Inhibitory effect of snake venom toxin on NF-κB activity prevents human cervical cancer cell growth via increase of death receptor 3 and 5 expression.

We previously found that snake venom toxin inhibits nuclear factor kappa B (NF-κB) activity in several cancer cells. NF-κB is implicated in cancer cell growth and chemoresistance. In our present study, we investigated whether snake venom toxin (SVT) inhibits NF-κB, thereby preventing human cervical cancer cell growth (Ca Ski and C33A). SVT (0-12 µg/ml) inhibited the growth of cervical cancer cells by the induction of apoptotic cell death. These inhibitory effects were associated with the inhibition of NF-κB activity. However, SVT dose dependently increased the expression of death receptors (DRs): DR3, DR5 and DR downstream pro-apoptotic proteins. Exploration of NF-κB inhibitor (Phenylarsine oxide, 0.1 µM) synergistically further increased SVT-induced DR3 and DR5 expressions accompanied with further inhibition of cancer cells growth. Moreover, deletion of DR3 and DR5 by small interfering RNA significantly abolished SVT-induced cell growth inhibitory effects, as well as NF-κB inactivation. Using TNF-related apoptosis-inducing ligand resistance cancer cells (A549 and MCF-7), we also found that SVT enhanced the susceptibility of chemoresistance of these cancer cells through down-regulation of NF-κB, but up-regulation of DR3 and DR5. In vivo study also showed that SVT (0.5 and 1 mg/kg) inhibited tumor growth accompanied with inactivation of NF-κB. Thus, our present study indicates that SVT could be applicable as an anticancer agent for cervical cancer, or as an adjuvant agent for chemoresistant cancer cells.

Bee venom ameliorates lipopolysaccharide-induced memory loss by preventing NF-kappaB pathway.

BACKGROUND: Accumulation of beta-amyloid and neuroinflammation trigger Alzheimer's disease. We previously found that lipopolysaccharide (LPS) caused neuroinflammation with concomitant accumulation of beta-amyloid peptides leading to memory loss. A variety of anti-inflammatory compounds inhibiting nuclear factor kappaB (NF-κB) activation have showed efficacy to hinder neuroinflammation and amyloidogenesis. We also found that bee venom (BV) inhibits NF-κB. METHODS: A mouse model of LPS-induced memory loss used administration of BV (0.8 and 1.6 µg/kg/day, i.p.) to ICR mice for 7 days before injection of LPS (2.5 mg/kg/day, i.p.). Memory loss was assessed using a Morris water maze test and passive avoidance test. For in vitro study, we treated BV (0.5, 1, and 2 µg/mL) to astrocytes and microglial BV-2 cells with LPS (1 µg/mL). RESULTS: We found that BV inhibited LPS-induced memory loss determined by behavioral tests as well as cell death. BV also inhibited LPS-induced increases in the level of beta-amyloid (Aß), ß-and γ-secretases activities, NF-κB and its DNA-binding activity and expression of APP, and BACE1 and neuroinflammation proteins (COX-2, iNOS, GFAP and IBA-1) in the brain and cultured cells. In addition, pull-down assay and molecular modeling showed that BV binds to NF-κB. CONCLUSIONS: BV attenuates LPS-induced amyloidogenesis, neuroinflammation, and therefore memory loss via inhibiting NF-κB signaling pathway. Thus, BV could be useful for treatment of Alzheimer's disease.

Bee venom inhibits growth of human cervical tumors in mice.

