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Introduction
Triptolide is a diterpenoid triepoxide extracted from the traditional Chinese
herb Tripterygium wilfordii Hook F (TWHF),
and exhibits potent immunosuppressive and anti-inflammatory properties, which are effectively used in treating autoimmune
diseases[1]. Recent data have shown that triptolide has a broad spectrum of activity against tumors.
In vitro, triptolide inhibited the proliferation of a number of tumor cell lines including H23 (non-small cell lung cancer, NSCLC), HT1080
(fibrosarcoma) and COLO205 (colon carcinoma) at extremely low concentrations (2_10 ng/mL). Likewise,
in vivo treatment of mice with triptolide inhibited the growth of xenografts formed by different tumor cell lines (B16 melanoma, MDA-435 breast
cancer, TSU bladder cancer, and MGC80-3 gastric carcinoma). Importantly, it is found that the antitumor effect of triptolide
was comparable or superior to that of conventional antitumor drugs, such as adriamycin, mitomycin, and cisplatin. Additionally,
tumor cells that were resistant to Taxol, attributable to the overexpre-ssion of the multidrug resistant gene 1, were still
sensitive to triptolide[2].
Chemokines are a group of small pro-inflammatory chemoattractant proteins that participate in homeostatic process,
including lymphocyte homing to secondary lymphoid organs, eg, lymph nodes and Peyer's patches under physiological
conditions, as well as in pathologic conditions such as inflammation, tumor growth and
metastasis[3,4]. Stromal cell-derived factor-1 (SDF-1), which binds only to CXC chemokine receptor 4 (CXCR4), belongs to the CXC chemo-kine subfamily. It is
produced mostly by bone marrow-, lymph node-, muscle-, and lung-derived stromal cells, endothelial cells and fibroblasts. In
humans there are two identified splice variant forms of SDF-1,
SDF-1a, and SDF-1b, derived from a single gene, and the former
is more abundant than the latter. This chemokine ligand/receptor axis plays an important biological role in homing
hematopoietic stem cells into the marrow
microenvironment[5]. Accumulating evidence suggested that functional CXCR4 was highly
expressed on a wide variety of tumor cells including those in breast cancer, NSCLC, prostate cancer, glioma, glioblastoma,
and so on. Moreover, it has been shown that these CXCR4 positive tumor cells can metastasize preferentially to the organs
that secrete SDF-1 (eg, bone marrow, lymph node, lung or
liver)[6_10]. Given the evidence that SDF-1/CXCR4 axis plays a key role
in regulating the metastatic behavior of several tumors, modulation of this axis may be important for inhibiting metastasis of
CXCR4 positive tumor cells.
Non-Hodgkin's lymphoma (NHL) is a heterogeneous group of malignancy of B cells or T cells that usually originate in the
lymph node, and are characterized by a lymph node metastatic pattern involving principally organs with large concentrations
of lymphatic tissue such as lymph nodes, tonsils, spleen and bone
marrow[11]. Regular radiotherapy and chemotherapy
simply target at killing tumor cells, the remission rate is limited. If a new antitumor agent is designed to focus on mechanisms
involved in tumor metastasis but not only to kill cells, a new field may be expanded in treatment of NHL. CXCR4 has been
reported to be also expressed on the majority of NHL cell lines and primary cells including those in diffuse large B-cell
lymphoma, peripheral blood T-cell lymphoma, lymphocytic lymphoma, follicular lymphoma and mantle cell lymphoma as well,
and CXCR4 is required for invasion of lymphoma cells into tissues and therefore essential for lymphoma
metastasis[12]. Because the SDF-1/CXCR4 axis is pivotal for modulating the trafficking of lymphoma cells, it can obviously become a new
target for the antitumor drug in the treatment of NHL.
In the present study, we chose B-NHL cell line Raji cells to investigate the antiproliferative effects of triptolide. Furthermore,
we chose stromal cells and lymphoma cells freshly isolated from the lymph nodes of patients with NHL as the target to study
the effects of triptolide on the lymph node metastasis and to explore the mechanism by which triptolide regulates
SDF-1/CXCR4 axis in NHL. We focused on the changes in the expression of SDF-1 and CXCR4, and observed the effects of triptolide
modulating the migration of primary lymphoma
cells in vitro.
