Objective To investigate the expression level of serine/threonine phosphoprotein phosphatase 4C(PPP4C)in gastric cancer,and analyze its relationship with prognosis and the underlying regulatory mechanism.Methods The clinical data of 104 gastric cancer patients admitted to the First Affiliated Hospital of Bengbu Medical College between January 2012 and August 2016 were collected.Immunohistochemical staining was employed to determine the expression levels of PPP4C and Ki-67 in the gastric cancer tissue.The gastric cancer cell lines BGC823 and HGC27 were cultured and transfected with the vector for PPP4C knockdown,the vector for PPP4C overexpression,and the lentiviral vector(control),respectively.The effects of PPP4C on the cell cycle and proliferation were analyzed and the possible regulatory mechanisms were explored.Results PPP4C was highly expressed in gastric cancer(P<0.001),and its expression promoted malignant progression of the tumor(all P<0.01).Univariate and Cox multivariate analysis clarified that high expression of PPP4C was an independent risk factor affecting the 5-year survival rate of gastric cancer patients(P=0.003).Gene ontology and Kyoto encyclopedia of genes and genomes enrichment analysis suggested that PPP4C may be involved in the cell cycle.The correlation analysis showed that the expression of PPP4C was positively correlated with that of Ki-67 in gastric cancer(P<0.001).The up-regulation of PPP4C expression increased the proportion of tumor cells in the S phase,alleviated the G2/M phase arrest,and promoted the proliferation of gastric cancer cells and the expression of cyclin D1 and cyclin-dependent kinase 6(CDK6)(all P<0.05).The down-regulation of PPP4C decreased the proportion of gastric cancer cells in the S phase,promoted G2/M phase arrest,and inhibited cell proliferation and the expression of cyclin D1,CDK6,and p53(all P<0.05).p53 inhibitors promoted the proliferation of BGC823 and HGC27 cells in the PPP4C knockdown group(P<0.001,P<0.001),while p53 activators inhibited the proliferation of BGC823 and HGC27 cells in the PPP4C overexpression group(P<0.001,P=0.002).Conclusions PPP4C is highly expressed in gastric cancer and affects the prognosis of the patients.It may increase the proportion of gastric cancer cells in the S phase and alleviate the G2/M phase arrest by inhibiting p53 signaling,thereby promoting cell proliferation.
Humans ; Stomach Neoplasms/genetics* ; Cyclin D1/metabolism* ; Tumor Suppressor Protein p53 ; Phosphoproteins/metabolism* ; Ki-67 Antigen ; Cell Line, Tumor ; Prognosis ; Cell Proliferation ; Phosphoprotein Phosphatases/metabolism* ; Threonine ; Serine

Phosphoprotein Phosphatases ; Kinetics ; Protein Phosphatase 2/metabolism*

Objective: To investigate the molecular mechanism of high phosphorylation levels of cofilin-1 (p-CFL-1) associated with paclitaxel resistance in epithelial ovarian cancer (EOC) cells. Methods: Cells displaying varying levels of p-CFL-1 and CFL-1 were created by plasmid transfection and shRNA interference. Cell inhibition rate indicating paclitaxel efficacy was assessed by Cell Counting Kit-8 (CCK-8) assay. Apoptosis was assessed by flow cytometry and protein levels were detected by western blotting. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure the expression levels of phosphokinases and phosphatases of CFL-1. Survival analysis evaluated the correlation between the prognosis of EOC patients and the levels of p-CFL-1 and slingshot-1 (SSH-1). Results: High levels of p-CFL-1 were observed in EOC cells that survived treatment with high doses of paclitaxel. SKOV3 cell mutants with upregulated p-CFL-1 showed impaired paclitaxel efficacy, as well as decreased apoptosis rates and pro-survival patterns of apoptosis-specific protein expression. Cytoplasmic accumulation of p-CFL-1 inhibited paclitaxel-induced mitochondrial apoptosis. SSH-1 silencing mediated CFL-1 phosphorylation in paclitaxel-resistant SKOV3 cells. Clinically, the high level of p-CFL-1 and the low level of SSH-1 in EOC tissues were closely related to chemotherapy resistance and poor prognosis in EOC patients. Conclusion The SSH-1/p-CFL-1 signaling pathway mediates paclitaxel resistance by apoptosis inhibition in EOC and is expected to be a potential prognostic predictor.
Antineoplastic Agents, Phytogenic/therapeutic use* ; Apoptosis ; Carcinoma, Ovarian Epithelial/metabolism* ; Cell Line, Tumor ; Cofilin 1/metabolism* ; Drug Resistance, Neoplasm ; Female ; Humans ; Ovarian Neoplasms/metabolism* ; Paclitaxel/therapeutic use* ; Phosphoprotein Phosphatases/metabolism* ; Phosphorylation