We studied whether bee venom (BV) inhibits cervical tumor growth through enhancement of death receptor (DR) expressions and inactivation of nuclear factor kappa B (NF-κB) in mice. In vivo study showed that BV (1 mg/kg) inhibited tumor growth. Similar inhibitory effects of BV on cancer growth in primary human cervical cancer cells were also found. BV (1-5 µg/ml) also inhibited the growth of cancer cells, Ca Ski and C33Aby the induction of apoptotic cell death in a dose dependent manner. Agreed with cancer cell growth inhibition, expression of death receptors; FAS, DR3 and DR6, and DR downstream pro-apoptotic proteins including caspase-3 and Bax was concomitantly increased, but the NF-κB activity and the expression of Bcl-2 were inhibited by treatment with BV in tumor mice, human cancer cell and human tumor samples as well as cultured cancer cells. In addition, deletion of FAS, DR3 and DR6 by small interfering RNA significantly reversed BV-induced cell growth inhibitory effects as well as NF-κB inactivation. These results suggest that BV inhibits cervical tumor growth through enhancement of FAS, DR3 and DR6 expression via inhibition of NF-κB pathway.

Growth Inhibitory Effect of (E)-2,4-bis(p-hydroxyphenyl)-2-Butenal Diacetate through Induction of Apoptotic Cell Death by Increasing DR3 Expression in Human Lung Cancer Cells.

The Maillard Reaction Products (MRPs) are chemical compounds which have been known to be effective in chemoprevention. Death receptors (DR) play a central role in directing apoptosis in several cancer cells. In our previous study, we demonstrated that (E)-2,4-bis(p-hydroxyphenyl)-2-butenal, a MRP product, inhibited human colon cancer cell growth by inducing apoptosis via nuclear factor-κB (NF-κB) inactivation and G2/M phase cell cycle arrest. In this study, (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate, a new (E)-2,4-bis(p-hydroxyphenyl)-2-butenal derivative, was synthesized to improve their solubility and stability in water and then evaluated against NCI-H460 and A549 human lung cancer cells. (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate reduced the viability in both cell lines in a time and dose-dependent manner. We also found that (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate increased apoptotic cell death through the upregulation of the expression of death receptor (DR)-3 and DR6 in both lung cancer cell lines. In addition to this, the transfection of DR3 siRNA diminished the growth inhibitory and apoptosis inducing effect of (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate on lung cancer cells, however these effects of (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate was not changed by DR6 siRNA. These results indicated that (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate inhibits human lung cancer cell growth via increasing apoptotic cell death by upregulation of the expression of DR3.

Inhibitory effect of esculetin on migration, invasion and matrix metalloproteinase-9 expression in TNF-α-induced vascular smooth muscle cells.

Esculetin, a potent non-competitive inhibitor of lipoxygenase, has been shown to inhibit vascular smooth muscle cell (VSMC) proliferation. However, the effect of esculetin on the matrix metalloproteinase-9 (MMP-9) regulation responsible for cell migration and invasion has not been previously investigated. The results of the present study showed the esculetin (12.5-25 µg/ml) induced the inhibition of migration and invasion in tumor necrosis factor-α (TNF-α)-treated VSMC, as demonstrated by a matrigel invasion assay and wound healing analysis. However, esculetin did not affect cell viability in TNF-α-treated VSMC under 0-25 µg/ml concentration conditions. In addition, both zymographic and immunoblot experiments showed that esculetin suppressed the TNF-α-induced expression of MMP-9 in VSMC in a dose-dependent manner. Furthermore, the treatment of cells with esculetin decreased the activity of the TNF-α-induced MMP-9 promoter, which was driven by a luciferase reporter. Finally, esculetin reduced the binding activities of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), which are cis-elements present in the promoter of the MMP-9 gene, in TNF-α-treated VSMC. Taken together, these results demonstrated that esculetin decreased the migration and invasion of cells by suppressing MMP-9 expression, which subsequently reduced the binding activities of NF-κB and AP-1 in TNF-α-treated VSMC. These novel findings provide basic information for effective therapeutic treatment with esculetin for atherosclerotic disease.

4-O-methylhonokiol attenuated ß-amyloid-induced memory impairment through reduction of oxidative damages via inactivation of p38 MAP kinase.

