Original Paper
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Acta Biochim Biophys
Sin 2007, 39: 89-95
doi:10.1111/j.1745-7270.2007.00254.x
Triptolide Inhibits
Cyclooxygenase-2 and Inducible Nitric Oxide Synthase Expression in Human Colon
Cancer and Leukemia Cells
Xiangmin TONG#,
Shuier ZHENG#, Jie JIN, Lifen ZHU, Yinjun LOU, and Hangping YAO*
Institute
of Hematologic Disease, Department of Hematology, First Affiliated Hospital,
Medical School, Zhejiang University, Hangzhou 310003, China
Received: October
16, 2006
Accepted: December
10, 2006
This work was supported
by the grants from National Natural Science Foundation of China (No.
30470746), Health Foundation of Zhejiang Province (No. 2006B041), and
Traditional Chinese Medicine Foundation of Zhejiang Province (No. 2005C066)
# These two authors
contributed equally to this study
*Corresponding
author: Tel, 86-571-87236582; Fax, 86-571-87236628; E-mail,
Abstract Triptolide (TP), a traditional Chinese medicine, has been reported
to be effective in the treatment of autoimmune diseases and exerting
antineoplastic activity in several human tumor cell lines. This study
investigates the antitumor effect of TP in human colon cancer cells (SW114) and
myelocytic leukemia (K562), and elucidates the possible molecular mechanism
involved. SW114 and K562 cells were treated with different doses of TP (0, 5,
10, 20, or 50 ng/ml). The cell viability was assessed by
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). Results
demonstrated that TP inhibited the proliferation of both tumor cell lines in a
dose-dependent manner. To further investigate its mechanisms, the products
prostaglandin E2 (PGE2) and nitric oxide (NO)
were measured by enzyme-linked immunosorbent assay (ELISA). Our data showed
that TP strongly inhibited the production of NO and PGE2.
Consistent with these results, the expression of inducible NO synthase (iNOS)
and cyclooxygenase-2 (COX-2) was up-regulated both at the mRNA level and the
protein expression level, as shown by real-time RT-PCR and Western blotting.
These results indicated that the inhibition of the inflammatory factor COX-2
and iNOS activity could be involved in the antitumor mechanisms of TP.
Key words triptolide; leukemia; colon cancer;
cyclooxygenase-2; inducible nitric oxide synthase
Inducible cyclooxygenase-2 (COX-2) and nitric oxide synthase (iNOS)
are important enzymes that mediate inflammatory processes [1]. Recent studies
have shown that improper up-regulation of COX-2 and/or iNOS has
been associated with pathophysiology of certain types of human cancers [2,3].
The expression of COX-2 and its induced product, prostaglandin E2 (PGE2), can be induced by various agents, including
inflammatory cytokines, mitogens, reactive oxygen intermediates and many other tumor
promoters. Increased expression of COX-2 and PGE2 has been reported in many colorectal tumors and adenocarcinomas
[4,5]. iNOS catalyzes the oxidative deamination of L-arginine to produce
NO, a potent pro-inflammatory mediator. NO has multifaceted roles in
mutagenesis and carcinogenesis [6–8].Triptolide (TP), a purified component of a traditional Chinese
medicine, is extracted from a shrub-like vine Tripterygium wilfordii
Hook F. It has been reported to be effective in the treatment of autoimmune diseases,
especially rheumatoid arthritis [9]. TP can also induce antineoplastic
activity on several human tumor cell lines. It was able to inhibit
transcriptional activation of NF-kB in Jurkat cells and human
bronchial epithelial cells [10,11]. Furthermore, TP has also been shown to
down-regulate the expression of various NF-kB-regulated
genes or to induce apoptosis [12,13]. It was also reported that TP inhibits
vascular endothelial growth factor expression, which is believed to play a role
in tumor angiogenesis [14].We have shown previously that the alleviation of rheumatoid
arthritis by TP might involve the inflammatory factors created through the
COX-2 and iNOS pathways [15]. In the present study, we show for the first time that TP suppression
on tumor proliferation is associated with inhibition of COX-2 and iNOS
activation in two different tumor cell lines, myelocytic leukemia cell line
K562 and human colon cancer cell line SW114. These results pave the way for a
comprehensive understanding of TP mechanisms.
