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Enhanced Burkitt 's lymphoma cell killing by the combination treatments of bortezomib with celecoxib and 2,5-dimethyl-celecoxib (DMC)

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Enhanced Burkitt 's lymphoma cell killing by the combination treatments of bortezomib with celecoxib and 2,5-dimethyl-celecoxib (DMC)

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Content ENHANCED BURKITT’S LYMPHOMA CELL KILLING BY THE COMBINATION
TREATMENTS OF BORTEZOMIB WITH CELECOXIB AND 2,5-DIMETHYL-
CELECOXIB (DMC)

by

Szu-Ting Chen
___________________________________________________

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

May 2008

Copyright 2008 Szu-Ting Chen

Table of Contents

List of Figures iii
Abstract iv
Introduction 1
Results 5
Discussion 17
Materials and Methods 21
References 25

ii

List of Figures

Figure 1: Chemical structures of celecoxib, UMC, and DMC 3
Figure 2: A significant Raji cell growth reduction is observed in the
treatments with bortezomib, UMC, celecoxib, DMC, and
staurosporine 6
Figure 3: Celecoxib and DMC induce CHOP/GADD153 and cleave
PARP-1 efficiently 9
Figure 4: The combinations of bortezomib with DMC and celecoxib
exert a synergistic effect 11
Figure 5: The combination treatments of bortezomib with DMC and
celecoxib stimulate CHOP/GADD153 and cleaved-PARP-1
expression 13
Figure 6: CHOP/GADD153 stimulation is not observed in the treatments
with staurosporine 15
Figure 7: CHOP/GADD153 induction is not observed in the combination
treatments of bortezomib with staurosporine 16

iii

iv
Abstract
Cell death induced by endoplasmic reticulum (ER) stress response (ESR)
mechanism has been implicated in many diseases. The proteasome inhibitor
bortezomib (Velcade
®
) is known to cause ESR via the accumulation of obsolete
and damaged proteins. The selective cycloxygenase-2 (COX-2) inhibitor,
celecoxib (Celebrex
®
) and its analog, 2,5-dimethyl-celecoxib (DMC) were
previously shown to trigger ER stress by inhibiting the sarcoplasmic/ER calcium
ATPase (SERCA), which allows leakage of calcium from the ER into the cytosol.
We hypothesized that the combination of two ER stress-triggering drugs,
bortezomib and DMC/celecoxib, would increase ESR substantially and greatly
enhance their anti-tumor efficiency in Burkitt’s lymphoma cell. With the use of
Raji cells, we have examined an elevated level of ER stress marker
CHOP/GADD153, apoptotic marker PARP-1, and a significant cell growth
reduction in the treatment of bortezomib with celecoxib/DMC. Hence, we propose
the treatments of bortezomib with DMC and celecoxib as effective combinations
to enhance Burkitt’s lymphoma cell killing in vitro.

Introduction
The proper functioning of the endoplasmic reticulum (ER) is vital for cell
physiology (12). Adaptive pathways such as the accumulation of unfolded
proteins, ER lipid or glycolipid, and imbalance or changes in the ionic condition of
the ER lumen can trigger an ER stress response (ESR). The primary purpose of
ESR is to restore ER homeostasis; however, in the presence of intense or
persistent ER stress, it would lead to apoptosis (12, 48). The correlation between
ER stress and apoptosis has been observed in diabetes, obesity,
neurodegenerative disorders, viral infection and a variety of ER storage diseases
(12, 32).
Bortezomib (Velcade
®
) is the first proteasome inhibitor to be evaluated in
clinical trials (1, 40). It was approved by the FDA for the treatment of multiple
myeloma in 2003 and mantle lymphoma, a rare type of non-Hodgkin’s lymphoma,
in 2006 (18). Bortezomib acts as a selective inhibitor of the 26S proteasome, a
multisubunit protein complex that functions to degrade ubiquitinated proteins.
Recent studies have shown that cell death by bortezomib is a result of the
accumulation of misfolded and/or damaged proteins, which lead to the activation
of the unfolded protein response (UPR) and then trigger ESR (31, 33). It is
believed that the continuous presence of bortezomib would overwhelm the
protective efforts of ESR and initiate cell death via the stimulation of
CHOP/GADD153, a critical executioner of the pro-apoptotic arm of ESR, and
other pro-apoptotic components of ESR (16).
Celecoxib (Celebrex
®
) is a selective inhibitor of cyclooxygenase-2 (COX-2)
1

