Title: Low levels of mycophenolic acid induce differentiation of human neuroblastoma cell lines
Abstract: In a previous report,1 we showed that mycophenolic acid (MPA), at concentrations readily attainable during immunosuppressive therapy (0.1–10 μM), using its mycophenolate mofetil prodrug, causes a decrease of intracellular levels of guanine nucleotides, G1 arrest and time- and dose-dependent death by apoptosis. The first effect appears to be the consequence of inosine monophosphate dehydrogenase (IMPDH) inhibition and to trigger the other effects through p53-mediated pathways. MPA fails to achieve high concentrations following in vivo administration because of rapid glucuronidation, which inactivates the drug.2 The morpholinoethyl ester of MPA (mycophenolate mofetil; CellCept, Roche, Milan, Italy) has improved bioavailability and is used as an immunosuppressant following solid-organ transplants.2 Mycophenolate mofetil is rapidly absorbed following oral administration and almost completely hydrolyzed to the active metabolite MPA, which is extensively and tightly bound to human albumin.2, 3, 4 Free MPA in most patients accounts for about 2% of the mean plasma drug level, which is usually maintained around 3 μg/ml.4 We previously reported apoptotic effects on human neuroblastoma cell lines obtained at MPA concentrations corresponding to the total plasma drug levels during immunosuppressive CellCept therapy.1 In the present study, we investigated the effects of this drug on human neuroblastoma cell lines at lower concentrations, corresponding to those of the active free drug readily attainable during therapy with mycophenolate mofetil. Three established human neuroblastoma cell lines (LAN5, IMR32 and SK-N-SH) were utilized in the current study. Exponentially growing cells were cultured at 37°C in a humidified atmosphere with 5% CO2 in RPMI-1640 supplemented with 10% FBS, 100 units/ml penicillin, 100 μg/ml streptomycin and 2 mM glutamine. Cells were treated with 50 nM MPA or not in the presence or absence of 0.1 mM guanosine for 3 and 6 days. Incubation media were changed every 3 days. Addition of 50 nM MPA led to a decrease of intracellular guanine nucleotide levels with respect to control cells cultured in the absence of drug after 3 or 6 days of incubation. In LAN5 cells, nucleotide extraction and chromatographic analysis performed as described1 evidenced a guanine nucleotide depletion (87% of GTP, 77% of GDP control values) after 3 days of incubation in the presence of drug. This effect was reversed, at least in part, by 0.1 mM guanosine, added simultaneously with the IMPDH inhibitor: after 6 days of incubation in the presence of guanosine, the reduction of guanine nucleotide levels with respect to control cells was less evident (GTP 92%, GDP 83% of control values). After 6 days of incubation in the presence of 50 nM MPA, neuroblastoma cells differentiated toward the neuronal phenotype with outgrowth of neurite-like processes. In IMR32 cells, the morphologic changes were less evident. Immunofluorescence or immunoperoxidase analysis using anti-NF200 (intermediate filaments), anti-GAP43 (growth-associated protein-43) or anti-MAP5 (microtubule-associated protein-5) monoclonal antibodies (MAbs) showed upregulation of the differentiation markers in MPA-treated cells (upper panel of Fig. 1). This effect was at least partially reversed by simultaneous addition of 0.1 mM guanosine. The differentiation-inducing potential of MPA has been previously reported in many cultured tumor cell lines, such as breast cancer,5 T-lymphoblastoid,6 erythroid7 and myeloid8 leukemia and melanoma.9 The mechanism whereby depletion of guanine nucleotides by MPA induces differentiation is unknown and might be different from that of physiologic differentiation inducers such as retinoic acid, vitamin D metabolites and cytokines acting through direct stimulation of the differentiation pathways by specific receptor-mediated signals.10 As shown in the lower panel of Figure 1, RT-PCR analysis performed after 6 days as described1 evidenced upregulation of p53, p21 and bax and downregulation of bcl-2 and survivin in MPA-treated cells with respect to controls incubated in the absence of drug. These effects were completely reversed by simultaneous addition of 0.1 mM guanosine. In cells treated with MPA, mRNA levels of p27 were not significantly different from control cells. It has been shown that 48 hr incubation of LAN5 cells in the presence of 100 nM MPA leads to upregulation of p53 protein, with its shuttling into the nucleus.1 Western blot analysis, performed as previously described,1 confirmed that incubation of LAN5 cells in the presence of 50 nM MPA for 3 or 6 days led to upregulation of p53 protein with respect to control cells incubated in the absence of drug. The same analysis evidenced (data not shown) that p27 protein levels were comparable to those of control cells, while at higher concentrations of the IMPDH inhibitor, a time- and dose-dependent decrease of p27 protein expression was observed.1 When neuroblastoma cells are treated with retinoic acid, they undergo growth and morphologic, biochemical and electrophysiologic changes that are characteristic of neuronal differentiation. These retinoic acid–induced changes are associated with a marked decrease in levels of p53 protein and mRNA11, 12 an increase in p2113, 14 and in Bcl-215, 16 expression and and a very large accumulation (>10-fold) of p27.17, 18 Bax expression is not significantly modified.15 Therefore, although both MPA and retinoic acid induce similar morphologic differentiation in neuroblastoma cells, the molecular mechanisms involved appear to be different. In conclusion, MPA, at concentrations corresponding to those of the active free drug readily attainable therapeutically using mycophenolate mofetil, induces neuroblastoma cells to differentiate toward the neuronal phenotype, confirming a potential role of CellCept in the management of neuroblastoma patients. Upper: Immunocytochemical analysis of differentiation marker expression in neuroblastoma cell lines after 6 days of treatment with 50 nM MPA (b,d,f,h) or control medium (a,c,e,g). Immunoperoxidase staining of LAN5 cells with anti-NF200 (a,b) and anti MAP5 (c,d). Immunofluorescent staining of SK-N-SH cells with anti-GAP43 (e,f) and of IMR32 cells with anti-NF200 (g,h).Lower: RT-PCR analysis of p53, p21, bax, bcl-2, survivin, p27 and β-actin expression in LAN5 cells treated for 6 days with control medium (lane 1), 50 nM MPA (lane 2) and 50 nM MPA + 0.1 mM guanosine (lane 3). Yours sincerely, Elisa Messina, Paola Gazzaniga, Vanna Micheli, Lucio Barile, Federico Lupi, Anna Maria Aglianò, Alessandro Giacomello