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Gecko proteins induce the apoptosis of bladder cancer 5637 cells by inhibiting Akt and activating the intrinsic caspase cascade.

Kim GY, Park SY, Jo A, Kim M, Leem SH, Jun WJ, Shim SI, Lee SC, Chung JW - BMB Rep (2015)

Bottom Line: Gecko proteins significantly reduced the viability of 5637 cells without any cytotoxic effect on normal cells.These proteins increased the Annexin-V staining and the amount of condensed chromatin, demonstrating that the Gecko proteinsinduced cell death was caused by apoptosis.Furthermore, Gecko proteins activated caspase 9 and caspase 3/7.

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiovascular and Rare Disease, Center for Biomedical Sciences, Korea National Institute of Health, Cheongju 28165, Korea.

ABSTRACT
Gecko proteins have long been used as anti-tumor agents in oriental medicine, without any scientific background. Although anti-tumor effects of Gecko proteins on several cancers were recently reported, their effect on bladder cancer has not been investigated. Thus, we explored the anti-tumor effect of Gecko proteins and its cellular mechanisms in human bladder cancer 5637 cells. Gecko proteins significantly reduced the viability of 5637 cells without any cytotoxic effect on normal cells. These proteins increased the Annexin-V staining and the amount of condensed chromatin, demonstrating that the Gecko proteinsinduced cell death was caused by apoptosis. Gecko proteins suppressed Akt activation, and the overexpression of constitutively active form of myristoylated Akt prevented Gecko proteins-induced death of 5637 cells. Furthermore, Gecko proteins activated caspase 9 and caspase 3/7. Taken together, our data demonstrated that Gecko proteins suppressed the Akt pathway and activated the intrinsic caspase pathway, leading to the apoptosis of bladder cancer cells. [BMB Reports 2015; 48(9): 531-536].

No MeSH data available.


Related in: MedlinePlus

GPE induces the 5637 cell death by suppressing Akt phosphorylation. (A) 5637 cells were treated with indicated concentrations of GPE for 48 h, the phosphorylation of Akt at Ser 473 was analyzed by immunoblotting. (B) Quantification of Akt phopshorylation that was normalized to the expression of total Akt. *P < 0.05, compared with no GPE treatment. (C) 5637 cells were seeded in 24-well cell culture plates at the density of 2 × 104 cells/well. Next day, cells were treated with 500 μg/ml GPE, with the pretreatment of Wortmannin (100 nM) for 2 h, where necessary. After 48 h incubation, cells were trypsinized and the number of viable cells was counted. *P < 0.05, compared with no treatment. #P < 0.05, compared with GPE treatment. ##P < 0.05, compared with Wortmannin treatment. (D) 5637 cells transfected with pcDNA3 or pcDNA3-myr-Akt, were incubated with 500 μg/ml GPE for 48 h, and viable cells were counted. *P < 0.05, compared with pcDNA3 treated with GPE.
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Figure 003: GPE induces the 5637 cell death by suppressing Akt phosphorylation. (A) 5637 cells were treated with indicated concentrations of GPE for 48 h, the phosphorylation of Akt at Ser 473 was analyzed by immunoblotting. (B) Quantification of Akt phopshorylation that was normalized to the expression of total Akt. *P < 0.05, compared with no GPE treatment. (C) 5637 cells were seeded in 24-well cell culture plates at the density of 2 × 104 cells/well. Next day, cells were treated with 500 μg/ml GPE, with the pretreatment of Wortmannin (100 nM) for 2 h, where necessary. After 48 h incubation, cells were trypsinized and the number of viable cells was counted. *P < 0.05, compared with no treatment. #P < 0.05, compared with GPE treatment. ##P < 0.05, compared with Wortmannin treatment. (D) 5637 cells transfected with pcDNA3 or pcDNA3-myr-Akt, were incubated with 500 μg/ml GPE for 48 h, and viable cells were counted. *P < 0.05, compared with pcDNA3 treated with GPE.

Mentions: Akt is the typical survival factor in various cancer cells, including bladder cancer (13-15), and Akt suppression was critical for exhibiting the anti-tumor effect of various bioactive peptides (14, 16). In addition, we had previously shown GPE induced cervical cancer cell death by inhibiting the Akt pathway (11). Thus, we examined the involvement of Akt pathway in GPE-induced apoptosis of 5637 cell. Cells were treated with GPE in a dose-dependent manner, and the levels of phosphorylated Akt were evaluated by immunoblotting (17). We found that Akt was phosphorylated at the basal level, and in consistence with our previous observations in cervical cancer cells (11), GPE suppressed the Akt phosphorylation (Fig. 3A, B). To show that suppression of Akt is critical for the GPE-induced cell death, we treated 5637 cells with either GPE, Wortmannin, or in combination, incubated cells for 48 h, and counted the number of viable cells. Interestingly, 100 nM Wortmannin showed a comparable cytotoxic effect as 500 μg/ml GPE, against the 5637 cells, co-treatment of these reagents further decreased the number of viable cells (Fig. 3C), implying that the Akt pathway is involved in GPE-induced death of 5637 cell. Furthermore, transfection of the constitutively active form of myristoylated Akt rescued the GPE-induced cell death (Fig. 3D). These results suggest that GPE induces 5637 cell death by inhibiting the Akt pathway.


