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Several natural compounds, especially plant products and
dietary constituents, have been found to have
chemopre-ventive activities in both in vitro
and in vivo model
systems[1]. Their mechanisms of action vary widely, with many suppressing
cell growth and modulating cell differentiation, and a few
inducing apoptosis. Deguelin, which is isolated from several
plant species, including Mundulea serice
(Leguminosae), has been shown to have cancer-chemopreventive effects in
models of both skin and mammary
tumorigenesis[2,3].
The transcription factor nuclear factor kappa B
(NF-kB) has been identified as a critical component of several signal
transduction pathways. One important function of
NF-kB is its ability to protect cells from
apoptosis[4]. NF-kB is a heterodimer comprising p50 and p65 subunits. It is sequestered in the cytoplasm by association with a binding protein
known as Ik Ba (NF-kB cytoplasmic inhibitor), which masks the nuclear localization signal of
NF-kB. A variety of external or internal signals modify
IkBa/NF-kB complexes by phosphorylating the serine residues of
IkBa and affect the subsequent degradation of
IkBa.
Raji human B lymphoma cells are resistant to nuclear apoptosis induced by various
stimuli[5]. Like other mature B cells,
Raji cells contain constitutive NF-kB-binding activity in the
nucleus[6]. The U937 human monocytic leukemia cell line was
derived from a patient with generalized ¡°histiocytic¡± lymphoma. This cell line is a well-established model for studying the
induction of apoptosis and differentiation[7]
. We demonstrated previously that deguelin inhibited the proliferation of human
Burkitt¡¯s lymphoma cells, such as Daudi cells, by regulating the expression of cyclin D1 and pRb
protein[8].
IkBa, similar to cyclin D1, has been shown to be regulated by
NF-kB. In the present study, we examined the effect of
deguelin on the expression of the IkBa gene product by immunoblotting and immunofluorescence assay.
We focus on changes in the expression of IkBa protein in Raji cells and U937 cells after deguelin treatment, and further explore the
anticancer molecular mechanisms of deguelin in
vitro.
Materials and methods
Drugs and reagents Deguelin (Sigma, St Louis, MO, USA) was initially dissolved in dimethylsulfoxide
(Me2SO, <1 %), stored at -20
oC and thawed before use. Tumor
necrosis factor a (TNF-a) was purchased from PeproTechEC (London, UK;
2¡Á107 U/mg). The Annexin V-FITC
Detection Kit II was purchased from BD Biosciences Pharmingen (San Jose, CA, USA),
and the RPMI-1640 medium and Me2SO were purchased from Sigma. Fetal calf serum (FCS),
anti-IkBa (SC-371), anti-p65 (SC-372) and
horseradish peroxidase (HRP)-conjugated secondary antibodies (goat IgG-HRP,
SC-2020) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Chemiluminescence (ECL) reagent kits were purchased from Pierce
Biotechnology (Rockford, IL, USA). The Raji and U937 cell lines were obtained from the China Center for Typical Culture
Collection (Wuhan, China). All cell groups were grown in an RPMI-1640 culture medium containing 10% FCS and
L-glutamine (2 mmol/L), penicillin (100 U/mL), and streptomycin (100
U/mL) at 37 oC in a humidified atmosphere containing 5%
CO2.
MTT assay The antiproliferative effect of deguelin against different group cells was determined by using the MTT dye
uptake method. Briefly, the final concentrations of deguelin were 5, 10, 20, 40 and 80 nmol/L. Each concentration of deguelin
was added to 6 wells, respectively. The plates were in the presence or absence of the indicated test samples for 24 h.
Thereafter, 20 µL MTT solution [5 mg/mL in phosphate-buffered saline (PBS)]was added to each well. After incubation for
4 h at 37 oC, the supernatant was removed and 150 µL
Me2SO was added. When the blue crystals were dissolved, the optical
density (OD) was detected in a microplate reader at a wavelength of 570 nm using a 96-well multiscanner autoreader (Biotech
Instruments µQuant, NY, USA). The following formula was used: cell proliferation inhibited
(%)=[1-(OD of the experimental samples/OD
of the control)]¡Á100% (n=6. Mean¡ÀSD).
