Naringenin ameliorates kainic acid-induced morphological alterations in the dentate gyrus in a mouse model of temporal lobe epilepsy.

Granule cell dispersion (GCD) in the dentate gyrus (DG) of the hippocampus is a morphological alteration characteristic of temporal lobe epilepsy. Recently, we reported that treatment with naringin, a flavonoid found in grapefruit and citrus fruits, reduced spontaneous recurrent seizures by inhibiting kainic acid (KA)-induced GCD and neuronal cell death in mouse hippocampus, suggesting that naringin might have beneficial effects for preventing epileptic events in the adult brain. However, it is still unclear whether the beneficial effects of naringin treatment are mediated by the metabolism of naringin into naringenin in the KA-treated hippocampus. To investigate this possibility, we evaluated whether intraperitoneal injections of naringenin could mimic naringin-induced effects against GCD caused by intrahippocampal KA injections in mice. Our results showed that treatment with naringenin delayed the onset of KA-induced seizures and attenuated KA-induced GCD by inhibiting activation of the mammalian target of rapamycin complex 1 in both neurons and reactive astrocytes in the DG. In addition, its administration attenuated the production of proinflammatory cytokines such as tumor necrosis tumor necrosis factor-α (TNFα) and interleukin-1ß (IL-1ß) from microglial activation in the DG following KA treatment. These results suggest that naringenin may be an active metabolite of naringin and help prevent the progression of epileptic insults in the hippocampus in vivo; therefore, naringenin may be a beneficial metabolite of naringin for the treatment of epilepsy.

Fermented Acanthopanax koreanum Root Extract Reduces UVB- and H2O2-Induced Senescence in Human Skin Fibroblast Cells.

The present study assessed the effects of an aqueous extract of Acanthopanax koreanum root (AE) and of AE following fermentation by lactic acid bacteria (Lactobacillus plantarum and Bifidobacterium bifidum) (AEF) on human skin fibroblast HS68 cells exposed to ultraviolet B (UVB) irradiation and oxidative stress. AEF effectively antagonized the senescence-associated ß-galactosidase staining and upregulation of p53 and p21(Cip1/WAF1) induced by UVB or H2O2 treatment in HS68 cells. It also exhibited excellent antioxidant activities in radical scavenging assays and reduced the intracellular level of reactive oxygen species induced by UVB or H2O2 treatment. The antioxidant and antisenescent activities of AEF were greater than those of nonfermented A. koreanum extract. AEF significantly repressed the UVB- or H2O2-induced activities of matrix metalloproteinase (MMP)-1 and -3, overexpression of MMP-1, and nuclear factor κB (NF-κB) activation. This repression of NF-κB activation and MMP-1 overexpression was attenuated by a mitogen-activated protein kinase activator, suggesting that this AEF activity was dependent on this signaling pathway. Taken together, these data indicated that AEF-mediated antioxidant and anti-photoaging activities may produce anti-wrinkle effects on human skin.

Melittin has a chondroprotective effect by inhibiting MMP-1 and MMP-8 expressions via blocking NF-κB and AP-1 signaling pathway in chondrocytes.

Bee venom is a natural ingredient produced by the honey bee (Apis mellifera), and has been widely used in China, Korea and Japan as a traditional medicine for various diseases such as arthritis, rheumatism, and skin diseases However, the regulation of the underlying molecular mechanisms of the anti-arthritis by bee venom and its major peptides is largely unknown. In this study, we investigated the potential molecular mechanisms underlying the anti-arthritis effect of bee venom and its major peptides, melittin and apamin, in tumor necrosis factor-α (TNF-α) responsive C57BL/6 mice chondrocyte cells. The bee venom and melittin significantly and selectively suppressed the TNF-α-mediated decrease of type II collagen expression, whereas the apamin had no effects on the type II collagen expression. We, furthermore, found that the bee venom and melittin inhibited the protein expression of matrix metalloproteinase (MMP)-1 and MMP-8, which suggests that the chondroprotective effect of bee venom may be caused by melittin. The inhibitory effects of melittin on the TNF-α-induced MMP-1 and MMP-8 protein expression were regulated by the inhibition of NF-kB and AP-1. In addition, melittin suppressed the TNF-α-induced phosphorylation of Akt, JNK and ERK1/2, but did not affect the phosphorylation of p38 kinase. These results suggest that melittin suppresses TNF-α-stimulated decrease of type II collagen expression by the inhibiting MMP-1 and MMP-8 through regulation of the NF-kB and AP-1 pathway and provision of a novel role for melittin in anti-arthritis action.

