University of Southern California Dissertations and Theses

Examining perillyl alcochol in glioblastoma treatment and relating its effect to endoplasmic reticulum stress response to further advance its usage in combination treatment with dimethyl-celecoxib

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Examining perillyl alcochol in glioblastoma treatment and relating its effect to endoplasmic reticulum stress response to further advance its usage in combination treatment with dimethyl-celecoxib

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Content
EXAMINING PERILLYL ALCOCHOL IN GLIOBLASTOMA TREATMENT AND
RELATING ITS EFFECT TO ENDOPLASMIC RETICULUM STRESS RESPONSE
TO FURTHER ADVANCE ITS USAGE IN COMBINATION TREATMENT WITH
DIMETHYL-CELECOXIB

by
Long-Chieh David Wang

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)

December 2009

Copyright 2009 Long-Chieh David Wang

ii
Table of Contents

List of Figures……………………………………………..………………...……………iii

Acknowledgements.…...……………………………………………………………….…iv

Abstract…………………………………………………………………………………....1

Introduction………………………………………………………………………………..2

Materials….……………………………………………………………………………….8

Methods……………………………………………………………………………………9

Results……………………………………………………………………………………12

Discussion………………………………………………………………………………..28

Numerical Bibliography.…………………………………………………………………32

Bibliography…...…………...……………...……………………………………..………34

iii
List of Figures

Figure 1. Chemical structure of perillyl alcohol and DMC…………………...................12

Figure 2. MTT assays of four glioblastoma cell lines…………………………………...14

Figure 3. Photos of glioblastoma cell-lines treated with POH for 24 hours…….……….15

Figure 4. Colony formation assay with U251 cells treated with POH………….………..17

Figure 5. Western blot of ER specific marker GRP78 on U251 cells…………………...18

Figure 6. Western blot of ER specific marker CHOP on U251 cells…………………....19

Figure 7. MTT assay of DMC/POH of four glioblastoma cell-lines…………………21-22

Figure 8. Colony formation assay on U251 cells treated with DMC, POH and DMC +
POH…………………………………………………………………………...24

Figure 9. Photos of U251 and U87 cells treated with single and dual treatment
after 18 hours………………………………………………………………….25

Figure 10. Western blot analysis of U251 cells treated with DMC and
POH alone and combined……………………………………………………27

iv
Acknowledgements

I would like to thank my advisor, Dr. Thomas Chen, for showing me the right
direction and giving me advice on the thesis and providing valuable critique and
feedbacks on all the experiments. Also, I would want to thank Dr. Encouse Golden and
Dr. Weijun Wang for instructions and providing technical support in the lab; Dr. Hee-
Yeon Cho for troubleshooting problems. Last but not least, I appreciate the Glioma
Research Group, Dr. Axel H. Schönthal, Dr. Stan Louie, and Dr. Florance Hofman for
their constant input and guidance to help me complete this thesis.

1
Abstract

Despite improvement in recent surgery, radiation, and chemotherapy, the
prognosis for patients with malignant glioma has not improved. Therefore, it’s even more
urgent to search for new chemotherapeutic drugs. The aim of this project is to investigate
the mechanism of perillyl alcohol (POH), an old chemotherapeutic agent, which has been
recently used as intra-nasal inhalation in malignant glioma treatment. In this thesis, I
document that POH alone, and combined with Dimethyl-celecoxib (DMC), shows
significant anti-tumor activity in-vitro and triggers the induction of Glucose Related
Protein 78 (GRP78), an anti-apoptotic protein that is elevated to deal with stress
response. In addition, I also show induction of CCAAT/enhancer binding protein, or
(CHOP), a pro-apoptotic protein signal for apoptosis, under the same treatments. These
findings imply that POH alone generates Endoplasmic reticulum (ER) stress and forces
the tumor cells to enter apoptosis, and when combined with DMC the effect is
synergistic
a
. This is particularly exciting due to the fact that POH can enter the brain via
nasal inhalation and thus providing the patients with an effective and yet non-invasive
treatment.

a
Synergism is said to occur when the combined effects of two agonists exceed that
predicted by the individual actions of these compounds (i.e., the resulting effect is more than
additive).
2
Introduction

Glioma is a type of cancer that starts in the brain or spine. It arises from glial
cells and the most common site of glioma is the brain. Glioblastoma is the most
malignant and results in most severe symptoms, including gait disturbance (61%),
headache (44%), weakness of the

limbs (42%) and diplopia (40%). Four patterns were
identified

on MRI, representing non-enhancing, diffusely infiltrative tumors

(50%),
contrast-enhancing localized masses (31%), isolated tectal

tumors (8%) and other patterns
(11%). Treatment consisted of

partial resection (8%), radiotherapy (94%) and
chemotherapy (Temozolomide)

(56%). Glioblastoma tumors are highly resistant to
treatment and the patients have a median survival time of 11.2 months.
1

ER stress and its relation to GRP78 and CHOP production.

The endoplasmic reticulum (ER) is an eukaryotic organelle that consists of
extensive membrane network held together by cytoskeleton. It serves many general
functions, including the facilitation of protein folding and the transport of synthesized
proteins. ER is vital to secretory and membrane protein synthesis and is responsible for
maintaining a balance level of proteins within human cells.
2

