Nigella sativa seed oil suppresses cell proliferation and induces ROS dependent mitochondrial apoptosis through p53 pathway in hepatocellular carcinoma cells

South African Journal of Botany 112 (2017) 70–78

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Nigella sativa seed oil suppresses cell proliferation and induces ROS dependent mitochondrial apoptosis through p53 pathway in hepatocellular carcinoma cells M.M. Al-Oqail a, E.S. Al-Sheddi a, S.M. Al-Massarani a, M.A. Siddiqui b,c, J. Ahmad b,c, J. Musarrat b,c, A.A. Al-Khedhairy b,c, N.N. Farshori a,⁎ a b c

Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia Al-Jeraisy Chair for DNA Research, Zoology Department, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia Zoology Department, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia

a r t i c l e

i n f o

Article history: Received 23 January 2017 Received in revised form 6 April 2017 Accepted 11 May 2017 Available online xxxx Edited by LJ McGaw Keywords: Nigella sativa Cytotoxicity Oxidative stress ROS generation Apoptosis Gene expression

a b s t r a c t Cancer is one of the life-threatening diseases and a leading cause of death worldwide. Herbal medicine has a potential of treating many diseases. Nigella sativa L. is a widely used plant in traditional systems of medicine. The cytotoxic potential of Nigella sativa seed oil (NSO) was assessed by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), neutral red uptake (NRU) assays and morphological alterations in HepG2, MCF-7, A-549 and HEK293 cell lines. Further, the influence of cytotoxic concentrations (50–250 μg/ml) of NSO on oxidative stress markers (GSH and LPO), reactive oxygen species (ROS) generation, mitochondrial membrane potential (MMP) and mRNA expression of apoptotic marker genes (p53, caspase-3, caspase-9, Bax, Bcl-2) were studied. The results exhibited significant decrease in the percentage cell viability of HepG2, MCF-7 and A-549 cells in a concentration-dependent manner. However, NSO showed higher cytotoxic response in HepG2 cells and less in HEK293 cells. Therefore, HepG2 cells were selected to further investigate the underlying mechanism(s) responsible for the cytotoxic response. NSO was found to induce oxidative stress in a concentration-dependent manner, which was indicated by induction of ROS and LPO along with decrease in reduction in GSH and MMP. Quantitative real-time PCR data showed that following the exposure of HepG2 cells to NSO, the level of mRNA expression of apoptotic marker genes (p53, caspase-3, caspase-9 and bax) was upregulated whereas, anti-apoptotic gene bcl-2 was down-regulated. The results demonstrated that NSO induced cytotoxicity and apoptosis in HepG2 cells via ROS generation, which is likely mediated through the p53 pathway. © 2017 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction Although there has been rapid advancement in biomedicine and associate domains, cancer remains a leading and growing cause of suffering and loss of life throughout the world (Stewart and Wild, 2014). Moreover, the incidence of many cancers, lung cancer, breast cancer, and liver cancer were the most common sites of cancer diagnosed in 2012 among men and women (Siegel et al., 2012). Cancer is differentiated by irregular growth of cell, which begin from a small number of inheritance or environmentally mutated genes (Renan, 1993). Each type of cancers is in need of a precise course of treatment that includes one or more modalities such as surgery, radiotherapy, and/or chemotherapy (Galaal et al., 2013; Shylasree et al., 2013). Even though there are limitations, but cancer patients are being successfully treated by the ⁎ Corresponding author at: Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia. E-mail address: [email protected] (N.N. Farshori).

http://dx.doi.org/10.1016/j.sajb.2017.05.019 0254-6299/© 2017 SAAB. Published by Elsevier B.V. All rights reserved.

surgery, radiation therapy and chemotherapy (Espinosa et al., 2003; Hoskin and Ramamoorthy, 2008; Liu, 2009). Although the desired goal of chemotherapy is to get rid of the tumor cells, diverse ranges of normal cell types are also affected, leading to many adverse side effects in multiple organ systems (Nicolson, 2005; Ahles and Saykin, 2007; Zhou et al., 2007; Constantinou et al., 2008; Han et al., 2008; Howes, 2009). Such kind of debilitating effects is a main clinical problem (Johnstone et al., 2002), whereas the toxicity often limits the usefulness of anticancer agents (Johnstone et al., 2002; Kovacic, 2007). Thus, there is a critical requirement to search for anti-cancer drugs with higher efficacy, less toxicity and at an affordable cost (Fadeyi et al., 2013). Natural products have been considered as a valuable source for the anticancer drug discovery (Cragg and Newman, 2005; Svejda et al., 2010; Khan et al., 2011; Randhawa and Alghamdi, 2011; Sharma et al., 2011; Al-Oqail et al., 2013; Farshori et al., 2013; Thoppil et al., 2013; Al-Sheddi et al., 2014). Nigella sativa L. (N. sativa), an annual herb that belongs to the Ranunculaceae family, is used as an important nutritional flavoring agent cultivated in many countries of the world in Southern Europe,

