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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 showed that the TFAM knockdown TE1-sh-TFAM(97) had the lowest TFAM (the first row), equal SDHA (the second row), highest LDH (the third row), highest E-cadherin (the fourth row) and lowest vimentin (the fifth row) protein expressions. The expression of beta-actin (the sixth row) was used as an internal control; (B) Illustrations are photographic (light microscopy) results of the trans-well migration activity among the parental TE1 cell, control TE1-NT cell, and TFAM knockdown TE1-sh-TFAM (97) cells. TE1-sh-TFAM(97) exhibited the lowest trans-well migration activity.
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f5-ijms-13-11228: (A) Western blot analysis showed that the TFAM knockdown TE1-sh-TFAM(97) had the lowest TFAM (the first row), equal SDHA (the second row), highest LDH (the third row), highest E-cadherin (the fourth row) and lowest vimentin (the fifth row) protein expressions. The expression of beta-actin (the sixth row) was used as an internal control; (B) Illustrations are photographic (light microscopy) results of the trans-well migration activity among the parental TE1 cell, control TE1-NT cell, and TFAM knockdown TE1-sh-TFAM (97) cells. TE1-sh-TFAM(97) exhibited the lowest trans-well migration activity.

Mentions: As shown in Table 2, the relative mtDNA copy number of TE1-sh-TFAM(97) was significantly lower than that of control TE1-NT (152.5% vs. 227.8%, p = 0.001) after the knockdown of TFAM. TE1-sh-TFAM(97) also exhibited a lower relative mRNA level of ND1(p = 0.050), a lower succinate-supported oxygen consumption rate (p = 0.065) and a lower intracellular level of ATP (p = 0.007) than those of the control TE1-NT. However, there were no significant differences between the parental TE1 and control TE1-NT among the above parameters. The mitochondrial bioenergetic function was significantly down-regulated in TE1-sh-TFAM(97). Because only 13 polypeptides of the respiratory enzyme complexes are encoded by mtDNA, it is considered that the knockdown of TFAM would not interfere with the expression of nDNA-encoded polypeptides of respiratory enzyme complexes. As illustrated in Figure 5A, there was no obvious difference in the SDHA expression among the parental TE1, control TE1-NT and the TFAM knockdown TE1-sh-TFAM(97) cells. This finding confirmed that the impaired mitochondrial bioenergetic function of TE1-sh-TFAM(97) cells was the consequence of the decrease in the mtDNA copy number and mtDNA-encoded polypeptides after the knockdown of TFAM.


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 showed that the TFAM knockdown TE1-sh-TFAM(97) had the lowest TFAM (the first row), equal SDHA (the second row), highest LDH (the third row), highest E-cadherin (the fourth row) and lowest vimentin (the fifth row) protein expressions. The expression of beta-actin (the sixth row) was used as an internal control; (B) Illustrations are photographic (light microscopy) results of the trans-well migration activity among the parental TE1 cell, control TE1-NT cell, and TFAM knockdown TE1-sh-TFAM (97) cells. TE1-sh-TFAM(97) exhibited the lowest trans-well migration activity.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5-ijms-13-11228: (A) Western blot analysis showed that the TFAM knockdown TE1-sh-TFAM(97) had the lowest TFAM (the first row), equal SDHA (the second row), highest LDH (the third row), highest E-cadherin (the fourth row) and lowest vimentin (the fifth row) protein expressions. The expression of beta-actin (the sixth row) was used as an internal control; (B) Illustrations are photographic (light microscopy) results of the trans-well migration activity among the parental TE1 cell, control TE1-NT cell, and TFAM knockdown TE1-sh-TFAM (97) cells. TE1-sh-TFAM(97) exhibited the lowest trans-well migration activity.
Mentions: As shown in Table 2, the relative mtDNA copy number of TE1-sh-TFAM(97) was significantly lower than that of control TE1-NT (152.5% vs. 227.8%, p = 0.001) after the knockdown of TFAM. TE1-sh-TFAM(97) also exhibited a lower relative mRNA level of ND1(p = 0.050), a lower succinate-supported oxygen consumption rate (p = 0.065) and a lower intracellular level of ATP (p = 0.007) than those of the control TE1-NT. However, there were no significant differences between the parental TE1 and control TE1-NT among the above parameters. The mitochondrial bioenergetic function was significantly down-regulated in TE1-sh-TFAM(97). Because only 13 polypeptides of the respiratory enzyme complexes are encoded by mtDNA, it is considered that the knockdown of TFAM would not interfere with the expression of nDNA-encoded polypeptides of respiratory enzyme complexes. As illustrated in Figure 5A, there was no obvious difference in the SDHA expression among the parental TE1, control TE1-NT and the TFAM knockdown TE1-sh-TFAM(97) cells. This finding confirmed that the impaired mitochondrial bioenergetic function of TE1-sh-TFAM(97) cells was the consequence of the decrease in the mtDNA copy number and mtDNA-encoded polypeptides after the knockdown of TFAM.

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