Synthesis and biological evaluation of bile carboxamide derivatives with pro-apoptotic effect on human colon adenocarcinoma cell lines.

We previously reported that the cinnamylpiperazinyl group in the side chain of the chenodeoxycholic acid showed apoptosis-inducing activity on multiple myeloma cancer cell line KMS-11. In the present study, we synthesized and tested the pro-apoptotic potency of fifteen new piperazinyl bile carboxamide derived from cholic, ursodeoxycholic, chenodeoxycholic, deoxycholic and lithocholic acids on human colon adenocarcinoma cell lines DLD-1, HCT-116, and HT-29. Cell viability was first measured using XTT assay. The most of the synthetic bile carboxamide derivatives decreased significantly cell viability in a dose-dependent manner. HCT-116 and DLD-1 cell lines were more sensitive than HT-29 to tested compounds. 9c, 9d showed the best in vitro results in term of solubility and dose-response effect on the three colon adenocarcinoma cell lines. Additionally, flow cytometric and Western-blotting analysis showed that 9c induced pro-apoptosis in DLD-1 and HCT-116 whereas 9d did not. We conclude that the benzyl group improved anti-proliferative activity and that the α-hydroxyl group was found to be more beneficial at the 7-position in steroid skeleton.

Up-regulation of type II collagen gene by 17ß-estradiol in articular chondrocytes involves Sp1/3, Sox-9, and estrogen receptor α.

UNLABELLED: The existence of a link between estrogen deprivation and osteoarthritis (OA) in postmenopausal women suggests that 17ß-estradiol (17ß-E2) may be a modulator of cartilage homeostasis. Here, we demonstrate that 17ß-E2 stimulates, via its receptor human estrogen receptor α 66 (hERα66), type II collagen expression in differentiated and dedifferentiated (reflecting the OA phenotype) articular chondrocytes. Transactivation of type II collagen gene (COL2A1) by ligand-independent transactivation domain (AF-1) of hERα66 was mediated by "GC" binding sites of the -266/-63-bp promoter, through physical interactions between ERα, Sp1/Sp3, Sox9, and p300, as demonstrated in chromatin immunoprecipitation (ChIP) and Re-Chromatin Immuno-Precipitation (Re-ChIP) assays in primary and dedifferentiated cells. 17ß-E2 and hERα66 increased the DNA-binding activities of Sp1/Sp3 and Sox-9 to both COL2A1 promoter and enhancer regions. Besides, Sp1, Sp3, and Sox-9 small interfering RNAs (siRNAs) prevented hERα66-induced transactivation of COL2A1, suggesting that these factors and their respective cis-regions are required for hERα66-mediated COL2A1 up-regulation. Our results highlight the genomic pathway by which 17ß-E2 and hERα66 modulate Sp1/Sp3 heteromer binding activity and simultaneously participate in the recruitment of the essential factors Sox-9 and p300 involved respectively in the chondrocyte-differentiated status and COL2A1 transcriptional activation. These novel findings could therefore be attractive for tissue engineering of cartilage in OA, by the fact that 17ß-E2 could promote chondrocyte redifferentiation. KEY MESSAGES: 17ß-E2 up-regulates type II collagen gene expression in articular chondrocytes. An ERα66/Sp1/Sp3/Sox-9/p300 protein complex mediates this stimulatory effect. This heteromeric complex interacts and binds to Col2a1 promoter and enhancer in vivo. Our findings highlight a new regulatory mechanism for 17ß-E2 action in chondrocytes. 17ß-E2 might be an attractive candidate for cartilage engineering applications.

N-substituted piperazinopyridylsteroid derivatives as abiraterone analogues inhibit growth and induce pro-apoptosis in human hormone-independent prostate cancer cell lines.

Nine new 17-(piperazin-1-yl)pyridin-5-yl)steroids as abiraterone analogues were synthesized. Compounds 5d and 5g showed selective activities against 17α-hydroxylase/C17,20-lyase (CYP17A1) and aromatase (CYP19), respectively. IC50 values of 5d were 5.09 and >50 µm, whereas these values for 5g were >50 µm and 7.40 µm, respectively, for CYP17A1 and CYP19. Molecular modelling highlighted that the inhibitor designed to bind cytochrome P450 haem iron is a necessary condition but not the only rationale to explain inhibitory activity. These abiraterone analogues were then evaluated on hormone-independent prostate cancer cell lines DU-145 and PC-3 and on hormone-dependent breast and prostate cancer cell lines MCF-7 and LNCaP, respectively. Compounds 5e, 5g and 5i have showed potent activities only on hormone-independent prostate cancer cell lines DU-145 and PC-3 with 60-85% inhibition of both cell viability and growth at 10 nm with pro-apoptotic mechanism as illustrated in PC-3 cells by DNA ladder assay and Western blotting of Bax, Casp-3 and its substrate, the poly (ADP-ribose) polymerase. We conclude that hybrid heterocycle steroids could be good lead compounds in the drug design especially against hormone-independent prostate cancer.