Materials and methods
Drugs and reagents Triptolide
(C20H24O6, molecular weight 360.4, Figure 1) was purchased from Sigma Chemical Company
(St Louis, MO, USA) and was initially dissolved in dimethylsulfoxide (DMSO), and stored at -20oC, and then thawed before use.
3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H
-tetrazolium (MTT) was purchased from Janssen Chimica Company (New
Brunswick, NJ, USA). Dulbecco's Modified Eagle's Medium (DMEM, low-glucose), RPMI-1640 culture medium, new-born
calf serum, fetal calf serum (FCS) and TRIzol reagent were purchased from Gibco, Invitrogen Corporation (Carlsbad, Califorlia,
USA). Me2SO was purchased from Sigma. RevertAid first-strand cDNA synthesis kit was purchased from MBI Fermentas
(Hanover, MD, USA). Trypsin was purchased from Amresco (Ohio, USA). Recombinant human
SDF-1a (rhSDF-1a) was purchased from PeproTech (Rocky Hill, NJ). Phycoerythrin (PE)-conjugated anti-human CXCR4 (Cat 12-9999, clone: 12G5)
and its isotype control PE-conjugated mouse IgG2a (Cat 12-4729) were purchased from eBioscience (CA, USA). Simultest
IMK-lymphocyte mouse anti-human CD3-fluorescein isothiocyanate (FITC)/CD19-PE and its isotype control IgG1/IgG2a
(Cat 340182) were purchased from Becton Dickinson Immunocyto-metry system (San Jose, CA, USA). Transwell (24-well cell
clusters, 6.5 mm diameter, 5.0 µm pore size) was purchased from Corning Incorporated Costar (NY, USA). The Raji cell line
was obtained from China Center for Typical Culture Collection (Wuhan, China), and was grown in RPMI-1640 culture medium
containing 10% FCS and 2 mmol/L L-glutamine at 37oC in a 5% CO2 incubator.
Patients After informed consent, cervical lymph nodes were obtained from newly diagnosed patients with NHL from the
Department of Hematology at Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. A
total of 7 patients included 5 cases of diffuse large B-cell lymphoma (3 of stage IV, 2 of stage III) and 2 cases of peripheral
T-cell lymphoma (1 of stage IV, 1 of stage II). The diagnosis and classification of patients were based on the Revised European
American Lymphoma Classification[13]. In addition, reactive hyperplasia lymph nodes in 3 cases of non-malignant diseases
were obtained as negative controls.
Lymph node stromal cells and lymphoma cells preparation
Single cell suspensions of lymph node specimens obtained
from patients with NHL were cultured in DMEM (low-glucose) supplemented with 10% heat-inactivated new-born calf serum
in plastic flasks at a concentration of
1×106cells/mL at 37oC in a humidified 5%
CO2 atmosphere for 3 d. Any non-adherent
cells were then removed and the adherent cells were cultured further until they reached confluency. Fresh medium was then
supplied twice a week depending on their growth and the cells were passaged every
2_3 weeks. The adherent cells prepared in this way were hereafter referred to as lymph node stromal cells, and early passage cells were used in the subsequent
studies. Cells were grouped as follows: the control group; and those treated with triptolide at concentrations of 12.5, 25, 50
nmol/L for 24 h, respectively. In addition, single cell suspensions freshly isolated from surgical lymph node specimens were
washed and resuspended in D-Hank's supplemented with 1% FCS. Trypan blue staining was used to assess the cell viability
to be more than 98%. The cells prepared were used as lymphoma cells and grouped as above.
