OBJECTIVES: To investigate the effect of borneol on cutaneous toxicity of gilteritinib and to explore possible compounds that can intervene with the cutaneous toxicity. METHODS: C57BL/6J male mice were given gilteritinib by continuous gavage for 28 d and the damage to keratinocytes in the skin tissues was observed with hematoxylin and eosin (HE) staining, TUNEL assay and immunohistochemistry. Human keratinocytes HaCaT were treated with gilteritinib, and cell death and morphological changes were examined by SRB staining and microscopy; apoptosis of HaCaT cells was examined by Western blotting, flow cytometry with propidium iodide/AnnexinⅤ double staining and immunofluorescence; the accumulation of cellular reactive oxygen species (ROS) was examined by flow cytometry with DCFH-DA. Compounds that can effectively intervene the cutaneous toxicity of gilteritinib were screened from a natural compound library using SRB method, and the intervention effect of borneol on gilteritinib cutaneous toxicity was further investigated in HaCaT cells and C57BL/6J male mice. RESULTS: In vivo studies showed pathological changes in the skin with apoptosis of keratinocytes in the stratum spinosum and stratum granulosum in the modeling group. Invitro studies showed apoptosis of HaCaT cells, significant up-regulation of cleaved poly (ADP-ribose) polymerase (c-PARP) and gamma-H2A histone family member X (γ-H2AX) levels, and increased accumulation of ROS in gilteritinib-modeled skin keratinocytes compared with controls. Screening of the natural compound library revealed that borneol showed excellent intervention effects on the death of HaCaT cells. In vitro, cell apoptosis was significantly reduced in the borneol+gilteritinib group compared to the gilteritinib control group. The levels of c-PARP, γ-H2AX and ROS in cells were significantly decreased. In vivo, borneol alleviated gilteritinib-induced skin pathological changes and skin cell apoptosis in mice. CONCLUSIONS Gilteritinib induces keratinocytes apoptosis by causing intracellular ROS accumulation, resulting in cutaneous toxicity. Borneol can ameliorate the cutaneous toxicity of gilteritinib by reducing the accumulation of ROS and apoptosis of keratinocytes in the skin tissue.
Male ; Humans ; Animals ; Mice ; Reactive Oxygen Species/metabolism* ; Poly(ADP-ribose) Polymerase Inhibitors/pharmacology* ; Mice, Inbred C57BL ; Apoptosis ; Poly(ADP-ribose) Polymerases/metabolism*

Adenosine diphosphate (ADP)-ribosylation is a unique post-translational modification that regulates many biological processes, such as DNA damage repair. During DNA repair, ADP-ribosylation needs to be reversed by ADP-ribosylhydrolases. A group of ADP-ribosylhydrolases have a catalytic domain, namely the macrodomain, which is conserved in evolution from prokaryotes to humans. Not all macrodomains remove ADP-ribosylation. One set of macrodomains loses enzymatic activity and only binds to ADP-ribose (ADPR). Here, we summarize the biological functions of these macrodomains in DNA damage repair and compare the structure of enzymatically active and inactive macrodomains. Moreover, small molecular inhibitors have been developed that target macrodomains to suppress DNA damage repair and tumor growth. Macrodomain proteins are also expressed in pathogens, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, these domains may not be directly involved in DNA damage repair in the hosts or pathogens. Instead, they play key roles in pathogen replication. Thus, by targeting macrodomains it may be possible to treat pathogen-induced diseases, such as coronavirus disease 2019 (COVID-19).
ADP-Ribosylation ; COVID-19/metabolism* ; DNA Repair/physiology* ; Evolution, Molecular ; Humans ; Models, Biological ; Models, Molecular ; N-Glycosyl Hydrolases/metabolism* ; Poly(ADP-ribose) Polymerases/metabolism* ; Protein Domains ; SARS-CoV-2/pathogenicity*

