Inhibition of Shc/Grb2 protein-protein interaction suppresses growth of B104-1-1 tumors xenografted in nude mice.

Actinomycin D was revealed as an inhibitor of Shc/Grb2 interaction in cell lines from our recent study. Shc and Grb2 proteins are important molecules in Ras signaling pathways leading to cellular differentiation and proliferation, which require dramatic morphological changes. It was detected by transmission electron microscopy that actinomycin D induced significant changes in cellular ultrastructures of B104-1-1 cells and confirmed that the changes were due to inhibition of Shc/Grb2 interaction by actinomycin D rather than its inhibitory effect on transcription. Because actinomycin D was dispersed mainly in cytoplasm and Shc peptide (synthetic 13 amino acid tyrosine phosphorylated polypeptide) successfully displaced actinomycin D binding to its cellular targets while the other polypeptide from PDGF receptor could not. We examined the effect of actinomycin D on growth of B104-1-1 tumor xenografted in nude mice. Tumor growth was inhibited in vivo after treatment with this inhibitor. Efficacy was correlated with a reduction in the levels of Shc/Grb2 binding in excised tumors. These results suggest that actinomycin D inhibited Shc/Grb2 interaction in B104-1-1 tumor xenografted in nude mice.

Investigation of cell cycle arrest effects of actinomycin D at G1 phase using proteomic methods in B104-1-1 cells.

Actinomycin D was previously reported as an inhibitor of Shc/Grb2 interaction in B104-1-1 cells. Actinomycin D arrested the cell cycle at the G1 phase at 1nM, which is about 10 times lower than the inhibition of Shc/Grb2 interactions in B104-1-1 cells. To evaluate other mechanisms of actinomycin D affected suppression of tumors and cell growth, except inhibition of Shc/Grb2 interactions, we examined the proteomic expression profile by proteomic technology. We found up-regulation of MEKK3 and down-regulation of Hsp70 expression from proteomic analysis, which is a very interesting observation because MEKK3 is strongly related with G1 arrest of cell cycle and Hsp70 is also involved in cell cycle regulation. These results indicate that the anti-tumor effects of actinomycin D is due to synergic effects of various proteins regulated by the compound including inhibition of the Shc/Grb2 interaction and other signaling pathways in the cytoplasm. Here we provide a mechanism-based explanation for growth inhibition by actinomycin D using proteomic technology. Thus, this approach may be a potentially useful method to reveal new mechanisms of active compounds or drugs with unknown cellular function.