Running title: hispolon induces human hepatoma cell apoptosis 1
2
Hispolon Induces Apoptosis and Cell Cycle Arrest of Human Hepatocellular
3Carcinoma Hep3B Cells by Modulating ERK Phosphorylation
45
Guan-Jhong Huang*†, Jeng-Shyan Deng , Shyh-Shyun Huang†, and Miao-Lin Hu*‡§ 6
7
†
School of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, College of Pharmacy, 8
China Medical University, Taichung, Taiwan; 9
Department of Health and Nutrition Biotechnology, Asia University, Taichung 413, Taiwan 10
‡
Department of Food Science and Biotechnology, National Chung Hsing University, Kuo-Kuang 11
Road, Taichung, Taiwan 12
§
Institute of Nutrition, China Medical University, Taichung, Taiwan 13
14
*Corresponding author: 15
Miao-Lin Hu, Ph.D., Department of Food Science and Biotechnology, National Chung Hsing 16
University, 250 Kuo-Kuang Road, Taichung 402, Taiwan 17
Tel.: +886 4 2281 2363; Fax: +886 4 2287 6211 18
E-mail address: mlhuhu@dragon.nchu.edu.tw 19
20 21 22 23
ABSTRACT 1
Hispolon is an active phenolic compound of Phellinus igniarius, a mushroom that has recently 2
been shown to have antioxidant, anti-inflammatory, and anticancer activities. In this study, we 3
investigated the antiproliferative effect of hispolon on human hepatocellular carcinoma Hep3B cells 4
by using the MTT assay, DNA fragmentation, DAPI (40, 6-diamidino-2-phenylindole 5
dihydrochloride) staining and flow cytometric analyses. Hispolon inhibited cellular growth of Hep3B 6
cells in a time-dependent and dose-dependent manner, through the induction of cell cycle arrest at S 7
phase measured using flow cytometric analysis and apoptotic cell death, as demonstrated by DNA 8
laddering. Hispolon-induced S-phase arrest was associated with a marked decrease in the protein 9
expression of cyclins A, and E and cyclin-dependent kinases (CDKs) 2, with concomitant induction 10
of p21waf1/Cip1 and p27Kip1. Exposure of Hep3B cells to Hispolon resulted in apoptosis as 11
evidenced by caspase activation, PARP cleavage, and DNA fragmentation. Hispolon treatment also 12
activated JNK, p38 MAPK and ERK expression. Inhibitors of ERK (PB98095), but not those of JNK 13
(SP600125) and p38 MAPK (SB203580), suppressed hispolon-induced S-phase arrest and apoptosis 14
in Hep3B cells. These findings establish a mechanistic link between MAPK pathway, and 15
hispolon-induced cell cycle arrest and apoptosis in Hep3B cells. 16
17
KEYWORKS: Phellinus igniarius; apoptosis; Hep3B; caspase; mitochondria 18
INTRODUCTION 19
Hepatocellular carcinoma (HCC) is a lethal and one of the four most prevalent 20
malignancies in adults in Taiwan, China, and Korea. Several etiologic factors, 21
including exposure to aflatoxin B1, and infection with hepatitis B virus and hepatitis 22
C virus, have been classified as high-risk factors associated with HCC (1). Apoptosis 23
is important in the control of cell quantity during development and proliferation. The 24
mechanism of apoptosis is conserved from lower eukaryotes to mammals and exhibits 25
a network of tightly ordered molecular events that finally converge into the enzymatic 26
fragmentation of chromosomal DNA, driving a cell to death (2). Apoptosis involves 27
the activation of a family of caspases, which cleave a variety of cellular substrates that 28
contribute to detrimental biochemical and morphological changes (3). At least two 29
pathways of caspase activation for apoptosis induction have been characterized. One 30
is mediated by the death receptor, Fas. Activation of Fas by binding with its natural 31
ligand (Fas ligand) induces apoptosis in sensitive cells (4). Fas ligand 32
characteristically initiates signaling via receptor oligomerization and recruitment of 33
specialized adaptor proteins followed by proteolysis and activation of procaspase-8. 34
Caspase-8 directly cleaves and activates caspase-3, which in turn cleaves other 35
caspases (e.g., caspase-6 and -7) for activation (5).The other pathway, driven by Bcl-2 36
family proteins, which may be anti-apoptotic (Bcl-2 and Bcl-XL) or pro-apoptotic
(Bax, Bak, and Bid), regulates cell death by controlling the permeability of 38
mitochondrial membrane during apoptosis (6). Upon apoptosis, pro-apoptotic proteins 39
translocate to the mitochondria and accelerate the opening of mitochondrial porin 40
channels, leading to release of cytochrome c and thereby triggering the cascade of 41
caspase activation (7). The induction of apoptosis by natural products on malignant 42
cells validates a promising strategy for human cancer chemoprevention (8). 43
Phellinus linteus (Berk. & M.A. Curt.) (PL) is a mushroom that belongs to the
44
genus Phellinus and is commonly called “Sangwhang” in Taiwan. It is popular in 45
oriental countries and has been traditionally used as food and medicine. PL contains 46
many bioactive compounds, and is known to improve health and to prevent and 47
remedy various diseases, such as gastroenteric disorders, lymphatic diseases, and 48
cancer (9). Recently, a few pharmacological actions of PL have been elucidated. For 49
instance, PL suppresses cellular proliferation and it induces apoptosis in lung and 50
prostate cancer cells (10). The anticancer effects of PL have been demonstrated by the 51
inhibition of invasive melanoma B16-BL6 cells (11). PL has been found to inhibit the 52
growth, angiogenesis and invasive behavior of breast cancer cells via the suppression 53
of AKT phosphorylation (12). We recently reported that hispolon, a phenol compound
54
isolated from PL, anti-inflammatory (13) and antimetastatic effects (14). Others have
55
also shown that hispolon has antiproliferative and immunomodulatory activities (15).
