Genetic analysis of the anti-mutagenic effect of genistein in Escherichia coli

Genistein, the main isoflavone in soy, has received considerable attention for its potential anti-carcinogenic properties. In a previous report, we investigated the possible role of genistein in anti-mutagenesis, using an Escherichia coli reversion assay system. Genistein reduced ENU-induced mutagenesis in a dose-dependent manner and the reduction of mutation frequency was differential among several categories of mutation. Most notable was a loss of transversion mutations, which require SOS functions. In this report, we further investigated the anti-mutagenic effect of genistein using a genetic approach. E. coli strains having alterations in genes involved in SOS-mutagenesis were examined, as were strains having defects in proteins that might serve as potential targets for genistein. The results showed that ENU-induced mutations produced in recA730 and lexA(Def) strains, both expressing a constitutive SOS response, were reduced by genistein to a lesser extent than in the wild-type strain. The effect of genistein was not entirely abolished, however. ENU mutagenesis in a umuC derivative, which reflects predominantly transition mutations, was unaffected by genistein. ENU-induced mutations in strains having defects in topA, gyrA, typA or uspA were not different than the wild-type, suggesting that these gene products were not involved in genistein's anti-mutagenic effect. In addition, we determined the distribution of genistein in various cellular fractions using HPLC. These studies revealed that genistein could be recovered from E. coli cells grown on agar media containing genistein; the intracellular concentration was similar to that in the agar plates. Further, most of the genistein recovered was associated with proteins in the cytosolic fraction and little partitioned in the membrane fraction. In vitro studies showed that genistein could be precipitated from a protein (BSA) containing solution. Finally, we examined the effect of genistein on formation of the RecA filament on ssDNA in vitro and observed an inhibition at high concentrations of genistein. In total, these results suggested that genistein may reduce SOS-dependent mutagenesis by reducing the interaction of RecA protein with ssDNA. As a consequence, genistein could cause a reduction in (1) the overall SOS response (confirmed using β-galactosidase assays) and (2) trans-lesion DNA synthesis by DNA polymerase V.     

Effect of protein tyrosine kinase inhibitors on the current through the Ca(V)3.1 channel

In the present study, we have investigated the effects of protein tyrosine kinase (PTK) inhibitors on the Ca(V)3.1 calcium channel stably transfected in HEK293 cells using the whole-cell configuration of the patch-clamp technique. We have tested two different tyrosine kinase inhibitors, genistein and tyrphostin AG213, and their inactive analogs, genistin and tyrphostin AG9. Bath application of genistein, but not genistin, decreased the T-type calcium current amplitude in a concentration-dependent manner with an IC(50) of 24.7+/-2.0 microM. This effect of genistein was accompanied by deceleration of channel activation and acceleration of channel inactivation. Intracellular application of neither genistein nor genistin had a significant effect on the calcium current. Extracellular application of 50 microM tyrphostin AG213 and its inactive analogue, tyrphostin AG9, did not affect the current through the Ca(V)3.1 channel. The effect of genistein on the channel was also not affected by the presence of catalytically active PTK, p60(c-src) inside the cell. We have concluded that genistein directly inhibited the channel. This mechanism does not involve a PTK-dependent pathway. The alteration of the channel kinetics by genistein suggests an interaction with the voltage sensor of the channel together with the channel pore occlusion.     

三羟异黄酮对HER-2/neu高表达乳腺癌细胞血管生成相关因子表达的影响

三羟异黄酮(genistein)是大豆中的一种非营养成分,其结构与黄酮化合物类似,能竞争性地与雌激素受体结合,故称之为植物雌激素(phytoestrogen)。它具有广泛的生物学作用,如抗肿瘤、抗病毒、抗真菌、抗氧化、抗突变、抗高血压、抗增生等,其中genistein抑制肿瘤的血管生成是当前研究的热点之一。肿瘤的血管生成是肿瘤进一步生长转移的基础,该过程受肿瘤细胞和血管内皮细胞分泌的血管生成相关因     

Inhibition of cellular proliferation by the genistein metabolite 5,7,3',4'-tetrahydroxyisoflavone is mediated by DNA damage and activation of the ATR signalling pathway

