OBJECTIVETumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance greatly limits the clinical therapeutic efficacy of TRAIL. Elucidating the molecular mechanism underlying TRAIL resistance will be fundamental to resolving this problem.

METHODSNuclear and cytoplasmic protein extraction and immuno？uorescence (IF) assay were used to detect changes in heterogeneous nuclear ribonucleoprotein K (hnRNPK) localization in H1299 cells. The evaluation of cell apoptosis in cells transfected with GFP-hnRNPK, GFP-hnRNPK S284/353A, or GFP-hnRNPK S284/353D mutant was performed using cleaved caspase-3 antibody. The gene expression of XIAP was tested by quantitative RT-PCR.

RESULTSPreviously, we reported that hnRNPK antagonized TRAIL-induced apoptosis through inhibition of PKC-mediated GSK3β phosphorylation. In this study, we further demonstrate that TRAIL treatment induces cytoplasmic accumulation of hnRNPK in H1299 cells. The hnRNPK localized in the cytoplasm has a higher capacity to antagonize TRAIL-induced apoptosis. Both ERK1/2 signaling inhibitor U0126 and ERK-phosphoacceptor-site mutant (GFP-hnRNPK S284/353A) diminish cytoplasmic accumulation of hnRNPK induced by TRAIL. Moreover, we show that XIAP is involved in hnRNPK-mediated TRAIL resistance in H1299 cells.

CONCLUSIONTaken together, these results give new insights into the understanding of the molecular mechanism associated with TRAIL resistance in lung adenocarcinoma.

Apoptosis ; physiology ; Cell Line, Tumor ; Gene Expression Regulation ; physiology ; Heterogeneous-Nuclear Ribonucleoprotein K ; genetics ; metabolism ; Humans ; Mitogen-Activated Protein Kinase 1 ; genetics ; metabolism ; Mitogen-Activated Protein Kinase 3 ; genetics ; metabolism ; TNF-Related Apoptosis-Inducing Ligand ; genetics ; metabolism ; Up-Regulation ; physiology ; X-Linked Inhibitor of Apoptosis Protein ; genetics ; metabolism

OBJECTIVETo investigate the effect of extracellular signal-regulated kinase (ERK) signaling pathway on the endothelial differentiation of periodontal ligament stem cells (PDLSC).

METHODSHuman PDLSC was cultured in the medium with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF) to induce endothelial differentiation. Endothelial inducing cells was incubated with U0126, a specific p-ERK1/2 inhibitor. PDLSC from one person were randomly divided into four groups: control group, endothelial induced group, endothelial induced+DMSO group and endothelial induced+U0126 group. The protein expression of the p-EKR1/2 was analyzed by Western blotting at 0, 1, 3, 6 and 12 hours during endonthelial induction. The mRNA expressions of CD31, VE-cadherin, and VEGF were detected by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) after a 7-day induction. The proportion of CD31(+) to VE-cadherin(+) cells was identified by flow cytometry, and the ability of capillary-like tubes formation was detected by Matrigel assay after a 14-day induction. The measurement data were statistically analyzed.

RESULTSPhosphorylated ERK1/2 protein level in PDLSC was increased to 1.24±0.12 and 1.03±0.24 at 1 h and 3 h respectively, during the endothelial induction (P<0.01). The mRNA expressions of CD31 and VEGF in induced+U0126 group were decreased to 0.09±0.18 and 0.49±0.17, which were both significantly different with those in induced group (P<0.05). The proportion of CD31(+) to VE-cadherin(+) cells of induced+U0126 group were decreased to 5.22±0.85 and 3.56±0.87, which were both significantly different with those in induced group (P<0.05). In Matrigel assay, the branching points, tube number and tube length were decreased to 7.0±2.7, 33.5±6.4, and (15 951.0±758.1) pixels, which were all significantly different with those in induced group (P<0.05).

CONCLUSIONSThe endothelial differentiation of PDLSC is positively regulated by ERK signaling pathway. Inhibition of ERK1/2 phosphorylation could suppress endothelial differentiation of PDLSC.

