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Toxicology in Vitro xxx (2011) xxx–xxx

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Contents lists available at ScienceDirect

Toxicology in Vitro

journal homepage: www.elsevier .com/locate / toxinvi t

Thymoquinone from Nigella sativa was more potent than cisplatin in eliminatingof SiHa cells via apoptosis with down-regulation of Bcl-2 protein

Wei Keat Ng a, Latifah Saiful Yazan a,b,⇑, Maznah Ismail a,c

a Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysiab Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysiac 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

252627282930313233343536

Article history:Received 28 January 2011Accepted 26 April 2011Available online xxxx

Keywords:ThymoquinoneNigella sativaCervical cancerApoptosisp53Bcl-2

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0887-2333/$ - see front matter � 2011 Published bydoi:10.1016/j.tiv.2011.04.030

⇑ Corresponding author at: Laboratory of MolecuBioscience, Universiti Putra Malaysia, 43400 UPM Serd+60 3 89472308; fax: +60 389472336.

E-mail address: latifah@medic.upm.edu.my (L.S. Y

Please cite this article in press as: Ng, W.K., et atosis with down-regulation of Bcl-2 protein. To

a b s t r a c t

Thymoquinone (TQ), the active constituent of Nigella sativa or black cumin exhibited cytotoxic effects inseveral 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 and9.33 ± 0.19 lg/mL as determined by MTT assay and trypan blue dye exclusion test, respectively, after72 h of incubation. TQ was more cytotoxic towards SiHa cells compared to cisplatin. Interestingly, TQwas less cytotoxic towards the normal cells (3T3-L1 and Vero). Cell cycle analysis performed by flowcy-tometer showed a significant increase in the accumulation of TQ-treated cells at sub-G1 phase, indicatinginduction 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 pro-tein was found in the treated cells, without any changes in the expression of the pro-apoptotic Bax pro-tein. In conclusion, thymoquinone from N. sativa was more potent than cisplatin in elimination of SiHacells via apoptosis with down-regulation of Bcl-2 protein.

� 2011 Published by Elsevier Ltd.

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1. Introduction

Although the mortality rate of cervical cancer has been gradu-ally reduced after the introduction of PAP-smear programme, it isstill one of the leading female malignancies worldwide (Liu et al.,2009). It was estimated that more than 500,000 new cases of cer-vical cancer were reported in the world during 2007 (AmericanCancer Society, 2008).

The problem of unacceptable adverse effects such as dose-re-lated toxicity, low specificity and the recurrence of patient tumorsdue to propagation of drug-resistant cells remains an inevitableobstacle to the achievement in anti-cancer chemotherapy (DeMesquita et al., 2009; Ferguson et al., 2009). Even though cis-diamminedichloroplatinum(II) or cisplatin for instance, is highlyeffective in treating cervical carcinoma (Fontanelli et al., 1992), ithas been reported to have neurotoxic effects upon the peripheralnervous system (PNS) and the central nervous system (CNS). Themost commonly observed side-effects include neurotoxicity, eme-sis, nephrotoxicity, ototoxicity and moderate myelosuppression.More rare side-effects include ophthalmological effects, seizuresand autonomic neuropathy (Mollman, 1999; Troy et al., 2000).

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lar Biomedicine, Institute ofang, Selangor, Malaysia. Tel.:

azan).

l. Thymoquinone from Nigella sxicol. in Vitro (2011), doi:10.10

Hence, the search for new chemotherapeutic agents has refocusedon natural products, which led to the finding of some new bioac-tive compounds.

Two preventive vaccines have recently been licensed for use:Gardasil and Cervarix. Gardasil is a quadrivalent vaccine containingrecombinant L1 VLPs for HPV genotypes 6, 11, 16, and 18 whereasthe bivalent vaccine Cervarix contains L1 VLPs for HPV-16 andHPV-18 (Bosch and Harper, 2006; Lin et al., 2010). However, thevaccines will reduce, but not eliminate, the risk of cervical cancer,as at present they only target HPV-6, -11, -16, and -18 oncogenicgenital types (Barr and Sings, 2008; Stanley, 2008). World HealthOrganization revealed that HPV vaccines do not cure cancer; theyprevent some, but not all, HPV-related cancers (World HealthOrganization, 2009), and 30% HPV-related cervical cancers typesare not covered by the vaccines (Torre et al., 2007). Hence, a callfor discovery of more effective agents to treat cancer is becomingincreasingly urgent (Jakopec et al., 2006).