Newly formed polypeptides marked by ER signaling peptide will travel to ER
from cytosol to be translated, modified, and translocated and become either secretory
proteins into the ER lumen or integrated as membranous proteins. Unfolded polypeptide
or random coil, form by a linear chain of amino acids, began to interact with each other
and the neighboring environment as they exist the ribosome. Protein folding is the
physical process by which a polypeptide folds into its characteristic and functional three-
dimensional structure from random coil through interactions of amino acid sequence.
3
The general trend is that the hydrophilic peptides are on the protein surface and the
hydrophobic peptides are buried internally in the protein. While these macromolecules
may be considered “self-folding” through their interaction within the amino acids and the
environment such as salt concentration and temperature, the process of folding also
depends heavily on the presence of molecular chaperones – GRP78.
GRP78, also known as Bip, is a central regulator of endoplasmic reticulum (ER)
function due to its roles in protein folding and assembly, targeting misfolded protein for
degradation, monitoring ER Ca
2+
-binding, and controlling the activation of trans-
membrane ER stress sensors. When the nucleotide-binding domain of GRP78 interacts
with ATP, its substrate-binding domain can bind with unfolded or misfolded protein.
Through the hydrolysis of ATP, the interaction between GRP78 and the proteins can
promote oxidation of disulfide bonds and rearrangement to ensure that the correct protein
conformation is established. As the level of misfolded proteins accumulates due to
hypoxia, low glucose or chemotherapeutic agents, the cells undergo ER stress and up-
regulate GRP78 to assist in proper protein folding.
3

Interestingly, when the cells experience prolonged elevated levels of GRP78
(indicating ER stress), the cells initiate programed cell death. ER stress associated
programed cell death is very specific.
4
Cleavage of caspase-4 followed by induction of
CCAAT/enhancer binding protein, or CHOP, is the precise pathway in ER stress
mediated cell death. CHOP is a pro-apoptotic protein that serves as a DNA binding
protein, suppressing the transcription of mRNA encoding for protein BCL-2, which is an
anti-apoptotic protein, thus favoring the cell to enter the pro-apoptotic pathway.
5

4
Due to ER’s crucial role in protein synthesis, generating ER stress has thought to
be favorable target in cancer therapy. From monitoring the level of GRP78 and the
production of CHOP, one can determine weather or not a cell is undergoing ER stress
after treating it with chemotherapeutic agent. By understanding how that
chemotherapeutic agent cause apoptosis in glioma cells, one can expand its usage and
efficiency.
Perillyl alcohol background
Perillyl alcohol (POH), also known as p-metha, 1,7-diene-6-ol or 4-isopropenyl-
cyclohexenecarbinol, is a monoterpene, which is isolated from the essential oils of
lavender, spearmint, cherries, peppermint, celery seeds, sage, cranberries, and various
other plants. In animal studies, POH has been demonstrated to cause tumor regression in
liver and pancreatic tumors.
6,7
Gould and Haag at the University of Wisconsin conducted
two separate studies in mice with mammary tumors induced by the carcinogen 7,12-
Dimethylbenz(a)anthracen (DMBA).
8
In the first study, POH diet (2.5% w/w) and a
control group were monitored for three weeks. At the end of the third week, 22 out of 27
mice (81%) in 2.5% POH diet resulted in complete regression of the primary tumors. In
the second study, the mice were randomly given 0.0, 0.5, 1.0, 1.5 or a 2.0 percent Perillyl
alcohol diet for 15 weeks with mammary tumors of approximate equal size (10mm in
diameter or greater). The 2% diet showed the most promising response with 10 of 20
with complete regression and 5 of 20 with partial regression. In addition to introducing
apoptosis in tumor cells, POH does not seem to exert toxicity on normal cells, and there
has been data showing that it can actually revert tumor cells back to a differentiated
5
state.
9
Its promising effect leads to possible application as a human chemotherapeutic
agent for neuroblastoma, and prostate and colon cancer.
In one phase I trial, a total of eighteen patients with various tumor types including
prostate, ovarian, and breast cancer, were treated with escalating doses of POH beginning
at 0.8 g/m
2
TID (Three times a day). Four, seven and seven patients completed three
months of oral POH therapy at the respective doses of 0.8, 1.6 and 2.4 g/m
2
TID. Two
patients at the highest dose experienced grade 3 nausea and vomiting, and two patients
experienced grade 2 nausea and vomiting. One patient at the highest dose experienced
grade 2 diarrhea. However, the medium and the low dose patients experience much
milder GI symptoms. In fact, only one patient out of eleven experienced grade 2 nausea
and vomiting.
9

The animals in various studies which had tumor regression were given food
mixed with 2% POH or greater and were allowed to feed ad libitum. A 2% POH diet
provides approximately 1.76 g POH/kg/day, which is equivalent to 20 g/day for a 70-kg
human adult. The nontoxic dose of 1.6 g/m
2
of POH administered five times a day to
simulate ad libitum provides about 20 g/day and may then yield results in humans similar
to the animal studies. Currently a phase II trial is being conducted in patients with
ovarian cancer using this dosing schedule to test the hypothesis.
8

However, POH’s biggest advantages is its ability to assist various existing known
chemotherapeutical agents, such as Dimethyl-celecoxib, and its application in intranasal
delivery, which is a practical and noninvasive approach that allows therapeutic agents
that do not cross the blood-brain barrier to enter the central nervous system, reducing
6
unwanted systemic side effects and increasing the existing effect of the previous known
treatments.
10

Phase I/II study had been conducted to evaluate the antitumoral activity of 0.3% of
POH, delivered intra-nasally in a 4 x daily schedule in patients with recurrent malignant
gliomas. A total of 29 patients enter the study with a medium follow-up of 48 weeks,
and the 6-month Progress Free Survival rate was 60%. 19 patients showed partial
regression and 10 other patients remain stable despite no sign of tumor regression. POH
intranasal delivery was well tolerated in all patients. Side effects were not commonly
reported at the dose given.
11

There has been some speculation on how POH works. Several studies show that
it appears to alter various cell parameters which include increasing mannose 6-
phosphate/insulin-like growth factor II receptors; increasing transforming growth factor
beta type I, II and II receptors; decreasing p21ras protein synthesis and increasing p21ras
catabolism; decreasing nuclear lamin B in actively replicating cells and finally inducing
Phase I and Phase II liver detoxification systems.
12
However, the exact mechanism of
POH is still unknown, thus preventing its potential to be fully appreciated. By
deciphering POH’s mechanism pathway, the possible usage of this chemical can be
expanded in more advanced combination therapy.
Specific Aims
The objectives of this paper are to investigate the effect and the dose responses of
POH in vitro on glioma cells, and to determine the mechanism in which POH induces
apoptosis in glioma cells while providing a possible combination treatment to enhance
anti-tumor effect. POH not only can be used as an innovate chemotherapeutic agent, but
7
also serve as a vehicle to deliver other drugs due to its ability to penetrate through the
blood brain barrier and reach the glioma tumors in patients. We hypothesize that POH
will mediate apoptosis through the ER stress mechanism, as previously shown with
DMC.
13,14
POH will be tested in a several glioblastoma cell lines in short and long term
treatment to compare its ER-mediated cell death and suppression of tumor formation.
Though monitoring important ER stress markers (GRP78 and CHOP)
15,16
, further
understanding the mechanism of POH can be achieved.

