ABSTRACT
This study investigated the mechanism by which icaritin (ICT) inhibits exosomes-induced lung metastasis of B16BL6 mouse melanoma cells. The culture supernatant of B16BL6 cells was collected for extraction of exosomes by ultracentrifugation and their characterization by transmission electron microscopy and Western blotting. Exosomal protein was quantified by BCA. A wound-healing assay was used to determine the effect of ICT on the migratory ability of B16BL6 cells induced by exosomes. After establishing an experimental melanoma lung metastasis model in C57BL/6 mice, we used H&E staining to study the ability of ICT to inhibit exosomes-induced melanoma metastasis. Animal experiments were approved by the Ethics Committee of Nanjing University of Chinese Medicine. ELISA and immunofluorescence were used to detect pro-inflammatory factors interleukin 6 (IL-6), S100 calcium-binding protein A8/A9 complex (S100A8/A9), serum amyloid A (SAA) and fibronectin in metastatic tumors. The expression of metastatic tissue-related proteins stimulator of interferon gene (STING), phospho-STING (p-STING), TANK-binding kinase 1 (TBK1) and phospho-TBK1 (p-TBK1) was detected by immunohistochemistry or Western blotting. The results showed that the particle size of exosomes was 149.33 ± 2.68 nm, the polydispersity index (PDI) was 0.192 ± 0.02, the zeta potential was -32.22 ± 0.50 mV, and the particles had classic tea tray-like membrane structure under TEM. The protein concentration of exosomes was measured to be 838.66 ± 62.14 μg·mL-1. The results of the cell scratch test showed that ICT can inhibit exosomes-induced migration of B16BL6 cells at a concentration of 5, 10, and 20 μmol·L-1. In vivo experimental results also showed that ICT can inhibit exosomes-induced metastasis of melanoma to the lungs and can significantly inhibit the expression of pro-inflammatory factors S100A8/A9, SAA and IL-6 in lung tissue, and inhibit the expression of p-STING and p-TBK1 in metastatic lung tissue. Taken together, these results indicated that ICT can significantly inhibit exosomes-induced tumor metastasis, and the inhibition is related to the inactivation of STING in metastatic foci.
ABSTRACT
Cyclic GMP-AMP synthase (cGAS)-stimulator of interferon gene (STING) signaling pathway as an essential immune response pathway in cytoplasm, can find cytoplasmic DNA to regulate the innate immune and adaptive immune response. Studies have shown that the signaling pathway can be activated by both tumor self-DNA and genomic instability, thus to promote or inhibit the development and metastasis of tumors. Therefore, the role of cGAS-STING in tumor genesis, development and metastasis will be systematically expounded from the structures, physiological functions, inhibitors and agonists of cGAS and STING as well as its pathway transduction regulation in this paper. The paper aims to offer theoretical basis and reference for targeting cGAS-STING anti-tumor drugs in clinical practice and cancer clinical and cancer research workers.
ABSTRACT
This study aimed to prepare an anti-metastatic diallyl trisulfide-exosome (DATS-Exo) drug delivery system. Exosomes in the supernatants of lung metastasis mouse melanoma B16BL6 cell line culture were extracted by ultracentrifugation. The quantity of exosomes was determined by transmission electron microscopy (TEM), Malvern particle size meter, and BCA assay. Expression levels of exosome-specific biomarkers CD9, TSG101, Flotillin 1 and lung organotropic biomarker α6 were detected by Western blot. The sonication method was used to load DATS into exosomes. The uptake of exosomes by B16BL6 cells and lung tissue was observed by laser confocal microscopy. Wound healing assay was used to evaluate the anti-migration effect of DATS-Exo in vitro. Experimental lung metastasis model was established to evaluate the anti-metastasis effect of DATS-Exo in vivo. Animal experiments have been approved by the Ethics Committee of Nanjing University of Chinese Medicine. The results showed that the particle size of DATS-Exo was 112.3 ± 1.98 nm, polydispersity index (PDI) was 0.24 ± 0.08, zeta potential was -24.33 ± 4.11 mV, and the particles have classic tea tray-like membrane structure under TEM. The protein concentration of DATS-Exo was measured to be 1 312.33 ± 6.27 μg·mL-1. The drug loading rate was about 0.33% ± 0.02%. The exosomes could be taken up by B16BL6 cells and the lung tissue. Compared with the free DATS group, DATS-Exo had a better inhibitory effect on tumor metastasis in vitro and in vivo. Taken together, these results indicate that exosomes derived from the lung metastasis tumor cells have lung organotropic characteristic and drug-loading properties. Using these kind of exosomes as carrier for anti-tumor drug delivery can be a novel and effective strategy of anti-metastatic therapy.