Nigella sativa extract chemoprevention in oral cancer: in vivostudy.
Unfortunately, the survival rate of oral cancer has remained 50% or less over the last three decades [1,2], although treatment at an early stage may increase the survival rate to above 80% . At the present, many researchers believe that medicinal plants are promising alternative therapeutics against cancer [4-6]. More studies should be performed within this context because a number of these plants are safe and promising anticancer agents. Nigella sativa (NS) or black cumin is an herb of the Ranunculaceae family that is commonly used as a spice and food preservative, particularly among Eastern countries [7,8]. The black seed composition is a mixture of proteins, carbohydrates, alkaloids, oils and other compounds. The active ingredients of NS were found to be effective against many diseases. A review of the literature revealed that extracts of NS and its active ingredient thymoquinone (TQ) have an anticancer effect using different mechanisms. However, these results were inconsistent and exhibited considerable controversy. In 2007, Ait et al. reported that both black seed oil and its ethyl extract demonstrated cytotoxic properties against a P815 cell line. Similarly, the extracts were tested on different cell lines [11-14], but the cytotoxic results varied according to the type of tumor cells. Moreover, diet supplementation with both honey and NS exhibited an anti-carcinogenesis effect in methylnitrosourea-induced lung, skin and colon cancers . However, Rooney and Ryan(2005)  failed to report a cytotoxic effect or apoptosis in lung carcinoma and larynx epidermoid carcinoma cells with [alpha]-hederinor thymoquinone (TQ), the active components of NS. Furthermore, an injection of the NS essential oil into the hrmor site of a DBA2/P815 (H2d) mouse model improved mouse survival by significantly inhibiting tumor development and the incidence of liver metastasis [9,10].
Studies investigating the effect of NS extract (NSE) or TQ on oral cancer have been limited. A recent shrdy demonstrated that TQ, orally taken at a dose of 30 mg/kg body weight, reduced tumor formation in 9,10-dimethyl-1,2- benzanthrancene (DMBA)-painted hamster buccal mucosa . Application of 0.5% DMBA on hamster cheek pouches 3 times weekly over a 14-week period is a well-known carcinogenesis protocol . However, 5-week DMBA application can be considered to be a premalignant stage of oral cancer, where it has been demonstrated to increase in tetraploidy or near-tetraploidy karyotypes starting from the second week of DMBA painting [16,17]. To the best of our knowledge, there has been no published study determining the effect of NS extract or TQ on the precancerous stage.
The combination of cancer treatments has received increased attention due to its potential to enhance the therapeutic effect and reduce toxicity by lowering the dose required for each agent . Cisplatin is a commonly used chemotherapeutic drug in oral cancer treatment. Unfortunately, its long-term use is associated with many side effects, complications and drug resistance . In the present study, we generated oral carcinogenesis in the hamster cheek pouch using DMBA to test the hypothesis that oral intake of NS extracts has a chemo-preventive effect in oral cancer and could inhibit malignant transformation at the premalignant stage. We also tested the hypothesis that the combination of cisplatin and NSE may result in a more pronounced in vivo anticancer effect in oral cancer growth compared to either agent alone.
Ethical approval for this research study was obtained from the ethical committee of King Abdulaziz University Faculty of Dentistry, Jeddah, Saudi Arabia.
Fifty 8- to 10-week-old golden Syrian male hamsters weighing 80 to 120 g were purchased from King Saud University Animal House in Riyadh and maintained in the King Fahad Research Center Animal House. The hamsters were kept under controlled conditions in cages and provided witha standard pellet diet and water ad libitum. The animals were randomly distributed into 10 groups with 5 hamsters per group.
Group 1: served as controls and received saline;
Group 2: received oral Nigella Sativa extract (NSE) (CarumCarvi extract. Batch no# DV8131,Elixir extract private limited, Kinfra Park, Nellad, India) at a dose of 30 mg/ kg body weight.
The animals in the remaining 8 groups were painted with 0.5% 9,10-dimethyl-1,2-benzanthracene (DMBA) (D3254-100 MG, Sigma Aldrich) in liquid paraffin on their left buccal pouches (using paint brush no. 4) three times weekly for 6 weeks (groups 3, 5, 7 and 9) or 14 weeks (groups 4, 6, 8 and 10).
Groups 3 and 4 received no further treatment in addition to DMBA;
Group 5: received DMBA for 6 weeks followed by oral NSE at 30 mg/ kg body weight 3 times/week;
Group 6: received DMBA for 14 weeks + oral NSE 30 mg/ kg body weight on alternating days.
Group 7: received DMBA for 6 weeks followed by intraperitoneal Cisplatin (CIS) injection (Ebewe, 50mg/100ml) at a dose of 35.0 pg/kg body weight weekly;
Group 8: received DMBA for 14 weeks +CIS intraperitoneal at a dose of 35.0 pg/ kg body weight weekly;
Group 9: received DMBA for 6 weeks followed by CIS 8.0 [micro]g/kg intraperitoneal once a week and oral NSE 30 mg/ kg body weight 3times/week;
Group 10: received DMBA for 14 weeks + oral NSE 30 mg/ kg body weight on alternating days + 8.0 [micro]g/kg body weight intraperitoneal CIS weekly.
The weights of all hamsters were recorded on a weekly basis throughout the experimental period. At the end of the study, a retro-orbital blood sample (3ml) was obtained from each animal in each group for complete blood counts, liver enzymes analysis (aspartate transaminase (AST) and alanine transaminase (ALT) using the IFCC method without pyridoxal phosphate (P-5'-P) (Kinetic UV) and kidney functions tests (Creatinine and Urea: Enzymatic- Colorimetric- Kinetic) using an ELITech- Flexor EL200 machine (Clinical Systems).
The experiment ended on the 14th week, and the animals were sacrificed by cervical dislocation. Their cheek pouches were excised for histopathological and immunohistochemical study.
