http://informahealthcare.com/gye ISSN: 0951-3590 (print), 1473-0766 (electronic) Gynecol Endocrinol, Early Online: 1–4 ! 2015 Informa UK Ltd. DOI: 10.3109/09513590.2015.1005593
ORIGINAL ARTICLE
Therapeutic effect of sunitinib on diabetes mellitus related ovarian injury: an experimental rat model study Oytun Erbas1, Halil Gursoy Pala2, Emel Ebru Pala3, Burcu Artunc Ulkumen2, Levent Akman4, Tulay Akman5, Fatih Oltulu6, Huseyin Aktug6, and Altug Yavasoglu6
Gynecol Endocrinol Downloaded from informahealthcare.com by University of Victoria on 02/26/15 For personal use only.
1
Physiology Department, Istanbul Bilim University School of Medicine, Istanbul, Turkey, 2Obstetrics and Gynecology-Perinatology Department, Celal Bayar University School of Medicine, Manisa, Turkey, 3Pathology Department, Tepecik Training and Research Hospital, Izmir, Turkey, 4Obstetrics and Gynecology Department, Ege University School of Medicine, Izmir, Turkey, 5Internal Diseases-Medical Oncology Department, Tepecik Training and Research Hospital, Izmir, Turkey, and 6Histology and Embryology Department, Ege University School of Medicine, Izmir, Turkey
Abstract
Keywords
The aim of our study is to investigate the effect of sunitinib on diabetes mellitus related-ovarian injury and fibrosis in rat models. An experimental diabetes mellitus model was created in 16 rats, and eight rats with normal blood glucose levels were included in control group (Group-1). The diabetic rats were divided into two groups:diabetic control group (water given) – Group-2 and sunitinib treatment group – Group-3. After four weeks, bilateral oophorectomy was performed and ovaries were examined histologically. The groups were compared by Student’s t-test, analysis of variance (ANOVA) and Mann–Whitney’s U-test. There was a significant increase in no-medication (water given) diabetic rat’s ovary (Group-2) in terms of follicular degeneration, stromal degeneration, stromal fibrosis and NF-kappaB immuneexpression compared with control group normal rats’ ovary (Group-1) (p50.0001). Stromal degeneration (p ¼ 0.04), stromal fibrosis (p ¼ 0.01), follicular degeneration (p ¼ 0.02), NF-kappaB immune-expression (p ¼ 0.001) significantly decreased in sunitinib-treated diabetic rat’s ovary (Group-3) when compared with no-medication (water given) diabetic rat’s ovary (Group-2) (p50.05). When we used sunitinib in the treatment of diabetic rats, ovarian injury, fibrosis and NF-kappaB immunoexpression decreased significantly. The effects of sunitinib in rat models give hope to the improved treatment of premature ovarian failure due to diabetes mellitus in humans.
Advanced glication end products, diabetes mellitus, NF-kappaB, premature ovarian failure, receptor of advanced glication end products, sunitinib
Introduction High levels of glucose causes tissue injury through five major mechanisms: (1) the polyol pathway, (2) the activation of protein kinase-C isoforms, (3) overactivity of the hexosamine pathway, (4) increased intracellular formation of advanced glycation end products (AGEs) and (5) increased expression of the receptor for AGEs and its activating ligands. Evidence of most studies indicate that all of these mechanisms are activated by mitochondrial overproduction of reactive oxygen species (ROS) [1]. AGEs are formed by the nonenzymatic reaction of glucose and other glycating components derived from glucose and fatty acid oxidation in arterial endothelial cells and heart with proteins [2,3]. In diabetes mellitus, AGEs increase in extracellular matrix [4,5]. Intracellular AGE precursors’ production can damage cells by three ways: (a) functional alterations,
Address for correspondence: Halil Gursoy Pala, Obstetrics and Gynecology-Perinatology Department, Celal Bayar University School of Medicine, Manisa 45210, Turkey. Tel: 902364652494, 905055252332. Fax: 902364652434. E-mail: [email protected]
History Received 26 March 2014 Revised 19 December 2014 Accepted 6 January 2015 Published online 23 February 2015
(b) abnormal interactions with other matrix components and integrins, (c) activation of the pleiotropic transcription factor nuclear factor (NF)-kappaB, by ROS. AGE receptors (RAGE) on cells, such as endothelial cells, vascular smooth muscle cells and macrophages, induce the production of ROS that leads to multiple pathological changes in gene expression. The activation of NF-kappaB pathway causes the production of vascular endothelial growth factor (VEGF) and inflammatory cytokines like TNF and IL-1 [6]. Like the other organ systems, glucose toxicity in ovary occurs through NF-kappaB pathway in diabetes mellitus [7]. Sunitinib is a receptor tyrosine kinase inhibitor in oral broad spectrum. It has been approved by the United States Food and Drug Administration (FDA) for the treatment of gastrointestinal stromal tumors and advanced stage renal cell carcinoma [8,9]. This drug was chosen for the investigation of adhesion prevention because it differs from other receptor tyrosine kinase inhibitors. It is more specific for VEGF receptors, although it also has an activity against platelet derived growth factor, stem cell factor receptor (C-kit), glial cell-line derived neutrophilic factor receptor (RET) and Fms-like tyrosine kinase-3 [10,11]. In this present work, our aim was to investigate the effect of sunitinib on diabetes mellitus related-ovarian injury and fibrosis in rat models.
