2-Hydroxy-3-methylanthraquinone inhibits homologous recombination repair in osteosarcoma through the MYC-CHK1-RAD51 axis.

BACKGROUND: Osteosarcoma is a malignant bone tumor that usually affects adolescents aged 15-19 y. The DNA damage response (DDR) is significantly enhanced in osteosarcoma, impairing the effect of systemic chemotherapy. Targeting the DDR process was considered a feasible strategy benefitting osteosarcoma patients. However, the clinical application of DDR inhibitors is not impressive because of their side effects. Chinese herbal medicines with high anti-tumor effects and low toxicity in the human body have gradually gained attention. 2-Hydroxy-3-methylanthraquinone (HMA), a Chinese medicine monomer found in the extract of Oldenlandia diffusa, exerts significant inhibitory effects on various tumors. However, its anti-osteosarcoma effects and defined molecular mechanisms have not been reported. METHODS: After HMA treatment, the proliferation and metastasis capacity of osteosarcoma cells was detected by CCK-8, colony formation, transwell assays and Annexin V-fluorescein isothiocyanate/propidium iodide staining. RNA-sequence, plasmid infection, RNA interference, Western blotting and immunofluorescence assay were used to investigate the molecular mechanism and effects of HMA inhibiting osteosarcoma. Rescue assay and CHIP assay was used to further verified the relationship between MYC, CHK1 and RAD51. RESULTS: HMA regulate MYC to inhibit osteosarcoma proliferation and DNA damage repair through PI3K/AKT signaling pathway. The results of RNA-seq, IHC, Western boltting etc. showed relationship between MYC, CHK1 and RAD51. Rescue assay and CHIP assay further verified HMA can impair homologous recombination repair through the MYC-CHK1-RAD51 pathway. CONCLUSION: HMA significantly inhibits osteosarcoma proliferation and homologous recombination repair through the MYC-CHK1-RAD51 pathway, which is mediated by the PI3K-AKT signaling pathway. This study investigated the exact mechanism of the anti-osteosarcoma effect of HMA and provided a potential feasible strategy for the clinical treatment of human osteosarcoma.

Hydroxygenkwanin Increases the Sensitivity of Liver Cancer Cells to Chemotherapy by Inhibiting DNA Damage Response in Mouse Xenograft Models.

Molecules involved in DNA damage response (DDR) are often overexpressed in cancer cells, resulting in poor responses to chemotherapy and radiotherapy. Although treatment efficacy can be improved with the concomitant use of DNA repair inhibitors, the accompanying side effects can compromise the quality of life of patients. Therefore, in this study, we identified a natural compound that could inhibit DDR, using the single-strand annealing yeast-cell analysis system, and explored its mechanisms of action and potential as a chemotherapy adjuvant in hepatocellular carcinoma (HCC) cell lines using comet assay, flow cytometry, Western blotting, immunofluorescence staining, and functional analyses. We developed a mouse model to verify the in vitro findings. We found that hydroxygenkwanin (HGK) inhibited the expression of RAD51 and progression of homologous recombination, thereby suppressing the ability of the HCC cell lines to repair DNA damage and enhancing their sensitivity to doxorubicin. HGK inhibited the phosphorylation of DNA damage checkpoint proteins, leading to apoptosis in the HCC cell lines. In the mouse xenograft model, HGK enhanced the sensitivity of liver cancer cells to doxorubicin without any physiological toxicity. Thus, HGK can inhibit DDR in liver cancer cells and mouse models, making it suitable for use as a chemotherapy adjuvant.

RAD51 supports DMC1 by inhibiting the SMC5/6 complex during meiosis.