The specimens were fixed in 10% formalin, embedded in paraffin blocks and cut into 4-[micro]m thick sections. Serial sections were stained with hematoxylin and eosin (H&E). Histopathological grading of epithelial dysplasia and squamous cell carcinoma(SCC) was performed according to 2005 classification .
Sections measuring 4 [micro]min thickness were mounted onto positively charged slides and deparaffinized by overnight incubation with xylene. Next, the sections were rehydrated in gradual descending concentrations of ethanol followed by a phosphate-buffered saline (PBS) wash. Blockade of endogenous peroxidase activity was performed using 3% hydrogen peroxide ([H.sub.2][O.sub.2]) for 5 minutes at room temperature. For antigen retrieval, tissue sections were placed into glass jars containing 0.01M sodium citrate buffer (pH 6.0) and boiled in a microwave oven twice for 5 minutes to enhance immunoreactivity (reserve the loss of antigenicity that occurred with some epitopes of formalin-fixed paraffin-embedded tissues).The slides were allowed to cool and rinsed with PBS, pH 7.2. Immunohistochemical staining for caspase-3 and ki-67 antibodies was performed according to the manufacturer's instructions (Thermo Scientific, USA).
Detection was performed using a universal kit (DAKO, Denmark) by washing the slides in PBS for 5 minutes and incubation with secondary antibody (biotinylated goat serum conjugated rabbit and mouse sera) for 30 minutes. The sections were then washed for 5 minutes in PBS followed by antigen-antibody visualization viadiaminobenzidine [DAB] in PBS containing 40% [H.sub.2][O.sub.2]. Sections were washed under running tap water for 10 minutes, counterstained with Mayer's hematoxylin, and then mounted.
Immunoreactivity for caspase-3 and ki67 was evaluated by estimating the area percentage of positive immune-stained cells in relationship to the area examined in each field using a Leica image analyzer computer system (Germany)controlled by Leica Qwin 500 software. The image analyzer wasautomatically calibratedto convert the measurement units pixels) produced by the image analyzer program into actual micrometer units. The area and area percentage of the reactive areas were measured with reference to a standard that measured the frame of the area 11434.9 [micro][m.sup.2] using a magnification (x 200). Using color detection, the reactive areas of positive immunostaining were masked by a blue binary color. Ten successive fields per slide were measured histomorphometrically. The mean values were obtained for each specimen for statistical analysis.
Statistical analysis was performed using SPSS (version 22). Scale variables were described by the mean, standard deviation (SD), standard error (SE), skewness, range (maximum--minimum) and 95% confidence interval for mean. Repeated measures tests were performed for statistical analysis of changes in weights. Normality tests were also performed such that the one-way analysis of variance (ANOVA) test could be used to compare the means of all groups. A Post-Hoc LSD-test was then performed to compare each group to the control group. In addition, the Kruskal-Wallis test was used to compare the medians of the ordinal variables of all groups. Significance was established at p-value < 0.05 (significant); while P < 0.01 and P < 0.001 were considered highly significant.
The growth rates of rats in all of the studied groups were constant during the experiment with no significant differences among the groups (P > 0.05). One hamster from each of the control group and Groups 6 and 9, as well as 2 animals from Groups 3 and 4 were moribund prior to the date of sacrifice. Thus, their data were not included in the study. Overt lesions were observed in DMBA-treated hamsters, whereas no lesions were observed in the control group.
Light Microscopic Analysis Results:
As shown in Figures (1 & 2):
Group 1 (control):
The epithelial lining of hamster buccal pouch (HBP) mucosa showed regular thickness of two to three cell layers. The connective tissue showed organized collagen bundles with a muscle layer and was free of inflammatory cells.
Group 2 (NSE):
The epithelial lining of HBP mucosa showed normal regular thickness with only few cells exhibiting apoptosis.
Group 3 (DMBA 6 weeks):
The epithelial lining of HBP mucosa showed either focal or diffuse hyperplasia with the formation of rete pegs. Marked supra-basal apoptosis was observed. Three cases showed focal atypia as basilar hyperplasia and hyperchromatism. The connective tissue revealed marked hyperemia.
Group 4 (DMBA 14 weeks):
The epithelial lining of HBP mucosa showed moderate to severe dysplasia with increased cellularity of dysplastic cells with obvious features of loss of adhesion of spinous cells, pleomorphic and hyperchromatic nuclei, abnormal mitosis and individual cell keratinization. Well-differentiated SCC with the invasion of islands of dysplastic epithelial and formation of keratin pearls was observed in 2 cases. Connective tissue showed a varying degree of hyperemia and the destruction of collagen bundles and muscle layer with inflammatory cell infiltration.
Group 5 (DMBA 6 weeks & NSE):
The epithelial lining of HBP mucosa showed focal areas of hyperplasia with marked suprabasal apoptosis. Two cases showed basilar cells atypia.
Group 6 (DMBA 14 weeks & NSE):
Generally, the epithelial lining of HBP mucosa among the animals in this group showed hyperplasia and mild dysplasia with basilar hyperplasia and pleomorphic hyperchromatic nuclei. Degeneration of connective tissue collagen fibers was observed. Only one animal within this group developed well-differentiated SCC.
Group 7 (DMBA 6 weeks & CIS):
The epithelial lining of HBP mucosa showed normal regular thickness with only focal areas of hyperplasia and suprabasal apoptosis.
Group 8 (DMBA 14 weeks & CIS):
The epithelial lining of HBP mucosa showed hyperplasia with mild dysplastic features as basilar hyperplasia and pleomorphic hyperchromatic nuclei, as well as marked suprabasal apoptosis.
Group 9 (DMBA 6 weeks & NSE + CIS):
The epithelial lining of HBP mucosa showed normal regular thickness with only focal areas of hyperplasia and suprabasal apoptosis.
Group 10 (DMBA 14 weeks & NSE + CIS):
The epithelial lining of HBP mucosa showed focal areas of hyperplasia, and 2 cases showed mild dysplastic changes and suprabasal apoptosis.