MTT assay The antiproliferative effects of triptolide against different cell groups were determined by the MTT dye
uptake method. A total of 40 000 Raji cells per well were incubated in triplicates in a 96-well plate. Different concentrations
of triptolide were added, and the final concentrations were 6.25, 12.5, 25, 50, and 100 nmol/L. The plates were in the presence
or absence of the indicated test samples for 0, 24, 36, 48, 60, and 72 h. The largest
Me2SO dissolved concentration group was
used as the control group. MTT solution 20 µL [5 g/L in phosphate-buffered saline (PBS)] was then added to each well. After
4 h at 37oC, the supernatant was removed and 150 µL DMSO was added. When the blue crystal was dissolved, the optical
density (OD) was detected in the microplate reader at 490 nm wavelength by a 96-well multiscanner autoreader (Biotech
Instruments, New York, USA). The following formula was used: cell proliferation inhibited
(%)=[1-(OD of the experimental samples/OD
of the control)]×100%.
Reverse transcriptase-polymerase chain reaction (RT-PCR) for
SDF-1a mRNA Oligonucleotide primers were
designed by premier 5.0 from human cDNA sequences available in GeneBank and synthesized by Shanghai
Sangon Biological Engineering & Technology Service (Shanghai, China) as follows:
SDF-1a, the sense primer was 5'-GCCATG-AACGCCAAGGTCGTGGT-3', and the antisense was 5'-CCT-CGAGTGGGTCTAGCGGAAAG-3';
β-actin, the sense primer was 5'-TACATGGCTGGGGTGTTGAA-3' and the antisense was 5'-AAGAGAGGCATCCTCACCCT-3'. Amplified fragments
were 317 bp for SDF-1a and 219 bp for β-actin. The lymph node stromal cells were digested by 0.25% trypsin. Total RNA was
isolated from the cells by TRIzol reagent. The reverse transcription reaction was performed using the RevertAid first-strand
cDNA synthesis kit and the synthesized cDNA was amplified by PCR. The 50 µL reaction mixture contained: 3 µL cDNA, 1
µL 10 mmol/L dNTPs, 3 µL 2.0 mmol/L
MgCl2, 5 µL 10×PCR buffer, 3 µL
of a pair of primer (10 pmol/µL), 1 µL
Taq polymerase (1 U/µL), and 31 µL
ddH2O. The PCR schedule consisted of a 5 min initial denaturation at 94oC, followed by 30 cycles of denaturation for 1 min at
94oC, annealing for 1 min at 60oC, polymerization for 1 min at 72oC, and finally extension for 10 min at 72oC. Amplification of β-actin was also performed as a RT-PCR control. A total of 6 µL of the PCR product was separated using 2% agarose gel. DNA
bands were visualized by UV light and documented by UVItec gel documentation and analysis system (Cambridge, UK). The
ratio between the target gene and β-actin gene band densities was used for quantitative evaluation.
Flow cytometric analysis of CXCR4
expression A total of 1×106 single cell suspension prepared from lymph node
specimens were stained with mouse anti-human CD3-FITC/CD19-PE firstly at 4oC for 30 min in the dark and washed twice with
PBS. Cells incubated with its isotype control IgG1/IgG2a were used as negative controls. The specimens contained more
than 80% of CD19 or CD3 positive cells were determined as lymphoma cells of B-NHL or T-NHL and used to detect the CXCR4
expression in the subsequent study. Then another total of
1×106 single cell suspension were
incubated with PE-conjugated anti-human CXCR4 monoanti-body for 30 min at 4oC in the dark and washed twice with PBS.
Cells incubated with PE-conjugated isotype mouse IgG2a were used as negative controls. Analysis was performed on a
FACSort flow cytometry (Becton Dickinson, NJ, USA). A minimum of 10 000 cells were analyzed in all specimens. The
percentage of positive cells was determined using the CellQuest software program (Becton Dickinson) and expressed as the
results.
