OBJECTIVE: To investigate the value of using MDM2 amplification probe and DDIT3 dual-color, break-apart rearrangement probe fluorescence in situ hybridization (FISH) technique in the diagnosis of liposarcoma. METHODS: In the study, 62 cases of liposarcoma diagnosed in Peking University First Hospital from January 2015 to December 2019 were analysed for clinicopathological information. Of these 62 cases of liposarcoma, all were analysed for MDM2 amplification and 48 cases were analysed for DDIT3 rearrangement using a FISH technique. Our study aimed to evaluate the status of MDM2 and DDIT3 by FISH in liposarcoma and correlate it with diagnosis of different subtypes of liposarcoma. The subtypes of liposarcoma were classified according to the FISH results, combined with the relevant clinicopathological features. RESULTS: The patients aged 31-89 years (mean: 59 years) with a 1.75:1 male to female ratio. Histologically, there were 20 cases of atypical lipomatous tumour/well-differentiated liposarcoma (ALT/WDLPS), 26 cases of dedifferentiated liposarcoma (DDLPS), 13 myxoid liposarcoma (MLPS) and 3 pleomorphic liposarcoma (PLPS). Tumors with DDLPS (23/26) and WDLPS (8/20) were localized retroperitoneally, while both tumours of MLPS and PLPS were localized extra-retroperitoneally, and the difference of sites among the four subtypes of liposarcoma was statistically significant (P < 0.05). Histologically, varied mucoid matrix could be observed in the four subtypes of liposarcoma, and the difference was statistically significant (P < 0.05). MDM2 gene amplification was demonstrated in all cases of ALT/WDLPS and DDLPS (100%, 20/20 and 26/26 respectively); DDIT3 gene rearrangement was noted only in MLPS (100%, 13/13); most cases of DDLPS (96.2%, 25/26) and ALT/WDLPS (83.3%, 5/6, 6 cases selected for detection) demonstrated the picture of amplification of the DDIT3 telomeric tag. According to the instructions of DDIT3 break-apart rearrangement probe, the 5' telomere probe and 3' centromere probe spanned but did not cover the DDIT3 gene itself, on the contrary, the 5' telomere probe covered the CDK4 gene, while the DDIT3 and CDK4 gene were located adjacent to each other on chromosome, therefore, when the amplification signal appeared on the telomeric tag of the DDIT3 rearrangement probe, it indeed indicated the CDK4 gene amplification rather than the DDIT3 gene rearrangement. Then the 10 cases with DDIT3 telomeric tag amplification were selected for CDK4 and DDIT3 gene amplification probe FISH tests, and all the cases showed CDK4 gene amplification (100%, 10/10) and two of the 10 cases demonstrated co-amplification of CDK4 and DDIT3 (20%, 2/10); DDIT3 polysomy detected by DDIT3 gene rearrangement probe was found in 1 case of DDLPS and 2 cases of PLPS (66.7%, 2/3) with morphology of high-grade malignant tumour and poor prognosis. CONCLUSION Our results indicate that a diagnosis of different subtype liposarcoma could be confirmed based on the application of MDM2 and DDIT3 FISH, combined with clinicopathological findings. It is also noteworthy that atypical signals should be correctly interpreted to guide correct treatment of liposarcomas.
Male ; Female ; Humans ; In Situ Hybridization, Fluorescence/methods* ; Cyclin-Dependent Kinase 4/metabolism* ; Liposarcoma/pathology* ; Lipoma/pathology* ; Gene Amplification ; Transcription Factor CHOP/genetics* ; Proto-Oncogene Proteins c-mdm2/metabolism*

OBJECTIVEPERK/eIF2α/CHOP is a major signaling pathway mediating endoplasmic reticulum (ER) stress related with atherosclerosis. Oxidized LDL (ox-LDL) also induces endothelial apoptosis and plays a vital role in the initiation and progression of atherosclerosis. The present study was conducted to explore the regulatory effect of ox-LDL on PERK/eIF2α/CHOP signaling pathway in vascular endothelial cells.

