The aim of this study was to examine the effects of endoplasmic reticulum (ER) stress on aldosterone (Aldo)-induced apoptosis of endothelial cells. Glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP, a hallmark of ER-associated apoptosis) were used to evaluate ER stress. Western blotting and real-time PCR were used to analyze indicators of ER molecule. Apoptosis was detected by annexin V/propidium iodide staining and flow cytometry. Human umbilical vein endothelial cells (HUVECs) were stimulated with different concentrations of Aldo for different durations. Aldo promoted apoptosis of HUVECs and induced ER stress, as evidenced by increased expression of GRP78 and CHOP. siRNA knockdown of CHOP attenuated Aldo-mediated apoptosis. These results indicate that ER stress may be involved in Aldo-induced apoptosis of HUVECs.
Aldosterone ; pharmacology ; Apoptosis ; drug effects ; Endoplasmic Reticulum Stress ; drug effects ; Gene Expression Regulation ; drug effects ; Heat-Shock Proteins ; biosynthesis ; Human Umbilical Vein Endothelial Cells ; cytology ; metabolism ; Humans ; Transcription Factor CHOP ; biosynthesis

OBJECTIVETo explore the feasibility of detecting FUS-CHOP fusion gene in formalin-fixed, paraffin-embedded tissue and its application in the diagnosis and differential diagnosis of myxoid/round cell liposarcomas (MRCLs).

METHODSForty-four formalin-fixed, paraffin-embedded MRCL samples and 60 control cases (atypical/well-differentiated liposarcoma, pleomorphic liposarcoma, low-grade myofibrosarcoma, etc.) retrieved from the archival files were studied. Nested reverse transcription-polymerase chain reaction (RT-PCR) technique was employed to detect the FUS-CHOP mRNA expression, followed by DNA sequencing confirmation of the PCR product. Housekeeping gene PGK was used to assess the quality of the mRNA templates.

RESULTSPGK mRNA was detected in 93 of 104 tumor cases (89.4%), including 39 MRCLs cases (39/44, 88.6%) and 90% of the negative control cases. Type II FUS-CHOP fusion transcript was successfully detected in 20 out of 39 (51.3%) MRCL cases. Type I FUS-CHOP fusion transcript was not detected in any MRCLs in this study. All 60 negative control cases were negative for the FUS-CHOP fusion gene transcripts.

CONCLUSIONS(1) Nested RT-PCR can be used to detect FUS-CHOP mRNA in formalin-fixed, paraffin-embedded tissues. (2) FUS-CHOP is considered a specific molecular and genetic hallmark for MRCLs. Nested RT-PCR is a sensitive and specific technique in detecting FUS-CHOP gene, and can be used in the diagnosis and differential diagnosis of MRCLs.

Adolescent ; Adult ; Aged ; Biomarkers, Tumor ; Diagnosis, Differential ; Female ; Humans ; Liposarcoma ; metabolism ; pathology ; Liposarcoma, Myxoid ; metabolism ; pathology ; Lower Extremity ; Male ; Middle Aged ; Oncogene Proteins, Fusion ; biosynthesis ; genetics ; Paraffin Embedding ; RNA, Messenger ; biosynthesis ; genetics ; RNA-Binding Protein FUS ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Soft Tissue Neoplasms ; metabolism ; pathology ; Transcription Factor CHOP ; biosynthesis ; genetics

Baicalein is one of the major flavonoids in Scutellaria baicalensis Georgi and possesses various effects, including cytoprotection and anti-inflammation. Because endoplasmic reticulum (ER) stress has been implicated in neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and cerebral ischemia, we investigated the effects of baicalein on apoptotic death of HT22 mouse hippocampal neuronal cells induced by thapsigargin (TG) and brefeldin A (BFA), two representative ER stress inducers. Apoptosis, reactive oxygen species (ROS) production, and mitochondrial membrane potential (MMP) were measured by flow cytometry. Expression level and phosphorylation status of ER stress-associated proteins and activation and cleavage of apoptosis-associated proteins were analyzed by Western blot. Baicalein reduced TG- and BFA-induced apoptosis of HT22 cells and activation and cleavage of apoptosis-associated proteins, such as caspase-12 and -3 and poly(ADP-ribose) polymerase. Baicalein also reduced the TG- and BFA-induced expression of ER stress-associated proteins, including C/EBP homologous protein (CHOP) and glucose-regulated protein 78, the cleavage of X-box binding protein-1 and activating transcription factor 6alpha, and the phosphorylation of eukaryotic initiation factor-2alpha and mitogen-activated protein kinases, such as p38, JNK, and ERK. Knock-down of CHOP expression by siRNA transfection and specific inhibitors of p38 (SB203580), JNK (SP600125), and ERK (PD98059) as well as anti-oxidant (N-acetylcysteine) reduced TG- or BFA-induced cell death. Baicalein also reduced TG- and BFA-induced ROS accumulation and MMP reduction. Taken together, these results suggest that baicalein could protect HT22 neuronal cells against ER stress-induced apoptosis by reducing CHOP induction as well as ROS accumulation and mitochondrial damage.
Animals ; *Apoptosis ; Brefeldin A/pharmacology ; Cell Line ; Cytoprotection ; DNA-Binding Proteins/metabolism ; Endoplasmic Reticulum/drug effects/*physiology ; Flavanones/*pharmacology ; Heat-Shock Proteins/biosynthesis ; Hippocampus/cytology ; Membrane Potential, Mitochondrial/drug effects ; Mice ; Mitogen-Activated Protein Kinases/metabolism ; Neurons/*drug effects/physiology ; Reactive Oxygen Species/*metabolism ; Signal Transduction/drug effects ; Thapsigargin/pharmacology ; Transcription Factor CHOP/*biosynthesis ; Transcription Factors/metabolism ; Unfolded Protein Response/drug effects