Opposite effects of two zinc(II) dithiocarbamates on NF-kB pathway

Inhibiting nuclear factor-kappaB (NF-kB) activation in anticancer and antiinflammatory therapy is of topical interest. Current research in molecular biology has dramatically advanced in the understanding of the cellular events involved in NF-kB induction. Dithiocarbamates, in particular diethyldithiocarbamate and pyrrolidinedithiocarbamate, have been known and widely used as strong inhibitors of NF-kB signaling pathway for more than ten years. Their activity is frequently thought to be due to chelating of zinc or copper present in serum supplemented in the culture medium. Zinc(II) diethyldithiocarbamate (Et2Zn) and zinc(II) dibenzyldithiocarbamate (Bz2Zn) were prepared by direct synthesis in aqueous millieu. They were structurally characterized by X-ray analysis (solid phase) and mass spectrometry (aqueous conditions). Et2Zn and Bz2Zn both in 20 micromolar concentration were applied to HeLa cells. The status of NF-kB signaling was assessed as nuclear translocation of p65 subunit. Surprisingly, Et2Zn activated NF-kB pathway, while TNF-dependent activation of NF-kB was inhibited by Bz2Zn. Our results are preliminary

Type IV fimbrial subunit protein ApfA contributes to protection against porcine pleuropneumonia

Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae accounts for serious economic losses in the pig farming industry worldwide. We examined here the immunogenicity and protective efficacy of the recombinant type IV fimbrial subunit protein ApfA as a single antigen vaccine against pleuropneumonia, or as a component of a multi-antigen preparation comprising five other recombinant antigens derived from key virulence factors of A. pleuropneumoniae (ApxIA, ApxIIA, ApxIIIA, ApxIVA and TbpB). Immunization of pigs with recombinant ApfA alone induced high levels of specific serum antibodies and provided partial protection against challenge with the heterologous A. pleuropneumoniae serotype 9 strain. This protection was higher than that engendered by vaccination with rApxIVA or rTbpB alone and similar to that observed after immunization with the tri-antigen combination of rApxIA, rApxIIA and rApxIIIA. In addition, rApfA improved the vaccination potential of the penta-antigen mixture of rApxIA, rApxIIA, rApxIIIA, rApxIVA and rTbpB proteins, where the hexa-antigen vaccine containing rApfA conferred a high level of protection on pigs against the disease. Moreover, when rApfA was used for vaccination alone or in combination with other antigens, such immunization reduced the number of pigs colonized with the challenge strain. These results indicate that ApfA could be a valuable component of an efficient subunit vaccine for the prevention of porcine pleuropneumonia

Rifampicin Does not Significantly Affect the Expression of Small Heterodimer Partner in Primary Human Hepatocytes

The small/short heterodimer partner (SHP, NR0B2) is a nuclear receptor corepressor lacking a DNA binding domain. SHP is induced by bile acid-activated farnesoid X receptor (FXR) resulting in CYP7A1 gene suppression. In contrast, Pregnane X receptor (PXR) activation by its ligands was recently suggested to inhibit SHP gene transactivation to maximize the induction of PXR target genes. However, there are also conflicting reports in literature whether PXR or rodent Pxr activation down-regulates SHP/Shp expression. Moreover, the PXR-mediated regulation of the SHP gene has been studied only at the SHP mRNA and transactivation (gene reporter assay) levels. In this study, we studied the effect of rifampicin, a prototype PXR ligand, on SHP mRNA, and protein expression in three primary human hepatocyte cultures. We found that SHP mRNA is not systematically down-regulated in hepatocyte in culture after 24 h treatment with rifampicin. Consistently, we did not observe down-regulation of SHP protein in primary human hepatocytes after 24 and 48 h of incubation with rifampicin. We can conclude that although we observed slight down-regulation of SHP mRNA and protein in several hepatocyte preparations, the phenomenon is unlikely critical for PXR-mediated induction of its target genes

