Triterpenoids are a large class of naturally occurring compounds, and some potentially interesting as anticancer agents have been found to target mitochondria. The objective of the present work was to investigate the mechanisms of mitochondrial toxicity induced by novel dimethylaminopyridine (DMAP) derivatives of pentacyclic triterpenes, which were previously shown to inhibit the growth of melanoma cells in vitro. MCF-7, Hs 578T and BJ cell lines, as well as isolated hepatic mitochondria, were used to investigate direct mitochondrial effects. On isolated mitochondrial hepatic fractions, respiratory parameters, mitochondrial transmembrane electric potential, induction of the mitochondrial permeability transition (MPT) pore and ion transport-dependent osmotic swelling were measured. Our results indicate that the DMAP triterpenoid derivatives lead to fragmentation and depolarization of the mitochondrial network in situ, and to inhibition of uncoupled respiration, induction of the permeability transition pore and depolarization of isolated hepatic mitochondria. The results show that mitochondrial toxicity is an important component of the biological interaction of DMAP derivatives, which can explain the effects observed in cancer cells.

Development of mitochondrially-targeted drugs is receiving increasing attention because of the central roles these organelles play in energy production, reactive oxygen generation, and regulation of cell death pathways. Previous studies have demonstrated that both natural and synthetic triterpenoids can disrupt mitochondrial structure and function. In this study, we tested the ability of a number of dimethylaminopyridine (DMAP) derivatives of lupane triterpenoids to target mitochochondria in two human melanoma cell lines and an untransformed normal fibroblast line. These compounds induced a striking fragmentation and depolarization of the mitochondrial network, along with an inhibition of cell proliferation. A range of potencies among these compounds was noted, which was correlated with the number, position, and orientation of the DMAP groups. Overall, the extent of proliferation inhibition mirrored the effectiveness of mitochondrial disruption. Thus, DMAP derivatives of lupane triterpenoids can be potent mitochondrial perturbants that appear to suppress cell growth primarily via their mitochondrial effects.Fifteen dimethylaminopyridine derivatives of betulin and betulinic acid were synthesized and found to disrupt mitochondrial structure and function. Effects on mitochondria include fragmentation and loss of membrane polarization.

Natural products represent a rich reservoir of potential small molecule inhibitors exhibiting antiproliferative and tumoricidal properties. An example is the isoquinoline alkaloid berberine, which is found in plants such as goldenseal (Hydrastis canadensis). Studies have shown that berberine is able to trigger apoptosis in different malignant cell lines, and can also lead to cell cycle arrest at sub-apoptotic doses. A particularly interesting feature of berberine is the fact that it is a fluorescent molecule, and its uptake and distribution in cells can be studied by flow cytometry and epifluorescence microscopy. To test the relationships between berberine uptake, distribution and cellular effect in melanoma cells, K1735-M2 mouse and WM793 human melanoma cells were treated with different concentrations of berberine, and alterations in cell cycle progression, DNA synthesis, cell proliferation, and cell death measured.Cell proliferation was measured by sulforhodamine B assays, cell death by flow cytometry, berberine uptake and distribution by laser scanning confocal microscopy and flow cytometry, cell cycle progression by flow cytometry, and DNA synthesis, M-phase, and mitochondrial effects by immunolabeling and epifluorescence microscopy methods.In these melanoma cell lines, berberine at low doses (12.5–50 μM) is concentrated in mitochondria and promotes G1 arrest. In contrast, higher doses (over 50 μM) result in cytoplasmic and nuclear berberine accumulation, and G2 arrest. DNA synthesis is not markedly affected by low doses of berberine, but 100 μM is strongly inhibitory. Even at 100 μM, berberine inhibits cell growth with relatively little induction of apoptosis.Berberine displays multiphasic effects in these malignant cell lines, which are correlated with the concentration and intracellular distribution of this alkaloid. These results help explain some of the conflicting information in the literature regarding the effects of berberine, and suggest that its use in clinical development may be more as a cytostatic agent than a cytotoxic compound.