EGCG inhibited bladder cancer SW780 cell proliferation and migration both in vitro and in vivo via down-regulation of NF-κB and MMP-9

Epigallocatechin-3-gallate (EGCG), the bioactive polyphenol in green tea, has been demonstrated to have various biological activities. Our study aims to investigate the antiproliferation and antimigration effects of EGCG against bladder cancer SW780 cells both in vitro and in vivo. Our results showed that treatment of EGCG resulted in significant inhibition of cell proliferation by induction of apoptosis, without obvious toxicity to normal bladder epithelium SV-HUC-1 cells. EGCG also inhibited SW780 cell migration and invasion at 25–100 μM. Western blot confirmed that EGCG induced apoptosis in SW780 cells by activation of caspases-8, -9 and -3, Bax, Bcl-2 and PARP. Besides, animal study demonstrated that EGCG [100 mg/kg, intraperitoneal (i.p.) injection daily for 3 weeks] decreased the tumor volume significantly in mice bearing SW780 tumors, as well as the tumor weight (decreased by 68.4%). In addition, EGCG down-regulated the expression of nuclear factor-kappa B (NF-κB) and matrix metalloproteinase (MMP)-9 in both protein and mRNA level in tumor and SW780 cells. When NF-κB was inhibited, EGCG showed no obvious effect in cell proliferation and migration. In conclusion, our study demonstrated that EGCG was effective in inhibition SW780 cell proliferation and migration, and presented first evidence that EGCG inhibited SW780 tumor growth by down-regulation of NF-κB and MMP-9.     

Berberine suppresses in vitro migration of human aortic smooth muscle cells through the inhibitions of MMP-2/9, u-PA,AP-1, and NF-κB

Berberine, a type of isoquinoline alkaloid isolated from Chinese medicinal herbs, has been reported to have various pharmacological activities. Studies have demonstrated that berberine has beneficial effects on vascular remodeling and alleviates restenosis after vascular injury. However, its mechanism of action on vascular smooth muscle cell migration is not fully understood. We therefore investigated the effect of berberine on human aortic smooth muscle cell (HASMC) migration. Boyden chamber assay was performed to show that berberine inhibited HASMC migration dosedependently. Real-time PCR and Western blotting analyses showed that levels of matrix metalloproteinase (MMP)-2, MMP-9, and urokinase-type plasminogen activator (u-PA) were reduced by berberine at both the mRNA and protein levels. Western blotting assay further confirmed that activities of c-Fos, c-Jun, and NF-κB were significantly attenuated. These results suggest that berberine effectively inhibited HASMC migration, possibly by down-regulating MMP-2, MMP-9, and u-PA; and interrupting AP-1 and NF-κB mediated signaling pathways. [BMB Reports 2014; 47(7): 388-392]     

Tat-DJ-1 enhances cell survival by inhibition of oxidative stress,NF-κB and MAPK activation in HepG2 cells

Objectives

To identify the protective effect of DJ-1 protein against oxidative stress-induced HepG2 cell death, we used cell-permeable wild type (WT) and a mutant (C106A Tat-DJ-1) protein.

Results

By using western blotting and fluorescence microscopy, we observed WT and C106A Tat-DJ-1 proteins were efficiently transduced into HepG2 cells. Transduced WT Tat-DJ-1 proteins increased cell survival and protected against DNA fragmentation and intracellular ROS generation levels in H₂O₂-exposed HepG2 cells. At the same time, transduced WT Tat-DJ-1 protein significantly inhibited NF-κB and MAPK (JNK and p38) activation as well as regulated the Bcl-2 and Bax expression levels. However, C106A Tat-DJ-1 protein did not show any protective effect against cell death responses in H₂O₂-exposed HepG2 cells.

Conclusions

Oxidative stress-induced HepG2 cell death was significantly reduced by transduced WT Tat-DJ-1 protein, not by C106A Tat-DJ-1 protein. Thus, transduction of WT Tat-DJ-1 protein could be a novel strategy for promoting cell survival in situations of oxidative stress-induced HepG2 cell death.

     

DA-9601 inhibits activation of the human mast cell line HMC-1 through inhibition of NF-κB

Mast cell-mediated allergic inflammation is involved in many diseases such as asthma, sinusitis, and rheumatoid arthritis. Mast cells induce synthesis and production of pro-inflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 with immune regulatory properties. The formulated ethanol extract of Artemisia asiatica Nakai (DA-9601) has been reported to have antioxidative and anti-inflammatory activities. In this report, we investigated the effect of DA-9601 on the expression of pro-inflammatory cytokines by the activated human mast cell line HMC-1 and studied its possible mechanisms of action. DA-9601 dose-dependently decreased the gene expression and production of TNF-α, IL-1β, and IL-6 on phorbol 12-myristate 13-acetate (PMA)- and calcium ionophore A23187-stimulated HMC-1 cells. In addition, DA-9601 attenuated PMA- and A23187-induced activation of NF-κB as indicated by inhibition of degradation of IκBα, nuclear translocation of NF-κB, NF-κB/DNA binding, and NF-κB-dependent gene reporter assay. Our in vitro studies provide evidence that DA-9601 might contribute to the treatment of mast cell-derived allergic inflammatory diseases.     