However, there have been no reports on the antiproliferative effects of hispolon in 57
liver cancer cells. In this study, we investigated the anticancer effects of hispolon on 58
three different hepatoma cell lines, including J5, HepG2, and Hep3B cells. A major 59
difference of these three hepatoma cell lines lies in their invasive activities, i.e., J5 > 60
HepG2 =Hep3B, based on their expression levels of thyroid hormone b1 nuclear 61
receptor and nm23-H1 (16); the latter is a tumor metastatic suppressor gene that has 62
been identified in murine and human cancer lines (17-19). The purpose of this study 63
was to investigate the anticancer effect of hispolon and to provide scientific rationales 64
for using hispolon as chemopreventive and/or chemotherapeutic agents against liver 65
cancer. 66
67
MATERIALS AND METHODS 68
Chemicals. Dulbecco’s modified Eagle’s medium (DMEM), 3-(4, 69
5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT), and other chemicals 70
were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Trypsin–EDTA, fetal 71
bovine serum (FBS) and penicillin/streptomycin were from Gibco Life Technologies, 72
Inc. (Paisley, UK). Cell culture supplies were purchased from Costar (Corning, Inc., 73
Cypress, CA, USA). The antibody against Bax, Bcl-2, Fas, FasL, Bid, caspase 3, 74
caspase 8, caspase 9, MAPK/extracellular signal–regulated kinase (ERK) 1/2, c-Jun 75
NH2-terminal kinase (JNK)/stress-activated protein kinase, and p38 MAPK proteins
76
and phosphorylated proteins were purchased from Cell Signaling Technology 77
(Beverly, MA). Anti-cyclin A, anti-cyclin E, anti-CDK 2, anti-p27, anti-p21, and 78
anti-PARP mouse monoclonal antibody and horseradish peroxidase-conjugated goat 79
anti-mouse IgG antibody were purchased from Santa Cruz Biotechnology Co. (Santa 80
Cruz, CA). 81
82
Isolation and Characterization of hispolon from Fruiting Body of PL. The fruiting 83
body of PL (about 1.0 kg, air dry weight) was powdered, and extracted with 95% 84
EtOH 6 L at room temperature (3 times, 72h each). Extracts were filtered and 85
combined together, and then evaporated at 40 °C (N-11, Eyela, Japan) to dryness 86
under reduced pressure to give a dark brown residue (40 g). The yield obtained for PL 87
is about 4 %. The crude extract was suspended in H2O (1 L), and then partitioned with
88
1 L n-hexane (× 2), 1 L EtOAc (× 2) and 1 L n-butanol (× 2), successively. 89
Hispolon (Fig.1A) was purified from the EtOAc soluble portion (8 g) by a 90
bioassay-guid separation. A portion of the active EtOAc fraction was subjected to 91
silica gel chromatography using stepwise CHCl3-MeOH (9:1, 8:2, 1:1 v/v) as eluent.
