Supernatant of bacterial fermented soybean induces apoptosis of
human hepatocellular carcinoma Hep 3B cells via activation of
caspase 8 and mitochondria
Chun-Li Su
a, Chao-Jung Wu
b, Fang-Nan Chen
c, Be-Jen Wang
d,
Shane-Rong Sheu
e, Shen-Jeu Won
f,*aDepartment of Nursing, Chang Jung Christian University, Tainan 711, Taiwan, ROC bInstitute of Medical Research, Chang Jung Christian University, Tainan 711, Taiwan, ROC cDepartment of Biotechnology, Institute of Nong Cyuan Hao, Puzih, Chaiyi 613, Taiwan, ROC
dDepartment of Food Science, National Chiayi University, Chiayi 600, Taiwan, ROC eBiotechnology Research Center, Far East College, Tainan 744, Taiwan, ROC
fDepartment of Microbiology and Immunology, Medical College, National Cheng Kung University, No. 1, Ta-Hsueh Road, Tainan 701, Taiwan, ROC
Received 25 November 2005; accepted 25 July 2006
Abstract
SC-1, the aqueous phase of soybean fermentation products by bacteria (Bacillus subtilis and Bacillus brevis), significantly inhibited the
growth and clonogenesity of human hepatocellular (Hep 3B), mouse hepatocellular (ML-1), and human colorectal (HCT 116 and
HT-29) carcinoma cells. Cytotoxicity of SC-1 in Hep 3B cells was through the process of apoptosis characterizing by increase in cell
population of sub-G1
phase, fragmentation of DNA, and change of nuclear morphology. Treatment of Hep 3B cells with SC-1 activated
caspase 8 and caspase 3. Elevation of nuclear DNA fragmentation factor 40 (DFF40) and cleavage form of poly(ADP-ribose)
polymer-ase (PARP) were also observed. SC-1 also activated intrinsic pathway via increpolymer-ase of pro-apoptotic (tBid, Bak and Bax) and decrepolymer-ase of
anti-apoptotic (Bcl-2 and Bcl-x
L) proteins on mitochondria, disruption of mitochondrial membrane potential, release of cytochrome c
and Smac (second mitochondria-derived activator of caspase/direct IAP binding protein with low PI) from mitochondria, and activation
of caspase 9. Inhibition on protein expression of Ku70 in cytosol and cyclooxygenase (COX)-2, but not COX-1, in whole cell lystes were
revealed in SC-1-treated Hep 3B cells. These results suggest caspase 8, Ku70 and mitochondria are involved in the antitumor mechanism
of SC-1 in Hep 3B cells.
2006 Elsevier Ltd. All rights reserved.
Keywords: Fermented soybean; Apoptosis; Mitochondria; Caspases; Cyclooxygenase
0278-6915/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2006.07.031
Abbreviations: Apaf-1, Apoptotic protease activating factor-1; ATCC, American type culture collection; B. brevis, Bacillus brevis; B. subtilis, Bacillus subtilis; COX, Cyclooxygenase; DFF, DNA fragmentation factor; DMEM, Dulbecco’s modified Eagle medium; DTT, Dithiothreitol; EDTA, Ethylen-ediamine-tetraacetic acid; EGTA, Ethylene glycol-bis(2-aminoethylether)-N,N,N0,N0-tetraacetic acid; FBS, Fetal bovine serum; HBS, HEPES buffer solution; HBV, Hepatitis B virus; HCC, Hepatocellular carcinoma; HEPES, N-2-hydroxyethylpiperazine-N0-2-ethane sulfonic acid; HRP, Horseradish peroxidase; MTT, 3-[4,5]-dimethylthiazol-2-yl-2,5-diphenyltetra-zolium bromide; PARP, Poly(ADP-ribose)polymerase; PBS, Phosphate buffered saline; PI, Propidium iodide; PMSF, Henylmethysufonyl fluoride; SC-1, The aqueous phase of soybean fermentation products by bacteria B. subtilis and B. brevis; SDS, Sodium dodecylsulphate; SDS-PAGE, SDS-polyacrylamide-gel electrophoresis; Smac, Second mitochondria-derived activator of caspase/ direct IAP binding protein with low PI; tBid, Truncated Bid; TBST, Tris buffer solution with tween; XIAP, X-linked apoptosis-inhibiting protein; Dwm,
Mitochondrial membrane potential.
*
Corresponding author. Tel.: +886 6 274 4435; fax: +886 6 2082705/2758808. E-mail address:[email protected](S.-J. Won).
1. Introduction
Soybean products such as tofu, soy milk (tonyu), miso
and soy sauce are Asian traditional food. Consumption
of soy foods, the predominant source of isoflavones, has
been reported to reduce breast, colon and prostate cancer
risk (
Birt et al., 2001
). Phosphatidyl inositol, saponins
and sphingolipids in soybeans also exhibit tumor
preven-tion properties in experimental animal model (
Fournier
et al., 1998
). Recently, soy fermentation products and their
ingredients are found to act as chemoprevention agents
(
Yang et al., 1997; Ohta et al., 2000; Chang et al., 2002
).
To determine the antitumor effect of a Taiwan traditional
remedy, the supernatant of fermented soybean products
by Bacillus subtilis (B. subtilis) and Bacillus brevis (B.
bre-vis) was used for evaluation of apoptotic efficacy.
Apoptosis (the programmed cell death) plays a pivotal
role
during
development,
homeostasis
and
immune
response in multicellular organisms (
Jacobson et al.,
1997; Earnshaw et al., 1999
). Cells fail to die of apoptosis
contribute to oncogenesis, autoimmunity and degenerative
disorders (
Williams, 1991; Vaux and Korsmeyer, 1999
).
Stimuli including radiation, tumor necrosis factor and
certain chemotherapeutic agents trigger apoptosis of tumor
cells (
Amarante-Mendes et al., 1998; Hu and Kavanagh,
2003
). Two major pathways have been reported. In
recep-tor-mediated pathway, interaction with death receptors
causes the activation of caspase 8, caspase 3 and their
downstream regulators (
Nothwehr and Martinou, 2003;
Belka et al., 2004
). In mitochondrial pathway, increase in
pro-apoptotic proteins of Bcl-2 family breakdowns
mito-chondrial membrane potential (Dw
m), causes the release
of cytochrome c and results in the activation of caspase 9
and caspase 3 (
Danial and Korsmeyer, 2004
). These two
mechanisms link at the activation of caspase 8 which
cleaves Bid and results in the release of apoptogenic
pro-teins from mitochondria (
Roth and Reed, 2002
).