The morbidity of neurodegenerative diseases are increased in recent years, however, the treatment is limited. Poly ADP-ribosylation (PARylation) is a post-translational modification of protein that catalyzed by poly(ADP-ribose) polymerase (PARP). Studies have shown that PARylation is involved in many neurodegenerative diseases such as stroke, Parkinson's diseases, Alzheimer's disease, amyotrophic lateral sclerosis and so on, by affecting intracellular translocation of protein molecules, protein aggregation, protein activity, and cell death. PARP inhibitors have showed neuroprotective efficacy for neurodegenerative diseases in pre-clinical studies and phase Ⅰ clinical trials. To find new PARP inhibitors with more specific effects and specific pharmacokinetic characteristics will be the new direction for the treatment of neurodegenerative diseases. This paper reviews the recent progress on PARylation in neurodegenerative diseases.
ADP-Ribosylation ; Humans ; Neurodegenerative Diseases ; physiopathology ; Poly Adenosine Diphosphate Ribose ; Poly(ADP-ribose) Polymerases ; metabolism

OBJECTIVETo explore the effect of a new emodin derivative E11 on proliferation and apoptosis of T lymphocytic leukemia cell line Molt-4 and its possible mechanisms.

METHODSMTT method was used to plot cell growth curve. Colony culture assay was performed for studying the effect of emodin derivative E11 on colony-formation of Molt-4. The fluorescent microscopy with DAPI staining was used to examine the cell morphological changes after E11 treatment. DNA fragmentation method was used to detect the inducing effect of emodin derivative E11 on cell apoptosis. Western blot was used to determine the expressions of apoptosis-related proteins including procaspase-9, procaspase-3, PARP and PI3K/AKT, MAPK signalling pathway.

RESULTSEmodin derivative E11 could strongly inhibit the growth of Molt-4 with the IC50 in 48 h at 1.381 ± 0.1552 µmol/L in dose-dependent manner. 0.1 µmol/L of E11 could inhibit cell colony formation. The typrical apopototic morphologic changes of Molt cells treated with E11 could be observed under fluorescence microscope with DAPI staining. DNA apoptotic ladder could be observed by DNA fragmentation.The expressions of procaspase -9, procaspase-3, PARP, p-MAPK, p-AKT, mTOR, p-mTOR, p-P70 and p-4BEP1 were down-regulated, while expressions of MAPK, AKT, 4EBP1 and P70 were not changed remarkably after Molt-4 were treated with E11 for 48 h.

CONCLUSIONE11 can remarkably inhibit the proliferation and induce the apoptosis of Molt-4 cells. The mechanism of apoptosis of Molt-4 cells may be related with the suppression of PI3K/AKT and MAPK signalling pathways.

Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Cell Line, Tumor ; drug effects ; Cell Proliferation ; Down-Regulation ; Emodin ; pharmacology ; Humans ; Leukemia, T-Cell ; pathology ; MAP Kinase Signaling System ; Phosphatidylinositol 3-Kinases ; metabolism ; Poly(ADP-ribose) Polymerases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; TOR Serine-Threonine Kinases ; metabolism

OBJECTIVETo investigate the apoptotic effects of Hsp90 selective inhibitor 17-AAG on human leukemia HL-60 and NB4 cells and analyse its possible mechanism.

METHODSCCK-8 assay was used to quantify the growth inhibition of cells after exposure to 17-AAG for 24 hours. Flow cytometrve with annexin V/propidium iodide staining was used to detect apoptosis of leukemia cells. Then Western blot was used to detect the activation of apoptosis related protein caspase-3 and PARP level. Gene expression profile of NB4 cells treated with 17-AAG was analyzed with real-time PCR arrays.

RESULTSThe inhibition of leukemia cell proliferation displayed a dose-dependent manner. Annexin V assay, cell cycle analysis and activation of PARP demonstrate that 17-AAG induced apoptosis leukemia cells. Real-time PCR array analysis showed that expression of 56 genes significantly up-regulated and expression of 23 genes were significantly down-regulated after 17-AAG treatment.

CONCLUSIONThe 17-AAG can inhibit the proliferation and induce the apoptosis of leukemia cells. After leukemia cells are treated with 17-AAG, the significant changes of apoptosis-related genes occured, and the cell apoptosis occurs via activating apoptosis related signaling pathway.

Apoptosis ; Benzoquinones ; pharmacology ; Caspase 3 ; metabolism ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Down-Regulation ; HL-60 Cells ; HSP90 Heat-Shock Proteins ; antagonists & inhibitors ; Humans ; Lactams, Macrocyclic ; pharmacology ; Leukemia ; metabolism ; Poly(ADP-ribose) Polymerases ; metabolism ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; Transcriptome

OBJECTIVETo explore whether MG-132 could enhance the anti-tumor activity of obatoclax against esophageal cancer cell line CaES-17.