8
Materials

(S)-(-)-Perillyl alcohol 96%, p-metha, 1,7-diene-6-ol or 4-isopropenyl-
cyclohexenecarbinol, is purchased from Sigma-Aldridge, St. Louis, MO. It is dissolved
in DMSO to make 10% stock solution, which equals to 63µM, and added to cell culture
medium that kept the final concentration of DMSO below its toxicity level (0.1%). DMC
is a close structural analog of celecoxib, 4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-
pyrazol-1-yl. The 5-aryl moiety had been altered by replacing 4-methylphenyl with 20,5-
dimethylphenyl, resulting in 4-[5-(2,5-dimethylphenyl)-3(trifluoromethyl)-1H-pyrazol-1-
yl] benezensulfonamide. This compound was synthesized in our laboratory according to
previously published procedures.
17

Cell lines and culture conditions

Dr. Frank B. Furnari and Dr. Webster K. Cavenee (Ludwig Institute of Cancer
Research, LA Jolla, CA) generously supplied human glioma cell lines (U251 and
LN229). U87 and A172 glioma cells were purchased from ATCC (Manassas, VA). All
cells were cultured in Dulbecoo’s modified Eagle’s medium (DMEM; GIBCO BRL,
Grand Island, NY) supplemented with 10% fetal bovine serum, 0.1mg/ml streptomycin
(p/s; GIBCO) and 100U/ml penicillin in a humidified incubator at 37°C and 5% CO
2
atmosphere.

9
Methods
Lysate preparation
After the cells were treated with specific drugs for various length of times to reach
the desired level of protein markers, cells were washed with cold PBS, collected by
scraping directly off the Petri dish, and centrifuged at 14,000 rpm. For the detection of
CHOP induction, both floating and adhering cells were harvested.
Total cellular lysates were prepared by harvesting cells in lysis buffer. Lysis
buffer contains 10 ml of RIPA (Radioimmunoprecipitation assay) buffer, 10 µl of PMSF
(phenylmethanesulphonylfluoride or phenylmethylsulphonyl fluoride), one tablet of
phosphatase cocktail and 100 µl of phosphatase inhibitor. The amount of lysis buffer
used depends on the size of the pellets determined visually. Lysed cells were then
vortexed three times for ten minutes to ensure complete cell lysis while being kept on ice.
Subsequently, cells were then centrifuged for fifteen minutes at 4°C, and the pellets were
discarded afterwards while the supernatants were the cell lysates. Bicinchoninic acid
(BCA) (Pierce, Rockford, IL) protein assay reagent was used to measure the protein
concentration in the lysates. A standard curve (concentration of BSA at 0, 5, 10, 15, 20,
and 30µg, where the R
2
value must be greater than 0.98) was plotted on Excel to
determine the volumes of each lysate sample so the loading concentration would be
50µg. A combination of 45µL lysates and RIPA buffer, and 5µL of LaemmLi sample
Dye was prepared for Western blot analysis. This mixture was well mixed and heated at
100°C for five minutes to ensure complete disruption of tertiary protein structure. The
mixture was then spun down and loaded onto a SDS-PAGE gel.

10
Chemiluminescence
The primary antibodies including, anti-GRP78 and anti-CHOP, as well as the
secondary antibodies including, anti-mouse, and anti-rabbit were purchased from Santa
Cruz Biotechnology, Inc. (Santa Cruz, CA). According to the manufacturer’s
instructions, primary antibodies were diluted 1:500 in 5% nonfat dry milk power in PBS-
Tween20 (0.1% v/v). After transferring proteins on the SDS-PAGE gel to the
membranes, non-specific binding was blocked with 5% nonfat dry milk power in PBS-
Tween20 (0.1% v/v) for 1 hour at room temperature. The membranes were then probed
with specific diluted antibodies and incubated on shaker in sealed bags at 4ºC overnight.
Primary-probed membranes were washed three times with PBS-Tween20 at ten minutes
intervals on a shaker. The amount of PBS-Tween20 depends on the size of the
membranes. Secondary antibodies were diluted 1:5000 in 5% nonfat dry milk power in
PBS-Tween20. Membranes were then incubated with secondary antibodies on shaker for
45-60 minutes at room temperature followed by three times PBS-Tween20 wash in the
same way described earlier.
In order to detect the signals, the secondary antibodies were coupled to
horseradish peroxidase. Signals were detected by chemiluminescence using the
SuperSignal West substrate from Pierce. The membranes were incubated with Pico
chemiluminescence solution (Thermo Scientific, IL, USA) first for two minutes before
exposure. The excess Pico solution was then wiped down with Kim wipes after the two
minutes of incubation. Membranes were then held between two pieces of plastic wrap
and placed in autoradiography cassette for exposure. In the dark room, CL-XPosure
films from Pierce were placed on top of the membranes to allow exposure. Various
11
exposure time periods were necessary to obtain clean and constant signals. If the signals
were too weak after developing, 5-15% of Femto chemiluminescence was added to
increase the sensitivity of the signals.
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay
The number of cells per unit volume was measured via hemocytometer.
Glioblastoma cell lines U251, LN229, U87 and A172 were seeded in 3.0 x 10
3
cells per
well in 96 well plates. The day after seeding, seeded cells were treated with media
containing various drugs at different concentration. One well of every plate was used for
background containing no cells with just media. After 48 hours, drugs were removed and
fresh medium was added to the cells before 10µl of MTT dye was added to all wells. The
reaction was then terminated by 100µl of 10% HCl solubilization solution after 4 hours.
The plates were read by an ELISA plate reader at 490nm. Samples were seeded in
triplicates to calculate the mean values with percent errors.
Colony formation assay
Cells were seeded into six-well plates at 200 cells per well. After complete cell
adherence, usually 24 hours, but this varies with different cell lines, the cells were
exposed to drug treatment for 48 hours. Thereafter, the drug was removed and fresh
medium was supplied. The cells were then kept for 10-12 days undisturbed during which
the surviving cells form colonies of proliferating cells. Colonies were stained for
visualization for 4 hours with 1% methylene blue dye and then counted.

