This study aims to explore the effect and mechanism of Danggui Buxue Decoction(DBD)-containing serum in alleviating the H9c2 cell injury caused by the exposure to intermittent low oxygen. H9c2 cells were assigned into five groups: control(CON) group, intermittent low oxygen(IH) group, intermittent low oxygen plus DBD-containing serum(IH+DBD) group, intermittent low oxygen plus the autophagy enhancer rapamycin(IH+RAPA) group, and intermittent low oxygen plus DBD-containing serum and the autophagy inhibitor 3-methyladenine(IH+DBD+3-MA) group. Monodansylcadaverine(MDC) staining was employed to detect the changes of autophagosomes. Cell counting kit-8(CCK-8) assay was employed to determine the activity of myocardial cells, and lactate dehydrogenase(LDH) and creatine kinase(CK) kits were used to measure the LDH and CK levels in the cell culture, which would reflect the degree of cell damage. TdT-mediated dUTP nick-end labeling(TUNEL) staining was used to detect the apoptosis of myocardial cells, and JC-1 fluorescence probe to detect the changes in mitochondrial membrane potential. Western blot was employed to determine the expression levels of the autophagy-related proteins microtubule-associated proteins light chain 3Ⅱ(LC3Ⅱ), microtubule-associated proteins light chain 3Ⅰ(LC3Ⅰ), P62, Parkin and apoptosis related proteins pro caspase-3, caspase-3, B-cell lymphoma-2(Bcl-2), Bcl-2-associated X(Bax). The results showed that compared with the CON group, the IH group showed decreased fluorescence intensity of MDC staining, decreased LC3Ⅱ/LC3Ⅰ ratio, down-regulated Parkin expression, and up-regulated expression of P62. In addition, the IH group showed decreased cell survival rate, increased content of LDH and CK in the culture medium, increased number of TUNEL positive cells, and decreased pro caspase-3/caspase-3 and Bcl-2/Bax ratios and mitochondrial membrane potential. Compared with the IH group, the IH+DBD and IH+RAPA groups showed increased fluorescence intensity of MDC staining, increased LC3Ⅱ/LC3Ⅰ ratio, up-regulated Parkin expression, and down-regulated P62 expression. In addition, the two groups showed increased cell survival rate, reduced content of LDH and CK in the culture medium, decreased number of TUNEL positive cells, and increased pro caspase-3/caspase-3 and Bcl-2/Bax ratios and mitochondrial membrane potential. The IH+DBD+3-MA and IH groups showed no significant differences in the above indicators. Compared with the IH+DBD group, the IH+DBD+3-MA group showed decreased fluorescence intensity of MDC staining, decreased LC3Ⅱ/LC3Ⅰ ratio, down-regulated Parkin expression, and up-regulated P62 expression. In addition, the group had decreased cell survival rate, increased content of LDH and CK in the culture medium, increased number of TUNEL positive cells, decreased pro caspase-3/caspase-3 and Bcl-2/Bax ratios, and declined mitochon-drial membrane potential. To sum up, DBD could promote the mitophagy, inhibit the apoptosis, and alleviated the injury of H9c2 cells exposed to low oxygen.
Oxygen ; bcl-2-Associated X Protein/metabolism* ; Caspase 3/genetics* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Apoptosis ; Autophagy ; Ubiquitin-Protein Ligases ; Microtubule-Associated Proteins

