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Proteomic Characterization of Annexin l (ANX1) and Heat Shock Protein 27 (HSP27) as Biomarkers for Invasive Hepatocellular Carcinoma Cells.

Wang RC, Huang CY, Pan TL, Chen WY, Ho CT, Liu TZ, Chang YJ - PLoS ONE (2015)

Bottom Line: In total, 106 and 55 proteins were successfully identified from the total cell lysate and the cytosolic, nuclear and membrane fractions in well-differentiated (HepG2) and poorly differentiated (SK-Hep-1) HCC clonal variants, respectively.Notably, Annexin 1 (ANX1), ANX-2, vimentin and stress-associated proteins, such as GRP78, HSP75, HSC-70, protein disulfide isomerase (PDI), and heat shock protein-27 (HSP27), were exclusively up-regulated in SK-Hep-1 cells.Knockdown of ANX1 or HSP27 in HCC cells resulted in a severe reduction in cell migration.

View Article: PubMed Central - PubMed

Affiliation: Tissue Bank, Chang Gung Memorial Hospital, Chiayi, Taiwan.

ABSTRACT
To search for reliable biomarkers and drug targets for management of hepatocellular carcinoma (HCC), we performed a global proteomic analysis of a pair of HCC cell lines with distinct differentiation statuses using 2-DE coupled with MALDI-TOF MS. In total, 106 and 55 proteins were successfully identified from the total cell lysate and the cytosolic, nuclear and membrane fractions in well-differentiated (HepG2) and poorly differentiated (SK-Hep-1) HCC clonal variants, respectively. Among these proteins, nine spots corresponding to proteins differentially expressed between HCC cell types were selected and confirmed by immunofluorescence staining and western blotting. Notably, Annexin 1 (ANX1), ANX-2, vimentin and stress-associated proteins, such as GRP78, HSP75, HSC-70, protein disulfide isomerase (PDI), and heat shock protein-27 (HSP27), were exclusively up-regulated in SK-Hep-1 cells. Elevated levels of ANX-4 and antioxidant/metabolic enzymes, such as MnSOD, peroxiredoxin, NADP-dependent isocitrate dehydrogenase, α-enolase and UDP-glucose dehydrogenase, were observed in HepG2 cells. We functionally demonstrated that ANX1 and HSP27 were abundantly overexpressed only in highly invasive types of HCC cells, such as Mahlavu and SK-Hep-1. Knockdown of ANX1 or HSP27 in HCC cells resulted in a severe reduction in cell migration. The in-vitro observations of ANX1 and HSP27 expressions in HCC sample was demonstrated by immunohistochemical stains performed on HCC tissue microarrays. Poorly differentiated HCC tended to have stronger ANX1 and HSP27 expressions than well-differentiated or moderately differentiated HCC. Collectively, our findings suggest that ANX1 and HSP27 are two novel biomarkers for predicting invasive HCC phenotypes and could serve as potential treatment targets.

No MeSH data available.


Related in: MedlinePlus

Protein classification.The functional classifications of the identified proteins.a: Cell cycle: 3%; b: chaperone/stress response: 9%; c: cytoskeleton/ cell mobility: 15%; d: DNA replication/gene regulation/cell proliferation: 12%; e: ion channels: 2%; f: membrane proteins: 4%; g: metabolic enzyme: 16%; h: protection and detoxification: 6%; i: protein synthesis and degradation: 8%; j: signal transduction: 8%; k: transport/binding proteins: 6%; l: intermediate filaments: 15%; and m: unannotated/ function inferred: 4%.
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pone.0139232.g002: Protein classification.The functional classifications of the identified proteins.a: Cell cycle: 3%; b: chaperone/stress response: 9%; c: cytoskeleton/ cell mobility: 15%; d: DNA replication/gene regulation/cell proliferation: 12%; e: ion channels: 2%; f: membrane proteins: 4%; g: metabolic enzyme: 16%; h: protection and detoxification: 6%; i: protein synthesis and degradation: 8%; j: signal transduction: 8%; k: transport/binding proteins: 6%; l: intermediate filaments: 15%; and m: unannotated/ function inferred: 4%.

