Tanta Dental Journal

: 2017  |  Volume : 14  |  Issue : 3  |  Page : 105--111

Flaxseed oil: an emerging nutraceutical decimates cisplatin-induced submandibular salivary glands damage in rats

Doaa A Labah1, Omneya M Wahba2,  
1 Department of Oral Biology, Faculty of Dentistry, Tanta University, Tanta, Egypt
2 Department of Oral Pathology, Faculty of Dentistry, Tanta University, Tanta, Egypt

Correspondence Address:
Doaa A Labah
19, Omar Ebn El Khatab Street, Villal El Gamah, El Zagazig


Objective The study aimed to investigate the capability of flaxseed oil (FXO) to alleviate the degenerative effect of chemotherapy with cisplatin (CP) on rats' submandibular salivary glands (SMGs). Materials and methods Rats were allocated equally into four groups: control, CP-treated, FXO-treated, and CP-FXO-treated groups. Rats of third and fourth groups were orally given a daily dose of FXO. After 10 days, rats in second and fourth groups were injected with a single dose of CP. After rat sacrifice, samples of SMGs were processed for histological, ultrastructural study beside immunohistochemical staining for Ki67 and vascular endothelial growth factor (VEGF) expressions. Analysis of variance was used to compare Ki67 and VEGF expressions between groups. Results The SMGs of CP group showed abnormal architecture when compared with control and FXO groups. CP group treated with FXO revealed architecture improvement in their SMGs. Immunohistochemical study revealed significant decrease in Ki67 and VEGF expressions in CP rats. However, CP group treated with FXO simulated control group in both Ki67 and VEGF expressions. Conclusion CP administration caused deleterious effects on rats' SMGs. However, dietary consumption of FXO was able to ameliorate CP effects. Hence, FXO intake is recommended simultaneously during chemotherapy with CP.

How to cite this article:
Labah DA, Wahba OM. Flaxseed oil: an emerging nutraceutical decimates cisplatin-induced submandibular salivary glands damage in rats.Tanta Dent J 2017;14:105-111

How to cite this URL:
Labah DA, Wahba OM. Flaxseed oil: an emerging nutraceutical decimates cisplatin-induced submandibular salivary glands damage in rats. Tanta Dent J [serial online] 2017 [cited 2017 Dec 15 ];14:105-111
Available from: http://www.tmj.eg.net/text.asp?2017/14/3/105/214327

Full Text


Cisplatin [cis-diamminedichloro-platinum II (CP)] is one of the most effective and widely used chemotherapeutic agents against human solid tumors, including head and neck, testicular, ovarian, cervical carcinomas, and germ-cell tumors [1]. The antitumor action of CP is attributed to its action on DNA synthesis as CP binds with DNA to form intrastrand cross-links and adducts that cause changes in the conformation of the DNA and affect DNA replication. Moreover, CP causes cell cycle arrest in the G2 phase and induces programmed cell death or apoptosis. Other mechanisms of CP cytotoxic effects on tumors cells include mitochondrial damage, decreased ATPase activity, and altered cellular transport mechanisms [2],[3],[4]. Although CP potent chemotherapeutic effects, some undesirable effects may be caused because the cytotoxic effect of CP is not selective for cancer cells but it also affects the normal tissues [1].

Patients with cancer chemotherapy, including platinum chemotherapy, complain from xerostomia, which associated with swallowing, speech and taste difficulties, oropharyngeal pain, and oral infections. These could be related to that chemotherapy causes imbalances and disturbances in salivary gland function and could induce apparent cytotoxic effect and structural changes in salivary glands [5],[6],[7].

Numerous studies have been focused on the ways for prevention of CP side effects through supplementation of preventive agents simultaneously [8]. Antioxidant thiols are used in platinum chemotherapy as chemoprotectants. Nevertheless, such chemoprotectants have had relatively limited clinical use due to concerns of impaired antitumor efficacy [9]. Recently, the approach of identifying naturally dietary sources and using as cytoprotectants provides a strategy that is of great interest for CP chemotherapy [10].

