Occupational exposure to respirable crystalline silica (cSiO2) has been etiologically linked to human autoimmunity. Intranasal instillation with cSiO2 triggers profuse inflammation in the lung and onset of autoimmunity in lupus-prone mice; however, dietary supplementation with the omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) abrogates these responses. Inflammasome activation, IL-1 cytokine release, and death in alveolar macrophages following cSiO2 exposure are early and critical events that likely contribute to triggering premature autoimmune pathogenesis by this particle. Here we tested the hypothesis that DHA suppresses cSiO2-induced NLRP3 inflammasome activation, IL-1 cytokine release, and cell death in the macrophage. The model used was the murine macrophage RAW 264.7 cell line stably transfected with the inflammasome adapter protein ASC (RAW-ASC). Following priming with LPS, both the canonical activator nigericin and cSiO2 elicited robust inflammasome activation in RAW-ASC cells, as reflected by IL-1β release and caspase-1 activation. These responses were greatly diminished or absent in wild-type RAW cells. In contrast to IL-1β, cSiO2 induced IL-1α release in both RAW-ASC and to a lesser extent in RAW-WT cells after LPS priming. cSiO2-driven effects in RAW-ASC cells were confirmed in bone-marrow derived macrophages. Pre-incubating RAW-ASC cells with 10 and 25 μM DHA for 24 h enriched this fatty acid in the phospholipids by 15- and 25-fold, respectively, at the expense of oleic acid. DHA pre-incubation suppressed inflammasome activation and release of IL-1β and IL-1α by nigericin, cSiO2, and two other crystals – monosodium urate and alum. DHA's suppressive effects were linked to inhibition of LPS-induced Nlrp3, Il1b, and Il1a transcription, potentially through the activation of PPARγ. Finally, nigericin-induced death was inflammasome-dependent, indicative of pyroptosis, and could be inhibited by DHA pretreatment. In contrast, cSiO2-induced death was inflammasome-independent and not inhibited by DHA. Taken together, these findings indicate that DHA suppresses cSiO2-induced inflammasome activation and IL-1 cytokine release in macrophages by acting at the level of priming, but was not protective against cSiO2-induced cell death.

The growth and development of non-small cell lung cancer (NSCLC) primarily depends on glutamine. Both glutamine and essential amino acids (EAAs) have been reported to upregulate mTOR in NSCLC, which is a bioenergetics sensor involved in the regulation of cell growth, cell survival, and protein synthesis. Seen as novel concepts in cancer development, ASCT2 and LAT transporters allow glutamine and EAAs to enter proliferating tumors as well as send a regulatory signal to mTOR. Blocking or downregulating these glutamine transporters in order to inhibit glutamine uptake would be an excellent therapeutic target for treatment of NSCLC. This study aimed to validate the metabolic dysregulation of glutamine and its derivatives in NSCLC using cellular 1H-NMR metabolomic approach while exploring the mechanism of delta-tocotrienol (δT) on glutamine transporters, and mTOR pathway. Cellular metabolomics analysis showed significant inhibition in the uptake of glutamine, its derivatives glutamate and glutathione, and some EAAs in both cell lines with δT treatment. Inhibition of glutamine transporters (ASCT2 and LAT1) and mTOR pathway proteins (P-mTOR and p-4EBP1) was evident in Western blot analysis in a dose-dependent manner. Our findings suggest that δT inhibits glutamine transporters, thus inhibiting glutamine uptake into proliferating cells, which results in the inhibition of cell proliferation and induction of apoptosis via downregulation of the mTOR pathway.