When the degree of dysplasia was analyzed in the studied groups, as shown in Figure (3), Groups (1 and 2), as well as the groups receiving DMBA for 6 weeks + CIS or + CIS + NSE (Groups 7 and 9), revealed no dysplasia. However, the groups receiving DMBA for 6 weeks without other treatment (Group 3) and DMBA for 14 weeks + CIS or + CIS + NSE (Groups 8 and 10), showed mild dysplasia in 100% of the included animals. In Group 5, where the animals received DMBA for 6 weeks + NSE, 40% of the animals showed no dysplastic changes, while 60% of the animals showed mild dysplasia. The severity of dysplasia was highest in Group 4 (DMBA for 14 weeks without any additional treatment) as 66.7% showed well-differentiated SCC development and the remaining 33.3% showed severe dysplasia. In Group 6 (DMBA for 14 weeks + NSE) only 1 hamster (representing 25% of surviving animals) developed SCC, while the remaining 75% had only mild dysplasia. When the Kruskal-Wallis test was applied, it demonstrated a highly significant difference for severity between the groups, with a p-value < 0.0001 being observed.
A. Caspase-3 iiiunostaining:
The epithelial lining of HBP mucosa of G1 showed cytoplasmic immunostaining of suprabasal cells. Similar results were obtained for mucosa of G2. While HBP mucosa of G3 showed positive immunostaining of all spinous cells but negative staining for basal cells, the connective tissue showed immunoreaction to caspase-3. The moderate and severe dysplastic epithelium of G4 showednegative basal and parabasal cell layers, while superficial cells showed cytoplasmic immune positivity. An immune reaction was found only in keratin pearls of well-differentiated SCC. Immuno-expression was also observed in connective tissue cells. HBP mucosa of G5 showed cytoplasmic immunostaining of suprabasal cells (Fig. 4).
HBP mucosa of G6 showed cytoplasmic immunostaining of basal and spinous cells, and an immune reaction in connective tissue cells was also observed. HBP mucosa of G7 showed cytoplasmic immunostaining of superficial spinous cells, and faint immune reaction was observed in connective tissue cells. HBP mucosa of G8 showed cytoplasmic immunostaining of spinous cells, while basal cells in certain areas exhibited negative immunostaining, and a weak immune reaction was observed in connective tissue cells. HBP mucosa of G9 exhibited intense cytoplasmic immunostaining of spinous cells. HBP mucosa of G10 demonstrated cytoplasmic immunostaining of spinous cells and connective tissue cells (Fig.5).
B. Ki-67 imumnostaining:
Normal HBP mucosa of G1 demonstrated nuclear immunostaining of a small number of basal cells, and similar findings were obtained with HBP mucosa of G2. Mild dysplasia of HBP mucosa of G3 exhibited nuclear immunostaining of basal and parabasal cells. Severe dysplasia of HBP mucosa of G4 showed nuclear immunostaining of basal and higher levels of spinous cells while SCC showed nuclear staining of spinous cells and invasive islands. HBP mucosa of G5 showed cytoplasmic immunostaining of few basal cells and parabasal cells (Fig.6). HBP mucosa of G6 showednuclear immunostaining of basal and parabasal cells, HBP mucosa of G7 showed nuclear immunostaining of basal and a few parabasal cells, HBP mucosa of G8 demonstratednuclear immunostaining of basal and a few parabasal cells, HBP mucosa of G9 exhibited nuclear immunostaining of a few basal cells, and HBP mucosa of G10 showed nuclear immunostaining of basal cells (Fig.7).
As shown in Table (1), the area % of caspase 3 immunostaining revealed a highly significant difference in the control group compared to the 6-week DMBA, DMBA 6+NSE, DMBA 6+CIS and DMBA 6+CIS +NSE groups (P < 0.001). However, when the 6-week DMBA group was compared with the last 3 groups, no significant difference was found (P > 0.05). When groups receiving DMBA for 14 weeks were compared (Table 1), the same results were obtained. However, when the area % of Ki-67 was considered, the comparison between DMBA for 6 weeks + CIS + NSE and NSE alone did not reveal a statistically significant difference. Conversely, differences between the DMBA 6 group and groups receiving treatment were statistically significant, thereby revealing a significant decrease in Ki-67 area % in DMBA 6 + CIS and DMBA 6 + NSE (P < 0.05) and a highly significant decrease in DMBA 6 + CIS + NSE (P < 0.001) (Table 2). When the Ki-67 area % was considered in groups receiving DMBA for 14 weeks, all DMBA groups, independent of treatment, showed a highly significant increase in Ki-67 area % compared to both the control group and group receiving NSE alone (P < 0.001). A comparison of the group receiving DMBA alone to groups receiving DMBA in addition to other treatment revealed a highly significant decrease in area % in DMBA 14 + NSE and DMBA 14 + CIS + NSE (P < 0.001), whereas DMBA 14 + CIS showed only a statistically significant decrease (P < 0.05) (Table 2).
Kidney and liver function analyses:
As shown in table (3), when all of the studied groups were statistically analyzed with regard to kidney and liver functions, the differences did not reach the level of significance. However, when the actual values were evaluated, we found that the highest levels of urea and creatinine were registered by groups receiving CIS, independent of DMBA administration for 6 or 14 weeks, whereas the NSE groups showed the lowest levels. In addition, when both were administered to the animals, the levels were lower than those for CIS alone. For example, the urea levels in DMBA 6 + CIS ranged from 147 to 245 units with a mean of 183.4 [+ or -] 47.29, whereas in DMBA 6 + NSE, it ranged from 105 to 149 units with a mean of 120.67 [+ or -] 24.58 and in DMBA 6 + CIS + NSE, it ranged from 88 to 173 units with a mean of 134 [+ or -] 42.93. In DMBA 14 + CIS, it ranged from 125 to 204 units with a mean of 164.5 [+ or -] 55.86. In DMBA 14 + NSE, it ranged from 113 to 141 with a mean of 125 [+ or -] 14.23. In DMBA 14 + CIS + NSE, it ranged from 147 to 162 units with a mean of 154 [+ or -] 7.55. Similarly, if liver function is considered, the aminotransferase levels registered with CIS were higher than those with NSE alone or combined with CIS. For example, ALT levels in groups DMBA 6 + CIS and DMBA 14 + CIS ranged from 71 to 100 units with a mean of 85[+ or -]11.14 and from 68 to 159 units with a mean of 100.6[+ or -]43.97, respectively. When NSE was added, ALT levels in Groups DMBA 6 + CIS +NSE and DMBA 14 + CIS + NSE ranged from 61 to 73 units with a mean of 66.67[+ or -]6.03 and from 66 to 114 units with a mean of 90[+ or -]33.94, respectively.
