Natural biflavonoid; matrix metalloproteinases (MMPs); pre-mRNA splicing

Hinokiflavone modulates splicing in cells. Hinokiflavone prevents assembly of the spliceosome B complex. Hinokiflavone blocks cell cycle progression. Hinokiflavone alters nuclear organization of a subset of splicing factors.Hinokiflavone promotes nuclear relocalization of SUMO.[1]

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Hinokiflavone reduced the proliferation, migration, and invasion and promoted the apoptosis in colorectal tumor cells in vitro. [2]

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Hinokiflavone inhibited the activity of ClpP of MRSA strain USA300 with an IC50 of 34.36 μg/mL. Further assays showed that hinokiflavone reduced the virulence of S. aureus by inhibiting multiple virulence factors expression. Results obtained from cellular thermal transfer assay (CETSA), thermal shift assay (TSA), local surface plasmon resonance (LSPR) and molecular docking (MD) assay enunciated that hinokiflavone directly bonded to ClpP with confirmed docking sites, including SER-22, LYS-26 and ARG-28. [3] Hinokiflavone increases levels of SUMOylated proteins. Hinokiflavone inhibits SENP1 activity. SUMOylated splicing factors accumulate in the insoluble fraction in cell extracts treated with hinokiflavone.

Treatment of hinokiflavone at a tolerable and safe dose (50 mg/kg) significantly suppressed tumor growth in mice bearing CT26 tumors by reducing tumor proliferation and metastasis and inducing apoptosis. Mechanically, treatment of hinokiflavone induced apoptosis by loss of mitochondrial transmembrane potential and increased reactive oxygen species generation. Conclusions: Hinokiflavone suppressed colorectal tumor cell proliferation, induced apoptosis via the reactive oxygen species-mitochondria-mediated apoptotic pathway, and inhibited tumor cell migration and invasion. Antitumor activity of hinokiflavone was also validated in mice model without observed toxicity. Our findings suggested that the plant-derived hinokiflavone could be used as an antitumor agent against colorectal cancer[2].

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In vivo, the evaluation of anti-infective activity showed that hinokiflavone in combination with vancomycin effectively protected mice from MRSA-induced fatal pneumonia, which was more potent than vancomycin alone. As mentioned above, hinokiflavone, as an inhibitor of ClpP, could be further developed into a promising adjuvant against S. aureus infections[3].

Cellular thermal shift assay[1]
To 1 ml of HeLa NE, either 50 µl of DMSO, or 50 µl of 10 mM hinokiflavone, was added and incubated at RT for 20 min. DMSO as well as hinokiflavone treated NE samples were then split into seven 100 µl aliquots. Each sample was incubated at a specific temperature (30°C, 33°C, 37°C, 40°C, 43°C, 47°C or 50°C) for 3 min, which was followed by 3 min incubation at RT. After ultracentrifugation at 35,000 rpm, 4°C for 20 min using a Optima MAX ultracentrifuge and a TLA 120.2 rotor, the supernatant was transferred to a new tube containing 25 µl 4x LDS buffer and boiled for 10 min at 95°C. The samples were subjected to western blot analysis with the indicated primary antibodies.

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Effect of Hinokiflavone on the protease activity of ClpP. [3]
A fluorescence resonance energy transfer (FRET)-based assay was used to screen for ClpP inhibitors based on the ability of ClpP to specifically cleave the fluorescent peptide substrate Suc-LY-AMC. The difference in RFU was used to define the enzymatic activity of ClpP and calculated the relative inhibitory activity. The compound was considered a potential inhibitor when the inhibitory activity was greater than 60%. The 100 μL reaction system consisting of 1 μΜ ClpP protein, various concentrations of hinokiflavone (0.5 to 256 μg/mL) and ClpP buffer (100 mM HEPES, 100 mM NaCl, pH = 7.0) were added to 96-well plates and incubated for 30 min, followed by the addition of the fluorescent substrate Suc-LY-AMC. After continuing incubation for 20 min, the activity of ClpP was determined by measuring excitation at 360 nm and emission at 465 nm using a microplate reader. Then the IC50 value of hinokiflavone was calculated. The reaction system containing an equal volume of DMSO was used as a negative control.

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Thermal shift assay (TSA). [3]
To determine the interaction between hinokiflavone and ClpP, a TSA assay was performed. ClpP protein (final concentration, 2 μM), SYPRO Orange, hinokiflavone and TSA buffer (150 mM NaCl, 10 mM HEPES, pH = 7.5) were added to a 96-well optical PCR plate. The mixture was heated at the rate of 1°C/min from 25 to 90°C by a real-time PCR instrument. This interaction was verified by observing the Tm shift after drug treatment. Tm was estimated as the temperature corresponding to 50% denaturation of the protein.