M.M. Al-Oqail et al. / South African Journal of Botany 112 (2017) 70–78

North Africa, Middle Eastern Mediterranean region, India and Saudi Arabia (Mozaffari et al., 2000). It is a natural health remedy, used for the treatment of numerous diseases in traditional folk medicinal systems of Ayurveda, Unani, Chinese and Arabic medicine since ancient times (Randhawa and Alghamdi, 2011). The seeds of N. sativa, commonly known as black seed or black cumin, are used in many food preparations while the oil prepared by compressing the seeds of N. sativa is used for cooking (Al-Khalaf and Ramadan, 2013). Many active ingredients of N. sativa seed have shown beneficial effects against various cancer diseases, including skin (Salomi et al., 1991), colon (Salim and Fukushima, 2003), blood (El-Mahdy et al., 2005), hepatic (Thabrew et al., 2005), fibrosarcoma (Awad, 2005), renal (Khan and Sultana, 2005), prostate (Yi et al., 2008), pancreatic (Chehl et al., 2009), cervical (Effenberger et al., 2010), and breast cancers (Farah and Begum, 2003; Ahmad et al., 2012). These studies demonstrated that N. sativa could be beneficial against the cancer cells. However, molecular mechanism(s) involved in N. sativa seed oil induced cancer cell death are not studied so far. Thus, the present investigation was aimed to explore the anticancer efficacy of N. sativa seed oil against human liver cancer (HepG2), human breast cancer (MCF-7), human lung cancer (A-549) and normal human embryonic kidney (HEK293) cell lines in vitro. 2. Materials and Methods 2.1. Cell culture Human liver cancer (HepG2), human breast cancer (MCF-7), human lung cancer (A-549) and normal human embryonic kidney (HEK293) cell lines obtained from American Type Culture Collection (ATCC; Manassas, VA, USA), were grown in Dulbecco's modified eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 0.2% sodium bicarbonate, and antibiotic/antimycotic solution (1 ml/100 ml of medium, Invitrogen, Life Technologies, USA). The cells were maintained in 5% CO2 and 95% atmosphere at 37 °C. Batches of cells showing more than 98% cell viability were used in the experiments. The cell viability was assessed by trypan blue dye exclusion assay following the reported protocol (Pant et al., 2001).

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mitochondrial membrane potential (MMP), mRNA expression of apoptotic marker genes (p53, caspase-3, caspase-9, Bax and Bcl-2) were studied. 2.5. Cytotoxicity assessment by MTT assay Percentage cell viability was assessed using the 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay following the reported protocol (Mosmann, 1983). Briefly, 10,000 cells were seeded in 96 well plates and were allowed to adhere in CO2 incubator at 37 °C for 24 h. Then, cells were exposed to different concentrations (10–250 μg/ml) of NSO for 24 h. After the exposure, 10 μl of MTT (5 mg/ml of stock) was added in each well and plates were incubated further for 4 h. The supernatant was discarded and 200 μl of DMSO was added in each well and mixed gently. The developed purple color was read at 550 nm. Untreated sets run under identical conditions served as control. 2.6. Cytotoxicity assessment by neutral red uptake (NRU) assay NRU assay was carried out following the protocol of Borenfreund and Puerner (1987). Briefly, 10,000 cells were seeded in 96 well plates and were allowed to adhere in CO2 incubator at 37 °C for 24 h. Then, cells were exposed to different concentrations (10–250 μg/ml) of NSO for 24 h. After the exposure, the medium was aspirated and cells were washed twice with PBS, and incubated for 3 h in a medium supplemented with neutral red (50 μg/ml). The medium was then washed off rapidly with a solution containing 0.5% formaldehyde and 1% calcium chloride. Cells were further incubated for 20 min at 37 °C in a mixture of acetic acid (1%) and ethanol (50%) to extract the dye. The plates were read at 550 nm. The values were compared with the control sets. 2.7. Morphological analysis by phase contrast microscope Morphological alterations in HepG2, MCF-7, A-549 and HEK293 cells exposed to NSO were observed under microscope. All the cells were exposed to 10–250 μg/ml of NSO for 24 h. The cell images were taken using an inverted phase contrast microscope at 20× magnification.