Human hepatocellular carcinoma (HCC) is the fifth
most frequent cancer worldwide, and is the third most
common cause of cancer-related death (
Llovet et al.,
2003
). In the present study, the anticancer efficacy of
SC-1 was determined on hepatitis B virus (HBV) related
HCC cells (Hep 3B) since chronic HBV infection further
increases the relative risk of developing HCC to a 100-fold
compared with non-infected individuals (
Beasley et al.,
1981
). Our results indicate that SC-1, the aqueous phase
of soybean fermentation products by bacteria (B. subtilis
and B. brevis), is a potent anticancer agent. Induction of
apoptosis via both caspase 8 and mitochondria is observed
in SC-1 treated Hep 3B cells.
2. Materials and methods
2.1. Reagents
Most of the chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) unless otherwise indicated. Dulbecco’s modified Eagle med-ium (DMEM) and fetal bovine serum (FBS) were purchased from GIBCO
BRL (Grand Island, NY). Penicillin and streptomycin were purchased form Y. F. Chemical Corp. (Taipei, Taiwan). Glycine and protein assay reagents were obtained from Bio-Rad Laboratories (Hercules, CA). Phosphate buffered saline (PBS), Na2HPO4Æ12H2O, NaH2PO4Æ2H2O,
Na4P2O7Æ10H2O, NaCl, methanol and ethanol were purchased from
Wako (Osaka, Japan). Triton X-100, RNase A, ethylenediamine-tetra-acetic acid (EDTA), NaF, sodium dodecylsulphate (SDS) and NaN3were
purchased from Merck (Darmstadt, Germany). Other reagents were obtained from the following sources: dithiothreitol (DTT, MDBio, Taipei, Taiwan); proteinase K (BD Biosciences Clontech, Palo Alto, CA); Tris– HCl (American Biorganics, Niagara Falls, NY); Tween-20 (SHOWA Chemical, Tokyo, Japan).
Antibodies to various proteins were obtained from the following sources: anti-caspase 3 mouse monoclonal and anti-second mitochondria-derived activator of caspase/direct IAP binding protein with low PI (Smac) rabbit polyclonal antibodies were purchased from IMGENEX (San Diego, CA); X-linked apoptosis-inhibiting protein (XIAP), anti-cytochrome c mouse monoclonal antibodies and anti-Bid, anti-poly(ADP-ribose) polymerase (PARP) rabbit polyclonal antibodies were purchased from BD Pharmingen (San Diego, CA); anti-caspase 9 mouse monoclonal antibody was purchased from Upstate (Lake Placid, NY); anti-caspase 8 mouse monoclonal antibody was purchased from Cell Signaling (Beverly, MA); anti-Ku70, anti-Bcl-2, anti-Bax, anti-Bcl-xL, anti-cyclooxygenase
(COX)-1 and COX-2 mouse monoclonal antibodies, Bak, anti-DNA fragmentation factor (DFF) 45 and anti-DFF40 rabbit polyclonal antibodies, goat anti-mouse and donkey anti-goat conjugated horseradish peroxidase (HRP) secondary antibodies were purchased from Santa Cruz Biotech (Santa Cruz, CA); goat anti-rabbit conjugated HRP secondary antibody was purchased from Amersham Pharmacia Biotech (Piscataway, NJ); anti-tBid rabbit polyclonal antibody was purchased from Biosource (Camarillo, CA); anti-b-actin was purchased from Sigma (St. Louis, MO).
2.2. Preparation of fermented soybean supernatant
Soybeans (1 kg) obtained from Dongshi Shiang, Chiayi, Taiwan were ground, boiled for 8 h and soaked in sterile water (10 l) for 10 days. After centrifugation at 1000· g, the supernatant was cultured with B. subtilis (105cells/ml) and B. brevis (105cells/ml) for 1 month at 37
C. Before the experiments, SC-1 was prepared by centrifuged this fermentation product at 15,000· g for 30 min, filtered through 0.22 lm filter (Corning, Corning, NY) to avoid the bacteria contamination, freeze-dried, and stored at 70 C until use. All solutions were prepared in pyrogen-free glassware that was heated for 5 h before use.
2.3. Cell culture
Human colorectal carcinoma (HCT 116 and HT-29) and HCC (Hep 3B) cells were obtained from American Type Culture Collection (ATCC, Rockville, MD). Mouse hepatoma ML-1 cells (Chen et al., 1992) were obtained from Dr. Huan-Yao Lei (Department of Microbiology and Immunology, Medical College, National Cheng Kung University). Cells were grown in DMEM supplemented with 10% FBS, 2 mM glutamine, 100 unit/ml penicillin and 100 lg/ml streptomycin. Cells were maintained at 37C in a 5% CO2humidified atmosphere.
2.4. Viability assay
The inhibition of cell viability was assessed by modified colorimetric assay (Mosmann, 1983). Cells (1.5· 103cells/well) were seeded in 96-well/
plates (Nunc, Denmark) for 12 h. After attachment to plates, the super-natants in each well were replaced very carefully with 100 ll of fresh DMEM containing different concentrations of SC-1. The plates were then incubated for 5 days. Cell viability was determined by adding 10 ll of 3-[4,5]-dimethylthiazol-2-yl-2,5-diphenyltetra-zolium bromide (MTT) at a final concentration of 0.5 mg/ml in PBS to each well. The MTT was taken up and converted to a purple formazan. After 4 h of incubation, 100 ll of 10% SDS in 0.01 N HCl was added to each well to dissolve the formazan
for overnight. The absorbance of each well was measured in a microplate reader at 590 nm on a Multiscan photometer (MRX II, Dynatech, McLean, VA). IC50was the concentration that achieved 50% cytotoxicity
against culture cells.
2.5. Colony formation assay
Cytotoxicity was determined to evaluate the ability of tumor cells to form colonies with SC-1 treatment. The assay was performed using a two-layer agar system in 6-well/plates (Nunc, Denmark). As described previ-ously (Wang et al., 2005), the base layer was 0.6% agar containing DMEM with 10% FBS, and the upper layer was 0.33% agar in DMEM supple-mented with 10% FBS and contained tumor cells (6· 104cells/well) and
various concentrations of SC-1. Colonies with diameter larger than 1 mm were counted 14 days later (Stewart et al., 1999).
