OBJECTIVE: To observe the neuroprotective effect of 6-shogaol (6-SH) in global cerebral ischemia/reperfusion injury (CIRI) following cardiac arrest (CA) and cardiopulmonary resuscitation (CPR) in rats. METHODS: Computer-aided molecular docking was used to determine whether 6-SH could spontaneously bind to death-associated protein kinase 1 (DAPK1). SPF-grade male SD rats were randomly divided into a sham group (n = 5), a CPR group (n = 7), and a CPR+6-SH group (n = 7). The CPR group and CPR+6-SH group were further divided into 12-, 24-, and 48-hour subgroups based on observation time points. A rat model of global CIRI after CA-CPR was established by asphyxiation. In the sham group, only tracheal and vascular intubation was performed without asphyxia and CPR induction. The CPR group was intraperitoneally injected with 1 mL of normal saline immediately after successful modeling. The CPR+6-SH group received an intraperitoneal injection of 20 mg/kg 6-SH (1 mL) immediately after successful modeling, followed by administration every 12 hours until the endpoint. Neurological Deficit Score (NDS) was recorded at each time point after modeling. After completion of observation at each time point, rats were anesthetized and sacrificed, and brain tissue specimens were collected. Histopathological changes of neurons were observed under light microscopy after hematoxylin-eosin (HE) staining. Ultrastructural changes of hippocampal neurons and autophagy were observed by transmission electron microscopy (TEM). Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect mRNA expression levels of DAPK1, vacuolar protein sorting 34 (VPS34), Beclin1, and microtubule-associated protein 1 light chain 3 (LC3) in brain tissues. Western blotting was used to detect protein expression levels of DAPK1, phosphorylated DAPK1 at serine 308 (p-DAPK1 ser308), VPS34, Beclin1, and LC3. Immunofluorescence was used to observe Beclin1 and LC3 expression in brain tissues under a fluorescence microscope. RESULTS: Molecular docking results indicated that 6-SH could spontaneously bind to DAPK1. Compared with the sham group, the NDS scores of the CPR group rats were significantly increased at all modeling time points; under light microscopy, disordered cell arrangement, widened intercellular spaces, and edema were observed in brain tissues, with pyknotic and necrotic nuclei in some areas; under TEM, mitochondria were markedly swollen with intact membranes, dissolved matrix, reduced or disappeared cristae, vacuolization, and increased autophagosomes. Compared with the CPR group, the NDS scores of the CPR+6-SH group rats were significantly decreased at all modeling time points; under light microscopy, local neuronal edema and widened perinuclear space were observed; under TEM, mitochondria were mostly mildly swollen with intact membranes, fewer autophagosomes, and alleviated injury. RT-qPCR results showed that compared with the sham group, mRNA expression levels of DAPK1, VPS34, Beclin1, and LC3 in brain tissues were significantly upregulated in all CPR subgroups, with the most pronounced changes at 24 hours. Compared with the CPR group, the CPR+6-SH group showed significantly lower mRNA expression of the above indicators at each time point [24 hours post-modeling (relative expression): DAPK1 mRNA: 3.41±0.68 vs. 4.48±0.62; VPS34 mRNA: 3.63±0.49 vs. 4.66±1.18; Beclin1 mRNA: 3.08±0.49 vs. 4.04±0.22; LC3 mRNA: 2.60±0.36 vs. 3.67±0.62; all P < 0.05]. Western blotting results showed that compared with the sham group, the protein expression levels of DAPK1, VPS34, Beclin1, and LC3 in all CPR subgroups were significantly increased, while the expression of p-DAPK1 ser308 was significantly decreased, with the most pronounced changes observed in the CPR 24-hour subgroup. Compared with the CPR group, the CPR+6-SH subgroups exhibited significantly reduced protein expression of DAPK1, VPS34, Beclin1, and LC3 [24-hour post-modeling: DAPK1/β-actin: 1.88±0.22 vs. 2.47±0.22; VPS34/β-actin: 2.55±0.06 vs. 3.46±0.05; Beclin1/β-actin: 2.12±0.03 vs. 2.87±0.03; LC3/β-actin: 2.03±0.24 vs. 3.17±0.23; all P < 0.05]. Conversely, the expression of p-DAPK1 ser308 was significantly upregulated in the CPR+6-SH group compared to the CPR group [24-hour post-modeling: p-DAPK1 ser308/β-actin: 0.40±0.02 vs. 0.20±0.07, P < 0.05]. Under the fluorescence microscope, fluorescence intensities of Beclin1 and LC3 in the CPR 24-hour group were significantly higher than those in the sham 24-hour group; compared with the CPR 24-hour group, the CPR+6-SH 24-hour group showed significantly reduced fluorescence intensities of Beclin1 and LC3. CONCLUSION 6-SH inhibited the expression of DAPK1, alleviated excessive autophagy after global CIRI following CA-CPR in rats, and exerted neuroprotective effects. The mechanism may be related to phosphorylation at the DAPK1 ser308 site.
Animals ; Rats, Sprague-Dawley ; Male ; Rats ; Cardiopulmonary Resuscitation ; Autophagy/drug effects* ; Heart Arrest/therapy* ; Death-Associated Protein Kinases/metabolism* ; Reperfusion Injury/metabolism* ; Disease Models, Animal ; Neuroprotective Agents/pharmacology* ; Brain Ischemia/metabolism*