2.2. Reagents and consumables

2.8. Lipid peroxidation (LPO)

All the chemicals, culture mediums, reagents, and kits were procured from Sigma Chemical Company Pvt. Ltd., St. Louis, MO, USA. Culture wares and other plastic consumables used in the study were procured from Nunc, Denmark.

Lipid peroxidation was performed using thiobarbituric acid-reactive substances (TBARS) protocol (Buege and Aust, 1978). Briefly, 1 × 105 cells were seeded in 6 well plates and were allowed to adhere in CO2 incubator at 37 °C for 24 h. Then, cells were exposed to cytotoxic concentrations (50–250 μg/ml) of NSO for 24 h. After the exposure, HepG2 cells were collected by centrifugation. Then, cells were sonicated in ice cold potassium chloride (1.15%) and centrifuged at 3000 × g for 10 min. Resulting supernatant (1 ml) was added to 2 ml of thiobarbituric acid (TBA) reagent (15% TCA, 0.7% TBA and 0.25NHCl) and was heated at 100 °C for 15 min in a boiling water bath. Samples were then placed in cold ice and were centrifuged at 1000 ×g for 10 min. The absorbance of the supernatant was measured at 550 nm.

2.3. Preparation of N. sativa seed oil The N. sativa seeds used in this study were obtained from the local market of Riyadh, Saudi Arabia. The seeds were identified by a taxonomist and a voucher specimen (#13987) was deposited in the Herbarium of the College of Pharmacy, King Saud University, Saudi Arabia. The seeds were screened manually to remove bad ones. The oil from N. sativa seeds was extracted by continuous extraction in Soxhlet apparatus for 12 h using petroleum ether (60–80 °C boiling range) as a solvent according to the method described by AOCS (Horwitz, 1980). At the end of the extraction the solvent was evaporated. The oil thus obtained was dried over anhydrous sodium sulfate and stored at 4 °C for further analysis. 2.4. Experimental design HepG2, MCF-7, A-549 and HEK293 cells were exposed to various NSO concentrations (10–250 μg/ml) for 24 h. Further, the effects of cytotoxic NSO concentrations (50, 100, and 250 μg/ml) on oxidative stress markers (GSH and LPO), reactive oxygen species (ROS) generation,

2.9. Glutathione (GSH) content Intracellular GSH content was estimated as described (Chandra et al., 2002). Briefly, 1 × 105 cells were seeded in 6 well plates and were allowed to adhere in a CO2 incubator at 37 °C for 24 h. Then, cells were exposed to 50–250 μg/ml of NSO for 24 h. After the exposure, HepG2 cells were collected by centrifugation. The cellular protein was precipitated by incubating 1 ml sonicated cell suspension with 1 ml TCA (10%) on ice for 1 h, followed by 10 min centrifugation at 3000 rpm. Then, supernatant was added to 2 ml of 0.4 M Tris buffer (pH 8.9) containing 0.02 M EDTA, followed by an addition of 0.01 M 5,5′-dithionitrobenzoic acid (DTNB) to a final volume of 3 ml. The

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tubes were incubated at 37 °C for 10 min in a shaking water bath. The absorbance of the yellow color developed was read at 412 nm.

the real-time PCR performed in triplicate and data were expressed as the mean of at least three independent experiments.

2.10. Reactive oxygen species (ROS) generation

2.13. Statistical analysis

ROS generation was assessed using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA; Sigma Aldrich, USA) dye as a fluorescence agent following the protocol described (Halliwell and Whiteman, 2004). Following the exposure of NSO (50–250 μg/ml) for 24 h, cells were washed with PBS and were incubated for 60 min in DCFH-DA (20 μM) containing incomplete culture medium in dark at 37 °C. Then, the cells were analyzed for intracellular fluorescence using fluorescence microscope.

