RESUMO
Camptothecin has been used in tumor therapy for a long time but its antitumor effect is rather limited due to the side effect and the drug resistance. FEN1, a major component of DNA repair systems, plays important roles in maintaining genomic stability via DNA replication and repair. Here we found that FEN1 inhibitor greatly sensitizes cancer cells to low-dose camptothecin. The combinative treatment of FEN1 inhibitor and 1 nM camptothecin induced a synthetic lethal effect, which synergistically suppressed cancer cell proliferation and significantly mediated apoptosis both in vitro and in vivo. Our study suggested that targeting FEN1 could be a potent strategy for tumor-targeting cancer therapy.
Assuntos
Camptotecina , Endonucleases Flap , Neoplasias , Apoptose , Camptotecina/farmacologia , Dano ao DNA , Endonucleases Flap/antagonistas & inibidores , Humanos , Mitocôndrias/metabolismoAssuntos
Proteínas de Ligação a DNA/genética , Endonucleases Flap/genética , Neoplasias/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Mutações Sintéticas Letais/genética , Antineoplásicos/química , Antineoplásicos/farmacologia , Reparo do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/química , Sistemas de Liberação de Medicamentos , Endonucleases Flap/antagonistas & inibidores , Recombinação Homóloga/efeitos dos fármacos , Humanos , Complexos Multiproteicos/genética , Neoplasias/genética , Parthanatos/efeitos dos fármacos , Poli(ADP-Ribose) Polimerase-1/genética , Saccharomyces cerevisiae/genética , Bibliotecas de Moléculas Pequenas/química , Mutações Sintéticas Letais/efeitos dos fármacosRESUMO
Ovarian cancer (OC) is one of the most lethal gynecological malignancies in the world. The aim of the present study was to examine the role of microRNA (miR)-134-3p in OC. Reverse transcription-quantitative PCR was used to measure the expression levels of miR-134-3p. Cell Counting Kit-8, TUNEL, flow cytometric and colony formation assays were performed to examine the effects of miR-134-3p on OC cell proliferation. Moreover, wound healing and Transwell assays were performed to examine the effects on migration and invasion. In addition, western blot analyses were used to assess protein expression. Finally, the target genes of miR-134-3p were analyzed by bioinformatics analysis and dual-luciferase reporter assay. The results revealed that miR-134-3p expression was low in OC cells compared with in normal ovarian cells. The overexpression of miR-134-3p decreased cell viability, facilitated cell apoptosis, inhibited cell proliferation and arrested the cell cycle in SKOV-3 and OVCAR-3 cells. Furthermore, transfection using a miR-134-3p mimic inhibited the migration and invasion of SKOV-3 and OVCAR-3 cells, and decreased the protein expression levels of cyclooxygenase-2, matrix metalloproteinase (MMP)2 and MMP9. Bioinformatics analysis indicated that one of the potential target genes of miR-134-3p was flap structure-specific endonuclease 1 (FEN1), which was confirmed by dual-luciferase reporter assay. Moreover, overexpression of miR-134-3p decreased the expression levels of FEN1 in SKOV-3 and OVCAR-3 cells. Additionally, overexpression of FEN1 reversed the effects of the miR-134-3p mimic on the proliferation, migration and invasion of SKOV-3 and OVCAR-3 cells. Overall, the findings of the present study demonstrated that miR-134-3p may inhibit OC cell proliferation, migration and invasion by directly targeting FEN1.
Assuntos
Endonucleases Flap/genética , MicroRNAs/fisiologia , Neoplasias Ovarianas/patologia , Apoptose , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Progressão da Doença , Feminino , Endonucleases Flap/antagonistas & inibidores , HumanosRESUMO
Synthetic lethality strategies for cancer therapy exploit cancer-specific genetic defects to identify targets that are uniquely essential to the survival of tumor cells. Here we show RAD27/FEN1, which encodes flap endonuclease 1 (FEN1), a structure-specific nuclease with roles in DNA replication and repair, and has the greatest number of synthetic lethal interactions with Saccharomyces cerevisiae genome instability genes, is a druggable target for an inhibitor-based approach to kill cancers with defects in homologous recombination (HR). The vulnerability of cancers with HR defects to FEN1 loss was validated by studies showing that small-molecule FEN1 inhibitors and FEN1 small interfering RNAs (siRNAs) selectively killed BRCA1- and BRCA2-defective human cell lines. Furthermore, the differential sensitivity to FEN1 inhibition was recapitulated in mice, where a small-molecule FEN1 inhibitor reduced the growth of tumors established from drug-sensitive but not drug-resistant cancer cell lines. FEN1 inhibition induced a DNA damage response in both sensitive and resistant cell lines; however, sensitive cell lines were unable to recover and replicate DNA even when the inhibitor was removed. Although FEN1 inhibition activated caspase to higher levels in sensitive cells, this apoptotic response occurred in p53-defective cells and cell killing was not blocked by a pan-caspase inhibitor. These results suggest that FEN1 inhibitors have the potential for therapeutically targeting HR-defective cancers such as those resulting from BRCA1 and BRCA2 mutations, and other genetic defects.