OBJECTIVETo investigate the expressions of DAPK3 and c-Myc and their prognostic value in endometrial carcinoma (EC).

METHODSThe expressions of DAPK3 and c-Myc were detected immunohistochemically in 132 surgical specimens. The relationship between DAPK3 and c-Myc protein expressions and the clinicopathological features of the patients was evaluated.

RESULTSImmunohistochemical analysis revealed low DAPK3 expression in 60.61% (80/132) and high c-Myc expression in 53.79% (71/132) of the specimens of EC. Both DAPK3 expression and c-Myc expression were significantly correlated with FIGO stage (P=0.034 and 0.015, respectively) and lymph node metastasis (P=0.022 and 0.031, respectively). DAPK3 expression was closely correlated with the histological grade (P=0.027) and depth of myometrial invasion (P=0.011). Kaplan-Meier survival analysis indicated that patients with low DAPK3 expressions had a shorter overall survival rate than those with high DAPK3 expressions (P=0.023), while patients with high c-Myc expressions had poorer prognoses than those with low c-Myc expressions (P=0.002). Spearman correlation analysis showed that DAPK3 and c-Myc expressions were negatively correlated (P<0.001, r=?0.310). Multivariate analysis identified a high c-Myc expression as the independent predictor of the prognosis of EC (P=0.007).

CONCLUSIONSA low expression of DAPK3 and a high expression of c-Myc are associated with aggressive and metastatic behaviors of EC, and their detection may help to predict the prognosis of the EC patients.

Death-Associated Protein Kinases ; metabolism ; Endometrial Neoplasms ; diagnosis ; metabolism ; Female ; Humans ; Kaplan-Meier Estimate ; Lymphatic Metastasis ; Prognosis ; Proto-Oncogene Proteins c-myc ; metabolism ; Survival Rate

OBJECTIVETo explore the effect of DAPK overexpression on the biological behaviors and caspase-3 expression in HL-60 cells.

METHODSThe expression of DAPK mRNA was detected by RT-PCR leukemia cell lines K562, Molt4, U937, and HL-60 cells. HL-60 cells were transfected by a eukaryotic expression vector pReceiver-M29-DAPK via LipofectamineTM 2000, and the impact of DAPK overexpression on cell apoptosis, cell cycle, cell differentiation and caspase-3 expression were analyzed.

RESULTSDAPK mRNA expression was positive in K562, Molt4 and U937 cells but negative in HL-60 cells. Significantly increased cell apoptosis was observed in pReceiver-M29-DAPK-transfected HL-60 cells by flow cytometry and Hoechst33342 staining. The cell cycle distribution and differentiation showed no significant changes after the transfection. The expression of caspase-3 was significantly increased in the cells after transfection.

