In glioblastoma cells (LN428, U87MG, LN2308, U251), the primary target of pinoresinol is the cellular FLICE-inhibitory protein long isoform (cFLIP_L) and survivin, leading to their downregulation at the post-translational level. Pinoresinol reduces cFLIP_L and survivin protein levels via proteasome-mediated degradation, thereby facilitating TRAIL DISC formation and caspase-8 activation. No IC50, Ki, EC50, or DC50 values for direct binding to cFLIP or survivin are reported. In ants (Formica exsectoides), pinoresinol acts as a feeding deterrent, but specific molecular targets are not identified. [1][2]

Pinoresinol induces TRAIL-resistant glioblastoma cells to construct death-inducing signaling complexes, which in turn activates the caspase-8-dependent apoptotic cascade [2].

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In TRAIL-resistant glioblastoma cells (LN428, U87MG, LN2308, U251), treatment with non-toxic doses of pinoresinol (0.2-1 μM) in combination with TRAIL (50 ng/mL) induced rapid apoptosis and caspase activation. Cell death began to appear from 9 hours after co-treatment and rapidly increased up to 24 hours (Fig. 2A). Pinoresinol alone (up to 1 μM for 24 h) showed only marginal growth inhibitory effects (<5% cell death). Combination treatment with 0.5 μM pinoresinol and 50 ng/mL TRAIL resulted in drastic increase in cell death (approx. 80-90% at 24 h) (Fig. 1D). The sensitization was observed in multiple glioblastoma cell lines (U87MG, LN2308, LN428, U251) but not in normal primary astrocytes (Fig. 2B). The cell death was caspase-dependent, as pre-treatment with pan-caspase inhibitor z-VAD-FMK (20 μM) or caspase-8 inhibitor z-IETD-fmk (50 μM) completely abrogated the cytotoxicity, whereas necrostatin-1 (30 μM, inhibitor of programmed necrosis) failed to protect (Fig. 2C,D). Annexin V/PI staining showed that pinoresinol plus TRAIL drastically increased the early apoptotic population (Annexin V+), prevented by z-VAD-FMK (Fig. 2E). Western blot analysis revealed that pinoresinol plus TRAIL caused activation of caspase-8 and caspase-3, and PARP cleavage from 6 h onwards; this was inhibited by z-IETD-fmk (Fig. 2F). Pinoresinol (0.5 μM) decreased protein levels of cFLIP_L and survivin in a time-dependent manner (starting at 4-6 h) without affecting death receptors (DR4/5), FADD, RIP1, TRAF2, cIAP1/2, XIAP, Bcl-x_SL, or Bid. In HT-29 cells, pinoresinol also downregulated cFLIP_S. mRNA levels of cFLIP_L and survivin were unchanged by pinoresinol (0.5-1 μM) as determined by RT-PCR (Fig. 4C). Pre-treatment with proteasome inhibitor MG132 (10 μM) prevented pinoresinol-induced downregulation of cFLIP_L and survivin (Fig. 4D). Overexpression of wild-type cFLIP_L significantly decreased cell death and caspase cascade activation induced by pinoresinol plus TRAIL; a cFLIP_L mutant (K167/195R) more profoundly abrogated apoptosis. Overexpression of survivin did not affect cell death (Fig. 5A,B). Immunoprecipitation assays showed that in LN428 cells, TRAIL alone led to recruitment of cFLIP_L to the DISC with weak detection of FADD and caspase-8/10; pre-treatment with pinoresinol (0.5 μM) increased DISC formation and procaspase-8/10 processing, decreased DISC-bound cFLIP_L, and led to complete processing of procaspase-8 to active p18 subunit (Fig. 5C). Pinoresinol did not affect NF-κB transcriptional activity at 0.5 μM (TNF or TRAIL induced activity was significantly decreased by pinoresinol pre-treatment, but overexpression of IκBα super-repressor or TPCA-1 did not alter TRAIL sensitization) (Fig. 3A-C). Pinoresinol sensitized TRAIL-induced cell death in both wild-type and p53-null HCT116 cells to a similar extent, and did not induce p53 or p21 (Fig. 3D,E). In cell-free in vitro transcription/translation assays, pinoresinol (0.1-1 μM) suppressed GFP production in a dose-dependent manner, similar to cycloheximide (CHX). Using in vitro synthesized EGFP mRNA, 1 μM pinoresinol completely interfered with EGFP protein production, comparable to 10 μM CHX (Fig. 6C,D). Pinoresinol (0.5 μM) also decreased cFLIP_L and survivin protein levels with similar kinetics to CHX (10 μM), and combined treatment did not accelerate downregulation (Fig. 6B). Pinoresinol treatment (0.5 μM for 4 h) in the presence of MG132 led to lower levels of ubiquitinated cFLIP_L compared to MG132 alone (Fig. 6A). In ants, pinoresinol applied topically to fruit flies at dosages of 0.001, 0.01, 0.1, 1.0, and 5.0 μg per fly was tested for deterrency. At ≥1 μg per fly, ants showed reduced carrying persistence (<3 min) and increased preening behavior (self-cleaning). For pinoresinol at 1 μg/fly, 100% of ants carried flies for less than 3 min and 100% engaged in preening; at 5 μg/fly, similar effects. The effect was significant (P < 0.001, χ²=27.45, df=5, n=66 for carrying; P<0.001, χ²=29.69, df=2, n=66 for preening). By comparison, mayolene-16 showed deterrency only at 5 μg/fly (carrying persistence: P<0.001, χ²=11.0, df=1, n=22). [1][2]

