Editorial Manager(tm) for Neurochemical Research Manuscript Draft
Manuscript Number: NERE2167R1
Title: Carnosic acid, a rosemary phenolic compound, induces apoptosis through reactive oxygen species-mediated p38 activation in human neuroblastoma IMR-32 cells
Article Type: Original
Keywords: Carnosic acid; apoptosis; reactive oxygen species; p38 kinase; human neuroblastoma IMR-32 cells
Corresponding Author: Chia-Wen Tsai
Corresponding Author's Institution: China Medical University First Author: Chia-Wen Tsai
Carnosic acid, a rosemary phenolic compound, induces apoptosis through reactive oxygen 1
species-mediated p38 activation in human neuroblastoma IMR-32 cells 2
3
Chia-Wen Tsai．Chia-Yuan Lin．Hui-Hsuan Lin．Jing-Hsien Chen 4
5
C. W. Tsai ()．C. Y. Lin 6
Department of Nutrition, China Medical University, 91, Hsueh-Shih Rd, Taichung 404, 7
Taiwan 8
e-mail address: [email protected] 9
10
H. H. Lin 11
School of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung, 12
Taiwan 13
Department of Medical Research, Chung Shan Medical University Hospital, Taichung, 14 Taiwan 15 16 J. H. Chen 17
Department of Nutrition, Chung Shan Medical University, Taichung, Taiwan 18
19
Running title: Carnosic acid induces apoptosis in neuroblastoma cells 20
Abbreviations CA, carnosic acid; JNK, c-Jun NH2-terminal kinase ; ERK, extracellular
21
signal-regulated kinase ; MAPK, mitogen-activated protein kinase; MTT, 3-(4, 22
5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolim bromide; NAC, N-acetylcysteine; PARP, 23
poly(ADP-ribose) polymerase; PI, propidium iodide; ROS, reactive oxygen species. 24
*Manuscript
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Abstract Carnosic acid (CA), a rosemary phenolic compound, has been shown to display
26
anti-cancer activity. We examined the apoptotic effect of CA in human neuroblastoma 27
IMR-32 cells and elucidated the role of the reactive oxygen species (ROS) and 28
mitogen-activated protein kinase (MAPK) associated with carcinogenesis. The result 29
indicated that CA decreased the cell viability in a dose-dependent manner. Further 30
investigation in IMR-32 cells revealed that cell apoptosis following CA treatment is the 31
mechanism as confirmed by flow cytometry, hoechst 33258, and caspase-3/-9 and 32
poly(ADP-ribose) polymerase (PARP) activation. Immunoblotting suggested a 33
down-regulation of anti-apoptotic Bcl-2 protein in the CA-treated cells. In flow cytometric 34
analysis, CA caused the generation of reactive oxygen species (ROS); however, pretreatment 35
with the antioxidant N-acetylcysteine (NAC) attenuated the CA-induced generation of ROS 36
and apoptosis. This effect was accompanied by increased activation of p38 and by decreased 37
activation of extracellular signal-regulated kinase (ERK) as well as activation of c-Jun 38
NH2-terminal kinase (JNK). Moreover, NAC attenuated the CA-induced phosphorylation of
39
p38. Silencing of p38 by siRNA gene knockdown reduced the CA-induced activation of 40
caspase-3. In conclusion, ROS-mediated p38 MAPK activation plays a critical role in 41
CA-induced apoptosis in IMR-32 cells. 42
Keywords Carnosic acid ˙ apoptosis ˙ reactive oxygen species ˙ p38 kinase˙human
43
neuroblastoma IMR-32 cells 44 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Introduction
45
Neuroblastoma, which is derived from cells of the sympathetic nervous system, is the most 46
common solid extracranial neoplasm in children [1]. Neuroblastoma is a pediatric tumor that 47
accounts for 15% of childhood cancer deaths and has a poor prognosis in children after 1 year 48
of age [2]. Despite aggressive multimodal therapies, advanced neuroblastoma often acquires 49
drug resistance and metastasizes [3]. Disruption of the apoptosis machinery plays an 50
important role in the drug resistance of neuroblastomas. Many chemopreventive agents take 51
effect by inducing apoptosis of neuroblastoma cells [4]. 52
