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Toxicology in Vitro xxx (2011) xxx–xxx 1
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Toxicology in Vitro
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Thymoquinone from Nigella sativa was more potent than cisplatin in eliminating of SiHa cells via apoptosis with down-regulation of Bcl-2 protein
Wei Keat Ng a, Latifah Saiful Yazan a,b,⇑, Maznah Ismail a,c
Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia c Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
a r t i c l e
i n f o
a b s t r a c t
Thymoquinone (TQ), the active constituent of Nigella sativa or black cumin exhibited cytotoxic effects in several cancer cell lines. In this study, the cytotoxicity of TQ in human cervical squamous carcinoma cells (SiHa) was investigated. TQ was cytotoxic towards SiHa cells with IC50 values of 10.67 ± 0.12 and 9.33 ± 0.19 lg/mL as determined by MTT assay and trypan blue dye exclusion test, respectively, after 72 h of incubation. TQ was more cytotoxic towards SiHa cells compared to cisplatin. Interestingly, TQ was less cytotoxic towards the normal cells (3T3-L1 and Vero). Cell cycle analysis performed by ﬂowcytometer showed a signiﬁcant increase in the accumulation of TQ-treated cells at sub-G1 phase, indicating induction of apoptosis by the compound. Apoptosis induction by TQ was further conﬁrmed by Annexin V/ PI and AO/PI staining. Signiﬁcant elevation of p53 and down-regulation of the anti-apoptotic Bcl-2 protein was found in the treated cells, without any changes in the expression of the pro-apoptotic Bax protein. In conclusion, thymoquinone from N. sativa was more potent than cisplatin in elimination of SiHa cells via apoptosis with down-regulation of Bcl-2 protein. Ó 2011 Published by Elsevier Ltd.
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Article history: Received 28 January 2011 Accepted 26 April 2011 Available online xxxx Keywords: Thymoquinone Nigella sativa Cervical cancer Apoptosis p53 Bcl-2
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1. Introduction Although the mortality rate of cervical cancer has been gradually reduced after the introduction of PAP-smear programme, it is still one of the leading female malignancies worldwide (Liu et al., 2009). It was estimated that more than 500,000 new cases of cervical cancer were reported in the world during 2007 (American Cancer Society, 2008). The problem of unacceptable adverse effects such as dose-related toxicity, low speciﬁcity and the recurrence of patient tumors due to propagation of drug-resistant cells remains an inevitable obstacle to the achievement in anti-cancer chemotherapy (De Mesquita et al., 2009; Ferguson et al., 2009). Even though cisdiamminedichloroplatinum(II) or cisplatin for instance, is highly effective in treating cervical carcinoma (Fontanelli et al., 1992), it has been reported to have neurotoxic effects upon the peripheral nervous system (PNS) and the central nervous system (CNS). The most commonly observed side-effects include neurotoxicity, emesis, nephrotoxicity, ototoxicity and moderate myelosuppression. More rare side-effects include ophthalmological effects, seizures and autonomic neuropathy (Mollman, 1999; Troy et al., 2000). Q1
⇑ Corresponding author at: Laboratory of Molecular Biomedicine, Institute of
Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. Tel.: +60 3 89472308; fax: +60 389472336. E-mail address: email@example.com (L.S. Yazan). 0887-2333/$ - see front matter Ó 2011 Published by Elsevier Ltd. doi:10.1016/j.tiv.2011.04.030
Hence, the search for new chemotherapeutic agents has refocused on natural products, which led to the ﬁnding of some new bioactive compounds. Two preventive vaccines have recently been licensed for use: Gardasil and Cervarix. Gardasil is a quadrivalent vaccine containing recombinant L1 VLPs for HPV genotypes 6, 11, 16, and 18 whereas the bivalent vaccine Cervarix contains L1 VLPs for HPV-16 and HPV-18 (Bosch and Harper, 2006; Lin et al., 2010). However, the vaccines will reduce, but not eliminate, the risk of cervical cancer, as at present they only target HPV-6, -11, -16, and -18 oncogenic genital types (Barr and Sings, 2008; Stanley, 2008). World Health Organization revealed that HPV vaccines do not cure cancer; they prevent some, but not all, HPV-related cancers (World Health Organization, 2009), and 30% HPV-related cervical cancers types are not covered by the vaccines (Torre et al., 2007). Hence, a call for discovery of more effective agents to treat cancer is becoming increasingly urgent (Jakopec et al., 2006). Nigella sativa, a dicotyledon of the Ranunculaceae family, is an amazing herb with a rich historical and religious background (Salem, 2005). Thymoquinone (TQ) or 2-isopropyl-5-methyle-1,4 benzoquinone (C10H12O2), with relative molecular mass of 164.2, is one of the bioactive compounds of N. sativa (Shoieb, 2003). It Q3 has been shown to exert anti-neoplastic, anti-oxidant, anti-inﬂammatory and anti-histamine effects (Gali-Muhtasib et al., 2006). In this study, the cytotoxicity of TQ from N. sativa towards human cervical squamous carcinoma cells (SiHa) was determined.
