title: Simultaneous induction of apoptosis and necroptosis by Tanshinone IIA in human hepatocellular carcinoma HepG2 cells.
abstract: Tanshinone IIA (Tan IIA), a constituent of the traditional medicinal plantSalvia miltiorrhizaBUNGE, has been reported to possess anticancer activity through induction of apoptosis in many cancer cells. Surprisingly, the present study finds that Tan IIA simultaneously causes apoptosis and necroptosis in human hepatocellular carcinoma HepG2 cells. We further find that apoptosis can be converted to necroptosis by pan-caspase inhibitor Z-VAD-fmk, and the two death modes can be blocked by necroptotic inhibitor necrostatin-1. The underlying mechanisms are revealed by analysis of the signaling molecules using western blotting. In control cells, FLICE inhibitory protein in short form (FLIPS) is expressed in relatively high levels and binds to caspase 8 in ripoptosome, which supposedly sustains cell survival. However, in Tan IIA-treated cells, FLIPSis down-regulated and may thus cause homodimer formation of cleaved caspase 8, cleavage of receptor-interacting serine/threonine-protein kinases 1, 3 (RIP1, RIP3), and mixed-lineage kinase domain-like (MLKL), in turn leads to cell apoptosis. In parallel, Tan IIA causes necroptosis by forming a suggested necrosomal complex composed of RIP1/RIP3. Regarding the inhibitors, z-VAD-fmk diminishes the cleaved caspase 8, RIP1, RIP3, and MLKL induced by Tan IIA, and reconstructs the ripoptosome complex, which marks cells moving from apoptosis to necroptosis. Nec-1 recovers the Tan IIA down-regulated FLIPS, consequently causes FLIPSto form heterodimer with caspase 8 and thus block apoptosis. Meanwhile, cleaved forms of RIP1 and RIP3 were observed preventing necroptosis. Intriguingly, the cytotoxicity of tumor necrosis factor-related apoptosis-inducing ligand to HepG2 cells is enhanced by Tan IIA in a pilot study, which may be attributed to low FLIPSlevels induced by Tan IIA. In short, Tan IIA simultaneously induces both Nec-1 inhibition and FLIPSregulation-mediated apoptosis/necroptosis, which has not been previously documented. Moreover, the involvement of the cleavage type of MLKL in executing necroptosis warrants further investigation.
authors.affiliation: Institute of Medical Sciences, Tzu Chi University , Hualien, Taiwan.
authors.forename: C-Y
authors.initials: CY
authors.lastname: Lin
authors.affiliation: Division of Crop Improvement, Hualien District Agricultural Research and Extension Station, Council of Agriculture , Hualien, Taiwan.
authors.forename: T-W
authors.initials: TW
authors.lastname: Chang
authors.affiliation: Department of Public Health, Tzu Chi University , Hualien, Taiwan.
authors.forename: W-H
authors.initials: WH
authors.lastname: Hsieh
authors.affiliation: Department of Medical Imaging and Radiological Sciences, Tzu Chi University of Science and Technology , Hualien, Taiwan.
authors.forename: M-C
authors.initials: MC
authors.lastname: Hung
authors.affiliation: School of Post-Baccalaureate Chinese Medicine, Tzu Chi University, Hualien, Taiwan; Department of Chinese Medicine, Buddhist Hualien Tzu Chi General Hospital, Hualien, Taiwan.
authors.forename: I-H
authors.initials: IH
authors.lastname: Lin
authors.affiliation: School of Post-Baccalaureate Chinese Medicine, Tzu Chi University, Hualien, Taiwan; Department of Pharmacy, Buddhist Hualien Tzu Chi General Hospital, Hualien, Taiwan.
authors.forename: S-C
authors.initials: SC
authors.lastname: Lai
authors.affiliation: Institute of Medical Sciences, Tzu Chi University, Hualien, Taiwan; Department of Molecular Biology and Human Genetics, Tzu Chi University, Hualien, Taiwan; Department of Life Science, Tzu Chi University, Hualien, Taiwan.
authors.forename: Y-J
authors.initials: YJ
authors.lastname: Tzeng