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Cellular thermal shift assay (CETSA). [3]
E. coli BL21(DE3) containing pET28a-ClpP plasmid was incubated until OD600 of 0.8, and 0.5 mM IPTG was added to induce ClpP protein expression. The supernatant was incubated with hinokiflavone and an equal volume of DMSO at 37°C for 1 h through centrifugation at 18,000 g for 20 min at 4°C. The reaction mixture was heated for 5 min at a temperature gradient of 25.0, 45.0, 51.2, 56.2, 61.4, and 65.0°C, respectively. It is worth noting that the mixture should be placed into ice water for 3 min immediately. Then gently centrifuged at 18,000g for 20 min to yield a supernatant and analyzed by SDS-PAGE. The samples were incubated with Coomassie brilliant blue G-250 stain, and the relative intensities of the indicated proteins were visualized using ImageJ software.

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Localized surface plasmon resonance (LSPR). [3]
The interaction between hinokiflavone and ClpP was performed by OpenSPR instruments. After a stable baseline is reached, PBS containing 1% DMSO (pH = 7.4) is first applied to enable activation of the chip surface. Following this, the ClpP protein was used as an analyte on the sensor chip. After stabilizing the reaction signal, and infusion of each concentration of hinokiflavone, the time for association and dissociation was 240 s, respectively. The results were finally analyzed by Trace Drawer and One To One.

Cell viability and cell colony formation assay[2]
For MTT assay, cells were seeded in 96-well culture plates and treated with hinokiflavone and then incubated with MTT solution. Formazan crystal was dissolved, and absorbance was measured by Spectra MAX M5 microplate spectrophotometer at 570 nm. For cell colony formation assay, cells were seeded in six-well plates and incubated with hinokiflavone for additional 12 days. The colonies were stained and counted under microscope.

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Hoechst staining and flow cytometry[2]
For Hoechst 33258 staining, cells were seeded in a six-well plate and incubated with hinokiflavone for 24 h and then fixed and stained with Hoechst 33258 solution. The nuclear morphology was observed and imagined by fluorescence microscopy). For flow cytometry (FCM), cells were harvested after incubated with hinokiflavone for 24 h and stained with Annexin V–fluorescein isothiocyanate/propidium iodide detection kit and then detected by flow cytometer.

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Mitochondrial membrane potential (ΔΨm) assay and reactive oxygen species detection[2]
Rh123 was used to determine the changes of mitochondrial transmembrane potential by FCM.24 Cells were harvested after incubated with hinokiflavone for 24 h, incubated with Rh123 solution, and then measured by FCM. To detect ROS level, cells were seeded and incubated with different concentration of hinokiflavone for 24 h, incubated with DCFH-DA, and then measured by FCM.25

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Boyden chamber migration and invasion assay[2]
Modified Boyden chamber migration and invasion assay was conducted as previously described.26, 27 For migration assay, cells in serum-free medium with hinokiflavone were added into the upper chamber, and medium containing 10% FBS and hinokiflavone was added at the bottom. For invasion assay, the upper surface of transwell membrane was coated with serum-free medium diluted with Matrigel, and then cells in serum-free medium containing hinokiflavone were added into the upper layer of the chamber, and the lower compartment of the chamber was filled with medium containing 10% FBS. Migrating and invasion cells were counted and photographed under a light microscope.[2]

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Determination of growth curves. [3]
The strain is kept in −20°C environment. Before experimenting, a single colony of USA300 and USA300 ΔclpP was picked for recovery after overnight incubation. The experiments are then carried out to ensure the activity of strains. Then the cultures were added to 2 mL of TSB medium at a ratio of 1:100, respectively. Hinokiflavone (64 μg/mL) or DMSO was also added. One hundred microliters of culture were used to measure absorbance values (OD600) at different time points.

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Determination of MIC. [3]
The MIC of hinokiflavone against S. aureus USA300 was determined by following the Clinical and Laboratory Standard Institute guidelines (CLSI). Briefly, 96-well plates containing hinokiflavone (0 to 512 μg/mL) and S. aureus USA300 (1 × 105 CFU) in 100 μL fresh CAMHB medium were incubated at 37°C for 16 to 20 h. The MIC was the lowest concentration at which no bacterium were found on visual observation.

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Cytotoxicity experiments. [3]
HEK293T cells were seeded into 96-well plates at 100 μL per well at a density of 5 × 104 cells/mL and cultured for 16 h. After adhering, various concentrations of hinokiflavone (0 to 256 μg/mL) were added to the 96-well plate. Cells were continued to grow in a 5% CO2 incubator at 37°C for 24 h. Subsequently, MTT solution was mixed to the medium at 10 μL/well and incubated for 4 h at 37°C. The culture medium was discarded, followed by the addition of 100 μL of DMSO. The absorbance at 490 nm was calculated using a microplate reader.