Chemotaxis assay Transwell was used to perform the chemotaxis assays. To examine the effect of triptolide on the
migration of primary lymphoma cells towards
rhSDF-1a in vitro, a total of
2×105 lymphoma cells freshly isolated were
pretreated with triptolide. Final concentrations were 0, 12.5, 25 and 50 nmol/L, respectively, in 100
mL RPMI-1640 supplemented with 10% FCS (referred to hereafter as the migration medium) at 37oC with 5% CO2 for 2 h. Cell suspension prepared
for each group was loaded into the upper chamber of each transwell filter. Migration medium 500 µL containing 200 ng/mL
rhSDF-1a was added to the lower chamber of each filter. The migration medium not containing
rhSDF-1a in the lower chambers were used as blank controls. There-after, the wells were incubated at 37oC for 4 h. The upper chambers were then
removed carefully and the cells in the lower chambers were collected and counted using the trypan blue exclusion method.
Cell number was counted in triplicates for each well and the following formula was used: cell migration rate(%)=(cell number
of the lower chamber/cell number of the total)×100%. Furthermore, to examine the effects of triptolide on the migration of
lymphoma cells towards cultured lymph node stromal cells
in vitro, 5×104 stromal cells in 500 µL DMEM (low-glucose) culture
medium were seeded into the lower chambers of each transwell filter at 37oC, in a 5% CO2 incubator for 3_5 d. When the
monolayers reached confluency, 100 µL migration medium contained
2×105 lymphoma cells pretreated with different
concentrations of triptolide (0, 12.5, 25, 50 nmol/L) for 2 h were added to the upper chambers and the wells were then incubated at 37oC for 4 h. The lower chambers without stromal cells cultured were used as blank controls. The cell migration rate was
counted as above. Stromal cells used in this study were prepared from lymph node lesions obtained from a patient with
diffuse large B-cell lymphoma.
Statistical analysis All data are expressed as mean±SD, and analyzed using SPSS 11.5 for windows XP. Paired-samples
t-test and independent-samples t-test was used for statistical analysis.
P values of less than 0.05 or 0.01 were considered to
be statistically significant.
Results
Effects of triptolide on the proliferation of Raji cells by MTT
Raji cells treated with different concentrations of triptolide
for 0, 24, 36, 48, 60, and 72 h resulted in the inhibition of cell proliferation, and the effects were dose- and time-dependent.
The OD value of triptolide-treated groups decreased significantly compared with the untreated group. The results show great
differences between the untreated group and the triptolide-treated groups (Figure 2). The
IC50 value of 24 h is 43.06 nmol/L, and the
IC50 value of 36 h is
25.08 nmol/L.
Effects of triptolide on the
SDF-1a mRNA expression in lymph node stromal
cells Extensive SDF-1a mRNA expression
was detected in lymph node stromal cells from all 7 patients with different types of NHL, whereas it was only expressed
weakly in the reactive hyperplasia lymph node stromal cells, the mRNA expression ratio for
SDF-1α/β-actin was 1.22±0.30 and 0.25±0.15, respectively
(P<0.01) (Figure 3). Lymph node stromal cells from patients with NHL were incubated with triptolide
for 24 h at different concentrations. The densities of DNA bands weakened accordingly as the treatment concentration
increased. The mRNA expression ratio for SDF-1α/β-actin were 1.15±0.33, 0.88±0.35, and
0.74±0.32 for stromal cells treated by 12.5, 25, 50 nmol/L triptolide, respectively. Compared with the untreated control cells,
triptolide (12.5 nmol/L) reduced the expression of
SDF-1a slightly, but the difference was not statistically significant
(P>0.05), whereas triptolide (25, 50 nmol/L) sharply inhibited the
SDF-1a expression and the inhibition was dose-related
(P<0.01) (Figure 4).
Effects of triptolide on the CXCR4 expression of lymphoma cells
Flow cytometry was performed to determine the expression of CXCR4 on the surface of lymphoma cells freshly isolated from lymph node lesions of patients with NHL. The
percentage of the lymphoma cell contained in the samples analyzed was more than 80% in all cases. In
untreated cells, the average expression rate of CXCR4 was 17.37%±7.21%. After treatment with 12.5, 25, 50 nmol/L triptolide
for 24 h, the CXCR4 expression rate of lymphoma cells were 11.13%±5.66%, 8.30%±4.61%, and 3.31%±2.35%, respectively,
and the expression decreased in a dose-dependent manner in comparison with the control. These data suggest that triptolide
could inhibit the expression of CXCR4 receptor on lymphoma cells (Figure 5).
