PPARγ; NF-κB

4-O-Mmethyl honokiol is a natural neolignan isolated from Magnolia officinalis, which can serve as a PPARγ agonist and decrease NF-κB activity. 4-O-methylhonokiol (20 μM) boosts PPARγ expression, transcriptional and DNA-binding activity, and nuclear translocation in prostate PC-3 and LNCap cells. 4-O-Mmethyl honokiol (0-30 μM) suppresses the growth of LNCaP and PC-3 cancer cells, producing G0/G1 phase arrest and promoting death, an action that can be reversed by PPARγ antagonists. 4-O-methylhonokiol can decrease NF-κB activity and cancer cell development, but this impact and its activation of PPARγ can be abolished by knocking down p21 [1]. 4-O-methylhonokiol (0.5, 1, and 2 μM) decreases LPS-induced release of NO, PGE2, ROS, TNF-α, and IL-1β in cultured astrocytes, as well as in cultured astrocytes and microglia. Amyloid synthesis in glial BV-2 cells [2] .

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In in vitro study, we also found that 4-O-methylhonokio suppressed the expression of iNOS and COX-2 as well as the production of reactive oxygen species, nitric oxide, prostaglandin E2, tumor necrosis factor-α, and interleukin-1β in the LPS-stimulated cultured astrocytes. 4-O-methylhonokio also inhibited transcriptional and DNA binding activity of NF-κB via inhibition of IκB degradation as well as p50 and p65 translocation into nucleus of the brain and cultured astrocytes. Consistent with the inhibitory effect on neuroinflammation, 4-O-methylhonokiol inhibited LPS-induced Aβ1-42 generation, β- and γ-secretase activities, and expression of amyloid precursor protein (APP), BACE1 and C99 as well as activation of astrocytes and neuronal cell death in the brain, in cultured astrocytes and in microglial BV-2 cells [2].

In SW620 and PC3 xenograft models, 4-O-methylhonokiol (40 or 80 mg/kg, intraperitoneally administered daily for 4 weeks) reduced the growth of SW620 and PC3 tumors. In tumor tissues, 4-O-methylhonokio dramatically raises p21 and PPARγ expression [1]. 4-O-methylhonokiol (0.5 or 1 mg/kg/day for 3 weeks) can prevent LPS-induced COX-2 and iNOS production in mice and dramatically alleviate LPS-induced memory impairment. In the brains of mice treated with lipopolysaccharide (LPS), 4-O-methyl honokiol also demonstrates inhibitory effect against Aβ1-42 formation and activates microglia and astrocytes [2].

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MH/4-O-methylhonokio inhibited the growth of SW620 and PC3 tumours in an in vivo xenograft model [1]
In PC3 xenograft studies, MH/4-O-methylhonokio was administered i.p. daily for 4 weeks to mice with tumours ranging from 100 to 300 mm3 in volume. On day 28, the final tumour weight was recorded. Tumour volumes in mice treated with MH at 40 and 80 mg·kg−1, and cisplatin at 10 mg·kg−1 were 71.0, 57.7 and 46.6% of the control group in PC3 tumour xenografts, respectively. Tumour weight in mice treated with MH/4-O-methylhonokio at 40 and 80 mg·kg−1 and cisplatin at 10 mg·kg−1 were 40.1, 30.9 and 22.1% of the control group in PC3 tumour xenografts, respectively (Figure 5A). Immunohistochemical analysis of tumour sections by H&E, and proliferation antigens against PCNA and Ki67 staining revealed that both 40 and 80 mg·kg−1 dose-dependently inhibited the growth of the tumour cells (Figure 5B). In addition, there was a trend towards decreased intensity of nuclear staining of p65 and p50 in MH-treated tumour tissue (Figure 5B). Moreover, PPARγ immunoreactivity against anti-PPARγ was also more intense in the tumours treated with MH than in untreated tumour tissues. Similar to its inhibitory effect in vitro, MH enhanced the DNA binding activity of PPARγ, but inhibited NF-κB activity in tumour tissue (Figure 5C). MH increased the expression of bax and cleaved caspases-3, but decreased the expression of bcl-2 in tumour tissue (Figure 5D). Immunohistochemical analysis also showed that the expression of cleaved caspases-3 positive cells was considerably increased in the MH-treated tumour tissue compared with those of the control group. Apoptotic cell death was also significantly increased in the MH-treated tumour tissue (Figure 5B). Moreover, MH significantly increased the expression of p21 and PPARγ in the tumour tissues (Figure 5B–D).

Cell growth assay [1]
Cells (5 × 104 cells per well) were plated onto 24-well plates. The cell growth inhibitory effect of 4-O-methylhonokio/MH was evaluated in cells treated with MH (0–30 μM) for 0–72 h, using an excluded trypan blue assay.