Nigella sativa, a dicotyledon of the Ranunculaceae family, is anamazing herb with a rich historical and religious background(Salem, 2005). Thymoquinone (TQ) or 2-isopropyl-5-methyle-1,4benzoquinone (C10H12O2), with relative molecular mass of 164.2,is one of the bioactive compounds of N. sativa (Shoieb, 2003). Ithas been shown to exert anti-neoplastic, anti-oxidant, anti-inflam-matory and anti-histamine effects (Gali-Muhtasib et al., 2006).

In this study, the cytotoxicity of TQ from N. sativa towards hu-man cervical squamous carcinoma cells (SiHa) was determined.

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The mode of cell death and involvement of p53, Bcl-2, and Baxwere also investigated.

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2. Materials and methods

2.1. Chemicals

Thymoquinone (TQ), cisplatin, tissue culture medium (EMEM),trypan blue powder, MTT powder, propidium iodide (PI), acridineorange (AO), and RNase were purchased from Sigma Chemicals(St. Louis, USA). RPMI-1640, penicillin/streptomycin antibiotic,Mycoplex™ foetal bovine serum (FBS) and trypsin–EDTA were pur-chased from PAA Laboratories (Linz, Austria). Human Annexin V-FITC kit, Human p53 ELISA kit and Human Bcl-2 ELISA kit were pur-chased from Bender MedSystem (Vienna, Austria). Human Bax ELI-SA kit was purchased from Assay Designs (USA).

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2.2. Cell culture

Human cervical squamous carcinoma cell (SiHa), Swiss mouseembryo fibroblast cells (3T3-L1) and African green monkey kidneyepithelial (Vero) were purchased from the American Type CultureCollection (ATCC), USA. SiHa cells were grown in EMEM, while3T3-L1 and Vero cells were maintained in RPMI-1640 culture med-ium. Both media were supplemented with 10% FBS and 1% antibi-otics (100 IU/mL penicillin and 100 lg/mL streptomycin). The cellswere maintained at 37 �C in a humidified atmosphere of 5% CO2.

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2.3. Determination of cytotoxicity of TQ by MTT assay and trypan bluedye exclusion test

The cells were treated with various concentrations of TQ or cis-platin, ranging from 1.0 to 30 lg/mL in a 96-well plate for 24, 48and 72 h. Control (without TQ or cisplatin) was also included(Shier, 1991). MTT solution (5 mg/mL) was added and the platewas incubated for 4 h. DMSO was then added to dissolve thedark-blue formazan crystals. The absorbance at 570 nm and thereference wavelength of 630 nm was measured with a microplatereader (Opsys MR, USA) (Mosmann, 1983).

For the trypan blue dye exclusion test, the treated and un-treated cell suspensions were mixed with 0.4% trypan blue dyegently at the ratio of 1:1 (Renzi et al., 1993). The number of viableand dead cell was counted with a haemocytometer under an in-verted light microscope (Nikon, Japan).

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2.4. Cell morphological studies

TQ-treated (1.0, 3.0, 10, and 30 lg/mL) and -untreated cellswere grown in a 6-well plate for 72 h. The changes in cell morphol-ogy were observed under an inverted light microscope (Nikon,Japan).

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2.5. Cell cycle analysis

TQ-treated and -untreated cells were harvested and washedtwice with ice-cold PBS. The cells were fixed with ice-cold 70% eth-anol and incubated at �20 �C for 2 h. The cells were again washedwith ice-cold PBS and the supernatants were discarded. The pelletswere resuspended in a solution containing with 425 lL of PBS,25 lL of PI (1 mg/mL) and 50 lL of RNase A (1 mg/mL), and incu-bated at 4 �C for 20 min. DNA content was analyzed by FACScanflowcytometer (CyAn ADP, Denmark). The population of cells ineach cell-cycle phase was determined by using the Submit V3.4software (CyAn ADP, Denmark).

