Sulindac Enhances the Killing of Cancer Cells Exposed to

Oxidative Stress
Louis Levin Andrew

INTRODUCTION RESULTS
Sulindac was one of the early non-steroidal anti-inflammatory drugs
(NSAIDs), which affect prostaglandin production by inhibiting
cyclooxygenases (COX) 1 and 2. Because of this attribute, Sulindac
has been of interest as a chemopreventive treatment for many
types of cancer, such as mouse urinary bladder cancer and colon
cancer. Sulindac is a pro-drug that must be converted to the
active COX-inhibitor, sulindac sulfide. It has been shown that
conversion of sulindac to sulindac sulfide can be catalyzed by
MsrA, one of the methionine sulfoxide reductase (Msr) group of
enzymes, and the Msr system plays an important role in protecting
cells against oxidative damage. As Sulindac is a substrate for MsrA,
oxidative damage is involved in the killing of tumor cells.

Figure 2 Figure 3

Figure 1: Structure of Sulindac

Materials and Methods

Experiment 1: the effect of Sulindac on the viability of cancer cells

in response to oxidative stress:
Lung cancer cells or colon cancer cells were incubated in the
presence or absence of 500 µM sulindac for 2 days. Cells were then Figure 5
cleaned to get rid of the free sulindac prior to incubation for 2 hr
with TBHP.
Figure 4
Experiment 2: the effect of Sulindac on the viability of cancer cells
in response to hydrogen peroxide:
Method similar to Experiment 1, only that TBHP is replaced with
hydrogen peroxide.

Experiment 3: the effect of other NSAIDs or sulindac sulfone on the

viability of lung cancer in response to oxidative stress:
Lung cancer cells were incubated in the presence or absence of
either 500 µM acetylsalicylic acid, 500 µM ibuprofen or 250 µM
sulindac sulfone for 2 days. Cells were then washed to remove the
free NSAID or sulindac sulfone prior to incubation for 2 hr with TBHP.
Experiment 4: Sulindac and sulindac sulfone protect normal lung
cells against oxidative stress:
Normal lung cells were incubated in TBHP for 48 hr in the presence
or absence of 500 µM sulindac or the presence or absence of 250
µM sulindac.
Experiment 5: Intracellular ROS levels in lung cancer cells
pretreated with sulindac followed by oxidative stress:
Lung cancer cells were treated with sulindac for 48 hours, exposed
to TBHP and then the intracellular ROS level was visualized using a
fluorescent dye
Experiment 6: Mitochondrial membrane potential as measured by
JC-1 distribution in lung cancer cells:
Lung cancer cells were treated with sulindac for 48 hours, exposed
to oxidative stress or treated with TBHP and then the disruption of
the disruption of mitochondrial disruption was visualized using JC-
1dye.
Figure 6 Figure 7
Each cancer cell line had a drop in cell viability of more than 80% in the
presence of TBHP after the pretreatment with sulindac (Figure 2A and 2B).
Each cancer cell line also had a drop in cell viability in the presence of
hydrogen peroxide following pretreatment with sulindac (Figure 3A and 3B).
Two NSAIDs, acetylsalicylic acid and ibuprofen did not raise the sensitivity of
cancer cells to oxidative stress (Figure 4A and 4B, respectively) whereas
pretreatment of lung cancer cells with sulindac sulfone (Figure 4C) did.
Since Incubation of normal lung cells with sulindac prior to exposure to TBHP
not only inhibited killing, but also sulindac protected it from oxidative stress
caused by TBHP (Figure 5A). This was also observed when the cells were
pretreated with sulindac sulfone (Figure 5B). Compared to untreated lung
cancer cells (Figure 6A), cells treated with sulindac alone (Figure 5B) or TBHP
alone (Figure 6C) showed a slight rise (in ROS levels based on appearance
of green fluorescence. However, lung cancer cells that were pretreated
with sulindac followed by an incubation with TBHP (Figure 6D) had a drastic
rise in intracellular green fluorescence compared to untreated cells. Relative
to untreated cells (Figure 7A) or cells treated with only sulindac (Figure 7B) or
TBHP alone (Figure 7C), sulindac pretreatment of lung cancer cells followed
by oxidative stress (Figure 7D) resulted in disruption of mitochondrial
membrane potential as evidenced by nearly a 20-fold increase in green
fluorescence.

Conclusion
Pretreatment of human colon and lung cancer cells with sulindac
improves killing by an oxidizing agent such as TBHP or hydrogen
peroxide. This effect does not involve cyclooxygenase (COX)
Citation
inhibition. However, under the conditions used, there is a significant
Marchetti M, Resnick L, Gamliel E, Kesaraju S, Weissbach H,
rise in reactive oxygen species (ROS) within the cancer cells and a
Binninger D (2009) Sulindac Enhances the Killing of Cancer Cells
loss of mitochondrial membrane potential, proving that cell death is
Exposed to Oxidative Stress. PLoS ONE 4(6): e5804.
due to apoptosis. In contrast, this improved killing was not observed
doi:10.1371/journal.pone.0005804
with normal lung or colon cells
Sulindac Enhances the Killing of Cancer Cells Exposed to
Oxidative Stress