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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

GA induces apoptosis. (A) The expression of caspase-3, −8, −9, and PARP were analyzed by Western blotting. HeLa cells were non treated (NT) or treated with 0.3 μg/ml of GA for 24 h. Effect of GA on the expression of (B) Bcl-2 family and (C) DR5 in HeLa cells. HeLa cells were treated with 0.3 μg/ml of GA for the indicated time points and analyzed by Western blotting. The results from representative experiments were expressed relative to the protein level at 0 h after normalization to β-actin signals.
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Fig5: GA induces apoptosis. (A) The expression of caspase-3, −8, −9, and PARP were analyzed by Western blotting. HeLa cells were non treated (NT) or treated with 0.3 μg/ml of GA for 24 h. Effect of GA on the expression of (B) Bcl-2 family and (C) DR5 in HeLa cells. HeLa cells were treated with 0.3 μg/ml of GA for the indicated time points and analyzed by Western blotting. The results from representative experiments were expressed relative to the protein level at 0 h after normalization to β-actin signals.

Mentions: To examine whether GA can induce apoptosis in HeLa cells, we examined the activation of caspases and Poly (ADP-ribose) polymerase (PARP), a substrate of caspase 3, by Western blotting. As shown in Figure 5A, GA reduced procaspase-8, −9 and −3 protein levels and increased active cleaved caspase-8, −9 and −3 and PARP cleavage. These results demonstrate that GA is involved in apoptosis induction in HeLa cells.Figure 5


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)

GA induces apoptosis. (A) The expression of caspase-3, −8, −9, and PARP were analyzed by Western blotting. HeLa cells were non treated (NT) or treated with 0.3 μg/ml of GA for 24 h. Effect of GA on the expression of (B) Bcl-2 family and (C) DR5 in HeLa cells. HeLa cells were treated with 0.3 μg/ml of GA for the indicated time points and analyzed by Western blotting. The results from representative experiments 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

Fig5: GA induces apoptosis. (A) The expression of caspase-3, −8, −9, and PARP were analyzed by Western blotting. HeLa cells were non treated (NT) or treated with 0.3 μg/ml of GA for 24 h. Effect of GA on the expression of (B) Bcl-2 family and (C) DR5 in HeLa cells. HeLa cells were treated with 0.3 μg/ml of GA for the indicated time points and analyzed by Western blotting. The results from representative experiments were expressed relative to the protein level at 0 h after normalization to β-actin signals.
Mentions: To examine whether GA can induce apoptosis in HeLa cells, we examined the activation of caspases and Poly (ADP-ribose) polymerase (PARP), a substrate of caspase 3, by Western blotting. As shown in Figure 5A, GA reduced procaspase-8, −9 and −3 protein levels and increased active cleaved caspase-8, −9 and −3 and PARP cleavage. These results demonstrate that GA is involved in apoptosis induction in HeLa cells.Figure 5

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