Please cite this article in press as: Ng, W.K., et al. Thymoquinone from Nigella stosis with down-regulation of Bcl-2 protein. Toxicol. in Vitro (2011), doi:10.10

2.6. 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 ofincubation, and washed with ice-cold PBS. The pellets were resus-pended in 5 lL of acridine orange (1 mg/mL) and 5 lL of propidiumiodide (1 mg/mL) (Cury-Boaventura et al., 2004). The morphologi-cal changes of the stained cells were then observed by using a fluo-rescence microscope (Leica, Germany).

2.7. Determination of mode of cell death by Annexin V/PI staining

Annexin V (AnnV) and PI staining was performed by using theHuman Annexin V-FITC Apoptosis Detection Kit (Bender Medsys-tems, Austria). After 72 h of incubation, TQ-treated and -untreatedcells were harvested and washed twice with ice-cold PBS andresuspended in 200 lL of 1� binding buffer containing AnnexinV and PI (1 mg/mL) for 10 min at 37 �C in the dark. The numberof viable, early apoptotic, late apoptotic and necrotic cells wasquantified by flowcytometer (CyAn ADP, USA) (Vermes et al.,1995).

2.8. Determination of the level of expression of p53, Bcl-2, and Bax

TQ-treated and -untreated cells were lysed. The level of expres-sion of p53, Bcl-2, and Bax was determined by colorimetric proce-dures by using Human p53 ELISA kit (Bender Medsystems, Austria),Human Bcl-2 ELISA kit (Bender Medsystems, Austria) and HumanBax ELISA kit (Assay Designs, USA), respectively, according to theinstructions provided by the manufacturer. Briefly, The p53, Bcl-2, or Bax protein from the cell lysate specifically bound to the pri-mary antibody and detected by Horseradish peroxidise (HRP) con-jugated secondary antibody. HRP-conjugated secondary antibodyprovided sensitive colorimetric. The expression of p53, Bcl-2, andBax was determined by measuring the absorbance at 450 nm andthe reference wavelength of 630 nm using a microplate reader(Opsys MR, USA).

2.9. Statistical analysis

Data were analyzed with one-way analysis of variance (ANOVA)and Duncan’s multiple range test (DMRT) using Statistical Packagefor Social Science (SPSS) version 17.0. All the data were expressedas mean ± standard error of mean (SEM). Probability of p < 0.05was considered significant.

3. Results

3.1. Cytotoxicity of TQ towards SiHa cells

The dose–response graph obtained from both MTT assay andtrypan blue dye exclusion test shows a significant decrease in thepercentage of cell viability (p < 0.05) of SiHa cells treated with TQor cisplatin at 1.0, 3.0, 10, and 30 lg/mL for 24, 48, and 72 h. Nev-ertheless, TQ was significantly less cytotoxic towards the normalcells (3T3-L1 and Vero) as compared to cisplatin (p < 0.05).

The IC50 values as determined by the MTT assay and trypanblue dye exclusion test are shown in Table 1.

3.2. Cell morphological studies

As shown in Fig. 1, TQ caused morphological changes in SiHacells. Reduction in the cell number was very obvious at higherconcentration of TQ (30 lg/mL), with majority of the cells weredetached from surface of the plate.

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Table 1IC50 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 NPVero 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 dwere 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 aninverted light microscope. Control (A) was also included (200� magnification).

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3.3. Cell cycle analysis

Cell cycle analysis showed that TQ induced a dose-dependentincrease in the hypoploid cells (sub-G1 cells). Fig. 2 shows the frac-tion of sub-G1 cells increased significantly (p < 0.05) from 6.72%(3.0 lg/mL) to 49.90% and 93.66% in treatment with, IC50 and30 lg/mL of TQ, respectively, as compared to the control (4.22%).

