RESUMO
The BRCA2-RAD51 interaction remains an intriguing target for cancer drug discovery due to its vital role in DNA damage repair mechanisms, which cancer cells become particularly reliant on. Moreover, RAD51 has many synthetically lethal partners, including PARP1-2, which can be exploited to induce synthetic lethality in cancer. In this study, we established a 19F-NMR-fragment based approach to identify RAD51 binders, leading to two initial hits. A subsequent SAR program identified 46 as a low micromolar inhibitor of the BRCA2-RAD51 interaction. 46 was tested in different pancreatic cancer cell lines, to evaluate its ability to inhibit the homologous recombination DNA repair pathway, mediated by BRCA2-RAD51 and trigger synthetic lethality in combination with the PARP inhibitor talazoparib, through the induction of apoptosis. Moreover, we further analyzed the 46/talazoparib combination in 3D pancreatic cancer models. Overall, 46 showed its potential as a tool to evaluate the RAD51/PARP1-2 synthetic lethality mechanism, along with providing a prospect for further inhibitors development.
Assuntos
Antineoplásicos , Neoplasias Pancreáticas , Humanos , Antineoplásicos/química , Proteína BRCA2/antagonistas & inibidores , Proteína BRCA2/metabolismo , Linhagem Celular Tumoral , Reparo do DNA , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Inibidores de Poli(ADP-Ribose) Polimerases/química , Rad51 Recombinase/antagonistas & inibidores , Rad51 Recombinase/metabolismo , Mutações Sintéticas LetaisRESUMO
OBJECTIVE: To describe the RAD51 response (DNA repair) to radiation-induced DNA damage in patient-derived vestibular schwannoma (VS) cells and investigate the utility of RAD51 inhibitor (RI-1) in enhancing radiation toxicity. STUDY DESIGN: Basic and translational science. SETTING: Tertiary academic facility. METHODS: VS tumors (n = 10) were cultured on 96-well plates and 16-well slides, exposed to radiation (0, 6, 12, or 18 Gy), and treated with RI-1 (0, 5, or 10 µM). Immunofluorescence was performed at 6 hours for γ-H2AX (DNA damage marker), RAD51 (DNA repair protein), and p21 (cell cycle arrest protein). Viability assays were performed at 96 hours, and capillary Western blotting was utilized to determine RAD51 expression in naïve VS tumors (n = 5). RESULTS: VS tumors expressed RAD51. In cultured VS cells, radiation initiated dose-dependent increases in γ-H2AX and p21 expression. VS cells upregulated RAD51 to repair DNA damage following radiation. Addition of RI-1 reduced RAD51 expression in a dose-dependent manner and was associated with increased γ-H2AX levels and decreased viability in a majority of cultured VS tumors. CONCLUSION: VS may evade radiation injury by entering cell cycle arrest and upregulating RAD51-dependent repair of radiation-induced double-stranded breaks in DNA. Although there was variability in responses among individual primary VS cells, RAD51 inhibition with RI-1 reduced RAD51-dependent DNA repair to enhance radiation toxicity in VS cells. Further investigations are warranted to understand the mechanisms of radiation resistance in VS and determine whether RI-1 is an effective radiosensitizer in patients with VS.
Assuntos
Neuroma Acústico , Rad51 Recombinase , Lesões por Radiação , Humanos , Linhagem Celular Tumoral , Dano ao DNA , Reparo do DNA , Rad51 Recombinase/antagonistas & inibidores , Células Tumorais Cultivadas/efeitos da radiaçãoRESUMO
Replication stress (RS) has a pivotal role in tumor initiation, progression, or therapeutic resistance. In this study, we depicted the mechanism of breast cancer stem cells' (bCSCs) response to RS and its clinical implication. We demonstrated that bCSCs present a limited level of RS compared with non-bCSCs in patient samples. We described for the first time that the spatial nuclear location of BMI1 protein triggers RS response in breast cancers. Hence, in bCSCs, BMI1 is rapidly located to stalled replication forks to recruit RAD51 and activate homologous-recombination machinery, whereas in non-bCSCs BMI1 is trapped on demethylated 1q12 megasatellites precluding effective RS response. We further demonstrated that BMI1/RAD51 axis activation is necessary to prevent cisplatin-induced DNA damage and that treatment of patient-derived xenografts with a RAD51 inhibitor sensitizes tumor-initiating cells to cisplatin. The comprehensive view of replicative-stress response in bCSC has profound implications for understanding and improving therapeutic resistance.