Phosphatidylcholine regulates NF-κB activation in attenuation of LPS-induced inflammation: evidence from in vitro study

Phosphatidylcholine (PC) has been demonstrated as anti-inflammatory and antioxidant/pro-oxidant molecules. In this study, we investigated the protective effects of PC on inflammatory bowel disease (IBD) caused by lipopolysaccharide (LPS)-induced injury in intestinal epithelia cells. The IEC-6 cells (intestinal epithelia cells) were stimulated with LPS (1 μg/mL) for 24 h with or without PC pretreatment, in the next steps: (1) the level of the inflammatory cytokine tumor necrosis factor (TNF)-α was measured with ELISA; (2) the nuclear translocation and phosphorylation of NF-κB was investigated with Western blot, EMSA, immunofluorence assay; (3) the protein phosporylation levels in MAPK signaling pathway were detected with Western blot method. The results showed: (1) compared with the normal group, 10 and 20?μg/mL of PC significantly inhibited the production and activation of TNF-α, (P < 0.01); (2) pretreatment with PC inhibited LPS-induced nuclear translocation and phosphorylation of p65 in IEC-6 cells; (3) pretreatment with PC inhibited the protein phosphorylation levels in MAPK signaling pathway. Our findings indicated that PC had the effect to protect IEC-6 cells from LPS-induced injury and this effect was exerted possibly through inhibiting the TNF-ɑ secretion, down-regulating nuclear translocation and phosphorylation of p65 and inhibiting MAPK signaling pathways.     

Oleanane-type triterpene saponins from the bark of Aralia elata and their NF-κB inhibition and PPAR activation signal pathway

Two new oleanane-type triterpene saponins, tarasaponin IV (1) and elatoside L (2), and four known; stipuleanoside R(2) (3), kalopanax-saponin F (4), kalopanax-saponin F methylester (5), and elatoside D (6) were isolated from the bark of Aralia elata. Kalopanax-saponin F methyl ester was isolated from nature for the first time. Their chemical structures were elucidated using the chemical and physical methods as well as good agreement with those of reported in the literature. Oleanane-type triterpene saponins are the main component of A. elata. All compounds were investigated the anti-inflammatory activity. We measured their inhibition of NF-κB and activation of PPARs activities in HepG2 cells using luciferase reporter system. As results, compounds 2 and 4 were found to inhibit NF-κB activation stimulated by TNFα in a dose-dependent manner with IC(50) values of 4.1 and 9.5 μM, respectively, when compared with that of positive control, sulfasalazine (0.9 μM). Compounds 2 and 4 also inhibited TNFα-induced expression of iNOS and COX-2 mRNA. Furthermore, compounds 1-6 were evaluated PPAR activity using PPAR subtype transactivation assays. Among of them, compounds 4-6 significantly increased PPARγ transactivation. However, compounds 4-6 did not activate in any other PPAR subtypes.     

Naringin abrogates osteoclastogenesis and bone resorption via the inhibition of RANKL-induced NF-κB and ERK activation

Osteolytic bone diseases including osteoporosis are commonly accompanied with enhanced osteoclast formation and bone resorption. Naringin, a natural occurring flavonoid has been found to protect against retinoic acid-induced osteoporosis and improve bone quality in rats. Here, we showed that naringin perturbs osteoclast formation and bone resorption by inhibiting RANK-mediated NF-κB and ERK signaling. Naringin suppressed gene expression of key osteoclast marker genes. Naringin was found to inhibit RANKL-induced activation of NF-κB by suppressing RANKL-mediated IκB-α degradation. In addition, naringin inhibited RANKL-induced phosphorylation of ERK. This study identifies naringin as an inhibitor for osteoclast formation and bone resorption, and provides evidence that natural compounds such as naringin might be beneficial as an alternative medicine for the prevention and treatment of osteolysis.     

