Paule Martel

Monoterpenes inhibit cell growth, cell cycle progression, and cyclin D1 gene expression in human breast cancer cell lines

Monoterpenes are found in the essential oils of many commonly consumed fruits and vegetables. These compounds have been shown to exert chemopreventive and chemotherapeutic activities in mammary tumor models and represent a new class of breast cancer therapeutic agents. In this study, we investigated the effects of limonene and limonene-related monoterpenes, perillyl alcohol and perillic acid, on cell growth, cell cycle progression, and expression of cyclin D1 cell cycle-regulatory gene in T-47D, MCF-7, and MDA-MB-231 breast cancer cell lines. Our results revealed that limonene-related monoterpenes caused a dose-dependent inhibition of cell proliferation. Of the three monoterpenes tested, perillyl alcohol was the most potent and limonene was the least potent inhibitor of cell growth. The enantiomeric composition of limonene and perillyl alcohol did not interfere with their effect on cell growth. Sensitivity of breast cancer cell lines to monoterpenes was in the following order: T-47D > MCF-7 > MDA-MB-231. Growth inhibition induced by perillyl alcohol and perillic acid was associated with a fall in the proportion of cells in the S phase and an accumulation of cells in the G1 phase of the cell cycle. Finally, we showed that the effects of limonene-related monoterpenes on cell proliferation and cell cycle progression were preceded by a decrease in cyclin D1 mRNA levels.

Flavonoids (apigenin, tangeretin) counteract tumor promoter-induced inhibition of intercellular communication of rat liver epithelial cells

We have shown previously that two flavonoids, apigenin and tangeretin, enhance gap junctional intercellular communication (GJIC) in rat liver epithelial cells, named REL cells. Here, we show that these two flavones also antagonize the inhibition of GJIC induced by tumor promoters like 12-O-tetradecanoyl-phorbol-acetate (TPA) and 3,5,di-tertio-butyl-4-hydroxytoluene (BHT). Their preventive effect is rapid. It does not seem to involve any change of the amount of the connexin expressed in REL cells, connexin 43 (Cx 43), and in its phosphorylation state. Other flavonoids tested including naringenin, myricetin, catechin and chrysin did not enhance GJIC nor counteract TPA-induced inhibition of GJIC.

Repetitive Treatments of Colon HT-29 Cells with Diallyl Disulfide Induce a Prolonged Hyperacetylation of Histone H3 K14

Abstract: Diallyl disulfide (DADS) is a sulfur compound derived from garlic. Several studies carried out in rodents have revealed protective effects of DADS against colon carcinogenesis. The antipromoting effects of DADS may be partly related to its ability to inhibit tumoral cell proliferation. In a previous study, we have shown that in two human colon tumor cell lines (HT‐29 and Caco‐2) seeded at a low density (0.2 × 106 cells/100‐mm petri dish), DADS antiproliferative effects were associated with a transient increase of histone H3 K14 acetylation. Moreover, DADS could inhibit nuclear histone deacetylase activity. Therefore, in the present study, we examined the possible effects of different experimental conditions (HT‐29 cells at high density, repetitive treatments with DADS) on the pattern of DADS‐induced histone hyperacetylation. Using HT‐29 cells seeded at a higher density (5 × 106 cells/100‐mm petri dish), we found that DADS induced histone H3 K14 hyperacetylation rapidly (3 h). When administrated as single treatments, the DADS effect on histone H3 K14 remained transient. In contrast, repetitive treatment with DADS resulted in a prolonged hyperacetylation of histone H3 K14. Whatever the cell culture conditions were, DADS had no effect on histone H4 acetylation. Thus, in vitro, the cell density and pattern of DADS treatment influenced the HT‐29 nuclear response to DADS. DADS belongs to food‐borne molecules that may play a role in chromatin remodeling and contribute to the nutritional modulation of gene expression.

Lack of tumor‐promoting effects of flavonoids: Studies on rat liver preneoplastic foci and on in vivo and in vitro gap junctional intercellular communication

Possible tumor-promoting activity of four flavonoids, quercetin (QC), tangeretin (TG), flavone (FO), and flavanone (FN), was examined in a rat liver short-term carcinogenesis assay as well as with in vivo and in vitro assays of inhibition of gap junctional intercellular communication (GJIC). Rat hepatocarcinogenesis was induced by aflatoxin B1 treatment followed by a selection phase (2-acetylaminofluorene treatment and partial hepatectomy), then treatment with or without test chemicals (in vivo studies of antipromotion were not performed). Using glutathione S-transferase placental form (GST-P)-positive foci, we compared the effects of flavonoids (at 1,000 ppm in the diet) with the effects of phenobarbital (PB) on the occurrence of liver preneoplastic lesions. In addition, we studied the effects of flavonoids on GJIC in the livers derived from these experiments and in two types of cultured cells. No significant difference in the number and area of GST-P-positive foci was found after one or three months of treatment between any flavonoid group and control group. In the positive control group, PB markedly increased the numbers and areas of preneoplastic lesions at three months. Whereas PB also decreased by 60% the average size of lucifer yellow dye spread in slices of liver parenchyma free of preneoplastic lesions among the different flavonoids, only TG decreased the dye transfer in vivo: by 30% at one month and 50% at three months. With the dye transfer assay applied to a rat liver epithelial cell line (REL) and the Chinese hamster V79 metabolic cooperation assay, none of the tested flavonoids (< or = 25 microM) inhibited GJIC. Conversely, protective properties were seen for some of the compounds in antipromotion in vitro studies, because TG and FN enhanced the dye transfer in REL cells and FO, TG, and QC partly prevented the inhibition of metabolic cooperation by 12-O-tetradecanoylphorbol-13-acetate. Thus, taken together, our results suggest that QC, FO, and FN do not show tumor-promoting activity. Concerning TG, some discrepancies in the in vivo data are observed. Some of them (GJIC inhibition in liver slices) are probably more relevant to promotion of hepatocarcinogenesis.

