Quantitative cardiotoxicity assessment of gambogic acid using multiple cellular phenotype analysis / 中国药理学与毒理学杂志

ABSTRACT

OBJECTIVE To evaluate the cardiotoxicity of a widen-spectrum antineoplastic drug, gambogic acid, through quantitative multiple cellular phenotypic characterization. METHODS H9c2 cell line was used as a model with doxorubicin (Dox) and amiodarone (Ami) as positive controls, hypaconi?tine as negative control and 0.1% DMSO as normal control. An optimized protocol was established to identify the morphology and function of cell nuclei. The effect of drugs on cell viability, nuclear area (Hoechst33342), mitochondria mass (MitoTracker Deep Red) and cytoplasmic calcium ion mobilization (Rhod2 AM)was studied. EC50 and Z′values were calculated to evaluate the degree of toxicology and to estimate the precision and false-positive rate, respectively. RESULTS Dose-response analysis indicated that EC50 of Dox on cell viability, nuclear area, mitochondrial mass was 0.72, 0.014 and 1.21μmol · L-1, respectively. On the other hand, EC50 of Ami on the parameters of cell viability, nuclear area and mitochon?drial mass was 14.83, 6.72 and 4.54μmol·L-1, respectively with Z′value above 0.5. Hypaconitine decreased the SER ridge of mitochondria. Gambogic acid caused significant mortality of H9c2 cells and induced nuclear shrinkage as Ami did. The EC50 values of cell viability and nuclear area were 0.24 and 1.16 μmol · L- 1. Meanwhile,gambogic acid disturbed the mitochondrial function as indicated by the increased mitochondrial area (EC50=0.44 μmol · L-1), abnormal SER Ridge(EC50=0.99 μmol · L-1) and decreased mitochondrial mass(EC50=1.21 μmol · L- 1). Cellular calcium mobilization was lower than normal (EC50=0.41 μmol · L-1). CONCLUSION The EC50 values of positive controls calculated from our assessment are similar those reported in literature. A multi-parameter and simultaneous evaluation enables a comprehensive analysis of the morphology of nuclei and mitochondria of cardiomyocytes and a preliminary assessment of the mechanisms of toxicity.