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Transfection and assay of luciferase activity [1]
Cells (1 × 105 cells per well) were plated in 24-well plates and transiently transfected with pNF-κB-Luc plasmid (5× NF-κB; Stratagene, La Jolla, CA, USA) or plasmid pFA-GAL4-PPARγ, using a mixture of plasmid and lipofectamine PLUS in OPTI-MEN according to manufacturer's specification. The transfected cells were treated with 4-O-methylhonokioMH in the absence (for assay of PPARγ activity) or presence (for assay of NF-κB activity) of TNF-α (10 ng·mL−1) for 8 h. To induce NF-κB luciferase activity, we co-treated the cells with TNF-α (10 ng·mL−1). Luciferase activity was measured by using the luciferase assay kit.

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Cell-cycle analysis by flow cytometry [1]
Subconfluent cells were treated with 4-O-methylhonokioMH (0–30 μM) in culture medium for 0–72 h. The analysis methods are as described elsewhere (Ban et al., 2009a).

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Pull-down assays [1]
MH/4-O-methylhonokio bead conjugation was prepared and a pull-down assay conducted as described previously (Shim et al., 2008). MH was conjugated with cyanogen bromide (CNBr)-activated Sepharose 4B. Briefly, MH (1 mg) was dissolved in 500 μL of coupling buffer (0.1 M NaHCO3 and 0.5 M NaCl, pH 6.0). The CNBr-activated Sepharose 4B was swelled and washed with 1 mM HCl, then washed with the coupling buffer. CNBr-activated Sepharose 4B beads were added to the MH-containing coupling buffer and incubated at 4°C for 24 h. The MH-conjugated Sepharose 4B was washed with three cycles of alternating pH wash buffers (buffer 1, 0.1 M acetate and 0.5 M NaCl, pH 4.0; buffer 2, 0.1 M Tris-HCl and 0.5 M NaCl, pH 8.0). MH-conjugated beads were then equilibrated with binding buffer (0.05 M Tris-HCl and 0.15 M NaCl, pH 7.5). The control unconjugated CNBr-activated Sepharose 4B beads were prepared as described earlier in the absence of MH. For the pull-down assay, PPARγ proteins or cell lysate from PC-3 prostate cancer cells were incubated with MH-Sepharose 4B beads in reaction buffer (50 mM Tris, pH 7.5, 5 mM EDTA, 150 mM NaCl, 1 mM dithiothreitol, 0.01% Nonidet P-40, 2 μg·mL−1 BSA, 0.02 mM PMSF, 1 × proteinase inhibitor). The beads were washed five times with buffer (50 mM Tris, pH 7.5, 5 mM EDTA, 150 mM NaCl, 1 mM dithiothreitol, 0.01% Nonidet P-40, 0.02 mM PMSF), and proteins bound to the beads were analysed by immunoblotting with the PPARγ antibodies or cell lysates.

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The cultured cells were treated simultaneously with LPS (1 μg/ml) and several concentrations (0.5, 1, 2 μM) of 4-O-methylhonokio dissolved in 0.05% ethanol, and the cells were harvested after 24 h. Western blotting was performed, and Aβ level and secretases activities were determined.[2]

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Nitric oxide and PGE2 determination [2]
Astrocytes were grown in 96-well plates and then incubated with or without LPS (1 μg/ml) in the absence or presence of various concentrations of 4-O-methylhonokio for 24 h. The nitrite accumulation in the supernatant was assessed by Griess reaction. Each 50 μl of culture supernatant was mixed with an equal volume of Griess reagent [0.1% N-(1-naphthyl)-ethylenediamine, 1% sulfanilamide in 5% phosphoric acid] and incubated at room temperature for 10 min. The absorbance at 540 nm was measured in a microplate absorbance reader, and a series of known concentrations of sodium nitrite was used as a standard. In the cultured supernatant of astrocytes, PGE2 concentration was determined using a PGE2 Enzyme Immunometric Assay (EIA) kit (R&D systems), according to the manufacturer's instructions.

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Reactive oxygen species (ROS) generation [2]
To monitor intracellular accumulation of ROS in cultured astrocytes, the fluorescent probe 2',7'-dichlorofluorescein diacetate (DCF-DA) was used. Following treatment with LPS (1 μg/ml) for 24 h in the presence or absence of 4-O-methylhonokiol (0.5, 1, 2 μM), the cells were washed in modified Kreb's buffer containing 145 mM NaCl, 5 mM potassium chloride (KCl), 1 mM magnesium chloride (MgCl2), 1 mM calcium chloride (CaCl2), 4 mM sodium hydrogen carbonate (NaHCO3), 5.5 mM glucose, 10 mM HEPES, pH 7.4. The cell suspension was transferred into plastic tubes. Measurement was started by an injection of 5 μM DCF-DA in the dark. After 30 min of incubation at 37°C, generation was determined by Fluorometer at Ex = 485 and Em = 538 nm.

