Cryptophycin-induced hyperphosphorylation of Bcl-2, cell cycle arrest and growth inhibition in human H460 NSCLC cells.
Bcl-2 has been described as a factor that can protect from apoptosis. The protective effect of Bcl-2 may be lost if the protein is phosphorylated. Bcl-2 phosphorylation can be induced by agents that affect microtubule depolymerization or prevent microtubule assembly. In 13 human tumor cell lines there was a high degree of heterogeneity in Bcl-2 protein expression. Human H460 non-small-cell lung carcinoma (NSCLC) cells expressed high levels of Bcl-2 and were selected for study. Western blot analysis for Bcl-2 phosphorylation was carried out after 4 h and 24 h of exposure to cryptophycin 52, cryptophycin 55, paclitaxel or vinblastine. Cryptophycin 52 and cryptophycin 55 were very potent inducers of Bcl-2 phosphorylation. After 4 h of exposure, Bcl-2 phosphorylation was evident with 0.05 nM cryptophycin 52, 0.25 nM cryptophycin 55, 5 nM vinblastine and 50 nM paclitaxel. The hyperphosphorylated form of Bcl-2 was evident after 24 h exposure of H460 cells to 0.25 nM cryptophycin 52 or cryptophycin 55 and 50 nM vinblastine or paclitaxel. The effects of the compounds on the cell cycle paralleled those on Bcl-2 phosphorylation. In H460 cells 90% cell killing was obtained with 0.13 nM cryptophycin 52, 0.2 nM cryptophycin 55, 20 nM paclitaxel and > 100 nM vinblastine after 24 h of exposure as determined by colony formation. In Bcl-2-negative Calu-6 NSCLC cells, 90% cell killing was obtained with 0.03 nM cryptophycin 52, 0.1 nM cryptophycin 55, 11 nM paclitaxel and 0.5 nM vinblastine using the same experimental design. Thus, cryptophycins are potent inducers of Bcl-2 phosphorylation. The cryptophycins were more potent cytotoxic agents in Bcl-2-negative Calu-6 cells than in Bcl-2-positive H460 cells indicating that pathways triggered by Bcl-2 phosphorylation are involved in cryptophycin-induced lethality.
Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285, USA.
K
K
Lu
J
J
Dempsey
R M
RM
Schultz
C
C
Shih
B A
BA
Teicher
D000970:Antineoplastic Agents / Q000494:pharmacology*; D015153:Blotting, Western; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy* / Q000473:pathology; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D047630:Depsipeptides; D016195:G2 Phase / Q000187:drug effects; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000473:pathology; D008938:Mitosis / Q000187:drug effects; D010456:Peptides, Cyclic / Q000494:pharmacology*; D010766:Phosphorylation; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D047630:Depsipeptides; D010456:Peptides, Cyclic; D019253:Proto-Oncogene Proteins c-bcl-2; C088419:cryptophycin