Antigens, CD ; genetics ; metabolism ; Butadienes ; pharmacology ; Cadherins ; genetics ; metabolism ; Cell Differentiation ; Endothelial Cells ; cytology ; physiology ; Enzyme Inhibitors ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; physiology ; Fibroblast Growth Factor 2 ; pharmacology ; Humans ; Mitogen-Activated Protein Kinase 3 ; antagonists & inhibitors ; metabolism ; Nitriles ; pharmacology ; Periodontal Ligament ; cytology ; metabolism ; Phosphorylation ; Platelet Endothelial Cell Adhesion Molecule-1 ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Random Allocation ; Signal Transduction ; Stem Cells ; cytology ; physiology ; Time Factors ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; pharmacology

OBJECTIVETo investigate the effects of silencing ADP-ribosylation factor 6 (Arf6) on the proliferation, migration, and invasion of prostate cancer cell line PC-3 and the possible molecular mechanisms.

METHODSThree Arf6-specific small interfering RNA (siRNA) were transfected into cultured prostate cancer cell line PC-3. Arf6 expression was examined by real-time PCR and Western blotting. MTT assay, wound healing assay, and Transwell migration and invasion assay were used to observe the effect of Arf6 silencing on the proliferation, migration, and invasion ability of PC-3 cells. The levels of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), ERK1/2, p-AKT, AKT and Rac1 were detected by Western blotting.

RESULTSTransfection of siRNA-3 resulted in significantly decreased Arf6 mRNA and protein expression with inhibition rates of (91.88±3.13)% and (86.37±0.57)%, respectively. Arf6 silencing by siRNA-3 markedly suppressed the proliferation, migration and invasion of PC-3 cells and reduced the expression levels of p-ERK1/2 and Rac1.

CONCLUSIONSilencing of Arf6 efficiently inhibits the proliferation, migration, and invasion of PC-3 cells in vitro, and the underlying mechanisms may involve the down-regulation of p-ERK1/2 and Rac1.

ADP-Ribosylation Factors ; genetics ; metabolism ; Cell Line, Tumor ; Cell Movement ; Down-Regulation ; Humans ; Male ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Neoplasm Invasiveness ; Prostatic Neoplasms ; pathology ; RNA Interference ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; Real-Time Polymerase Chain Reaction ; Transfection ; Wound Healing ; rac1 GTP-Binding Protein ; metabolism

BACKGROUND/AIMS: 5'-Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a cellular energy sensor that monitors intracellular AMP/adenosine triphosphate (ATP) ratios and is a key regulator of the proliferation and survival of diverse malignant cell types. In the present study, we investigated the effect of activating AMPK by 5-aminoimidazole-4-carboxamide-ribonucleotide (AICAR) in thyroid cancer cells. METHODS: We used FRO thyroid cancer cells harboring the BRAF(V600E) mutation to examine the effect of AICAR on cell proliferation and cell survival. We also evaluated the involvement of mitogen-activated protein kinase (MAPK) pathways in this effect. RESULTS: We found that AICAR treatment promoted AMPK activation and suppressed cell proliferation and survival by inducing p21 accumulation and activating caspase-3. AICAR significantly induced activation of p38 MAPK, and pretreatment with SB203580, a specific inhibitor of the p38 MAPK pathway, partially but significantly rescued cell survival. Furthermore, small interfering RNA targeting AMPK-alpha1 abolished AICAR-induced activation of p38 MAPK, p21 accumulation, and activation of caspase-3. CONCLUSIONS: Our findings demonstrate that AMPK activation using AICAR inhibited cell proliferation and survival by activating p38 MAPK and proapoptotic molecules in FRO thyroid cancer cells. These results suggest that the AMPK and p38 MAPK signaling pathways may be useful therapeutic targets to treat thyroid cancer.
AMP-Activated Protein Kinases/genetics/metabolism ; Aminoimidazole Carboxamide/*analogs & derivatives/pharmacology ; Antineoplastic Agents/*pharmacology ; Caspase 3/metabolism ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Cell Survival/drug effects ; Cyclin-Dependent Kinase Inhibitor p21/metabolism ; Dose-Response Relationship, Drug ; Enzyme Activation ; Enzyme Activators/pharmacology ; Humans ; Mutation ; Protein Kinase Inhibitors/pharmacology ; Proto-Oncogene Proteins B-raf/genetics ; RNA Interference ; Ribonucleotides/*pharmacology ; Signal Transduction/*drug effects ; Thyroid Neoplasms/*enzymology/genetics/pathology ; Time Factors ; Transfection ; p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors/*metabolism

