The study aimed to investigate the effect of inhibition of poly(ADP-ribose) polymerase-1 (PARP-1) activity on tau phosphorylation in HEK293/tau441 cells and its mechanism. HEK293/tau441 cells were treated with 3-aminobenzamide (3-AB), a PARP-1 inhibitor, at different doses (0.5, 1, 2, 4 mmol/L). After 24 h, the cell morphology was observed under phase contrast microscope, tau phosphorylation level in different sites (tau-1, tau-5, Thr231) and the activity of glycogen synthase kinase 3 (GSK-3) were detected by Western blotting. The results showed: (1) 3-AB at different doses failed to change the morphology of cells; (2) The 3-AB-induced decrease in activity of PARP-1 resulted in increase of unphosphorylation level in tau-1(Ser195/198/199/202) sites; (3) The phosphorylation of tau was decreased in Thr231 site, while the total tau was slightly changed after 3-AB treatment; (4) With the increased phosphorylation of GSK-3 at Ser9 site, the activity of GSK-3 was decreased after 3-AB treatment. The results suggest that the inhibition of PARP-1 by 3-AB could decrease tau phosphorylation in HEK293/tau441 cells probably through decreasing GSK-3 activity.
Benzamides ; pharmacology ; Depression, Chemical ; Glycogen Synthase Kinase 3 ; metabolism ; HEK293 Cells ; Humans ; Phosphorylation ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerase Inhibitors ; Poly(ADP-ribose) Polymerases ; metabolism ; tau Proteins ; metabolism

To construct the pharmacophore model of the poly (ADP-ribose) polymerase-1 inhibitor and to investigate the possible inhibitory mechanisms, ten pharmacophore models of PARP-1 inhibitor were established from the training set of thirty-eight PARP-1 inhibitors with conformer analysis and pharmacophore mapping by using the Catalyst software. Based on the mechanism of action and the known structure-activity relationship of PARP-1 inhibitor, an optimal pharmacophore model including two hydrogen-bonding acceptors and two aromatic hydrophobic core was confirmed. The reliability of the optimal pharmacophore model is preferably with RMS = 0.46, Correl = 0.91, Weight = 2.06, and Config = 15.97. This pharmacophore model not only provided some information about the interaction between enzyme and compound, but also showed excellent forecast ability and contributes to design the PARP-1 inhibitors with undiscovered structure.
Computer-Aided Design ; Drug Design ; Enzyme Inhibitors ; chemistry ; pharmacology ; Models, Molecular ; Molecular Structure ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerase Inhibitors ; Poly(ADP-ribose) Polymerases ; chemistry ; Protein Conformation

PARP1 is an important part of DNA repair machinery. In recent years, PARP1 as novel anti-cancer therapeutic target has been broadly explored. In this study, we expressed hPARP1 enzyme in the baculovirus system and tested its activity. We inserted hPARP1 gene into the pFastBac1, a baculovirus transfer vector and then transformed it into DH10Bac containing a shuttle vector of Bacmid. After co-transfecting the recombinant plasmid into Sf9 insect cells, the expressed hPARP1 was purified by 3-aminobezamide affinity chromatography. The expression of hPARPI was visualized by SDS-PAGE and Western blotting; the activity of expressed and purified hPARP1 was confirmed by the reaction of consumption of NAD+ by hPARP1 in vitro. After the purification by 3-aminobezamide affinity column, 3.2 mg protein was obtained and its specific activity was 1.988 nmol/(min x microg).
Animals ; Baculoviridae ; genetics ; Blotting, Western ; Electrophoresis, Polyacrylamide Gel ; Genetic Vectors ; Humans ; Insecta ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; biosynthesis ; Recombinant Proteins ; Sf9 Cells ; Transfection

PARP [poly(ADP-ribose)polymerase] represents a novel potential target in cancer therapy. It is involved in a DNA repair process by catalyzing the transfer of ADP-ribose units from NAD to a number of its substrate proteins. In this work, a series of novel azaindole derivatives was designed and synthesized. Moreover, 16 target molecules were screened and 8 compounds displayed inhibitory activity against PARP-1. It has been demonstrated that these azaindoles bearing cycloamine substituents at 2-position were active to both PARP-1 and PARP-2.
Antineoplastic Agents ; chemical synthesis ; chemistry ; pharmacology ; Aza Compounds ; chemical synthesis ; chemistry ; pharmacology ; Indoles ; chemical synthesis ; chemistry ; pharmacology ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; metabolism

OBJECTIVETo investigate DNA methylation variation in human cells induces by B(a)P, and to explore the role of PARP1 during this process.

