Simvastatin enhances the effects of 5-fluorouracil (5-FU) on human colorectal cancer cell lines. In vitro studies found that pretreatment with simvastatin sensitizes two colorectal cancer cell lines to 5-FU-induced apoptosis in a dose-dependent manner. The combination of simvastatin and 5-FU was more effective at inhibiting cell growth and inducing apoptosis than either treatment alone. This effect involves downregulation of anti-apoptotic proteins Mcl-1 and Bcl-2 as well as increased reactive oxygen species production. Further research is recommended to study the molecular mechanisms and potential in vivo effects.

1. in vitro Effects of Simvastatin and
5-Fluorouracil on
Human Colorectal Cancer Cell Lines
Maysa’a O. Al-Bataineh

2. Colorectal Cancer (CRC)
• Is a type of cancer that form in the tissue of the colon,
the rectum, or both.
• Globally, CRC is the third most common cancer and
the fourth most common cancer cause of death,
Whereas in Jordan, CRC is the second most common
cancer type after breast cancer.
• The median age at diagnose is 61 yrs for both genders.

3. CRC risk
factors
Old age.
Personal history of adenomatous polyps
or inflammatory bowel disease.
Family history of CRC or
adenomatous polyps.
Inherited disorders
(like lynch syndrome).
Environmental factors,
which includes:
Diet
Smoking
low physical
activity
obesity

4. Pic from “http://iwandahnial.wordpress.com/2010/11/16/understanding-colorectal-cancer/”
CRC stages
Tumor stage Spreading
Stage0 (carcinoma in situ) Cancer found in the innermost lining of the colon only
Stage I
Cancer has grown through the mucosa and invaded the
muscular layer
Stage II Tumor extended through the wall of the colon
Stage III Spreading of the cancer to nearby lymph nodes
Stage IV Cancer spreading to other parts like lung or liver

5. CRC
treatment
Surgery
Chemotherapy
FOLFOX
FOLFIRI
Capecitabine
irinnotecan
5-FU with
Leucovorine
Radiation
Targeted therapy Like, Cetuximab
and Bevacizumab
Combination of
one or more

6. 5-Flurouracil (5-FU)
• 5-FU is an analogue of uracil with a fluorine atom at the
C5 position.
• Plays an important role in the treatment of many cancer
types including CRC.
• Clinical use of 5-FU alone in cancer treatment became
limited due to drug resistance.
(Valeriote and Santelli, 1984).

7. Simvastain (Sim)
• Sim is a member of the statin drug family.
• Statins are 3-hydroxy-3-methylglutaryl-coenzyme A
(HMG-CoA) reductase inhibtors
• It is currently widely used as safe and well tolerated
drug for the treatment of :
Hypercholesterolemia
Atherosclerosis
Coronary heart disease and stroke

8. Epidemiological and meta analyses reports on statin use
showed that either is:
1. No helpful or harmful effect on the risk of cancer.
2. A reduction in cancer incidence among statin users.
3. Increases in cancer incidents.
• Whereas in vitro study on Sim and other statins
showed anti-tumor effect against a wide range of
cancer cell lines including:
- breast - prostate - bladder - CRC
- hepatic - leukemia - pancreas - skin - lung

9. Rationale
 5-FU is still used in the treatment of CRC since its
discovery in 1957, but it has many drawbacks:
The relative toxicity of 5-FU,
lack of selectivity towards tumors,
 its action efficiency limitation due to its short half life,
and the resistance of some tumors,
 So thus, research is focused on findings ways to
overcome these obstacles or diminish its effect, and one
way to accomplish this is by using other compound that
can enhance the activity of 5-FU; i.e. show synergism
with 5-FU.

10. Objective
• Investigate the effect of simvastatin in
combination with 5-FU in stimulating apoptotic
response in CRC cells in vitro.
• If there was a response whether it will be dose
dependent or not.
• Studying the molecular mechanism underlying
the response of the combined treatment
(simvastatin and 5-FU).

11. Methodology

12. Cell harvest and count
HCT116 or SW480 cell
culture
Harvest the cells by
trypsinization
after 72hrs (70-80%)
Cells is ready to be
use in the tests
Determine cell concentration in
the sample using hemocytometer

13. Cell viability assay
5000 cells well of HCT116 or SW480
with DMEM
• Resulting formazan
solubilized with 100µl DMSO
•Absorbance read by ELISA
microplate reader at 540nm.
72 hr incubation with
the treatment
•Remove media.
•Add 100µl MTT dye
solution
2 hr incubation
37 ̊ C

14. Apoptosis assay
1.5*105 cells well
HCT116 or SW480 with 1ml DMEM
Media
removed
Wash
with PBS
1 ml propidium iodide stain
30 min.
incubation
Pipette off cells
and stain
Incubation overnight then analyzed by
flowcytometry .
excitation at 488 nm and emission at
617nm
72 hr incubation with
the treatment

15. Caspase dependency
1.5*105 cells well
HCT116 or SW480 with 1ml DMEM
1 ml propidium iodide stain
30 min.
incubation
Pipette off cells
and stain
Incubation overnight then analyzed by
flowcytometry .
excitation at 488 nm and emission at
617nm
72 hr incubation with
the treatment
Z-VAD added 2hr at 20 µM before
each treatment