The expression of hypoxia-inducible factor (HIF) is influenced by reactive oxygen species (ROS). Effect of bilirubin on HIF-1 expression in proximal tubular cells was investigated under physiological oxygen concentration, which is relative hypoxic condition mimicking oxygen content in the medulla of renal tissue. The human kidney (HK2) cells were cultured in 5% oxygen with or without bilirubin. HIF-1alpha protein expression was increased by bilirubin treatment at 0.01-0.2 mg/dL concentration. The messenger RNA expression of HIF-1alpha was increased by 1.69+/-0.05 folds in the cells cultured with 0.1 mg/dL bilirubin, compared to the control cells. The inhibitors of PI3K/mTOR, PI3K/AKT, and ERK 1/2 pathways did not attenuate increased HIF-1alpha expression by bilirubin. HIF-1alpha expression decreased by 10 microM exogenous hydrogen peroxide (H2O2); scavenger of ROS with or without bilirubin in the HK2 cells increased HIF-1alpha concentration more than that in the cells without bilirubin. Exogenous H2O2 decreased the phosphorylation of P70S6 kinase, which was completely reversed by bilirubin treatment. Knockdown of NOX4 gene by small interfering RNA (siRNA) increased HIF-1alpha mRNA expression. In coonclusion, bilirubin enhances HIF-1alpha transcription as well as the up-regulation of HIF-1alpha protein translation through the attenuation of ROS and subunits of NADPH oxidase.
Bilirubin/*pharmacology ; Cell Line ; Epithelial Cells/cytology/metabolism ; Humans ; Hydrogen Peroxide/toxicity ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics/*metabolism ; Kidney Tubules, Proximal/cytology ; Mitogen-Activated Protein Kinase 1/metabolism ; Mitogen-Activated Protein Kinase 3/metabolism ; NADPH Oxidase/antagonists & inhibitors/genetics/metabolism ; Oxygen/*pharmacology ; Phosphatidylinositol 3-Kinases/metabolism ; Phosphorylation/drug effects ; Proto-Oncogene Proteins c-akt/metabolism ; RNA Interference ; Ribosomal Protein S6 Kinases, 70-kDa/metabolism ; Signal Transduction/drug effects ; TOR Serine-Threonine Kinases/metabolism ; Transcriptional Activation/*drug effects ; Up-Regulation/drug effects

To investigate the role of the extracellular signal-regulated kinase (ERK1/2) and PI3K/AKT/ mTOR signal pathway inducing bone marrow mesenchymal stem cells (BMSCs) differentiation into neural cells, mouse bone marrow-derived mesenchymal stem cell lines D1 cells were used as research object. And they were divided into control groups and salidroside (SD) groups. Different concentrations (5, 25, 50, 100 and 200 microg x mL(-1) of SD were used and SD (100 microg x mL(-1)) was used to induce at different time (0.5, 1, 3, 6, 9, 12, 24, 48 and 72 h). The immunofluorescence staining chemical technology, real-time PCR and Western blotting were used to detect the positive rates of NSE, MAP2, beta-Tubulin III, NES, GFAP and the expression levels of beta-Tubulin III, NSE, ERK1/2, AKT. The expression of ERK1/2 and NSE was detected when the ERK1/2 and PI3K/AKT/ mTOR signal pathway was blocked by PD98059 and LY294002. It indicated that the positive rates of NSE, MAP2, beta-Tubulin III, NES and GFAP were gradually enhanced with time increased. The expression level of NSE and beta-Tubulin III protein were significantly higher than those in control groups (P < 0.01). The expression of ERK1/2, AKT mRNA and protein were higher with concentration and time increased. When the ERK1/2 and PI3K/AKT/mTOR signal pathway were blocked, the expression levels of NSE, NES and beta-Tubulin III mRNA and NSE protein were inhibited significantly. It points out that SD can stimulate the ERK1/2 and PI3K/AKT/mTOR signal pathway to promote BMSCs differentiation into neural cells.
Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Chromones ; pharmacology ; Enzyme Inhibitors ; pharmacology ; Flavonoids ; pharmacology ; Glial Fibrillary Acidic Protein ; metabolism ; Glucosides ; antagonists & inhibitors ; isolation & purification ; pharmacology ; MAP Kinase Signaling System ; drug effects ; Mesenchymal Stromal Cells ; cytology ; Mice ; Microtubule-Associated Proteins ; metabolism ; Mitogen-Activated Protein Kinase 1 ; genetics ; metabolism ; Mitogen-Activated Protein Kinase 3 ; genetics ; metabolism ; Morpholines ; pharmacology ; Nestin ; metabolism ; Neurons ; cytology ; metabolism ; Phenols ; antagonists & inhibitors ; isolation & purification ; pharmacology ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphopyruvate Hydratase ; genetics ; metabolism ; Plants, Medicinal ; chemistry ; Protein Kinase Inhibitors ; pharmacology ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Rhodiola ; chemistry ; Signal Transduction ; drug effects ; TOR Serine-Threonine Kinases ; metabolism ; Tubulin ; metabolism