β-Caryophyllene ameliorates the Mycoplasmal pneumonia through the inhibition of NF-κB signal transduction in mice

BackgroundPneumonia is a frequent infectious disease that mainly affects the children and the global death rate is nearly 19% among children at the below 5 age. β-caryophyllene is an active compound, mainly occurs in the spices and it possesses immense biological activities.ObjectiveThis investigation deliberated to scrutinize the beneficial actions of β-caryophyllene against the M. pneumoniae induced pneumonia.MethodsThe pneumonia was stimulated to the BALB/c mice by infecting them with 100 µl of M. pneumonia for 2 days via nasal drops with the concomitant treatment with 20 mg/kg of β-caryophyllene. The total cells in the BALF of test mice were counted by using the Neuber chamber. The total protein and the pro-inflammatory cytokines status were examined by using the commercial ELISA kits. The PCR technique was used to measure the M. pneumoniae bacterial load. The NF-?B expression was investigated using western blotting. The lung tissues were analyzed microscopically.ResultsThe β-caryophyllene notably diminished the total protein status, total cell count, and bacterial load in the pneumonia provoked mice. The marked reduction in the status of pro-inflammatory regulators was seen in the β-caryophyllene supplemented pneumonia mice. β-caryophyllene also down-regulated the expression of NF-?B thereby reduced the lung inflammation and tissue damages as seen in the result of histological analysis.ConclusionThese findings were confirmed the therapeutic potential of β-caryophyllene against the M. pneumoniae-activated pneumonia in animals.     

Antitumor activity of IL-32β through the activation of lymphocytes,and the inactivation of NF-κB and STAT3 signals

Cytokine and activation of lymphocytes are critical for tumor growth. We investigated whether interleukin (IL)-32β overexpression changes other cytokine levels and activates cytotoxic lymphocyte, and thus modify tumor growth. Herein, IL-32β inhibited B16 melanoma growth in IL-32β-overexpressing transgenic mice (IL-32β mice), and downregulated the expressions of anti-apoptotic proteins (bcl-2, IAP, and XIAP) and cell growth regulatory proteins (Ki-67 antigen (Ki-67) and proliferating cell nuclear antigen (PCNA)), but upregulated the expressions of pro-apoptotic proteins (bax, cleaved caspase-3, and cleaved caspase-9). IL-32β also inhibited colon and prostate tumor growth in athymic nude mice inoculated with IL-32β-transfected SW620 colon or PC3 prostate cancer cells. The forced expression of IL-32β also inhibited cell growth in cultured colon and prostate cancer cells, and these inhibitory effects were abolished by IL-32 small interfering RNA (siRNA). IL-10 levels were elevated, but IL-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) levels were reduced in the tumor tissues and spleens of IL-32β mice, and athymic nude mice. The number of cytotoxic T (CD8⁺) and natural killer (NK) cells in tumor tissues, spleen, and blood was significantly elevated in IL-32β mice and athymic nude mice inoculated with IL-32β-transfected cancer cells. Constituted activated NF-κB and STAT3 levels were reduced in the tumor tissues of IL-32β mice and athymic nude mice, as well as in IL-32β-transfected cultured cancer cells. These findings suggest that IL-32β inhibits tumor growth by increasing cytotoxic lymphocyte numbers, and by inactivating the NF-κB and STAT3 pathways through changing of cytokine levels in tumor tissues.     

Anti-inflammatory potential of an ethyl acetate fraction isolated from Justicia gendarussa roots through inhibition of iNOS and COX-2 expression via NF-κB pathway

Justicia gendarussa Burm.f. (J. gendarussa) is a plant used as traditional medicine in different parts of India and China to treat inflammatory disorders like rheumatoid arthritis. But its mechanism of anti-inflammatory action is still unclear. Hence in this context, the objective of our study is to reveal the mechanism of anti-inflammatory activity of J. gendarussa which would form an additional proof to the traditional knowledge of this plant. The anti-inflammatory function and mechanism(s) of action was studied in an ethyl acetate fraction isolated from methanolic extract of J. gendarussa roots (EJG). Anti-inflammatory studies were conducted on rats using partitioned fractions isolated from methanolic extract of J. gendarussa roots. In carrageenan-induced rat paw edema, ethyl acetate fraction brought about 80% and 93% edema inhibition at 3rd and 5th hour at a dose of 50 mg/kg, when compared to other extracts and Voveran. We investigated whether EJG inhibits the release of cycloxygenase (COX), 5-lipoxygenase (5-LOX), interleukin-6 (IL-6) and nuclear factor kappa B (NF-κB) in LPS stimulated human peripheral blood mononuclear cells (hPBMCs). Results shows that EJG dose dependently inhibited LPS-activated COX, 5-LOX, IL-6, and NF-κB in hPBMCs. EJG also reduced LPS induced levels of iNOS and COX-2 mRNA expression in hPBMCs. This study provides an insight into the probable mechanism(s) underlying the anti-inflammatory activity of EJG and therefore, we report the first confirmation of the anti-inflammatory potential of this traditionally employed herbal medicine in vitro.