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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 apoptosis with down-regulation of Bcl-2 protein. Toxicol. in Vitro (2011), doi:10.1016/j.tiv.2011.04.030
and washed with ice-cold PBS. Reduction in the cell number was very obvious at higher concentration of TQ (30 lg/mL). Japan). 2. propidium iodide (PI).0. and Bax was determined by colorimetric procedures by using Human p53 ELISA kit (Bender Medsystems. Chemicals Thymoquinone (TQ). 48 and 72 h. in Vitro (2011). Bcl-2. Denmark).7. Toxicol. Determination of mode of cell death by Annexin V/PI staining Annexin V (AnnV) and PI staining was performed by using the Human Annexin V-FITC Apoptosis Detection Kit (Bender Medsystems. The number of viable and dead cell was counted with a haemocytometer under an inverted light microscope (Nikon. The p53.4% trypan blue dye gently at the ratio of 1:1 (Renzi et al. tissue culture medium (EMEM). Human Bcl-2 ELISA kit (Bender Medsystems. or Bax protein from the cell lysate speciﬁcally bound to the primary antibody and detected by Horseradish peroxidise (HRP) conjugated secondary antibody. et al. and 30 lg/mL for 24. ranging from 1. and Bax was determined by measuring the absorbance at 450 nm and the reference wavelength of 630 nm using a microplate reader (Opsys MR. Determination of mode of cell death by acridine orange (AO)/ propidium iodide (PI) dual staining TQ-treated and -untreated cells were harvested after 72 h of incubation.0. and 30 lg/mL) and -untreated cells were grown in a 6-well plate for 72 h. Bcl2. For the trypan blue dye exclusion test. Control (without TQ or cisplatin) was also included (Shier.05 was considered signiﬁcant.. The population of cells in each cell-cycle phase was determined by using the Submit V3. 10. USA). 1983). Human p53 ELISA kit and Human Bcl-2 ELISA kit were purchased from Bender MedSystem (Vienna. cisplatin.4 software (CyAn ADP.K. with majority of the cells were detached from surface of the plate. 139 140 141 142 143 144 145 146 88 89 90 91 92 93 94 95 96 97 98 147 148 149 150 151 152 153 154 155 156 99 100 101 102 103 104 105 106 107 157 158 159 160 161 162 163 164 165 166 167 168 169 170 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 171 172 173 174 175 176 123 124 125 126 127 177 178 179 180 181 182 183 184 185 186 128 129 130 131 132 133 134 135 136 137 138 187 188 189 190 191 Please cite this article in press as: Ng. TQ caused morphological changes in SiHa cells.1016/j. All the data were expressed as mean ± standard error of mean (SEM). Human Annexin VFITC kit.0 to 30 lg/mL in a 96-well plate for 24. The absorbance at 570 nm and the reference wavelength of 630 nm was measured with a microplate reader (Opsys MR. 1993). The pellets were resuspended in a solution containing with 425 lL of PBS. and Bax TQ-treated and -untreated cells were lysed. 2. acridine orange (AO). 2004). Model 5G The mode of cell death and involvement of p53. 2.. Austria).2. Bcl-2. / Toxicology in Vitro xxx (2011) xxx–xxx No. The morphological changes of the stained cells were then observed by using a ﬂuorescence microscope (Leica. the treated and untreated cell suspensions were mixed with 0. 1995). late apoptotic and necrotic cells was quantiﬁed by ﬂowcytometer (CyAn ADP. Thymoquinone from Nigella sativa was more potent than cisplatin in eliminating of SiHa cells via apoptosis with down-regulation of Bcl-2 protein. RPMI-1640. Cell morphological studies TQ-treated (1. 