Assuntos
Neoplasias da Mama/patologia , Núcleo Celular/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos dos fármacos , Complexo Repressor Polycomb 1/metabolismo , Rad51 Recombinase/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Cisplatino/farmacologia , Dano ao DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Recombinação Homóloga , Humanos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Complexo Repressor Polycomb 1/antagonistas & inibidores , Rad51 Recombinase/antagonistas & inibidoresRESUMO
RAD51 is the central protein in DNA repair by homologous recombination (HR), involved in several steps of this process. It is shown that overexpression of the RAD51 protein is correlated with increased survival of cancer cells to cancer treatments. For the past decade, RAD51 overexpression-mediated resistance has justified the development of targeted inhibitors. One of the first molecules described to inhibit RAD51 was the 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) molecule. This small molecule is effective in inhibiting different functions of RAD51, however its mode of action and the chemical functions involved in this inhibition have not been identified. In this work, we used several commercial molecules derived from DIDS to characterize the structural determinants involved in modulating the activity of RAD51. By combining biochemical and biophysical approaches, we have shown that DIDS and two analogs were able to inhibit the binding of RAD51 to ssDNA and prevent the formation of D-loop by RAD51. Both isothiocyanate substituents of DIDS appear to be essential in the inhibition of RAD51. These results open the way to the synthesis of new molecules derived from DIDS that should be greater modulators of RAD51 and more efficient for HR inhibition.
Assuntos
Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/análogos & derivados , Rad51 Recombinase/química , Rad51 Recombinase/metabolismo , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/administração & dosagem , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico/administração & dosagem , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico/farmacologia , DNA de Cadeia Simples/metabolismo , Relação Dose-Resposta a Droga , Rad51 Recombinase/antagonistas & inibidoresRESUMO
The anthracycline derivative doxorubicin (Doxo) induces DNA double-strand breaks (DSBs) by inhibition of DNA topoisomerase type II. Defective mismatch repair (MMR) contributes to Doxo resistance and has been reported for colon and mammary carcinomas. Here, we investigated the outcome of pharmacological inhibition of various DNA repair-related mechanisms on Doxo-induced cytotoxicity employing MMR-deficient HCT-116 colon carcinoma cells. Out of different inhibitors tested (i.e. HDACi, PARPi, MRE11i, RAD52i, RAD51i), we identified the RAD51-inhibitor B02 as the most powerful compound to synergistically increase Doxo-induced cytotoxicity. B02-mediated synergism rests on pleiotropic mechanisms, including pronounced G2/M arrest, damage to mitochondria and caspase-driven apoptosis. Of note, B02 also promotes the cytotoxicity of oxaliplatin and 5-fluoruracil (5-FU) in HCT-116 cells and, furthermore, also increases Doxo-induced cytotoxicity in MMR-proficient colon and mammary carcinoma cells. Summarizing, pharmacological inhibition of RAD51 is suggested to synergistically increase the cytotoxic efficacy of various types of conventional anticancer drugs in different tumor entities. Hence, pre-clinical in vivo studies are preferable to determine the therapeutic window of B02 in a clinically oriented therapeutic regimen.