Effects of four inorganic lead compounds on the proliferation and junctional coupling of cultured REL liver cells

BACKGROUND Chronic, low-level exposure to inorganic lead (Pb) has been involved in a number of human diseases, including tumors. In this study, the effect of four different inorganic Pb compounds (acetate, chloride, monoxide, and sulfate) was evaluated, in vitro, on liver-derived REL cells, known to be very sensitive to tumor promoters. METHODS Cytotoxicity and effects on intercellular communication (GJIC) were evaluated, respectively, by cell- density/proliferation and dye-transfer assays. Pb concentration in the media solutions used for each treatment was quantified by atomic absorption spectroscopy-electrothermal atomization. RESULTS Each of the Pb compounds we tested showed a typical dose- and time-related effect on REL cell proliferation, this effect not being related to the free metal concentration. Contrary to classical tumor promoters, none of the compounds significantly affected REL GJIC (1-hour treatment). CONCLUSIONS Our results are indicative of specificity in the effects of the different Pb compounds. The mechanism(s) of their action need further investigations.

Constitutive overexpression of c-fos protein in rat liver epithelial cells decreases TGF-β synthesis and increases TGF-β1 receptors

We have previously shown that rat liver epithelial cells were more sensitive to TGF-beta 1 when they were transfected with c-fos cDNA. We analyzed the production of TGF-beta and TGF-beta 1 binding proteins in transfected and parental cells. TGF-beta-like activity released in the medium was reduced in c-fos expressing cells. TGF-beta 1 binding sites were more numerous in transfected cells (x3). Cross-linking studies confirmed that c-fos transfected cells showed increased binding of 125I-TGF-beta 1 to membrane binding sites corresponding to type I, II and III receptors. Transfected cells internalized and degraded 125I-TGF-beta 1 more rapidly than parental cells. TGF-beta 1 incubation rapidly down-regulated the receptors. In parental cells, the down-regulation was total, while in transfected cells, a few binding proteins could still be detected. The c-fos cell line is an interesting tool in analysing the mechanism of action of TGF-beta.

Effect of sodium butyrate on the stimulation of casein gene expression by prolactin

Sodium butyrate, but not isobutyrate, inhibits prolactin action on the induction of casein synthesis and casein mRNA accumulation in rabbit mammary explants. Sodium butyrate specifically prevents the generation of the prolactin relay which can be released from isolated membranes incubated with prolactin and which stimulates directly casein gene transcription when added to isolated mammary nuclei. This indicates that sodium butyrate exerts its inhibitory action essentially at the membrane level.

TGF‐β receptors are diminished after retinoid exposure in rat liver epithelial cells

When rat liver epithelial cells were exposed to retinoic acid or retinol for 24 hr, the levels of transforming growth factor‐β (TGF‐β) receptors were reduced in a dose‐dependent way. The decrease appeared after 12 hr of incubation with the retinoids and binding levels remained low until 24 hr after the removal of the molecules. Retinoid treatment induced a fourfold enhancement of transglutaminase (TGase) activity in the cell membranes, and cystamine, an inhibitor of TGase, prevented the decrease of the receptors. Neutralization of TGF‐β by a monoclonal antibody did not suppress the decrease of the binding levels, indicating that decreased TGF‐β binding capacity was not due merely to the internalization of ligand‐bound receptors promoted by a stimulation of TGF‐β synthesis. Thus, retinoid treatment resulted in an intense disappearance of the functional receptors from the membranes that seemed to be mediated by increased TGase activity. This phenomenon can represent a strong signal attenuation for TGF‐β following retinoid exposure.

Altered response to growth factors in rat epithelial liver cells overexpressing human c-Fos protein

Human c‐fos cDNA was transfected into normal rat liver epithelial (REL) cells to identify cellular modifications associated with high expression of c‐Fos protein. Responses to EGF and TGFβ were examined in the different cell lines, under anchorage‐dependent and ‐independent conditions. Sensitivity to both factors was modified in transfected cells. While parental cells in monolayer did not respond to EGF, c‐fos containing cells growth was stimulated by this factor. Overexpression of c‐Fos protein led to an enhanced TGFβ‐induced growth inhibition under anchorage dependent conditions, and TGFβ abolished spontaneous growth in soft agar of the cell lines containing c‐fos oncogene. The mechanisms underlying, the increased sensitivity to TGFβ in c‐fos transfected cells are still to be determined.