Effects of triptolide on the migration of lymphoma
cells in vitro Chemotaxis assays showed that lymphoma cells isolated
from the lymph node of patients with NHL in the upper chambers could not only migrate to the lower chambers contained
rhSDF-1a, but also effectively migrate to the lower chambers seeded cultured lymph node stromal cells. After pretreatment
with 0, 12.5, 25, 50 nmol/L triptolide for
2 h, when lower chambers contained rhSDF-1a, the cell
migration rates were 75.4%±15.8%, 59.4%±17.3%, 30.3%±
13.8%, and 21.4%±9.9%, respectively; whereas when lower chambers seeded the cultured stromal cells derived from the
patient with diffuse large B-cell lymphoma, the cell migration rates were 61.1%±15.5%, 53.4%±17.3%, 20.8%±7.4%, and
15.4%±6.3%, respectively. Our results reveal that compared with the control groups, lymphoma cell migration towards either
rhSDF-1a or the cultured lymph node stromal cells was markedly inhibited by the addition of
triptolide in vitro, and the inhibitions were dose-dependent (Figure 6).
Discussion
The interest in exploiting traditional medicines for prevention or treatment of tumors is increasing. The Chinese herb
TWHF has been used in traditional Chinese medicine for more than two thousand years. However, its potential value was
recognized by Western medicine only after investigators observed the effectiveness of TWHF in the treatment of leprosy
and rheumatoid arthritis. Triptolide is purified from TWHF, and has been identified as the major component of TWHF
responsible for immunosuppressive and anti-inflammatory effects. It has been confirmed that triptolide is effective for the
treatment of a variety of autoimmune diseases and for the prevention of allograft rejection and graft-versus-host disease, and
it also possesses a male anti-fertility
effect[1]. Currently, multiple studies
in vitro and in vivo have shown that triptolide was
cytotoxic to various types of tumor cells and significant anti-proliferative and apoptotic
effects were observed in tumor cells treated by
triptolide[14_17]. In the study of
Yinjun et al[14], triptolide inhibited the proliferation of multiple myeloma cell lines
(RPMI-8226 and U266) in a dose-dependent manner (10_80 ng/mL), and induced the apoptosis in MM cells through
activation of the cystein protease caspase 8, 9 and 3, and subsequent cleavage of the DNA repair enzyme poly (ADP-ribose)
polymerase. Moreover, triptolide downregulated nuclear factor (NF)-kappaB activity in MM cell lines, and also induced
chemosensitivity to doxorubicin and suppressed cell proliferation of fresh MM cells.
Lou et al[15] demonstrated that triptolide
inhibited chronic myelogenous leukemia cell line K562 cell proliferation and induced apoptosis in a dose- and time-dependent
manner. The growth-inhibitory IC50 value for triptolide treatment was 40 ng/mL. Obviously, triptolide-induced apoptosis of
K562 cells was associated with a decline in the expression of bcr-abl, which was the hybrid gene of the Philadelphia
chromosome t (9;22). Triptolide was able to decrease the expression of bcr-abl down to 50%, 30%, and 20% of the basal value at the
concentrations of 20 ng/mL, 40 ng/mL, and 80 ng/mL, respectively, after 72 h. Another study by Chang
et al[16] found that triptolide enhanced chemotherapy-induced apoptosis in fibrosarcoma cell line (HT1080) and NSCLC cell line (A549). Triptolide
induced p53, but inhibited p21waf1/cip1 expression. They also found that triptolide induced accumulation of cells in S phase and
blocked p21-mediated accumulation of cells in
G2/M. In addition, it has been shown that triptolide inhibited the proliferation
of glioma cells in vitro, and could also promote the expression of Bax and inhibit the expression of Bcl-2 and therefore
accelerate cell apoptosis in glioma cell
lines[17]. In this study, we found that triptolide could inhibit the proliferation of B-NHL
cell line Raji cells in a dose- and time-dependent manner. The 24-h
IC50 value was 43.06 nmol/L, and the 36-h
IC50 value was 25.08 nmol/L. Therefore, it seems that triptolide is cytotoxic to tumor cells at a relatively low concentration. The mechanism
of its antiproliferative effect and the activity to initiate apoptosis are related to downregulation of NF-kappaB, activation of
caspase proteins, and regulation of the expression of several oncogenes and anti-oncogenes which are important regulator
of apoptosis and cell cycle. Apart from this, it was found triptolide inhibited experimental metastasis of melanoma cell line
B16F10 cells to the lungs and spleens of mice, which suggested that
triptolide possessed the activity to inhibit tumor metastasis
[12]. However, its mechanism has not been fully
elucidated.