Assuntos
Neoplasias do Colo/tratamento farmacológico , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Rad51 Recombinase/genética , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Caspases/genética , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Dano ao DNA/efeitos dos fármacos , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Reparo de Erro de Pareamento de DNA/genética , Doxorrubicina/efeitos adversos , Sinergismo Farmacológico , Fluoruracila/farmacologia , Células HCT116 , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Oxaliplatina/farmacologia , Rad51 Recombinase/antagonistas & inibidoresRESUMO
The genetic principle of synthetic lethality has most successfully been exploited in therapies engaging Poly-ADP-ribose-polymerase (PARP) inhibitors to treat patients with homologous recombination (HR)-defective tumors. In this work, we went a step further following the idea of a local molecular cooperation and designed hybrid compounds M1-M3. The drug conjugates M1-M3 combine Olaparib, the first PARP inhibitor approved for clinical use, with Cpd 1, an inhibitor of RAD51 that blocks its HR functions and yet permits RAD51 nucleoprotein filament formation on single-stranded DNA. While in M2 and M3, the parental drugs are linked by -CO-(CH2)n-CO-spacers (n = 2 and 4, respectively), they are directly merged omitting the piperazine ring of Olaparib in M1. Monitoring anti-survival effects of M1-M3 in six breast cancer cell lines of different molecular subtypes showed that in each cell line, at least one of the drug conjugates decreased viability by one to two orders of magnitude compared with parental drugs. While triple-negative breast cancer (TNBC) cells with frequent BRCA1 pathway dysfunction were sensitive to spacer-linked hybrid compounds M1 and M2 regardless of their HR capacities, non-TNBC cells were responsive to the merged drug conjugate M1 only, suggesting different spatial requirements for dual inhibition in these two groups of cell lines. These results demonstrate that, depending on chemical linkage, dual PARP1-RAD51 inhibitory drugs can either sensitize non-TNBC and re-sensitize TNBC cells, or discriminate between these groups of cells.
Assuntos
Antineoplásicos , Proteínas de Neoplasias/antagonistas & inibidores , Ftalazinas , Piperazinas , Inibidores de Poli(ADP-Ribose) Polimerases , Rad51 Recombinase/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Feminino , Humanos , Células MCF-7 , Proteínas de Neoplasias/metabolismo , Ftalazinas/química , Ftalazinas/farmacologia , Piperazinas/química , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/síntese química , Inibidores de Poli(ADP-Ribose) Polimerases/química , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Rad51 Recombinase/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Targeting DNA repair proteins with small-molecule inhibitors became a proven anti-cancer strategy. Previously, we identified an inhibitor of a major protein of homologous recombination (HR) RAD51, named B02. B02 inhibited HR in human cells and sensitized them to chemotherapeutic drugs in vitro and in vivo. Here, using a medicinal chemistry approach, we aimed to improve the potency of B02. We identified the B02 analog, B02-isomer, which inhibits HR in human cells with significantly higher efficiency. We also show that B02-iso sensitizes triple-negative breast cancer MDA-MB-231 cells to the PARP inhibitor (PARPi) olaparib.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Recombinação Homóloga/efeitos dos fármacos , Quinazolinonas/farmacologia , Rad51 Recombinase/antagonistas & inibidores , Antineoplásicos/química , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Inibidores Enzimáticos/química , Humanos , Simulação de Acoplamento Molecular , Ftalazinas/farmacologia , Piperazinas/farmacologia , Ligação Proteica , Quinazolinonas/química , Rad51 Recombinase/química , Rad51 Recombinase/metabolismoRESUMO
