Elevated ZIPK is required for TNF-α-induced cell adhesion molecule expression and leucocyte adhesion in endothelial cells.

Leucocyte adhesion to the vascular endothelium is a critical event in the early inflammatory response to infection and injury. This process is primarily regulated by the expression of cell adhesion molecules (CAMs) in endothelial cells. It has been well documented that tumor necrosis factor alpha (TNF-α) is a key regulator of CAM expression within this process, but its regulatory mechanism remains controversial. To investigate the scenario within this process, we assessed the role of zipper-interacting protein kinase (ZIPK), a serine/threonine kinase with multiple substrates, in CAM expression. We used TNF-α as inflammatory stimulator and found that ZIPK was integrated into the signaling regulation of TNF-α-mediated CAM expression. In human umbilical vein endothelial cells (HUVECs), TNF-α exposure led to significantly increased expression of both intercellular CAM-1 (ICAM-1) and vascular CAM-1 (VCAM-1), along with an increase in the adhesion of THP-1 monocytes to HUVECs. Simultaneously, ZIPK gene was also up-regulated at the transcription level. These effects were clearly inhibited by the ZIPK-specific inhibitor Tc-DAPK6 or small interfering RNA (siRNA) capable of specifically inhibiting ZIPK expression. We thus suggest that both ZIPK activation and ZIPK gene expression are necessary for TNF-α-mediated CAM expression and leucocyte adhesion. Interestingly, ZIPK inhibition also significantly suppressed TNF-α-induced nuclear factor kappa B (NF-κB) activation, indicating that TNF-α-mediated ZIPK expression functions upstream of NF-κB and CAM expression. We thus propose a TNF-α/ZIPK/NF-κB signaling axis for CAM expression that is necessary for leucocyte adhesion to endothelial cells. Our data in this study revealed a potential molecular target for exploring anti-inflammation drugs.

Death-associated Protein Kinase 1 Mediates Ventilator-induced Lung Injury in Mice by Promoting Alveolar Epithelial Cell Apoptosis.

BACKGROUND: Alveolar epithelial cell apoptosis is implicated in the onset of ventilator-induced lung injury. Death-associated protein kinase 1 (DAPK1) is associated with cell apoptosis. The hypothesis was that DAPK1 participates in ventilator-induced lung injury through promoting alveolar epithelial cell apoptosis. METHODS: Apoptosis of mouse alveolar epithelial cell was induced by cyclic stretch. DAPK1 expression was altered (knockdown or overexpressed) in vitro by using a small interfering RNA or a plasmid, respectively. C57/BL6 male mice (n = 6) received high tidal volume ventilation to establish a lung injury model. Adeno-associated virus transfection of short hairpin RNA and DAPK1 inhibitor repressed DAPK1 expression and activation in lungs, respectively. The primary outcomes were alveolar epithelial cell apoptosis and lung injury. RESULTS: Compared with the control group, the 24-h cyclic stretch group showed significantly higher alveolar epithelial cell apoptotic percentage (45 ± 4% fold vs. 6 ± 1% fold; P < 0.0001) and relative DAPK1 expression, and this group also demonstrated a reduced apoptotic percentage after DAPK1 knockdown (27 ± 5% fold vs. 53 ± 8% fold; P < 0.0001). A promoted apoptotic percentage in DAPK1 overexpression was observed without stretching (49 ± 6% fold vs. 14 ± 3% fold; P < 0.0001). Alterations in B-cell lymphoma 2 and B-cell lymphoma 2-associated X are associated with DAPK1 expression. The mice subjected to high tidal volume had higher DAPK1 expression and alveolar epithelial cell apoptotic percentage in lungs compared with the low tidal volume group (43 ± 6% fold vs. 4 ± 2% fold; P < 0.0001). Inhibition of DAPK1 through adeno-associated virus infection or DAPK1 inhibitor treatment appeared to be protective against lung injury with reduced lung injury score, resolved pulmonary inflammation, and repressed alveolar epithelial cell apoptotic percentage (47 ± 4% fold and 48 ± 6% fold; 35 ± 5% fold and 34 ± 4% fold; P < 0.0001, respectively). CONCLUSIONS: DAPK1 promotes the onset of ventilator-induced lung injury by triggering alveolar epithelial cell apoptosis through intrinsic apoptosis pathway in mice.