Cell migration along chemokine gradients involves a
number of steps including the establishment of cell polarity, directional cell locomotion through cytoskeletal
rearrange-ments, and adhesive interactions with extracellular
matrix[18]. Metastasis, which is the major cause of morbidity and mortality
in the majority of tumors, shares many similarities with normal circulating
cells[5]. As mentioned above, SDF-1/CXCR4 axis is
an important mechanism regulating tumor cell migration.
Muller et al[6] were the first to provide direct evidence to support the
notion that the SDF-1/CXCR4 axis could actually mediate human tumor metastasis
in vivo. They
reported that CXCR4 was highly expressed on human breast cancer cell lines, malignant breast tumors, and metastases, but
not on normal breast tissue. Furthermore, SDF-1 mRNA exhibited peak levels of expression in lymph nodes, lung and bone
marrow, where breast cancer cells metastasize preferen-tially. Signaling through CXCR4 in breast cancer cells mediated actin
polymerization and pseudopodia formation, and subsequently induced chemotactic and invasive responses at the local
level. Finally, in vivo neutralization with a specific monoclonal antibody against CXCR4 effectively inhibited the metastasis
of breast cancer cells in an organ-specific manner. The research on NSCLC also found that CXCR4 was significantly
expressed on tumor cells in freshly isolated tumor specimens from patients,
and in vitro SDF-1 stimulation of CXCR4 on NSCLC cells could lead to chemo-taxis, calcium mobilization, and activation of mitogen-activated protein kinase p42/44
(extracellular signal-related kinase, ERK-1/2). Using a severe combined immunodeficiency (SCID) mouse system of
heterotopic or orthotopic xenoengraftment of human NSCLC cells, they found that
SDF-1 protein levels were significantly higher in organs that
are known to be highly susceptible to human NSCLC metastases, as compared with either the primary tumor or
plasma levels, suggesting that a chemotactic gradient could be established between the site of the primary tumor and those
organs that develop NSCLC tumor metastases. Moreover, in this model system, it appeared that NSCLC cells expressing
CXCR4 had a selective advantage for metastasizing to these organs. Furthermore,
in vivo neutralization of SDF-1 in model system of spontaneous metastasis of human NSCLC resulted in marked attenuation of NSCLC metastases to several organs
including the adrenal glands, liver, lung, brain, and bone
marrow[7].
