Bergamottin (5-Geranoxypsoralen)

Kinase experiment:

CYP1A1 Supersomes and different concentrations of 7-ethoxyresorufin (ER) are used to determine CYP1A1 enzymatic kinetics. A typical Michaelis-Menten curve is found. The final reaction mixture contains: 1 pmol CYP1A1, 0.5 mM NADPH, different ER concentrations and 0, 4, 8 and 16 nM of Bergamottin (BG). The reaction is started with the addition of NADPH. Kinetic constants are obtained by a nonlinear regression analysis of experimental data fitted to Michaelis-Menten equation with competitive-type inhibition. Kinetic analysis is also shown by using the Lineweaver-Burk, Dixon and replot of the slopes of the Dixon plot[1].

Cell experiment:

Inhibition of cell proliferation by Bergamottin is measured by the MTT assay. Briefly, the human lung adenocarcinoma cancer A549 cells are plated in 96-well culture plates (1×105 cells/well). After 24 h of incubation, the cells are treated with Bergamottin (0, 5, 10, 25, 50, 75 and 100 μM) for 24 and 48 h, MTT solution (10 mg/mL) is then added to each well. After a 4-h incubation, the formazan precipitate is dissolved in 100 μL DMSO, and then the absorbance is measured in an automated microplated reader at 570 nm. The cell viability ratio is calculated. Cytotoxicity is expressed as the concentration of Bergamottin needed to inhibit cell growth by 50% (IC50 value)[2].

Animal experiment:

Mice[2]Female BALB/c nude mice (six weeks old) (a total of 20 are obtained) are maintained with water and food ad libitum in a pathogen-free environment with a 12 h light and 12 h dark cycle in an animal care facility. Human non‑small cell lung carcinoma A549 cells (2×106 cells/mouse) are injected into the right axilla of the nude mice (5 mice/group) to create tumors in the mice. Subsequent to tumor development, the mice are divided into 4 groups and treated with Bergamottin injected intraperitoneally. The control group in the study is treated with an equal amount of PBS while the other three groups are treated with 25, 50 and 100 mg/kg of Bergamottin. Afterwards, the mice are sacrificed after 18 days, and the tumor weight and volume of each mouse are evaluated. Tumor length and width are measured using a Vernier caliper and the tumor volume (TV) is calculated[2].

References:

[1]. Olguín-Reyes S, et al. Bergamottin is a competitive inhibitor of CYP1A1 and is antimutagenic in the Ames test. Food Chem Toxicol. 2012 Sep;50(9):3094-9.
[2]. Wu HJ, et al. Bergamottin isolated from Citrus bergamia exerts in vitro and in vivo antitumor activity in lung adenocarcinoma through the induction of apoptosis, cell cycle arrest, mitochondrial membrane potential loss and inhibition of cell migration and invasion. Oncol Rep. 2016 Jul;36(1):324-32.