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

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 phenanthrenequinone 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 GSDMEdependent pyroptosis were attenuated by si RNA-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.

A high-throughput Gaussia luciferase reporter assay for screening potential gasdermin E activators against pancreatic cancer

It is discovered that activated caspase-3 tends to induce apoptosis in gasdermin E(GSDME)-deficient cells,but pyroptosis in GSDME-sufficient cells.The high GSDME expression and apoptosis resistance of pancreatic ductal adenocarcinoma(PDAC)cells shed light on another attractive strategy for PDAC treatment by promoting pyroptosis.Here we report a hGLuc-hGSDME-PCA system for high-throughput screening of potential GSDME activators against PDAC.This screening system neatly quantifies the oligomerization of GSDME-N to characterize whether pyroptosis occurs under the stimulation of chemotherapy drugs.Based on this system,ponatinib and perifosine are screened out from the FDA-approved anti-cancer drug library containing 106 compounds.Concretely,they exhibit the most potent luminescent activity and cause drastic pyroptosis in PDAC cells.Further,we demonstrate that perifosine suppresses pancreatic cancer by promoting pyroptosis via caspase-3/GSDME pathway both in vitro and in vivo.Collectively,this study reveals the great significance of hGLuc-hGSDME-PCA in identifying compounds triggering GSDME-dependent pyroptosis and developing promising therapeutic agents for PDAC.

Expression, Purification and Characterization of C-FADD

FADD is an important proapoptotic adaptor in death receptor-induced apoptosis. Recently, FADD has been found to participate in a variety of non-apoptotic processes, such as development, cell cycle progression and survival. Its non-apoptotic activities were regulated by the phosphorylated status of the serine residue located at the C-terminal region, a domain distinct from the proapoptotic function related DED and DD domains. However, due to the difficulties in expression and crystallization of natural FADD, by far the molecuiar structures of all FADD variants did not contain the C-terminal region. To elucidate the structure-function relationship of C-terminal region, we need to obtain a FADD variant that containing C-terminal region. In this study, mouse FADD （80-205） containing DD domain and C-terminal region, designated as C-FADD, was expressed in E. coli with His-tag at the N-terminus and purified by Ni^2＋ affinity chromatography. The purified protein existed as a homogenous monomer in glutaraldehyde cross-linking analysis and exhibited a typical a-helix spectrum in CD （circular dichroism） assay. In vitro His-tag pull-down assay demonstrated that the purified C-FADD possessed the CK Ia-binding activity which was important for its non-apoptotic function.