Inhibition of cytokinesis and akt phosphorylation by chaetoglobosin K in ras-transformed epithelial cells.
OBJECTIVE
Chaetoglobosin K (ChK), a bioactive natural product previously shown to have anti-tumor promoting activity in glial cells and growth inhibitory effects in ras-transformed fibroblasts, inhibited anchorage-dependent and anchorage-independent growth in ras-transformed liver epithelial cells. The purpose of this study was to identify cellular targets of ChK that mediate its anti-tumor effects.


METHODS
Anchorage-independent cell growth assays, using soft agar-coated dishes, and anchorage-dependent growth assays were performed on transformed WB- ras1 cells. Phase/contrast and fluorescent microscopy were used to visualize cell morphological changes and DAPI-stained nuclei. Analyses of p21 Ras membrane versus cytosolic forms, p44/42 mitogen-activated protein kinase (MAPK) phosphorylation, Akt kinase phosphorylation and connexin 43 phosphorylation were performed by Western blotting. Gap junction-mediated cellular communication was measured by fluorescent dye transfer.


RESULTS
Treatment of WB- ras1 cells with a non-cytotoxic dose of ChK inhibited both anchorage-dependent and anchorage-independent growth. Inhibited cells were generally larger and less spindle-shaped in morphology than vehicle-treated cells, many of which were multinucleate. Removal of ChK induced cytokinesis and a return to predominantly single-nucleate cells, suggesting that ChK inhibits cytokinesis. The proportion of membrane-associated versus cytosolic forms of p21 Ras was unchanged by ChK treatment, suggesting that ChK does not act as a farnesylation inhibitor. ChK treatment decreased the level of phosphorylation of Akt kinase, a key signal transducer of the Phosphatidylinositol 3-kinase pathway. In contrast, ChK had no effect on phosphorylation of p44/42 MAPK, which mediates the MAPK/ERK Ras effector pathway. Phosphorylation of the gap junction protein, connexin 43, shown previously to increase following treatment with other anti-Ras compounds, was also not altered by ChK, which correlated with its lack of effect on gap junction-mediated cellular communication.


CONCLUSIONS
Our results demonstrate that ChK inhibits Akt kinase phosphorylation and cytokinesis in ras-transformed cells, which likely contribute to its ability to inhibit tumorigenic growth.
Matesic
Diane F
DF
Department of Pharmaceutical Sciences, Mercer University, 3001 Mercer University Drive, Atlanta, GA 30341, USA. matesic_df@mercer.edu
Villio
Kimberly N
KN
Folse
Stacey L
SL
Garcia
Erin L
EL
Cutler
Stephen J
SJ
Cutler
Horace G
HG
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002461:Cell Line, Transformed; D049109:Cell Proliferation / Q000187:drug effects*; D048749:Cytokinesis / Q000187:drug effects; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D026121:Indole Alkaloids / Q000494:pharmacology*; D008099:Liver; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics* / Q000378:metabolism; D051381:Rats
D000970:Antineoplastic Agents; D026121:Indole Alkaloids; C019290:chaetoglobosins; D051057:Proto-Oncogene Proteins c-akt; D016283:Proto-Oncogene Proteins p21(ras)