Laurence Gamet-Payrastre

Relationship between flavonoid structure and inhibition of phosphatidylinositol 3-kinase: A comparison with tyrosine kinase and protein kinase C inhibition

Depending on their structure, flavonoids display more or less potent inhibitory effects on the growth and proliferation of certain malignant cells in vitro, and these effects are thought to be due to inhibition of various enzymes. We investigated the inhibitory action of fourteen flavonoids of different chemical classes on phosphatidylinositol 3-kinase alpha (PI 3-kinase alpha) activity, an enzyme recently shown to play an important role in signal transduction and cell transformation. Of the fourteen flavonoids tested, myricetin was the most potent PI 3-kinase inhibitor (IC50 = 1.8 microM), while luteolin and apigenin were also effective inhibitors, with IC50 values of 8 and 12 microM, respectively. Fisetin and quercetin, as previously reported, were also found to significantly inhibit PI 3-kinase activity. The same flavonoids were also analyzed for inhibition of epidermal growth factor receptor (EGF-R), intrinsic tyrosine kinase and bovine brain protein kinase C (PKC). At elevated doses, some of these flavonoids were found to also cause significant inhibition of PKC and tyrosine kinase activity of EGF-R. A structure-activity study indicated that the position, number and substitution of the hydroxyl group of the B ring, and saturation of the C2-C3 bond are important factors affecting flavonoid inhibition of PI 3-kinase. They may also play a significant role in specificity of inhibition and could help to provide a basis for the further design of specific inhibitors of this lipid kinase. Finally, possible relationships between the antitumoral properties of these flavonoids and their biological activities are discussed.

Flavonoids and the inhibition of PKC and PI 3-kinase

Flavonoids provide a large number of interesting natural compounds that are consumed daily and exhibit more or less potent and selective effects on some signaling enzymes as well as on the growth and proliferation of certain malignant cells in vitro. Among the identified signal transducers, phosphoinositide 3-kinase (PI 3-kinase) and protein kinase C (PKC) are now considered key players in many cellular responses including cell multiplication, apoptosis, and transformation. Despite their lack of strict specificity, some flavonoids provide valuable bases for the design of analogues that could be used to specifically block particular isoforms of PI 3-kinase or PKC and their downstream-dependent cellular responses.

Occupational exposure to pesticides and risk of hematopoietic cancers: meta-analysis of case-control studies

ObjectiveIn this study we conducted a meta-analysis of 13 case–control studies that examined the occurrence of hematopoietic cancers in pesticide related occupations in order to undertake a qualitative and quantitative evaluation of a possible relationship.MethodsPubmed databases were searched for case–control studies published between 1990 and 2005 investigating the relation between hematopoietic cancers and occupational exposure to pesticides. Fixed and random effect meta-analysis models were used depending on the presence of heterogeneity between studies.ResultsThe overall meta-odds ratio obtained after pooling 44 ORs from 13 studies was 1.3 (95% CI: 1.3–1.5). We realized stratified analysis on three different types of hematopoietic cancers (non-Hodgkin lymphoma (NHL), leukemia and multiple myeloma). A significant increased risk of NHL was found (OR = 1.35; 95% CI = 1.2–1.5). Moreover, increased risks of Leukemia (OR = 1.35; 95% CI = 0.9–2) and multiple myeloma (OR = 1.16; 95% CI = 0.99–1.36) were also detected but these results were not statistically significant. Significant heterogeneity existed among the different studies and a publication bias was detected. Therefore, a meta-regression was carried out. Our results showed that a long period of exposure (more than 10 years) provided an increase in the risk of all hematopoietic cancers and for NHL by fractions of 2.18 (95% CI = 1.43–3.35) and 1.65 (95% CI = 1.08–2.51), respectively. Conclusions: The overall meta-odds ratio suggests that there is a significantly positive association between occupational exposure to pesticides and all hematopoietic cancers as well as NHL. A major limitation of our meta-analysis is the lack of sufficient data about exposure information and other risk factors for hematopoietic cancer (genetic predisposition, ethnic origin, immunodepression…). In addition, data concerning specific subtypes of hematopoietic cancers are often confusing. Thus, future epidemiological studies should undertake a major effort to assess the identity and the level of pesticides exposure and should control for the most likely potential confounders.