Gecko proteins induce the apoptosis of bladder cancer 5637 cells by inhibiting Akt and activating the intrinsic caspase cascade.

Kim GY, Park SY, Jo A, Kim M, Leem SH, Jun WJ, Shim SI, Lee SC, Chung JW - BMB Rep (2015)

GPE induces the 5637 cell death by suppressing Akt phosphorylation. (A) 5637 cells were treated with indicated concentrations of GPE for 48 h, the phosphorylation of Akt at Ser 473 was analyzed by immunoblotting. (B) Quantification of Akt phopshorylation that was normalized to the expression of total Akt. *P < 0.05, compared with no GPE treatment. (C) 5637 cells were seeded in 24-well cell culture plates at the density of 2 × 104 cells/well. Next day, cells were treated with 500 μg/ml GPE, with the pretreatment of Wortmannin (100 nM) for 2 h, where necessary. After 48 h incubation, cells were trypsinized and the number of viable cells was counted. *P < 0.05, compared with no treatment. #P < 0.05, compared with GPE treatment. ##P < 0.05, compared with Wortmannin treatment. (D) 5637 cells transfected with pcDNA3 or pcDNA3-myr-Akt, were incubated with 500 μg/ml GPE for 48 h, and viable cells were counted. *P < 0.05, compared with pcDNA3 treated with GPE.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 003: GPE induces the 5637 cell death by suppressing Akt phosphorylation. (A) 5637 cells were treated with indicated concentrations of GPE for 48 h, the phosphorylation of Akt at Ser 473 was analyzed by immunoblotting. (B) Quantification of Akt phopshorylation that was normalized to the expression of total Akt. *P < 0.05, compared with no GPE treatment. (C) 5637 cells were seeded in 24-well cell culture plates at the density of 2 × 104 cells/well. Next day, cells were treated with 500 μg/ml GPE, with the pretreatment of Wortmannin (100 nM) for 2 h, where necessary. After 48 h incubation, cells were trypsinized and the number of viable cells was counted. *P < 0.05, compared with no treatment. #P < 0.05, compared with GPE treatment. ##P < 0.05, compared with Wortmannin treatment. (D) 5637 cells transfected with pcDNA3 or pcDNA3-myr-Akt, were incubated with 500 μg/ml GPE for 48 h, and viable cells were counted. *P < 0.05, compared with pcDNA3 treated with GPE.
Mentions: Akt is the typical survival factor in various cancer cells, including bladder cancer (13-15), and Akt suppression was critical for exhibiting the anti-tumor effect of various bioactive peptides (14, 16). In addition, we had previously shown GPE induced cervical cancer cell death by inhibiting the Akt pathway (11). Thus, we examined the involvement of Akt pathway in GPE-induced apoptosis of 5637 cell. Cells were treated with GPE in a dose-dependent manner, and the levels of phosphorylated Akt were evaluated by immunoblotting (17). We found that Akt was phosphorylated at the basal level, and in consistence with our previous observations in cervical cancer cells (11), GPE suppressed the Akt phosphorylation (Fig. 3A, B). To show that suppression of Akt is critical for the GPE-induced cell death, we treated 5637 cells with either GPE, Wortmannin, or in combination, incubated cells for 48 h, and counted the number of viable cells. Interestingly, 100 nM Wortmannin showed a comparable cytotoxic effect as 500 μg/ml GPE, against the 5637 cells, co-treatment of these reagents further decreased the number of viable cells (Fig. 3C), implying that the Akt pathway is involved in GPE-induced death of 5637 cell. Furthermore, transfection of the constitutively active form of myristoylated Akt rescued the GPE-induced cell death (Fig. 3D). These results suggest that GPE induces 5637 cell death by inhibiting the Akt pathway.

Bottom Line: Gecko proteins significantly reduced the viability of 5637 cells without any cytotoxic effect on normal cells.These proteins increased the Annexin-V staining and the amount of condensed chromatin, demonstrating that the Gecko proteinsinduced cell death was caused by apoptosis.Furthermore, Gecko proteins activated caspase 9 and caspase 3/7.

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiovascular and Rare Disease, Center for Biomedical Sciences, Korea National Institute of Health, Cheongju 28165, Korea.

ABSTRACT
Gecko proteins have long been used as anti-tumor agents in oriental medicine, without any scientific background. Although anti-tumor effects of Gecko proteins on several cancers were recently reported, their effect on bladder cancer has not been investigated. Thus, we explored the anti-tumor effect of Gecko proteins and its cellular mechanisms in human bladder cancer 5637 cells. Gecko proteins significantly reduced the viability of 5637 cells without any cytotoxic effect on normal cells. These proteins increased the Annexin-V staining and the amount of condensed chromatin, demonstrating that the Gecko proteinsinduced cell death was caused by apoptosis. Gecko proteins suppressed Akt activation, and the overexpression of constitutively active form of myristoylated Akt prevented Gecko proteins-induced death of 5637 cells. Furthermore, Gecko proteins activated caspase 9 and caspase 3/7. Taken together, our data demonstrated that Gecko proteins suppressed the Akt pathway and activated the intrinsic caspase pathway, leading to the apoptosis of bladder cancer cells. [BMB Reports 2015; 48(9): 531-536].

No MeSH data available.


Related in: MedlinePlus