Melittin inhibits TGF-ß-induced pro-fibrotic gene expression through the suppression of the TGFßRII-Smad, ERK1/2 and JNK-mediated signaling pathway.

Renal fibrosis is characterized by the excessive accumulation of extracellular matrix (ECM) proteins such as type I collagen, fibronectin, and by the increased expression of PAI-1. This study evaluated the anti-fibrotic effect of bee venom and its major compounds (melittin and apamin) on TGF-ß-induced pro-fibrotic gene expression. Bee venom and melittin significantly suppressed type I collagen, fibronectin, and PAI-1 protein expression in the TGF-ß-treated kidney fibroblast. However, apamin only inhibited the expression of fibronectin and type I collagen. These results indicated that the inhibitory effects of bee venom on TGF-ß-induced pro-fibrotic gene expression are caused by melittin. Moreover, we attempted to elucidate mechanisms underlying the anti-fibrotic effect of melittin. Melittin dramatically inhibited the phosphorylation of TGFßRII and Smad2/3. Also, melittin inhibited the phosphorylation of ERK1/2 and JNK, but not the phosphorylation of PI3K, Akt, and p38. These results suggested that melittin inhibits TGF-ß-induced pro-fibrotic genes expression through the suppression of TGFßR-Smad2/3, ERK1/2, and JNK phosphorylation, and melittin can be used as a clinical drug for the treatment of fibrosis associated with renal diseases.

Coumestrol induces senescence through protein kinase CKII inhibition-mediated reactive oxygen species production in human breast cancer and colon cancer cells.

An inhibitor of the protein kinase CKII (CKII) was purified from leaves of Glycine max (L.) Merrill and was identified as coumestrol by structural analysis. Coumestrol inhibited the phosphotransferase activity of CKII toward ß-casein, with an IC50 of about 5 µM. It acted as a competitive inhibitor with respect to ATP as a substrate, with an apparent Ki value of 7.67 µM. Coumestrol at 50µM resulted in 50% and 30% growth inhibition of human breast cancer MCF-7 and colorectal cancer HCT116 cells, respectively. Coumestrol promoted senescence through the p53-p21(Cip1/WAF1) pathway by inducing reactive oxygen species (ROS) production in MCF-7 and HCT116 cells. The ROS scavenger N-acetyl-l-cysteine (NAC), NADPH oxidase inhibitor apocynin and p22(phox) siRNA almost completely abolished this event. Overexpression of CKIIα antagonised cellular senescence mediated by coumestrol, indicating that this compound induced senescence via a CKII-dependent pathway. Since senescence is an important tumour suppression process in vivo, these results suggest that coumestrol can function by inhibiting oncogenic disease, at least in part, through CKII inhibition-mediated cellular senescence.

Caffeic acid and quercitrin purified from Houttuynia cordata inhibit DNA topoisomerase I activity.

A methanol extract of Houttuynia cordata showed an inhibitory effect on mammalian DNA topoisomerase I. Two topoisomerase I inhibitory compounds were purified and identified as caffeic acid and quercitrin. Caffeic acid and quercitrin inhibited the activity of topoisomerase I with IC(50) values of about 0.15 and 0.05 mM, respectively. A concentration of 45 µM caffeic acid caused 50% growth inhibition in human leukaemia U937 cells, but not on those of normal fibroblast NIH3T3 cells. However, quercitrin mysteriously stimulated proliferation of U937 and NIH3T3 cells. Caffeic acid-induced cell death was characterised with the cleavage of poly (ADP-ribose) polymerase and procaspase-3, indicating that this inhibitor triggered apoptosis. The apoptotic induction by caffeic acid was also confirmed using flow cytometry analysis. Because DNA topoisomerase I is an important target for tumour chemotherapy, the present study suggests that caffeic acid, but not quercitrin, may function by suppressing oncogenic disease through the inhibition of cellular topoisomerase I activity.