In insect behavioral assays using ants (Formica exsectoides), pinoresinol demonstrated feeding deterrent activity. Individual worker ants were offered single fruit flies (Drosophila melanogaster, vestigial wing strain) treated topically with pinoresinol in methanol solution (1 μL) at dosages of 0.001, 0.01, 0.1, 1.0, and 5.0 μg per fly. Control flies received 1 μL methanol. Ants that received pinoresinol-treated flies at ≥1 μg per fly showed a decreased tendency to carry the flies for more than 3 minutes and increased preening (antennal wiping with forelegs). At 1 μg/fly, 0% of ants carried flies for >3 min (vs. 100% in control), and 100% engaged in preening (vs. 0% in control). The effect was dose-dependent. Pinoresinol was more potent than mayolene-16, which only showed deterrency at 5 μg/fly. [1]

Human glioblastoma cells (LN428, U87MG, LN2308, U251MG) were cultured in DMEM with 10% FBS, 2 mmol/L glutamine, and 100 U/mL penicillin/streptomycin. Normal primary astrocytes were prepared from neonatal rats and cultured in MEM with 10% FBS, 2 mmol/L glutamine, and 100 U/mL penicillin/streptomycin. For cell viability assessment, cells were seeded in 96-well plates and treated with pinoresinol (0.2-1 μM) for 30 min followed by TRAIL (50 ng/mL) for 24 h. Cell death was quantified using Cell Titer-glo Luminescent Cell Viability Assay kit (luminescence measured by plate reader). Viability rates calculated as (1 − treated/control) × 100%. Representative images were taken with an inverted microscope. For Annexin V/PI staining, cells were harvested after treatment, double-stained with FITC-conjugated annexin V and propidium iodide in HEPES buffer (pH 7.4) according to kit instructions, and analyzed by flow cytometry (FACScan). For cell cycle analysis, cells were fixed in 70% ethanol, washed with PBS, and stained with PI solution (100 μg/mL PI with 50 μg/mL RNase) for 30 min at room temperature, then analyzed by flow cytometry. For immunoblotting, cells were lysed in ice-cold M2 buffer (20 mM Tris pH 7.6, 0.5% NP-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 2 mM DTT, 0.5 mM PMSF, 20 mM β-glycerol phosphate, 1 mM sodium vanadate, 1 µg/mL leupeptin). Lysates were resolved on 8-12% SDS-PAGE, transferred to PVDF membranes, incubated with primary antibodies (1:1000 dilution) and secondary antibodies (1:2000 dilution), and visualized by enhanced chemiluminescence. For immunoprecipitation, cell lysates were incubated with anti-caspase-8 antibody and protein A-agarose beads at 4°C overnight; immunoprecipitants washed three times with M2 buffer and analyzed by immunoblotting. For RT-PCR, total RNA isolated with ReliaPrep RNA Miniprep kit, cDNA prepared using M-MLV reverse transcriptase. PCR primers: cFLIP_L (5′-CTGGTTGCCCCAGATCAACT-3′ and 5′-CCCAGGGAAGTGAAGGTGTC-3′); survivin (5′-TGACGACCCCATAGAGGAACA-3′ and 5′-TCAATCCATGGCAGCCAGC-3′); GAPDH (5′-CACCATCTTCCAGGAGCGAG-3′ and 5′-GATGGCATGGACTGTGGTCA-3′). For transfection and luciferase assay, LN428 cells were transfected with p2xNF-κB-Luc and pRSV-β-galactosidase using Lipofectamine, then treated with TNF (30 ng/mL) or TRAIL (50 ng/mL) for 6 h; luciferase activity measured with kit and normalized to β-galactosidase activity. For cFLIP_L overexpression, cells were transfected with pCA-flag-cFLIP_L (wild-type or K167/195R mutant) or pCA-flag-survivin for 24 h, then treated with TRAIL (50 ng/mL) plus pinoresinol (1 μM) for 12 h. [2]

