AIM： To elucidate the localization of vasodilator stimulated phosphoprotein （VASP）, a cytoskeletal organizing protein and a substrate of protein kinases A and G in mitotic gastric cancer cells. METHODS： Immunofluo...AIM： To elucidate the localization of vasodilator stimulated phosphoprotein （VASP）, a cytoskeletal organizing protein and a substrate of protein kinases A and G in mitotic gastric cancer cells. METHODS： Immunofluorescence microscopy was used to observe the localization of α-tubulin, VASP and Ser157 phosphorylated VASP （p-VASP） in interphase of mitotic gastric cancer of the cell line SGC-7901. RESULTS： Immunofluorescence staining showed that p-VASP but not VASP was co-localized with α-tubulin on spindle poles and fibers in prophase, metaphase and anaphase of the mitotic process of the gastric cancer cell line SGC-7901. H89, an inhibitor of protein kinases A and G, had no effect on the localization of p-VASP on the spindles. CONCLUSION： VASP may play a role in assembling and stabilizing the mitotic spindle of cells, and phosphorylation of the protein is the precondition for it to exert this function.展开更多
Aim:Extracellular matrix(ECM)-adhesions and their interaction with actin cytoskeleton are fundamental for hepatocellular carcinoma(HCC).Fascin-1,an actin-bundling protein,is correlated with poor HCC prognosis,and is k...Aim:Extracellular matrix(ECM)-adhesions and their interaction with actin cytoskeleton are fundamental for hepatocellular carcinoma(HCC).Fascin-1,an actin-bundling protein,is correlated with poor HCC prognosis,and is known regarding the molecular mechanism of its action.In this study,the authors investigated Fascin-1 basic molecular mechanism and cellular properties in HCC cells.Methods:Fascin-1 was silenced by small interfering RNA and the expression of actin.The ECM-adhesion-related proteins were assessed along with the cells’adhesion capacity in two cell lines that differ in terms of aggressiveness;the hepatoma cell line PLC/PRF/5(Alexander)and the highly invasive HCC cell line HepG2.Results:This study shows that Fascin-1 is upregulated in HepG2 cells compared to Alexander cells and when silenced leads to increased cell adhesion only in HepG2,while at the same time is associated with reduced migfilin and vasodilator-stimulated phosphoprotein(VASP)expression.Conclusion:This is the first study to show that Fascin-1 contributes to a more aggressive phenotype in HCC cells and acts through migfilin and VASP.展开更多
Trastuzumab resistance is one of the causes of poor prognosis in patients with human epidermal growth factor receptor 2(HER2)-positive(HER2+)breast cancer(BC).The truncated isoform of dopamine-and cAMPregulated phosph...Trastuzumab resistance is one of the causes of poor prognosis in patients with human epidermal growth factor receptor 2(HER2)-positive(HER2+)breast cancer(BC).The truncated isoform of dopamine-and cAMPregulated phosphoprotein(t-DARPP)has been reported to be involved in trastuzumab therapy resistance and promoting tumor progression.To evaluate the t-DARPP expression in BC,paired tumors and surrounding normal tissues were analyzed by real-time polymerase chain reaction and confirmed higher DARPP-32 kDa family mRNA expression in HER2+BC tumor tissues.We established 2 patient-derived xenografts(PDX)mice models to test the efficacy of trastuzumab,named model 1(non-responder)and model 2(responder).t-DARPP and p95-HER2 protein-protein interactions were detected in PDX tumor tissue from non-responders using Förster resonance energy transfer assays.Instead,there is no response from the responder.Furthermore,mechanistic studies using transwell and western blot assays demonstrated that t-DARPP could upregulate epithelial-mesenchymal transition signaling proteins,enhance p95-HER2 expression and promote cell migration.We found that quercetin effectively reduced t-DARPP expression in HER2+BC cells.In t-DARPP ShRNA-suppressed cells,quercetin synergistically enhanced trastuzumab-induced apoptotic cell death and G2/M phase arrest.In conclusion,the combination of quercetin and trastuzumab treatment by targeting t-DARPP in HER2+BC patients has the potential as a biomarker for mitigating drug resistance.展开更多