2.6. Flow cytometry analysis for cell cycle distribution
The measurement was assessed by the reported method (Di Leonardo et al., 1994). As described previously (Lee et al., 2005), cells (2· 105
cells/ well) were seeded into 6-well/plates (Nunc, Denmark). After treated with different concentrations of SC-1 for 48 or 72 h, cells were trypsinized and resuspended in HEPES buffer solution (HBS). After centrifugation at 800· g for 10 min at 4 C, cells were resuspended in 70% ethanol at 4 C for overnight. The samples were washed and resuspended in HBS con-taining 40 lg/ml of propidium iodide (PI) and 100 mg/ml of RNase A in the dark for 30 min. Cell cycles (G0/G1, S and G2/M) were determined by
DNA histograms obtained by flow cytometry (Becton Dickinson Immunocytometry system). Results were analyzed with the Windows Multiple Document Interface software for Flow Cytometry (WinMDI 2.8, Scripps Research Institute, San Diego, CA).
2.7. DNA fragmentation assay
As described previously (Wang et al., 2005), SC-1 treated or untreated cells (1· 106
cells/well) were incubated with lysis buffer containing 10 mM Tris–HCl [pH 7.6], 1 mM EDTA and 1% IGEPAL CA-630 (NP-40) at 37C for 20 min. Cell lysates were centrifuged and the supernatants were incubated with the solution that contained 0.5% of SDS and 4 mg/ml of RNase A at 56C for 2 h. After incubation, 40 ll of proteinase K (20 mg/ ml) was added to a final concentration of 1 mg/ml and the mixtures were incubated at 37C for overnight. DNA was precipitated with 0.1 volume of ammonium acetate (10 M) and 2.5 volume of ethanol at20 C for overnight. Pellets were obtained after centrifugation at 13,000· g for 15 min. DNA samples were stained with ethidium bromide and separated on 1% agarose gel. The fragments of DNA were examined by exposing to UV light (Sandstrom and Buttke, 1993).
2.8. Determination of nuclear morphologic changes
Cells (1· 105cells/well) on 6-well/plates (Nunc, Denmark) were incubated without or with SC-1 for various time periods (0–72 h). After trypsinization and washing with PBS, cells were fixed with 4% parafor-maldehyde in PBS for 5 min. Following washing three times with PBS, cells were spun on glass microscope slides and permeabilized in PBS containing 0.1% Triton X-100 and 0.05% Tween-20. Staining was carried out in the dark by incubating cells with 0.5 lg/ml of Hoechst 33258 in PBS at 37C for 30 min. Cells were washed three times and mounted with 3 ll of rapid mounting media (Merck, Darmstadt, Germany). Changes of nuclear morphology were visualized under fluorescence microscope (Leica DMRBE microscope).
2.9. Fractionation of cell proteins
Cells (5· 105cells/well) were treated without or with SC-1 for various
time periods (0–72 h). The methods of protein extraction were performed
as previously described (Feng and Lo, 1999; Watabe et al., 2000; Lee et al., 2005). Briefly, whole cells were lysed with 200 ll lysis buffer containing 10 mM Tris–HCl [pH 7.9], 0.15 M NaCl, 1% (w/v) Triton X-100, 5 mM EDTA, 10 mM NaF, 10 mM NaN3, 5 mM Na2HPO4Æ12 H2O, 5 mM
NaH2PO4Æ2H2O, 5 mM Na4P2O7Æ10H2O, and 1 tablet of complete
protease inhibitor cocktail (Boehringer, Mannheim, Germany). Cell mix-tures were centrifuged at 15,000· g for 10 min and the resulting super-natants were used as the whole cell lysates for immunoblotting.
Cytosolic and mitochondrial proteins were prepared as described previously (Earnshaw et al., 1999; Watabe et al., 2000). Briefly, cells (5· 105cells/well) were washed with PBS and harvested by centrifugation
at 800· g for 10 min at 4 C. The pellets were washed twice with ice-cold PBS and were then resuspended in TSE buffer (10 mM Tris, 0.25 M sucrose, 0.1 mM EDTA, pH 7.4). Cell suspensions were transferred to a Dounce homogenizer (Glas-Col, Terre Haute, IN) and broken with 10 strokes of a Teflon pestle. The homogenates were centrifuged at 750· g at 4C for 30 min. The supernatants were centrifuged at 12,000 · g for 30 min at 4C. The lysed solutions were centrifuged at 100,000 · g for 1 h and their resulting supernatants were used for cytosolic fractions. The obtaining pellets were incubated with 100 ll lysis buffer containing 10 mM Tris–HCl [pH 7.9], 0.15 M NaCl, 1% (w/v) Triton X-100, 5 mM EDTA, 10 mM NaF, 10 mM NaN3, 5 mM Na2HPO4Æ12 H2O, 5 mM NaH
2-PO4Æ2H2O, 5 mM Na4P2O7Æ10H2O, and 1 tablet of complete protease
inhibitor cocktail (Boehringer, Mannheim, Germany). The lysed solutions were used as mitochondrial fractions for immunoblotting.
Nuclear fractions were prepared as previous described (Feng and Lo, 1999). Cells (5· 105 cells/well) were isolated by centrifugation, washed
twice with ice-cold PBS, lysed in 400 ll of buffer A (10 mM HEPES [pH 7.9], 5 mM MgCl2, 10 mM KC1, 3 mM Na3VO4, 10 mM NaF, 0.5 mM
DTT, 0.5 mM phenylmethysufonyl fluoride (PMSF), 2 lg/ml leupeptin and 2 lg/ml pepstatin A), and incubated on ice for 20 min. After centri-fugation at 11,000· g at 4 C for 10 s, the pellets were resuspended in 60 ll of buffer B (20 mM HEPES [pH 7.9], 1.5 mM MgCl2, 420 mM NaCl,
0.2 mM EDTA, 25% glycerol, 3 mM Na3VO4, 10 mM NaF, 0.5 mM DTT,
0.5 mM PMSF, 2 lg/ml leupeptin and 2 lg/ml pepstatin A) and incubated for 15 min on ice with occasional mixing. Debris was removed by centri-fugation at 12,000· g for 15 min at 4 C. The obtained nuclear proteins were used for immunoblotting.
