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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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The experimental flow-chart for two-dimensional difference in-gel electrophoresis (2D-DIGE).
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Figure 10: The experimental flow-chart for two-dimensional difference in-gel electrophoresis (2D-DIGE).

Mentions: The 2D-DIGE experimental flow-chart was shown (Figure10). Briefly, Mitochondrial proteins were labeled with fluorescent cyanine dyes following the Instruction of Amersham CyDye DIGE Fluors (minimal dyes) for Ettan DIGE Product Booklet. An amount (50 μg) of proteins were labeled with 400 pmol of dyes (dark; on ice; 30 min) and then quenched (10 min) with 1 μl of 10 mM lysine. Two sets of metastatic 5-8F cell proteins were labeled with Cy3 and the third set with Cy5. Two sets of nonmetastatic 6-10B cell proteins were labeled with Cy5 and the third set with Cy3. Each set of above sample contained 50 μg proteins. The internal standard (75 μg of metastatic 5-8F cell proteins plus 75 μg of nonmetastatic 6-10B cell proteins) was labeled with Cy2. One set (50 μg) of Cy3-labeled proteins was combined with one set (50 μg) of Cy5-labeled proteins and combined again with one third (50 μg) of Cy2-labeled internal standard proteins. The combined sample was added in equal volume of 2 × sample buffer (8 M urea, 130 mM DTT, 4% CHAPS, and 2% Pharmalyte 3-10), and then supplied with rehydration buffer (8 M urea, 130 mM DTT, 4% CHAPS, and 1% pharmalyte 3-10) to the total volume of 450 μl. The mixed sample (450 μl) was applied to an immobiline DryStrip gel; and three analytic gels and one preparative gel were simultaneously focused on an IPGphor Unit (GH Healthcare) with a total of 80,000 Vhr. The focused proteins in the immobiline DryStrips were equilibrated in a solution containing 65 mM DTT (15 min), and in another solution containing 135 mM iodacetamide (15 min). The equilibrated proteins in the immobiline DryStrip were separated by 12.5% SDS-PAGE gels in an Ettan DALT II system (GE Healthcare). After electrophoresis, each gel was scanned on a Typhoon 9410 scanner (GE Healthcare) at appropriate wavelengths that were specific for Cy2 (488/520 nm), Cy3 (532/580 nm), and Cy5 (633/670 nm). In total 9 protein-spot image maps were generated. Each gel-spot density was normalized by the total density in each protein-spot image map. The DeCyder differential in-gel analysis (DIA) module was used for pair-wise comparison of each 5-8F and 6-10B protein to the internal standard in each gel. The DeCyder biological variation analysis (BVA) module was then used to simultaneously match all 9 protein-spot image maps. The normalized Cy3/normalized Cy2 and normalized Cy5/normalized Cy2 DIA ratios were used to calculate average abundance changes. The average of three DIA ratios from metastatic cell 5-8F mitochondria represents the relative expression level of a protein in metastatic cell mitochondria, and the average of three DIA ratios from nonmetastatic cell 6-10B mitochondria represents the relative expression level of a protein in nonmetastatic cell mitochondria. The paired Student’s t-test p-values were used for the variance of these ratios for each protein pair across all samples (all 9 protein-spot image maps) to determine each DEP-contained gel-spot. Each DEP-contained gel-spot was excised for protein identification.


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)

The experimental flow-chart for two-dimensional difference in-gel electrophoresis (2D-DIGE).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 10: The experimental flow-chart for two-dimensional difference in-gel electrophoresis (2D-DIGE).
Mentions: The 2D-DIGE experimental flow-chart was shown (Figure10). Briefly, Mitochondrial proteins were labeled with fluorescent cyanine dyes following the Instruction of Amersham CyDye DIGE Fluors (minimal dyes) for Ettan DIGE Product Booklet. An amount (50 μg) of proteins were labeled with 400 pmol of dyes (dark; on ice; 30 min) and then quenched (10 min) with 1 μl of 10 mM lysine. Two sets of metastatic 5-8F cell proteins were labeled with Cy3 and the third set with Cy5. Two sets of nonmetastatic 6-10B cell proteins were labeled with Cy5 and the third set with Cy3. Each set of above sample contained 50 μg proteins. The internal standard (75 μg of metastatic 5-8F cell proteins plus 75 μg of nonmetastatic 6-10B cell proteins) was labeled with Cy2. One set (50 μg) of Cy3-labeled proteins was combined with one set (50 μg) of Cy5-labeled proteins and combined again with one third (50 μg) of Cy2-labeled internal standard proteins. The combined sample was added in equal volume of 2 × sample buffer (8 M urea, 130 mM DTT, 4% CHAPS, and 2% Pharmalyte 3-10), and then supplied with rehydration buffer (8 M urea, 130 mM DTT, 4% CHAPS, and 1% pharmalyte 3-10) to the total volume of 450 μl. The mixed sample (450 μl) was applied to an immobiline DryStrip gel; and three analytic gels and one preparative gel were simultaneously focused on an IPGphor Unit (GH Healthcare) with a total of 80,000 Vhr. The focused proteins in the immobiline DryStrips were equilibrated in a solution containing 65 mM DTT (15 min), and in another solution containing 135 mM iodacetamide (15 min). The equilibrated proteins in the immobiline DryStrip were separated by 12.5% SDS-PAGE gels in an Ettan DALT II system (GE Healthcare). After electrophoresis, each gel was scanned on a Typhoon 9410 scanner (GE Healthcare) at appropriate wavelengths that were specific for Cy2 (488/520 nm), Cy3 (532/580 nm), and Cy5 (633/670 nm). In total 9 protein-spot image maps were generated. Each gel-spot density was normalized by the total density in each protein-spot image map. The DeCyder differential in-gel analysis (DIA) module was used for pair-wise comparison of each 5-8F and 6-10B protein to the internal standard in each gel. The DeCyder biological variation analysis (BVA) module was then used to simultaneously match all 9 protein-spot image maps. The normalized Cy3/normalized Cy2 and normalized Cy5/normalized Cy2 DIA ratios were used to calculate average abundance changes. The average of three DIA ratios from metastatic cell 5-8F mitochondria represents the relative expression level of a protein in metastatic cell mitochondria, and the average of three DIA ratios from nonmetastatic cell 6-10B mitochondria represents the relative expression level of a protein in nonmetastatic cell mitochondria. The paired Student’s t-test p-values were used for the variance of these ratios for each protein pair across all samples (all 9 protein-spot image maps) to determine each DEP-contained gel-spot. Each DEP-contained gel-spot was excised for protein identification.

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