Extract
Note: Please read the complete
full text with Figures and Tables at
Introduction
The tumor necrosis factor-related apoptosis-inducing
ligand (TRAIL), a member of the TNF family, is considered a
promising anticancer agent due to its ability to induce
apoptosis in various tumor cell types, but is not cytotoxic to
many normal cell types in vitro or in
vivo[1,2]. Cellular sensitivity to TRAIL is dependent on the expression of cell
membrane TRAIL receptors and
caspase-8[3,4]. Caspase-8 is
activated in response to TRAIL and released cytochrome
c, where it initiates a protease cascade that activates effector
caspases, including caspase-3 and
caspase-9[5]. Although the majority of breast cancer cell lines are resistant in TRAIL-induced
apoptosis, TRAIL with chemotherapeutic drugs resulted in
the synergistic induction of cell
death[6]. A previous study reported that more than 250 ng/mL TRAIL induces 30%
apoptosis in MDA-MB-231 cells[7]. These results suggest
that anticancer agents can be used in synergistic treatment
with TRAIL to sensitize resistant cells to TRAIL-mediated
apoptosis[8]. A better understanding of the molecular
mechanisms underlying identification of the sensitizing agents
capable of overcoming TRAIL resistance may facilitate the
establishment of TRAIL-based combination regimens for the
improved treatment of breast cancers.
Phytosterols or plant sterols are the counterparts of
animal cholesterol[9] and are not synthesized endogenously in
humans, but are derived solely from the diet through
intestinal absorption[10,11]. Among them,
β-sitosterol (SITO) is one of the most common dietary phytosterols in the blood. Previous
studies revealed that SITO reduces carcinogen-induced
cancer of the colon in rats[12] and exhibits
anti-inflammatory[13,14],
anti-angiogenic[15], and immune-modulating
properties[16,17]. SITO also induces apoptosis in hormone-insensitive and
metastatic MDA-MB-231 human breast cancer cells through
the activation of caspases[18_21]. When the cells are treated
with high dosages of SITO (¡Ý16 µmol/L) or long time points
(more than 3 d), the cells can become more sensitive to
SITO-induced apoptosis. To overcome these problems, the
synergistic treatment of chemopreventive agents and TRAIL is a
promising approach to selectively induce apoptosis and
regression of breast cancer with minimal toxicity to normal
cells[6,7].
In the present study, we first investigated whether SITO
is a potent sensitizer for TRAIL-induced apoptosis in
TRAIL-resistant MDA-MB-231 breast cancer cells. Moreover, we
determined the first evidence that synergistic treatment with
SITO and TRAIL regulates the activation of caspases and
pro-apoptotic proteins, leading to a significant induction of
TRAIL-mediated signaling and cell death in MDA-MB-231
human breast cancer cells.
Materials and methods
Reagents SITO (Cat No: S9889),
2-hydroxypropyl-cyclodextrin (CD), 4,6-diamidino-2-phenylindole (DAPI), and
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide (MTT) were obtained from Sigma (St Louis, MO, USA).
Caspase activity assay kits were obtained from R&D
Systems (Minneapolis, MN, USA). An enhanced
chemiluminescence (ECL) kit was purchased from Amersham (Arlington
Heights, IL, USA). Pan-caspase inhibitor (z-VAD-fmk) was
obtained from Calbiochem (San Diego, CA, USA).
RPMI-1640 medium and fetal bovine serum (FBS) were purchased
from Invitrogen (Carlsbad, CA, USA) and GIBCO (Gaithersburg, MD, USA), respectively. Soluble
recombinant TRAIL (Cat No: K0112977; Koma Biotech, Seoul, Korea)
was purchased from R&D Systems (USA). SITO was
complexed with 5 mmol/L CD in order to solubilize it; 5
mmol/L CD was used as a vehicle control.
Antibodies Antibodies against Bax, Bad, Bcl-2,
Bcl-XL, Fas, FasL, cIAP-1, cIAP-2, XIAP, DFF40/CAD, DFF40/ICAD,
poly(ADP-ribose) polymerase [PARP], lamin A, capase-3,
capase-8, and capase-9 were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA, USA). The antibody against
â-actin came from Sigma. Peroxidase-labeled donkey antirabbit
and sheep antimouse immunoglobulin were purchased from
Amersham.