CONCLUSIONDAPK gene overexpression promotes apoptosis of HL-60 cells without affecting the cell cycle and differentiation. Caspase-3 may be involved in the regulation of cell apoptosis.

Apoptosis ; Caspase 3 ; metabolism ; Cell Cycle ; Cell Differentiation ; Cell Line, Tumor ; Death-Associated Protein Kinases ; metabolism ; HL-60 Cells ; Humans ; RNA, Messenger ; metabolism ; Transfection ; U937 Cells

OBJECTIVETo investigate the effects of telocinobufagin on viability and apoptosis of colorectal cancer (CRC) cells and explore the mechanism of telocinobufagin-induced apoptosis.

METHODSMTT assay was performed to detect the viability of CRC cells exposed to telocinobufagin. Nuclear staining with Hoechst 33342 and flow cytometry were used to analyze the cell death of CRC cells. Expressions of proteins related with cell apoptosis and oxidative stress were determined with Western blotting.

RESULTSTelocinobufagin decreased the viability of CRC cells in a time- and dose-dependent manner. The presence of karyopycnosis and apoptotic bodies together with the results of flow cytometry suggested that telocinobufagin induced cell apoptosis to cause cell death. Western blotting showed that telocinobufagin exposure of the cells resulted in upregulated p53 and Bax protein expressions and promoted cleavage of caspase 9 and PARP. Telocinobufagin induced phosphorylation of Bad and PARP cleavage, and suppressed phosphorylation of IKBα and TAK1 and expression of survivin in the cells.

CONCLUSIONTelocinobufagin can decrease the viability of CRC cells by inducing cell apoptosis, which involves p53-mediated Bax activation and inhibition of the IAP pathway.

Apoptosis ; Bufanolides ; pharmacology ; Caspase 9 ; metabolism ; Cell Survival ; Colorectal Neoplasms ; pathology ; Humans ; MAP Kinase Kinase Kinases ; metabolism ; NF-KappaB Inhibitor alpha ; metabolism ; Oxidative Stress ; Poly (ADP-Ribose) Polymerase-1 ; metabolism ; Tumor Cells, Cultured ; Tumor Suppressor Protein p53 ; metabolism ; bcl-2-Associated X Protein ; metabolism ; bcl-Associated Death Protein ; metabolism

OBJECTIVETo investigate the role of death associated protein kinase(DAPK) in colon cancer drug-resistance.

METHODSImmunohistochemistry was used to detect DAPK expression in colon carcinoma tissues of 61 cases and adjacent tissues of 32 cases. 5-fluorouracil (5-FU)-induced drug-resistant colon cancer cell lines HCT116/5-FU model was established. DAPK-siRNA was transfected into cells to down-regulate the DAPK gene expression (DAPK-siRNA grouyp), FAM-siRNA was transfected as control group, and DAPK over-expression plasmid vectors were constructed to up-regulate the DAPK gene expression(DAPK over-expression group). Real-time quantitative PCR and Western blotting were used to examine the mRNA and protein expression levels of DAPK, multidrug resistance protein (MRP) and P- glycoprotein (P-gp). MTT and flow cytometry were used to detect cell proliferation and apoptosis for cells treated with 5-FU (8 mg/L) and cells without treatment of 5-FU in 3 groups respectively.

RESULTSPositive expression rate of DAPK in colon cancer tissues was significantly lower than that in adjacent normal tissues [18.0% (11/61) vs. 90.6% (29/32), P < 0.05]. Compared with FAM-siRNA group, DAPK mRNA and protein expression levels were significantly lower in DAPK-siRNA group, but significantly higher in DAPK over-expression group (P<0.05). After treatment of 5-FU, cell proliferation was significantly inhibited, but cell apoptosis was significantly increased in DAPK over-expression group compared to FAM-siRNA group (P < 0.05). Cell proliferation and apoptosis were not significantly different between DAPK siRNA and FAM-siRNA groups (all P < 0.05). Compared with FAM-siRNA group, DAPK over-expression could significantly reduce the mRNA and protein levels of MRP and P-gp, whereas DAPK siRNA had no obvious such effects.

