Detection of the Calcium and ATP Role in Apoptosis of Retinoblastoma Culture Cells through Caspase-3 Expression

Abstract

Retinoblastoma is a malignant retinal tumor associated with apoptotic deregulation. Retinoblastoma cells are sensitive to NK cells. These cells can stimulate apoptosis. Apoptosis in retinoblastoma occurs in the early phase and is paradoxical. The increasing amounts of Bcl-2 and Caspase-3 as apoptotic executors are inversely proportional to apoptosis. Calcium and ATP as second messengers and signaling molecules play role in mediating cell responses including retinoblastoma cell development and death. Apoptosis requires sufficient energy from ATP and its mediated by Calcium. This study aimed to perceive the effects of Calcium and ATP in the process of retinoblastoma cell death through Caspase-3 pathway. The subjects were poorly differentiated retinoblastoma cell cultures treated with NK cells (treatment group) compared to those which were not exposed to NK cells (control group). Through the cell flowcytometry test that expresses Caspase-3 and apoptosis is calculated, meanwhile the levels of Calcium and ATP activity produced during the apoptosis process are quantitatively calculated. Examination of ATP activity uses a colorimetric method while the calcium content is calculated using a clinical chemistry system. Calcium and ATP were negatively correlated at 27.4% (p <0.05). ATP also showed a very significant negative correlation of 75.8% (p <0.01) against Caspase-3 and is significantly positively correlated with apoptosis of 46.8% (p,0.05). Whereas Caspase-3 is negatively correlated with apoptosis by 46.6% (p <0.05). In the Caspase-3 pathway, allogeneic NK cell administration in retinoblastoma cells increases the level of Calcium which plays a role in the early phase apoptosis process, whereas ATP which was formed is insufficient to cause maximal apoptosis due to extracellular calcium entry into NK cells. Extracellular ATP does not play a role in the induction of apoptosis in retinoblastoma cells treated with NK cells.