Abstract
The Wnt pathway is critical for the proliferation and cell fate determination of many cell types, including B cells of chronic lymphocytic leukemia (CLL). Wnt proteins act on target cells by binding to the Frizzled (Fz)/low-density lipoprotein (LRP) complex at the cell membrane. In the hematopoietic system, Wnt/β-catenin signaling pathway has been shown to be important in the control of the survival, proliferation and differentiation of hematopoietic cells.
Nanotechnology, a new and promising field, may be of use in medicine and the pharmaceutical industry. Dendrimers are nanoparticles of dendritic architecture. We have already proved the influence of PPI-G4-OS-M3 dendrimers in cultures in vitro on CLL cells apoptosis. CLL lymphocytes are characterized by the failure in the apoptosis pathway, but it is also proved that they manifest increased proliferation.
Herein, the objective was to evaluate how MEC-1 cells survival and proliferation in vitro is affected by blockage of Wnt pathway by PPI-G4-OS-M3 dendrimer comparing to FA.
Material and methods
Dendrimer, in which approximately 35% of peripheral amino groups, was coated with maltotriose have been defined as PPI-G4-OS-M3 and was used in concentration of 8 mg/ml (the IC50 value for this dendrimer). 'OS' abbreviation stands for the open shell structure of carbohydrate-modified dendrimers. The molar mass of this PPI dendrimer was 31000 g/mol.
Fludarabine (FA, Genzyme) in concentration of 1.6 µ M, based on previous studies, was used.
MEC-1 (DSMZ no. ACC 497) was used as a homogenous cell line with del(17p)(11q).
In cultures the percentage of apoptotic cells was verified using AnnV and PI by means of flow cytometer. Cells predominated in the early stage of apoptosis.
A microarray gene expression (Agilent SurePrint Technologies) was performed. Samples were hybridized to a whole human genome microarray 8x60K. Arrays were scanned on Agilent DNA Microarray Scanner. Data were deposited at Gene Expression Omnibus (GEO) (accession number GSE68094).
Analysis of differential expression of genes was done with the limma method (Smyth, G. K., 2004) as implemented in R/Bioconductor software. We used the FDR multiple testing adjustment. We declared as differentially expressed the genes with FDR-adjusted p-value <0.1, which means that 10% of genes declared as DE are expected to be false positives.
Results
PPI-G4-OS-M3 dendrimer depicts the ability to inhibit the proliferation increases with the rise in dendrimer concentration (MethoCultTM Assay, Stemcell Technology).
Microarray data analysis pointed 7 out of 100 members of Wnt genes whose expression was significantly important. Details concerning genes description and expression are collected in the table 1.
Table 1. Wnt genes expression in MEC-1 cells under influence of dendrimers and FA in 4-hour-cultures. PPI-G4-OS-M3 FA Probe set Gene symbol Gene full name logFC adj P value logFC adj P value P102117 WNT10A Wingless-type MMTV integration site family, member 10A -0.59 0.04 -0.56 0.03 P81103 SFRP2 Secreted frizzled-related protein 2 0.96 0.04 -0.59 0.07 P65518 DACT1 Dishevelled-binding antagonist of beta-catenin 1 0.66 0.05 0.81 0.03 P206359 CDH1 Cadherin 1 type 1 -0.71 0.05 -0.99 0.03 P119916 WNT6 Wingless-type MMTV integration site family, member 6 -1.93 0.1 -0.23 0.8 (NS) P1505 LRP5 Low density lipoprotein receptor-related protein 5 0.39 0.1 -0.28 0.1 P117029 LDLR Low density lipoprotein receptor 0.21 0.1 -0.49 0.03
Conclusion: Our results show significant changes and differences in some of Wnt/β-catenin pathway genes expression in CLL influenced by glycodendrimer and FA treatment. The downregulation in WNT10A, WNT6, SFRP2 expression results in β-catenin less phosphorylation and is subjected to proteosomal degradation. LRP5 and LDLR genes expression is also weak thus a reaction cascade is blocked and transcription process is suppressed. The loss of Wnt signals by dendrimers and FA treatment induces a reduction in the proliferation and survival of treatment resistant cell line MEC-1.
To summarize, the PPI-G4-OS-M3 dendrimer demonstrated inhibition of proliferation beside higher cytotoxicity towards CLL cells. Its potency is similar to FA widely used in CLL therapy. Thus, dendrimers are a potential tool for CLL treatment.
The study was partially supported by Grant No. DEC-2011/01/B/NZ5/01371from the National Science Centre, Poland
Disclosures
Robak: Novartis: Consultancy, Honoraria, Research Funding; GlaxoSmithKline: Research Funding.
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