oa Involvement Of Intracellular Calcium [ca2+]i In The Treatment Of Human Neuroblastoma With Cisplatin (cddp) And Topotecan (topo)

Neuroblastoma is a solid malignant embryonic tumor. It is usually diagnosed in the infancy and rarely found after the age of 10 years. In the current treatment options, CDDP and TOPO are included. Methods: Neuroblastoma (SH SY-5Y) cell death was investigated after treatment with CDDP and TOPO (0.1nM-1μM) using Trypan Blue Cytotoxicity Test and Total Cytotoxicity and Apoptosis Detection Kit (FACS). These data are compared to the drug triggered increase of the intracellular calcium ([Ca2+]i) concentration using live calcium imaging with the calcium sensitive dye Fluo-4 AM. Furthermore, we analyzed changes of gene expression of selected [Ca2+]i signaling related genes such as the calcium binding protein S100A6, IP3 receptors, Ryanodine Receptors, Calmodulin (CALM) and the Calmoduling Binding Transcriptor Activator (CAMTA1). Results: With increasing concentration of either of the two substances the amount of viable cells was reduced after 24h of application. The tests revealed a higher cytotoxicity of TOPO (about 25% cell survival at 0.1μM) compared to CDDP (more than 80% survival). With increasing concentrations the percentage of cells which were in the “late apoptosis” stage increased for both drugs but were more pronounced for TOPO. A similar observation was made when the incubation time for the two drugs was prolonged to 48h or 72h when the amount of viable cells was further reduced. Individual (but not all) neuroblastoma cells increased their [Ca2+]i after the application of either CDDP or TOPO (both 1 μM). Over the period of three to four hours the fluorescence increased about 40%. The increase was time and concentration dependent for CDDP and TOPO, while a concentration of 0.01μM was most efficient to increase [Ca2+]i. CDDP and TOPO influence the gene expression of SHSY-5Y cells, targeting specific calcium signaling related genes. In this context, a concentration and time dependent increase in the expression of S100A6 was observed in the cells treated with CDDP. TOPO showed a more pronounced effect than CDDP in increasing the mRNA expression of S100A6. The expression of ITPR1 and ITPR3 genes encoding the type 1 and 3 of the IP3R were found deregulated in the neuroblastoma cells treated to CDDP and TOPO. The 1μM CDDP exposure for 72h was responsible of ITPR1 and ITPR3 down regulation however; the same concentration applied for 24h transiently increased the mRNA expression of ITPR3. The application of TOPO did diminished concentration and time dependently the mRNA expression of ITPR1 after 24h and 72h exposure and of ITPR3 after 72h exposure, however 24h exposure to TOPO did transiently increased the mRNA expression of ITPR3 for the lower concentrations tested. The expression of Ryanodine Receptor genes RYR1 and RYR3 was found deregulated in the neuroblastoma cells treated to CDDP and TOPO. Conclusion: Time and concentration dependently, the treatment of SHSY-5Y cells with TOPO and CDDP increased the cell death, at clinical relevant concentration. That correlates with increased [Ca2+]i levels and with the deregulation in gene expression of calcium signaling related genes suggesting that [Ca2+]i regulation is involved in the anticancer effects of TOPO and CDDP.