OBJECTIVE: To investigate the effect of the tripartite motif containing 31 (TRIM31) gene silencing on the proliferation and apoptosis of multiple myeloma cells and its possible mechanism. METHODS: The normal bone marrow plasma cells (nPCs) were selected as control, and the mRNA and protein expression levels of TRIM31 in human multiple myeloma cell lines (U266, RPMI-8226, NCI-H929 and KMS-11) were detected by RT-qPCR and Western blot. Recombinant lentivirol vector containing shRNA-TRIM31 and its negative control were used to infect U266 cells respectively, and the mRNA expression level of TRIM31 in infected cells was detected by RT-qPCR. Then cell proliferation, colony forming and apoptosis were analyzed by CCK-8, soft agar assay, and flow cytometry, respectively. The protein expression levels of TRIM31, cleaved-caspase-3, BCL-2, Bax, p-Akt (Ser473), Akt and PI3K (p110α) were evaluated by Western blot. In addition, the PI3K/Akt signaling pathway-specific inhibitor LY294002 and TRIM31-shRNA lentivirus were used to interfere with U266 cells, and the cell proliferation, apoptosis, and protein expression of p-Akt (Ser473) and Akt were detected by CCK-8, flow cytometry and Western blot, respectively. RESULTS: Compared with nPCs, the expression levels of TRIM31 mRNA and protein in U266, RPMI-8226, NCI-H929 and KMS-11 cells were significantly increased (P<0.001), especially in U266 cells. After lentivirus infection, the levels of TRIM31 mRNA and protein in U266 cells were significantly decreased (P<0.001). TRIM31 silencing significantly inhibited the proliferation of U266 cells (P<0.05), attenuated the ability of cell cloning, improved cell apoptosis, up-regulated the protein expressions of cleaved-caspase-3 and Bas as well as down-regulated expressions of BCL-2, p-Akt (Ser473) and PI3K (p110α). There was no significant effect on Akt protein. Intervention of LY294002 significantly enhanced the inhibition on cell proliferation and the promotion on apoptosis mediated by TRIM31 gene silencing in U266 cells. CONCLUSION TRIM31 gene silencing can inhibit U266 cell proliferation and promote its apoptosis, which may be closely related to inhibition of PI3K/Akt signaling pathway.
Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Gene Silencing ; Humans ; Multiple Myeloma ; Phosphatidylinositol 3-Kinases/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Tripartite Motif Proteins/genetics* ; Ubiquitin-Protein Ligases/genetics*

BACKGROUND: Ubiquitin-conjugating enzyme E2C (UBE2C) has been shown to be associated with the occurrence of various cancers and involved in many tumorigenic processes. This study aimed to investigate the specific molecular mechanism through which UBE2C affects breast cancer (BC) proliferation. METHODS: BC-related datasets were screened according to filter criteria in the Gene Expression Omnibus (GEO) database and The Cancer Genome Atlas (TCGA) database. Then differentially expressed genes (DEGs) were identified using Venn diagram analysis. By using DEGs, we conducted the following analyses including Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), protein-protein interaction (PPI), and survival analysis, and then validated the function of the hub gene UBE2C using quantitative reverse transcription-polymerase chain reaction (RT-qPCR), cell counting kit-8 (CCK-8) assay, transwell assay, and Western blot assay. RESULTS: In total, 151 DEGs were identified from the GEO and TCGA databases. The results of GO analysis demonstrated that the DEGs were significantly enriched with mitotic nuclear division, lipid droplet, and organic acid-binding. KEGG analysis showed that the peroxisome proliferators-activated receptor (PPAR) signaling pathway, regulation of lipolysis in adipocytes, and proximal tubule bicarbonate reclamation were significantly enriched in the signal transduction pathway category. The top three hub genes that resulted from the PPI network were FOXM1, UBE2C, and CDKN3. The results of survival analysis showed a close relationship between UBE2C and BC. The results of CCK-8 and transwell assays suggested that the proliferation and invasion of UBE2C knockdown cells were significantly inhibited (P < 0.050). The results of Western blot assay showed that the level of phosphorylated phosphatase and tensin homology deleted on chromosome 10 (p-PTEN) was obviously increased (P < 0.050), whereas the levels of phosphorylated protein kinase B (p-AKT), phosphorylated mammalian target of rapamycin (p-mTOR), and hypoxia-inducible factor-1 alpha (HIF-1α) were dramatically decreased (P < 0.050) in the UBE2C knockdown cell. CONCLUSION UBE2C can promote BC proliferation by activating the AKT/mTOR signaling pathway.
Biomarkers, Tumor ; Breast Neoplasms/pathology* ; Cell Proliferation/genetics* ; Computational Biology ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Proto-Oncogene Proteins c-akt/genetics* ; Signal Transduction/genetics* ; TOR Serine-Threonine Kinases/genetics* ; Ubiquitin-Conjugating Enzymes/metabolism*