Western blot analysis
Preparation of nuclear extracts for NF-kB The nuclear extracts were prepared according to the method described by
Schreiber et al[9]. Briefly,
2¡Á106 cells were washed with cold PBS
and suspended in 0.4 mL hypotonic lysis buffer containing
protease inhibitors for 30 min. The cells were then lysed with
12.5 µL 10% Nonidet P-40. The homogenate was centrifuged,
and supernatant containing the cytoplasmic extracts was stored at -80 ºC
. The nuclear pellet was resuspended in 25 µL
ice-cold nuclear extraction buffer. After 30 min with intermittent mixing, the extract
was centrifuged, and supernatants containing
nuclear extracts were obtained. The protein content was measured by using the Bradford
method. If the nuclear extracts were
not used immediately, they were stored at -80
oC.
Preparation of whole-cell lysates Cells were harvested and lysed in 100 µL of lysis buffer by incubation on ice for 30
min, and the extracts were centrifuged at 18
000¡Ág for 15 min to remove cell
debris. Protein concentrations were determined
by using the Bio-Rad protein assay (Bio-Rad). After the addition of 5¡Áloading buffer,
the samples were incubated at 95
oC for 5 min and then resolved
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were
transferred onto nitrocellulose membranes and probed with
anti-IkBa and anti-p65 antibody (at a 1:1500 dilution). The
antigen-antibody complexes were incubated for 1 h at room temperature with HRP-conjugated secondary antibodies at a final dilution
of 1:1500 in Western washing solution. After the mixture was washed 3 times with
Tris-buffered saline, antibody binding was
visualized by using enhanced chemiluminescence
and autoradiography. Quantification of the bands was carried out using
the Quantity One densitometric analysis software (Bio-Rad).
Indirect immunofluorescence U937 cells were fixed with 4% paraformaldehyde for 5 min and permeabilized with 0.2%
Triton X-100 for 5 min at room temperature. To block nonspecific antibody binding, slides were incubated with 3% milk/PBS
at room temperature for 30 min. Then slides were incubated with rabbit polyclonal antihuman
IkBa antibody (dilution, 1:50 in 1% milk/PBS). After overnight incubation, the slides were washed and then incubated with fluorescein isothiocyanate
(FITC)-labeled goat anti-rabbit secondary antibodies (1:50 in 1% milk/PBS) at 37
oC for 1 h. Finally, slides were washed 3 times
for 5 min each with PBS and covered with glycerol (1:9 in PBS). Slides were examined by using an fluorescence microscope
(Olympus, Tokyo, Japan). Photographs were taken with a camera, and images were created using a software package.
Annexin V/PI double-labeled cytometry For detection of apoptotic cells, expression of Annexin-V-FITC and exclusion of
PI were simultaneously detected by using two-color flow cytometry. The U937 cells treated or untreated with deguelin (40
or 80 nmol/L) for 24 h were washed with PBS and resuspended in binding buffer containing 5 µL of FITC-labeled
anti-Annexin-V antibody and 10 µL of 20 µg/mL PI. After incubation for 10 min at room temperature in a light protected area, all
specimens were quantified on the FACS can (Becton Dickinson, CA, USA)
Statistical analysis All data are expressed as mean¡ÀSD, and were analyzed using SPSS 10.0 for Windows 98. The linear
t-test was used for statistical analysis, and
P<0.05 was considered to be statistically significant.
Results
Effects of deguelin on proliferation of U937 and Raji
cells U937 and Raji cells were treated with different concentrations
of deguelin (5, 10, 20, 40 and 80 nmol/L) for 24 h, resulted in the inhibition of cell proliferation in a dose- dependent manner.