BACKGROUNDOvarian serous adenocarcinoma can be divided into low- and high-grade tumors, which exhibit substantial differences in pathogenesis, clinicopathology, and prognosis. This study aimed to investigate the differences in the PH domain leucine-rich repeat protein phosphatase (PHLPP), forkhead homeobox type O 3a (FoxO3a), and RAD51 protein expressions, and their associations with prognosis in patients with low- and high-grade ovarian serous adenocarcinomas.

METHODSThe PHLPP, FoxO3a, and RAD51 protein expressions were examined in 94 high- and 26 low-grade ovarian serous adenocarcinomas by immunohistochemistry. The differences in expression and their relationships with pathological features and prognosis were analyzed.

RESULTSIn high-grade serous adenocarcinomas, the positive rates of PHLPP and FoxO3a were 24.5% and 26.6%, while in low-grade tumors, they were 23.1% and 26.9%, respectively (P < 0.05 vs. the control specimens; low- vs. high-grade: P > 0.05). The positive rates of RAD51 were 70.2% and 65.4% in high- and low-grade serous adenocarcinomas, respectively (P < 0.05 vs. the control specimens; low- vs. high-grade: P > 0.05). Meanwhile, in high-grade tumors, Stage III/IV tumors and lymph node and omental metastases were significantly associated with lower PHLPP and FoxO3a and higher RAD51 expression. The 5-year survival rates of patients with PHLPP- and FoxO3a-positive high-grade tumors (43.5% and 36.0%) were significantly higher than in patients with PHLPP-negative tumors (5.6% and 7.2%, respectively; P< 0.05). Similarly, the 5-year survival rate of RAD51-positive patients (3.0%) was significantly lower than in negative patients (42.9%; P< 0.05). In low-grade tumors, the PHLPP, FoxO3a, and RAD51 expressions were not significantly correlated with lymph node metastasis, omental metastasis, Federation of Gynecology and Obstetrics stage, or prognosis.

CONCLUSIONSAbnormal PHLPP, FoxO3a, and RAD51 protein expressions may be involved in the development of high- and low-grade ovarian serous adenocarcinomas, suggesting common molecular pathways. Decreased PHLPP and FoxO3a and increased RAD51 protein expression may be important molecular markers for poor prognosis, and RAD51 may be an independent prognosis factor, of high-grade, but not low-grade, ovarian serous adenocarcinomas.

Adult ; Aged ; Biomarkers, Tumor ; metabolism ; Cystadenocarcinoma, Serous ; metabolism ; pathology ; Female ; Forkhead Box Protein O3 ; metabolism ; Humans ; Immunohistochemistry ; Lymphatic Metastasis ; Middle Aged ; Neoplasm Staging ; Nuclear Proteins ; metabolism ; Ovarian Neoplasms ; metabolism ; pathology ; Phosphoprotein Phosphatases ; metabolism ; Prognosis ; Rad51 Recombinase ; metabolism

The 26S proteasome at the center of the ubiquitin-proteasome system (UPS) is essential for virtually all cellular processes of eukaryotes. A common misconception about the proteasome is that, once made, it remains as a static and uniform complex with spontaneous and constitutive activity for protein degradation. Recent discoveries have provided compelling evidence to support the exact opposite insomuch as the 26S proteasome undergoes dynamic and reversible phosphorylation under a variety of physiopathological conditions. In this review, we summarize the history and current understanding of proteasome phosphorylation, and advocate the idea of targeting proteasome kinases/phosphatases as a new strategy for clinical interventions of several human diseases.
Animals ; Humans ; Phosphoprotein Phosphatases ; genetics ; metabolism ; Phosphorylation ; genetics ; Proteasome Endopeptidase Complex ; genetics ; metabolism ; Protein Kinases ; genetics ; metabolism

OBJECTIVETo study the effect of hypoxia on Slingshot protein expression in human intestinal epithelial cell and its relation with changes in barrier function of the cells.

