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. 2020 Aug;10(8):1397-1413.
doi: 10.1016/j.apsb.2020.06.015. Epub 2020 Jul 2.

Miltirone induces cell death in hepatocellular carcinoma cell through GSDME-dependent pyroptosis

Xiaowei Zhang  1   2 , Ping Zhang  1   2 , Lin An  1   2 , Ningyuan Sun  1   2 , Liying Peng  1   2 , Weiwei Tang  1   2 , Dingyuan Ma  3 , Jun Chen  1   2
Affiliations

Miltirone induces cell death in hepatocellular carcinoma cell through GSDME-dependent pyroptosis

Xiaowei Zhang et al. Acta Pharm Sin B. 2020 Aug.

Abstract

Pyroptosis is a form of programmed cell death, and recently described as a new molecular mechanism of chemotherapy drugs in the treatment of tumors. Miltirone, a derivative of phenanthrene-quinone isolated from the root of Salvia miltiorrhiza Bunge, has been shown to possess anti-cancer activities. Here, we found that miltirone inhibited the cell viability of either HepG2 or Hepa1-6 cells, and induced the proteolytic cleavage of gasdermin E (GSDME) in each hepatocellular carcinoma (HCC) cell line, with concomitant cleavage of caspase 3. Knocking out GSDME switched miltirone-induced cell death from pyroptosis to apoptosis. Additionally, the induction effects of miltirone on GSDME-dependent pyroptosis were attenuated by siRNA-mediated caspase three silencing and the specific caspase three inhibitor Z-DEVD-FMK, respectively. Miltirone effectively elicited intracellular accumulation of reactive oxygen species (ROS), and suppressed phosphorylation of mitogen-activated and extracellular signal-regulated kinase (MEK) and extracellular regulated protein kinases 1/2 (ERK1/2) for pyroptosis induction. Moreover, miltirone significantly inhibited tumor growth and induced pyroptosis in the Hepa1-6 mouse HCC syngeneic model. These results provide a new insight that miltirone is a potential therapeutic agent for the treatment of HCC via GSDME-dependent pyroptosis VSports手机版. .

Keywords: 7-AAD, 7-aminoactinomycin D; AKT, AKT serine/threonine kinase, also known as protein kinase B; ANOVA, analysis of variance; BAX, BCL2-associated X; CCK-8, cell counting kit-8; CRISPR, clustered regularly interspaced short palindromic repeats; Cas9, caspase 9; Cell death; DCFH-DA, dye 2,7-dichlorofluoresce diacetate; DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethyl sulfoxide; ECL, enhanced chemiluminescence; ERK1/2, extracellular regulated protein kinases 1/2; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSDMD, gasdermin D; GSDME; GSDME, gasdermin E; H&E, hematoxylin and eosin; HCC, hepatocellular carcinoma; HRP, horseradish peroxidase; HepG2; Hepa1-6; Hepatocellular carcinoma; IC50, the half maximal inhibitory concentration; IgG (H + L), immunoglobulin G (heavy chain + light chain); KO, knockout; LDH, lactic dehydrogenase; MEK, mitogen-activated and extracellular signal-regulated kinase; MEM, minimum essential medium; MMP, mitochondrial membrane potential; MS, mass spectrum; Miltirone; N-GSDME, N-terminal GSDME; NAC, N-acetyl cysteine; NC, negative control; NMR, nuclear magnetic resonance; NS, no significance; PARP, poly ADP-ribose polymerase; PBS, phosphate-based buffer; PI, propidium iodide; PI3K, phosphatidylinositol 3-kinase; Pyroptosis; RIPA, radioimmunoprecipitation assay; ROS, reactive oxygen species; SD, standard deviation; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; TBST, Tris-buffered saline with Tween solution; TCGA, the Cancer Genome Atlas; VEGF, vascular endothelial growth factor; gRNA, guide RNA; i. p V体育安卓版. , intraperitoneal; i. v. , intravenous; mTOR, mammalian target of rapamycin; p-AKT, phosphorylated-AKT; p-ERK1/2, phosphorylated-ERK1/2; p-MEK, phosphorylated-MEK. .