Melatonin directly binds and inhibits death-associated protein kinase 1 function in Alzheimer's disease.

Death-associated protein kinase 1 (DAPK1) is upregulated in the brains of human Alzheimer's disease (AD) patients compared with normal subjects, and aberrant DAPK1 regulation is implicated in the development of AD. However, little is known about whether and how DAPK1 function is regulated in AD. Here, we identified melatonin as a critical regulator of DAPK1 levels and function. Melatonin significantly decreases DAPK1 expression in a post-transcriptional manner in neuronal cell lines and mouse primary cortical neurons. Moreover, melatonin directly binds to DAPK1 and promotes its ubiquitination, resulting in increased DAPK1 protein degradation through a proteasome-dependent pathway. Furthermore, in tau-overexpressing mouse brain slices, melatonin treatment and the inhibition of DAPK1 kinase activity synergistically decrease tau phosphorylation at multiple sites related to AD. In addition, melatonin and DAPK1 inhibitor dramatically accelerate neurite outgrowth and increase the assembly of microtubules. Mechanistically, melatonin-mediated DAPK1 degradation increases the activity of Pin1, a prolyl isomerase known to play a protective role against tau hyperphosphorylation and tau-related pathologies. Finally, elevated DAPK1 expression shows a strong correlation with the decrease in melatonin levels in human AD brains. Combined, these results suggest that DAPK1 regulation by melatonin is a novel mechanism that controls tau phosphorylation and function and offers new therapeutic options for treating human AD.

Expression and Functional Relevance of Death-Associated Protein Kinase in Human Drug-Resistant Epileptic Brain: Focusing on the Neurovascular Interface.

Death-associated protein kinase (DAPK) is a key player in various cell death signaling pathways. Prolonged seizures induce neuronal stress; thus, we studied DAPK expression in resected brain tissues from patients with refractory epilepsy and the pathophysiological relevance of neurovascular DAPK. We used brain resections from temporal lobe epilepsy (TLE), tumor (BT), arteriovenous malformation (AVM), and autopsy, and isolated human endothelial cells (EPI-ECs) and glial cells (EPI-Astro) from epileptic brains compared to control brain endothelial cells (HBMECs) and astrocytes. DAPK and phosphorylated DAPK (p-DAPK) expression was evaluated by immunohistochemistry and western blot. Subcellular localization of DAPK in epileptic brain was explored; DAPK mRNA/protein levels in EPI-ECs/EPI-Astro were evaluated. We assessed DAPK localization with hypoxic inducible factor (HIF-1α) and vascular endothelial growth factor (VEGF) in epilepsy, BT, and AVM. We found DAPK overexpression across neurons, microcapillaries, and astrocytes in TLE vs controls; DAPK and p-DAPK levels significantly increased only in microsomal fractions of epileptic brain. DAPK mRNA remained unchanged, although increased DAPK and p-DAPK protein expression was observed in EPI-ECs. DAPK inhibition reduced p-DAPK, HIF-1α, and VEGF expression, but increased cytotoxicity and decreased cell viability in EPI-ECs and EPI-astro vs. controls. DAPK staining in TLE resembled BT and AVM, with predominant DAPK/p-DAPK expression in neurons and vasculature. Taken together, these findings suggest DAPK could be a potential molecular target in neuronal death and vascular changes in epilepsy. Increased brain endothelial and astrocytic DAPK in epilepsy, identified for the first time, may have relevance to angiogenesis, hypoxia, and cell survival in pathological conditions.

Epigenetic silencing of the tumor suppressor genes SPI1, PRDX2, KLF4, DLEC1, and DAPK1 in childhood and adolescent lymphomas.