METHODSThe human intestinal epithelial cell line Caco-2 was used to reproduce monolayer-cells. One portion of the monolayer-cell specimens were divided into six parts according to the random number table, and they were respectively exposed to hypoxia for 0 (without hypoxia), 1, 2, 6, 12, and 24 h. Transepithelial electrical resistance (TER) was determined with an ohmmeter. Another portion of the monolayer-cell specimens were exposed to hypoxia as above. Western blotting was used to detect the protein expressions of zonula occludens 1 (ZO-1), occludin, claudin-1, Slingshot-1, Slingshot-2, and Slingshot-3. The remaining portion of the monolayer-cell specimens were also exposed to hypoxia as above. The content of fibrous actin (F-actin) and globular actin (G-actin) was determined by fluorescence method. The sample number of above-mentioned 3 experiments was respectively 10, 10, and 18 at each time point. Data were processed with one-way analysis of variance and Dunnett test.

RESULTS(1) Compared with that of cells exposed to hypoxia for 0 h, TER of cells exposed to hypoxia for 1 to 24 h was significantly reduced (P values below 0.01). (2) Compared with those of cells exposed to hypoxia for 0 h (all were 1.00), the protein expressions of ZO-1, occludin, and claudin-1 of cells exposed to hypoxia for 1 to 24 h were generally lower, especially those of cells exposed to hypoxia for 12 h or 24 h (respectively 0.69 ± 0.20, 0.47 ± 0.15, and 0.47 ± 0.22, P<0.05 or P<0.01). Compared with those of cells exposed to hypoxia for 0 h, the protein expressions of Slingshot-1 and Slingshot-3 of cells exposed to hypoxia for 1 to 24 h were not obviously changed (P values above 0.05). The protein expression of Slingshot-2 of cells was decreased at first and then gradually increased from hypoxia hour 1 to 24. The protein expression of Slingshot-2 of cells exposed to hypoxia for 24 h (1.54 ± 0.57) was significantly higher than that of cells exposed to hypoxia for 0 h (1.00, P<0.05). (3) Compared with those of cells exposed to hypoxia for 0 h, the content of F-actin of cells exposed to hypoxia for 1, 6, 12, and 24 h was significantly decreased, whereas the content of G-actin of cells exposed to hypoxia for 6-24 h was significantly increased, P<0.05 or P<0.01; the content of F-actin and G-actin of cells exposed to hypoxia for the other time points was not obviously changed (P values above 0.05).

CONCLUSIONSHypoxia may cause cofilin activation after dephosphorylation and the depolymerization of F-actin by inducing Slingshot-2 protein expression, which in turn affects the tight junction of human intestinal epithelial cells, thus leading to deterioration of barrier function of these cells.

Actins ; metabolism ; Blotting, Western ; Caco-2 Cells ; Cell Hypoxia ; Claudin-1 ; metabolism ; Epithelial Cells ; cytology ; metabolism ; Humans ; Intestines ; cytology ; Occludin ; metabolism ; Phosphoprotein Phosphatases ; metabolism ; Tight Junctions ; metabolism ; Zonula Occludens-1 Protein ; metabolism

OBJECTIVEThe protozoan Toxoplasma gondii expresses large amounts of a 37 kDa Type 2C serine-threonine phosphatase, the so-called TgPP2C which has been suggested to contribute to parasite growth regulation. Ectopic expression in mammalian cells also indicated that the enzyme could regulate growth and survival. In this study, we aimed to investigate the interaction of TgPP2C with human SSRP1 (structure-specific recognition protein 1) and the effects of TgPP2C on cell viability.

METHODSThe yeast two hybrid system, His-tag pull-down and co-immunoprecipitation assays were used to confirm the interaction of TgPP2C with SSRP1 and determine the binding domain on SSRP1. The evaluation of cell apoptosis was performed using cleaved caspase-3 antibody and Annexin-V/PI kit combined with flow cytometry.

RESULTSWe identified human SSRP1 as an interacting partner of TgPP2C. The C-terminal region of SSRP1 including the amino acids 471 to 538 was specifically mapped as the region responsible for interaction with TgPP2C. The overexpression of TgPP2C down-regulated cell apoptosis and negatively regulated apoptosis induced by DRB, casein kinase II (CKII) inhibitor, through enhanced interaction with SSRP1.

CONCLUSIONTgPP2C may be a parasitic factor capable of promoting cell survival through interaction with the host protein SSRP1, thereby creating a favorable environment for parasite growth.