BACKGROUND Perianal fistulae are either primary(idiopathic)or secondary[commonly associated with Crohn’s disease,(CD)].It is assumed,although not proven,that the pathophysiology differs.AIM To systematically compare ...BACKGROUND Perianal fistulae are either primary(idiopathic)or secondary[commonly associated with Crohn’s disease,(CD)].It is assumed,although not proven,that the pathophysiology differs.AIM To systematically compare the clinical phenotypes,cytokine and phosphoprotein profiles of idiopathic and CD-related perianal fistulae.METHODS Sixty-one patients undergoing surgery for perianal fistula were prospectively recruited(48 idiopathic,13 CD)into a cohort study.Clinical data,including the Perineal Disease Activity Index(PDAI)and EQ-5D-5L were collected.Biopsies of the fistula tract,granulation tissue,internal opening mucosa and rectal mucosa were obtained at surgery.Concentrations of 30 cytokines and 39 phosphoproteins were measured in each biopsy using a magnetic bead multiplexing instrument and a chemiluminescent antibody array respectively.Over 12000 clinical and 23500 laboratory measurements were made.RESULTS The PDAI was significantly higher(indicating more active disease)in the CD group with a mean difference of 2.40(95%CI:0.52-4.28,P=0.01).Complex pathoanatomy was more prevalent in the CD group,namely more multiple fistulae,supralevator extensions,collections and rectal thickening.The IL-12p70 concentration at the internal opening specimen site was significantly higher(median difference 19.7 pg/mL,99%CI:0.2-40.4,P=0.008)and the IL-1RA/IL-1βratio was significantly lower in the CD group at the internal opening specimen site(median difference 15.0,99%CI=0.4-50.5,P=0.008).However in the remaining 27 cytokines and all 39 of the phosphoproteins across the four biopsy sites,no significant differences were found between the groups.CONCLUSION CD-related perianal fistulae are more clinically severe and anatomically complex than idiopathic perianal fistulae.However,overall there are no major differences in cytokine and phosphoprotein profiles.展开更多
A sensitive method based on the fluorescence quenching effect of the Tb^3＋-Tiron complex is proposed for the determination of alkali-labile phosphoprotein phosphorus （ALP） released from fish plasma. The detection l...A sensitive method based on the fluorescence quenching effect of the Tb^3＋-Tiron complex is proposed for the determination of alkali-labile phosphoprotein phosphorus （ALP） released from fish plasma. The detection limit was 5.4 ng/ml （S/N=2）, and the relative standard deviation of the quenching effect （6 replicates） was 4.6%. The results obtained by the proposed method were in good agreement with those obtained by the colorimetric assay. The advantages of the present method are its relatively simple detection procedure, the lack of toxic organic solvents, and high sensitivity.展开更多
AIM:To determine the effect and molecular mechanism of ezrin-radixin-moesin-binding phosphoprotein-50(EBP50) in hepatocellular carcinoma(HCC).METHODS:Three human HCC cell lines,i.e.,SMMC7721,HepG2 and Hep3B,were used....AIM:To determine the effect and molecular mechanism of ezrin-radixin-moesin-binding phosphoprotein-50(EBP50) in hepatocellular carcinoma(HCC).METHODS:Three human HCC cell lines,i.e.,SMMC7721,HepG2 and Hep3B,were used.We transfected the Pbk-CMV-HA-EBP50 plasmid into SMMC7721 cells with Lipofectamine 2000 to overexpress EBP50.Western blotting were performed to determine the effects of the plasmid on EBP50 expression and to detect the expression of β-catenin and E-cadherin before and after the transfection of the plasmid into SMMC7721 cells.In vitro cell proliferation was assessed with a Cell Counting Kit-8(CCK-8) assay.Cell cycle distribution was assessed with flow cytometry.Invasion and migration ability of before and after the transfection were determined with a transwell assay.Cell apoptosis was demonstrated with Annexin V-FITC.The effect of EBP50 overexpressing on tumor growth in vivo was performed with a xenograft tumor model in nude mice.RESULTS:The transfection efficiency was confirmed with Western blotting(1.36 ± 0.07 vs 0.81 ± 0.09,P < 0.01).The CCK8 assay demonstrated that the growth of cells overexpressing EBP50 was significantly lower than control cells(P < 0.01).Cell cycle distribution showed there was a G0/G1 cell cycle arrest in cells overexpressing EBP50(61.3% ± 3.1% vs 54.0% ± 2.4%,P < 0.05).The transwell assay showed that cell invasion and migration were significantly inhibited in cells overexpressing EBP50 compared with control cells(5.8 ± 0.8 vs 21.6 ± 1.3,P < 0.01).Annexin V-FITC revealed that apoptosis was significantly increased in cells overexpressing EBP50 compared with control cells(14.8% ± 2.7% vs 3.4% ± 1.3%,P < 0.05).The expression of β-catenin was downregulated and E-cadherin was upregulated in cells overexpressing EBP50 compared with control cells(0.28 ± 0.07 vs 0.56 ± 0.12,P < 0.05;0.55 ± 0.08 vs 0.39 ± 0.07,P < 0.05).In vivo tumor growth assay confirmed that up-regulation of EBP50 could obviously slow the growth of HCC derived from SMMC7721 cells(28.9 ± 7.2 vs 70.1 ± 7.2,P < 0.01).CONCLUSION:The overexpression of EBP50 could inhibit the growth of SMMC7721 cells and promote apoptosis by modulating β-catenin,E-cadherin.EBP50 may serve asa potential therapeutic target in HCC.展开更多