METHODSMTT assay was used to determine the cytotoxicity of obatoclax and MG-132 in CaES-17 cells. The IC(50) of obatoclax and MG-132 were used to determine the molar ratio (1:2.4) of the two drugs for combined treatment of the cells. The concentrations of obatoclax and MG-132 ranged from 1/8 IC(50) to 4 IC(50) after serial dilution, and their combination index (CI) was calculated using CompuSyn software. The expression of ubiquitin and the cleavage of PARP, caspase-9, phospho-histone H3 and phospho-aurora A/B/C in the exposed cells were examined with Western blotting; the cell apoptosis was measured by flow cytometry with Annexin V staining, and the percentage of cells in each cell cycle phase was also determined by flow cytometry.

RESULTSThe CI of obatoclax and MG-132 was 0.296 for a 50% inhibition of Caes-17 cells and was 0.104 for a 95% inhibition. The cells treated with obatoclax or MG-132 alone showed increased expression of ubiquitin and cleavage of PARP and caspase-9. Compared with the cells treated with obatoclax or MG-132 alone, the cells with a combined treatment exhibited significantly increased expression of ubiquitin, cleavage of PARP and caspase-9, and expression of phospho-Histone H3 (P<0.05). The combined treatment of the cells also resulted in significantly increased expression of phospho-Aurora A/B/C compared with obatoclax treatment alone. The cells with the combined treatment showed significantly higher percentages of apoptotic cells and cells in sub-G(1) and G(2)/M phases compared with the cells treated with either of the drugs (P<0.05).

CONCLUSIONObatoclax combined with MG-132 shows a significant synergistic anti-tumor effect against esophageal cancer CaES-17 cells by inducing apoptosis and cell cycle arrest.

Apoptosis ; Caspase 9 ; metabolism ; Cell Cycle Checkpoints ; Cell Line, Tumor ; drug effects ; Esophageal Neoplasms ; pathology ; Histones ; metabolism ; Humans ; Leupeptins ; pharmacology ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; metabolism ; Pyrroles ; pharmacology

The molecular mechanism of DNA damage induced by hydroquinone (HQ) remains unclear. Poly(ADP-ribose) polymerase-1 (PARP-1) usually works as a DNA damage sensor, and hence, it is possible that PARP-1 is involved in the DNA damage response induced by HQ. In TK6 cells treated with HQ, PARP activity as well as the expression of apoptosis antagonizing transcription factor (AATF), PARP-1, and phosphorylated H2AX (γ-H2AX) were maximum at 0.5 h, 6 h, 3 h, and 3 h, respectively. To explore the detailed mechanisms underlying the prompt DNA repair reaction, the above indicators were investigated in PARP-1-silenced cells. PARP activity and expression of AATF and PARP-1 decreased to 36%, 32%, and 33%, respectively, in the cells; however, γ-H2AX expression increased to 265%. Co-immunoprecipitation (co-IP) assays were employed to determine whether PARP-1 and AATF formed protein complexes. The interaction between these proteins together with the results from IP assays and confocal microscopy indicated that poly(ADP-ribosyl)ation (PARylation) regulated AATF expression. In conclusion, PARP-1 was involved in the DNA damage repair induced by HQ via increasing the accumulation of AATF through PARylation.
Antioxidants ; toxicity ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Cell Line ; DNA Damage ; drug effects ; Gene Expression Regulation ; drug effects ; Gene Silencing ; Histones ; genetics ; metabolism ; Humans ; Hydroquinones ; toxicity ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; genetics ; metabolism ; Protein Transport ; Repressor Proteins ; genetics ; metabolism

OBJECTIVETo evaluate the anti-tumor effect of the combination of suberoylanilide hydroxamic acid(SAHA) with statins(lovastatin or simvastatin) on non-small cell lung carcinoma(NSCLC) cells.

METHODSHuman NSCLC A549 cells were treated with SAHA in combination of lovastatin or simvastatin. The cell growth was analyzed by SRB method, and the apoptosis of A549 cells was assessed by flow cytometer. The expression of cleaved poly-ADP-ribose polymerase(cleaved-PARP) and p21 protein was analyzed by Western-blotting when A549 cells were challenged with 2.5μmol/L SAHA and 5μmol/L lovastatin.

