediated end joining (MMEJ). Using this method, we show that patient-derived glioblastoma (GBM) samples with acquired temozolomide (TMZ) resistance display elevated HR and MMEJ activity, suggesting that these pathways contribute to treatment resistance. We screen clinically relevant small molecules for DSBR inhibition with the aim of identifying improved GBM combination therapy regimens. We identify the ATM kinase inhibitor, AZD1390, as a potent dual HR/MMEJ inhibitor that suppresses radiation-induced phosphorylation of DSBR proteins, blocks DSB end resection, and enhances the cytotoxic effects of TMZ in treatment-naïve and treatment-resistant GBMs with TP53 mutation. We further show that a combination of G2/M checkpoint deficiency and reliance upon ATM-dependent DSBR renders TP53 mutant GBMs hypersensitive to TMZ/AZD1390 and radiation/AZD1390 combinations. This report identifies ATM-dependent HR and MMEJ as targetable resistance mechanisms in TP53-mutant GBM and establishes an approach for simultaneously measuring multiple DSBR pathways in treatment selection and oncology research.

Asunto(s)

Proteínas de la Ataxia Telangiectasia Mutada , Roturas del ADN de Doble Cadena , Glioblastoma , Temozolomida , Proteína p53 Supresora de Tumor , Humanos , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/genética , Glioblastoma/genética , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Glioblastoma/patología , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Roturas del ADN de Doble Cadena/efectos de los fármacos , Temozolomida/farmacología , Línea Celular Tumoral , Mutación , Resistencia a Antineoplásicos/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/metabolismo , Animales , Reparación del ADN por Unión de Extremidades/efectos de los fármacos , Ratones , Fosforilación/efectos de los fármacos

RESUMEN

Fascaplysin is a red cytotoxic pigment with anticancer properties isolated from the marine sponge Fascaplysinopsis sp. Recently, structure-activity relationship analysis reported by our group suggested that selective cytotoxicity of fascaplysin derivatives towards tumor cells negatively correlates with their ability to intercalate into DNA. To validate this hypothesis, we synthesized 6- and 7-tert-butylfascaplysins which reveal mitigated DNA-intercalating properties. These derivatives were found to be strongly cytotoxic to drug-resistant human prostate cancer cells, albeit did not demonstrate improved selectivity towards cancer cells when compared to fascaplysin. At the same time, kinome analysis suggested an activation of CHK1/ATR axis in cancer cells shortly after the drug exposure. Further experiments revealed induction of replication stress that is eventually converted to the toxic DNA double-strand breaks, resulting in caspase-independent apoptosis-like cell death. Our observations highlight new DNA-targeting effect of some fascaplysin derivatives and indicate more complex structure-activity relationships within the fascaplysin family, suggesting that cytotoxicity and selectivity of these alkaloids are influenced by multiple factors. Furthermore, combination with clinically-approved inhibitors of ATR/CHK1 as well as testing in tumors particularly sensitive to the DNA damage should be considered in further studies.

Asunto(s)

Antineoplásicos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Humanos , Línea Celular Tumoral , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Indoles/farmacología , Indoles/química , Apoptosis/efectos de los fármacos , Relación Estructura-Actividad , Masculino , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , ADN/metabolismo , Animales , Roturas del ADN de Doble Cadena/efectos de los fármacos , Compuestos de Amonio Cuaternario , Carbolinas , Indolizinas

RESUMEN

The molecular nanomachine, human DNA topoisomerase IIα, plays a crucial role in replication, transcription, and recombination by catalyzing topological changes in the DNA, rendering it an optimal target for cancer chemotherapy. Current clinical topoisomerase II poisons often cause secondary tumors as side effects due to the accumulation of double-strand breaks in the DNA, spurring the development of catalytic inhibitors. Here, we used a dynamic pharmacophore approach to develop catalytic inhibitors targeting the ATP binding site of human DNA topoisomerase IIα. Our screening of a library of nature-inspired compounds led to the discovery of a class of 3-(imidazol-2-yl) morpholines as potent catalytic inhibitors that bind to the ATPase domain. Further experimental and computational studies identified hit compound 17, which exhibited selectivity against the human DNA topoisomerase IIα versus human protein kinases, cytotoxicity against several human cancer cells, and did not induce DNA double-strand breaks, making it distinct from clinical topoisomerase II poisons. This study integrates an innovative natural product-inspired chemistry and successful implementation of a molecular design strategy that incorporates a dynamic component of ligand-target molecular recognition, with comprehensive experimental characterization leading to hit compounds with potential impact on the development of more efficient chemotherapies.