Meiosis is a fundamental process for sexual reproduction in most eukaryotes and the evolutionarily conserved recombinases RADiation sensitive51 (RAD51) and Disrupted Meiotic cDNA1 (DMC1) are essential for meiosis and thus fertility. The mitotic function of RAD51 is clear, but the meiotic function of RAD51 remains largely unknown. Here we show that RAD51 functions as an interacting protein to restrain the Structural Maintenance of Chromosomes5/6 (SMC5/6) complex from inhibiting DMC1. We unexpectedly found that loss of the SMC5/6 partially suppresses the rad51 knockout mutant in terms of sterility, pollen inviability, and meiotic chromosome fragmentation in a DMC1-dependent manner in Arabidopsis thaliana. Biochemical and cytological studies revealed that the DMC1 localization in meiotic chromosomes is inhibited by the SMC5/6 complex, which is attenuated by RAD51 through physical interactions. This study not only identified the long-sought-after function of RAD51 in meiosis but also discovered the inhibition of SMC5/6 on DMC1 as a control mechanism during meiotic recombination.

Real-time tracking reveals catalytic roles for the two DNA binding sites of Rad51.

During homologous recombination, Rad51 forms a nucleoprotein filament on single-stranded DNA to promote DNA strand exchange. This filament binds to double-stranded DNA (dsDNA), searches for homology, and promotes transfer of the complementary strand, producing a new heteroduplex. Strand exchange proceeds via two distinct three-strand intermediates, C1 and C2. C1 contains the intact donor dsDNA whereas C2 contains newly formed heteroduplex DNA. Here, we show that the conserved DNA binding motifs, loop 1 (L1) and loop 2 (L2) in site I of Rad51, play distinct roles in this process. L1 is involved in formation of the C1 complex whereas L2 mediates the C1-C2 transition, producing the heteroduplex. Another DNA binding motif, site II, serves as the DNA entry position for initial Rad51 filament formation, as well as for donor dsDNA incorporation. Our study provides a comprehensive molecular model for the catalytic process of strand exchange mediated by eukaryotic RecA-family recombinases.

The effect of thermal dose on hyperthermia-mediated inhibition of DNA repair through homologous recombination.

Hyperthermia has a number of biological effects that sensitize tumors to radiotherapy in the range between 40-44 °C. One of these effects is heat-induced degradation of BRCA2 that in turn causes reduced RAD51 focus formation, which results in an attenuation of DNA repair through homologous recombination. Prompted by this molecular insight into how hyperthermia attenuates homologous recombination, we now quantitatively explore time and temperature dynamics of hyperthermia on BRCA2 levels and RAD51 focus formation in cell culture models, and link this to their clonogenic survival capacity after irradiation (0-6 Gy). For treatment temperatures above 41 °C, we found a decrease in cell survival, an increase in sensitization towards irradiation, a decrease of BRCA2 protein levels, and altered RAD51 focus formation. When the temperatures exceeded 43 °C, we found that hyperthermia alone killed more cells directly, and that processes other than homologous recombination were affected by the heat. This study demonstrates that optimal inhibition of HR is achieved by subjecting cells to hyperthermia at 41-43 °C for 30 to 60 minutes. Our data provides a guideline for the clinical application of novel combination treatments that could exploit hyperthermia's attenuation of homologous recombination, such as the combination of hyperthermia with PARP-inhibitors for non-BRCA mutations carriers.

Applying a Weight-of-Evidence Approach to Evaluate Relevance of Molecular Landscapes in the Exposure-Disease Paradigm.

Information on polymorphisms, mutations, and epigenetic events has become increasingly important in our understanding of molecular mechanisms associated with exposures-disease outcomes. Molecular landscapes can be developed to illustrate the molecular characteristics for environmental carcinogens as well as associated disease outcomes, although comparison of these molecular landscapes can often be difficult to navigate. We developed a method to organize these molecular data that uses a weight-of-evidence approach to rank overlapping molecular events by relative importance for susceptibility to an exposure-disease paradigm. To illustrate the usefulness of this approach, we discuss the example of benzene as an environmental carcinogen and myelodysplastic syndrome (MDS) as a causative disease endpoint. Using this weight-of-evidence method, we found overlapping polymorphisms in the genes for the metabolic enzymes GST and NQO1, both of which may infer risk of benzene-induced MDS. Polymorphisms in the tumor suppressor gene, TP53, and the inflammatory cytokine gene, TNF-α, were also noted, albeit inferring opposing outcomes. The alleles identified in the DNA repair gene RAD51 indicated an increased risk for MDS in MDS patients and low blood cell counts in benzene-exposed workers. We propose the weight-of-evidence approach as a tool to assist in organizing the sea of emerging molecular data in exposure-disease paradigms.

