RESUMO
Prostate cancer (PCa) is a common cancer and a major cause of cancer-related death worldwide in men, necessitating novel targets for cancer therapy. High expression of Bloom's syndrome protein (BLM) helicase is associated with the occurrence and development of PCa. Therefore, the identification and development of new BLM inhibitors may be a new direction for the treatment of PCa. Here, we identified a novel inhibitor by molecular docking and put it to systematic evaluation via various experiments, AO/854, which acted as a competitive inhibitor that blocked the BLM-DNA interaction. Cellular evaluation indicated that AO/854-suppressed tumor growth and metastasis in PC3 cells by enhancing DNA damage, phosphorylating Chk1/Chk2, and altering the p53 signaling pathway. Collectively, the study highlights the potential of BLM as a therapeutic target in PCa and reveals a distinct mechanism by which AO/854 competitively inhibits the function of BLM.
Assuntos
Síndrome de Bloom , Neoplasias da Próstata , Humanos , Masculino , Síndrome de Bloom/genética , Síndrome de Bloom/metabolismo , Simulação de Acoplamento Molecular , RecQ Helicases/genética , RecQ Helicases/metabolismo , DNA/metabolismo , Dano ao DNA , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genéticaRESUMO
Using standard DNA-damaging medicines with DNA repair inhibitors is a promising anticancer tool to achieve better therapeutic responses and reduce therapy-related side effects. Cell viability assay, neutral comet assay, western blotting (WB), and cell cycle and apoptosis analysis were used to determine the synergistic effect and mechanism of ML216, a Bloom syndrome protein (BLM) helicase inhibitor, and cisplatin (CDDP), a DNA-crosslinking agent, in PCa cells. Based on the online database research, our findings revealed that BLM was substantially expressed in PCa, which is associated with a bad prognosis for PCa patients. The combination of ML216 and CDDP improved the antiproliferative properties of three PCa cell lines. As indicated by the increased production of γH2AX and caspase-3 cleavage, ML216 significantly reduced the DNA damage-induced high expression of BLM, making PC3 more susceptible to apoptosis and DNA damage caused by CDDP. Furthermore, the combination of ML216 and CDDP increased p-Chk1 and p-Chk2 expression. The DNA damage may have triggered the ATR-Chk1 and ATM-Chk2 pathways simultaneously. Our results demonstrated that ML216 and CDDP combination therapy exhibited synergistic effects, and combination chemotherapy could be a novel anticancer tactic.
Assuntos
Antineoplásicos , Cisplatino , Humanos , Cisplatino/farmacologia , Antineoplásicos/farmacologia , RecQ Helicases/genética , Apoptose , Dano ao DNA , DNA/farmacologia , Linhagem Celular TumoralRESUMO
G-quadruplex DNA (G4DNA) structure, which widely exists in the chromosomal telomeric regions and oncogenic promoter regions, plays a pivotal role in extending telomeric DNA with the help of telomerase in human cells. Bloom (BLM) helicase, a crucial member of the family of genome surveillance proteins, plays an essential role in DNA metabolic and repair pathways, including DNA replication, repair, transcription, recombination during chromosome segregation, and assuring telomere stability. The unwinding of G4DNA requires the participation of DNA helicase, which is crucial for maintaining chromosomal stability in cancer cells. Using fluorescence polarization and the electrophoretic mobility shift assay (EMSA), this study aimed to investigate the DNA-binding and unwinding properties of BLM helicase, cloned and purified from prostate cancer cells, toward G4DNA. The results revealed that BLM helicase derived from prostate cancer cells could bind and unwind G4DNA. The molecular affinity of bond between G4DNA and the helicase was dependent on the single-stranded DNA (ssDNA) terminals in G4DNA; the helicase was effectively bound to the G4DNA when the helicase monomer sufficiently covered approximately 10 nucleotides at the 3' or 5' ssDNA tail of G4DNA. For the unwinding of G4DNA, there was an apparent requirement of a 3' ssDNA tail and ATP; a G4DNA with only a 3' ssDNA tail was identified to be the most suitable substrate to be unwound by BLM helicase and required 3' ssDNA tails of at least 10 nt in length for efficient unwinding. Besides, BLM helicase was loosely bound and partly unwound the blunt-ended G4DNA. Although further mechanistic studies are warranted, the experimental results presented in this study are beneficial to further our understanding of the functional implication of BLM helicase in prostate cancer cells.
Assuntos
DNA/química , Quadruplex G , Neoplasias da Próstata/metabolismo , RecQ Helicases/metabolismo , Telômero/metabolismo , DNA/genética , DNA/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Humanos , Cinética , Masculino , Modelos Moleculares , Conformação de Ácido Nucleico , Células PC-3 , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Ligação Proteica , RecQ Helicases/genética , Especificidade por Substrato , Telômero/genéticaRESUMO
Aging has been considered a phenomenon that can be only applied to cells or organisms. Here, we show that RecQ helicase from E. coli displays an aging phenomenon: this macromolecular motor loses its structure and function after hydrolyzing a certain number of ATP molecules. The aging process was only triggered by repeated catalytic cycles. These observations lead to a new concept: macromolecule aging.
