In vitro toxicity evaluation in the development of new anticancer drugs—genistein glycosides

Abstract 

In vitro cytotoxicity tests currently in use applied in the developmental stages of anticancer drug discovery are able to select the most potent compounds, but are not predictive of their potential toxicity. In this study, we have demonstrated the applicability of neutral red uptake assay using mouse fibroblasts Balb/c 3T3 cell line (3T3 NRU assay) for in vitro toxicity testing of newly synthesized genistein glycosides, the compounds that appear to show anticancer activity. We have also proven the compatibility of in-house 3T3 NRU assay with the prediction model for acute rodent oral toxicity testing, endorsed by NIEHS-ICCVAM workshop. The combined results from the cytotoxicity and the in vitro toxicity tests facilitated the selection of the most promising genistein derivatives, compounds G21 and G23, which were the most active and selective towards cancer cells. The comparison of predicted LD50 values revealed that almost all genistein derivatives are at least two-fold less toxic than the chemotherapeutics currently used in cancer therapy, which is very promising for this new group of compounds.

Keywords: Toxicity in vitro, HTS, Anticancer agents, NRU assay, Balb/c 3T3 cells, Genistein glycosides

Abbreviations:: 3T3 NRU assay, neutral red uptake assay using Balb/c 3T3 cells, DMEM, Dullbecco's modified Eagle's medium, HTS, high throughput screening, MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, NIEHS-ICCVAM, US National Institute of Environmental Health Sciences—Interagency Coordinating Committee on the Validation of Alternative Methods, OD, optical density, RC, register of cytotoxicity, SX, selectivity index