Melatonin sensitizes human breast cancer cells to ionizing radiation by downregulating proteins involved in double-strand DNA break repair.

Radiation and adjuvant endocrine therapy are nowadays considered a standard treatment option after surgery in breast cancer. Melatonin exerts oncostatic actions on human breast cancer cells. In the current study, we investigated the effects of a combination of radiotherapy and melatonin on human breast cancer cells. Melatonin (1 mm, 10 µm and 1 nm) significantly inhibited the proliferation of MCF-7 cells. Radiation alone inhibited the MCF-7 cell proliferation in a dose-dependent manner. Pretreatment of breast cancer cells with melatonin 1 wk before radiation led to a significantly greater decrease of MCF-7 cell proliferation compared with radiation alone. Melatonin pretreatment before radiation also decreased G2 -M phase arrest compared with irradiation alone, with a higher percentage of cells in the G0 -G1 phase and a lower percentage of cells in S phase. Radiation alone diminished RAD51 and DNA-protein kinase (PKcs) mRNA expression, two main proteins involved in double-strand DNA break repair. Treatment with melatonin for 7 days before radiation led to a significantly greater decrease in RAD51 and DNA-PKcs mRNA expression compared with radiation alone. Our findings suggest that melatonin pretreatment before radiation sensitizes breast cancer cells to the ionizing effects of radiation by decreasing cell proliferation, inducing cell cycle arrest and downregulating proteins involved in double-strand DNA break repair. These findings may have implications for designing clinical trials using melatonin and radiotherapy.

The RAD51 135G>C polymorphism is related to the effect of adjuvant therapy in early breast cancer.

PURPOSE: RAD51, a central player in the response to DNA damage, has been suspected to contribute to tumour resistance to therapy. A single-nucleotide polymorphism, RAD51 135G>C, in the untranslated region of the RAD51 gene elevates breast cancer risk among BRCA2 carriers. In this study, it was investigated whether this polymorphism is related to prognosis of breast cancer and RAD51 protein expression and whether it is indicative of resistance to radiotherapy or cyclophosphamide/methotrexate/5-fluorouracil (CMF) chemotherapy. PATIENTS AND METHODS: We genotyped 306 patients with early breast cancer, who were randomised to receive post-operative radiotherapy or CMF chemotherapy, for the RAD51 135G>C polymorphism. RAD51 protein expression was evaluated with immunohistochemistry. RESULTS: 15.4 % of the patients had at least one C-allele (three were C homozygotes). There was no correlation between genotype and protein expression. Patients who were G homozygotes benefitted from radiotherapy with decreased risk of local recurrences (RR = 0.32, 95 % C.I. 0.16-0.64, p = 0.001). CMF chemotherapy reduced the risk of distant recurrence for patients carrying at least one C-allele (RR = 0.29, 95 % C.I. 0.10-0.88, p = 0.03), whereas G homozygotes had no benefit from chemotherapy. There was a significant interaction between chemotherapy and genotype (p = 0.02). CONCLUSION: The results suggest that the RAD51 135G>C polymorphism predicts CMF chemotherapy effect in early breast cancer.

Structure of Yin Yang 1 oligomers that cooperate with RuvBL1-RuvBL2 ATPases.

Yin Yang 1 (YY1) is a transcription factor regulating proliferation and differentiation and is involved in cancer development. Oligomers of recombinant YY1 have been observed before, but their structure and DNA binding properties are not well understood. Here we find that YY1 assembles several homo-oligomeric species built from the association of a bell-shaped dimer, a process we characterized by electron microscopy. Moreover, we find that YY1 self-association also occurs in vivo using bimolecular fluorescence complementation. Unexpectedly, these oligomers recognize several DNA substrates without the consensus sequence for YY1 in vitro, and DNA binding is enhanced in the presence of RuvBL1-RuvBL2, two essential AAA+ ATPases. YY1 oligomers bind RuvBL1-RuvBL2 hetero-oligomeric complexes, but YY1 interacts preferentially with RuvBL1. Collectively, these findings suggest that YY1-RuvBL1-RuvBL2 complexes could contribute to functions beyond transcription, and we show that YY1 and the ATPase activity of RuvBL2 are required for RAD51 foci formation during homologous recombination.

