Deoxycholate promotes survival of breast cancer cells by reducing the level of pro-apoptotic ceramide.
BACKGROUND
At physiologic concentration in serum, the bile acid sodium deoxycholate (DC) induces survival and migration of breast cancer cells. Here we provide evidence of a novel mechanism by which DC reduces apoptosis that is induced by the sphingolipid ceramide in breast cancer cells.


METHODS
Murine mammacarcinoma 4T1 cells were used in vitro to determine apoptosis and alteration of sphingolipid metabolism by DC, and in vivo to quantify the effect of DC on metastasis.


RESULTS
We found that DC increased the number of intestinal metastases generated from 4T1 cell tumors grafted into the fat pad. The metastatic nodes contained slowly dividing cancer cells in immediate vicinity of newly formed blood vessels. These cells were positive for CD44, a marker that has been suggested to be expressed on breast cancer stem cells. In culture, a subpopulation (3 +/- 1%) of slowly dividing, CD44+ cells gave rise to rapidly dividing, CD44- cells. DC promoted survival of CD44+ cells, which was concurrent with reduced levels of activated caspase 3 and ceramide, a sphingolipid inducing apoptosis in 4T1 cells. Z-guggulsterone, an antagonist of the farnesoid-X-receptor, obliterated this anti-apoptotic effect, indicating that DC increased cell survival via farnesoid-X-receptor. DC also increased the gene expression of the vascular endothelial growth factor receptor 2 (Flk-1), suggesting that DC enhanced the initial growth of secondary tumors adjacent to blood vessels. The Flk-1 antagonist SU5416 obliterated the reduction of ceramide and apoptosis by DC, indicating that enhanced cell survival is due to Flk-1-induced reduction in ceramide.


CONCLUSIONS
Our findings show, for the first time, that DC is a natural tumor promoter by elevating Flk-1 and decreasing ceramide-mediated apoptosis of breast cancer progenitor cells. Reducing the level or effect of serum DC and elevating ceramide in breast cancer progenitor cells by treatment with Z-guggulsterone and/or vascular endothelial growth factor receptor 2/Flk-1 antagonists may thus be a promising strategy to reduce breast cancer metastasis.
Krishnamurthy
Kannan
K
Institute of Molecular Medicine and Genetics, School of Medicine, Medical College of Georgia, 15th Street, Augusta, GA 30912, USA.
Wang
Guanghu
G
Rokhfeld
Dmitriy
D
Bieberich
Erhard
E
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002470:Cell Survival / Q000187:drug effects; D002518:Ceramides / Q000378:metabolism*; D002756:Cholagogues and Choleretics / Q000494:pharmacology*; D002855:Chromatography, Thin Layer; D003840:Deoxycholic Acid / Q000494:pharmacology*; D005260:Female; D005434:Flow Cytometry; D018960:Hyaluronan Receptors; D007124:Immunoenzyme Techniques; D008325:Mammary Neoplasms, Experimental / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D008807:Mice, Inbred BALB C; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013107:Sphingolipids / Q000378:metabolism; D014407:Tumor Cells, Cultured; D040301:Vascular Endothelial Growth Factor Receptor-2
C476399:Cd44 protein, mouse; D002518:Ceramides; D002756:Cholagogues and Choleretics; D018960:Hyaluronan Receptors; D012333:RNA, Messenger; D013107:Sphingolipids; D003840:Deoxycholic Acid; D040301:Vascular Endothelial Growth Factor Receptor-2