Complete blood count (hemogram) results:
As shown in table (3), no statistically significant difference was observed in any of the blood elements studied in the different groups.
The Syrian golden hamster buccal pouch (HBP) carcinogenesis model was selected for the present investigation because it is considered to be the closest animal model that mimics the events occurring during the development of carcinogenesis in human oral tissues. Furthermore, this hamster represents a unique model for squamous cell carcinomas . According to Morris (1961) , the cheek pouch mucosa of younger hamsters is more responsive to the effects of carcinogenic agents than those of older animals, thereby recommending the use of hamsters ranging in age from 3 to 9 weeks. However, as previously described by the same author, hamsters are difficult to manipulate before five weeks of age. Thus, the hamsters used in the present study were 8 to 10 weeks old. Moreover, the concentration of carcinogen (9,10-dimethyl-1,2-benzanthracene; DMBA) used was 0.5% in mineral oil, which is the optimal concentration suggested for rapid carcinogenesis in the hamster cheek pouch. This concentration has been reported to generate the maximum tumorigenic effect without a prolonged latent period or toxic effects on the experimental animals. It was applied 3 times/week to ensure a shorter latent period for tumor development compared to animals receiving the carcinogen twice weekly . DMBA, a potent organ and site-specific carcinogen, is known to induce its carcinogenic effect in a sequence of definitive steps, starting with hyperplasia and followed by dysplasia, thereby mimicking the progression of tumors arising in oral cancer patients . Hyperplastic changes have been identified within a period of 1 to 4 weeks, and dysplasia appears at 6 to 8 weeks followed by papillomatous lesion formation at approximately 8 to 10 weeks. Early invasive carcinomas are evident at 11-13 weeks, whereas SCCs are well-developed by the 14th to 16th weeks. The tumors then gradually invade the surrounding tissues and metastasize to regional lymph nodes after 16 weeks of the carcinogen application. Consequently, DMBA-induced hamster buccal pouch carcinogenesis serves as an ideal model for the study of chemoprevention in oral cancer [24, 25].
In the present investigation, when the severity of dysplastic changes, as revealed by histopathological examination, was compared among the studied groups, the group receiving DMBA for 6 weeks showed mild dysplasia in 100% of cases, whereas the addition of NSE to the same DMBA protocol resulted in only 60% mild dysplasia and 40% without dysplasia, thereby revealing the protective or chemo-preventive effect of NSE against oral cancer development. NSE has been previously shown to prevent cancer development. It has been reported that its topical application inhibited the progression of two-stage initiation of skin cancer by dimethylbenzo[a] anthracenecroton oil in mice . They also found that intraperitoneal injections of NSE significantly reduced the incidence of sarcoma development after 30 days of subcutaneous 20-methylcholanthrene (MCA) injections. Moreover, the present results also showed that the worst results were observed among the group receiving DMBA for 14 weeks, where 66.7% carcinoma and 33.3% severe dysplasia was observed. When NSE was administered in addition to the same DMBA protocol, there was only 25% carcinoma and 75% mild dysplasia. Indeed, previous in vitro studies have suggested that NSE, via its constituents TQ and [alpha]-hederin, may exhibit anti-neoplastic activity [13, 27]. TQ has been reported to be active against acute lymphoblastic leukemia cells in vitro, which has been attributed to effects on specific epigenetic mechanisms that act as causative and maintenance factors for cancer development . TQ has been successful against other various cancer models in vitro and in vivo . Furthermore, [alpha]-hederin has been isolated in one study and was determined to be a significant contributor to the anticancer activity of NSE . Thabrew et al. (2005)  reported that aqueous NSE was active against hepatocellular carcinoma cells in vitro. More recently, Al-Sheddi et al. (2014)  demonstrated a significant inhibitoiy effect for NSE against human lung cancer cells. Furthermore, in addition to its direct antineoplastic activity against various types of cancer cells, aqueous NSE can also enhance the anticancer activity of natural killer (NK) cells , thereby upgrading immunity against cancer in vivo. Moreover, TQ has been reported to exhibit an anti-angiogenic effect via inhibition of the NF-Kappa B pathway and suppression of AKT and ERK signaling pathways, hindering tumor growth via angiogenesis limitation .
A synergistic effect appeared in the combination between NSE and CIS, where the group receiving DMBA for 6 weeks in addition to 8.0 [micro]g/kg CIS + NSE had 100% lack of dysplasia, equivalent to the group receiving DMBA for 6 weeks in addition to 35 [micro]g/kg CIS alone. Also, in the groups receiving DMBA for 14 weeks, adding NSE to 8.0 [micro]g/kg CIS gave the same results (100% mild dysplasia) as when adding 35 [micro]g/kg CIS alone. Thus, the results of the present study provide an in vivo confirmation of the previously mentioned synergism between TQ and cisplatin, where Attoub et al. (2013)  found that such combination caused greater inhibition of LNM35 lung cancer cell viability compared to each drug administered alone.
