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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 ER stress via up-regulation of GRP78, CHOP, ERdj4 and GADD34 mRNA. HeLa cells were treated with 10 μg/ml of tunicamycin (Tm) or 0.3 μg/ml of GA for 0, 4 and 8 h. Cells were collected and mRNA was extracted and evaluated by real-time RT-PCR. Each mRNA was normalized with the mRNA of GAPDH. Data are the results of three independent experiments, expressed as the mean ± SD, n = 3. *p < 0.05; **p < 0.01 shows significant difference compared with 0 h; #p < 0.05, is significantly different as compared between 4 h and 8 h.
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Fig3: GA induces ER stress via up-regulation of GRP78, CHOP, ERdj4 and GADD34 mRNA. HeLa cells were treated with 10 μg/ml of tunicamycin (Tm) or 0.3 μg/ml of GA for 0, 4 and 8 h. Cells were collected and mRNA was extracted and evaluated by real-time RT-PCR. Each mRNA was normalized with the mRNA of GAPDH. Data are the results of three independent experiments, expressed as the mean ± SD, n = 3. *p < 0.05; **p < 0.01 shows significant difference compared with 0 h; #p < 0.05, is significantly different as compared between 4 h and 8 h.

Mentions: We further examined whether GA induced ER stress. The mRNA expression levels of ER stress-associated molecules (GRP78, CHOP, endoplasmic reticulum-localized DnaJ homologues (ERdj4) and growth arrest and DNA damage-inducible protein (GADD34)) were investigated by real -time PCR. CHOP, also known as GADD153, encodes a member of the CCAAT/enhancer-binding protein family and acts as an inhibitor or activator of transcription, leading to apoptosis [20]. ERdj4, which is a chaperone protein localized in the ER, is a downstream gene of XBP1, while GADD34 is downstream gene of eIF2α. Figure 3 showed GRP78 was slightly increased at 8 h in cells treated with GA. On the other hand, Tm showed obviously increased GRP78 expression and the highest level at 8 h. In addition, there was a large increase in CHOP and ERdj4 level between 4 and 8 h following exposure to Tm and GA. It is noticed that GA increased GADD34 expression, while it had very little effect in cells treated with Tm. All of these observations strongly imply that GA induced ER stress in HeLa cells.Figure 3


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 ER stress via up-regulation of GRP78, CHOP, ERdj4 and GADD34 mRNA. HeLa cells were treated with 10 μg/ml of tunicamycin (Tm) or 0.3 μg/ml of GA for 0, 4 and 8 h. Cells were collected and mRNA was extracted and evaluated by real-time RT-PCR. Each mRNA was normalized with the mRNA of GAPDH. Data are the results of three independent experiments, expressed as the mean ± SD, n = 3. *p < 0.05; **p < 0.01 shows significant difference compared with 0 h; #p < 0.05, is significantly different as compared between 4 h and 8 h.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: GA induces ER stress via up-regulation of GRP78, CHOP, ERdj4 and GADD34 mRNA. HeLa cells were treated with 10 μg/ml of tunicamycin (Tm) or 0.3 μg/ml of GA for 0, 4 and 8 h. Cells were collected and mRNA was extracted and evaluated by real-time RT-PCR. Each mRNA was normalized with the mRNA of GAPDH. Data are the results of three independent experiments, expressed as the mean ± SD, n = 3. *p < 0.05; **p < 0.01 shows significant difference compared with 0 h; #p < 0.05, is significantly different as compared between 4 h and 8 h.
Mentions: We further examined whether GA induced ER stress. The mRNA expression levels of ER stress-associated molecules (GRP78, CHOP, endoplasmic reticulum-localized DnaJ homologues (ERdj4) and growth arrest and DNA damage-inducible protein (GADD34)) were investigated by real -time PCR. CHOP, also known as GADD153, encodes a member of the CCAAT/enhancer-binding protein family and acts as an inhibitor or activator of transcription, leading to apoptosis [20]. ERdj4, which is a chaperone protein localized in the ER, is a downstream gene of XBP1, while GADD34 is downstream gene of eIF2α. Figure 3 showed GRP78 was slightly increased at 8 h in cells treated with GA. On the other hand, Tm showed obviously increased GRP78 expression and the highest level at 8 h. In addition, there was a large increase in CHOP and ERdj4 level between 4 and 8 h following exposure to Tm and GA. It is noticed that GA increased GADD34 expression, while it had very little effect in cells treated with Tm. All of these observations strongly imply that GA induced ER stress in HeLa cells.Figure 3

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