Benzoxazole Derivative K313 Induces Cell Cycle Arrest, Apoptosis and Autophagy Blockage and Suppresses mTOR/p70S6K Pathway in Nalm-6 and Daudi Cells.

Benzoxazole derivative K313 has previously been reported to possess anti-inflammatory effects in lipopolysaccharide-induced RAW264.7 macrophages. To date, there have been no related reports on the anticancer effects of K313. In this study, we found that K313 reduced the viability of human B-cell leukemia (Nalm-6) and lymphoma (Daudi) cells in a dose-dependent manner without affecting healthy peripheral blood mononuclear cells (PBMCs) and induced moderate cell cycle arrest at the G0/G1 phase. Meanwhile, K313 mediated cell apoptosis, which was accompanied by the activation of caspase-9, caspase-3, and poly ADP-ribose polymerase (PARP). Furthermore, cells treated with K313 showed a significant decrease in mitochondrial membrane potential (MMP), which may have been caused by the caspase-8-mediated cleavage of Bid, as detected by Western blot analysis. We also found that K313 led to the downregulation of p-p70S6K protein, which plays an important role in cell survival and cell cycle progression. In addition, treatment of these cells with K313 blocked autophagic flux, as reflected in the accumulation of LC3-II and p62 protein levels in a dose- and time-dependent manner. In conclusion, K313 decreases cell viability without affecting normal healthy PBMCs, induces cell cycle arrest and apoptosis, reduces p-p70S6K protein levels, and mediates strong autophagy inhibition. Therefore, K313 and its derivatives could be developed as potential anticancer drugs or autophagy blockers in the future.

K313, a novel benzoxazole derivative, exhibits anti-inflammatory properties via inhibiting GSK3ß activity in LPS-induced RAW264.7 macrophages.

Benzoxazole and its derivatives have been widely studied in recent years due to their various biological properties. A previous study has demonstrated that K313 (1H-indole-2,3-dione 3-(1,3-benzoxazol-2-ylhydrazone)), a novel benzoxazole derivative, inhibits T cell proliferation to yield immunosuppressive effects. However, there are no related reports about its anti-inflammatory effects. In the present study, we investigated the anti-inflammatory properties and the underlying molecular mechanism of K313 in lipopolysaccharide (LPS)-induced RAW264.7 macrophages. K313 dose-dependently (5, 10, and 20 µM) inhibited LPS-stimulated nitric oxide (NO), interleukin (IL)-6, tumor necrosis factor (TNF)-α, and 3-nitrotyrosine (3-NT) production and significantly decreased the gene transcription levels of inducible nitric oxide (iNOS), IL-6, and TNF-α. In addition, the results showed that the inflammatory cytokines suppressed by K313 were not regulated by p65 NF-κB, ERK1/2, AKT, or p38 MAPK. Instead, K313 increased phosphorylation of glycogen synthase kinase-3 beta (GSK-3ß) (Ser9) resulting in GSK-3ß deactivation. Moreover, in LPS-stimulated RAW264.7 macrophages, K313 and lithium chloride (LiCl) had a synergistic effect on the anti-inflammatory response. These results indicated that K313 exhibited anti-inflammatory properties and revealed the potential mechanism. K313 can increase GSK-3ß (Ser9) phosphorylation to decrease GSK-3ß activation in LPS-induced RAW264.7 macrophages.

Regulatory Dendritic Cells Induced by K313 Display Anti-Inflammatory Properties and Ameliorate Experimental Autoimmune Encephalitis in Mice.

As a GSK-3ß inhibitor reported by our group, K313 is a novel benzoxazole derivative and displays anti-inflammatory properties in RAW264.7 macrophages without cytotoxicity. The activity of GSK-3ß affects the differentiation and maturation of bone marrow-derived dendritic cells (DCs). This study aims to investigate whether K313 can be used to induce regulatory/tolerogenic dendritic cells (DCregs), and the therapeutic effects of DCregs induced by K313 in the autoimmune model of experimental autoimmune encephalitis (EAE). The results show that compared with LPS stimulated mature DCs, K313-treated bone marrow-derived DCs display obvious tolerogenic characteristics with decreased expression of co-stimulatory molecules, downregulated secretions of pro-inflammatory cytokines and unregulated secretion of anti-inflammatory cytokine IL-10. The above characteristics conform to the typical phenotypes of DCregs. Moreover, K313-modified DCregs inhibit antigen-specific T cell responses in vitro. Furthermore, by adoptive transfer, K313 modified DCregs to the EAE mice, and the development of disease was ameliorated to some extent. In addition, treatment with K313-modified DCregs also significantly reduced the percentages of splenetic Th1 and Th17 cells and increased the percentage of regulatory T cells in EAE mice. In conclusion, K313-modified DCregs show anti-inflammatory properties in vitro and have a significant positive effect on the EAE disease in vivo. Our data indicate that K313-induced DCregs pulsed with auto-antigen might have potential use as a therapeutic approach for autoimmune inflammation of the central nervous system.