The aim of the study was to investigate the expression and methylation status of seven distinctive genes with tumor suppressing properties in childhood and adolescent lymphomas. A total of 96 patients with Hodgkin Lymphoma (HL, n = 41), Non-Hodgkin Lymphoma (NHL, n = 15), and reactive lymphoid hyperplasia (RLH, n = 40, as controls) are included in the research. The expression status of CDKN2A, SPI1, PRDX2, DLEC1, FOXO1, KLF4 and DAPK1 genes were measured with QPCR method after the RNA isolation from paraffin blocks of tumor tissue and cDNA conversion. DNA isolation was performed from samples with low gene expression followed by methylation PCR study specific to promoter regions of these genes. We found that SPI1, PRDX2, DLEC1, KLF4, and DAPK1 genes are significantly less expressed in patient than the control group (p = 0.0001). However, expression of CDKNA2 and FOXO1 genes in the patient and control groups were not statistically different. The methylation ratios of all genes excluding the CDKN2A and FOXO1 were significantly higher in the HL and NHL groups than the controls (p = 0.0001). We showed that SPI1, PRDX2, DLEC1, KLF4 and DAPK1 genes are epigenetically silenced via hypermethylation in the tumor tissues of children with HL and NHL. As CDKN2A gene was not expressed in both patient and control groups, we conclude that it is not specific to malignancy. As FOXO1 gene was similarly expressed in both groups, its relationship with malignancy could not be established. The epigenetically silenced genes may be candidates for biomarkers or therapeutic targets in childhood and adolescent lymphomas.

MicroRNA-141-3p targets DAPK1 and inhibits apoptosis in rat ovarian granulosa cells.

The polycystic ovary syndrome (PCOS) is a complex and heterogeneous endocrine disorder. MicroRNAs negatively regulate the expression of target genes at posttranscriptional level by binding to the 3' untranslated region of target genes. Our previous study showed that miR-141-3p was dramatically decreased in the ovaries of rat PCOS models. In this study, we aimed to characterize the target of miR-141-3p in rat ovarian granulosa cells. 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay showed that cell viability was dramatically increased when miR-141-3p was overexpressed but was decreased when miR-141-3p was interfered. Flow cytometry showed that cell apoptotic rate was dramatically decreased when miR-141-3p was overexpressed but was increased when miR-141-3p was interfered. Bioinformatics analysis predicted that death-associated protein kinase 1 (DAPK1) might be the target gene of miR-141-3p because the 3' untranslated region of DAPK1 contains sequences complementary to microRNA-141-3p. Transfection with miR-141-3p mimics and inhibitor into granulosa cells showed that both DAPK1 mRNA and protein levels were negatively correlated with miR-141-3p level. Dual-luciferase reporter assay established that DAPK1 was the target of miR-141-3p. Taken together, our data indicate that miR-141-3p may inhibit ovarian granulosa cell apoptosis via targeting DAPK1 and is involved in the etiology of PCOS.

Comparison of Selected Protein Levels in Tumour and Surgical Margin in a Group of Patients with Oral Cavity Cancer.

Oral cavity cancer belongs to head-and-neck squamous cell carcinoma group. The purpose of the study was to assess the levels of certain proteins in a tumour and surgical margin in a group of patients with oral cavity cancer. The levels of DAPK1, MGMT, CDH1, SFRP1, SFRP2, RORA, TIMP3, p16, APC and RASSF1 proteins were measured by ELISA in tissue homogenates. The protein levels of DAPK1, MGMT, CDH1, SFRP2 and RASSF1 were significantly higher in tumour tissue than in the margin, contrary to TIMP3 which was lower in the tumour itself. DAPK1 level in the tumour was significantly higher in females than in males, the MGMT and p16 levels were lower in the tumours with lymph node metastasis (N1 + N2) than in N0 samples. The CDH1 expression was higher in a group with smoking habits, whereas TIMP3 was lower in this group. Changes in the levels of proteins in tumour and surgical margin may be either reflective of tumour occurrence and development, or they might be also responsible for the progress and reoccurrence of the disease. Levels of the studied proteins might be good prognostic factors; however, further studies are required.

Characterization of DAPK1 as a novel transcriptional target of BRMS1.

