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Repression of PKR mediates palmitate-induced apoptosis in HepG2 cells through regulation of Bcl-2.

Yang X, Chan C - Cell Res. (2009)

Bottom Line: Palmitate down-regulates the activity of PKR and thereby decreases the level of Bcl-2 protein, mediated in part by reduced activation of the NF-kappaB transcription factor.The decrease in the phosphorylation of Bcl-2 at Ser70 upon exposure to palmitate is mediated by inhibition of PKR and possibly by c-Jun N-terminal kinase (JNK), whereas the phosphorylation of Bcl-2 at Ser87 is unaffected by palmitate or PKR.In summary, PKR mediates the regulation of the protein level and the phosphorylation status of Bcl-2, providing a novel mechanism of palmitate-induced apoptosis in HepG2 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, MI 48824, USA.

ABSTRACT
The present study shows that double-stranded RNA-dependent protein kinase (PKR) regulates the protein expression level and phosphorylation of Bcl-2 and plays an anti-apoptotic role in human hepatocellular carcinoma cells (HepG2). In various types of cells, saturated free fatty acids (FFAs), such as palmitate, have been shown to induce cellular apoptosis by several mechanisms. Palmitate down-regulates the activity of PKR and thereby decreases the level of Bcl-2 protein, mediated in part by reduced activation of the NF-kappaB transcription factor. In addition to the level of Bcl-2 protein, the phosphorylation of Bcl-2 at different amino acid residues, such as Ser70 and Ser87, is also important in regulating cellular apoptosis. The decrease in the phosphorylation of Bcl-2 at Ser70 upon exposure to palmitate is mediated by inhibition of PKR and possibly by c-Jun N-terminal kinase (JNK), whereas the phosphorylation of Bcl-2 at Ser87 is unaffected by palmitate or PKR. In summary, PKR mediates the regulation of the protein level and the phosphorylation status of Bcl-2, providing a novel mechanism of palmitate-induced apoptosis in HepG2 cells.

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Related in: MedlinePlus

Effects of palmitate on cytotoxicity and apoptosis of HepG2 cellsHepG2 cells were cultured in regular media until reaching approximately 90% confluency, and then exposed to different levels of palmitate or 0.7 mM oleate for 24 hours. The vehicle for the FFAs (0.7 mM BSA) was used as the control (i.e., regular media with BSA) (A, B). LDH release (A) and caspase-3 activity (B) were measured after treating with palmitate. Data expressed as averages of nine samples ± SD from three independent experiments. One-way ANOVA with Tukey’s post hoc method was used for analyzing the differences between treatment groups. ★, significantly higher than control, i.e., regular media with BSA, p<0.01.
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Figure 1: Effects of palmitate on cytotoxicity and apoptosis of HepG2 cellsHepG2 cells were cultured in regular media until reaching approximately 90% confluency, and then exposed to different levels of palmitate or 0.7 mM oleate for 24 hours. The vehicle for the FFAs (0.7 mM BSA) was used as the control (i.e., regular media with BSA) (A, B). LDH release (A) and caspase-3 activity (B) were measured after treating with palmitate. Data expressed as averages of nine samples ± SD from three independent experiments. One-way ANOVA with Tukey’s post hoc method was used for analyzing the differences between treatment groups. ★, significantly higher than control, i.e., regular media with BSA, p<0.01.

Mentions: Previous work in our lab showed that palmitate induced cytotoxicity in HepG2 cells (30, 58), while unsaturated FFAs, e.g. oleate and linoleate, were not cytotoxic (30, 58). In a separate study, we found upon exposure to palmitate the HepG2 cells stained for Annexin-V (data not shown), which indicates phosphatidylserine externalization, a sign of early stage apoptosis. In the present study, we further found that palmitate increased LDH released and caspase-3 activity of HepG2 cells in a dose dependent manner, while oleate did not have a significant effect on LDH release and caspase-3 activity (Fig. 1 A and B), supporting that palmitate induces cytotoxicity and apoptosis in HepG2 cells. It has been confirmed also in the literature, that palmitate induces apoptosis of HepG2 cells (31, 32).