Flaxseed oil (FXO), also known as linseed oil, is edible oil obtained from the dried, ripened seeds of flax plant (Linum usitatissimum). Flaxseed contains massive beneficial constituents and is considered as an important functional food ingredient because it is one of the richest plant dietary sources of omega-3 polyunsaturated fatty acids especially α-linolenic acid [11].

A number of investigations have demonstrated that diet supplemented with FXO has profound beneficial health effects as it has antioxidant, hypolipidemic and antidiabetic effects [12],[13],[14]. Moreover, FXO acts as a potent anticarcinogen, which inhibits the development, growth, and metastasis of cancer [15],[16],[17]. Additionally, dietary FXO consumption provides a great promise for modulating inflammatory diseases and plays important role in reducing cardiovascular and kidney diseases [18],[19],[20]. Interestingly, dietary FXO supplementation ameliorated CP-induced hepatotoxicity and nephrotoxicity [10],[21].

Considering the potential clinical use of CP and the benefits of FXO, it is of prime importance to investigate the capability of FXO to alleviate CP-induced changes in rats' submandibular salivary glands (SMGs).

 Materials and Methods


Thirty-two male Sprague-Dawley rats (∼150–200 g in weight) were selected for this study. The animals were housed at Faculty of Medicine, Zagazig University, in separate cages and received a standard diet for rodents and tap water ad libitum. Room temperature and humidity were maintained at 23°C and 60%, respectively, and normal photoperiod was kept (12 h dark and light). All animal experiments were carried out in accordance with the guidelines of the National Institutes of Health (NIH) for the care and use of laboratory animals (NIH Publication, number 85-23, Revised 1985). After 1 week acclimatization period, the animals were randomly divided into four equal groups (each compromised eight rats): group I (control), group II (CP-treated), group III (FXO-treated), and group IV (CP-FXO-treated). Animals were fed only on normal diet in both control and CP groups while were fed on normal diet and were orally given FXO (Imtenan Health Co., Egypt) in a dose of 500 mg/kg body weight [22] in both CP-FXO and FXO groups. After 10 days, rats in CP and CP-FXO groups were intraperitoneally injected with a single dose (6 mg/kg body weight in 0.9% saline) of CP [21] (Sigma Chemical Co., USA). Animals in the control and FXO groups were injected with an equivalent amount of normal saline.

Animal euthanasia and samples collections

Four days after CP administration, rats were anesthetized with ketamine at a dose of 50 mg/kg body weight and the SMGs of each animal were dissected carefully. The right glands were prepared for light microscopic examination and immunohistochemical study while the left ones were prepared for electron microscopic examination. The animals were then scarified with overdose of anesthetic, according to the ethical guidelines, confirmed with cervical dislocation.

Light microscopy

The specimens from the right SMGs were fixed with 4% buffered formalin solution then dehydrated by a graded ethanol series and the whole gland was embedded in paraffin. Five-micrometer sections were cut and representative sections were stained with hematoxylin and eosin for conventional histological assessment using light microscope (Leica ICC50 HD; Leica).

Electron microscopy

The left SMGs were trimmed into 1-mm cubes and fixed for 2 h at room temperature, in a mixture of 4% paraformaldehyde and 1% glutaraldehyde. Then, they were rinsed in buffer and postfixed in 1% osmium tetroxide, following fixation, samples were dehydrated in a graded series of ethanol, placed in absolute acetone, and embedded in Epon. Ultrathin sections were cut, stained with uranyl acetate and lead citrate and examined with Jeol 100 CX transmission electron microscope (Jeol) in Electron Microscope Unit at Faculty of Medicine, Tanta University.


Cell proliferation was evaluated by Ki67 immunohistochemistry while angiogenesis was assessed by vascular endothelial growth factor (VEGF) immunoreactivity. For immunohistochemical staining, 4 μm sections were prepared from each paraffin block and then were deparaffinized in xylene and dehydrated in graded alcohol series. To block the internal peroxidase activity they were placed in 3% hydrogen peroxide in PBS. Antigen retrieval was done in a microwave oven (Panasonic 1380W; Panasonic) for 10 min, under the pressure of 2 atm in 120°C. Further incubations with prediluted ready to use primary mouse monoclonal antibody (anti-Ki67, clone MIB-5; Dako, Glostrup, Denmark) and anti-VEGF (Sigma-Aldrich Inc.) were used as the primary antibody for 30 min and was incubated in a moist chamber at room temperature (24 h) with a working dilution 1:50 and 1:100, respectively, followed by the application of secondary antibody (for 15 min), DAB (to produce brown staining), and Meyer's hematoxylin (for background staining). The samples were placed in PBS after each mentioned step. Kidney and skin were the positive control for VEGF and Ki67, respectively, according to the manufacturer's instructions. The negative control was obtained by the replacement of primary antibody with PBS.