Results are expressed as mean ± standard error of three experiments. Statistical analysis was performed using one-way analysis of variance (ANOVA) and post hoc Dunnett's test was applied to compare values between control and treated groups. The values depicting p b 0.05 were considered, as statistically significant. 3. Results 3.1. Cytotoxicity assessment by MTT assay

2.11. Mitochondrial membrane potential (MMP) MMP was measured following the protocol of Zhang et al. (2011). In brief, control and cells exposed to 50–250 μg/ml of NSO for 24 h were washed twice with PBS. Then cells were further treated with 10 μg/ml of Rhodamine-123 fluorescent dye for 1 h at 37 °C in dark. Cells were then washed twice with PBS. The fluorescence intensity of Rhodamine123 was measured using fluorescence microscope by grabbing the images at 20× magnification. 2.12. Quantitative real-time PCRq analysis of apoptotic marker genes Alterations in the mRNA expression of apoptosis marker genes (p53, caspase-3, caspase-9, Bax, and Bcl-2) were studied following the protocol (Kashyap et al., 2010). In brief, cells (1 × 106) were allowed to grow in 6-well culture plates. Then cells were exposed to 50 μg/ml of NSO for 24 h. After the exposure, total RNA was isolated from both experimental and control sets using RNeasy mini Kit (Qiagen) according to the manufacturer's instructions. Concentration of the extracted RNA was determined using Nanodrop 8000 spectrophotometer (Thermo-Scientific) and the integrity of RNA was visualized on 1% agarose gel using gel documentation system (Universal Hood II, BioRad). The first-strand cDNA was synthesized from 1 μg of total RNA by Reverse Transcriptase using M-MLV (Promega) and oligo (dT) primers (Promega) according to the manufacturer's protocol. Quantitative real-time PCR (RT-PCRq) was performed by Light Cycler®480 instrument (Roche Diagnostics, Rotcreuz, Switzerland). Two microliter of template cDNA was added to the final volume of 20 μl of reaction mixture. Real-time PCR cycle parameters included 10 min at 95 °C followed by 40 cycles involving denaturation at 95 °C for 15 s, annealing at 60 °C for 20 s, and elongation at 72 °C for 20 s. The sequences of the specific sets of primer for p53, caspase-3, caspase-9, Bax, Bcl-2 and β-actin used in this study are given in Table 1. Expressions of selected genes were normalized to β-actin gene, which was used as an internal housekeeping control. All

The key results obtained by MTT assay in HepG2, MCF-7, A-549 and HEK293 cells are presented in Fig. 1. A concentration-dependent decrease in cell viability was observed in HepG2, MCF-7, and A-549 cells after NSO exposure for 24 h. The cell viability at 50, 100, and 250 μg/ml NSO concentrations was measured as 44%, 28%, and 18% in HepG2 cells, 46%, 32%, and 24% in MCF-7 cells, 89%, 64% and 49% in A-549 cells and 99%, 95% and 89% in HEK293 cells, respectively. NSO was found less cytotoxic to A-459 and MCF-7 cells as compared to HepG2 cells and no significant cytotoxicity was found in HEK293 cells except at a dose of 250 μg/ml. The IC50 values of NSO obtained by MTT assay were 46.2 μg/ml for MCF-7, 44.6 μg/ml for HepG2, 245 μg/ml for A-549 and 1136 μg/ml for HEK293 cell lines (Fig. 1). 3.2. Cytotoxicity assessment by NRU assay The results obtained using the NRU assay are summarized in Fig. 2. A concentration-dependent decrease in the cell viability of HepG2, MCF-7 and A-549 cells was also observed following 24 h exposure. The percentage cell viability at 50, 100, and 250 μg/ml NSO concentrations was measured as 48%, 33%, and 22% in HepG2 cells, 52%, 39% and 28% in MCF-7 cells, 90%, 68% and 50% in A-549 cells, respectively. However, in HEK293 cells, the percentage cell viability was found as 88% at 250 μg/ml NSO concentration. As in the MTT assay, NSO was found less cytotoxic to A-459 and MCF-7 cells as compared to HepG2 cells and no significant cytotoxicity was found in HEK293 cells except

Cell Lines

MCF-7

HepG2

A-549

HEK293

IC50 Values obtained

46.2 µg/ml

44.6 µg/ml

245 µg/ml

1136 µg/ml

120

MCF-7

HepG2

A-549

HEK293

100 *

*

Table 1 Sequences of primers used for quantitative real-time PCR. Target gene

Forward

p53 5′-CCCAGCCAAAGAAGAAACCA-3′ Caspase-3 5′-ACATGGCGTGTCATAAAAT ACC-3′ Caspase-9 5′-CCAGAGATTCGCAAACCAG AGG-3′ Bax 5′-TGCTTCAGGGTTTCATCCAG-3′ Bcl-2 5′-AGGAAGTGAACATTTCGGT GAC-3′ β-Actin 5′-TCACCCACACTGTGCCCATCT ACGA-3′

