BACKGROUND: Invasive nonfunctioning pituitary adenomas (NFPAs) remain challenging due to their high complication rate and poor prognosis. We aimed to identify the distinctive molecular signatures of invasive NFPAs, compared with noninvasive NFPAs, using gene expression profiling by RNA sequencing. METHODS: We obtained frozen fresh tissue samples from 14 patients with NFPAs who underwent primary transsphenoidal surgery. Three non-invasive and 11 invasive NFPAs were used for RNA sequencing. The bioinformatics analysis included differential gene expression, gene ontology analysis, and pathway analysis. RESULTS: A total of 700 genes were differentially expressed (59 up-regulated and 641 down-regulated genes) between invasive and non-invasive NFPAs (false discovery rate <0.1, and |fold change| ≥2). Using the down-regulated genes in invasive NFPAs, gene ontology enrichment analyses and pathway analyses demonstrated that the local immune response was attenuated and that transforming growth factor-β (TGF-β) RII-initiated TGF-β signaling was down-regulated in invasive NFPAs. The overexpression of claudin-9 (CLDN9) and the down-regulation of insulin-like growth factor-binding protein 5 (IGFBP5), death-associated protein kinase 1 (DAPK1), and tissue inhibitor of metalloproteinase-3 (TIMP3) may be related with invasiveness in NFPAs. CONCLUSION: Invasive NFPAs harbor different gene expression profiles relative to noninvasive NFPAs. In particular, local suppression of the immune response and TGF-β signaling can make PAs prone to invasiveness.
Computational Biology ; Death-Associated Protein Kinases ; Down-Regulation ; Gene Expression ; Gene Expression Profiling ; Gene Ontology ; Humans ; Pituitary Neoplasms ; Prognosis ; Sequence Analysis, RNA ; Tissue Inhibitor of Metalloproteinase-3 ; Transcriptome

The apoptotic effects of shikonin (5,8-dihydroxy-2-[(1R)-1-hydroxy-4-methylpent-3-enyl]naphthalene-1,4-dione) on the human colon cancer cell line SNU-407 were investigated in this study. Shikonin showed dose-dependent cytotoxic activity against SNU-407 cells, with an estimated IC50 value of 3 µM after 48 h of treatment. Shikonin induced apoptosis, as evidenced by apoptotic body formation, sub-G1 phase cells, and DNA fragmentation. Shikonin induced apoptotic cell death by activating mitogen-activated protein kinase family members, and the apoptotic process was mediated by the activation of endoplasmic reticulum (ER) stress, leading to activation of the PERK/elF2α/CHOP apoptotic pathway, and mitochondrial Ca2+ accumulation. Shikonin increased mitochondrial membrane depolarization and altered the levels of apoptosis-related proteins, with a decrease in B cell lymphoma (Bcl)-2 and an increase in Bcl-2-associated X protein, and subsequently, increased expression of cleaved forms of caspase-9 and -3. Taken together, we suggest that these mechanisms, including MAPK signaling and the ER-and mitochondria-mediated pathways, may underlie shikonin-induced apoptosis related to its anticancer effect.
Apoptosis ; bcl-2-Associated X Protein ; Caspase 9 ; Cell Death* ; Cell Line ; Colon* ; Colonic Neoplasms* ; DNA Fragmentation ; Endoplasmic Reticulum* ; Extracellular Vesicles ; Humans* ; Inhibitory Concentration 50 ; Lymphoma, B-Cell ; Mitochondria ; Mitochondrial Membranes ; Protein Kinases

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 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 screen the differential methylation patterns of tumor suppressor gene DAPK and evaluate its value as a biomarker for the diagnosis of leukemia.

METHODSThe methylation status of DAPK gene promoter's CpG island was analyzed in the genomes of normal human white blood cells and HL-60, U937 and Jurkat cell lines by bisulfite sequencing PCR (BSP). The effectiveness of differential methylation patterns of DAPK gene for diagnosis of leukemia was verified in the leukemia cell lines and peripheral blood samples by methylation specific PCR (MSP).

RESULTSThe methylation pattern of DAPK gene in different cell genomes displayed that the degree of unmethylation in normal cell genome was higher than that of leukemia cell lines. The differential CpG sites were found and could be used to differentiate HL-60 and the other 3 cell lines by MSP. Meanwhile, the differential methylation patterns in clinical specimens could distinguish acute non-lymphocytic leukemia (ANLL) and other types of leukemia by MSP. The diagnostic sensitivity, specificity and accuracy were 59.1%, 100% and 82.7% respectively. No relationship was found between MSP diagnosis results and clinical pathological typing.

CONCLUSIONThe differential methylation patterns of DAPK gene as potential tumor biomarker for diagnosis of leukemia can enrich the means of diagnosis of leukemia, provide idea and basis for finding all kinds of tumor's DNA methylation biomarkers in the future.

