Depletion of p185erbB2, Raf-1 and mutant p53 proteins by geldanamycin derivatives correlates with antiproliferative activity.
OBJECTIVE
Recently, it has been shown that geldanamycin (GA), a benzoquinone ansamycin, is able to deplete mutant p53, p185erbB2 and Raf-1 proteins in cancer cells. However, the relationship between these activities of GA and its antiproliferative activity is not clear. Here we investigated the effects of 28 GA derivatives in SKBr3, a human breast cancer cell line.


METHODS
We performed Western blot analysis of Raf-1, p185erbB2 and mutant p53 proteins following drug treatment and correlated these findings with the cytotoxicity of the various GA derivatives.


RESULTS
We found that downregulation of Raf-1, p185erbB2 and mutant p53 proteins was correlated. Thus, a drug that was active against one oncoprotein was equally active against the two others. Inactive derivatives were identified by their inability to downregulate these oncoproteins, even at a high dose (2 microM). These inactive drugs also had no or minimal antiproliferative activity (IC50 > 3 microM). All other analogs (at a concentration of 2 microM) downregulated p53, p185erbB2, and Raf-1, and also displayed cytotoxicity (IC50 in the range 6-600 nM). This category of drugs was further divided into more- and less-active agents by testing at lower doses (40 nM). The drugs that remained active against their molecular targets had an IC50 for antiproliferative activity of less than 40 nM. Maximal effects on mutant p53, p185erbB2 and Raf-1 were observed at doses that were 4-5 times greater than the cytotoxic IC50.


CONCLUSIONS
These findings suggest that GA and its derivatives are cytostatic/cytotoxic at concentrations that also downregulate Raf-1, p185erbB2 and mutant p53, and raise the possibility that depletion of these proteins and the antiproliferative activities of GA have a common mechanism.
An
W G
WG
Clinical Pharmacology Branch, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
Schnur
R C
RC
Neckers
L
L
Blagosklonny
M V
MV
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D016227:Benzoquinones; D015536:Down-Regulation; D005260:Female; D006801:Humans; D047029:Lactams, Macrocyclic; D009154:Mutation; D017346:Protein Serine-Threonine Kinases / Q000032:analysis*; D011518:Proto-Oncogene Proteins / Q000032:analysis*; D019908:Proto-Oncogene Proteins c-raf; D011809:Quinones / Q000494:pharmacology*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000032:analysis*; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000032:analysis*
D000903:Antibiotics, Antineoplastic; D016227:Benzoquinones; D047029:Lactams, Macrocyclic; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D019908:Proto-Oncogene Proteins c-raf; D011809:Quinones; D018719:Erb-b2 Receptor Tyrosine Kinases; D016159:Tumor Suppressor Protein p53; C001277:geldanamycin