A number of "suicide genes" have been developed as safety switches for gene therapy vectors or as potential inducible cytotoxic agents for hyperproliferative disorders, such as cancer or restenosis. However, most of these approaches have relied on foreign proteins, such as HSV thymidine kinase, that primarily target rapidly dividing cells. In contrast, novel artificial death switches based on chemical inducers of dimerization (CIDs) and endogenous proapoptotic molecules function efficiently in both dividing and nondividing cells. In this approach, lipid-permeable, nontoxic CIDs are used to conditionally cross-link target proteins that are fused to CID-binding domains (CBDs), thus activating signaling cascades leading to apoptosis. In previous reports, CID-regulated Fas and caspases 1, 3, 8, and 9 were described. Since the maximum efficacy of these artificial death switches requires low basal and high specific activity, we have optimized these death switches for three parameters: (1) extent of oligomerization, (2) spacing between CBDs and target proteins, and (3) intracellular localization. We describe improved conditional Fas and caspase 1, 3, 8, and 9 alleles that function at subnanomolar levels of the CID AP1903 to trigger apoptosis. Further, we demonstrate for the first time that oligomerization of the death effector domain of the Fas-associated protein, FADD, is sufficient to trigger apoptosis, suggesting that the primary function of FADD, like that of Apaf-1, is oligomerization of associated caspases. Finally, we demonstrate that nuclear-targeted caspases 1, 3, and 8 can trigger apoptosis efficiently, implying that the cleavage of nuclear targets is sufficient for apoptosis.
Background: The heterogeneous course of moderate-to-severe atopic dermatitis necessitates treatment flexibility.Objective: We evaluated the maintenance of abrocitinib-induced response with continuous abrocitinib treatment, dose reduction or withdrawal, and response to treatment reintroduction following flare (JAK1 Atopic Dermatitis Efficacy and Safety [JADE] REGIMEN: National Clinical Trial 03627767).Methods: Patients with moderate-to-severe atopic dermatitis responding to open-label abrocitinib 200 mg monotherapy for 12 weeks were randomly assigned in a 1:1:1 ratio to blinded abrocitinib (200 or 100 mg) or placebo for 40 weeks. Patients experiencing flare received rescue treatment (abrocitinib 200 mg plus topical therapy).Results: Of 1233 patients, 798 responders to induction (64.7%) were randomly assigned. The flare probability during maintenance was 18.9%, 42.6%, and 80.9% with abrocitinib 200 mg, abrocitinib 100 mg, and placebo, respectively. Among patients with flare in the abrocitinib 200 mg, abrocitinib 100 mg, and placebo groups, 36.6%, 58.8%, and 81.6% regained investigator global assessment 0/1 response, respectively, and 55.0%, 74.5%, and 91.8% regained eczema area and severity index response, respectively, with rescue treatment. During maintenance, 63.2% and 54.0% of patients receiving abrocitinib 200 and 100 mg, respectively, experienced adverse events.Limitations: The definition of protocol-defined flare was not established, limiting the generalizability of findings.
Rac1 GTPase regulates a variety of signaling pathways that are implicated in malignant phenotypes. Here, we show that selective inhibition of Rac1 activity by the pharmacologic inhibitor NSC23766 suppressed cell growth in a panel of human breast cancer cell lines, whereas it had little toxicity to normal mammary epithelial cells. NSC23766 elicits its cytotoxicity via two distinct mechanisms in a cell line-dependent manner: induction of G 1 cell cycle arrest in cell lines (MDA-MB-231, MCF7, and T47D) that express retinoblastoma (Rb) protein or apoptosis in Rb-deficient MDA-MB-468 cells. In MDA-MB-231 cells, Rac1 inhibition induced G 1 cell cycle arrest through downregulation of cyclin D1 and subsequent dephosphorylation/inactivation of Rb. By contrast, MDA-MB-468 cells underwent substantial apoptosis that was associated with loss of antiapoptotic proteins survivin and X-linked inhibitor of apoptosis protein (XIAP). Rac1 knockdown by RNAi interference confirmed the specificity of NSC23766 and requirement for Rac1 in the regulation of cyclin D1, survivin, and XIAP in breast cancer cells. Further, NF-κB, but not c-Jun NH 2 -terminal kinase or p38 pathways, mediates the survival signal from Rac1. Overall, our results indicate that Rac1 plays a central role in breast cancer cell survival through regulation of NF-κB-dependent gene products. Mol Cancer Ther; 9(6); 1657-68. ©2010 AACR.
The fungal toxin gliotoxin induces apoptotic cell death in a variety of cells. Apoptosis induced in thymocytes by gliotoxin is rapid, and DNA fragmentation is observable within 4 h treatment. Apoptosis induced by gliotoxin is calcium-independent and unaffected by protein synthesis inhibitors. We have previously shown that gliotoxin results in phosphorylation of a 16.3-kDa protein within 10 min treatment of thymocytes. Here we show that this protein is histone H3 and phosphorylation occurs on Ser-10. Cyclic AMP levels and activity of protein kinase A (PKA) are raised in cells treated with gliotoxin. Apoptosis is inhibited by genistein which also inhibits PKA and histone H3 phosphorylation. Apoptosis is also inhibited by a number of specific inhibitors of PKA suggesting apoptosis induced by gliotoxin is modulated by this kinase. The agents forskolin and cholera toxin do not induce rapid phosphorylation of H3 although some increase in phosphorylation of H3 does occur after 8 h with these agents. Forskolin and cholera toxin also induce apoptosis but over a longer time course than gliotoxin. In all cases levels of apoptosis correlate with degree of H3 phosphorylation. Cells treated with gliotoxin show an early sensitivity to micrococcal nuclease and DNase I digestion indicating a functional relationship between DNA fragmentation and H3 phosphorylation.
Purpose. The present study is an attempt to develop a vitamin E loaded naringenin (NRG) Nanoemulsion (NE) for direct nose-to-brain delivery for better management of Parkinson's disease (PD). Methods. The optimized NE was evaluated for efficacy in PD using multiple behavioral studies (including narrow beam test, muscular coordination test, grip strength test, forced swimming test, and akinesia test) in a rat model. Optimized formulation was evaluated for droplet size, polydispersity index (PDI), refractive index, transmittance, zeta potential, and viscosity. Results. Optimized NE had a droplet size of 38.70 ± 3.11nm, PDI of 0.14 ± 0.0024, refractive index of 1.43 ± 0.01, transmittance of 98.12 ± 0.07 %, zeta potential of − 27.4 ± 0.14 mV, and viscosity of 19.67 ± 0.25 Pa s. Behavioral studies showed that 6-OHDA induced PD in rats were successfully reversed when administered with NRG NE intranasally along with the levodopa. While the levels of GSH and SOD were significantly higher, levels of MDA were significantly lower in the group treated with NRG NE via intranasal route along with levodopa. Conclusion. Encouraging results from current study provide evidence for possible efficacy of a novel noninvasive intranasal delivery system of NRG for management of PD related symptoms.
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