Reverse 5′-TTCCAAGGCCTCATTCAGCT-3′ 5′-CACAAAGCGACTGGATGAAC-3′

% Cell Viability

80 *

60 *

**

*

40 ** **

20

** **

0 5′-GAGCACCGACATCACCAAA TCC-3′ 5′-GGCGGCAATCATCCTCTG-3′ 5′-GCTCAGTTCCAGGACCAGGC-3′ 5′-AGCGGAACCGCTCATTGCCAA TGG-3′

Control

10 µg/ml

25 µg/ml

50 µg/ml

100 µg/ml

250 µg/ml

Concentrations Fig. 1. Cytotoxicity assessments by MTT assay in MCF-7, HepG2, A-549, and HEK-293 cells. All the cells were exposed to different concentrations of Nigella sativa seed oil (NSO) for 24 h. Values are the mean ± SE of three independent experiments. *p b 0.05 and **p b 0.01 versus Control.

M.M. Al-Oqail et al. / South African Journal of Botany 112 (2017) 70–78 MCF-7

HepG2

A-549

HEK293

IC50 Values obtained

52 µg/ml

48 µg/ml

250 µg/ml

1041.6 µg/ml

MCF-7

HepG2

A-549 C

100.0

% Cell Viability

3.4. Glutathione depletion

Cell Lines

120.0

HEK293

*

*

80.0 *

60.0

*

3.5. Lipid peroxidation

** **

20.0

**

**

0.0 Control

10 µg/ml

25 µg/ml

50 µg/ml

Fig. 4A summarizes the decrease in the level of intracellular glutathione in HepG2 cells treated with 50–250 μg/ml concentrations of NSO. The results indicate that NSO decreased the glutathione level in a concentration-dependent manner. The decrease in GSH level was observed as 22%, 42%, and 64% at 50, 100, and 250 μg/ml NSO concentrations, respectively as compared to the control.

**

**

40.0

73

100 µg/ml 250 µg/ml

Concentrations Fig. 2. Cytotoxicity assessments by neutral red uptake (NRU) assay in MCF-7, HepG2, A-549, and HEK-293 cells. All the cells were exposed to different concentrations of Nigella sativa seed oil (NSO) for 24 h. Values are the mean ± SE of three independent experiments. *p b 0.05 and **p b 0.01, ***p b 0.001 versus Control.

at a dose of 250 μg/ml. The IC50 values of NSO obtained by NRU assay were 52 μg/ml for MCF-7, 48 μg/ml for HepG2, 250 μg/ml for A-549 and 1041.6 μg/ml for HEK293 cell lines (Fig. 2).

Fig. 4B summarizes the effect on lipid peroxidation level induced by NSO in HepG2 cells. A concentration dependent statistically significant increase in lipid peroxidation was observed. An increase of 14%, 32%, and 47% was recorded at 50, 100, and 250 μg/ml NSO concentrations, respectively.

3.6. ROS generation The results obtained from ROS generation are summarized in Fig. 5. A statistically significant (p b 0.01) concentration-dependent ROS generation was observed in HepG2 cells treated to 50–250 μg/ml NSO concentrations for 24 h. An increase of 145 ± 6.7%, 190 ± 10.5%, and 225 ± 15.7% was observed in ROS generation at 50, 100 and 250 μg/ml NSO concentrations, respectively, as compared to control.

3.3. Morphological changes Fig. 3 shows morphological changes observed in HepG2, MCF-7 and A-549 cells exposed to different concentrations of NSO for 24 h. Morphological changes were observed using phase contrast inverted microscope. The cells exposed to higher doses of NSO lost their normal morphology and shape; these cells appeared more rounded and less adherent than control. The HepG2 and MCF-7 cells indicate the most prominent effects after the exposure of NSO as compared to A-549 cells. However, HEK293 cells exposed to NSO did not cause any observable change in the normal morphology of the cells.

The change observed in the level of MMP are summarized in Fig. 6. The effect of NSO exposure on MMP in HepG2 cells was evaluated. A concentration dependent statistically significant (p b 0.01) decrease in the level of MMP was also observed in HepG2 cells after NSO exposure for 24 h. The MMP level was observed to be 78 ± 6.4%, 54 ± 4.5% and 35 ± 2.8% at 50, 100, and 250 μg/ml NSO concentrations, respectively as compared to untreated control.

