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TIV 2639 21 May 2011Toxicology in Vitro xxx (2011) xxxxxx 1

No. of Pages 7, Model 5G

Contents lists available at ScienceDirect

Toxicology in Vitrojournal homepage: www.elsevier.com/locate/toxinvit

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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 proteinWei Keat Ng a, Latifah Saiful Yazan a,b,, Maznah Ismail a,ca

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, Malaysiab

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a b s t r a c tThymoquinone (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 owcytometer showed a signicant 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 conrmed by Annexin V/ PI and AO/PI staining. Signicant 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. 2011 Published by Elsevier Ltd.25 26 27 28 29 30 31 32 33 34 35 36 37 38

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 specicity 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 ofBioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. Tel.: +60 3 89472308; fax: +60 389472336. E-mail address: latifah@medic.upm.edu.my (L.S. Yazan). 0887-2333/$ - see front matter 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 nding 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-inammatory 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

TIV 2639 21 May 20112 86 87 W.K. Ng et al. / Toxicology in Vitro xxx (2011) xxxxxx

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The mode of cell death and involvement of p53, Bcl-2, and Bax were also investigated. 2. Materials and methods 2.1. Chemicals Thymoquinone (TQ), cisplatin, tissue culture medium (EMEM), trypan blue powder, MTT powder, propidium iodide (PI), acridine orange (AO), and RNase were purchased from Sigma Chemicals (St. Louis, USA). RPMI-1640, penicillin/streptomycin antibiotic, Mycoplex foetal bovine serum (FBS) and trypsinEDTA were purchased from PAA Laboratories (Linz, Austria). Human Annexin VFITC kit, Human p53 ELISA kit and Human Bcl-2 ELISA kit were purchased from Bender MedSystem (Vienna, Austria). Human Bax ELISA kit was purchased from Assay Designs (USA). 2.2. Cell culture Human cervical squamous carcinoma cell (SiHa), Swiss mouse embryo broblast cells (3T3-L1) and African green monkey kidney epithelial (Vero) were purchased from the American Type Culture Collection (ATCC), USA. SiHa cells were grown in EMEM, while 3T3-L1 and Vero cells were maintained in RPMI-1640 culture medium. Both media were supplemented with 10% FBS and 1% antibiotics (100 IU/mL penicillin and 100 lg/mL streptomycin). The cells were maintained at 37 C in a humidied atmosphere of 5% CO2. 2.3. 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, ranging from 1.0 to 30 lg/mL in a 96-well plate for 24, 48 and 72 h. Control (without TQ or cisplatin) was also included (Shier, 1991). MTT solution (5 mg/mL) was added and the plate was incubated for 4 h. DMSO was then added to dissolve the dark-blue formazan crystals. The absorbance at 570 nm and the reference wavelength of 630 nm was measured with a microplate reader (Opsys MR, USA) (Mosmann, 1983). For the trypan blue dye exclusion test, the treated and untreated cell suspensions were mixed with 0.4% trypan blue dye gently at the ratio of 1:1 (Renzi et al., 1993). The number of viable and dead cell was counted with a haemocytometer under an inverted light microscope (Nikon, Japan). 2.4. Cell morphological studies TQ-treated (1.0, 3.0, 10, and 30 lg/mL) and -untreated cells were grown in a 6-well plate for 72 h. The changes in cell morphology were observed under an inverted light microscope (Nikon, Japan). 2.5. Cell cycle analysis TQ-treated and -untreated cells were harvested and washed twice with ice-cold PBS. The cells were xed with ice-cold 70% ethanol and incubated at 20 C for 2 h. The cells were again washed with ice-cold PBS and the supernatants were discarded. The pellets were 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 incubated at 4 C for 20 min. DNA content was analyzed by FACScan owcytometer (CyAn ADP, Denmark). The population of cells in each cell-cycle phase was determined by using the Submit V3.4 software (CyAn ADP, Denmark).

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 of incubation, and washed with ice-cold PBS. 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., 2004). The morphological changes of the stained cells were then observed by using a uorescence 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 the Human Annexin V-FITC Apoptosis Detection Kit (Bender Medsystems, Austria). After 72 h of incubation, TQ-treated and -untreated cells were harvested and washed twice with ice-cold PBS and resuspended in 200 lL of 1 binding buffer containing Annexin V and PI (1 mg/mL) for 10 min at 37 C in the dark. The number of viable, early apoptotic, late apoptotic and necrotic cells was quantied by owcytometer (C

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