Genetic and Enzymatic Characteristics of CYP2A13 in Relation to Lung Damage

Cytochrome P450 2A13 is an omitted brother of CYP2A6 that has an important role in the drug metabolism of liver. Due to extrahepatic expression, it has gained less attention than CYP2A6, despite the fact that it plays a significant role in toxicant-induced pulmonary lesions and, therefore, lung cancer. The purpose of this mini-review is to summarize the basic knowledge about this enzyme in relation to the substrates, inhibitors, genetic polymorphisms, and transcriptional regulation that are known so far (September 2021)

The Impact of Indoles Activating the Aryl Hydrocarbon Receptor on Androgen Receptor Activity in the 22Rv1 Prostate Cancer Cell Line

The activation of the aryl hydrocarbon receptor (AhR) by xenobiotic compounds was demonstrated to result in the degradation of the androgen receptor (AR). Since prostate cancer is often dependent on AR, it has become a significant therapeutic target. As a result of the emerging concept of bacterial mimicry, we tested whether compounds with indole scaffolds capable of AhR activation have the potential to restrict AR activity in prostate cancer cells. Altogether, 22 indolic compounds were tested, and all of them activated AhR. However, only eight decreased DHT-induced AR luciferase activity. All indoles, which met the AhR-activating and AR-suppressing criteria, decreased the expression of DHT-inducible AR target genes, specifically KLK3 and FKBP5 mRNAs. The reduced AR binding to the KLK3 promoter was confirmed by a chromatin immunoprecipitation (ChIP) assay. In addition, some indoles significantly decreased AR protein and mRNA level. By using CRISPR/Cas9 AhR knockout technology, no relationship between AhR and AR, measured as target gene expression, was observed. In conclusion, some indoles that activate AhR possess AR-inhibiting activity, which seems to be related to the downregulation of AR expression rather than to AR degradation alone. Moreover, there does not seem to be a clear relationship that would connect AhR activation with AR activity suppression in 22Rv1 cells

Effects of microtubule-interfering agents on aryl hydrocarbon receptor signalling

