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Role of annexin a5 on mitochondria-dependent apoptosis induced by tetramethoxystilbene in human breast cancer cells.

Hong M, Park N, Chun YJ - Biomol Ther (Seoul) (2014)

Bottom Line: Moreover, we found that the cytosolic release of apoptogenic factors such as cytochrome c, or apoptosis-inducing factor (AIF) in mitochondria was markedly increased.Annexin A5 depletion by siRNA led to decreased proapoptotic factors such as Bax, Bak, and annexin A5.Taken together, our results indicate that annexin A5 may play an important role in TMS-mediated mitochondrial apoptosis through the regulation of proapoptotic proteins and VDAC1 expression.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea.

ABSTRACT
We have previously shown that 2,4,3',5'-tetramethoxystilbene (TMS), a trans-stilbene analogue, induces apoptosis in human cancer cells. However, the detailed mechanisms of mitochondria-dependent apoptosis induced by TMS are not fully understood. In the present study, the possible roles of annexin A5 in TMS-mediated apoptosis were investigated in MCF7 human breast cancer cells. Quantitative real-time PCR analysis and Western blot analysis showed that the expression of annexin A5 was strongly increased in TMS-treated cells. TMS caused a strong translocation of annexin A5 from cytosol into mitochondria. Confocal laser scanning microscopic analysis clearly showed that TMS induced translocation of annexin A5 into mitochondria. TMS increased the expression and oligomerization of voltage-dependent anion channel (VDAC) 1, which may promote mitochondria-dependent apoptosis through disruption of mitochondrial membrane potential. When cells were treated with TMS, the levels of Bax, and Bak as well as annexin A5 were strongly enhanced. Moreover, we found that the cytosolic release of apoptogenic factors such as cytochrome c, or apoptosis-inducing factor (AIF) in mitochondria was markedly increased. Annexin A5 depletion by siRNA led to decreased proapoptotic factors such as Bax, Bak, and annexin A5. Taken together, our results indicate that annexin A5 may play an important role in TMS-mediated mitochondrial apoptosis through the regulation of proapoptotic proteins and VDAC1 expression.

No MeSH data available.


Related in: MedlinePlus

TMS induces VDAC1 expression and oligomerization. (A) Western blot analysis. MCF-7 cells were treated with various concentrations of TMS (0, 1, 5, or 10 μM) for 48 h. After incubation, cells were harvested and extracted proteins were resolved by SDS-PAGE (10%) and Western blot analysis was conducted. GAPDH level was determined as loading controls. (B) VDAC oligomerization. Cells were treated with TMS (0, 1, 5, or 10 μM) for 48 h. Cells were then harvested and incubated with sulfo-EGS (250 μM) for 20 min at 30°C. After proteins were resolved by SDS-PAGE (8%), VDAC1 proteins were measured using Western blot analysis. A 33-kDa band represents VDAC monomers, while a 65-kDa band represents VDAC dimers. (C) Mitochondrial membrane potential. After cells were incubated with TMS for 48 h, cells were labeled with 100 nM TMRM for 20 min. After washing, cells were analyzed by flow cytometry. TMRM was monitored as log FL2-H (x axis, 574 nM) versus relative cell number (y axis) in the histogram. The data shown are representative of three independent experiments.
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f3-bt-22-519: TMS induces VDAC1 expression and oligomerization. (A) Western blot analysis. MCF-7 cells were treated with various concentrations of TMS (0, 1, 5, or 10 μM) for 48 h. After incubation, cells were harvested and extracted proteins were resolved by SDS-PAGE (10%) and Western blot analysis was conducted. GAPDH level was determined as loading controls. (B) VDAC oligomerization. Cells were treated with TMS (0, 1, 5, or 10 μM) for 48 h. Cells were then harvested and incubated with sulfo-EGS (250 μM) for 20 min at 30°C. After proteins were resolved by SDS-PAGE (8%), VDAC1 proteins were measured using Western blot analysis. A 33-kDa band represents VDAC monomers, while a 65-kDa band represents VDAC dimers. (C) Mitochondrial membrane potential. After cells were incubated with TMS for 48 h, cells were labeled with 100 nM TMRM for 20 min. After washing, cells were analyzed by flow cytometry. TMRM was monitored as log FL2-H (x axis, 574 nM) versus relative cell number (y axis) in the histogram. The data shown are representative of three independent experiments.

Mentions: To elucidate the mechanism of TMS-induced apoptosis, we focused on the role of VDAC1 in mitochondria. MCF-7 cells were treated with TMS (1, 5, or 10 μM) for 48 h and VDAC1 protein expression and formation of VDAC dimer and oligomers were determined. TMS clearly increased the expression of VDAC in a concentration-dependent manner. Moreover, the formation of VDAC dimer and oligomers was significantly enhanced in TMS-treated cells (Fig. 3A, B). To determine the effect of VDAC1 on mitochondrial depolarization, cells were treated with TMS (1, 5, or 10 μM) for measuring mitochondrial membrane potential. TMS caused a significant decrease in FL2-H fluorescence, indicating TMS may induce disruption of mitochondrial membrane potential through inducing VDAC1 expression and oligomerization.


