CYP2D6 expression is cumulatively up regulated in multidrug treated hepatoma cells: a predective pharmacogenetics in vitro model

Cancer and many other diseases require concomitant treatments with combinations of many drugs.Debrisoquine 4-Hydroxylayase (CYP2D6) is microsomal enzyme involved in phase I metabolism of a long list of drugs. Also, it is a marker of inter-individual variability in drug responsiveness. This study was designated to explore the regulation of CYP2D6 in hepatoma cells exposed to combinations of anticancer and epigenetic modifying drugs. HepG2 cell were treated with combinations of anticancer drug (Taxol), glucocorticoid (dexamethasone, DEX) and epigenetic modifiers: Trichostatin A (TSA) and 5 aza-deoxycytidine (5 aza-dC). The expression of CYP2D6 was determined by quantitative RT-PCR and compared to other CYPs and the corresponding cumulative apoptotic effect was determined by flow cytometry. The obtained results revealed thatUnder non-induced conditions, CYP2D6 was stably expressed and sub micromolar concentration of DEX mildly increased its expression. Individual treatments as DEX, Taxol, TSA and 5-azadC induced 2-6-fold increase in the transcript level, where the TSA was the most potent inducer. Combinations of 2 drugs as (Taxol+DEX), (Taxol+TSA), (Taxol+5-aza-dC) and (TSA+5-aza-dC). led to 3-10-fold increase (average 6.2), whereas cocktails of 3 drugs as (Taxol+DEX+5-aza-dC), (Taxol+DEX+TSA) and (Taxol+TSA+5-aza-dC) led to further up regulatory effect (11-27-fold). The highest increment (28-fold) was observed when cells were treated with 4 drugs as (Taxol+TSA+5-aza-dC+DEX). The progressive induction of CYP2D6 was correlated with the cumulative apoptotic effect (r=0.79). Conclusively, the data suggest, for the first time, that anticancer, epigenetic regulatory factors and dexamethasone cumulatively enhanced the baseline expression of CYP2D6

Phosphorylation-dependent Akt-Inversin interaction at the basal body of primary cilia

A primary cilium is a microtubule-based sensory organelle that plays an important role in human development and disease. However, regulation of Akt in cilia and its role in ciliary development has not been demonstrated. Using yeast two-hybrid screening, we demonstrate that Inversin (INVS) interacts with Akt. Mutation in the INVS gene causes nephronophthisis type II (NPHP2), an autosomal recessive chronic tubulointerstitial nephropathy. Co-immunoprecipitation assays show that Akt interacts with INVS via the C-terminus. In vitro kinase assays demonstrate that Akt phosphorylates INVS at amino acids 864-866 that are required not only for Akt interaction, but also for INVS dimerization. Co-localization of INVS and phosphorylated form of Akt at the basal body is augmented by PDGF-AA. Akt-null MEF cells as well as siRNA-mediated inhibition of Akt attenuated ciliary growth, which was reversed by Akt reintroduction. Mutant phosphodeador NPHP2-related truncated INVS, which lack Akt phosphorylation sites, suppress cell growth and exhibit distorted lumen formation and misalignment of spindle axis during cell division. Further studies will be required for elucidating functional interactions of Akt-INVS at the primary cilia for identifying the molecular mechanisms underlying NPHP2

The nephroprotective effects of allicin and ascorbic acid against cisplatin-induced toxicity in rats

