Nonconventional Gene Expression within the NF-κb Signaling Pathway Induced By Poly(propylene)Imine Glycodendrimers in Chronic Lymphocytic Leukemia Cells

Introduction

The nuclear factor kappa-light-chain enhancer of activated B-cells (NF-κB) signaling pathway is constitutively active in a variety of cancers, including chronic lymphocytic leukemia (CLL). The importance of this signaling pathway identifies it as a prime therapeutic target, however the complexity and potential side effects of inhibiting NF-κB have thus far made the clinical use of NF-κB inhibitors a relatively unexplored resource in this disease. There are a few combined therapies available for the treatment of CLL includes chemotherapy with agents such as chlorambucil, cyclophosphamide, fludarabine and bendamustine, along with immunotherapy including rituximab and alemtuzumab. None available therapy for CLL is curative.

Nanotechnology, a new and promising field of scientific research, may be of use in medicine and the pharmaceutical industry. Dendrimers, nanoparticles of dendritic architecture, can interact effectively and specifically with cell components. We have already proved the influence of PPI-G4-OS-M3 dendrimers in cultures in vitro on CLL cells apoptosis.Herein, the objective was to evaluate how MEC-1 cells survival in vitro is affected by influence on NF-κB 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 µM, 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

Dendrimer induced expression of REL, RELB and NFKBIB genes. In contrast, FA monotherapy resulted in significant differences in gene expression of cellular pathway-dependent transcription factor NF-kB. The most significant differences in the function of the FA and dendrimer are reflected in different levels of expression of three genes: NFKBIA, BCL3 and CHUK.

Conclusion

Constitutive NF-κB signaling contributes to cell growth, proliferation and survival. CLL cells have high basal levels of NF-κB compared with normal B cells. The activity is variable in CLL patients, correlates with in vitro cell survival, and importantly, increased levels of

NF-κB activity enhanced resistance to the purine analogues FA (del17p). Therefore, disruption of NF-κB signaling and downstream target genes either promoted or repressed is an important strategy to pursue to disrupt drug resistance in CLL. The study indicates that the use of PPI dendrimers modified maltotriose may be the key to developing therapies deliberates CLL.

The study was partially supported by Grant No. DEC-2011/01/B/NZ5/01371from the National Science Centre, Poland.