50 µg/ml

100 µg/ml

MCF-7 Cells

HepG2 Cells

A-549 Cells

Control

3.7. Mitochondrial membrane potential (MMP)

Fig. 3. Representative images of morphological changes in MCF-7, HepG2 and A-549 cells exposed to NSO at 50 and 100 μg/ml for 24 h. Images were taken using an inverted phase contrast microscope at 20× magnification.

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120

(A)

(B) 180 160

*

80

**

60 40

**

20

% Change in LPO levels

% Change in GSH levels

100

** **

140 120

*

100 80 60 40 20 0

0 Control

Control

50 µg/ml 100 µg/ml 250 µg/ml

50 µg/ml 100 µg/ml 250 µg/ml

Concentrations

Concentrations

Fig. 4. Nigella sativa seed oil (NSO) induced oxidative stress in HepG2 cells exposed for 24 h. [A] Glutathione depletion and [B] Lipid peroxidation level. Values are mean ± SE of three independent experiments. *p b 0.05 and **p b 0.01 versus Control.

3.8. Transcriptional changes in apoptosis markers. The alterations in the levels of mRNA expression of selected apoptosis marker genes in HepG2 cells exposed to N. sativa for 24 h are presented in Figs. 7A–E. A significant (p b 0.05) upregulation in the expression of pro-apoptotic genes, i.e., p53 (2.05 fold), caspase-3 (1.95 fold), caspase-9 (2.2) and Bax (1.85 fold) was observed at 50 μg/ml concentration, whereas, anti-apoptotic gene Bcl-2 (0.45 fold) was significantly downregulated at 50 μg/ml NSO concentrations.

4. Discussion Though many researches have been explored for the advancement to protect cancer diseases, still there is a need to develop new drugs to improve the efficacy. Nigella sativa is one of the famous medicinal plant that has been investigated for its beneficial effects in several experimental and clinical studies (Al-Naggar et al., 2003; Boskabady et al., 2011; Sayeed et al., 2013; Sayeed et al., 2014). It is a grassy plant with larger leaves above and blue or white flowers which bear small black seeds (Datau et al., 2010; Asgary et al., 2015). These seeds of N. sativa can be used as an additive with high nutritional value in several food products such as tea, coffee, and bread. A plethora of literature evidences numerous pharmacological potential of N. sativa and its active ingredient (Elujoba et al., 2005; Cragg and Newman, 2013; Solowey et al., 2014; Tomlinson and Akerele, 2015). These includes, antioxidant

(Burits and Bucar, 2000; Abdel-Wahhab and Aly, 2005), antiinflammatory (Ghannadi et al., 2005; Alemi et al., 2013), hypoglycemic (Salama, 2011; Shafiee-Nick et al., 2012), antidiabetic (Kanter et al., 2009), antihypertensive (Dehkordi and Kamkhah, 2008), antimicrobial and antifungal (Khan et al., 2003; Bakathir and Abbas, 2011) activities. The beneficial effects of N. sativa in the treatment of cardiovascular diseases (Shabana et al., 2013), epilepsy (Ezz et al., 2011), gastrointestinal disorders (Abdel-Sater, 2009), hepatoprotective effects (Meral et al., 2001; Coban et al., 2010), and cancer (Randhawa and Alghamdi, 2011; Al-Sheddi et al., 2014) have also been documented. It reported that the use of conventional cancer preventive drugs that typically target cancer cells is often associated with deleterious side-effects caused by inadvertent drug-induced damage to normal healthy cells (Cassidy and Misset, 2002; Hoskin and Ramamoorthy, 2008). The development of a new anticancer drug that lacks the toxicity of conventional chemotherapeutic agents would be a major advancement in cancer treatment. Bearing this fact in mind, we aimed to examine the anticancer potential of N. sativa against three human cancer cell lines, i.e. liver cancer (HepG2), breast cancer (MCF-7), lung cancer (A-549), and one normal human embryonic kidney (HEK293) cells. Further, oxidative stress (GSH and LPO), reactive oxygen species (ROS) generation, mitochondrial membrane potential (MMP), and mRNA expression of apoptotic marker genes were also studied in liver cancer (HepG2) cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide; MTT and neutral red uptake, and colorimetric assays are commonly used endpoints for the assessment of cell

[A]

(iii)

(ii)

(iv)

300

% Induction in ROS generation

(i)

[B] 250

** **

200

*

150

100

50

0 Control

50 µg/ml

100 µg/ml 250 µg/ml

Concentrations Fig. 5. (A) Nigella sativa seed oil (NSO) induced ROS generation in HepG2 cells. ROS generation was studied using dichlorofluorescindiacetate (DCFH-DA) dye after the exposure of NSO for 24 h. (i) Control; (ii) 50 μg/ml; (iii) 100 μg/ml; and (iv) 250 μg/ml of NSO. (B) Percentage induction in ROS generation in HepG2 cells following the exposure of various concentrations of NSO for 24 h. (*p b 0.01, **p b 0.001 vs Control).