12
Results

Figure 1. shows the actual chemical structures and properties of Perillyl alcohol
and DMC. Perillyl alcochol, a monoterpene is isolated from essential oils of lavender
and several other plants. Monoterpenes are formed in the mevalonic acid pathway in
plants, which assist in increasing level of liver enzymes involved in detoxifying
xenobiotics.
18
DMC (2,5-dimethyl-celecoxib), an extra methyl group is added to
celecoxib, making 2 methyl groups at positions 2 and 5 of the phenyl ring. In comparison
to celecoxib, DMC lacks the COX-2 inhibitory activity but is able to potently promote
the anti-tumor effect by inhibiting certain signal transduction pathways, blocking cell
proliferation, and inducing apoptosis.
19

Perillyl Alcohol DMC
Figure 1. Chemical structures of perillyl alcohol and DMC.
16

13
In order to assess the cell growth and survival in response to POH, four different
glioblastoma cell lines that were treated with increasing POH concentration and cell
viability was determined by MTT assays (Figure 2). In all four cell-lines, POH greatly
decreases cell viability and the IC50 is in the range from 945 µM to 1260 µM. Out of all
four cell-lines, U87 shows more resistance while the other three cell-lines seem to be
more sensitive to POH. None of the cell lines reach complete cell death even at
concentration of POH greater than 1260 µM; the cell viability plateaus at about 20% and
no more cell death is observed even when POH concentration reached 6300 µM.
In addition, three cell-lines, U87, LN229, A172 were treated with increasing POH
for 24 hour to show physiological effect of the drug in figure 3. The glioma cells show
decrease in proliferation and cell death as the concentration of the POH increases. The
results are very similar in all three cell-lines. There is very little difference between the
control (0 µM POH) and 315 µM POH treated group. However, as the concentration
reached the IC 50 of POH at 630 µM-1260 µM, there is a significant decrease in cell
proliferation in all three cell-lines.

14

Figure 2. MTT assays of four glioblastoma cell lines.
Four glioblastoma cell-lines, U251, LN227, U87 and A172 are treated with increasing
concentration of perillyl alcohol. Cell proliferation and survival is measured after 48
hours by MTT assay. MTT dye is added after 48 hours of incubation with POH and
additional 4 hours of incubation with MTT dye is needed before adding the solubilization
solution to terminate the reaction. Cell viability is determined with an ELIZA plate
reader at 490 nm. The experiment is repeated three times with triplicates each time.

15

Control 315 µM 630 µM 1260 µM

Figure 3. Photos of glioblastoma cell-lines treated with POH for 24 hours.
From top to bottom, respectively, U87, LN229 and A172 are treated with increasing
concentration of 0, 315, 630,and 1260 µM of POH for 24 hours. As the concentration of
POH reaches or exceeds the IC50 of the glioma cells (greater than or equal to 630 µM),
the reduction in cell proliferation became more visible.

16
Colony formation assay is used to further investigate the long-term effect of
perillyl alcohol on glioma cells to enable a more in depth understanding of POH in Figure
4. To obtain a quantitative figure for the colony formation assay, 200 of U251 and A172
glioblastoma cells were seeded and increasing concentration of POH was administered.
The final cell colony count was obtained after removing the drug and adding fresh
medium for another 10 days. The critical decrease in cell colony occurs at 1260 µM
POH for both U251 and A172 cells, which shows that 1260 µM POH reduced cell
proliferation. Complete cell death is reached at 2520 µM, which doesn’t occur in MTT
assay. This observation could indicate that POH’s has long half-life, which allows it to
remain in the cell. Therefore, the more potent effect can be seen at latter time point.
After demonstrating the effect of POH on glioma cells both long and short term,
the next step is to determine the mechanism behind its toxicity. As previously reported
that increasing ER stress is often the result for many drugs, ER stress-specific markers
GRP78 and CHOP were investigated at sixteen and eighteen-hour time period via
Western Blot in Figure 5 and Figure 6, respectively. At sixteen hour time period, earlier
events in ER stress response can be detected. GRP78 induction indicates this is a
defensive mechanism cells activate to compensate for ER stress. The level of GRP78
increased as the concentration of POH increased, which indicated that the anti-tumor
effect of POH does correlate with the drug’s ability to induce ER stress.
20

17

Figure 4. Colony formation assay with U251 and A172 cells.

U251 and A172 cells are treated with increasing concentration of perillyl alcohol to show
the long-term effect of POH. There is a significant drop of cell colony formation at the
concentration 1260 µM) compare to the IC 50 of 630 µM shown in MTT assay which
indicate that higher dosage is needed for the long term treatment to become more
effective.

18

GRP78

Actin

Figure 5. Western blot of ER specific marker GRP78 on U251 cells.
U251 cells are treated with increasing concentration of POH for 16 hours. Total cell
lysates for Western blot analysis are prepared with specific antibodies against GRP78 and
Actin. Standard GRP78 is just a control to ensure the positive identification of the
correct protein marker.

