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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.

Methods for determination of genistein and its four new analogues in culture media have been developed to support studies on their potential anticancer activities. The investigated compounds were extracted from the media using liquid-liquid extraction with appropriate solvent. After evaporation of organic solvents each of the dry extracts was reconstituted in appropriate mobile phase. Reversed-phase HPLC was applied to quantitative determining of tested compounds. The methods are specific, sensitive and technically simple. They were used to evaluate concentration level of investigated compounds in experiments with human promyelocytic leukemia cells (HL-60 cell line).

Genistein, the principal soy isoflavone, is a molecule of great interest as an innovative chemotherapeutic agent or as a lead-compound in anticancer drug design. To enhance intrinsic activity of genistein and to explore its pharmacophoric potential, its glycosidic derivatives were synthesized. On the basis of structural features and calculated lipophilicity coefficient (ClogP) the derivatives were classified as hydrophilic (i.e. those containing free sugar moiety) or lipophilic (i.e. those with alkylated or acylated sugar hydroxyls). The in vitro cytostatic and cytotoxic studies showed hydrophilic glycosides to be practically inactive against human cancer cell lines when compared to the free aglycone. On the contrary, lipophilic glycosides were significantly more active than the parent isoflavone although the correlation between ClogP and the activity was not clear. On the basis of GI50 and LC50 values two of the most active glycosides were found to be several times more potent in their cytostatic and cytotoxic effect than genistein. Additionally all lipophilic glycosides were revealed to exhibit different mode of action in comparison to genistein. It may suggest that these compounds do not undergo rapid biodegradation, either in culture media or inside cells, and exert their biological effects primarily as intact molecules.

Genistein, a principal soy isoflavone, has recently aroused interest in medical research owning to its numerous biochemical properties such as: inhibition of the activity of tyrosine-specific protein kinases and topoisomerase II, estrogenic and antioxidant activity as well as antiproliferative and antiangiogenic effects. Therefore, genistein is extensively investigated as a novel anticancer drug. To improve physicochemical properties of genistein (e.g., water solubility) we have synthesized its complexes with amines. Genistein-piperazine complex (GP) has been then examined whether it exhibits anticancer action against human promyelocytic leukemia cell line (HL-60) cultured in vitro. The parallel study with pure genistein has also been undertaken. Cell proliferation, viability, apoptosis and cell cycle kinetics have been assayed for various drugs concentrations (10-40 microM) and periods of exposure (1-6 days). GP reduced proliferation rate, decreased cell viability and induced apoptotic cell death, in a dose- and time-dependent manner. Flow-cytometric analysis of cell cycle distribution revealed a progressive and sustained accumulation of cells in the G2/M phase that was accompanied by unperturbed protein synthesis. The measured anticancer effects of GP and genistein were qualitatively and quantitatively similar, indicating that genistein-amine complex does not loose the activity of the parent compound.

Genistein--a soy derived isoflavone has recently attracted much attention of the medical scientific community. This compound was found to be a potent agent in both prophylaxis and treatment of cancer as well as other chronic diseases. The great interest that has focused on genistein led to the identification of numerous intracellular targets of its action in the live cell. At the molecular level, genistein inhibits the activity of ATP utilizing enzymes such as: tyrosine-specific protein kinases, topoisomerase II and enzymes involved in phosphatidylinositol turnover. Moreover, genistein can act via an estrogen receptor-mediated mechanism. At the level one step higher, i.e., at the cellular level, genistein induces apoptosis and differentiation in cancer cells, inhibits cell proliferation, modulates cell cycling, exerts antioxidant effects, inhibits angiogenesis, and suppresses osteoclast and lymphocyte functions. These activities make genistein a promising innovative agent in the treatment of cancer. Additionally, genistein health beneficial effects have been shown in osteoporosis, cardiovascular diseases and menopause. Genistein was also successfully used as an immunosuppressive agent both in vitro and in vivo. All these effects at the three biological levels of action need varied genistein concentrations and only some of them are relevant in people consuming soy-rich diet. The others would occur after purified genistein administration at higher doses. The main genistein advantage as a potential drug is its multidirectional action in the live cell and its very low toxicity.