The inflammasome adaptor protein, ASC, contributes to both innate immune responses and inflammatory diseases via self-oligomerization, which leads to the activation of the protease, caspase-1. Here, we report that the cytosolic tyrosine kinases, FAK and Pyk2, are differentially involved in NLRP3 and AIM2 inflammasome activation. The inhibition of FAK and Pyk2 with RNA interference or chemical inhibitors dramatically abolished ASC oligomerization, caspase-1 activation, and IL-1β secretion in response to NLRP3 or AIM2 stimulation. Pyk2 is phosphorylated by the kinase Syk and relocalizes to the ASC specks upon NLRP3 inflammasome activation. Pyk2, but not FAK, could directly phosphorylate ASC at Tyr146, and only the phosphorylated ASC could participate in speck formation and trigger IL-1β secretion. Moreover, the clinical-trial-tested Pyk2/FAK dual inhibitor PF-562271 reduced monosodium urate-mediated peritonitis, a disease model used for studying the consequences of NLRP3 activation. Our results suggest that although Pyk2 and FAK are involved in inflammasome activation, only Pyk2 directly phosphorylates ASC and brings ASC into an oligomerization-competent state by allowing Tyr146 phosphorylation to participate ASC speck formation and subsequent NLRP3 inflammation.
Platonin is a photosensitizer used for photodynamic therapy. In this study, we tested the effect of platonin on human leukemic cells. Treatment with platonin in the dark markedly reduced cell membrane integrity, and induced significant G 0 /G 1 arrest of a panel of human leukemic cell lines, including U937, HL-60, K562, NB4 and THP-1. Development of hypodiploid cells was not evident in these cell lines within 24 h, but was noted in U937, HL-60 and NB4 cells after 24 h. No myeloid differentiation of these cells was noted after five-day treatment. Intriguingly, exposure of monoblastic U937 cells to platonin caused changes characteristic of autophagy, including appearance of cytoplasmic membranous vacuoles and formation of acidic vesicular organelles (AVO) in more than 95% of cells. The platonin-induced autophagy was accompanied by localization of microtubule-associated protein 1 light chain 3 to autophagosomes. Pretreatment with pancaspase inhibitor Z-VAD-fmk abrogated the platonin-induced hypodiploidity, but had no effect on growth inhibition and formation of AVO, indicating a caspase-independent autophagy-associated cell death. Pretreatment of cells with 3-methyladenine attenuated platonin-mediated growth inhibition and formation of AVO. Platonin augmented the expression of BNIP3 in both U937 and K562 cells, whereas had an opposite effect on phosphorylation of mTOR downstream molecule p70S6K. Platonin, at the condition inducing autophagy, induced the mitochondrial membrane permeation. These results suggest that the platonin is capable of inhibiting growth as well as inducing cell death, mainly autophagy-associated, in leukemic cells via a mitochondria-mediated and caspase-independent pathway. A markedly less viability inhibition was noted to human monocytes, the normal counterpart of these myeloid leukemic cells. Platonin, other than a photodynamic agent, may offer significant promise as a therapeutic agent against leukemia.
The combined administration of programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) inhibitors might be considered as a treatment for poorly responsive cancer. We report a patient with brain metastatic lung adenocarcinoma in whom fatal myocarditis developed after sequential use of PD-1 and PD-L1 inhibitors. This finding was validated in syngeneic tumor-bearing mice. The mice bearing lung metastases of CT26 colon cancer cells treated with PD-1 and/or PD-L1 inhibitors showed that the combination of anti-PD-1 and anti-PD-L1, either sequentially or simultaneously administered, caused myocarditis lesions with myocyte injury and patchy mononuclear infiltrates in the myocardium. A significant increase of infiltrating neutrophils in myocytes was noted only in mice with sequential blockade, implying a role for the pathogenesis of myocarditis. Among circulating leukocytes, concurrent and subsequent treatment of PD-1 and PD-L1 inhibitors led to sustained suppression of neutrophils. Among tumor-infiltrating leukocytes, combinatorial blockade increased CD8+ T cells and NKG2D+ T cells, and reduced tumor-associated macrophages, neutrophils, and natural killer (NK) cells in the lung metastatic microenvironment. The combinatorial treatments exhibited better control and anti-PD-L1 followed by anti-PD-1 was the most effective. In conclusion, the combinatory use of PD-1 and PD-L1 blockade, either sequentially or concurrently, may cause fulminant cardiotoxicity, although it gives better tumor control, and such usage should be cautionary.
6-(methylsulfinyl) hexyl isothiocyanate (6-MITC) is a naturally occurring compound isolated from Wasabia japonica (wasabi). The synthetic derivatives, 6-(methylsulfenyl) hexyl isothiocyanate (I7447) and 6-(methylsulfonyl) hexyl isothiocyanate (I7557), were derived from 6-MITC with the deletion and addition of oxygen, respectively. We aimed to evaluate the effect of these synthetic compounds on human oral cancer cells, SAS and OECM-1. All three compounds (I7447, 6-MITC, and I7557) inhibited the viability of SAS and OECM-1 cells using MTT assay. Morphological observations showed various proportions of mitotic arrest and apoptosis in cells treated with these compounds. Cell cycle analysis revealed relatively abundant G2/M arrest in 6-MITC and I7557-treated cells, whereas sub-G1 accumulation was found in I7447-treated cells. In using phosphorylated histone H3 as a marker for mitosis, the addition of 6-MITC and I7557 (excluding I7447) could be shown to arrest cells during mitosis. In contrast, I7447 induced more prominent apoptosis than the 6-MITC or I7557 compounds. The down-regulated expression of the phosphorylated form of CHK1 and Cdc25c was noted in 6-MITC and I7557-treated cells. I7557 could sensitize SAS cells to death by radiation. The wasabi compound, 6-MITC, and its chemical derivatives with different numbers of oxygen may have differential pharmacological effects on human oral cancer cells.
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