Control 315 µM 630 µM 1260 µM Standard GRP78
19
At eighteen hours, CHOP induction became detectable. CHOP induction
indicates that the cell is undergoing apoptosis, therefore this specific protein marker
shows up later than GRP78, which tries to adapt to increasing ER stress. However, when
cells undergo high level of GRP78, CHOP induction occurs to promote cell apoptosis.
CHOP induction in U251 cells showed very similar pattern in which the higher the POH
concentration, the stronger the CHOP induction. CHOP induction is relatively weak at
315 µM, and the signal only becomes more prominent at 630 µM and 1260 µM.

Figure 6. Western blot of ER specific marker CHOP on U251 cells.

U251 cells are treated with increasing concentration of POH for 18 hours. Total cell
lysates for Western blot analysis are prepared for antibodies against CHOP and Actin.

Control 315 µM 630 µM 1260 µM
CHOP
Actin
20
DMC is a known drug to cause ER stress and trigger apoptosis in glioblastoma
cells. In addition, POH has also been shown to have similar effects in this study.
Therefore, combining PHO and DMC could potentially enhance the anti-tumor effect
against glioblastoma and provide an innovative way of delivering drugs into the brain via
nasal inhalation. MTT assay on DMC alone with four glioblastoma cell-lines is needed to
establish a concentration for this combination treatment to proceed. Simultaneously,
MTT assay for combination of POH and DMC is also established in order to compare the
difference between single and dual treatment. In figure 7, three out of four glioblastoma
cell-lines, U251, LN229 and U87 all show synergistic effect of the combination treatment
versus the single treatment of DMC alone. (P<0.05)

21

0
20
40
60
80
100
120
0 20 30 40
U251
DMC
DMC+POH (630 µM)

0
20
40
60
80
100
120
0 10 20 30
A172
% Cell survival
DMC (µM)
% Cell survival
DMC (µM)
A.
B.
22

0 30 40 50

Figure 7. MTT assay of DMC/POH on four glioblastoma cell-lines.
Four glioblastoma cell-lines, U251, LN227, U87 and A172 are treated with increasing
concentration of DMC and with combination of POH 630 µM. Cell survival is measured
after 48 hours by MTT assay. Cell viability is determined with an ELIZA plate reading at
490 nm. Stars indicate there is a statistical significance between (P<0.05) the cell
viability in single (DMC) versus combination (DMC + POH) treatment. Single treatment
of POH at 630 µM can be seen in Figure 2.
0
20
40
60
80
100
120
LN229
% Cell survival
0
20
40
60
80
100
120
140
0 30 40 50
U87
DMC (µM)
% Cell survival
DMC (µM)
D.
C.
23
Cell colony formation of the combination treatment is also performed to study the
long-term effect of the combination treatment on U251 cells (Figure 8). As the data
suggested, DMC 20 µM and DMC 30 µM hardly affected the glioma cells, POH at 630
µM actually eliminated half of the cell colony for both U251 and A172 cells compared to
the control. However, combination of DMC 30 µM + POH 630 µM reduced the colony
formation to 20% which is a statically significant reduction and thus total cellular lysates
for cells in these condition was collected for Western blot analysis against GRP78 and
CHOP protein to observe ER stress level.

24

Figure 8. Colony Formation Assay on U251 and A172 cells treated with DMC,
POH, and DMC+POH.

200 of U251 and A172 cells were seeded in six well plates and treated with DMC alone
at 20 µM, 30 µM and POH at 630 µM and combination of DMC 20 µM + POH 630 µM
and DMC 30 µM + POH 630 µM to monitor the long-term effect of these drugs. Stars
indicate that there is a statistically significant (P<0.05) difference in cell colony
formation between the combination treatment (DMC 30 µM + POH 630 µM) than the
single treatment of DMC or POH.

25
In figure 9, photos of the single versus combination treatments are taken for cell-
lines U251 and U87 to provide visual evidence. The combination treatment shows cell
debris and dead cells, which confirms the MTT and cell colony formation results that the
combination treatment is far more potent than the single treatment alone.

Figure 9. Photos of U251 and U87 cells treated with single and dual treatments after
18 hours.

There wasn’t much of a difference between the control and the single treatments at least
morphologically. However, with the dual treatment, there were presences of dead cells
and cell debris in both U251 and U87 cell-lines, indicating cell death and also significant
reduction of cell proliferation could be observed. Arrows point to the dead cells and/or
cell debris.

Control DMC 30µM POH 630µM DMC 30µM +
POH 630µM
26
Whole cellular lysates of U251 cells after combination treatment were prepared
for two different time periods at sixteen hours and eighteen hours; GRP78 and CHOP are
the specific protein markers at respective time point to indicate elevated ER stress and
apoptosis. GRP78 level was quite consistently low throughout the single treatment of
DMC and POH and even at DMC 20 µM and POH 630 µM combination treatment.
However, there is a drastic increase of GRP78 induction under the combination treatment
of DMC 30 µM and POH 630 µM, which is consistent with the MTT assay, cell colony
formation and the cell culture evidence mentioned earlier. In addition, CHOP induction
was absence in the control and treatment of DMC alone; CHOP is only detectable in
treatment by POH 630 µM and in both combination treatment. However, the CHOP
induction in the combination treatments is definitely stronger than the POH treatment
alone (Figure 10).

27

Figure 10. Western blot analysis of U251 cells treated with DMC and POH alone
and combined.