The OD value of the deguelin-treated group was significantly lower than that of the untreated group (Figure 1). The
IC50 values after 24 h treatment for the U937 cells and Raji cells were 21.61 and 45.37 nmol/L, respectively.
Effect of deguelin on IkB-a degradation in Raji
cells IkBa protein level in Raji cells was reduced after 24 h treatment with
deguelin at concentrations of 5, 10, 20, 40 and 80 nmol/L or after treatment with deguelin at a concentration of 40 nmol/L for
0, 2, 8, 12, 24, and 48 h. This indicates that deguelin promotes
IkBa protein degradation in Raji cells in a concentration- and
time-dependent manner (Figure 2A, 2B, Figure 4).
Effect of deguelin and TNF-a on IkBa
degradation in U937 cells IkBa protein level in U937 cells was reduced after 24
h treatment with deguelin at concentrations of 5, 10, 20, 40 and 80 nmol/L or after treatment with deguelin at a concentration
of 20 nmol/L for 0, 2, 8, 12, 24 and 48 h. These findings indicate that deguelin promotes
IkBa protein degradation in U937 cells in a concentration- and time-dependent manner (Figure 3A, 3B, Figure 4).
IkBa protein showed abrupt and complete depletion within 48 h after treatment with deguelin. On the other hand,
IkBa protein showed only a partial decline in a
dose-dependent manner compared with the control cells.
After treatment with TNF-a (10 ng/mL) or TNF-a (10
ng/mL) plus deguelin (20 nmol/L) for 15 min, the
IkBa protein level was decreased. Nuclear extracts for
NF-kB/p65 were prepared and imunoblotting revealed that there was p65 protein
expression in the nucleus after treatment with
TNF-a or TNF-a plus deguelin for 15 min, but no p65 protein was found in
control and deguelin-treated groups (Figure 3C, 3D).
Effect of deguelin on the subcellular localization of
IkBa in U937 cells After treatment with TNF-a
(10 ng/mL) or deguelin (20 nmol/L) plus TNF-a (10 ng/mL) for 15 min, there was a substantial reduction in the amount of
IkBa protein in the cytoplasm compared with controls. After treatment with deguelin 20 nmol/L for 4, 24, or 48 h,
IkBa protein expression was gradually reduced in a time-dependent manner compared with control cells (Figure 5).
Effect of deguelin on apoptosis of U937
cells There was little binding of annexin V-FITC to untreated U937 cells
(0.07%). Binding of annexin V-FITC was increased by 14.4% and 40.3% following treatment with deguelin at concentrations
of 40 and 80 nmol/L for 24 h, respectively. The
P value for the test for interaction was 0.003 (Figure 6).
Discussion
Activation of the NF-kB signaling pathway has been linked to resistance to chemotherapeutic drugs, and its downregulation,
by means of NF-kB inhibitors, lowers resistance to various types of therapy in tumor cell lines. The
IkBa protein is ubiquitously expressed as an NF-kB cytoplasmic inhibitor. In response to various stimuli,
IkBa is rapidly degraded to allow NF-kB nuclear translocation and
NF-kB target gene transcription. Because IkBa itself is the product of an
NF-kB-regulated gene, it is rapidly resynthesized and can then migrate to the nucleus to terminate the first phase of
NF-kB activation. Because the IkBa gene is an
NF-kB target gene, it has been shown to be regulated by
NF-kB. The expression of IkBa was downregulated in Raji and U937 cells treated with deguelin. Our results indicate that deguelin induced apoptosis in U937 cells and inhibited
the expression of IkBa protein in U937 and Raji cells. The anti-proliferative activity of deguelin is related to the signaling
pathway of NF-kB.