Apoptosis is characterized by morphological changes such as cell membrane blebbing, 53
cell shrinkage, nuclear condensation, and formation of apoptotic bodies [5]. Activation of 54
caspase is generally considered a hallmark of apoptotic cell death. The active caspase-9 55
recruits and activates procaspase-3, generating a fragment that activates the mitochondrial 56
pathway. The DNA repair enzyme poly(ADP)-ribose polymerase (PARP) is shown to be 57
cleaved by caspase-3 and as a result becomes incapable of responding to DNA damage during 58
apoptosis [6, 7]. 59
Recent studies have suggested that reactive oxygen species (ROS) may play an important 60
role during apoptosis induction [8]. Many stimulants such as cigarette smoke, anticancer 61
drugs, UV irradiation, and chemopreventive agents prompt cells to produce ROS. ROS induce 62
a number of events in mediating apoptosis, including mitogen-activated protein kinases 63
(MAPKs) signal transduction pathways [9]. Activated MAPKs play key roles in activating 64
transcription factors and downstream kinases, leading to the induction of immediate-early 65
gene expression and subsequent changes in other cellular processes [10]. The MAPKs are 66
composed of several subfamilies, including the c-Jun NH2-terminal kinase (JNK),
67
extracellular signal-regulated kinase (ERK), and p38 kinase. ERK and JNK are activated 68
through receptor-mediated signaling stimuli and are associated with cell proliferation, 69 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
differentiation, and survival [11-13]. The p38 pathway is generally activated by stress agents 70
and is implicated as a key regulator of stress-induced apoptosis in different cell types [14]. 71
Rosemary (Rosmarinus officinalis), a commonly herb or spice, has been reported to 72
possess a number of therapeutic applications in folk medicines. The rosemary phenolic 73
compounds, in particular carnosic acid (CA) , carnosol, and rosemarinic acid, have some 74
biological properties such as antiinflammatory, antioxidative, antiviral, and anticarcinogenic 75
activities [15-18]. CA has been shown to inhibit lipid peroxidation [16] and to protect red 76
cells against oxidative hemolysis [19]. Recently, interest has been growing in the 77
anticarcinogenic properties of CA. Evidence has suggested that the arresting of human 78
colonic adenocarcinoma Caco-2 cells in the G2/M phase by CA was shown to be caused by 79
reduction of cyclin A [20]. In 7,12-dimethylbenz(a)anthracene (DMBA)-induced hamster 80
buccal pouch carcinogenesis model, the chemopreventive potential of CA is probably due to 81
its modulating effect on carcinogen detoxification enzyme [21]. In addition, CA was shown to 82
cause apoptosis and enhance the anticancer activity of vitamin D3 in HL-60 human leukemia 83
cells [22, 23]. Moreover, the combined effect of CA and curcumin on apoptosis in acute 84
myeloid leukemia cells is associated with activation of caspase-8, caspase-9, and caspase-3 85
and the proapoptotic protein Bid [24]. Although CA is considered to be an anti-cancer agent, 86
its particular effects on neuroblastoma IMR-32 cells and the mechanisms involved remain 87
unknown. In this study, we investigated the apoptosis effects of CA in human neuroblastoma 88
IMR-32 cells. Moreover, we determined the involvement of ROS generation and the MAPK 89
pathway in these processes. 90 91 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Materials and methods
92
Chemical 93
Carnosic acid, leupeptin, aprotinin, Hoechst 33258 solution, paraformaldehyde, 94
phosphatase inhibitor, HEPES, sodium bicarbonate, EDTA, glycerol, Triton X-100, 95
dimethylsulfoxide (DMSO), sodium pyruvate, 3-(4, 5-dimethylthiazol-2-yl)-2,5- 96
diphenyltetrazolium bromide (MTT), rotenone, ascorbate, and N-acetylcysteine (NAC) were 97
obtained from Sigma Chemical Company (St. Louis, MO). MEM medium, L-glutamine, 98
nonessential amino acids, trypsin, sodium bicarbonate, and penicillin-streptomycinsolution 99
