Partial Inhibition of Multidrug Resistance by Safingol Is Independent of Modulation of P-glycoprotein Substrate Activities and Correlated with Inhibition of Protein Kinase C

Abstract

Safingol is a lysosphingolipid protein kinase C (PKC) inhibitor that competitively interacts at the regulatory phorbol binding domain of PKC. We investigated the effects of safingol on antineoplastic drug sensitivity and PKC activity of MCF-7 tumor cell lines. Safingol treatment of 32 P-labeled MCF-7 WT and MCF-7 DOX R cells inhibited phosphorylation of the myristoylated alaninerich protein kinase C substrate in both cell lines, suggesting inhibition of cellular PKC. However, only in MCF-7 DOX R cells did safingol treatment increase accumulation of [ 3 H]vinblastine and enhance toxicity of Vinca alkaloids and anthracyclines. Drug accumulation changes in MCF-7 DOX R cells treated with safingol were accompanied by inhibition of basal and phorbol 12,13-dibutyrate-stimulated phosphorylation of P-glycoprotein (P-gp). Expression of P-gp and levels of mdr1 message in MCF-7 DOX R cells were not altered by safingol treatment alone or in combination with vinblastine. Treatment of MCF-7 DOX R cell membranes with safingol did not inhibit [ 3 H]vinblastine binding or [ 3 H]azidopine photoaffinity labeling of P-gp. Furthermore, safingol did not stimulate P-gp ATPase activity in membranes prepared from MCF-7 DOX R cells. We conclude that enhanced drug accumulation and sensitivity in MCF-7 DOX R cells treated with safingol are correlated with inhibition of PKC rather than competitive interference with P-gp drug binding through direct interaction with P-glycoprotein.Exposure of cancer cells to a single natural product antineoplastic drug can give rise to cells that also exhibit crossresistance to other unrelated natural cytotoxins. This phenotype is known as multidrug resistance and is characterized by the reduced intracellular accumulation of these drugs, due to drug efflux by P-glycoprotein (P-gp), 1 a 150 -180-kDa plasma membrane transport ATPase encoded by the human mdr1 gene (reviewed in Refs. 1 and 2)). A number of agents have been shown to inhibit multidrug resistance (3). Most modulators of multidrug resistance inhibit specific binding of drugs to P-gp in membranes derived from MDR cells (4 -7) and photoaffinity labeling of P-gp by photoactive drug analogs (4,8,9). These results suggest that competitive inhibition of P-gp drug binding is a mechanism by which P-gp function and multidrug resistance may be inhibited.P-gp is subject to phosphorylation (10, 11) and changes in P-gp phosphorylation have been associated with inhibition of P-gp drug transport by modulators of the MDR phenotype (12, 13). Although other kinases have been shown to phosphorylate P-gp in vitro (11,14), there is considerable experimental evidence that suggests that phosphorylation of P-gp by protein kinase C (PKC) may play a role in regulating multidrug resistance.PKC is a family of serine/threonine kinases, composed of calcium-dependent and calcium-independent isotypes, which are dependent upon phospholipid for activation (15). In vitro, P-gp is phosphorylated by PKC (13, 16) at serine residues (13, 17) primarily in the linker region of P-gp (18,19). ...

Sphingosine 1-phosphate as a therapeutic agent

“…68 An analog of sphingosine, L-threo-dihydrosphingosine (known as safingol), which also acts as a sphingosine kinase inhibitor, enhanced doxorubicin accumulation and sensitivity of MCF-7 drug-resistant cells. 69 Because DMS and L-threodihydrosphingosine induce apoptosis regardless of P-glycoprotein expression, they may provide a new strategy for the treatment of anticancer drug-resistant cancers. Indeed, in pilot clinical phase I trials with safingol, which also potentiated the tumor-inhibiting effect of doxorubicin in tumor-bearing animals, it was found that safingol can be given safely with doxorubicin.…”

AKAP150 signaling complex promotes suppression of the M-current by muscarinic agonists

“…Pretreatment with calphostin C was carried out under dim light in the incubator. For treatment with L-threo-dihydrosphingosine (safingol), ethanol stock solution of safingol (Sigma) was diluted into α-MEM containing 1 mM BSA, sonicated briefly, and then incubated at room temperature for 1 h to form complexes as described 39 . The cells were then treated with either 10 μM safingol or vehicle for 4-6 h before recording.…”

“…5a,c). In contrast, calphostin C and safingol, which act on the diacylglycerol binding site 38,39 , reduced the sensitivity to Oxo-M (Fig. 5a,b).…”

Co-ordinate loss of protein kinase C and multidrug resistance gene expression in revertant MCF-7/Adr breast carcinoma cells

“…PKC activators also increased drug accumulation and decreased drug sensitivity in MCF-7/Adr (Fine et al, 1988) and KM12L4a cells (Dong et al, 1991), and induced MDRJ gene expression in normal human lymphocytes (Chaudhary and Roninson, 1992). Conversely, a variety of PKC inhibitors, such as staurosporine (Sato et al, 1990), the staurosporine analogues CGP 41251 (Utz et al, 1994; Budworth et al, 1996) and GF 109203X , calphostin C (Bates et al, 1993) and safingol (Sachs et al, 1995), reversed P-gp-mediated mdr. However, reversal of drug resistance caused by staurosporine analogues is probably associated with direct interaction with P-gp rather than with PKC inhibition (Smith and Zilfou, 1995;Gekeler et al, 1996;Goodfellow et al, 1996;Budworth et al, 1996).…”