It has been reported that TNF-a induces IkBa
degradation[10,11]. Our data also indicate that
IkBa degradation is induced by treatment with
TNF-a for 15 min in U937 cells. Recently, it has been reported that deguelin is a powerful inhibitor of
PI3K[12]. Chun et al investigated whether deguelin could enhance sensitivity to chemotherapeutic drugs of human U937 leukemia
cells and acute myeloid leukemia (AML) blasts with an activated PI3K/Akt
network[12]. They found that deguelin might
be useful in the future for increasing sensitivity to therapeutic treatments of leukemia cells with an active PI3K/Akt signaling
network. Crowell et al found that deguelin decreased cancer-related increased AKT activity via the PI3K pathway in lung
carcinogenesis[13]. Deguelin effectively inhibited AKT in premalignant cells. Because activated AKT can affect numerous
cellular functions via intermediary molecules, including mTOR,
NF-kB, and p53, which control cell survival, growth and
proliferation, effective inhibition of PI3K/AKT by deguelin means it can affect the activation of
NF-kB.
Although deguelin decreases the expression of
IkBa, p65 is released for translocation from the cytoplasm into the
nucleus, where it interacts with p50 and binds specifically to the
NF-kB target DNA sequence. Following specific binding to
DNA, transcriptional activation of NF-kB is regulated through specific phosphorylation of p65 at several distinct sites. At
present, we do not know the precise mechanisms of transcriptional inhibition by deguelin in the nucleus. We hypothesize
that deguelin inhibits IkBa protein expression through transcriptional inhibition of the
IkBa gene, prob
ably via NF-kB targets such as triptolide. Triptolide potently inhibits the expression of
IkBa mRNA and protein, so more p65 is released for translocation into the nucleus and specific binding to DNA. Triptolide almost completely blocked
TNF-a-induced NF-kB activation by inhibiting p65 transactivation, but not DNA binding. Curcumin is an upstream inhibitor of
NF-kB and prevents the activation of inhibitory B
(IkB) kinase (IKK), an enzyme required for the activation of
NF-kB. Triptolide inhibits NF-kB activity further downstream by interfering with the
NF-kB-mediated transcription process. We hypothesize
that deguelin inhibits NF-kB activity also further downstream. So
NF-kB would not be activated and thereby an increase in
apoptosis was caused by deguelin.
Numerous studies in animals have demonstrated that deguelin has potent chemopreventive activity against a wide
variety of different tumors. Deguelin has cancer chemo-preventive effects in skin and mammary tumorigenesis models, and
additional studies are warranted to characterize the cancer chemopreventive or chemotherapeutic potential of this substance
more fully[14]. Deguelin inhibits the growth of colon cancer cells through the induction of apoptosis and cell cycle arrest.
Deguelin inhibits the proliferation of human Burkitt¡¯s lymphoma cells by regulating the expression of cyclin D1 and pRb
protein[8]. IkBa, Bcl-2,
Bcl-xL, interleukin-6, and cyclin D1 are all
NF-kB target genes, and are regulated by
NF-kB[15,16].
Overall, our results indicate that deguelin can inhibit the expression of
IkBa protein in U937 and Raji cells in a dose- and
time-dependent manner. Deguelin might inhibit
IkBa protein expression through transcriptional inhibition of the
IkBa gene, but the precise mechanisms of transcriptional inhibition by deguelin are unknown. Our findings suggest that the
anti-proliferative activity of deguelin is related to the signal pathway of
NF-kB.
Acknowledgement
This work was supported in part by the Institute of Occupational Medicine, Tongji Medical College, Huazhong
University of Science and Technology, Wuhan, China. We thank Dr Tang-chun WU and Dr Jian-hua YU for expert assistance with
cell culture, the nuclear protein extracts and Western blotting, Dr Hao TANG and Dr Xiao-bo YANG for their assistance with
indirect immunofluorescence micro-scopy, and Dr Tang-chun WU for critical review of the manuscript.
References
1 Cline JM, Hughes CL Jr. Phytochemicals for the prevention of breast and endometrial cancer. Cancer Treat Res 1998; 94: 107-34.