Assuntos
Antineoplásicos/farmacologia , Endonucleases Flap/antagonistas & inibidores , Recombinação Homóloga/efeitos dos fármacos , Neoplasias/genética , Animais , Proteína BRCA1/deficiência , Proteína BRCA1/genética , Proteína BRCA2/deficiência , Proteína BRCA2/genética , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Endonucleases Flap/genética , Instabilidade Genômica/genética , Humanos , Camundongos , Neoplasias/tratamento farmacológico , RNA Interferente Pequeno/farmacologia , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Mutações Sintéticas Letais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Studies show that patients with hepatocellular carcinoma (HCC) have poor prognosis, particularly when patients are diagnosed at late stages of the disease development. The flap endonuclease 1 (FEN1) gene is overexpressed in multiple malignant tumors and may promote tumor aggressiveness. However, its expression profile and functional roles in HCC are still unclear. Here, we evaluated the molecular mechanisms of FEN1 in HCC. METHODS: The expression of FEN1 in HCC was evaluated using HCC mRNA expression data from TCGA and GEO databases. The expression of FEN1 was also confirmed by immunohistochemistry (IHC) using a tissue microarray (TMA) cohort with a total of 396 HCC patients. Kaplan-Meier analysis and univariate and multivariate Cox regression analyses were used to determine the correlation between FEN1 expression and survival rate of HCC patients. The molecular mechanism and biological functions of FEN1 in HCC were predicted using functional and pathway enrichment analysis in vitro experiments. RESULTS: FEN1 was overexpressed in multiple HCC cohorts at both mRNA and protein levels. The receiver operating characteristic (ROC) curve showed that FEN1 can serve as a diagnostic predictor of HCC. Meanwhile, patients with high FEN1 expression levels showed lower overall survival (OS) and relapse-free survival (RFS) rates than those with low FEN1 expression. More importantly, we found that FEN1 elevation was an independent prognostic factor for OS and RFS in HCC patients based on univariate and multivariate analyses, indicating that FEN1 might be a potential prognostic marker in HCC. Furthermore, knocking down FEN1 resulted in suppressed cell proliferation and migration in vitro. This could have been due to regulation expressions of c-Myc, survivin, and cyclin D1 genes, indicating that FEN1 may function as an oncogene through its role in the cell cycle and DNA replication pathway. CONCLUSION: Our study indicated that high FEN1 expression might function as a biomarker for diagnosis and prognosis. In addition, the study confirms that FEN1 is an oncogene in HCC progression.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/genética , Endonucleases Flap/genética , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/genética , Adulto , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Proliferação de Células , Estudos de Coortes , Ciclina D1/genética , Ciclina D1/metabolismo , Diagnóstico Tardio , Progressão da Doença , Feminino , Endonucleases Flap/antagonistas & inibidores , Endonucleases Flap/metabolismo , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Modelos de Riscos Proporcionais , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Curva ROC , Survivina/genética , Survivina/metabolismoRESUMO
Cross-species complementation can be used to generate humanized yeast, which is a valuable resource with which to model and study human biology. Humanized yeast can be used as an in vivo platform to screen for chemical inhibition of human protein drug targets. To this end, we report the systematic complementation of nonessential yeast genes implicated in chromosome instability (CIN) with their human homologs. We identified 20 human-yeast complementation pairs that are replaceable in 44 assays that test rescue of chemical sensitivity and/or CIN defects. We selected a human-yeast pair (hFEN1/yRAD27), which is frequently overexpressed in cancer and is an anticancer therapeutic target, to perform in vivo inhibitor assays using a humanized yeast cell-based platform. In agreement with published in vitro assays, we demonstrate that HU-based PTPD is a species-specific hFEN1 inhibitor. In contrast, another reported hFEN1 inhibitor, the arylstibonic acid derivative NSC-13755, was determined to have off-target effects resulting in a synthetic lethal phenotype with yRAD27-deficient strains. Our study expands the list of human-yeast complementation pairs to nonessential genes by defining novel cell-based assays that can be utilized as a broad resource to study human drug targets.
