Anti-proliferative effect by cyclic nucleotides in human glioblastoma and neuroblastoma cell lines

IntroductionGlioblastoma is the most aggressive and common malignant primary brain tumor of adolescence. Neuroblastoma is the most common malignant extracranial tumor of infancy. We have already demonstrated the anti-proliferative effect of second messengers cGMP, cAMP, cCMP, cUMP in various cancer cell lines. To expand our research we analyzed that effect in human glioblastoma and neuroblastoma cell lines.

MethodsWe used the alamarBlue assay to investigate proliferation in HEK293, U373 (glioblastoma), and SK-N- BE(2) (neuroblastoma) cells. To imitate the functions of cNMPs, acetoxymethyl ester analogues (AMs) were used. Multidrug resistant proteins (MRPs) were inhibited by probenecid and phosphodiesterases (PDEs) by IBMX to alter effects. With qRT-PCR analysis the expression of MRPs, PDEs, soluble adenylyl cyclase (sAC), and soluble guanylyl cyclases (sGC) was analyzed.

ResultsAll cNMP-AMs showed a time- and concentration-dependent anti-proliferative effect in all three cell lines. Highest reduction of proliferation in HEK293 cells was after treatment with cAMP-AM, cGMP-AM, and cCMP-AM, in U373 cells with cAMP-AM, and cCMP-AM, in SK-N-BE(2) cells with cCMP-AM. Treatment with cNMP-AMs after preincubating with probenecid and IBMX showed an increased effect in HEK293 and SK-N-BE(2) cells after 48 h and in U373 cells after 72 h. MRP4, MRP5 and PDE isoforms are expressed in all three cell lines, the expression of some sGC- isoforms is higher than sAC.

ConclusionsEfflux and degradation of cNMPs is important. When inhibited with probenecid and IBMX, cNMPs are accumulating and anti-proliferative effect is increased. IBMX alone showed no further decrease of proliferation, therefore MRPs may be more important in the cNMP-metabolism.