were obtainedfrom Gibco Laboratory (Grand Island, NY). Fetal bovine serum was purchased 100
from Hyclone (Logan, UT). 101
102
Cell culture 103
Human neuroblastoma IMR-32 cells were purchased from Bioresources Collection and 104
Research Center (BCRC, Taiwan). IMR-32 cells were grown in MEM medium supplemented 105
with 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 0.1 mM nonessential amino acids, 1.0 106
mM sodium pyruvate, 1105 unit/L penicillin, 100 mg/L streptomycin, and 10% fetal bovine 107
serum. Cells were incubated at 37C in a humidified atmosphere of 5% CO2 and 95% air. For
108
all studies, cells between passages 3 and 10 were used. IMR-32 cells were plated on 35-mm 109
plastic tissueculture dishes (Corning, NY) at a density of 0.7×106 cells per dish or on 60-mm 110
plastic tissueculture dishes at a density of 2.5×106 cells per dish for Western blot analysis, 111
and the dishes weretreated until 70% confluence was reached. Cells were changed to fresh 112
culture medium containing 2.5% fetal bovine serum for 12 h before CA treatment. Different 113
concentrations of CA in 2.5% fetal bovine serum culture medium were then added, and the 114
cells were incubated for the indicated times. Cells treated with 0.1% DMSO alone were 115
regarded as controls. For antioxidant treatments, NAC at a concentration 2 mM and ascorbate 116 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
at a concentration 1 mM were added 1 h before CA treatment. 117
Cell viability 118
IMR-32 cells were plated on 35-mm plastic tissueculture dishes at a density of 0.7×106 cells 119
per dish.Cell viability was determined by the MTT assay. Cells were stimulated with 5, 10, 120
20, 30, and 40 μM of CA for 24 h. MTT solution (5 mg/mL) was added to each dish and the 121
dishes were incubated for 2 h. The formazan product was dissolved by the addition of 1 mL 122
isopropanol to each dish with shaking for 10 min. Absorbance was detected at 570 nm by use 123
of a microplate reader (Bio Rad, Japan). 124
125
Hoechst 33258 staining 126
IMR-32 cells were treated with 5, 10, 20, 30, and 40 μM CA for 24 h. After being washed 127
with phosphate-buffered saline, the cells were fixed with 3.7% paraformaldehyde (pH 7.4) for 128
50 min. Subsequently, Hoechst 33258 nuclear dye was added to a final concentration 5 μg/mL 129
for 1 h at 25C in the dark. Morphological changes were observed by using a fluorescence 130
microscope. 131
132
Annexin V and propidium iodide (PI) staining 133
IMR-32 cells were exposed to 0.1% DMSO or 30 μM CA for 12, 24, 36, 48, and 60 h. The 134
Annexin V-FITC apoptosis detection kit (Becton Dickinson, San Diego, CA) was used 135
according to the manufacturer’s instructions. Following treatment, cells were harvested by 136
trypsinization and washed with warm phosphate-buffered saline, centrifuged at 1,500 x g for 5 137
min at 25C, and resuspended in 100 μl of 1X binding buffer [10 mM HEPES/NaOH (pH 7.4), 138
140 mM NaCl, and 2.5 mM CaCl2]. Then Annexin-V FITC and PI were added for 15 min in
139
the dark and finally 400 μL of 1X binding buffer was added. Samples were then immediately 140
analyzed by use of a flow cytometer (Becton Dickinson, Heidelberg, Germany). Acquisition 141 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
gates of the cells and a minimum of 10,000 events were collected for each sample. 142
Western blot analysis 143
IMR-32 cells were washed with cold phosphate-buffered saline and were then harvested in 144
lysis buffer (25 mM Tris-HCl, 150 mM NaCl, 0.5 % Triton X-100, 10% glycerol, 2 mM 145
EDTA, 1 mM PMSF, 1 μg/mL leupeptin, 1 μg/mL aprotinin, and phosphatase inhibitor). 146
Lysates were centrifuged at 14,000 x g for20 min at 4°C. Protein concentrations were 147
measured with a Coomassie plus protein assay reagent kit (Pierce, Rockford, IL). Thirty 148
micrograms of protein from each sample was appliedto 12.5% SDS-PAGE gels and was 149
