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. 2017 Apr 18;8(16):26992-27006.
doi: 10.18632/oncotarget.15935.

Hispidin Induces Autophagic and Necrotic Death in SGC-7901 Gastric Cancer Cells Through Lysosomal Membrane Permeabilization by Inhibiting Tubulin Polymerization

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Hispidin Induces Autophagic and Necrotic Death in SGC-7901 Gastric Cancer Cells Through Lysosomal Membrane Permeabilization by Inhibiting Tubulin Polymerization

Long-Xian Lv et al. Oncotarget. .
Free PMC article

Abstract

Hispidin and its derivatives are widely distributed in edible mushrooms. Hispidin is more cytotoxic to A549, SCL-1, Bel7402 and Capan-1 cancer cells than to MRC5 normal cells; by contrast, hispidin protects H9c2 cardiomyoblast cells from hydrogen peroxide-induced or doxorubicin-induced apoptosis. Consequently, further research on how hispidin affects normal and cancer cells may help treat cancer and reduce chemotherapy-induced side effects. This study showed that hispidin caused caspase-independent death in SGC-7901 cancer cells but not in GES-1 normal cells. Hispidin-induced increases in LC3-II occurred in SGC-7901 cells in a time independent manner. Cell death can be partially inhibited by treatment with ATG5 siRNA but not by autophagy or necroptosis inhibitors. Ultrastructural evidence indicated that hispidin-induced necrotic cell death involved autophagy. Hispidin-induced lysosomal membrane permeabilization (LMP) related to complex cell death occurred more drastically in SGC-7901 cells than in GES-1 cells. Ca2+ rather than cathepsins from LMP contributed more to cell death. Hispidin induced microtubule depolymerization, which can cause LMP, more drastically in SGC-7901 cells than in GES-1 cells. At 4.1 μM, hispidin promoted cell-free tubulin polymerization but at concentrations higher than 41 μM, hispidin inhibited polymerization. Hispidin did not bind to tubulin. Alterations in microtubule regulatory proteins, such as stathmin phosphorylation at Ser16, contributed to hispidin-induced SGC-7901 cell death. In conclusion, hispidin at concentrations higher than 41 μM may inhibit tubulin polymerization by modulating microtubule regulatory proteins, such as stathmin, causing LMP and complex SGC-7901 cell death. This mechanism suggests a promising novel treatment for human cancer.

Keywords: autophagy; hispidin; lysosomal membrane permeabilization; microtubule depolymerization; necrotic cell death.

Conflict of interest statement

CONFLICTS OF INTEREST

There is no conflicts of interest.