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Image 1
Graphical abstract
Figure 1
Figure 1
Miltirone inhibited the viability of HepG2 and Hepa1-6 cells in dose- and time-dependent manners. (A) Chemical structure of miltirone. (B) and (C) HepG2 and Hepa1-6 cells were treated with miltirone (0–80 μmol/L) or sorafenib (60 μmol/L) for 24 h, cell viability was analyzed by CCK-8 assay and expressed as mean ± SD (n = 3). (D) HepG2 and Hepa1-6 cells were treated with 40 μmol/L miltirone at indicated time, cell viability was analyzed by CCK-8 assay and expressed as mean ± SD (n = 3). ∗∗P < 0.01 vs. control.
Figure 2
Figure 2
Miltirone triggered pyroptosis in HCC cells (HepG2 and Hepa1-6). (A) HepG2 and Hepa1-6 cells were treated with miltirone (12 h) or sorafenib (24 h), and microscopic imaging was performed. Arrowheads indicate ballooned cell membrane characteristic of pyroptotic cells, scale bar = 20 μm. (B) HepG2 and Hepa1-6 cells were treated with miltirone or sorafenib at indicated concentrations for 24 h, LDH-release was analyzed using LDH assay kit and expressed as mean ± SD (n = 3). (C) HepG2 and Hepa1-6 cells were treated with miltirone (40 μmol/L) for 6, 12, and 24 h. LDH-release was analyzed using LDH assay kit and expressed as mean ± SD (n = 3). (D) The percentage of PI (red) positive cells were increased in HepG2 and Hepa1-6 cells after the treatment with miltirone for 24 h. Scale bar = 100 μm. (E) The percentage of PI (red) positive cells were increased in HepG2 and Hepa1-6 cells after treatment with miltirone (40 μmol/L) for 6, 12, and 24 h. Scale bar = 100 μm. (F) and (G) HepG2 and Hepa1-6 cells were treated with miltirone or necrostatin-1 for 24 h, cell viability was analyzed by CCK-8 assay, and LDH-release was analyzed using LDH assay kit and expressed as mean ± SD (n = 3). NS, no significance, P < 0.01 vs. miltirone alone.
Figure 3
Figure 3
GSDMD is not involved in miltirone-induced cell death in HepG2 or Hepa1-6 cells. (A) HepG2 and Hepa1-6 cells were treated with miltirone (0–40 μmol/L) for 24 h, total cellular extracts were prepared and subjected to Western blotting analyses using antibodies against GSDMD and GADPH. (B) Hepa1-6 cells were transfected with siRNA targeting Gsdmd (Gsdmd-siRNA-1/2/3) or control siRNA (NC, negative control) for 24 h, total cellular extracts were prepared and subjected to Western blotting analyses using antibodies against GSDMD and GADPH (n = 3). (C) and (D) Hepa1-6 cells were transfected with siRNA targeting Gsdmd (Gsdmd-siRNA-1/2/3) or control siRNA (NC) for 24 h, cell viability was analyzed by CCK-8 assay, and LDH-release was analyzed using LDH assay kit and expressed as mean ± SD (n = 3). NS vs. NC. (E) and (F) HepG2 and Hepa1-6 cells were treated with miltirone (0–40 μmol/L) for 24 h, or treated with miltirone (40 μmol/L) for 6, 12, and 24 h, total cellular extracts were prepared and subjected to Western blotting analyses using antibodies against caspase 3, PARP, GSDME, and GADPH. Protein levels are expressed as mean ± SD (n = 3). ∗∗P < 0.05, ∗∗P < 0.01 vs. control.
Figure 4
Figure 4
GSDME mediates pyroptosis in HCC cells in response to miltirone. (A) Gsdme wild-type (WT) and Gsdme KO Hepa1-6 cells were treated with miltirone (40 μmol/L) for 24 h, total cellular extracts were prepared and subjected to Western blotting analyses by using antibodies against caspase 3, PARP, GSDME, and GADPH (n = 3). (B) Gsdme WT and Gsdme KO Hepa1-6 cells were treated with miltirone (40 μmol/L) for 12 h, and microscopic imaging was performed. Arrowheads indicate ballooned cell membrane characteristic of pyroptotic cells, scale bar = 20 μm. (C) and (D) Gsdme WT and Gsdme KO Hepa1-6 cells were treated with miltirone (40 μmol/L) for 6, 12, and 24 h. Cell viability was analyzed by CCK-8 assay, and LDH-release was analyzed by LDH assay and expressed as mean ± SD (n = 3). ∗∗P < 0.01 vs. GSDME WT. (E) and (F) Gsdme WT and Gsdme KO Hepa1-6 cells were treated with miltirone (40 μmol/L) for 1–6 h, stained by annexin V-FITC and PI, and analyzed by flow cytometry (n = 3). ∗P < 0.05, ∗∗P < 0.01 vs. Gsdme WT.
Figure 5
Figure 5