OBJECTIVE: To study the effects of the overexpression of autophagy-related gene 3 (ATG3) on autophagy and salinomycin-induced apoptosis in breast cancer cells and explore the underlying mechanisms. METHODS: We used the lentivirus approach to establish a breast cancer cell line with stable overexpression of ATG3. Western blotting, immunofluorescence staining and transmission electron microscopy were used to analyze the effect of ATG3 overexpression on autophagy in breast cancer MCF-7 cells. Using the AKT/mTOR agonists SC79 and MHY1485, we analyzed the effect of AKT/mTOR signal pathway activation on ATG3 overexpression-induced autophagy. Western blotting and flow cytometry were used to analyze the effect of autophagy on apoptosis of the ATG3-overexpressing cells treated with salinomycin and 3-MA (an autophagy inhibitor). RESULTS: In ATG3-overexpressing MCF-7 cells, ATG3 overexpression obviously promoted autophagy, inhibited the AKT/mTOR signaling pathway, significantly weakened salinomycin-induced apoptosis ( < 0.01), caused significant reduction of the levels of the pro-apoptotic proteins cleaved-caspase 3 ( < 0.01) and Bax ( < 0.05), and enhanced the expression of the anti-apoptotic protein Bcl-2 ( < 0.05). The inhibition of autophagy obviously weakened the inhibitory effect of ATG3 overexpression on salinomycin-induced apoptosis. CONCLUSIONS ATG3 overexpression promotes autophagy possibly by inhibiting the AKT/mTOR signaling pathway to decrease salinomycin-induced apoptosis in MCF-7 cells, suggesting that autophagy induction might be one of the mechanisms of drug resistance in breast cancer cells.
Acetates ; pharmacology ; Apoptosis ; drug effects ; genetics ; Autophagy ; drug effects ; Autophagy-Related Proteins ; metabolism ; Benzopyrans ; pharmacology ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Drug Resistance, Neoplasm ; Female ; Gene Expression Regulation ; Humans ; MCF-7 Cells ; Morpholines ; pharmacology ; Proto-Oncogene Proteins c-akt ; antagonists & inhibitors ; metabolism ; Pyrans ; pharmacology ; TOR Serine-Threonine Kinases ; antagonists & inhibitors ; metabolism ; Triazines ; pharmacology ; Ubiquitin-Conjugating Enzymes ; metabolism

OBJECTIVETo explore the effects of silencing HERC4 on the proliferation, apoptosis, and migration of cervical cancer cell line Hela and the possible molecular mechanisms.

METHODSThree HERC4-specific small interfering RNAs (siRNAs) were transfected into Hela cells, and HERC4 expression in the cells was examined with Western blotting. CCK-8 assay, annexin V-FITC/PI assay, and wound healing assay were used to assess the effect of HERC4 silencing on the proliferation, apoptosis and migration ability of Hela cells. The expression levels of cyclin D1 and Bcl-2 in the cells were detected using Western blotting.

RESULTSTransfection of siRNA-3 resulted in significantly decreased HERC4 protein expression (P<0.01). HERC4 silencing by siRNA-3 markedly suppressed the proliferation and migration of Hela cells, increased the apoptosis rate (P<0.01) and reduced the expression levels of cyclin D1 and Bcl-2 (P<0.01).

CONCLUSIONSilencing of HERC4 efficiently inhibits the proliferation, migration, and invasion of Hela cells in vitro, and the underlying mechanisms may involve the down-regulation of cyclin D1 and Bcl-2.

