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. 2017 Aug:12:198-207.
doi: 10.1016/j.redox.2017.02.017. Epub 2017 Feb 24.

Importance of ROS-mediated autophagy in determining apoptotic cell death induced by physapubescin B

Jian Xu  1 , Yihua Wu  1 , Guang Lu  2 , Shujun Xie  1 , Zhongjun Ma  3 , Zhe Chen  4 , Han-Ming Shen  5 , Dajing Xia  6
Affiliations

Importance of ROS-mediated autophagy in determining apoptotic cell death induced by physapubescin B

Jian Xu et al. Redox Biol. 2017 Aug.

Abstract

Physapubescin B, a steroidal compound extracted from the plant Physalis pubescens L. (Solanaceae), has been reported to possess anti-cancer potential, whereas the molecular mechanism remains elusive. In this study, we first demonstrated that physapubescin B induced autophagy in human cancer cells based on the evidence that physapubescin B increased lipidation of microtubule-associated protein 1 light chain 3 (LC3) as well as number of GFP-LC3 puncta. We further examined the molecular mechanisms and found that physapubescin B enhanced the autophagic flux through promotion of reactive oxygen species (ROS)-mediated suppression of mammalian target of rapamycin complex I (mTORC1), the key negative regulator of autophagy. Additionally, excessive ROS caused by physapubescin B also induced p53-dependent apoptotic cell death. Furthermore, we provided evidence that inhibition of autophagy either by a chemical inhibitor or gene silencing promoted physapubescin B-induced apoptotic cell death, indicating that autophagy serves as a cell survival mechanism to protect cell death. Thus, our data provide a clue that inhibition of autophagy would serve as a novel strategy for enhancing the anti-cancer potential of physapubescin B.

Keywords: Apoptotic cell death; Autophagy; MTORC1; Physapubescin B; ROS.

