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Annonaceous acetogenin mimic AA005 induces cancer cell death via apoptosis inducing factor through a caspase-3-independent mechanism.

Han B, Wang TD, Shen SM, Yu Y, Mao C, Yao ZJ, Wang LS - BMC Cancer (2015)

Bottom Line: AA005 treatment also reduced expression of mitochondrial Complex I components, and leads to accumulation of intracellular reactive oxygen species (ROS) at the early stage.Blocking ROS formation significantly suppresses AA005-induced cell death in SW620 cells.AA005 may trigger the cell death via mediated by AIF through caspase-3 independent pathway.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China. hb054080108@126.com.

ABSTRACT

Background: Annonaceous acetogenins are a family of natural products with antitumor activities. Annonaceous acetogenin mimic AA005 reportedly inhibits mammalian mitochondrial NADH-ubiquinone reductase (Complex I) and induces gastric cancer cell death. However, the mechanisms underlying its cell-death-inducing activity are unclear.

Methods: We used SW620 colorectal adenocarcinoma cells to study AA005 cytotoxic activity. Cell deaths were determined by Trypan blue assay and flow cytometry, and related proteins were characterized by western blot. Immunofluorescence and subcellular fractionation were used to evaluate AIF nuclear translocation. Reactive oxygen species were assessed by using redox-sensitive dye DCFDA.

Results: AA005 induces a unique type of cell death in colorectal adenocarcinoma cells, characterized by lack of caspase-3 activation or apoptotic body formation, sensitivity to poly (ADP-ribose) polymerase inhibitor Olaparib (AZD2281) but not pan-caspase inhibitor Z-VAD.fmk, and dependence on apoptosis-inducing factor (AIF). AA005 treatment also reduced expression of mitochondrial Complex I components, and leads to accumulation of intracellular reactive oxygen species (ROS) at the early stage. Blocking ROS formation significantly suppresses AA005-induced cell death in SW620 cells. Moreover, blocking activation of RIP-1 by necroptosis inhibitor necrotatin-1 inhibits AIF translocation and partially suppresses AA005-induced cell death in SW620 cells demonstrating that RIP-1 protein may be essential for cell death.

Conclusions: AA005 may trigger the cell death via mediated by AIF through caspase-3 independent pathway. Our work provided new mechanisms for AA005-induced cancer cell death and novel clues for cancer treatment via AIF dependent cell death.

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Related in: MedlinePlus

AA005 induces cell death through caspase-3-independent mechanisms. (A) SW620 cells were treated with or without 1 μM AA005 for the indicated times. Treatment with 20 μM camptothecin for 36 h was used as positive control. Proteins PARP-1, and caspase-3 were detected by western blot, standardized to actin. ΔPARP-1, and ΔCaspase-3 indicate cleaved 85-kDa fragment of PARP-1 and active Caspase-3 fragment proteins, respectively. (B) SW620 cells were preincubated with 100 μM Z-VAD.fmk for 1 h and then treated with or without 1 μM AA005 for 48 h, and with 20 μM camptothecin for 36 h. Annexin-V/PI double-stained cells and cell death were measured by flow cytometry. Results show mean ± S.D. *P < 0.05, versus camptothecin-treated group. (C, D) NB4 cells were preincubated with 40 μM Z-VAD.fmk for 1 h and then treated with or without 1 μM camptothecin (C), or 1 μM AA005 (D), for indicated times. Annexin-V/PI double-stained cells and dead cells were measured on flow cytometry. Values show mean ± S.D. of triplicates in an independent experiment.
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Fig3: AA005 induces cell death through caspase-3-independent mechanisms. (A) SW620 cells were treated with or without 1 μM AA005 for the indicated times. Treatment with 20 μM camptothecin for 36 h was used as positive control. Proteins PARP-1, and caspase-3 were detected by western blot, standardized to actin. ΔPARP-1, and ΔCaspase-3 indicate cleaved 85-kDa fragment of PARP-1 and active Caspase-3 fragment proteins, respectively. (B) SW620 cells were preincubated with 100 μM Z-VAD.fmk for 1 h and then treated with or without 1 μM AA005 for 48 h, and with 20 μM camptothecin for 36 h. Annexin-V/PI double-stained cells and cell death were measured by flow cytometry. Results show mean ± S.D. *P < 0.05, versus camptothecin-treated group. (C, D) NB4 cells were preincubated with 40 μM Z-VAD.fmk for 1 h and then treated with or without 1 μM camptothecin (C), or 1 μM AA005 (D), for indicated times. Annexin-V/PI double-stained cells and dead cells were measured on flow cytometry. Values show mean ± S.D. of triplicates in an independent experiment.

Mentions: To elucidate the mechanisms of AA005-induced cell death, we analyzed the involvement of caspase-3, an important executing caspase of apoptosis. Camptothecin treatment was used as a positive control. AA005 treatment failed to trigger cleavage of caspase-3 and its substrate PARP-1, even when cell death was as high as 55% (Figure 3A). Additionally, Z-VAD.fmk, a broad-spectrum caspase inhibitor, greatly blocked camptothecin-induced cell death, but showed no effect on AA005-induced cell death (Figure 3B). To further investigate whether caspase-3 played a role in AA005-induced cell death, we also tested caspase-3 activation in NB4 cells. Similarly, Z-VAD.fmk blocked most cell death triggered by camptothecin (Figure 3C), but had no effect on the cell death induced by AA005 in NB4 cells (Figure 3D). Taken together, these results suggested that AA005 induced cell death through caspase-3 independent mechanisms.Figure 3


Annonaceous acetogenin mimic AA005 induces cancer cell death via apoptosis inducing factor through a caspase-3-independent mechanism.