Similar studies regarding the role of SDF-1/CXCR4 axis were also investigated on malignant hematological diseases. It
has been shown that CXCR4 was expressed on malignant B lymphocytes recovered from patients with different B-cell
malignancies including acute lymphoblastic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, mantle cell lymphoma,
marginal zone B-cell lymphoma, small lymphocytic lymphoma, follicular cell lymphoma, and diffuse large B-cell lymphoma,
and migration assays showed that SDF-1 was able to trigger migration of malignant B lympho-
cytes[12,19,20]. Moreover CXCR4 has been shown to be expressed in a subset of T-cell NHLs such as anaplastic large cell
lymphoma, angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma, which suggests that this chemokine
receptor may have a role in the spread and
metastasis of T-cell neoplasms as
well[12,21]. Arai J et
al[22]
revealed that SDF-1 was expressed in the lymph node stromal cells present in lymphoma lesions, and most B-lymphoma cells
expressed CXCR4 on their surface. CXCR4 positive primary B-lymphoma cells and B-lymphoma cell lines migrated towards
rhSDF-1 and cultured stromal cells derived from the lymph node of patients with follicular or diffuse lymphoma, and these
migrations were almost completely inhibited by the addition of anti-CXCR4 or anti-SDF-1 neutralizing antibody. Their data
strongly suggested that the CXCR4 expressed on malignant B-lymphoma cells were functionally active, and that
SDF-1/CXCR4 axis was the major chemokine system involved in the chemotactic interaction between B-lymphoma cells and lymph
node stromal cells. Likewise, in a nonobese diabetes/severe combined immunodeficiency (NOD/SCID) mouse model of
kine
human high-grade NHL, CXCR4 neutralization by monoclonal antibodies had impressive
in vitro effects on NHL cells including inhibition of transendo-thelial/stromal migration, enhanced apoptosis, decreased proliferation, and inhibition of
pseudopodia formation[12]. In the present study, we examined the expression of SDF-1 and CXCR4 in lymph nodes of patients
with NHL, and explored the possibility that metastatic lymphoma cells were responsive to stimulation of rhSDF-1 or primary
lymph node stromal cells. It was found that SDF-1 transcript was strongly expressed in lymph node stromal cells from all the
patients with different types of NHL, and CXCR4 was detected on primary lymphoma cells isolated from the lymph nodes of
NHL patients. Chemotaxis assays revealed that primary lymphoma cells could effectively migrate towards to rhSDF-1 or
cultured primary lymph node stromal cells. The results suggested that this chemokine ligand and receptor pair could mediate
the metastasis of NHL cells. These results were consistent with that of other
researchers[12,19_22]. Therefore, the
SDF-1/CXCR4 axis might be a potential subject for therapeutic interventions in NHL.
Few data was available regarding the effect of triptolide on the lymph node metastasis in NHL in the published literature.
In this report we reveal that, first, triptolide inhibited the expression of
SDF-1a mRNA in lymph node stromal cells obtained from patients with NHL. Although the difference of
SDF-1a expression between the group treated by triptolide (12.5 nmol/L)
and the control was not significant, triptolide (25, 50 nmol/L) inhibited the
SDF-1a expression and the inhibition was dose-related. Second, after a different dose of triptolide for 24 h, the expression of CXCR4 on the surface of primary lymphoma cells
isolated from lymph nodes of patients with NHL decreased gradually in a dose-dependent manner. Third, chemotaxis assays
showed that the migration of freshly isolated lymphoma cells towards either rhSDF-1 or cultured lymph node stromal cells
was markedly inhibited by the addition of triptolide
in vitro, and the inhibition was dose-dependent. Compared with the
concentration to produce a marked effect on several other tumors as mentioned above, 12.5_50 nmol/L was relatively low, and
might have low toxicities in renal, cardiac, hematopoietic and reproductive systems in human. In short, we concluded that
triptolide was able to inhibit the migration of lymphoma cells via lymph nodes and the underlying mechanism might be related
to the blockage of SDF-1/CXCR4 axis.
Furthermore, the SDF-1/CXCR4 axis has an effect on cell motility and directed chemotaxis, it also plays an important role
in cell secretion. Interactions of CXCR4 with SDF-1 lead to secretion of some angiopoietic factors such as vascular
endothelial growth factor (VEGF) in tumor
cells[5]. These factors secreted by target cells are involved in tumor vasculo-genesis and
in crosstalk between CXCR4 positive cells and
endothelial[5]. In a previous study, we found that triptolide could suppress the
VEGF expression on Raji cells[23]. Given the evidence that triptolide can not only block the SDF/CXCR4 axis, but also
suppress the expression of VEGF in NHL, these mechanisms may be invovled together in regulating tumor metastasis by
triptolide in NHL. In conclusion, the inhibitory effect both on proliferation and tumor metastasis of triptolide showed that
triptolide, purified by a traditional Chinese medicine, might be developed into a new anti-tumor drug in the treatment of NHL.
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