Antitumour activity study in in vivo xenograft animal model [1]
All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (Kilkenny et al., 2010; McGrath et al., 2010). SW620 and PC3 cells were injected s.c. (1 × 107 cells in 0.1 mL PBS per animal) into the lower right flanks of mice. After 20 days, when the tumours had reached an average volume of 300–400 mm3 or about 50 mm3 (for prostate), the tumour-bearing nude mice were i.p. injected with MH/4-O-methylhonokio (40 and 80 mg·kg−1 dissolved in 0.1% DMSO) twice per week for 3 weeks. Cisplatin (10 mg·kg−1) was also i.p. injected once a week as a positive control. The group treated with 0.1% DMSO was designated as the control. The tumour volumes were measured with vernier calipers and calculated by the following formula: (A × B2)/2, where A is the larger and B is the smaller of the two dimensions.

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Dosage (0.5 and 1 mg/kg/day) of 4-O-methylhonokio in this study was used by referring to our previous studies. 4-O-methylhonokio (15 and 30 μg/mouse) was added to drinking water (5 ml of average water consumption of mouse per day) and mice were allowed access for 3 weeks ad libitum before induction of memory impairment as shown in Figure 1B.[2]

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Lipopolysaccharide-induced memory impairment mouse model [2]
All mice were housed in a room that was automatically maintained at 21-25°C and relative humidity (45-65%) with a controlled light-dark cycle. Several researchers reported that repeated i.p. injection of LPS induced cognitive impairment like AD in mice. We therefore used this method as an AD mice model. The LPS (final concentration of 0.1 mg/ml) was dissolved, and aliquots in saline were stored at -20°C until use. The i.p. injection (250 μg/kg) of LPS or control (saline) was daily administered for 7 days. Subsequently, the behavioral tests of learning and memory capacity were assessed using two separate tests (water maze and passive avoidance test). One day interval was given between tests for adaptation of new circumstances as shown in Figure 1B.

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Water maze test [2]
The water maze test is also a widely accepted method for memory test, and we performed this test as described by Morris et al. Maze testing was performed by the SMART-CS program and equipment. A circular plastic pool (height: 35 cm, diameter: 100 cm) was filled with milky water kept at 22-25°C. An escape platform (height: 14.5 cm, diameter: 4.5 cm) was submerged 0.5-1 cm below the surface of the water in position. On training trials, the mice were placed in a pool of water and allowed to remain on the platform for 10 s and were then returned to the home cage during the second-trial interval. The mice that did not find the platform within 60 s were placed on the platform for 10 s at the end of trial. They were allowed to swim until they sought the escape platform. These trials were performed in single platform and in three starting positions of rotational starting. Escape latency, escape distance, swimming speed and swimming pattern of each mouse was monitored by a camera above the center of the pool connected to a SMART-LD program.

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Probe test [2]
A probe trial in order to assess memory consolidation was performed 24 h after the 5-day acquisition tests. In this trial, the platform was removed from the tank, and the mice were allowed to swim freely. For these tests, the percentage time in the target quadrant and target site crossings within 60 s was recorded. The time spent in the target quadrant is taken to indicate the degree of memory consolidation that has taken place after learning. The time spent in the target quadrant was used as a measure of spatial memory. Swimming pattern of each mouse was monitored by a camera above the center of the pool connected to a SMART-LD program described above.

[1]. 4-O-methylhonokiol, a PPARγ agonist, inhibits prostate tumour growth: p21-mediated suppression of NF-κB activity. Br J Pharmacol. 2013 Mar;168(5):1133-45.

[2]. Inhibitory effect of 4-O-methylhonokiol on lipopolysaccharide-induced neuroinflammation, amyloidogenesis and memory impairment via inhibition of nuclear factor-kappaB in vitro and in vivo models. J Neuroinflammation. 2012 Feb 19;9:35.

4-O-Methylhonokiol has been reported in Magnolia officinalis, Magnolia virginiana, and Magnolia obovata with data available.

