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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 XBP1 mRNA splicing in HeLa cells. HeLa cells were treated with 10 μg/ml of tunicamycin (Tm) or 0.3 μg/ml of GA for 0, 4 and 8 h. mRNA was extracted and subjected to the RT-PCR. The active form was normalized to the inactive form at 0 h. Results are mean values ± SD of three independent experiments (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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Fig2: GA induces XBP1 mRNA splicing in HeLa cells. HeLa cells were treated with 10 μg/ml of tunicamycin (Tm) or 0.3 μg/ml of GA for 0, 4 and 8 h. mRNA was extracted and subjected to the RT-PCR. The active form was normalized to the inactive form at 0 h. Results are mean values ± SD of three independent experiments (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: In response to ER stress, IRE1 splices a 26 nucleotide long intron of inactive unspliced XBP1 mRNA (XBP1u), generating an active and stable transcription factor XBP1s. To access whether GA triggers ER stress, we analyzed XBP1 mRNA splicing in HeLa cells. The XBP1 cDNA was amplified by RT-PCR. Tunicamycin (Tm), a well-recognized inducer of ER stress, served as a positive control in these tests. The result showed GA induced XBP1 splicing at 4 h and the XBP1 mRNA was elevated by approximately 6 fold compared to the level observed in control cells (0 h) (Figure 2). In contrast to Tm, the expression of XBP1 splicing was decreased following treatment with GA for 4 h to 8 h.Figure 2


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 XBP1 mRNA splicing in HeLa cells. HeLa cells were treated with 10 μg/ml of tunicamycin (Tm) or 0.3 μg/ml of GA for 0, 4 and 8 h. mRNA was extracted and subjected to the RT-PCR. The active form was normalized to the inactive form at 0 h. Results are mean values ± SD of three independent experiments (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

Fig2: GA induces XBP1 mRNA splicing in HeLa cells. HeLa cells were treated with 10 μg/ml of tunicamycin (Tm) or 0.3 μg/ml of GA for 0, 4 and 8 h. mRNA was extracted and subjected to the RT-PCR. The active form was normalized to the inactive form at 0 h. Results are mean values ± SD of three independent experiments (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: In response to ER stress, IRE1 splices a 26 nucleotide long intron of inactive unspliced XBP1 mRNA (XBP1u), generating an active and stable transcription factor XBP1s. To access whether GA triggers ER stress, we analyzed XBP1 mRNA splicing in HeLa cells. The XBP1 cDNA was amplified by RT-PCR. Tunicamycin (Tm), a well-recognized inducer of ER stress, served as a positive control in these tests. The result showed GA induced XBP1 splicing at 4 h and the XBP1 mRNA was elevated by approximately 6 fold compared to the level observed in control cells (0 h) (Figure 2). In contrast to Tm, the expression of XBP1 splicing was decreased following treatment with GA for 4 h to 8 h.Figure 2

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