CHARACTERIZATION OF THE DE NOVO CERAMIDE METABOLIC PATHWAY IN THE BCL-2 AND BCL-XL-DEPENDENT RESPONSE TO TNF-α-INDUCED APOPTOSIS IN MCF-7 CELLS

Abstract

Several challenges remain in the treatment of breast cancer, the world’s most prevalent cancer in women. One of these challenges, resistance to chemotherapeutic drugs, has been associated with the upregulation in the expression of Bcl-2 and Bcl-xL in several models. Bcl-2 and Bcl-xL, members of the Bcl-2 family, prevent apoptosis. They inhibit TNF-α-induced apoptosis in different cellular models. On the other hand, ceramide, a signaling sphingolipid that controls different cellular processes, induces apoptosis. Bcl-2 and Bcl-xL interaction with ceramide was previously studied in the TNF-α-induced response in MCF-7 breast cancer cell line where it was shown that Bcl-xL acts upstream of ceramide while Bcl-2 functions downstream of this sphingolipid. Both Bcl-2 and Bcl-xL inhibited ceramide-induced death in response to TNF-α. However, Bcl-2 did not inhibit ceramide accumulation in response to TNF-α and displayed a protective effect against ceramide-induced death. Although Bcl-xL, and to a lesser degree Bcl-2, were shown by previous literature to interfere with ceramide generation following TNF-α treatment in MCF-7 cells, disclosing regulatory mechanisms controlling this interaction remains to be essential. In the current work, we aimed to characterize the metabolic pathways that control ceramide accumulation, particularly through de novo synthesis and salvage pathways, in response to TNF-α treatment in MCF-7 cells overexpressing Bcl-2 or Bcl-xL. We hypothesized that Bcl-2 and Bcl-xL regulate the de novo ceramide synthesis pathway and might be involved in the activation of sphingosine kinase 1 (SphK1) of the salvage pathway, thus contributing to resistance to apoptosis following TNF-α treatment. We first validated the resistance of MCF-7 Bcl-2 and MCF-7 Bcl-xL to TNF-α-induced cytotoxicity by MTT. We then studied the transcriptional regulation of DEGS1, CerS2, CerS4, and CerS6, enzymes of the de novo synthesis pathway, by qRT-PCR and revealed that expression of Bcl-xL produced a downregulation in the mRNA expression of DEGS1 and CerS4 after 8h of TNF-α treatment whereas no change was detected with Bcl-2 expression. SphK1 mRNA expression was also not affected in both cell lines, and this was confirmed by the inability of SK1-I to sensitize the resistant cells to TNF-α by MTT. We then aimed to interfere with the de novo pathway using 2 synthetic retinoids: 4-HPR and ST1926. Whereas both modulators are activators of the de novo synthesis pathway, 4-HPR inhibits DEGS1 enzyme leading to the accumulation of the ceramide precursor, dihydroceramide. Pre-treating the MCF-7 Vector control cells with 4-HPR partially inhibited TNF-α induced cell death. In addition, ST1926 was able to re-sensitize MCF-7 Bcl-xL to TNF-α induced apoptotic cell death evidenced by the expression of cleaved PARP-1. MCF-7 Bcl-2, however, displayed resistance to the effect of ST1926 and nearly total inhibition of PARP-1 cleavage in response to TNF-α with or without ST1926. Further investigation of the death response induced by TNF-α with or without ST1926 revealed that ST1926 is not likely to function through p53. Interestingly, MCF-7 Vector cells displayed a significant upregulation of p53 upon TNF-α treatment at 48h suggesting the involvement of p53 in TNF-α induced apoptosis. While no change in the protein expression of p53 was detected in MCF-7 Bcl-xL at 48h, MCF-7 Bcl-2 displayed a significant increase in p53, which, in the absence of apoptosis, suggests that cells may be induced to undergo cycle arrest. Although sharing similar function, it appears that the anti-apoptotic proteins Bcl-xL and Bcl-2 function at sites that can modulate different responses to TNF-α. Understanding the exact mechanism by which these proteins resist TNF-α-induced apoptosis may help to discover new therapeutic approaches for breast cancer.