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Figures

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Graphical abstract
Fig. 1.
Fig. 1
Physapubescin B increases autophagic flux. (A) The chemical structure of physapubescin B. (B) HCT116 (D) HeLa cells were treated with the indicated doses of physapubescin B with or without chloroquine (CQ, 50 μM) for 3 h. Cell lysates were collected and subjected to western blot for detecting LC3 conversion. β-actin was used as a loading control. (C) HCT116 (E) HeLa proteins expression from (B) and (D) was evaluated by ImageJ, means±SD were presented (*, P<0.05; **, P<0.01; ns, P>0.05; Student's t-test). (F) HCT116 cells were treated with physapubescin B (20 μM) for the indicated time points with or without CQ (50 μM). Cell lysates were collected and subjected to western blot for detecting LC3 conversion. β-actin was used as a loading control, and (G) the proteins expression was evaluated by ImageJ, means±SD were presented (**, P<0.01; ns, P>0.05; Student's t-test). (H) HeLa cells stably expressing GFP-LC3 were incubated with the indicated concentrations of physapubescin B with or without CQ (50 μM) for 3 h. Autophagosomes were inferred by the presence of GFP-LC3 puncta under confocal microscopy (×600). Scale bar, 5 µm. Amino acid starvation (AA-) was used as a positive control for induction of autophagy. (I) The mean fluorescence intensity (MFI) was analyzed by ImageJ, means±SD were presented (*, P<0.05; **, P<0.01, Student's t-test).
Fig. 2.
Fig. 2
Physapubescin B inhibits mTORC1 activity. (A) HCT116 cells were treated with the indicated concentrations of physapubescin B for 3 h. Cell lysates were collected and subjected to western blot. Amino acid-free DMEM (AA-, 3 h) was used as a positive control for mTORC1 suppression. And (B) proteins expression was evaluated by ImageJ, means±SD were presented (*, P<0.05; **, P<0.01; ns, P>0.05; Student's t-test). (C) HCT116 cells were treated with physapubescin B (20 μM) for various time points as indicated or amino acid-free DMEM (AA-) for 3 h. Cell lysates were collected for western blot analysis. And (D) proteins expression was evaluated by ImageJ, means±SD were presented (**, P<0.01; ns, P>0.05; Student's t-test).
Fig. 3.
Fig. 3
Physapubescin B suppresses mTORC1 and induces autophagy through ROS generation. (A) HCT116 cells were treated with physapubescin B (20 μM) with or without NAC (5 mM) for 4 h followed by incubation with CM-H2DCFDA. The cells were then observed under a fluorescence microscope (x100). Scale bar, 200 µm. (B) HCT116 cells were treated as in (A). The fluorescence intensity was analyzed by flow cytometry. (C) Statistical analysis of the fluorescence intensity of HCT116 cells as treated in (A). Data (mean±SD) are representative of three independent experiments. (**, P<0.01, Student's t-test). (D) HCT116 cells were treated as in (A). Cell lysate was harvested and subjected to western blot, and (E) proteins expression was evaluated by ImageJ, means±SD were presented (**, P<0.01, Student's t-test).
Fig. 4.
Fig. 4
Physapubescin B induces apoptotic cell death. (A) HCT116 and HeLa cells were treated with physapubescin B (0 5 10 20 μM) for 24 h. After treatment, the cells were then followed by propidium iodide (PI, 5 μg/ml) live cell exclusion staining for quantification of cell death. Data (mean±SD) are representative of three independent experiments. (*, P<0.05; **, P<0.01; ns, P>0.05; Student's t-test). (B) HCT116 cells were treated with physapubescin B (0 5 10 20 μM) for 24 h. The cells were then harvested and subjected to western blot analysis, and (C) proteins expression was evaluated by ImageJ, means±SD were presented (*, P<0.05; **, P<0.01, Student's t-test). (D) HCT116 cells were harvested after treatment with physapubescin B (20 μM) for the indicated periods of time, western blot was then performed for the detection of various apoptosis markers. β-actin was used as a loading control. And (E) proteins expression was evaluated by ImageJ, means±SD were presented (*, P<0.05; **, P<0.01, Student's t-test). (F) HCT116 cells were treated with physapubescin B (20 μM) alone or combined with zVAD (20 μM) for 24 h followed by western blot to detect apoptosis markers, and (G) proteins expression was evaluated by ImageJ (**, P<0.01, Student's t-test). (H) HCT116 cells were treated as in (F) and the cell death was quantified by flow cytometry after PI staining (*, P<0.05, Student's t-test).
Fig. 5.
Fig. 5
Physapubescin B-induced cell death is partly dependent on ROS-p53 axis. (A) HCT116 cells were incubated with physapubescin B (0 5 10 20 μM) for 24 h, western blot analysis was performed to detect p53, p21 levels, and (B) proteins expression was evaluated by ImageJ, means±SD were presented (*, P<0.05; **, P<0.01, Student's t-test). (C) HCT116 cells were treated with physapubescin B (20 μM) with or without NAC (5 mM) for 24 h. Protein levels of p53, p21 and β-actin were evaluated by western blot, and (D) proteins expression was evaluated by ImageJ, means±SD were presented (**, P<0.01, Student's t-test). (E) HCT116 p53+/+(WT) and p53-/- (KO) cells were treated as indicated, cell lysate was collected and subjected to western blot, and (F) proteins expression was evaluated by ImageJ, means±SD were presented (*, P<0.05; **, P<0.01; ns, P>0.05; Student's t-test). (G) HCT116 p53+/+(WT) and p53-/- (KO) cells were treated with physapubescin B (20 μM) for 24 h. Cell morphology was observed by a microscope (x200). Scale bar, 100 µm. (H) Cell viability of (G) was quantified by flow cytometry combined with PI staining. Data (mean±SD) are representative of three independent experiments (*, P<0.05, Student's t-test).
Fig. 6.
Fig. 6
Physapubescin B-induced autophagy acts as a cell survival mechanism. (A) HeLa cells were treated with the indicated doses of physapubescin B with or without CQ (50 μM) for 24 h. Cell morphology was observed under a microscope. Scale bar, 50 µm. (B) Shown are representative dot-plots of flow cytometry data of the PI exclusion assay. HeLa cells were incubated in physapubescin B (20 μM) with or without CQ (50 μM) for 24 h. (C) Quantification of the cell viability data from panel A is shown. Data were presented as the means±SD of three independent experiments (*, P<0.05; **, P<0.01, Student's t-test). (D) HeLa cells were treated as in (A), cell lysate was collected for western blot analysis, and (E) proteins expression was evaluated by ImageJ, means±SD were presented (**, P<0.01, Student's t-test). (F) HCT116 cells were transiently transfected with a nonspecific siRNA or Atg7-targeted siRNA for 48 h and treated with physapubescin B (20 μM) for the indicated time periods. Cell lysate was harvested and subjected to western blot, and (G) proteins expression was evaluated by ImageJ, means±SD were presented (*, P<0.05; **, P<0.01, Student's t-test).
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