Mechanism of Sulforaphane-Induced Cell Cycle Arrest and Apoptosis in Human Colon Cancer Cells

Sulforaphane (SFN) is a natural micronutrient found in cruciferous vegetables that has been shown to possess antitumoral properties in carcinogen-treated rats. In vitro, SFN regulates phase II enzymes, cell cycle, and apoptosis. In the present study, we investigated the relationship between SFN induction of apoptosis and cell cycle arrest in HT29 human colon carcinoma cells. In previously published data, a significant increase in the G2/M phase of the cell cycle has been observed in SFN-treated cells that was associated with increased cyclin B1 protein levels. In the present study, our results show that SFN induced p21 expression. Moreover, preincubation of HT29 cells with roscovitine, a specific cdc2 kinase inhibitor, blocked the G2/M phase accumulation of HT29 cells treated with SFN and abolished its apoptotic effect (22.2 ± 4 of floating cells in SFN-treated cells vs. 6.55 ± 2 in cells treated with both SFN and roscovitine). These results suggest that the cdc2 kinase could be a key target for SFN in the regulation of G2/M block and apoptosis. Moreover, in SFN-treated cells the retinoblastoma tumor suppressor protein (Rb) is highly phosphorylated. Inhibition of the cdc2 kinase by roscovitine did not change the phosphorylation status of Rb in SFN-treated cells, suggesting that this cyclin-dependent kinase may not be involved. In our study, we did not observe any significant change in the proteasomal activity between control and SFN-treated cells. Moreover, inhibition of proteasomal activity through the use of MG132 diminished SFN-induced HT29 cell death, suggesting that the apoptotic effect of SFN requires a functional proteasome-dependent degradation system. In summary, we have elucidated part of the mechanism of action of SFN in the concomitant regulation of intestinal cell growth and death.

SELECTIVE CYTOSTATIC AND CYTOTOXIC EFFECTS OF GLUCOSINOLATES HYDROLYSIS PRODUCTS ON HUMAN COLON CANCER CELLS IN VITRO

Glucosinolates hydrolysis products are attracting increasing attention since many studies have suggested that they may be involved in the anticarcinogenic property of cruciferous vegetables. In this study, we show that diindolylmethane (DIM) and sulforaphane, produced during the hydrolysis of glucobrassicin and glucoraphanin, respectively, exert a dose-dependent cytotoxicity on human colon adenocarcinoma HT29 cells. Moreover, these products are able to inhibit quiescent cells to re-enter the cell cycle. Interestingly, our results clearly show that low doses of DIM and sulforaphane, although very effective on undifferentiated intestinal HT29 cells, do not affect the viability of the differentiated CaCo2 cells. The reversibility of their effects has also been tested and is discussed.

Exposure to pesticides and risk of childhood cancer: a meta-analysis of recent epidemiological studies.

Objectives The authors performed a meta-analysis of case–control and cohort studies to clarify the possible relationship between exposure to pesticides and childhood cancers. Methods Two cohort and 38 case–control studies were selected for the first meta-analysis. After evaluating homogeneity among studies using the Cochran Q test, the authors calculated a pooled meta-OR stratified on each cancer site. The authors then constructed a list of variables believed to play an important role in explaining the relation between parental exposure to pesticide and childhood cancer, and performed a series of meta-analyses. The authors also performed a distinct meta-analysis for three cohort studies with RR data. Results Meta-analysis of the three cohort studies did not show any positive links between parental pesticide exposure and childhood cancer incidence. However, the meta-analysis of the 40 studies with OR values showed that the risk of lymphoma and leukaemia increased significantly in exposed children when their mother was exposed during the prenatal period (OR=1.53; 95% CI 1.22 to 1.91 and OR=1.48; 95% CI 1.26 to 1.75). The risk of brain cancer was correlated with paternal exposure either before or after birth (OR=1.49; 95% CI 1.23 to 1.79 and OR=1.66; 95% CI 1.11 to 2.49). The OR of leukaemia and lymphoma was higher when the mother was exposed to pesticides (through household use or professional exposure). Conversely, the incidence of brain cancer was influenced by the fathers exposure (occupational activity or use of household or garden pesticides). Conclusion Despite some limitations in this study, the incidence of childhood cancer does appear to be associated with parental exposure during the prenatal period.

Comparative effects of flavonoids on the growth, viability and metabolism of a colonic adenocarcinoma cell line (HT29 cells).

The aim of the present study was to compare the effect of five structural classes of flavonoids on the viability and metabolism of a colonic adenocarcinoma cell line (HT29 cells). The most prominent structural features of flavonoids favoring both their cytotoxic activity and their capacity to inhibit lactate release appear to be the desaturation of the 2, 3 bond and the position of attachment of the B ring. Indeed, flavonol and flavone are the most potent and, in both classes, the order of potency can be modulated by hydroxyl or methoxyl substituents. On the other hand, in our model, we did not find any correlation between flavonoid structure and their capacity to modulate cAMP level. This last point is discussed.