2. 3.3. penicillin/streptomycin antibiotic. respectively. Materials and methods 2. Bcl-2.TIV 2639 21 May 2011 2 86 87 W. and RNase were purchased from Sigma Chemicals (St. 10. and incubated at 4 °C for 20 min. USA). The number of viable. The expression of p53. 2. After 72 h of incubation. 2. and Bax were also investigated. SiHa cells were grown in EMEM.1. Statistical analysis Data were analyzed with one-way analysis of variance (ANOVA) and Duncan’s multiple range test (DMRT) using Statistical Package for Social Science (SPSS) version 17.2011. early apoptotic. while 3T3-L1 and Vero cells were maintained in RPMI-1640 culture medium. 48. HRP-conjugated secondary antibody provided sensitive colorimetric. of Pages 7.04. Swiss mouse embryo ﬁbroblast cells (3T3-L1) and African green monkey kidney epithelial (Vero) were purchased from the American Type Culture Collection (ATCC). and 72 h. W. trypan blue powder.05). MTT solution (5 mg/mL) was added and the plate was incubated for 4 h. 1991). USA) (Vermes et al. Ng et al. The cells were ﬁxed with ice-cold 70% ethanol and incubated at À20 °C for 2 h.K. Both media were supplemented with 10% FBS and 1% antibiotics (100 IU/mL penicillin and 100 lg/mL streptomycin).6. 3. Results 3. Germany). TQ was signiﬁcantly less cytotoxic towards the normal cells (3T3-L1 and Vero) as compared to cisplatin (p < 0.030 . Brieﬂy.05) of SiHa cells treated with TQ or cisplatin at 1. The IC50 values as determined by the MTT assay and trypan blue dye exclusion test are shown in Table 126.96.36.199. DMSO was then added to dissolve the dark-blue formazan crystals.. 2. Cytotoxicity of TQ towards SiHa cells The dose–response graph obtained from both MTT assay and trypan blue dye exclusion test shows a signiﬁcant decrease in the percentage of cell viability (p < 0. The changes in cell morphology were observed under an inverted light microscope (Nikon. TQ-treated and -untreated cells were harvested and washed twice with ice-cold PBS and resuspended in 200 lL of 1Â binding buffer containing Annexin V and PI (1 mg/mL) for 10 min at 37 °C in the dark. USA. Austria) and Human Bax ELISA kit (Assay Designs. doi:10. Austria). Denmark). 2. The level of expression of p53. Mycoplex™ foetal bovine serum (FBS) and trypsin–EDTA were purchased from PAA Laboratories (Linz. Determination of cytotoxicity of TQ by MTT assay and trypan blue dye exclusion test The cells were treated with various concentrations of TQ or cisplatin. Probability of p < 0. Bcl-2.1.9. Austria). USA) (Mosmann.4. according to the instructions provided by the manufacturer. Cell culture Human cervical squamous carcinoma cell (SiHa). The cells were again washed with ice-cold PBS and the supernatants were discarded.0. Nevertheless. 25 lL of PI (1 mg/mL) and 50 lL of RNase A (1 mg/mL). Cell cycle analysis TQ-treated and -untreated cells were harvested and washed twice with ice-cold PBS. Human Bax ELISA kit was purchased from Assay Designs (USA).0. 3. 2. The cells were maintained at 37 °C in a humidiﬁed atmosphere of 5% CO2. DNA content was analyzed by FACScan ﬂowcytometer (CyAn ADP. MTT powder. Determination of the level of expression of p53. USA). Louis. 3.tiv.2. Japan). Cell morphological studies As shown in Fig. The pellets were resuspended in 5 lL of acridine orange (1 mg/mL) and 5 lL of propidium iodide (1 mg/mL) (Cury-Boaventura et al.. Austria). 1.