METHODSThe changes of DNA methylation of 16HBE and its PARP1-deficient cells exposed to B(a)P (1.0, 2.0, 5.0, 10.0, 15.0, 30.0 µmol/L) were investigated by immunofluorescence and high performance capillary electrophoresis, and simultaneously, the expression level of PARP 1 and DNMT 1 were monitored dynamically.

RESULTSThe percentage of methylated DNA of overall genome (mCpG%) in 16HBE and 16HBE-shPARP1 cells were separately (4.04 ± 0.08)% and (9.69 ± 0.50)%. After being treated by 5-DAC for 72 hours, mCpG% decreased to (3.15 ± 0.14)% and (6.07 ± 0.54)%. After both being exposed to B(a)P for 72 hours, the mCpG% in 16HBE group (ascending rank) were separately (5.10 ± 0.13), (4.25 ± 0.10), (3.91 ± 0.10), (4.23 ± 0.27), (3.70 ± 0.15), (3.08 ± 0.07); while the figures in 16HBE-shPARP1 group (ascending rank) were respectively (10.63 ± 0.60), (13.08 ± 0.68), (9.75 ± 0.55), (7.32 ± 0.67), (6.90 ± 0.49) and (6.27 ± 0.21). The difference of the results was statistically significant (F values were 61.67 and 60.91, P < 0.01). For 16HBE group, expression of PARP 1 and DNMT 1 were 141.0%, 158.0%, 167.0%, 239.0%, 149.0%, 82.9% and 108.0%, 117.0%, 125.0%, 162.0%, 275.0%, 233.0% comparing with the control group, whose difference also has statistical significance (t values were 11.45, 17.32, 32.24, 33.44, 20.21 and 9.87, P < 0.01). For 16HBE-shPARP1 group, expression of PARP 1 and DNMT 1 were 169.0%, 217.0%, 259.0%, 323.0%, 321.0%, 256.0% and 86.0%, 135.0%, 151.0%, 180.0%, 229.0%, 186.0% comparing with the control group, with statistical significance (t values were 9.06, 15.92, 22.68, 26.23, 37.19 and 21.15, P < 0.01). When the dose of B(a)P reached 5.0 µmol/L, the mRNA expression of DNMT 1 in 16HBE group (ascending rank) were 125.0%, 162.0%, 275.0%, 233.0% times of it in control group, with statistical significance (t values were 12.74, 24.92, 55.11, 59.07, P < 0.01); while the dose of B(a)P reached 2.0 µmol/L, the mRNA expression of DNMT 1 in 16HBE-shPARP1 group were 135.0%, 151.0%, 180.0%, 229.0%, 186.0% of the results in control group, and the differences were statistically significant (t values were 23.82, 40.17, 32.69, 74.85, 46.76, P < 0.01).

CONCLUSIONThe hypomethylation of 16HBE cells induced by B(a)P might be one important molecular phenomenon in its malignant transformation process. It suggests that PARP1 could regulate DNA methylation by inhibiting the enzyme activity of DNMT1, and this effect could be alleviated by PARP1-deficiency.

Benzo(a)pyrene ; adverse effects ; Cell Line ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; genetics ; metabolism ; DNA Damage ; DNA Methylation ; Epithelial Cells ; drug effects ; metabolism ; Humans ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; genetics ; metabolism

OBJECTIVETo investigate the DNA methylation changes induced by hydroquinone (HQ) in human bronchial epithelial cells and to explore the role of poly (ADP-ribose) polymerase-l (PARP-l) in this process.