A novel phosphoprotein separation material was developed, which is constructed by a magnetic mesoporous Fe3 O4@TiO2（Fe3 O4@mTiO2） microsphere and a 5-aminoisophthalic acid（AIPA） monolayer that provides additional ...A novel phosphoprotein separation material was developed, which is constructed by a magnetic mesoporous Fe3 O4@TiO2（Fe3 O4@mTiO2） microsphere and a 5-aminoisophthalic acid（AIPA） monolayer that provides additional binding sites toward phosphate groups. The results of characteristic experiments demonstrated that Fe3 O4@mTiO2-AIPA had good dispersability, high magnetic susceptibility, and satisfactory grafting ratio of AIPA, ascribed to the large specific surface area of the inorganic substrate. Taking advantages of these features, Fe3 O4@mTiO2-AIPA was successfully utilized to separate α-casein（a typical phosphoprotein） and bovine serum albumin（BSA, a typical non-phosphoprotein） from their mixtures（molar ratio = 1:2）. Through adjusting pH and polarity of solutions, the BSA and α-casein were respectively enriched in washing fraction and elution fraction. This result displays the good potential of Fe3 O4@mTiO2-AIPA for application in phosphoprotein enrichment.展开更多
Objective:To investigate the potential role of human cytomegalovirus lower matrix phosphoprotein 65(HCMV-pp65) in murine systemic lupus erythematosus(SLE).Methods:The prokaryotic plasmid pET-28b-pp65 was constructed t...Objective:To investigate the potential role of human cytomegalovirus lower matrix phosphoprotein 65(HCMV-pp65) in murine systemic lupus erythematosus(SLE).Methods:The prokaryotic plasmid pET-28b-pp65 was constructed to express the HCMVpp65 protein.BXSB mice and C57BL/6 mice were inoculated with pp65 eukarvotic plasmid pcDNA3.0-pp65 intramuscularly 5 times at 2-week intervals,and then the blood of the mice was subsequently collected via the retro-orbital vein.Indirect ELISAs were used to evaluate the concentration of anti-pp65 immunoglobulin G,anti-double-stranded DNA and antinuclear antibodies.lnterleukin-1β and tumor necrosis factor-α were also determined by competitive ELISA.At the same time,3 major SLE-related circulating microRNAs were examined by quantitative RT-PCR.Results:The early production of autoantibodies was observed in pp65-immunized male BXSB as well as C57BL/6 mice.Overexpression of interleukin-1β and tumor necrosis factor-a were detected in pp65-immunized male BXSB mice.Quantitative RT-PCR analyses showed that three SLE related microRNAs(microRNA-126,microRNA-125 a,and microRKA-146a) were dovvnrcgulatcd in peripheral blood mononuclear cells of pp65-immunizcd mice.Conclusions:Our findings indicate that HCMV-pp65 immunization strongly triggers the development and progression of" SLE-like disease in both BXSB and C57BL/6 mice,which indicates that the immune responses induced by HCMV-pp65 may be involved in the development of SLE.展开更多
Prostate cancer is a major public health concern world-wide, being one of the most prevalent cancers in men. Great improvements have been made both in terms of early diagnosis and therapeutics. However, there is still...Prostate cancer is a major public health concern world-wide, being one of the most prevalent cancers in men. Great improvements have been made both in terms of early diagnosis and therapeutics. However, there is still an urgent need for reliable biomarkers that could overcome the lack of cancer-specifcity of prostate-specifc antigen, as well as alternative therapeutic targets for advanced metastatic cases. Reversible phosphorylation of proteins is a post-translational modifcation critical to the regulation of numerous cellular processes. Phosphoprotein phosphatase 1 （PPP1） is a major serine/threonine phos-phatase, whose specifcity is determined by its interacting proteins. These interactors can be PPP1 substrates, regulators, or even