92
Final purification was achieved by preparative HPLC (Spherisorb ODS-2 RP18, 5 μm 93
(Promochem), 250×25 mm, acetonitrie-H2O (83: 17 v/v), at a flow rate of 10 mL/min
94
and UV detection at 375nm). The identification of hispolon was performed by 95
comparing their physical spectral data with literature values (20). 96
97
Cell Culture. The hepatocarcinoma J5, HepG2, and Hep3B cell was purchased from 98
the Bioresources Collection and Research Center (BCRC) of the Food Industry 99
Research and Development Institute (Hsinchu, Taiwan). Cells were cultured in plastic 100
dishes containing Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 101
10% fetal bovine serum (FBS) in a CO2 incubator (5% CO2 in air) at 37 °C and
102
subcultured every 2 days at a dilution of 1:5 using 0.05% trypsin–0.02% EDTA in 103
Ca2+-, Mg2+- free phosphate-buffered saline (DPBS). 104
105
Assay of Cell Viability. The cells (2 x 105) were cultured in 96-well plate containing 106
DMEM supplemented with 10% FBS for 1 day to become nearly confluent. Then 107
cells were cultured with hispolon for 24, 48, and 72 h. Then, the cells were washed 108
twice with DPBS and incubated with 100 L of 0.5 mg/mL MTT for 2 h at 37°C 109
testing for cell viability. The medium was then discarded and 100 L 110
dimethylsulfoxide (DMSO) was added. After 30-min incubation, absorbance at 570 111
nm was read by a microplate reader. At least three repeats were done for each sample 112
to determine cell proliferation. Decolorization was plotted against the concentration of 113
the sample extracts, and the amount of test sample necessary to decrease 50% 114
absorbance of MTT (IC50) was calculated.
115
116
Assay of DNA Fragmentation. Apoptosis was determined by the presence of 117
internucleosomal DNA fragmentation (DNA laddering) after cell treated with 118
increasing dose of hispolon for 48 h, or treated with 45 M for 24, 48, and 72 h. Hep 119
3B cells were cultured in 24-well microtiter plates at a density of 2 x 106 cells/well (1 120
mL final volume). To extract genomic DNA, cells were harvested, washed with cold 121
10 mM Tris–HCl, pH 7.5, 100 mM NaCl, 2 mM EDTA, and lysed by adding 0.5% 122
SDS. Cell lysates were then incubated at 56C for 3 h in the presence of 100 g/mL 123
of proteinase K. DNA was purified by successive phenol/chloroform extractions and 124
the resultant aqueous phase was mixed with 3M sodium acetate (pH 5.2) and absolute 125
ethanol. The mixture was incubated at -20C overnight and the ethanol-precipitated 126
DNA was washed with 70% ethanol. Purified DNA was resuspended in 10 mM 127
Tris–HCl, pH 7.5, 1 mM EDTA and treated with 50 g/mL DNase-free RNase A for 128
1 h. Samples were resolved on a 1% agarose gel and stained with 0.5 g/mL ethidium 129
bromide before DNA was visualized with ultraviolet light (21). 130
131
DAPI (40, 6-diamidino-2-phenylindole dihydrochloride) Staining. Cells were 132
seeded onto a 12-well plate at a density of 5 x 104 cells/well before treating with drugs. 133
Hep3B cells were cultured with vehicle alone or 45 M hispolon in DMEM medium 134
for 24, 48, and 72 h. After the treatment, cells were fixed with 3.7% formaldehyde for 135
15 min, permeabilized with 0.1% Triton X-100 and stained with 1 g/mL DAPI for 5 136
min at 37 ºC. The cells were then washed with PBS and examined by fluorescence 137
microscopy (Nikon, Tokyo, Japan). 138
139
Flow Cytometric Analysis for Cell Cycle Distribution. Human hepatocellular 140
carcinoma Hep3B cells (1 x 106 cells) were suspended in a hypotonic solution (0.1% 141
Triton X-100, 1 mM Tris-HCl (pH 8.0), 3.4 mM sodium citrate, and 0.1 mM EDTA) 142
and stained with 50 g/mL of propidium iodide (PI). DNA content was analyzed with 143
a FACScan (Becton Dickinson, San Jose, CA). The population of cells in each phase 144
of cell cycle was determined using CellQuest PRO software (Becton Dickinson, San 145
Jose, CA). 146
147
Assay of Cell Apoptosis. Quantitative assessment of apoptosis was analyzed by an 148
Annexin V-FITC assay kit (BD Biosciences, San Jose, CA). Briefly, cells grown in 10 149
cm Petri dishes were harvested with trypsin and washed in PBS. Cells were then 150
resuspended in a binding buffer (10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl, 2.5 151
mM CaCl2) and stained with Annexin V-FITC and PI at room temperature for 15 min
152
in the dark. Cells were analyzed in an EPICS flow cytometer (Coulter Electronics) 153
within 1 h after staining. Data from 10,000 cells were detected for each data file. 154
Early apoptotic cells were defined as Annexin V-FITC-positive and PI-negative cells 155
(Annexin V+/PI- fraction) and late apoptosis or necrotic cells were defined as annexin 156
V+/PI+ cells. 157