16. ROS production
1.5*105 cells well
HCT116 or SW480 with 1ml DMEM
Harvested by trypsinization
Supernatant discarded.
500µl PBS and 10µl of dichloro-dihydro-
fluorescein diacetate (DCFH-DA)
30 min.
incubation
24, 48, 72 hr
incubation with the
treatment
Centrifugation
5 min
1700 rmp
500µl PBS
Centrifugation
5 min
1700 rmp
Supernatant discarded.
500µl PBS added
Intensity measured by Flowcytometry
(exc. 485 nm, emission 525 nm)

17. Western and Immunoblotting
4.5*105 cellswell in 6-well
plate
24 hr after
treatment
Harvest by trypsinization
Cells lysed with
triton X-100 based
lyses buffer
Protein concentration by
Bradford assay
Protein samples electrophoresed
on SDS-PAGE (2.30 hr, 130 V)
Transfer to nitro-
cellulose membrane
Block for 1hr with
blocking buffer (5%
BSA)

18. Membrane washed with TTBS
buffer after the blocking
Incubated with
primary antibodies
over night at 4 ̊C
Wash 5
times with
TTBS
Incubated with secondary
antibodies for 1 hr
Wash 3
times with
TTBSVisualize with diaminobenzidine
(DAB) and Nickel (II) chloride (NiCl2 )

19. The results

20. Simvastatin inhibits CRC cells growth
50
60
70
80
90
100
110
-ve S10 S25 S50 S75
CellViability%
Treatment
HCT 116
SW480
*
*
•-ve ; control
•S10, 25, 50, 75; simvastatin 10µM, 25 µM, 50 µM, 75 µM

21. Simvastatin sensitized CRC cells to 5-FU
induced growth inhibition
Concentration
Cell viability %
HCT116 SW480
Control 100 100
5-FU 35 65
Simvastatin 10 µM + 5-FU 29.4 54.4
Simvastatin 25 µM + 5-FU 26.5 53.2
Simvastatin 50 µM + 5-FU 20.3* 17.8*
Simvastatin 75 µM + 5-FU 20.3* 18*
* indicate that the change was highly significant p-value <0.0001

22. Simvastatin sensitizes CRC cells to 5-FU
by induction of apoptosis
0%
10%
20%
30%
40%
50%
60%
70%
control 5-FU S50 S50 +5-FU
%ofapoptoiccells
Treatment
SW480
HTC116
**
* *
*
* *
**
* Significant ( p value < 0.05 – 0.01 ) ** p value < 0.007
5-FU; 5-flurouracil
S50; simvastatin
50µM

23. Sensitization of CRC cells to 5-FU-induced
apoptosis by simvastatin is caspase-independent
0%
10%
20%
30%
40%
50%
60%
70%
80%
Apoptoticcells%
HT116*
*

24. Simvastatin sensitized CRC cells to 5-FU
induced cytotoxicity by induction of ROS
species
0
0.5
1
1.5
2
0 24 hrs 48 hrs 72 hrs
MFI
Hours after addition of 5-FU
HCT 116
Fluorescent intensity (MFI)
was shown as the ratio of
geometric mean fluorescence
intensity of the test sample
and the corresponding
control.

25. Simvastatin down regulates Mcl-1 and Bcl-2
Mcl-1 40KDa
Bcl-2 25KDa
Bax 23KDa
Bak 23KDa
GAPDH 37KDa
Simvastatin - + - + - + - +
5-FU - - + + - - + +
HCT116 SW480

26. 0 0.2 0.4 0.6 0.8 1 1.2 1.4
Bak
Bax
Bcl2
Mcl2
HCT116 cell line
0 0.3 0.6 0.9 1.2 1.5 1.8
Bak
Bax
Bcl2
Mcl2
Relative density
SW480 cell line
Done by Image Studio Lite
version 5.0

27. Conclusion
• To the best of my knowledge, this is the first time to provide evidence that
simvastatin pretreatment sensitizes CRC cells to 5-FU induced apoptosis.
• The results of the study strongly suggest that down-regulation of Mcl-1 and
Bcl-2 by simvastatin and 5-FU combination leads to sensitize CRC to
apoptosis.
• Our results showed the involvement of ROS generation in sensitizing
HCT116 cell line to 5-FU induced cytotoxicity by simvastatin pretreatment.

28. Future Recommendation
• Study the effect of simvastatin and 5-FU on the expression of other proteins
important in cell survival signaling like GRP-78.
• Study the expression of Mcl-1 and Bcl-2 with other proteins at transcriptional
level using real-time PCR.
• Using animal model to study the effect of simvastatin and 5-FU combined
treatment in vivo.
• Study the effect of simvastatin and 5-FU combined treatment on CRC cells
ability of invasion and metastasization.
• Study the effect of other statin, like cervastatin and fluvastatin, in
combination with 5-FU-induced apoptosis on CRC cells.

29. Acknowledgment
• I express my heartiest gratitude and indebtness to my Co-
advisor Dr.Nizar Mhaidat.
• My gratitude and thanks to my main supervisor Dr. Emad
Malkawi for all his help.
• I'm extremely thankful for my colleagues, Aseel Mhaidat
and Ahmed Al-Zyoud, big thanks for your efforts; I
wouldn't have been able to complete this project without
your presence
• my friends Esraa Khader and Aseel Rataan and Hussien
Alyosef.
• My thanks also to my friends Ala’a, Noura, Sewar, Ruba,
and Yasmine.

30. Thank you for listening