and is commonly prescribed for the relief of symptoms of osteoarthritis and
rheumatoid arthritis; it was also approved as an adjunct to the standard care for
patients with familial adenomatous polyposis (FAP). In experimental results,
celecoxib has demonstrated its effective anti-cancer activity in various animal
tumor models, and it is believed that celecoxib might be a possible
chemotherapeutic agent for colorectal and other types of cancer (14, 25, 30).
However, the molecular mechanisms that celecoxib undertakes to exert its
anti-tumor effects are still controversial, primarily due to an increasing number of
reports demonstrating potent anti-proliferative and pro-apoptotic effects of
celecxoib without any involvement of COX-2 (3, 19, 21, 26, 42, 45, 50). One of
the evidences that excludes the potential contribution of COX-2 comes from the
utilization of 2,5-dimethylcelecoxib (DMC), a close structural analog of celecoxib
that lacks the capability to inhibit COX-2 activity (24). Despite its inability to
hinder COX-2 activity, DMC mimics all of celecoxib’s numerous anti-tumor effects
more potently both in vitro and in vivo, including the reduction of
neovascularization and the inhibition of experimental tumor growth in various
animal tumor models (7, 23, 24, 26, 29, 36, 38, 43, 51).
Recent reports have shown that DMC and celecoxib induce tumor cell death
in vitro and in vivo through ESR (46, 47). The initiating event appears to be the
inhibition of the sarcoplasmic/ER calcium ATPase (SERCA), which results in
extensive leakage of calcium into the cytosol; the calcium imbalance induces
severe ER stress within seconds of the addition of celexcoxib or DMC to
cultured cells (20, 36, 44).
2

Figure 1: Chemical Structures of Celecoxib, UMC, and DMC
Celecoxib has a methyl group at the C-4 (p) position of its terminal phenyl ring.
UMC lacks a methyl group at the C-4 (p) position. DMC has two methyl groups,
which are located at the 2- and 5- positions.

3

Burkitt’s lymphoma is a mature B-cell lymphoma characterized by a rapid
proliferative rate and an inclination for extranodal sites of involvement, such as
gastrointestinal tracts and the central nervous system (11). Aggressive multiagent
chemotherapy is the current treatment regimen for Burkitt’s lymphoma (8).
We hypothesized that the combination treatments of bortezomib with
celecoxib and DMC would exert a potent anti-proliferative effect via ER stress
mechanism on Burkitt’s lymphoma cell.

4

Results
Non-Hodgkin’s lymphoma (NHL) has become the most common
hematologic malignancy with an estimated 56,390 new cases and 19,200 deaths
in the U.S. in 2005 (2). Burkitt’s lymphoma is a rare and aggressive form of NHL
for which an optimal treatment regimen still remains unknown (9). The clinical
efficacy of bortezomib against multiple myeloma provided the rationale for using
bortezomib to treat patients with Burkitt’s lymphoma. To investigate whether or
not bortezomib could be a potent anti-proliferative compound for Raji cell, we
have treated Raji cells with increasing concentrations of bortezomib. Ramos, a
Burkitt’s lymphoma cell line, T47D, a breast tumor cell line, and U266 and
RPMI/8226, multiple myeloma cell lines, were also treated in the same condition
for drug-sensitivity comparison. Ramos and Raji were both responsive to
bortezomib with 50% inhibition of cell growth (IC
50
) at 10 nM and 8 nM
respectively. Raji was as sensitive to bortezomib as RPMI/8226, a human
multiple myeloma cell line, with an IC
50
of 10 nM bortezomib (Figure 2A).
Dexamethasone is a glucocorticosteroid hormone and a potent and
commonly used agent to treat lymphoid malignancies (15). It is also one of the
first-line chemotherapeutic regimens for relapsed mantle lymphoma (28).
However, in figure 2B, Raji cells show resistance to dexamethasone from
treatments with various concentrations of the drug. 100 µM dexamethasone
achieved approximately 25% cell growth inhibition. The result illustrated that the
sensitivity to bortezomib observed in Raji cell was specific, and it supported our
rationale to investigate this drug as a potential Burkitt’s lymphoma
5

chemotherapeutic agent.

