Microspectrofluorometry of the protonation state of ellipticine, an antitumor alkaloid, in single cells

Abstract

The protonation state and intracellular distribution of ellipticine were investigated in single human mammary T47D cells by confocal laser microspectrofluorimetry. In the cell nucleus, only the protonated form of ellipticine was detected as a direct consequence of its apparent pK increase upon DNA binding. Both protonated and neutral forms were present in the aqueous cytoplasm, where the pH is close to the drug pK. When cells were incubated in high concentrations of K+, a condition that depolarizes the plasma membrane potential, ellipticine cellular accumulation was reduced. In the cytoplasm, ellipticine was mainly bound to mitochondria, and its protonation equilibrium was shifted toward the neutral form. The fluorescence spectrum of ellipticine bound to mitochondria was insensitive to valinomycin, whereas it was markedly shifted toward the protonated form after carbonyl cyanide p-trifluoromethoxy-phenylhydrazone or nigericin addition. Similar studies with ellipticine bound to isolated mitochondria suggest that it behaves as a fluorescent probe of mitochondrial pH in both isolated mitochondria and single living cells.

Membrane Perturbations Induced by New Analogs of Neocryptolepine

“…DIMIQ, possessing simultaneously at this pH the neutral and anionic forms, binds to a substantially larger extent (up to 45%). As reported by Sureau et al 43) in the presence of neutral micelles or liposomes, the pK a of ellipticine was reduced, as a result of reduced polarity at the interface. The same mode of action is presumably present in our experiment.…”

“…Moreover, the increase of pK a should also be considered as a consequence of increased proton activity at the anionicwater interface, as observed for ellipticine. 43,44) Binding to the membranes, apart from the hemolytic effect, as will be discussed below, can cause an increase in membrane surface area and therefore osmotic fragility. Overall, observations with hydrophobic model systems might suggest that the protonation equilibrium of tested chemicals can be shifted in response to its interactions with different biological macromolecules in vivo as a consequence of the local pH environment.…”

Ellipticine derivative NSC 338258 represents a potential new antineoplastic agent for the treatment of multiple myeloma

“…This radical not only tremendously improves the hydrophilicity of the compound, allowing rapid cellular uptake, but also prevents intracellular metabolism by the hydroxylation at C-9 of ellipticine (14). Although initially categorized as a topo II inhibitor that localizes to the nucleus, ellipticine has more recently been shown to localize in the cytoplasm and accumulate in mitochondria (26). Similarly, EPED3, which displays intrinsic fluorescence identical to ellipticine, was found to rapidly accumulate in the mitochondria in this study.…”

Protonophoric Activity of Ellipticine and Isomers across the Energy-transducing Membrane of Mitochondria

“…Tetraphenylphosphonium (TPP ϩ ) was purchased from Merck. Mitochondria Isolation-Rat liver mitochondria from male Wistar albino rats were isolated by differential centrifugation as described previously (6).…”

“…However, good evidence lacks when it comes to correlating in vivo activities with a specific DNA binding mode (5). Data based on microspectrofluorometric analyses have shown that an important fraction of ellipticine accumulates in mitochondria (6). The possibility that these organelles rather than the nucleus might be a privileged pharmacological target has been suggested.…”