Cells (the human pancreatic cell lines, Mia PaCa-2, BxPC-3, AsPC-1, PANC-1, PL-45, and normal pancreatic ductal epithelial cells, hTERT-HPNE cells) are seeded into 96-well plates (3000 cells/well) in triplicate. After overnight incubation, the medium is changed and cells are treated with I3C and/or NBMPR for 24 h. The medium is changed again and cells are cultured in medium containing different concentrations of Gemcitabine in the presence or absence of the same concentrations of I3C and/or NBMPR for 48 h. The cells are then subjected to CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) as per the manufacturer's instructions. Absorbance at 490 nm is measured 2 h after the addition of 20 μL of MTS reagent/well^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[2]
At 1 month of age, LSL-Kras^G12D/+; LSL-Trp53^R172H; Pdx-1-Cre mice are randomized into treatment groups (placebo, DMAPT, Gemcitabine, DMAPT/Gemcitabine). Placebo (vehicle=hydroxylpropyl methylcellulose, 0.2% Tween 80 [HPMT]) and DMAPT (40 mg/kg body weight in HPMT) are administered by oral gastric lavage once daily. Gemcitabine (50 mg/kg body weight in PBS) is administered by intraperitoneal injection twice weekly. Mouse weight is monitored weekly. Treatment is continued until mice show signs of lethargy, abdominal distension or weight loss at which time they are sacrificed. Successful excision-recombination events are confirmed in the pancreata of mice by detecting the presence of a single LoxP site.
Rats^[3]
The study is conducted in 80 female Wistar rats, with an initial weight of approximately 250 g. Animals are identified by ear mark and divided into groups as follows. Forty rats are divided into five groups of eight: four groups had lung delivery of Gemcitabine via endotracheal spray at doses of 2, 4, 6, and 8 mg/kg, respectively (groups LD2, LD4, LD6, LD8), and one group receive a spray administration of the 0.9% saline vehicle solution (group LDv). The remaining 40 rats are divided into five groups of eight: four groups have oral delivery of Gemcitabine via gavage at doses of 2, 4, 6, and 8 mg/kg, respectively (groups OD2, OD4, OD6, OD8), and one group receive an identical volume of 0.9% saline via gavage (group ODv). The protocol includes nine sessions separated by 1-week intervals. Between sessions, the animals are kept under standard laboratory conditions and housed by groups of four animals in each cage with litter and free access to pellet food and tap water. Cages are placed in a closed chamber connected to an aspiration system.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Wang H, et al. Enhanced efficacy of Gemcitabine by indole-3-carbinol in pancreatic cell lines: the role of human equilibrativenucleoside transporter 1. Anticancer Res. 2011 Oct;31(10):3171-80.

  [2]. Yip-Schneider MT, et al. Dimethylaminoparthenolide and Gemcitabine: a survival study using a genetically engineered mouse model of pancreatic cancer. BMC Cancer. 2013 Apr 17;13:194.

  [3]. Gagnadoux F, et al. Safety of pulmonary administration of gemcitabine in rats. J Aerosol Med. 2005 Summer;18(2):198-206

  [4]. Lou M, et al. Physical interaction between human ribonucleotide reductase large subunit and thioredoxin increases colorectal cancer malignancy. J Biol Chem. 2017 Jun 2;292(22):9136-9149.

  [5]. Wang Y, et al. Licoricidin enhances gemcitabine-induced cytotoxicity in osteosarcoma cells by suppressing the Akt and NF-κB signal pathways. Chem Biol Interact. 2018 May 18;290:44-51.

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95058-81-4

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