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3.4. Mode of cell death induced by TQ determined by AO/PI staining

The mode of cell death induced by TQ was also investigated byusing the AO/PI fluorescence microscopy double staining. In thecontrol and low concentration groups (1.0 and 3.0 lg/mL of TQ)(Fig. 4b and c), uniformly green live cells with normal and large nu-cleus were observed. In addition, reduction in the cell number wasnoted with the increase in TQ concentration. At higher concentra-tion groups (IC50 and 30.0 lg/mL of TQ) (Fig. 4e and f), orange lateapoptotic cells with nuclear condensation were seen.

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3.5. Mode of cell death induced by TQ determined by Annexin V/PI

The percentage of apoptotic cells (AnnV+/PI� for early apopto-sis and AnnV+/PI+ for late apoptosis) increased significantly aftertreatment with TQ (p < 0.05) at the concentration of 3.0, 10.0,IC50, and 30.0 lg/mL for 72 h. On the other hand, the percentageof viable cells decreased significantly (p < 0.05) at 10.0, IC50 and30.0 lg/mL of TQ treatment after 72 h of incubation (Fig. 3). Inall treatment, the percentage of necrotic cells was lower than theone of apoptotic cells.

Please cite this article in press as: Ng, W.K., et al. Thymoquinone from Nigella stosis with down-regulation of Bcl-2 protein. Toxicol. in Vitro (2011), doi:10.10

3.6. The level of expression of p53, Bcl-2 and Bax

Significant increase (p < 0.05) in the p53 expression was ob-served in the cells treated with TQ at 1.0, 3.0, 10.0, IC50, and30 lg/mL after 72 h of incubation (Fig. 5).

Bcl-2 expression in the treatment with 1.0, 3.0, 10.0, IC50, and30 lg/mL of TQ after 72 h of incubation decreased significantly(p < 0.05) as compared to the control. Activities of the anti-apopto-tic protein Bcl-2 decreased with the increase in the concentrationof TQ (Fig. 6). Nevertheless, there were no significant changes(p > 0.05) in the regulation of pro-apoptotic protein Bax in thetreatment with 1.0, 3.0, 10.0, IC50, and 30 lg/mL of TQ after 72 hof incubation (Fig. 6).

4. Discussion

Table 1 shows that the IC50 values determined by both trypanblue dye exclusion test and MTT assay decrease with the increasingincubation time. In short, Both MTT assay and trypan blue dyeexclusion test showed that TQ was cytotoxic towards SiHa cellsin a dose- and time-dependent manner.

TQ has previously shown significant cytotoxicity against severalcancer cell lines such as human cervical adenocarcinoma (HeLa)cells (Latifah et al., 2009), canine osteosarcoma (COS31), its cis-platin-resistant variant (COS31/rCDDP), human breast adenocarci-noma (MCF7), and human ovarian adenocarcinoma (BG-1) cells(Shoieb et al., 2003).

Even though both MTT assay and trypan blue dye exclusion testcan be used for the determination of cytotoxicity, there was a

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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 alsoincluded. 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 fluorescencemicroscope. Control (A) was also included. (200� magnification).

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significant difference in the IC50 values obtained. It has been re-ported that different methods often yield considerably differentvalues of cytotoxicity due to their different principles (Lee et al.,2005). Formazan accumulation in MTT assay directly reflectedthe mitochondrial activity in live cell, which was an indirect mea-surement for the cell viability (Mosmann, 1983). However, the try-pan blue dye exclusion test was based on the interaction of trypanblue dye with the cell if the cell membrane is damaged, which indi-cates the cell viability directly (Cho et al., 2008). The study showsthat the IC50 values as determined by trypan blue dye exclusiontest were significantly lower than MTT assay. It may due to thecondition that the cells are more vulnerable to damage when tryp-sinization was performed to count the stained and unstained cells

Please cite this article in press as: Ng, W.K., et al. Thymoquinone from Nigella stosis with down-regulation of Bcl-2 protein. Toxicol. in Vitro (2011), doi:10.10

(Seth et al., 2005). Hence, it is believed that the use of multiple dif-ferent endpoint assays to define cytotoxicity can be more usefuland informative.