b.0. a. and 30. 2009).0.191 NP NP Cisplatin 21.41⁄.0.0 lg/mL of TQ treatment after 72 h of incubation (Fig. orange late apoptotic cells with nuclear condensation were seen.37 ± 0. 4.10 ± 0. Both MTT assay and trypan blue dye exclusion test showed that TQ was cytotoxic towards SiHa cells in a dose.0. (D) 10 lg/mL (E) IC50 (10. 6).0.0.05) in the regulation of pro-apoptotic protein Bax in the treatment with 1. IC50. Cell cycle analysis Cell cycle analysis showed that TQ induced a dose-dependent increase in the hypoploid cells (sub-G1 cells). (C) 3. 2 shows the fraction of sub-G1 cells increased signiﬁcantly (p < 0. IC50 and 30.2011. c. Control (A) was also included (200Â magniﬁcation). not performed.30b 10.05) as compared to the control. 3.05) in the p53 expression was observed in the cells treated with TQ at 1.b 3. / Toxicology in Vitro xxx (2011) xxx–xxx No. IC50. and 30 lg/mL after 72 h of incubation (Fig.c 12.07⁄. 3.12d 14.90 ± 0. At higher concentration groups (IC50 and 30.37 ± 0. The level of expression of p53. b. 5). reduction in the cell number was noted with the increase in TQ concentration. Fig. the percentage of viable cells decreased signiﬁcantly (p < 0. On the other hand.e 18. W.00 ± 0. Ã were signiﬁcant as compared to TQ while a.0. Mode of cell death induced by TQ determined by AO/PI staining The mode of cell death induced by TQ was also investigated by using the AO/PI ﬂuorescence microscopy double staining.33 ± 0. Even though both MTT assay and trypan blue dye exclusion test can be used for the determination of cytotoxicity. Discussion Table 1 shows that the IC50 values determined by both trypan blue dye exclusion test and MTT assay decrease with the increasing incubation time. Ng et al. 3).22⁄.27 ± 0.. In addition. 1. 4b and c).72% (3. Thymoquinone from Nigella sativa was more potent than cisplatin in eliminating of SiHa cells via apoptosis with down-regulation of Bcl-2 protein.0. c... Bcl-2 expression in the treatment with 1. as compared to the control (4. uniformly green live cells with normal and large nucleus were observed.0.K.0 lg/mL. 2003).0 and 3.05). 4e and f).7 lg/mL). 3. Morphological changes of SiHa cells treated with (B) 1. 1. In the control and low concentration groups (1. et al.73 ± 0.1016/j.62c Cisplatin 24. Bcl-2 and Bax Signiﬁcant increase (p < 0.05) from 6.05) at 10.092 9.tiv.66% in treatment with.3.0 lg/mL for 72 h.2 NP NP 3T3-L1 Vero Values were the means of three replicate samples.03 ± 0. e. human breast adenocarcinoma (MCF7).3 11.67 ± 0. canine osteosarcoma (COS31). its cisplatin-resistant variant (COS31/rCDDP). 6). Cell line Incubation time (hour) IC50 values (lg/mL) MTT assay TQ SiHa 24 48 72 72 72 19.15a 12. there was a 228 229 230 231 232 233 234 235 236 237 238 239 240 241 207 208 209 210 211 212 213 214 215 Please cite this article in press as: Ng. and human ovarian adenocarcinoma (BG-1) cells (Shoieb et al. The data were presented as mean ± SEM.030 .04.0 lg/mL) to 49.67 ± 0.90% and 93.0. In short. In all treatment.05) at the concentration of 3.d Trypan blue dye exclusion test TQ 17. doi:10.83 ± 0. NP.13 ± 0. 10. respectively. and 30 lg/mL of TQ after 72 h of incubation decreased signiﬁcantly (p < 0.15⁄.5 15. IC50. Model 5G 3 Table 1 IC50 values of TQ and cisplatin towards SiHa. 192 193 194 195 196 197 3.and time-dependent manner. 2. 216 217 218 219 220 221 222 223 224 225 226 227 198 199 200 201 202 203 204 205 206 4. TQ has previously shown signiﬁcant cytotoxicity against several cancer cell lines such as human cervical adenocarcinoma (HeLa) cells (Latifah et al. 3. the percentage of necrotic cells was lower than the one of apoptotic cells. of Pages 7. 