ICACT, FIFTEENTH INTERNATIONAL CONGRESS ON ANTI-CANCER TREATMENT. February 9th-12th, 2004, Paris-France.

Pegylated-colloidal with linked anti-DNMTI/HDAC2 bispecific single-chain
Fv molecules (bs-ScFv) and encapsulated vinorelbine induces apoptosis in chemoresistant infiltrating (or invasive) ductal carcinoma of the breast (IDC) characterised by HDAC2 overexpression and 5'CpG island hypermethylation of growth regulators, signal transducers, tumour suppressor genes, invasion/metastasis suppressor genes,DNA repair geneshormone and kinase receptors, angiogenesis inhibitors, tumour antigens, GTP proteins and apoptotic genes.

Giannios J., Dept.of Clinical Oncology, GSHA, Michailakis E., Dept.of Clinical Oncology, GSHA, Alexandropoulos N., Dept.of Clinical Biochemistry and Genetics IH, Kononas T., Dept.of Surgery, IH and Xepapadakis G., Dept.of Surgical Oncology, MH, GR, EU.

IDC is characterised by intrinsic resistance to chemotherapy and mechanisms of this clinical resistance are poorly known and understood. Faure to trigger apoptotic cell death due to epigenomic modifications including changes in DNA methylation and histone acetylation which cause gene silencing is a contributing factor in chemoresistance of human breast malignancies. More analytically, DNMTI interacts with HDAC2 to repress transcription of growth regwators,signal transducers, tumor suppressor genes, inva- sion/ metastasis suppressors, DNA repair genes,angiogenesis suppressors,hormone kinase receptors, GTP proteins and tumor antigens interfering with therapeutic and diagnostic procedures.Our cohort consists of 126 samples of late stages III and IV of IDC surgically removed from chemoresistant patients. Samples were analysed by DMH n-iicroarrays for simultaneous analysis of many CpG island loci on one tumor at a timemethylation-spedfic PCRCHIP assayPCR- based LOH,RT-PCR,Northem blot and IHC.WE detected LOH at FHIT and promoter CpG island hypermethylation of the following genes: BRCAI, p73, RASSFIA, PACE4, NES- I/KLKIO, HSPA2, p33/INGIb, HMLHI, LOTI (PLAGLI/ZACI), TRAIL, HICI, MINT25, MINT31, hTR, pl6 (INK4A/MTSI/CDKN2), mda-7/il-24, TP53, SRP68, EVPL, ACOXI, FOXJ 1, CDK3 PRPSAP 1, TSP- 1, p I 4/ARF, ARHI, Nm23, Kissl, KAII, CADI, BRMSI, MKK4, RAR-02, APC, CDHI-E-cadherin, 14-3-3-a (stratifin), Twist, MAGE- 1, Cyclin D2 and MDGI. There was overexpression of DNMTI and HDAC2 suggesting a link between histone deacetylation,cytosine methylationjocal chromatin conden- sation with subsequent transcriptional repression of all the above mentioned genes. Concurrent methylation at the 5'- end of the regulatory region of multiple genes and other loci was seen in the IDC samples defining them as CpG island methylator phenotype (CIMP) positive. We treated IDC ceus with pegylated-liposomal formulation which had linked anti-DNMTI/HDAC2 bispecific single-chain Fv molecules (bS-SCFV) and encapsulated vinorelbine-tartrate which was protected from biological milieu interactions. This formula- tion was termed as inimunokposomal vinorelbine (ILV). Post-treatment, inhibition of HDAC2 and DNMTI block- ing the 5'CpG island methylation of hypermethylated genes resulted to transcriptional activation by upregulation of their MRNA. Re-expression of ARHI induced p2i Wafl/Cip I. Furthermore,there was histone hyperacetylation opening chromatin structure in which DNA is more loosely wrapped around the histones making it more receptive to interaction with transcription factors.Turnor suppressor gene re-expression combined with the microtubule depoly merizing action of vinorelbine inhibited metabolic activity and DNA synthesis of tumor ceus according to MTT and BrdU assays, respectively. There was TRAIL-induced apop- tosis which upregwated the expression levels of caspase-8 and r-aspase-3 and the pro-apoptotic genes such as Bid, Bim, Bax and Bakln contrast,anti-apoptotic genes such as Mcl- 1, Bcl-xl, XLAP were downregulated. There were changes in niitor-hondrial membrane permeability enhancing release of Smac/DIABLO and cyt-c. Apoptosis was confirmed with TdT-mediated-DUTP-biotin nick end labeling (TUNEL). TEM exhibited irreversible D2-apoptotic signs such as dis- integration of tumor cells to membrane bound small bodies which were phagocytosed by adjacent tumor cells leading to a bystander killing effect. Concluding, this therapeutic approach may revolutionize IDC treatment adding significantly to the current clinical armamentarium.