both. Deregulation of this protein-protein interaction network alters cell dynamics and underlies the development of several cancer hallmarks. Therefore, the identification of PPP1 interactome in specific cellular context is of crucial importance. The knowledge on PPP1 complexes in prostate cancer remains scarce, with only 4 holoenzymes characterized in human prostate cancer models. However, an increasing number of PPP1 interactors have been identifed as expressed in human prostate tissue, including the tumor suppressors TP53 and RB1. Efforts should be made in order to identify the role of such proteins in prostate carcinogenesis, since only 26 have yet well-recognized roles. Here, we revise literature and human protein databases to provide an in-depth knowledge on the biological significance of PPP1 complexes in human prostate carcinogenesis and their potential use as therapeutic targets for the development of new therapies for prostate cancer.展开更多
BACKGROUND Dopamine and cyclic adenosine monophosphate(cAMP)-regulated phosphop-rotein with an apparent Mr of 32000(DARPP-32)is a protein that is involved in regulating dopamine and cAMP signaling pathways in the brai...BACKGROUND Dopamine and cyclic adenosine monophosphate(cAMP)-regulated phosphop-rotein with an apparent Mr of 32000(DARPP-32)is a protein that is involved in regulating dopamine and cAMP signaling pathways in the brain.However,recent studies have shown that DARPP-32 is also expressed in other tissues,including colorectal cancer(CRC),where its function is not well understood.AIM To explore the effect of DARPP-32 on CRC progression.METHODS The expression levels of DARPP-32 were assessed in CRC tissues using both quantitative polymerase chain reaction and immunohistochemistry assays.The proliferative capacity of CRC cell lines was evaluated with Cell Counting Kit-8 and 5-ethynyl-2’-deoxyuridine assays,while apoptosis was measured by flow cytometry.The migratory and invasive potential of CRC cell lines were deter-mined using wound healing and transwell chamber assays.In vivo studies involved monitoring the growth rate of xenograft tumors.Finally,the underlying molecular mechanism of DARPP-32 was investigated through RNA-sequencing and western blot analyses.RESULTS DARPP-32 was frequently upregulated in CRC and associated with abnormal clinicopathological features in CRC.Overexpression of DARPP-32 was shown to promote cancer cell proliferation,migration,and invasion and reduce apoptosis.DARPP-32 knockdown resulted in the opposite functional effects.Mechanistically,DARPP-32 may regulate the phosphoinositide 3-kinase(PI3K)/AKT signaling pathway in order to carry out its biological function.CONCLUSION DARPP-32 promotes CRC progression via the PI3K/AKT signaling pathway.展开更多
Reversible protein phosphorylation is a central regulatory mechanism of cell function. Deregulation of the balanced actions of protein kinases and phosphatases has been frequently associated with several pathological ...Reversible protein phosphorylation is a central regulatory mechanism of cell function. Deregulation of the balanced actions of protein kinases and phosphatases has been frequently associated with several pathological conditions, including cancer. Many studies have already addressed the role of protein kinases misregulation in cancer. However, much less is known about protein phosphatases influence. Phosphoprotein Phosphatase 1 (PPP1) is one of the major serine/threonine protein phosphatases who has three catalytic isoforms: PPP1CA, PPP1CB, and PPP1CC. Its function is achieved by binding to regulatory subunits, known as PPP1-interacting proteins (PIPs), which may prefer a catalytic isoform. Also, some inhibitors/enhancers may exhibit isoform specificity. Here we show that, prodigiosin (PG), a molecule with anticancer properties, promotes the formation of PPP1CA-AKT complex and not of PPP1CC-MAPK complex. Both, AKT and MAPK, are well-known PIPs from two pathways that crosstalk and regulate melanoma cells survival. In addition, the analysis performed using surface plasmon resonance (SPR) technology indicates that PPP1 interacts with obatoclax (OBX), a drug that belongs to the same family of PG. Overall, these results suggest that PG might, at least in part, act through PPP1C/PIPs. Also, this study is pioneer in demonstrating PPP1 isoform-specific modulation by small molecules.展开更多