2.10. Immunoblotting
Protein contents of whole cell, cytosolic, mitochondria and nuclear fractions were determined by protein assay kit (Bio-Rad, Hercules, CA). All isolated proteins were stored at 80 C before use. Proteins were resolved using 10–12% SDS polyacrylamide gel electrophoresis (PAGE) with running buffer (25 mM Tris, 192 mM glycine, 3.5 mM SDS, pH 8.3) and subsequently transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA) as described previously (Chendil et al., 2002). Membranes were blocked by incubating in TBST (20 mM Tris, 137 mM NaCl, 0.05% Tween-20, pH 7.4) containing 5% skim milk for 2 h at room temperature. Follow by probed the membrane with a appropriate first antibody, a secondary probe with HRP-labeled goat anti-mouse (1:5000), goat anti-rabbit (1:5000) or donkey anti-goat (1:5000) antibody was visualized by exposure to X-ray film (Kodak, PerkinElmer, Rochester, NY) after staining with chemiluminescence reagents (PerkinElmer, Boston, MA).
2.11. Determination of Dwm
by confocal microscopy
Dwm was measured by using a laser scanning confocal microscope
(Leica TCS-SP2, Germany) as described previously (Yang et al., 1997; Lee et al., 2005). Briefly, cells (1· 105cells/well) were plated in 6-well/plates
(Nunc, Denmark) containing methanol-sterilized glass cover slips. After overnight incubation, cells were treated or untreated with various con-centrations of tested agent for the indicated time periods and then stained with 5 lM rhodamine 123 at 37C for 30 min. After washing with PBS, cells were fixed with 4% paraformaldehyde for 15 min, mounted onto
poly-L-lysine coated glass microscope slides (Menzel-Glaser, Germany) and examined immediately. Samples were viewed in the dark with a Leica TCSNT laser scanning confocal imaging system coupled to a Leica DMRBE microscope, using a Leica 630 fluotar objective. The stained cells were excited with the 488-nm lines of a 25-mW laser. The laser was set to the optimum power that could produce a fluorescent signal. Rhodamine 123 fluorescence was visualized with a BF530/30 emission filter combi-nation. Optical sections close to the middle of the cells were chosen for visual evaluation of experimental treatments.
3. Results
3.1. SC-1 inhibits the growth of tumor cells
To examine whether the supernatant of the fermented
soybeans would alter the malignant proliferation, the
inhibitory effects of SC-1 on the growth of tumor cells were
determined by a modified MTT colorimetric assay. As
shown in
Table 1
, the IC
50value (50% cell growth
inhibi-tory concentration) for human HCC (Hep 3B), mouse
hep-atoma cells (ML-1) and human colorectal carcinoma (HCT
116 and HT-29) cells were 53.7, 58.7, 54.5 and 48.4 lg/ml
of SC-1, respectively. A time (0–5 days) and dose (20–
165 lg/ml) related inhibition of the cell growth by SC-1
was also observed (data not shown). To further analyze
the ability of SC-1 treated tumor cells on
anchorage-inde-pendent growth properties, the effect of SC-1 on clonogenic
survival was evaluated by colony formation assay. After 14
days of incubation, SC-1 decreased the number of colonies
to 50% at the concentrations of 47.7, 65.9, 75.2 and
55.2 lg/ml for Hep 3B, ML-1, HCT 116 and HT-29 cells,
respectively (
Table 1
). The data demonstrate that these
tumor cells are comparably sensitive to SC-1 on the
sup-pression of cancerous growth and colony formation.
3.2. Induction of apoptosis by SC-1
Induction of apoptosis has been reported to be a
poten-tially promising approach for cancer therapy (
Green,
2000
). Exhibition of apoptotic phenomena (cell cycle
redis-tribution, DNA fragmentation, and chromatin
condensa-tion) represents the proceeding of apoptosis (
Nicoletti
et al., 1991; Danial and Korsmeyer, 2004
). To examine
the possible anti-proliferation mechanism of SC-1 in
HCC cells, cell cycle distributions of Hep 3B cells cultured
with SC-1 (0–662.5 lg/ml) for 48 or 72 h were analyzed by
flow cytometry. After staining the DNA with PI, the
per-cent of hypodipliod DNA content was determined. As
shown in
Fig. 1
A, a dose-related accumulation of cells at
sub-G
1phase was observed. At the concentration of 0,
41.4, 82.8, 165.6, 331.23 and 662.5 lg/ml, SC-1 increased
the percentage of cells at sub-G
1phase from 0.7 to 1.5
(2.1-fold), 1.6 (2.3-fold), 2.7 (3.9-fold), 37.7 (53.9-fold)
and 57.7% (82.4-fold) at 48 h, and from 1.2 to 3.0
(1.8-fold), 3.3 (2.8-(1.8-fold), 5.1 (4.3-(1.8-fold), 40.7 (33.9-fold) and
60.3% (50.3-fold) at 72 h (
Fig. 1
A). The data indicate that
within the range of 0–662.5 lg/ml and over the incubation
time of 0–72 h, SC-1 increases in the percentage of cells at
the sub-G
1phase in a dose- and time-related manner. For
detection of DNA fragmentation, agarose gel
electrophore-sis was performed. After 72 h incubation, a dose-related
(0–662.5 lg/ml) increase in DNA ladders was displayed
(
Fig. 1
B). A time-related effect (24, 48 and 72 h of
incuba-tion time) of SC-1 on DNA fragmentaincuba-tion was also
observed (data not shown). For nuclear morphology assay,
Hep 3B cells were cultured with 165.6 lg/ml of SC-1 for
various time periods (0–72 h). Chromatin condensation
was visualized by staining cells with Hoechst 33258. As
shown in
Fig. 1
C, control cells appeared to be
morpholog-ically normal with intact DNA. As incubation time
increased, the number of cells with condensed chromatin
increased especially at the time of 48 or 72 h. These results
demonstrate that SC-1 induces apoptosis of Hep 3B cells
in vitro in a SC-1 dose and/or incubation time-related
manner.