RESULTSLovastatin and simvastatin synergized SAHA in the inhibition of A549 cells. SAHA induced apoptosis was also enhanced by lovastatin. Treatment with 2.5μmol/L SAHA significantly up-regulated the expression of p21 protein in 48 h, while the protein expression was reduced in combined treatment with 5μmol/L lovastatin.

CONCLUSIONStatins can synergize the anti-tumor effect of SAHA in human NSCLC cells through a p21-dependent way.

Antineoplastic Agents ; pharmacology ; Apoptosis ; Carcinoma, Non-Small-Cell Lung ; pathology ; Cell Line, Tumor ; drug effects ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Humans ; Hydroxamic Acids ; pharmacology ; Hydroxymethylglutaryl-CoA Reductase Inhibitors ; pharmacology ; Poly(ADP-ribose) Polymerases ; metabolism

OBJECTIVETo investigate the anticancer effect and its mechanism of SN-38 combined with sorafenib on hepatocellular cancer cell lines HepG-2 and BEL-7402.

METHODSSRB colorimetry was employed to measure the viability of HepG-2 and BEL-7402 cells after the treatment of SN-38 with sorafenib. Propidium iodide flow cytometric assay and DAPI staining were used to evaluate the apoptosis of HCC cells. Western blotting was conducted to detect the expression level of apoptosis-related and DNA damage-related proteins.

RESULTSSRB colorimetry showed the synergistic anticancer activities of SN-38 combined with sorafenib, with a combination index of <0.9. The apoptotic rates of HepG-2 cells in control, 60 nmol/L SN-38, 2.5μmol/L sorafenib and combination groups were 4.25%±2.45%, 28.95%±10.75%, 3.49%±2.49% and 53.19%±11.21%, respectively(P<0.05). Western blotting showed that the combination of these two drugs increased the enzymolysis of PARP, Caspase-8 and Caspase-3, and promoted the expression levels of p53, p21 and γ-H2AX significantly.

CONCLUSIONSN-38 and sorafenib have synergistic anticancer activity on hepatocellular carcinoma cells in vitro with the augmentation of apoptosis.

Apoptosis ; Camptothecin ; analogs & derivatives ; pharmacology ; Carcinoma, Hepatocellular ; pathology ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Cell Line, Tumor ; drug effects ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Histones ; metabolism ; Humans ; Liver Neoplasms ; pathology ; Niacinamide ; analogs & derivatives ; pharmacology ; Phenylurea Compounds ; pharmacology ; Poly(ADP-ribose) Polymerases ; metabolism ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVETo investigate the effect of small interfering RNA（siRNA）for MSI-2 on the growth, apoptosis and NUMB expression of THP-1 cells.

METHODSThree siRNA for MSI-2 gene was designed and transfected into THP- 1 cells. The cell inhibition, colony formation and apoptosis were determined. The protein expression of NUMB, caspase- 3 and PARP were detected by Western blotting.

RESULTSAfter MSI- 2 expression of THP- 1 cells was down- regulated for 24 hours, cell inhibition of siRNA MSI-2 group was（47.89±7.64）%, obviously higher than that of negative control group（P=0.005）. After 9 days, cell colony count of siRNA MSI-2 group was 7.50±1.53, also lower than that of negative control group（35.75±7.46, P<0.001）. In addition, apoptotic rates of siRNA MSI- 2 group at 24 hours ［（15.22±1.52）%］and 48 hours［（33.83±3.96）%］were significantly higher than those of negative control group（P=0.008 and P=0.001, respectively）. Accordingly, activations of caspase-3 and PARP and increased NUMB were observed in siRNA MSI- 2 group.

CONCLUSIONsiRNA for MSI- 2 gene could increase the expressions of NUMB to inhibit the proliferation and induce apoptosis of THP-1 cells.

Apoptosis ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Down-Regulation ; Humans ; Membrane Proteins ; genetics ; metabolism ; Nerve Tissue Proteins ; genetics ; metabolism ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; metabolism ; RNA Interference ; RNA, Small Interfering ; RNA-Binding Proteins ; genetics ; metabolism ; Transfection