METHODS AND RESULTS: TQRF was extracted from N. sativa seeds using supercritical fluid extraction. The regulatory effects of TQRF at 80 microg/ml and TQ at 2 microg/ml on LDLR and HMGCR gene expression were investigated in HepG2 cells using quantitative real-time PCR. The TQ content in TQRF was 2.77% (w/w) and was obtained at a temperature of 40 degrees C and a pressure of 600 bar. Treatment of cells with TQRF and TQ resulted in a 7- and 2-fold upregulation of LDLR mRNA level, respectively, compared with untreated cells. The mRNA level of HMGCR was downregulated by 71 and 12%, respectively, compared with untreated cells.
CONCLUSION: TQRF and TQ regulated genes involved in cholesterol metabolism by two mechanisms, the uptake of low-density lipoprotein cholesterol via the upregulation of the LDLR gene and inhibition of cholesterol synthesis via the suppression of the HMGCR gene.
Materials and methods: 3D-mediated inhibition on cell viability was evaluated by MTT and real-time cell proliferation was measured by xCelligence RTDP instrument. Western blotting was used to measure pro-apoptotic, anti-apoptotic proteins and JAK2-STAT3 phosphorylation. Flow cytometry was used to measure ROS production and apoptosis.
Results: Our study revealed that 3D treatment significantly reduced the viability of human CRC cells HT-29 and SW620. Furthermore, 3D treatment induced the generation of reactive oxygen species (ROS) in human CRC cells. Confirming our observation, N-acetylcysteine significantly inhibited apoptosis. This is further evidenced by the induction of p53 and Bax; release of cytochrome c; activation of caspase-9, caspase-7 and caspase-3; and cleavage of PARP in 3D-treated cells. This compound was found to have a significant effect on the inhibition of antiapoptotic proteins Bcl2 and BclxL. The results further demonstrate that 3D inhibits JAK2-STAT3 pathway by decreasing the constitutive and IL-6-induced phosphorylation of STAT3. 3D also decreases STAT3 target genes such as cyclin D1 and survivin. Furthermore, a combination study of 3D with doxorubicin (Dox) also showed more potent effects than single treatment of Dox in the inhibition of cell viability.
Conclusion: Taken together, these findings indicate that 3D induces ROS-mediated apoptosis and inhibits JAK2-STAT3 signaling in CRC.
METHODS: Anti-retroviral therapy (ART) naïve HIV-seropositive individuals (progressors, n=16) and long-term non-progressors (LTNPs, n=10) were recruited for this study. We employed multi-color flow cytometry on frozen peripheral blood mononuclear cells (PBMCs) to determine iNKT subset frequencies, the levels of co-inhibitory 2B4 expression, and intracellular IFN-γ production. CD1d tetramer was used to characterize iNKT cells.
RESULTS: We report significantly lower level of 2B4 expression on bulk LTNPs iNKT cells as well as on their CD4 subsets compared to HIV progressors. Furthermore, the iNKT cells from LTNPs produced higher amount of IFN-γ than HIV progressors as detected by intracellular cytokine staining. Interestingly, the frequency of 2B4iNKT cells of progressors but not LTNPs significantly correlates with CD4 T cell count, HIV viral load and IFNγ production by iNKT cells.
CONCLUSION: Our results suggest that in addition to suppressed HIV replication, diminished 2B4 expression and associated co-inhibitory signaling, and substantial production of IFN-γ could contribute to preserved iNKT cell phenotype in LTNPs.