Rasfonin

The underlying mechanism by which growth factor receptor-bound protein 2 (Grb2) regulates necroptosis remains unexplored. In the present study, we found that rasfonin, a fungal natural product and an activator of necroptosis, enhanced Grb2 binding to receptor-interacting serine/threonine kinase 1 (RIP1), which plays a critical role in regulating programmed necrosis. Moreover, we observed that SQSTM/p62 (p62), a protein that can form necrosomes with RIP1, increased its interaction with Grb2 upon rasfonin challenge. Although it has been used as an activator of autophagy in our previous study, here we found that a high dose of rasfonin was able to inhibit autophagic process. Inhibition of RIP1 either chemically or genetically reversed the inhibition of rasfonin on autophagy, whereas knockdown of Grb2 markedly reduced rasfonin-induced necrosis. Additionally, we found that the compound failed to upregulate the expression of RIP1 in Grb2-deprived cells. In summary, our data revealed that Grb2 actively participated in rasfonin-induced necroptosis by interacting with the components of necrosome and mediating their expression.

Objective: To investigate the effects of rasfonin, a fungal secondary metabolite, on the proliferation and migration of osteosarcoma 143B cells. Methods: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay was performed to examine 143B cell viability following treatment of rasfonin. Using dimethyl sulfoxide (DMSO) group as control, cell viability was detected when 143B cells were treated with rasfonin (3 μmol/L and 6 μmol/L) for 12 or 24 hours. The effect of rasfonin on colony forming ability was detected by clone formation assay. 143B cells treated with DMSO or rasfonin (3 μmol/L) for one week, and the number of clones formed in the two groups was counted. Wound healing and transwell assay were employed to analyze cell invasion and migration upon rasfonin challenge. The DMSO group was used as control while rasfonin (3 μmol/L) was used for 24 hours. The wound healing rate and the number of invasive cells were compared between the two groups. The intracellular autophagosomes were monitored by transmission electron microscopy when 143B cells were treated with DMSO or rasfonin (3 μmol/L) for 4 hours. The expression of p62, microtubule-associated protein 1 light chain 3 fusion protein (LC3) and poly (ADP-ribose) polymerase-1 (PARP-1) in response to rasfonin were detected by immunoblotting assay. Results: Rasfonin reduced the viability of 143B cells in a dose-dependent manner (12 h: F=31.36, P<0.01; 24 h: F=67.07, P<0.01). Rasfonin (3 μmol/L) completely inhibited the clonal formation of 143B cells (P<0.01). The wound healing result revealed that rasfonin significantly decreased migratory ability of 143B cells (33.91%±0.83% vs. 65.11%±0.94%, P<0.01), whereas its treatment significantly reduced the number of 143B cells penetrating through Matrigel-containing basement membrane (21.33±1.45 vs. 49.33±2.40, P<0.01). Compared with the control group, rasfonin markedly increased the number of autophagic vacuoles. The immunoblotting results revealed that rasfonin increased LC3-II accumulation and decreased p62 levels. Choloroquine (CQ), an often used autophagic inhibitor, further accumulated rasfonin-induced LC3-II. In addition, rasfonin appeared to cause the cleavage of PARP-1. Conclusion: Rasfonin induced autophagy and activated caspase-dependent apoptosis in 143B cells concurring with suppressing the proliferation and migration of the cells; these results provide an experimental basis for rasfonin as a potential therapeutic agent for osteosarcoma.

Both Raptor and Rictor are the key components in the complexes of mammalian target of rapamycin (mTOR), which play a vital role in mediating autophagy. Unlike mTOR, the regulatory role of either Raptor or Rictor in the regulation of autophagic process is relatively less explored. In present study, we found that rasfonin, which isolated from Talaromyces sp. 3656-A1 and was a fungal natural product, activated both caspase-dependent apoptosis and autophagy in ACHN, a renal carcinoma cell line. Knockdown of Raptor decreased both rasfonin-induced autophagic flux and PARP-1 cleavage, and in contrast, Rictor silencing increased apoptosis concomitantly enhancing rasfonin-induced autophagy. Unexpectedly, API-2, which was widely used as an inhibitor of Akt, promoted rasfonin-dependent autophagy in Raptor-depleted but not Rictor-deprived cells. Collectively, these results demonstrated that Raptor and Rictor could play a distinctly regulatory role in rasfonin-enhanced autophagy and apoptosis.