Asunto(s)

ADN-Topoisomerasas de Tipo II , Inhibidores de Topoisomerasa II , Humanos , ADN-Topoisomerasas de Tipo II/metabolismo , Inhibidores de Topoisomerasa II/farmacología , Inhibidores de Topoisomerasa II/química , Línea Celular Tumoral , Descubrimiento de Drogas/métodos , Antineoplásicos/farmacología , Antineoplásicos/química , Simulación del Acoplamiento Molecular , Relación Estructura-Actividad , Imidazoles/farmacología , Imidazoles/química , Roturas del ADN de Doble Cadena/efectos de los fármacos , Antígenos de Neoplasias/metabolismo

RESUMEN

DNA damage plays a significant role in the tumorigenesis and progression of the disease. Abnormal DNA repair affects the therapy and prognosis of cancer. In this study, it is demonstrated that the deubiquitinase USP25 promotes non-homologous end joining (NHEJ), which in turn contributes to chemoresistance in cancer. It is shown that USP25 deubiquitinates SHLD2 at the K64 site, which enhances its binding with REV7 and promotes NHEJ. Furthermore, USP25 deficiency impairs NHEJ-mediated DNA repair and reduces class switch recombination (CSR) in USP25-deficient mice. USP25 is overexpressed in a subset of colon cancers. Depletion of USP25 sensitizes colon cancer cells to IR, 5-Fu, and cisplatin. TRIM25 is also identified, an E3 ligase, as the enzyme responsible for degrading USP25. Downregulation of TRIM25 leads to an increase in USP25 levels, which in turn induces chemoresistance in colon cancer cells. Finally, a peptide that disrupts the USP25-SHLD2 interaction is successfully identified, impairing NHEJ and increasing sensitivity to chemotherapy in PDX model. Overall, these findings reveal USP25 as a critical effector of SHLD2 in regulating the NHEJ repair pathway and suggest its potential as a therapeutic target for cancer therapy.

Asunto(s)

Roturas del ADN de Doble Cadena , Ubiquitina Tiolesterasa , Animales , Ratones , Roturas del ADN de Doble Cadena/efectos de los fármacos , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismo , Humanos , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Modelos Animales de Enfermedad , Reparación del ADN/genética , Reparación del ADN por Unión de Extremidades/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Motivos Tripartitos/genética , Proteínas de Motivos Tripartitos/metabolismo , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Neoplasias del Colon/tratamiento farmacológico , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo

RESUMEN

The emergence of chemoresistance poses a significant challenge to the efficacy of DNA-damaging agents in cancer treatment, in part due to the inherent DNA repair capabilities of cancer cells. The Ku70/80 protein complex (Ku) plays a central role in double-strand breaks (DSBs) repair through the classical non-homologous end joining (c-NHEJ) pathway, and has proven to be one of the most promising drug target for cancer treatment when combined with radiotherapy or chemotherapy. In this study, we conducted a high-throughput screening of small-molecule inhibitors targeting the Ku complex by using a fluorescence polarization-based DNA binding assay. From a library of 11,745 small molecules, UMI-77 was identified as a potent Ku inhibitor, with an IC50 value of 2.3 µM. Surface plasmon resonance and molecular docking analyses revealed that UMI-77 directly bound the inner side of Ku ring, thereby disrupting Ku binding with DNA. In addition, UMI-77 also displayed potent inhibition against MUS81-EME1, a key player in homologous recombination (HR), demonstrating its potential for blocking both NHEJ- and HR-mediated DSB repair pathways. Further cell-based studies showed that UMI-77 could impair bleomycin-induced DNA damage repair, and significantly sensitized multiple cancer cell lines to the DNA-damaging agents. Finally, in a mouse xenograft tumor model, UMI-77 significantly enhanced the chemotherapeutic efficacy of etoposide with little adverse physiological effects. Our work offers a new avenue to combat chemoresistance in cancer treatment, and suggests that UMI-77 could be further developed as a promising candidate in cancer treatment.