METHODSThe effects of ox-LDL on PERK and p-eIF2α protein expression of primary human umbilical vein endothelial cells (HUVECs) were investigated by Western blot analysis. PERK gene silencing and selective eIF2α phosphatase inhibitor, salubrinal were used to inhibit the process of ox-LDL induced endothelial cell apoptosis, caspase-3 activity, and CHOP mRNA level.

RESULTSOx-LDL treatment significantly increased the expression of PERK, PERK-mediated inactivation of eIF2α phosphorylation, and the expression of CHOP, as well as the caspase-3 activity and apoptosis. The effects of ox-LDL were markedly decreased by knocking down PERK with stable transduction of lentiviral shRNA or by selective eIF2α phosphatase inhibitor, salubrinal.

CONCLUSIONThis study provides the first evidence that ox-LDL induces apoptosis in vascular endothelial cells mediated largely via the PERK/eIF2α/CHOP ER-stress pathway. It adds new insights into the molecular mechanisms underlying the pathogenesis and progression of atherosclerosis.

Apoptosis ; Endoplasmic Reticulum Stress ; Eukaryotic Initiation Factor-2 ; genetics ; metabolism ; Human Umbilical Vein Endothelial Cells ; metabolism ; Humans ; Lipoproteins, LDL ; genetics ; metabolism ; Signal Transduction ; Transcription Factor CHOP ; genetics ; metabolism ; eIF-2 Kinase ; genetics ; metabolism

OBJECTIVETo explore the possible mechanism of curcumin in inducing the apoptosis of breast cancer cell MDA-MB-231.

METHODCurcumin of different concentrations at 0, 10 25, 50, 100, 150, 200 micromol x L(-1) were used to intervene breast cancer cells MDA-MB-231 for 24 hours. MTT was used to observe its effect on the proliferation of breast cancer cells. The flow cytometry was used to detect its effect on the cell apoptosis. The real-time quantitative PCR and Western blot was used to assess the expression levels of GRP78 and CHOP in breast cancer cells.

RESULTCurcumin could inhibit the proliferative ability of breast cancer cells by inducing them in a concentration-dependent manner. Curcumin could significantly increase the expression levels of GRP78 and CHOP in breast cancer cells.

CONCLUSIONCurcumin could induce the apoptosis of breast cancer cells MDA-MB-231 by activating endoplasmic reticulum stress.

Apoptosis ; drug effects ; Breast Neoplasms ; drug therapy ; genetics ; metabolism ; physiopathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Curcumin ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Endoplasmic Reticulum Stress ; drug effects ; Female ; Heat-Shock Proteins ; genetics ; metabolism ; Humans ; Transcription Factor CHOP ; genetics ; metabolism

Oxidative stress is critical for causing cardiac injuries during ischemia-reperfusion (IR), yet the molecular mechanism for this remains unclear. In the present study, we observe that hypoxia and reoxygenation, a component of ischemia, effectively induces apoptosis in the cardiac myocytes from neonatal rats and it concomitantly leads to induction of GADD153, an apoptosis-related gene. Furthermore, IR injury of rat heart showed a GADD153 overexpression in the ischemic area where the TUNEL reaction was positive. A downregulation of cardiac ankyrin repeat protein (CARP) was also observed in this ischemic area. Promoter deletion and reporter analysis revealed that hypoxia transcriptionally activates a GADD153 promoter through the AP-1 element in neonatal cardiomyocytes. Ectopic overexpression of GADD153 resulted in the downregulation of CARP expression. Accordingly, the induction of GADD153 mRNA were followed by the CARP down-regulation in an in vivo rat coronary ischemia/reperfusion injury model. These results suggest that GADD153 over-expression and the resulting downregulation of CARP may have causative roles in apoptotic cell death during cardiac IR injury.
Animals ; Animals, Newborn ; Anoxia ; Apoptosis/physiology ; Cells, Cultured ; Humans ; Male ; *Myocardial Reperfusion Injury/metabolism/pathology ; *Myocardium/metabolism/pathology ; Myocytes, Cardiac/cytology/metabolism ; Nuclear Proteins/genetics/*metabolism ; Promoter Regions, Genetic ; Rats ; Rats, Sprague-Dawley ; Repressor Proteins/genetics/*metabolism ; Transcription Factor AP-1/genetics/metabolism ; Transcription Factor CHOP/genetics/*metabolism

