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Mitochondrial proteomics of nasopharyngeal carcinoma metastasis.

Liu J, Zhan X, Li M, Li G, Zhang P, Xiao Z, Shao M, Peng F, Hu R, Chen Z - BMC Med Genomics (2012)

Bottom Line: The functional enrichment analyses of DEPs discovered five significant biological processes including cellular response to reactive oxygen species, hydrogen peroxide metabolic process, regulation of mitochondrial membrane potential, cell redox homeostasis and oxidation reduction, and five significant molecular functions including oxidoreductase activity, caspase inhibitor activity, peroxiredoxin activity, porin activity and antioxidant activity.Those 5-8F cells with suppression of PRDX3 showed an increased mobility potential.These data suggest that those mitochondrial DEPs are potential biomarkers for NPC metastasis, and their dysregulation would play important roles in mitochondria oxidative stress-mediated NPC metastatic process.

View Article: PubMed Central - HTML - PubMed

Affiliation: Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, 87 Xiangya Road, Changsha, Hunan, 410008, PR China.

ABSTRACT

Background: Mitochondrial proteomic alterations of nasopharyngeal carcinoma metastasis remain unknown. Our purpose is to screen mitochondrial proteins for the elucidation of the molecular mechanisms of nasopharyngeal carcinoma metastasis and the discovery of metastasis-related biomarkers.

Methods: Mitochondria were isolated from nasopharyngeal carcinoma metastatic (5-8F) and nonmetastatic (6-10B) cell lines, respectively. After characterization of isolated mitochondria, mitochondrial differentially expressed proteins (DEPs) were quantified by two-dimensional difference in-gel electrophoresis (2D-DIGE), and identified by peptide mass fingerprint (PMF) and tandem mass spectrometry (MS/MS). A functional enrichment analysis and a protein-protein interaction sub-network analysis for DEPs were carried out with bioinformatics. Furthermore, siRNAs transient transfections were used to suppress expressions of some up-regulated DEPs in metastatic cells (5-8F), followed by Transwell Migration assay.

Results: Sixteen mitochondrial DEPs including PRDX3 and SOD2 were identified. Those 5-8F cells with suppression of PRDX3 showed an increased mobility potential. The functional enrichment analyses of DEPs discovered five significant biological processes including cellular response to reactive oxygen species, hydrogen peroxide metabolic process, regulation of mitochondrial membrane potential, cell redox homeostasis and oxidation reduction, and five significant molecular functions including oxidoreductase activity, caspase inhibitor activity, peroxiredoxin activity, porin activity and antioxidant activity. A protein-protein interaction sub-network of DEPs was generated with literature data. Ten mitochondrial DEPs including PRDX3, PRDX6, SOD2, ECH1, SERPINB5, COX5A, PDIA5, EIF5A, IDH3B, and PSMC4 were rationalized in the tumor-stroma co-evolution model that mitochondrial oxidative stress directly contributes to tumor metastasis.

Conclusions: Sixteen mitochondrial DEPs were identified with mass spectrometry and ten of them were rationalized in the tumor-stroma co-evolution model. Those 5-8F cells with suppression of PRDX3 showed an increased mobility potential. These data suggest that those mitochondrial DEPs are potential biomarkers for NPC metastasis, and their dysregulation would play important roles in mitochondria oxidative stress-mediated NPC metastatic process.

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Characterization of isolated mitochondria from cells. A. Western blotting images of 6 marker proteins in the whole-cell protein samples (left column) and the isolated mitochondrial protein samples (right column). A total of 60 μg proteins was loaded and isolated by 12% SDS-PAGE, and transferred to PVDF membrane and incubated with primary antibodies (dilution 1:500) of each marker protein and second antibodies (dilution 1:2,000). B. Electron micrograph of isolated mitochondrial samples from cells (20,000 ×).
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Figure 1: Characterization of isolated mitochondria from cells. A. Western blotting images of 6 marker proteins in the whole-cell protein samples (left column) and the isolated mitochondrial protein samples (right column). A total of 60 μg proteins was loaded and isolated by 12% SDS-PAGE, and transferred to PVDF membrane and incubated with primary antibodies (dilution 1:500) of each marker protein and second antibodies (dilution 1:2,000). B. Electron micrograph of isolated mitochondrial samples from cells (20,000 ×).

Mentions: The purity of isolated mitochondria was assessed by Western blotting and electron microscopic observations. Figure1A showed Western blotting images of extracted proteins from isolated mitochondrial samples against known marker proteins from cytoplasm (LDH), mitochondria (COX IV), nucleus (PCNA), endoplasmic reticulum (GRP 78), peroxisome (catalase), and lysosome (Cathepsin D). Mitochondrial protein COX IV was specifically detected in isolated mitochondrial sample and this sample lacked any detectable contamination of abundant cytosolic (LDH), nuclear (PCNA), and endoplasmic reticulum proteins (GRP78), except for detectable contamination of catalase from peroxisome and cathepsin D from lysosome. Figure1B showed electron microscopic observation of isolated mitochondrial samples, where mitochondria had typical membrane architecture. All the results showed that isolated mitochondrial samples were full of intact mitochondria, though little contaminated by other sub-cellular compartments.