The cellular actions of genistein, and its in vivo metabolites, are believed to mediate the decreased risk of breast cancer associated with high soy consumption. The genistein metabolite, 5,7,3′,4′-tetrahydroxyisoflavone (THIF), induced G2-M cell cycle arrest in T47D tumorigenic breast epithelial cells via a mechanism involving the activation of ataxia telangiectasia and Rad3-related kinase (ATR) via its phosphorylation at Ser⁴²⁸. This activation of ATR appeared to result from THIF-induced increases in intracellular oxidative stress, a depletion of cellular GSH and an increase in DNA strand breakage. THIF treatment also led to an inhibition of cdc2, which was accompanied by the phosphorylation of both p53 (Ser¹⁵) and Chk1 (Ser²⁹⁶) and the de-activation of cdc25C phosphatase. We suggest the anti-proliferative actions of THIF may be mediated by initial oxidative DNA damage, activation of ATR and downstream regulation of the p53 and Chk1 pathways leading to cell cycle arrest in G2-M. This may represent one mechanism by which genistein exerts its cellular activity in vivo.     

Genistein induces the metastasis suppressor kangai-1 which mediates its anti-invasive effects in TRAMP cancer cells

Previous studies demonstrated a direct correlation with loss of kangai-1 (KAI1), a metastasis suppressor, and poor prognosis in human prostate and other cancers. In this study, we have characterized the age-dependent downregulation of KAI1 in the TRAMP model which was reversed when mice were fed a genistein-enriched diet. We demonstrated here that doses of genistein (5 and 10 microM)--achievable by supplement intake--significantly induced the expression of KAI1, both at the mRNA and protein levels (up to 2.5-fold), and decreased the invasiveness of TRAMP-C2 cells >2.0-fold. We have pinpointed KAI1 as the invasion suppressor, since its knockdown by siRNA restored the invasive potential of genistein-treated TRAMP-C2 cells to control levels. This work provides the first evidence that genistein treatment may counteract KAI1 downregulation, which is observed in many cancer types and therefore, could be used in anti-metastatic therapies.     

Two-signal electrochemical method for evaluation suppression and proliferation of MCF-7 cells based on intracellular purine

Two electrochemical signals ascribed to xanthine/guanine and hypanthine/adenine in MCF-7 cells were detected at 0.726 and 1.053 V, respectively. Based on the intensity of signals, the genistein-induced proliferation and suppression of MCF-7 cells could be evaluated. The results showed that with the increase of genistein dose at the range of 10⁻⁹ to 10⁻⁶ M, the two electrochemical signals of MCF-7 cell suspension increased due to the proliferation, whereas the tendency at the high dosage range of more than 10⁻⁵ M was decreased. The proliferation and cytotoxicity obtained by the electrochemical method were in agreement with those obtained by cell counting and the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] method. Thus, the two-signal electrochemical method is an effective way to evaluate the effect of drugs on cell activity based on purine metabolism.     

Over-expression of FoxM1 leads to epithelial-mesenchymal transition and cancer stem cell phenotype in pancreatic cancer cells

FoxM1 is known to play important role in the development and progression of many malignancies including pancreatic cancer. Studies have shown that the acquisition of epithelial-to-mesenchymal transition (EMT) phenotype and induction of cancer stem cell (CSC) or cancer stem-like cell phenotypes are highly inter-related, and contributes to drug resistance, tumor recurrence, and metastasis. The molecular mechanism(s) by which FoxM1 contributes to the acquisition of EMT phenotype and induction of CSC self-renewal capacity is poorly understood. Therefore, we established FoxM1 over-expressing pancreatic cancer (AsPC-1) cells, which showed increased cell growth, clonogenicity, and cell migration. Moreover, over-expression of FoxM1 led to the acquisition of EMT phenotype by activation of mesenchymal cell markers, ZEB1, ZEB2, Snail2, E-cadherin, and vimentin, which is consistent with increased sphere-forming (pancreatospheres) capacity and expression of CSC surface markers (CD44 and EpCAM). We also found that over-expression of FoxM1 led to decreased expression of miRNAs (let-7a, let-7b, let-7c, miR-200b, and miR-200c); however, re-expression of miR-200b inhibited the expression of ZEB1, ZEB2, vimentin as well as FoxM1, and induced the expression of E-cadherin, leading to the reversal of EMT phenotype. Finally, we found that genistein, a natural chemo-preventive agent, inhibited cell growth, clonogenicity, cell migration and invasion, EMT phenotype, and formation of pancreatospheres consistent with reduced expression of CD44 and EpCAM. These results suggest, for the first time, that FoxM1 over-expression is responsible for the acquisition of EMT and CSC phenotype, which is in part mediated through the regulation of miR-200b and these processes, could be easily attenuated by genistein.