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Retinitis pigmentosa (RP) is a group of inherited
disorders that cause degeneration of retinal photoreceptor cells.
Animal studies indicate that apoptosis, or programmed cell death,
may be involved in such cell degeneration. Bile acids, such as tauroursode-oxycholic
acid (TUDCA), have been shown to block apoptosis in a number of
other neurogenic disorders. Therefore, it is reasonable to assume
that TUDCA may also prevent apoptosis in RP. In this project, the
effect of TUDCA was examined on apoptosis induced by various pro-apoptotic
agents in human WERI-Rb-1 retinoblastoma cells, which are considered
to be photoreceptor-like. The hypothesis was that TUDCA will have
a significant inhibitory effect on the induction of apoptosis in
human WERI-Rb-1 retinoblastoma cells. Two different pro-apoptotic
agents were used in this project: tert-butyl hydroperoxide (tBH)
and camptothecin. Human retinoblastoma cells preincubated with different
concentrations of TUDCA were treated with either tBH or camptothecin.
Apoptosis was assessed by a fluorometric TdT-mediated dUTP Nick-End
Labeling (TUNEL) system. Preincubation of cells with increasing
concentrations of TUDCA resulted in decreasing numbers of apoptotic
cells, regardless of which apoptotic agent was used. The optimal
concentration of TUDCA with the maximal protective effect was found
to be 100uM in both tBH- and camptothecin-treated cells. The hypothesis
of this project is accepted as TUDCA is found to have a significant
dosage-dependent inhibitory effect on the induction of apoptosis
in human WERI-Rb-1 retinoblastoma cells. The mechanism of action
of TUDCA is not specific to its antioxidant effect, as it also blocks
the apoptotic effect of camptothecin. Unlike tBH, camptothecin is
not an oxidant. Rather, its mechanism of action is through the inhibition
of DNA topoisomerase during DNA replication. TUDCA is relatively
inexpensive, nontoxic, and easy to administer. It is also FDA-approved
for the treatment of Primary Biliary Cirrhosis. Therefore, the potential
of TUDCA as a pharmacological agent to treat RP is relatively high.
Many animal model studies suggest that apoptosis,
characterized by cellular shrinking, nuclear condensation and DNA
fragmentation, and eventual cellular break-up, is an end-stage event
in photoreceptor cell death in human retinitis pigmentosa (RP).
Both mitochondrial (Van Loo et al. 2002) and non-mitochondrial (Mathiasen
and Jaattela 2002) apoptotic pathways have been proposed. TUDCA
prevents neurodegeneration in a number of animal model as well as
neuronal cell studies of Huntington's Disease (Keene et al. 2002),
Parkinson's Disease (Duan et al. 2002), and acute stroke (Rodrigues
et al. 2002). In these studies, TUDCA appears to act at several
points in the apoptotic pathway. It directly inhibits mitochondrial
permeability transition, prevents alterations in DeltaPsi(m), and
suppresses mitochondrial release of cytochrome c. TUDCA blocks reactive
oxygen intermediate production and, thererfore, acts as an antioxidant.
In addition, TUDCA blocks caspase activation, nuclear enzyme PARP
activation, and the translocation of the pro-apoptotic molecule
BAX from the cytosol to the mitochondria. Therefore, if the apoptotic
pathway of neural cells in RP is similar to that in other neuronal
tissues, it is reasonable to propose that TUDCA may also prevent
apoptosis of human retinoblastoma cells involved in RP.
Apoptosis was induced by using two different agents
in separate experiments: tert-butyl hydroperoxide (tBH) and camptothecin.