158
Preparation of Whole-Cell Lysates. Hep3B cells (1 × 105 cells) were plated in a 159
100-mm Petri dish and were treated with various concentrations of hispolon. Hep3B 160
cells were washed twice with PBS and were scraped into a microcentrifuge tube. The 161
cells were centrifuged at 1,250g for 5 min, and the pellet was lysed with iced-cold 162
RIPA (Radio-Immunoprecipitation Assay) buffer (1% NP-40, 50 mM Tris-base, 0.1% 163
SDS, 0.5% deoxycholic acid, 150 mM NaCl, pH 7.5), to which was added freshly 164
prepared phenylmethylsulfonyl fluoride (10 mg/mL), leupeptin (17 mg/mL), and 165
sodium orthovanadate (10 mg/mL). After incubation for 5 min on ice, the samples 166
were centrifuged at 10,000g for 10 min, and then the supernatants were collected as 167
whole-cell lysates. The lysates were denatured and subjected to SDS-PAGE and 168
Western blotting. The protein content was determined with Bio-Rad protein assay 169
reagent using BSA as a standard. 170
171
Western Blotting Analysis. Whole-cell lysates proteins (30-50 μg of partially 172
purified protein) were mixed with an equal volume of electrophoresis sample buffer, 173
and the mixture was then boiled for 10 min. Then, an equal protein content of total 174
cell lysate from control, 0.2% DMSO, and hispolon-treated sample were resolved on 175
10-12% SDS-PAGE gels. Proteins were then transferred onto nitrocellulose 176
membranes (Millipore, Bedford, MA) by electroblotting using an electroblotting 177
apparatus (Bio-Rad). Nonspecific binding of the membranes was blocked with 178
Tris-buffered saline (TBS) containing 1% (w/v) nonfat dry milk and 0.1% (v/v) 179
Tween-20 (TBST) for more than 2 h. Membranes were washed with TBST three 180
times each for 10 min and then incubated with an appropriate dilution of specific 181
primary antibodies in TBST overnight at 4 °C. The membranes were washed with 182
TBST and then incubated with an appropriate secondary antibody (horseradish 183
peroxidase-conjugated, goat antimouse, or antirabbit IgG) for 1 h. After washing the 184
membrane three times for 10 min in TBST, the bands were visualized using ECL 185
reagents (Millipore, Billerica, MA). Band intensity on scanned films was quantified 186
using Kodak Molecular imaging (MI) software and expressed as relative intensity 187
compared with control. 188
189
Statistical Analysis. Values are expressed as means ± SD and analyzed using 190
one-way ANOVA followed by LSD Test for comparisons of group means. All 191
statistical analyses were performed using SPSS for Windows, version 10 (SPSS, Inc.); 192
a P value <0.05 is considered statistically significant. 193
194
RESULTS 195
Isolation of hispolon from PL and its Structural characterization. PL was isolated 196
via extensive chromatographic purification of the ethyl acetate-soluble fraction of the 197
dried fruiting body. The chemical structure of the purified yellow powder was 198
elucidated by NMR spectroscopy and mass spectrometry studies and was identified as 199
hispolon. 200
201
Inhibitory Effects of Hispolon on Tumor Cell Growth. To examine whether 202
hispolon would alter malignant proliferation, inhibitory effects on the growth of J5, 203
HepG2, and Hep3B tumor cells were determined by a MTT colorimetric assay. As 204
shown in Fig. 1, hispolon inhibited cellular growth of J5, HepG2, and Hep3B cells in 205
a time-dependent and dose-dependent manner, and treatment for 24, 48 and 72 h 206
induced marked inhibition of cellular growth. The IC50 values (50% cell growth
207
inhibitory concentration) at 72 h for human hepatoma cancer cells J5, HepG2, and 208
Hep3B cells were 54.53 ± 0.63, 87.59 ± 1.42, and 35.90 ± 1.10 M, respectively 209
(Table 1). As compared to J5 and HepG2 cells, hispolon seemed to have a stronger 210
death effect toward Hep3B liver cancer cells. The results indicate that hispolon was 211
more cytotoxic to Hep3B cells. 212
213
Effects of Hispolon on Nuclear DNA Fragmentation of Hep3B Cells. We assessed 214
the effect of hispolon on the induction of apoptosis in Hep3B cells by DNA 215
fragmentation assay. Hep3B cells treated with 22.5, 45, and 66.5 M hispolon for 48 216
h, or treated with 45 M for 24, 48 and 72 h showed that hispolon treatment resulted 217
in the formation of DNA fragments. Nucleosomal DNA fragmentation was observed 218
in cells treated with 45 M of hispolon for 0, 24, 48, and 72 h or treated with 22.5, 45, 219
and 66.5 M hispolon for 48 h (Fig. 2A). The profile for hispolon-induced apoptosis 220
closely correlated with its growth suppressive effect. Thus, growth suppression 221
induced by hispolon in Hep3B cells may be related to the induction of apoptosis. 222
Effects of Hispolon on Phenotypic Changes in Cell Nucleus. This study further 224
