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

Xiaowei Zhang; Ping Zhang; Lin An; Ningyuan Sun; Liying Peng; Weiwei Tang; Dingyuan Ma; Jun Chen

doi:10.1016/j.apsb.2020.06.015

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

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

Authors

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 Ma³, Jun Chen^{1

2}

Affiliations

¹ State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China.
² Department of Pharmacognosy, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 210009, China.
³ Department of Prenatal Diagnosis, Nanjing Maternity and Child Health Care Hospital, Women's Hospital of Nanjing Medical University, Nanjing 210004, China.

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.

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., intraperitoneal; i.v., intravenous; mTOR, mammalian target of rapamycin; p-AKT, phosphorylated-AKT; p-ERK1/2, phosphorylated-ERK1/2; p-MEK, phosphorylated-MEK.