Differential effects of glyphosate and roundup on human placental cells and aromatase.

Roundup is a glyphosate-based herbicide used worldwide, including on most genetically modified plants that have been designed to tolerate it. Its residues may thus enter the food chain, and glyphosate is found as a contaminant in rivers. Some agricultural workers using glyphosate have pregnancy problems, but its mechanism of action in mammals is questioned. Here we show that glyphosate is toxic to human placental JEG3 cells within 18 hr with concentrations lower than those found with agricultural use, and this effect increases with concentration and time or in the presence of Roundup adjuvants. Surprisingly, Roundup is always more toxic than its active ingredient. We tested the effects of glyphosate and Roundup at lower nontoxic concentrations on aromatase, the enzyme responsible for estrogen synthesis. The glyphosate-based herbicide disrupts aromatase activity and mRNA levels and interacts with the active site of the purified enzyme, but the effects of glyphosate are facilitated by the Roundup formulation in microsomes or in cell culture. We conclude that endocrine and toxic effects of Roundup, not just glyphosate, can be observed in mammals. We suggest that the presence of Roundup adjuvants enhances glyphosate bioavailability and/or bioaccumulation.

Synthesis and evaluation of benzoxazolinonic imidazoles and derivatives as non-steroidal aromatase inhibitors.

New compounds were tested in vitro on aromatase activity in human placental and equine testicular microsomes. Equine aromatase, very well characterized biochemically, is used as a comparative model to understand the mechanism of aromatase inhibition. Among 15 molecules screened, 5 of them (11-15) strongly inhibit human and equine aromatases with IC50 values ranging from 13-85nM and from 23-103nM respectively. These results were corroborated by Ki/Km values. Moreover, spectral studies showed a type II spectrum with both enzymes, which is characteristic of an interaction between the nitrogen atom of the molecule and the heme of the cytochrome P450. Compound 12, which has the lowest IC50 and Ki/Km ratio, inactivates aromatase in a dose and time-dependent manner. This might be very important for the treatment of estrogen-dependent diseases such as breast cancer. Finally, MTT assays on E293 cells revealed that the molecules were not cytotoxic.

Molecular characterization and expression of equine testicular cytochrome P450 aromatase.

We characterized testicular equine aromatase and its expression. A 2707 bp cDNA was isolated, it encoded a polypeptide of 503 residues with a deduced molecular mass of 57.8 kDa. The sequence features were those of a cytochrome P450 aromatase, with a 78% polypeptide identity with the human counterpart. The gene has a minimal length of 74 kb comprising at least 9 exons and expresses a 2.8 kb mRNA in the testis. Transient cDNA transfections in E293 cells and in vitro translations in a reticulocyte lysate system allowed aromatase protein and activity detections. The activity increased with androstenedione as substrate in a dose-dependent manner. The isolation of testicular aromatase by a new immunoaffinity method demonstrated that the protein could exist either glycosylated or not with a 2 kDa difference. All these results taken together allow new structural studies to progress in the understanding of this cytochrome P450.

Free and conjugated estrogens and androgens in stallion semen.

The steroid content of semen from a total of 11 mature fertile stallions was studied during two breeding seasons and one winter. The levels of free and conjugated substrates (testosterone and androstenedione), and products (estradiol and estrone), of aromatase were measured by radioimmunoassay with a validated method. The results were seasonally and monthly highly variable with characteristic peaks. The concentrations of free and conjugated estrogens were always higher in the gel-free ejaculate than in the gel except in one subfertile stallion used as comparison. Furthermore, the steroid production and the maximal resulting aromatase activity, estimated by the estrogens/androgens ratio, peaked in April-May and June. The breeding season (spring and summer) presents a clear estrogenic profile with estrogens/androgens ratios higher in contrast to the nonbreeding period (autumn and winter). The involvement of estrogens in the regulation of reproduction and equine spermatogenesis is discussed, and estrogens production and thus equine aromatase is proposed as a strong marker of testicular endocrine function.