Annexin A1 (ANXA1) is a kind of endogenous scaffold protein. Previous research showed that ANXA1 could increase markedly with multiple increase of drug resistance in K562/imatinib cell lines in vitro. Here the stable transfection cell strains K562-pEGFP-N1 which was the native control and K562-pEGFP-N1-ANXA1 which can stably express ANXA1 were established using the Lipofectamine 2000 in order to find whether ANXA1 involved in the drug resistance. Cell growth inhibition experiment via MTT and cell proliferation experiment via MTS showed that K562-pEGFP-N1-ANXA1 cell strain was more sensitive to imatinib than the K562-pEGFP-N1 cell strain, and however the ability of proliferation of K562-pEGFP-N1-ANXA1 cell strain did not change compared with the negative control. Western blotting results showed that the expression of proteins in Annexin family did not change; drug resistance proteins, Bcr-Abl/p-Bcr-Abl (Tyr245), Src family kinase for example, did not change; proteins related with cell proliferation and cell cycle, such as ERK1/2MAPK, p-38MAPK, CDK1 and Wee 1, did not change either in the K562-pEGFP-N1-ANXA1 cell strain compared with the negative control. The co-immunoprecipitation result showed that the interaction between ANXA1 and beta-actin in the K562-pEGFP-N1-ANXA1 cell strain increased markedly. The deduction was that ANXA1 may make the K562-pEGFP-N1-ANXA1 cell strain more sensitive to imatinib due to the increased uptake of imatinib via the increase of ANXA1 and the interaction between ANXA1 and beta-actin in the K562-pEGFP-N1-ANXA1 cell strain in vitro.
Actins ; metabolism ; Annexin A1 ; genetics ; metabolism ; Antineoplastic Agents ; pharmacology ; Cell Proliferation ; drug effects ; Drug Resistance, Neoplasm ; Fusion Proteins, bcr-abl ; metabolism ; Humans ; Imatinib Mesylate ; pharmacology ; Immunoprecipitation ; K562 Cells ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Transfection ; src-Family Kinases ; metabolism

Taxol has clinical efficacy on many malignant tumors, thus having a good market prospects. The anti-cancer mechanism of taxol is to arrest the cell cycle of tumor cells, leading to apoptosis. Based on our research over the years, we reviewed the latest developments in signaling pathways, the effects of related genes and proteins on apoptosis during paclitaxel-induced apoptosis process, including the paclitaxel-producing gene in microbial fermentation process, cyclin, telomerase of apoptosis.
Animals ; Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; genetics ; Cell Cycle Checkpoints ; drug effects ; Humans ; MAP Kinase Signaling System ; drug effects ; NF-kappa B ; metabolism ; Neoplasms ; drug therapy ; pathology ; Paclitaxel ; pharmacology ; Phosphatidylinositol 3-Kinases ; metabolism ; Signal Transduction ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo investigate the effect of the selective PI3K inhibitor and MEK inhibitor on KRAS and PTEN co-mutated non-small cell lung cancer cell line NCI-H157 and the relevant mechanisms.

METHODSNCI-H157 was cultured routinely and treated with different concentrations of the two inhibitors. Cell proliferation was detected by MTT cell cycle assay. Based on the MTT results the cells were divided into four groups: the control group, PI3K inhibitor group (GDC-0941, 0.5 and 5.0 µmol/L), combination group I (0.5 µmol/L AZD6244 + 0.5 µmol/L GDC-0941) and combination group II (5.0 µmol/L AZD6244 + 5.0 µmol/L GDC-0941). Colony formation assay was performed to detect colony formation efficiency. The cell cycle and apoptosis were analyzed by flow cytometry. The expression of protein related to apoptosis was tested with Western blot.

