Caspase-3; Caspase-10
Kelch-like ECH-associated protein 1 (Keap1, a sensor protein for phase 2 inducers) - Acts as a covalent modifier/reactant.
Phase 2 response / Keap1-Nrf2-ARE pathway (CD value for NQO1 induction = 0.15 μM in murine hepatoma Hepa1c1c7 cells and wild-type L1210 cells) [1]

The specific activities of NQO1 and glutathione reductase increase by 6 and 1.5 fold, respectively, when L1210 cells are exposed to 0.6 M 2-HBA (bis(2-hydroxy-benzylidene)acetone). Additionally, a 2.4-fold coordinate induction of the total cellular glutathione content occurs. In a more in-depth investigation, it was discovered that 2-HBA induces NQO1 in a concentration-dependent manner. Treatments with 2-HBA induce cell cycle arrest and apoptosis in L1210 wild type cells and their Y8 drug-resistant counterparts in a concentration-dependent manner. Caspase-3 and Caspase-10 can both be triggered by 2-HBA[1].

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1. In murine leukemia L1210 wild-type cells, treatment with 2-HBA at submicromolar concentrations (e.g., 0.6 μM for 48 hours) induced the phase 2 response, marked by a 6-fold increase in NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1) activity, a 1.5-fold increase in glutathione reductase activity, and a 2.4-fold increase in total cellular glutathione levels compared to untreated controls. No cytotoxicity was observed at these concentrations. [1]
2. The concentration required to double NQO1 specific activity (CD value) of 2-HBA in wild-type L1210 cells was 0.15 μM, consistent with previous data in murine hepatoma cells. [1]
3. In contrast, the deoxyadenosine-resistant L1210 Y8 mutant cells, which lack functional p53 protein expression, showed no induction of NQO1 activity upon treatment with 2-HBA. The basal NQO1 activity in Y8 cells was about 50% of that in wild-type cells. [1]
4. At higher (micromolar) concentrations, 2-HBA caused cell cycle arrest and apoptosis in both wild-type and Y8 L1210 cells. In wild-type cells, treatment (e.g., 5-15 μM for 24 hours) caused a G2/M cell cycle block. In Y8 cells, treatment (e.g., 0.5-2.5 μM for 24 hours) led to increases in both G0/G1 and G2/M populations. [1]
5. 2-HBA induced apoptosis in a concentration-dependent manner, as assessed by Annexin-V-FLUOS/propidium iodide flow cytometry. The Y8 mutant cells were more sensitive to the apoptotic effects of 2-HBA. For example, at 2.5 μM, 2-HBA induced apoptosis in 56.6% of Y8 cells, compared to requiring 15 μM to induce apoptosis in 37.8% of wild-type cells. Necrosis was minimal. [1]
6. Treatment of L1210 cells with 2-HBA (15 μM for wild-type, 2.5 μM for Y8 cells, for 6 hours) markedly elevated the activities of caspase-3 (the terminal executioner caspase) and caspase-10 (an initiator caspase) in cell-free extracts. The activities of caspase-1, -6, and -8 were not significantly affected. The activation was more pronounced in Y8 cells. [1]
7. The apoptosis induced by 2-HBA is p53-independent, as neither L1210 wild-type (which expresses mutant p53) nor Y8 cells (which lack p53 expression) have functional p53 protein. [1]

In 96-well plates, cells are grown for 24 hours before being exposed to 2-HBA (bis(2-hydroxybenzylidene)acetone) for either 24 or 48 hours, depending on whether glutathione levels are to be measured or the activities of enzymes. After the exposure time has passed, cells are collected by centrifugation (1500 g for 15 min at 4 °C), then cleaned with DPBS before being lysed in 0.08% digitonin. For protein analysis, an aliquot (25 μL) is used. Using the Prochaska test, NQO1 activity is evaluated[1].