Apoptosis ; Blotting, Western ; DNA-Binding Proteins ; genetics ; metabolism ; Flow Cytometry ; HeLa Cells ; High Mobility Group Proteins ; genetics ; metabolism ; Humans ; Immunoprecipitation ; Phosphoprotein Phosphatases ; genetics ; metabolism ; Protein Phosphatase 2C ; Toxoplasma ; enzymology ; Transcriptional Elongation Factors ; genetics ; metabolism ; Two-Hybrid System Techniques

BACKGROUNDAdvances in the understanding of cardiovascular pathogenesis have highlighted that inflammation plays a central role in atherosclerotic coronary heart disease. Therefore, exploring pharmacologically based anti-inflammatory treatments to be used in cardiovascular therapeutics is worthwhile to promote the discovery of novel ways of treating cardiovascular disorders.

METHODSThe myocardial cell line H9c2(2-1) was exposed to lipopolysaccharide (LPS) in culture and resulted in a cellular pro-inflammation status. miR-21 microRNA levels were detected using quantitative real-time polymerase chain reaction (Q-RT-PCR). The influence of lovastatin on miR-21 under normal and pro-inflammatory conditions was tested after being added to the cell culture mixture for 24 hours. Conditional gene function of two predicted cardiovascular system relevant downstream targets of miR-21, protein phosphatase 1 regulatory subunit 3A (PPP1R3A) and signal transducer and activator of transcription 3 (STAT3), were analyzed with immunoblotting.

RESULTSForty-eight hours of LPS treatment significantly increased the miR-21 to 170.71%± 34.32% of control levels (P = 0.002). Co-treatment with lovastatin for 24 hours before harvesting attenuated the up-regulation of miR-21 (P = 0.013). Twenty-four hours of lovastatin exposure up-regulated PPP1R3A to 143.85%± 21.89% of control levels in cardiomyocytes (P = 0.023). Lovastatin up-regulated the phosphorylation level of STAT3 compared to the background LPS pretreatment (P = 0.0077), this effect was significantly (P = 0.018) blunted when miR-21 was functionally inhibited.

CONCLUSIONSmiR-21 plays a major role in the regulation of the cellular anti-inflammation effects of lovastatin.

Blotting, Western ; Cell Line ; Humans ; Lipopolysaccharides ; pharmacology ; Lovastatin ; pharmacology ; MicroRNAs ; genetics ; Myocardium ; metabolism ; Myocytes, Cardiac ; drug effects ; metabolism ; Phosphoprotein Phosphatases ; metabolism ; Phosphorylation ; STAT3 Transcription Factor ; metabolism

This study was aimed to investigate the effect of AG490, a JAK2 inhibitor, on expression of PHLPP and p-Akt in K562. K562 cells were treated with different concentrations of AG490. The proliferation of K562 cells was examined by WST-1 assay and apoptosis of K562 cells was detected by flow cytometry with Annexin V-FITC/PI double staining. The expressions of PHLPP, phosphorate-Akt (p-Akt) and total Akt protein were detected by Western blot. The results indicated that AG490 inhibited the proliferation of K562 cells in concentration-and time-dependent manners, with the IC(50) 338.0 µmol/L in 48 h. AG490 100 µmol/L also induced apoptosis of K562 cells in a time-dependent manner. AG490 100 µmol/L time-dependently down-regulated the protein expression of p-Akt and PHLPP, but without significant effect on expression of total Akt. It is concluded that AG490 can inhibit proliferation and induce apoptosis of K562 cells through down-regulation of p-Akt expression, but inhibiting efficacy of AG490 on K562 proliferation also may be limited due to the down-regulation of p-Akt regulatory protein PHLPP expression.
Cell Proliferation ; drug effects ; Down-Regulation ; Humans ; K562 Cells ; Nuclear Proteins ; metabolism ; Phosphoprotein Phosphatases ; metabolism ; Tyrphostins ; pharmacology

Programmed necrosis, also known as necroptosis, has recently drawn great attention. As an important cellular regulation mechanism, knowledge of its signaling components is expanding. Necroptosisis demonstrated to be regulated by the RIP1 and RIP3 kinases, and its pathophysiological importance has been confirmed in a number of disease models. Here we review the new members of this necroptosis pathway, MLKL, PGAM5, Drp1 and DAI, and discuss some of their possible applications according to recent findings.
Animals ; Carrier Proteins ; metabolism ; DNA-Binding Proteins ; metabolism ; GTP Phosphohydrolases ; metabolism ; Humans ; Microtubule-Associated Proteins ; metabolism ; Mitochondrial Proteins ; metabolism ; Necrosis ; Phosphoprotein Phosphatases ; Protein Kinases ; chemistry ; metabolism ; Receptor-Interacting Protein Serine-Threonine Kinases ; metabolism ; Signal Transduction ; Tumor Necrosis Factors ; metabolism