Sulindac derivatives inhibit cell growth and induce apoptosis in primary cells from malignant peripheral nerve sheath tumors of NF1-patients

Abstract

BACKGROUND: Malignant peripheral nerve sheath tumors (MPNSTs) are neoplasms leading to death in most cases. Patients with Neurofibromatosis type 1 have an increased risk of developing this malignancy. The metabolites of the inactive prodrug Sulindac, Sulindac Sulfide and Sulindac Sulfone (Exisulind) are new chemopreventive agents that show promising results in the treatment of different cancer types. In this study we examined the antineoplastic effect of these compounds on primary cells derived from two MPNSTs of Neurofibromatosis type 1 patients. RESULTS: Exisulind and Sulindac Sulfide showed a dramatic time- and dose-dependent growth inhibitory effect with IC50-values of 120 microM and 63 microM, respectively. The decrease in viability of the tested cells correlated with induction of apoptosis. Treatment with 500 microM Exisulind and 125 microM Sulindac Sulfide for a period of 2 days increased the rate of apoptosis 21-27-fold compared to untreated cells. Reduced expression of RAS-GTP and phosphorylated ERK1/2 was detected in treated MPNST cells. Moreover, elevated levels of phosphorylated SAPK/JNK were found after drug treatment, and low activation of cleaved caspase-3 was seen. CONCLUSIONS: Our results suggest that this class of compounds may be of therapeutic benefit for Neurofibromatosis type 1 patients with MPNST.

Figure 1

Growth inhibition of MPNST cell lines S462 and S520 Cells were plated at a density of 15000/well on 12 mm coverslips and treated with indicated concentrations of Exisulind and Sulindac Sulfide for 48 h and 96 h. Cell growth was measured by BrdU-incorporation. The final DMSO concentration used here did not exceed 0.2% and had no effect on cell growth. The values represent the means and standard deviations of triplicates.

Figure 2

Correlation of growth inhibition (reduction of viable cell number) and apoptosis The increase of apoptotic cells is paralleled by reduction of viable cell number at the corresponding concentrations. A-B: Viability of cell lines S462 and S520 were measured by their ability to reduce XTT metabolically to a purple formazan product after 48 h of treatment with Exisulind and Sulindac Sulfide at different concentrations. The percentage of viable cells was determined setting the absorbance of cells treated with the vehicle as 100%. C-D: Percentages of apoptotic cells were determined by photometric quantification of DNA- and histone-fragmentation after 48 h of drug treatment. The absorbance mean values of treated samples (triplicates) were normalized to the corresponding control of untreated cells and the maximal absorbance from the test compound included in the assay was used as positive control (100% apoptosis).

Figure 3

TUNEL assay of cell line S462 showing time dependent induction of apoptosis Cells were either treated with 0.2% DMSO for 48 h (A, D), 500 μM Exisulind (Exi) for 24 h (B) and 48 h (C) or 125 μM Sulindac Sulfide (s. s.) for 24 h (E) and 48 h (F). The percentage of gated cells (upper box) represents the ratio of apoptotic cells (FITC-BrdU) to the total number of cells (PI), values are presented beneath each display.

Figure 4

Changes in cell morphology characteristic for apoptosis Morphologic changes became evident after 48 h of treatment with 500 μM Exisulind or 125 μM Sulindac Sulfide. A-C: Phase contrast photomicrographs of S462 cells before (A) and after Exisulind (B) treatment. Cell shrinkage, nuclear condensation and formation of apoptotic bodies shown on immunocytochemically labeled cell nuclei with PI after Sulindac Sulfide treatment (C) are classical characteristics of apoptosis and not necrosis.

Figure 5

Inhibition of RAS-GTP and phosphorylated ERK1/2 in human MPNST cells exposed to Sulindac derivatives A: Cell lines S462 and S520 were grown in DMEM with 10% serum and treated at 80–90% confluency with either 0.2% DMSO, 500 μM Exisulind (Exi) or 125 μM Sulindac Sulfide (s. s.). Cells were lysed after 24 h and RAS was immunoprecipitated and detected by western blotting with an anti-RAS antibody, the same lysates were blotted for phosphorylated (phospho-) and basal (total-) ERK1/2. B: Upregulation of phosphorylated ERK1/2 and AKT for treated and untreated cells after addition of EGF, whereas the RAS-GTP level remains unchanged at all conditions. Cell line S462 was starved over night and then Sulindac metabolites were supplemented at concentrations mentioned above in DMEM containing 0.1% serum. EGF was added after 24 h of treatment, cells were lysed 15 min later and after western blotting incubated with phosphorylated and unphosphorylated ERK1/2, phospho-AKT and RAS-antibodies.

Figure 6

Activation of SAPK/JNK but not caspase-3 after treatment with Sulindac metabolites The activation of phospho-SAPK/JNK (dimer: 46 kD + 54 kD) reached the highest level after 24 h of treatment with Exisulind (Exi) and Sulindac Sulfide (s. s.), but no effect was seen on cleaved caspase-3 activation. DHA did not have any effect on neither JNK nor caspase-3 activation. Cell line S462 was grown in DMEM with 10% serum and treated at 80–90% confluency with 0.2% DMSO, 500 μM Exisulind (Exi) or 125 μM Sulindac Sulfide (s. s.) and blots were incubated with either phosphorylated SAPK/JNK or cleaved caspase-3 antibody. The positive control for caspase activation is a mouse MPNST cell line treated 7 h with 30 μM DHA.