Oxidative stress induced neuronal cell death by accumulation of ß-amyloid (Aß) is a critical pathological mechanism of Alzheimer's disease (AD). Intracerebroventrical infusion of Aß(1-42) (300 pmol/day per mouse) for 14 days induced neuronal cell death and memory impairment, but pre-treatment of 4-O-methylhonokiol (4-O-MH), a novel compound extracted from Magnolia officinalis for 3 weeks (0.2, 0.5 and 1.0 mg/kg) prior to the infusion of Aß(1-42) and during the infusion dose dependently improved Aß(1-42)-induced memory impairment and prevented neuronal cell death. Additionally, 4-O-MH reduced Aß(1-42) infusion-induced oxidative damages of protein and lipid but reduced glutathione levels in the cortex and hippocampus. Aß(1-42) infusion-induced activation of astrocytes and p38 mitogenic activated protein (MAP) kinase was also prevented by 4-O-MH in mice brains. In further study using culture cortical neurons, p38 MAP kinase inhibitor abolished the inhibitory effect of 4-O-MH (10 µM) on the Aß(1-42) (5 µM)-induced reactive oxidative species generation and neuronal cell death. These results suggest that 4-O-MH might prevent the development and progression of AD through the reduction of oxidative stress and neuronal cell death via inactivation of p38 MAP kinase pathway.

Signaling pathway for TNF-alpha-induced MMP-9 expression: mediation through p38 MAP kinase, and inhibition by anti-cancer molecule magnolol in human urinary bladder cancer 5637 cells.

We investigated the molecular mechanisms involved in the expression of matrix metalloproteinase-9 (MMP-9) and the inhibition of MMP expression by magnolol in 5637 human urinary bladder cancer cells. Tumor necrosis factor-alpha (TNF-alpha) stimulated the secretion of MMP-9 in 5637 cells, as shown by zymography and promoter assay. The transcription factor nuclear factor kappaB (NF-kappaB) binding site was identified by gel-shift assay to be a cis-element for TNF-alpha activation of the MMP-9 promoter. Our results also demonstrated that TNF-alpha stimulates MMP-9 expression via the p38 MAP kinase signaling pathway in 5637 cells. Moreover, p38 MAP kinase-mediated MMP-9 gene regulation in response to TNF-alpha is involved in the NF-kappaB response element in 5637 cells. In addition, magnolol inhibited TNF-alpha-induced expression of the MMP-9, as determined by zymography and immunoblot, in 5637 cells. The TNF-alpha-induced invasion and migration of cells was inhibited by magnolol, as assessed by a modified boyden chamber and wound-healing assays, respectively. Finally, magnolol blocked MMP-9 expression, at least in part, by decreasing the binding of transcription factor NF-kappaB to DNA. In conclusion, TNF-alpha induced MMP-9 expression in 5637 cells by activating the transcription factor NF-kappaB, which is involved in the p38 MAP kinase-mediated control of MMP-9 regulation. Magnolol inhibited MMP-9 expression through the transcription factor NF-kappaB in TNF-alpha-induced 5637 cells.

Anti-proliferate and pro-apoptotic effects of 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyranone through inactivation of NF-kappaB in human colon cancer cells.

Many natural compounds have been shown to prevent cancer cell growth through the redox regulation of transcription factors. NF-kappaB, a redox transcription factor, has been implicated in the apoptotic cell death of several cancer cells. This study examined whether or nor 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyranone (DDMP) isolated from onions can modulate the activity of NF-kappaB, thereby induce the apoptotic cell death of colon cancer cells. Treatment with different DDMP concentrations (0.5-1.5 mg/mL) for various periods (0-48 h) inhibited the growth of colon cancer cells (SW620 and HCT116) followed by the induction of apoptosis in a dose dependent manner. It was also found that DDMP modulated tumor necrosis factor-alpha (TNF-alpha) and tetradeanoyl phorbol acetate (TPA)-induced NF-kappaB transcriptional and DNA binding activity. Moreover, DDMP suppressed the NF-kappaB target anti-apoptotic genes (Bcl-2), whereas it induced the expression of the apoptotic genes (Bax, cleaved caspase-3 and cleaved PARP). These results suggest that DDMP from onions inhibit colon cancer cell growth by inducing apoptotic cell death through the inhibition of NF-kappaB.