3. Activities of the anti-apoptotic protein Bcl-2 decreased with the increase in the concentration of TQ (Fig. Nevertheless. Fig. there were no signiﬁcant changes (p > 0. and (F) 30 lg/mL of TQ for 72 h viewed under an inverted light microscope.K.0 lg/mL. 10. and 30 lg/mL of TQ after 72 h of incubation (Fig.09⁄.b 2.5.23 ± 0.254 12. 3. 3T3-L1. 10.0. 3.09⁄. Toxicol. 5. and d were signiﬁcantly different (p < 0. d.0 lg/mL of TQ) (Fig.93 ± 0.6.12a 18.13 ± 0.4.29⁄. IC50.TIV 2639 21 May 2011 W. in Vitro (2011). IC50 and 30 lg/mL of TQ.0.0 lg/mL of TQ) (Fig. and Vero cells at various incubation times as determined by using MTT assay and trypan blue dye exclusion test. Mode of cell death induced by TQ determined by Annexin V/PI The percentage of apoptotic cells (AnnV+/PIÀ for early apoptosis and AnnV+/PI+ for late apoptosis) increased signiﬁcantly after treatment with TQ (p < 0. 10.22%).13⁄.
However. It is important for a potential anticancer lead to exhibit cytotoxicity but such property should be speciﬁc for cancer cell only (Lai et al.TIV 2639 21 May 2011 4 W.2011. Ã were signiﬁcantly different from the control (p < 0. Normal cell lines such as primary mouse keratinocytes and Madin–Darby canine kidney (MDCK) cell lines are 255 256 257 258 259 260 261 262 263 264 265 266 267 Please cite this article in press as: Ng. 3. 2008). Each sample was run in triplicate. The ability of TQ to kill several types of tumors effectively without signiﬁcant cytotoxicity to normal cells has been shown previously.7 lg/mL). in Vitro (2011). and (D) 30 lg/mL of TQ for 72 h as determined by ﬂowcytometer. which was an indirect measurement for the cell viability (Mosmann.1016/j. Thymoquinone from Nigella sativa was more potent than cisplatin in eliminating of SiHa cells via apoptosis with down-regulation of Bcl-2 protein.030 .. Hence. Model 5G Fig. et al. MTT assay and trypan blue dye exclusion test indicated that TQ was more potent towards SiHa cells as compared to cisplatin. Control (A) was also included. The study shows that the IC50 values as determined by trypan blue dye exclusion test were signiﬁcantly lower than MTT assay. which indicates the cell viability directly (Cho et al. 242 243 244 245 246 247 248 249 250 251 252 253 254 signiﬁcant difference in the IC50 values obtained. it is believed that the use of multiple different endpoint assays to deﬁne cytotoxicity can be more useful and informative.tiv. 2008). of Pages 7. viewed under a ﬂuorescence microscope.0 lg/mL.0 lg/mL. 2005). and (F) 30 lg/mL of TQ for 72 h. It has been reported that different methods often yield considerably different values of cytotoxicity due to their different principles (Lee et al. W. / Toxicology in Vitro xxx (2011) xxx–xxx No. Ng et al. Control (A) was also included. (D) 10 lg/mL. Nevertheless. (200Â magniﬁcation). AO/PI staining of SiHa cells treated with (B) 1. the trypan blue dye exclusion test was based on the interaction of trypan blue dye with the cell if the cell membrane is damaged. (E) IC50 (10. 1983). Formazan accumulation in MTT assay directly reﬂected the mitochondrial activity in live cell.04.. normal cells (3T3-L1 and Vero) were less sensitive towards TQ. doi:10. It may due to the condition that the cells are more vulnerable to damage when trypQ4 sinization was performed to count the stained and unstained cells (Seth et al. Fig. 2005).K. (C) 3. Cell cycle analysis of SiHa cells treated with (B) 10 lg/mL (C) IC50 (10. Toxicol.05)..7 lg/mL). 2...K. Data were presented as mean ± SEM.