Assuntos
Endonucleases Flap/genética , Proteínas Fúngicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Instabilidade Cromossômica/efeitos dos fármacos , Instabilidade Cromossômica/genética , Desenvolvimento de Medicamentos/métodos , Endonucleases Flap/antagonistas & inibidores , Proteínas Fúngicas/antagonistas & inibidores , Teste de Complementação Genética , Humanos , Mutação/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidoresRESUMO
BACKGROUND: Cervical cancer is one of the most common causes of cancer-associated mortality among affected women in the world. At present, treatment with weekly cisplatin plus ionizing radiation (IR) therapy is the standard regimen for cervical cancer, especially for locally advanced cervical cancer. The purpose of this study is to determine whether FEN1 inhibitors could enhance the therapeutic effect of IR therapy. METHODS: Western blot was applied to determine the expression of FEN1- and apoptosis-related proteins. Cell growth inhibition assay and colony formation assay were used to determine the effects of FEN1 inhibitor and IR exposure for Hela cells in vitro. CRISPR technology was used to knockdown FEN1 expression level of 293T cells, and tumor xenograft in nude mice was employed to determine the effects of FEN1 inhibitor and IR exposure on tumor growth in vivo. RESULTS: Our data revealed that FEN1 is overexpressed in HeLa cell and can be upregulated further by IR. We also demonstrated that FEN1 inhibitor enhances IR sensitivity of cervical cancer in vitro and in vivo. CONCLUSION: FEN1 inhibitor SC13 could sensitize radiotherapy of cervical cancer cell.
Assuntos
Inibidores Enzimáticos/farmacologia , Endonucleases Flap/antagonistas & inibidores , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Modelos Animais de Doenças , Feminino , Endonucleases Flap/genética , Endonucleases Flap/metabolismo , Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Células HeLa , Humanos , Camundongos , Radiação Ionizante , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Flap endonuclease 1 (FEN1) is recognized as a pivotal factor in DNA replication, long-patch excision repair, and telomere maintenance. Excessive FEN1 expression has been reported to be closely associated with cancer progression, but the specific mechanism has not yet been explored. In the present study, we demonstrated that FEN1 promoted breast cancer cell proliferation via an epigenetic mechanism of FEN1-mediated up-regulation of DNA methyltransferase (DNMT)1 and DNMT3a. FEN1 was proved to interact with DNMT3a through proliferating cell nuclear antigen (PCNA) to suppress microRNA (miR)-200a-5p expression mediated by methylation. Furthermore, miR-200a-5p was identified to repress breast cancer cell proliferation by inhibiting the expression of its target genes, hepatocyte growth factor (MET), and epidermal growth factor receptor (EGFR). Overall, our data surprisingly demonstrate that FEN1 promotes breast cancer cell growth via the formation of FEN1/PCNA/DNMT3a complex to inhibit miR-200a expression by DNMT-mediated methylation and to recover the target genes expression of miR-200a, MET, and EGFR. The novel epigenetic mechanism of FEN1 on proliferation promotion provides a significant clue that FEN1 might serve as a predictive biomarker and therapeutic target for breast cancer.-Zeng, X., Qu, X., Zhao, C., Xu, L., Hou, K., Liu, Y., Zhang, N., Feng, J., Shi, S., Zhang, L., Xiao, J., Guo, Z., Teng, Y., Che, X. FEN1 mediates miR-200a methylation and promotes breast cancer cell growth via MET and EGFR signaling.
