A Novel Kinase Inhibitor AX-0085 Inhibits Interferon-γ-Mediated Induction of PD-L1 Expression and Promotes Immune Reaction to Lung Adenocarcinoma Cells.

In this study, we describe a novel kinase inhibitor AX-0085 which can suppress the induction of PD-L1 expression by Interferon-γ (IFN-γ) in lung adenocarcinoma (LUAD) cells. AX-0085 effectively blocks JAK2/STAT1 signaling initiated by IFN-γ treatment and prevents nuclear localization of STAT1. Importantly, we demonstrate that AX-0085 reverses the IFN-γ-mediated repression of T cell activation in vitro and enhances the anti-tumor activity of anti-PD-1 antibody in vivo when used in combination. Finally, transcriptomic analyses indicated that AX-0085 is highly specific in targeting the IFN-γ-pathway, thereby raising the possibility of applying this reagent in combination therapy with checkpoint inhibitor antibodies. It may be particularly relevant in cases in which PD-L1-mediated T cell exhaustion leads to immunoevasive phenotypes.

Quantitative biochemical characterization and biotechnological production of caspase modulator, XIAP: Therapeutic implications for apoptosis-associated diseases.

BACKGROUND: Regulating apoptosis is a common and essential therapeutic strategy for cancer and neurodegenerative disorders. Based on basic studies of apoptotic mechanisms, various researches have attempted to overcome the pathogenesis of such diseases by activating or inhibiting apoptosis. Generally, the biochemical characteristics of the target molecules should be evaluated along with understanding of their mechanisms of action during drug development. Among apoptotic regulators, XIAP serves as a potent negative regulator to block apoptosis through the inhibition of caspase (CASP)-9 and -3/7. Although XIAP is an attractive target with such apoptotic-modulating property, biochemical and biophysical studies of XIAP are still challenging. METHODS: In this study, the CASP-9 and -3/7 inhibitors XIAP, 242Δ and Δ230 were prepared using the pGEX expression system and biochemically characterized. RESULTS: These inhibitors were expressed in Escherichia coli at a concentration of ≥20 mg/L culture under a native condition with 0.01 mM IPTG induction. Notably, using a simple and rapid affinity purification technique, these CASP-9 and -3/7 inhibitors have been purified, yielding ≥5 mg/L culture at approximately 90% purity. CONCLUSIONS: We have determined that HtrA2 specifically binds to the BIR2 and BIR3 of XIAP at a 1:1 molecular ratio. Moreover, in vitro cell-free CASP-9 and -3/7 activation-apoptosis assays have demonstrated that these purified XIAP proteins dramatically inhibit CASP-9 and -3/7 action. GENERAL SIGNIFICANCE: Our system is suitable for biochemical studies, such as quantitation of the number of molecules acting on the apoptosis regulation, and provides a basis and insights that can be applied to the development of therapeutic agents for neurodegenerative disorders and cancer.

Harmless effects of argon plasma on caudal fin regeneration and embryogenesis of zebrafish: novel biological approaches for safe medical applications of bioplasma.

The argon plasma jet (Ar-PJ) is widely used in medical fields such as dermatology and dentistry, and it is considered a promising tool for cancer therapy. However, the in vivo effects of Ar-PJ for medical uses have not yet been investigated, and there are no biological tools to determine the appropriate clinical dosages of Ar-PJ. In this study, we used the caudal fin and embryo of zebrafish as novel in vivo tools to evaluate the biosafety of Ar-PJ. Typically, Ar-PJ is known to induce cell death in two-dimensional (2D) cell culture systems. By contrast, no detrimental effects of Ar-PJ were shown in our 3D zebrafish systems composed of 2D cells. The Ar-PJ-treated caudal fins grew by an average length of 0.7 mm, similar to the length of the normally regenerating fins. Remarkably, Ar-PJ did not affect the expression patterns of Wnt8a and ß-Catenin, which play important roles in fin regeneration. In the embryo system, 85% of the Ar-PJ-treated embryos hatched, and the lateral length of these embryos was ~3.3 mm, which are equivalent to the lengths of normal embryos. In particular, vasculogenesis, which is the main cellular process during tissue regeneration and embryogenesis, occurred normally under the Ar-PJ dose used in this study. Therefore, our biosafety evaluation tools that use living model systems can be used to provide an experimental guideline to determine the clinically safe dosage of Ar-PJ.

A novel link between the conformations, exposure of specific epitopes, and subcellular localization of α-synuclein.

BACKGROUND: Genetic studies and the abundance of alpha-synuclein (α-Syn) in presynaptic terminals suggest that α-Syn plays a critical role in maintaining synaptic vesicle pools. However, there are still few experimental tools for elucidating its physiological roles. METHODS: Unexpectedly, we detected various cellular distribution patterns of endogenous α-Syn by immunofluorescence assays (IFAs). To provide new molecular insights into α-Syn research, we identified associations between epitopes, conformations, and subcellular localization of α-Syn and categorized them. RESULTS: The α-Syn exposing Y125 was found to coexist with F-actin at the edge of the cells, including the plasma membrane. α-Syn conformations exposing P128 or both F94 and K97 were partly localized to the mitochondria. These results indicate that various conformations of α-Syn are associated with specific subcellular localizations. Intriguingly, we demonstrate for the first time that the phosphorylated α-Syn at Ser129, also known as a Parkinson's disease (PD)-causing form, is targeted to the mitochondria. CONCLUSIONS: Our study showed that different subcellular distribution patterns of α-Syn reflect the existence of various α-Syn conformations under normal conditions. GENERAL SIGNIFICANCE: This study provides novel clues for deciphering the physiological function of α-Syn in connection with subcellular localization. Dissecting the specific α-Syn conformations may lead to useful strategies in PD therapy and diagnosis.