3.3. SC-1 activates caspases in Hep 3B cells
Apoptosis can be carried out by the activation of
casp-ases in which caspase-2, -8 and -9 are initiator caspcasp-ases
and caspase-3 is classified into effecter caspases (
Li and
Yuan, 1999
). To determine the signal pathway in SC-1
treated Hep 3B cells, cells were incubated with 165.6 lg/
ml of SC-1 for 0–72 h. Whole cell lysates and nuclear
proteins were obtained for immunoblotting. SC-1 increased
the expression of activated cleavage form of caspase 8 at
12 h (
Fig. 2
A), and the activation of its downstream
cas-pase 3 was also observed at 12 h (
Fig. 2
B). In contrast,
acti-vation of caspase 2 was not detected (data not shown). The
substrates of activated caspase-3 include PARP (
Chinnai-yan et al., 1995
) and DFF45 (
Enari et al., 1998
). The
pres-ent results exhibit that in the presence of SC-1 (165.6 lg/
ml), nuclear full-length PARP (116 kDa) decreased and
its cleavage form (89 kDa) increased at 24 h (
Fig. 2
C
and E). SC-1 also significantly decreased total DFF45
and DFF35, and increased nuclear DFF40 expressions
(7.1, 11.9 and 29.8-fold at 12, 24 and 72 h) (
Fig. 2
D and
E). The data suggest that SC-1 may act through the
initia-tor caspase-8 and then executioner caspase-3 to increase
both cleavage form of PARP and nuclear DFF40 for
DNA fragmentation.
Table 1
IC50of cell growth and colony formation on human and mouse tumor cell
lines
Cell line Growth inhibition
(lg/ml) Clonogenic cell inhibition (lg/ml) Hep 3B 53.7 ± 4.6a,b 47.7 ± 1.2 ML-1 58.7 ± 0.8 65.9 ± 6.6 HCT 116 54.5 ± 4.5 75.2 ± 9.4 HT-29 48.4 ± 2.8 55.2 ± 4.4 a
Control cells were treated with DMEM. Experiments were repeated three times and each concentration of samples was conducted in eight replicates.
b
3.4. Involvement of mitochondria in SC-1 induced
apoptosis
Depolarization of mitochondrial membrane, regulated
by the members of Bcl-2 family, has been reported could
be an early event in apoptosis (
Marchetti et al., 1996; Danial
and Korsmeyer, 2004
). To investigate if mitochondria
anticipate in the process of SC-1 induced apoptosis, changes
of Dw
mwere determined by staining cells with
mitochon-drial specific dye rhodamine 123, and the fluorescence
Fig. 1. Induction of apoptosis by SC-1 in Hep 3B cells. (A) Effect of SC-1 on cell cycle progression. Hep 3B cells (2· 105cells/well) were treated with theindicated concentrations of SC-1. At 48 and 72 h, cells were harvested and stained with PI for DNA content analysis. Apoptosis were measured by the accumulation of sub-G1DNA contents in the cells. The percentage in the figure indicates the proportion of apoptotic cells. (B) DNA fragmentation by
SC-1. After treating with the indicated concentrations (0–662.5 lg/ml) of SC-1 for 72 h, Hep 3B cells (1· 106
cells/well) were lyzed. DNA fragments were analyzed by 1% agarose gel electrophoresis. M, DNA molecular weight marker; lane 1, 0 lg/ml of SC-1; lane 2, 41.4 lg/ml of SC-1; lane 3, 82.8 lg/ml of SC-1; lane 4, 165.6 lg/ml of SC-1; lane 5, 331.3 lg/ml of SC-1; lane 6, 662.5 lg/ml of SC-1. (C) Change of nuclear morphology by SC-1. Hep 3B cells (1· 105
cells/well) were treated with 165.6 lg/ml of SC-1 for 0–72 h. After staining with Hoechst 33258, morphology of the cells was analyzed by fluorescence microscopy. Arrowhead indicates apoptotic cells with condensed and segmented DNA. Dilutions were made by mixing SC-1 with DMEM. Control cells were treated with DMEM. Results are representative of three independent experiments.
intensity was analyzed using confocal microscopy. As
shown in
Fig. 3
, a time-related decrease in the intensity
of rhodamine 123 staining was observed in the
mitochon-dria of Hep 3B cells treated with SC-1 (165.6 lg/ml). The
decrease in Dw
mwas observed as early as 12 h and reached
the lowest level at 72 h (
Fig. 3
).
Expressions of Bcl-2 family proteins following SC-1
treatment were also evaluated. As shown in
Fig. 4
A and
B, the increase in caspase 8-mediated cleavage of Bid into
truncated Bid (tBid) was first observed at 12 h (1.4-fold)
which is similar to the activation time of caspase 8
(
Fig. 2
A). The increase of mitochondrial tBid was peaked
at 72 h of SC-1 (165.6 lg/ml) treatment (5.4-fold).
Mito-chondrial Bax was elevated at 48 h (2.0-fold) and kept
increased at 72 h (3.0-fold) (
Fig. 4
A and B). Profoundly
increase in mitochondrial Bak was observed as early as
12 h (37.7-fold) and peaked at 48–72 h (about 180-fold)
in SC-1 treated Hep 3B cells (
Fig. 4
A and C). In contrast,
SC-1 decreased anti-apoptotic Bcl-2 and Bcl-x
Lin
mito-chondria at 24 h (0.6-fold) and 12 h (0.8-fold), respectively
(
Fig. 4
A and B). Ku70 is also an important factor for
apop-tosis (
Mancinelli et al., 2006
). Decrease in Ku70 has been
reported to enhance Bax-mediated apoptosis (
Nothwehr
and Martinou, 2003
), while increase in Ku70 inhibits the
process (
Sawada et al., 2003
). In
Fig. 4
D and E, SC-1
sup-pressed Ku70 in cytosol at 4–24 h. However, SC-1 did not
change the expression of nuclear Ku70 (
Fig. 4
D). Reports
also indicate that change of Dw
mtriggers the release of
apoptogenic proteins such as cytochrome c and Smac
(
Hengartner, 2000; Roth and Reed, 2002
). Complex of
cytochrome c, apoptotic protease activating factor-1
(Apaf-1) and caspase-9 activates caspase-9 itself and thence
caspase-3 (
Hengartner, 2000
). Smac also promotes
caspase-9 activation by binding with XIAP to neutralize its
anti-apoptotic activity (
Shi, 2001
). As expected, the release of
cytochrome c (1.5, 2.0 and 4.0-fold at 12, 24 and 48 h,
respectively) and Smac (1.5-fold at 48 and 1.9-fold at
72 h)
from
mitochondria
to
cytosol
was
observed
(
Fig. 4
F and G). Decrease in XIAP (0.4-fold) was displayed
at 12 h and reached the lowest level (0.1-fold) at 72 h in
SC-1 (SC-165.6 lg/ml) treated Hep 3B cells (
Fig. 4
F and G). After
having shown that mitochondria were affected by SC-1,
activation of caspase 9 was determined. As shown in
Fig. 4
H, expression of full length caspase 9 decreased and
the activation form of cleaved caspase 9 increased in
SC-1 (SC-165.6 lg/ml) treated Hep 3B cells.