electrophoretically transferred to polyvinylidene fluoridemembranes (Millipore, Bedford, 150
MA). The nonspecific binding sites on the membranes were blocked at 4°C overnight with 50 151
g/L nonfat dry milkin 25 mM Tris/150 mM NaCl buffer, pH 7.4. The blots were then 152
incubated with primary antibodies against procaspase-3, and -9 or cleaved caspase-3, and -9 153
or cleaved PARP ( all purchased from Cell Signaling Technology, Beverly, MA); β-tubulin 154
(purchased from Sigma Chemical Company, Louis, MO); JNK1, ERK1/2, phospho-JNK1, or 155
phospho-ERK1/2 (all from Santa Cruz Biotechnology, Inc., Santa Cruz, CA); p38 (purchased 156
from Cell Signaling Technology, Beverly, MA); or phospho-p38 (purchased from Abcam, 157
Cambridge, UK) overnight at 4°C and were subsequently incubated with horseradish 158
peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG. The bands were detected 159
by using an enhanced chemiluminescence kit (all purchased from Perkin Elmer Life Science, 160
Boston, MA). 161
162
Measurement of ROS generation 163
Measurement of intracellular ROS production was made by using the peroxide-sensitive 164
fluorescent probe 2,7-dichlorofluorescin diacetate (DCF-DA) (Molecular Probes Inc., Eugene, 165
OR) as described previously [25].In addition, mitochondrial was measured using MitoSOX 166 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
Red (Invitrogen, Carlsbad, CA). After reaching 90% confluence, cells were changed to fresh 167
culture medium containing 2.5% fetal bovine serum for 12 h before CA treatment. Cells were 168
changed to fresh culture medium containing 2.5% fetal bovine serum and 30 μM CA for 1, 3, 169
6, and 9 h. For examining the antioxidant effect, cells were pretreated with 2 mM NAC or 170
1mM ascorbate for 1 h and were then co-cultured with 30 μM CA for 6 h. An amount of 5 171
µM DCF-DA or 5 μM MitoSOXTM red were then added to the medium for 45 min before the 172
termination of CA treatment. DCF and MitoSOXRedfluorescence were measured in a flow 173
cytometer (Becton Dickinson, Heidelberg, Germany). 174
175
Transient transfection of small RNA interference 176
IMR-32 cells were seeded at a density of 0.7 ×106 cells/dish in a 35-mm plastic tissueculture 177
dish. When 80% confluence was reached, for p38 small interfering RNA (siRNA) transfection, 178
the cells were transfected with p38-siRNA (100 nM) or nontargeting control siRNA by using 179
the DharmaFECT® siRNA transfection reagent according to the manufacturer’s instruction 180
(all from Thermo Fisher Scientific, Lafayette, CO) for 12 h. The sense sequences of these p38 181
siRNAs were as follows: 1) 5’-GGACCUCCUUAUAGACGAA-3’, 2) 182
5’-GCACACUGAUGACGAAAUG-3’, 3) 5’-ACACUCGGCUGACAUAAUC-3’, and 4) 183
5’-GAAUGUGAUUGGUCUGUUG-3’. Twelve hours after transfection, the cells were 184
changed to fresh culture medium containing 2.5% fetal bovine serum and 30 μM CA for 12 h 185
or 24 h and protein expression was examined by Western blot analysis. 186
187
Statistical analysis 188
Statistical analysis was performed with commercially available software (SAS Institute 189
Inc,Cary, NC). Data were analyzed by means of one-way ANOVA, and the significant 190
difference among treatment means was assessed by use of Tukey’s test. Differences between 2 191 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
groups were assessed using Student’s t test. Differences were considered significant at P 192 <0.05. 193 194 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
Results
195
CA inhibited cell viability in IMR-32 cells 196
The chemical structure of CA is shown in Fig. 1. First, we investigated the effect of CA 197
treatment on the viability of human neuroblastoma IMR-32 cells. In cells exposed to 5, 10, 20, 198
30, and 40 μM CA for 24 h, cell viability was reduced in a dose-dependent manner (P <0.05) 199
(Fig. 2). CA exhibited potent cytotoxic activity against neuroblastoma IMR-32 cells, with an 200
IC50 value of approximately 30 μM.