The results of the present study showed a highly significant difference in % area of caspase-3 immunostaining between control and treated groups, independent of the application of DMBA (for 6 or 14 weeks) alone or in combination with NSE, CIS or NSE+ CIS, indicating a significantly elevated rate of apoptosis in the treated groups. When the groups receiving DMBA alone (for 6 or 14 weeks) were compared with the corresponding groups receiving NSE, CIS or NSE+ CIS, there was no significant difference between the groups. Apoptosis is a genetically programmed form of cell death, of which caspases are central components  Apoptosis is transduced by two major pathways- the death receptor or extrinsic pathway, and the mitochondrial or intrinsic pathway, resulting in the activation of caspases that eventually execute a coordinated proteolysis program to destroy critical cell structures [36,37]. In addition, inhibitors of apoptosis proteins (IAPs), including survivin and X chromosome-linked IAP (XIAP), also regulate apoptosis  Development of HBP tumors is associated with increased cell proliferation, as well as reduced apoptosis of genetically damaged cells; however, due to the increase in cell number, these apoptotic figures are present at higher levels compared to the normal epithelium  Blanc et al. (2000) showed that caspase-3 was essential for cisplatin-induced apoptosis. In addition, Chu et al. (2014)  found that TQ induced cell death in oral cancer cells via two different mechanisms, one of which is apoptosis. Paramasivam et al. (2012)  and Peng et al. (2013)  reported that TQ present in NSE induced apoptosis via caspase-3 activation and down-regulation of XIAP. A similar finding was reported by Alhazmi et al. (2014)  when they tested the effect of NSE on breast cancer cell line. Consequently, all treated groups had an underlying cause for the elevated apoptosis, resulting in the lack of statistical significance. Conversely, the results obtained using the proliferation marker Ki-67 yielded different results. Although there was a statistically significant difference between the DMBA-treated groups and each of the groups receiving NSE, CIS or NSE + CIS in addition to the carcinogen, the synergism between NSE and CIS was evident, where the level of significance was higher in the group receiving NSE + CIS compared to the group receiving CIS alone. These results are also consistent with findings obtained by Jafri et al. (2010)  who reported that TQ (one of the main constituents of NSE) enhanced the inhibitoiy effect of CIS on NCI-H460 lung cancer cell proliferation, as well as tumor growth. Thus, the synergistic effect between NSE and CIS seems to reside in their anti-proliferative activities, not their apoptotic effects.
One of the most important results in the present investigation is that the administration of NSE to hamsters did not affect liver or renal functions or the animals' hemogram. Jafri et al. (2010)  previously demonstrated that TQ does not induce mortality or any pathological changes in the lung, heart, or kidneys. Furthermore, Badaiy et al. (1997)  reported that TQ, which is a major ingredient in NSE, reduced the toxic effects of the anticancer drug cisplatin. It was reported to prevent cisplatin-induced nephrotoxicity via a decrease in MDA, 8- isoprostane, multidrug resistance-associated proteins and increasing organic cation transporters.  In addition, administration of NSE concomitant with cisplatin administration, was found to minimize its nephrotoxic effects as demonstrated by a clear reduction in the biochemical and physiological indices of nephrotoxicity . The protective action of NSE was attributed to its antioxidant action, which helps to buffer the free radicals generated by the drug, which are harmful to the kidneys . This finding was consistent with our present results, where tests on kidney function revealed lower values in the NSE + CIS-treated group compared to the CIS-treated group; however, this difference did not reach statistical significance. Good hepatic function of experimental animals receiving NSE observed in the present study was also consistent with previous reports described by Zafeer et al. (2012) . Mariod et al.(2009)  also reported that NSE was found to protect the liver from oxidative stress by increasing the activity of some enzymes, including myeloperoxidase, glutathione-S- transferase and adenosine deaminase, as well as decreasing hepatic lipid peroxidation. Hassan et al. (2013)  revealed that there was no significant change in liver hmction tests as a result of supplementing the rats' diet for 28 days with powdered NS. Histopathological studies have shown minimal and mild changes of liver fatty degeneration in normal and high doses (1 g/kg) in NS-treated groups, while inflammation and necrosis were not observed. However, treatment of rats with NSE for up to 12 weeks has been reported to induce changes in their blood picture, in the form of increased packed cell volume (PCV) and hemoglobin (Hb) . However, in the present investigation, treatment with NSE did not produce any significant changes in blood picture.
On the basis of our experimental findings, we propose that NSE might have a clinical benefit as chemopreventive agent in oral cancer, particularly when combined with some chemotherapeutic agents, such as cisplatin, where it canprevent the progress of mild dysplasia to frank SCC. It also significantly potentiates the anticancer activities of the concomitantly administered drug, helpsto reduce its dose and nullifiesits toxic side effects on normal body organs and cells.
The authors declare that no competing financial interests exist.
Received 5 August 2015
Accepted 20 September 2015
Available online 30 September 2015
The authors would like to express their deep gratitude to Abdulaziz Mohammed Banasser, Khabbab Khalid Bakhsh, Ali Sulaiman Arab, Ammar Talaljijawi, Faculty of Dentistty, KAU, for their help during the experimental work. The work was done in Stem Cell Unit- King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia and was funded through scientific research council of King AbdulAziz University, Project Code 2-165-35-RG. .
 Speight, P.M. and P.R. Morgan, 1993. The natural history and pathology of oral cancer and precancer. Community Dent Health, 10(suppl 1), 31-41.
 The Oral Cancer foundation 2015. Oral Cancer Facts. 2015; Available at: http://www.oralcancerfoundation.org/facts/. Accessed: February 2015.
 Warnakulasuriya, S., 2009. Global epidemiology of oral and oropharyngeal cancer. Oral Oncology, 45: 309-16.
 Pratheeshkumar, P., C. Sreekala, Z. Zhang, et al., 2012. Cancer Prevention with Promising Natural Products: Mechanisms of Action and Molecular Targets. Anti-Cancer Agents in Medicinal Chemistry, 12: 1-26.