Asunto(s)

Antineoplásicos , Autoantígeno Ku , Humanos , Autoantígeno Ku/metabolismo , Animales , Línea Celular Tumoral , Ratones , Antineoplásicos/farmacología , Antineoplásicos/química , Daño del ADN/efectos de los fármacos , Simulación del Acoplamiento Molecular , Ensayos Antitumor por Modelo de Xenoinjerto , Reparación del ADN por Unión de Extremidades/efectos de los fármacos , Etopósido/farmacología , Descubrimiento de Drogas , Roturas del ADN de Doble Cadena/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos

RESUMEN

Tert-butyl hydroperoxide (t-BuOOH) is an organic hydroperoxide widely used as a model compound to induce oxidative stress. It leads to a plethora of cellular damage, including lipid peroxidation, DNA double-strand breaks (DNA DSBs), and breakdown of the mitochondrial membrane potential (MMP). We could show in several cell lines that t-BuOOH induces ferroptosis, triggered by iron-dependent lipid peroxidation. We have further revealed that not only t-BuOOH-mediated ferroptosis, but also DNA DSBs and loss of MMP are prevented by cell-cell contacts. The underlying mechanisms are not known. Here, we show in murine fibroblasts and a human colon carcinoma cell line that t-BuOOH (50 or 100 µM, resp.) causes an increase in intracellular Ca2+, and that this increase is key to lipid peroxidation and ferroptosis, DNA DSB formation and dissipation of the MMP. We further demonstrate that cell-cell contacts prevent t-BuOOH-mediated raise in intracellular Ca2+. Hence, we provide novel insights into the mechanism of t-BuOOH-triggered cellular damage including ferroptosis and propose a model in which cell-cell contacts control intracellular Ca2+ levels to prevent lipid peroxidation, DNA DSB-formation and loss of MMP. Since Ca2+ is a central player of toxicity in response to oxidative stress and is involved in various cell death pathways, our observations suggest a broad protective function of cell-cell contacts against a variety of exogenous toxicants.

Asunto(s)

Calcio , Roturas del ADN de Doble Cadena , Ferroptosis , Peroxidación de Lípido , Potencial de la Membrana Mitocondrial , terc-Butilhidroperóxido , Ferroptosis/efectos de los fármacos , Calcio/metabolismo , Humanos , terc-Butilhidroperóxido/toxicidad , Animales , Peroxidación de Lípido/efectos de los fármacos , Ratones , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Comunicación Celular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Línea Celular Tumoral

RESUMEN

Acute myeloid leukemia (AML) is a hematological malignancy that is characterized by an expansion of immature myeloid precursors. Despite therapeutic advances, the prognosis of AML patients remains poor and there is a need for the evaluation of promising therapeutic candidates to treat the disease. The objective of this study was to evaluate the efficacy of duocarmycin Stable A (DSA) in AML cells in vitro. We hypothesized that DSA would induce DNA damage in the form of DNA double-strand breaks (DSBs) and exert cytotoxic effects on AML cells within the picomolar range. Human AML cell lines Molm-14 and HL-60 were used to perform 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), DNA DSBs, cell cycle, 5-ethynyl-2-deoxyuridine (EdU), colony formation unit (CFU), Annexin V, RNA sequencing and other assays described in this study. Our results showed that DSA induced DNA DSBs, induced cell cycle arrest at the G2M phase, reduced proliferation and increased apoptosis in AML cells. Additionally, RNA sequencing results showed that DSA regulates genes that are associated with cellular processes such as DNA repair, G2M checkpoint and apoptosis. These results suggest that DSA is efficacious in AML cells and is therefore a promising potential therapeutic candidate that can be further evaluated for the treatment of AML.