M.M. Al-Oqail et al. / South African Journal of Botany 112 (2017) 70–78

75

[B]

[A] (ii)

(i)

120

% Decrease in MMP level

100

(iv)

(iii)

*

80 60

**

40

**

20 0 Control

50 µg/ml 100 µg/ml 250 µg/ml

Concentrations Fig. 6. (A) Nigella sativa seed oil (NSO) induced reduction in the intensity of mitochondrial membrane potential (MMP) in HepG2 cells exposed for 24 h. MMP was studied using Rh123 fluorescent dye (i) Control; (ii) 50 μg/ml; (iii) 100 μg/ml; and (iv) 250 μg/ml of NSO. (B) Percentage decrease in MMP level in HepG2 cells following the exposure of various concentrations of NSO for 24 h. (*p b 0.01, **p b 0.001 vs Control).

viability. The MTT assay indicates the mitochondrial function based on the enzymatic reduction of a tetrazolium salt by the mitochondrial dehydrogenase of viable cells (Mosmann, 1983) and the neutral red uptake is a measure of lysosomal integrity since it reflects the capacity of viable cells to incorporate vital dye into these organelles (Borenfreund and Puerner, 1987). Since, MTT and NRU assays evaluate different aspects of cellular functions, therefore, they can be useful endpoints to examine the potential cytotoxic effects of natural products. Our results demonstrated, a concentration-dependent decrease in the percentage cell viability in HepG2 and MCF-7 cells after NSO exposure for 24 h. The cytotoxic effects of alcoholic and aqueous extracts of Nigella sativa seed alone or in combination with doxorubicin have also been reported in MCF-7 (Mahmoud and Torchilin, 2013). However, less cytotoxicity in A-549 cells and non-cytotoxic activity towards HEK293 cells. The

[A]

[B] 2.5

*

Fold change

2

Fold change

[C]

2.5

1.5 1

0.5 0 Control

50 µg/ml

1.5

2

1.5

1

1

0.5

0.5

0

0 Control

Control

50 µg/ml

[D]

50 µg/ml

[E]

2.5

1.2 *

1 Fold change

2

Fold change

*

*

2

Fold change

2.5

cytotoxic response of NSO was found more in HepG2 cells, followed by MCF-7 and A-549 cells. These kinds of differential cytotoxic response in various cancer cells has been previously reported (Mishra et al., 2013; Sak, 2014), showing this effects due to the specificity of plant towards different cancer cells (Al-Oqail et al., 2016). These cytotoxic responses of NSO showed promising anti-cancer activity. Since, the highest cytotoxic response was found in HepG2 cells, we selected HepG2 cells to further investigate the underlying mechanism (s) responsible for the cytotoxic response. Previous studies showed that the treatment of natural products promotes oxidative stress in the cells, such as induction in lipid peroxidation and depletion in glutathione levels (Grigutytė et al., 2009; Abdullah et al., 2015). In this study, a concentration dependent increase in lipid peroxidation and depletion in glutathione level have been observed after 24 h NSO exposure in HepG2 cells. The results

1.5 1

0.5

0.8 0.6

*

0.4 0.2

0 Control

50 µg/ml

0 Control

50 µg/ml

Fig. 7. Real Time-PCR analysis of alterations in the mRNA expression of various apoptosis genes in HepG2 cells exposed to Nigella sativa seed oil for 24 h. (A) p53, (B) Caspase-3, (C) Caspase-9, (D) Bax and (E) Bcl-2. The data provided are mean ± SE from three separate experiments. *p b 0.05 versus control.

