UNISP

Evaluation of New Pharmacological Approaches in the Treatment of MYCN-AMPLIFIED NEUROBLASTOMA

 

Neuroblastoma is a malignant neoplasm of the autonomic nervous system that frequently consider as the most common solid extracranic tumor in children. Originates from neural crest cells of neuroectodermal abnormalities, which being a primitive embryonic structure located at the edges of the paraxial channel promotes neural, upon closure of the neural tube, a migration of cells that leads to the formation of the sympathetic ganglia and the adrenal Medulla. One of the forms of neuroblastoma that is often diagnosticated in patients is characterized by MYCN oncogene amplification. In general, it has been seen that most children suffering from MYCN-amplified neuroblastoma has a low response to chemotherapy drugs. This resistance to conventional therapy is due to the action of two genes (ABCC1 and ABCC4), which are at the origin of the mechanism of Multidrug resistance. During this process of resistance, genes ABCC4 and ABCC1 encodes for channel proteins (P-glycoproteins) that can function as efflux pump of ATP for facilitating the escape of chemotherapy drugs.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

The aim of the thesis is to show at first as the combination of chemotherapy drugs and PNA anti- MYCN can be an important tool for the treatment of MYCN-amplified neuroblastoma; later, it carried out an evaluation of the therapeutic effect given exclusively by inhibition of the MYCN oncogene in some animal models.

To evaluate the effect of the combination of cytotoxic chemotherapy drugs and PNA anti-MYCN, we first and foremost effectuate a Real-Time PCR with the purpose to study the variation of the gene expression of Multidrug resistance (ABCC1 and ABCC4) chased to a treatment with PNA anti-MYCN. For this study we used three representative of cell lines MYCN-amplified neuroblastoma (IMR-32; Kelly and SKNBE 2 c). The results obtained reveal a correlation between genes ABCC1 and ABCC4 with MYCN. In particular, the reduction of transcript of MYCN leads to a consequent inhibition of the production of mRNA of ABCC1 and ABCC4.

On the basis of the previous results were carried out two series of vitality assays on two different cell lines (Kelly and SKNBE 2 c): the first series made it possible to determine the cellular IC50 of single drugs (PNA anti-MYCN and chemotherapeutic agents), while the second series, held on the basis of the results obtained in the first, made it possible to evaluate the therapeutic effect of the combination of chemotherapy drugs/PNA anti-MYCN. The results obtained were entered into the Compusyn software, which is used to study the combination of drugs. Under this program, the PNA anti-MYCN shows a synergic effect with three chemotherapy drugs(vincristin, Etoposide and Cisplatin). This synergy is probably favored by the injection of anti-gene molecule, whose action involves the gene silencing of the MYCN oncogene resulting in inhibition of cell proliferation processes (regulated by MYCN) and the mechanism of the resistance (regulated by ABCC1and ABCC4 genes), giving a cancer cell more vulnerable to treatment with conventional chemotherapy.

 

 

 

 

 

 

 

 

 

 

 

 

The results obtained in vitro experiments have generated an interest on the probable therapeutic activity of PNA anti-MYCN. It was then carried out an evaluation study of the possible therapeutic activity of PNA anti-MYCN on a ectotopic mice model inoculated with IMR-32 cell Luc. Four mice were used in which two controls and two treated with PNA anti-MYCN 5 m/Kg/day. From the collected data shows a reduction of 64% of the treated tumor compared to that of the control. This effect may be due to a possible therapeutic activity of PNA anti-MYCN. Giving that in neuroblastoma, MYCN acts as a transcription factor in the regulation of several cellular processes (proliferation, differentiation and cell growth), his silence could result in a blockage of tumor progression in vivo. The latter consideration remains yet to be confirmed because the experiment was set up using a very small number of mice.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

In conclusion, the PNA anti-MYCN is a molecule that could be used as a medication in combination with chemotherapeutic agents in the treatment against MYCN-amplified neuroblastoma. Its use in treatment could not only facilitate the activity of chemotherapeutic drugs already in therapy but also limit the resistance mechanisms present in some patients.

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Dr. Mbiandjeu Serge Cedrick

Institute for Pharmacological Research Mario Negri

 UNISP Team Research