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1.
Mol Oncol ; 18(8): 1958-1965, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-38520049

RESUMEN

Mammalian cells replicate ~ 3 × 109 base pairs per cell cycle. One of the key molecules that slows down the cell cycle and prevents excessive DNA damage upon DNA replication stress is the checkpoint kinase ataxia-telangiectasia-and-RAD3-related (ATR). Proteolysis-targeting-chimeras (PROTACs) are an innovative pharmacological invention to molecularly dissect, biologically understand, and therapeutically assess catalytic and non-catalytic functions of enzymes. This work defines the first-in-class ATR PROTAC, Abd110/Ramotac-1. It is derived from the ATR inhibitor VE-821 and recruits the E3 ubiquitin-ligase component cereblon to ATR. Abd110 eliminates ATR rapidly in human leukemic cells. This mechanism provokes DNA replication catastrophe and augments anti-leukemic effects of the clinically used ribonucleotide reductase-2 inhibitor hydroxyurea. Moreover, Abd110 is more effective than VE-821 against human primary leukemic cells but spares normal primary immune cells. CRISPR-Cas9 screens show that ATR is a dependency factor in 116 myeloid and lymphoid leukemia cells. Treatment of wild-type but not of cereblon knockout cells with Abd110 stalls their proliferation which verifies that ATR elimination is the primary mechanism of Abd110. Altogether, our findings demonstrate specific anti-leukemic effects of an ATR PROTAC.


Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada , Proliferación Celular , Replicación del ADN , Leucemia , Humanos , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Replicación del ADN/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Leucemia/patología , Leucemia/tratamiento farmacológico , Leucemia/metabolismo , Leucemia/genética , Línea Celular Tumoral , Proteolisis/efectos de los fármacos , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Hidroxiurea/farmacología , Hidroxiurea/análogos & derivados , Daño del ADN/efectos de los fármacos
2.
Eur J Med Chem ; 267: 116167, 2024 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-38308949

RESUMEN

The Ataxia telangiectasia and RAD3-related (ATR) kinase is a key regulator of DNA replication stress responses and DNA-damage checkpoints. Several potent and selective ATR inhibitors are reported and four of them are currently in clinical trials in combination with radio- or chemotherapy. Based on the idea of degrading target proteins rather than inhibiting them, we designed, synthesized and biologically characterized a library of ATR-targeted proteolysis targeting chimera (PROTACs). Among the synthesized compounds, the lenalidomide-based PROTAC 42i was the most promising. In pancreatic and cervix cancer cells cancer cells, it reduced ATR to 40 % of the levels in untreated cells. 42i selectively degraded ATR through the proteasome, dependent on the E3 ubiquitin ligase component cereblon, and without affecting the associated kinases ATM and DNA-PKcs. 42i may be a promising candidate for further optimization and biological characterization in various cancer cells.


Asunto(s)
Ataxia Telangiectasia , Femenino , Humanos , Quimera Dirigida a la Proteólisis , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteolisis , Daño del ADN
3.
J Adv Res ; 2023 Jul 17.
Artículo en Inglés | MEDLINE | ID: mdl-37467961

RESUMEN

INTRODUCTION: Posttranslational modification of proteins by reversible acetylation regulates key biological processes. Histone deacetylases (HDACs) catalyze protein deacetylation and are frequently dysregulated in tumors. This has spurred the development of HDAC inhibitors (HDACi). Such epigenetic drugs modulate protein acetylation, eliminate tumor cells, and are approved for the treatment of blood cancers. OBJECTIVES: We aimed to identify novel, nanomolar HDACi with increased potency over existing agents and selectivity for the cancer-relevant class I HDACs (HDAC1,-2,-3,-8). Moreover, we wanted to define how such drugs control the apoptosis-autophagy interplay. As test systems, we used human leukemic cells and embryonic kidney-derived cells. METHODS: We synthesized novel pyrimidine-hydroxamic acid HDACi (KH9/KH16/KH29) and performed in vitro activity assays and molecular modeling of their direct binding to HDACs. We analyzed how these HDACi affect leukemic cell fate, acetylation, and protein expression with flow cytometry and immunoblot. The publicly available DepMap database of CRISPR-Cas9 screenings was used to determine sensitivity factors across human leukemic cells. RESULTS: Novel HDACi show nanomolar activity against class I HDACs. These agents are superior to the clinically used hydroxamic acid HDACi SAHA (vorinostat). Within the KH-series of compounds, KH16 (yanostat) is the most effective inhibitor of HDAC3 (IC50 = 6 nM) and the most potent inducer of apoptosis (IC50 = 110 nM; p < 0.0001) in leukemic cells. KH16 though spares embryonic kidney-derived cells. Global data analyses of knockout screenings verify that HDAC3 is a dependency factor in 115 human blood cancer cells of different lineages, independent of mutations in the tumor suppressor p53. KH16 alters pro- and anti-apoptotic protein expression, stalls cell cycle progression, and induces caspase-dependent processing of the autophagy proteins ULK1 and p62. CONCLUSION: These data reveal that HDACs are required to stabilize autophagy proteins through suppression of apoptosis in leukemic cells. HDAC3 appears as a valid anti-cancer target for pharmacological intervention.