RESUMO
OBJECTIVE: To study the effect of fleroxacin (FLRX) on biological properties of Bloom (BLM) helicase catalytic core (BLM642-1290 helicase) in vitro and the molecular mechanism of interaction between the two molecules. METHODS: DNA-binding and unwinding activities of BLM642-1290 helicase were assayed by fluorescence polarization and gel retardation assay under conditions that the helicase was subjected to different concentrations of FLRX. Effect of FLRX on helicase ATPase activity was analyzed by phosphorus-free assay based on a colorimetric estimation of ATP hydrolysis-produced inorganic phosphate. Molecular mechanism of interaction between the two molecules was assayed by ultraviolet and fluorescence spectra. RESULTS: The DNA unwinding and ATPase activities of BLM642-1290 helicase were inhibited whereas the DNA-binding activity was promoted in vitro. A BLM-FLRX complex was formed through one binding site, electrostatic and hydrophobic interaction force. Moreover, the intrinsic fluorescence of the helicase was quenched by FLRX as a result of non-radioactive energy transfer. The biological activity of helicase was affected by FLRX, which may be through an allosteric mechanism and stabilization of enzyme conformation in low helicase activity state, disruption of the coupling of ATP hydrolysis to unwinding, and blocking helicase translocation on DNA strands. CONCLUSION: FLRX may affect the biological activities and conformation of BLM642-1290 helicase, and DNA helicase may be used as a promising drug target for some diseases.
Assuntos
Fleroxacino/farmacologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , RecQ Helicases/antagonistas & inibidores , DNA/metabolismo , RecQ Helicases/metabolismo , Espectrometria de Fluorescência , Espectrofotometria UltravioletaRESUMO
Bloom's syndrome (BS) is a rare human autosomal recessive disorder characterized by a strong predisposition to a wide range of cancers commonly affecting the general population. Understanding the functioning mechanism of the BLM protein may provide the opportunity to develop new effective therapy strategies. In this work, we studied the DNA unwinding kinetic mechanism of the helicase core of the BLM protein using various stopped-flow assays. We show that the helicase core of BLM unwinds duplex DNA as monomers even under conditions strongly favoring oligomerization. An unwinding rate of approximately 20 steps per second and a step size of 1 bp have been determined. We have observed that the helicase has a very low processivity. From dissociation and inhibition experiments, we have found that during its ATP hydrolysis cycle in DNA unwinding the helicase tends to dissociate from the DNA substrate in the ADP state. The experimental results imply that the BLM helicase core may unwind duplex DNA in an inchworm manner.
Assuntos
DNA/química , RecQ Helicases/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Síndrome de Bloom/enzimologia , Síndrome de Bloom/metabolismo , DNA/metabolismo , Dimerização , Humanos , Hidrólise , Cinética , RecQ Helicases/químicaRESUMO
The RecQ helicase family is highly conserved from bacteria to men and plays a conserved role in the preservation of genome integrity. Its deficiency in human cells leads to a marked genomic instability that is associated with premature aging and cancer. To determine the thermodynamic parameters for the interaction of Escherichia coli RecQ helicase with DNA, equilibrium binding studies have been performed using the thermodynamic rigorous fluorescence titration technique. Steady-state fluorescence anisotropy measurements of fluorescein-labeled oligonucleotides revealed that RecQ helicase bound to DNA with an apparent binding stoichiometry of 1 protein monomer/10 nucleotides. This stoichiometry was not altered in the presence of AMPPNP (adenosine 5'-(beta,gamma-imido) triphosphate) or ADP. Analyses of RecQ helicase interactions with oligonucleotides of different lengths over a wide range of pH, NaCl, and nucleic acid concentrations indicate that the RecQ helicase has a single strong DNA binding site with an association constant at 25 degrees C of K=6.7 +/- 0.95 x 10(6) M(-1) and a cooperativity parameter of omega=25.5 +/- 1.2. Both single-stranded DNA and double-stranded DNA bind competitively to the same site. The intrinsic affinities are salt-dependent, and the formation of DNA-helicase complex is accompanied by a net release of 3-4 ions. Allosteric effects of nucleotide cofactors on RecQ binding to DNA were observed only for single-stranded DNA in the presence of 1.5 mM AMPPNP, whereas both AMPPNP and ADP had no detectable effect on double-stranded DNA binding over a large range of nucleotide cofactor concentrations.
Assuntos
Adenosina Trifosfatases/química , DNA Helicases/química , DNA/química , Escherichia coli/enzimologia , Adenosina Trifosfatases/metabolismo , Sítio Alostérico , Anisotropia , Sítios de Ligação , Ligação Competitiva , DNA Helicases/metabolismo , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/química , Relação Dose-Resposta a Droga , Concentração de Íons de Hidrogênio , Cinética , Oligonucleotídeos/química , Ligação Proteica , RecQ Helicases , Sefarose/química , Temperatura , Termodinâmica , Fatores de TempoRESUMO
The RecQ helicases belong to an important family of highly conserved DNA helicases that play a key role in chromosomal maintenance, and their defects have been shown to lead to several disorders and cancer in humans. In this work, the conformational and functional properties of the Escherichia coli RecQ helicase have been determined using a wide array of biochemical and biophysical techniques. The results obtained clearly indicate that E. coli RecQ helicase is monomeric in solution up to a concentration of 20 microM and in a temperature range between 4 and 37 degrees C. Furthermore, these properties are not affected by the presence of ATP, which is strictly required for the unwinding and translocating activity of the protein, or by its nonhydrolyzable analogue 5'-adenylyl-beta,gamma-imidodiphosphate. Consistent with the structural properties, functional analysis shows that both DNA unwinding activity and single-stranded DNA-stimulated ATPase specific activity were independent of RecQ concentration. The monomeric state was further confirmed by the ATPase-deficient mutants of RecQ protein. The rate of unwinding was unchanged when the wild type RecQ helicase was mixed with the ATPase-deficient mutants, indicating that nonprotein-protein interactions were involved in the unwinding processes. Taken together, these results indicate that RecQ helicase functions as a monomer and provide new data on the structural and functional properties of RecQ helicase that may help elucidate its mechanism action.