Breast cancer metastasis suppressor 1 (BRMS1) can specifically regulate tumor metastasis in many cancers. Our previous studies have demonstrated that BRMS1 can promote cell apoptosis through regulating osteopontin (OPN) expression in hepatocellular carcinoma (HCC) cells. However, the transcriptional targets of BRMS1 have not been thoroughly studied. In this study, death-associated protein kinase 1 (DAPK1), a tumor suppressor gene with multiple roles in regulating cell death, was identified as a potential transcriptional target of BRMS1 in the whole genome expression microarray. Quantitative real-time PCR and western blot analysis of HCC cells overexpressing BRMS1 further confirmed the transcriptional regulation relationship between BRMS1 and DAPK1. Moreover, DAPK1 expression was frequently decreased or even lost in HCC tissue samples by comparison with neighboring pathologically normal liver tissue, which was consistent with the decreased BRMS1 expression pattern. To unravel the molecular mechanism of BRMS1 in regulating DAPK1, a series of deletion mutants of DAPK1 promoter was subjected to luciferase assay. The luciferase units of -200 to -80 bp region, with two tandem putative NF-κB binding sites, were specifically enhanced by BRMS1 expression. Site-directed mutants of NF-κB binding sites blocked the transcriptional activation effect. In addition, the binding capability of BRMS1 and the putative NF-κB binding sites were demonstrated in the chromatin immunoprecipitation (ChIP) assay. In conclusion, our study characterized DAPK1 as a novel transcriptional target of BRMS1. Transcriptional activation of DAPK1 might be another important mechanism accounting for the metastasis suppressive activity of BRMS1.

YCl3 Promotes Neuronal Cell Death by Inducing Apoptotic Pathways in Rats.

The pollutants rare earth elements (REEs) have posed great threats to human health. To investigate the cytotoxicity of yttrium (Y), a model that rats have free access to water containing YCl3 for 6 months is utilized. The results showed that YCl3 treatment promoted neuronal cell apoptosis by upregulating the proapoptotic factors Bax, caspase-3, Cyto c, and DAPK and by downregulating the antiapoptotic factors Bcl-2 and XIAP at both mRNA and protein levels. Conclusively, YCl3 exhibited cytotoxicity and promoted neuronal cell death by the induction of apoptotic pathways.

Placental DAPK1 and autophagy marker LC3B-II are dysregulated by TNF-α in a gestational age-dependent manner.

Autophagy, a cell-survival process responsible for degradation of protein aggregates and damaged organelles, is increasingly recognized as another mechanism essential for human placentation. A substantial body of experiments suggests inflammation and oxidative stress as the underlying stimuli for altered placental autophagy, giving rise to placenta dysfunction and pregnancy pathologies. Here, the hypothesis is tested whether or not pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α are able to influence the expression profile of autophagy genes in human first-trimester villous placenta. Autophagy-focused qPCR arrays identified substantial downregulation of death-associated protein kinase 1 (DAPK1) in first-trimester placental explants in response to IL-6 and TNF-α, respectively. Immunohistochemistry of placental explants detected considerable DAPK1 staining in placental macrophages, villous cytotrophoblasts and less intense in the syncytiotrophoblast. Both immunohistochemistry and Western blot showed decreased DAPK1 protein in TNF-α-treated placental explants compared to control. On cellular level, DAPK1 expression decreased in SGHPL-4 trophoblasts in response to TNF-α. Observed changes in the expression profile of autophagy-related genes were reflected by significantly decreased lipidation of autophagy marker microtubule-associated protein light chain 3 beta (LC3B-II) in first trimester placental explants in response to TNF-α. Analysis of TNF-α-treated term placental explants showed decreased DAPK1 protein, whereas in contrast to first-trimester LC3B expression and lipidation increased. Immunohistochemistry of placental tissues from early-onset preeclampsia (PE) showed less DAPK1 staining, when compared to controls. Accordingly, DAPK1 mRNA and protein were decreased in primary trophoblasts isolated from early-onset PE, while LC3B-I and -II were increased. Results from this study suggest that DAPK1, a regulator of apoptosis, autophagy and programmed necrosis, decreases in human placenta in response to elevated maternal TNF-α, irrespective of gestational age. In contrast, TNF-α differentially regulates levels of autophagy marker LC3B in human placenta over gestation.

Death-associated Protein Kinase-1 Expression and Autophagy in Chronic Lymphocytic Leukemia Are Dependent on Activating Transcription Factor-6 and CCAAT/Enhancer-binding Protein-ß.

Expression of DAPK1, a critical regulator of autophagy and apoptosis, is lost in a wide variety of tumors, although the mechanisms are unclear. A transcription factor complex consisting of ATF6 (an endoplasmic reticulum-resident factor) and C/EBP-ß is required for the IFN-γ-induced expression of DAPK1 IFN-γ-induced proteolytic processing of ATF6 and phosphorylation of C/EBP-ß are obligatory for the formation of this transcriptional complex. We report that defects in this pathway fail to control growth of chronic lymphocytic leukemia (CLL). Consistent with these observations, IFN-γ and chemotherapeutics failed to activate autophagy in CLL patient samples lacking ATF6 and/or C/EBP-ß. Together, these results identify a molecular basis for the loss of DAPK1 expression in CLL.