Endoplasmic reticulum (ER) stress occurs in macrophage-rich areas of advanced atherosclerotic lesions and contributes to macrophage apoptosis and subsequent plaque necrosis. The purpose of the present study was to investigate the effects of caveolin-1 (Cav-1) on ER stress-induced apoptosis in cultured macrophages and the underlying mechanisms. RAW264.7 cells were incubated with thapsigargin (TG) to establish ER stress model. And Cav-1 expression was detected by Western blot. After being pretreated with filipin(III), a caveolae inhibitor, RAW264.7 cells were assayed with flow cytometry and confocal laser scanning microscopy to detect cell apoptosis. Moreover, p38 mitogen-activated protein kinase (MAPK) phosphorylation and C/EBP homologous protein (CHOP) expression were detected with Western blot. The results showed that Cav-1 expression was markedly increased at early stage of TG treatment (P < 0.05) and then decreased with prolonged or high dose TG treatments. The increasing of Cav-1 expression induced by TG in RAW264.7 cells was abolished under inhibition of caveolae by filipin(III) (P < 0.05). The effect of TG on apoptosis of RAW264.7 cells was further augmented after pretreatment with filipin(III) (P < 0.05). Western blotting showed that MAPK phosphorylation induced by TG was inhibited by filipin(III) in RAW264.7 cells (P < 0.05), whereas CHOP remained unchanged (P > 0.05). These results suggest that Cav-1 may play a critical role in suppressing ER stress-induced macrophages apoptosis in vitro, and one of the mechanisms may be correlated with the activation of p38 MAPK prosurvival pathway.
Animals ; Apoptosis ; drug effects ; Caveolin 1 ; genetics ; metabolism ; Cell Line ; Endoplasmic Reticulum Stress ; physiology ; Filipin ; pharmacology ; MAP Kinase Signaling System ; Macrophages ; cytology ; drug effects ; Mice ; Thapsigargin ; pharmacology ; Transcription Factor CHOP ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism

The human CCR4-NOT deadenylase complex consists of at least nine enzymatic and non-enzymatic subunits. Accumulating evidence suggests that the non-enzymatic subunits are involved in the regulation of mRNA deadenylation, although their precise roles remain to be established. In this study, we addressed the function of the CNOT1 subunit by depleting its expression in HeLa cells. Flow cytometric analysis revealed that the sub G(1) fraction was increased in CNOT1-depleted cells. Virtually, the same level of the sub G1 fraction was seen when cells were treated with a mixture of siRNAs targeted against all enzymatic subunits, suggesting that CNOT1 depletion induces apoptosis by destroying the CCR4-NOT-associated deadenylase activity. Further analysis revealed that CNOT1 depletion leads to a reduction in the amount of other CCR4-NOT subunits. Importantly, the specific activity of the CNOT6L immunoprecipitates-associated deadenylase from CNOT1-depleted cells was less than that from control cells. The formation of P-bodies, where mRNA decay is reported to take place, was largely suppressed in CNOT1-depleted cells. Therefore, CNOT1 has an important role in exhibiting enzymatic activity of the CCR4-NOT complex, and thus is critical in control of mRNA deadenylation and mRNA decay. We further showed that CNOT1 depletion enhanced CHOP mRNA levels and activated caspase-4, which is associated with endoplasmic reticulum ER stress-induced apoptosis. Taken together, CNOT1 depletion structurally and functionally deteriorates the CCR4-NOTcomplex and induces stabilization of mRNAs, which results in the increment of translation causing ER stress-mediated apoptosis. We conclude that CNOT1 contributes to cell viability by securing the activity of the CCR4-NOT deadenylase.
Apoptosis ; Caspases, Initiator ; genetics ; metabolism ; Cell Survival ; Endoplasmic Reticulum ; enzymology ; Enzyme Activation ; Flow Cytometry ; HEK293 Cells ; HeLa Cells ; Humans ; Protein Subunits ; genetics ; metabolism ; RNA Stability ; RNA, Messenger ; analysis ; RNA, Small Interfering ; genetics ; metabolism ; Ribonucleases ; metabolism ; Stress, Physiological ; Transcription Factor CHOP ; genetics ; metabolism ; Transcription Factors ; genetics ; metabolism ; Transfection