Despite advances in surgery, chemotherapy, and radiation, there are limited treatment options for advanced head and neck squamous cell carcinoma (HNSCC) and survival remains very poor. Therefore, effective therapies are desperately needed. Recently, selective exploitation of DNA damage and replication stress responses has become a novel approach for cancer treatment. Wee1 kinase and Rad51 recombinase are two proteins involved in regulating replication stress and homologous recombination repair in cancer cells. In this study, we investigated the combined effect of Rad51 inhibitor (B02) and Wee1 inhibitor (AZD1775) in vitro and in vivo in various HNSCC cell lines. Clonogenic survival assays demonstrated that B02 synergized with AZD1775 in vitro in all HNSCC cell lines tested. The synergy between these drugs was associated with forced CDK1 activation and reduced Chk1 phosphorylation leading to induction of excessive DNA damage and replication stress, culminating in aberrant mitosis and apoptosis. Our results showed that elevated Rad51 mRNA expression correlated with worse survival in HNSCC patients with HPV-positive tumors. The combination of B02 and AZD1775 significantly inhibited tumor growth in vivo in mice bearing HPV-positive HNSCC tumors as compared to HPV-negative HNSCC. This differential sensitivity appears to be linked to HPV-positive tumors having more in vivo endogenous replication stress owing to transformation by E6 and E7 oncogenes. Furthermore, addition of B02 radiosensitized the HPV-negative HNSCC tumors in vitro and in vivo In conclusion, our data implicate that a novel rational combination with Rad51 and Wee1 inhibitors holds promise as synthetic lethal therapy, particularly in high-risk HPV-positive HNSCC.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/antagonistas & inibidores , Dano ao DNA/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Rad51 Recombinase/antagonistas & inibidores , Animais , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Biologia Computacional/métodos , Reparo do DNA/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Perfilação da Expressão Gênica , Recombinação Homóloga , Humanos , Camundongos , Pirazóis/farmacologia , Pirimidinonas/farmacologia , Radiossensibilizantes/farmacologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/etiologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
R-loops are three-stranded structures generated by annealing of nascent transcripts to the template DNA strand, leaving the non-template DNA strand exposed as a single-stranded loop. Although R-loops play important roles in physiological processes such as regulation of gene expression, mitochondrial DNA replication, or immunoglobulin class switch recombination, dysregulation of the R-loop metabolism poses a threat to the stability of the genome. A previous study in yeast has shown that the homologous recombination machinery contributes to the formation of R-loops and associated chromosome instability. On the contrary, here, we demonstrate that depletion of the key homologous recombination factor, RAD51, as well as RAD51 inhibition by the B02 inhibitor did not prevent R-loop formation induced by the inhibition of spliceosome assembly in human cells. However, we noticed that treatment of cells with B02 resulted in RAD51-dependent accumulation of R-loops in an early G1 phase of the cell cycle accompanied by a decrease in the levels of chromatin-bound ORC2 protein, a component of the pre-replication complex, and an increase in DNA synthesis. Our results suggest that B02-induced R-loops might cause a premature origin firing.
Assuntos
Instabilidade Cromossômica/efeitos dos fármacos , DNA/biossíntese , Inibidores Enzimáticos/farmacologia , Fase G1/efeitos dos fármacos , Estruturas R-Loop , Rad51 Recombinase , Linhagem Celular Tumoral , Humanos , Complexo de Reconhecimento de Origem/metabolismo , Rad51 Recombinase/antagonistas & inibidores , Rad51 Recombinase/metabolismoRESUMO
Break-induced replication (BIR) is essential for the repair of DNA double-strand breaks (DSBs) with single ends. DSBs-induced microhomology-mediated BIR (mmBIR) and template-switching can increase the risk of complex genome rearrangement. In addition, DSBs can also induce the multi-invasion-mediated DSB amplification. The mmBIR-induced genomic rearrangement has been identified in cancer cells and patients with rare diseases. However, when and how mmBIR is initiated have not been fully and deeply studied. Furthermore, it is not well understood about the conditions for initiation of multi-invasion-mediated DSB amplification. In the G2 phase oocyte of mouse, we identified a type of short-scale BIR (ssBIR) using the DNA replication indicator 5-ethynyl-2'-deoxyuridine (EdU). These ssBIRs could only be induced in the fully grown oocytes but not the growing oocytes. If the DSB oocytes were treated with Rad51 or Chek1/2 inhibitors, both EdU signals and DSB marker γH2A.X foci would decrease. In addition, the DNA polymerase inhibitor Aphidicolin could inhibit the ssBIR and another inhibitor ddATP could reduce the number of γH2A.X foci in the DSB oocytes. In conclusion, our results showed that DNA DSBs in the fully grown oocytes can initiate ssBIR and be amplified by Rad51 or DNA replication.