The Effect of Cisplatin on the Sodium/Potassium Pump in Caco-2 Cells

Abstract

Cancer is one of the main health concerns in the world. It prevents cells from undergoing apoptosis and sends them into an uncontrolled replication path. Over the years, many chemotherapeutic agents have been used to fight cancer, and cisplatin was one of the most commonly used ones. Cisplatin targets DNA in the cancer cells and damages it, reverting the cells to the apoptotic route. A decrease in the activity of the Na+/K+ ATPase or Na+/K+ pump is one of the hallmarks of apoptosis. The aim of this work was to investigate if cisplatin targets and affects the ATPase activity, using Caco-2 cells as a model. The activity of the pump was assayed by measuring the amount of inorganic phosphate liberated in presence and absence of ouabain, a specific inhibitor of the Na+/K+ ATPase. Cisplatin was found at sub-toxic concentrations to exert a significant inhibitory effect on the pump that was most prominent at 8M and 24 hrs but had no effect on a purified Na+/K+ ATPase indicating that it acts indirectly on the ATPase via activation of various intermediate molecules. Fluorescence imaging revealed that this inhibition is due to a decrease in the abundance of the pump at the membrane. Among the mediators identified to be involved in the effect of cisplatin is JNK. Its mere inhibition resulted in a reduced activity of the pump and no further increase was observed in the simultaneous presence of cisplatin, implying that the drug inhibits JNK which is active at basal levels and needed for the activation of the Na+/K+ ATPase. Additional mediators identified were p38MAPK, PKA and calcium, since the effect of the drug disappeared when the kinases were inhibited or when calcium was chelated with BAPTA-AM but reappeared in the simultaneous presence of a JNK inhibitor implying that JNK is the most downstream mediator. PKA activation in presence of a p38MAPK inhibitor maintained the effect of cisplatin indicating that PKA is upstream JNK and downstream p38MAPK. Calcium is suspected to be the one leading to p38MAPK activation, however this still needs to be confirmed. It was concluded that cisplatin inhibits the Na+/K+ ATPase by increasing intracellular calcium which activates sequentially p38MAPK and PKA. The latter inhibits JNK leading to an inhibition of the pump.