CONCLUSIONDAPK can inhibit the proliferation and promote the apoptosis in drug-resistant colon cancer cells, and it probably enhances the sensitivity of cancer cells to drugs by down-regulating the mRNA and protein levels of MRP and P-gp.

ATP-Binding Cassette, Sub-Family B, Member 1 ; Antineoplastic Agents ; Apoptosis ; Cell Proliferation ; Colorectal Neoplasms ; drug therapy ; enzymology ; Death-Associated Protein Kinases ; metabolism ; Drug Resistance, Neoplasm ; Fluorouracil ; Genetic Vectors ; HCT116 Cells ; Humans ; RNA, Messenger ; RNA, Small Interfering ; Transfection

Neural stem cells (NSCs) have been suggested as a groundbreaking solution for stroke patients because they have the potential for self-renewal and differentiation into neurons. The differentiation of NSCs into neurons is integral for increasing the therapeutic efficiency of NSCs during inflammation. Apoptosis signal-regulating kinase 1 (ASK1) is preferentially activated by oxidative stress and inflammation, which is the fundamental pathology of brain damage in stroke. ASK1 may be involved in the early inflammation response after stroke and may be related to the differentiation of NSCs because of the relationship between ASK1 and the p38 mitogen-activated protein kinase pathway. Therefore, we investigated whether ASK1 is linked to the differentiation of NSCs under the context of inflammation. On the basis of the results of a microarray analysis, we performed the following experiments: western blot analysis to confirm ASK1, DCX, MAP2, phospho-p38 expression; fluorescence-activated cell sorting assay to estimate cell death; and immunocytochemistry to visualize and confirm the differentiation of cells in brain tissue. Neurosphere size and cell survival were highly maintained in ASK1-suppressed, lipopolysaccharide (LPS)-treated brains compared with only LPS-treated brains. The number of positive cells for MAP2, a neuronal marker, was lower in the ASK1-suppressed group than in the control group. According to our microarray data, phospho-p38 expression was inversely linked to ASK1 suppression, and our immunohistochemistry data showed that slight upregulation of ASK1 by LPS promoted the differentiation of endogenous, neuronal stem cells into neurons, but highly increased ASK1 levels after cerebral ischemic damage led to high levels of cell death. We conclude that ASK1 is regulated in response to the early inflammation phase and regulates the differentiation of NSCs after inflammatory-inducing events, such as ischemic stroke.
Animals ; Cell Death ; Infarction, Middle Cerebral Artery/*metabolism ; Lipopolysaccharides/pharmacology ; MAP Kinase Kinase Kinase 5/genetics/*metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Microtubule-Associated Proteins/genetics/metabolism ; Neural Stem Cells/cytology/drug effects/*metabolism ; *Neurogenesis ; Neuropeptides/genetics/metabolism ; p38 Mitogen-Activated Protein Kinases/genetics/metabolism

OBJECTIVE: To study the inhibitory effect of Arsenic Trioxide (As2O3) combined with diamminedichloroplatinum (DDP) on the growth of human nasopharyngeal carcinoma cell strain CNE-2Z xenograft in nude mice, and to explore the possible effect mechanisms of the antitumor. METHOD: The models of human poorly differentiated nasopharyngeal carcinoma in nude mice were established and randomly divided into four groups, control group, As2O3 group, DDP group and As2O3 + DDP group. The effect of antitumor on each group was studied. The specimen obtained from the mice were detected by optical microscope and tdt-mediated dutp rock end labeling (tunel) method. Expression of DAPK was detected by real time-PCR and immunohistochemistry. RESULT: As2O3 group and AS2O3 + DDP group could obviously inhibit the growth of tumor, induce the apoptosis of human naso pharyngeal carcinoma cell and up-regulate the expression of RASSF1A. CONCLUSION As2O3 can greatly inhibit the growth of human nasopharyngeal carcinoma cell strain CNE-2Z xenograft in nude mice, which were related to the induced apoptosis of human nasopharyngeal carcinoma cell and up-regulated expression of DAPK Combination of As2O3 with DDP seem to be more effective.
Animals ; Apoptosis ; drug effects ; Arsenic Trioxide ; Arsenicals ; administration & dosage ; pharmacology ; Carcinoma ; Cell Line, Tumor ; Cisplatin ; administration & dosage ; pharmacology ; Death-Associated Protein Kinases ; metabolism ; Gene Expression Regulation, Neoplastic ; Humans ; Mice ; Mice, Nude ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; metabolism ; pathology ; Oxides ; administration & dosage ; pharmacology ; Xenograft Model Antitumor Assays