e.030 .. doi:10. 5. Meanwhile. a. Thymoquinone from Nigella sativa was more potent than cisplatin in eliminating of SiHa cells via apoptosis with down-regulation of Bcl-2 protein. which further conﬁrmed that TQ induced late apoptosis in SiHa cells.. Up-regulation of the expression of p53 in SiHa cells treated with TQ. Figs. late 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 Please cite this article in press as: Ng. Ng et al. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) to access basal cytotoxicity (Lai et al.05).04. The percentage of cell distribution of SiHa cells induced by TQ after 72 h incubation time as determined by Annexin V/PI staining. d. Cell cycle analysis of TQ-treated SiHa cells showed accumulation of the cell population in the sub-G1 phase. et al. which is an indication of the cleavage of nuclear DNA into multiple fragments and caused apoptosis (Han and Park.K. In this study. Both tests indicated that TQ was cytotoxic towards SiHa cells through a mechanism that involves apoptosis. Liao et al. variations and adventitious agent contamination of the primary cultures (Carosati et al. Ã and ÃÃ were signiﬁcantly different as compared to control ⁄p < 0. b.01) and a were signiﬁcantly different as compared to viable cells (p < 0. Values are the mean of three independent experiments ±SEM.. analyzed by ﬂowcytometer. / Toxicology in Vitro xxx (2011) xxx–xxx No. Model 5G 5 Fig.2011. of Pages 7. but upon induction of apoptosis it is translocated to the outer membrane leaﬂet and becomes available for Annexin V binding.. Swiss mouse embryo ﬁbroblast cells (3T3-L1) and African green monkey kidney epithelial (Vero) were used to assess the cytotoxicity of TQ towards normal cells. cell treated with IC50 and 30 lg/mL showed the morphology of orange color nuclei (Fig. The data were presented as mean ± SEM. and 30 lg/mL of TQ.05).TIV 2639 21 May 2011 W. and f were signiﬁcant different (p < 0.K. Annexin V/propidium iodide (AnnV/PI) staining is based on ability of the protein Annexin V to bind to phosphatidylserine (PS) exposed on the outer membrane leaﬂet in apoptotic cells. W. 2010). Vero cells are homologous with human body cells as it shares a common embryonic origin (mesoderm) with cells from human genital tract. 2010). allowing to culture cells for longer than normal cell line (Ferrari et al. 4e and f). 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 reported to be resistant to the cytotoxic effects of TQ (Shoieb et al. in the AO/PI analysis. The indication of apoptosis was further conﬁrmed by the Annexin V-FITC/PI and AO/PI. the rationale behind the use of 3T3-L1 and Vero cells rather than primary cervical cancer cells is that these normal cells have be banked and well characterized. early apoptotic (AnnV+/PIÀ).tiv. Ã were signiﬁcantly different as compared to control (p < 0. thus. PS is located in the inner membrane leaﬂet. 4. 2–4 revealed that TQ induced apoptosis in SiHa cells in a dose-dependent manner. 3T3-Li cell line is recommended by US National Institute of Environmental Health Sciences (NIEHS).. 2003). avoiding the issue of lot-by-lot viability. and this line is non-tumorigenic but immortalized. Values were the means of three replicate samples.1016/j. Annexin V/PI analysis revealed that majority of the cells underwent apoptosis when treated with 10.. Toxicol. In viable cells. 2008). 2009).05). IC50. Fig.05 and ⁄⁄p < 0. In addition. c. in Vitro (2011). 2009. The addition of PI enabled viable (AnnVÀ/PIÀ).