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1. NQO1 Activity Assay (Prochaska Test): L1210 cells are seeded in 96-well plates (20,000 cells per well) and grown for 24 hours. They are then exposed to serial dilutions of 2-HBA for 48 hours. After exposure, cells are collected by centrifugation, washed with Dulbecco's phosphate-buffered saline (DPBS), and lysed in 0.08% digitonin. An aliquot of the lysate is used for protein determination. NQO1 activity is measured directly in the microtiter plate wells. The assay typically involves monitoring the enzyme-catalyzed reduction of a suitable quinone substrate coupled to a colorimetric or fluorescent indicator. The specific activity is calculated, and the concentration required to double the enzyme activity (CD value) is determined from the dose-response curve. [1]
2. Glutathione Reductase Activity Assay: Cell lysates are prepared from 2-HBA-treated and control L1210 cells as described above. Glutathione reductase activity is measured according to a standard enzymatic procedure, which typically involves monitoring the NADPH-dependent reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) by following the decrease in absorbance of NADPH at 340 nm. [1]

After exposure to 2-HBA (bis(2-hydroxybenzylidene)acetone) for 24 h, duplicate aliquots of cells (1×106) are collected by centrifugation and washed with cold DPBS. The Annexin-V-FLUOS assay is used to determine apoptosis, and propidium iodide uptake is used to determine the necrotic fraction at the same time[1].

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1. Cell Cycle Analysis by Flow Cytometry: L1210 cells (wild-type and Y8 mutant) are exposed to increasing concentrations of 2-HBA for 24 hours. Cells are then collected, washed with cold DPBS, and resuspended in a sodium citrate solution. Propidium iodide is added, and cells are incubated on ice for 30 minutes. RNase is then added, and after a 30-minute incubation at room temperature, cells are kept overnight at 4°C. Before analysis, cell suspensions are passed through a fine needle to ensure single cells. Aliquots of 1×10⁶ cells are analyzed on a flow cytometer using an argon ion laser (488 nm excitation) and detection at 560 nm. DNA content is measured, and cell cycle distribution (G0/G1, S, G2/M phases) and the sub-G0/G1 apoptotic population are determined using appropriate analysis software. [1]
2. Apoptosis and Necrosis Assessment by Annexin-V/PI Staining: After a 24-hour exposure to 2-HBA, duplicate aliquots of L1210 cells (1×10⁶ cells) are collected by centrifugation and washed with cold DPBS. Apoptosis is determined using a fluorescent Annexin-V conjugate assay kit. Cells are resuspended in a binding buffer containing Annexin-V-fluorescein and propidium iodide (PI) and incubated. Cells are then analyzed by flow cytometry using 488 nm excitation. Fluorescein (Annexin-V) fluorescence is detected with a 515 nm emission filter, and PI fluorescence is detected with a 560 nm filter. This allows differentiation of viable cells (Annexin-V negative, PI negative), early apoptotic cells (Annexin-V positive, PI negative), and late apoptotic/necrotic cells (Annexin-V positive, PI positive). [1]
3. Caspase Activity Assay in Cell-Free Extracts: Wild-type and Y8 L1210 cells are exposed to 2-HBA (15 μM and 2.5 μM, respectively) for 6 hours. Aliquots of 1×10⁶ cells are used to prepare cell-free extracts, which are stored frozen until analysis. Caspase activities are determined in a 96-well plate format using specific fluorogenic peptide substrates and inhibitors for each caspase. For caspase-3, the substrate Ac-DEVD-AMC and inhibitor Ac-DEVD-CHO are used; for caspase-6, Ac-VEID-AFC and Ac-VEID-CHO; for caspase-8, Ac-IETD-AFC and Ac-IETD-CHO; for caspase-10, Z-AEVD-AFC and Z-AEVD-FMK. Cell extract is incubated with the substrate (and substrate plus inhibitor for control) at 37°C for 1.5 hours. The release of the fluorescent product (AMC or AFC) is measured fluorimetrically using a plate reader with appropriate excitation/emission filters (e.g., excitation at 400 nm, emission at 505 nm). Caspase activity is calculated from the fluorescence signal. [1]
4. Total Glutathione Determination: L1210 cells treated with 2-HBA (e.g., 0.6 μM for 24 hours) are lysed. An aliquot of the lysate is treated with ice-cold metaphosphoric acid in EDTA to precipitate proteins. After centrifugation, the supernatant is transferred to another plate and neutralized with sodium phosphate buffer (pH 7.5) containing EDTA. Total cellular glutathione (GSH + GSSG) is determined by a recycling assay. This assay typically involves the cyclic reduction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) by glutathione reductase in the presence of NADPH, generating a yellow product (5-thio-2-nitrobenzoic acid, TNB) whose rate of formation, measured spectrophotometrically at 412 nm, is proportional to the total glutathione concentration. [1]