3.5. Changes of COX protein expression in
SC-1 treated Hep 3B cells
Overexpression of COX-2 has been found in many types
of cancer, and expression of HBV surface protein further
increases the expression of COX-2 (
Hung et al., 2004
). As
shown in
Fig. 5
A, COX-1 protein expressions in SC-1
trea-ted Hep 3B cells were not affectrea-ted. However, the
expres-sions of COX-2 were significantly inhibited by the
treatment of SC-1 (165.6 lg/ml) in a time-related manner
43 41 57 Time (h) 0 12 24 48 72 kDa
SC-1
Caspase 8 Cleaved caspase 8 β-Actin 46 Caspase 3SC-1
Cleaved caspase 3 32 17 12 Time (h) 0 12 24 48 72 kDa β-Actin 46SC-1
PARP Cleaved PARP 116 89 Time (h) 0 12 24 48 72 kDa Nuclear β-Actin 46 Nuclear DFF40SC-1
Time (h) 0 12 24 48 72 kDa 45 β-Actin Total DFF45 Total DFF35 35 46 40 β-Actin 46 time (h) 0 12 24 48 72 Fold o f control 0 20 40 60 80 100 120 140 160 180 Nuclear DFF40 Nuclear PARP Cleaved PARPA
B
C
D
E
Fig. 2. Activation of caspases and their target proteins by SC-1. (A) caspase 8, (B) caspase 3, (C) PARP and (D) DFF expressions were determined in Hep 3B cells (5· 105cells/well) treated with 165.6 lg/ml of
SC-1. At the indicated time periods (0–72 h), whole cell lysates or nuclear proteins were subjected for western blot analysis. Anti-caspase 8, anti-caspase 3, anti-PARP, anti-DFF45 (for total DFF) and anti-DFF40 (for nuclear DFF) antibodies were served as probes. b-actin was used as loading control. The intensity of individual nuclear PARP, cleaved PARP (C) and nuclear DFF40 (D) protein signal was quantified by densitometry. Each signal was normalizing to that of b-actin. The densities of nuclear PARP, cleaved PARP and DFF40 in the control condition were designated as 1, and the levels of the remaining samples were expressed as fold of the control (E). Results are representative of three independent experiments.
(0.8, 0.5, 0.1-fold at 12, 24 and 48 h, respectively) (
Fig. 5
B
and C).
4. Discussion
SC-1 effectively inhibits the growth and clonogenecity of
human HCC (Hep 3B), mouse hepatoma cells (ML-1) and
human colorectal carcinoma (HCT 116 and HT-29) cells
(
Table 1
). Since colony formation is an in vitro assay used
to examine anchorage-independent growth which strongly
correlates with tumorgenicity and invasiveness of tumor
cells (
Moore et al., 1998
), the data in
Table 1
indicate
SC-1 both affects dependent and
anchorage-independent growth of these tumor cells. Cytotoxicity of
SC-1 on HBV-related human HCC Hep 3B cells is further
investigated. The results in
Fig. 1
A–C reveal that SC-1
induces apoptosis in Hep 3B cells. Since the percentage
of hypodiploid DNA pick represents the percentage of
reduced DNA content of apoptotic nuclei which can be
used to measure the percentage of cells with hypodiploid
DNA content (apoptotic cells) (
Nicoletti et al., 1991
), the
decrease in the accumulation of cells at sub-G
1phase
(
Fig. 1
A) suggests that the SC-1 treated Hep 3B cells is
undergoing apoptotic process. Upon apoptotic induction,
chromatin becomes condensed, apoptotic body forms,
and activated endonucleases cleave DNA at the linker
regions between nucleosomes to produce 180 bp
oligonu-cleosome (DNA ladder) (
Wyllie, 1980; Compton, 1992
).
Ladders of DNA and condensation of chromatin in SC-1
treated Hep 3B cells (
Fig. 1
B and C) provide further signs
of apoptosis.
A cascade of proteolytic activity is involved in the
pro-cess of apoptosis, much of which is performed by caspases
(
Earnshaw et al., 1999
). Activation of caspase 8 can be
trig-gered by death receptor-independent apoptotic stimuli
including ionizing radiation, chemotherapeutic drugs and
viruses (
Green, 1998; Slee et al., 1999; Borner, 2003
).
Acti-vated caspase 8 can directly activate its downstream effecter
caspases, such as caspase 3 (
Danial and Korsmeyer, 2004
).
PARP, a substrate of caspase 3, is reported to play a
piv-otal role in DNA repair mechanism (
Sakahira et al.,
1998; Soldani et al., 2001
). DFF45 has also been reported
to mediate genomic DNA fragmentation during apoptosis
and can be degraded by activated caspase 3 to allow
DFF40 entering the nucleus to execute DNA
fragmenta-tion (
Chen et al., 2000
). In the present study, both caspase
8 and 3 are activated at 12 h in SC-1 treated Hep 3B cells
(
Fig. 2
A and B). The activated caspase 3 causes the release
of DFF40 to enter the nucleus at 12 h (
Fig. 2
D and E), and
also cleaves PARP at 24 h (
Fig. 2
C and E). The
combina-tion of the increase in nuclear cleaved PARP and DFF40
(
Fig. 2
C–E) may contribute to the fragmentation of
DNA in SC-1 treated Hep 3B cells (
Fig. 1
B).
