Endocrine Abstracts (2005) 9 P150

Antisense chemoradioimmunotherapy consisting of antiEGFscFv linked onto high energy radioisotopes,vinorelbine and 21-nucleotide double stranded siRNA targeted to DNMT1 induce apoptosis in anaplastic thyroid CCCa characterised by hypermethylated oncosuppressor Gadd45 promoter CpG islands and overexpression of bcl-2,VEGF and Raf-1

J Giannios1, P Lambrinos2, E Michailakis1, E Maragudakis3 & N Alexandropoulos4

1Department of Clinical Oncology, GSHA, Athens, Greece; 2Department of Oncology, PF, Athens, Greece; 3Department of Radiotherapeutic Oncology, IASO, Athens, Greece; 4Department of Clinical Biochemistry, IH, Athens, Greece.

Introduction:There is a great need for targeted and tailored treatment strategies in anaplastic thyroid cancer patients.The current conventional treatment strategies consisting of surgery,chemo- or radiotherapy have a palliative effect leading to short survival periods.The failure of tumor cells to undergo apoptosis may cause resistance to chemoradiological therapies due to overexpression of antiapoptotic oncogenes and transcriptionally repressed apoptotic tumour suppressor genes (TSGs) due to aberrant promoter methylation which was associated with CIMP+.

Methods:We obtain tumour cells from an anaplastic thyroid cancer patients characterised by radio- and chemoresistance.MS-PCR detected methylation of Gadd45 promoter.Quantitative IHc,WB,SB and PCR exhibited overexpression of DNMT1,bcl-2,EGF,VEGF and Raf1.We treated the aplastic thyroid Ca cells with anti-EGFscFv attached to high energy radioisotopes,vinorelbine-tartrate and 21-nucleotide double stranded siRNA segment generated against DNMT1.

Results:Post-treatment,we detected re-expression of oncosuppressor Gadd45 after inhibition of DNMT1mRNA,downregulation of antiapoptotic EGF due to targeted scFv and inactivation of bcl-2,Raf1 and VEGF due to phosphorylation by vinorelbine.Furthermore,we detected upregulation of p21Waf1,p27kip and Bak.The high energy radioisotopes induced DNA double strand breaks in tumour cells arresting synergistically with MT,depolymerizing vinorelbine their growth at the G2/M transition according to flow cytometry analysis.We detected externalisation of PS,depolarization of mitochondrial transmembrane potential ,activation of caspase-3,caspase-9,bax and DNA fragmentation.TEM exhibited irreversible D2 apoptotic signs forming apoptotic bodies which were phagocytosed by adjacent tumor cells leading to a bystander killing effect (BKE).BrdU and MTT exhibited inhibition of DNA synthesis and metabolic activity of treated tumour cells compared to untreated controls.

Conclusion:We have achieved to eradicate anaplastic thyroid Ca with combined chemoradioimmunotherapy after circumvention of chemo-and radioresistant mechanisms such as hypermethylation of oncosuppressor Gadd45 promoter and overexpression of antiapoptotic oncogenes such as bcl-2,Raf-1,EGF and angiogenic VEGF.