Upregulation of DAPK contributes to homocysteine-induced endothelial apoptosis via the modulation of Bcl2/Bax and activation of caspase 3.

Hyperhomocysteinemia is characterized by an abnormally high level of homocysteine (Hcy) in the blood and is associated with cardiovascular diseases such as atherosclerosis. Endothelial dysfunction may lead to the pro-atherogenic effects associated with hyperhomocysteinemia. Endothelial dysfunction induced by Hcy has been previously investigated; however, the underlying molecular mechanism remains to be fully elucidated. The present study investigated whether death-associated protein kinase (DAPK) is involved in Hcy­induced apoptosis in human umbilical vein endothelial cells (HUVECs). It was determined that Hcy treatment upregulated the mRNA and protein expression levels of DAPK in HUVECs. Additionally, it was identified that the knockdown of DAPK using small interfering RNA may attenuate the Hcy-induced apoptosis and dissipation of mitochondrial membrane potential. DAPK inhibition may also reverse the effect of Hcy by the upregulation of B cell leukemia/lymphoma 2 (Bcl2) and poly ADP­ribose polymerase, and the downregulation of Bcl2­associated X protein (Bax) and of caspase 3. In conclusion, the present study demonstrated that DAPK contributed to the Hcy­induced endothelial apoptosis via modulation of Bcl2/Bax expression levels and activation of caspase 3.

Inhibition of DNA methylation promotes breast tumor sensitivity to netrin-1 interference.

In a number of human cancers, NTN1 upregulation inhibits apoptosis induced by its so-called dependence receptors DCC and UNC5H, thus promoting tumor progression. In other cancers however, the selective inhibition of this dependence receptor death pathway relies on the silencing of pro-apoptotic effector proteins. We show here that a substantial fraction of human breast tumors exhibits simultaneous DNA methylation-dependent loss of expression of NTN1 and of DAPK1, a serine threonine kinase known to transduce the netrin-1 dependence receptor pro-apoptotic pathway. The inhibition of DNA methylation by drugs such as decitabine restores the expression of both NTN1 and DAPK1 in netrin-1-low cancer cells. Furthermore, a combination of decitabine with NTN1 silencing strategies or with an anti-netrin-1 neutralizing antibody potentiates tumor cell death and efficiently blocks tumor growth in different animal models. Thus, combining DNA methylation inhibitors with netrin-1 neutralizing agents may be a valuable strategy for combating cancer.

MicroRNA-520g promotes epithelial ovarian cancer progression and chemoresistance via DAPK2 repression.

The lack of efficient tumor progression and chemoresistance indicators leads to high mortality in epithelial ovarian cancer (EOC) patients. Dysregulated miR-520g expression is involved in these processes in hepatic and colorectal cancers. In this study, we found that miR-520g expression gradually increased across normal, benign, borderline and EOC tissues. High miR-520g expression promoted tumor progression and chemoresistance to platinum-based chemotherapy, and reduced survival in EOC patients. miR-520g upregulation increased EOC cell proliferation, induced cell cycle transition and promoted cell invasion, while miR-520g downregulation inhibited tumor-related functions. In vivo, overexpression or downregulation of miR-520g respectively generated larger or smaller subcutaneous xenografts in nude mice. Death-associated protein kinase 2 (DAPK2) was a direct target of miR-520g. In 116 EOC tissue samples, miR-520g expression was significantly lower following DAPK2 overexpression. DAPK2 overexpression or miR-520g knockdown reduced EOC cell proliferation, invasion, wound healing and chemoresistance. This study suggests that miR-520g contributes to tumor progression and drug resistance by post-transcriptionally downregulating DAPK2, and that miR-520g may be a valuable therapeutic target in patients with EOC.

miR-520h is crucial for DAPK2 regulation and breast cancer progression.

MicroRNAs (miRNAs) are small RNAs that suppress gene expression by their interaction with 3'untranslated region of specific target mRNAs. Although the dysregulation of miRNAs has been identified in human cancer, only a few of these miRNAs have been functionally documented in breast cancer. Thus, defining the important miRNA and functional target involved in chemoresistance is an urgent need for human breast cancer treatment. In this study, we, for the first time, identified a key role of miRNA 520h (miR-520h) in drug resistance. Through protecting cells from paclitaxel-induced apoptosis, expression of miR-520h promoted the drug resistance of human breast cancer cells. Bioinformatics prediction, compensatory mutation and functional validation further confirmed the essential role of miR-520h-suppressed Death-associated protein kinase 2 (DAPK2) expression, as restoring DAPK2 abolished miR-520h-promoted drug resistance, and knockdown of DAPK2 mitigated cell death caused by the depletion of miR-520h. Furthermore, we observed that higher level of miR-520h is associated with poor prognosis and lymph node metastasis in human breast cancer patients. These results show that miR-520h is not only an independent prognostic factor, but is also a potential functional target for future applications in cancer therapeutics.

Activation of death-associated protein kinase in human peritumoral tissue: A potential therapeutic target.

To further understand the molecular mechanisms of N-methyl-D-aspartate receptor 2B (NR2B) phosphorylation and its contribution to glioma-related seizures, we investigated the expression of death-associated protein kinase-1 (DAPK1), which is a kinase known to phosphorylate NR2B at S1303 in glioma and peritumoral tissue. The molecular mechanisms leading to glioma-associated seizures are poorly understood. We recently discovered that NR2B is phosphorylated at S1303 in glioma peritumoral tissue. NR2B is an excitatory glutamate receptor, suggesting that glutamate released from glioma tumor cells may excite the neurons in the peritumoral tissue and contribute to glioma-associated epileptogenesis. DAPK1 levels were assessed in an intracranial mouse model of human glioma and in primary patient peritumoral and glioma tissues using immunohistochemistry. DAPK1 is highly expressed in the peritumoral region, but is poorly expressed in glioma tissues in both a mouse model of human glioma and in the primary patient glioma. In our previous report, we found that NR2B is also highly phosphorylated in the same region. Upregulation of DAPK1 in the peritumoral tissues suggests that DAPK1 can phosphorylate NR2B, increase its excitability, lead to glioma-induced seizures, and could potentially be an important therapeutic target. Furthermore, the xenograft model offers an opportunity to develop and test therapeutic approaches that can block DAPK1 activity in vivo.

The clinicopathological significance of miR-1307 in chemotherapy resistant epithelial ovarian cancer.

BACKGROUND: We aimed to examine the expression of miR-1307 in chemosensitive and chemoresistant epithelial ovarian cancer tissues and cell lines and to analyze the clinicopathological significance of miR-1307 in ovarian cancer. METHODS: MicroRNA microarray was used to screen differentially expressed microRNAs between the chemosensitive and chemoresistant epithelial ovarian cancer tissues. RT-PCR was used to validate the candidate microRNA. The potential target genes and their enriched biological pathways of microRNA were also analyzed. Dual Luciferase Reporter Gene Assay was conducted to validate the regulation of miRNA-1307 on the 3'-UTR of DAPK3. RESULTS: miRNA-1307 was up-regulated in the chemoresistant epithelial ovarian cancer tissues compared to the chemosensitive counterparts. The up-regulation of miRNA-1307 was not associated with menopause, tumor differentiation state, clinical stage, and lymph node metastasis of ovarian cancer. Gene ontology analysis of miR-1307 candidate target genes indicated that miR-1307 candidate target genes were enriched in the processes of cell proliferation and differentiation, nucleotide synthesis and metabolism, and lymphocytes activation. CONCLUSION: Our results suggest that miRNA-1307 may play a role in the development of chemoresistance in ovarian cancer.

Differential hypermethylation of death-associated protein kinase promoter in central neurocytoma and oligodendroglioma.