201 202
Effect of CA on cell morphology of IMR-32 cells 203
To determine whether the reduced cell viability was due to apoptosis, IMR-32 cells were 204
stained with Hoechst 33258. In the control group, the IMR-32 cells were homogeneously 205
stained (Fig. 3). Nuclear condensation and fragmentation were significantly increased in the 206
cells treated with 30 and 40 μM CA for 24 h. In addition, cells treated with CA were shown to 207
have apoptotic bodies by phase-contrast microscopy. 208
209
CA induced apoptosis in IMR-32 cells 210
To further confirm the apoptosis, the cells were examined by flow cytometric analysis using 211
double staining of Annexin V-FITC and PI. As shown in Fig. 4, the apoptotic cells were 212
observed in IMR-32 cells treated with CA for 24 h. In the cells treated with CA, the apoptotic 213
population increased gradually throughout the culture period. CA at 60 h increased the 214
apoptotic population by 3.5-fold compared with that of the control cells. 215
216
CA induced the expression of apoptosis regulatory proteins 217
To clarify the mechanism of CA-induced apoptosis, we examined changes in the caspase 218
family proteins and anti-apoptotic protein Bcl-2 by Western blot analysis. CA significantly 219 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
reduced procaspase-9 and -3 but markedly increased the cleaved forms of caspase-9, -3, and 220
PARP in a dose-dependent manner (Fig. 5). The ratio of cleaved to procaspase-9 and -3 was 221
increased in the CA-treated group (P <0.05). However, caspase-8 protein was not expressed 222
after cells were treated with CA (data not shown). The level of anti-apoptotic Bcl-2 protein 223
was reduced in cells treated with 30 and 40 μM CA. These results suggested that the induction 224
of cell death by CA mainly involved activation of the apoptotic mitochondrial pathway. 225
226
Generation of ROS in CA-induced apoptosis 227
We next explored whether ROS generation was involved in the CA-induced apoptosis of 228
IMR-32 cells. The results of the flow cytometry analysis using DCF-DA as a fluorescent ROS 229
indicator showed thatintracellular ROS level was gradually increased and reached a 230
maximum at 6 h and then decreased in the presence of CA (Fig. 6A). Compared with the 231
control group, there was a 1.6-fold increase at 6 h. Pretreatment with NAC reduced 232
CA-induced ROS generation by 39%. In addition, we further used the mitochondrial targeted 233
ROS probe-MitoSOXRedto confirm the ROS production.Increases of 2.3 and 2.5 foldin 234
MitoSOX Red fluorescence intensity were noted in cells cultured with CA androtenone (a 235
mitochondrial inhibitor), respectively, as compared with the control cells. Pretreatment with 236
ascorbate reduced CA-induced mitochondrial ROS generation by 21% (Fig. 6 B). 237
Immunoblots also revealed that CA induced the cleavage of caspase-9, caspase-3, and PARP 238
protein and reduced procaspase-9 and -3 proteins (Fig. 6 C).In contrast, both NAC and 239
ascorbate decreased the CA-induced cleavage of caspase-9, caspase-3, and PARP protein (Fig. 240
6 C). These findings suggested that the generation of ROS may play an important role in the 241
CA-induced apoptosis in IMR-32 cells. 242
243
Role of MAPKs in CA-mediated apoptosis 244 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
Activation of the MAPK cascades is considered to play a crucial role as a regulator of 245
apoptotic signaling pathways [9]. Therefore, we attempted to determine whether CA-induced 246
apoptosis was regulated by JNK1, ERK, and p38 kinase. As shown in Fig. 7 A, the activation 247
of p38 was notably increased after the cells were treated with CA for 12 and 24 h, and 248
returned to basal level after 36 and 48 h. However, the phosphorylation of ERK and JNK1 249
was decreased in a time-dependent manner (Fig. 7 B). Cells pretreatment with NAC 250
attenuated the activation of p38 by CA, and had little effect on the phosphorylation of ERK 251
and JNK1. These results suggested that the activation of p38 was mainly involved in the 252
CA-induced ROS generation. 253
To confirm the involvement of p38 activation in the CA-induced apoptosis, we used 254
