digitonin and sanguinarine

P-glycoprotein (P-gp or MDR1) is an ATP-binding cassette (ABC) transporter. It is involved in the efflux of several anticancer drugs, which leads to chemotherapy failure and multidrug resistance (MDR) in cancer cells. Representative secondary metabolites (SM) including phenolics (EGCG and thymol), terpenoids (menthol, aromadendrene, β-sitosterol-O-glucoside, and β-carotene), and alkaloids (glaucine, harmine, and sanguinarine) were evaluated as potential P-gp inhibitors (transporter activity and expression level) in P-gp expressing Caco-2 and CEM/ADR5000 cancer cell lines. Selected SM increased the accumulation of the rhodamine 123 (Rho123) and calcein-AM (CAM) in a dose dependent manner in Caco-2 cells, indicating that they act as competitive inhibitors of P-gp. Non-toxic concentrations of β-carotene (40μM) and sanguinarine (1μM) significantly inhibited Rho123 and CAM efflux in CEM/ADR5000 cells by 222.42% and 259.25% and by 244.02% and 290.16%, respectively relative to verapamil (100%). Combination of the saponin digitonin (5μM), which also inhibits P-gp, with SM significantly enhanced the inhibition of P-gp activity. The results were correlated with the data obtained from a quantitative analysis of MDR1 expression. Both compounds significantly decreased mRNA levels of the MDR1 gene to 48% (p<0.01) and 46% (p<0.01) in Caco-2, and to 61% (p<0.05) and 1% (p<0.001) in CEM/ADR5000 cells, respectively as compared to the untreated control (100%). Combinations of digitonin with SM resulted in a significant down-regulation of MDR1. Our findings provide evidence that the selected SM interfere directly and/or indirectly with P-gp function. Combinations of different P-gp substrates, such as digitonin alone and together with the set of SM, can mediate MDR reversal in cancer cells.

Topics: Alkaloids; Antineoplastic Agents, Phytogenic; ATP Binding Cassette Transporter, Subfamily B, Member 1; Benzophenanthridines; beta Carotene; Caco-2 Cells; Colonic Neoplasms; Digitonin; Dose-Response Relationship, Drug; Drug Combinations; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Drug Synergism; Fluoresceins; Humans; Isoquinolines; Leukemia; Phenols; Phytochemicals; Phytotherapy; Plant Extracts; Rhodamine 123; RNA, Messenger; Terpenes

In phytotherapy, extracts from medicinal plants are employed which contain mixtures of secondary metabolites. Their modes of action are complex because the secondary metabolites can react with single or multiple targets. The components in a mixture can exert additive or even synergistic activities. In this study, the cytotoxicity of some phytochemicals, including phenolics (EGCG and thymol), terpenoids (menthol, aromadendrene, β-sitosterol-O-glucoside, and β-carotene) and alkaloids (glaucine, harmine, and sanguinarine) were investigated alone or in combination with the cytotoxic monodesmosidic steroidal saponin digitonin in Caco-2, MCF-7, CEM/ADR5000, and CCRF-CEM cells. Digitonin was combined in non-toxic concentrations (5μM in each cell line; except in MCF-7 the concentration was 2μM), together with a selection of phenolics, terpenoids, and alkaloids to evaluate potential synergistic or additive effects. An enhanced cytotoxicity was observed in most combinations. Even multi-drug resistant (MDR) cells (such as CEM/ADR5000 cells), with a high expression of P-glycoprotein, were responsive to combinations. Sanguinarine was the most cytotoxic alkaloid against CEM/ADR5000, MCF-7, and CCRF-CEM cells alone and in combination with digitonin. As compared to sanguinarine alone, the combination was 44.53-, 15.38-, and 6.65-fold more toxic in each cell line, respectively. Most combinations synergistically increased the cytotoxicity, stressing the importance of synergy when using multi-target drugs and mixtures in phytotherapy.

Topics: Alkaloids; Antineoplastic Agents, Phytogenic; ATP Binding Cassette Transporter, Subfamily B, Member 1; Benzophenanthridines; Caco-2 Cells; Digitalis; Digitonin; Drug Resistance, Multiple; Drug Synergism; Female; Humans; Isoquinolines; MCF-7 Cells; Neoplasms; Phenols; Phytotherapy; Plant Extracts; Terpenes

We determined the ability of some phytochemicals, including alkaloids (glaucine, harmine, and sanguinarine), phenolics (EGCG and thymol), and terpenoids (menthol, aromadendrene, β-sitosterol-O-glucoside, and β-carotene), alone or in combination with the saponin digitonin to reverse the relative multi-drug resistance of Caco-2 and CEM/ADR5000 cells to the chemotherapeutical agent doxorubicin. The IC(50) of doxorubicin in Caco-2 and CEM/ADR5000 was 4.22 and 44.08μM, respectively. Combination of non-toxic concentrations of individual secondary metabolite with doxorubicin synergistically sensitized Caco-2 and CEM/ADR5000 cells, and significantly enhanced the cytotoxicity of doxorubicin. Furthermore, three-drug combinations (secondary metabolite+digitonin+doxorubicin) were even more powerful. The best synergist was the benzophenanthridine alkaloid sanguinarine. It reduced the IC(50) value of doxorubicin 17.58-fold in two-drug combinations (sanguinarine+doxorubicin) and even 35.17-fold in three-drug combinations (sanguinarine+digitonin+doxorubicin) in Caco-2 cells. Thus synergistic drug combinations offer the possibility to enhance doxorubicin efficacy in chemotherapy.

Topics: Alkaloids; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Benzophenanthridines; Caco-2 Cells; Digitonin; Doxorubicin; Drug Resistance, Neoplasm; Drug Synergism; Humans; Inhibitory Concentration 50; Isoquinolines; Neoplasms; Phenols; Phytotherapy; Plant Extracts; Plants; Terpenes