2 Gerhäuser C, Lee SK, Kosmeder JW, Moriarty RM, Hamel E, Mehta
RG, et al. Regulation of ornithine decarboxylase induction by deguelin,
a natural product cancer chemopreventive agent. Cancer Res 1997; 57: 429-35.
3 Udeani GO, Gerhauser C, Thomas CF, Moon RC, Kosmeder JW, Kinghorn AD,
et al. Cancer chemopreventive activity mediated by
deguelin, a naturally occurring rotenoid. Cancer Res 1997; 57: 3424-8.
4 Antwerp DJ, Martin SJ, Kafri T, Green DR, Verma IM. Suppression of
TNF-a-induced apoptosis by NF-kB. Science 1996; 274: 787-9.
5 Kawabata Y, Hirokawa M, Kitabayashi A, Horiuchi T, Kuroki J,
Miura AB. Defective apoptotic signal transduction pathway downstream
of caspase-3 in human B-lymphoma cells: a novel mechanism of nuclear apoptosis resistance. Blood 1999; 94: 3523-30.
6 Goldfeld AE, Strominger JL, Doyle C. Human tumor necrosis factor alpha gene regulation in phorbol ester stimulated T and B cell lines.
J Exp Med 1991; 174: 73-81.
7 Hu X, Janssen WE, Moscinski LC, Bryington M, Dangsupa A, Rezai-Zadeh
N, et al. An IkappaBalpha inhibitor causes leukemia cell death
through a p38 MAP kinase-dependent, NF-kappaB-independent mechanism. Cancer Res 2001; 61: 6290-6.
8 Liu HL, Chen Y, Cui GH, Wu QL, He J. Experimental study on regulating expressions of cyclin D1, pRb, and anticancer effects of deguelin
on human Burkitt¡¯s lymphoma Daudi cells in vitro.
Acta Pharmacol Sin 2005; 26: 873-80.
9 Schreiber E, Matthias P, Muller MM, Schaffner W. Rapid detection of octamer binding proteins with ¡®mini-extracts¡¯, prepared from a
small number of cells. Nucleic Acids Res 1989; 17: 6419.
10 Gallois C, Habib A, Tao J, Moulin S, Maclouf J, Mallat
A, et al. Role of NF-kappaB in the antiproliferative effect of
endothelin-1 and tumor necrosis factor-alpha in human hepatic stellate cells. Involvement of cyclooxygenase-2. J Biol Chem 1998; 273: 23183-90.
11 Nasuhara Y, Adcock IM, Catley M, Barnes PJ, Newton R. Differential IkappaB kinase activation and IkappaBalpha degradation by
interleukin-1beta and tumor necrosis factor-alpha in human U937 monocytic cells: evidence for additional regulatory steps in
kappaB-dependent transcription. J Biol Chem 1999; 274: 19965-72.
12 Chun KH, Kosmeder JW 2nd, Sun S, Pezzuto JM, Lotan R, Hong WK,
et al. Effects of deguelin on the phosphatidylinositol 3-kinase/Akt
pathway and apoptosis in premalignant human bronchial epithelial cells. J Natl Cancer Inst 2003; 95: 291-302
13 Crowell JA, Steele VE. AKT and the phosphatidylinositol 3-kinase/AKT pathway: important molecular targets for lung cancer prevention
and treatment. J Natl Cancer Inst 2003; 95: 252-3.
14 Udeani GO, Gerhauser C, Thomas CF, Moon RC, Kosmeder JW, Kinghorn AD,
et al. Cancer chemopreventive activity mediated by
deguelin, a naturally occurring rotenoid. Cancer Res 1997; 57: 3424-8.
15 Shishodia S, Aggarwal BB. Nuclear factor-kappa B activation: a question of life and death. J Biochem Mol Biol 2002; 35: 28-40.
16 Pahl HL. Activators and target genes of Rel/NF-kappaB transcription factors. Oncogene 1999; 18: 6853-66.
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