Open AccessSynthesis of saporin–antibody conjugates for targeting EpCAM positive tumour cells
Author(s) -
Athyala Prasanna Kumar,
Chitipothu Srujana,
Kanwar Jagat Rakesh,
Krishnakumar Subramanian,
Narayanan Janakiraman
Publication year - 2019
Publication title -
iet nanobiotechnology
Language(s) - English
Resource type - Journals
ISSN - 1751-875X
DOI - 10.1049/iet-nbt.2018.5222
Subject(s) - microbiology and biotechnology , flow cytometry , chemistry , apoptosis , conjugate , biology , biochemistry , mathematical analysis , mathematics
Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein involved in cell proliferation and differentiation. Ribosomal inactivating proteins derived from plants specifically target ribosomes and irreversibly inhibit protein synthesis. EpCAM antibody and saporin were conjugated using 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide hydrochloride/ N ‐hydroxysuccinimide chemistry. The mass of the conjugates were characterised using matrix‐assisted laser desorption ionisation (MALDI). The saporin–EpCAM (SAP–EpAB) conjugates were tested in‐vitro against MCF‐7 (breast cancer cells), WERI‐Rb1 (retinoblastoma) cells. The flow cytometry and fluorescence microscopy were performed to show the binding efficiency of SAP–EpAB conjugate. Whole transcriptome changes of sap‐conjugate treated cells were studied using affymetrix microarrays. MALDI‐TOF analysis and polyacrylamide gel electrophoresis confirmed the conjugation of SAP with EpCAM antibody. Flow cytometry and fluorescent microscopy analysis revealed the binding of SAP–EpAB conjugates to the MCF‐7, WERI‐Rb1 cells. Apoptosis assay by annexin‐V has shown an increased apoptotic and necrotic population in conjugate treated cells. MTT assay confirmed the tumour cell death and had shown the IC 50 value of 0.8 µg for conjugate in MCF‐7 (breast cancer cells), and 1 µg for WERI‐Rb1 (retinoblastoma) cells. The microarray analysis revealed downregulation of the tumourigenic genes and upregulation of pro‐apoptotic genes leading to apoptosis of tumour cells.