 Menon, K.C., 2014. Optimizing nutrition support in cancer care. Asian Pac J Cancer Prev., 15: 2933-4.
 Rabe, S.T., S.A. Emami, M. Iranshahi, et al., 2015. Anti-cancer Properties of a Sesquiterpene Lactone-bearing Fraction from Artemisia khorassanica. Asian Pac J Cancer Prev, 16: 863-8.
 Elsayed, S.I., 2010. Potent cancer chemoprevention of Nigella sativa oil in the rat.Oncology Letters,1m 913-24.
 Rooney, S and M.F. Ryan, 2005. Modes of action of alpha-hederin and thymoquinone, active constituents of Nigella sativa, against HEp-2 cancer cells. Anticancer Res., 25: 4255-9.
 Padhye, S., S. Banerjee, A. Ahmad, R. Mohammad and F.H. Sarkar, 2008. From here to eternity--the secret of Pharaohs: Therapeutic potential of black cumin seeds and beyond. Cancer Ther, 6: 495-510.
 Ait, M.L., M.H. Ait, N. Elabbadi, M. Bensalah, A. Gamouh, R. Aboufatima, A. Benharref, A. Chait, M. Kamal, A. Dalal, A. Zyad, 2007. Anti-tumor properties of black seed (Nigella sativa L.) extracts. Braz J Med Biol Res, 40: 839- 47.
 Khan, A., H. Chen, M. Tania, et al., 2011. Anticancer activities of nigella sativa (black cumin). Afr J Tradit Complement Altern Med, 8(S): 226-32.
 Dilshad, A.,O. Abulkhair, D. Nemenqani, et al. 2012. Antiproliferative Properties of Methanolic Extract of Nigella sativa against MDA-MB-231 Cancer Cell Line.Asian Pacific J Cancer Prev,13(11): 5839-42.
 Chu, S-C., Y-S. Hsieh, C-C. Yu, et al. 2014. Thymoquinone Induces Cell Death in Human Squamous Carcinoma Cells via Caspase Activation-Dependent Apoptosis and LC3-II Activation-Dependent Autophagy. PLoS ONE 2014;9 (7), e101579.
 Al-Sheddi, E.S., N.N. Farshori, M.M. Al-Oqail, et al, 2014.Nigella Sativa Seed Extracts Against Lung Cancer Cells.Asian Pac J Cancer Prev., 15(2): 983-7.
 Mabrouk, G.M., S.S. Moselhy, S.F. Zohny, E.M. Ali, T.E. Helal, A.A. Amin and A.A. Khalifa, 2002. Inhibition of methylnitrosourea (MNU) induced oxidative stress and carcinogenesis by orally administered bee honey and Nigella grains in Sprague Dawely rats. J ExpClin Cancer Res., 21: 341-46.
 Rajkamal, G., K. Suresh, G. Sugunadevi, M.A. Vijayaanand and K. Rajalingam, 2010. Evaluation of chemopreventive effects of Thymoquinone on cell surface glycoconjugates and cytokeratin expression during DMBA induced hamster buccal pouch carcinogenesis, BMB Rep., 43: 664-9.
 Bhuvaneswari, V., K.V.P. Chandra Mohan, S. Nagini, 2004. Combination chemoprevention by tomato and garlic in the hamster buccal pouch carcinogenesis model. Nutr Res., 24: 133-46.
 Agagrwal, B.B., H. Ichikawa, P. Garodia, 2006. From traditional Ayurvedic medicine to modern medicine: identification of therapeutic targets for suppression of inflammation and cancer. Expert OpinTher Targets, 10: 87-118.
 In, L.L., N.M. Arshad, H. Ibrahim, M.N. Azmi, K. Awang and N.H. Nagoor, 2012. 1'-Acetoxychavicol acetate inhibits growth of human oral carcinoma xenograft in mice and potentiates cisplatin effect via pro-inflammatory microenvironment alterations. BMC Complement Altern Med, 12, 179-86. Also Available at: http://www.biomedcentral.com/1472-6882/12/179.
 Barnes, L., J.W. Eveson, P. Reichart, D. Sidransky (Eds), 2005. Pathology and Genetics of Head and Neck Tumors.IARC Press, Lyon.
 Nagini, S.P., 2009. Of Humans and Hamsters: The Hamster Buccal Pouch Carcinogenesis Model as a Paradigm for Oral Oncogenesis and Chemoprevention. Anticancer Agents Med Chem, 9: 843-52.
 Morris, A.L., 1961. Factors influencing experimental carcinogenesis in the hamster cheek pouch; J D Res, 40: 3-15.
 Ohnishi, M., N. Yoshimi, T. Kawamori, N. Ino, Y. Hirose, T. Tanaka, J. Yamahara, H. Mlyata and H. Mori, 1997. Inhibitory effects of dietary protocatechuic acid and costunolide on 7, 12-dimethylbenz(a)anthracene induced hamster cheek pouch carcinogenesis. Jpn J Cancer Res.,88: 111-19.
 Gimenez-Conti, I.B., 1993. The Hamster Cheek Pouch Carcinogenesis Model.J Cell Biochem, Supplement 17F: 83-90.
 Shklar, G., 1999. Development of experimental oral carcinogenesis and its impact on current oral cancer research. J Dent Res., 78: 1768-72.
 Salomi, N.J., S.C. Nair, K.K. Jayawardhanan, C.D. Varghese, L.R. Panikkar, 1992. Anti-tumor principles from Nigella sativa and saffron (Crocus sativus) on chemical carcinogenesis in mice. Nutr Cancer,16: 67-72.
 Badary, O.A., M.N. Nagi, O.A. al-Shabanah, H.A. al-Sawaf, M.O. al-Sohaibani, A.M. al-Bekairi, 1997. Thymoquinone ameliorates the nephrotoxicity induced by cisplatin in rodents and potentiates its antitumor activity. Can J PhysiolPharmacol., 75: 1356-61.