Assessment of immunohistochemistry-stained sections

Presence of brown colored reaction localized to the nucleus or the cytoplasm was considered as positive reaction. The intensity of the immunostaining was classified as negative, weak or strong from three fields in blinded analysis performed by two independent pathologists using a conventional diagnostic microscope (Eclipse 80i; Nikon, Tokyo, Japan) in Faculty of Medicine, Tanta University, and further image analysis was done with the Image J software (version 4.10.03; Nikon).

Statistical analysis

For statistical analysis, all measurement data were presented as mean ± SD. All statistical analyses were performed using one-way analysis of variance to reveal the significant difference among groups followed by Dunnett's post-hoc test to compare all groups with CP group. Values of P less than 0.05 indicated a statistically significant difference. All statistics were performed with SPSS (version 11.0; SPSS Inc., Chicago, Illinois, USA).


Light microscopic results

The SMG of control group revealed the presence of numerous normally appeared seromucous acini and intralobular ducts. At higher magnification, the acinar cells were roughly pyramidal in shape with basally located nuclei. The striated duct was lined with simple columnar epithelium with basal nuclei ([Figure 1]a and [Figure 1]b). In CP group, the SMG exhibited separation between the acini and intralobular ducts in some areas. In addition, some ducts showed stagnation of the secretory material while destructed ducts were obvious in some areas. At higher magnification signs of acini degeneration represented by loss of normal architecture of the secretory portions and destruction of cytoplasm. Some of the secretory elements as well as ductal elements revealed complete degeneration and were completely missed leaving large vacuoles. Nuclei of the acinar cells revealed different sizes and shape. Some of the acinar cells appeared to be binucleated while others seem to lack their nuclei ([Figure 1]c and [Figure 1]d). SMG of FXO group resemble that of control group. The acini were uniform in shape and regularly structured. Intralobular ducts showed normal outline with its cells properly aligned ([Figure 1]e and [Figure 1]f).{Figure 1}

SMG of CP group treated with FXO showed significant improvement in acinar and ductal cells compared to CP group. The acinar and ductal cells appeared almost normal ([Figure 1]g and [Figure 1]h).

Electron microscopic results

Electron microscopic examination of the SMG of control group showed no pathological changes. The acinar portion appeared to be formed from roughly pyramidal cells with basally situated nuclei. Also, the striated duct was lined with simple columnar epithelium with basal nuclei and basal striations appeared to be due to packing of numerous mitochondria arranged vertically between basal in-folding of plasma membrane ([Figure 2]a and [Figure 2]b). The SMG of CP group revealed abnormal architecture of seromucous aciniar cells and abnormal organization of cytoplasmic organelles. Rarefaction and vacuolization of the cytoplasm could be recognized in some cells. Moreover, RER appeared dilated and abnormally distributed. The nuclei appeared of different size and shape with some of them appeared pyknotic and others were detected with destructed chromatin. Additionally, the striated duct showed irregular arrangement of cells and contained atypical nuclei. Mitochondria was rarely seen ([Figure 2]c and [Figure 2]d). The SMG of FXO group revealed almost normal appearance of the seromucous acinar cells and intralobular ducts. The intercalated duct appeared to be normally lined with low cuboidal cells and centrally located nuclei. Also, normal GCT could be detected with electron dense granules and basally situated nucleus ([Figure 2]e and [Figure 2]f). The SMG of CP group treated with FXO showed almost normally appeared seromucous acini and striated ducts. Although, there were cytoplasmic vacuolization in some areas of the gland ([Figure 2]g and [Figure 2]h).{Figure 2}