Activation of caspase-8 can also be stimulated by death
receptor-dependent pathway. Fas has been reported to
control programmed cell death in hepatocytes (
Natoli
et al., 1995
) and play a essential role in the pathogenesis
of liver diseases including hepatitis, cirrhosis and HCC
(
Galle et al., 1995
). Upregulation of membrane Fas and
Fas-L expression and induction of Fas-dependent
apopto-sis by chemotherapeutic drugs is revealed (
Muller et al.,
1997
). In the present study, the expression of Fas and
Fas-L was not affected by SC-1 in Hep 3B cells (data not
shown). The percentages of cells accumulated at the
sub-G
1phase were not changed by an anti-Fas neutralizing
ZB-4 antibody (data not shown). These results suggest
the induction of apoptosis by SC-1 in Hep 3B cells is
Fas-independent.
Accumulating evidences also suggest that caspase 8 acts
as an upstream caspase to induce caspase 8-mediated
cleav-age of Bid into tBid, which triggers the activation of
mito-chondrial pathway and subsequently causes the release of
cytochrome c to induce apoptotic signal (
Wieder et al.,
2001; Wajant, 2002
). In the mitochondrial pathway,
apop-togenic factors such as cytochrome c and Smac are released
from the intermembrane space of mitochondria into the
cytoplasm (
Liu et al., 1996; Kroemer et al., 1997; Green,
1998; Du et al., 2000
). Members of Bcl-2 family can either
induce (Bid, Bax, Bad, Bik and Bak) or inhibit (2,
Bcl-w, Mcl-1 and Bcl-x
L) apoptosis through mitochondria
con-trolling mechanism (
Adams and Cory, 1998
). Activation of
Bid by caspase 8 links intrinsic and extrinsic apoptotic
pathways through mitochondrial damages to activate the
downstream caspases (
Green and Reed, 1998; Li et al.,
1998; Newmeyer and Ferguson-Miller, 2003
). In addition,
tBid triggers the oligomerization of pro-apoptotic Bak or
Bax and results in the release of apoptogenic factors from
mitochondria (
Wei et al., 2001; Tsujimoto, 2003
).
How-ever, Ku70 binds to pro-apoptotic protein Bax, prevents
Bax translocate to the mitochondria and therefore inhibits
apoptosis (
Sawada et al., 2003
). In the present study, the
activated caspase 8 cleaves Bid into tBid at 12 h of SC-1
Fig. 3. Change in mitochondrial membrane potential (Dwm). Hep 3B cells (1· 105cells/well) were treated with 165.6 lg/ml of SC-1 for 0–72 h. After15 SC-1 Cytosol Bid Mito tBid 22 Time (h) 0 12 24 48 72 kDa Mito Bcl-2 29 Mito Bcl-xL 32 Mito Bak 23 Mito Bax 28 time (h) 0 12 24 48 72 Fold of control 0 1 2 3 4 5 6 Cytosol Bid Mito tBid Mito Bcl-2 Mito Bcl-xL Mito Bax time (h) 0 12 24 48 72 Fold of control 0 50 100 150 200 Mito Bak
A
B
C
D
time (h) 0 2 4 6 12 24 fold o f con trol 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Cytosol Ku70E
Mito Smac SC-1 Time (h) 0 12 24 48 72 kDa Total XIAP 15 Cytosol Smac Cytosol cyt c 57 Mito cyt c 15 25 25F
time (h) 0 12 24 48 72 Fold of control 0 1 2 3 4 5 Cytosol cyt c Mito cyt c Cytosol Smac Mito Smac Total XIAPG
Caspase 9 SC-1 Cleaved caspase 9 46 35 Time (h) 0 12 24 48 72 kDa β-Actin 46 37H
SC-1 Time (h) 0 2 4 6 12 24 kDa 70 Cytosol Ku70 Nuclear Ku70 β-Actin 70Fig. 4. Expressions of mitochondria-related proteins. (A) Distribution of Bid, tBid, Bcl-2, Bcl-xL, Bak and Bax, (D) Expression of Ku70, (F) Distribution
of cytochrome c (cyt c), Smac and XIAP, and (H) Expression of caspase 9 were determined by western blot analysis. Hep 3B cells (5· 105cells/well) were
treated with 165.6 lg/ml of SC-1. At the indicated time periods (0–72 h), whole cell, mitochondrial (Mito), cytosolic and nuclear fractions were subjected for western blot analysis. Anti-Bid, anti-Bcl-2, anti-Bcl-xL, anti-Bak, anti-Bax, anti-Ku70, anti-cytochrome c, anti-Smac, anti-XIAP and anti-caspase 9
antibodies were served as probes. b-actin was used as loading control. The intensity of each protein band in (A), (D) and (F) was quantified by densitometry. The densities of these proteins in the control condition were designated as 1, and the levels of the remaining samples were expressed as fold of the control (B), (C), (E) and (G). Results are representative of three independent experiments.
treatment (
Fig. 4
A and B). The translocated tBid on the
mitochondria (
Fig. 4
A and B) may form a complex with
pro-apoptotic Bax or Bak (
Fig. 4
A–C) to disrupt Dw
mand in turn trigger cytochrome c and Smac release from
the mitochondria to the cytosol. Moreover, a time- and
dose-related elevation of mitochondrial Bak (37.7-fold at
12 h) and Bax (2.0-fold at 48 h) indicates Bak is more
important on the changes of Dw
mcompared with Bax
(
Fig. 4
A–C). The significant decrease in cytosolic Ku70
(
Fig. 4
D and E) by SC-1 also suggests the increase in
mito-chondrial Bax (
Fig. 4
A) is via decrease in cytosolic Ku70.
The loss of mitochondrial staining by SC-1 (
Fig. 3
)
indi-cates the disruption of Dw
m, which may result from the
changes in permeability of inner mitochondrial membrane
(
Kroemer et al., 1997
). Colocalization of Bcl-2 family
proteins at the surface of mitochondria has been reported
to regulate the movement of cytochrome c (
Hengartner,
2000
). The release of cytochrome c and Smac is observed
at 12 and 48 h (
Fig. 4
F and G). Since Smac can antagonize
the anti-apoptotic function of XIAP, decrease in XIAP
(0.4-fold at 12 h) may further promote the process of
SC-1 induced apoptosis (
Fig. 4
F and G). Cytochrome c has
been reported to bind and activate Apaf-1, which then
aggregates caspase 9 to form apoptosome and thus
acti-vates caspase 9 and its downstream caspases such as
cas-pase 3 (
Nicholson, 2001; Roth and Reed, 2002
). In the
present study, the released cytochrome c may form
apopto-somes to activate caspase 9 at 24 h (
Fig. 4
H). Activated
caspase 9 further promotes the activation of caspase 3
(
Fig. 2
B) and its downstream regulators DFF40 and PARP
(
Fig. 2
C–E). Since Bcl-x
Lcan bind to Bax and prevent
the insertion of Bax into the outer membrane of
mitochon-dria (
Desagher and Martinou, 2000
), the decrease of
mito-chondrial anti-apoptotic Bcl-2 at 24 h and Bcl-x
Lat 12 h
further supports the process of programmed cell death
(
Fig. 4
A–C).
