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1

Contents lists available at ScienceDirect

Toxicology in Vitro
journal homepage: www.elsevier.com/locate/toxinvit

2 Thymoquinone from Nigella sativa was more potent than cisplatin in eliminating
3 of SiHa cells via apoptosis with down-regulation of Bcl-2 protein
4 Wei Keat Ng a, Latifah Saiful Yazan a,b,⇑, Maznah Ismail a,c
5 a
Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
6 b
Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
7 c
Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

8
9
a r t i c l e i n f o a b s t r a c t
1
2 1
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12 Article history: Thymoquinone (TQ), the active constituent of Nigella sativa or black cumin exhibited cytotoxic effects in 25
13 Received 28 January 2011 several cancer cell lines. In this study, the cytotoxicity of TQ in human cervical squamous carcinoma cells 26
14 Accepted 26 April 2011 (SiHa) was investigated. TQ was cytotoxic towards SiHa cells with IC50 values of 10.67 ± 0.12 and 27
15 Available online xxxx
9.33 ± 0.19 lg/mL as determined by MTT assay and trypan blue dye exclusion test, respectively, after 28
72 h of incubation. TQ was more cytotoxic towards SiHa cells compared to cisplatin. Interestingly, TQ 29
16 Keywords: was less cytotoxic towards the normal cells (3T3-L1 and Vero). Cell cycle analysis performed by flowcy- 30
17 Thymoquinone
tometer showed a significant increase in the accumulation of TQ-treated cells at sub-G1 phase, indicating 31
18 Nigella sativa
19 Cervical cancer
induction of apoptosis by the compound. Apoptosis induction by TQ was further confirmed by Annexin V/ 32
20 Apoptosis PI and AO/PI staining. Significant elevation of p53 and down-regulation of the anti-apoptotic Bcl-2 pro- 33
21 p53 tein was found in the treated cells, without any changes in the expression of the pro-apoptotic Bax pro- 34
22 Bcl-2 tein. In conclusion, thymoquinone from N. sativa was more potent than cisplatin in elimination of SiHa 35
23 cells via apoptosis with down-regulation of Bcl-2 protein. 36
Ó 2011 Published by Elsevier Ltd. 37

38
39
40 1. Introduction Hence, the search for new chemotherapeutic agents has refocused 60
on natural products, which led to the finding of some new bioac- 61
41 Although the mortality rate of cervical cancer has been gradu- tive compounds. 62
42 ally reduced after the introduction of PAP-smear programme, it is Two preventive vaccines have recently been licensed for use: 63
43 still one of the leading female malignancies worldwide (Liu et al., Gardasil and Cervarix. Gardasil is a quadrivalent vaccine containing 64
44 2009). It was estimated that more than 500,000 new cases of cer- recombinant L1 VLPs for HPV genotypes 6, 11, 16, and 18 whereas 65
45 vical cancer were reported in the world during 2007 (American the bivalent vaccine Cervarix contains L1 VLPs for HPV-16 and 66
46 Cancer Society, 2008). HPV-18 (Bosch and Harper, 2006; Lin et al., 2010). However, the 67
47 The problem of unacceptable adverse effects such as dose-re- vaccines will reduce, but not eliminate, the risk of cervical cancer, 68
48 lated toxicity, low specificity and the recurrence of patient tumors as at present they only target HPV-6, -11, -16, and -18 oncogenic 69
49 due to propagation of drug-resistant cells remains an inevitable genital types (Barr and Sings, 2008; Stanley, 2008). World Health 70
50 obstacle to the achievement in anti-cancer chemotherapy (De Organization revealed that HPV vaccines do not cure cancer; they 71
51 Mesquita et al., 2009; Ferguson et al., 2009). Even though cis- prevent some, but not all, HPV-related cancers (World Health 72
52 diamminedichloroplatinum(II) or cisplatin for instance, is highly Organization, 2009), and 30% HPV-related cervical cancers types 73
53 effective in treating cervical carcinoma (Fontanelli et al., 1992), it are not covered by the vaccines (Torre et al., 2007). Hence, a call 74
54 has been reported to have neurotoxic effects upon the peripheral for discovery of more effective agents to treat cancer is becoming 75
55 nervous system (PNS) and the central nervous system (CNS). The increasingly urgent (Jakopec et al., 2006). 76
56 most commonly observed side-effects include neurotoxicity, eme- Nigella sativa, a dicotyledon of the Ranunculaceae family, is an 77
57 sis, nephrotoxicity, ototoxicity and moderate myelosuppression. amazing herb with a rich historical and religious background 78
58 More rare side-effects include ophthalmological effects, seizures (Salem, 2005). Thymoquinone (TQ) or 2-isopropyl-5-methyle-1,4 79
59 and autonomic neuropathy (Mollman, 1999; Troy et al., 2000). benzoquinone (C10H12O2), with relative molecular mass of 164.2, 80
is one of the bioactive compounds of N. sativa (Shoieb, 2003). It Q3 81