Bee venom suppresses PMA-mediated MMP-9 gene activation via JNK/p38 and NF-kappaB-dependent mechanisms.

ETHNOPHARMACOLOGICAL RELEVANCE: Bee venom has been used for the treatment of inflammatory diseases such as rheumatoid arthritis and for the relief of pain in traditional oriental medicine. AIM OF THE STUDY: The purpose of this study is to elucidate the effects of bee venom on MMP-9 expression and determine possible mechanisms by which bee venom relieves or prevents the expression of MMP-9 during invasion and metastasis of breast cancer cells. We examined the expression and activity of MMP-9 and possible signaling pathway affected in PMA-induced MCF-7 cells. MATERIAL AND METHODS: Bee venom was obtained from the National Institute of Agricultural Science and Technology of Korea. Matrigel invasion assay, wound-healing assay, zymography assay, western blot assay, electrophoretic mobility shift assay and luciferase gene assay were used for assessment. RESULTS: Bee venom inhibited cell invasion and migration, and also suppressed MMP-9 activity and expression, processes related to tumor invasion and metastasis, in PMA-induced MCF-7 cells. Bee venom specifically suppressed the phosphorylation of p38/JNK and at the same time, suppressed the protein expression, DNA binding and promoter activity of NF-kappaB. The levels of phosphorylated ERK1/2 and c-Jun did not change. We also investigated MMP-9 inhibition by melittin, apamin and PLA(2), representative single component of bee venom. We confirmed that PMA-induced MMP-9 activity was significantly decreased by melittin, but not by apamin and phospholipase A(2). These data demonstrated that the expression of MMP-9 was abolished by melittin, the main component of bee venom. CONCLUSION: Bee venom inhibits PMA-induced MMP-9 expression and activity by inhibition of NF-kappaB via p38 MAPK and JNK signaling pathways in MCF-7 cells. These results indicate that bee venom can be a potential anti-metastatic and anti-invasive agent. This useful effect may lead to future clinical research on the anti-cancer properties of bee venom.

Bee venom suppresses LPS-mediated NO/iNOS induction through inhibition of PKC-alpha expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Bee venom (BV) is a traditional Korean medicine that has been widely used with satisfactory results in the treatment of some immune-related diseases, especially rheumatoid arthritis. AIM OF THE STUDY: The purpose of this study is to elucidate the molecular mechanism underlying the anti-inflammatory effects of BV, which is used in the treatment of various inflammatory diseases in traditional Korean medicine. We evaluated the anti-inflammatory effect of BV on NO generation and iNOS expression by LPS in rat C6 glioma cells. MATERIAL AND METHODS: BV was obtained from the National Institute of Agricultural Science and Technology (NIAST) of Korea. Nitrite measurement, Immuno blot analysis, Reverse transcriptase-PCR and Electrophoretic mobility shift assay (EMSA) were used for assessment. RESULTS: BV suppressed the LPS-induced NO generation and iNOS expression, and it also inhibited the expressions of LPS-induced pro-inflammatory molecules including Cox-2 and IL-1 beta in rat C6 glioma cells. Then, BV inhibited LPS-induced expression of PKC-alpha and MEK/ERK, not p38 and JNK. Moreover, inhibition of LPS-induced iNOS expression by BV was dependent on transcriptional activities of AP-1/NF-kappaB through MEK/ERK pathway. CONCLUSION: These results indicate that BV suppresses LPS-induced iNOS activation through regulation of PKC-alpha. Accordingly, BV exerts a potent suppressive effect on pro-inflammatory responses in rat C6 glioma cells.

Apoptotic cell death through inhibition of protein kinase CKII activity by 3,4-dihydroxybenzaldehyde purified from Xanthium strumarium.

