In vitro activity: Pirarubicin is rapidly taken up by M5076 cells and the intracellular concentration of pirarubicin reaches more than 2.5-fold that of doxorubicin. Pirarubicin is more effective than doxorubicin in terms of the 50% cell growth-inhibitory concentration in vitro. Pirarubicin causes G0/G1 cell cycle arrest in MG-63 cells. Pirarubicin suppresses the expression of PCNA, cyclin D1, cyclin E and Bcl-2, and increases Bax expression in MG-63 cells. Pirarubicin markedly relaxes contractions induced by noradrenaline (0.1 μM) in the aorta with endothelium, but not in that without endothelium. Pirarubicin-induced relaxation is inhibited by methylene blue (5 μM), hydroquinone (100 μM), phenidone (50 μM), haemoglobin (1 μM) and p-bromophenacyl bromide (50 μM), but not by indomethacin (25 μM). Pirarubicin is approximately 2-5 times more potent than Adriamycin in SKUT1B, HEC1A, and BG1 cell lines. Pirarubicin also displays a reverse dose-response pattern of G2 block so that at high dose, cell cycle kinetics would mirror those of untreated controls.

Cell Assay: MTS is used to analyze cell survival. Briefly, cells are plated in 96-well plates in triplicate at 2 × 103 cells per well and cultured in growth medium. Then cells are treated with pirarubicin at different concentrations (2.5 μg/mL, 5 μg/mL, 10 μg/mL) for 24 h. MTS reagent (5 mg/mL) is added and incubated at 37°C for 4 h. The absorbance is monitored at 490 nm using a microplate reader.