Arabidopsis meiotic crossover hot spots overlap with H2A.Z nucleosomes at gene promoters.

PRDM9 directs human meiotic crossover hot spots to intergenic sequence motifs, whereas budding yeast hot spots overlap regions of low nucleosome density (LND) in gene promoters. To investigate hot spots in plants, which lack PRDM9, we used coalescent analysis of genetic variation in Arabidopsis thaliana. Crossovers increased toward gene promoters and terminators, and hot spots were associated with active chromatin modifications, including H2A.Z, histone H3 Lys4 trimethylation (H3K4me3), LND and low DNA methylation. Hot spot-enriched A-rich and CTT-repeat DNA motifs occurred upstream and downstream, respectively, of transcriptional start sites. Crossovers were asymmetric around promoters and were most frequent over CTT-repeat motifs and H2A.Z nucleosomes. Pollen typing, segregation and cytogenetic analysis showed decreased numbers of crossovers in the arp6 H2A.Z deposition mutant at multiple scales. During meiosis, H2A.Z forms overlapping chromosomal foci with the DMC1 and RAD51 recombinases. As arp6 reduced the number of DMC1 or RAD51 foci, H2A.Z may promote the formation or processing of meiotic DNA double-strand breaks. We propose that gene chromatin ancestrally designates hot spots within eukaryotes and PRDM9 is a derived state within vertebrates.

Haploid meiosis in Arabidopsis: double-strand breaks are formed and repaired but without synapsis and crossovers.

Two hallmark features of meiosis are i) the formation of crossovers (COs) between homologs and ii) the production of genetically-unique haploid spores that will fuse to restore the somatic ploidy level upon fertilization. In this study we analysed meiosis in haploid Arabidopsis thaliana plants and a range of haploid mutants to understand how meiosis progresses without a homolog. Extremely low chiasma frequency and very limited synapsis occurred in wild-type haploids. The resulting univalents segregated in two uneven groups at the first division, and sister chromatids segregated to opposite poles at the second division, leading to the production of unbalanced spores. DNA double-strand breaks that initiate meiotic recombination were formed, but in half the number compared to diploid meiosis. They were repaired in a RAD51- and REC8-dependent manner, but independently of DMC1, presumably using the sister chromatid as a template. Additionally, turning meiosis into mitosis (MiMe genotype) in haploids resulted in the production of balanced haploid gametes and restoration of fertility. The variability of the effect on meiosis of the absence of homologous chromosomes in different organisms is then discussed.

Radiosensitization effects of berberine on human breast cancer cells.

Berberine, an isoquinoline derivative alkaloid, has recently been shown to have antitumor activity. The present study aimed to investigate the effects of the concomitant administration of berberine and radiation on breast cancer. The effects of berberine on the radiosensitivity of MCF-7 and MDA-MB-468 cells were evaluated by using cell clonogenic assays. Cells pre-treated with berberine or dimethyl sulfoxide (DMSO) for 24 h were irradiated using a Faxitron Cabinet X-ray System to deliver the indicated doses (0, 1, 2, 3 and 4 Gy). Changes in cell cycle distribution were determined by flow cytometry. γ-H2AX foci were detected by immunofluorescence staining. The levels of Ku70, Ku86 and RAD51 proteins were evaluated by western blot analysis. We observed that berberine increased the MCF-7 and MDA-MB-468 cell radiosensitivity with cell clonogenic assays. the radiation-induced G2/M cell cycle delay was reduced in the MCF-7 cells pre-teated with berberine. Berberine pre-treatment prolonged the persistence of DNA double-strand breaks in the MCF-7 cell line. In comparison with the control cells, the protein levels of RAD51 were decreased in the MCF-7 and MDA-MB-468 cells treated with berberine, and in the cells pre-treated with 15 µM berberine for 24 h, the level of RAD51 protein decreased significantly at the indicated time-points (0, 2, 6 and 24 h) following X-ray exposure. In conclusion, berberine sensitizes human breast cancer cells to ionizing radiation by inducing cell cycle arrest and the downregulation of the homologous recombination repair protein, RAD51. Berberine may be a promising radiosensitizer for the treatment of breast cancer.

