Abstract
Tanshinone IIA (Tan IIA), a constituent of the traditional medicinal plant Salvia miltiorrhiza BUNGE, 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, FLIPS is 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 FLIPS to 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 FLIPS levels induced by Tan IIA. In short, Tan IIA simultaneously induces both Nec-1 inhibition and FLIPS regulation-mediated apoptosis/necroptosis, which has not been previously documented. Moreover, the involvement of the cleavage type of MLKL in executing necroptosis warrants further investigation.
Highlights
Apoptosis, a kind of cell death, is a natural way to prevent the development of cancer
Some corresponding complexes vary in terms of initiator, modulator, or effecter components depending on different cell types,[1] the well-known composition of ripoptosome is caspase 8, Fas-associated death domain protein (FADD), and two receptor-interacting serine/ threonine-protein kinases RIPK1 and RIPK3.2 Caspase 8 is an apoptosis effector, FADD is an adaptor, receptor-interacting protein 1 (RIP1) and receptor-interacting protein 3 (RIP3) are necroptotic effectors, and FLICE inhibitory protein (FLIP) is a modulator
This study shows that Tan Tanshinone IIA (IIA) causes apoptosis and necroptosis simultaneously in HepG2 cells. z-VAD-fmk converts the apoptosis to necroptosis, and both modes of cell death can be inhibited by Nec-1
Summary
A kind of cell death, is a natural way to prevent the development of cancer. (i.e., a necrosome) dissociates from the ripoptosome and makes apoptosis results in increasing necroptosis This suggestion was necroptosis available.[5,6] RIP3 of the necrosome is phosphorylated supported by elevated expression levels of necroptotic markers and in turn recruits mixed-lineage kinase domain-like (MLKL) and cyclophilin A and HMGB1 observed in the cells treated with Tan phosphorylate it, resulting in it being oligomerized, translocated to plasma membrane and eventually forming a calcium influxmediated pore.[7]. We thereafter determined that Tan IIA-induced LDH activity was diminished by Nec-1 (Figure 1b), and the expression levels of necrotic markers cyclophilin A and HMGB1 were likewise significantly reduced by Nec-1 (Figure 2a and b). We analyzed the signaling pathway responsible for cell survival, apoptosis, and necroptosis by means of western blotting and IP
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