BACKGROUND: Central neurocytoma and oligodendroglioma are rare tumors of the central nervous system. However, diagnosis between these two types of tumors is challenging due to their many cytological and histological similarities. Death-associated protein kinase (DAPK) is a calcium/calmodulin-regulated serine/threonine protein kinase involved in many apoptosis pathways, and repressed expression of DAPK by promoter hypermethylation has been found in a variety of human cancers. The purpose of this study was to assess DAPK protein expression and promoter hypermethylation in central neurocytoma and oligodendroglioma. METHOD: Central neurocytoma and oligodendroglioma samples were obtained from age- and sex-matched patients. DAPK protein expression was performed using immunohistochemical assays in formalin-fixed, paraffin-embedded sections. DAPK promoter hypermethylation was carried out using bisulfite-modified genomic DNA in methylation-specific PCR followed by separation in agarose gels. FINDINGS: A statistically significant difference (P = 0.021) in DAPK promoter hypermethylation between central neurocytoma (76.9%) and oligodendroglioma (20%) was observed. High levels of DAPK protein expression were generally found in oligodendroglioma (90%), compared with 38.5% in central neurocytoma (P = 0.054; not statistically significant). There was an inverse correlation between DAPK protein expression and DAPK promoter hypermethylation in the cohort of 23 patients (P = 0.002). CONCLUSIONS: The results show that DAPK promoter hypermethylation and repressed expression of DAPK protein were more common in central neurocytoma than in oligodendroglioma. Thus, DAPK promoter hypermethylation could be useful for differential diagnosis between these two types of tumors, whereas DAPK protein expression might be less predictive. The role of DAPK promoter hypermethylation in the pathogenesis of central neurocytoma warrants further study.

Knockdown of S100P by lentiviral-mediated RNAi promotes apoptosis and suppresses the colony-formation ability of gastric cancer cells.

S100P is a putative candidate oncogene in several types of human tumors. However, expression of S100P, its potential role and its clinical significance in gastric cancer remain unclear. In the present study, S100P expression was examined by immunohistochemistry using a tissue microarray. Positive staining for S100P was noted in 77.1% of the cases while 22.9% were negative. In two gastric cancer cell lines, MGC-803 and SGC-7901, S100P expression was knocked down by a lentiviral short hairpin delivery system. The RNA interference-mediated downregulation of S100P expression markedly promoted cell apoptosis and inhibited cell colony-formation ability of the gastric cancer cells. In addition, knockdown of S100P significantly regulated the expression of 12 apoptosis-associated genes with a >1.5-fold change compared with the negative control. Among them, FOS, DDIT3 and FN1 were significantly upregulated, while FASLG, DAPK1, CTNNB1 and CASP2 were notably downregulated following S100P silencing. These results suggest that S100P acts as an oncogenic factor in gastric cancer and is a potential molecular target for gastric cancer gene therapy.

Promoter methylation of DAPK1, FHIT, MGMT, and CDKN2A genes in cervical carcinoma.

BACKGROUND AND OBJECTIVES: Aberrant DNA methylation contributes to the malignant phenotype in virtually all types of human cancer. This study explored the relationship between promoter methylation and inactivation of the DAPK1, FHIT, MGMT, and CDKN2A genes in cervical cancer. METHODS: The promoter methylation of DAPK1, FHIT, MGMT, and CDKN2A was investigated by using a methylation-specific polymerase chain reaction in 53 specimens of cervical cancer (42 squamous cell carcinoma, 11 adenocarcinoma), 22 specimens of intraepithelial neoplasia tissues, and 24 control normal cervical tissue specimens. The correlation of promoter methylation with the clinicopathological features of cervical cancer was analyzed. The expressions of DAPK1, FHIT, MGMT, and CDKN2A were detected by measuring relative mRNA levels. RESULTS: The promoter methylation of DAPK1, FHIT, MGMT, and CDKN2A in cervical cancer vs. intraepithelial neoplasia vs. normal cervical tissue was 75.5 vs. 31.8 vs. 4.2 % (p < 0.0001), 66.0 vs. 59.1 vs. 25.0 % (p = 0.0033), 34.0 vs. 27.3 vs. 20.8 % (p = 0.76), and 17.0 vs. 31.8 vs. 8.3 % (p = 0.11), respectively. The methylation of the promoter region significantly decreased the expression of only DAPK1 (p = 0.03). The methylation rate of the DAPK1 gene promoter was significantly higher in cervical cancer tissues than in cervical intraepithelial neoplasia and normal cervical tissues. CONCLUSION: Promoter methylation may therefore lead to the inactivation of the DAPK1 gene, and may be related to the progression of cervical oncogenesis.