knockdown of p38 by siRNA transfection. Immunoblots revealed that CA increased the 255
activation of p38 and caspase-3 (Fig. 8 A). With p38 siRNA, the cellular p38 level was 256
decreased (vs. si-control), which resulted in alleviation of the phosphorylation of p38 by CA. 257
The activation of caspase-3 expression by CA was then suppressed (Fig. 8 B). 258 259 260 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Discussion
261
Rosemary extracts have been widely investigated for their antiproliferativeand 262
anticarcinogenic properties [26, 27]. Application of rosemary extracts was shown to prevent 263
DNA damage and tumor formation by 7,12-dimethylbenz[a]anthracene in mouse skin and rat 264
mammary gland [26, 28]. The accumulated evidence supportsthat rosemary extracts inhibit 265
benzo[a]pyrene-induced genotoxicity in human bronchialcells, and the components of 266
rosemary, such as CA, carnosol, and rosmarinic acid, were responsible for this effect [29]. 267
Carnosol displays growth-inhibitory effects in human prostate cancer PC3 cells by G2-phase 268
cell cycle arrest [17]. Rosmarinic acid induces apoptosis and inhibits the proliferation of 269
human HCT115 colorectal cells via MAPK/ERK pathway [30]. In human colon 270
adenocarcinoma COLO 205 cells, the rosmanol extracted from rosemary is capable of 271
inducing apoptosis through both a mitochondria-mediated pathway and a receptor-mediated 272
pathway [31]. Recent studies have suggested that CA inhibits the proliferation of Caco-2 cells 273
by causing cell cycle arrest at the G2/M phase and induces apoptosis in human promyelocytic 274
leukemia HL-60 cells [20, 23]. Moreover, the combinatorial effect of CA and curcumin on 275
apoptosis in acute myeloid leukemia cells was associated with activation of caspase-9 and 276
caspase-3 and the pro-apoptotic protein Bid [24]. The results of the present study suggest that 277
CA induced the apoptosis of IMR32 neuroblastoma cells via the mitochondrial pathway. We 278
suggest that the generation of ROS by CA leads to activation of the p38 pathway, which 279
results in apoptosis. 280
Caspases are a family of cysteine proteases that play a central role during the executional 281
phase of apoptosis. Several chemotherapeutic drugs induce cell death through the 282
caspase-mediated apoptosis pathways. Activation of caspase-8 is via the extrinsic apoptosis 283
pathway, which is induced by triggering of the death receptors pathway. Our results indicated 284
that caspase-8 protein was not expressed after cells were treated with CA for 24 h (data not 285 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
shown). This finding is supported by the findings of others that some neuroblastoma cell lines 286
such as IMR-32 do not express caspase-8 protein [32]. Moreover, loss of caspase-8 287
expression has been reported in patients with highly malignant neuroblastoma [33]. In the 288
intrinsic apoptosis pathway, upon apoptotic stimulation, initiator caspases such as caspase-9 289
are cleaved and activated. The activated upstream caspases further process the downstream 290
executioner caspases, such as caspase-3 and -7, by cleaving them into large and small 291
subunits, thereby initiating a caspase cascade leading to apoptosis [7]. The Bcl-2 family 292
proteins, the pro-apoptotic Bax and the anti-apoptotic Bcl-2, regulate cell death by controlling 293
mitochondrial membrane permeability during apoptosis [34]. A decrease in the levels of Bcl-2 294
leads to the loss of mitochondrial transmembrane potential, a key event in the induction of 295
apoptosis, and opens mitochondrial permeability transition pores [35]. Isobavachalcone, a 296
chalcone constituent of Angelica keiskei, induces apoptotic cell death with caspase-3 and -9 297
activation and Bax upregulation in neuroblastoma IMR-32 and NB-39 cells [32]. Zn 298
deficiency triggers IMR-32 apoptotic death associated with the intrinsic pathway, which can 299
be a consequence of ERK inhibition and caspase-3 activation [36]. However, xanthoangelol, 300
another chalcone constituent of Angelica keiskei, induces apoptotic cell death by activation of 301
