• antimutagenesis
• antitumor
• apoptosis
• cytotoxicity
• genotoxicity
• human cells
• luciferase assay
• saponins

Medicinal plants have been used for the cure of different illnesses, including cancer, for thousands of years. Large numbers of medicinal plants having anticancer properties are available in nature but they are not fully phytochemically characterized. Among natural chemicals used as cancer chemo-preventive and/or chemotherapeutic agents, saponins are one of the most promising and interesting family of compounds. Recently, special attention is paid to combinations of saponins with other anticancer drugs, since their potential to enhance the antitumor activity of clinical drugs.
In the present study, we assessed the biological effects of the combination of certain saponins, namely saponin fractions (SFs 6-11), obtained from in vitro cultures of Astragalus verrucosus Moris. In this regard, little is known about this species. Particularly, we tested the activity of these SFs alone or in combination with conventional chemotherapeutic drugs, in different cell systems such as human tumor cells, the yeast Saccharomyces cerevisiae (as a model cell system), and human lymphocytes (as healthy human cells).
Interestingly, SF (8) showed a strongly inhibitory effect (GI50 of 7.92±0.3 µg/mL) on the proliferation of human colon adenocarcinoma cell line (HCT116) via activation of a p53-dependent apoptotic pathway. Thus, the lack of cytotoxicity exerted by the individual saponins contained in SF (8), Astraverrucin VI and VII, Astrailienin A and Cicloaralosyde D (GI50 higher than 100 µg/mL), corroborates our hypothesis that the qualitative/quantitative combination of saponins present in SF (8) is required to inhibit the cell growth of HCT116 cell line and to induce apoptosis.
The p53 activity is ubiquitously lost in cancers. Therefore, the restoration of p53 activity by inhibiting the negative effect of its endogenous regulators represents an appealing therapeutic strategy for many wt p53 tumors. SFs, when tested alone, did not show significant alterations of p53 transactivation, as reported by luciferase assay in HCT116 cells. Furthermore, the pharmacological activation of the p53 pathway can be exploited to work in combination with other therapeutic agents either to protect from side effects of chemotherapy or to enhance the antitumor impact. Particularly, very low concentrations of SF (6) showed a p53-dependent synergistic effect with doxorubicin greater than exerted by the drug alone, leading to the successful activation of p53 and downstream cell signaling. These results indicated that SF (6) sensitizes HCT116 tumor cells to the effects of chemotherapeutic drug. Using a functional assay developed in yeast, we also confirmed the ability of SF (6) combined with doxorubicin to led p53 transactivation, via inhibition the p53-MDM2 interaction,.
In addition, all SFs and saponins displayed neither genotoxic nor cytostatic/cytotoxic activity in human peripheral lymphocytes (up to a concentration of 100 µg/ml). Interestingly, some SFs were able to counteract undesirable outcomes of DNA damaging inducing cancer drugs, demonstrating their effectiveness in cancer chemoprevention.
Concluding, this study is the first aimed to investigate biological activities of saponin fractions obtained from in vitro cultures of A. verrucosus and may pave the way for further investigations on extracts containing secondary metabolites belonging to a single chemical class. This study also highlights the superior activity of certain extracts compared to their chemical constituents, and, although further studies are required to clarify the mechanisms of action of selected SFs, provides evidences of the wide biological potential of this species.