cytochrome-c-t and gadolinium-chloride

Gadolinium (Gd) compounds have important applications as MRI contrast and potential anticancer agents. The present study investigated the mechanisms of the proapoptotic effect of gadolinium chloride (GdCl(3)) on hepatoblastoma cell line (Hep G2) tumor cells. The experimental results indicated that GdCl(3) induced apoptosis of Hep G2 at high concentration and with long time incubation; however, unlike the actions on normal cell lines, GdCl(3) did not cause any oxidative stress on tumor cells. Cytochrome c (Cyt c) and apoptosis inducing factor release, Bax translocation, collapse of mitochondria membrane potential, caspase 3 and 8 activation, and Bid cleavage were observed along with a sustained activation of extracellular signal-regulated kinase (ERK) and c-Jun NH2 terminal kinase (JNK). Addition of ERK and JNK inhibitor attenuated the effect of GdCl(3) induced apoptosis and Cyt c release. All the results suggested a novel mechanism that GdCl(3) induced Hep G2 cell death through intrinsic and external death pathways without significant elevation of reactive oxygen species generation. The present work provided new insight to understand the mechanisms of the biological effects of GdCl(3) and implications for the development of anticancer Gd agents.

Topics: Apoptosis; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Blotting, Western; Caspase 3; Caspase 8; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Extracellular Signal-Regulated MAP Kinases; Flow Cytometry; Gadolinium; Hep G2 Cells; Humans; JNK Mitogen-Activated Protein Kinases; Membrane Potential, Mitochondrial; Microscopy, Confocal; Mitochondria; Models, Biological; Reactive Oxygen Species; Signal Transduction; Time Factors

The calcium-sensing receptor (CaR) is a seven-transmembrane G-protein coupled receptor, which activates intracellular effectors, for example, it causes inositol phosphate (IP) accumulation to increase the release of intracellular calcium. Although intracellular calcium overload has been implicated in the cardiac ischemia/reperfusion (I/R)-induced apoptosis, the role of CaR in the induction of apoptosis has not been fully understood. This study tested the hypothesis that CaR is involved in I/R cardiomyocyte apoptosis by increasing [Ca2+]i. The isolated rat hearts were subjected to 40-min ischemia followed by 2 h of reperfusion, meanwhile GdCl3 was added to reperfusion solution. The expression of CaR increased at the exposure to GdCl3 during I/R. By laser confocal microscopy, it was observed that the intracellular calcium was significantly increased and exhibited a Deltapsim, as monitored by 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolcarbocyanine iodide (JC-1) during reperfusion with GdCl3. Furthermore, the number of apoptotic cells was significantly increased as shown by TUNEL assay. Typical apoptotic cells were observed with transmission electron microscopy in I/R with GdCl3 but not in the control group. The expression of cytosolic cytochrome c and activated caspase-9 and caspase-3 was significantly increased whereas the expression of mitochondrial cytochrome c significantly decreased in I/R with GdCl3 in comparison to the control. In conclusion, these results suggest that CaR is involved in the induction of cardiomyocyte apoptosis during ischemia/reperfusion through activation of cytochrome c-caspase-3 signaling pathway.

Topics: Animals; Apoptosis; Calcium; Calcium Channels, L-Type; Caspase 3; Caspase 9; Caspases; Cytochromes c; Gadolinium; Male; Microscopy, Confocal; Mitochondria, Heart; Myocardial Reperfusion Injury; Myocytes, Cardiac; Rats; Receptors, Calcium-Sensing