International audienceCisplatin (CDDP) may induce nephrotoxicity through oxidative stress, DNA damage, and inflammation. This study was performed to evaluate the antioxidant and anti-inflammatory effects of allicin and ascorbic acid (AA) and investigate the nephroprotective efficacy of their combination against CDDP-induced intoxication. Rats were divided into seven groups: control, allicin (10 mg/kg for 14 days), AA (20 mg/kg for 14 days), CDDP (7 mg/kg as a single dose on the seventh experimental day), CDDP-allicin, CDDP-AA, and CDDP-allicin-AA (at the aforementioned doses). The administration of CDDP induced marked body weight loss and renal damage, manifested by significant increases (p < 0.05) in serum creatinine, urea, and uric acid levels and significant reductions in serum Na, Ca, and phosphorus concentrations, in addition to severe alterations in serum and renal tissue levels of tumor necrosis factor-α in comparison with control rats. Moreover, CDDP-intoxicated rats exhibited significantly (p < 0.05) higher lipid peroxidation, as well as lower levels of reduced glutathione and activities of glutathione peroxidase,superoxide dismutase, and catalase enzymes in the renal tissue, compared with control rats. The administration of allicin or AA significantly reduced (p < 0.05) the CDDP-induced changes in all the aforementioned parameters. Interestingly, allicin achieved comparable nephroprotection to AA in most assessed parameters; however, the restoration of normal serum and renal tissue concentrations of these parameters was more frequent in the CDDP-AA group. In conclusion, both allicin and AA showed significant nephroprotective effects against CDDP intoxication and their combination exhibited better protection than either agent alone. These results are probably mediated by their antioxidant and anti-inflammatory activities

Identification of RNA aptamer which specifically interacts with PtdIns(3)P

The phosphinositide Ptdlns(3)P plays an important role in autophagy; however, the detailed mechanism of its activity remains unclear. Here, we used a Systematic Evolution of Ligands by EXponential enrichment (SELEX) screening approach to identify an RNA aptamer of 40 nucleotides that specifically recognizes and binds to intracellular lysosomal Ptdlns(3)P. Binding occurs in a magnesium concentration- and pH-dependent manner, and consequently inhibits autophagy as determined by LC3II/I conversion, p62 degradation, formation of LC3 puncta, and lysosomal accumulation of Phafin2. These effects in turn inhibited lysosomal acidification, and the subsequent hydrolytic activity of cathepsin D following induction of autophagy. Given the essential role of Ptdlns(3)P as a key targeting molecule for autophagy induction, identification of this novel Ptdlns(3)P RNA aptamer provides new opportunities for investigating the biological functions and mechanisms of phosphoinositides. (C) 2019 The Authors. Published by Elsevier Inc

Functional characterization of lysosomal interaction of Akt with VRK2

Serine-threonine kinase Akt (also known as PKB, protein kinase B), a core intracellular mediator of cell survival, is involved in various human cancers and has been suggested to play an important role in the regulation of autophagy in mammalian cells. Nonetheless, the physiological function of Akt in the lysosomes is currently unknown. We have reported previously that PtdIns (3)P-dependent lysosomal accumulation of the Akt-Phafin2 complex is a critical step for autophagy induction. Here, to characterize the molecular function of activated Akt in the lysosomes in the process of autophagy, we searched for the molecules that interact with the Akt complex at the lysosomes after induction of autophagy. By time-of-flight-mass spectrometry (TOF/MS) analysis, kinases of the VRK family, a unique serine-threonine family of kinases in the human kinome, were identified. VRK2 interacts with Akt1 and Akt2, but not with Akt3; the C terminus of Akt and the N terminus of VRK2 facilitate the interaction of Akt and VRK2 in mammalian cells. The kinase-dead form of VRK2A (KD VRK2A) failed to interact with Akt in coimmunoprecipitation assays. Bimolecular fluorescence complementation (BiFC) experiments showed that, in the lysosomes, Akt interacted with VRK2A but not with VRK2B or KD VRK2A. Immunofluorescent assays revealed that VRK2 and phosphorylated Akt accumulated in the lysosomes after autophagy induction. WT VRK2A, but not KD VRK2A or VRK2B, facilitated accumulation of phosphorylated Akt in the lysosomes. Downregulation of VRK2 abrogated the lysosomal accumulation of phosphorylated Akt and impaired nuclear localization of TFEB; these events coincided to inhibition of autophagy induction. The VRK2-Akt complex is required for control of lysosomal size, acidification, bacterial degradation, and for viral replication. Moreover, lysosomal VRK2-Akt controls cellular proliferation and mitochondria) outer-membrane stabilization. Given the roles of autophagy in the pathogenesis of human cancer, the current study provides a novel insight into the oncogenic activity of VRK2-Akt complexes in the lysosomes via modulation of autophagy