MTT assay and trypan blue dye exclusion test indicated that TQwas more potent towards SiHa cells as compared to cisplatin. Nev-ertheless, normal cells (3T3-L1 and Vero) were less sensitive to-wards TQ.

It is important for a potential anticancer lead to exhibit cytotox-icity but such property should be specific for cancer cell only (Laiet al., 2008). The ability of TQ to kill several types of tumors effec-tively without significant cytotoxicity to normal cells has beenshown previously. Normal cell lines such as primary mouse kerat-inocytes and Madin–Darby canine kidney (MDCK) cell lines are

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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. Valueswere 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 awere 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 ascompared to control (p < 0.05). a, b, c, d, e, and f were significant different (p < 0.05).

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reported to be resistant to the cytotoxic effects of TQ (Shoieb et al.,2003). In this study, Swiss mouse embryo fibroblast cells (3T3-L1)and African green monkey kidney epithelial (Vero) were used toassess the cytotoxicity of TQ towards normal cells. 3T3-Li cell lineis recommended by US National Institute of Environmental HealthSciences (NIEHS), Interagency Coordinating Committee on the Val-idation of Alternative Methods (ICCVAM) to access basal cytotoxic-ity (Lai et al., 2008). Vero cells are homologous with human bodycells as it shares a common embryonic origin (mesoderm) withcells from human genital tract, and this line is non-tumorigenicbut immortalized, allowing to culture cells for longer than normalcell line (Ferrari et al., 2009; Liao et al., 2010). In addition, the ratio-nale behind the use of 3T3-L1 and Vero cells rather than primarycervical cancer cells is that these normal cells have be bankedand well characterized, thus, avoiding the issue of lot-by-lot viabil-ity, variations and adventitious agent contamination of the primarycultures (Carosati et al., 2010).

Figs. 2–4 revealed that TQ induced apoptosis in SiHa cells in adose-dependent manner. Cell cycle analysis of TQ-treated SiHa

Please cite this article in press as: Ng, W.K., et al. Thymoquinone from Nigella stosis with down-regulation of Bcl-2 protein. Toxicol. in Vitro (2011), doi:10.10

cells showed accumulation of the cell population in the sub-G1phase, which is an indication of the cleavage of nuclear DNA intomultiple fragments and caused apoptosis (Han and Park, 2009).The indication of apoptosis was further confirmed by the AnnexinV-FITC/PI and AO/PI. Both tests indicated that TQ was cytotoxic to-wards SiHa cells through a mechanism that involves apoptosis. An-nexin V/PI analysis revealed that majority of the cells underwentapoptosis when treated with 10, IC50, and 30 lg/mL of TQ. Mean-while, in the AO/PI analysis, cell treated with IC50 and 30 lg/mLshowed the morphology of orange color nuclei (Fig. 4e and f),which further confirmed that TQ induced late apoptosis in SiHacells.

Annexin V/propidium iodide (AnnV/PI) staining is based onability of the protein Annexin V to bind to phosphatidylserine(PS) exposed on the outer membrane leaflet in apoptotic cells. Inviable cells, PS is located in the inner membrane leaflet, but uponinduction of apoptosis it is translocated to the outer membraneleaflet and becomes available for Annexin V binding. The additionof PI enabled viable (AnnV�/PI�), early apoptotic (AnnV+/PI�), late

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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 meanof 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).

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apoptotic (AnnV+/PI+) and necrotic (AnnV�/PI+) cells to be distin-guished (Baskic et al., 2006). Whereas, in the AO/PI staining,DNA-binding dye acridine orange (AO) and propidium iodide (PI)were used for the morphological detection of apoptotic and necro-tic cells. AO intercalates into the DNA giving it a green appearance.Thus, viable cells have a bright green nucleus. PI is only taken up bynon-viable cells. This dye also intercalates into DNA, making it ap-pears orange. The live cells with intact membranes have a uniformgreen color in their nuclei. Early apoptotic cells have chromatincondensation with bright green colored nuclei. Late apoptotic cellshave bright orange areas of condensed chromatin in the nucleusthat distinguish them from necrotic cells, which have a uniform or-ange color (Cury-Boaventura et al., 2004).

