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

Microbial-Derived Toll-like Receptor Agonism in Cancer Treatment and Progression

Toll-like receptors (TLRs) are typical transmembrane proteins, which are essential pattern recognition receptors in mediating the effects of innate immunity. TLRs recognize structurally conserved molecules derived from microbes and damage-associated molecular pattern molecules that play an important role in inflammation. Since the first discovery of the Toll receptor by the team of J. Hoffmann in 1996, in Drosophila melanogaster, numerous TLRs have been identified across a wide range of invertebrate and vertebrate species. TLR stimulation leads to NF-κB activation and the subsequent production of pro-inflammatory cytokines and chemokines, growth factors and anti-apoptotic proteins. The expression of TLRs has also been observed in many tumors, and their stimulation results in tumor progression or regression, depending on the TLR and tumor type. The anti-tumoral effects can result from the activation of anti-tumoral immune responses and/or the direct induction of tumor cell death. The pro-tumoral effects may be due to inducing tumor cell survival and proliferation or by acting on suppressive or inflammatory immune cells in the tumor microenvironment. The aim of this review is to draw attention to the effects of TLR stimulation in cancer, the activation of various TLRs by microbes in different types of tumors, and, finally, the role of TLRs in anti-cancer immunity and tumor rejection

Expression of Immunomodulatory Checkpoint Molecules in Drug-Resistant Neuroblastoma: An Exploratory Study

Neuroblastoma is a common childhood cancer with poor prognosis when at its advanced stage. Checkpoint molecule inhibition is successful in treating multiple advanced adult cancers. We investigated PD-L1 and other checkpoint molecule expression to determine their roles in drug resistance and usefulness as targets for drug therapy. We developed three doxorubicin-resistant (DoxR) cell lines from parental cell lines. Matrigel in vitro invasion assays were used to compare invasiveness. Western blot assays were used to compare PD-L1 expression. Immuno-oncology checkpoint protein panels were used to compare concentrations of 17 checkpoint molecules both cellular and soluble. PD-L1 and 12 other checkpoint molecules were present in all cell lysates of each cell line without significantly different levels. Three were solubilized in the media of each cell line. PD-L1 is expressed in all DoxR and parental neuroblastoma cells and may be a potential target for drug therapy although its role in drug resistance remains unclear. Benchmarking checkpoint molecules provides the basis for future studies identifying targets for directed therapy and biomarkers for cancer detection or prognosis

Fertility and hormone preservation and restoration for female children and adolescents receiving gonadotoxic cancer treatments: A systematic review.

The purpose of this systematic review by the American Pediatric Surgical Cancer Committee was to summarize evidence from the current medical literature regarding fertility restoration and hormone replacement for female children and adolescents treated with gonadotoxic treatments.info:eu-repo/semantics/publishe

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

Expression of selected DRGs using qRT-PCR.

<p>Expression of endothelial PAS domain protein 1 (EPAS1), somatostatin (SST) and paraoxanase 2 (PON2) in (a) SK-N-SH and (b) SK-N-Be(2)C WT-v, DoxR and DoxR-v cells relative to their parental lines. Results were normalized to GAPDH by qRT-PCR and expressed as mean ± standard deviation for three biological repeats.</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

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

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