Figure 2: A significant Raji lymphoma cell growth reduction is observed in
the treatments with bortezomib, UMC, celecoxib, DMC, and staurosporine
Cell viability was analyzed through MTT assay. All cell lines were treated
concentration-dependently for 48 hours. A) Burkitt’s lymphoma cell lines, Raji
and Ramos, human multiple myeloma cell lines, U266 and RPMI/8226, and a
breast tumor cell line, T47D, were treated with increasing concentrations of
6

bortezomib (BZM). All cell lines except T47D were sensitive to bortezomib. B)
Raji cells and MM. 1S were treated with various concentration of dexamethasone
(DEX). The drug efficiently reduced cell growth in MM. 1S but Raji cells were
resistant to dexamethasone. C) Raji cells were treated with various
concentrations of 2,5-dimethyl-celecoxib (DMC), unmethylated-celecoxib (UMC),
and celecoxib. DMC exhibited the most potent anti-tumor effect among the three
drugs, whereas UMC was the least effective anti-proliferative compound. DMC
needed to reach a threshold (approximately 20 µM) in vitro to exert its anti-
proliferative effect. D) Raji cells were treated with increasing concentrations of
staurosporine (STS), and a decrease in cell viability was observed with the IC
50

at 400 nM. For the purpose of comparison to the known anti-multiple myeloma
effects of bortezomib and dexamethasone, Ramos, U266, RPMI/8226, and MM.
1S were included in (A) and (B).

7

In addition, we have established IC
50
s for celecoxib, DMC, and
unmethylated-celecoxib (UMC) in Raji cell. UMC harbors 20% greater COX-2-
inhibitory potency than celecoxib, but it exhibited the least potency among the
three drugs with an IC
50
greater than 100µM. On the other hand, DMC, a non-
COX-2 inhibitor, displayed the strongest anti-proliferative effect with an IC
50
of
approximately 50µM. Celecoxib showed an IC
50
of 60 µM in Raji cell (Figure 2C).
The observation exemplified that the lymphoma cell death observed in vitro is
COX-2 independent.
To determine the abilities of UMC, DMC, and celecoxib to induce ESR in
Burkitt’s lymphoma cell, Raji cells were treated with various concentrations of
each drug for 18 hours and analyzed via western blot. CHOP/GADD153, a basic
leucine zipper transcription factor associated with ER stress-induced apoptosis
(33), was employed as an indicator of ESR. In figure 3A, CHOP/GADD153
induction was observed in the treatments with 35 µM, 50 µM DMC and 75 µM
celecoxib. Among all the treatments, DMC exhibited the strongest
CHOP/GADD153 expression, which suggested COX-2 inhibition was not
involved in the activation of ESR. In figure 3B, poly (ADP-ribose) polymerase-1
(PARP-1), one of the main substrates of activated caspase pathways and a well-
established indicator of apoptotic cell death, was more effectively cleaved in the
treatment with 50 µM DMC, but there was no PARP-1 cleavage in the treatment
with 100 µM UMC (Figure3B). This result implies that UMC is not potent
enough to generate a sufficiently large amount of ESR to overwhelm the
protective effort of ESR and activate its apoptotic pathway.
8

Figure 3: Celecoxib and DMC induce CHOP/GADD153 and cleave PARP-1
efficiently.
A) Raji cells were treated with various concentrations of UMC, celecoxib, and
DMC for 18 hours. All the lysates were analyzed by western blot with specific
antibodies that recognized CHOP/GADD153, pro-PARP-1 and cleaved-PARP-1.
Actin was used as a loading control. CHOP/GADD153 is an indicator of ESR.
DMC-treated cells exhibited a strong CHOP/GADD153 expression at a
concentration lower than celecoxib and UMC, and it correlated with the
capability of cleaving PARP-1, an indicator for apoptosis, in (B).
9

Previous studies have demonstrated that DMC and celecoxib induced ESR
via the inhibition of SERCA, which leads to increased levels of intracellular
calcium (20, 36, 44). Hence, we hypothesized that combining drugs that trigger
UPR and ion imbalance would increase ESR substantially and hence would more
efficiently induce lymphoma cell death. In figure 4A, treating Raji with bortezomib,
DMC, UMC and celecoxib alone displayed approximately 40%, 45%, 10%, and
45% cell-growth inhibition respectively, whereas the combination of bortezomib
with celecoxib reduced cell viability by approximately 80%. Cells treated with
bortezomib and DMC combination exerted a slightly more potent anti-proliferative
effect, with 85% cell growth inhibition.
To confirm our hypothesis that the synergistic effect observed in Burkitt’s
lymphoma cell is associated with the elevation of ESR, we treated the Raji cells
with different drug combinations for 18 hours and analyzed via western blot.
Strong CHOP/GADD153 was observed from the combinations of 10 nM
bortezomib with 35 µM DMC and 50 µM celecoxib as well as cleaved-PARP
expression (Figure 5). The result verified our premise that the synergistic effect
observed from MTT assay is correlated with the level of ESR (Figure 4A and
Figure 5).

10

Figure 4: The combinations of bortezomib with DMC and celecoxib exert a
synergistic effect.
Raji cells were cultured with 5 nM of bortezomib, 35 µM DMC, 50 µM celecoxib,
and 75 µM UMC for 48 hours in (A). Cell viability was analyzed by MTT assay.
11

A synergy was observed from the combinations of bortezomib with DMC and
celecoxib with 85% and 80% reduction in cell viability respectively. However, no
synergistic activity was observed in cells treated with the combination of
bortezomib with staurosporine in (B); cell viability decreased only 10% more in
the combination treatments than in the single-drug treatments.

12

Figure 5

Figure 5: The combination treatments of bortezomib with DMC and
celecoxib induce CHOP/GADD153 and cleaved-PARP-1 expression.
Raji cells underwent treatments with 10 nM bortezomib, 70 µM UMC, 35 µM
DMC, and 50 µM celecoxib for 18 hours and were analyzed by western blot with
specific antibodies, CHOP/GADD153, pro-PARP-1 and cleaved-PARP-1. Actin
was used as a loading control. Cells that were treated with combinations of
bortezomib with UMC, DMC, and celecoxib exerted CHOP/GADD153 and
cleaved-PARP expression, but more prominent effects were observed from the
bortezomib with DMC and celecoxib combinations.

13

To further demonstrate that the treatment of bortezomib with
celecoxib/DMC is a novel combination treatment for Burkitt’s lymphoma, Raji cell
was treated with a combination of staurosporine and bortezomib. Staurosporine
inhibits in vitro phosphorlyation of PKC-specific substrate, myelin basic protein
fragment 4-14 (13). Treatments with staurosporine displayed an anti-proliferative
effect in Raji cell with an IC
50
of 400 nM (Figure 2D). In figure 6,
CHOP/GADD153 was absent in treatments with various concentrations of
staurosporine, and PARP-1 was efficiently cleaved. Treatments with 0.2 µM and
0.6 µM staurosporine alone inhibited cell growth by approximately 22% and 65%
respectively; nonetheless, there was no significant increase in its anti-
proliferative effect in the combination treatments (Figure 4B). The result
illustrated that although each drug alone caused cell death, it did not necessarily
mean that a synergy would be present in combination treatments. Moreover, the
absence of CHOP/GADD153 in the staurosporine and bortezomib combination
treatments correlated with the absence of its synergistic activity (Figure 7). Thus,
these results indicated that the combination of bortezomib with DMC/celecoxib is
an effective and unique treatment for Burkitt’s lymphoma.

14

Figure 6: CHOP/GADD153 stimulation is not observed in the treatments
with staurosporine
Raji cells were treated with 0.5 µM, 0.75 µM, and 1.0 µM of staurosporine for 18
hours and were analyzed by western blot. Bortezomib, a known ESR-inducer,
was included as a positive control. CHOP induction was not examined from all
three of the staurosporine treatments. However, it was expected because
15

staurosporine was known to induce apoptosis via protein kinase C inhibition

Figure 7: CHOP/GADD153 induction is not observed in the combination
treatments of bortezomib with staurosporine
.
Raji cells were cultured with 10 nM bortezomib, 0.2 µM, and 0.6 µM
staurosporine for 18 hours. The lysate, which consisted of cells treated with a
combination of bortezomib and DMC, was included in this experiment as a
positive control. None of the single-drug or combination treatments exhibited
CHOP/GADD153 induction. CHOP/GADD153 expression was not observed from
the single-drug bortezomib treatment, primarily due to the short incubation time
period.

16

Discussion
Burkitt’s lymphoma was once thought to be incurable in adults due to its
high proliferative rates (9). Although the current treatment of Burkitt’s lymphoma
is short-duration intensive combination chemotherapy, the optimal therapeutic
strategy for Burkitt’s lymphoma still remains unknown.
Glucocorticoids such as dexamethasone have played an important role in
the treatment of hematologic malignancies for more than 50 years. Sustained
exposure to pharmacological glucocorticoid concentrations inhibits lymphocyte
proliferation and ultimately culminates in cell death (49). However, when applied
dexamethasone dose-dependently to Raji cells, there was no significant increase
in anti-proliferative inhibition (Figure 2B). Thus, our data has illustrated that
Burkitt’s lymphoma cells are resistant to dexamethasone and an alternative
chemotherapeutic agent is needed.
A large body of literature has established the correlation between ESR and
many disease states, especially cancer (4-6, 34, 39, 41). Cells respond to ER
stress by activating survival pathways and pro-apoptotic pathways through UPR.
Survival pathways would up-regulate Ca-binding molecular chaperone, glucose-
regulated protein 78 (GRP78/BiP) in ER. GRP78 plays an important role in
protein folding and assembly, targeting misfolded proteins for degradation, ER
Ca2+ binding, and controlling the activation of transmembrane ER stress
sensors in response to ER stress (27, 37). However, if the ER stress becomes
too intense, ESR switches from its protective function to its pro-apoptotic
17

properties and triggers cell death. Previous studies have demonstrated that
aggravating ER stress via the combination of two ER stress-triggering drugs
would lead to glioblastoma cell death in vitro and vivo (22). Thus, we wanted to
investigate whether a similar effect could be observed in Burkitt’s lymphoma cells
via the simultaneous application of two ER stress-triggering drugs.
In our experiments, we have chosen three different drugs that were known to
trigger ER stress and cause tumor cell death. Bortezomib induces ESR through
proteasome inhibition, which results in the accumulation of damaged and/or
unfolded proteins (41). Celecoxib and its structural analog, DMC, trigger ESR
from the inhibition of SERCA, which causes leakage of calcium to cytoplasm (20,
36). Although DMC, a non-COX-2 inhibitor, is not a FDA-approved drug, it
potently mimics the anti-tumor effect observed in its parental compound in vitro
and in vivo (7, 20, 23, 24, 26, 43, 51). Moreover, another structural analog, UMC,
a potent COX-2 inhibitor, was utilized to allow further investigation of a potential
role for COX-2 in anti-tumor effect.
In our studies, single-drug treatment with bortezomib on different multiple
myeloma and Burkitt’s lymphoma cell lines impeded cell growth effectively with
IC
50
s equal to or less than 10nM (Figure 2A). In figure 6, CHOP/GADD153
expression was not observed from 10nM bortezomib, due primarily to a shorter
treatment time (18 hours). In the concentration-dependent treatments of DMC,
celecoxib, and UMC, our result is consistent with the previously published data
indicating that DMC is a more potent anti-proliferative compound than celecoxib.
Intriguingly, UMC, which maintains a more effective COX-2 inhibitory function,
18

exhibited less anti-tumor effect. This finding further supports recent studies (10,
26, 42, 45, 50, 51) including from our lab (21, 24) that the antitumor effect
observed from celecoxib is COX-2-independent.
When we combined bortezomib with either celecoxib or DMC, a strong
cytotoxicity was observed (Figure 4A), and this correlates with an increase in
ESR (Figure 5). To verify that the combination of two different ER stress-
triggering drugs would lymphoma cell killing, we have included the combination
of staurosporine with bortezomib in our study. Staurosporine, an indolo [2,3-
alpha] cabazole, is a protein kinase C (PKC) inhibitor. The combination of two
drugs neither exerted a synergistic effect (Figure 4B) nor stimulated ESR (Figure
7). From a pharmacological perspective, using minimal dosage to achieve the
effect has always been a priority. Although both combinations of 5 nM bortezomib
with 35 µM DMC or 600 nM staurosporine reduced cell viability by 85% and 80%
respectively, much lower concentrations of DMC are required to achieve the
same effect in vivo (26, 36). Moreover, induction of ER stress in cell culture
requires celecoxib or DMC concentrations of > 40 or >30 µM respectively, but
both drugs can trigger ER stress in animal models (36, 46, 47), where drug
concentrations measured in the serum are below 10 µM (26, 36). Thus, our result
has illustrated the combination treatment of bortezomib with DMC/celecoxib is a
novel drug combination for Burkitt’s lymphoma.
In summary, from our experimental data, we have illustrated a positive
correlation between ESR and cell death in the combination treatments of
bortezomib with DMC and celecoxib. Moreover, we used another close
19

structural analogue of celecoxib, UMC, which is 20% more effective in COX-2
inhibition than celecoxib (35), to further demonstrate that an anti-tumor effect
does not necessarily involve the contribution of COX-2 inhibition. Previous
experiments done in our laboratory have shown that in the case of Burkitt’s
lymphoma, DMC and celecoxib were effective in reducing tumor growth in vivo
(24). Thus, further investigation should be carried out to study the effect of the
combination treatments in animal models. Moreover, up-regulation of ER
chaperones such as GRP78 has been examined in varieties of cancer cell lines
and human cancer specimens, including breast cancer, lung cancer, liver cancer,
and prostate cancer, and been associated with malignancy, metastasis and drug
resistance (32). Down-regulation of GRP78 by siRNA was able to sensitize
various cancer cells to chemotherapeutic agents (37). Hence, experiments
should be done to examine the level of GRP78 in the Burkitt’s lymphoma cells
treated with combination of bortezomib with celecoxib and DMC.

20

Materials and Methods

Materials
Celecoxib is 4-[5-(4 methylphenyl)-3-(trifluoromethyl)-1 H-pyrazol-1-
yl]benzenesulfonamide. DMC and UMC are structural analogs of celecoxib. DMC
has 4-methylphenyl replaced by 2,5-dimethylphenyl, resulting in 4-[5-(2,5-
dimethylphenyl)-3-(trifluoromethyl)-1 H –pyrazol-1-yl]benzenesulfonamide and
UMC is an unmethlyated form of celecoxib, which results in 4-[3-(trifluoromethyl)-
1 H –pryzaol-1-yl]benzenesulfonamide. All three of them were synthesized in our
laboratory. The procedures can be found in previously published papers (24, 35).
In addition, Celecoxib, DMC, and UMC were dissolved in DMSO to a stock
concentration of 100 mM and added to the cell culture medium to keep the final
concentration of solvent below 0.1%. Velcade (Bortezomib
®
) was obtained from
the pharmacy with 3.5mg suspended in 3.5ml saline (Millennium
Pharmaceuticals, Cambridge MA). 1.0 mM staurosporine was purchased from
Sigma-Aldrich, and dexamethasone was acquired from Millennium
Pharmaceuticals with a concentration of 10mg/ml.

Cell Lines and Culture Conditions
Raji and Ramos Burkitt’s lymphoma; U266 and RPMI/8226 multiple myeloma;
T47D breast carcinoma were obtained from the American Tissue Culture
Collection, (Manassas, VA) and were cultured in RPMPI (Cellgro, Herndon, VA)
for lymphoma and multiple myeloma and in DMEM (Cellgro, Herndon, VA) for
21

T47D. The culture media were further supplemented with10% fetal bovine serum,
100 U/ml penicillin, and 0.1 mg/ml streptomycine. Cell cultures were kept in
incubators at 37°C and 5% CO
2.
MM. 1S multiple myeloma was acquired from
Nancy Krett (Northwestern University, Chicago, IL.) and was cultured and kept in
the same condition as other multiple myeloma cell lines.

MTT Assay
The 2.0-2.5 x 10
4
suspension

cells (lymphoma and multiple myeloma) or 3.0-8.0 x
10
3
adherent cells (T47D) were seeded into 96-well plates in a volume of 50 µL in
each well. The cells were treated with 50 µL of medium containing various
concentrations of drugs. After 48 hours of treatment time, 10 µL of thiazolyl blue
tetrazolium bromide (methylthiazoletetrazolium, MTT; Sigma-Aldrich, St Louis,
MO) was added to each well (stock solution of MTT is 5 mg/mL in phosphate-
buffered saline (PBS)). 100 µL of solubilization solution (10% sodium dodecyl
sulfate [SDS] in 0.01 M hydrochloric acid [HCl] ) was added after 4 hours of
incubation with methylthiazoletetrazolium. After an overnight incubation, an
enzyme-linked immunosorbent assay (ELISA) reader was used to read the plates
at single wavelength 490 nM optical density (O.D.). The background was
subtracted by a blank, which contains 100 µL of medium, 10 µL of
methylthiazoletetrazolium, and 100 µL of solubilization solution.

Western Blot Amplification
The amplification was used with PARP-1 (Cell Signaling) antibody; 25 µg of
22

protein was loaded onto 7% acrylamide gel. Protein was transferred to a
nitrocellulose blotting membrane (Life Science) via a semi-dry transfer machine
(Bio-Rad) for 80 minutes at 11V. The primary antibody contained 1µL of PARP-1
antibody in 5 ml of 5% albumin from bovine serum, minimum 98%
electrophoresis (BSA). The membrane was left in primary antibody overnight. An
Ultra-Sensitive ABC Rabbit IgG Staining Kit (PIERCE) was used for amplification.
A mixture of 10µL standard ultra-reagent A and 10µL standard ultra-reagent B in
10ml of 1X Tris-Buffered Saline (TBS) was made 30 minutes prior to use. The
membrane was incubated in 10ml of 1X TBS with 10µL of biotinylated affinity
purified goat anti-rabbit IgG for 30 minutes at room temperature. Then it was
washed three times with Tris-Buffered Saline Tween-20 (TBST) and incubated in
the ultra-reagent A and B mixture for 30 minutes at room temperature follow by
three washes with TBST. The signal was detected by using SuperSignal West
Pico Stable Peroxide Solution and SuperSignal West Pico Lumino/Enhancer
Solution (PIERCE) with SuperSignal West Femto Maximum Sensitivity Substrate
(PIERCE) and following the manufactory’s protocol.

Western Blot
50µg of protein was loaded onto 10% acrylamide gel. Protein was transferred to
a nitrocellulose blotting membrane (Life Science) via a semi-dry transfer machine
(Bio-Rad) for 80 minutes at 11V. A 500-fold dilution with CHOP/GADD-153
mouse monoclonal IgG (Santa Cruz Biotechnology)/Actin rabbit polyclonal IgG
(Santa Cruz Biotechnology) was used as the primary antibody. The membrane
23

was blocked in 5% milk for two hours and incubated in the primary antibody
overnight at 4ºC on a shaker. Then, the membrane was washed three times with
TBST and soaked in secondary antibody for 45 minutes followed by three final
washes with TBST. The signal was detected by using mixture of SuperSignal
West Pico Stable Peroxide Solution and SuperSignal West Pico
Lumino/Enhancer Solution (PIERCE) with SuperSignal West Femto Maximum
Sensitivity Substrate (PIERCE) and following the manufactory’s protocol.

24

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Abstract (if available)

Abstract Cell death induced by endoplasmic reticulum (ER) stress response (ESR) mechanism has been implicated in many diseases. The proteasome inhibitor bortezomib (Velcade®) is known to cause ESR via the accumulation of obsolete and damaged proteins. The selective cycloxygenase-2 (COX-2) inhibitor, celecoxib (Celebrex®) and its analog, 2,5-dimethyl-celecoxib (DMC) were previously shown to trigger ER stress by inhibiting the sarcoplasmic/ER calcium ATPase (SERCA), which allows leakage of calcium from the ER into the cytosol. We hypothesized that the combination of two ER stress-triggering drugs, bortezomib and DMC/celecoxib, would increase ESR substantially and greatly enhance their anti-tumor efficiency in Burkitt 's lymphoma cell. With the use of Raji cells, we have examined an elevated level of ER stress marker CHOP/GADD153, apoptotic marker PARP-1, and a significant cell growth reduction in the treatment of bortezomib with celecoxib/DMC. Hence, we propose the treatments of bortezomib with DMC and celecoxib as effective combinations to enhance Burkitt 's lymphoma cell killing in vitro.

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Asset Metadata

Creator Chen, Szu-Ting (author)

Core Title Enhanced Burkitt 's lymphoma cell killing by the combination treatments of bortezomib with celecoxib and 2,5-dimethyl-celecoxib (DMC)

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Degree Conferral Date 2008-05

Publication Date 04/16/2008

Defense Date 03/28/2008

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

Tag 2,5-dimethyl-celecoxib,Burkitt's lymphoma,celecoxib,OAI-PMH Harvest

Language English

Advisor Tokes, Zoltan A. (committee chair), Hong, Young Kwon (committee member), Schonthal, Axel (committee member)

Creator Email szutingc@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m1137

Unique identifier UC1406256

Identifier etd-Chen-20080416 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-57293 (legacy record id),usctheses-m1137 (legacy record id)

Legacy Identifier etd-Chen-20080416.pdf

Dmrecord 57293

Document Type Thesis

Rights Chen, Szu-Ting

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