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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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AA005-induced cell death significantly decreases inAIFknockdown cells. (A) SW620 cells were transfected with scrambled negative control (NC) or siRNAs against AIF (A3 or A5); absence of AIF expression was confirmed by western blot analysis, standardized to actin. (B–D)AIF knockdown SW620 cells and controls were treated with or without 1 μM AA005 for 48 h (B), 500 μM MNNG for 8 h (C), and 20 μM camptothecin for 36 h (D). Annexin-V/PI double stained cells and cell death were measured on flow cytometry. All experiments were repeated 3 times with the same results. Results show mean ± S.D. **P < 0.01, versus AA005- or MNNG-treated NC group.
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Fig5: AA005-induced cell death significantly decreases inAIFknockdown cells. (A) SW620 cells were transfected with scrambled negative control (NC) or siRNAs against AIF (A3 or A5); absence of AIF expression was confirmed by western blot analysis, standardized to actin. (B–D)AIF knockdown SW620 cells and controls were treated with or without 1 μM AA005 for 48 h (B), 500 μM MNNG for 8 h (C), and 20 μM camptothecin for 36 h (D). Annexin-V/PI double stained cells and cell death were measured on flow cytometry. All experiments were repeated 3 times with the same results. Results show mean ± S.D. **P < 0.01, versus AA005- or MNNG-treated NC group.

Mentions: To assess involvement of AIF, SW620 cells were transfected with retrovirus harboring NC or siRNAs against AIF (designated as A3 and A5; Figure 5A). Absence of AIF expression was confirmed by western blot analysis (Figure 5A). Furthermore, AIF knockdown almost completely blocked the cell death induced by AA005 (Figure 5B). We also confirmed that AIF knockdown inhibited the cell death induced by MNNG, the action of which is reportedly mediated by AIF (Figure 5C) [20], but had no effect on camptothecin-induced cell death, which is caspase-dependent (Figure 5D). Together, these results indicate that AA005 promote AIF nuclear translocation and trigger AIF-dependent cell death.Figure 5


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-induced cell death significantly decreases inAIFknockdown cells. (A) SW620 cells were transfected with scrambled negative control (NC) or siRNAs against AIF (A3 or A5); absence of AIF expression was confirmed by western blot analysis, standardized to actin. (B–D)AIF knockdown SW620 cells and controls were treated with or without 1 μM AA005 for 48 h (B), 500 μM MNNG for 8 h (C), and 20 μM camptothecin for 36 h (D). Annexin-V/PI double stained cells and cell death were measured on flow cytometry. All experiments were repeated 3 times with the same results. Results show mean ± S.D. **P < 0.01, versus AA005- or MNNG-treated NC group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4379763&req=5

Fig5: AA005-induced cell death significantly decreases inAIFknockdown cells. (A) SW620 cells were transfected with scrambled negative control (NC) or siRNAs against AIF (A3 or A5); absence of AIF expression was confirmed by western blot analysis, standardized to actin. (B–D)AIF knockdown SW620 cells and controls were treated with or without 1 μM AA005 for 48 h (B), 500 μM MNNG for 8 h (C), and 20 μM camptothecin for 36 h (D). Annexin-V/PI double stained cells and cell death were measured on flow cytometry. All experiments were repeated 3 times with the same results. Results show mean ± S.D. **P < 0.01, versus AA005- or MNNG-treated NC group.
Mentions: To assess involvement of AIF, SW620 cells were transfected with retrovirus harboring NC or siRNAs against AIF (designated as A3 and A5; Figure 5A). Absence of AIF expression was confirmed by western blot analysis (Figure 5A). Furthermore, AIF knockdown almost completely blocked the cell death induced by AA005 (Figure 5B). We also confirmed that AIF knockdown inhibited the cell death induced by MNNG, the action of which is reportedly mediated by AIF (Figure 5C) [20], but had no effect on camptothecin-induced cell death, which is caspase-dependent (Figure 5D). Together, these results indicate that AA005 promote AIF nuclear translocation and trigger AIF-dependent cell death.Figure 5

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