Ki67 immunohistochemical findings

In normal SMG, there was moderate nuclear staining of ductal cells with anti-Ki67 antibody however acinar cells showed weak nuclear staining ([Figure 3]a). In CP group, ductal cells showed weak staining with some negative cells. Almost all acinar cells were negative except few acini showed faint positivity ([Figure 3]b). In FXO group, there was strong nuclear staining of ductal cells and moderate staining was detected in some acinar cells ([Figure 3]c). In CP group treated with FXO, moderate staining of some ductal and acinar cells were detected ([Figure 3]d).{Figure 3}

Statistical analysis demonstrated a significant difference in Ki67 expression between CP group and other groups (namely, control, FXO, and CP-FXO groups). On the other hand, CP group treated with FXO revealed Ki67 expression simulate that of control group ([Table 1] and [Figure 4]).{Figure 4}{Table 1}

Vascular endothelial growth factor immunohistochemical findings

In normal SMG, there was moderate to strong cytoplasmic staining of ductal cells with anti-VEGF antibody and weak staining in acinar cells ([Figure 5]a). In CP group, weak cytoplasmic staining of ductal cells was detected and acinar cells showed faint staining ([Figure 5]b). In FXO group, there was strong cytoplasmic staining of ductal and acinar cells ([Figure 5]c). In CP group treated with FXO, ductal cells showed moderate staining while acinar cells showed moderate to faint staining ([Figure 5]d).{Figure 5}

Statistical analysis demonstrated a significant difference in VEGF expression between CP group and other groups (namely, control, FXO, and CP-FXO groups). Conversely, CP group treated with FXO revealed VEGF expression similar to that of control group (no significant difference) ([Table 2] and [Figure 6]).{Figure 6}{Table 2}


The present study attempted to clarify the degenerative effects of chemotherapy on rat's SMGs and the putative role of FXO to alleviate these effects. The SMG was considered an excellent model to study such effects since it produces about 60% of saliva and the time for a drug required to reach the maximum concentration in submandibular saliva was shorter than that in parotid saliva [23],[24].

In the current study, CP injection had adversely affected the histological structure of the rat SMGs. Signs of acini degeneration represented by disfigured lobular structure and loss of normal architecture of the secretory portions were seen. These results were coincided with the findings of Oktay et al. [25] who demonstrated that cancer chemotherapy could induce salivary gland acinar degeneration in the form of acinar and ductal cell vacuolization, apoptosis in the acinar cells with pyknosis in the nuclei and a reduction in secretion granules. These cytotoxic effects of CP on glandular tissues could associated with the capability of CP on the induction of oxidative stress and accumulation of reactive oxygen species and nitric oxide, which exert direct cytotoxic effects by favoring the opening of the permeability transition pore complex [26],[27]. Interestingly in the present study, animals treated with FXO and injected with CP showed marked improvement in SMGs architecture that appeared to be almost normal. This improvement in SMGs could be explained by the beneficial antioxidant activity of FXO, which could be attributed to its high content of omega-3 polyunsaturated fatty acid as reported by Khan et al. [28]. The omega-3 fatty acids suppress the antioxidant enzymes activities including superoxide dismutase, catalase, and glutathione peroxidase enzymes thereby raising the efficiency of antioxidant defense system [29],[30]. For this reason, Rubiolo et al. [31] concluded that it is important to enrich our diet with antioxidants to protect against many chronic diseases related to oxidative damage.

