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High mitochondrial DNA copy number and bioenergetic function are associated with tumor invasion of esophageal squamous cell carcinoma cell lines.

Lin CS, Lee HT, Lee SY, Shen YA, Wang LS, Chen YJ, Wei YH - Int J Mol Sci (2012)

Bottom Line: Among them, TE1 had the highest relative mtDNA copy number of 240.7%.These findings indicate that TE1 exhibited the highest bioenergetic function of mitochondria.A decline in mitochondrial bioenergetic function was observed in TE1-sh-TFAM(97).

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Medicine, National Yang-Ming University, Taipei 112, Taiwan; E-Mails: doc2765c@ms59.hinet.net (C.-S.L.); htlee1228@gmail.com (H.-T.L.); yjchen@ym.edu.tw (Y.-J.C.) ; Faculty of Medicine, National Yang-Ming University, Taipei 112, Taiwan ; Division of Thoracic Surgery, Department of Surgery, Taipei Hospital, Department of Health, Executive Yuan, New Taipei City 242, Taiwan.

ABSTRACT
We previously reported a gradual increase of relative mitochondrial DNA (mtDNA) copy number during the progression of esophageal squamous cell carcinoma (ESCC). Because mitochondria are the intracellular organelles responsible for ATP production, we investigated the associations among mtDNA copy number, mitochondrial bioenergetic function, tumor invasion and the expression levels of epithelial mesenchymal transition (EMT) markers in a series of seven ESCC cell lines, including 48T, 81T, 146T, TE1, TE2, TE6 and TE9. Among them, TE1 had the highest relative mtDNA copy number of 240.7%. The mRNA of mtDNA-encoded ND1 gene (2.80), succinate-supported oxygen consumption rate (11.21 nmol/min/10(6) cells), ATP content (10.7 fmol/cell), and the protein level of mitochondrial transcription factor A (TFAM) were the highest and the lactate concentration in the culture medium (3.34 mM) was the lowest in TE1. These findings indicate that TE1 exhibited the highest bioenergetic function of mitochondria. Furthermore, TE1 showed the highest trans-well migration activity of 223.0 cells/field, the highest vimentin but the lowest E-cadherin protein expression levels, which suggest that TE1 had the highest invasion capability. We then conducted a knockdown study using pLKO.1-based lentiviral particles to infect TE1 cells to suppress the expression of TFAM. Molecular analyses of the parental TE1, control TE1-NT and TFAM knockdown TE1-sh-TFAM(97) cells were performed. Interestingly, as compared to the control TE1-NT, TE1-sh-TFAM(97) exhibited lower levels of the relative mtDNA copy number (p = 0.001), mRNA of mtDNA-encoded ND1 gene (p = 0.050), succinate-supported oxygen consumption rate (p = 0.065), and ATP content (p = 0.007), but had a higher lactate concentration in the culture medium (p = 0.010) and higher protein level of lactate dehydrogenase. A decline in mitochondrial bioenergetic function was observed in TE1-sh-TFAM(97). Significantly, compared to the control TE1-NT, TE1-sh-TFAM(97) had a lower trans-well migration activity (p < 0.001), a higher E-cadherin level but a lower vimentin protein level, which indicates a decrease of invasiveness. Taken together, we suggest that high relative mtDNA copy number and bioenergetic function of mitochondria may confer an advantage for tumor invasion of ESCC.

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Related in: MedlinePlus

(A) Western blot analysis of the TFAM expression among the parental TE1, control TE1-NT, and TFAM knockdown TE1-sh-TFAM(96) and TE1-sh-TFA(97) cells; (B) The growth kinetic curves of the parental TE1, control TE1-NT, and TFAM knockdown TE1-sh-TFAM(96) and TE1-sh-TFA(97) cells, respectively, were plotted by SPSS 12.0 software (SPSS Inc, Chicago, Ill). Data are shown in percentage when the cell number of each cell line on day 1 was defined as 100.0%.
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f4-ijms-13-11228: (A) Western blot analysis of the TFAM expression among the parental TE1, control TE1-NT, and TFAM knockdown TE1-sh-TFAM(96) and TE1-sh-TFA(97) cells; (B) The growth kinetic curves of the parental TE1, control TE1-NT, and TFAM knockdown TE1-sh-TFAM(96) and TE1-sh-TFA(97) cells, respectively, were plotted by SPSS 12.0 software (SPSS Inc, Chicago, Ill). Data are shown in percentage when the cell number of each cell line on day 1 was defined as 100.0%.

Mentions: As shown in Figure 4A, the TFMA expression levels of parental TE1, control TE1-NT, and TFAM knockdown TE1-sh-TFAM(96) and TE1-sh-TFAM(97) were examined. There was no obvious difference in the TFAM expression between the parental TE1 and the control TE1-NT cells. However, a significant knock-down efficiency was achieved in TE1-sh-TFAM(96) and TE1-sh-TFAM(97) cells, with a higher knockdown efficiency in TE1-sh-TFAM(96) cells. As shown in Figure 4B, there was an obvious growth arrest in TE1-sh-TFAM(96) cells, and no significant difference was noted between the parental TE1 and the control TE1-NT cells. Thus, we only focused on parental TE1, control TE1-NT and TE1-sh-TFAM(97) cells in the following experiments.