caspase-3 in neuroblastoma IMR-32 cells through a mechanism that does not involve 302
Bax/Bcl-2 signal transduction [37]. In the present study, both CA and rotenone induced 303
apoptotic cell death with Bcl-2 downregulation and caspase-9 and caspase-3 activation, 304
resulting in cleavage of PARP in IMR32 neuroblastoma cells (Fig. 5). Taken together, these 305
results indicate that the CA-induced cell death involved activation of the apoptotic 306
mitochondrial pathway. 307
Recent studies have indicated that cancer chemopreventive agents induce apoptosis in 308
part by the generation of ROS and the disruption of redox homeostasis [38]. The generation of 309
ROS induces mitochondrial cytochrome c release, in which sequential activation of caspase-9 310 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
and -3 occurs [39]. The induction of apoptosis by garlic diallyl disulfide is associated with the 311
production of ROS and activation of caspase-3 in Ca Ski cells [40]. In addition, surfactin 312
induces apoptosis in human breast cancer MCF-7 cells through a ROS-mediated 313
mitochondrial/caspase pathway [41]. In the present study, we monitored the change in cellular 314
redox status by the DCF-DA and MitoSOX Red cytofluorimetric assay. With CA treatment, 315
ROS production gradually increased and reached a maximum at 6 h and then decreased (Fig. 316
6 A) Moreover, this effect was reduced by pretreatment with NAC and ascorbate. This 317
suggests that the activation of the apoptosis caspase cascade can be explained, at least in part, 318
by a change in redox states caused by CA (Fig. 6 C). 319
MAPKs control many cellular events, including differentiation, proliferation, and 320
apoptosis [12, 14, 42]. JNK regulates serotonin-mediated proliferation and migration in 321
pulmonary artery smooth muscle cells [12]. Treatment of IMR-32 cells with CdSe-core 322
induces mitochondrial-dependent apoptotic processes by inhibiting ERK survival signaling 323
[13]. Xavier and co-workers presented that romarinic acid induces apoptosis and inhibits the 324
proliferation of human HCT115 colorectal cells via inhibition of the ERK pathway [30]. In 325
particular, p38 is known to play a critical role in the transmission of apoptotic signals [43]. 326
Indole ethyl isothiocyanate is thought to inhibit the cell proliferation and cell viability of 327
neuroblastoma SMS-KCNRthrough activation of p38 signaling [14]. The p38 MAPK 328
pathway is also critical for 5,5'-dibromodiindolylmethane-induced apoptosis to prevent oral 329
squamous carcinoma cells [42]. These findings agree with our results that IMR-32 cells 330
treated with CA activated p38 protein and down-regulated ERK1/2 and JNK protein (Fig. 7A). 331
Furthermore, the CA-induced activation of p38 through a ROS-dependent mechanism was 332
evidenced by inhibition of p38 phosphorylation by NAC (Fig. 7 B). Pretreatment with p38 333
siRNA attenuated the activation of p38 and caspase-3 by CA (Fig. 8 A and 8 B). These data 334
suggest that the p38 pathway played an important role in the generation of ROS by CA, which 335 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
induced apoptosis of IMR-32 cells. This explanation is similar by the finding that the 336
activation of the p38 signaling pathway by arachidonic acid and the resulting induction of 337
human leukemia U937 cell apoptosis are prevented by NAC [38]. 338
In conclusion, the results of the present study indicate that CA induces apoptotic cell 339
death though the mitochondrial pathway in human neuroblastoma IMR-32 cells. Moreover, 340
ROS-mediated phosphorylation of p38 could play a critical role in CA-induced apoptosis. 341 342 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Acknowledgment
343
This work was supported by China Medical University (CMU) (grant no. CMU97-246). 344 345 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
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466
Fig. 1 Chemical structure of carnosic acid (CA).
467
Fig. 2 Carnosic acid (CA) inhibited cell growth in human neuroblastoma IMR-32 cells.
