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. 2012;13(9):11228-11246.
doi: 10.3390/ijms130911228. Epub 2012 Sep 10.

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

Chen-Sung Lin  1   2   3 , Hui-Ting Lee  1   2   4 , Shu-Yu Lee  3   5 , Yao-An Shen  5   6 , Liang-Shun Wang  7 , Yann-Jang Chen  1   6 , Yau-Huei Wei  1   2   5   8
Affiliations

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

Chen-Sung Lin et al. Int J Mol Sci. 2012.

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.

Keywords: bioenergetic function; epithelial mesenchymal transition (EMT); esophageal squamous cell carcinoma (ESCC); invasion; mitochondrial DNA (mtDNA).

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Figures

Figure 1
Figure 1
Western blot analysis shows that TE1 had the highest mitochondrial transcription factor A (TFAM) (the first row); relative higher succinate dehydrogenase A (SDHA) (the second row); medium lactate dehydrogenase (LDH) (the third row); lowest E-cadherin (the fourth row) and highest vimentin (the fifth row) protein expression. The expression of beta-actin (the sixth row) was used as an internal control.
Figure 2
Figure 2
Illustration of the quantitative (bar, cells/field) and photographic (light microscopy) results of the assay of trans-well migration activity among the seven cell lines. TE1 exhibited the highest trans-well migration activity.
Figure 3
Figure 3
Growth kinetics of the seven ESCC cell lines. Data are shown in percentage when the cell number on day 1 is defined as 100.0%. The graph was plotted by using the SPSS 12.0 software (SPSS Inc, Chicago, IL, USA).
Figure 4
Figure 4
(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%.
Figure 4
Figure 4
(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%.
Figure 5
Figure 5
(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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