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.