The CKII inhibitory compound was purified from the fruit of Xanthium strumarium by organic solvent extraction and silica gel chromatography. The inhibitory compound was identified as 3,4-dihydroxybenzaldehyde by analysis with FT-IR, FAB-Mass, EI-Mass, (1)H-NMR and (13)C-NMR. 3,4-dihydroxybenzaldehyde inhibited the phosphotransferase activity of CKII with IC(50) of about 783 microM. Steady-state studies revealed that the inhibitor acts as a competitive inhibitor with respect to the substrate ATP. A value of 138.6 microM was obtained for the apparent K(i). Concentration of 300 microM 3,4-dihydroxybenzaldehyde caused 50% growth inhibition of human cancer cell U937. 3,4-dihydroxybenzaldehyde-induced cell death was characterised with the cleavage of poly(ADP-ribose) polymerase and procaspase-3. Furthermore, the inhibitor induced the fragmentation of DNA into multiples of 180 bp, indicating that it triggered apoptosis. This induction of apoptosis by 3,4-dihydroxybenzaldehyde was also confirmed by using flow cytometry analysis. Since CKII is involved in cell proliferation and oncogenesis, these results suggest that 3,4-dihydroxybenzaldehyde may function by inhibiting oncogenic disease, at least in part, through the inhibition of CKII activity.

Cudraflavanone A purified from Cudrania tricuspidata induces apoptotic cell death of human leukemia U937 cells, at least in part, through the inhibition of DNA topoisomerase I and protein kinase C activity.

A chloroform extract of the root bark of Cudrania tricuspidata showed an inhibitory effect on mammalian DNA topoisomerase I. The topoisomerase I inhibitory compound was purified and identified as 2S-2',5,7-trihydroxy-4',5'-(2,2-dimethylchromeno)-6-prenyl flavanone (cudraflavanone A). Cudraflavanone A was shown to inhibit the activity of topoisomerase I with approximately 0.4 mmol/l 50% inhibitory concentration. A concentration of 6 micromol/l cudraflavanone A caused a 50% growth inhibition of human cancer cell U937. Cudraflavanone A-induced cell death was characterized by the cleavage of poly(ADP-ribose) polymerase and pro-caspase-3. Furthermore, cudraflavanone A induced the fragmentation of DNA into multiples of 180 bp (an apoptotic DNA ladder), indicating that the inhibitor triggered apoptosis. This induction of apoptosis by cudraflavanone A was also confirmed using flow-cytometry analysis. In addition, this compound inhibited protein kinase C activity with approximately 150 micromol/l 50% inhibitory concentration. Taken together, these results suggest that cudraflavanone A may function by inhibiting oncogenic disease, at least in part, through the inhibition of protein kinase C and topoisomerase I activity.

2',5,7-Trihydroxy-4',5'-(2,2-dimethylchromeno)-8-(3-hydroxy-3-methylbutyl) flavanone purified from Cudrania tricuspidata induces apoptotic cell death of human leukemia U937 cells.

Cellular DNA topoisomerase I is an important target in cancer chemotherapy. A chloroform extract of the root barks of Cudrania tricuspidata showed an inhibitory effect on mammalian DNA topoisomerase I. The topoisomerase I inhibitory compound was purified and identified as 2',5,7-trihydroxy-4',5'-(2,2-dimethylchromeno)-8-(3-hydroxy-3-methylbutyl) flavanone. The compound, temporarily designated as PKH-3, was shown to inhibit the activity of topoisomerase I with IC50 about 1.0 mM. Concentration of 10 microM PKH-3 caused 50% growth inhibition of human cancer cell U937. PKH-3-induced cell death was characterized with the cleavage of poly(ADP-ribose) polymerase (PARP) and pro-caspase 3. Furthermore, PKH-3 induced the fragmentation of DNA into multiples of 180 b.p. (an apoptotic DNA ladder), indicating that the inhibitor triggered apoptosis. This induction of apoptosis by PKH-3 was also confirmed using flow cytometry analysis. Taken together, these results suggest that PKH-3 may function by inhibiting oncogenic disease, at least in part, through the inhibition of topoisomerase I activity.