Midkine mediates intercellular crosstalk between drug-resistant and drug sensitive neuroblastoma cells in vitro and in vivo

Resistance to cytotoxic agents has long been known to be a major limitation in the treatment of human cancers. Although many mechanisms of drug resistance have been identified, chemotherapies targeting known mechanisms have failed to lead to effective reversal of drug resistance, suggesting that alternative mechanisms remain undiscovered. Previous work identified midkine (MK) as a novel putative survival molecule responsible for cytoprotective signaling between drug-resistant and drug-sensitive neuroblastoma, osteosarcoma and breast carcinoma cells in vitro. In the present study, we provide further in vitro and in vivo studies supporting the role of MK in neuroblastoma cytoprotection. MK overexpressing wild type neuroblastoma cells exhibit a cytoprotective effect on wild type cells when grown in a co-culture system, similar to that seen with doxorubicin resistant cells. siRNA knockdown of MK expression in doxorubicin resistant neuroblastoma and osteosarcoma cells ameliorates this protective effect. Overexpression of MK in wild type neuroblastoma cells leads to acquired drug resistance to doxorubicin and to the related drug etoposide. Mouse studies injecting various ratios of doxorubicin resistant or MK transfected cells with GFP transfected wild type cells confirm this cytoprotective effect in vivo. These findings provide additional evidence for the existence of intercellular cytoprotective signals mediated by MK which contribute to chemotherapy resistance in neuroblastoma

The Effect of Vorinostat on the Development of Resistance to Doxorubicin in Neuroblastoma

<div><p>Histone deacetylase (HDAC) inhibitors, especially vorinostat, are currently under investigation as potential adjuncts in the treatment of neuroblastoma. The effect of vorinostat co-treatment on the development of resistance to other chemotherapeutic agents is unknown. In the present study, we treated two human neuroblastoma cell lines [SK-N-SH and SK-N-Be(2)C] with progressively increasing doses of doxorubicin under two conditions: with and without vorinsotat co-therapy. The resultant doxorubicin-resistant (DoxR) and vorinostat-treated doxorubicin resistant (DoxR-v) cells were equally resistant to doxorubicin despite significantly lower P-glycoprotein expression in the DoxR-v cells. Whole genome analysis was performed using the Ilumina Human HT-12 v4 Expression Beadchip to identify genes with differential expression unique to the DoxR-v cells. We uncovered a number of genes whose differential expression in the DoxR-v cells might contribute to their resistant phenotype, including hypoxia inducible factor-2. Finally, we used Gene Ontology to categorize the biological functions of the differentially expressed genes unique to the DoxR-v cells and found that genes involved in cellular metabolism were especially affected.</p> </div

Altered biological processes based on the genes with differential expression (fold change>1.5; adjusted p<0.1) unique to the DoxR-v cell lines as mapped by Gene Ontology.

<p>The number of genes annotated to each biological process category by Gene Ontology is shown in the third column. The number of actual differentially expressed genes (DEG) in each category is compared to the expected number of DEGs. P values were calculated by the Fischer exact test.</p

Relative expression of known drug-resistance genes in doxorubicin resistant (DoxR) and vorinostat-treated doxorubicin-resistant (DoxR-v) cells compared to the parental lines.

<p>Results are expressed as a fold-change (all p<0.1). N.D. indicates no difference in gene expression (fold-change<1.5 and/or p>0.1). The following genes had no significant difference in any comparison: ABCC1 (MRP1), ABCC2 (MPR2), ABCC6 (mrp6), ABCC8 (mrp8), ABCC10 (mrp10), ABCC11 (mrp11), ABCC12 (mrp12), ABCC13 (mrp13), MGMT, SOD, HDAC1-8.</p

Drug resistance in the SK-N-SH and SK-N-Be(2)C cells.

<p>Doxorubicin resistance was assessed by MTT cell proliferation assay in the (a) SK-N-SH and (b) SK-N-Be(2)C cell lines. WT and WT-v proliferation was reduced by increasing concentrations of doxorubicin whilst the DoxR and DoxR-v cells were equally resistant to doxorubicin therapy. (c) Cross resistance to etoposide was likewise confirmed in the SK-N-SH cell line. (d) Western immunoblotting demonstrated significantly greater upregulation of P-glycoprotein (P-gp) in the DoxR than the DoxR-v cells. (e) Likewise in the SK-N-Be(2)C cell line, western blot revealed a pattern of upregulation in hypoxia inducible factor -1α (HIF-1α) similar to that observed with P-gp.</p

Genes with a potential role in doxorubicin-resistance following vorinostat treatment.

<p>There were 405 unique genes differentially expressed (fold change >1.5, adjusted p<0.1) in both SK-N-SH and SK-N-Be(2)C DoxR-v cells, but not in the DoxR or WT-v lines. The complete list of all 405 unique DEGs is available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040816#pone.0040816.s004" target="_blank">Table S1</a>, while the subset of those genes with the greatest differential expression (fold change>2) is listed here.</p

Relative expression of genes differentially expressed in doxorubicin-resistant compared to wild-type cells.

<p>Heat map for the SK-N-SH and SK-N-Be(2)C cell lines showing relative expression of the subset of the 405 DEGs unique to the DoxR-V versus WT comparison with a fold change >2.</p