Role of annexin a5 on mitochondria-dependent apoptosis induced by tetramethoxystilbene in human breast cancer cells.

Hong M, Park N, Chun YJ - Biomol Ther (Seoul) (2014)

TMS induces VDAC1 expression and oligomerization. (A) Western blot analysis. MCF-7 cells were treated with various concentrations of TMS (0, 1, 5, or 10 μM) for 48 h. After incubation, cells were harvested and extracted proteins were resolved by SDS-PAGE (10%) and Western blot analysis was conducted. GAPDH level was determined as loading controls. (B) VDAC oligomerization. Cells were treated with TMS (0, 1, 5, or 10 μM) for 48 h. Cells were then harvested and incubated with sulfo-EGS (250 μM) for 20 min at 30°C. After proteins were resolved by SDS-PAGE (8%), VDAC1 proteins were measured using Western blot analysis. A 33-kDa band represents VDAC monomers, while a 65-kDa band represents VDAC dimers. (C) Mitochondrial membrane potential. After cells were incubated with TMS for 48 h, cells were labeled with 100 nM TMRM for 20 min. After washing, cells were analyzed by flow cytometry. TMRM was monitored as log FL2-H (x axis, 574 nM) versus relative cell number (y axis) in the histogram. The data shown are representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4256031&req=5

f3-bt-22-519: TMS induces VDAC1 expression and oligomerization. (A) Western blot analysis. MCF-7 cells were treated with various concentrations of TMS (0, 1, 5, or 10 μM) for 48 h. After incubation, cells were harvested and extracted proteins were resolved by SDS-PAGE (10%) and Western blot analysis was conducted. GAPDH level was determined as loading controls. (B) VDAC oligomerization. Cells were treated with TMS (0, 1, 5, or 10 μM) for 48 h. Cells were then harvested and incubated with sulfo-EGS (250 μM) for 20 min at 30°C. After proteins were resolved by SDS-PAGE (8%), VDAC1 proteins were measured using Western blot analysis. A 33-kDa band represents VDAC monomers, while a 65-kDa band represents VDAC dimers. (C) Mitochondrial membrane potential. After cells were incubated with TMS for 48 h, cells were labeled with 100 nM TMRM for 20 min. After washing, cells were analyzed by flow cytometry. TMRM was monitored as log FL2-H (x axis, 574 nM) versus relative cell number (y axis) in the histogram. The data shown are representative of three independent experiments.
Mentions: To elucidate the mechanism of TMS-induced apoptosis, we focused on the role of VDAC1 in mitochondria. MCF-7 cells were treated with TMS (1, 5, or 10 μM) for 48 h and VDAC1 protein expression and formation of VDAC dimer and oligomers were determined. TMS clearly increased the expression of VDAC in a concentration-dependent manner. Moreover, the formation of VDAC dimer and oligomers was significantly enhanced in TMS-treated cells (Fig. 3A, B). To determine the effect of VDAC1 on mitochondrial depolarization, cells were treated with TMS (1, 5, or 10 μM) for measuring mitochondrial membrane potential. TMS caused a significant decrease in FL2-H fluorescence, indicating TMS may induce disruption of mitochondrial membrane potential through inducing VDAC1 expression and oligomerization.

Bottom Line: Moreover, we found that the cytosolic release of apoptogenic factors such as cytochrome c, or apoptosis-inducing factor (AIF) in mitochondria was markedly increased.Annexin A5 depletion by siRNA led to decreased proapoptotic factors such as Bax, Bak, and annexin A5.Taken together, our results indicate that annexin A5 may play an important role in TMS-mediated mitochondrial apoptosis through the regulation of proapoptotic proteins and VDAC1 expression.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea.

ABSTRACT
We have previously shown that 2,4,3',5'-tetramethoxystilbene (TMS), a trans-stilbene analogue, induces apoptosis in human cancer cells. However, the detailed mechanisms of mitochondria-dependent apoptosis induced by TMS are not fully understood. In the present study, the possible roles of annexin A5 in TMS-mediated apoptosis were investigated in MCF7 human breast cancer cells. Quantitative real-time PCR analysis and Western blot analysis showed that the expression of annexin A5 was strongly increased in TMS-treated cells. TMS caused a strong translocation of annexin A5 from cytosol into mitochondria. Confocal laser scanning microscopic analysis clearly showed that TMS induced translocation of annexin A5 into mitochondria. TMS increased the expression and oligomerization of voltage-dependent anion channel (VDAC) 1, which may promote mitochondria-dependent apoptosis through disruption of mitochondrial membrane potential. When cells were treated with TMS, the levels of Bax, and Bak as well as annexin A5 were strongly enhanced. Moreover, we found that the cytosolic release of apoptogenic factors such as cytochrome c, or apoptosis-inducing factor (AIF) in mitochondria was markedly increased. Annexin A5 depletion by siRNA led to decreased proapoptotic factors such as Bax, Bak, and annexin A5. Taken together, our results indicate that annexin A5 may play an important role in TMS-mediated mitochondrial apoptosis through the regulation of proapoptotic proteins and VDAC1 expression.

No MeSH data available.


Related in: MedlinePlus