Materials and Methods
Cell culture and triptolide
preparation
Human myelogenous leukemia cell line, K562 (ATCC), and human colon
cancer cell line, SW114 (ATCC), were grown in RPMI 1640 medium (Gibco,
California, USA) supplemented with 10% heat inactivated fetal calf serum
(Gibco), 100 U/ml penicillin, 100 mg/ml streptomycin (Gibco) and 2 mM L-glutamine
(Gibco). All cell lines were kept under sterile conditions at 37 ?C with 5% CO2. TP (Sigma, New York, USA) was diluted to various concentrations in
serum-free culture medium. K562 and SW114 cells were treated with various
concentrations of TP (0, 5, 10, 20, or 50 ng/ml) for 24 h.
Assay for PGE2 and NO
production
The levels of PGE2 in culture supernatants were
determined by competitive enzyme-linked immunosorbent assay (ELISA) kit
(R&D Systems) according to the manufacturer’s instructions. The lower limit
of detection was 36.2 pg/ml.NO levels in culture supernatants were measured as its oxidized
product nitrate. The kits were purchased from R&D Systems, and the lower
limit of detection was 1.35 mM.
RNA isolation and real-time
reverse transcription-polymerase chain reaction (RT-PCR)
RNA isolation and real-time
reverse transcription-polymerase chain reaction (RT-PCR)
Total cellular RNA in the treated K562 and SW114 cells were isolated
with Trizol reagent (Gibco) in accordance with the manufacturer’s instructions.
Complementary DNA (cDNA) was prepared by RT of 2 mg total RNA using oligo dT18 and 200 U superscript II reverse transcriptase (Invitrogen,
California, USA) at 42 ?C for 70 min according to the manufacturer’s
instructions.Quantitative RT-PCR was carried out by LightCycler technology (Roche
Molecular Biochemicals, Mannheim, Germany) using SYBR Green I detection. In all
assays, cDNA was amplified using a standardized program (10 min for the denaturing
step; 55 cycles of 5 s at 95 ?C, 15 s at 65 ?C, and 15 s at 72 ?C; melting
point analysis in 0.1 ?C steps, the final cooling step). Each LightCycler
capillary was loaded with 1.5 ml DNA Master mix, 1.8 ml MgCl2 (25
mM), 10.1 ml H2O and 0.4 ml of each primer (10 mM). The final
amount of cDNA per reaction corresponded to the 2.0 ng of RNA used for RT.
Relative quantification of target gene expression was carried out using a
mathematical model, which was also recommended by Roche Molecular Biochemicals.
The following primers were use for the experiment: COX-2 (product size:
756 bp), sense, 5‘-CAGCACTTCACGCATCAGTT-3‘, antisense, 5‘-TCTGGTCAATGGAAGCCT-3‘;
iNOS (product size: 237 bp), sense, 5‘-TCTTGGTCAAAGCTGTGCTG-3‘,
antisense, 5‘-CATTGCCAAACGTACTGGTC-3‘; b-actin (product size: 619 bp), sense, 5‘-CGCTGCGCTGGTCGTCGACA-3‘,
antisense, 5‘-GTCACGCACGATTTCCCGCT-3‘.
Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and Western blotting analysis
Cell lysates were prepared for Western blotting analysis of iNOS and
COX-2 by using whole cellular protein extraction kits (Active Motif,
California, USA). The concentration of protein in each cell lysate was
determined using a BCA protein assay it (Pierce, Rockford, USA) with bovine
serum albumin (BSA) as the standard. An identical amount of protein (40 mg) from each sample was loaded onto a 10% sodium dodecyl
sulfate-polyacrylamide gel and transferred to nitrocellulose membranes (0.45 mm; S&S, Dassel, Germany). Nitrocellulose membranes were blocked
with 5% BSA (Sigma) in TBS (25 mM Tris-HCl, 150 mM sodium chloride, pH 7.2)
for 1 h at room temperature. Blots were incubated with anti-COX-2, anti-iNOS or
anti-b-actin specific rabbit polyclonal IgG primary antibody (Santa Cruz
Biotechnology, Santa Cruz, USA) at 1:500 dilution at 37 ?C for 2 h. Blots were
washed three times then incubated in horseradish peroxidase (HRP)-conjugated
goat anti-rabbit antibody (1:2000 dilution) for 2 h at room temperature. All blots
were developed using enhanced chemoluminescence reagents (Super signal dura
kit; Pierce) following the manufacturer’s instructions. Blots were scanned and
analyzed for the measurement of the band intensities. Results were calculated
as relative ratios of a specific band and the b-actin
one.
Cell viability
In vitro, the growth inhibition effect
of TP on K562 and SW114 cells was determined by measuring
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) dye
absorbance of living cells. Briefly, cells (1?105 cells per well) were seeded in 96-well
microtiter plates. After exposure to the drug (0, 5, 10, 20, or 50 ng/ml) for
48 h, 20 ml of MTT solution (5 mg/ml in PBS) was added to each well and the
plates were incubated for an additional 4 h at 37 ?C. MTT solution in medium
was aspirated off. To achieve solubilization of the formazan crystal formed in
viable cells, 200 ml of dimethyl sulfoxide (DMSO) was added to
each well before absorbance at 570 nm was measured. Each assay was carried out
three times.
Results
Effects of TP on PGE2 and NO
production
The treatment of K562 and SW114 cells with the presence of TP
reduced both the PGE2 and NO production. As it was shown in Table
1, the response was dose-dependent, and the effect was significant when the
concentration of TP was above 20 ng/ml.
Effects of TP on the mRNA
transcription level of COX-2 and iNOS
As described previously, PGE2 is
synthesized by COX-2, and NO is synthesized by iNOS. The results of the
specific RT-PCR analysis corresponded well to the level of PGE2 and NO in supernatants (Figs. 1 and 2). The
inhibition pattern between COX-2 and iNOS did not show any significant
difference. It was observed that TP markedly inhibited COX-2 and iNOS
mRNA expression in K562 cells at 20 ng/ml and 50 ng/ml, respectively. In
contrast, marked inhibition of expression in SW114 cells was observed at 5
ng/ml.
Effects of TP on COX-2 and
iNOS protein expression
As expected, COX-2 and iNOS expression in tumor cells
at the protein level, under different doses, was parallel with that at the mRNA
level, suggesting that no post-translational modifications of the mRNA
transcript are necessary to account for the effect. The inhibition effect of TP
on tumor cell proliferation was dose-dependent (Figs. 3 and 4).
We observed that TP markedly reduced the expression of COX-2 and iNOS protein
in K562 and SW114 cells at concentrations of 10 ng/ml and 5 ng/ml, respectively.
Cell viability assay of SW114
and K562 cells
The cytotoxic effect of TP on SW114 and K562 cells was examined by
exposing the cells to different concentrations of TP for 48 h. The resulting
growth curves (Fig. 5) show that TP has a concentration-dependent
inhibitory effect. The inhibitory effects of TP were more pronounced at higher
doses. The TP IC50 was approximately 25–35 ng/ml for SW114 and 40–50 ng/ml for
K562. The results show that SW114 was more sensitive than K562 to the treatment
by TP. To determine if the inhibition effect of TP on tumor cell proliferation
was associated with the COX-2 and iNOS pathways, we studied its effect in
RPMI-8226 cells (low COX-2 and iNOS expression) and found a
similar cytotoxic effect (data not shown).
