Download Conjugation of aptamer-toxin for targeting tumour cells. Athyala

Conjugation of aptamer-toxin for targeting tumour cells.
Athyala Prasanna Kumar1, Subramanian Krishnakumar
Narayanan Janakiraman1.
2,1,
Jagat R. Kanwar3
1. Department of Nano Biotechnology, Vision Research Foundation, Sankara
Nethralaya, Chennai, India.
2. Larsen and Turbo Department of Ocular Pathology, Vision Research
Foundation, Sankara Nethralaya, Chennai, India.
3. Nanomedicine Laboratory of Immunology and Molecular Biomedical Research
(N-LIMBR), School of Medicine (SoM), Molecular and Medical Research
(MMR) Strategic Research centre, Faculty of Health, Deakin University,
Geelong Technology Precinct (GTP), Geelong, Victoria, Australia.
(Funded by Department of Biotechnology, Government of India, New Delhi.
BT/PR2285/MED/31/128/2011)
PURPOSE: Epithelial cell adhesion molecule (EpCAM) is a transmembrane
glycoprotein involved in cell signalling, migration, proliferation and differentiation. It
also serves as diagnostic marker for various cancers due to its role
in tumorogenesis and metastasis of carcinomas. In a conventional treatment of
cancer, the chemotherapic drugs and toxin molecules not only affect the cancer cells
but also the normal cells on their way. By conjugating the drug or toxin to the specific
tumor targeting ligand, helps in targeted therapy of tumor cells alone. In the present
study we have chosen a bacterial toxin Neocarzinostatin (NCS) which is a potential
DNA damaging agent and a plant toxin Saporin which acts on ribosomes leading to
the ribosomal inactivation, to conjugate with EpCAM RNA aptamer.
METHODS: We have found the IC50 value for native toxins molecules on
retinoblastoma and breast cancer cell lines using Resazurin (7-Hydroxy-3Hphenoxazin-3-one 10-oxide) reagent. Toxin molecule is being conjugated with the
aptamer using hetero-bifunctional cross linker (Sulfo-LC-SPDP). The conjugate was
further characterized with Native PAGE, MALDI-TOF, and UV-Vis spectrometry.
Binding affinity of native Aptamer and the conjugate was evaluated on cancer cell
lines with FACS and Fluorescent microscopy. Apoptosis, cell cycle assay was
performed to study the effect of targeted delivery on cancer cells.
RESULTS: Aptamer-toxin conjugate was characterized with Native PAGE, and it
revealed the shift in the gel with respect to native protein. MALDI-TOF analysis
showed increase mass for conjugate by 3.5 KDa with respect to native protein.
Conjugate as native Aptamer showed similar binding towards the EpCAM positive
cells, and the conjugates were able to target the EpCAM positive cells and shown
the effect on cell cycle arrest and Apoptosis.
CONCLUSION: In conclusion, we report that toxin-aptamer conjugates showed
improved targeting of cancer cells than the native toxins alone.