RESULTSCell growth was inhibited by the two inhibitors. Combination groups led to stronger cell proliferation inhibition: combination group Ishowed synergistic effect of their actions and combination group II showed an additive effect; in both groups, there were decreased colony number [(77.2 ± 1.54)/well vs (61.50 ± 2.12)/well, P < 0.01] and [(51.00 ± 4.00)/ well vs (22.50 ± 3.53)/well, P < 0.01]; and enhanced apoptotic ratios [(18.30 ± 0.82)% vs (21.32 ± 0.56)%, P < 0.01] and [(27.14 ± 1.58)% vs (42.45 ± 4.42)%, P < 0.01]. In addition, compared to the PI3K inhibitor alone group, the NCI-H157 cells in the combination groups showed increased G0/G1 phase and decreased S phase (P < 0.01). Western blotting showed that the combination groups demonstrated significantly decreased expression of cyclin D1 and cyclin B1, increased p21 and cleaved PARP and decreased bcl-2/bax ratio, compared to the PI3K inhibitor only group.

CONCLUSIONThe combined inhibition of PI3K (AZD6244) and MEK (GDC-0941) has synergistic effects on the proliferation of NCI-H157 cells, but such effects appear to be in a dose-dependent manner.

Apoptosis ; drug effects ; Benzimidazoles ; administration & dosage ; pharmacology ; Carcinoma, Non-Small-Cell Lung ; genetics ; pathology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cyclin B1 ; metabolism ; Cyclin D1 ; metabolism ; Dose-Response Relationship, Drug ; Drug Synergism ; Humans ; Indazoles ; administration & dosage ; pharmacology ; Lung Neoplasms ; genetics ; pathology ; Mitogen-Activated Protein Kinase Kinases ; antagonists & inhibitors ; metabolism ; Mutation ; PTEN Phosphohydrolase ; genetics ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors ; metabolism ; Poly(ADP-ribose) Polymerases ; metabolism ; Proto-Oncogene Proteins ; genetics ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Proto-Oncogene Proteins p21(ras) ; metabolism ; Signal Transduction ; Sulfonamides ; administration & dosage ; pharmacology ; bcl-2-Associated X Protein ; metabolism ; ras Proteins ; genetics

The differentiation of periodontal ligament (PDL) progenitor cells is important for maintaining the homeostasis of PDL tissue and alveolar bone. Vitamin C (VC), a water-soluble nutrient that cannot be biosynthesized by humans, is vital for mesenchymal stem cells differentiation and plays an important role in bone remodeling. Therefore, the objective of this study was to determine the function and mechanism of VC in PDL progenitor cells osteogenic differentiation at the molecular level. We demonstrated that VC could induce the osteogenic differentiation and maturation of PDL progenitor cell without other osteogenic agents. During the process, VC preferentially activated ERK1/2 but did not affect JNK or p38. Co-treatment with ERK inhibitor effectively decreased the Vitamin C-induced expression of Runx2. ERK inhibitor also abrogated Vitamin C-induced the minimized nodules formation. PELP1, a nuclear receptor co-regulator, was up-regulated under VC treatment. PELP1 knockdown inhibited ERK phosphorylation. The overexpression of PELP1 had a positive relationship with Runx2 expression. Taken together, we could make a conclude that VC induces the osteogenic differentiation of PDL progenitor cells via PELP1-ERK axis. Our finding implies that VC may have a potential in the regeneration medicine and application to periodontitis treatment.
Ascorbic Acid ; pharmacology ; Butadienes ; pharmacology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Co-Repressor Proteins ; antagonists & inhibitors ; genetics ; metabolism ; Core Binding Factor Alpha 1 Subunit ; genetics ; metabolism ; Humans ; MAP Kinase Signaling System ; drug effects ; Mitogen-Activated Protein Kinase 1 ; antagonists & inhibitors ; metabolism ; Mitogen-Activated Protein Kinase 3 ; antagonists & inhibitors ; metabolism ; Nitriles ; pharmacology ; Periodontal Ligament ; cytology ; Phosphorylation ; drug effects ; RNA Interference ; RNA, Small Interfering ; metabolism ; Stem Cells ; cytology ; Transcription Factors ; antagonists & inhibitors ; genetics ; metabolism ; Up-Regulation ; drug effects