Han B, Wang TD, Shen SM, Yu Y, Mao C, Yao ZJ, Wang LS - BMC Cancer (2015)

AA005 induces cell death through caspase-3-independent mechanisms. (A) SW620 cells were treated with or without 1 μM AA005 for the indicated times. Treatment with 20 μM camptothecin for 36 h was used as positive control. Proteins PARP-1, and caspase-3 were detected by western blot, standardized to actin. ΔPARP-1, and ΔCaspase-3 indicate cleaved 85-kDa fragment of PARP-1 and active Caspase-3 fragment proteins, respectively. (B) SW620 cells were preincubated with 100 μM Z-VAD.fmk for 1 h and then treated with or without 1 μM AA005 for 48 h, and with 20 μM camptothecin for 36 h. Annexin-V/PI double-stained cells and cell death were measured by flow cytometry. Results show mean ± S.D. *P < 0.05, versus camptothecin-treated group. (C, D) NB4 cells were preincubated with 40 μM Z-VAD.fmk for 1 h and then treated with or without 1 μM camptothecin (C), or 1 μM AA005 (D), for indicated times. Annexin-V/PI double-stained cells and dead cells were measured on flow cytometry. Values show mean ± S.D. of triplicates in an independent experiment.
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Related In: Results  -  Collection

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Fig3: AA005 induces cell death through caspase-3-independent mechanisms. (A) SW620 cells were treated with or without 1 μM AA005 for the indicated times. Treatment with 20 μM camptothecin for 36 h was used as positive control. Proteins PARP-1, and caspase-3 were detected by western blot, standardized to actin. ΔPARP-1, and ΔCaspase-3 indicate cleaved 85-kDa fragment of PARP-1 and active Caspase-3 fragment proteins, respectively. (B) SW620 cells were preincubated with 100 μM Z-VAD.fmk for 1 h and then treated with or without 1 μM AA005 for 48 h, and with 20 μM camptothecin for 36 h. Annexin-V/PI double-stained cells and cell death were measured by flow cytometry. Results show mean ± S.D. *P < 0.05, versus camptothecin-treated group. (C, D) NB4 cells were preincubated with 40 μM Z-VAD.fmk for 1 h and then treated with or without 1 μM camptothecin (C), or 1 μM AA005 (D), for indicated times. Annexin-V/PI double-stained cells and dead cells were measured on flow cytometry. Values show mean ± S.D. of triplicates in an independent experiment.
Mentions: To elucidate the mechanisms of AA005-induced cell death, we analyzed the involvement of caspase-3, an important executing caspase of apoptosis. Camptothecin treatment was used as a positive control. AA005 treatment failed to trigger cleavage of caspase-3 and its substrate PARP-1, even when cell death was as high as 55% (Figure 3A). Additionally, Z-VAD.fmk, a broad-spectrum caspase inhibitor, greatly blocked camptothecin-induced cell death, but showed no effect on AA005-induced cell death (Figure 3B). To further investigate whether caspase-3 played a role in AA005-induced cell death, we also tested caspase-3 activation in NB4 cells. Similarly, Z-VAD.fmk blocked most cell death triggered by camptothecin (Figure 3C), but had no effect on the cell death induced by AA005 in NB4 cells (Figure 3D). Taken together, these results suggested that AA005 induced cell death through caspase-3 independent mechanisms.Figure 3

Bottom Line: AA005 treatment also reduced expression of mitochondrial Complex I components, and leads to accumulation of intracellular reactive oxygen species (ROS) at the early stage.Blocking ROS formation significantly suppresses AA005-induced cell death in SW620 cells.AA005 may trigger the cell death via mediated by AIF through caspase-3 independent pathway.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China. hb054080108@126.com.

ABSTRACT

Background: Annonaceous acetogenins are a family of natural products with antitumor activities. Annonaceous acetogenin mimic AA005 reportedly inhibits mammalian mitochondrial NADH-ubiquinone reductase (Complex I) and induces gastric cancer cell death. However, the mechanisms underlying its cell-death-inducing activity are unclear.

Methods: We used SW620 colorectal adenocarcinoma cells to study AA005 cytotoxic activity. Cell deaths were determined by Trypan blue assay and flow cytometry, and related proteins were characterized by western blot. Immunofluorescence and subcellular fractionation were used to evaluate AIF nuclear translocation. Reactive oxygen species were assessed by using redox-sensitive dye DCFDA.

Results: AA005 induces a unique type of cell death in colorectal adenocarcinoma cells, characterized by lack of caspase-3 activation or apoptotic body formation, sensitivity to poly (ADP-ribose) polymerase inhibitor Olaparib (AZD2281) but not pan-caspase inhibitor Z-VAD.fmk, and dependence on apoptosis-inducing factor (AIF). AA005 treatment also reduced expression of mitochondrial Complex I components, and leads to accumulation of intracellular reactive oxygen species (ROS) at the early stage. Blocking ROS formation significantly suppresses AA005-induced cell death in SW620 cells. Moreover, blocking activation of RIP-1 by necroptosis inhibitor necrotatin-1 inhibits AIF translocation and partially suppresses AA005-induced cell death in SW620 cells demonstrating that RIP-1 protein may be essential for cell death.

Conclusions: AA005 may trigger the cell death via mediated by AIF through caspase-3 independent pathway. Our work provided new mechanisms for AA005-induced cancer cell death and novel clues for cancer treatment via AIF dependent cell death.

Show MeSH
Related in: MedlinePlus