A toxin conjugate containing transforming growth factor-alpha and ricin A specifically inhibits growth of A431 human epidermoid cancer cells.

Abstract

The inhibitory effect of human epidermoid cancer cells A431 caused by conjugate toxin containing transforming growth factor (TGF-alpha) and ricin A was studied. TGF-alpha is a protein with 50 amino acids that specifically binds and stimulates phosphorylation of cell surface epidermal growth factor receptor (EGFR) and, subsequently, triggers cell proliferation. TGF-alpha as a ligand for EGFR is internalized upon binding and decomposed within lysosome. Lectin ricin is contained in the castor bean plant. The lectin consists of two subunits, ricin A and B. Toxic ricin A binds to ribosome and inhibits protein synthesis of target cells. In view of the abundance of EGFR in human cancer cells, the receptor-mediated endocytosis with the conjugate toxin composed of TGF-alpha and ricin A was synthesized, purified and tested for growth inhibition in both normal and tumor cells. The cytotoxicity of the conjugate was studied within the range of 10(-12) and 10(-8) M and IC50 was found to be 10(-10) M for human A431 epidermoid cells that over-express EGFR. Compared to A431 cells, the brain metastatic variant of human Non-Small Cell Lung Cancer (NSCLC) H226Br squamous cells showed a reduced inhibitory effect. On the other hand, no inhibitory effect was found with other NSCLC cells studied and normal human lung cells because of the fewer available EGF binding sites on the surface of the cells. These results indicated that the amount of the available EGFR contributes to the cytotoxic effect on human cancer cells, thereby demonstrating involvement of the receptor-mediated endocytosis of the conjugate. The result from 12labeled EGF-mediated competition assay further demonstrated the specific inhibition activity conferred by TGF-alpha and ricin A conjugation. Due to poor recovery of the chemical conjugation, modification in the form of a recombinant toxin is needed for further in-depth studies.