 Alhosin, M., A. Abusnina, M. Achour, 2010. Induction of apoptosis by thymoquinone in lymphoblastic leukemia Jurkat cells is mediated by a p73-dependent pathway which targets the epigenetic integrator UHRF1. BiochemPharmacol, 79: 1251- 60.
 Schneider-Stock, R., I.H. Fakhoury, A.M. Zaki, C.O. El-Baba and H.U. Gali-Muhtasib, 2014. Thymoquinone: Fifty years of success in the battle against cancer models. Drug Discovery Today, 19: 18-30.
 Kumara, S.S.M., B.T.K. Huat, 2001. Extraction, isolation and characterisation of antitumor principle, hederin, from the seeds of Nigella sativa.Planta Med., 67: 29-32.
 Thabrew, M.I., R.R. Mitry, M.A. Morsy, R.D. Hughes, 2005. Cytotoxic effects of a decoction of Nigella sativa, HemidesmusindicusandSmilax glabraon human hepatoma HepG2 cells. Life Sci., 77: 1319-30.
 Majdalawieh, A.F., R. Hmaidan, R.I. Carr, 2010. Nigella sativa modulates splenocyte proliferation, Th1/Th2 cytokine profile, macrophage function and NK anti-tumor activity. J Ethnopharmacol., 131(2): 268-75.
 Yi, T., S. Cho, Z. Yi, X. Pang, M. Rodriguez, Y. Wang, G. Sethi, B. Aggarwal, M. Liu, 2008. Thymoquinone inhibits tumor angiogenesis and tumor growth through suppressing AKT and extracellular signal-regulated kinase signaling pathways. Molecular Cancer Therapeutics,7: 1789-96.
 Attoub, S., O. Sperandiob, H. Razac, 2013. Thymoquinone as an anticancer agent: evidence from inhibition of cancer cells viability and invasion in vitro and tumor growth in vivo. FundamClinPharmacol., 27: 557-69.  Coutinho- Camillo, C.M., S.V. Lourenco, I.N. Nishimoto, L.P. Kowalski, F.A. Soares, 2011. Caspase expression in oral squamous cell carcinoma. Head Neck., 33(8): 1191-8.
 Rupinder, S.K., A.K. Gurpreet, S. Manjeet, 2007. Cell suicide and caspases. VasculPharmacol, 46: 383-93.
 Skommer, J., D. Wlodkowic, A. Deptala, 2007. Larger than life: Mitochondria and the Bcl-2 family. Leuk Res., 31: 277-86.
 Hunter, A.M., E.C. La Casse, R.G. Korneluk, 2007. The inhibitors of apoptosis (IAPs) as cancer targets.Apoptosis, 12: 1543-68.  Blanc, C., Q.L. Deveraux, S. Krajewski, R.U. Janicke, A.G. Porter, J.C. Reed, R. Jaggi, A. Marti, 2000. Caspase-3 is essential for procaspase-9 processing and cisplatin-induced apoptosis of MCF-7 breast cancer cells. Cancer Res., 60: 4386-90.
 Paramasivam, A., S. Sambantham, J. Shabnam, 2012. Anti-cancer effects of thymoquinone in mouse neuroblastoma (Neuro-2a) cells through caspase-3 activation with down-regulation of XIAP. Toxicol Lett, 213: 151-9.
 Peng, L., A. Liu, Y. Shen, et al. 2013. Antitumor and anti-angiogenesis effects of thymoquinone on osteosarcoma through the NF-?B pathway. Oncol Rep., 29: 571-8.
 Alhazmi, M.I., T.N. Hasan, G. Shafi, et al 2014. Roles of p53 and Caspases in Induction of Apoptosis in MCF-7 Cells by a Methanolic Extract of Nigella sativa Seeds.Asian Pac J Cancer Prev., 15(22): 9655-60
 Jafri, S.H., J. Glass, R. Shi, S. Zhang, M. Prince, H. Kleiner-Hancock, 2010. Thymoquinone and cisplatin as a therapeutic combination in lung cancer: in vitro and in vivo. J ExpClin Cancer Res., 29: 87-94.
 Ulu, R., A. Dogukan, M. Tuzcu, H. Gencoglu, M. Ulas, N. Ilhan, I. Muqbil, R.M. Mohammad, O. Kucuk, K. Sahin, 2012. Regulation of renal organic anion and cation transporters by thymoquinone in cisplatin induced kidney injury. Food ChemToxicol.,50: 1675-9.
 El Daly, E.S., 1998. Protective effect of cysteine and vitamin E, Crocus sativusandNigella sativa extracts on cisplatin induced toxicity in rats. J Pharm Bel, 53: 87-95.
 Ragheb, A., A. Attia, W.S. Eldin, F. Elbarbry, S. Gazarin and A. Shoker, 2009. The protective effect of thymoquinone, an antioxidant and anti-inflammatory agent, against renal injury: a review. Saudi J Kidney Dis Transpl, 20: 741-52.
 Zafeer, M.F., M. Waseem, S. Chaudhary, et al. 2012. Cadmium-induced hepatotoxicity and its abrogation by thymoquinone. J BiochemMolToxicol.,26: 199-205.
 Mariod, A.A., R.M. Ibrahim, M. Ismail, N. Ismail, 2009. Antioxidant activity and phenolic content of phenolic rich fractions obtained from black cumin (Nigella sativa) seedcake. Food Chem, 116: 306-12.
 Hassan, M.H., L. Abdul Latiff, S. Parhizkar, M.A. Dollah, 2013. Toxicity Effect of Nigella Sativa on the Liver Function of Rats. Advanced Pharmaceutical Bulletin, 3(1): 97-102.
 Zaoui, A., Y.Cherrah, K.Alaoui, N.Mahassine, H.Amarouch, M.Hassar, 2002.Effects of Nigella sativa fixed oil on blood homeostasis in rat. J Ethnopharmacol., 79: 23-26.
