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Nigella sativa extract chemoprevention in oral cancer: in vivostudy.

INTRODUCTION

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% [3]. 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.[9] 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. [10]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 [15]. However, Rooney and Ryan(2005) [8] 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 [16]. Application of 0.5% DMBA on hamster cheek pouches 3 times weekly over a 14-week period is a well-known carcinogenesis protocol [17]. 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 [18]. 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 [19]. 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.

Methods:

Ethical approval for this research study was obtained from the ethical committee of King Abdulaziz University Faculty of Dentistry, Jeddah, Saudi Arabia.

Animals:

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 [20].

Immunohistochemical analysis:

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.

Histomorphometric analysis:

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:

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.

Results:

General observations:

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.

Inunohistocheiical Results:

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).

Histomorphometric Results:

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.

Discussion:

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 [21]. According to Morris (1961) [22], 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 [22]. 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 [23]. 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 [26]. 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 [28]. TQ has been successful against other various cancer models in vitro and in vivo [29]. Furthermore, [alpha]-hederin has been isolated in one study and was determined to be a significant contributor to the anticancer activity of NSE [30]. Thabrew et al. (2005) [31] reported that aqueous NSE was active against hepatocellular carcinoma cells in vitro. More recently, Al-Sheddi et al. (2014) [14] 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 [32], 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 [33].

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) [34] 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 [35] 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 [38] 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 [21] Blanc et al. (2000) [39]showed that caspase-3 was essential for cisplatin-induced apoptosis. In addition, Chu et al. (2014) [13] found that TQ induced cell death in oral cancer cells via two different mechanisms, one of which is apoptosis. Paramasivam et al. (2012) [40] and Peng et al. (2013) [41] 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) [42] 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) [43] 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) [43] previously demonstrated that TQ does not induce mortality or any pathological changes in the lung, heart, or kidneys. Furthermore, Badaiy et al. (1997) [27] 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. [44] 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 [45]. 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 [46]. 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) [47]. Mariod et al.(2009) [48] 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) [49] 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) [50]. However, in the present investigation, treatment with NSE did not produce any significant changes in blood picture.

Conclusions:

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.

Disclosure Statement

The authors declare that no competing financial interests exist.

ARTICLE INFO

Article history:

Received 5 August 2015

Accepted 20 September 2015

Available online 30 September 2015

ACKNOWLEDGMENTS

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. .

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(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: fatheyazahran@gmail.com

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.
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Author:Attas, Safia A. Al-; Munshi, Anjana; Noorwali, Abdul-Wahab; Algrigri, Mehal A.; Abohager, Eman A.; Z
Publication:Advances in Environmental Biology
Article Type:Report
Date:Sep 1, 2015
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