Asunto(s)

Apoptosis , Proliferación Celular , Duocarmicinas , Leucemia Mieloide Aguda , Humanos , Apoptosis/efectos de los fármacos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/metabolismo , Proliferación Celular/efectos de los fármacos , Duocarmicinas/farmacología , Línea Celular Tumoral , Roturas del ADN de Doble Cadena/efectos de los fármacos , Células HL-60 , Antineoplásicos/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos

RESUMEN

BACKGROUND: Bleomycin, a potent antitumor agent, is limited in clinical use due to the potential for fatal pulmonary toxicity. The accelerated DNA damage and senescence in alveolar epithelial cells (AECs) is considered a key factor in the development of lung pathology. Understanding the mechanisms for bleomycin-induced lung injury is crucial for mitigating its adverse effects. METHODS: Human lung epithelial (A549) cells were exposed to bleomycin and subsequently assessed for cellular senescence, DNA damage, and double-strand break (DSB) repair. The impact of Rad51 overexpression on DSB repair and senescence in AECs was evaluated in vitro. Additionally, bleomycin was intratracheally administered in C57BL/6 mice to establish a pulmonary fibrosis model. RESULTS: Bleomycin exposure induced dose- and time-dependent accumulation of senescence hallmarks and DNA lesions in AECs. These effects are probably due to the inhibition of Rad51 expression, consequently suppressing homologous recombination (HR) repair. Mechanistic studies revealed that bleomycin-mediated transcriptional inhibition of Rad51 might primarily result from E2F1 depletion. Furthermore, the genetic supplement of Rad51 substantially mitigated bleomycin-mediated effects on DSB repair and senescence in AECs. Notably, decreased Rad51 expression was also observed in the bleomycin-induced mouse pulmonary fibrosis model. CONCLUSIONS: Our works suggest that the inhibition of Rad51 plays a pivotal role in bleomycin-induced AECs senescence and lung injury, offering potential strategies to alleviate the pulmonary toxicity of bleomycin.

Asunto(s)

Bleomicina , Senescencia Celular , Reparación del ADN , Recombinasa Rad51 , Bleomicina/efectos adversos , Recombinasa Rad51/metabolismo , Recombinasa Rad51/genética , Animales , Senescencia Celular/efectos de los fármacos , Senescencia Celular/genética , Humanos , Ratones , Reparación del ADN/efectos de los fármacos , Ratones Endogámicos C57BL , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Células A549 , Daño del ADN/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de los fármacos , Factor de Transcripción E2F1/metabolismo , Factor de Transcripción E2F1/genética , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/efectos de los fármacos

RESUMEN

The clinical use of the DNA damaging anticancer drug doxorubicin (DOX) is limited by irreversible cardiotoxicity, which depends on the cumulative dose. The RAS-homologous (RHO) small GTPase RAC1 contributes to DOX-induced DNA damage formation and cardiotoxicity. However, the pathophysiological relevance of other RHO GTPases than RAC1 and different cardiac cell types (i.e., cardiomyocytes, non-cardiomyocytes) for DOX-triggered cardiac damage is unclear. Employing diverse in vitro and in vivo models, we comparatively investigated the level of DOX-induced DNA damage in cardiomyocytes versus non-cardiomyocytes (endothelial cells and fibroblasts), in the presence or absence of selected RHO GTPase inhibitors. Non-cardiomyocytes exhibited the highest number of DOX-induced DNA double-strand breaks (DSB), which were efficiently repaired in vitro. By contrast, rather low levels of DSB were formed in cardiomyocytes, which however remained largely unrepaired. Moreover, DOX-induced apoptosis was detected only in non-cardiomyocytes but not in cardiomyocytes. Pharmacological inhibitors of RAC1 and CDC42 most efficiently attenuated DOX-induced DNA damage in all cell types examined in vitro. Consistently, immunohistochemical analyses revealed that the RAC1 inhibitor NSC23766 and the pan-RHO GTPase inhibitor lovastatin reduced the level of DOX-induced residual DNA damage in both cardiomyocytes and non-cardiomyocytes in vivo. Overall, we conclude that endothelial cells, fibroblasts and cardiomyocytes contribute to the pathophysiology of DOX-induced cardiotoxicity, with RAC1- and CDC42-regulated signaling pathways being especially relevant for DOX-stimulated DSB formation and DNA damage response (DDR) activation. Hence, we suggest dual targeting of RAC1/CDC42-dependent mechanisms in multiple cardiac cell types to mitigate DNA damage-dependent cardiac injury evoked by DOX-based anticancer therapy.