METHODSHuman bronchial epithelial 16HBE cells and PARP-l-deficient 16HBE cells (16HBE-shPARP-l cells) were exposed to HQ (10, 20, 40, 60, and 80 µmol/L) for 48h, while control cells were treated with an equal volume of PBS solution. The changes in genomic DNA methylation were investigated by high-performance capillary electrophoresis, and the expression levels of PARP-l and DNA methyltransferase 1 (DNMT1) were measured.

RESULTSThe percentages of methylated DNA of overall genome (mCpG%) in 16HBE and 16HBE-shPARP-l cells were 4.89%±0.07% and 9.53%±0.51%, respectively; after treatment with 5-aza-2'-deoxycytidine for 72 h, mCpG% decreased to 3.07±0.12% and 6.34%±0.3%, respectively. The one-way analysis of variance revealed significant differences in mCpG% between the cells exposed to different concentrations of HQ in both 16HBE and 16HBE-shPARP-l groups (F = 61.25, P < 0.01; F = 60.36, P < 0.01). For 16HBE cells treated with HQ (10, 20, 40, 60, and 80 µmol/L), the mRNA expression levels of PARP-1 were 145.0%, 159.0%, 169.0%, 215.0%, and 236.0%, respectively, compared with those in the control group, with significant differences (P < 0.01 for all); for 16HBE-shPARP-l cells treated with HQ (10, 20, 40, 60, and 80 µmol/L), the mRNA expression levels of PARP-l were 170.0%, 223.0%, 264.0%, 327.0%, and 320.0%, respectively, compared with those in the control group, with significant differences (P < 0.01 for all). When the dose of HQ reached 20, 40, 60, and 80 µmol/L, the mRNA expression levels of DNMT1 in 16HBE group were 114.0%, 126.0%, 136.0%, and 162.0%, respectively, compared with those in the control group, with significant differences (P < 0.01 for all); when the dose of HQ reached 10, 20, 40, 60, and 80 µmol/L, the mRNA expression levels of DNMT1 in the 16HBE-shPARP-l group were 141.0%, 165.2%, 186.9%, 202.1%, and 217.3%, respectively, compared with those in the control group, with significant differences (P < 0.01 for all).

CONCLUSIONHQ can induce hypomethylation in 16HBE cells, and PARP-1 can regulate DNA methylation in 16HBE cells by influencing the expression and activity of DNMT1.

Cells, Cultured ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; metabolism ; DNA Damage ; DNA Methylation ; Epithelial Cells ; metabolism ; Humans ; Hydroquinones ; toxicity ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; metabolism

OBJECTIVETo study hPARP1 genetic polymorphism in southern Chinese Han and Miao populations.

METHODSBlood samples from 187 and 210 southern healthy Han and Miao populations were collected. The mutations of exons 12,13,16 and 17 of hPARP1 gene were investigated by PCR-single-strand conformation polymorphism(SSCP).

RESULTSFragments of 253 bp,313 bp,175 bp,362 bp within exons 12,13,16 and 17 respectively of hPARP1 gene were amplified by multiple PCR. An SSCP variant in exons 12,13,16 and 17 of PARP1 gene in 187 healthy Han and 210 healthy Miao individuals was identified. Seven single-base substitutions compared with the sequence of PARP1 gene were identified: a T to C transition in exon 12 (Phe548Ser), a G to T transition in exon 13 (Ala683Ser), a G to T transition in exon 16 (Asp798Tyr), and a A to G transition in exon 17 (His808Arg).

CONCLUSIONThere were polymorphism sites in exons 12,13,16,17 of hPARP1 gene in southern Chinese Han and Miao populations; these results may be useful for the establishment of PARP1 genotyping, and these newly described PARP1 alleles would be advantageous indicators for population studies.

Adult ; Alleles ; China ; Exons ; genetics ; Female ; Gene Frequency ; Genotype ; Humans ; Male ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; genetics ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide ; genetics ; Polymorphism, Single-Stranded Conformational

OBJECTIVETo study the effect of silicon dioxide nanoparticles on the expression and promoter region CpG islands methylation of (Poly [ADP-ribose] polymerase 1, PARP-1) gene in human HaCaT Cell.