elucidated whether hispolon also induces DNA fragmentation and chromatin 225
condensation in Hep3B cells. Treatment with hispolon resulted in changes in nuclear 226
morphology, as demonstrated by DAPI staining. Condensation and fragmentation 227
were seen in cells 24, 48, and 72 h after 45 M of hispolon treatment (Fig. 2B). The 228
phenotypic characteristic of hispolon-treated Hep3B cells was also evaluated by 229
microscopic inspection of overall morphology. Apoptotic bodies were observed after 230
Hep3B cells were treated with hispolon for 24 h. Based on the above data, DNA 231
condensation and formation of apoptotic bodies indicated that hispolon-induced 232
Hep3B cell death was a typical apoptotic cell death. 233
234
Effects of Hispolon on Cell Cycle Distribution. Induction of apoptosis has been 235
reported to be a potentially promising approach for cancer therapy. Exhibition of the 236
biological phenomena (cell cycle redistribution, DNA fragmentation, and chromatin 237
condensation) represents the proceeding of apoptosis (22). The apoptotic effect of 238
hispolon was confirmed by flow cytometric analysis. As shown in Fig. 3A, 239
concomitant with the growth inhibitory effect, hispolon treatment induced a strong 240
S-phase arrest in a time-dependent manner. When Hep3B cells were incubated with 241
45 M of hispolon for 0, 6, 12, and 24 h, the relative percentage of cells staying at the 242
S phase were 17.31%, 43.95%, 70.98% and 70.89%, respectively (Fig. 3B). This 243
increase in the S-phase cell population was accompanied by a concomitant decrease in 244
the G0/G1 and G2/M phase cell populations. Meanwhile, the sub-G1 population was 245
slightly increased in cells exposed to 45 μM hispolon. These results indicated that 246
hispolon caused cell cycle arrest at the S phase, followed by apoptosis. 247
248
Effects of Hispolon on Cell Apoptosis. To further confirm and quantify the 249
apoptosis of Hep3B cells triggered by hispolon, cells were stained with both Annexin 250
V-FITC and PI, and subsequently analyzed by flow cytometry (5). Fig. 4 shows the 251
annexin V-FITC/PI analysis of Hep3B cells cultured with 45 M of hispolon for 0, 6, 252
12 and 24 h. Annexin V positive cells were considered as the relative amount of 253
apoptoic cells. Early apoptotic cells appeared in the annexin V+/PI− fraction, whereas 254
cells damaged by scraping appeared in the annexin V−/PI+ fraction, and late 255
apoptosis or necrotic cells were evident in the annexin V+/PI+ fraction. After 256
treatment with 45 M of hispolon for 0, 6, 12, and 24 h, the corresponding quantities 257
of necrosis and apoptosis were 1.2%, 5.4%, 14.4%, and 19.6%, respectively (Annexin 258
V+/PI+ fraction). 259
260
Hispolon Induces Apoptosis via Intrinsically- and Extrinsically-Mediated 261
Pathways. The effects of hispolon on the protein expression of Fas, FasL, 262
pro-caspase-8, and Bid in Hep3B cells are shown in Fig. 5A and 5B. Treatment of 263
Hep3B cells with hispolon (45 μM) for 0, 3, 6, 12, and 24 h resulted in significant 264
increases in the levels of Fas and FasL expression. Treatment with 45 μM hispolon for 265
24 h significantly decreased the expression levels of pro-caspase-8 and Bid by 48% 266
and 56%, respectively, as compared to those of the control. 267
The effects of hispolon on the protein expression of the Bcl-2 family and cytosolic 268
cytochrome c in Hep3B cells are shown in Fig. 6A and 6B. After treatment with 45 269
μM hispolon for 24 h, the level of pro-apoptotic protein expression of Bax was 270
increased by 187.7%, in comparison to the control. Hispolon treatment at 45 μM for 271
24 h significantly decreased the level of Bcl-2 (antiapoptotic protein) expression by 272
38% in comparison with the control. Cytochrome c release in the cytosolic fraction 273
following hispolon treatment was then investigated. Treatment with hispolon (45 μM, 274
24 h) resulted in a significant increase in the level of cytosolic cytochrome c 275
expression by 177%, as comparison to the control. A significant time-dependent shift 276
in the ratio of Bax to Bcl-2 was observed after hispolon treatment at 45 μM for 0-24 h 277
(Fig. 6B). 278
The effects of hispolon on the protein expression of pro-caspase-3, caspase-9, 279
and poly (ADP-ribose) polymerase (PARP) in Hep3B cells are shown in Fig.7A and 280
7B. The results show that exposure of Hep3B cells to hispolon (45 μM, 24 h) caused 281
the degradation of pro-caspase-3 and caspase-9, which generated a fragment of 282
caspase-9 and caspase-3. Hispolon treatment at 45 μM for 24 h significantly increased 283
the level of expression of cleaved PARP by 138%, as comparison to the control. The 284