Ki67 protein is a cellular marker for proliferation, which is involved in the early steps of polymerase I-dependent ribosomal RNA synthesis and hence that protein has an important function in cell division [32],[33]. In the current study, normal SMGs showed moderate nuclear staining of ductal cells and weak nuclear staining of acinar cells with anti-Ki67 antibodies. This is in disagreement with the results of Tadbir et al. [34] who stated that normal salivary gland did not express Ki67. Moreover, the present study showed marked reduction in Ki67 expression after CP dose with significant P value less than 0.001 (mean ± SD = 2.46 ± 0.76). The decrease in cell proliferation after CP may explain the salivary impairment with decreasing the whole salivary flow rate after anticancer therapy. Decreased Ki67 value following chemotherapy was reported by Nishimura et al. [35] who suggested that the Ki67 value before neoadjuvant chemotherapy of breast cancer is a strong predictive factor for the effectiveness of the therapy and the lower Ki67 values indicate a low chance for pathological complete response but a better prognosis. In contrary to our results, Dowsett et al. [36] showed higher Ki67 labeling index after 2 weeks of neoadjuvant treatment. In the present study, the improvement in the structure of SMG after coadministration of FXO and CP was accompanied with increasing the Ki67 expression (mean ± SD = 6.46 ± 0.58, P < 0.001, significant). This is in contrary to the results found on cancer cells by Lilian et al. [37] who concluded that daily intake of FXO can significantly reduce cell proliferation, increase apoptosis, and affect cell signaling by reducing c-erbB2 expression of human breast cancer cells.

VEGF is a potent endothelial cell mitogen and key regulator of physiological and pathological angiogenesis [38]. In the present study, SMGs of the control group exhibited moderate to strong cytoplasmic staining of ductal cells with anti-VEGF antibody and weak cytoplasmic staining in acinar cells with mean ± SD of 4.92 ± 1.61. This is in accordance with Pammer et al. [39] who detected the expression of VEGF in endocrine cells such as in the salivary gland and clarified that the presence of considerable quantities of VEGF in normal human saliva could explain its important role in the maintenance of the homeostasis of mucous membranes. Moreover, Li et al. [40] explained that the important biological activity of VEGF is its ability to induce endothelial fenestration of postcapillary and muscular venules and capillaries. Furthermore, the fenestration in the capillaries of salivary glands may be due to locally produced VEGF [41]. Thus, acinar cell-derived VEGF may participate in regulation of saliva production by altering the permeability of glandular capillaries and increasing the fluid supply to the secretory cells [42]. In the present study, after CP dose there was significant decrease in VEGF expression in SMG (P< 0.002, mean ± SD = 2.45 ± 0.62), which could be related to the destructive effect of the chemotherapeutic drug on the salivary gland tissue. Similarly, Zhong et al. [43] reported that CP inhibited VEGF expression in human ovarian cancer cells. Interestingly in this study, coadministration of FXO and CP increased VEGF expression in SMG with significant P value less than 0.013. In this regard, the increased VEGF expression following FXO intake could be explained by the results of Lemay et al. [44] who reported that flaxseed is the richest dietary source of lignans, a type of phytoestrogen, which is a plant nutrient similar to the female hormone estrogen. Additionally, Mueller et al. [45] stated that the estrogenic effects on endometrial angiogenesis are mediated indirectly via production of VEGF that stimulates capillary endothelial mitogenesis and morphogenesis.


CP, though is an essential chemotherapeutic used to treat several cancers, its frequent side effects could harm salivary glands architecture and functions. Due to its substantial beneficial ingredients, FXO has the ability to alleviate the degenerative effects caused by CP, which consider it a prospective treatment for salivary gland complications in patients under chemotherapy medication.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