OBJECTIVETo establish a model of ER stress-induced apoptosis with tunicamycin and to examine whether Bim is dysregulated and its potential role in resistance of melanoma cells to apoptosis under endoplasmic reticulum (ER) stress.

METHODSA model of ER stress-induced apoptosis was established with tunicamycin. Apoptotic cells were quantitated using the annexin V/propidium iodide method by flow cytometry. Hoechst staining was also used to confirm the apoptotic cell death. Western blotting was used to measure the activation of caspase-3 and -9, and the expression of Bim, GRP78, CHOP, and Foxo1 at the protein level. The expression of Bim, CHOP and Foxo1 at the mRNA level was quantitated by qPCR. The siRNA technique was used to inhibit the expression of Bim.

RESULTSTreatment of the melanoma cells with tunicamycin did not induce significant apoptosis and activation of caspase cascade, whereas it caused marked activation of caspase-3 and -9, and apoptosis in HEK293 cells which were used as a control. With exposure to tunicamycin (3 µmol/L) for 12, 24, 36 hours the Bim protein levels were not increased in Mel-RM and MM200 cells. Its mRNA levels were 0.37 ± 0.05, 0.13 ± 0.02 and 0.02 ± 0.01 in Mel-RM cells, while 0.41 ± 0.06, 0.16 ± 0.04 and 0.21 ± 0.03 in MM200 cells, respectively. The expression of Bim mRNA was significantly reduced compared with that in the control groups of the two cell lines (P < 0.01). siRNA knockdown of Bim protected HEK293 cells against activation of caspase-3. The cell apoptosis of Bim siRNA group was (5.69 ± 0.38)%, significantly lower than that of the siRNA control group (40.32 ± 1.64)% and blank control group (35.46 ± 2.01)% (P < 0.01). In the melanoma cells after exposure to tunicamycin (3 µmol/L) for 6, 12, 24, and 36 hours the transcription factor CHOP at mRNA level were significantly increased and the expressions at protein level were also up-regulated. The expressions of another transcription factor Foxo1 at mRNA level significantly decreased and the expressions at protein level were down-regulated, too.

CONCLUSIONSThe lack of Bim up-regulation contributes to the resistance of melanoma cells to ER stress-induced apoptosis and may be a mechanism by which melanoma cells adapt to ER stress conditions. Transcription factors CHOP and Foxo1 may be responsible for the dysregulation of Bim in melanoma cells upon ER stress.

Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Bcl-2-Like Protein 11 ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Cell Line, Tumor ; Endoplasmic Reticulum Stress ; drug effects ; Forkhead Box Protein O1 ; Forkhead Transcription Factors ; genetics ; metabolism ; HEK293 Cells ; Heat-Shock Proteins ; metabolism ; Humans ; Melanoma ; genetics ; metabolism ; pathology ; Membrane Proteins ; genetics ; metabolism ; Proto-Oncogene Proteins ; genetics ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Transcription Factor CHOP ; genetics ; metabolism ; Tunicamycin ; pharmacology

BACKGROUNDPrevious studies have indicated that endoplasmic reticulum stress participates in and mediates liver injury and apoptosis in brain-dead (BD) rats. In this study, we observed the effect of salubrinal (Sal, Sigma, USA) on liver cells in BD rats and explored its relevant mechanisms.