High mitochondrial DNA copy number and bioenergetic function are associated with tumor invasion of esophageal squamous cell carcinoma cell lines.

Lin CS, Lee HT, Lee SY, Shen YA, Wang LS, Chen YJ, Wei YH - Int J Mol Sci (2012)

(A) Western blot analysis of the TFAM expression among the parental TE1, control TE1-NT, and TFAM knockdown TE1-sh-TFAM(96) and TE1-sh-TFA(97) cells; (B) The growth kinetic curves of the parental TE1, control TE1-NT, and TFAM knockdown TE1-sh-TFAM(96) and TE1-sh-TFA(97) cells, respectively, were plotted by SPSS 12.0 software (SPSS Inc, Chicago, Ill). Data are shown in percentage when the cell number of each cell line on day 1 was defined as 100.0%.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3472741&req=5

f4-ijms-13-11228: (A) Western blot analysis of the TFAM expression among the parental TE1, control TE1-NT, and TFAM knockdown TE1-sh-TFAM(96) and TE1-sh-TFA(97) cells; (B) The growth kinetic curves of the parental TE1, control TE1-NT, and TFAM knockdown TE1-sh-TFAM(96) and TE1-sh-TFA(97) cells, respectively, were plotted by SPSS 12.0 software (SPSS Inc, Chicago, Ill). Data are shown in percentage when the cell number of each cell line on day 1 was defined as 100.0%.
Mentions: As shown in Figure 4A, the TFMA expression levels of parental TE1, control TE1-NT, and TFAM knockdown TE1-sh-TFAM(96) and TE1-sh-TFAM(97) were examined. There was no obvious difference in the TFAM expression between the parental TE1 and the control TE1-NT cells. However, a significant knock-down efficiency was achieved in TE1-sh-TFAM(96) and TE1-sh-TFAM(97) cells, with a higher knockdown efficiency in TE1-sh-TFAM(96) cells. As shown in Figure 4B, there was an obvious growth arrest in TE1-sh-TFAM(96) cells, and no significant difference was noted between the parental TE1 and the control TE1-NT cells. Thus, we only focused on parental TE1, control TE1-NT and TE1-sh-TFAM(97) cells in the following experiments.

Bottom Line: Among them, TE1 had the highest relative mtDNA copy number of 240.7%.These findings indicate that TE1 exhibited the highest bioenergetic function of mitochondria.A decline in mitochondrial bioenergetic function was observed in TE1-sh-TFAM(97).

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Medicine, National Yang-Ming University, Taipei 112, Taiwan; E-Mails: doc2765c@ms59.hinet.net (C.-S.L.); htlee1228@gmail.com (H.-T.L.); yjchen@ym.edu.tw (Y.-J.C.) ; Faculty of Medicine, National Yang-Ming University, Taipei 112, Taiwan ; Division of Thoracic Surgery, Department of Surgery, Taipei Hospital, Department of Health, Executive Yuan, New Taipei City 242, Taiwan.

ABSTRACT
We previously reported a gradual increase of relative mitochondrial DNA (mtDNA) copy number during the progression of esophageal squamous cell carcinoma (ESCC). Because mitochondria are the intracellular organelles responsible for ATP production, we investigated the associations among mtDNA copy number, mitochondrial bioenergetic function, tumor invasion and the expression levels of epithelial mesenchymal transition (EMT) markers in a series of seven ESCC cell lines, including 48T, 81T, 146T, TE1, TE2, TE6 and TE9. Among them, TE1 had the highest relative mtDNA copy number of 240.7%. The mRNA of mtDNA-encoded ND1 gene (2.80), succinate-supported oxygen consumption rate (11.21 nmol/min/10(6) cells), ATP content (10.7 fmol/cell), and the protein level of mitochondrial transcription factor A (TFAM) were the highest and the lactate concentration in the culture medium (3.34 mM) was the lowest in TE1. These findings indicate that TE1 exhibited the highest bioenergetic function of mitochondria. Furthermore, TE1 showed the highest trans-well migration activity of 223.0 cells/field, the highest vimentin but the lowest E-cadherin protein expression levels, which suggest that TE1 had the highest invasion capability. We then conducted a knockdown study using pLKO.1-based lentiviral particles to infect TE1 cells to suppress the expression of TFAM. Molecular analyses of the parental TE1, control TE1-NT and TFAM knockdown TE1-sh-TFAM(97) cells were performed. Interestingly, as compared to the control TE1-NT, TE1-sh-TFAM(97) exhibited lower levels of the relative mtDNA copy number (p = 0.001), mRNA of mtDNA-encoded ND1 gene (p = 0.050), succinate-supported oxygen consumption rate (p = 0.065), and ATP content (p = 0.007), but had a higher lactate concentration in the culture medium (p = 0.010) and higher protein level of lactate dehydrogenase. A decline in mitochondrial bioenergetic function was observed in TE1-sh-TFAM(97). Significantly, compared to the control TE1-NT, TE1-sh-TFAM(97) had a lower trans-well migration activity (p < 0.001), a higher E-cadherin level but a lower vimentin protein level, which indicates a decrease of invasiveness. Taken together, we suggest that high relative mtDNA copy number and bioenergetic function of mitochondria may confer an advantage for tumor invasion of ESCC.

Show MeSH
Related in: MedlinePlus