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , Replicação do DNA/fisiologia , Animais , Afidicolina/farmacologia , Células Cultivadas , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/metabolismo , Nucleotídeos de Desoxiadenina/farmacologia , Didesoxinucleotídeos/farmacologia , Feminino , Fase G2 , Indóis/farmacologia , Camundongos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Oócitos , Cultura Primária de Células , Rad51 Recombinase/antagonistas & inibidores , Rad51 Recombinase/metabolismo , Tetra-Hidroisoquinolinas/farmacologiaRESUMO
BRCA2 controls RAD51 recombinase during homologous DNA recombination (HDR) through eight evolutionarily conserved BRC repeats, which individually engage RAD51 via the motif Phe-x-x-Ala. Using structure-guided molecular design, templated on a monomeric thermostable chimera between human RAD51 and archaeal RadA, we identify CAM833, a 529 Da orthosteric inhibitor of RAD51:BRC with a Kd of 366 nM. The quinoline of CAM833 occupies a hotspot, the Phe-binding pocket on RAD51 and the methyl of the substituted α-methylbenzyl group occupies the Ala-binding pocket. In cells, CAM833 diminishes formation of damage-induced RAD51 nuclear foci; inhibits RAD51 molecular clustering, suppressing extended RAD51 filament assembly; potentiates cytotoxicity by ionizing radiation, augmenting 4N cell-cycle arrest and apoptotic cell death and works with poly-ADP ribose polymerase (PARP)1 inhibitors to suppress growth in BRCA2-wildtype cells. Thus, chemical inhibition of the protein-protein interaction between BRCA2 and RAD51 disrupts HDR and potentiates DNA damage-induced cell death, with implications for cancer therapy.
Assuntos
Proteína BRCA2/antagonistas & inibidores , Rad51 Recombinase/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína BRCA2/química , Proteína BRCA2/metabolismo , Morte Celular/efeitos dos fármacos , Cristalografia por Raios X , Dano ao DNA , Humanos , Modelos Moleculares , Conformação Molecular , Ligação Proteica/efeitos dos fármacos , Rad51 Recombinase/química , Rad51 Recombinase/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Células Tumorais CultivadasRESUMO
Cisplatin, a representative of platinum-based drug, is clinically and widely used in the treatment of various types of malignant cancer. However, its non-selectivity to almost all the cell lines and resistance in long-term use severely limit its scope of use. As biotin-specific uptake systems are overexpressed in many types of tumors but rarely occur in normal tissues, making biotin a promising target for cancer treatment. In the study, we synthesized the Pt(II) complex C2 and determined its biological activities. The existence of biotin enhanced the ability of the complex to target tumors, while the introduction of a naphthalimide compound makes it possible to diagnose tumors and monitor their progress. We have also introduced a known Pt(II) complex DN604, which not only retains the excellent cytotoxicity of platinum drugs, but also inhibits the expression of DNA double-strand breaks (DSBs) repair-related NHEJ protein Ku70 and HR protein Rad51. In summary, we report a novel trifunctional Pt(II) complex that could target tumor cells, monitor tumor progression, and reverse DSBs repair-induced cisplatin-resistance.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Autoantígeno Ku/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Compostos Organoplatínicos/farmacologia , Rad51 Recombinase/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cisplatino/química , Cisplatino/farmacologia , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Autoantígeno Ku/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Estrutura Molecular , Compostos Organoplatínicos/síntese química , Compostos Organoplatínicos/química , Rad51 Recombinase/metabolismo , Relação Estrutura-AtividadeRESUMO