Asunto(s)

Aminoquinolinas , Doxorrubicina , Células Endoteliales , Fibroblastos , Miocitos Cardíacos , Pirimidinas , Proteína de Unión al GTP cdc42 , Proteína de Unión al GTP rac1 , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rac1/antagonistas & inhibidores , Proteína de Unión al GTP rac1/genética , Animales , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Miocitos Cardíacos/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Proteína de Unión al GTP cdc42/metabolismo , Doxorrubicina/toxicidad , Doxorrubicina/efectos adversos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Células Endoteliales/metabolismo , Cardiotoxicidad , Antibióticos Antineoplásicos/toxicidad , Ratones , Apoptosis/efectos de los fármacos , Masculino , Humanos , Ratones Endogámicos C57BL , Roturas del ADN de Doble Cadena/efectos de los fármacos , Neuropéptidos/metabolismo , Daño del ADN/efectos de los fármacos , Células Cultivadas

RESUMEN

Homologous recombination (HR)-related gene alterations are present in a significant subset of prostate, breast, ovarian, pancreatic, lung, and colon cancers rendering these tumors as potential responders to specific DNA damaging agents. A small molecule acylfulvene prodrug, LP-184, metabolizes to an active compound by the oxidoreductase activity of enzyme prostaglandin reductase 1 (PTGR1), which is frequently elevated in multiple solid tumor types. Prior work demonstrated that cancer cell lines deficient in a spectrum of DNA damage repair (DDR) pathway genes show increased susceptibility to LP-184. Here, we investigated the potential of LP-184 in targeting multiple tumors with impaired HR function and its mechanism of action as a DNA damaging agent. LP-184 induced elevated DNA double-strand breaks in HR deficient (HRD) cancer cells. Depletion of key HR components BRCA2 or ataxia telangiectasia mutated (ATM) in cancer cells conferred up to 12-fold increased sensitivity to the LP-184. LP-184 showed nanomolar potency in a diverse range of HRD cancer models, including prostate cancer organoids, leiomyosarcoma cell lines, and patient-derived tumor graft models of lung, pancreatic, and prostate cancers. LP-184 demonstrated complete, durable tumor regression in 10 patient-derived xenograft (PDX) models of HRD triple-negative breast cancer (TNBC) including those resistant to PARP inhibitors (PARPi). LP-184 further displayed strong synergy with PARPi in ovarian and prostate cancer cell lines as well as in TNBC PDX models. These preclinical findings illustrate the potential of LP-184 as a pan-HRD cancer therapeutic. Taken together, our results support continued clinical evaluation of LP-184 in a large subset of HRD solid tumors. SIGNIFICANCE: New agents with activity against DDR-deficient solid tumors refractory to standard-of-care therapies are needed. We report multiple findings supporting the potential for LP-184, a novel alkylating agent with three FDA orphan drug designations, to fill this void clinically: strong nanomolar potency; sustained, durable regression of solid tumor xenografts; synthetic lethality with HR defects. LP-184 adult phase IA trial to assess safety in advanced solid tumors is ongoing.

Asunto(s)

Antineoplásicos , Recombinación Homóloga , Ensayos Antitumor por Modelo de Xenoinjerto , Humanos , Animales , Ratones , Línea Celular Tumoral , Recombinación Homóloga/efectos de los fármacos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Femenino , Roturas del ADN de Doble Cadena/efectos de los fármacos , Masculino , Reparación del ADN/efectos de los fármacos