CHICAGOAmifostine (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.
like amifostine, turns on NFkB leading to an enhancement of a number of
"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
"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."