468
IMR-32 cells were treated with 0.1% dimethylsulfoxide (DMSO) alone (control, －) or with 469
10, 20, 30, or 40 μM of CA for 24 h. Cell viability was assessed by using the 470
3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The level in the 471
control cells was set at 100%. Values are shown as the means±SD of four independent 472
experiments. Means without a common letter differ, P <0.05. 473
474
Fig. 3 Carnosic acid (CA) induces nuclear morphology changes in human neuroblastoma
475
IMR-32 cells. Nuclei were visualized with Hoechst 33258 staining. Cells were treated with 476
0.1% dimethylsulfoxide (DMSO) alone (control, －) or with 5, 10, 20, 30, or 40 μM of CA 477
for 24 h. Upper panels show the phase contrast image and lower panels show the fluorescent 478
image. Phase contrast and fluorescent images were obtained from the same view 479
(magnification, 200 x). Arrows indicate apoptotic cells. One representative image out of four 480
independent experiments is shown. 481
482
Fig. 4 Carnosic acid (CA) induces apoptosis in human neuroblastoma IMR-32 cells. Cells
483
were exposed to the medium with 0.1% dimethylsulfoxide (DMSO) alone (control, －) or 484
with 30 μM CA for 12, 24, 36, 48, and 60 h. Cell distribution was analyzed by using Annexin 485
V-FITC binding and propidium iodide (PI) uptake as described in the Materials and Methods. 486
FITC and PI fluorescence were measured by flow cytometry. In these dot graphs, Q1-1 487
indicates necrotic cells (Annexin V-/PI+), Q2-1 indicates late apoptotic cells (Annexin V+/PI+), 488
Q3-1 indicates viable cells (Annexin V-/PI-), and Q4-1 indicates early apoptotic cells 489 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
(Annexin V+/PI-). Bars are the percentage of early and late apoptotic cells. Values are 490
expressed as the means±SD of three representative experiments. Groups not sharing a 491
common letter differ significantly, P <0.05. 492
493
Fig. 5 Carnosic acid (CA) dose-dependently increased apoptotic regulatory proteins in human
494
neuroblastoma IMR-32 cells.Cells were treated with 0.1% dimethylsulfoxide (DMSO) alone 495
(control, －) or with 10, 20, 30, or 40 μM CA for 24 h to determine the protein levels. The 496
cleaved caspase/procaspase ratio relative to the control group (mean±SD) is shown. 497
Normalization of Western blots was ensured by β-tubulin. The level in control cells was 498
regarded as 1. Means without a common letter differ, P <0.05. One representative 499
immunoblot out of four independent experiments is shown. 500
501
Fig. 6 Carnosic acid (CA)-induced apoptosis is associated with the generation of intracellular
502
reactive oxygen species (ROS) in human neuroblastoma IMR-32 cells. Cells were cultured 503
with 0.1% dimethylsulfoxide (DMSO) alone (control, －) or with 30 μM of CA for 1, 3, 6, 504
and 9 h, or with 50μM of rotenone for 6 h . For examining the antioxidant effect, cells were 505
pretreated with 2 mM NAC or 1 mM ascrobate for 1 h and then co-cultured with CA for 6 h. 506
(a) DCF fluorescence and (b) MitoSOX Red fluorescence were measured by flow cytometry. 507
The level in the control cells was set at 1. Values are shown as the means±SD of four 508
independent experiements. Means without a common letter differ significantly, P <0.05. 509
*Different from CA or rotenone alone in control group, P <0.05. #Different from CA 510
co-cultured with ascrobate in CA alone group, P <0.05. (c) The expression of indicated 511
proteins was analyzed by Western blotting. One representative immunoblot out of four 512
independent experiments is shown. 513 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
514
Fig. 7 Effect of carnosic acid (CA) on the activation of ERK1/2, JNK1, and p38 in human
515
neuroblastoma IMR-32 cells. Cells were cultured with 0.1% dimethylsulfoxide (DMSO) 516
alone (control, －) or with 30 μM of CA for 12, 24, 36, and 48 h. (a) Activation of ERK1/2, 517
JNK1, and p38 was assessed by immunoblot analysis of the phosphorylated forms (P-) of the 518
mitogen-activated protein kinases in whole cell lysates. β-Tubulin was used as the loading 519
control. (b) The expression of indicated proteins was analyzed after incubation with CA for 12 520
h in the presence or absence of NAC, which was added to cells 1 h before CA treatment. One 521
representative immunoblot out of three independent experiments is shown. 522
523
Fig. 8 Carnosic acid (CA)-induced activation of caspase-3 was inhibited by p38-siRNA in
524
human neuroblastoma IMR-32 cells. Cells were transfected with p38-siRNA (si-p38) or 525
nontargeting control siRNA (si-control) for 12 h. The transfected cells were then treated with 526
30 μM of CA for 12 and 24 h. The activation of p38 and capase-3 were measured by Western 527
blotting. β-tubulin was used as the loading control. One representative immunoblot out of 528
three independent experiments is shown. 529 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
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