Multidrug resistance (MDR) in
tumor cells is generally associated with increased efflux of the cytotoxic compounds, due to the activation of mechanisms of intracellular transport and to the overexpression of
surface proteins, such as
P-glycoprotein (Pgp), which act as
ATP-dependent molecular pumps. In a previous study,
voacamine, a bisindolic
alkaloid from Peschiera fuchsiaefolia, was examined for its possible capability of enhancing the cytotoxic effect of
doxorubicin (DOX) on resistant human
osteosarcoma cells. The effects of
voacamine on the cell survival and on accumulation of DOX were investigated on both the parental cell line, U-2 OS-WT, and its resistant counterpart, U-2 OS-R. A differential effect between sensitive and resistant cells on the intracellular DOX concentration and distribution was revealed. In particular,
voacamine induced a significant increase of
drug retention and intranuclear location in resistant cells. Moreover, the cell survival analysis and the electron microscopic observations revealed an enhancement of the cytotoxic effect of DOX induced by the
plant extract. In the present study, a panel of
monoclonal antibodies (MAbs), recognizing different and specific structural and functional state of Pgp, was used. By flow cytometry and immunofluorescence confocal microscopy, a dose-dependent increase of the reactivity of Pgp with MAb UIC2, which specifically recognizes an
epitope of the
drug transporter in its functional conformation, was detected in
voacamine-treated U-2 OS-R cells. Conversely, the expression of the
epitope recognized by MAb MC57 was downregulated while MAb MM4.17 did not change its binding level to treated and untreated MDR cells. These data suggest that the
plant extract reacts with Pgp producing conformational changes with consequent
epitope modulation. Taken together, our observations seem to demonstrate that
voacamine is a substrate for Pgp and, therefore, interferes with the Pgp-mediated
drug export, acting as a competitive antagonist of
cytotoxic agents.