Regulation of apoptosis can also be made by inhibitors
of COX proteins (
Belka et al., 2004
). Two major isoforms
of COX were reported (
Smith et al., 1996
). COX-1 is
expressed constitutively, while COX-2 can be induced at
site of inflammation (
Dubois et al., 1998
) and contributes
to carcinogenesis and resistance of apoptosis (
Tsujii and
DuBois, 1995
). Increase in COX-2 is also related to
increase metastasis of tumor cells (
Jiang et al., 2001
). In
the present study, the protein levels of COX-2 in
HBV-related Hep 3B cells are significantly inhibited by SC-1 in
a time-related manner (
Fig. 5
B and C). Integration of
HBV DNA into the genome of hepatocytes may cause
transformation of the cells (
Bruix and Llovet, 2003
).
X protein of HBV may induce COX-2 gene expression in
HBV-infected liver cells and therefore increases risk of
liver cancer (
Cheng et al., 2004
). Induction of apoptosis
plus inhibition of COX-2 expression in Hep 3B cells offers
SC-1 a good chance on chemoprevention and
chemo-therapy of HBV associated chronic liver disease and
HBV-related HCC.
Interesting enough, all of the tested tumors are affected
by the treatment of SC-1. Our previous experiment
indi-cated that SC-1 (132.5 lg/ml) does not cause mutagenicity
on Salmenella typhimurium tested strains (TA97, TA98,
TA100, TA102 and TA1535) by Ames test, or
clastogenic-ity in Chinese hamster ovary cells by in vitro chromosome
aberration assay (unpublished data). In addition, no
observed toxic effects are found in our parallel in vivo
study, in which the soybean fermentation products were
fed to BALB/c mice for 75 days and the tumor size was
significantly reduced (unpublished data). Even though
soy-bean products are thought in general to be relatively safe,
the toxicity of SC-1 on normal cells awaits further
assess-ment. The possible ingredients of soybean that account
for cancer chemoprevention include isoflavones (genistein,
genistin, daidzein and biochanin A), phytosterols, soy
phy-tates, protease inhibitors and saponins (
Chang et al., 2002
).
In primary human food, soybean is the predominant
source of isoflavonoids, and which can only occur by
die-tary intake in mammals (
Birt et al., 2001
). Isoflavonoids,
mostly present in foods as glycosidic conjugates, require
enzymatic cleavage of the sugar moiety by mammalian or
microbial glucosidases before absorption (
Ohta et al.,
COX-1
SC-1
72 Time (h) 0 12 24 48 72 kDa ββ -Actin 46 COX-2 72 Time (h) kDa β-Actin 46 time (h) 0 12 24 48 72 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Fold of controlSC-1
0 12 24 48 72A
B
C
Fig. 5. Expressions of COX-1 and COX-2. (A) COX-1 and (B) COX-2 expressions were determined in Hep 3B cells (5· 105
cells/well) treated with 165.6 lg/ml of SC-1. At the indicated time periods (0–72 h), whole cell lysates were subjected for western blot analysis. Anti-COX-1 and anti-COX-2, antibodies were served as probes. b-actin was used as loading control. The intensity of individual COX-2 (B) protein signal was quantified by densitometry. Each signal was normalizing to that of b-actin. The densities of COX-2 in the control condition were designated as 1, and the levels of the remaining samples were expressed as fold of the control (C).
2000
). Fermentation of soybean by microorganisms may
alter the structure of the active compounds and therefore
increase the availability of isoflavones in soy (
Hutchins
et al., 1995
). SC-1 is a soybean product fermented with
B. subtilis and B. brevis. So far, Bacillus species are still
dominant bacteria in industrial fermentations, and some
of them are on the Food and Drug Administration’s
GRAS (generally regarded as safe) list (
Green et al.,
1976
). Possible products secreted by Bacillus species
include enzymes, heterologous proteins, antibiotics, purine
nucleotides, poly-c-glutamic acid and D-ribose (
Schallmey
et al., 2004
). Recently, a saturated branched-chain fatty
acid, 13-methyltetradecanoic acid, other than isoflavonoids
and other known compounds is isolated from soy
fermen-tation products, and it exhibits significant antitumor effects
by induction of apoptosis (
Yang et al., 2000
). Our previous
experiment revealed that SC-1 contains total phenolic
com-pounds (35.7 mg gallic acid equivalents/g dw), folic acid
(8.4 ng/g dw), pantothenic acid (0.4 ng/g dw), vitamin B
6(0.9 mg/g dw) and flavonoids (9.8 lg/g dw). In addition
to the protective effects of flavonoids and phenolic
com-pounds against carcinogenesis, the chemopreventive
abili-ties of folic acid (
Levin, 1999; Cao et al., 2005
) and
vitamin B6 (
Komatsu et al., 2001; Komatsu et al., 2002
)
have recently been reported.
In conclusion, SC-1 serves as antitumor agents to inhibit
the growth and clonogenesity of Hep 3B cells by induction
of apoptosis. Molecular mechanism includes activation of
caspase 8 and caspase 3, and increase of nuclear cleaved
PARP and DFF40 expression. Increase of tBid, Bak and
Bax on the mitochondria, decrease of mitochondrial
anti-apoptotic Bcl-2 and Bcl-x
Lproteins, disruption of Dw
m,
release of mitochondrial apoptogenic proteins (cytochrome
c and Smac), and the activation of caspase 9 in
mitochon-drial pathway also participate in the process of SC-1
induced programmed cell death. Decrease of Ku70 and
COX-2 expression in Hep 3B cells further assists the
process of apoptosis.
Acknowledgement
This research was supported by a grant from National
Cheng Kung University Research and Development
Foun-dation (No.: 91S03).
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