From top to bottom, the bands represent GRP78, Action and CHOP, respectively. Whole
cellular lysates of U251 cells are prepared for 16 hours to detect GRP78 and 18 hours for
CHOP. U251 cells are treated with single DMC or POH, and combination of both.
GRP78 and CHOP signals increase suggest that combination treatment greatly induce ER
stress and ER stress mediated cell apoptosis. Actin is the marker to ensure equal loading
in all samples to avoid bias. The standard protein markers are used to indicate the bands
correlate with the desired protein markers and serve as a reference to ensure quality
control.
21

Control DMC20µM DMC30µM POH630µM DMC20µM
+
POH630µM
DMC30µM
+
POH 630µM
Standard
protein
markers
28
Discussion

The mechanism that perillyl alcohol uses to cause apoptosis in tumor cells is still
not proven. However, without significant insight on the mechanism of action in this
promising chemotherapeutic agent, its benefit is limited and underappreciated. In many
previous studies, there was much evidence that supports the notion of POH being able to
suppress tumor growth and therefore it is worthwhile to further investigate POH’s full
potential in cancer treatment.
22
POH has great blood brain barrier penetration
23
, and could
serve as a solvent to many existing drugs with low solubility and poor blood brain barrier
penetration, such as DMC, which automatically make POH an excellent candidate for
treating glioblastoma. Glioblastoma is the most common and most aggressive type of
primary brain tumor in humans, and despite recent advances in medicine, the survival
rate for these patients is between 12-15 months.
To demonstrate the short-term effect of POH, MTT assays were performed to
compare 48 hours treatment in four different glioblastoma cell-lines. U251, LN229, U87
and A172 are monitored for cell proliferation. While U87 has the highest tolerance, the
other three cell-lines have IC50 at around 630 µM, with U251 being the most sensitive
cells to POH. One other interesting observation is that in all four cell-lines, there was no
complete cell death even after the concentration of POH reached 6300 µM. The cells
reach a plateau at 2520 µM with +/- 20% cell viability. One hypothesis is that there may
be a specific binding site for POH in the cells and at higher concentration, all the
available binding site are already occupied and thus leaving 20% of the cells surviving
despite excessive concentration. Further experiment could be carried out with C-14 POH,
in which the cells will be treated with the radioactive POH and then monitor the
29
radioactivity to identify specific binding sites on cell and provide more information on its
mechanism. Furthermore, saturation and affinity of POH to the specific binding site can
be calculated and studied, which can lead to creating an even more potent analog if the
specific property is found to be responsible for tighter binding.
For long-term POH effect, cell colony assay was performed on U251 cell-line due
to its higher sensitivity towards POH. The IC 50 for the U251 from the MTT assay was
shown to be at 630 µM, however, in the cell colony assay, its IC50 increased to be more
than 630 µM. The reason behind this discrepancy could be that the POH has a more
potent immediate effect, and thus in long-term treatment, a stronger dose of POH is
needed to achieve desirable results. However, in cell colony assay, complete cell death
was reach at 2520 µM, which can be explained by long half-life of POH within the cells.
POH might not be susceptible to degradation due to its benzene ring structure, and thus
allows itself to remain in the cells. This hypothesis can also be tested by tracing C-14
POH and its metabolites through a time course and reveal its mechanism and half-life.
There were also photos of the cells that were taken after the cells were incubated
with increasing concentration of POH for 24 hours. The evidence was clear that as POH
concentration increases, the cells exhibit signs of reduction in proliferation and even cell-
death.
After determining the effect of POH on glioma cells, U251 cells were treated with
increasing concentration of POH and the whole cellular lysates were prepared to analyze
the specific protein markers GRP78 and CHOP to reveal the pathway behind POH.
24
The
result shows that there is intensification in both GRP78 and CHOP induction as the
concentration of POH increases. Since GRP78 is a protective protein that binds to
30
misfolded or unfolded proteins to revert ER stress, the increase in GRP78 expression
suggests that cells are under elevated ER stress, which indicates that POH increases ER
stress. To further substantiate this point, CHOP, a pro-apoptotic protein that is specific
for ER and apoptosis, was also analyzed. The increased expression of CHOP is
consistent with GRP78; as the POH concentration rise, the higher the CHOP induction
becomes in U251 cells. These results imply that the cells are undergoing ER stress,
which is accompanied by ER-mediated apoptosis pathway.
In prior studies, DMC was found to show similar trend as POH.
13
With this
knowledge at hand, combining POH with DMC could prove to become a highly effective
treatment. First of all, MTT assays for DMC alone and combination of DMC and POH
were done to establish a basic dosage reference for the drugs. Three out of the four
glioblastoma cell-lines, U251, U87, and LN229 all showed statistically significant
difference in cell proliferation when treated with combination of DMC (20 or 30 µM) and
POH (630 µM). U251 was once again the most sensitive cell-line, and even at DMC 30
µM with POH 630 µM, the cells showed a dramatic decrease in proliferation. Cell
colony formation assay was thus performed on U251 cells to verify the effect for long-
term treatment. The result was promising, with the combination of DMC 30 µM and
POH 630 µM, U251 cells only have than 20% cell colony compared to the control and
also statistically significantly lower cell colony than any of the other single drug
treatments (DMC 20 µM, DMC 30 µM and POH 630 µM).
Western blot analysis of GRP78 and CHOP was also used to verify the pathway
of the combination drug treatment. The purpose in combination treatment is to use the
minimal level of the drugs, while achieving better results. For this reason, U251 cells
31
were treated with DMC 30 µM and POH 630 µM; both lower than the IC50 obtained by
the MTT assays and colony formation assays. However, in the Western blot analysis, the
GRP78 and CHOP induction showed a greater increase when U251 cells are treated with
DMC 30 µM and POH 630 µM compared to any single treatment (DMC 20 µM, DMC
30 µM, POH 630 µM) and the control. This not only indicate that this combination
treatment causes ER stress and ER stress-mediated apoptosis, but also implies that there
must be synergistic effect (p<0.05) in the combination treatment.
To further investigate perillyl alcohol in the future, studies on mice bearing
subcutaneous U251 tumors will provide a great model to test the dosage in which POH
would work in-vivo. POH will be directly injected into the tumor and tumor size will the
measured daily. In addition, intracranial U251 tumor model could be used in rats to
compare POH + DMC with DMC alone and gain prospective on whether or not POH
would enhance the effect of DMC in-vivo as well. POH, DMC and POH + DMC
mixtures will be given through nasal inhalation. If POH proves to not only be a great
anti-tumor agent, but also a great vehicle for other drugs, the possibilities are endless.
These studies wouldn’t be meaningful before determining that POH actually does cause
ER stress and generate ER-stress mediated apoptosis. Therefore, the in-vivo study can
now be done with more insight and understanding and POH can be utilized to its full
potential.

