image: (A) NeoL activates STING-TBK1 pathways. Jurkat and Hut102 cells were treated with 0–4 μmol/L of NeoL for 4 h, and the expression of p-TBK1, TBK1, p-STING, and STING was determined by western blotting, with β-actin as a loading control. p-STING and p-TBK1 were increased at 1 and 2 μmol/L but declined at 4 μmol/L (blue arrowheads).
(B) Quantification of total expression of STING in Jurkat and Hut102 cells, showing significant reduction of total STING and p-STING (blue arrowheads) after 4 h of NeoL treatment at 4 μmol/L, suggesting accelerated degradation of p-STING under 4 μmol/L of NeoL (n = 3).
(C) STING-Ubiquitin co-precipitation. Jurkat cells were treated with 20 μmol/L of 2',3'-cGAMP for 12 h or 4 μmol/L of NeoL for 2 h. STING served as a loading control for immunoprecipitation. For input, β-actin served as a loading control, showing increased ubiquitin after STING activation.
(D) The interaction between STING and NeoL was revealed by molecular docking.
(E) The interaction between STING and NeoL was confirmed by DARTS assay. Cell lysates were incubated with DMSO, NeoL, or IsoL, and digested with pronase in 1:4800 or 1:2400 dilution. Immunoblotting demonstrated increased STING after NeoL incubation. GAPDH was used as a loading control.
(F) The interaction between STING and NeoL was also confirmed by CETSA assay. Cells were incubated with 4 μmol/L of NeoL or DMSO for 30 min, divided into seven pairs, and treated at the indicated temperatures for 3 min. Soluble proteins were extracted by freezing/thawing in liquid nitrogen, blotted for STING or β-actin as a loading control. Increased STING was detected in lysate at 47 °C, 49 °C, and 51 °C.
(G) The Jurkat and Hut102 cells were treated without or with 4 μmol/L of NeoL for 2 h and immune-labelled with anti-STING (green) and anti-LAMP1 for lysosomes (red). NeoL treatment increased the colocalization of lysosomes and STING. The coincidence of fluorescence curves was analyzed by Image J software. P values were indicated in the graph, and P < 0.05 indicates statistical significance.
Credit: Po Hu, Yuxuan Huang, Zipeng Yu, Xiaolong Zhang, Guangming Yang, Yang Pan
A recent study at Nanjing University of Chinese Medicine, published in Genes & Diseases, reported that neoliensisnine (NeoL)—a tribenzylisoquinoline alkaloid isolated from Plumula nelumbinis (the embryo of Nelumbo nucifera Gaertn.)—exerts potent anti-T-CM activity and elucidated its mechanism of action.
Preliminary findings revealed that NeoL has a strong cytotoxic effect on T-CM cells. Previous studies have shown that the stimulator of interferon genes (STING) pathway is essential in T lymphocytes and plays a critical role in antitumor immunity. In this study, the authors observed high STING expression in T-CM cells, which was reduced upon treatment with NeoL. Molecular docking, cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS) analyses showed that NeoL binds directly to STING with high affinity and thereby exerts a stronger inhibitory effect, suggesting that NeoL induces T-CM cell death by promoting STING degradation.
Research over the years has shown that STING is degraded via the lysosomal pathway through ESCRT-driven microautophagy. In the current study, the authors demonstrated that NeoL, by regulating TUBA1B—an important microtubule isoform involved in cytoskeleton formation—promotes STING degradation via the microtubule–lysosome pathway. Furthermore, TUBA1B mediates NeoL-induced lysosomal trafficking of STING by disrupting lysosomal cholesterol homeostasis.
Additionally, treatment with NeoL induces lysophagy and lysosomal membrane permeabilization, which lead to lysosomal damage and lysosome-dependent cell death in T-CM. The authors also showed that STING, microtubules, and cholesterol accumulation play critical roles in mediating NeoL-induced lysosomal dysfunction. The inhibitory effect of NeoL on T-CM was also validated in vivo.
In conclusion, this study shows that NeoL is a safe and efficient candidate drug that, by promoting lysosome-dependent cell death via the TUBA1B/STING/cholesterol axis, inhibits T-CM—thus positioning it as a promising therapeutic agent to improve treatment outcomes for T-CMs.
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