1. At submicromolar concentrations (e.g., 0.6 μM), 2-HBA did not show cytotoxicity in L1210 cells, as evidenced by cell morphology, and the total protein content was the same in the treated and control groups. [1]
2. At higher micromolar concentrations, 2-HBA induced apoptosis, mainly manifested as apoptosis with very low necrosis. Annexin-V/PI double staining results showed that most affected cells were in the early apoptotic stage (Annexin-V positive, PI negative). [1]

[1]. Bis(2-hydroxybenzylidene)acetone, a potent inducer of the phase 2 response, causes apoptosis in mouse leukemia cells through a p53-independent, caspase-mediated pathway. Cancer Lett. 2007 Jan 8;245(1-2):341-9.

See also: 1,5-bis(2-hydroxyphenyl)pentan-1,4-dien-3-one (note moved to).

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1. Background and compound class: 2-HBA is a dual Michael response receptor belonging to the bis(benzylidene) ketone class. Michael response receptors are the main class of phase II response inducers, characterized by their ability to react with thiol groups. The ortho-hydroxyl group on the aromatic ring of 2-HBA is crucial for its high inducibility and thiol reactivity. [1] 2. Dual concentration-dependent effects: The biological effects of 2-HBA exhibit a clear concentration-dependent dichotomy. At low concentrations (sub-micromolar levels), it effectively induces a protective phase II response. At high concentrations (micromolar levels), 2-HBA induces p53-independent apoptosis. In L1210 cells, the chemopreventive index (apoptosis IC50 / induction CD value) of 2-HBA was approximately 100 (15 μM / 0.15 μM). This pattern of low-dose induction of phase II apoptosis and high-dose induction of apoptosis appears to be a common phenomenon among various phase II apoptosis inducers. [1] 3. Potential synergistic effects in chemoprotection: 2-HBA can both enhance the cell defense mechanisms of normal/precancerous cells (inducing phase II apoptosis) and eliminate genetically abnormal cells through apoptosis, making it an ideal candidate drug for chemopreventive strategies targeting multiple steps of the carcinogenic process. [1] 4. Significance of model systems: Using p53-deficient L1210 and Y8 cell lines, it was shown that 2-HBA can induce apoptosis independently of p53 status, which is significant given the high frequency of p53 mutations in human cancers. [1]

C17H14O3

266.29126

266.094

C, 76.68; H, 5.30; O, 18.02

131359-24-5

131359-24-5

5472867

Light yellow to yellow solid powder

1.3±0.1 g/cm3

495.5±40.0 °C at 760 mmHg

267.6±23.8 °C

0.0±1.3 mmHg at 25°C

1.704

4.91

2

3

4

20

338

0

O=C(/C=C/C1=CC=CC=C1O)/C=C/C2=CC=CC=C2O

RFRXIWQYSOIBDI-UHFFFAOYSA-N

InChI=1S/C17H14O3/c1-2-14-16(13-5-3-4-6-15(13)20-14)17(19)11-7-9-12(18)10-8-11/h3-10,18H,2H2,1H3

(2-ethyl-1-benzofuran-3-yl)-(4-hydroxyphenyl)methanone

2-HBA

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 (~375.5 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.39 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 (9.39 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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 (Please use freshly prepared in vivo formulations for optimal results.)