Effect of Pectin on the Expression of Proteins Associated with Mitochondrial Biogenesis and Cell Senescence in HT29-Human Colorectal Adenocarcinoma Cells

Abstract

Mitochondria dynamic is regulated by different proteins, maintaining a balance between fission and fusion. An imbalance towards mitochondrial fission has been associated with tumor cell proliferation. The aim of this study was to analyze whether pectin modifies the viability of human colon cancer cells and the expression of proteins involved in mitochondrial fusion and fission. The human colon carcinoma cell line HT29 cells was growth in 10% fetal bovine serum in the absence and presence of pectin. Pectin reduced HT29 cell viability in a concentration-dependent manner, reaching a plateau at 150~300 μmol/L pectin. The presence of 200 μmol/L pectin reduced the expression of dynamin-related protein-1 and increased expression of the mitochondrial fusion-associated proteins mitofusin-1 and 2. Expression of cyclin B1, a protein involved in G2/M transition, was found decreased in pectin-incubated HT29 cells. Moreover, expression of p53 protein, the amount of p53 in the nucleous and β-galactosidase activity, which are all biomarkers for cellular senescence, were significantly higher in pectin-incubated HT29 cells than in HT29 cells incubated without pectin. Expression of the protein B-cell lymphoma 2 (Bcl-2) homologous antagonist/killer was increased in response to incubation with pectin. However, incubation with pectin did not affect expression of Bcl-2-associated X protein or Bcl-2, or the caspase-3 activity. Overall, we concluded that pectin reduces the viability of human HT29 colon cancer cells, which is accompanied with a shift in the expression of proteins associated with mitochondrial dynamics towards mitochondrial fusion. Moreover, incubation with pectin favors cellular senescence over apoptosis in HT29 cells.

Keywords: cell proliferation; cellular senescence; colon cancer; mitochondria biogenesis; pectin.

Fig. 1

(A) The viability index of HT29 cells incubated in culture medium containing 0.5% or 10% fetal bovine serum (FBS). Results are represented as mean±SEM of three experimental repeats. *P <0.05 with respect to 0.5% FBS. (B) Effects of increased pectin concentrations on HT29 cell viability. Results are expressed as mean±SEM of six experimental repeats. *P <0.05 with respect to HT29 incubated without pectin.

Fig. 2

Representative Western blots showing the expression of proteins associated with mitochondrial fission, dynamin-related protein 1 (Drp-1), and mitochondrial fusion, mitofusin (Mfn)-1 and Mfn-2 in HT29 cells incubated with and without pectin (200 μmol/L). The expression of β-actin was used as loading control. Bar graphs show the densitometric analysis of all the Western blots in arbitrary units (AU). Results are presented as mean±SEM of six experimental repeats. *P <0.05 with respect to HT29 cells incubated without pectin.

Fig. 3

Activity of mitochondrial citocrome C oxidase in HT29 cells incubated with and without pectin (200 μmol/L). Results are presented as mean±SEM of six experimental repeats. *P < 0.05 between curves.

Fig. 4

Representative Western blots of the expression of proteins associated with cellular senescence, p53 and cyclin B1, in HT29 cells incubated with and without pectin (200 μmol/L). The expression of β-actin was used as a loading control. Representative dot blots showing the content of nuclear p53 in HT29 cells incubated with and without pectin (200 μmol/L). Bar graphs show the densitometric analysis of all Western blots and dot blots in arbitrary units (AU). Results are presented as mean±SEM of six experimental repeats. *P <0.05 with respect to HT29 cells incubated without pectin.

Fig. 5

Representative Western blots to determine the expression of proteins associated with cellular apoptosis, B-cell lymphoma 2 (Bcl-2)-antagonist/killer (Bak), Bcl-2-associated X protein, and Bcl-2, in HT29 cells incubated with and without pectin (200 μmol/L). The expression of β-actin was used as loading control. Bar graphs show the densitometric analysis of all the Western blots in arbitrary units (AU). Results are presented as mean±SEM of six experimental repeats. *P <0.05 with respect to HT29 cells incubated without pectin.

Fig. 6

Caspase-3 activity was determined in HT29 cells incubated with and without pectin (200 μmol/L). Results are presented as mean±SEM of six experimental repeats. NS: not significant.