METHODSThirty Sprague-Dawley rats were equally randomized into three groups: BD group, Sal group, and DMSO group. The BD models were established by increasing intracranial pressure in a modified, slow, and intermittent way. In the drug groups, Sal was administered 1 h before the induction of BD. After modeling was completed, the blood and liver samples were harvested. CHOP and Caspase-12 mRNA expression was detected using quantitative polymerase chain reaction. PKR-like ER kinase (PERK), P-eukaryotic translation initiation factor 2α (eIF2α), eIF2α, CHOP and caspase-12 expression was detected using western blotting (WB). CHOP and caspase-12 distribution and expression in liver tissues were determined using immunohistochemistry (IHC). Alanine aminotransferase and aspartate aminotransferase level were detected using an automatic biochemical analyzer. Hepatic cell apoptosis was detected using TUNEL. The results were analyzed using Quantity-one v4.62 software (Bio-Rad, USA).

RESULTSCHOP and caspase-12 expression and PERK, eIF2α, and P-eIF2α protein expression showed no significant difference between BD group and DMSO group. Compared with BD group, Sal group had a significantly higher P-eIF2C level and a lower P-PERK level 2 h and 6 h after BD (P < 0.05). However, eIF2α expression showed no significant difference (P > 0.05). After the Sal treatment, CHOP and caspase-12 mRNA expression significantly decreased 4 h after BD (P < 0.05). WB and IHC indicated that CHOP and caspase-12 expression also significantly decreased after Sal treatment. Sal was associated with improved liver function and decreased hepatic cell apoptosis.

CONCLUSIONSSal can significantly reduce apoptosis in hepatic cells of BD rats. This protective effect may be achieved via the PERK-eIF2α signaling pathway.

Animals ; Apoptosis ; drug effects ; Blotting, Western ; Brain Death ; metabolism ; Caspase 12 ; genetics ; metabolism ; Cinnamates ; Disease Models, Animal ; Endoplasmic Reticulum Stress ; drug effects ; Eukaryotic Initiation Factor-2 ; genetics ; metabolism ; Immunohistochemistry ; Liver ; drug effects ; injuries ; Male ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Thiourea ; analogs & derivatives ; Transcription Factor CHOP ; genetics ; metabolism

Tumor necrosis factor-related apoptosis-induced ligand (TRAIL) induces apoptosis selectively in cancer cells while sparing normal cells. However, many cancer cells are resistant to TRAIL-induced cell death. Here, we report that paxilline, an indole alkaloid from Penicillium paxilli, can sensitize various glioma cells to TRAIL-mediated apoptosis. While treatment with TRAIL alone caused partial processing of caspase-3 to its p20 intermediate in TRAIL-resistant glioma cell lines, co-treatment with TRAIL and subtoxic doses of paxilline caused complete processing of caspase-3 into its active subunits. Paxilline treatment markedly upregulated DR5, a receptor of TRAIL, through a CHOP/GADD153-mediated process. In addition, paxilline treatment markedly downregulated the protein levels of the short form of the cellular FLICE-inhibitory protein (c-FLIPS) and the caspase inhibitor, survivin, through proteasome-mediated degradation. Taken together, these results show that paxilline effectively sensitizes glioma cells to TRAIL-mediated apoptosis by modulating multiple components of the death receptor-mediated apoptotic pathway. Interestingly, paxilline/TRAIL co-treatment did not induce apoptosis in normal astrocytes, nor did it affect the protein levels of CHOP, DR5 or survivin in these cells. Thus, combined treatment regimens involving paxilline and TRAIL may offer an attractive strategy for safely treating resistant gliomas.
Antineoplastic Agents/*pharmacology ; Apoptosis/*drug effects ; Astrocytes/metabolism ; CASP8 and FADD-Like Apoptosis Regulating Protein/genetics/*metabolism ; Caspase 3/metabolism ; Cell Line, Tumor ; Drug Discovery ; Flow Cytometry ; Glioma/*metabolism/pathology ; Humans ; Indoles/*pharmacology ; Inhibitor of Apoptosis Proteins/metabolism ; RNA, Small Interfering ; Receptors, TNF-Related Apoptosis-Inducing Ligand/genetics/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; TNF-Related Apoptosis-Inducing Ligand/metabolism/*pharmacology ; Transcription Factor CHOP/analysis

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