Cell culture Human breast cancer MDA-MB-231 cells
were obtained from the American Type Culture Collection
(Rockville, MD, USA). The cells were cultured at 37 °C in a
5% CO2 humidified incubator and maintained in RPMI-1640
culture medium containing 10% heat-inactivated FBS.
Cell viability and growth The cells were treated with the
indicated concentrations of SITO, TRAIL, or synergistic
treatment (SITO+TRAIL). Control cells were supplemented
with 5 mmol/L CD for 48 h. Following the treatment, cell
viability was determined by MTT assay.
Nuclear staining After treatment with the indicated
compounds for 48 h, the cells were harvested, washed in ice-cold
phosphate-buffered saline (PBS), and fixed with 3.7%
paraformaldehyde for 10 min at room temperature. The fixed
cells were permeabilized with 0.5% Triton X-100 and stained
with DAPI solution for 10 min at room temperature. The
nuclear morphology of the cells was examined by
fluorescence microscopy.
Annexin V analysis For the analysis of apoptosis, the cells
were stimulated with the indicated compounds, and apoptosis
was analyzed over time by the staining of
phosphatidylserine translocation with fluorescein-isothiocyanate-Annexin V
(PharMingen, SinDiego, CA, USA) according to the manufacturer's instructions.
Protein extraction and Western blot analysis
The cells were harvested, washed once with ice-cold PBS, and gently
lysed for 10 min in 80 µL ice-cold lysis buffer (20 mmol/L
sucrose, 1 mmol/L EDTA, 20 µmol/L Tris-Cl [pH 7.2], 1
mmol/L dithiothreitol, 10 mmol/L KCl, 1.5 mmol/L
MgCl2, 5 µg/mL pepstatin A, 10 µg/mL leupeptin, and 2 µg/mL aprotinin).
The supernatants were collected and the protein
concentrations were determined using a Bio-Rad protein assay kit
(Bio-Rad, Hercules, CA, USA). The samples were stored at _80
°C or immediately used for the Western blot analysis.
Aliquots containing 50 µg of total protein were separated on
SDS_PAGE and transferred to nitrocellulose membranes
(Schleicher & Schuell, Keene, NH, USA) for the Western
blot analysis using the indicated primary antibodies.
Peroxidase-conjugated secondary antibodies were detected using
an ECL detection system.
Lactate dehydrogenase assay To determine plasma
membrane integrity loss, lactate dehydrogenase (LDH) release
into the extracellular medium was measured using the
cyto-tox96 nonradioactive assay from Promega (Madison, WI,
USA) in order to determine cytotoxicity. This assay
measures the formation of a red formazan product after the
conversion of lactate and nicotinamide adenine dinucleotide to
pyruvate and NADH. The assay was used according to the
manufacturer's instructions. Briefly, the maximum release of
LDH was obtained by adding 100 µL of 2% Triton X-100 to
the untreated cells. In total, 100 mL of each sample were
incubated with 100 µL of LDH assay reagents for 10 min, and
the absorbance of the samples was measured at 490 nm. The
percentage of LDH release was determined by dividing
the amount of LDH released by the cells under each condition
by the maximum amount of LDH release, and then
multiplying the fraction by 100.
Determination of caspase activity Caspase activities were
determined by colorimetric assays using caspases-3, -8, and
-9 activation kits according to the manufacturer's protocol.
Briefly, the cells were lysed in the supplied lysis buffer. The
supernatants were collected and incubated with the
supplied reaction buffer containing dithiothreitol and substrates
at 37 °C. The caspase activities were determined by
measuring changes in absorbance at 405 nm using the microplate
reader.
Statistical analysis All data from the MTT assays, FACS
analyses, Western blot analysis, and caspase activity
experiments were derived from at least 3 independent
experiments. The images were visualized with Chemi-Smart
2000 (Vilber Lourmat, Marine, Cedex, France). The images
were captured using Chemi-Capt (Vilber Lourmat, France)
and transported into Photoshop. All data are presented as
mean±SD. Significant differences among the groups were
determined using one-way ANOVA with Scheffe's test. A
value of P<0.05 was accepted as an indication of statistical
significance.