32
Numerical Bibliography

1. Guillamo JS, Monjour A, Taillandier L, Devaux B, Varlet P, Haie-Meder C,
Defer GL, Maison P, Mazeron JJ, Cornu P, Delattre JY. 2001. Brainstem gliomas
in adults: prognostic factors and classification. Brain. 124:2528-39.

2. Borgese, N., Francolni, M. & Snapp, E. 2006. Endoplasmic reticulum
architecture: structures in flux. Curr Opin Cell Biol 18:258-364.

3. Li, J. & Lee, A.S. 2006. Stress induction of GRP78/BiP and its role in cancer.
Curr Mol Med 6:45-54.

4. Boyce, M & Yuan, J. 2006. Cellular response to endoplasmic reticulum stress; a
matter of life and death. Cell Death Differ 13:363-373.

5. Oyadomari, S. & Mori, M. 2004. Roles of CHOP/GADD153 in endoplasmic
reticulum stress. Cell Death Differ 11:381-389.

6. Gould MN. 1997. Cancer chemoprevention and therapy by monotrepenes.
Environ Health Prospect 105:977-9

7. Crowell PL. 1996. Antitumor effect of limonene and perillyl alcohol against
pancreatic and breast cancer. In: American Institute for Cancer Research.
Dietary Phytochemicals in Cancer Prevention and Treatment. New York: Plenum
Press.

8. Haag JD, Gould MN. 1994. Mammary carcinoma regression induced by perillyl
alcohol, a hydroxylated analog of limonene. Cancer Chemother Pharmacol
34:477-483.

9. Belanger JT. 1998. Perillyl alcohol: application in oncology. Altern Med Rev
3:448-57.

10. Schönthal, A.H. 2006. Antitumor properties of dimethyl-celecoxib, a derivative of
celecoxib that does not inhibit cyclooxygenase-2; implication for glioma therapy.
Neurosurg Focus 20:E21.

11. Fernandes J, Da Fonseca CO, Teixeira A, Gattass CR. 2007. Phase I/II study of
perillyl alcohol intranasal administration in adults with recurrent malignant
gliomas. Neuro-Oncology 13: 943-7.

12. Jirtle RL, Gould MN. 1993. Increased mannose 6-phosphate/insulin-like growth
factor II receptor and transforming growth factor beta 1 levels during
monotrepene-induced regression of mammary tumors. Cancer Res 53:3849-3852.

33
13. Kardosh, A., et al. 2005. Multitarget inhibition of drug-resistant multiple
myeloma cell lines by dimethyl-celecoxib (DMC), a non-COX2 inhibitory analog
of celecoxib. Blood 106:4330-4338.

14. Pyrko, P., Schönthal, A.H., Hofman, F.M., Chen, T.C. & Lee, A.S. 2007.The
unfolded protein reponse regulator GRP78/BiP as a novel target for increasing
chemosensitivity in malignant gliomas. Cancer Res 67:9808-9816.

15. Lee E., et al. 2006. GRP78 as a novel predictor of responsiveness to
chemotherapy in breast cancer. Cancer Res 66:7849-7853.

16. Kim, S.H., et al. 2007. GADD153 mediated celecoxib-induced apoptosis in
cervical cancer cells. Carcinogenesis 28:223-231.

17. Schönthal, A.H., Chen, T.C.. Hofman, F.M., Louie, S.G. & Petasis, N.A. 2008.
Celecoxib analogs that lack COX-2 inhibitory fuction: preclinical development of
novel anticancer drugs. Expert Opin Investig Drugs 17:197-208.

18. Kelloff GJ, Boone CW, Crowell JA, et al. 1996. New agents for cancer
chemoprevention. Cellular Biochem 26S:1-28.

19. Schönthal, A.H. 2007. Direct non-cyclooxygenase-2 targets of celecoxib and their
potential relevance for cancer therapy. Br J Cancer 97:1465-1466.

20. Virrey, J.J, et al. 2008. Stress chaperon GRP78/BiP confers chemoresistance to
tumor-associated endothelial cells. Mol Cancer Res 6:1268-1275.

21. Liu, Y.Y., Kardosh, A., Cooc, J. & Schönthal, A.H. 2006. Potential
misidentification of cycloxygenase-2 by Western blot analysis and prevention
through the inclusion of appropriate controls. Mol Biotechnol 34:329-335.

22. Jeffers L. 1995. The effect of perillyl alcohol on the proliferation of human
prostatic cell-lines. Proc Am Assoc Cancer Res 36:303.

23. Ripple GH, Gould MN, Stewart JA, et al. 1998. Phase I clinical trial of perillyl
alcohol administered daily. Clin Cancer Res 4:1159-1164.

24. Gonzalez-Gronow, M., et al. 2006. Prostate cancer cell proliferation in vitro is
modulated by antibodies against glucose-regulated protein 78 isolated from
patient serum. Cancer Res 66:11424-11431.

34

Bibliography

Belanger JT. 1998. Perillyl alcohol: application in oncology. Altern Med Rev 3:448-57.

Borgese, N., Francolni, M. & Snapp, E. 2006. Endoplasmic reticulum architecture:
structures in flux. Curr Opin Cell Biol 18:258-364.

Boyce, M & Yuan, J. 2006. Cellular response to endoplasmic reticulum stress; a matter
of life and death. Cell Death Differ 13:363-373.

Crowell PL. 1996. Antitumor effect of limonene and perillyl alcohol against pancreatic
and breast cancer. In: American Institute for Cancer Research. Dietary Phytochemicals
in Cancer Prevention and Treatment. New York: Plenum Press.

Fernandes J, Da Fonseca CO, Teixeira A, Gattass CR. 2007. Phase I/II study of perillyl
alcohol intranasal administration in adults with recurrent malignant gliomas. Neuro-
Oncology 13: 943-7.

