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8‑bromo‑7‑methoxychrysin induces apoptosis by regulating Akt/FOXO3a pathway in cisplatin‑sensitive and resistant ovarian cancer cells.

Ding Q, Chen Y, Zhang Q, Guo Y, Huang Z, Dai L, Cao S - Mol Med Rep (2015)

Bottom Line: 8‑bromo‑7‑methoxychrysin (BrMC), a novel chrysin analog, was reported to have anti‑cancer activities.BrMC‑induced apoptotic cell death occurred mainly by the activation of Akt, which was accompanied by the overexpression of transcription factor FOXO3a, with a concomitant increase in the expression levels of Bim.Silencing Bim expression by using small interfering RNA, attenuated the induction of apoptosis by BrMC treatment.

View Article: PubMed Central - PubMed

Affiliation: Hunan University of Traditional Chinese Medicine, Changsha, Hunan 410208, P.R.China.

ABSTRACT
8‑bromo‑7‑methoxychrysin (BrMC), a novel chrysin analog, was reported to have anti‑cancer activities. The aim of the present study was to investigate the molecular mechanism of 8‑bromo‑7‑methoxychrysin (BrMC)‑induced apoptosis via the Akt/forkhead box O3a (FOXO3a) pathway in cisplatin (DDP)‑sensitive and ‑resistant ovarian cancer cells. The human ovarian cancer cell lines A2780 and A2780/DDP were cultured in vitro. Various molecular techniques were used to assess the expression of FOXO3a and B cell lymphoma 2 (Bcl‑2)‑interacting mediator of cell death (Bim) in cisplatin‑sensitive and ‑resistant ovarian cancer cells. Different concentrations of BrMC induced apoptosis in cisplatin‑sensitive and ‑resistant ovarian cancer cells. BrMC‑induced apoptotic cell death occurred mainly by the activation of Akt, which was accompanied by the overexpression of transcription factor FOXO3a, with a concomitant increase in the expression levels of Bim. Silencing Bim expression by using small interfering RNA, attenuated the induction of apoptosis by BrMC treatment. The results indicated that BrMC‑induced apoptosis in cisplatin‑sensitive and ‑resistant ovarian cancer cells may occur via the regulation of Akt/FOXO3a, leading to Bim transcription.

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Caspase-3 regulates the phosphorylation of Akt associated with PP2A. (A and B) A2780 and A2780/DDP cells were pre-treated with indicated concentrations of okadaic acid for 1 h and exposed to BrMC for 24 h, then detected for Akt and p-Akt (Ser473) levels by western blot analysis. β-Actin was used as a control. (C) To determine the Akt phosphorylation. A2780/DDP cells were pre-treated with or without various doses of caspase 3 inhibitor z-DEVD-fmk (20 or 80 µM) for 1 h and exposed to BrMC (5 µM) for 24 h, then p-Akt (Ser473) levels were detected using western blot analysis. (D) The apoptotic rate of the A2780 and A2780/DDP cells following pre-treatment with different doses of z-DEVD-fmk. All data are depicted graphically as the mean ± standard error of the mean of at least three independent experiments. *P<0.05, **P<0.01, vs. apoptotic rate of the A2780 and A2780/DDP cells treated with BrMC (5 µm) alone; #P<0.05, vs. treatment with BrMC (5 µm) and z-DEVD-fmk (20 µm). (E and F) Detection of the binding of Akt with PP2A. Cells were incubated with or without BrMC for 48 h, 20 µM of z-DEVD-fmk was added 1 h prior to treatment with the drug. The cells were lysed with lysis buffer for immunoprecipitation with anti-Akt antibody followed by immunoblot assay with anti-PP2A/C and anti-Akt antibodies. Data are representative of at least three independent experiments. p-Akt, phosphorylated Akt; CF, cleaved form of PP2A; PP2A, protein phosphotase 2A; BrMC, 8-bromo-7-methoxychrysin; DDP, cisplatin; z-DEVD-fmk, Caspase-3 specific inhibitor Z-Asp-Glu-Val-Asp-CH2F.
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f5-mmr-12-04-5100: Caspase-3 regulates the phosphorylation of Akt associated with PP2A. (A and B) A2780 and A2780/DDP cells were pre-treated with indicated concentrations of okadaic acid for 1 h and exposed to BrMC for 24 h, then detected for Akt and p-Akt (Ser473) levels by western blot analysis. β-Actin was used as a control. (C) To determine the Akt phosphorylation. A2780/DDP cells were pre-treated with or without various doses of caspase 3 inhibitor z-DEVD-fmk (20 or 80 µM) for 1 h and exposed to BrMC (5 µM) for 24 h, then p-Akt (Ser473) levels were detected using western blot analysis. (D) The apoptotic rate of the A2780 and A2780/DDP cells following pre-treatment with different doses of z-DEVD-fmk. All data are depicted graphically as the mean ± standard error of the mean of at least three independent experiments. *P<0.05, **P<0.01, vs. apoptotic rate of the A2780 and A2780/DDP cells treated with BrMC (5 µm) alone; #P<0.05, vs. treatment with BrMC (5 µm) and z-DEVD-fmk (20 µm). (E and F) Detection of the binding of Akt with PP2A. Cells were incubated with or without BrMC for 48 h, 20 µM of z-DEVD-fmk was added 1 h prior to treatment with the drug. The cells were lysed with lysis buffer for immunoprecipitation with anti-Akt antibody followed by immunoblot assay with anti-PP2A/C and anti-Akt antibodies. Data are representative of at least three independent experiments. p-Akt, phosphorylated Akt; CF, cleaved form of PP2A; PP2A, protein phosphotase 2A; BrMC, 8-bromo-7-methoxychrysin; DDP, cisplatin; z-DEVD-fmk, Caspase-3 specific inhibitor Z-Asp-Glu-Val-Asp-CH2F.