Results
High dosages of SITO (¡Ý 16 µmol/L) induces apoptosis
in MDA-MB-231 breast cancer cells To investigate whether
SITO decreases cell viability, the MDA-MB-231 cells were
stimulated with the various concentrations of SITO for 48 h
and then subjected to MTT assay. As shown in Figure 1A,
the inhibition of cell viability was dramatically observed at
16 µmol/L (65%±5%), which in turn decreased to
39%±5% when the cells were exposed to 32 µmol/L SITO at 48 h. CD
(5 mmol/L), used as a vehicle control, did not affect cell
proliferation and viability. We next examined whether
SITO-induced cell death was followed by morphological features.
As shown in Figure 1B, 16 or 32 µmol/L SITO induced
distinct morphological change and the formation of the apoptotic
body of the cells (white arrow), while the vehicle control
displayed intact morphology and nuclear structure.
Additionally, SITO (16 or 32 µmol/L) resulted in a significant
increase of LDH release to 18%±2% or 35%±4%,
respectively (Figure 1C). These results indicate that a high dose of
SITO (¡Ý 16 µmol/L) inhibits cell proliferation and induces cell
death in MDA-MB-231 cells.
SITO induces apoptosis through upregulation of
caspases and pro-apoptotic proteins, and downregulation of
anti-apoptotic proteins In order to investigate whether
caspases are likely to be involved in the apoptotic response
observed in Figure 1, the cells were treated with the
indicated concentrations of SITO for 48 h, and the caspase
activity from the cell lysates was determined. As shown in
Figure 2A, caspases-3, -8, and -9 were activated at
concentrations greater than 16 µmol/L SITO. As shown by the
Western blot analysis, SITO significantly increased the cleavage
of PARP and the expression of DFF40/CAD with the downregulation of DFF40/ICAD at concentrations of 16 or
32 µmol/L SITO. These results indicate that treatment with
more than 16 µmol/L SITO increases the activation of
caspases-3, -8, and -9 followed by increases in PARP
cleavage and DFF40/CAD expression (Figure 2B). In a parallel
experiment, to determine the expression of anti- and
pro-apoptotic proteins in SITO-mediated apoptosis, we analyzed
the expressions of Bcl-2 and inhibitor of apoptosis (IAP)
family proteins by Western blot analysis. As shown in
Figure 2C, SITO dose dependently increased the Bax level and
slightly decreased the Bcl-2 level. We further revealed that
more than 16 µmol/L SITO decreased IAP family proteins,
such as XIAP, but not cIAP-1 and cIAP-2 in the
MDA-MB-231 cells. The SITO-treated cells resulted in a
dose-dependent downregulation of IAP family members. These results
suggest that the apoptotic actions of SITO are closely
related with the upregulation of the Bax/Bcl-2 ratio, the
downregulation of the IAP family, and caspase activation.
Non-toxic dose of SITO significantly triggers
TRAIL-mediated apoptosis in a low dose of TRAIL-resistant
MDA-MB-231 cells To investigate the synergistic effects, the
MDA-MB-231 cells were treated with subtoxic
concentration of SITO and TRAIL for 48 h and the cell viability was
assessed using the MTT assay. As shown in Figure 3A,
treatment with SITO (8 µmol/L) or TRAIL alone (30 ng/mL)
for 48 h was not able to reduce cell viability in the
MDA-MB-231 cells. Notably, the MDA-MB-231 cells treated with a
synergistic treatment of 8 µmol/L SITO and 30 ng/mL TRAIL
significantly reduced cell viability (42%±6%), which was
similar to treatment with more than 32 µmol/L SITO alone. For an
assessment of apoptosis, we analyzed
chromatin condensation and apoptotic bodies. Treatment with SITO (8
μmol/L) or TRAIL (30 ng/mL) alone did not induce any
morphological features or apoptotic bodies indicative of cell death
(Figure 3B). However, the synergistic treatment with SITO
and TRAIL induced significant decreased membrane shrinkage, cell rounding, and the appearance of apoptotic
bodies in MDA-MB-231 cells. Additionally, LDH release
was also increased more than 4 times than that of the control
in the synergistic treatment with SITO and TRAIL (Figure
3C). Next, we analyzed the apoptotic effects using flow
cytometric analysis to detect Annexin
V+ cells. The synergistic treatment resulted in a markedly increased
accumulation of Annexin V+ cells, whereas treatment with SITO or
TRAIL alone did not (Figure 3D). We also examined whether
SITO could sensitize various resistant tumor cells to the
apoptotic effects of TRAIL. The treatment of MCF-7, A549,
HepG2, and Hep3B cells with 30 ng/mL and 8 µmol/L SITO
reduced cell viability in all of the tested cells, but the effects
were weaker than the MDA-MB-231 cells. TRAIL alone or
SITO alone had no significant effect on the viability (Figure
4). Taken together, these results indicate that SITO
significantly sensitizes TRAIL-induced apoptosis in a low dose of
TRAIL-resistant MDA-MB-231 cells.