Programmed cell death (cell suicide) or apoptosis plays a vitalrole in many physiological and developmental stages, as well astissue homeostasis to control the cell number (Arepalli et al.,2009). Disruption in apoptosis caused a major manifestation in dis-eases such as cancer (Ray et al., 2006). Effect on the regulation ofapoptosis and ability to manipulate the mechanism of pro-grammed cell death may lead to new possible agents for cancertherapy (Tamatani et al., 2007). Therefore, induction of cell apopto-sis and targeting apoptotic pathways has emerged as an attractiveapproach for treatment of cancer (Li et al., 2007). Many FDA ap-proved clinical available anti-cancer drugs such as paclitaxel (acompound extracted from the Pacific yew tree, Taxus brevifolia),camptothecin (an alkaloid isolated from the Chinese tree, Camptot-heca acuminate) and genistein (a soy-derived isoflavone and phyto-estrogen) have been found to induce apoptosis (Ding et al., 2009).Perhaps, the ability of TQ to induce apoptosis in SiHa cells suggeststhat TQ may be a potentially effective chemotherapeutic agentagainst cervical cancer.

Cytotoxicity of TQ in SiHa cells involved an elevation of the levelof the p53 activity and the Bax to Bcl-2 ratio, due to the significantdown-regulation of Bcl-2. Nevertheless, the Bax activity in TQ-trea-ted cells was not changes even at the highest concentration of30 lg/mL.

Previous reports showed that TQ triggered human pancreaticadenocarcinoma, uterine sarcoma, and leukemic cell lines to

Please cite this article in press as: Ng, W.K., et al. Thymoquinone from Nigella stosis with down-regulation of Bcl-2 protein. Toxicol. in Vitro (2011), doi:10.10

undergo apoptosis that is associated with increase in the geneand protein expression of p53 and inhibition of the anti-apoptoticBcl-2 protein. This indicates that the cytotoxic effect of TQ is med-iated by pro-apoptotic effects which are modulated by Bcl-2 pro-tein, and is linked to and dependent on p53 (Worthen et al., 1998).

Tumor suppressor gene p53 has been reported to be able to reg-ulate the expression of a number of downstream proteins, such asBax and Bcl-2 in response to DNA damage (Coutts and La Thangue,2006; Roos and Kaina, 2006). High ratio of Bax to Bcl-2 can causethe permeabilization of the outer mitochondrial membrane, result-ing in the release of cytochrome c. Cytochrome c forms an apopto-some that is composed of Apaf-1 and procaspase-9, resulting in theactivation of caspase-9. Caspase-9 activates the effector procaspas-es including caspase-3, -6, and -7 to carry out the process of apop-tosis (Han and Park, 2009). Therefore, it is presumed that TQinduces DNA damage in SiHa cells, leading to the increase in the le-vel of p53 expression, and resulting in the regulation of the Bax toBcl-2 ratio.

In conclusion, the findings demonstrate that cytotoxicity of TQtowards SiHa cells is associated with its ability to induce apoptosisin the cells. The apoptosis induction by TQ in SiHa cells was in ap53-dependent manner, via down-regulation of anti-apoptoticBcl-2 protein. Interestingly, the cytotoxicity of TQ was more selec-tive towards cancerous cells, SiHa and less cytotoxic towards thenormal cells (3T3-L1 and Vero) as compared to cisplatin, suggest-ing TQ may be a potential agent for the management of cervicalcancer in future.

Conflict of interest

None declared.

Acknowledgment

This study was partly supported by the Research UniversityGrant Scheme (04/01/07/0136RU), Universiti Putra Malaysia.

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