Mentions: Approximately 450 μg of the total protein obtained from total cell lysates of either HepG2 or SK-Hep–1 cells was focused by IEF on pH 3–10 nonlinear IPG strips before being separated on a 12% polyacrylamide gel (PAGE). The spots were visualized by Coomassie blue G250 staining. Among the 60 spots that were identified, 46 were associated with HepG2 cells, and 14 spots belonged to SK-Hep–1 cells. Protein spots of interest were analyzed using MALDI-TOF and the MALDI spectra followed by identification using the MASCOT search engine. The MALDI-TOF identification of the protein spots from the lysates of HCC sublines is presented in Fig 1A and Table 1. We also isolated proteins from various cellular compartments of HepG2 and SK-Hep–1 cells. Cytosolic (500 μg), nuclear (250 μg) and membrane (250 μg) proteins were then subjected to IEF followed by PAGE. Protein spots were identified by MALDI-TOF, as indicated in Fig 1B, 1C and 1D and Tables 2, 3 and 4. Among the abundantly expressed protein spots on the 2-DE map (161 spots), a total of 106 spots (64%) belonged to the well-differentiated HepG2 cells, with the following order of distribution in each cellular compartment: total cell lysate (46; 46%)>cytosol (35; 33%)>membrane (13; 12%)>nucleus (12; 11%) (Fig 2 and Tables 2, 3 and 4). Conversely, a total of 55 spots (36%) belonged to the poorly differentiated SK-Hep–1 cells, with the following order of abundance in each cellular compartment: nucleus (18; 33%)>cytosol (16; 29%)>total cell lysate (14; 25%)>membrane (7; 13%) (Fig 2 and Tables 2, 3 and 4). Notably, the well-differentiated HCC cells overexpressed antioxidant/metabolic enzymes, such as MnSOD, peroxiredoxin (Prdx), NADP-dependent isocitrate dehydrogenase (ICDH), α-enolase and UDP-glucose dehydrogenase. In contrast, poorly differentiated HCC cells exhibited high levels of stress-associated proteins, such as Grp78, HSP75, HSC–70, protein disulfide isomerase (PDI), and HSP27 (Tables 5 and 6).


Proteomic Characterization of Annexin l (ANX1) and Heat Shock Protein 27 (HSP27) as Biomarkers for Invasive Hepatocellular Carcinoma Cells.

Wang RC, Huang CY, Pan TL, Chen WY, Ho CT, Liu TZ, Chang YJ - PLoS ONE (2015)

Protein classification.The functional classifications of the identified proteins.a: Cell cycle: 3%; b: chaperone/stress response: 9%; c: cytoskeleton/ cell mobility: 15%; d: DNA replication/gene regulation/cell proliferation: 12%; e: ion channels: 2%; f: membrane proteins: 4%; g: metabolic enzyme: 16%; h: protection and detoxification: 6%; i: protein synthesis and degradation: 8%; j: signal transduction: 8%; k: transport/binding proteins: 6%; l: intermediate filaments: 15%; and m: unannotated/ function inferred: 4%.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4592234&req=5