Inhibition of cell growth and induction of apoptosis via inactivation of NF-kappaB by a sulfurcompound isolated from garlic in human colon cancer cells.

Compounds such as S-allylmercaptocysteine, diallyl disulfide, and S-trityl-L-cysteine isolated from garlic have been known to be effective in chemoprevention. Nuclear transcription factor-kappaB (NF-kappaB) has been known to be an implicated factor in apoptotic cell death of several cancer cells. In this study, we investigated whether a sulfurcompound (named thiacremonone) isolated from garlic could modulate NF-kappaB activity and thereby induce apoptotic cell death of colon cancer cells. Treatment with different concentrations (30 - 150 microg/ml) of thiacremonone for various periods (0 - 48 h) inhibited colon cancer cell (SW620 and HCT116) growth followed by induction of apoptosis in a dose-dependent manner. We also found that thiacremonone modulated tumor necrosis factor-alpha (TNF-alpha) and tetradeanoyl phorbol acetate (TPA)-induced NF-kappaB transcriptional and DNA binding activity. Moreover, thiacremonone suppressed NF-kappaB target anti-apoptotic genes (Bcl-2, cIAP1/2, and XIAP) and inflammatory genes (iNOS and COX-2), whereas it induced apoptotic genes (Bax, cleaved caspse-3, and cleaved PARP) expression. These results suggest that a novel sulfurocompound from garlic inhibited colon cancer cell growth through induction of apoptotic cell death by modulating of NF-kappaB.

Inhibitory role of magnolol on proliferative capacity and matrix metalloproteinase-9 expression in TNF-alpha-induced vascular smooth muscle cells.

Magnolol, an active component extracted from Magnolia officinalis, has been reported to inhibit the development of atherosclerotic disease. However, it is not known whether magnolol exerts similar cardioprotective effects in cells treated with TNF-alpha. In the present study, magnolol treatment was found to show potent inhibitory effects on cell proliferation in cultured VSMC in the presence of TNF-alpha. These inhibitory effects were associated with reduced extracellular signal-regulated kinase (ERK) 1/2 activity and G1 cell cycle arrest. Magnolol treatment strongly induced the expression of p21WAF1, but resulted in a decrease in cyclin-dependent kinases (CDKs) and cyclins involved in G1 progression. In addition to G1 cell cycle arrest and growth inhibition in VSMC, magnolol also caused the strong inhibition of TNF-alpha-induced matrix metalloproteinase-9 (MMP-9) expression in a dose-dependent manner as determined by zymography and immunoblot. Moreover, magnolol treatment strongly decreased MMP-9 promoter activity in response to TNF-alpha. We further demonstrated that magnolol reduced the transcriptional activity of NF-kappaB and activation protein-1 (AP-1), two important nuclear transcription factors that are involved in MMP-9 expression. Collectively, these results show that magnolol inhibits cell proliferation, G1 to S phase cell cycle progress and MMP-9 expression through the transcription factors NF-kappaB and AP-1 in TNF-alpha-induced VSMC. The findings of the present study reveal a potential mechanism that explains the anti-atherogenic activity of magnolol.

Furfural from Pine Needle Extract Inhibits the Growth of a Plant Pathogenic Fungus, Alternaria mali.

The antifungal effect of pine needle extract prepared by a distinguishable extraction method and the dry distillation method, was examined. The effect of this extract itself was insignificant. The chemical components of pine needle extract were then investigated by gas chromatographic analysis, and four chemical components, acetol, furfural, 5-methyl furfural, and terpine-4-ol, were identified. The antifungal effects of those four chemical components against Alternaria mali (A. mali), an agent of Alternaria blotch of apple, were then examined. It was observed that the minimum inhibitory concentrations (MICs) were 6.25, 0.78, 0.78, and 12.5 (mg/ml) of acetol, furfural, 5-methyl furfural, and terpine-4-ol, respectively. MICs of furfural and 5-methyl furfural had the same order of magnitude as that of an antifungal agrochemical, chlorothalonil. Although furfural itself can not be completely substituted for an antifungal agrochemical, a partial mixture of furfural and antifungal agrochemical may be used as a substitute. The use of agrochemicals for the prevention of plant disease caused by pathogenic fungus such as A. mali could be partially reduced by the application of this mixture.