Mice model and toxicity evaluation[2]
Female BALB/c mice (6–8 weeks of age) were maintained in a specific pathogen-free condition facility. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. CT26 cells (0.5 × 106) were injected into the right flank of mice. When the tumor volume reached ~60mm3, mice were randomly divided and received intraperitoneally injection of hinokiflavone 25 and 50 mg/kg or vehicle once daily. Tumor volumes were calculated by the formula 0.52 × length × width2. Tumor inhibition rates (TIs) are calculated by TI = (Vcontrol − Vexperiment)∕Vcontrol. Internal organs and blood samples were collected after final treatment, and tumors were also collected and weighed (on day 24 after tumor cell inoculation). For toxicity assessment, related indices including body weight, diarrhea, anorexia, and other clinical symptoms were monitored continuously. Tissue samples (tumor, heart, liver, spleen, lung, and kidney) were examined by hematoxylin and eosin (HE) staining, and blood samples were subjected to biochemical and blood routine analyses. Pneumonia model experiment.[3]
Hinokiflavone was evaluated in vivo using a mouse model of pneumonia infection. Liaoning Changsheng Biologicals provided the mice as 6 weeks old male SPF grade C57BL/6J mice, weight 18 to 22 g. The mouse model of S. aureus pneumonia was constructed as previously described.[3]
To assess survival rates, mice were randomly divided into 7 groups of 10 mice. Each group was infected with 2 × 108 CFU/30 μL S. aureus. One hour after inoculating, hinokiflavone (100 mg/kg/d) was injected subcutaneously every 12 h to assess the survival rate at 96 h. After 2 h of infection, a specific dosage of hinokiflavone (100 mg/kg·d−1), vancomycin (100 mg/kg·d−1), or the combination of hinokiflavone (100 mg/kg·d−1) and vancomycin (100 mg/kg·d−1) was administered through the subcutaneous injection route for hinokiflavone and the intraperitoneal route for the antibiotic.

[1]. Characterisation of the biflavonoid hinokiflavone as a pre-mRNA splicing modulator that inhibits SENP. Elife. 2017 Sep 8;6. pii: e27402.

Hinokiflavone is a biflavonoid that is apigenin substituted by a 4-(5,7-dihydroxy-4-oxo-4H-chromen-2-yl)phenoxy group at position 6. A diflavonyl ether, it is isolated from Rhus succedanea and has been found to possess significant cytotoxic potential. It has a role as a neuroprotective agent, an antineoplastic agent and a metabolite. It is a biflavonoid, an aromatic ether and a hydroxyflavone. It is functionally related to an apigenin.
Hinokiflavone has been reported in Garcinia multiflora, Rhus punjabensis, and other organisms with data available.

C30H18O10

538.4579

538.09

C, 66.92; H, 3.37; O, 29.71

19202-36-9

5281627

Light yellow to yellow solid

1.6±0.1 g/cm3

841.5±65.0 °C at 760 mmHg

353-355ºC

284.8±27.8 °C

0.0±3.2 mmHg at 25°C

1.771

4.33

5

10

4

40

1020

0

O1C(C2C([H])=C([H])C(=C([H])C=2[H])O[H])=C([H])C(C2C(=C(C(=C([H])C1=2)O[H])OC1C([H])=C([H])C(=C([H])C=1[H])C1=C([H])C(C2=C(C([H])=C(C([H])=C2O1)O[H])O[H])=O)O[H])=O

WTDHMFBJQJSTMH-UHFFFAOYSA-N

InChI=1S/C30H18O10/c31-16-5-1-14(2-6-16)24-12-21(35)28-26(40-24)13-22(36)30(29(28)37)38-18-7-3-15(4-8-18)23-11-20(34)27-19(33)9-17(32)10-25(27)39-23/h1-13,31-33,36-37H

6-[4-(5,7-dihydroxy-4-oxochromen-2-yl)phenoxy]-5,7-dihydroxy-2-(4-hydroxyphenyl)chromen-4-one

4',6''-O-Biapigenin; Hinokiflavone; 19202-36-9; 4',6''-O-Biapigenin; GFF5VYC4NB; 4H-1-benzopyran-4-one, 6-[4-(5,7-dihydroxy-4-oxo-4H-1-benzopyran-2-yl)phenoxy]-5,7-dihydroxy-2-(4-hydroxyphenyl)-; 6-[4-(5,7-dihydroxy-4-oxochromen-2-yl)phenoxy]-5,7-dihydroxy-2-(4-hydroxyphenyl)chromen-4-one; CHEBI:5721; 4H-1-Benzopyran-4-one, 6-(4-(5,7-dihydroxy-4-oxo-4H-1-benzopyran-2-yl)phenoxy)-5,7-dihydroxy-2-(4-hydroxyphenyl)-; Hinokiflavone

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 : ~50 mg/mL (~92.86 mM)
Ethanol : ~2 mg/mL (~3.71 mM)

Solubility in Formulation 1: ≥ 0.83 mg/mL (1.54 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 8.3 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 + to the above solution and mix evenly; then add 450 μL of 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: 10 mg/mL (18.57 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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