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.
The ribosomal acidic P0 protein, an essential component of the eukaryotic ribosomal stalk, was found to interact with the helix-loop-helix protein human Grap2 and cyclin D interacting protein (GCIP)/D-type cyclin-interacting protein 1/human homolog of MAID protein. Using in vivo and in vitro binding assays, we show that P0 can interact with the N and C termini of GCIP via its N-terminal 39-114 amino-acid residues. Although the P0-GCIP complex was detected mainly in cytoplasmic fraction, polysome profile analysis indicated that the P0-GCIP complex did not coelute with either polysomes or 60S ribosomes, suggesting that GCIP associates with the free form of P0 in the cytoplasm. Transfection of GCIP into MCF-7 cells resulted in decreased levels of pRb phosphorylation. Cotransfection of P0 with GCIP, however, resulted in GCIP-mediated reduction of pRb phosphorylation level which was repressed by P0. Furthermore, overexpression of P0 in breast cancer and hepatocellular cancer cell lines promoted cell growth and colony formation compared to control transfectants. Overexpression of P0 also increased cyclin D1 expression and phosphorylation of pRb at Ser780. Interestingly, P0 mRNA was overexpressed in 12 of 20 pairs of breast cancer/ normal breast specimens (60%). Together, these data indicate that P0 overexpression may cause tumorigenesis in breast and liver tissues at least in part by inhibiting GCIP-mediated tumor suppression.
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