Role of P-glycoprotein inhibitors in ceramide-based therapeutics for treatment of cancer

Abstract

The anticancer properties of ceramide, a sphingolipid with potent tumor-suppressor properties, can be dampened via glycosylation, notably in multidrug resistance wherein ceramide glycosylation is characteristically elevated. Earlier works using the ceramide analog, C6-ceramide, demonstrated that the antiestrogen tamoxifen, a first generation P-glycoprotein (P-gp) inhibitor, blocked C6-ceramide glycosylation and magnified apoptotic responses. The present investigation was undertaken with the goal of discovering non-anti-estrogenic alternatives to tamoxifen that could be employed as adjuvants for improving the efficacy of ceramide-centric therapeutics in treatment of cancer. Herein we demonstrate that the tamoxifen metabolites, desmethyltamoxifen and didesmethyltamoxifen, and specific, high-affinity P-gp inhibitors, tariquidar and zosuquidar, synergistically enhanced C6-ceramide cytotoxicity in multidrug resistant HL-60/VCR acute myelogenous leukemia (AML) cells, whereas the selective estrogen receptor antagonist, fulvestrant, was ineffective. Active C6-ceramide-adjuvant combinations elicited mitochondrial ROS production and cytochrome c release, and induced apoptosis. Cytotoxicity was mitigated by introduction of antioxidant. Effective adjuvants markedly inhibited C6-ceramide glycosylation as well as conversion to sphingomyelin. Active regimens were also effective in KG-1a cells, a leukemia stem cell-like line, and in LoVo human colorectal cancer cells, a solid tumor model. In summary, our work details discovery of the link between P-gp inhibitors and the regulation and potentiation of ceramide metabolism in a pro-apoptotic direction in cancer cells. Given the active properties of these adjuvants in synergizing with C6-ceramide, independent of drug resistance status, stemness, or cancer type, our results suggest that the C6-ceramide-containing regimens could provide alternative, promising therapeutic direction, in addition to finding novel, off-label applications for P-gp inhibitors.

Inhibitors of Ceramide- and Sphingosine-Metabolizing Enzymes as Sensitizers in Radiotherapy and Chemotherapy for Head and Neck Squamous Cell Carcinoma

“…The antiestrogen tamoxifen, a first-generation P-gp inhibitor, blocked C6-ceramide glycosylation and magnified apoptotic responses. Specific high-affinity P-gp inhibitors, tariquidar and zosuquidar, synergistically enhanced C6-ceramide cytotoxicity in multidrug resistant leukemia cells [128]. A drug cocktail of GluCS inhibitor, CDase inhibitor, and imipramine that disturbs lipid turnover in biological membranes was reported to induce a marked increase in ceramide levels in radio-resistant HNSCC cells, corresponding with marked effects on radiation sensitivity [129]…”

Chemotherapy selection pressure alters sphingolipid composition and mitochondrial bioenergetics in resistant HL-60 cells

“…Cell viability was determined by fluorescence measurement as previously described (22). Briefly, cells were seeded in black-wall 96-well plates in RPMI-1640 medium containing 5% FBS (cell numbers given in figure legends).…”

“…GCS activity was measured in intact HL-60 wt and in drugresistant counterparts using C6-NBD-ceramide complexed to BSA as previously described (22,24). The GCS assays were conducted in the absence of the chemotherapy drugs.…”

Silencing GnT‑V reduces oxaliplatin chemosensitivity in human colorectal cancer cells through N‑glycan alteration of organic cation transporter member 2

“…The possible involvement of another substrate, P-glycoprotein (P-gp), a membrane glycoprotein known to regulate drug sensitivity in cancer cells was also evaluated ( 22 , 23 ). Cells were treated for 24 h with the P-gp inhibitor zosuquidar in combination with oxaliplatin.…”