OBJECTIVETo explore the amplification and expression status of DAPK1 and CD147 in esophageal squamous cell carcinoma (ESCC) and their relationship with the prognosis of ESCC.

METHODSImmunohistochemical staining and RT-PCR were used to detect the expression and amplification of DAPK1 and CD147 in esophageal squamous carcinoma tissue and normal esophageal mucosa. Statistical analysis of the clinocopathological data was performed with SPSS 11.5 software package.

RESULTSThe positive rates of expression of DAPK1 protein and CD147 protein in the specimens of esophageal carcinoma were 31.3% and 58.5%, and in normal esophageal mucosa 57.5% and 25.0%, respectively, with a statistically significant difference (P < 0.001). The expressions of DAPK1 and CD147 were significant correlated with invasion depth, lymph node metastasis, TNM stage and the degree of cancer differentiation. (P < 0.05). The negative expression of DAPK1 and positive expression of CD147 indicated a poor prognosis. In 52 ESCC cases, the expression of DAPK1 in cancer tissues was 0.236 ± 0.049, and 0.395 ± 0.058 in normal esophageal mucosa, while that of CD147 mRNA expression was 0.942 ± 0.204 and 0.821 ± 171, respectively, statistically both with a very significant difference (P < 0.01). There was a higher expression level of DAPK1 mRNA in the cancer tissue in patients with no lymph node metastasis, well differentiation, and earlier pathological stage, and a higher expression level of CD147 mRNA in the cancer tissues in patients with lymph node metastasis, poor differentiation, and later pathological stage.

CONCLUSIONSThe expression of DAPK1 and CD147 proteins is closely correlated with the clinicopathological characteristics of ESCC. The genes DAPK1 and CD147 may participate in the metastasis and apoptosis of ESCC. The expression of DAPK1 and CD147 may be used as important prognostic predictors in ESCC.

Adult ; Aged ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Basigin ; genetics ; metabolism ; Biomarkers, Tumor ; metabolism ; Calcium-Calmodulin-Dependent Protein Kinases ; genetics ; metabolism ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Death-Associated Protein Kinases ; Esophageal Neoplasms ; metabolism ; pathology ; Female ; Humans ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Invasiveness ; Neoplasm Staging ; RNA, Messenger ; metabolism ; Risk Factors ; Survival Rate

OBJECTIVETo investigate the methylation levels of death-associated protein kinase (DAPK) in Uyghur female patients with different cervical lesions in Xinjiang, and to discuss the relationship of the expression and significance of DAPK in normal cervix, chronic cervicitis, cervical intraepithelial neoplasia (CINI, CIN II/III) and invasive cervical squamous cell carcinoma.

METHODS30 cases of normal cervix and chronic cervicitis, 30 cases of CINI, 30 cases of CINII/III and 30 cases of cervical squamous cell carcinoma were tested by methylation specific PCR (MSP). Expressions of DAPK in 30 cases of normal cervix and chronic cervicitis, 30 cases of CINI, 30 cases of CINII/III and 30 cases of cervical squamous cell carcinoma were assayed using immunohistochemical SP staining.

RESULTSThe methylation rate of DAPK gene in normal cervix and chronic cervicitis was 3.33%, 10% in cervical intraepithelial neoplasia CINI, 36.7% in CINII/III, and 63.3% in invasive cervical squamous cell carcinoma. The methylation rate of DAPK in the SCC group was significantly higher than that in the other groups (P < 0.05). Aberrant promoter methylation of the DAPK gene was positively correlated with the degree of cervical lesions. The positive rate of DAPK protein in normal cervix and chronic cervicitis was 93.3%, 83.3% in cervical intraepithelial neoplasia CINI, 60.0% in CINII/III, and 33.3% in invasive cervical squamous cell carcinoma. The expression of DAPK in the SCC group was significantly lower than that in the other groups (P < 0.05). The positive rate of DAPK protein was negatively correlated with the degree of cervical lesions (r(s) = -0.603, P < 0.001).