Mitochondrial proteomics of nasopharyngeal carcinoma metastasis.

Liu J, Zhan X, Li M, Li G, Zhang P, Xiao Z, Shao M, Peng F, Hu R, Chen Z - BMC Med Genomics (2012)

Characterization of isolated mitochondria from cells. A. Western blotting images of 6 marker proteins in the whole-cell protein samples (left column) and the isolated mitochondrial protein samples (right column). A total of 60 μg proteins was loaded and isolated by 12% SDS-PAGE, and transferred to PVDF membrane and incubated with primary antibodies (dilution 1:500) of each marker protein and second antibodies (dilution 1:2,000). B. Electron micrograph of isolated mitochondrial samples from cells (20,000 ×).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539862&req=5

Figure 1: Characterization of isolated mitochondria from cells. A. Western blotting images of 6 marker proteins in the whole-cell protein samples (left column) and the isolated mitochondrial protein samples (right column). A total of 60 μg proteins was loaded and isolated by 12% SDS-PAGE, and transferred to PVDF membrane and incubated with primary antibodies (dilution 1:500) of each marker protein and second antibodies (dilution 1:2,000). B. Electron micrograph of isolated mitochondrial samples from cells (20,000 ×).
Mentions: The purity of isolated mitochondria was assessed by Western blotting and electron microscopic observations. Figure1A showed Western blotting images of extracted proteins from isolated mitochondrial samples against known marker proteins from cytoplasm (LDH), mitochondria (COX IV), nucleus (PCNA), endoplasmic reticulum (GRP 78), peroxisome (catalase), and lysosome (Cathepsin D). Mitochondrial protein COX IV was specifically detected in isolated mitochondrial sample and this sample lacked any detectable contamination of abundant cytosolic (LDH), nuclear (PCNA), and endoplasmic reticulum proteins (GRP78), except for detectable contamination of catalase from peroxisome and cathepsin D from lysosome. Figure1B showed electron microscopic observation of isolated mitochondrial samples, where mitochondria had typical membrane architecture. All the results showed that isolated mitochondrial samples were full of intact mitochondria, though little contaminated by other sub-cellular compartments.

Bottom Line: The functional enrichment analyses of DEPs discovered five significant biological processes including cellular response to reactive oxygen species, hydrogen peroxide metabolic process, regulation of mitochondrial membrane potential, cell redox homeostasis and oxidation reduction, and five significant molecular functions including oxidoreductase activity, caspase inhibitor activity, peroxiredoxin activity, porin activity and antioxidant activity.Those 5-8F cells with suppression of PRDX3 showed an increased mobility potential.These data suggest that those mitochondrial DEPs are potential biomarkers for NPC metastasis, and their dysregulation would play important roles in mitochondria oxidative stress-mediated NPC metastatic process.

View Article: PubMed Central - HTML - PubMed

Affiliation: Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, 87 Xiangya Road, Changsha, Hunan, 410008, PR China.

ABSTRACT

Background: Mitochondrial proteomic alterations of nasopharyngeal carcinoma metastasis remain unknown. Our purpose is to screen mitochondrial proteins for the elucidation of the molecular mechanisms of nasopharyngeal carcinoma metastasis and the discovery of metastasis-related biomarkers.

Methods: Mitochondria were isolated from nasopharyngeal carcinoma metastatic (5-8F) and nonmetastatic (6-10B) cell lines, respectively. After characterization of isolated mitochondria, mitochondrial differentially expressed proteins (DEPs) were quantified by two-dimensional difference in-gel electrophoresis (2D-DIGE), and identified by peptide mass fingerprint (PMF) and tandem mass spectrometry (MS/MS). A functional enrichment analysis and a protein-protein interaction sub-network analysis for DEPs were carried out with bioinformatics. Furthermore, siRNAs transient transfections were used to suppress expressions of some up-regulated DEPs in metastatic cells (5-8F), followed by Transwell Migration assay.

Results: Sixteen mitochondrial DEPs including PRDX3 and SOD2 were identified. Those 5-8F cells with suppression of PRDX3 showed an increased mobility potential. The functional enrichment analyses of DEPs discovered five significant biological processes including cellular response to reactive oxygen species, hydrogen peroxide metabolic process, regulation of mitochondrial membrane potential, cell redox homeostasis and oxidation reduction, and five significant molecular functions including oxidoreductase activity, caspase inhibitor activity, peroxiredoxin activity, porin activity and antioxidant activity. A protein-protein interaction sub-network of DEPs was generated with literature data. Ten mitochondrial DEPs including PRDX3, PRDX6, SOD2, ECH1, SERPINB5, COX5A, PDIA5, EIF5A, IDH3B, and PSMC4 were rationalized in the tumor-stroma co-evolution model that mitochondrial oxidative stress directly contributes to tumor metastasis.

Conclusions: Sixteen mitochondrial DEPs were identified with mass spectrometry and ten of them were rationalized in the tumor-stroma co-evolution model. Those 5-8F cells with suppression of PRDX3 showed an increased mobility potential. These data suggest that those mitochondrial DEPs are potential biomarkers for NPC metastasis, and their dysregulation would play important roles in mitochondria oxidative stress-mediated NPC metastatic process.

Show MeSH
Related in: MedlinePlus