BackgroundDelta-tocotrienol (δT), an isomer of vitamin E, exhibits anticancer properties in different cancer types including non-small-cell lung cancer (NSCLC). Yet, anti-invasive effects of δT and its underlying cellular mechanism in NSCLC have not been fully explored. Matrix metalloproteinase 9 (MMP-9)-based cell migration and invasion are critical cellular mechanisms in cancer development. The current evidence indicates that MMP-9 is upregulated in most patients, and the inhibition of MMPs is involved in decreasing invasion and metastasis in NSCLC. Therefore, its suppression is a promising strategy for attenuating cell invasion and metastasis processes in NSCLC.PurposeThe aim of this study was to evaluate the possibility of MMP-9 inhibition as the underlying mechanism behind the antimetastatic properties of δT on NSCLC cells.MethodsThe effects of δT on cell proliferation, migration, invasion, adhesion, and aggregation capabilities were investigated using different cell-based assays. An inhibitory effect of MMP-9 enzyme activity with δT was also identified using gel zymography. Using real-time PCR and Western blot analysis, a number of cellular proteins, regulatory genes, and miRNA involved in the Notch-1 and urokinase-type plasminogen activator (uPA)-mediated MMP-9 pathways were examined.ResultsThe study found that δT inhibited cell proliferation, cell migration, invasion, aggregation, and adhesion in a concentration-dependent manner and reduced MMP-9 activities. Real-time PCR and Western blot analysis data revealed that δT increased miR-451 expressions and downregulated Notch-1-mediated nuclear factor-κB (NF-κB), which led to the repressed expression of MMP-9 and uPA proteins.ConclusionδT attenuated tumor invasion and metastasis by the repression of MMP-9/uPA via downregulation of Notch-1 and NF-κB pathways and upregulation of miR-451. The data suggest that δT may have potential therapeutic benefit against NSCLC metastasis.

Workplace exposure to respirable crystalline silica dust (cSiO2) has been etiologically linked to the development of lupus and other human autoimmune diseases. Lupus triggering can be recapitulated in female NZBWF1 mice by four weekly intranasal instillations with 1 mg cSiO2. This elicits inflammatory/autoimmune gene expression and ectopic lymphoid structure (ELS) development in the lung within 1 week, ultimately driving early onset of systemic autoimmunity and glomerulonephritis. Intriguingly, dietary supplementation with docosahexaenoic acid (DHA), an ω-3 polyunsaturated fatty acid (PUFA) found in fish oil, beginning 2 week prior to cSiO2 challenge, prevented inflammation and autoimmune flaring in this novel model. However, it is not yet known how ω-3 PUFA intervention influences established autoimmunity in this murine model of toxicant-triggered lupus. Here we tested the hypothesis that DHA intervention after cSiO2-initiated intrapulmonary autoimmunity will suppress lupus progression in the NZBWF1 mouse. Six-week old NZWBF1 female mice were fed purified isocaloric diet for 2 weeks and then intranasally instilled with 1 mg cSiO2 or saline vehicle weekly for 4 consecutive weeks. One week after the final instillation, which marks onset of ELS formation, mice were fed diets supplemented with 0, 4, or 10 g/kg DHA. One cohort of mice (n = 8/group) was terminated 13 weeks after the last cSiO2 instillation and assessed for autoimmune hallmarks. A second cohort of mice (n = 8/group) remained on experimental diets and was monitored for proteinuria and moribund criteria to ascertain progression of glomerulonephritis and survival, respectively. DHA consumption dose-dependently increased ω-3 PUFA content in the plasma, lung, and kidney at the expense of the ω-6 PUFA arachidonic acid. Dietary intervention with high but not low DHA after cSiO2 treatment suppressed or delayed: (i) recruitment of T cells and B cells to the lung, (ii) development of pulmonary ELS, (iii) elevation of a wide spectrum of plasma autoantibodies associated with lupus and other autoimmune diseases, (iv) initiation and progression of glomerulonephritis, and (v) onset of the moribund state. Taken together, these preclinical findings suggest that DHA supplementation at a human caloric equivalent of 5 g/d was an effective therapeutic regimen for slowing progression of established autoimmunity triggered by the environmental toxicant cSiO2.