2-Methylhydroquinone: Anti-inflammatory and Anti-angiogenic Properties

2-Methylhydroquinone, the secondary metabolite of marine algae symbiotic microorganism,inhibits angiogenesis in HUVECs Angiogenesis.

2-Methylhydroquinone from Aspergillus sp. suppress inflammatory mediators

The study of marine environment has detected consistently novel bioactive products known as secondary metabolites. Many diseases are treated by chemotherapeutic agents developed from these secondary metabolites. 2-Methylhydroquinone is one of the secondary metabolites from Aspergillus sp. It has been reported with an anti-oxidative effect, anti-angiogenesis effect on Human Umbilical Vine Endothelial Cells (HUVECs) and antibacterial effect against the methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant S. aureus, but other bioactivities have not been investigated. In this study, we investigated anti-inflammatory effects of toluhydroquinone in LPS-stimulated RAW264.7 cells. In 2-Methylhydroquinone-treated RAW264.7 cells, the expression of iNOS and cyclooxyganse-2 (COX-2) was abolished, and in turn, production of NO and PGE2 was reduced, respectively. Inflammatory cytokines, including TNF-α, IL-1β and IL-6, were also down-regulated by 2-Methylhydroquinone. These inflammatory mediators were regulated through NF-κB, Akt and MAPKs. Our results proved that toluhydroquinone has inhibitory effects on the production of inflammatory mediators via regulating NF-κB, Akt and MAPKs pathways in LPS-induced RAW264.7 cells.[1]

Secondary metabolites, which are known as natural products, are produced by many endophytic fungi. 2-Methylhydroquinone is one of the secondary metabolites, derived from Aspergillus sp., an endophytic fungus. Toluhydroquinone has been studied for anti-oxidant effects, anti-angiogenesis effect on HUVECs, and anti-bacterial effect against MRSA and multidrug-resistant S. aureus but other biological effects of toluhydroquinone are rarely known. Therefore, we aimed to study anti-inflammatory effects of 2-Methylhydroquinone in LPS-induced RAW264.7 cells. NO is produced by iNOS and plays a role as an inflammatory mediator, regulating host defence and immune response. Increase of NO production is mediated to many diseases, such as carcinogenesis, circulatory shock and inflammation. Thus, inhibition of NO production can be a resolution for inflammatory diseases. iNOS inhibitors, which are inhibitors of NO synthesis, have been studied to treat inflammatory diseases on arthritis and colitis. Our results showed that toluhydroquinone abolished NO production via inhibition of iNOS expression in LPS-stimulated RAW264.7 cells. These results suggest that 2-Methylhydroquinone could be a potential inflammatory therapeutic agent by inhibition of excessive NO production.

PGE2 is one of the inflammatory mediators and is generated by COX-2, an inducible form of cyclooxygenase. Inflammatory stimuli trigger the conversion of arachidonic acid to PGE2 by COX-2, resulting in amplifying pro-inflammatory reactions. In arthritis, increase of COX-2 expression elicits excessive PGE2 production, facilitating pro-inflammatory cytokines production And fever is known as one of the inflammatory symptom. To relieve fever in inflammatory responses, COX-2 inhibition is an effective strategy. Therefore, regulation of COX-2 expression can be a key of anti-inflammatory treatments. In our research, toluhydroquinone reduced PGE2 and COX-2 expression in a concentration-dependent manner. Based on these results, 2-Methylhydroquinone may relieve inflammatory responses through suppression of PGE2 production and COX-2 expression.TNF-α, IL-1β and IL-6 are members of pro-inflammatory cytokines inducing inflammatory responses, produced by phosphorylation of NF-κB and degradation of IκBα. Because of inducible effects of the pro-inflammatory cytokines on inflammation, regulation of inflammatory cytokines production is a crucial treatment to ameliorate inflammatory diseases. In our results, toluhydroquinone significantly inhibited the expression of TNF-α, IL-1β and IL-6 in LPS-induced RAW264.7 cells. In accordance with the regulatory effect of toluhydroquinone on pro-inflammatory cytokines, we expect that 2-Methylhydroquinone could be used as a source of medicine for regulating inflammatory cytokine expression. In this research, we demonstrated that the anti-inflammatory effects of toluhydroquinone, a secondary metabolite, from Aspergillus sp., in LPS-induced RAW264.7 cells. Toluhydroquione inhibited the expression of iNOS, COX-2, TNF-α, IL-1β and IL-6 at protein level, resulting in reduction of levels of NO, PGE2 and the secreted pro-inflammatory cytokines. Also, t 2-Methylhydroquinone inhibited phosphorylation of NF-κB and IκBα, and translocation of NF-κB. In the same manner, toluhydroquinone suppressed LPS-induced phosphorylation of Akt, MAPK kinases and MAPKs. With our findings, we expect that 2-Methylhydroquinone may possess potential as a therapeutic agent for anti-inflammatory medicines.