Assuntos
Neoplasias da Mama/metabolismo , Endonucleases Flap/metabolismo , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Epigênese Genética , Receptores ErbB/metabolismo , Feminino , Endonucleases Flap/antagonistas & inibidores , Endonucleases Flap/genética , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Células MCF-7 , Masculino , Metilação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Mutação , Antígeno Nuclear de Célula em Proliferação/metabolismo , Mapas de Interação de Proteínas , Transdução de SinaisRESUMO
PURPOSE: Flap endonuclease 1 (FEN1) is up-regulated by estrogen (17ß-estradiol, E2) and related to cisplatin resistance of human breast cancer cells. Letrozole, an aromatase inhibitor, suppresses the change of testosterone into estrogen and is frequently used to treat breast cancer. However, the effects of letrozole on FEN1 expression and cisplatin sensitivity in breast cancer cells overexpressing aromatase have not been revealed. METHODS: The expression of FEN1 and the proteins in ERK/Elk-1 signaling were evaluated by RT-PCR and Western blot. Cisplatin sensitivity was explored through CCK-8 and flow cytometry analysis, respectively. FEN1 siRNAs and FEN1 expression plasmid were transfected into cells to down-regulate or up-regulate FEN1 expression. The promotor activity of FEN1 was detected using luciferase reporter assay. RESULTS: FEN1 down-regulation improved cisplatin sensitivity of breast cancer cells overexpressing aromatase. Letrozole down-regulated FEN1 expression and increased cisplatin sensitivity. The sensitizing effect of letrozole to cisplatin was dependent on FEN1 down-regulation. FEN1 overexpression could block the sensitizing effect of letrozole to cisplatin. Testosterone up-regulated the promotor activity, protein expression of FEN1, and phosphorylation of ERK/Elk-1, which could be eliminated by both letrozole and MEK1/2 inhibitor U0126. Letrozole down-regulated FEN1 expression in an ERK/Elk-1-dependent manner. CONCLUSIONS: Our findings clearly demonstrate that letrozole improves cisplatin sensitivity of breast cancer cells overexpressing aromatase via down-regulation of FEN1 and suggest that a combined use of letrozole and cisplatin may be a potential treatment protocol for relieving cisplatin resistance in human breast cancer.
Assuntos
Aromatase/metabolismo , Neoplasias da Mama/tratamento farmacológico , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Endonucleases Flap/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Letrozol/farmacologia , Antineoplásicos/farmacologia , Aromatase/química , Inibidores da Aromatase/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células , Regulação para Baixo , Feminino , Endonucleases Flap/genética , Endonucleases Flap/metabolismo , Humanos , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Human flap endonuclease 1 (hFEN1) is instrumental in DNA replication and repair. It is able to cleave the 5' single-stranded protrusion (also known as 5' flap) resulting from strand displacement reactions. In light of its crucial functions, hFEN1 is now deemed as a nontrivial target in the DNA damage response system for anticancer drug development. Herein, we report that myricetin and some natural flavonoids are able to inhibit hFEN1. Structure-activity relationship, inhibitory mechanisms, molecular docking, and cancer cell-based assays have been performed. Our original findings expand the activity of flavonoids and may pave the way for flavonoid-assisted targeted cancer therapy.
Assuntos
Neoplasias do Colo/enzimologia , Inibidores Enzimáticos/química , Endonucleases Flap/antagonistas & inibidores , Flavonoides/química , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Inibidores Enzimáticos/farmacologia , Endonucleases Flap/genética , Endonucleases Flap/metabolismo , Flavonoides/farmacologia , Células HT29 , Humanos , Simulação de Acoplamento MolecularRESUMO
Hepatitis B virus (HBV) is one of the major etiological pathogens for liver cirrhosis and hepatocellular carcinoma. Chronic HBV infection is a key factor in these severe liver diseases. During infection, HBV forms a nuclear viral episome in the form of covalently closed circular DNA (cccDNA). Current therapies are not able to efficiently eliminate cccDNA from infected hepatocytes. cccDNA is a master template for viral replication that is formed by the conversion of its precursor, relaxed circular DNA (rcDNA). However, the host factors critical for cccDNA formation remain to be determined. Here, we assessed whether one potential host factor, flap structure-specific endonuclease 1 (FEN1), is involved in cleavage of the flap-like structure in rcDNA. In a cell culture HBV model (Hep38.7-Tet), expression and activity of FEN1 were reduced by siRNA, shRNA, CRISPR/Cas9-mediated genome editing, and a FEN1 inhibitor. These reductions in FEN1 expression and activity did not affect nucleocapsid DNA (NC-DNA) production, but did reduce cccDNA levels in Hep38.7-Tet cells. Exogenous overexpression of wild-type FEN1 rescued the reduced cccDNA production in FEN1-depleted Hep38.7-Tet cells. Anti-FEN1 immunoprecipitation revealed the binding of FEN1 to HBV DNA. An in vitro FEN activity assay demonstrated cleavage of 5'-flap from a synthesized HBV DNA substrate. Furthermore, cccDNA was generated in vitro when purified rcDNA was incubated with recombinant FEN1, DNA polymerase, and DNA ligase. Importantly, FEN1 was required for the in vitro cccDNA formation assay. These results demonstrate that FEN1 is involved in HBV cccDNA formation in cell culture system, and that FEN1, DNA polymerase, and ligase activities are sufficient to convert rcDNA into cccDNA in vitro.