Mentions: It was reported that caspase-3 regulated the phosphorylation of Akt through the cleavage of the regulatory A subunit of (PP2A/A), increasing PP2A activity (38). In the caspase inhibitor assay, cells were pretreated with caspase inhibitors (20 µM ZVAD-CHO) for 1 h prior to the addition of the agents tested. Results of the present study demonstrated that Bim was involved in the regulation of Akt dephosphorylation (Fig. 4E). Therefore, it was proposed that Bim may regulate Akt phosphorylation by caspase-3-mediated PP2A activation in ovarian cells. To validate this hypothesis, it was first determined whether PP2A mediated Akt phosphorylation. Pretreatment of the cells with okadaic acid (OA), an inhibitor of PP2A, resulted in reversal of BrMC-induced Akt dephosphorylation in A2780 cells (Fig. 5A). At the same time, OA also rescued Akt phosphorylation in A2780/DDP cells during BrMC treatment (Fig. 5B).


8‑bromo‑7‑methoxychrysin induces apoptosis by regulating Akt/FOXO3a pathway in cisplatin‑sensitive and resistant ovarian cancer cells.

Ding Q, Chen Y, Zhang Q, Guo Y, Huang Z, Dai L, Cao S - Mol Med Rep (2015)

Caspase-3 regulates the phosphorylation of Akt associated with PP2A. (A and B) A2780 and A2780/DDP cells were pre-treated with indicated concentrations of okadaic acid for 1 h and exposed to BrMC for 24 h, then detected for Akt and p-Akt (Ser473) levels by western blot analysis. β-Actin was used as a control. (C) To determine the Akt phosphorylation. A2780/DDP cells were pre-treated with or without various doses of caspase 3 inhibitor z-DEVD-fmk (20 or 80 µM) for 1 h and exposed to BrMC (5 µM) for 24 h, then p-Akt (Ser473) levels were detected using western blot analysis. (D) The apoptotic rate of the A2780 and A2780/DDP cells following pre-treatment with different doses of z-DEVD-fmk. All data are depicted graphically as the mean ± standard error of the mean of at least three independent experiments. *P<0.05, **P<0.01, vs. apoptotic rate of the A2780 and A2780/DDP cells treated with BrMC (5 µm) alone; #P<0.05, vs. treatment with BrMC (5 µm) and z-DEVD-fmk (20 µm). (E and F) Detection of the binding of Akt with PP2A. Cells were incubated with or without BrMC for 48 h, 20 µM of z-DEVD-fmk was added 1 h prior to treatment with the drug. The cells were lysed with lysis buffer for immunoprecipitation with anti-Akt antibody followed by immunoblot assay with anti-PP2A/C and anti-Akt antibodies. Data are representative of at least three independent experiments. p-Akt, phosphorylated Akt; CF, cleaved form of PP2A; PP2A, protein phosphotase 2A; BrMC, 8-bromo-7-methoxychrysin; DDP, cisplatin; z-DEVD-fmk, Caspase-3 specific inhibitor Z-Asp-Glu-Val-Asp-CH2F.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4581817&req=5