Repression of PKR mediates palmitate-induced apoptosis in HepG2 cells through regulation of Bcl-2.

Yang X, Chan C - Cell Res. (2009)

Effects of palmitate on cytotoxicity and apoptosis of HepG2 cellsHepG2 cells were cultured in regular media until reaching approximately 90% confluency, and then exposed to different levels of palmitate or 0.7 mM oleate for 24 hours. The vehicle for the FFAs (0.7 mM BSA) was used as the control (i.e., regular media with BSA) (A, B). LDH release (A) and caspase-3 activity (B) were measured after treating with palmitate. Data expressed as averages of nine samples ± SD from three independent experiments. One-way ANOVA with Tukey’s post hoc method was used for analyzing the differences between treatment groups. ★, significantly higher than control, i.e., regular media with BSA, p<0.01.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2664847&req=5

Figure 1: Effects of palmitate on cytotoxicity and apoptosis of HepG2 cellsHepG2 cells were cultured in regular media until reaching approximately 90% confluency, and then exposed to different levels of palmitate or 0.7 mM oleate for 24 hours. The vehicle for the FFAs (0.7 mM BSA) was used as the control (i.e., regular media with BSA) (A, B). LDH release (A) and caspase-3 activity (B) were measured after treating with palmitate. Data expressed as averages of nine samples ± SD from three independent experiments. One-way ANOVA with Tukey’s post hoc method was used for analyzing the differences between treatment groups. ★, significantly higher than control, i.e., regular media with BSA, p<0.01.
Mentions: Previous work in our lab showed that palmitate induced cytotoxicity in HepG2 cells (30, 58), while unsaturated FFAs, e.g. oleate and linoleate, were not cytotoxic (30, 58). In a separate study, we found upon exposure to palmitate the HepG2 cells stained for Annexin-V (data not shown), which indicates phosphatidylserine externalization, a sign of early stage apoptosis. In the present study, we further found that palmitate increased LDH released and caspase-3 activity of HepG2 cells in a dose dependent manner, while oleate did not have a significant effect on LDH release and caspase-3 activity (Fig. 1 A and B), supporting that palmitate induces cytotoxicity and apoptosis in HepG2 cells. It has been confirmed also in the literature, that palmitate induces apoptosis of HepG2 cells (31, 32).

Bottom Line: Palmitate down-regulates the activity of PKR and thereby decreases the level of Bcl-2 protein, mediated in part by reduced activation of the NF-kappaB transcription factor.The decrease in the phosphorylation of Bcl-2 at Ser70 upon exposure to palmitate is mediated by inhibition of PKR and possibly by c-Jun N-terminal kinase (JNK), whereas the phosphorylation of Bcl-2 at Ser87 is unaffected by palmitate or PKR.In summary, PKR mediates the regulation of the protein level and the phosphorylation status of Bcl-2, providing a novel mechanism of palmitate-induced apoptosis in HepG2 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, MI 48824, USA.

ABSTRACT
The present study shows that double-stranded RNA-dependent protein kinase (PKR) regulates the protein expression level and phosphorylation of Bcl-2 and plays an anti-apoptotic role in human hepatocellular carcinoma cells (HepG2). In various types of cells, saturated free fatty acids (FFAs), such as palmitate, have been shown to induce cellular apoptosis by several mechanisms. Palmitate down-regulates the activity of PKR and thereby decreases the level of Bcl-2 protein, mediated in part by reduced activation of the NF-kappaB transcription factor. In addition to the level of Bcl-2 protein, the phosphorylation of Bcl-2 at different amino acid residues, such as Ser70 and Ser87, is also important in regulating cellular apoptosis. The decrease in the phosphorylation of Bcl-2 at Ser70 upon exposure to palmitate is mediated by inhibition of PKR and possibly by c-Jun N-terminal kinase (JNK), whereas the phosphorylation of Bcl-2 at Ser87 is unaffected by palmitate or PKR. In summary, PKR mediates the regulation of the protein level and the phosphorylation status of Bcl-2, providing a novel mechanism of palmitate-induced apoptosis in HepG2 cells.

Show MeSH
Related in: MedlinePlus