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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 inhibits cell growth in HeLa cells. (A) Formula of Gambogic acid (GA). (B) Effect of GA on cell viability in HeLa cells. Time- and dose-dependent effect of GA was performed when HeLa cells were treated with various concentrations of GA at different time points and their viability was determined by trypan blue exclusion assay. Results are mean values ± SD of three independent experiments (n = 3).
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Fig1: GA inhibits cell growth in HeLa cells. (A) Formula of Gambogic acid (GA). (B) Effect of GA on cell viability in HeLa cells. Time- and dose-dependent effect of GA was performed when HeLa cells were treated with various concentrations of GA at different time points and their viability was determined by trypan blue exclusion assay. Results are mean values ± SD of three independent experiments (n = 3).

Mentions: Garcinia hanburyi was collected from Laem-ngob, Trat province, Thailand in Jan 2012. A voucher specimen (Montree Kurukitkoson No. 001) was deposited at the Faculty of Medicine, Srinakharinwirot University, Bangkok, Thailand. GA (Figure 1A) was isolated from gamboge (G. Hanburyi). The extraction and separation method was as followed: dried resin of gamboge (1 g) was grounded into a powder and extracted with acetone: dH20 (1:1, 1 L), followed by ethyl acetate (1:1). After evaporation, the extract yielded as a yellowish solid (0.6 g). A portion of extract (0.3 g) was subjected to silica gel column chromatography (Silica gel 60, 60–230 μm, Merck, Darmstadt, Germany) using chloroform/methanol stepwise system and yielded the major compound, GA, including other minor compounds. Repeated purification of these column fractions was performed on a SSC-1311 recycling HPLC system equipped with a SSC-5410 UV–vis detector and SSC-3462 pump (Senshu Scientific, Japan). The column was Capcell Pak C18, type UG80 (20 mm id. × 250 mm, 5 μm, Shiseido, Japan). The mobile phase was 75% acetonitrile at a flow rate of 10 ml/min. The UV detection wavelength was set at 360 nm. The chemical structures of GA were then identified by comparing their 1H NMR and 13C NMR spectra (JNM-ECA500 NMR, JEOL, Japan) with the literature data [22]. GA was dissolved and diluted in methanol (Wako, Japan) at the desired concentration for assays.Figure 1


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 inhibits cell growth in HeLa cells. (A) Formula of Gambogic acid (GA). (B) Effect of GA on cell viability in HeLa cells. Time- and dose-dependent effect of GA was performed when HeLa cells were treated with various concentrations of GA at different time points and their viability was determined by trypan blue exclusion assay. Results are mean values ± SD of three independent experiments (n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: GA inhibits cell growth in HeLa cells. (A) Formula of Gambogic acid (GA). (B) Effect of GA on cell viability in HeLa cells. Time- and dose-dependent effect of GA was performed when HeLa cells were treated with various concentrations of GA at different time points and their viability was determined by trypan blue exclusion assay. Results are mean values ± SD of three independent experiments (n = 3).
Mentions: Garcinia hanburyi was collected from Laem-ngob, Trat province, Thailand in Jan 2012. A voucher specimen (Montree Kurukitkoson No. 001) was deposited at the Faculty of Medicine, Srinakharinwirot University, Bangkok, Thailand. GA (Figure 1A) was isolated from gamboge (G. Hanburyi). The extraction and separation method was as followed: dried resin of gamboge (1 g) was grounded into a powder and extracted with acetone: dH20 (1:1, 1 L), followed by ethyl acetate (1:1). After evaporation, the extract yielded as a yellowish solid (0.6 g). A portion of extract (0.3 g) was subjected to silica gel column chromatography (Silica gel 60, 60–230 μm, Merck, Darmstadt, Germany) using chloroform/methanol stepwise system and yielded the major compound, GA, including other minor compounds. Repeated purification of these column fractions was performed on a SSC-1311 recycling HPLC system equipped with a SSC-5410 UV–vis detector and SSC-3462 pump (Senshu Scientific, Japan). The column was Capcell Pak C18, type UG80 (20 mm id. × 250 mm, 5 μm, Shiseido, Japan). The mobile phase was 75% acetonitrile at a flow rate of 10 ml/min. The UV detection wavelength was set at 360 nm. The chemical structures of GA were then identified by comparing their 1H NMR and 13C NMR spectra (JNM-ECA500 NMR, JEOL, Japan) with the literature data [22]. GA was dissolved and diluted in methanol (Wako, Japan) at the desired concentration for assays.Figure 1

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