Deguelin

Kinase experiment:

Caspase 3 activity is determined using Caspase-Glo-3 assays. This assay provides luminogenic substrate in a buffer system optimized for each specific caspase activity. The caspase cleavage of the substrate is followed by generation of a luminescent signal. The signal generated is proportional to the amount of caspase activity present in the sample. Protein (10 µg) from the cell samples is diluted in water to a final volume of 50 µL and added to a white 96-well microtitre plate, followed by 50 µL of Caspase-Glo-3 reagent. The plate is sealed and gently mixed at 300-500 rpm for 30 s and incubated at room temperature for 30 min. Luminescence is measured in a microplate reader (TECAN Infinite 200).

Cell experiment:

Breast cancer cells are incubated with increasing concentration of Deguelin ranging from 31 nM to 500 nM for 24, 48 and 72 h. At the termination the cells are trypsinized and cell proliferation is evaluated by counting cells using Z-series Coulter counter. Data are presented as Mean±SE percent of control.

Animal experiment:

Six to seven weeks old female athymic mice (nu/nu) are housed in a barrier free environment under 24±2°C temperature, 50±10% relative humidity, and 12-hour light/12-hour dark cycle. Mice are provided with sterile mouse chow and water ad libitum. MDA-MB-231 cells (3.0 million cells/animal) are suspended in sterile PBS and then injected subcutaneously into the dorsal flank region using 23 g hypodermic needle. Animals are observed daily for the growth of palpable tumor at the site of injection. Once the tumor (approximately 50 mm3) appears, the mice are randomized in to three groups, animals receiving either 1) vehicle as a control 2) Deguelin treatment at 2 mg/kg bodyweight dose or 3) Deguelin at 4 mg/kg body weight. Each group consists of 10 animals. Vehicle or Deguelin is administered through i.p. injection daily for 21 days. Animals are monitored daily for the signs of drug/vehicle associated toxicity and weighed once weekly. Growth of tumor at the site of cell injection is monitored every alternate day and of tumor size is measured using calipers. Tumor volume is calculated by using the well-established formula: tumor volume (mm3)=π/6 length×width×depth. Data represent the mean tumor volume+SE (mm3) in each group. The animals are sacrificed at the indicated time unless they appear to be moribund or tumors show sign of necrosis. At termination, the tumor is excised, freed from connective tissue and other organs, a small piece is fixed in 10% buffered formalin and remaining tumor is snap frozen for future biochemical analysis. Liver, lung, kidney and spleen are excised and weighed.

References:

[1]. Mehta R, et al. Deguelin action involves c-Met and EGFR signaling pathways in triple negative breast cancer cells. PLoS One. 2013 Jun 10;8(6):e65113.
[2]. Yang YL, et al. Deguelin induces both apoptosis and autophagy in cultured head and neck squamous cell carcinoma cells. PLoS One. 2013;8(1):e54736.
[3]. Li Z, et al. Deguelin, a natural rotenoid, inhibits mouse myeloma cell growth in vitro via induction of apoptosis. Oncol Lett. 2012 Oct;4(4):677-681.
[4]. Kang HW, et al. Deguelin, an Akt inhibitor, down-regulates NF-κB signaling and induces apoptosis in colon cancer cells and inhibits tumor growth in mice. Dig Dis Sci. 2012 Nov;57(11):2873-82.