Assuntos
DNA Circular/metabolismo , DNA Viral/metabolismo , Endonucleases Flap/metabolismo , Vírus da Hepatite B/genética , Hepatite B/genética , Vírion/genética , DNA Circular/genética , DNA Viral/genética , Inibidores Enzimáticos/farmacologia , Endonucleases Flap/antagonistas & inibidores , Endonucleases Flap/genética , Células Hep G2 , Hepatite B/enzimologia , Hepatite B/virologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Vírion/enzimologia , Replicação ViralRESUMO
Studies on cervical cancer are urgently required to improve clinical outcomes. As a major anticancer drug for cervical cancer, paclitaxel has been used for many years in clinical therapy but its therapeutic efficacy is limited by common obstacle from cancer cells. The enhanced DNA repair pathways of cancer cells have been proved to survive DNA damage induced by chemotherapeutic drug. Inhibitors of specific DNA repair pathway can sensitize cancer cells to the treatment of chemotherapeutic drugs. In this paper we found that the effect of paclitaxel can be significantly improved when used in combination with FEN1 inhibitor SC13, suggesting a synergistic mechanism between the two compounds. Our studies suggest that FEN1 inhibition could be a novel strategy of tumor-targeting therapy for cervical cancer. Our work also revealed that paclitaxel demonstrates stronger synergistic effect with SC13 than other common used chemical drugs such as doxorubicin, carboplatin or camptothecin on cervical cancer cells.
Assuntos
Antineoplásicos/farmacologia , Endonucleases Flap/antagonistas & inibidores , Paclitaxel/farmacologia , Neoplasias do Colo do Útero/tratamento farmacológico , Animais , Antineoplásicos/uso terapêutico , Camptotecina/farmacologia , Camptotecina/uso terapêutico , Carboplatina/farmacologia , Carboplatina/uso terapêutico , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Sinergismo Farmacológico , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Paclitaxel/uso terapêutico , Neoplasias do Colo do Útero/enzimologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Human Flap endonuclease1 (FEN1) is an enzyme that is indispensable for DNA replication and repair processes and inhibition of its Flap cleavage activity results in increased cellular sensitivity to DNA damaging agents (cisplatin, temozolomide, MMS, etc.), with the potential to improve cancer prognosis. Reports of the high expression levels of FEN1 in several cancer cells support the idea that FEN1 inhibitors may target cancer cells with minimum side effects to normal cells. In this study, we used large publicly available, high-throughput screening data of small molecule compounds targeted against FEN1. Two machine learning algorithms, Support Vector Machine (SVM) and Random Forest (RF), were utilized to generate four classification models from huge PubChem bioassay data containing probable FEN1 inhibitors and non-inhibitors. We also investigated the influence of randomly selected Zinc-database compounds as negative data on the outcome of classification modelling. The results show that the SVM model with inactive compounds was superior to RF with Matthews's correlation coefficient (MCC) of 0.67 for the test set. A Maybridge database containing approximately 53 000 compounds was screened and top ranking 5 compounds were selected for enzyme and cell-based in vitro screening. The compound JFD00950 was identified as a novel FEN1 inhibitor with in vitro inhibition of flap cleavage activity as well as cytotoxic activity against a colon cancer cell line, DLD-1.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Endonucleases Flap/antagonistas & inibidores , Aprendizado de Máquina , Antineoplásicos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Colo/efeitos dos fármacos , Colo/enzimologia , Colo/patologia , Bases de Dados de Compostos Químicos , Descoberta de Drogas , Inibidores Enzimáticos/química , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Endonucleases Flap/genética , Endonucleases Flap/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Concentração Inibidora 50 , Naftoquinonas/química , Naftoquinonas/farmacologia , Especificidade de ÓrgãosRESUMO