METHODSHaCaT Cells were treated with nm-SiO₂at 0, 2.5, 5 and 10 µg/mL and micro-SiO₂at 10 µg/ml for 24 h and DAC treatment was given at 10 µg/ml group for 48 h. Real-time PCR and western blot assay was used to detect the expression of PARP-1 mRNA and protein. BSP (Bisulfite Pyrosequence, BSP) assay was used to detect the promoter region CpG islands methylation status of PARP-1 gene.

RESULTSAfter exposure to nano-SiO₂particles, compared to CTRL group, the mRNA and protein expression of PARP-1 in micro-SiO₂and 2.5 µg/ml group unchanged, but he mRNA and protein expression of PARP-1 in 5, 10 µg/ml as well as DAC group was down-regulated and there are statistical significance between CTRL group and 5, 10 µg/ml as well as DAC group and the PARP-1 promoter region CpG islands showed methylation.

CONCLUSIONnano-SiO₂can down-regulate PARP-1 expression in HaCaT Cell and this is associated with the change in the methylation of PARP-1 gene promoter region CpG islands induced by nano-SiO₂particles.

Cell Line, Tumor ; CpG Islands ; DNA Methylation ; Humans ; Nanoparticles ; adverse effects ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; metabolism ; Promoter Regions, Genetic ; RNA, Messenger ; metabolism ; Silicon Dioxide ; adverse effects

This study was purposed to explore the inhibitory effect of Curcumin on growth of retinoic acid-resistant acute promyelocytic leukemia (APL) cells and its mechanism. The NB4-R1, an APL cell line resistant to retinoic acid, was used as a model. The growth level of NB4-R1 was detected by MTT assay, the morphologic features of cells were observed by light microscopy, the mitochondrial transmembrane potential was determined by flow cytometry, the expressions of apoptosis-related proteins procaspase 3, caspase 3, PARP and BCL-XL were measured by Western blot. The results indicated that the sensitivity of NB4-R1 to Curcumin was consistent with NB4 though NB4-R1 was resistant to retinoic acid, Curcumin displayed inhibitory effect on growth of NB4-R1 in time-and concentration-dependent manners. The morphologic observation showed existence of apoptotic bodies in NB4-R1 cells treated with 20 micromol/L of Curcumin. The flow cytometry indicated that the mitochondrial transmembrane potential in NB4-R1 cells treated with 20 micromol/L of Curcumin obviously decreased. The Western blot detection revealed that expressions of pro-caspase 3 and BCL-XL were down-regulated, expressions of caspase 3 and sheared PAPP were up-regulated in NB4-R1 cells treated with 20 micromol/L of Curcumin. It is concluded that the Curcumin can inhibit the growth and induce the apoptosis of NB4-R1.
Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Curcumin ; pharmacology ; Humans ; Leukemia, Promyelocytic, Acute ; metabolism ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; metabolism ; Tretinoin ; pharmacology ; bcl-X Protein ; metabolism

Poly(ADP-ribose) polymerase-1 (PARP-1) has emerged as a promising anticancer drug target due to its key role in the DNA repair process. It can polymerize ADP-ribose units on its substrate proteins which are involved in the regulation of DNA repair. In this work, a novel series of para-substituted 1-benzyl-quinazoline-2, 4 (1H, 3H)-diones was designed and synthesized, and the inhibitory activities against PARP-1 of compounds 7a-7e, 8a-8f, 9a-9c and 10a-10c were evaluated. Of all the tested compounds, nine compounds displayed inhibitory activities with IC50 values ranging from 4.6 to 39.2 micromol x L(-1). In order to predict the binding modes of the potent molecules, molecular docking was performed using CDOCKER algorithm, and that will facilitate to further develop more potent PARP-1 inhibitors with a quinazolinedione scaffold.
Antineoplastic Agents ; chemical synthesis ; chemistry ; pharmacology ; Drug Design ; Enzyme Inhibitors ; chemical synthesis ; chemistry ; pharmacology ; Molecular Docking Simulation ; Molecular Structure ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; Quinazolinones ; chemical synthesis ; chemistry ; pharmacology ; Structure-Activity Relationship