Cell experiment:

Briefly, cells are seeded into 96-well plates at a density of 1 × 105 cells/well and incubated for 24 h. The cells are treated with Troglitazone in the presence or absence of other chemicals for a further 24 h using FBS-free medium. The assay utilizes the conversion of alamar blue reagent to fluorescent resorufin by metabolically active cells. The resorufin signal is measured at an excitation wavelength of 530 nm and an emission wavelength of 580 nm. The 50% growth inhibitory concentrations (IC50) are calculated according to the sigmoid inhibitory effect model E = IC50 γ/(IC50 γ + Cγ), where E represents the surviving fraction (% of control), C represents the drug concentration in the medium, and γ represents the Hill coefficient. For co-exposure studies, the Troglitazone dosage is set to approximately the IC50 value for each cell line[2].

Animal experiment:

Balb/c male mice (4 weeks old) are subcutaneously inoculated in the back with MIA Paca2 cells (5 × 106 cells/100 μL in PBS) 14 days prior to starting Troglitazone administration. Mice are then orally administered 200 mg/kg Troglitazone in 0.5% methylcellulose solution or vehicle daily for 5 weeks. Tumor size is measured bi-dimensionally and the volume is calculated using the formula (length × width2) × 0.5. Body weights of mice are also monitored throughout the experiment[2].

Peroxisome proliferator-activated receptor gamma (PPARγ) agonists and PPARg/a dual agonists are used in the treatment of type 2 diabetes mellitus. Troglitazone is a prototypical sarcomagenic PPARg agonist.

In vitro: Troglitazone PPARγ ligands showed potent inhibitory effect on proliferation, and could induce RCC cell apoptosis, suggesting that the PPARγ ligands have potential antitumor effects on renal carcinoma cells [1].

In vivo: Troglitazone increased EC proliferation in brown and white adipose tissue and liver in mice at sarcomagenic doses (400 and 800 mg/kg) after four weeks of treatment [2].

Clinical trial: Several clinical trials have been conducted to investigate the efficacy, safety and pharmacokinetics of troglitazone in the liposarcoma and diabtes mellitus patients [3].

References:
[1] Yang FG, Zhang ZW, Xin DQ, Shi CJ, Wu JP, Guo YL, Guan YF.  Peroxisome proliferator-activated receptor gamma ligands induce cell cycle arrest and apoptosis in human renal carcinoma cell lines. Acta Pharmacol Sin. 2005;26(6):753-61.
[2] Kakiuchi-Kiyota S, Arnold LL, Yokohira M, Koza-Taylor P, Suzuki S, Varney M, Pennington KL, Cohen SM.  Evaluation of direct and indirect effects of the PPARγ agonist troglitazone on mouse endothelial cell proliferation. Toxicol Pathol. 2011;39(7):1032-45.