Therefore. 373 374 375 Please cite this article in press as: Ng. SiHa and less cytotoxic towards the normal cells (3T3-L1 and Vero) as compared to cisplatin. making it appears orange. and resulting in the regulation of the Bax to Bcl-2 ratio.030 . Perhaps. suggesting TQ may be a potential agent for the management of cervical cancer in future. 2004).K. 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 Conﬂict of interest None declared. The expression of Bcl-2 and Bax protein in SiHa cells treated with TQ. Caspase-9 activates the effector procaspases including caspase-3. AO intercalates into the DNA giving it a green appearance. Thus. respectively. Therefore. the ﬁndings demonstrate that cytotoxicity of TQ towards SiHa cells is associated with its ability to induce apoptosis in the cells. as well as tissue homeostasis to control the cell number (Arepalli et al. via down-regulation of anti-apoptotic Bcl-2 protein. c. PI is only taken up by non-viable cells.2011. viable cells have a bright green nucleus.. W. due to the signiﬁcant down-regulation of Bcl-2. induction of cell apoptosis and targeting apoptotic pathways has emerged as an attractive approach for treatment of cancer (Li et al. evaluated by human Bcl-2 ELISA kit and human Bax ELISA kit. DNA-binding dye acridine orange (AO) and propidium iodide (PI) were used for the morphological detection of apoptotic and necrotic cells.K. This dye also intercalates into DNA. Tumor suppressor gene p53 has been reported to be able to regulate the expression of a number of downstream proteins. The live cells with intact membranes have a uniform green color in their nuclei.. e. Previous reports showed that TQ triggered human pancreatic adenocarcinoma. Cytochrome c forms an apoptosome that is composed of Apaf-1 and procaspase-9. the cytotoxicity of TQ was more selective towards cancerous cells.. the ability of TQ to induce apoptosis in SiHa cells suggests that TQ may be a potentially effective chemotherapeutic agent against cervical cancer. Universiti Putra Malaysia. 371 372 Acknowledgment This study was partly supported by the Research University Grant Scheme (04/01/07/0136RU). This indicates that the cytotoxic effect of TQ is mediated by pro-apoptotic effects which are modulated by Bcl-2 protein. 6.. et al. uterine sarcoma. Early apoptotic cells have chromatin condensation with bright green colored nuclei.1016/j.. -6. Toxicol. b. Programmed cell death (cell suicide) or apoptosis plays a vital role in many physiological and developmental stages. 2007). Ã were signiﬁcantly different as compared to control (p < 0. Effect on the regulation of apoptosis and ability to manipulate the mechanism of programmed cell death may lead to new possible agents for cancer therapy (Tamatani et al. High ratio of Bax to Bcl-2 can cause the permeabilization of the outer mitochondrial membrane.TIV 2639 21 May 2011 6 W. and leukemic cell lines to undergo apoptosis that is associated with increase in the gene and protein expression of p53 and inhibition of the anti-apoptotic Bcl-2 protein. Thymoquinone from Nigella sativa was more potent than cisplatin in eliminating of SiHa cells via apoptosis with down-regulation of Bcl-2 protein. The apoptosis induction by TQ in SiHa cells was in a p53-dependent manner. Roos and Kaina. Cytotoxicity of TQ in SiHa cells involved an elevation of the level of the p53 activity and the Bax to Bcl-2 ratio. Disruption in apoptosis caused a major manifestation in diseases such as cancer (Ray et al. in Vitro (2011). d. and 1 were signiﬁcant different (p < 0..05). it is presumed that TQ induces DNA damage in SiHa cells. 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 apoptotic (AnnV+/PI+) and necrotic (AnnVÀ/PI+) cells to be distin´ guished (Baskic et al. resulting in the release of cytochrome c. the Bax activity in TQ-treated cells was not changes even at the highest concentration of 30 lg/mL.. Late apoptotic cells have bright orange areas of condensed chromatin in the nucleus that distinguish them from necrotic cells... 2009). Ng et al. / Toxicology in Vitro xxx (2011) xxx–xxx No. In conclusion. doi:10. and is linked to and dependent on p53 (Worthen et al. which have a uniform orange color (Cury-Boaventura et al.04.05). Nevertheless.tiv. 2006). 2009). 2007). 2006. Whereas. 2009). Interestingly. 2006). and -7 to carry out the process of apoptosis (Han and Park. of Pages 7. 2006). Camptotheca acuminate) and genistein (a soy-derived isoﬂavone and phytoestrogen) have been found to induce apoptosis (Ding et al. camptothecin (an alkaloid isolated from the Chinese tree. f. Many FDA approved clinical available anti-cancer drugs such as paclitaxel (a compound extracted from the Paciﬁc yew tree. resulting in the activation of caspase-9. Values are the mean of three independent experiments ±SEM. Model 5G Fig. 1998). Taxus brevifolia). such as Bax and Bcl-2 in response to DNA damage (Coutts and La Thangue. a. in the AO/PI staining. leading to the increase in the level of p53 expression.
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