Miltirone activates the mitochondrial intrinsic apoptotic pathway to elicit GSDME-dependent pyroptosis. (A) Hepa1-6 cells were treated with miltirone (40 μmol/L) in the absence or presence of Z-DEVD-FMK (20 μmol/L) for 24 h, total cellular extracts were prepared and subjected to Western blotting analyses using antibodies against caspase 3, GSDME, and GADPH (n = 3). (B) Hepa1-6 cells were treated with miltirone (40 μmol/L) in the absence or presence of Z-DEVD-FMK (20 μmol/L) for 6, 12, and 24 h, LDH-release was analyzed using LDH assay kit and expressed as mean ± SD (n = 3). ∗∗P < 0.01, vs. miltirone alone at each indicated time. Hepa1-6 cells were transfected with siRNA targeting caspase 3 (siRNA-casp3-1/2/3) or control siRNA and then treated with miltirone for 24 h. (C) Total cellular extracts were prepared and subjected to Western blotting analyses using antibodies against caspase 3, GSDME, and GADPH (n = 3). (D) LDH-release was analyzed by LDH assay and expressed as mean ± SD (n = 3). ∗∗P < 0.01, vs. miltirone alone at each indicated time. (E) Annexin V-FITC and PI stained cells were analyzed by flow cytometry.
Figure 6
Figure 6
Miltirone induced HCC cells pyroptosis is mediated through RAF/MEK/ERK1/2 signaling pathway. (A)–(C) Hepa1-6 cells were treated with miltirone (0–40 μmol/L) for 24 h, total cellular extracts were prepared and subjected to Western blotting analyses using AKT, p-AKT, MEK, p-MEK, ERK1/2, p-ERK1/2, and GADPH antibodies. Protein levels were expressed as mean ± SD (n = 3). (D) Hepa1-6 cells were treated with miltirone (20 μmol/L) in the absence or presence of ceramide C6 (10 μmol/L) for 6, 12, and 24 h, LDH release were measured by LDH assay and expressed as mean ± SD (n = 3). ∗P < 0.05, ∗∗P < 0.01, vs. miltirone alone at each indicated time. (E) Hepa1-6 cells were treated with miltirone (20 μmol/L) in the absence or presence of ceramide C6 (10 μmol/L) for 24 h, total cellular extracts were prepared and subjected to Western blotting analyses using antibodies against ERK1/2, p-ERK1/2, caspase 3, GSDME, and GADPH (n = 3).
Figure 7
Figure 7
ROS–MEK/ERK1/2 signaling pathway participates in miltirone-induced pyroptosis in HCC cells. (A) Hepa1-6 cells were treated with miltirone and/or NAC for 2 h, intracellular ROS content was measured with confocal microscopy. Scale bar = 40 μm Hepa1-6 cells were treated with miltirone and/or NAC for 24 h. (B) and (C) Cell viability and LDH release were measured, respectively, and expressed as mean ± SD (n = 3). (D)–(I) Total cellular extracts were prepared and subjected to Western blotting analyses using antibodies against BAX, caspase 9, caspase 3, GSDME, MEK, p-MEK, ERK1/2, p-ERK1/2, and GADPH. ∗∗P < 0.01 vs. control; ##P < 0.01, vs. miltirone alone.
Figure 8
Figure 8
Miltirone inhibits the tumor growth and induces HCC cells pyroptosis in vivo. Hepa1-6 cells were inoculated into mice to establish tumor model as described in the Material and methods. Mice bearing tumors were randomly grouped and administered vehicle (Vehicle), 1 mg/kg body weight of miltirone (1 mg/kg), 3 mg/kg body weight of miltirone (3 mg/kg), 6 mg/kg body weight of miltirone (6 mg/kg), or 10 mg/kg weight of sorafenib (Sorafenib), respectively. (A) The tumor volume measurement was proceeded every other day, presented as mean ± standard error of mean (SEM), n = 7. (B) The tumors were dissected and weighted (presented as mean ± SD, n = 7). (C) Representative photographs of isolated tumors at Day 27 after treatment. (D) Body weight of mice during the 27 days (presented as mean ± SD, n = 7). (E) The release of serum LDH in the mice was measured by LDH assay (presented as mean ± SD, n = 7). (F) and (G) Western blotting analyses of GSDME, N-GSDME, caspase 3, cleaved-caspase 3, caspase 9, cleaved-caspase 9, and BAX expression in treated tumor tissues. Protein levels were expressed as mean ± SD (n = 3). (H) Histological analysis of tumors, kidneys, and livers at the end of experiment. Scale bar = 50 μm. ∗P < 0.05, ∗∗P < 0.01 vs. vehicle treatment; #P < 0.05 vs. indicated treatment.
Figure 9
Figure 9
A schematic summary of this study, showing that through regulating ROS/ERK1/2 pathway, miltirone can elicit BAX–caspase–GSDME-dependent pyroptosis.
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