In our previous studies,
voacamine, a bisindolic
alkaloid extracted from Peschiera fuchsiaefolia, was examined for its possible capability of enhancing the cytotoxic effect of
doxorubicin (DOX) on multidrug resistant (MDR) human
osteosarcoma cells (U-2 OS-R).
Voacamine induced in resistant cells a significant increase of
drug retention and intranuclear location which became comparable to those observed in the parental sensitive counterparts (U-2 OS-WT). In the present study, the cell survival analysis and the electron microscopic observations confirmed the evident cytotoxicity of DOX on MDR cells after pre-treatment with the
plant extract. Moreover, an increase of the reactivity of
P-glycoprotein (P-gp) with the
monoclonal antibody UIC2, which recognizes an
epitope of the
drug transporter in its functional conformation, was revealed, demonstrating that
voacamine is a substrate of P-gp, thus acting as a competitive antagonist of the
cytotoxic agent. Moreover, to investigate if the enhancement of the cytotoxic effect induced by
voacamine could be due to an apoptotic process, we carried out the analysis of cell morphology after Hoechst staining and the quantification of apoptosis by
Annexin V-FITC assay. These evaluations showed a very low rate of apoptosis in U-2 OS-R cells treated with
voacamine and DOX given in association. In addition, the combined treatment induced ultrastructural modifications suggestive of autophagic cell death. In particular, transmission electron microscopy observations revealed the presence of numerous lysosomes and the formation of a large number of autophagosomes containing residual digested material. In conclusion, these findings seem to indicate that
voacamine is capable of enhancing the cytotoxic effect of DOX on MDR cells by favouring a lethal autophagic process.