Biomarkers, Tumor ; genetics ; CpG Islands ; DNA Methylation ; Death-Associated Protein Kinases ; genetics ; HL-60 Cells ; Humans ; Jurkat Cells ; Leukemia ; diagnosis ; genetics ; Polymerase Chain Reaction ; Promoter Regions, Genetic

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 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

This study was purpose to investigate the expression of death-associated protein kinase (DAPK) gene in acute leukemia (AL) patients and the methylation status of its promoter region through experiments of DAPK methylation and expression, and to analyze the relation between them. The expression of DAPK gene in leukemia cells and normal bone marrow cells was detected by RT-PCR; the methylation status of DAPK gene promoter region in cells from AL patients and leukemia cell lines HL-60 and U937 was detected by nested methylation specific PCR (n-MSP); 2 randomly primers selected from randomly amplified products of second round nMS-PCR were cloned and sequenced in professional company. The results showed that the DAPK gene expressed in bone marrow specimens of all 10 normal controls, with average value of expression 0.92 ± 0.18, while the average value of DAPK expression in bone marrow specimens of AL patients was 0.61 ± 0.40 which was lower than that in normal controls (P < 0.05). The low or deletion of DAPK mRNA expression were found in bone marrow specimens of 9/17 (52.94%) cases of ALL and 42/102 (41.18%) cases of AML. The cell line U937 showed normal expression of DAPK gene, while cell line HL-60 showed the expression detection of DAPK gene. The methylation of DAPK promoter region existed in 33 out of bone marrow specimens of 102 AML patients and in 8 out of bone marrow specimens of 17 ALL patients, the methylation rates were 32.4% (33/102) and 47% respectively. The DAPK promoter region in bone marrow of 7 normal controls was unmethylated, while DAPK promoter region in U937 cells and HL-60 cells were unmethylated and methylated respectively. The DAPK mRNA expression in ALL and AML patients significantly negatively correlated with the methylation of its promoter region (r = -0.855, P < 0.05, in AML patients and r = -0.343, P < 0.05, in AML patients) suggesting the close relationship between them. It is concluded that the methylation of DAPK gene promoter region relates with abnormal expression or detection of DAPK mRNA in AL patients.
Acute Disease ; Case-Control Studies ; DNA Methylation ; Death-Associated Protein Kinases ; genetics ; HL-60 Cells ; Humans ; Leukemia ; genetics ; Promoter Regions, Genetic ; RNA, Messenger ; genetics

This study was aimed to investigate the effect of methylation transferase inhibitor 5-aza-2'-deoxycytidine (5-aza-2dC) of different concentrations on the apoptosis of human acute myeloid leukemia (AML) cell line HL-60 and the expression of DAPK gene in HL-60 cells, as well as to explore the possible anti-AML mechanism of 5-aza-2dC. HL-60 cells were treated by 5-aza-2dC of different concentrations. The effect of 5-aza-2dC on the HL-60 cell morphology was observed by Wright's staining. The effect of 5-aza-2dC on HL-60 cell apoptosis and DAPK mRNA expression was detected by flow cytometry and reverse transcription-polymerize chain reaction (RT-PCR) respectively. The results showed that the 5-aza-2dC induced the apoptosis of HL-60 cells in a concentration-dependent manner; the 5-aza-2dC increased the expression levels of DAPK mRNA in HL-60 cells in a concentration-dependent manner. It is concluded that the apoptosis rate of HL-60 cells and DAPK mRNA expression level displayed a rising trend with 5-aza-2dC concentration increasing. Therefore, DAPK gene may participate in HL-60 cell apoptosis induced by 5-aza-2dC.
Apoptosis ; drug effects ; Death-Associated Protein Kinases ; genetics ; Deoxycytidine ; pharmacology ; Gene Expression ; drug effects ; HL-60 Cells ; Humans

OBJECTIVETo study the effect of triptolide (TP) on the methylation status of human promyelocytic leukemia cells (HL-60) and explore a preliminary demethylation mechanism.

METHODSNormal HL-60 cells as control group, the cell proliferation level of HL-60 cells was detected by MTT assay, being treated by different concentration TP (3.125, 6.25, 12.5, 25 nmol/L) for 24 h or 48 h respectively; Choosing the 3.125 nmol/L and the 6.25 nmol/L TP affected HL-60 cells for 48 h, the cell apoptosis rate and cell cycle were determined by flow cytometry, the expressions of death-associated protein kinase 1 (DAPK-1) and methyltransferase DNMT1, DNMT3B mRNA were measured by real time-PCR (RT-PCR), LINE-1, DAPK-1 genes'methylation variations were analyzed by methylation specific PCR (MSP).

RESULTSCompared with control group, the different concentration TP could significantly inhibit the proliferation of HL-60 in a time-dose dependent manner (P<0.05, P<0.01). After being treated by TP for 48 h, the cell early apoptosis rate of control group and 6.250 nmol/L TP group were (2.07 ± 1.91)%, (9.77 ± 3.52)%, respectively (P<0.05); When the TP concentration increased, DAPK-1mRNA expression increased (P<0.01), DNMT1, DNMT3B mRNA expression significantly dampened (P<0.01); the promoter of LINE-1, DAPK-1 genes were hypermethylation state in the control group, after being treated by TP for 48 h, the brightness of LINE-1, DAPK-1 genes'methylation strips weakened, and the non-methylation strips enhanced all in a dose-dependent manner.

CONCLUSIONTP could down-regulate the transcriptional expression of methyltransferase DNMT1/3B genes, indirect action to reduce the degree of DAPK-1, LINE-1 genes mathylation, thus promote DAPK-1 gene expression level and inhibit the HL 60 cell growth.

DNA (Cytosine-5-)-Methyltransferases ; DNA Methylation ; drug effects ; Death-Associated Protein Kinases ; Diterpenes ; pharmacology ; Epoxy Compounds ; pharmacology ; HL-60 Cells ; Humans ; Phenanthrenes ; pharmacology ; Promoter Regions, Genetic ; RNA, Messenger