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obtained from this study suggest that oxidative stress may be the primary mechanism of the cell death in HepG2 cells. Our results also support previous findings, which suggest the role of oxidative stress in the cell death (Tor et al., 2014: Farshori et al., 2015). Induction in the reactive oxygen species (ROS) generation is one of the common causes of apoptotic cell death in cancer cells (Liou and Storz, 2010). Our results showed an increase of 145 ± 6.7%, 190 ± 10.5%, and 225 ± 15.7% in ROS generation at 50, 100 and 250 μg/ml, respectively, as compared to control. These findings are well supported by previous reports, where the induction in the ROS generation have been documented due to the exposure of natural products (Karimi et al., 2010; Ghali et al., 2014). A concentration dependent statistically significant (p b 0.01) decrease in the level of MMP was also observed in HepG2 cells after the exposure of NSO for 24 h. It is reported that that high level of reactive oxygen species generation can lead to mitochondrial damage, which can result in cell damage (Siddiqui et al., 2015). The decrease in the MMP level indicate the role of oxidative stress and ROS generation in cell death of HepG2 cells treated with N. sativa seed oil for 24 h. Mitochondria-dependent apoptotic pathway is also known to be involved in cell death (Wang et al., 2011; Djafarzadeh et al., 2012). The mitochondrial pathway has also been implicated in the function of a majority of anticancer drugs (Fogg et al., 2011; Indran et al., 2011; Goh et al., 2014). Alterations in the p53 gene are the most frequent genetic change in human cancers and plays an important role in the regulation of apoptosis by the interaction of p53 protein with mitochondria and promote mitochondrial membrane permeabilization (Moll et al., 2005; Yu and Zhang, 2005). The Bax gene has been found to be a transcriptional target of p53 and could be up-regulated in response to a variety of p53- dependent apoptosis triggers (McGill and Fisher, 1997). The up-regulation in p53 promote induction in the expression of pro-apoptotic Bax, whereas down-regulation in the expression of antiapoptotic Bcl-2 protein. Therefore, this imbalance in the ratio of Bax/Bcl-2 lead the dysfunction in the mitochondria and release cytochrome-c in cytosol, activate caspase-9 which, triggers the activation of caspase-3 and/or caspase-7 (Cain et al., 1999; Zou et al., 1999). In the current study, we measured the mRNA expression of various mitochondrial apoptosis genes at transcriptional level. Our results of mRNA expression of apoptosis genes indicate upregulation of p53, Bax, caspase-3 and caspase-9 and downregulation in Bcl-2 genes. All these results suggest that NSO induced apoptosis in HepG2 cells through mechanisms involving mitochondriadependent pathway. The results reported in this study have proven the crucial role of mitochondrial apoptotic pathway in antitumor efficacy of various natural products in experimental hepatocarcinogenesis (Bishayee and Dhir, 2009; Bishayee et al., 2011; Bhatia et al., 2013). Similar kind of mitochondrial mediated cell death pathway has been also observed with the various phytoconstituents (Haridas et al., 2001), including HepG2 cells (Bhatia et al., 2015).

5. Conclusion In conclusion, the present study demonstrated that Nigella sativa seed oil suppresses cell proliferation of cancer cells in a concentrationdependent manner. NSO was also found to induce ROS dependent mitochondrial apoptosis through p53 pathway in hepatocellular carcinoma cells. Apoptosis was found to be associated with oxidative stress and mitochondrial membrane potential. The mRNA expression of apoptotic gens (p53, caspase-3, caspase-9 and Bax) were up-regulated and the anti-apoptotic gene Bcl-2 was down-regulated. This study provides differential cytotoxic responses of NSO against HepG2, MCF-7 and A-549 cells. Our results suggest that N. sativa seed oil has promising anticancer potential and could be considered as a promising chemotherapeutic agent to treat the cancer.

Conflict of interest The authors hereby declare that there are no conflicts of interest. Author contributions NNF and MAS conceived of and designed the experiments. MMA, NNF, JA and MAS performed the experiments. MAS, NNF, ESA, SMA and JM analyzed the data. ESA and AAA contributed reagents/ materials/analysis tools. NNF and MAS wrote the paper. All authors read and approved the final manuscript. Acknowledgement The authors are grateful to the Deanship of Scientific Research, King Saud University for funding through Vice Deanship of Scientific Research Chairs. References Abdel-Sater, K.A., 2009. Gastroprotective effects of Nigella sativa oil on the formation of stress gastritis in hypothyroida rats. International Journal of Physiology, Pathophysiology and Pharmacology 1, 143–149. Abdel-Wahhab, M., Aly, S., 2005. Antioxidant property of Nigella sativa (black cumin) and Syzygium aromaticum (clove) in rats during aflatoxicosis. Journal of Applied Toxicology 25, 218–223. 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