⇑ Corresponding author at: Laboratory of Molecular Biomedicine, Institute of


has been shown to exert anti-neoplastic, anti-oxidant, anti-inflam- 82
Q1
Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. Tel.:
matory and anti-histamine effects (Gali-Muhtasib et al., 2006). 83
+60 3 89472308; fax: +60 389472336. In this study, the cytotoxicity of TQ from N. sativa towards hu- 84
E-mail address: latifah@medic.upm.edu.my (L.S. Yazan). man cervical squamous carcinoma cells (SiHa) was determined. 85

0887-2333/$ - see front matter Ó 2011 Published by Elsevier Ltd.


doi:10.1016/j.tiv.2011.04.030

Please cite this article in press as: Ng, W.K., et al. Thymoquinone from Nigella sativa was more potent than cisplatin in eliminating of SiHa cells via apop-
tosis with down-regulation of Bcl-2 protein. Toxicol. in Vitro (2011), doi:10.1016/j.tiv.2011.04.030
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86 The mode of cell death and involvement of p53, Bcl-2, and Bax 2.6. Determination of mode of cell death by acridine orange (AO)/ 139
87 were also investigated. propidium iodide (PI) dual staining 140

TQ-treated and -untreated cells were harvested after 72 h of 141


88 2. Materials and methods
incubation, and washed with ice-cold PBS. The pellets were resus- 142
pended in 5 lL of acridine orange (1 mg/mL) and 5 lL of propidium 143
89 2.1. Chemicals
iodide (1 mg/mL) (Cury-Boaventura et al., 2004). The morphologi- 144
cal changes of the stained cells were then observed by using a fluo- 145
90 Thymoquinone (TQ), cisplatin, tissue culture medium (EMEM),
rescence microscope (Leica, Germany). 146
91 trypan blue powder, MTT powder, propidium iodide (PI), acridine
92 orange (AO), and RNase were purchased from Sigma Chemicals
93 (St. Louis, USA). RPMI-1640, penicillin/streptomycin antibiotic, 2.7. Determination of mode of cell death by Annexin V/PI staining 147

94 Mycoplex™ foetal bovine serum (FBS) and trypsin–EDTA were pur-


95 chased from PAA Laboratories (Linz, Austria). Human Annexin V- Annexin V (AnnV) and PI staining was performed by using the 148

96 FITC kit, Human p53 ELISA kit and Human Bcl-2 ELISA kit were pur- Human Annexin V-FITC Apoptosis Detection Kit (Bender Medsys- 149

97 chased from Bender MedSystem (Vienna, Austria). Human Bax ELI- tems, Austria). After 72 h of incubation, TQ-treated and -untreated 150

