Andrew B. Adams and Dr. Steven G. Wood, Chemistry and Biochemistry
Recently more emphasis has been placed on developing anti-cancer agents with greater specificity and fewer side effects. Immunotoxins and their relatives, mitotoxins, have arisen as possible answers. Mitotoxins are composed of mitogens coupled to plant or bacterial toxins. The mitotoxin under investigation is the EGF-Pyrularia Thionin combination.
Pyrularia Thionin (P-Tox) is a 47 amino acid peptide toxin isolated from the nut of Pyrularia pubera, a parasitic tree indigenous to the Southeastern United States. P-Tox is a member of the thionin family, which also includes other natural toxins isolated from wheat, barley, rye, and cabbage. This family of toxins, including P-Tox, is known to attach to the cell membrane and disrupt its integrity thereby affecting its permeability.
Epidermal growth factor (EGF) is a growth factor that binds to a specific receptor on the outside of the membrane and stimulates the cell internally to divide and grow. Cancer cells are known to have a higher number of receptors for EGF than non-cancer cells; thus there is the possibility of selective disruption of the cancer cell membrane and/or disruption of cancer cell signal transduction through the use of a mitotoxin composed of EGF and P-Tox.
The conjugate of EGF and P-Tox was made without difficulty using the commercially available linker N-Succinimidyl-39[2-pyridyldithio] propionate (SPDP). The progress of the conjugation reaction was followed and characterized using U.V. spectroscopy techniques.
The MCF-7 and MDA-MB-468 cell lines were used in evaluation of the proposed mitotoxin. The MCF-7 cell line has a mitogenic response to EGF where as the MDA-MB- 468 cell line has an apoptotic response to EGF. Both cell lines were evaluated using a standard MTT bioassay for their response to EGF, P-Tox, EGF-P-Tox conjugate, and EGF/P-Tox unconjugated at varying concentrations. A standard 3H-Thymidine incorporation assay was used to evaluate the apoptotic response of the MDA-MB-468 cell line to EGF and the EGF-P-Tox conjugate.
A MTT bioassay was performed on the MCF-7 cell line. The results indicated that the conjugate did eliminate the mitogenic signal of EGF. The results also revealed that the conjugate was toxic (conc 100 ng/ml), but no more than P-Tox alone. Upon assaying the MCF-7 cell line for disruption of the EGF mitogenic signal, significant morphological differences were found in the method of death. The death caused by P-Tox alone appeared to be necrotic in contrast to the type of death induced by the conjugate which appeared to be possibly apoptotic in nature. In order to inspect this phenomena more closely the MDAMB-468 cell line was purchased.A 3H-Thymidine incorporation assay was performed to evaluate changes in DNA synthesis in hopes of describing the apoptotic event triggered by EGF in the MDA-MB-468 cell line and comparing it to the EGF-P-Tox conjugate.
The results were intriguing but not definitive. The results indicate that EGF halts 3HThymidine incorporation between 60 and 80 hours. The cells subjected to the EGF-P-Tox conjugate seemed to continue to incorporate the radioactive precursor even at 100 hrs. There was not enough evidence to support the theory that the conjugate disrupted the apoptotic signal of EGF.
We are confident that we successfully chemically linked EGF to P-Tox, unfortunately it appears that the conjugate is no more toxic than P-Tox alone. This correlates with the findings of others who successfully fabricated an immunotoxin composed of P-Tox and an CD5 antibody but found that it was no more toxic than P-Tox alone. This does however raise some interesting questions regarding the method of death of P-Tox. Whether death is density-dependent or dependent simply upon a critical concentration. Still questions remain unanswered concerning where the conjugate ends up, is it endocytosed with the EGF receptor or does it remain on the outside of the cell membrane? We plan to answer these questions and others by using fluorescent microscopy and attempting to follow the movement of the conjugate as it binds to the receptor.