(1) Safia A Al-Attas, (2) Anjana Munshi, (3) Abdul-Wahab Noorwali, (3) Mehal A Algrigri, (4) Eman A Abohager, (5) Fat'heya M Zahran
(1) Oral Diagnostic Sciences Department, Faculty of Dentistry, King Abdulaziz University, Jeddah, Saudi Arabia. (2) Center for Human Genetics, School of Health Sciences, Central University of Punjab, India. (3) Stem Cell Unit- King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia. (4) Department of Oral and Dental Pathology, Faculty of Oral Medicine for Girls, Al-Azhar University, Egypt. (5) Oral Diagnostic Sciences Department, Faculty of Dentistry, King Abdulaziz University, Jeddah, Saudi Arabia and Oral Medicine and Periodontology Department, Faculty of Oral and Dental Medicine, Cairo University, Egypt.
Corresponding Author: Fat'heya M Zahran, Oral Diagnostic Sciences Department, Faculty of Dentistry, King Abdulaziz University, Jeddah, Saudi Arabia. E-mail: firstname.lastname@example.org
Table 1: One way ANOVA test results for caspase-3 area% pair-wise comparisons among studied groups. Group Mean SD P-value Control 6.82 0.85 DMBA 6 +NSE 25.05 4.15 0 DMBA 6 +CIS 24.17 2.58 0 DMBA 6 +CIS +NSE 23.52 3.74 0 NSE alone 8.38 1.72 DMBA 6+NSE 25.05 4.15 0 DMBA 6+CIS 24.17 2.58 0 DMBA 6+CIS +NSE 23.52 3.74 0 DMBA 6 26.02 2.98 DMBA 6+NSE 25.05 4.15 0.682 DMBA 6+CIS 24.17 2.58 0.276 DMBA 6 +CIS +NSE 23.52 3.74 0.276 Group Mean SD P-value Control 6.82 0.85 DMBA 14+NSE 21.31 1.59 0 DMBA 14+CIS 21.31 1.47 0 DMBA 14+CIS +NSE 21.46 1.74 0 NSE alone 8.38 1.72 DMBA 14+NSE 21.31 1.59 0 DMBA 14+CIS 21.31 1.47 0 DMBA 14+CIS +NSE 21.46 1.74 0 DMBA 14 19.16 8.52 DMBA 14+NSE 21.31 1.59 0.594 DMBA 14+CIS 21.31 1.47 0.593 DMBA 14+CIS +NSE 21.46 1.74 057 P< 0.05 = significant P< 0.01 = highly significant P> 0.05 = non significant Table 2: One way ANOVA testresults for Ki-67 area% pair-wise comparisons among studied groups. Group Mean SD P-value Control 1.12 0.23 DMBA 6 +NSE 2.47 0.55 0.003 DMBA 6 +CIS 2.2 0.62 0.014 DMBA 6+CIS +NSE 1.77 0.28 0.004 NSE alone 1.33 0.34 DMBA 6 +NSE 2.47 0.55 0.006 DMBA 6+CIS 2.2 0.62 0.033 DMBA 6+CIS +NSE 1.77 0.28 0.057 DMBA 6 5.09 1.89 DMBA 6+NSE 2.47 0.55 0.018 DMBA 6+CIS 2.2 0.62 0.012 DMBA 6+CIS +NSE 1.77 0.28 0.005 Group Mean SD P-value Control 1.12 0.23 DMBA 14+NSE 4.81 0.21 0 DMBA 14+CIS 4.62 1.55 0.007 DMBA 14+CIS +NSE 3.93 1.3 0.007 NSE alone 1.33 0.34 DMBA 14+NSE 4.81 0.21 0 DMBA 14+CIS 4.62 1.55 0.008 DMBA 14+CIS +NSE 3.93 1.3 0.002 DMBA 14 6.62 0.95 DMBA 14+NSE 4.81 0.21 0.003 DMBA 14+CIS 4.62 1.55 0.04 DMBA 14+CIS +NSE 3.93 1.3 0.006 P< 0.05 = significant P< 0.01 = highly significant P> 0.05 = non significant Table 3: Statistical analysis for results obtained from the liver and kidney function tests and hemogram in the studied groups. Tested item F P Liver function AST 1.102 0.397 ALT 0.735 0.673 Kidney function Creatinine 1.108 0.393 Urea 1.413 0.237 Complete Hb 1.423 0.223 blood count RBCs count 1.396 0.235 MCV 1.027 0.443 MCH 0.868 0.563 WBCs (total) 0.697 0.706 Granulocytes 2.088 0.063 Lymphocytes 2.135 0.058 Monocytes 1.214 0.323 Platelets 0.527 0.843 P < 0.05 = significant P < 0.01 = highly significant P > 0.05 = non significant Fig. 3: Bar chart showing severity of dysplasia in histopathologic specimenin different groups Control -ve no dysplasia 100.0 Control + ve NSE no dysplasia 100.0 DMBA 6 mild 100.0 DMBA 14 severe 33.3 carcinoma 66.7 DMBA 6+NSE no dysplasia 40.0 mild 60.0 DMBA 14+NSE mild 75.0 carcinoma 25.0 DMBA 6+CIS no dysplasia 100.0 DMBA 14+CIS mild 100.0 DMBA 6+CIS +NSE no dysplasia 100.0 DMBA 14+CIS +NSE mild 100.0 Note: Table made from bar graph.
|Printer friendly Cite/link Email Feedback|
|Author:||Attas, Safia A. Al-; Munshi, Anjana; Noorwali, Abdul-Wahab; Algrigri, Mehal A.; Abohager, Eman A.; Z|
|Publication:||Advances in Environmental Biology|
|Date:||Sep 1, 2015|
|Previous Article:||Zero-discharge technology in the crystal glass industry.|
|Next Article:||Sprouted barley grains on olive cake and Barley straw mixture as goat diets in Sinai.|