The rice RAD51C gene is required for the meiosis of both female and male gametocytes and the DNA repair of somatic cells.

The RecA/RAD51 family of rice (Oryza sativa) consists of at least 13 members. However, the functions of most of these members are unknown. Here the functional characterization of one member of this family, RAD51C, is reported. Knockout (KO) of RAD51C resulted in both female and male sterility in rice. Transferring RAD51C to the RAD51C-KO line restored fertility. Cytological analyses showed that the sterility of RAD51C-KO plants was associated with abnormal early meiotic processes in both megasporocytes and pollen mother cells (PMCs). PMCs had an absence of normal pachytene chromosomes and had abnormal chromosome fragments. The RAD51C-KO line showed no obvious difference from wild-type plants in mitosis in the anther wall cells, which was consistent with the observation that the RAD51C-KO line did not have obviously abnormal morphology during vegetative development. However, the RAD51C-KO line was sensitive to different DNA-damaging agents. These results suggest that RAD51C is essential for reproductive development by regulating meiosis as well as for DNA damage repair in somatic cells.

Effects of emodin extracted from Chinese herbs on proliferation of non-small cell lung cancer and underlying mechanisms.

To aim of this was to observe emodin-mediated cytotoxicity and its influence on Rad51 and ERCC1 expressionin non-small cell lung cancer (NSCLC). NSCLC cells were cultured in vitro with emodin at various concentrations (0, 25, 50, 75 and 100 µmol/L) for 48 h and the proliferation inhibition rate was determined by the MTT method. Then, NSCLC were treated with emodin (SK-MES-1 40 µmol/L, A549 70 µmol/L) or 20 µmol/L U0126 (an ERK inhibitor) for 48 h, or with various concentrations of emodin for 48 h and the protein and mRNA expressions of ERCC1 and Rad51 were determined by RT-PCR and Western blot assay, respectively. Emodin exerted a suppressive effect on the proliferation of NSCLC in a concentration dependent manner. Protein and mRNA expression of ERCC1 and Rad51 was also significantly decreased with the dose. Vacuolar degeneration was observed in A549 and SK-MES-1 cell lines after emodin treatment by transmission electron microscopy. Emodin may thus inhibited cell proliferation in NSCLC cells by downregulation ERCC1 and Rad51.

Selective inhibition of BRCA2-deficient mammary tumor cell growth by AZD2281 and cisplatin.

PURPOSE: To assess efficacy of the novel, selective poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor AZD2281 against newly established BRCA2-deficient mouse mammary tumor cell lines and to determine potential synergy between AZD2281 and cisplatin. EXPERIMENTAL DESIGN: We established and thoroughly characterized a panel of clonal cell lines from independent BRCA2-deficient mouse mammary tumors and BRCA2-proficient control tumors. Subsequently, we assessed sensitivity of these lines to conventional cytotoxic drugs and the novel PARP inhibitor AZD2281. Finally, in vitro combination studies were done to investigate interaction between AZD2281 and cisplatin. RESULTS: Genetic, transcriptional, and functional analyses confirmed the successful isolation of BRCA2-deficient and BRCA2-proficient mouse mammary tumor cell lines. Treatment of these cell lines with 11 different anticancer drugs or with gamma-irradiation showed that AZD2281, a novel and specific PARP inhibitor, caused the strongest differential growth inhibition of BRCA2-deficient versus BRCA2-proficient mammary tumor cells. Finally, drug combination studies showed synergistic cytotoxicity of AZD2281 and cisplatin against BRCA2-deficient cells but not against BRCA2-proficient control cells. CONCLUSION: We have successfully established the first set of BRCA2-deficient mammary tumor cell lines, which form an important addition to the existing preclinical models for BRCA-mutated breast cancer. The exquisite sensitivity of these cells to the PARP inhibitor AZD2281, alone or in combination with cisplatin, provides strong support for AZD2281 as a novel targeted therapeutic against BRCA-deficient cancers.