人核仁磷酸化蛋白1(Nucleolar and coiledbody phosphoprotein 1,NOLC1)在癌症的发生发展过程中起着至关重要的调控作用,为探讨NOLC1对肺癌细胞的作用,本研究通过Gateway系统构建重组NOLC1腺病毒载体,成功包装NOLC1腺病毒后,分别感染正...人核仁磷酸化蛋白1(Nucleolar and coiledbody phosphoprotein 1,NOLC1)在癌症的发生发展过程中起着至关重要的调控作用,为探讨NOLC1对肺癌细胞的作用,本研究通过Gateway系统构建重组NOLC1腺病毒载体,成功包装NOLC1腺病毒后,分别感染正常人类胚胎肺细胞(HEL)和非小细胞肺癌细胞(A549细胞),过表达NOLC1。通过MTT实验、AnnexinV-APC/PI双染法和线粒体膜电位实验,证明与HEL细胞相比,NOLC1的过表达对A549细胞的活性降低、凋亡增加、线粒体膜电位下降影响较为显著;通过Real-time PCR检测Caspase家族、TNF与受体家族和BCL2家族基因的表达,发现过表达NOLC1明显上调了A549细胞中促凋亡基因的表达,下调了抗凋亡基因的表达,其中两种重要的促凋亡蛋白CASP8和BAX均显著上调,但是在HEL细胞中这种影响不明显。研究结果表明过表达NOLC1蛋白通过对线粒体通路和死亡受体通路的共同作用,对非小细胞癌具有显著的抗肿瘤活性。展开更多
基金Supported by National Natural Science Foundation of China, No. 30340036 and 30470891 Startup Grant from Jiangsu University, and Grant of Zhenjiang Key Institute of Clinical Laboratory Medicine (SH2006066)
文摘AIM： To elucidate the localization of vasodilator stimulated phosphoprotein （VASP）, a cytoskeletal organizing protein and a substrate of protein kinases A and G in mitotic gastric cancer cells. METHODS： Immunofluorescence microscopy was used to observe the localization of α-tubulin, VASP and Ser157 phosphorylated VASP （p-VASP） in interphase of mitotic gastric cancer of the cell line SGC-7901. RESULTS： Immunofluorescence staining showed that p-VASP but not VASP was co-localized with α-tubulin on spindle poles and fibers in prophase, metaphase and anaphase of the mitotic process of the gastric cancer cell line SGC-7901. H89, an inhibitor of protein kinases A and G, had no effect on the localization of p-VASP on the spindles. CONCLUSION： VASP may play a role in assembling and stabilizing the mitotic spindle of cells, and phosphorylation of the protein is the precondition for it to exert this function.
基金supported by the European Association for the Study of the Liver Sheila Sherlock fellowship 2012.
文摘Aim:Extracellular matrix(ECM)-adhesions and their interaction with actin cytoskeleton are fundamental for hepatocellular carcinoma(HCC).Fascin-1,an actin-bundling protein,is correlated with poor HCC prognosis,and is known regarding the molecular mechanism of its action.In this study,the authors investigated Fascin-1 basic molecular mechanism and cellular properties in HCC cells.Methods:Fascin-1 was silenced by small interfering RNA and the expression of actin.The ECM-adhesion-related proteins were assessed along with the cells’adhesion capacity in two cell lines that differ in terms of aggressiveness;the hepatoma cell line PLC/PRF/5(Alexander)and the highly invasive HCC cell line HepG2.Results:This study shows that Fascin-1 is upregulated in HepG2 cells compared to Alexander cells and when silenced leads to increased cell adhesion only in HepG2,while at the same time is associated with reduced migfilin and vasodilator-stimulated phosphoprotein(VASP)expression.Conclusion:This is the first study to show that Fascin-1 contributes to a more aggressive phenotype in HCC cells and acts through migfilin and VASP.
基金The National Science and Technology Council of Taiwan funded this study.
文摘Trastuzumab resistance is one of the causes of poor prognosis in patients with human epidermal growth factor receptor 2(HER2)-positive(HER2+)breast cancer(BC).The truncated isoform of dopamine-and cAMPregulated phosphoprotein(t-DARPP)has been reported to be involved in trastuzumab therapy resistance and promoting tumor progression.To evaluate the t-DARPP expression in BC,paired tumors and surrounding normal tissues were analyzed by real-time polymerase chain reaction and confirmed higher DARPP-32 kDa family mRNA expression in HER2+BC tumor tissues.We established 2 patient-derived xenografts(PDX)mice models to test the efficacy of trastuzumab,named model 1(non-responder)and model 2(responder).t-DARPP and p95-HER2 protein-protein interactions were detected in PDX tumor tissue from non-responders using Förster resonance energy transfer assays.Instead,there is no response from the responder.Furthermore,mechanistic studies using transwell and western blot assays demonstrated that t-DARPP could upregulate epithelial-mesenchymal transition signaling proteins,enhance p95-HER2 expression and promote cell migration.We found that quercetin effectively reduced t-DARPP expression in HER2+BC cells.In t-DARPP ShRNA-suppressed cells,quercetin synergistically enhanced trastuzumab-induced apoptotic cell death and G2/M phase arrest.In conclusion,the combination of quercetin and trastuzumab treatment by targeting t-DARPP in HER2+BC patients has the potential as a biomarker for mitigating drug resistance.
