Because of rapid degradation of IκB with stimulation, it could be a target to analyze the effect of drugs on the NF–κB signaling pathway [8
]. Activation of the NF-κB can be detected by green fluorescent protein-tagged IκB degradation in living cells [9
]. In this study, we first used the IκB–EGFP protein as an indicator to screen microbial metabolites and demonstrated that the effective substance selected from the screening system can inhibit activity of NF-κB by inhibition of IκB degradation. Mechanisms for regulation of NF-κB activity comprise inhibiting the activation of IKK complex, inhibiting the phosphorylation of IκB, preventing the degradation of IκB, blocking the migration of NF-κB to nucleus, and decreasing the DNA binding activity of NF-κB. For activation of NF-κB, IκB must be phosphorylated to release itself from the complex of NF-κB and process of degradation. In this study, the results revealed that the degradation of IκB is exactly a good reference to relate the activity of NF-κB. Thus, utilization of IκB degradation as an indicator to judge intracellular NF-κB activity can be established as a drug screen system for selecting microbial metabolites.
The degradation of endogenous IκB regulates NF-κB activation, and IκB can be degraded rapidly within 2 h to prevent the accumulation of IκB. Thus, the stability of cellular IκB is low. In this system, we find that the changes in the amounts of IκB or phosphorylated IκB are similar to endogenous IκB or IκB–EGFP, which is dependent on the stimulation with the NF-κB activator, such as TNF-α or PMA. In addition, the half-life of IκB is greatly shortened to 5–30 min with TNFα-induced IκB degradation. Therefore, we proposed that the endogenous IκB could not influence the expression of IκB–EGFP.
Plasmid pIκB–EGFP was transfected into different cell lines (Huh 7 and 293 cells) to establish cell-based drug screening system, and the results showed that the pIκB–EGFP-transfected 293 cells have better response in fluorescent expression after HQ treatment than Huh7 cell (Figures and ). Therefore, the sensitivity of IκB degradation-based drug screening system is related to cell type. The cells that have stronger intracellular NF-κB activity may be more proper to develop the screening system.
The most common active form of NF-κB is p50/p65 heterodimer, whereas c-Rel, Rel-B, or p52 also has the ability to form a heterodimer with p50. Except for heterodimers, the homodimers of NF-κB including p50/p50, p52/p52, and RelA/RelA also existed and have activities in cells. These NF-κB dimers have different affinities to NF-κB binding sequence in promoter [10
]. In addition, the expressions of identical gene are regulated with different NF-κB dimers in different cells. For example, the vascular cell adhesion molecule-1 (VCAM-1) gene of endothelial cells is regulated by p50/p65; however, the VCAM-1 gene of Hela cell is regulated by p65/p65 but not p50/p65 [11
]. It can be concluded that the members of IκB family—IκB-α, IκB-β, IκB-γ, and Bcl-3 proteins—all have the ability to suppress NF-κB translocation into cell nucleus. Whereas different IκB have their own specific target molecules, such as IκB-α that prefers binding to p50/p65 and p65/p65, nevertheless it inefficiently binds to p50/p50 [12
]. In addition, IκB-β specifically binds to p65 and c-Rel but not p50, and IκB-γ and Bcl-3 specifically bind to p50 [13
]. Thus, different cells have various NF-κB existences and regulate the activity of NF-κB by different IκB members. Because of the widespread importance of NF-κB, it has been difficult to develop NF-κB inhibitors that act specifically in a specific cell to employ in therapy of diseases. Therefore, certain specific IκB inhibitors may be found according to our model presented here and inhibit specific NF-κB activity in certain cells without affecting the NF-κB signal pathway in other cells.
HQ was chosen from this system, and the previous literatures showed that it could decrease the secretion of cytokines [14
]. HQ exists in extract of tobacco and inhibits human lymphocytes to produce IL-1β, IL-2, and TNF-α [16
]. HQ has also been reported to reduce the expression of CD19 by decreasing the intracellular activity of NF-κB, and its mechanism is not due to the inhibition of the DNA binding activity of NF-κB [17
]. In this study, we first reveal that HQ inhibits the activity of NF-κB by inhibiting the degradation of IκB.
In this study, we have demonstrated that the plasmid pIκB–EGFP transfectants (Huh 7 and 293 cells) were used as a drug screening system to seek NF-κB inhibitor, and we did obtain HQ of microbial origin, which have been shown to have a significant activity against NF-κB activity, from the screening program. NF-κB target genes have been studied and are involved in immunity, inflammation, cell proliferation, apoptosis, and cell migration [18
]. A close connection between inflammation and cancer has also been suspected. NF-κB pathway is also involved in cell adhesion. The cell adhesion molecule, such as integrin, has been correlated with the cell differentiation or adhesion. HQ, a NF-κB inhibitor, can block upstream signaling for both NF-κB activation and cell adhesion. HQ also inhibits NF-κB activation through suppressing integrin expression for cell adhesion [19
]. HQ exposure affected cell proliferation and delayed cell growth and attachment in a dose-dependent manner [19
]. In addition, HQ diminishes surface levels of CD29 and CD18 and suppresses CD29-mediated cell–cell adhesion in monocyte [20
]. Therefore, such downregulation with HQ treatment may be an inhibitory mechanism for cell adhesion. Therefore, the system may provide a good method to develop a new drug for inflammation and cancer therapy (Figure ).
Propolis was used to determine whether this screening system is sensitive enough to identify the effective substance from a natural extracted mixture. The results showed that propolis increased the fluorescence of pIκB–EGFP-transfected cells compared to negative control groups. Different kinds of propolis were extracted by 80% ethanol, and the results showed that the effective substance for inhibiting the IκB degradation is favorable in ethanol (Figure ). The previous evidences revealed that the effective substance should be caffeic acid phenethyl ester which is hydrophobic and can inhibit NF-κB activity [21
]. In addition, red propolis or green propolis was harvested respectively in winter or summer, and the results also showed different propolises harvested in different seasons have different activities to inhibit the IκB degradation (Figure ). Propolis is the product after bees harvested the resin from the tree, and different seasons would affect what kind of tree resin was harvested by the bee. Thus, the propolises have different activities to IκB degradation that is reasonable. This study showed that this assay could determine the activity of natural product for IκB degradation.
In this study, an assay was established to monitor the activities of substances for IκB degradation in the cell. The results showed that the assay could be applied to pure chemical and natural mixture. After selection by this assay, the chosen candidate exactly has the ability to cause the change in the NF-κB activity via regulating the IκB and further regulate the expression related with NF-κB. Therefore, the system may provide a good method to develop a new drug for certain diseases that are related to NF-κB activities such as inflammation and cancer.