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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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Akt is involved in BrMC-induced apoptosis. (A and B) Expression levels of t-Akt and p-Akt (Ser473) were detected by western blot analysis in A2780cells and A2780/DDP cells. Cells were treated with BrMC (2.5 µM) for 24 h, then lysed and assayed for individual protein levels by western blot. β-Actin was used as a control. p-Akt and Akt levels were measured by densitometric analysis of the western blots and compared to actin levels. (C) Detection of the role of LY294002 on apoptosis of cells. Cells were treated with BrMC and/or 20 µM LY294002 for 24 h, and then individual protein levels were detected by western blotting. (D) Apoptotic rate of the A2780 and A2780/DDP cells. All data are depicted graphically as the mean ± standard error of the mean for at least three independent experiments. **P<0.01; #P<0.05, vs. treatment with BrMC (5 µm) and LY294002 (20 µm). p/t-Akt, phosphorylated/total Akt; BrMC, 8-bromo-7-methoxychrysin; DDP, cisplatin.
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f3-mmr-12-04-5100: Akt is involved in BrMC-induced apoptosis. (A and B) Expression levels of t-Akt and p-Akt (Ser473) were detected by western blot analysis in A2780cells and A2780/DDP cells. Cells were treated with BrMC (2.5 µM) for 24 h, then lysed and assayed for individual protein levels by western blot. β-Actin was used as a control. p-Akt and Akt levels were measured by densitometric analysis of the western blots and compared to actin levels. (C) Detection of the role of LY294002 on apoptosis of cells. Cells were treated with BrMC and/or 20 µM LY294002 for 24 h, and then individual protein levels were detected by western blotting. (D) Apoptotic rate of the A2780 and A2780/DDP cells. All data are depicted graphically as the mean ± standard error of the mean for at least three independent experiments. **P<0.01; #P<0.05, vs. treatment with BrMC (5 µm) and LY294002 (20 µm). p/t-Akt, phosphorylated/total Akt; BrMC, 8-bromo-7-methoxychrysin; DDP, cisplatin.

Mentions: Recent studies have indicated that Akt modulates Bim activation either directly or indirectly (34,35). Moreover, chrysin induced apoptosis in leukemia cells by decreasing Akt activity and expression in cells (36). Based on these observations, it was speculated that Akt may be involved in BrMC-induced apoptosis by regulating Bim expression. Analysis was performed to determine whether Akt was involved in BrMC-induced apoptosis in ovarian cancer cells. As shown in Fig. 3A, BrMC induced the downregulation of Akt phosphorylation at Ser 473 in cisplatin-sensitive A2780 cells in a time-dependent manner. However, expression levels of total Akt protein exhibited no change. Similarly, BrMC induced the reduction of p-Akt in A2780/DDP cells (Fig. 3B), regardless of differences in chemo-sensitivity. To validate the role of Akt in BrMC-induced apoptosis, LY294002, a phosphoinositide 3-kinase inhibitor, was used to inhibit the phosphorylation of Akt by upstream signaling molecules and the pro-apoptotic effect of BrMC in ovarian cancer cells was examined. BrMC alone downregulated p-Akt and induced apoptosis, while LY294002 alone was not sufficient to induce apoptosis, though it partially decreased p-Akt. Further experiments proved that the combination of LY294002 and BrMC completely eliminated p-Akt and enhanced the induction of apoptosis in A2780 and A2780/DDP cells (Fig. 3C and D). This indicated that the effect of the combination of LY294002 and BrMC on p-Akt and apoptosis was more significant than that of LY294002 alone, and that the Akt signaling pathway was involved in BrMC-induced apoptosis in ovarian cancer cells.


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)

Akt is involved in BrMC-induced apoptosis. (A and B) Expression levels of t-Akt and p-Akt (Ser473) were detected by western blot analysis in A2780cells and A2780/DDP cells. Cells were treated with BrMC (2.5 µM) for 24 h, then lysed and assayed for individual protein levels by western blot. β-Actin was used as a control. p-Akt and Akt levels were measured by densitometric analysis of the western blots and compared to actin levels. (C) Detection of the role of LY294002 on apoptosis of cells. Cells were treated with BrMC and/or 20 µM LY294002 for 24 h, and then individual protein levels were detected by western blotting. (D) Apoptotic rate of the A2780 and A2780/DDP cells. All data are depicted graphically as the mean ± standard error of the mean for at least three independent experiments. **P<0.01; #P<0.05, vs. treatment with BrMC (5 µm) and LY294002 (20 µm). p/t-Akt, phosphorylated/total Akt; BrMC, 8-bromo-7-methoxychrysin; DDP, cisplatin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3-mmr-12-04-5100: Akt is involved in BrMC-induced apoptosis. (A and B) Expression levels of t-Akt and p-Akt (Ser473) were detected by western blot analysis in A2780cells and A2780/DDP cells. Cells were treated with BrMC (2.5 µM) for 24 h, then lysed and assayed for individual protein levels by western blot. β-Actin was used as a control. p-Akt and Akt levels were measured by densitometric analysis of the western blots and compared to actin levels. (C) Detection of the role of LY294002 on apoptosis of cells. Cells were treated with BrMC and/or 20 µM LY294002 for 24 h, and then individual protein levels were detected by western blotting. (D) Apoptotic rate of the A2780 and A2780/DDP cells. All data are depicted graphically as the mean ± standard error of the mean for at least three independent experiments. **P<0.01; #P<0.05, vs. treatment with BrMC (5 µm) and LY294002 (20 µm). p/t-Akt, phosphorylated/total Akt; BrMC, 8-bromo-7-methoxychrysin; DDP, cisplatin.
Mentions: Recent studies have indicated that Akt modulates Bim activation either directly or indirectly (34,35). Moreover, chrysin induced apoptosis in leukemia cells by decreasing Akt activity and expression in cells (36). Based on these observations, it was speculated that Akt may be involved in BrMC-induced apoptosis by regulating Bim expression. Analysis was performed to determine whether Akt was involved in BrMC-induced apoptosis in ovarian cancer cells. As shown in Fig. 3A, BrMC induced the downregulation of Akt phosphorylation at Ser 473 in cisplatin-sensitive A2780 cells in a time-dependent manner. However, expression levels of total Akt protein exhibited no change. Similarly, BrMC induced the reduction of p-Akt in A2780/DDP cells (Fig. 3B), regardless of differences in chemo-sensitivity. To validate the role of Akt in BrMC-induced apoptosis, LY294002, a phosphoinositide 3-kinase inhibitor, was used to inhibit the phosphorylation of Akt by upstream signaling molecules and the pro-apoptotic effect of BrMC in ovarian cancer cells was examined. BrMC alone downregulated p-Akt and induced apoptosis, while LY294002 alone was not sufficient to induce apoptosis, though it partially decreased p-Akt. Further experiments proved that the combination of LY294002 and BrMC completely eliminated p-Akt and enhanced the induction of apoptosis in A2780 and A2780/DDP cells (Fig. 3C and D). This indicated that the effect of the combination of LY294002 and BrMC on p-Akt and apoptosis was more significant than that of LY294002 alone, and that the Akt signaling pathway was involved in BrMC-induced apoptosis in ovarian cancer cells.

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