基金Supported by Bowel and Cancer Research charity,No.MMBG1J3R
文摘BACKGROUND Perianal fistulae are either primary(idiopathic)or secondary[commonly associated with Crohn’s disease,(CD)].It is assumed,although not proven,that the pathophysiology differs.AIM To systematically compare the clinical phenotypes,cytokine and phosphoprotein profiles of idiopathic and CD-related perianal fistulae.METHODS Sixty-one patients undergoing surgery for perianal fistula were prospectively recruited(48 idiopathic,13 CD)into a cohort study.Clinical data,including the Perineal Disease Activity Index(PDAI)and EQ-5D-5L were collected.Biopsies of the fistula tract,granulation tissue,internal opening mucosa and rectal mucosa were obtained at surgery.Concentrations of 30 cytokines and 39 phosphoproteins were measured in each biopsy using a magnetic bead multiplexing instrument and a chemiluminescent antibody array respectively.Over 12000 clinical and 23500 laboratory measurements were made.RESULTS The PDAI was significantly higher(indicating more active disease)in the CD group with a mean difference of 2.40(95%CI:0.52-4.28,P=0.01).Complex pathoanatomy was more prevalent in the CD group,namely more multiple fistulae,supralevator extensions,collections and rectal thickening.The IL-12p70 concentration at the internal opening specimen site was significantly higher(median difference 19.7 pg/mL,99%CI:0.2-40.4,P=0.008)and the IL-1RA/IL-1βratio was significantly lower in the CD group at the internal opening specimen site(median difference 15.0,99%CI=0.4-50.5,P=0.008).However in the remaining 27 cytokines and all 39 of the phosphoproteins across the four biopsy sites,no significant differences were found between the groups.CONCLUSION CD-related perianal fistulae are more clinically severe and anatomically complex than idiopathic perianal fistulae.However,overall there are no major differences in cytokine and phosphoprotein profiles.
基金Project supported by the Hi-Tech Research and Development Program (863) of China (No. 2006AA06Z424) the National Basic Research Program (973) of China (No. 2003CB415001)the Pilot Project of Knowledge Innovation Program of Chinese Academy of Sciences (No. KJCX2-SW-H06).
文摘A sensitive method based on the fluorescence quenching effect of the Tb^3＋-Tiron complex is proposed for the determination of alkali-labile phosphoprotein phosphorus （ALP） released from fish plasma. The detection limit was 5.4 ng/ml （S/N=2）, and the relative standard deviation of the quenching effect （6 replicates） was 4.6%. The results obtained by the proposed method were in good agreement with those obtained by the colorimetric assay. The advantages of the present method are its relatively simple detection procedure, the lack of toxic organic solvents, and high sensitivity.
基金Supported by Fundamental Research Funds for the Central Universities of China,No.201130202020005
文摘AIM:To determine the effect and molecular mechanism of ezrin-radixin-moesin-binding phosphoprotein-50(EBP50) in hepatocellular carcinoma(HCC).METHODS:Three human HCC cell lines,i.e.,SMMC7721,HepG2 and Hep3B,were used.We transfected the Pbk-CMV-HA-EBP50 plasmid into SMMC7721 cells with Lipofectamine 2000 to overexpress EBP50.Western blotting were performed to determine the effects of the plasmid on EBP50 expression and to detect the expression of β-catenin and E-cadherin before and after the transfection of the plasmid into SMMC7721 cells.In vitro cell proliferation was assessed with a Cell Counting Kit-8(CCK-8) assay.Cell cycle distribution was assessed with flow cytometry.Invasion and migration ability of before and after the transfection were determined with a transwell assay.Cell apoptosis was demonstrated with Annexin V-FITC.The effect of EBP50 overexpressing on tumor growth in vivo was performed with a xenograft tumor model in nude mice.RESULTS:The transfection efficiency was confirmed with Western blotting(1.36 ± 0.07 vs 0.81 ± 0.09,P < 0.01).The CCK8 assay demonstrated that the growth of cells overexpressing EBP50 was significantly lower than control cells(P < 0.01).Cell cycle distribution showed there was a G0/G1 cell cycle arrest in cells overexpressing EBP50(61.3% ± 3.1% vs 54.0% ± 2.4%,P < 0.05).The transwell assay showed that cell invasion and migration were significantly inhibited in cells overexpressing EBP50 compared with control cells(5.8 ± 0.8 vs 21.6 ± 1.3,P < 0.01).Annexin V-FITC revealed that apoptosis was significantly increased in cells overexpressing EBP50 compared with control cells(14.8% ± 2.7% vs 3.4% ± 1.3%,P < 0.05).The expression of β-catenin was downregulated and E-cadherin was upregulated in cells overexpressing EBP50 compared with control cells(0.28 ± 0.07 vs 0.56 ± 0.12,P < 0.05;0.55 ± 0.08 vs 0.39 ± 0.07,P < 0.05).In vivo tumor growth assay confirmed that up-regulation of EBP50 could obviously slow the growth of HCC derived from SMMC7721 cells(28.9 ± 7.2 vs 70.1 ± 7.2,P < 0.01).CONCLUSION:The overexpression of EBP50 could inhibit the growth of SMMC7721 cells and promote apoptosis by modulating β-catenin,E-cadherin.EBP50 may serve asa potential therapeutic target in HCC.