2-Methylhydroquinone, the secondary metabolite of marine algae symbiotic microorganism

As novel bioactive secondary metabolites isolated from marine microorganisms have anti-tumor, anti-virus and other bioactive properties, investigation of secondary metabolites from marine microorganisms has been an attractive field to search potent drug candidates. This is the first report that investigates anti-angiogenesis effects of 2-Methylhydroquinone, derived from marine algae symbiotic fungus, Aspergillus sp. To treat cancer, there are marine microorganism-derived secondary metabolites developed as chemotherapeutic agents. In this study, we used toluhydroquinone (2-methyl-1,4-hydroquinone), one of the secondary metabolites isolated from marine algae symbiotic fungus, Aspergillus sp. We examined the effects of 2-Methylhydroquinone on angiogenesis using HUVECs. We identified that toluhydroquinone inhibited the activity of β-catenin and down-regulated Ras/Raf/MEK/ERK signaling which are crucial components during angiogenesis. In addition, the expression and activity of MMPs are reduced by the treatment of toluhydroquinone. In conclusion, we confirmed that toluhydroquinone has inhibitory effects on angiogenic behaviors of human endothelial cells, HUVECs. Our findings suggest that 2-Methylhydroquinone can be proposed as a potent anti-angiogenesis drug candidate to treat cancers.[2]

Because a strength of cell-substratum adhesion determines whether the cells can migrate on a given substratum or not, we tested the effects of 2-Methylhydroquinone in cell adhesion ability. As a result, toluhydroquinone weakened the adhesion force between HUVECs and substratum , which means that HUVECs are not able to adhere strongly to extracellular matrix. Endothelial cells can express several integrins, hetero-dimer cell surface receptors combined with unique α and β subunit, which serve endothelial cells to anchor their specific matrix, including fibronectin and collagen during angiogenesis. In addition to, as many reports have demonstrated that endothelial cell adhesion is tightly associated with endothelial cell migration and invasion, we may think that 2-Methylhydroquinone has a capability to block endothelial cell migration or invasion.

At the initial stage of new blood vessel formation, endothelial cell invasion must occur, breaking down ECM to induce cell migration toward where angiogenesis occurs. We found that 2-Methylhydroquinone inhibited VEGF-induced endothelial cell invasion, then we expected that toluhydroquinone may affect MMPs, essential proteolytic enzymes for digestion of ECM. We found that toluhydroquinone suppressed expression of MMP-9 and MMP-2, involved in initiation and promotion of angiogenesis, particularly. We expected that the down-regulated MMPs cause decreasing in proteolytic activities of these enzymes. 2-Methylhydroquinone reduced proteolytic activities of MMP-9. It implies that toluhydroquinone can inhibit endothelial cell invasion through an attenuation of MMP activities by reducing MMP expression. We next focused on identifying molecular targets of 2-Methylhydroquinone in HUVECs. Endothelial cells require the reorganization of the cytoskeleton for migration. The small G protein of Rac1 plays an important role in cytoskeletal reorganization, regulating membrane extensions in lamellipodia.

2-Methylhydroquinone down-regulated the expression of Rac1, suggesting that toluhydroquinone can inhibit cell migration through suppressing Rac1 in HUVECs. In addition to, endothelial cells require Ras activity to induce vessel sprouting in the presence of angiogenic growth factors such as VEGF and endothelial cell proliferation is indispensable to branching morphogenesis through Ras/MAPK pathway. Thus we thought that an inhibition of Ras/MAPK signaling can lead to retraction of sprouting vessels, which is consistent with decrease in the number of branching points of tubule structures. Therefore, the destruction for tubule formation by 2-Methylhydroquinone seems to result from its down-regulatory effects on Ras/MAPK pathway. However, toluhydroquinone did not represent anti-proliferative activities at certain concentrations which can interrupt tube formation, we suggest that 2-Methylhydroquinone can obstruct tubule formation of HUVECs through regulating Ras/MAPK pathway, but it is not enough to affect cell survival. In short, toluhydroquinone interrupts cell migration and tubule formation by suppressing the small G proteins, including Rac1 and Ras without influencing cell proliferation.

References

[1]Hwang, Dukhyun et al. “Toluhydroquinone from Aspergillus sp. suppress inflammatory mediators via nuclear factor-κB and mitogen-activated protein kinases pathways in lipopolysaccharide-induced RAW264.7 cells.” The Journal of pharmacy and pharmacology vol. 67,9 (2015): 1297-305. doi:10.1111/jphp.12421

[2]Kim, Nan-Hee et al. “Toluhydroquinone, the secondary metabolite of marine algae symbiotic microorganism, inhibits angiogenesis in HUVECs.” Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie vol. 70 (2015): 129-39. doi:10.1016/j.biopha.2015.01.004