1Rosenberg B. Fundamental studies with cisplatin. Cancer 1985; 55:2303–2315.
2Crul M, Schellens JHM, Beijnen JH, Maliepaard M. Cisplatin resistance and DNA repair. Cancer Treat Rev 1997; 23:341–366.
3Chu G. Cellular responsestocisplatin. TherolesofDNA- binding proteins and DNA repair. JBiol Chem 1994;269:787–790.
4Siddik ZH. Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene2003; 22:7265–7279
5Jensen SB, Pedersen AM, Reibel J, Nauntofte B. Xerostomia and hypofunction of the salivary glands in cancer therapy. Support Care Cancer 2003; 11:207–225
6Mahmoud EF, Mahmoud MF, Haleem MAA. Royal jelly ameliorates oxidative stress and tissue injury in submandibular salivary gland of methotrexate treated rabbits: immunohistochemical study. J Am Sci 2012; 8:501–508.
7Al-Refai AS, Khaleel AK, Ali S. The effect of green tea extract on submandibular salivary gland of methotrexate treated albino rats: immunohistochemical study. J Cytol Histol 2014; 5:212–220.
8Liao Y, Lu X, Lu C, Li G, Jin Y, Tang H. Selection of agents for prevention of cisplatin-induced hepatotoxicity. Pharmacol Res 2008; 57:125–131.
9Wu YJ, Muldoon LL, Neuwelt EA. The chemoprotective agent N-acetylcysteine blocks cisplatin-induced apoptosis through caspase signaling pathway. J Pharmacol Exp Ther 2005; 312:424–431.
10Naqshbandi A, Rizwan S, Khan F. Dietary supplementation of flaxseed oil ameliorates the effect of cisplatin on rat kidney. J Funct Foods 2013; 5:316–326.
11Gebauer SK, Psota TL, Harris WS, Kris-Etherton PM. n-3 Fatty acid dietary recommendations and food sources to achieve essentiality and cardiovascular benefits. Am J Clin Nutr 2006; 83:1526s–1535s.
12Newairy AS, Abdou HM. Protective role of flax lignans against lead acetate induced oxidative damage and hyperlipidemia in rats. Food Chem Toxicol 2009; 47:813–818.
13Shahidi F. Antioxidant factors in plant foods and selected oilseeds. Biofactors 2000; 13:179–185.
14Kelley DS, Vemuri M, Adkins Y, Gill SHS, Fedor D, Mackey BE. Flaxseed oil prevents trans-10, cis-12-conjugated linoleic acid-induced insulin resistance in mice. Br J Nutr 2009; 101:701–708.
15Williams DS, Verghese M, Walker LT, Boateng J, Shackelford LA, Guyton M, et al. Chemopreventive effects of flaxseed oil and flax seed meal on azoxymethane-induced colon tumors in fisher 344 male rats. IJCR 2008; 2:28–40.
16Lin X, Gingrich JR, Bao WJ, Haroon ZA, Demark-Wahnefried W. Effect of flaxseed supplementation on prostatic carcinoma in transgenic mice. Urol 2002; 60:919–924.
17Chen J, Stavro PM, Thompson LU. Dietary flaxseed inhibits human breast cancer growth and metastasis and down regulates expression of insulin-like growth factor and epidermal growth factor receptor. Nutr Cancer 2002; 43:187–192.
18Chilton FH, Rudel LL, Parks JS, Arm JP, Seeds MC. Mechanisms by which botanical lipids affect inflammatory disorders. Am J Clin Nutr 2008; 87:498s–503s.
19Rodriguez-Leyva D, Dupasquier CM, McCullough R, Pierce GN. The cardiovascular effects of flaxseed and its omega-3 fatty acid, alpha-linolenic acid. Can J Cardiol 2010; 26:489–496.
20Baggio B, Musacchio E, Priante G. Polyunsaturated fatty acids and renal fibrosis: pathophysiologic link and potential clinical implications. J Nephrol 2005; 18:362–367.
21Naqshbandi A, Khan W, Rizwan S, Khan F. Studies on the protective effect of flaxseed oil on cisplatin-induced hepatotoxicity. Hum Exp Toxicol 2012; 31:364–375.
22Wahba HM, Ibrahim TA. Protective effect of flaxseed oil and vitamin E on potassium bromate-induced oxidative stress in male rats. Int J Curr Microbiol App Sci 2013; 2:299–309.
23Dodds MW, Johnson DA, Yeh CK. Health benefits of saliva: a review. J Dent 2005; 33:223–233.
24Ibrahim SH, Soliman ME, Yehia NM. Effect of ciprofloxacin on the submandibular salivary gland of adult male albino rat: a light and electron microscope study. Egypt J Histol 2004; 27:339–354.