基金Funded by the National Natural Science Foundation of China(Nos.51473131,21275114,51533007 and 51521001)the Major State Basic Research Development Program of China(973 Program)(No.2013CB933002)+1 种基金Hubei Provincial Department of Education for Financial Assistance Through the “Chutian Scholar” ProgramHubei Provincial Natural Science Foundation of China(No.2014CFA039)
文摘A novel phosphoprotein separation material was developed, which is constructed by a magnetic mesoporous Fe3 O4@TiO2（Fe3 O4@mTiO2） microsphere and a 5-aminoisophthalic acid（AIPA） monolayer that provides additional binding sites toward phosphate groups. The results of characteristic experiments demonstrated that Fe3 O4@mTiO2-AIPA had good dispersability, high magnetic susceptibility, and satisfactory grafting ratio of AIPA, ascribed to the large specific surface area of the inorganic substrate. Taking advantages of these features, Fe3 O4@mTiO2-AIPA was successfully utilized to separate α-casein（a typical phosphoprotein） and bovine serum albumin（BSA, a typical non-phosphoprotein） from their mixtures（molar ratio = 1:2）. Through adjusting pH and polarity of solutions, the BSA and α-casein were respectively enriched in washing fraction and elution fraction. This result displays the good potential of Fe3 O4@mTiO2-AIPA for application in phosphoprotein enrichment.
基金Supported by National High Technology Research and Development Program of China(863 program No.2011AA02A116)
文摘Objective:To investigate the potential role of human cytomegalovirus lower matrix phosphoprotein 65(HCMV-pp65) in murine systemic lupus erythematosus(SLE).Methods:The prokaryotic plasmid pET-28b-pp65 was constructed to express the HCMVpp65 protein.BXSB mice and C57BL/6 mice were inoculated with pp65 eukarvotic plasmid pcDNA3.0-pp65 intramuscularly 5 times at 2-week intervals,and then the blood of the mice was subsequently collected via the retro-orbital vein.Indirect ELISAs were used to evaluate the concentration of anti-pp65 immunoglobulin G,anti-double-stranded DNA and antinuclear antibodies.lnterleukin-1β and tumor necrosis factor-α were also determined by competitive ELISA.At the same time,3 major SLE-related circulating microRNAs were examined by quantitative RT-PCR.Results:The early production of autoantibodies was observed in pp65-immunized male BXSB as well as C57BL/6 mice.Overexpression of interleukin-1β and tumor necrosis factor-a were detected in pp65-immunized male BXSB mice.Quantitative RT-PCR analyses showed that three SLE related microRNAs(microRNA-126,microRNA-125 a,and microRKA-146a) were dovvnrcgulatcd in peripheral blood mononuclear cells of pp65-immunizcd mice.Conclusions:Our findings indicate that HCMV-pp65 immunization strongly triggers the development and progression of" SLE-like disease in both BXSB and C57BL/6 mice,which indicates that the immune responses induced by HCMV-pp65 may be involved in the development of SLE.