pone.0139232.g002: Protein classification.The functional classifications of the identified proteins.a: Cell cycle: 3%; b: chaperone/stress response: 9%; c: cytoskeleton/ cell mobility: 15%; d: DNA replication/gene regulation/cell proliferation: 12%; e: ion channels: 2%; f: membrane proteins: 4%; g: metabolic enzyme: 16%; h: protection and detoxification: 6%; i: protein synthesis and degradation: 8%; j: signal transduction: 8%; k: transport/binding proteins: 6%; l: intermediate filaments: 15%; and m: unannotated/ function inferred: 4%.
Mentions: Approximately 450 μg of the total protein obtained from total cell lysates of either HepG2 or SK-Hep–1 cells was focused by IEF on pH 3–10 nonlinear IPG strips before being separated on a 12% polyacrylamide gel (PAGE). The spots were visualized by Coomassie blue G250 staining. Among the 60 spots that were identified, 46 were associated with HepG2 cells, and 14 spots belonged to SK-Hep–1 cells. Protein spots of interest were analyzed using MALDI-TOF and the MALDI spectra followed by identification using the MASCOT search engine. The MALDI-TOF identification of the protein spots from the lysates of HCC sublines is presented in Fig 1A and Table 1. We also isolated proteins from various cellular compartments of HepG2 and SK-Hep–1 cells. Cytosolic (500 μg), nuclear (250 μg) and membrane (250 μg) proteins were then subjected to IEF followed by PAGE. Protein spots were identified by MALDI-TOF, as indicated in Fig 1B, 1C and 1D and Tables 2, 3 and 4. Among the abundantly expressed protein spots on the 2-DE map (161 spots), a total of 106 spots (64%) belonged to the well-differentiated HepG2 cells, with the following order of distribution in each cellular compartment: total cell lysate (46; 46%)>cytosol (35; 33%)>membrane (13; 12%)>nucleus (12; 11%) (Fig 2 and Tables 2, 3 and 4). Conversely, a total of 55 spots (36%) belonged to the poorly differentiated SK-Hep–1 cells, with the following order of abundance in each cellular compartment: nucleus (18; 33%)>cytosol (16; 29%)>total cell lysate (14; 25%)>membrane (7; 13%) (Fig 2 and Tables 2, 3 and 4). Notably, the well-differentiated HCC cells overexpressed antioxidant/metabolic enzymes, such as MnSOD, peroxiredoxin (Prdx), NADP-dependent isocitrate dehydrogenase (ICDH), α-enolase and UDP-glucose dehydrogenase. In contrast, poorly differentiated HCC cells exhibited high levels of stress-associated proteins, such as Grp78, HSP75, HSC–70, protein disulfide isomerase (PDI), and HSP27 (Tables 5 and 6).

Bottom Line: In total, 106 and 55 proteins were successfully identified from the total cell lysate and the cytosolic, nuclear and membrane fractions in well-differentiated (HepG2) and poorly differentiated (SK-Hep-1) HCC clonal variants, respectively.Notably, Annexin 1 (ANX1), ANX-2, vimentin and stress-associated proteins, such as GRP78, HSP75, HSC-70, protein disulfide isomerase (PDI), and heat shock protein-27 (HSP27), were exclusively up-regulated in SK-Hep-1 cells.Knockdown of ANX1 or HSP27 in HCC cells resulted in a severe reduction in cell migration.

View Article: PubMed Central - PubMed

Affiliation: Tissue Bank, Chang Gung Memorial Hospital, Chiayi, Taiwan.

ABSTRACT
To search for reliable biomarkers and drug targets for management of hepatocellular carcinoma (HCC), we performed a global proteomic analysis of a pair of HCC cell lines with distinct differentiation statuses using 2-DE coupled with MALDI-TOF MS. In total, 106 and 55 proteins were successfully identified from the total cell lysate and the cytosolic, nuclear and membrane fractions in well-differentiated (HepG2) and poorly differentiated (SK-Hep-1) HCC clonal variants, respectively. Among these proteins, nine spots corresponding to proteins differentially expressed between HCC cell types were selected and confirmed by immunofluorescence staining and western blotting. Notably, Annexin 1 (ANX1), ANX-2, vimentin and stress-associated proteins, such as GRP78, HSP75, HSC-70, protein disulfide isomerase (PDI), and heat shock protein-27 (HSP27), were exclusively up-regulated in SK-Hep-1 cells. Elevated levels of ANX-4 and antioxidant/metabolic enzymes, such as MnSOD, peroxiredoxin, NADP-dependent isocitrate dehydrogenase, α-enolase and UDP-glucose dehydrogenase, were observed in HepG2 cells. We functionally demonstrated that ANX1 and HSP27 were abundantly overexpressed only in highly invasive types of HCC cells, such as Mahlavu and SK-Hep-1. Knockdown of ANX1 or HSP27 in HCC cells resulted in a severe reduction in cell migration. The in-vitro observations of ANX1 and HSP27 expressions in HCC sample was demonstrated by immunohistochemical stains performed on HCC tissue microarrays. Poorly differentiated HCC tended to have stronger ANX1 and HSP27 expressions than well-differentiated or moderately differentiated HCC. Collectively, our findings suggest that ANX1 and HSP27 are two novel biomarkers for predicting invasive HCC phenotypes and could serve as potential treatment targets.

No MeSH data available.


Related in: MedlinePlus