CONCLUSIONSMethylation of DAPK is involved in the cervical carcinogenesis and DAPK gene promoter methylation occurs in the early development of cervical cancer in Uyghur women in Xinjiang. Detection of DAPK gene methylation may provide a basis for use in early detection of cervical cancer. DAPK protein expression is decreasing even disappears along with the progression of cervical lesions.

Adult ; Aged ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Calcium-Calmodulin-Dependent Protein Kinases ; genetics ; metabolism ; Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Cervical Intraepithelial Neoplasia ; genetics ; metabolism ; pathology ; China ; ethnology ; DNA Methylation ; Death-Associated Protein Kinases ; Disease Progression ; Female ; Humans ; Middle Aged ; Promoter Regions, Genetic ; genetics ; Uterine Cervical Neoplasms ; genetics ; metabolism ; pathology ; Uterine Cervicitis ; genetics ; metabolism ; pathology

OBJECTIVETo analyze the promoter methylation levels of p15, CDH1, DAPK and HICI genes of patients with myelodysplastic syndrome (MDS) and explore the relationship between the level of methylation and clinical features.

METHODSDNA methylation levels of p15, CDH1, DAPK and HICI in peripheral blood (PB) or bone marrow (BM) samples from 52 MDS patients were detected by real-time quantitative PCR. The correlation of the methylation level with clinical features and hematological findings was analyzed. 38 de novo AML patients and 46 normal individuals served as controls.

RESULTSThe methylation levels of p15, CDH1, DAPK and HICI were 16.23 ± 21.69, 6.59 ± 9.39, 0.14 ± 0.11 and 7.81 ± 9.70 in BM, and 14.96 ± 20.16, 6.00 ± 9.26, 0.12 ± 0.14 and 6.74 ± 9.72 in PB, respectively from 18 MDS patients, and the difference between BM and PB was not statistically significant (P > 0.05). The methylation levels of p15 (14.70 ± 18.17) and CDH1 (6.61 ± 8.79) genes in high risk (RAEBI/II) MDS were significantly higher than in low risk (RCMD/RARS/5q-, p15: 1.99 ± 1.59, CDH1: 1.23 ± 1.14 and RCMD, p15: 3.02 ± 3.42, CDH1:1.53 ± 2.06) MDS or control (p15: 1.69 ± 1.82, CDH1: 1.01 ± 1.12) (P < 0.05). The methylation levels of DAPK gene had no difference among subtypes of MDS, and that of HIC1 gene only differed between RAEB I/II (9.16 ± 11.95) and control (2.49 ± 2.26) (P = 0.042). The difference of methylation levels of p15, CDH1, DAPK and HICI in BM was statistically significant among subtypes of MDS (P = 0.001, 0.003, 0.039, 0.023, respectively). And so did of p15 and DAPK in PB (P = 0.013, 0.006, respectively). The methylation level of p15 and CDH1 was significantly correlated with IPSS classification and blasts percentage in BM.

CONCLUSIONSp15 and CDH1 genes are special hypermethylation genes in MDS. Methylation level of HIC1 gene showed an upward tendency from low risk to high risk MDS.

Adult ; Aged ; Aged, 80 and over ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Cadherins ; genetics ; metabolism ; Calcium-Calmodulin-Dependent Protein Kinases ; genetics ; metabolism ; Case-Control Studies ; Cyclin-Dependent Kinase Inhibitor p15 ; genetics ; metabolism ; DNA Methylation ; Death-Associated Protein Kinases ; Female ; Humans ; Kruppel-Like Transcription Factors ; genetics ; metabolism ; Male ; Middle Aged ; Myelodysplastic Syndromes ; genetics ; metabolism ; Promoter Regions, Genetic ; Real-Time Polymerase Chain Reaction ; Young Adult