Amifostine May Mimic Antitumor Gene Therapy

CHICAGO—Amifostine (Ethyol) has a number of effects on transcription factors and may also mimic antitumor gene therapy by upregulating expression of manganese superoxide dismutase (MnSOD), according to David J. Grdina, MBA, PhD, professor of radiation and cellular oncology at the University of Chicago.

Transcription factors, such as NFkB, reside in the cytoplasm of the cell, Dr. Grdina explained. When the cell is stressed with oxidative damage or radiation, the transcription factor migrates from the cytoplasm into the nucleus, binds to certain DNA sequences, and turns genes on. Dr. Grdina said that recent studies have shown that reducing agents, which add electrons to a compound, can also activate NFkB. N-acetylcysteine (NAC), like amifostine, turns on NFkB leading to an enhancement of a number of genes.

"You need millimolar amounts of other thiols to induce NFkB, but with amifostine you get a very nice induction at 40 µmol/L, and with only a 30 minute exposure. This is a level that’s achieved in the blood stream," Dr. Grdina said.

Cell Survival Cascade

NFkB is important in the inflammatory cascade and in the cell survival cascade. Turning on a transcription factor, however, does not necessarily mean turning on all the genes in that transcription factor’s domain. "We used DNA chip technology to identify between 55 and 60 genes that have NFkB responsive elements. Only four were activated by amifostine. One of those four (the c-myc oncogene) was actually repressed," he said.

One key gene that NFkB turns on is the intracellular antioxidant MnSOD. Gene therapy researchers have transfected MnSOD and achieved protection of normal cells from irradiation or oxidative stress.

"In normal human microvascular endothelial cells, we found that MnSOD gene expression increases 14 hours following exposure to amifostine. It seems to peak at 20 hours and continues to about 24 hours," Dr. Grdina said.

"In these same cells," he continued, "protein expression increases between five- and tenfold. Amifostine effectively increases MnSOD gene expression in normal cells by about twofold and protein levels between five- and tenfold. Gene therapy increases MnSOD expression by one and one half to twofold and protein levels between three- and five-fold. Gene therapy based on MnSOD is duplicated with amifostine and leaves a legacy of MnSOD gene expression and protein levels at 14 and 24 hours after exposure to the drug."

Tumor Suppressor Gene

There is some evidence suggesting that MnSOD is acting as a tumor suppressor gene. "As cells go from normal into malignancy, they lose MnSOD gene expression and protein levels. When you increase MnSOD in these cells, they lose their malignant phenotype and their ability to metastasize. Human squamous cell carcinoma and prostate cancer transfected with MnSOD start to lose their malignant phenotype. Mouse tumor cells lose their ability to metastasize," Dr. Grdina said.

This may explain why amifostine was able to inhibit spontaneous metastases in three mouse tumor lines. "Amifostine treatment was associated with a significant reduction in the proportion of mice developing metastases and also with a lower average number of metastases in the mice that develop them," Dr. Grdina said.

"My conclusions are that NFkB is activated in both normal and malignant cells by the thiol and disulfide forms of amifostine. Neither catalase nor pyruvate inhibits this activation, suggesting that hydrogen peroxide (H₂O₂) is not involved in this process," Dr. Grdina said.

"Not all genes that have NFkB responsive elements in their promoter regions will be turned on, as is shown by an increase in expression of MnSOD with no change in ICAM-1 expression. Furthermore, amifostine does not affect either E-selectin or human vascular endothelial growth factor (VEGF) protein levels secreted by exposed cells," he added. "We think that NFkB activation may represent an important biomarker for use in the evaluation of novel cytoprotective agents."