Rad51 is an essential factor of the homologous recombination DNA repair pathway and therefore plays an important role in maintaining genomic stability. We show that RAD51 and other homologous recombination repair genes are overexpressed in metastatic melanoma cell lines and in melanoma patient samples, which correlates with reduced survival of melanoma patients. In addition, Rad51 expression in melanoma cells was regulated on a transcriptional level by the MAPK signaling pathway with Elk1 as the main downstream transcriptional effector. Most strikingly, melanoma cells which developed resistance towards MAPK inhibitors could be efficiently targeted by Rad51 inhibitors similar to their sensitive counterparts, leading to DNA damage, G2/M arrest and apoptosis. Furthermore, the treatment of MAPK inhibitor resistant cells with Rad51 inhibitors enhances the susceptibility of these cells for MAPK inhibitor treatment in vitro and in vivo. These data indicate that Rad51 plays a critical role in the survival of metastatic melanoma cells and is a promising target for the therapy of melanoma irrespective of its MAPK inhibitor resistance status.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Sistema de Sinalização das MAP Quinases , Melanoma/enzimologia , Melanoma/patologia , Terapia de Alvo Molecular , Inibidores de Proteínas Quinases/farmacologia , Rad51 Recombinase/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Modelos Biológicos , Rad51 Recombinase/antagonistas & inibidores , Proteínas Elk-1 do Domínio ets/genética , Proteínas Elk-1 do Domínio ets/metabolismoRESUMO
OBJECTIVE: Osteosarcoma is a malignant bone tumor and is generally treated with radiotherapy combined with radiosensitizers. The aim of the present study was to investigate the radiosensitization effects of berberine on osteosarcoma cells and the role of Rad51 in radiosensitivity by berberine. MATERIALS AND METHODS: Cells from the human osteosarcoma cell line MG-63 were exposed to γ-ray irradiation (0, 2, 4, 6, and 8 Gy) and berberine (20 µM). Radiosensitivity was evaluated by determining cell viability using an MTT assay. Flow cytometry was used to determine cell cycle and apoptosis. Real-time PCR and western blot were performed to analyze the mRNA and protein expressions of Rad51. The protein levels of E-cadherin and vimentin were also measured to evaluate the epithelial-mesenchymal transition (EMT) process. Tumor invasion was determined by the Boyden chamber assay. RESULTS: Berberine exacerbated the decline in viability of MG-63 cells exposed to γ-rays irradiation at various concentrations (25, 50, 75, and 100 µmol/L) and induced cell cycle arrest in the G2/M phase as well as apoptosis. The mRNA and protein expressions of Rad51 were significantly decreased by berberine in MG-63 cells. Inhibition of Rad51 by B02 enhanced the radiosensitivity of MG-63 cells. Berberine inhibited their invasive capability as well as increased E-cadherin and decreased vimentin protein levels; this indicated that berberine suppressed the EMT process in MG-63 cells exposed to γ-rays irradiation. CONCLUSION: Berberine enhances the radiosensitivity of MG-63 osteosarcoma cells. Rad51 is a potential target of berberine in the radiosensitization of osteosarcoma.
Assuntos
Berberina/farmacologia , Pontos de Checagem do Ciclo Celular , Sobrevivência Celular , Transição Epitelial-Mesenquimal , Osteossarcoma/radioterapia , Rad51 Recombinase/antagonistas & inibidores , Radiossensibilizantes/farmacologia , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Neoplasias Ósseas/radioterapia , Linhagem Celular Tumoral , Humanos , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Rad51 Recombinase/metabolismoRESUMO
DNA double-strand breaks (DSBs) are deleterious and tumorigenic but could also be essential for DNA-based processes. Yet the landscape of physiological DSBs and their role and repair are still elusive. Here, we mapped DSBs at high resolution in cancer and non-tumorigenic cells and found a transcription-coupled repair mechanism at oncogenic super-enhancers. At these super-enhancers the transcription factor TEAD4, together with various transcription factors and co-factors, co-localizes with the repair factor RAD51 of the homologous recombination pathway. Depletion of TEAD4 or RAD51 increases DSBs at RAD51/TEAD4 common binding sites within super-enhancers and decreases expression of related genes, which are mostly oncogenes. Co-localization of RAD51 with transcription factors at super-enhancers occurs in various cell types, suggesting a broad phenomenon. Together, our findings uncover a coupling between transcription and repair mechanisms at oncogenic super-enhancers, to control the hyper-transcription of multiple cancer drivers.