Melittin inhibits inflammatory target gene expression and mediator generation via interaction with IkappaB kinase.

We previously found that bee venom (BV) and melittin (a major component of BV) has anti-inflammatory effect by reacting with the sulfhydryl group of p50 of NF-kappaB. Since the sulfhydryl group is present in IkappaB kinase (IKKalpha and IKKbeta), anti-inflammatory effect of melittin via interaction with IKKs was investigated. We first examined binding of melittin to IKKs using surface plasmon resonance analyzer. Melittin binds to IKKalpha (K(d) = 1.34 x 10(-9) M) and IKKbeta (K(d) = 1.01 x 10(-9) M). Consistent with the high binding affinity, melittin (5 and 10 microg/ml) and BV (0.5, 1 and 5 microg/ml) suppressed sodium nitroprusside, TNF-alpha and LPS induced-IKKbeta and IKKbeta activities, IkappaB release, and NF-kappaB activity as well as the expressions of iNOS and COX-2, and the generation of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) in Raw 264.7 mouse macrophages and synoviocytes obtained from rheumatoid arthritis patients. The binding affinities of melittin to mutant IKKs, was reduced, and the inhibitory effect of melittin on IKK and NF-kappaB activities, and NO and PGE(2) generation were abrogated by the reducing agents or in Raw 264.7 transfected with mutant plasmid IKKalpha (C178A) or IKKbeta (C179A). These results suggest that melittin binding to the sulfhydryl group of IKKs resulted in reduced IKK activities, IkappaB release, NF-kappaB activity and generation of inflammatory mediators, indicating that IKKs may be also anti-inflammatory targets of BV.

Mutant presenilin 2 causes abnormality in the brain lipid profile in the development of Alzheimer's disease.

Mutation in the presenilin 2 (PS2mt) is known to be one of factors involved in the development of Alzheimer's disease (AD). It was recently revealed that an abnormality of lipid metabolism is a phenomenon occurring in AD. Therefore, the aim of this study was to investigate the potential relationship between the mutation of PS2 and alterations of the lipid profile within the brain. The results showed there increases in the levels of cholesterol, low density lipoprotein and triglyceride, but a decrease in the level of high density lipoprotein in brain tissues expressing mutant PS2. These findings indicated that PS2mt is involved in the abnormalities of the lipid profile, which could cause or result in the development of AD.

Dietary vitamin E protects the fathead minnow, Pimephales promelas, against noise exposure.

The fathead minnow (Pimephales promelas) was employed to examine if dietary vitamin E supplementation could protect the inner ear from the deleterious effects of noise. Fish were fed one of the three experimental diets containing either: (1) low vitamin E content (14.5 mg/kg diet as alpha-tocopheryl acetate), (2) an adequate amount of vitamin E (50 mg/kg), or (3) high vitamin E content (450 mg/kg). After 4 weeks on the diet, fish were exposed to either 2 or 24 h of intense white noise (142 dB re: 1 microPa, bandwidth 0.3-4.0 kHz). Auditory thresholds were measured, using the auditory brainstem response (ABR) technique, within 0.5 days following noise exposure or within a recovery period of 1.5 days. Additionally, liver samples were analyzed for vitamin E content. Increased vitamin E supplementation was dose-dependently associated with a reduction in statistically significant threshold shifts after noise exposure and an enhancement of recovery (i.e., more complete recovery over a shorter period) for fish exposed to either 2 or 24 h of noise. The results obtained suggest that dietary vitamin E affords protection against noise exposure in a cyprinid fish.