4.
Cancers (Basel) ; 15(14)2023 Jul 17.
Artículo en Inglés | MEDLINE | ID: mdl-37509312

RESUMEN

Epigenetic modifiers of the histone deacetylase (HDAC) family are often dysregulated in cancer cells. Experiments with small molecule HDAC inhibitors (HDACi) have proven that HDACs are a vulnerability of transformed cells. We evaluated a novel hydroxamic acid-based HDACi (KH16; termed yanostat) in human pancreatic ductal adenocarcinoma (PDAC) cells, short- and long-term cultured colorectal cancer (CRC) cells, and retinal pigment epithelial cells. We show that KH16 induces cell cycle arrest and apoptosis, both time and dose dependently in PDAC and CRC cells. This is associated with altered expression of BCL2 family members controlling intrinsic apoptosis. Recent data illustrate that PDAC cells frequently have an altered expression of the pro-apoptotic BH3-only protein NOXA and that HDACi induce an accumulation of NOXA. Using PDAC cells with a deletion of NOXA by CRISPR-Cas9, we found that a lack of NOXA delayed apoptosis induction by KH16. These results suggest that KH16 is a new chemotype of hydroxamic acid HDACi with superior activity against solid tumor-derived cells. Thus, KH16 is a scaffold for future research on compounds with nanomolar activity against HDACs.

5.
Methods Mol Biol ; 2589: 337-344, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36255635

RESUMEN

Reactive oxygen species (ROS) are induced by several chemotherapeutics. In this protocol, we describe a flow cytometry-based method for the analysis of the intracellular levels of ROS in vital leukemic cells in response to the histone deacetylase inhibitor vorinostat. This measurement of ROS using the cell-permeable dye CM-H2DCFDA indicates intracellular oxidative stress.


Asunto(s)
Inhibidores de Histona Desacetilasas , Estrés Oxidativo , Inhibidores de Histona Desacetilasas/farmacología , Especies Reactivas de Oxígeno , Vorinostat/farmacología , Línea Celular Tumoral , Apoptosis , Ácidos Hidroxámicos/farmacología
6.
Nutr Cancer ; 74(6): 2152-2173, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-34590505

RESUMEN

Most current larynx cancer therapies are generally aimed at the global mass of tumor, targeting the non-tumorigenic cells, and unfortunately sparing the tumorigenic cancer stem cells (CSCs) that are responsible for sustained growth, metastasis, and chemo- and radioresistance. Phytochemicals and herbs have recently been introduced as therapeutic sources for eliminating CSCs. Therefore, we assessed the anti-tumor effects of two herbal ingredients, the green tea extract "Epigallocatechin-3-gallate (EGCG)" and Honokiol (HNK), on parental cells or CD44high CSCs of the human laryngeal squamous cell carcinoma cell line HEp-2. Results revealed that EGCG had a preeminent apoptotic potential on HEp-2 laryngeal CSCs. HNK conferred higher cytotoxic impacts on parental cells mostly by necrosis induction, especially with higher doses, but apoptosis induction with lower doses was also observed. The Notch signaling pathway genes were more potently suppressed by EGCG than HNK. However, HNK surpassed EGCG in downregulating the ß-catenin and the Sonic Hedgehog signaling pathways genes. On a genetic basis, both agents engaged the BCL-2 family-regulated and caspase-dependent intrinsic apoptotic pathway, but EGCG and HNK triggered apoptosis via p53-independent and p53-dependent pathways, respectively. Taken together, EGCG and HNK eradicated HEp-2 human larynx cancer cells through targeting multiple self-renewal pathways and activating diverse cell death modalities.