基金Supported by Fundao para a Ciência e Tecnologia(FCT)(PTDC/QUI-BIQ/118492/2010)Fundo Europeu de Desenvolvimento Regional(FEDER)(FCOMP-01-0124-FEDER-020895),Portugal
文摘Prostate cancer is a major public health concern world-wide, being one of the most prevalent cancers in men. Great improvements have been made both in terms of early diagnosis and therapeutics. However, there is still an urgent need for reliable biomarkers that could overcome the lack of cancer-specifcity of prostate-specifc antigen, as well as alternative therapeutic targets for advanced metastatic cases. Reversible phosphorylation of proteins is a post-translational modifcation critical to the regulation of numerous cellular processes. Phosphoprotein phosphatase 1 （PPP1） is a major serine/threonine phos-phatase, whose specifcity is determined by its interacting proteins. These interactors can be PPP1 substrates, regulators, or even both. Deregulation of this protein-protein interaction network alters cell dynamics and underlies the development of several cancer hallmarks. Therefore, the identification of PPP1 interactome in specific cellular context is of crucial importance. The knowledge on PPP1 complexes in prostate cancer remains scarce, with only 4 holoenzymes characterized in human prostate cancer models. However, an increasing number of PPP1 interactors have been identifed as expressed in human prostate tissue, including the tumor suppressors TP53 and RB1. Efforts should be made in order to identify the role of such proteins in prostate carcinogenesis, since only 26 have yet well-recognized roles. Here, we revise literature and human protein databases to provide an in-depth knowledge on the biological significance of PPP1 complexes in human prostate carcinogenesis and their potential use as therapeutic targets for the development of new therapies for prostate cancer.
基金Supported by Chongqing Key Diseases Research and Application Demonstration Program,No.2019ZX003General Project of Chongqing Nature Science Foundation,No.cstc2021jcyj-msxmX0283.
文摘BACKGROUND Dopamine and cyclic adenosine monophosphate(cAMP)-regulated phosphop-rotein with an apparent Mr of 32000(DARPP-32)is a protein that is involved in regulating dopamine and cAMP signaling pathways in the brain.However,recent studies have shown that DARPP-32 is also expressed in other tissues,including colorectal cancer(CRC),where its function is not well understood.AIM To explore the effect of DARPP-32 on CRC progression.METHODS The expression levels of DARPP-32 were assessed in CRC tissues using both quantitative polymerase chain reaction and immunohistochemistry assays.The proliferative capacity of CRC cell lines was evaluated with Cell Counting Kit-8 and 5-ethynyl-2’-deoxyuridine assays,while apoptosis was measured by flow cytometry.The migratory and invasive potential of CRC cell lines were deter-mined using wound healing and transwell chamber assays.In vivo studies involved monitoring the growth rate of xenograft tumors.Finally,the underlying molecular mechanism of DARPP-32 was investigated through RNA-sequencing and western blot analyses.RESULTS DARPP-32 was frequently upregulated in CRC and associated with abnormal clinicopathological features in CRC.Overexpression of DARPP-32 was shown to promote cancer cell proliferation,migration,and invasion and reduce apoptosis.DARPP-32 knockdown resulted in the opposite functional effects.Mechanistically,DARPP-32 may regulate the phosphoinositide 3-kinase(PI3K)/AKT signaling pathway in order to carry out its biological function.CONCLUSION DARPP-32 promotes CRC progression via the PI3K/AKT signaling pathway.
基金supported by grants from Fundacao para a Ciencia e Tecnologia(FCT)of the Portuguese Ministry of Science and Higher Education(PTDC/DTP-PIC/0460/2012)by FEDER through Eixo I do Programa Operacional Fatores de Competitividade(POFC)(FCOMP-01-0124-FEDER-028692)co-funded by QREN
文摘Reversible protein phosphorylation is a central regulatory mechanism of cell function. Deregulation of the balanced actions of protein kinases and phosphatases has been frequently associated with several pathological conditions, including cancer. Many studies have already addressed the role of protein kinases misregulation in cancer. However, much less is known about protein phosphatases influence. Phosphoprotein Phosphatase 1 (PPP1) is one of the major serine/threonine protein phosphatases who has three catalytic isoforms: PPP1CA, PPP1CB, and PPP1CC. Its function is achieved by binding to regulatory subunits, known as PPP1-interacting proteins (PIPs), which may prefer a catalytic isoform. Also, some inhibitors/enhancers may exhibit isoform specificity. Here we show that, prodigiosin (PG), a molecule with anticancer properties, promotes the formation of PPP1CA-AKT complex and not of PPP1CC-MAPK complex. Both, AKT and MAPK, are well-known PIPs from two pathways that crosstalk and regulate melanoma cells survival. In addition, the analysis performed using surface plasmon resonance (SPR) technology indicates that PPP1 interacts with obatoclax (OBX), a drug that belongs to the same family of PG. Overall, these results suggest that PG might, at least in part, act through PPP1C/PIPs. Also, this study is pioneer in demonstrating PPP1 isoform-specific modulation by small molecules.