Synergistic treatment with SITO and TRAIL upregulates
pro-apoptotic proteins and downregulates anti-apoptotic
proteins Caspases are known to act as important
mediators of apoptosis and contribute to the overall apoptotic
morphology by cleavage of various cellular substrates. Therefore,
we investigated the activation and expression of
caspases-3, -8, and -9 in MDA-MB-231 cells treated with SITO and
TRAIL for 48 h. Cell lysates containing equal amounts of
total protein from cells treated with SITO and TRAIL were
assayed for in vitro caspase activity. As shown in Figure
5A, treatment with TRAIL alone slightly increased caspase
activity; however, the synergistic treatment with SITO (8
ìmol/L) and TRAIL (30 ng/mL) significantly increased the
activity of caspases-3, -8, and -9 in MDA-MB-231 cells. The
Western blot analysis also revealed that the synergistic
treatment markedly induced cleavage of caspases in
MDA-MB-231 cells (Figure 5B). To evaluate the expression of anti- and
pro-apoptotic family proteins in the synergistic treatment,
we analyzed the expression of these proteins using Western
blot analysis. As shown in Figure 5C, the pro-apoptotic Bax
expression was significantly induced in the synergistic
treatment, whereas the levels of anti-apoptotic Bcl-2 and
Bcl-XL remained unchanged. One of the IAP family members,
anti-apoptotic XIAP was markedly downregulated, and the
expressions of Fas and FasL were significantly augmented
by the synergistic treatment. As shown in Figure 5D,
synergistic treatment-induced apoptosis was significantly
suppressed in the presence of pan-caspase inhibitor z-VAD-fmk,
indicating that the TRAIL-induced apoptosis sensitized by
SITO was mediated through caspase activation in the
MDA-MB-231 cells. These results indicate that caspases are
crucial regulators of apoptosis by the combined treatment of
SITO and TRAIL in MDA-MB-231 cells.
Discussion
TRAIL has aroused great interest in cancer therapy
because it can selectively induce cancer cells, and transformed
cells can undergo apoptosis with no toxicity to normal
cells[1,2]. These observations indicate that TRAIL may prove to
be a safe and effective biological agent for cancer therapy in
humans. However, recent studies have shown that many
cancer cells, including human breast carcinoma cells, are
resistant to the apoptotic effects of
TRAIL[18_20]. Recent studies have also reported that SITO, as a chemotherapeutic
agent, can induce apoptosis in metastatic MDA-MB-231
breast cancer cells[21_24]. If the breast cancer cells are treated
with high concentrations of SITO for more than 2 or 3 d,
SITO can significantly induce apoptosis. To overcome these
problems, in the present study, we investigated whether
synergistic treatment with SITO and TRAIL triggers
apoptosis in MDA-MB-231 breast cancer cells that are
normally resistant to either agent alone. The synergistic
treatment induced cell death followed by cell shrinkage and
apoptotic body formation, which are hallmark features of
apoptosis. Our results also revealed that the synergistic
treatment with SITO and TRAIL enhances apoptosis through the activation of caspases in MDA-MB-231 cells.
This synergistic chemotherapy is very efficient to sensitize
TRAIL-induced apoptosis in TRAIL-resistant MDA-MB-231
breast cancer cells.
Recently, several researchers reported that some
chemopreventive agents sensitize TRAIL-mediated apoptosis through the activation of caspase-3 in
TRAIL-resistant cancer cells[25,26]. Caspases belong to a family of
cysteine proteases that are integral parts of the apoptotic
pathway. In particular, activated caspases have many
cellular targets, that when severed and/or activated, produce the
morphological features of
apoptosis[27]. Many studies have
determined that a variety of chemotherapeutic agents
induce apoptosis through the activation of caspases and the
degradation of PARP and lamin A[28,29]. Our findings indicate
that caspases are critical protease mediators of apoptosis
triggered by the synergistic treatment of TRAIL and SITO.
