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Apoptosis induction associated with the ER stress response through up-regulation of JNK in HeLa cells by gambogic acid.

Krajarng A, Imoto M, Tashiro E, Fujimaki T, Shinjo S, Watanapokasin R - BMC Complement Altern Med (2015)

Bottom Line: Our results indicated a time- and dose-dependent decrease of cell viability by GA.Apoptosis cell death detected by increased DR5, caspase-8, -9, and -3 expression as well as increased cleaved-PARP, while decreased Bcl-2 upon GA treatment.These results suggest that GA induce apoptosis associated with the ER stress response through up-regulation of p-JNK and down-regulation of p-ERK in HeLa cells.

View Article: PubMed Central - PubMed

Affiliation: Chulabhorn International College of Medicine, Thammasart University, Pratumthani, Thailand. krajarng@yahoo.com.

ABSTRACT

Background: Gambogic acid (GA) was extracted from the dried yellow resin of gamboge (Garcinia hanburyi) which is traditionally used as a coloring material for painting and cloth dying. Gamboge has been also used as a folk medicine for an internal purgative and externally infected wound. We focused on the mechanisms of apoptosis induction by GA through the unfold protein response (ER stress) in HeLa cells.

Methods: The cytotoxic effect of GA against HeLa cells was determined by trypan blue exclusion assay. Markers of ER stress such as XBP-1, GRP78, CHOP, GADD34 and ERdj4 were analyzed by RT-PCR and Real-time RT-PCR. Cell morphological changes and apoptotic proteins were performed by Hoechst33342 staining and Western blotting technique.

Results: Our results indicated a time- and dose-dependent decrease of cell viability by GA. The ER stress induction is determined by the up-regulation of spliced XBP1 mRNA and activated GRP78, CHOP, GADD34 and ERdj4 expression. GA also induced cell morphological changes such as nuclear condensation, membrane blebbing and apoptotic body in Hela cells. Apoptosis cell death detected by increased DR5, caspase-8, -9, and -3 expression as well as increased cleaved-PARP, while decreased Bcl-2 upon GA treatment. In addition, phosphorylated JNK was up-regulated but phosphorylated ERK was down-regulated after exposure to GA.

Conclusions: These results suggest that GA induce apoptosis associated with the ER stress response through up-regulation of p-JNK and down-regulation of p-ERK in HeLa cells.

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Related in: MedlinePlus

Effect of GA on MAPK pathway in HeLa cells. HeLa cells were treated with 0.3 μg/ml of GA for the indicated time points. The expression of phosphorylated and total ERK1/2, JNK and p38 were analyzed by Western blotting and were expressed relative to the protein level at 0 h after normalization to β-actin signals.
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Fig6: Effect of GA on MAPK pathway in HeLa cells. HeLa cells were treated with 0.3 μg/ml of GA for the indicated time points. The expression of phosphorylated and total ERK1/2, JNK and p38 were analyzed by Western blotting and were expressed relative to the protein level at 0 h after normalization to β-actin signals.

Mentions: MAPKs are important signaling members of serine/threonine protein kinase family that control cellular proliferation, differentiation, survival and apoptosis. Three major mammalian MAPK subfamilies, extracellular regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) and p38, were activated through a specific phosphorylation cascade. In HeLa cells, GA decreased the level of phosphorylated ERK1/2 but not the total form (Figure 6). On the other hand, GA increased the level of phosphorylated JNK but decreased the level of total JNK protein after treatment with the compounds. In addition, GA did not affect p38 activity. The results suggested that GA may induce ER stress and apoptosis through the ERK and JNK MAPK pathways.Figure 6


Apoptosis induction associated with the ER stress response through up-regulation of JNK in HeLa cells by gambogic acid.

Krajarng A, Imoto M, Tashiro E, Fujimaki T, Shinjo S, Watanapokasin R - BMC Complement Altern Med (2015)

Effect of GA on MAPK pathway in HeLa cells. HeLa cells were treated with 0.3 μg/ml of GA for the indicated time points. The expression of phosphorylated and total ERK1/2, JNK and p38 were analyzed by Western blotting and were expressed relative to the protein level at 0 h after normalization to β-actin signals.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4340837&req=5

Fig6: Effect of GA on MAPK pathway in HeLa cells. HeLa cells were treated with 0.3 μg/ml of GA for the indicated time points. The expression of phosphorylated and total ERK1/2, JNK and p38 were analyzed by Western blotting and were expressed relative to the protein level at 0 h after normalization to β-actin signals.
Mentions: MAPKs are important signaling members of serine/threonine protein kinase family that control cellular proliferation, differentiation, survival and apoptosis. Three major mammalian MAPK subfamilies, extracellular regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) and p38, were activated through a specific phosphorylation cascade. In HeLa cells, GA decreased the level of phosphorylated ERK1/2 but not the total form (Figure 6). On the other hand, GA increased the level of phosphorylated JNK but decreased the level of total JNK protein after treatment with the compounds. In addition, GA did not affect p38 activity. The results suggested that GA may induce ER stress and apoptosis through the ERK and JNK MAPK pathways.Figure 6

Bottom Line: Our results indicated a time- and dose-dependent decrease of cell viability by GA.Apoptosis cell death detected by increased DR5, caspase-8, -9, and -3 expression as well as increased cleaved-PARP, while decreased Bcl-2 upon GA treatment.These results suggest that GA induce apoptosis associated with the ER stress response through up-regulation of p-JNK and down-regulation of p-ERK in HeLa cells.

View Article: PubMed Central - PubMed

Affiliation: Chulabhorn International College of Medicine, Thammasart University, Pratumthani, Thailand. krajarng@yahoo.com.

ABSTRACT

Background: Gambogic acid (GA) was extracted from the dried yellow resin of gamboge (Garcinia hanburyi) which is traditionally used as a coloring material for painting and cloth dying. Gamboge has been also used as a folk medicine for an internal purgative and externally infected wound. We focused on the mechanisms of apoptosis induction by GA through the unfold protein response (ER stress) in HeLa cells.

Methods: The cytotoxic effect of GA against HeLa cells was determined by trypan blue exclusion assay. Markers of ER stress such as XBP-1, GRP78, CHOP, GADD34 and ERdj4 were analyzed by RT-PCR and Real-time RT-PCR. Cell morphological changes and apoptotic proteins were performed by Hoechst33342 staining and Western blotting technique.

Results: Our results indicated a time- and dose-dependent decrease of cell viability by GA. The ER stress induction is determined by the up-regulation of spliced XBP1 mRNA and activated GRP78, CHOP, GADD34 and ERdj4 expression. GA also induced cell morphological changes such as nuclear condensation, membrane blebbing and apoptotic body in Hela cells. Apoptosis cell death detected by increased DR5, caspase-8, -9, and -3 expression as well as increased cleaved-PARP, while decreased Bcl-2 upon GA treatment. In addition, phosphorylated JNK was up-regulated but phosphorylated ERK was down-regulated after exposure to GA.

Conclusions: These results suggest that GA induce apoptosis associated with the ER stress response through up-regulation of p-JNK and down-regulation of p-ERK in HeLa cells.

Show MeSH
Related in: MedlinePlus