Gender-linked haematopoietic and metabolic disturbances induced by a pesticide mixture administered at low dose to mice

Defining the impact on health of exposure to a low-dose pesticide mixture via food intake is a topical question since epidemiological studies suggest that this may increase the risk of pathologies and particularly haematopoietic malignancies. Here we investigated on the haematopoietic system of mice, the effect of a mixture of six pesticides frequently ingested through the intake of fruits and vegetables produced in France (alachlor, captan, diazinon, endosulfan, maneb, mancozeb). The mixture was administered repeatedly by gavage to mice for 4 weeks at levels derived from the human Acceptable Daily Intake (ADI) level adapted to the mean weight of mice. Using a NMR-based metabonomic approach, we show that this treatment led to specific gender-linked variations in the level of hepatic metabolites involved in oxidative stress and in the regulation of glucose metabolism, indicating a metabolic signature for this repeated administration. Interestingly, exposure to the low-dose pesticide mixture induced significant changes in the blood cell counts with modifications in the clonogenic and differentiating capacities of haematopoietic progenitors showing abnormalities in the granulocytic and monocytic lineages in female and male mice, respectively. From a molecular point of view, the changes induced by the pesticide treatment correlated with modifications of the PI 3-kinase/Akt signalling pathway, the tyrosine kinase Pyk2 and the c-Myc transcription factor, which are involved in the balance between self-renewal and differentiation of haematopoietic stem cells. Our results point to a significant effect of a very low dose of a mixture of commonly used pesticides on mice metabolism and haematopoietic system with major differences between males and females.

Cytostatic effect of polyethylene glycol on human colonic adenocarcinoma cells

Polyethylene glycol (PEG 8000) is a potent cancer chemopreventive agent. This osmotic laxative polymer markedly suppresses colon cancer in rats. To explain the mechanism, we have tested the in vitro effect of PEG on four human cell lines. Two poorly differentiated adenocarcinoma lines (HT29 and COLO205), a fetal mucosa line (FHC) and a differentiated line (post‐confluent Caco‐2) were incubated with various PEG concentrations for 2–5 days. Results show that PEG markedly and dose‐dependently inhibited HT29 and COLO205 cell growth. This cytostatic effect was associated with a blocking of the cell cycle in G0/G1 phase. In addition, PEG decreased the viability of HT29 and COLO205 adenocarcinoma cells. In contrast, post‐confluent intestinal‐like Caco‐2 cells and normal FHC cells were, respectively, not or little affected by PEG. Moreover, the lactate concentration increased twofold in the medium of PEG‐treated HT29 cells compared with untreated cells. Microscopic observations showed that PEG induced cell shrinking, membrane blebbing and the condensation of nuclear chromatin. However, because no DNA ladder and no annexin staining were detected, we presume that PEG did not induce apoptosis. PEG increased the osmotic pressure of the culture medium. Hyperosmotic media with added NaCl or sorbitol also inhibited HT29 cell growth, and increased lactate release. These results suggest that PEG may be selectively cytostatic for proliferating cancer cells. This growth inhibition may be due to the high osmotic pressure induced by PEG in vitro. Because the osmotic pressure is high in feces of PEG‐fed rats, it may explain the suppression of colon carcinogenesis by PEG.

In vitro aromatic bioactivation of the weak estrogen E2α and genesis of DNA adducts

Abstract Specific A-ring hydroxylated metabolites of 17β-estrogens are known to be endogenous pro-carcinogens, more particularly the 4-hydroxylated forms of estrogens produced by cytochrome P4501B1. In this study, we investigated whether estradiol-17α, the main hepatic residue of estradiol-17β in cattle treated for anabolic purposes with estradiol containing implants, could be significantly metabolized by human cells, and whether its aromatic metabolites could induce the formation of DNA adducts as estradiol-17β and estrone do. First, using a human kidney adenocarcinoma cell line, which expresses specifically the cytochrome P4501B1, we showed that estradiol-17α is bioactivated into a mixture of 2- and 4-catechol estrogens leading to the corresponding methoxyestrogens unambiguously identified by LC–APCI–MS/MS. We then demonstrated that the 2- and 4-hydroxylated derivatives of estradiol-17α incubated under oxidative conditions with calf thymus DNA gave stable DNA adducts and abasic sites, respectively. From these results, we can consider that human cells expressing CYP1B1-dependent hydroxylation activities metabolize estradiol-17α at the same magnitude as estradiol-17β and estrone, and that in oxidative conditions, the resulting aromatic metabolites can lead to the formation of both stable and unstable DNA adducts.