Assuntos
Reparo do DNA , Rad51 Recombinase/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Replicação do DNA , DNA Topoisomerases Tipo I/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Estradiol/farmacologia , Humanos , Proteínas Musculares/antagonistas & inibidores , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Rad51 Recombinase/antagonistas & inibidores , Rad51 Recombinase/genética , Radiação Ionizante , Fatores de Transcrição de Domínio TEA , Fator de Transcrição AP-1/química , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
RAD51, is a key homologous recombination protein that repairs DNA damage and maintains gene diversity and stability. Previous studies have demonstrated that the overexpression of RAD51 is associated with chemotherapy resistance of tumor cells to chemotherapy, and enhanced activity of DNA damage repair (DDR) systems contributes to resistance of adult Tcell leukemialymphoma (ATL) resistance to chemotherapy. Thus, targeting RAD51 is a potential strategy for the sensitization of ATL cells to chemotherapeutic drugs by inducing DNA damage. In general, cells can repair minor DNA damage through DDR; however, serious DNA damage may cause cell toxicity in cells which cannot be restored. In the present, down regulation of RAD51 by shRNA and imatinib sensitized Jurkat cells to etoposide by decreasing the activity of homologous recombination (HR). We found that the suppression of RAD51 by shRNA inhibited tumor cells proliferation and enhanced apoptosis of Jurkat cells after etoposide treatment. Importantly, downregulation of RAD51 by imatinib obviously increased the apoptosis of Jurkat cell after etoposide treatment. These results demonstrated that RAD51 may be of great value to as a novel target for the clinical treatment of adult Tcell leukemialymphoma (ATL), and it may improve the survival of leukemia patients.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Leucemia-Linfoma de Células T do Adulto/tratamento farmacológico , Rad51 Recombinase/antagonistas & inibidores , Reparo de DNA por Recombinação/efeitos dos fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Quebras de DNA de Cadeia Dupla , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Etoposídeo/farmacologia , Etoposídeo/uso terapêutico , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Mesilato de Imatinib/farmacologia , Mesilato de Imatinib/uso terapêutico , Células Jurkat , Leucemia-Linfoma de Células T do Adulto/genética , Leucemia-Linfoma de Células T do Adulto/patologia , RNA Interferente Pequeno/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismoRESUMO
DNA damaging chemotherapies are successful in cancer therapy, however, the damage can be reversed by DNA repair mechanisms that may be up-regulated in cancer cells. We hypothesized that inhibiting RAD51, a protein involved in homologous recombination DNA repair, would block DNA repair and restore the effectiveness of DNA damaging chemotherapy. We used phage-display to generate a novel synthetic antibody fragment that bound human RAD51 with high affinity (KD = 8.1 nM) and inhibited RAD51 ssDNA binding in vitro. As RAD51 is an intracellular target, we created a corresponding intrabody fragment that caused a strong growth inhibitory phenotype on human cells in culture. We then used a novel cell-penetrating peptide "iPTD" fusion to generate a therapeutically relevant antibody fragment that effectively entered living cells and enhanced the cell-killing effect of a DNA alkylating agent. The iPTD may be similarly useful as a cell-penetrating peptide for other antibody fragments and open the door to numerous intracellular targets previously off-limits in living cells.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Reparo do DNA/efeitos dos fármacos , Fragmentos de Imunoglobulinas/uso terapêutico , Rad51 Recombinase/antagonistas & inibidores , Peptídeos Penetradores de Células/farmacologia , Peptídeos Penetradores de Células/uso terapêutico , Células Cultivadas , Dano ao DNA/efeitos dos fármacos , Humanos , Fragmentos de Imunoglobulinas/farmacologia , Biblioteca de PeptídeosRESUMO
Although improvements in radiation therapy were made over the years, radioresistance is still a major challenge. Cancer cells are often deficient for DNA repair response, a feature that is currently exploited as a new anti-cancer strategy. In this context, combination of inhibitors targeting complementary pathways is of interest to sensitize cells to radiation. In this work, we used PARP (Olaparib) and RAD51 (B02) inhibitors to radiosensitize cancer cells to proton and X-ray radiation. More particularly, Olaparib and B02 were used at concentration leading to limited cytotoxic (alone or in combination) but increasing cell death when the cells were irradiated. We showed that, although at limited concentration, Olaparib and B02 were able to radiosensitize different cancer cell lines, i.e. lung and pancreatic cancer cells. Antagonistic, additive or synergistic effects were observed and correlated to cell proliferation rate. The inhibitors enhanced persistent DNA damage, delayed apoptosis, prolonged cell cycle arrest and senescence upon irradiation. These results demonstrated that radiation-induced synthetic lethality might widen the therapeutic window, hence extending the use of PARP inhibitors to patients without BRCAness.