f5-mmr-12-04-5100: Caspase-3 regulates the phosphorylation of Akt associated with PP2A. (A and B) A2780 and A2780/DDP cells were pre-treated with indicated concentrations of okadaic acid for 1 h and exposed to BrMC for 24 h, then detected for Akt and p-Akt (Ser473) levels by western blot analysis. β-Actin was used as a control. (C) To determine the Akt phosphorylation. A2780/DDP cells were pre-treated with or without various doses of caspase 3 inhibitor z-DEVD-fmk (20 or 80 µM) for 1 h and exposed to BrMC (5 µM) for 24 h, then p-Akt (Ser473) levels were detected using western blot analysis. (D) The apoptotic rate of the A2780 and A2780/DDP cells following pre-treatment with different doses of z-DEVD-fmk. All data are depicted graphically as the mean ± standard error of the mean of at least three independent experiments. *P<0.05, **P<0.01, vs. apoptotic rate of the A2780 and A2780/DDP cells treated with BrMC (5 µm) alone; #P<0.05, vs. treatment with BrMC (5 µm) and z-DEVD-fmk (20 µm). (E and F) Detection of the binding of Akt with PP2A. Cells were incubated with or without BrMC for 48 h, 20 µM of z-DEVD-fmk was added 1 h prior to treatment with the drug. The cells were lysed with lysis buffer for immunoprecipitation with anti-Akt antibody followed by immunoblot assay with anti-PP2A/C and anti-Akt antibodies. Data are representative of at least three independent experiments. p-Akt, phosphorylated Akt; CF, cleaved form of PP2A; PP2A, protein phosphotase 2A; BrMC, 8-bromo-7-methoxychrysin; DDP, cisplatin; z-DEVD-fmk, Caspase-3 specific inhibitor Z-Asp-Glu-Val-Asp-CH2F.
Mentions: It was reported that caspase-3 regulated the phosphorylation of Akt through the cleavage of the regulatory A subunit of (PP2A/A), increasing PP2A activity (38). In the caspase inhibitor assay, cells were pretreated with caspase inhibitors (20 µM ZVAD-CHO) for 1 h prior to the addition of the agents tested. Results of the present study demonstrated that Bim was involved in the regulation of Akt dephosphorylation (Fig. 4E). Therefore, it was proposed that Bim may regulate Akt phosphorylation by caspase-3-mediated PP2A activation in ovarian cells. To validate this hypothesis, it was first determined whether PP2A mediated Akt phosphorylation. Pretreatment of the cells with okadaic acid (OA), an inhibitor of PP2A, resulted in reversal of BrMC-induced Akt dephosphorylation in A2780 cells (Fig. 5A). At the same time, OA also rescued Akt phosphorylation in A2780/DDP cells during BrMC treatment (Fig. 5B).

Bottom Line: 8‑bromo‑7‑methoxychrysin (BrMC), a novel chrysin analog, was reported to have anti‑cancer activities.BrMC‑induced apoptotic cell death occurred mainly by the activation of Akt, which was accompanied by the overexpression of transcription factor FOXO3a, with a concomitant increase in the expression levels of Bim.Silencing Bim expression by using small interfering RNA, attenuated the induction of apoptosis by BrMC treatment.

View Article: PubMed Central - PubMed

Affiliation: Hunan University of Traditional Chinese Medicine, Changsha, Hunan 410208, P.R.China.

ABSTRACT
8‑bromo‑7‑methoxychrysin (BrMC), a novel chrysin analog, was reported to have anti‑cancer activities. The aim of the present study was to investigate the molecular mechanism of 8‑bromo‑7‑methoxychrysin (BrMC)‑induced apoptosis via the Akt/forkhead box O3a (FOXO3a) pathway in cisplatin (DDP)‑sensitive and ‑resistant ovarian cancer cells. The human ovarian cancer cell lines A2780 and A2780/DDP were cultured in vitro. Various molecular techniques were used to assess the expression of FOXO3a and B cell lymphoma 2 (Bcl‑2)‑interacting mediator of cell death (Bim) in cisplatin‑sensitive and ‑resistant ovarian cancer cells. Different concentrations of BrMC induced apoptosis in cisplatin‑sensitive and ‑resistant ovarian cancer cells. BrMC‑induced apoptotic cell death occurred mainly by the activation of Akt, which was accompanied by the overexpression of transcription factor FOXO3a, with a concomitant increase in the expression levels of Bim. Silencing Bim expression by using small interfering RNA, attenuated the induction of apoptosis by BrMC treatment. The results indicated that BrMC‑induced apoptosis in cisplatin‑sensitive and ‑resistant ovarian cancer cells may occur via the regulation of Akt/FOXO3a, leading to Bim transcription.

Show MeSH
Related in: MedlinePlus