Furthermore, downstream in the TRAIL-induced apoptotic
pathway, mutations to the pro-apoptotic protein Bax and
increased expression of IAP family members such as XIAP
and cIAP, can contribute to the resistance to
TRAIL-mediated apoptosis[30_32]. In the present study, we found that the
synergistic treatment increases the expression of the
pro-apoptotic Bax, Fas, and FasL protein and results in a
decrease of the anti-apoptotic XIAP, but not cIAP-1 and
cIAP-2. The ability to induce apoptosis makes synergistic
treatment with SITO and TRAIL a potentially effective
preventive and therapeutic agent to combat malignant breast
cancer cells.
Thus, the results of this study suggest that SITO
sensitizes TRAIL-mediated apoptosis by the upregulation of
apoptotic proteins, including Bax and caspases. Therefore,
the use of TRAIL with low concentrations of SITO may
provide an effective therapeutic strategy for safely treating
TRAIL-resistant breast cancer cells.
References
1 Ashkenazi A, Pai RC, Fong S, Leung S, Lawrence DA, Marsters
SA, et al. Safety and antitumor activity or recombinant soluble
Apo2 ligand. J Clin Invest 1999; 104: 155_62.
2 Walczak H, Miller RE, Ariail K, Gliniak B, Griffith TS, Kubin M,
et al. Tumoricidal activity of tumor necrosis factor-related
apoptosis-inducing ligand in vivo. Nat Med 1999; 5: 157_63.
3 Pan G, Ni J, Wei YF, Yu G, Gentz R, Dixit VM. An antagonist
decoy receptor and a death domain-containing receptor for
TRAIL. Science 1997; 277: 815_8.
4 Pan G, O'Rourke K, Chinnaiyan AM, Gentz R, Ebner R, Ni J,
et al. The receptor for the cytotoxic ligand TRAIL. Science 1997;
276: 111_3.
5 Srivastava RK. TRAIL/Apo-2L: mechanisms and clinical
applications in cancer. Neoplasia 2001; 3: 535_46.
6 Singh TR, Shankar S, Chen X, Asim M, Srivastava RK.
Synergistic interactions of chemotherapeutic drugs and tumor necrosis
factor-related apoptosis-inducing ligand/Apo-2 ligand on
apoptosis and on regression of breast carcinoma
in vivo. Cancer Res 2003; 63: 5390_400.
7 Shao R, Lee DF, Wen Y, Ding Y, Xia W, Ping B,
et al. E1A sensitizes cancer cells to TRAIL-induced apoptosis through
enhancement of caspase activation. Mol Cancer Res 2005; 3:
219_26.
8 Butler LM, Liapis V, Bouralexis S, Welldon K, Hay S, Thai LM,
et al. The histone deacetylase inhibitor, suberoylanilide
hydroxamic acid, overcomes resistance of human breast cancer
cells to Apo2L/TRAIL. Int J Cancer 2006; 119: 944_54.
9 Awad AB, Fink CS. Phytosterols as anticancer dietary components:
evidence and mechanism of action. J Nutr 2000; 130: 2127_30.
10 Vanhanen HT, Miettinen TA. Effects of unsaturated and
saturated dietary plant sterols on their serum contents. Clin Chim
Acta 1992; 205: 97_107.
11 Muti P, Awad AB, Schunemann H, Fink CS, Hovey K, Freudenheim
JL, et al. A plant food-based diet modifies the serum
β-sitosterol concentration in hyperandrogenic postmenopausal women. J
Nutr 2003; 133; 4252_55.
12 Raicht RF, Cohen BI, Fazzini EP, Sarwal AN, Takahashi M.
Protective effect of plant sterols against chemically induced colon
tumors in rats. Cancer Res 1980; 40: 403_5.
13 Gupta MB, Nath R, Srivastava N, Shanker K, Kishor K, Bhargava
KP. Anti-inflammatory and antipyretic activities of
β-sitosterol. Planta Med 1980; 39: 157_63.
14 Garcia MD, Saenz MT, Gomez MA, Fernandez MA. Topical
anti-inflammatory activity of phytosterols isolated from
Eryngium foetidum on chronic and acute inflammation models.
