Restoration of TNF-α-induced ceramide generation and apoptosis in resistant human leukemia KG1a cells by the P-glycoprotein blocker PSC833

Abstract

Tumor necrosis factor (TNF-alpha) is a cytokine with antitumor activity against several cellular models. TNF-alpha-induced apoptosis seems to be mediated by a signaling pathway termed 'sphingomyelin-ceramide' pathway, which consists of the hydrolysis of sphingomyelin and the production of its breakdown product ceramide. Our study shows that KG1a cells, which are inherently resistant to TNF-alpha and do not produce ceramide upon cytokine stimulation, can be sensitized by the use of the P-glycoprotein inhibitor PSC833. Coincubation with 1 microM of this cyclosporin derivative restored the apoptotic potential of 10 ng/ml TNF-alpha. This effect was associated with the restoration of ceramide generation (315%) and activation of neutral, but not acid sphingomyelinase activity (143%). Furthermore, we demonstrate that treatment of KG1a cells with 1 microM PSC833 led to a threefold increase in inner plasma membrane sphingomyelin content and basal neutral sphingomyelinase activity. These results support the hypothesis whereby resistance to TNF-alpha-mediated apoptosis of certain leukemic cells is linked to the disposability of the sphingomyelin pool. These data also suggest a role for P-glycoprotein in sphingomyelin transverse plasma membrane asymmetry.

β2-microglobulin induces apoptosis in HL-60 human leukemia cell line and its multidrug resistant variants overexpressing MRP1 but lacking Bax or overexpressing P-glycoprotein

“…To con®rm our Western blot results that caspase-8 is not activated, we also performed Western blot analysis of the pro-apoptotic protein Bid in untreated and b 2 m-treated cells and found that all three cell lines contain full-length Bid, but were not able to detect degradation of the p15 form of truncated Bid (tBid) (Rastegar and Safa, unpublished results). Another line of evidence that caspases-3 and -8 are not involved in b 2 m-induced apoptosis in HL-60/VCR is that, as we have shown, these cells overexpress P-gp, and several reports showed that cells induced to overexpress P-gp either by drug selection or by gene transfer experiments are resistant to a variety of apoptotic stimuli including serum starvation, Fas ligand, tumor necrosis factor and UVirradiation (Robinson, et al, 1997;Bezombes, et al, 1998;Smyth, et al, 1998;Pallis and Russell, 2000) since caspases-3 and -8, but not caspase-9, activations are inhibited in P-gp expressing cells (Smyth et al, 1998;Ruepi et al, 2000). Interestingly, P-gp cannot inhibit caspaseindependent apoptosis (Ruepi et al, 2000).…”

Induction of Mdr1b expression by tumor necrosis factor-α in rat liver cells is independent of p53 but requires NF-κB signaling

“…Also drug-selected cell lines have been shown to be less sensitive to apoptosis, but the cytotoxic compounds used to select resistant cells may induce other changes besides up-regulation of Mdr1 proteins. [39][40][41] In these studies PSC833 is often used as a Mdr1 inhibitor, but may itself increase apoptosis. 42 Regarding the mechanism involved, it has been observed that increased expression of MDR protein lowers the sensitivity of the cell to caspase-dependent apoptosis by decreasing the caspase-3 activity.…”

NF-κB transcription factor induces drug resistance through MDR1 expression in cancer cells

“…Moreover, TNF-a activates a sphingomyelinase that induces apoptosis through the generation of ceramides from sphingomyelin (Kolesnick, 1992;Hannun, 1994;Kolesnick and Golde, 1994). P-gp-dependent sphingomyelin excretion may inhibit TNF-a-induced ceramide production and subsequent apoptosis (Bezombes et al, 1998).…”