For ant deterrency assays, Formica exsectoides ants were collected from a natural site and housed in a large aquarium. Fruit flies (Drosophila melanogaster, vestigial wing strain) from a laboratory culture were immobilized by cooling. Pinoresinol was dissolved in methanol and applied topically to the flies at dosages of 0.001, 0.01, 0.1, 1.0, and 5.0 μg in 1 μL volume using a micropipette. Mayolene-16 was tested at 0.1 and 5 μg/fly. Control flies received 1 μL methanol. After application, flies were allowed 10-30 minutes for methanol evaporation (flies regained full mobility). Each test consisted of offering a single treated fly to an individual ant confined in a Petri dish, and events were videotaped for 10 minutes. Eleven tests were performed per dosage and for controls, with fresh ants and flies each time. Parameters recorded: (i) length of time ants carried the fly in their mandibles, and (ii) whether ants engaged in self-cleaning (preening) behavior. [1]

In glioblastoma cells, pinoresinol alone at concentrations up to 1 μM for 24 hours showed only marginal growth inhibitory effects (<5% cell death), indicating non-toxic doses. In normal primary astrocytes, pinoresinol (0.5 μM) plus TRAIL (50 ng/mL) did not induce cell death, suggesting selectivity for cancer cells. [1][2]

[1]. Pinoresinol: A lignol of plant origin serving for defense in a caterpillar. Proc Natl Acad Sci U S A. 2006 Oct 17;103(42):15497-501.

[2]. Accelerated degradation of cFLIPL and sensitization of the TRAIL DISC-mediated apoptotic cascade by pinoresinol, a lignan isolated from Rubia philippinensis. Sci Rep. 2019 Sep 18;9(1):13505.

(+)-Pinoresinol is the enantiomer of pinoresinol, possessing the (+)-1S,3aR,4S,6aR- configuration. It has hypoglycemic effects and is a plant metabolite and phytoestrogen. Pinoresinol has been reported to be found in tea (Camellia sinensis), Disynaphia multicrenulata, and other organisms with relevant data. See also: Acai berry pulp (partial).

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Pinoresinol is a lignan dimer of coniferyl alcohol, widely distributed in plants. In Pieris rapae caterpillars, it is sequestered from the food plant Brassica oleracea; the caterpillar secretes it as part of a defensive mixture (mayolenes:pinoresinol ≈ 96:4). The absolute configuration in the caterpillar was determined to be (-)-pinoresinol with 94% enantiomeric purity. Feeding experiments on an artificial wheat-germ-based diet (cabbage-free) showed that secretion lacked pinoresinol; supplementation with (±)-pinoresinol (0.1% of dry ingredients) resulted in its presence in the secretion, indicating sequestration. Cabbage leaves did not contain free pinoresinol or simple glycosides, but acid hydrolysis of cabbage extract yielded pinoresinol (~0.1 mg from two plants) along with epi-pinoresinol, suggesting polymer-bound forms. In cancer biology, pinoresinol acts as a TRAIL sensitizer by inhibiting de novo protein synthesis (similar to cycloheximide), leading to rapid turnover of short-lived anti-apoptotic proteins cFLIP_L and survivin via proteasomal degradation, thereby facilitating DISC-mediated caspase-8 activation and apoptosis in TRAIL-resistant glioblastoma cells. It does not act via NF-κB or p53 pathways. Pinoresinol also induced G2/M arrest (increased G2 population) in LN428 cells (Supplementary Fig. 34). Additionally, pinoresinol has been reported to suppress efflux via P-glycoprotein (P-gp) and exhibit anti-inflammatory properties through NF-κB inhibition at higher concentrations (≥10 μM). [1][2]

C20H22O6

358.3851

358.141

C, 67.03; H, 6.19; O, 26.78

487-36-5

73399

White to off-white solid powder

1.3±0.1 g/cm3

556.5±50.0 °C at 760 mmHg

121 °C

290.4±30.1 °C

0.0±1.6 mmHg at 25°C

1.598

1.54

2

6

4

26

431

4

COC1=C(C=CC(=C1)[C@@H]2[C@H]3CO[C@@H]([C@H]3CO2)C4=CC(=C(C=C4)O)OC)O

HGXBRUKMWQGOIE-AFHBHXEDSA-N

InChI=1S/C20H22O6/c1-23-17-7-11(3-5-15(17)21)19-13-9-26-20(14(13)10-25-19)12-4-6-16(22)18(8-12)24-2/h3-8,13-14,19-22H,9-10H2,1-2H3/t13-,14-,19+,20+/m0/s1

4-[(3S,3aR,6S,6aR)-6-(4-hydroxy-3-methoxyphenyl)-1,3,3a,4,6,6a-hexahydrofuro[3,4-c]furan-3-yl]-2-methoxyphenol

NSC 35444; (+)-Pinoresinol

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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 (~279.03 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.98 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.98 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.)