results indicate that hispolon treatment causes a significant increase in the activity of 285
caspase-9 and caspase-3 and hispolon may have acted through initiator caspase-8 and 286
then executioner caspase-3 to increase the cleavage form of PARP. 287
288
Effects of Hispolon on the Expression of Cell Cycle Regulators Involved in 289
S-Phase Arrest. As shown by immunoblot analysis in Fig. 8A, hispolon (45 μM, 24 h) 290
treatment caused a time-dependent decrease in the expression levels of cell cycle 291
regulators including cyclin A, cyclin E and cyclin-dependent kinases CDK 2, which 292
may contribute to the cell cycle progression from G0/G1 to S-phase. Hispolon 293
treatment at 45 μM for 24 h significantly decreased the level of expression of cyclin A, 294
cyclin E, and CDK 2 by 48.3%, 61.2% and 42.2%, respectively, in comparison with 295
the control. Binding of cyclins to CDKs would form active kinase complexes, which 296
are regulated and inhibited by various CKDIs and growth suppressor genes such as 297
p21waf1/Cip1 and p27Kip1. As shown by immunoblot analysis in Fig. 8A, the 298
expression levels of p21waf1/Cip1 and p27Kip1 were up-regulated in a 299
time-dependent manner by hispolon treatment. Hispolon treatment at 45 μM for 24 h 300
significantly increased the level of expression of p21waf1/Cip1 and p27Kip1 by 156% 301
and 144% in comparison with the control. 302
303
Effects of Hispolon on MAPK Signaling Pathway. Studies have shown that the 304
MAPK signaling pathway plays an important role in the action of chemotherapeutic 305
drugs (23). Therefore, we determined whether the MAPKs were activated in 306
hispolon-treated Hep3B cells by Western blot analysis using specific antibodies 307
against the phosphorylated (activated) forms of the kinases. It was found that hispolon 308
treatment induced differential phosphorylation of JNK, ERK, and p38 MAPK in cells 309
exposed to 45 M hispolon (Fig. 9A and 9B). Phosphorylation of ERK was detected 310
as a sustained activation from 0 to 24 h, which decreased thereafter and reached the 311
control level at 24 h. Activation of p38 by hispolon was also observed as early as 3 h 312
after hispolon treatment, which peaked at approximately 24 h. A time course study 313
showed that JNK activation displayed a rapid onset after 3h of treatment, followed by 314
a progressive decline, returning to the basal level after 24 h. 315
To study the role of MAPK activation in hispolon-induced growth inhibition, we 316
examined the effects of specific MAPK inhibitors on overall cell death. The results of 317
the MTT assay showed that pretreatment with SP600125 (a JNK inhibitor) or 318
SB203580 (a p38 inhibitor) had no effect on hispolon-induced cell death (Fig. 9C), 319
although these inhibitors reduced the phosphorylation of their target kinases. 320
The results suggested that JNK and p38 did not play important roles in regulating 321
cell death in Hep3B cells induced by hispolon. However, pretreatment with PD98059 322
(an ERK inhibitor) significantly decreased the extent of cell death induced by 323
hispolon (Fig. 9C). Only the ERK inhibitor PD98059 significantly blocked 324
hispolon-mediated cell death. These contradictory results imply that the drug actions 325
of hispolon indeed result from the complex interaction of many compounds and many 326
targeted molecules. These results suggested that activation of the ERK pathway was 327
involved in the apoptotic cell death of Hep3B cells induced by hispolon. 328
329
330
Discussion
331In the present study, we investigated the apoptosis of human hepatocellular 332
carcinoma cells induced by hispolon. Our data revealed that hispolon, a phenol 333
compound, acts directly on human hepatocellular carcinoma cancer cells to induce 334
cytotoxicity in a manner that causes apoptosis (Table 1). In breast and bladder cancer 335
cells, Lu et al. have reported that hispolon treatment for 72 h inhibits the cell viability 336
at IC50 values ranging from 20 M-40 M (24) as well as inhibits the growth of
human gastric cancer cells cells in a dose- and time-dependent manner, with an IC50
338
of 30 M at 72 h of incubation (25). 339
A flow cytometic analysis of PI -labeled cells shows that treating Hep3B cells 340
with hispolon (45 M) induced significant accumulation of cells in the S phase (Fig. 341
3A). The ratio of G0/G1 to S to G2/M phase in Hep3B cells at 0, 6, 12, and 24 h varied
342
significantly in the presence of 45 M (Fig. 3B). The G0/G1 cell population increased
343
to 70.89% in Hep3B cells treated with 45 M of hispolon for 24 h. Moreover, a 344
characteristic hypodiploid DNA content peak (sub-G1) was easily detected after
345
treatment with hispolon 45 M for 0, 6, 12, and 24 h. A significant increase in sub-G1