Asunto(s)
Catequina , Neoplasias Laríngeas , Apoptosis , Compuestos de Bifenilo , Catequina/análogos & derivados , Catequina/metabolismo , Catequina/farmacología , Línea Celular Tumoral , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/farmacología , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Neoplasias Laríngeas/tratamiento farmacológico , Neoplasias Laríngeas/genética , Neoplasias Laríngeas/metabolismo , Lignanos , Células Madre Neoplásicas , Proteína p53 Supresora de Tumor/metabolismo
7.
Saudi Dent J ; 31(4): 395-416, 2019 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-31700218

RESUMEN

Head and neck squamous cell carcinomas (HNSCCs) arise in the mucosal linings of the upper aerodigestive tract and are heterogeneous in nature. Risk factors for HNSCCs are smoking, excessive alcohol consumption, and the human papilloma virus. Conventional treatments are surgery, radiotherapy, chemotherapy, or a combined modality; however, no international standard mode of therapy exists. In contrast to the conventional model of clonal evolution in tumor development, there is a newly proposed theory based on the activity of cancer stem cells (CSCs) as the model for carcinogenesis. This "CSC hypothesis" may explain the high mortality rate, low response to treatments, and tendency to develop multiple tumors for HNSCC patients. We review current knowledge on HNSCC etiology and treatment, with a focus on CSCs, including their origins, identifications, and effects on therapeutic options.

8.
Med Oncol ; 35(9): 124, 2018 Aug 04.
Artículo en Inglés | MEDLINE | ID: mdl-30078069

RESUMEN

The efficacy of cisplatin (CIS) and 5-fluorouracil (5-FU) against squamous cell carcinomas of the head and neck (SCCHN) remains restricted due to their severe toxic side effects on non-cancer (normal) tissues. Recently, the broccoli extract sulforaphane (SF) was successfully tested as a combination therapy to target cancer cells. However, the effect of lower doses of CIS or 5-FU combined with SF on SCCHN remained unknown. This study tested the chemotherapeutic efficacies of SF combined with much lower doses of CIS or 5-FU against SCCHN cells aiming to reduce cytotoxicity to normal cells. Titrations of SF standalone or in combination with CIS and 5-FU were tested on SCCHN human cell lines (SCC12 and SCC38) and non-cancerous human cells (fibroblasts, gingival, and salivary cells). Concentrations of SF tested were comparable to those found in the plasma following ingestion of fresh broccoli sprouts. The treatment effects on cell viability, proliferation, DNA damage, apoptosis, and gene expression were measured. SF reduced SCCHN cell viability in a time- and dose-dependent manner. SF-combined treatment increased the cytotoxic activity of CIS by twofolds and of 5-FU by tenfolds against SCCHN, with no effect on non-cancerous cells. SF-combined treatment inhibited SCCHN cell clonogenicity and post-treatment DNA repair. SF increased SCCHN apoptosis and this mechanism was due to a down-regulation of BCL2 and up-regulation of BAX, leading to an up-regulation of Caspase3. In conclusion, combining SF with low doses of CIS or 5-FU increased cytotoxicity against SCCHN cells, while having minimal effects on normal cells.


Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Carcinoma de Células Escamosas/patología , Neoplasias de Cabeza y Cuello/patología , Isotiocianatos/farmacología , Extractos Vegetales/farmacología , Anciano , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cisplatino/farmacología , Daño del ADN/efectos de los fármacos , Fluorouracilo/farmacología , Expresión Génica/efectos de los fármacos , Humanos , Masculino , Persona de Mediana Edad , Carcinoma de Células Escamosas de Cabeza y Cuello , Sulfóxidos
9.
Metab Brain Dis ; 33(2): 583-587, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-29080085

RESUMEN

Tauopathy comprises a group of disorders caused by abnormal aggregates of tau protein. In these disorders phosphorylated tau protein tends to accumulate inside neuronal cells (soma) instead of the normal axonal distribution of tau. A suggested therapeutic strategy for tauopathy is to induce autophagy to increase the ability to get rid of the unwanted tau aggregates. One of the key controllers of autophagy is mTOR. Blocking mTOR leads to stimulation of autophagy. Recently, unravelling molecular structure of mTOR showed that it is formed of two subunits: mTORC1/C2. So, blocking both subunits of mTOR seems more attractive as it will explore all abilities of mTOR molecule. In the present study, we report using pp242 which is a dual mTORC1/C2 blocker in cellular model of tauopathy using LUHMES cell line. Adding fenazaquin to LUHMES cells induced tauopathy in the form of increased phospho tau aggregates. Moreover, fenazaquin treated cells showed the characteristic somatic redistribution of tau. PP242 use in the present tauopathy model reversed the pathology significantly without observable cellular toxicity for the used dosage of 1000 nM. The present study suggests the possible use of pp242 as a dual mTOR blocker to treat tauopathy.


Asunto(s)
Indoles/farmacología , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Diana Mecanicista del Complejo 2 de la Rapamicina/antagonistas & inhibidores , Purinas/farmacología , Proteínas tau/metabolismo , Adolescente , Adulto , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Inhibidores de Proteínas Quinasas/farmacología , Serina-Treonina Quinasas TOR/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Adulto Joven
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