98 SA kit was purchased from Assay Designs (USA). cells were harvested and washed twice with ice-cold PBS and 151
resuspended in 200 lL of 1 binding buffer containing Annexin 152
V and PI (1 mg/mL) for 10 min at 37 °C in the dark. The number 153
99 2.2. Cell culture of viable, early apoptotic, late apoptotic and necrotic cells was 154
quantified by flowcytometer (CyAn ADP, USA) (Vermes et al., 155
100 Human cervical squamous carcinoma cell (SiHa), Swiss mouse 1995). 156
101 embryo fibroblast cells (3T3-L1) and African green monkey kidney
102 epithelial (Vero) were purchased from the American Type Culture
2.8. Determination of the level of expression of p53, Bcl-2, and Bax 157
103 Collection (ATCC), USA. SiHa cells were grown in EMEM, while
104 3T3-L1 and Vero cells were maintained in RPMI-1640 culture med-
TQ-treated and -untreated cells were lysed. The level of expres- 158
105 ium. Both media were supplemented with 10% FBS and 1% antibi-
sion of p53, Bcl-2, and Bax was determined by colorimetric proce- 159
106 otics (100 IU/mL penicillin and 100 lg/mL streptomycin). The cells
dures by using Human p53 ELISA kit (Bender Medsystems, Austria), 160
107 were maintained at 37 °C in a humidified atmosphere of 5% CO2.
Human Bcl-2 ELISA kit (Bender Medsystems, Austria) and Human 161
Bax ELISA kit (Assay Designs, USA), respectively, according to the 162
108 2.3. Determination of cytotoxicity of TQ by MTT assay and trypan blue instructions provided by the manufacturer. Briefly, The p53, Bcl- 163
109 dye exclusion test 2, or Bax protein from the cell lysate specifically bound to the pri- 164
mary antibody and detected by Horseradish peroxidise (HRP) con- 165
110 The cells were treated with various concentrations of TQ or cis- jugated secondary antibody. HRP-conjugated secondary antibody 166
111 platin, ranging from 1.0 to 30 lg/mL in a 96-well plate for 24, 48 provided sensitive colorimetric. The expression of p53, Bcl-2, and 167
112 and 72 h. Control (without TQ or cisplatin) was also included Bax was determined by measuring the absorbance at 450 nm and 168
113 (Shier, 1991). MTT solution (5 mg/mL) was added and the plate the reference wavelength of 630 nm using a microplate reader 169
114 was incubated for 4 h. DMSO was then added to dissolve the (Opsys MR, USA). 170
115 dark-blue formazan crystals. The absorbance at 570 nm and the
116 reference wavelength of 630 nm was measured with a microplate 2.9. Statistical analysis 171
117 reader (Opsys MR, USA) (Mosmann, 1983).
118 For the trypan blue dye exclusion test, the treated and un- Data were analyzed with one-way analysis of variance (ANOVA) 172
119 treated cell suspensions were mixed with 0.4% trypan blue dye and Duncan’s multiple range test (DMRT) using Statistical Package 173
120 gently at the ratio of 1:1 (Renzi et al., 1993). The number of viable for Social Science (SPSS) version 17.0. All the data were expressed 174
121 and dead cell was counted with a haemocytometer under an in- as mean ± standard error of mean (SEM). Probability of p < 0.05 175
122 verted light microscope (Nikon, Japan). was considered significant. 176

123 2.4. Cell morphological studies 3. Results 177

124 TQ-treated (1.0, 3.0, 10, and 30 lg/mL) and -untreated cells 3.1. Cytotoxicity of TQ towards SiHa cells 178
125 were grown in a 6-well plate for 72 h. The changes in cell morphol-
126 ogy were observed under an inverted light microscope (Nikon, The dose–response graph obtained from both MTT assay and 179
127 Japan). trypan blue dye exclusion test shows a significant decrease in the 180
percentage of cell viability (p < 0.05) of SiHa cells treated with TQ 181
128 2.5. Cell cycle analysis or cisplatin at 1.0, 3.0, 10, and 30 lg/mL for 24, 48, and 72 h. Nev- 182
ertheless, TQ was significantly less cytotoxic towards the normal 183
129 TQ-treated and -untreated cells were harvested and washed cells (3T3-L1 and Vero) as compared to cisplatin (p < 0.05). 184
130 twice with ice-cold PBS. The cells were fixed with ice-cold 70% eth- The IC50 values as determined by the MTT assay and trypan 185
131 anol and incubated at 20 °C for 2 h. The cells were again washed blue dye exclusion test are shown in Table 1. 186
132 with ice-cold PBS and the supernatants were discarded. The pellets
133 were resuspended in a solution containing with 425 lL of PBS, 3.2. Cell morphological studies 187
134 25 lL of PI (1 mg/mL) and 50 lL of RNase A (1 mg/mL), and incu-
135 bated at 4 °C for 20 min. DNA content was analyzed by FACScan As shown in Fig. 1, TQ caused morphological changes in SiHa 188
136 flowcytometer (CyAn ADP, Denmark). The population of cells in cells. Reduction in the cell number was very obvious at higher 189
137 each cell-cycle phase was determined by using the Submit V3.4 concentration of TQ (30 lg/mL), with majority of the cells were 190
138 software (CyAn ADP, Denmark). detached from surface of the plate. 191

Please cite this article in press as: Ng, W.K., et al. Thymoquinone from Nigella sativa was more potent than cisplatin in eliminating of SiHa cells via apop-
tosis with down-regulation of Bcl-2 protein. Toxicol. in Vitro (2011), doi:10.1016/j.tiv.2011.04.030
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Table 1
IC50 values of TQ and cisplatin towards SiHa, 3T3-L1, and Vero cells at various incubation times as determined by using MTT assay and trypan blue dye exclusion test.