Phytother Res 1999; 13: 78_80.
15 Choi S, Kim KW, Choi JS, Han ST, Park YI, Lee SK,
et al. Angiogenic activity of β-sitosterol in the
ischemia/reperfusion-damaged brain of Mongolian
gerbil. Planta Med 2002; 68: 330_5.
16 Bouic PJ, Lamprecht JH. Plant sterols and sterolins: a review of
their immune-modulating properties. Altern Med Rev 1999; 4:
170_7.
17 Bouic PJ. The role of phytosterols and phytosterolins in
immune modulation: a review of the past 10 years. Curr Opin Clin
Nutr Metab Care 2001; 4: 471_5.
18 Clodi K, Wimmer D, Li Y, Goodwin R, Jaeger U, Mann G,
et al. Expression of tumor necrosis factor (TNF)-related
apoptosis-inducing ligand (TRAIL) receptors and sensitivity to
TRAIL-induced apoptosis in primary B-cell acute lymphoblastic
leukemia cells. Br J Haematol 2000; 111: 580_6.
19 Fulda S, Jeremias I, Debatin KM. Cooperation of betulinic acid
and TRAIL to induce apoptosis in tumor cells. Oncogene 2004;
23: 7611_20.
20 MacFarlane M, Harper N, Snowden RT, Dyer MJ, Barnett GA,
Pringle JH, et al. Mechanisms of resistance to TRAIL-induced
apoptosis in primary B cell chronic lymphocytic leukemia.
Oncogene 2002; 21: 6809_18.
21 Awad AB, Roy R, Fink CS. β-sitosterol, a plant sterol, induces
apoptosis and activates key caspases in MDA-MB-231 human
breast cancer cells. Oncol Rep 2003; 10: 497_500.
22 Awad AB, Williams H, Fink CS. Phytosterols reduce
in vitro metastatic ability of MDA-MB-231 human breast caner cells.
Nutr Cancer 2001; 40: 157_64.
23 Awad AB, Downie A, Fink CS, Kim U. Dietary phytosterol
inhibits the growth and metastasis of MDA-MB-231 human breast
cancer cells grown in SCID mice. Anticancer Res 2000; 20:
821_4.
24 Awad AB, Downie AC, Fink CS. Induction of growth and
stimulation of apoptosis by β-sitosterol treatment of MDA-MB-231
human breast cancer cells in culture. Int J Mol Med 2000; 5:
541_45.
25 Sprick MR, Weigand MA, Rieser E, Rauch CT, Juo P, Blenis J,
et al. FADD/MORT1 and caspase-8 are recruited to TRAIL
receptors 1 and 2 are essential for apoptosis mediated by TRAIL
receptor 2. Immunity 2000; 12: 599_609.
26 Bratton SB, MacFarlane M, Cain K, Cohen GM. Protein
complexes activate distinct caspase cascades in death receptor and
stress-induced apoptosis. Exp Cell Res 2000; 256: 27_33.
27 Cohen GM. Caspases: the executioners of apoptosis. Biochem J
1997; 326: 1_16.
28 Stennicke HR, Salvesen GS. Properties of the caspases. Biochim
Biophys Acta 1998; 1387: 17_31.
29 Earnshaw WC, Martins LM, Kaufmann SH. Mammalian caspases:
structure, activation, substrates, and functions during apoptosis.
Annu Rev Biochem 1999; 68: 383_424.
30 Yang LQ, Fang DC, Wang RQ, Yang SM. Effect of
NF-κB, survivin, Bcl-2 and caspase3 on apoptosis of gastric cancer cells
induced by tumor necrosis factor related apoptosis inducing ligand.
World J Gastroenterol 2004; 10: 22_5.
31 LeBlanc H, Lawrence D, Varfolomeev E, Totpal K, Morlan J,
Schow P, et al. Tumor-cell resistance to death receptor-induced
apoptosis through mutational inactivation of the proapoptotic
Bcl-2 homolog Bax. Nat Med 2002; 8: 274_81.
32 Kim EH, Kim SU, Shin DY, Choi KS. Roscovitine sensitizes
glioma cells to TRAIL-mediated apoptosis by downregulation of
survivin and XIAP. Oncogene 2004; 23: 446_56.
|
