Withangulatin A is a newly identified in vitro
topoisomerase II inhibitor isolated from the Chinese antitumor herb Physalis angulata. In vivo, it was found to be cytotoxic, capable of suppressing general
protein synthesis and of inducing the synthesis of a small set of
proteins including those generated by heat-shock treatment. The 70 kDa
protein generated by
withangulatin A was unequivocally identified as the
heat-shock protein 70 (HSP70) since both
proteins migrated to the same position on two-dimensional
polyacrylamide gels, could be recognized by a
monoclonal antibody to human HSP70, and exhibited identical
peptide maps. The induction of
protein synthesis by
withangulatin A was regulated at the transcriptional level since it was aborted in cells pre-treated with
actinomycin D. However, the initiation of this process did not require de novo
protein synthesis since it was not affected by
cycloheximide. Other cellular effect of
withangulatin A was alterations of
protein phosphorylation including an enhancement of phosphorylation of a 65 kDa
protein which was also detected in the heat-shocked cells. Moreover, this process was observed within 7.5 min after the initial heat treatment which is much faster than the onset of HSP synthesis. Therefore, increased phosphorylation of the 65 kDa
protein may represent one of the earliest signals generated by both heat-shock and withangluatin A and may be involved in the upstream regulation of heat-shock response in cells.