Cell line Incubation time (hour) IC50 values (lg/mL)


MTT assay Trypan blue dye exclusion test
TQ Cisplatin TQ Cisplatin
SiHa 24 19.27 ± 0.12d 24.10 ± 0.15⁄,e 17.90 ± 0.254 21.13 ± 0.22⁄,5
48 14.67 ± 0.30b 18.73 ± 0.09⁄,c 12.03 ± 0.092 15.93 ± 0.41⁄,3
72 10.67 ± 0.12a 12.37 ± 0.13⁄,b 9.33 ± 0.191 11.13 ± 0.29⁄,2
3T3-L1 72 18.23 ± 0.15a 2.83 ± 0.07⁄,b NP NP
Vero 72 12.00 ± 0.62c 3.37 ± 0.09⁄,d NP NP

Values were the means of three replicate samples. The data were presented as mean ± SEM.  were significant as compared to TQ while a, b, c, d, e, 1, 2, 3, 4, 5, a, b, c, and d
were significantly different (p < 0.05). NP, not performed.

Fig. 1. Morphological changes of SiHa cells treated with (B) 1.0 lg/mL, (C) 3.0 lg/mL, (D) 10 lg/mL (E) IC50 (10.7 lg/mL), and (F) 30 lg/mL of TQ for 72 h viewed under an
inverted light microscope. Control (A) was also included (200 magnification).

192 3.3. Cell cycle analysis 3.6. The level of expression of p53, Bcl-2 and Bax 216

193 Cell cycle analysis showed that TQ induced a dose-dependent Significant increase (p < 0.05) in the p53 expression was ob- 217
194 increase in the hypoploid cells (sub-G1 cells). Fig. 2 shows the frac- served in the cells treated with TQ at 1.0, 3.0, 10.0, IC50, and 218
195 tion of sub-G1 cells increased significantly (p < 0.05) from 6.72% 30 lg/mL after 72 h of incubation (Fig. 5). 219
196 (3.0 lg/mL) to 49.90% and 93.66% in treatment with, IC50 and Bcl-2 expression in the treatment with 1.0, 3.0, 10.0, IC50, and 220
197 30 lg/mL of TQ, respectively, as compared to the control (4.22%). 30 lg/mL of TQ after 72 h of incubation decreased significantly 221
(p < 0.05) as compared to the control. Activities of the anti-apopto- 222
tic protein Bcl-2 decreased with the increase in the concentration 223
198 3.4. Mode of cell death induced by TQ determined by AO/PI staining
of TQ (Fig. 6). Nevertheless, there were no significant changes 224
(p > 0.05) in the regulation of pro-apoptotic protein Bax in the 225
199 The mode of cell death induced by TQ was also investigated by
treatment with 1.0, 3.0, 10.0, IC50, and 30 lg/mL of TQ after 72 h 226
200 using the AO/PI fluorescence microscopy double staining. In the
of incubation (Fig. 6). 227
201 control and low concentration groups (1.0 and 3.0 lg/mL of TQ)
202 (Fig. 4b and c), uniformly green live cells with normal and large nu-
203 cleus were observed. In addition, reduction in the cell number was
4. Discussion 228
204 noted with the increase in TQ concentration. At higher concentra-
205 tion groups (IC50 and 30.0 lg/mL of TQ) (Fig. 4e and f), orange late
Table 1 shows that the IC50 values determined by both trypan 229
206 apoptotic cells with nuclear condensation were seen.
blue dye exclusion test and MTT assay decrease with the increasing 230
incubation time. In short, Both MTT assay and trypan blue dye 231
207 3.5. Mode of cell death induced by TQ determined by Annexin V/PI exclusion test showed that TQ was cytotoxic towards SiHa cells 232
in a dose- and time-dependent manner. 233
208 The percentage of apoptotic cells (AnnV+/PI for early apopto- TQ has previously shown significant cytotoxicity against several 234
209 sis and AnnV+/PI+ for late apoptosis) increased significantly after cancer cell lines such as human cervical adenocarcinoma (HeLa) 235
210 treatment with TQ (p < 0.05) at the concentration of 3.0, 10.0, cells (Latifah et al., 2009), canine osteosarcoma (COS31), its cis- 236
211 IC50, and 30.0 lg/mL for 72 h. On the other hand, the percentage platin-resistant variant (COS31/rCDDP), human breast adenocarci- 237
212 of viable cells decreased significantly (p < 0.05) at 10.0, IC50 and noma (MCF7), and human ovarian adenocarcinoma (BG-1) cells 238
213 30.0 lg/mL of TQ treatment after 72 h of incubation (Fig. 3). In (Shoieb et al., 2003). 239
214 all treatment, the percentage of necrotic cells was lower than the Even though both MTT assay and trypan blue dye exclusion test 240
215 one of apoptotic cells. can be used for the determination of cytotoxicity, there was a 241