In the first set of experiments, human WERI-Rb-1 retinoblastoma
cells preincubated with different concentrations of TUDCA (25uM,
50uM, and 100uM) for 30 minutes at 37 degrees Celsius were treated
with a fixed concentration of tBH (200uM). These starting concentrations
were based on the values from the literature (Keene et al. 2002;
Rodrigues et al. 2002). In the second experimental set, cells preincubated
with the same concentrations of TUDCA were treated with 200mM of
camptothecin. Following a 16-hour incubation period with either
tBH or camptothecin, the levels of apoptosis for all of the sets
of experiments were measured by a fluorometric TdT-mediated dUTP
Nick-End Labeling (TUNEL) system. The TUNEL system measures the
fragmented DNA of apoptotic cells by catalytically incorporating
fluorescein-12-dUTP at 3'-OH DNA ends using the Terminal Deoxynucleotidyl
Transferase, Recombinant, enzyme (rTdT). Apoptotic (stained yellow)
and non-apoptotic (stained red) cells were visualized with a confocal
microscope and quantitated by using NIH Image software. After performing
all of these experimental sets, the effect of TUDCA on apoptosis
induced by tBH or camptothecin in human retinoblastoma cells was
observed. In addition, the TUDCA concentration needed to obtain
protection against apoptosis was optimized. Cell counts were averaged
for each group,and simple ANOVAs were performed with Student-Newman-Keuls
post hoc analysis.
Untreated human WERI-Rb-1 retinoblastoma cells had
approximately 77% non-apoptotic cells. When three different concentrations
of TUDCA (25uM, 50uM, and 100uM) alone were added to these cells,
there was not a significant (p > 0.05) change in the mean percentage
of non-apoptotic cells (75%-82% of non-apoptotic cells; Figure 2).
The induction of apoptosis by tBH led to a significant (p < 0.001)
decrease in the percentage of non-apoptotic cells (40% of non-apoptotic
cells; Figure 2). However, preincubating these cells with increasing
concentrations of TUDCA increased the mean percentage of non-apoptotic
cells in a dose-dependent manner (39%, 58%, and 73% of non-apoptotic
cells, respectively; Figure 2). Camptothecin-treated retinoblastoma
cells exhibited similar findings. There was a significant (p <
0.001) decrease in the percentage of non-apoptotic cells in the
group treated with camptothecin alone (45% of non-apoptotic cells;
Figure 2). However, preincubating these cells with increasing concentrations
of TUDCA increased the mean percentage of non-apoptotic cells in
a dose-dependent manner (55%, 66%, and 79% of non-apoptotic cells,
respectively; Figure 2).
1. The hypothesis of this project is accepted as TUDCA has a significant
inhibitory effect on the induction of apoptosis in human WERI-Rb-1
retinoblastoma cells.
2. Increasing concentrations of TUDCA result in increasing prevention
of apoptosis.
3. The mechanism of action of TUDCA is not specific to its antioxidant
effect, as it also blocks the apoptotic effect of camptothecin.
Unlike tBH, camptothecin is not an oxidant, but rather acts by inhibiting
the action of DNA topoisomerase during DNA replication.
4. TUDCA is relatively inexpensive, nontoxic, and easy to administer.
It is FDA-approved for the treatment of Primary Biliary Cirrhosis.
Therefore, the potential of TUDCA as a pharmacological agent to
treat RP is relatively high.
Future Studies:
- Explore the effects of even more different concentrations of TUDCA
on the induction of apoptosis in human WERI-Rb-1 retinoblastoma
cells as well as other cell lines.
- Investigate the effects of other apoptosis induction modalities.
- Determine whether TUDCA has the ability to arrest, rather than
prevent, the induction of apoptosis in human retinoblastoma cells.
- Explore the effects of TUDCA in primary cell cultures in an in
vivo animal model.
Supported by the Student Inquiry Research Experience award, the
Howard Hughes Medical Institute, NIH NEI Core Grant EY06360, Emory's
Center for Alternative Medicine, and the Foundation Fighting Blindness.
Apoptosis is a process of cell death. This may be involved in many
human eye disorders. A bile acid, TUDCA, has been shown to prevent
apoptosis in many other human diseases. If the apoptosis in human
eye disorders is similar to that in other diseases, then TUDCA may
stop apoptosis in human eye disorders. Based on the data collected
from the experiments, TUDCA prevents apoptosis in human eye cells
involved in many disorders. Increasing amounts of TUDCA cause greater
protection of the cells from apoptosis. The exact mechanism of action
of TUDCA is not known at this time.
TdT-mediated dUTP Nick-End Labeling (TUNEL) system, confocal microscope,
NIH Image software.
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