Flap endonuclease 1 (FEN1) is a structure selective endonuclease required for proficient DNA replication and the repair of DNA damage. Cellularly active inhibitors of this enzyme have previously been shown to induce a DNA damage response and, ultimately, cell death. High-throughput screens of human cancer cell-lines identify colorectal and gastric cell-lines with microsatellite instability (MSI) as enriched for cellular sensitivity to N-hydroxyurea series inhibitors of FEN1, but not the PARP inhibitor olaparib or other inhibitors of the DNA damage response. This sensitivity is due to a synthetic lethal interaction between FEN1 and MRE11A, which is often mutated in MSI cancers through instabilities at a poly(T) microsatellite repeat. Disruption of ATM is similarly synthetic lethal with FEN1 inhibition, suggesting that disruption of FEN1 function leads to the accumulation of DNA double-strand breaks. These are likely a result of the accumulation of aberrant replication forks, that accumulate as a consequence of a failure in Okazaki fragment maturation, as inhibition of FEN1 is toxic in cells disrupted for the Fanconi anemia pathway and post-replication repair. Furthermore, RAD51 foci accumulate as a consequence of FEN1 inhibition and the toxicity of FEN1 inhibitors increases in cells disrupted for the homologous recombination pathway, suggesting a role for homologous recombination in the resolution of damage induced by FEN1 inhibition. Finally, FEN1 appears to be required for the repair of damage induced by olaparib and cisplatin within the Fanconi anemia pathway, and may play a role in the repair of damage associated with its own disruption.
Assuntos
Reparo do DNA/efeitos dos fármacos , Endonucleases Flap/metabolismo , Hidroxiureia/toxicidade , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/toxicidade , DNA/efeitos dos fármacos , DNA/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/efeitos dos fármacos , Endonucleases Flap/antagonistas & inibidores , Endonucleases Flap/genética , Humanos , Hidroxiureia/química , Proteína Homóloga a MRE11 , Instabilidade de Microssatélites/efeitos dos fármacos , Mutação , Ftalazinas/toxicidade , Piperazinas/toxicidade , Inibidores de Poli(ADP-Ribose) Polimerases/toxicidade , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Rad51 Recombinase/genéticaRESUMO
Lung cancer is one of the leading causes of cancer mortality worldwide. The therapeutic effect of chemotherapy is limited due to the resistance of cancer cells, which remains a challenge in cancer therapeutics. In this work, we found that flap endonuclease 1 (FEN1) is overexpressed in lung cancer cells. FEN1 is a major component of the base excision repair pathway for DNA repair systems and plays important roles in maintaining genomic stability through DNA replication and repair. We showed that FEN1 is critical for the rapid proliferation of lung cancer cells. Suppression of FEN1 resulted in decreased DNA replication and accumulation of DNA damage, which subsequently induced apoptosis. Manipulating the amount of FEN1 altered the response of lung cancer cells to chemotherapeutic drugs. A small-molecule inhibitor (C20) was used to target FEN1 and this enhanced the therapeutic effect of cisplatin. The FEN1 inhibitor significantly suppressed cell proliferation and induced DNA damage in lung cancer cells. In mouse models, the FEN1 inhibitor sensitized lung cancer cells to a DNA damage-inducing agent and efficiently suppressed cancer progression in combination with cisplatin treatment. Our study suggests that targeting FEN1 may be a novel and efficient strategy for a tumor-targeting therapy for lung cancer.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Endonucleases Flap/metabolismo , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Células A549 , Animais , Antineoplásicos/farmacologia , Apoptose , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Proliferação de Células , Cisplatino/farmacologia , Dano ao DNA , Reparo do DNA , Replicação do DNA , Progressão da Doença , Endonucleases Flap/antagonistas & inibidores , Endonucleases Flap/genética , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Camundongos Endogâmicos BALB C , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
DNA flap endonuclease 1 (FEN1) plays critical roles in maintaining genome stability and integrity by participating in both DNA replication and repair. Suppression of FEN1 in cells leads to the retardation of DNA replication and accumulation of unrepaired DNA intermediates, resulting in DNA double strand breaks (DSBs) and apoptosis. Therefore, targeting FEN1 could serve as a potent strategy for cancer therapy. In this study, we demonstrated that FEN1 is overexpressed in breast cancers and is essential for rapid proliferation of cancer cells. We showed that manipulating FEN1 levels in cells alters the response of cancer cells to chemotherapeutic drugs. Furthermore, we identified a small molecular compound, SC13 that specifically inhibits FEN1 activity, thereby interfering with DNA replication and repair in vitro and in cells. SC13 suppresses cancer cell proliferation and induces chromosome instability and cytotoxicity in cells. Importantly, SC13 sensitizes cancer cells to DNA damage-inducing therapeutic modalities and impedes cancer progression in a mouse model. These findings could establish a paradigm for the treatment of breast cancer and other cancers as well.