Please cite this article in press as: Ng, W.K., et al. Thymoquinone from Nigella sativa was more potent than cisplatin in eliminating of SiHa cells via apop-
tosis with down-regulation of Bcl-2 protein. Toxicol. in Vitro (2011), doi:10.1016/j.tiv.2011.04.030
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Fig. 2. Cell cycle analysis of SiHa cells treated with (B) 10 lg/mL (C) IC50 (10.7 lg/mL), and (D) 30 lg/mL of TQ for 72 h as determined by flowcytometer. Control (A) was also
included. Each sample was run in triplicate. Data were presented as mean ± SEM.  were significantly different from the control (p < 0.05).

Fig. 3. AO/PI staining of SiHa cells treated with (B) 1.0 lg/mL, (C) 3.0 lg/mL, (D) 10 lg/mL, (E) IC50 (10.7 lg/mL), and (F) 30 lg/mL of TQ for 72 h, viewed under a fluorescence
microscope. Control (A) was also included. (200 magnification).

242 significant difference in the IC50 values obtained. It has been re- (Seth et al., 2005). Hence, it is believed that the use of multiple dif- 255
243 ported that different methods often yield considerably different ferent endpoint assays to define cytotoxicity can be more useful 256
244 values of cytotoxicity due to their different principles (Lee et al., and informative. 257
245 2005). Formazan accumulation in MTT assay directly reflected MTT assay and trypan blue dye exclusion test indicated that TQ 258
246 the mitochondrial activity in live cell, which was an indirect mea- was more potent towards SiHa cells as compared to cisplatin. Nev- 259
247 surement for the cell viability (Mosmann, 1983). However, the try- ertheless, normal cells (3T3-L1 and Vero) were less sensitive to- 260
248 pan blue dye exclusion test was based on the interaction of trypan wards TQ. 261
249 blue dye with the cell if the cell membrane is damaged, which indi- It is important for a potential anticancer lead to exhibit cytotox- 262
250 cates the cell viability directly (Cho et al., 2008). The study shows icity but such property should be specific for cancer cell only (Lai 263
251 that the IC50 values as determined by trypan blue dye exclusion et al., 2008). The ability of TQ to kill several types of tumors effec- 264
252 test were significantly lower than MTT assay. It may due to the tively without significant cytotoxicity to normal cells has been 265
253 condition that the cells are more vulnerable to damage when tryp- shown previously. Normal cell lines such as primary mouse kerat- 266
254 Q4 sinization was performed to count the stained and unstained cells inocytes and Madin–Darby canine kidney (MDCK) cell lines are 267

Please cite this article in press as: Ng, W.K., et al. Thymoquinone from Nigella sativa was more potent than cisplatin in eliminating of SiHa cells via apop-
tosis with down-regulation of Bcl-2 protein. Toxicol. in Vitro (2011), doi:10.1016/j.tiv.2011.04.030
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Fig. 4. The percentage of cell distribution of SiHa cells induced by TQ after 72 h incubation time as determined by Annexin V/PI staining, analyzed by flowcytometer. Values
were the means of three replicate samples. The data were presented as mean ± SEM.  and  were significantly different as compared to control ⁄p < 0.05 and ⁄⁄p < 0.01) and a
were significantly different as compared to viable cells (p < 0.05).