Apoptosis ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Cyclin D1 ; metabolism ; Down-Regulation ; Female ; HeLa Cells ; Humans ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection ; Ubiquitin-Protein Ligases ; genetics ; metabolism ; Uterine Cervical Neoplasms ; pathology

OBJECTIVE: To investigate the effects of LCL161, a Smac mimetic, on the proliferation and apoptosis in hepatocellular carcinoma cells and the underlying mechanisms. 
 METHODS: The effect of LCL161 on the cell viability of HepG2 and SMMC7721 cells was measured by MTT assay. The effect of LCL161 at lower concentrations on the proliferation in hepatocellular carcinoma (HCC) cells was detected by colony formation assay. Apoptosis was assessed by flow cytometry with PI staining. The mitochondrial membrane potential was measured by JC-1 staining. The expression of PARP, p-Akt, cIAP1 and XIAP protein was analyzed by Western blot.
 RESULTS: LCL161 displayed notable antiproliferative activity on HCC cells at the concentrations of 1-16 μmol/L (P<0.01), with IC50 values of 4.3 and 4.9 μmol/L for HepG2 and SMMC7721 cells, respectively, after treatment for 48 h. LCL161 at lower concentrations obviously inhibited the colony formation of HCC cells. LCL161 induced significant apoptosis in HCC cells (P<0.01), and resulted in the apoptotic rate at (1.5±0.8)% or (1.8±0.6)% , (15.2±2.8)% or (12.2±2.4)%, (28.7±3.0)% or (22.4±2.7)%, (34.6±2.3)% or (30.2±2.4)% for HepG2 cells or SMMC7721 cells at the concentration of 0, 2, 4 or 8 μmol/L, respectively. The result of JC-1 staining indicated that the mitochondrial membrane potential of HCC cells was reduced by LCL161. In addition, LCL161 promoted the cleavage of PARP, down-regulated the protein expression of p-Akt, and degraded cIAP1.
 CONCLUSION LCL161 possesses significant anti-proliferative activity and pro-apoptotic action in HepG2 and SMMC7721 cells, which might be correlated with reduction in mitochondrial membrane potential, down-regulation of p-Akt and degradation of cIAP1.
Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; drug therapy ; genetics ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; Down-Regulation ; Hep G2 Cells ; Humans ; Inhibitor of Apoptosis Proteins ; metabolism ; Liver Neoplasms ; Membrane Potential, Mitochondrial ; drug effects ; Proto-Oncogene Proteins c-akt ; genetics ; Thiazoles ; pharmacology ; Ubiquitin-Protein Ligases ; metabolism ; X-Linked Inhibitor of Apoptosis Protein

SUMOylation is recently found to function as a targeting signal for the degradation of substrates through the ubiquitin-proteasome system. RNF4 is the most studied human SUMO-targeted ubiquitin E3 ligase. However, the relationship between SUMO proteases, SENPs, and RNF4 remains obscure. There are limited examples of the SENP regulation of SUMO2/3-targeted proteolysis mediated by RNF4. The present study investigated the role of SENP3 in the global protein turnover related to SUMO2/3-targeted ubiquitination and focused in particular on the SENP3 regulation of the stability of Sp1. Our data demonstrated that SENP3 impaired the global ubiquitination profile and promoted the accumulation of many proteins. Sp1, a cancer-associated transcription factor, was among these proteins. SENP3 increased the level of Sp1 protein via antagonizing the SUMO2/3-targeted ubiquitination and the consequent proteasome-dependent degradation that was mediated by RNF4. De-conjugation of SUMO2/3 by SENP3 attenuated the interaction of Sp1 with RNF4. In gastric cancer cell lines and specimens derived from patients and nude mice, the level of Sp1 was generally increased in parallel to the level of SENP3. These results provided a new explanation for the enrichment of the Sp1 protein in various cancers, and revealed a regulation of SUMO2/3 conjugated proteins whose levels may be tightly controlled by SENP3 and RNF4.
Animals ; Cysteine Endopeptidases ; genetics ; metabolism ; Gene Expression Regulation, Neoplastic ; Humans ; Immunoenzyme Techniques ; Immunoprecipitation ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Prognosis ; Protein Processing, Post-Translational ; Proteolysis ; RNA, Messenger ; genetics ; Real-Time Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction ; Small Ubiquitin-Related Modifier Proteins ; antagonists & inhibitors ; genetics ; metabolism ; Sp1 Transcription Factor ; genetics ; metabolism ; Stomach Neoplasms ; genetics ; metabolism ; pathology ; Sumoylation ; Tumor Cells, Cultured ; Ubiquitination ; Ubiquitins ; antagonists & inhibitors ; genetics ; metabolism ; Xenograft Model Antitumor Assays