Discussion
Elevated PGE2 production can stimulate
epithelial cell growth and promote cellular survival. A number of previous
experimental studies support a role for products of COX and iNOS activity in the
pathogenesis of cancer [16,17]. NO mediates DNA damage or hinders DNA repair,
and is thus potentially carcinogenic. NO can stimulate tumor growth and
metastasis by promoting migratory, invasive, and angiogenic abilities of tumor
cells, which might also be triggered by activation of COX-2 [18]. Up-regulation
of both PGE2 and NO has been reported in a variety of different malignancies [19–22], including
colorectal cancer and leukemia. Our results revealed that TP indeed inhibited
the production of PGE2 and NO in SW114 and K562 cells in a
dose-dependent manner, assessed by ELISA. These studies suggest the reduced
products of NO or PGE2 might contribute to the inhibition effects of
TP on tumor growth.The up-regulation of PGE2 and NO results from the
production of COX-2 and iNOS. COX-2 and iNOS products have been implicated in
the regulation of the immune system, in tumor cell apoptosis, and involved in
many human carcinogeneses, including chronic myeloid leukaemic cells [23–25]. Ozel et
al. [26] demonstrated that iNOS expression might act in the first
steps of carcinogenesis, whereas COX-2 expression was seen in more
advanced tumors. Cianchi et al. [27] showed a prominent role of NO in
stimulating COX-2 activity in colorectal cancer. For further study, we therefore
decided to test the effects of TP on two types of cell lines to see if this
compound would suppress the expression of COX-2 and iNOS.The major focus of this study was to investigate the chemopreventive
efficacy of TP as a possible inhibitor of COX-2 and iNOS
expression using SW114 and K562 cells. Selection of TP for study as a
chemopreventive agent was, in part, based on the evidence that TP has an
inhibitory effect on arachidonic acid-induced inflammation and on its
inhibition of arachidonic acid metabolism through the inhibition of
cyclooxygenase. The outcome of this study is significant as it clearly
emphasizes that TP has the potential to specifically inhibit the expression of COX-2
and iNOS at the mRNA and protein level in SW114 and K562 cells. The
inhibitory effect of TP is concentration-dependent. Furthermore, our results
also revealed that SW114 was more sensitive than K562 in the inhibition of COX-2
and iNOS expression by TP. We also addressed the possibility that the down-regulated PGE2 and NO by TP might be the result of the suppression of COX-2 and
iNOS instead of the inhibition of tumor cell proliferation. We have found a
similar growth inhibitory effect in RPMI-8226 cells (low COX-2 and iNOS
expression) with TP treatment (data not shown) [28]. The results of this study confirm that TP is an inhibitor of COX-2
and iNOS and their products PGE2 and NO in SW114 and K562
cells. Because COX-2- or iNOS-dependent mechanisms are involved in
carcinogenesis and tumor progression [29], these findings provide a new
uncovering mechanism about antitumor effects of TP.However, TP does have one major drawback as an antitumor agent,
namely its toxicity. Pyatt et al. [30] demonstrated that therapeutic
concentrations of TP exerted a significant hematotoxic effect by inhibiting
growth factor response in CD34+ bone marrow cells. However, it
should be pointed out that our clinical data showed different results. As early
as the 1980s, our clinical department and other departments in China have used
TP to treat acute leukemia in clinical trials [31]. The effective dose is 30 mg/kg for day 1–7. No toxicity of heart, liver, kidney, or gastrointestinal tract
were observed, and the hematological toxicity was also mild.In conclusion, we have demonstrated that TP could inhibit COX-2 and
iNOS activity, highlighting the potential clinical value of TP in the treatment
of colon cancer and leukemia. These data suggest that further evaluation of the
pharmacological effect of TP is needed to develop a new therapeutic strategy
for treating cancer.
Acknowledgement
We would like to thank Dr. Licheng WU (The Chicago University,
Chicago, USA) for reviewing this manuscript.
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