Valproic acid inhibits adhesion of vincristine- and cisplatin-resistant neuroblastoma tumour cells to endothelium

Abstract

Drug resistance to chemotherapy is often associated with increased malignancy in neuroblastoma (NB). In pursuit of alternative treatments for chemoresistant tumour cells, we tested the response of multidrug-resistant SKNSH and of vincristine (VCR)-, doxorubicin (DOX)-, or cisplatin (CDDP)-resistant UKF-NB-2, UKF-NB-3 or UKF-NB-6 NB tumour cell lines to valproic acid (VPA), a differentiation inducer currently in clinical trials. Drug resistance caused elevated NB adhesion (UKF-NB-2(VCR), UKF-NB-2(DOX), UKF-NB-2(CDDP), UKF-NB-3(VCR), UKF-NB-3(CDDP), UKF-NB-6(VCR), UKF-NB-6(CDDP)) to an endothelial cell monolayer, accompanied by downregulation of the adhesion receptor neural cell adhesion molecule (NCAM). Based on the UKF-NB-3 model, N-myc proteins were enhanced in UKF-NB-3(VCR) and UKF-NB-3(CDDP), compared to the drug naïve controls. p73 was diminished, whereas the p73 isoform deltaNp73 was upregulated in UKF-NB-3(VCR) and UKF-NB-3(CDDP). Valproic acid blocked adhesion of UKF-NB-3(VCR) and UKF-NB-3(CDDP), but not of UKF-NB-3(DOX), and induced the upregulation of NCAM surface expression, NCAM protein content and NCAM coding mRNA. Valproic acid diminished N-myc and enhanced p73 protein level, coupled with downregulation of deltaNp73 in UKF-NB-3(VCR) and UKF-NB-3(CDDP). Valproic acid also reverted enhanced adhesion properties of drug-resistant UKF-NB-2, UKF-NB-6 and SKNSH cells, and therefore may provide an alternative approach to the treatment of drug-resistant NB by blocking invasive processes.

Figure 1

Valproic acid treatment causes adhesion blockade in CDDP- and VCR-resistant NB cells. The figure depicts adhesion capacity of parental UKF-NB-3 (control) vs VCR- (UKF-NB-3^VCR), CDDP- (UKF-NB-3^CDDP) or DOX-resistant NB subpopulations (UKF-NB-3^DOX) vs resistant cell lines treated with 1 mM VPA for 3 (VPA3) or 5 days (VPA5). Neuroblastoma cells were added at a density of 0.5 × 10⁶ cells/well to HUVEC monolayers for 60 min. Non-adherent tumour cells were washed off in each sample, the remaining cells were fixed and counted in five different fields (5 × 0.25 mm²) using a phase contrast microscope. Adhesion capacity is depicted as tumour cell adhesion mm⁻² (mean±s.d.; n=6).

Figure 2

Valproic acid treatment enhances NCAM surface expression in CDDP- and VCR-resistant NB cells. Histograms plots show NCAM surface expression on untreated vs VPA-treated UKF-NB-3^CDDP, UKF-NB-3^VCR and UKF-NB-3^DOX. IgG isotype controls are also included. The complete experimental data set (MFU±s.d.; n=6) is given below the histograms. Tumour cells were disaggregated mechanically in each experiment and washed in blocking solution. An FITC-conjugated monoclonal antibody anti-CD56, clone 16.2, was used to detect the NCAM 120, 140 and 180 kDa isoform. A mouse IgG1-FITC served as the isotype control (IgG). Fluorescence was analysed using a FACScan flow cytometer, and a histogram plot (FL1-Height) was generated to show FITC fluorescence.

Figure 3

Western blot analysis of NCAM from the proteins of UKF-NB-3, UKF-NB-3^CDDP UKF-NB-3^VCR UKF-NB-3^DOX and resistant subpopulations treated with VPA for 3 (VPA3) or 5 days (VPA5). Cell lysates were subjected to SDS–PAGE and blotted on the membrane incubated with anti-NCAM (clone 16.2) monoclonal antibodies. β-Actin served as the internal control. The figure shows one representative from three separate experiments.

Figure 4

RT–PCR analysis of NCAM 140 and 180 kDa RNA in UKF-NB-3, UKF-NB-3^CDDP UKF-NB-3^VCR UKF-NB-3^DOX and resistant subpopulations treated with VPA for 3 (VPA3) or 5 days (VPA5). Ribonucleic acid were extracted, reverse-transcribed and submitted to semiquantitative RT–PCR using gene-specific primers as indicated in Materials and Methods. The internal control for the RT–PCR reaction was performed by running parallel reaction mixtures with the housekeeping gene GAPDH. The figure shows one representative from three separate experiments.

Figure 5

Valproic acid alters N-myc expression in CDDP- and VCR-resistant UKF-NB-3 cells. The right part of the figure depicts results from flow cytometry analysis. To allow intracellular staining, tumour cells were fixed and permeabilised by methanol–acetone before antibodies were added. To identify N-myc, the monoclonal anti-N-myc antibody clone NCM II 100 was used. Primary antibodies were labelled with goat anti-mouse IgG-FITC. Background staining was evaluated by goat anti-mouse IgG-FITC. The histograms plots show N-myc expression level in untreated vs VPA-treated UKF-NB-3^CDDP, UKF-NB-3^VCR and UKF-NB-3^DOX. IgG isotype controls are also included. The complete experimental data set (MFU±s.d.; n=6) is given alongside. The left part of the figure shows Western blot analysis of N-myc from the proteins of UKF-NB-3, UKF-NB-3^CDDP UKF-NB-3^VCR UKF-NB-3^DOX and resistant subpopulations treated with VPA for 3 (VPA3) or 5 days (VPA5). Cell lysates were subjected to SDS–PAGE and blotted on the membrane incubated with anti-N-myc monoclonal antibodies. β-Actin served as the internal control. The figure shows one representative from three separate experiments.