25Oktay O, Abdullah A, Fethullah K, Fatma A, Ramazan S, Mustafa D, et al. Histopathologic changes in the rabbit submandibular gland after 5-fluorouracil chemotherapy. Turk J Med Sci 2010; 40:213–220.
26Masuda H, Tanaka T, Takahama U. Cisplatin generates superoxide anion by interaction with DNA in a cell-free system. Biochem Biophys Res Commun 1994; 203:1175–1180.
27Brenner C, Grimm S. The permeability transition pore complex in cancer cell death. Oncogene 2006; 25:4744–4756.
28Khan MW, Privamvada S, Khan S, Naqshbandi A, Khan S, Yusufi A. Protective effect of 3-polyunsaturated fatty acids (PUFAs) on sodium nitroprusside-induced nephrotoxicity and oxidative damage in rat kidney. Hum Exp Toxicol 2012; 31:1035–1049.
29Priyamvada S, Khan SA, Khan MW. Studies on the protective effect of dietary fish oil on uranyl nitrate-induced nephrotoxicity and oxidative damage in rat kidney. Prostaglandins Leukot Essent Fatty Acids 2010; 82:35–44.
30Khan MW, Priyamvada S, Khan SA, Khan S, Gangopadhyay A, Yusufi ANK. Fish/flaxseed oil protect against nitric oxide-induced hepatotoxicity and cell death in the rat liver. Hum Exp Toxicol 2016; 35:302–311.
31Rubiolo LA, Mithieux G, Vega F. Resveratrol protects primary rat hepatocytes against oxidative stress damage. Activation of the Nrf2 transcription factor and augmented activities of antioxidant enzymes. Eur J Pharmacol 2008; 591:66–72.
32Scholzen T, Gerdes J. The Ki-67 protein: from the known and the unknown. J Cell Physiol 2000; 182:311–322.
33Rahmanzadeh R, Huttmann G, Gerdes J, Scholzen T. Chromophore-assisted light inactivation of pKi67 leads to inhibition of ribosomal RNA synthesis. Cell Prolif 2007; 40:422–430.
34Tadbir AA, Pardis S, Ashkavandi ZJ, Najvani AD, Ashraf MJ, Taheri A, et al. Expression of Ki67 and CD105 as proliferation and angiogenesis markers in salivary gland tumors. Asian Pac J Cancer Prev 2012; 13:5155–5159.
35Nishimura R, Osako T, Okumura Y. Clinical significance of Ki-67 in neoadjuvant chemotherapy for primary breast cancer as a predictor for chemosensitivity and for prognosis. Breast Cancer 2010; 17:269–275.
36Dowsett M, Smith IE, Ebbs SR. Prognostic value of Ki-67 expression after short-term presurgical endocrine therapy for primary breast cancer. J Nat Cancer Inst 2007; 99:167–170.
37Lilian U, Jian M, Tong L, Kathrin S, Paul E. Dietary flaxseed alters tumor biological markers in postmenopausal breast cancer. Clin Cancer Res 2005; 11:3828–3835.
38Ferrara N. Role of vascular endothelial growth factor in regulation of physiological angiogenesis. Am J Physiol Cell Physiol2001; 280:C1358–C1366.
39Pammer J, WeningerW, Mildner M, Burian M, Wojta J, Tschachler E. Vascular endothelial growth factor is constitutively expressed in normal human salivary glands and is secreted in the saliva of healthy individuals. J Pathol 1998; 186:186–191.
40Li XF, Gregory J, Ahmed A. Immunolocalization of vascular endothelial cell growth in human endometrium. Growth Factors 1994; 11:277–282.
41Watanabe I, Koriyama Y, Yamada E. High-resolution scanning electron microscopic study of the mouse submandibular salivary gland. Acta Anat 1992; 143:59–66.
42Roberts WG, Palade GE. Increased microvascular permeability and endothelial fenestration induced by vascular endothelial growth factor. J Cell Sci 1995; 108:2369–2379.
43Zhong XS, Liu LZ, Skinner HD, Cao Z, Ding M, Jiang BH. Mechanism of vascular endothelial growth factor expression mediated by cisplatin in human ovarian cancer cells. Biochem Biophys Res Commun 2007; 358:92–98.
44Lemay A, Dodin S, Kadri N, Forest J. Flaxseed dietary supplement versus hormone replacement therapy in hypercholesterolemic menopausal women. Obstet Gynecol 2002; 100:495–504.
45Mueller M, Vigne J, Minchenko A, Lebovic D, Leitman D, Taylor R. Regulation of vascular endothelial growth factor (VEGF) gene transcription by oestrogen receptors A and B. Proc Natl Acad Sci USA 2000; 97:10972–10977.