Anti-inflammatory activities of
thymoquinone (TQ) have been demonstrated in in vitro and in vivo studies. However, the precise mechanism(s) of TQ in these anti-inflammatory activities is not well understood. Using a newly developed assay to detect
sialidase activity in live macrophage cells (Glycoconj J doi: 10.1007/s10719-009-9239-8 ), here we show that TQ has no inhibitory effect on
endotoxin lipopolysaccharide (LPS) induced
sialidase activity in live BMC-2 macrophage cells. In contrast, the parent black seed oil (BSO) and another constituent of BSO para-
cymene (p-CY) completely block LPS induced
sialidase activity. All of these compounds had no effect on cell viability. On the other hand, TQ induces a vigorous
sialidase activity in live BMC-2 macrophage cells in a dose dependent manner as well in live DC-2.4 dendritic cells, HEK-TLR4/MD2, HEK293, SP1 mammary
adenocarcinoma cells, human WT and 1140F01 and WG0544 type I
sialidosis fibroblast cells.
Tamiflu (
oseltamivir phosphate) inhibits TQ-induced
sialidase activity in live BMC-2 cells with an IC(50) of 0.0194 microM compared to an IC(50) of 19.1 microM for
neuraminidase inhibitor DANA (2-deoxy-2,3-dehydro-N-acetylneuraminic acid). Anti-Neu1, -2 and -3
antibodies have no inhibition of TQ-induced
sialidase activity in live BMC-2 and human THP-1 macrophage cells but anti-Neu4
antibodies completely block this activity. There is a vigorous
sialidase activity associated with TQ treated live primary bone marrow (BM) macrophage cells derived from WT and hypomorphic
cathepsin A mice with a secondary
Neu1 deficiency (NeuI KD), but not from Neu4 knockout (Neu4 KO) mice.
Pertussis toxin (PTX), a specific inhibitor of Galphai
proteins of
G-protein coupled receptor (GPCR) and the broad range inhibitors of
matrix metalloproteinase (
MMP)
galardin and
piperazine applied to live BMC-2, THP-1 and primary BM macrophage cells completely block TQ-induced
sialidase activity. These same inhibitory effects are not observed with the
GM1 ganglioside specific
cholera toxin subunit B (CTXB) as well as with CTX,
tyrosine kinase inhibitor K252a, and the broad range GPCR inhibitor
suramin. The specific inhibitor of MMP-9, anti-MMP-9 antibody and anti-Neu4 antibody, but not the specific inhibitor of MMP-3 completely block TQ-induced
sialidase activity in live THP-1 cells, which express Neu4 and MMP-9 on the cell surface. Neu4
sialidase activity in cell lysates from TQ-treated live THP-1 cells desialylates natural
gangliosides and
mucin substrates. RT-PCR and western blot analyses reveal no correlation between
mRNA and
protein values for Neu3 and Neu4 in human monocytic THP-1 cells, suggesting for the first time a varied post-transcriptional mechanism for these two mammalian sialidases independent of TQ activation. Our findings establish an unprecedented activation of Neu4
sialidase on the cell surface by
thymoquinone, which is derived from the nutraceutical black cumin oil. The potentiation of GPCR-signaling by TQ via membrane targeting of Galphai subunit
proteins and
matrix metalloproteinase-9 activation may be involved in the activation process of Neu4
sialidase on the cell surface.