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Background and purpose: The effects of 4-O-methylhonokio (MH), a constituent of Magnolia officinalis, were investigated on human prostate cancer cells and its mechanism of action elucidated.
Experimental approach: The anti-cancer effects of MH were examined in prostate cancer and normal cells. The effects were validated in vivo using a mouse xenograft model.
Key results: MH increased the expression of PPARγ in prostate PC-3 and LNCap cells. The pull-down assay and molecular docking study indicated that MH directly binds to PPARγ. MH also increased transcriptional activity of PPARγ but decreased NF-κB activity. MH inhibited the growth of human prostate cancer cells, an effect attenuated by the PPARγ antagonist GW9662. MH induced apoptotic cell death and this was related to G(0) -G(1) phase cell cycle arrest. MH increased the expression of the cell cycle regulator p21, and apoptotic proteins, whereas it decreased phosphorylation of Rb and anti-apoptotic proteins. Transfection of PC3 cells with p21 siRNA or a p21 mutant plasmid on the cyclin D1/ cycline-dependent kinase 4 binding site abolished the effects of MH on cell growth, cell viability and related protein expression. In the animal studies, MH inhibited tumour growth, NF-κB activity and expression of anti-apoptotic proteins, whereas it increased the transcriptional activity and expression of PPARγ, and the expression of apoptotic proteins and p21 in tumour tissues.
Conclusions and implication: MH inhibits growth of human prostate cancer cells through activation of PPARγ, suppression of NF-κB and arrest of the cell cycle. Thus, MH might be a useful tool for treatment of prostate cancer. [1]

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Background: Neuroinflammation is important in the pathogenesis and progression of Alzheimer disease (AD). Previously, we demonstrated that lipopolysaccharide (LPS)-induced neuroinflammation caused memory impairments. In the present study, we investigated the possible preventive effects of 4-O-methylhonokio, a constituent of Magnolia officinalis, on memory deficiency caused by LPS, along with the underlying mechanisms. Methods: We investigated whether 4-O-methylhonokiol (0.5 and 1 mg/kg in 0.05% ethanol) prevents memory dysfunction and amyloidogenesis on AD model mice by intraperitoneal LPS (250 μg/kg daily 7 times) injection. In addition, LPS-treated cultured astrocytes and microglial BV-2 cells were investigated for anti-neuroinflammatory and anti-amyloidogenic effect of 4-O-methylhonkiol (0.5, 1 and 2 μM).
Results: Oral administration of 4-O-methylhonokiol ameliorated LPS-induced memory impairment in a dose-dependent manner. In addition, 4-O-methylhonokiol prevented the LPS-induced expression of inflammatory proteins; inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) as well as activation of astrocytes (expression of glial fibrillary acidic protein; GFAP) in the brain. In in vitro study, we also found that 4-O-methylhonokiol suppressed the expression of iNOS and COX-2 as well as the production of reactive oxygen species, nitric oxide, prostaglandin E2, tumor necrosis factor-α, and interleukin-1β in the LPS-stimulated cultured astrocytes. 4-O-methylhonokiol also inhibited transcriptional and DNA binding activity of NF-κB via inhibition of IκB degradation as well as p50 and p65 translocation into nucleus of the brain and cultured astrocytes. Consistent with the inhibitory effect on neuroinflammation, 4-O-methylhonokiol inhibited LPS-induced Aβ1-42 generation, β- and γ-secretase activities, and expression of amyloid precursor protein (APP), BACE1 and C99 as well as activation of astrocytes and neuronal cell death in the brain, in cultured astrocytes and in microglial BV-2 cells.
Conclusion: These results suggest that 4-O-methylhonokiol inhibits LPS-induced amyloidogenesis via anti-inflammatory mechanisms. Thus, 4-O-methylhonokiol can be a useful agent against neuroinflammation-associated development or the progression of AD.[2]

C19H20O2

280.3609

280.146

C, 81.40; H, 7.19; O, 11.41

68592-15-4

155160

Orange to red viscous liquid

1.054g/cm3

396.5ºC at 760 mmHg

176.2ºC

1.569

4.524

1

2

6

21

339

0

O(C([H])([H])[H])C1C([H])=C([H])C(=C([H])C=1C([H])([H])C([H])=C([H])[H])C1=C(C([H])=C([H])C(C([H])([H])C([H])=C([H])[H])=C1[H])O[H]

OQFHJKZVOALSPV-UHFFFAOYSA-N

InChI=1S/C19H20O2/c1-4-6-14-8-10-18(20)17(12-14)15-9-11-19(21-3)16(13-15)7-5-2/h4-5,8-13,20H,1-2,6-7H2,3H3

2-(4-methoxy-3-prop-2-enylphenyl)-4-prop-2-enylphenol

4-O-Methylhonokiol; 68592-15-4; 4-O-Methyl honokiol; METHYLHONOKIOL; 4-methoxyhonokiol;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~356.68 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.92 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.92 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)