Fig. 5. Up-regulation of the expression of p53 in SiHa cells treated with TQ. Values are the mean of three independent experiments ±SEM.  were significantly different as
compared to control (p < 0.05). a, b, c, d, e, and f were significant different (p < 0.05).

268 reported to be resistant to the cytotoxic effects of TQ (Shoieb et al., cells showed accumulation of the cell population in the sub-G1 287
269 2003). In this study, Swiss mouse embryo fibroblast cells (3T3-L1) phase, which is an indication of the cleavage of nuclear DNA into 288
270 and African green monkey kidney epithelial (Vero) were used to multiple fragments and caused apoptosis (Han and Park, 2009). 289
271 assess the cytotoxicity of TQ towards normal cells. 3T3-Li cell line The indication of apoptosis was further confirmed by the Annexin 290
272 is recommended by US National Institute of Environmental Health V-FITC/PI and AO/PI. Both tests indicated that TQ was cytotoxic to- 291
273 Sciences (NIEHS), Interagency Coordinating Committee on the Val- wards SiHa cells through a mechanism that involves apoptosis. An- 292
274 idation of Alternative Methods (ICCVAM) to access basal cytotoxic- nexin V/PI analysis revealed that majority of the cells underwent 293
275 ity (Lai et al., 2008). Vero cells are homologous with human body apoptosis when treated with 10, IC50, and 30 lg/mL of TQ. Mean- 294
276 cells as it shares a common embryonic origin (mesoderm) with while, in the AO/PI analysis, cell treated with IC50 and 30 lg/mL 295
277 cells from human genital tract, and this line is non-tumorigenic showed the morphology of orange color nuclei (Fig. 4e and f), 296
278 but immortalized, allowing to culture cells for longer than normal which further confirmed that TQ induced late apoptosis in SiHa 297
279 cell line (Ferrari et al., 2009; Liao et al., 2010). In addition, the ratio- cells. 298
280 nale behind the use of 3T3-L1 and Vero cells rather than primary Annexin V/propidium iodide (AnnV/PI) staining is based on 299
281 cervical cancer cells is that these normal cells have be banked ability of the protein Annexin V to bind to phosphatidylserine 300
282 and well characterized, thus, avoiding the issue of lot-by-lot viabil- (PS) exposed on the outer membrane leaflet in apoptotic cells. In 301
283 ity, variations and adventitious agent contamination of the primary viable cells, PS is located in the inner membrane leaflet, but upon 302
284 cultures (Carosati et al., 2010). induction of apoptosis it is translocated to the outer membrane 303
285 Figs. 2–4 revealed that TQ induced apoptosis in SiHa cells in a leaflet and becomes available for Annexin V binding. The addition 304
286 dose-dependent manner. Cell cycle analysis of TQ-treated SiHa of PI enabled viable (AnnV /PI ), early apoptotic (AnnV+/PI ), late 305

Please cite this article in press as: Ng, W.K., et al. Thymoquinone from Nigella sativa was more potent than cisplatin in eliminating of SiHa cells via apop-
tosis with down-regulation of Bcl-2 protein. Toxicol. in Vitro (2011), doi:10.1016/j.tiv.2011.04.030
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Fig. 6. The expression of Bcl-2 and Bax protein in SiHa cells treated with TQ, evaluated by human Bcl-2 ELISA kit and human Bax ELISA kit, respectively. Values are the mean
of three independent experiments ±SEM.  were significantly different as compared to control (p < 0.05). a, b, c, d, e, f, and 1 were significant different (p < 0.05).