Figures

Figure 1
Figure 1. Hispidin induces caspase-independent cell death in SGC-7901 cells
(A) Chemical structure of hispidin. Cells were incubated with hispidin (41, 82 or 122 μM) or 0.1% DMSO for 12, 24, 48 or 72 h. The viability of SGC-7901 (B) and GES-1 (C) cells was determined using the MTT assay. (D) Cells were incubated with hispidin or 0.1% DMSO and then were assayed for phosphatidyl serine externalization and PI permeability. (E) SGC-7901 cells were treated with 41, 81, or 122 μM hispidin; 0.1% DMSO; or 3.4 μM Adriamycin for 6 h. Then, caspase activity was examined. (F) SGC-7901 cells were treated with hispidin or Adriamycin. Then, caspase-3, caspase-8 and caspase-9 were detected by Western blotting using β-actin as an internal control. (G) SGC-7901 cells were preincubated in the presence or absence of 25 or 50 μM z-VAD-FMK for 2 h before being treated with 122 μM hispidin. Then, the cells were examined for PI permeability.
Figure 2
Figure 2. Hispidin induces necrotic cell death involving autophagy in SGC-7901 cells
(A) SGC-7901 cells were treated with hispidin or rapamycin. Then, LC3-I and LC3-II were detected by Western blotting using β-actin as an internal control. (B) SGC-7901 cells were transiently transfected with mCherry-GFP-LC3B and treated with hispidin, 3-MA, or rapamycin containing medium. The colocalization of GFP and mCherry signals was analyzed. Quantitation represents the number of autophagosomes and autolysosomes per cell (n = 20). (C) After preincubation with either 10 mM 3-Methyladenine (3-MA), 100 nM wortmannin, 100 μM hydroxychloroquine, 100 nM bafilomycin A1, 10 μg/mL pepstatin A or 10 μg/mL E64d SGC-7901 cells were treated with 122 μM hispidin and assayed for cell viability. (D and E) SGC-7901 cells were transfected with scrambled RNA and ATG5 RNA for 48 h, and ATG levels were determined by Western blot; cell viability was assayed using MTT. (F) SGC-7901 cells were preincubated in the presence or absence of 25, 50 or 100 nM necrostatin-1 for 2 h before being treated with 122 μM hispidin. Then, the cells were examined for viability. (G) Transmission electron microscopy of SGC-7901 cells. Cells were treated with 122 μM hispidin.
Figure 3
Figure 3. Hispidin induces lysosomal membrane permeabilization (LMP)
(A) SGC-7901 and GES-1 cells were treated with 122 μM hispidin at different time points and then tested for LMP with acridine orange and LysoTracker red. (B) After being incubated with 122 μM hispidin or 0.1% DMSO for 0.5, 1, 2, or 3 h, SGC-7901 and GES-1 cells were assayed for Fluo-3 fluorescence. (C) SGC-7901 cells were treated with 122 μM hispidin for 30 min and examined for Fluo-3 (Ca2+) and the LysoTracker, ER-Tracker and MitoTracker signals. After preincubation with BAPTA AM (D) or cathepsin inhibitor 1 (F) for 2 h, SGC-7901 cells were treated with or without 122 μM hispidin for 24 h and assayed for cell viability using MTT.
Figure 4
Figure 4. Hispidin induces LMP-related nitric oxide (NO) production, contributing to SGC-7901 cancer cell death
(A) After being incubated with 122 μM hispidin or 0.1% DMSO for 0.5, 1, 2, or 3 h, SGC-7901 and GES-1 cells were assayed for DAF fluorescence. (B) After preincubation with either 20 μM carboxy-PTIO or 20 μM hemoglobin for 2 h, SGC-7901 cells were treated with 122 μM hispidin and assayed for PI permeability. (C) After being incubated with 122 μM hispidin or 0.1% DMSO, SGC-7901 and GES-1 cells were assayed for ATP. (D) After preincubation with L-NMMA, L-NAME and febuxostat for 2 h, SGC-7901 cells were treated with 122 μM hispidin or 0.1% DMSO for 24 h and assayed for cell viability using the MTT assay. (E) After preincubation with 0, 5, 10, 20 μM febuxostat for 2 h, SGC-7901 cells were stained with DAF-FM-DA, incubated with 122 μM hispidin, and assayed for DAF fluorescence (NO) for 90 min with a Microplate (F) After preincubation with 0, 20 μM BAPTA AM for 2 h, SGC-7901 cells were stained with DAF-FM-DA, incubated with 122 μM hispidin, and assayed for DAF fluorescence (NO) for 90 min with a Microplate Reader.
Figure 5
Figure 5. LMP-related redox system destruction increases oxidative stress in SGC-7901 cancer cells
(A) After being incubated with 122 μM hispidin or 0.1% DMSO for 0.5, 1, 2, or 3 h, SGC-7901 and GES-1 cells were assayed for DCFH fluorescence. (B) After preincubation with either 2000 U catalase or 10 mM NAC for 2 h, SGC-7901 cells were treated with 122 μM hispidin and assayed for PI permeability. (C) After being incubated with 122 μM hispidin or 0.1% DMSO for 0.5, 1, 2, or 3 h, SGC-7901 and GES-1 cells were assayed for GSH. *P < 0.05 vs GES-1 samples treated under the same conditions. (D) SGC-7901 and GES-1 cells were incubated with 122 μM hispidin or 0.1% DMSO and assayed for GCS activity. The GCS activity of GES-1 cells at 0 h was used as the basic standard for both SGC-7901 and GES-1 cells.
Figure 6
Figure 6. Hispidin induces microtubule depolymerization and is able to cause LMP
(A) SGC-7901 cells were preincubated with 5 μM BAPTA AM, 20 μM carboxy PITO, 2000 U catalase or 10 mM NAC. Then, the cells were treated with 122 μM hispidin and stained with acridine Orange (AO). (B) SGC-7901 and GES-1 cells were incubated with 250 nM tubulin Tracker Green and 122 μM hispidin and assayed for fluorescence at different time points. (C) SGC-7901 and GES-1 cells were incubated with hispidin or 0.1% DMSO. Then, the α-tubulin in the cells was detected by Western blotting. (D) MAP-rich tubulin in a reaction buffer was incubated at 37°C in the presence of DMSO, hispidin, paclitaxel, and nocodazole. The polymerization of tubulin was determined by measuring the increase in absorbance over time at 340 nm. (E) Enlargement of Figure D in the early five min.
Figure 7
Figure 7. STMN1 phosphorylation and dephosphorylation is involved in hispidin-induced microtubule depolymerization
(A and B) Tubulin was purified, cross-linked and used as a receptor in hispidin and paclitaxel binding assays. (C) SGC-7901 and GES-1 cells were incubated with hispidin or 0.1% DMSO. Then, STMN1 phosphorylation at ser16 was detected by Western blotting using β-actin as an internal control. (D) SGC-7901 cells were transfected with scrambled RNA and STMN1 RNA for 48 h, and STMN1 levels were determined by Western blot. (E) The transfected cells were treated with 122 μM hispidin and assayed for viability.

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