Les cytochromes P450 1A (CYP1A) sont impliqués dans le métabolisme des hydrocarbures polycycliques aromatiques et la formation de métabolites réactifs à partir de ces molécules. Le contrôle transcriptionnel de l'expression de ces gènes dépend en partie du récepteur des hydrocarbures polycycliques aromatiques (AhR). Les fonctions de ce récepteur sont affectées par toute une série de stimuli physiopathologiques, incluant notamment les agents perturbateurs du réseau de microtubules (MIAs). Toutefois, le rôle du cytosquelette sur la voie de signalisation dépendante du AhR n'est pas totalement élucidé. Dans ce travail, je me suis intéressé aux effets de divers MIAs présentant des structures chimiques différentes et souvent utilisés dans le traitement de cancers (colchicine, nocodazole, taxol, vincristine, vimblastine) sur la fonction du AhR. Dans la première partie de la thèse, j'ai travaillé sur les cellules HepG2 (carcinome hépatocellulaire humain) et les hépatocytes de rat en culture primaire (RH). Dans les deux types cellulaires, colchine et nocodazole produisent un effondrement du réseau des microtubules, analysé en microscopie et immunofluorescence utilisant des anticorps anti-b-tubuline. Dans les cellules HepG2, la colchicine et le nocodazole provoquent une diminution dose- et temps-dépendante de l'induction du CYP1A1 (mRNA, protéine et activité) en réponse à la dioxine (TCDD). Des résultats similaires sont observés dans les RH avec la colchicine, mais pas avec le nocodazole. Des analyses de liaison de ligand ont révélé qu'aucune de ces deux molécules n'est antagoniste du AhR de souris. La colchicine diminue la translocation nucléaire d'une construction chimérique AhR-GFP en réponse au TCDD dans des cellules HeLa. L'analyse par FACS des cellules HepG2 et des RH exposés pendant 24 heures à la colchicine ou au nocodazole montre une augmentation très importante de la population des cellules HepG2 en G2/M par rapport aux contrôles, alors que la distribution des populations de RH reste inchangée. En conclusion de cette partie du travail, l'intégrité du réseau des microtubules est importante pour la transduction du signal dépendant du AhR dans les cellules HepG2 et les RH. Cet effet n'est pas exclusivement dû à la perturbation du cycle cellulaire (observée sur les cellules HepG2) comme le montrent les résultats obtenus avec les RH. Dans la deuxième partie de ma thèse, j'ai étudié l'effet des MIAs sur la fonction du AhR dans les hépatocytes humains en culture primaire (HH). A l'exception du taxol (comme attendu), tous les MIAs testés produisent après 6 et 24 heures de traitement un effondrement dose-dépendant du réseau des microtubules, analysé en microscopie et immunofluorescence utilisant des anticorps anti-b-tubuline. Par contre, seuls le nocodazole et la vimblastine provoquent une diminution significative de l'induction du CYP1A1 mRNA en réponse au TCDD. Tous les MIAs produisent une diminution dose- et temps-dépendante significative de l'expression du CYP1A2 mRNA en réponse au TCDD. De même, après 16 heures de traitement, tous les MIAs (taxol inclus) provoquent une inhibition de la translocation nucléaire du Ahr en réponse au TCDD, mesurée par analyse en western blot des extraits nucléaires. Les MIAs seuls ou en association avec TCDD produisent une augmentation d'accumulation de la protéine AhR et du mRNA. En conclusion, les MIAs affectent la transduction de signal du AhR, notamment l'expression inductible des CYP1A1 et 1A2. Cependant ces effets ne sont pas directement corrélés à l'impact des MIAs sur le réseau des microtubules et semblent être dépendants des types cellulaires (HH versus RH, ou HepG2 versus HH) et des espèces (HH versus RH). Ces effets pourraient être dépendants de la dynamique du réseau des microtubules plutôt que de son intégrité, comme le suggèrent le fait que les résultats obtenus avec le taxol sont identiques à ceux obtenus avec les autres molécules. Le lien entre réseau des microtubules et cycle cellulaire observé sur les cellules HepG2 ne tient pas avec les RH et HH qui, dans nos conditions de culture, sont des cellules en G0.MONTPELLIER-BU Médecine UPM (341722108) / SudocPARIS-BIUP (751062107) / SudocMONTPELLIER-BU Médecine (341722104) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Different regulation of aryl hydrocarbon receptor-regulated genes in response to dioxin in undifferentiated and neuronally differentiated human neuroblastoma SH-SY5Y cells

Some environmental pollutants derived from industrial processes have been suggested to be responsible for neurological impairment in children, especially in heavily polluted areas. Since these compounds are usually activators of aryl hydrocarbon receptor (AhR), it would be important to better understand the molecular pathways downstream of AhR leading to neural deficits. To this purpose, appropriate in vitro human neural model is much needed. Here we have investigated whether undifferentiated and neuronally differentiated human neuroblastoma cells, SH-SY5Y cells, can provide a suitable model for monitoring AhR activity induced by environmental pollutants, focusing on 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD), a known activator of AhR. Further characterization of differentiated SH-SY5Y showed an increase in AhRR (aryl hydrocarbon receptor repressor), no change in ARNT1 (AhR nuclear translocator 1), and a decrease in ARNT2 expression with differentiation; in contrast, AhR was undetectable in both undifferentiated and differentiated cells. Nonetheless, treatment of parental as well as differentiated SH-SY5Y cells with TCDD resulted in the induction of AhR-regulated genes, CYP1A1 and CYP1B1; AhRR expression was also affected, but to a much smaller extent. These results indicate that undifferentiated SH-SY5Y are less sensitive to TCDD than neuronally differentiated ones, suggesting a higher resistance of the undifferentiated tumor cells to toxic insults. They also suggest that TCDD in these cells may not act via direct activation of AhR that is undetectable in SH-SY5Y as well as in differentiated neurons. Hence, these cells do not provide an appropriate model for studying ligand-mediated activation of AhR