346
phase is indicative of induction of apoptosis. Uncontrolled cell proliferation is the 347
hallmark of cancer, and tumor cells have typically acquired mutations in genes that 348
directly regulate their cell cycle (26-27). 349
Inhibition of deregulated cell cycle progression in cancer cells is an effective 350
strategy to halt tumor growth (28). Cyclins, CDKs, and CDKIs play essential roles in 351
the regulation of cell cycle progression. CDKIs, such as p21waf1/Cip1 and p27Kip1, 352
are tumor suppressor proteins that downregulate the cell cycle progression by binding 353
with active cyclin-CDK complexes and thereby inhibiting their activities (29). 354
Chemopreventive agents usually cause apoptosis or cell cycle arrest at the G0/G1 or 355
G2/M phases. Relatively little is known about mechanisms that control progress 356
within the S-phase. It has been reported that hispolon elicits cell-cycle arrest at G2-M 357
phases in human breast and bladder cancer cells through the induction of CDKIs and 358
the inhibition of cyclins and CDKs (24). Although these results offer much insight for 359
the cell cycle arrest action of hispolon, the detailed molecular mechanisms remain to 360
be clarified. It has been reported that S-phase cell cycle arrest occurs with the loss of 361
Cdk2 activity due to reduced formation of active complex cyclin E/Cdk2 kinase (30). 362
We demonstrate here that hispolon-induced cell-cycle arrest was accompanied by 363
down-regulating the protein levels of cyclin A, cyclin E, and CDK2 and up-regulation 364
of p21 and p27 in Hep3B cells. It has been reported that S phase cell-cycle arrest 365
occurs with the loss of Cdk2 activity due to up-regulation of p21 and reduced 366
formation of active complex cyclin E/Cdk2 kinase (23). Our findings that hispolon 367
down-regulated cyclin A, cyclin E and CDK2 but up-regulated p21 and p27 suggest 368
that S-phase arrest is responsible for the cell-cycle-arresting effect of hispolon in 369
Hep3B cells. 370
Hispolon-induced apoptosis in Hep3B cells was also indicated by DNA laddering 371
(Fig. 2A) and DAPI positive staining (Fig. 2B). The induction of apoptosis stimulates 372
endonucleases, which catalyze the breakage of double-stranded DNA to form 373
fragments with oligonucleosome-length, resulting in a typical DNA electrophoresis 374
ladder that signifies apoptotic cell death (26). The apoptosis-inducing effect of 375
hispolon on Hep3B cells appeared to be directly proportional to its concentration. In 376
addition, the apoptosis-inducing efficacy of hispolon was found to be similar to its 377
anti-porliferative activity toward Hep3B cells. Furthermore, using annexin V−FITC to 378
identify apoptotic cells by binding to phosphatidyl serine and a red-fluorescent PI to 379
bind to nucleic acids of necrotic cells, the present study further demonstrated that 380
hispolon induced a significant and dose-dependent increase of annexin V+/PI+ 381
apoptotic cells (Fig. 4). 382
Caspases are believed to play crucial roles in mediating various apoptotic 383
responses. A model involving two different caspases (caspase-8 and -9) in the 384
mediation of distinct typesof apoptotic stimuli has been proposed (31). The cascade 385
led by caspase-8is involved in death receptor-mediated apoptosis such as theone 386
triggered by Fas.Ligation of Fas by Fas ligand results in sequential recruitmentof 387
FADD (Fas-associateddeath domain) and procaspase-8 to the death domain of Fas to 388
formthe death-inducing signaling complex, leading to cleavage ofprocaspase-8, with 389
the consequent generation of active caspase-8. Active caspase-8 in turn activates 390
downstream effecter caspases throughthe cleavage of Bid, committing the cell to 391
apoptosis (27). The present results suggest that hispolon may act through the initiator 392
caspase-8 and then the executioner caspase-3 to increase the cleavage form of PARP 393
for DNA fragmentation (Fig. 5A and 7A). 394
Many reports have pointed out that the ability of anticancer agents in inducing 395
apoptosis of tumor cells (such as Taxol) correlates with the ability of decreasing the 396
expression of Bcl-2 (32). In the present study, we showed that the expression of Bcl-2 397
decreased as the concentration of hispolon and the percentage of apoptotic Hep3B 398
cells increased. This inverse proportional relationship suggested that Bcl-2 may play a 399
preventive role in hispolon-mediated apoptosis of Hep3B cells. 400
Mitochondrial-dependent apoptosis is often through the activation of a pro-apoptotic 401
factor in the Bcl-2 family. Thus, one possible role of Bcl-2 in the prevention of 402