Assuntos
Neoplasias/radioterapia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Rad51 Recombinase/antagonistas & inibidores , Radiossensibilizantes/uso terapêutico , Mutações Sintéticas Letais , Células A549 , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Compostos de Boro/uso terapêutico , Compostos de Boro/toxicidade , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Humanos , Ftalazinas/uso terapêutico , Ftalazinas/toxicidade , Piperazinas/uso terapêutico , Piperazinas/toxicidade , Poli(ADP-Ribose) Polimerases/metabolismo , Prótons , Radiação Ionizante , Fatores de TempoRESUMO
RAD51 is the central protein in homologous recombination (HR) repair, where it first binds ssDNA and then catalyzes strand invasion via a D-loop intermediate. Additionally, RAD51 plays a role in faithful DNA replication by protecting stalled replication forks; this requires RAD51 to bind DNA but may not require the strand invasion activity of RAD51. We previously described a small-molecule inhibitor of RAD51 named RI(dl)-2 (RAD51 inhibitor of D-loop formation #2, hereafter called 2 h), which inhibits D-loop activity while sparing ssDNA binding. However, 2 h is limited in its ability to inhibit HR inâ vivo, preventing only about 50 % of total HR events in cells. We sought to improve upon this by performing a structure-activity relationship (SAR) campaign for more potent analogues of 2 h. Most compounds were prepared from 1-(2-aminophenyl)pyrroles by forming the quinoxaline moiety either by condensation with aldehydes, then dehydrogenation of the resulting 4,5-dihydro intermediates, or by condensation with N,N'-carbonyldiimidazole, chlorination, and installation of the 4-substituent through Suzuki-Miyaura coupling. Many analogues exhibited enhanced activity against human RAD51, but in several of these compounds the increased inhibition was due to the introduction of dsDNA intercalation activity. We developed a sensitive assay to measure dsDNA intercalation, and identified two analogues of 2 h that promote complete HR inhibition in cells while exerting minimal intercalation activity.
Assuntos
Inibidores Enzimáticos/síntese química , Quinoxalinas/síntese química , Rad51 Recombinase/antagonistas & inibidores , Sobrevivência Celular , Reação de Cicloadição , DNA/química , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Inibidores Enzimáticos/metabolismo , Células HEK293 , Humanos , Estrutura Molecular , Oxirredução , Ligação Proteica , Pirróis/química , Quinoxalinas/metabolismo , Relação Estrutura-AtividadeRESUMO
Double-strand breaks (DSBs), the most serious lesions of DNA, often induce chromosomal aberrations and are intimately associated with oncogenesis. A normal DNA damage response (DDR) network contains two major repair pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). Studies of DSB repair-associated molecules have focused mainly on DNA repair proteins. However, non-coding RNAs also play important roles in the process of DSB repair. Over the past two decades, microRNAs (miRNAs) have been extensively investigated. Our previous work showed that miR-34c-5p overexpression results in suppression of RAD51 and upregulation of γH2AX. In accordance with this, we confirmed that miR-34s family overexpression increased endogenous DSB levels to different extents, an effect that was further confirmed to be associated with the decreased efficiency of HR repair. In addition, miR-34s overexpression also induced G1 arrest, inhibited proliferation and promoted apoptosis. As a central molecule in the process of HR pathway, RAD51 expression was strongly repressed in cells transfected with the miR-34a/b/c-5p mimic. Finally, we demonstrated that miR-34a/b/c-5p directly targets the RAD51 mRNA 3'-UTR or indirectly inhibits RAD51 expression via the p53 signaling pathway. Taken together, our results indicate that miR-34s overexpression depresses the efficiency of HR repair and induces DSBs by downregulating RAD51 expression. Our findings highlight a novel mechanism of HR pathway regulation via the miR-34s/p53/RAD51 axis.