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Inibidores Enzimáticos/farmacologia , Endonucleases Flap/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Proliferação de Células , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Modelos Animais de Doenças , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/genética , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Feminino , Endonucleases Flap/química , Endonucleases Flap/genética , Endonucleases Flap/metabolismo , Expressão Gênica , Humanos , Camundongos , Modelos Moleculares , Conformação Molecular , Terapia de Alvo Molecular , Gradação de Tumores , Estadiamento de Neoplasias , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The structure-specific nuclease human flap endonuclease-1 (hFEN1) plays a key role in DNA replication and repair and may be of interest as an oncology target. We present the crystal structure of inhibitor-bound hFEN1, which shows a cyclic N-hydroxyurea bound in the active site coordinated to two magnesium ions. Three such compounds had similar IC50 values but differed subtly in mode of action. One had comparable affinity for protein and protein-substrate complex and prevented reaction by binding to active site catalytic metal ions, blocking the necessary unpairing of substrate DNA. Other compounds were more competitive with substrate. Cellular thermal shift data showed that both inhibitor types engaged with hFEN1 in cells, and activation of the DNA damage response was evident upon treatment with inhibitors. However, cellular EC50 values were significantly higher than in vitro inhibition constants, and the implications of this for exploitation of hFEN1 as a drug target are discussed.
Assuntos
Inibidores Enzimáticos/farmacologia , Endonucleases Flap/antagonistas & inibidores , Endonucleases Flap/metabolismo , Domínio Catalítico/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Endonucleases Flap/química , Humanos , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , TemperaturaRESUMO
Flap structure-specific endonuclease 1 (FEN1) is one of the enzymes that involve in Eukaryotic DNA replication and repair. Recent studies have proved that FEN1 is highly over-expressed in various types of cancer cells and is a drug target. However, a limited number of FEN1 inhibitors has been identified and approved. Herein, we investigate the catalytic activity of FEN1, and propose a substrate-based inhibitor. As a consequence, one of the phosphorothioate-modified substrates is proved to exhibit the most efficient inhibitory effect in our in vitro examinations. A novelly-designed substrate-based FEN1 inhibitor was accordingly constructed and determined a remarkable IC50 value.
Assuntos
Endonucleases Flap/metabolismo , Endonucleases Flap/antagonistas & inibidores , Humanos , Especificidade por SubstratoRESUMO
Flap endonuclease-I (FEN-1) is involved in DNA repair and considered to be a novel target for the development of anticancer agents. N-hydroxy urea derivatives have been reported as FEN-1 inhibitors. To derive in vitro and in silico correlation, we have performed 2D-quantitative structure activity relationship (QSAR) analysis and docking studies on these compounds. 2D-QSAR models were developed using multiple linear regression (MLR) analysis and cross-validation using leave one out (LOO) method. The best model displayed R(2) of 0.806 and Q(2) of 0.607. Docking study revealed key interactions with desired amino acids and compare well with the in vitro potency of the reported compounds. Both studies reveal a link between FEN-1 inhibition and physicochemical descriptors or interactions with amino acids in active site. The information generated is first of its kind and may be helpful in the design of novel FEN-1 inhibitors.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Endonucleases Flap/antagonistas & inibidores , Hidroxiureia/química , Hidroxiureia/farmacologia , Endonucleases Flap/metabolismo , Humanos , Modelos Lineares , Simulação de Acoplamento Molecular , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Relação Quantitativa Estrutura-AtividadeRESUMO
A high throughput screen allowed the identification of N-hydroxyimide inhibitors of ERCC1-XPF endonuclease activity with micromolar potency, but they showed undesirable selectivity profiles against FEN-1. A scaffold hop to a hydroxypyrimidinone template gave compounds with similar potency but allowed selectivity to be switched in favour of ERCC1-XPF over FEN-1. Further exploration of the structure-activity relationships around this chemotype gave sub-micromolar inhibitors with >10-fold selectivity for ERCC1-XPF over FEN-1.