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Treatment of cancer cells with celecoxib led to a significant increase of C16:0-Cer via the specific activation of ceramide synthase 6 (CerS6). The increase in C16:0-Cer contributes in part to the proapoptotic effect of celecoxib.Ceramides serve as bioactive molecules with important roles in cell proliferation and apoptosis. Ceramides (Cer) with different N-acyl side chains (C14:0-Cer–C26:0-Cer) possess distinctive roles in cell signaling and are differentially expressed in HCT-116 colon cancer cells. Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, exhibiting antiproliferative effects, activates the sphingolipid pathway. To elucidate the mechanism, HCT-116 cells were treated with 50 μM celecoxib leading to a significant increase of C16:0-Cer. Interestingly, 50 μM celecoxib resulted in a 2.8-fold increase of ceramide synthase (CerS) activity as measured by a cell-based activity assay. siRNA against several CerSs revealed that CerS6 was predominantly responsible for the increase of C16:0-Cer in HCT-116 cells. Moreover, the silencing of CerS6 partially protected HCT-116 cells from the toxic effects induced by celecoxib. Treatment of cells with celecoxib and fumonisin B1 (inhibitor of CerSs) or myriocin (inhibitor of l-serine palmitoyl transferase) or desipramine (inhibitor of acid sphingomyelinase and acid ceramidase) revealed that the increase of C16:0-Cer results predominantly from activation of the salvage pathway. Using the nude mouse model we demonstrated that celecoxib induces also in vivo a significant increase of C16:0-Cer in stomach, small intestine and tumor tissue. In conclusion, celecoxib causes a specific increase of C16:0-Cer by activating CerS6 and the salvage pathway, which contribute to the toxic effects of celecoxib.