apoptosis is to block the release of cytochrome c from mitochondria (Fig. 6A). On the 403
contrary, increases in the expression of Bax and cytochrome c release were observed 404
during hispolon treatment in the present study. Bax is a pro-apoptotic protein that has 405
also been shown to induce cytochrome c release and caspase activation recently (33). 406
The above findings suggest that hispolon induces apoptosis in Hep3B cells through a 407
mitochondria-mediated pathway. 408
Several protein kinase pathways have been known to regulate cell proliferation 409
and survival. MAPKs, a family of serine-threonine protein kinases, have been 410
implicated in apoptosis and cell cycle regulation signaling in diverse cell models (23). 411
In general, JNK and p38 are activated by diverse stimuli such as oxidative stress, UV 412
irradiation, and osmotic shock and required for the induction of apoptosis. ERK plays 413
vital roles in cell growth and division and is generally considered to be a survival 414
mediator (34). In human HCC cell lines, multiple anticancer effects such as inhibition 415
of cellular proliferation as well as induction of cell cycle arrest and apoptosis have 416
been achieved by blocking ERK signaling (35). ERK inactivation observed in this 417
study may contribute to the S phase cell cycle arresting and apoptotic activities of 418
hispolon, which need to be investigated further. In addition, different MAPK 419
signaling pathways can be coordinately manipulated to enhance the efficacy of 420
anticancer drug. Cotreatment of anticancer drugs with ERK inhibitors has been found 421
to enhance anticancer effects. In our experiments, as shown in Fig. 9A, hispolon 422
markedly elevated the phosphorylated forms of JNK and p38 and reduced the 423
phosphorylated form of ERK1/2 in a dose dependent manner. Therefore, 424
hispolon-induced apoptosis in Hep3B involves mitochondria caspase pathways, 425
activation of JNK and P38 and inhibition of the ERK MAPK signaling. The use of 426
specific inhibitors revealed that JNK and p38 did not play important roles in 427
regulating cell death induced by hispolon in Hep3B cells. In this study, the MTT
428
method was used to examine the effects of specific MAPK inhibitors, as the same
429
method has often been used to examine the effects of specific MAPK inhibitors (23,
430
36, 37). In our previous reports, we also used the same approach to show that hispolon
431
modulates ERK phosphorylation (20, 23).
Our observations that hispolon induced S-phase arrest and p21 overexpression
433
are in agreement with those of a previous report which shows that the transduction of
434
the p21 gene results in S-phase arrest (40). P21, an inhibitor of CDKs, directly inhibits
435
CDK2, CDK3, CDK4, and CDK6 activity. Overexpression of p21 usually leads to G1
436
or G2 arrest by inhibiting CDK activity. P21 can also directly inhibit DNA synthesis
437
by binding to proliferating cell nuclear antigen (PCNA) (41). The expression of p21
438
can be regulated at the transcriptional, post-transcriptional, or post-translational levels
439
by p53-dependent and -independent mechanisms (5). Indeed, we found that
440
suppression of ERK activation attenuated hispolon-mediated induction of p21
441
expression and S-phase arrest. Although ERK and p21 are likely to play a role in
442
hispolon-mediated S-phase arrest, it is possible that some other molecules that were
443
not examined here may also be involved in hispolon-mediated S-phase arrest.
444
In conclusion, this study has provided mechanistic insights into how hispolon 445
regulates the components of cell cycle progression and apoptotic machinery to delay S 446
to G2/M transition and induces apoptosis in Hep3B cells. Our data imply the potential 447
of hispolon as a chemotherapeutic agent because many antbicancer drugs are known 448
to achieve their anticancer function by inducing apoptosis and/or cell cycle arrest in 449
susceptible cells. 450
ABBREVIATIONS USED 452
FBS, fetal bovine serum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium 453
bromide; PARP, poly(ADP-ribose) polymerase; MAPK, mitogen-activated protein 454
kinase; ERK, extracellular signaling-regulating kinase; JNK/SAPK, c-Jun N-terminal 455
kinase/ stress-activated protein kinase; 456
457
ACKNOWLEDGEMENTS 458
The authors want to thank the financial supports from the National Science Council 459
(NSC 97-2313-B-039 -001 -MY3), China Medical University (CMU) (CMU97-232 460
and CMU99-S-29) and and Taiwan Department of Health Clinical Trial and Research 461
Center of Excellence (DOH100-TD-B-111-004) and the Cancer Research Center of 462 Excellence (DOH100-TD-C-111-005). 463 464 LITERATURE CITED 465
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