Gonzalez-Gronow, M., et al. 2006. Prostate cancer cell proliferation in vitro is modulated
by antibodies against glucose-regulated protein 78 isolated from patient serum. Cancer
Res 66:11424-11431.

Gould MN. 1997. Cancer chemoprevention and therapy by monotrepenes. Environ
Health Prospect 105:977-9

Guillamo JS, Monjour A, Taillandier L, Devaux B, Varlet P, Haie-Meder C, Defer GL,
Maison P, Mazeron JJ, Cornu P, Delattre JY. 2001. Brainstem gliomas in adults:
prognostic factors and classification. Brain. 124:2528-39.

Haag JD, Gould MN. 1994. Mammary carcinoma regression induced by perillyl alcohol,
a hydroxylated analog of limonene. Cancer Chemother Pharmacol 34:477-483.

Jeffers L. 1995. The effect of perillyl alcohol on the proliferation of human prostatic cell-
lines. Proc Am Assoc Cancer Res 36:303.

Jirtle RL, Gould MN. 1993. Increased mannose 6-phosphate/insulin-like growth factor II
receptor and transforming growth factor beta 1 levels during monotrepene-induced
regression of mammary tumors. Cancer Res 53:3849-3852.

Kardosh, A., et al. 2005. Multitarget inhibition of drug-resistant multiple myeloma cell
lines by dimethyl-celecoxib (DMC), a non-COX2 inhibitory analog of celecoxib. Blood
106:4330-4338.

Kelloff GJ, Boone CW, Crowell JA, et al. 1996. New agents for cancer chemoprevention.
Cellular Biochem 26S:1-28.
35

Kim, S.H., et al. 2007. GADD153 mediated celecoxib-induced apoptosis in cervical
cancer cells. Carcinogenesis 28:223-231.

Lee E., et al. 2006. GRP78 as a novel predictor of responsiveness to chemotherapy in
breast cancer. Cancer Res 66:7849-7853.

Li, J. & Lee, A.S. 2006. Stress induction of GRP78/BiP and its role in cancer. Curr Mol
Med 6:45-54.

Liu, Y.Y., Kardosh, A., Cooc, J. & Schönthal, A.H. 2006. Potential misidentification of
cycloxygenase-2 by Western blot analysis and prevention through the inclusion of
appropriate controls. Mol Biotechnol 34:329-335.

Oyadomari, S. & Mori, M. 2004. Roles of CHOP/GADD153 in endoplasmic reticulum
stress. Cell Death Differ 11:381-389.

Pyrko, P., Schönthal, A.H., Hofman, F.M., Chen, T.C. & Lee, A.S. 2007.The unfolded
protein reponse regulator GRP78/BiP as a novel target for increasing chemosensitivity in
malignant gliomas. Cancer Res 67:9808-9816.

Ripple GH, Gould MN, Stewart JA, et al. 1998. Phase I clinical trial of perillyl alcohol
administered daily. Clin Cancer Res 4:1159-1164.

Schönthal, A.H. 2006. Antitumor properties of dimethyl-celecoxib, a derivative of
celecoxib that does not inhibit cyclooxygenase-2; implication for glioma therapy.
Neurosurg Focus 20:E21.

Schönthal, A.H. 2007. Direct non-cyclooxygenase-2 targets of celecoxib and their
potential relevance for cancer therapy. Br J Cancer 97:1465-1466.

Schönthal, A.H., Chen, T.C.. Hofman, F.M., Louie, S.G. & Petasis, N.A. 2008.
Celecoxib analogs that lack COX-2 inhibitory fuction: preclinical development of novel
anticancer drugs. Expert Opin Investig Drugs 17:197-208.

Virrey, J.J, et al. 2008. Stress chaperon GRP78/BiP confers chemoresistance to tumor-
associated endothelial cells. Mol Cancer Res 6:1268-1275.

Abstract (if available)

Abstract Despite improvement in recent surgery, radiation, and chemotherapy, the prognosis for patients with malignant glioma has not improved. Therefore, it’s even more urgent to search for new chemotherapeutic drugs. The aim of this project is to investigate the mechanism of perillyl alcohol (POH), an old chemotherapeutic agent, which has been recently used as intra-nasal inhalation in malignant glioma treatment. In this thesis, I document that POH alone, and combined with Dimethyl-celecoxib (DMC), shows significant anti-tumor activity in-vitro and triggers the induction of Glucose Related Protein 78 (GRP78), an anti-apoptotic protein that is elevated to deal with stress response. In addition, I also show induction of CCAAT/enhancer binding protein, or (CHOP), a pro-apoptotic protein signal for apoptosis, under the same treatments. These findings imply that POH alone generates Endoplasmic reticulum (ER) stress and forces the tumor cells to enter apoptosis, and when combined with DMC the effect is synergistic . This is particularly exciting due to the fact that POH can enter the brain via nasal inhalation and thus providing the patients with an effective and yet non-invasive treatment.

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Asset Metadata

Creator Wang, Long-Chieh David (author)

Core Title Examining perillyl alcochol in glioblastoma treatment and relating its effect to endoplasmic reticulum stress response to further advance its usage in combination treatment with dimethyl-celecoxib

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Degree Conferral Date 2009-12

Publication Date 10/15/2009

Defense Date 08/28/2009

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag Dimethyl-celecoxib,ER stress,glioma treatment,OAI-PMH Harvest,perillyl alcohol

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Tokes, Zoltan A. (committee chair), Chen, Thomas C. (committee member), Markland, Francis (committee member), Swenson, Stephen (committee member)

Creator Email longchieh@hotmail.com,longchiw@usc.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m2671

Unique identifier UC1155585

Identifier etd-Wang-3284 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-266114 (legacy record id),usctheses-m2671 (legacy record id)

Legacy Identifier etd-Wang-3284.pdf

Dmrecord 266114

Document Type Thesis

Rights Wang, Long-Chieh David

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu