Enhanced Antitumor Effect of CombinedT riptolide and
Wei Wang,1,2Shanmin Yang,1Ying Su,1Zhenyu Xiao,1Chunyou Wang,3Xinfeng Li,2Ling Lin,2
BruceM. Fenton,1Scott F. Paoni,1Ivan Ding,1PeterKeng,1Paul Okunieff,1and Lurong Zhang1
Purpose:The lackof effective treatment for pancreatic cancer results in a very low survival rate.
Thisstudyexplorestheenhancementof thetherapeuticeffectonhumanpancreaticcancer viathe
combinationof triptolide andionizing radiation (IR).
Experimental Design: In vitro AsPC-1human pancreatic cancer cells were treated with
triptolide alone, IRalone, or triptolideplus IR. Cellproliferationwas analyzed withsulforhodamine
B (SRB) method and clonogenic survival; comparison of apoptosis induced by the above
treatment was analyzed by annexin V ^ propidium iodide (PI) staining. Furthermore, the
expression of apoptotic pathway intermediates was measured by the assay of caspase activity
andWesternblot. Mitochondrial transmembrane potential was determinedbyJC-1assay. Invivo,
AsPC-1xenografts were treated with 0.25 mg/kg triptolide, 10 Gy IR, or triptolide plus IR.
The tumors were measured for volume and weight at the end of the experiment.Tumor tissues
were tested for terminal nucleotidyl transferase ^ mediated nick end labeling (TUNEL) and
Results: The combination of triptolide plus IR reduced cell survival to 21% and enhanced
apoptosis, compared with single treatment. In vivo, tumor growth of AsPC-1xenografts was
reduced further in the group treated with triptolide plus IR compared with single treatment.
TUNEL and immunohistochemistry of caspase-3 cleavage in tumor tissues indicated that the
combination of triptolide plus IR resulted in significantly enhanced apoptosis compared with
Conclusions:Triptolide in combination with ionizing radiation produced synergistic antitumor
effects on pancreatic cancer both in vitro and in vivo and seems promising in the combined
modality therapy of pancreatic cancer.
Pancreatic adenocarcinoma remains one of the most lethal of
malignancies. The incidence of pancreatic cancer has steadily
increased over the past four decades (1). Satisfactory treatment
is available only for the minority of patients who present with
very early-stage disease. Despite recent research and improve-
ments in imaging, efforts to detect tumors at an earlier stage
or augment standard therapy have done little to change the
dismal prognosis. The 5-year survival rate is <5% (1), ranking
this cancer as the fourth leading cause of cancer death (2).
Importantly, at the time of diagnosis, the majority of patients
(80-90%) already have locally advanced, metastatic, or
inoperable tumors. Radiation therapy alone or in combina-
tion with chemotherapy has shown only modest efficacy in
local control and palliation (3, 4). A new therapeutic strategy
is urgently needed to control this aggressive cancer.
Triptolide, a diterpenoid triepoxide (MW, 360) derived
from the herb Tripterygium wilfordii, has been used as a natural
medicine in China for hundreds of years (5). Several recent
papers have evaluated triptolide as an antitumor agent (6–8).
Among its actions, triptolide slows proliferation of a variety of
cell types. Slowly proliferating cells accumulate more genetic
damage per cell cycle during a course of radiation therapy
than more rapidly dividing cells. This heightened accumula-
tion of DNA damage might overcome repopulation, the
process in which cells continue to divide during a protracted
course of therapy. Triptolide is also reported to be an effective
inducer of apoptosis in solid cancer cells, including breast,
prostate, and lung cancer (9). Although the mechanism is not
well elucidated, it is suggested that triptolide might induce
apoptosis by altering pathways involving p21 and p53 (10).
Several studies have also shown that triptolide induces DNA
damage (11, 12). In this study, we explore the effect of
Cancer Therapy: Preclinical
Medical Center, Rochester, NewYork;2Department of Surgery, Second Affiliated
Hospital of Fujian Medical University, Quanzhou, Fujian, China; and3Pancreas
Surgery Center, Union HospitalofTongji Medical College,Wuhan, Hubei, China
Received 2/20/07; revised 4/5/07; accepted 5/24/07.
Grant support: U.S. Army Medical Research and Materiel Command (DAMD17-
00-1-0081and DAMD17-01-1-0708) and Susan G. Komen Foundation (L. Zhang),
the DAMD17-01-1-0708 (S.Yang), and the National Cancer Institute/Institute of
The costs of publicationof this articlewere defrayedinpart by the paymentof page
charges.This article must therefore be hereby marked advertisement in accordance
with18 U.S.C. Section1734 solely toindicatethis fact.
Requests for reprints: Paul Okunieff, Department of Radiation Oncology,
University of Rochester Medical Center, 601Elmwood Avenue, Rochester, NY
14642-8647. Phone: 585-275-2985; Fax: 585-275-1531 ; E-mail: Paul___Okunieff@
F2007 American Association for Cancer Research.
www.aacrjournals.org Clin Cancer Res 2007;13(16) August15, 20074891
triptolide in combination with IR in an attempt to evaluate
the underlying mechanisms by which this new therapeutic
approach controls pancreatic cancer.
Materials and Methods
Cells and reagents.
obtained from the American Type Culture Collection. The cells were
maintained as monolayer cultures in DMEM supplemented with 10%
fetal bovine serum, 100 Ag/mL of streptomycin, and 100 units/mL
of penicillin. The cells were incubated at 37jC in a humidified
atmosphere of 5% CO2.
Triptolide with 99.9% purity was purchased from the Institute of
Medical Research (Fuzhou, China); sulforhodamine B (SRB) was
purchased from Sigma; caspase-3 assay kits were purchased from
Molecular Probes; caspase-8 and caspase-9 assay kits were purchased
from BioVision; JC-1, annexin V, and propidium iodide (PI) were
purchased from Molecular Probes; cytochrome c, cleaved caspase-3,
and poly(ADP-ribose) polymerase (PARP) polyclonal antibodies were
purchased from Cell Signaling Technology, Inc.; and ApopTag in situ
apoptosis detection kits were purchased from Chemicon International,
Cell viability assay. Viability of AsPC-1 after different treatments
was evaluated by SRB assay (13). Briefly, AsPC-1 cells (1 ? 103cells per
well) were plated in triplicate in 96-well plates overnight and changed
to fresh media. The cells were treated with different concentrations
(0, 12.5, 25, or 50 nmol/L) of triptolide alone or followed by 4 Gy IR at
a dose rate of 280 cGy/min delivered by a Cs-137 Mark I irradiator. The
control cells were treated with the same concentration of vehicle
(0.01% DMSO) or mock IR. Forty-eight hours later, 50 AL of 10%
trichloroacetic acid was added to fix cells at 4jC for 2 h, stained with
70 AL of 0.3% SRB for 30 min, color developed with 200 AL Tris base
(10 mmol/L; pH, 10.5), and read at A490.
Clonogenic survival assay. AsPC-1 cells were treated with vehicle
(DMSO) alone, triptolide alone (0, 3.125, 6.25, 12.5, or 25 nmol/L),
triptolide plus IR at a dose of 0, 2, 4, 6, or 8 Gy, or IR alone and plated
in 60-mm dishes at different densities based on the stringency of
treatments. The number of cells was adjusted to generate 50 to 200
colonies per dish at each radiation dose. After 21 days, the colonies
(containing z50 cells) were stained with crystal violet, and the numbers
of colonies were counted with FluorChem SP (Alpha Innotech). The
surviving fraction (SF) was calculated as a ratio of the number of
colonies to the number of cells plated (plating efficiency) divided by the
same ratio calculated for the nonirradiated group. D0(the incremental
dose required for reducing the fraction of colonies to 37%, indicative of
single-event killing) was calculated using the formula of the single hit
multitarget (SHMT) model [SF = 1 - (1 - e-(D/D0))n; ref. 14]. SF2 is the
surviving fraction of exponentially growing cells when irradiated at
the clinically relevant dose of 2 Gy.
Flow-cytometric analysis of apoptosis.
with triptolide (25 nmol/L) alone, IR alone (4 Gy), or triptolide plus IR,
cells were harvested, stained with annexin V for 30 min and then with
PI, immediately followed by flow-cytometric analysis according to the
manufacturer’s instructions. The percentage of cells that were annexin V
positive but PI negative was compared among the different treatment
groups. For the cell cycle assay, the harvested cells were immediately
fixed in 75% alcohol overnight, then treated with 1% RNase A for
30 min at room temperature, and stained with PI for 10 min; samples
were then measured by flow cytometry (FACS, Becton Dickinson). The
data were analyzed with CellQuest software.
JC-1 analysis for mitochondria membrane potential.
membrane potential was measured by flow cytometry using JC-1
staining. About 2 Ag of JC-1 in 30 AL of saline was added to 100 AL of
single cell suspension that had been treated with 25 nmol/L triptolide
alone (48 h exposure) or 4 Gy IR alone, or both in combination (IR
AsPC-1, a human pancreatic cancer cell line, was
Exactly 48 h after treatment
followed immediately by triptolide treatment). After 10 min, cells were
washed twice with PBS and immediately subjected to flow-cytometric
analysis. The percentage of cells in the high-red region or low-red and
high-green region was measured under the different treatments.
Assays for activities of caspase-3, caspase-8, and caspase-9.
activity of caspase-3 was measured with fluorescent substrate assay
according to the manufacturer’s instructions (Molecular Probes).
Briefly, 1 ? 106cells treated with either vehicle alone, triptolide alone
(25 nmol/L), IR alone (4 Gy), or both in combination (IR followed
immediately by triptolide treatment) for 24, 48, and 72 h were collected
and resuspended in cold lysis buffer. About 50 AL of 2? reaction buffer
was added to 50 AL of cell lysate and incubated for 2 h at 37jC with
DEVD-R110, a caspase-3 substrate, which released fluorescence after its
cleavage. Similarly, caspase-8 and caspase-9 activities were measured
using kits purchased from BioVision following the manufacturer’s
Western blot analysis. For detection of released cytochrome c, AsPC-
1 cells were treated with 25 nmol/L triptolide alone, 4 Gy IR once, or
both in combination. Exactly 48 h later, cells were washed with PBS and
harvested in a buffer [20 mmol/L HEPES (pH, 7.5), 10 mmol/L KCl,
1.5 mmol/L MgCl2, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L
DTT, 250 mmol/L sucrose, 1 mmol/L phenylmethylsulfonyl fluoride,
1 mg/mL aprotinin, and 1 mg/mL pepstatin A] for 20 min at 4jC
and then homogenized using a Dounce homogenizer (40 strokes) and
centrifuged at 10,000 rpm for 15 min at 4jC for the cytosol portion.
Cytosol protein (50 Ag) was subjected to 12% SDS-PAGE, transferred to
immunoblotting membrane, stained with monoclonal antibody against
cytochrome c, followed by antimouse secondary antibody conjugated
to horseradish peroxidase (HRP), and then visualized with enhanced
chemiluminescence (ECL; Amersham). The cleavage of caspase-3 and
PARP is a characteristic marker of apoptosis. For the detection of this,
AsPC-1 cells in 100-mm dishes at 80% confluence were treated with
vehicle alone, 25 nmol/L triptolide alone, 4 Gy IR alone, or triptolide
and IR in combination for 48 h, and then harvested with 1 mL of lysis
buffer (1% Triton X-100, 0.5% Na deoxycholate, 0.5 Ag/mL leupeptin,
1 mmol/L EDTA, 1 Ag/mL pepstatin, and 0.5 mmol/L phenyl-
methylsulfonyl fluoride). The protein concentration of the lysate was
determined by the bicinchoninic acid method (Pierce). About 30 Ag of
protein was loaded onto a 10% SDS-PAGE, electrophoresed, and
transferred to a nitrocellulose membrane. The loading and transferring
of equal amounts of protein were confirmed by staining the membrane
with a Ponceau S solution (Sigma). The membranes were blocked with
5% fat-free milk in PBS (pH, 7.4) for 30 min and then incubated
overnight with 0.2 Ag/mL of anticleaved caspase-3 or PARP polyclonal
antibodies separately. After washing, the membranes were incubated
with HRP-labeled secondary antibodies for 1 h followed with ECL
exposure. For reprobing glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), the blots were stripped with a buffer containing 50 mmol/L
Tris-HCl (pH, 6.8), 2% SDS, and 0.1 mol/L h-mercaptoethanol.
Xenografts in nude mice.AsPC-1 cells were grown in 15-cm2dishes
to 80% confluence, harvested with 10 mmol/L EDTA, and resuspended
in PBS for 107cells/mL. A suspension of 2 ? 106cells in 0.2 mL PBS
was injected s.c. into the hind leg of athymic nude mice using a 27.5-
gauge needle. Tumors were allowed to grow for 7 days before treatment.
Thirty-two nude mice with established tumors (all f100 mm3) were
divided into four groups and treated with (a) vehicle (PBS) alone; (b) a
single dose of 10 Gy IR; (c) 0.25 mg/kg of triptolide twice weekly for
4 weeks; or (d) triptolide plus IR (first dose of triptolide administered
immediately after IR). The IR treatment of tumors grown in hind legs
was carried out at a dose rate of 128 cGy/min with a Cs-137 Mark I
irradiator on day 7 (initiation of treatment). Tumor size was measured
thrice per week with Vernier calipers. The tumor volume was
determined according to the formula: (length ? width2)/2. The body
weight of each mouse in each group was recorded once per week.
Growth delay time (GD) was calculated as the time for treated tumors
to reach z400 mm3in volume minus the time for control tumors to
reach z400 mm3in volume. Most tumors were harvested on day 38;
Cancer Therapy: Preclinical
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T reatment Effectof CombinedT riptolide and Radiation
www.aacrjournals.orgClin Cancer Res 2007;13(16) August15, 20074899