306 apoptotic (AnnV+/PI+) and necrotic (AnnV /PI+) cells to be distin- undergo apoptosis that is associated with increase in the gene 344
307 guished (Baskić et al., 2006). Whereas, in the AO/PI staining, and protein expression of p53 and inhibition of the anti-apoptotic 345
308 DNA-binding dye acridine orange (AO) and propidium iodide (PI) Bcl-2 protein. This indicates that the cytotoxic effect of TQ is med- 346
309 were used for the morphological detection of apoptotic and necro- iated by pro-apoptotic effects which are modulated by Bcl-2 pro- 347
310 tic cells. AO intercalates into the DNA giving it a green appearance. tein, and is linked to and dependent on p53 (Worthen et al., 1998). 348
311 Thus, viable cells have a bright green nucleus. PI is only taken up by Tumor suppressor gene p53 has been reported to be able to reg- 349
312 non-viable cells. This dye also intercalates into DNA, making it ap- ulate the expression of a number of downstream proteins, such as 350
313 pears orange. The live cells with intact membranes have a uniform Bax and Bcl-2 in response to DNA damage (Coutts and La Thangue, 351
314 green color in their nuclei. Early apoptotic cells have chromatin 2006; Roos and Kaina, 2006). High ratio of Bax to Bcl-2 can cause 352
315 condensation with bright green colored nuclei. Late apoptotic cells the permeabilization of the outer mitochondrial membrane, result- 353
316 have bright orange areas of condensed chromatin in the nucleus ing in the release of cytochrome c. Cytochrome c forms an apopto- 354
317 that distinguish them from necrotic cells, which have a uniform or- some that is composed of Apaf-1 and procaspase-9, resulting in the 355
318 ange color (Cury-Boaventura et al., 2004). activation of caspase-9. Caspase-9 activates the effector procaspas- 356
319 Programmed cell death (cell suicide) or apoptosis plays a vital es including caspase-3, -6, and -7 to carry out the process of apop- 357
320 role in many physiological and developmental stages, as well as tosis (Han and Park, 2009). Therefore, it is presumed that TQ 358
321 tissue homeostasis to control the cell number (Arepalli et al., induces DNA damage in SiHa cells, leading to the increase in the le- 359
322 2009). Disruption in apoptosis caused a major manifestation in dis- vel of p53 expression, and resulting in the regulation of the Bax to 360
323 eases such as cancer (Ray et al., 2006). Effect on the regulation of Bcl-2 ratio. 361
324 apoptosis and ability to manipulate the mechanism of pro- In conclusion, the findings demonstrate that cytotoxicity of TQ 362
325 grammed cell death may lead to new possible agents for cancer towards SiHa cells is associated with its ability to induce apoptosis 363
326 therapy (Tamatani et al., 2007). Therefore, induction of cell apopto- in the cells. The apoptosis induction by TQ in SiHa cells was in a 364
327 sis and targeting apoptotic pathways has emerged as an attractive p53-dependent manner, via down-regulation of anti-apoptotic 365
328 approach for treatment of cancer (Li et al., 2007). Many FDA ap- Bcl-2 protein. Interestingly, the cytotoxicity of TQ was more selec- 366
329 proved clinical available anti-cancer drugs such as paclitaxel (a tive towards cancerous cells, SiHa and less cytotoxic towards the 367
330 compound extracted from the Pacific yew tree, Taxus brevifolia), normal cells (3T3-L1 and Vero) as compared to cisplatin, suggest- 368
331 camptothecin (an alkaloid isolated from the Chinese tree, Camptot- ing TQ may be a potential agent for the management of cervical 369
332 heca acuminate) and genistein (a soy-derived isoflavone and phyto- cancer in future. 370
333 estrogen) have been found to induce apoptosis (Ding et al., 2009).
334 Perhaps, the ability of TQ to induce apoptosis in SiHa cells suggests
335 that TQ may be a potentially effective chemotherapeutic agent Conflict of interest 371
336 against cervical cancer.
337 Cytotoxicity of TQ in SiHa cells involved an elevation of the level None declared. 372
338 of the p53 activity and the Bax to Bcl-2 ratio, due to the significant
339 down-regulation of Bcl-2. Nevertheless, the Bax activity in TQ-trea-
340 ted cells was not changes even at the highest concentration of Acknowledgment 373
341 30 lg/mL.
342 Previous reports showed that TQ triggered human pancreatic This study was partly supported by the Research University 374
343 adenocarcinoma, uterine sarcoma, and leukemic cell lines to Grant Scheme (04/01/07/0136RU), Universiti Putra Malaysia. 375

Please cite this article in press as: Ng, W.K., et al. Thymoquinone from Nigella sativa was more potent than cisplatin in eliminating of SiHa cells via apop-
tosis with down-regulation of Bcl-2 protein. Toxicol. in Vitro (2011), doi:10.1016/j.tiv.2011.04.030
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Please cite this article in press as: Ng, W.K., et al. Thymoquinone from Nigella sativa was more potent than cisplatin in eliminating of SiHa cells via apop-
tosis with down-regulation of Bcl-2 protein. Toxicol. in Vitro (2011), doi:10.1016/j.tiv.2011.04.030