Vacuolar-ATPase-mediated intracellular sequestration of ellipticine contributes to drug resistance in neuroblastoma cells

Abstract

Neuroblastoma is the most common cancer in infants and the fourth most common cancer in children. Aggressive cell growth and chemoresistance are notorious obstacles in neuroblastoma therapy. Exposure to the anticancer drug ellipticine inhibits efficiently growth of neuroblastoma cells and induces apoptosis in these cells. However, ellipticine induced resistance in these cells. The upregulation of a vacuolar (V)-ATPase gene is one of the factors associated with resistance development. In accordance with this finding, we found that levels of V-ATPase protein expression are higher in the ellipticine-resistant UKF-NB-4ELLI line than in the parental ellipticine-sensitive UKF-NB-4 cell line. Treatment of ellipticine-sensitive UKF-NB-4 and ellipticine-resistant UKF-NB-4ELLI cells with ellipticine-induced cytoplasmic vacuolization and ellipticine is concentrated in these vacuoles. Confocal microscopy and staining of the cells with a lysosomal marker suggested these vacuoles as lysosomes. Transmission electron microscopy and no effect of an autophagy inhibitor wortmannin ruled out autophagy. Pretreatment with a V-ATPase inhibitor bafilomycin A and/or the lysosomotropic drug chloroquine prior to ellipticine enhanced the ellipticine‑mediated apoptosis and decreased ellipticine-resistance in UKF-NB-4ELLI cells. Moreover, pretreatment with these inhibitors increased formation of ellipticine-derived DNA adducts, one of the most important DNA-damaging mechanisms responsible for ellipticine cytotoxicity. In conclusion, resistance to ellipticine in the tested neuroblastoma cells is associated with V-ATPase-mediated vacuolar trapping of this drug, which may be decreased by bafilomycin A and/or chloroquine.

The Function of V-ATPases in Cancer

“…Combining V-ATPase inhibition and cytotoxic drugs overcomes the protective change in pH and causes synergistic killing (70,96,131,167,199,227). The combination is effective in resistant lines, but does not enhance drug efficacy in sensitive lines (96).…”

“…Some cancer stem cells are highly drug resistant and are reported to express high levels of V-ATPase genes (167). V-ATPase expression increases in response to drug treatment, which is accompanied by an increase in cellular pH and lysosomal drug accumulation (70,131,199). In vitro binding studies found that cisplatin binds DNA with much higher affinity at lower pH (131).…”

Cluster of differentiation 147 mediates chemoresistance in breast cancer by affecting vacuolar H+-ATPase expression and activity

“…The plasma membrane V-ATPase is critical for the invasion and migration of MDA-MB-231 breast cancer cells in vitro (37). V-ATPase expression was elevated in the ellipticine-resistant UKF-NB-4ELLI cell line and mediated its ellipticine resistance via the sequestration of ellipticine into the subcellular compartments (38). García-García et al (39) also demonstrated the overexpression of the V-ATPase subunit C gene in cisplatin-resistant tumors.…”

Establishment of IL-7 Expression Reporter Human Cell Lines, and Their Feasibility for High-Throughput Screening of IL-7-Upregulating Chemicals

“…However, EL also induced the expression of several other proinflammatory cytokine genes, indicating that EL is not an IL-7-specific inducer. Since EL is a prodrug and its effect on cell physiology depends on the expression/function of multiple proteins such as cytochrome P450, peroxidases, and vacuolar (V)-ATPases, the mechanism by which the prodrug upregulates the expression of IL-7 and other interleukins remains unclear [37, 40, 41]. …”