This study is aimed at gaining insights into the brain site-specific proteomic senescence signature while comparing physiologically aged brains with aging-related dementia brains (for example, Alzheimer's disease (AD)). Our study of proteomic differences within the hippocampus (Hp), parietal cortex (pCx) and cerebellum (Cb) could provide conceptual insights into the molecular mechanisms involved in aging-related neurodegeneration. Using an isobaric tag for relative and absolute quantitation (iTRAQ)-based two-dimensional liquid chromatography coupled with tandem mass spectrometry (2D-LC-MS/MS) brain site-specific proteomic strategy, we identified 950 proteins in the Hp, pCx and Cb of AD brains. Of these proteins, 31 were significantly altered. Most of the differentially regulated proteins are involved in molecular transport, nervous system development, synaptic plasticity and apoptosis. Particularly, proteins such as Gelsolin (GSN), Tenascin-R (TNR) and AHNAK could potentially act as novel biomarkers of aging-related neurodegeneration. Importantly, our Ingenuity Pathway Analysis (IPA)-based network analysis further revealed ubiquitin C (UBC) as a pivotal protein to interact with diverse AD-associated pathophysiological molecular factors and suggests the reduced ubiquitin proteasome degradation system (UPS) as one of the causative factors of AD.
Aged, 80 and over ; Alzheimer Disease/*metabolism ; Brain/*metabolism ; Female ; Gelsolin/genetics/metabolism ; Humans ; Male ; Membrane Proteins/genetics/metabolism ; Neoplasm Proteins/genetics/metabolism ; Organ Specificity ; Proteome/genetics/*metabolism ; Tenascin/genetics/metabolism ; Ubiquitin C/genetics/metabolism

Mdm2 and Mdm4 are two key negative regulators of the tumor suppressor p53. Deletion of either Mdm2 or Mdm4 induces p53-dependent early embryonic lethality in knockout mouse models. The tissue-specific deletion of Mdm2 induces p53-dependent apoptosis, whereas the deletion of Mdm4 induces both p53-dependent apoptosis and cell cycle arrest. Compared to Mdm4 deletion, Mdm2 deletion causes more severe phenotypic defects. Disrupting the Mdm2 and Mdm4 interaction using knockin mice models causes embryonic lethality that can be completely rescued by the concomitant loss of p53, suggesting that Mdm2 and Mdm4 heterodimerization is critical to inhibit p53 activity during embryogenesis. Overexpression of Mdm2 and Mdm4 in mice induces spontaneous tumorigenesis, which clearly indicates that Mdm2 and Mdm4 are bona fide oncogenes. Studies from these mouse models strongly suggest that blocking Mdm2- and Mdm4-mediated p53 inhibition is an appealing therapeutic strategy for cancer patients with wild-type p53 alleles.
Animals ; Apoptosis ; Cell Cycle Checkpoints ; Mice ; Mice, Knockout ; Models, Animal ; Proto-Oncogene Proteins ; genetics ; metabolism ; Proto-Oncogene Proteins c-mdm2 ; genetics ; metabolism ; Tumor Suppressor Protein p53 ; antagonists & inhibitors ; genetics ; metabolism ; Ubiquitin-Protein Ligases ; genetics ; metabolism

Adenocarcinoma ; metabolism ; pathology ; Apoptosis ; drug effects ; Baculoviral IAP Repeat-Containing 3 Protein ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Chromones ; pharmacology ; Enzyme Inhibitors ; pharmacology ; Female ; Humans ; Inhibitor of Apoptosis Proteins ; genetics ; metabolism ; Morpholines ; pharmacology ; Ovarian Neoplasms ; metabolism ; pathology ; Phosphatidylinositol 3-Kinases ; genetics ; metabolism ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Signal Transduction ; Ubiquitin-Protein Ligases ; X-Linked Inhibitor of Apoptosis Protein ; genetics ; metabolism