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 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
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
目的探究雄黄主要成分二硫化二砷(As_(2)S_(2))对三阴性乳腺癌(triple negative breast cancer,TNBC)的作用及表观遗传调控机制。方法采用CCK-8、平板克隆形成和细胞划痕实验探究As_(2)S_(2)对人正常乳腺上皮细胞MCF-10A及TNBC细胞增殖...目的探究雄黄主要成分二硫化二砷(As_(2)S_(2))对三阴性乳腺癌(triple negative breast cancer,TNBC)的作用及表观遗传调控机制。方法采用CCK-8、平板克隆形成和细胞划痕实验探究As_(2)S_(2)对人正常乳腺上皮细胞MCF-10A及TNBC细胞增殖和迁移的影响;4D-label free定量蛋白质组学分析挖掘As_(2)S_(2)抗TNBC的潜在干预靶点酸性核磷蛋白家族成员32A(acidic nuclear phosphoprotein family member 32A,ANP32A);慢病毒感染法构建ANP32A过表达敲低细胞株,探究潜在靶点ANP32A对As_(2)S_(2)抗TNBC作用的影响;蛋白质免疫共沉淀和Western blot实验探究As_(2)S_(2)是否通过ANP32A调控TNBC细胞H3乙酰化。结果As_(2)S_(2)对人正常乳腺上皮细胞MCF-10A影响甚微,但显著抑制TNBC细胞增殖和迁移,且呈剂量依赖性;4D-lable free定量蛋白质组学分析结果显示,促癌因子ANP32A被As_(2)S_(2)显著下调,且ANP32A表达影响As_(2)S_(2)在TNBC中的抗增殖和迁移效果。As_(2)S_(2)能下调ANP32A的蛋白水平,抑制乙酰转移酶抑制剂复合物亚基的招募,增加H3乙酰化水平。结论As_(2)S_(2)通过下调ANP32A蛋白调控TNBC细胞中H3乙酰化,抑制TNBC细胞增殖和迁移。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
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 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.
文摘目的探究雄黄主要成分二硫化二砷(As_(2)S_(2))对三阴性乳腺癌(triple negative breast cancer,TNBC)的作用及表观遗传调控机制。方法采用CCK-8、平板克隆形成和细胞划痕实验探究As_(2)S_(2)对人正常乳腺上皮细胞MCF-10A及TNBC细胞增殖和迁移的影响;4D-label free定量蛋白质组学分析挖掘As_(2)S_(2)抗TNBC的潜在干预靶点酸性核磷蛋白家族成员32A(acidic nuclear phosphoprotein family member 32A,ANP32A);慢病毒感染法构建ANP32A过表达敲低细胞株,探究潜在靶点ANP32A对As_(2)S_(2)抗TNBC作用的影响;蛋白质免疫共沉淀和Western blot实验探究As_(2)S_(2)是否通过ANP32A调控TNBC细胞H3乙酰化。结果As_(2)S_(2)对人正常乳腺上皮细胞MCF-10A影响甚微,但显著抑制TNBC细胞增殖和迁移,且呈剂量依赖性;4D-lable free定量蛋白质组学分析结果显示,促癌因子ANP32A被As_(2)S_(2)显著下调,且ANP32A表达影响As_(2)S_(2)在TNBC中的抗增殖和迁移效果。As_(2)S_(2)能下调ANP32A的蛋白水平,抑制乙酰转移酶抑制剂复合物亚基的招募,增加H3乙酰化水平。结论As_(2)S_(2)通过下调ANP32A蛋白调控TNBC细胞中H3乙酰化,抑制TNBC细胞增殖和迁移。
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.