2
O. Erbas et al.
Materials and methods Animals In this study, 24 female Sprague Dawley albino mature rats of 8 weeks, weighing 200–220 g were used. Animals were fed ad libitum and housed in pairs in steel cages having a temperaturecontrolled environment (22 ± 2 C) with 12-hour light/dark cycles. The experimental procedures were approved by the Committee for Animal Research of Gaziosmanpasa University. All animal studies are strictly conformed to the animal experiment guidelines of the Committee for Human Care.
Gynecol Endocrinol Downloaded from informahealthcare.com by University of Victoria on 02/26/15 For personal use only.
Experimental protocol Diabetes was induced by intraperitoneal (i.p.) injection of streptozocin (STZ) (Sigma-Aldrich, Inc.; Saint Louis, MO) (60 mg/kg in 0.9% NaCl, adjusted to a pH 4.0 with 0.2 M sodium citrate) for 16 rats. No drug was administered to the remaining rats whose blood glucose levels were under 120 mg/dl (n ¼ 8) (control group, Group-1). Diabetes was verified after 24 h by evaluating blood glucose levels with the use of glucose oxidase reagent strips (Boehringer–Mannheim, Indianapolis, IN). The rats with blood glucose levels 250 mg/dl and higher were included in this study as diabetic rat group (n ¼ 16). We waited 21 days for the development of diabetes-related microvascular complications. Then, 16 diabetic rats were randomly divided into 2 groups: Group-2 (diabetic control group, 8 rats) were given 4 ml/per day tap water by oral gavage and the rats in Group 3 (sunitinib group, 8 rats) were given 1 mg/kg/day of oral sunitinib for four weeks. Capsul containing 50 mg sunitinib (Sutent, Pfizer Inc, NYC, NY) was crushed and suspended in tap water to yield a concentration of 10 mg/ml. According to the weight of each rat, suspended drug solution was completed to 4 ml with tap water. The medications were given via orogastric tubes. Then, the animals were euthanized and blood samples were collected by cardiac puncture for biochemical analysis and bilateral oophorectomy was performed for histopathological examination. Histopathological examination of ovary Formalin-fixed/paraffin embedded ovary sections (4 mm) were stained with hematoxylen & eosine (HE). All sections were photographed with Olympus C-5050 digital camera mounted on Olympus BX51 microscope (Olympus, Tokyo, Japan). Follicular degeneration, stromal degeneration, and stromal fibrosis were scored by three histologists (FO, HA, AY) and one pathologist (EEP) from 0 to 3 according to the severity of injury, where 0 represented no pathologic findings, and 1, 2 and 3 represented pathologic findings of less than 33%, 33% to 66%, and more than 66% of the ovarian section, respectively. NF-kappaB immunoexpression For immunohistochemistry, sections were incubated with H2O2 (10%) for 30 min to eliminate endogenous peroxidase activity and blocked with 10% normal goat serum (Invitrogen) for 1 hour at room temperature. Subsequently, sections were incubated with primary antibodies (NF-kappaB, Bioss Inc.; dilution 1/100) for 24 h at 4 C. Antibody detection was performed with the Histostain-Plus Bulk kit (Bioss Inc, Woburn, MA) against rabbit IgG, and 3,30 diaminobenzidine (DAB) was used to visualise the final product. All sections were washed in PBS and photographed with an Olympus C-5050 digital camera attached to Olympus BX51 microscope. Brown cytoplasmic staining in oocyte was scored positive for NF-kappaB. The number of NF-kappaB positive cells was assessed systematically by scoring at least 100 ovarian stromal cells per
Gynecol Endocrinol, Early Online: 1–4
10 fields of tissue sections at 100 magnification by three histologists (FO, HA, AY) and one pathologist (EEP). Statistical analysis Data analyses were performed using SPSS version 15.0 for Windows (Chicago, IL). The groups of parametric variables were compared by Student’s t-test and analysis of variance (ANOVA). The groups of nonparametric variables were compared by the Mann–Whitney U-test. Results were given as mean ± standard error of mean (SEM). A value of p50.05 was accepted as statistically significant. p50.001 was accepted as statistically highly significant.
Results The comparison of follicular degeneration, stromal degeneration, stromal fibrosis and NF-kappaB immune-expression scores of normal control group (Group 1), no-medication (water given) group (Group 2) and sunitinib-medication group (Group 3) are summarized in Table 1. There was a significant increase in follicular degeneration, stromal degeneration, stromal fibrosis and NF-kappaB immuneexpression in no-medication (water given) diabetic rat’s ovary (Group-2) when compared with control group normal rats (Group-1) (p50.0001). Stromal degeneration (p ¼ 0.04), stromal fibrosis (p ¼ 0.01), follicular degeneration (p ¼ 0.02), NF-kappaB immune-expression (p ¼ 0.001) were significantly decreased in sunitinib-treated diabetic rat’s ovary (Group-3) when compared with no-medication (water given) diabetic rat’s ovary (Group-2). In diabetic rat’s ovary (Group-2), the morphological examination showed degeneration and stromal inflammation. Hematoxylen & Eosine and immunohistochemical staining results of these 3 groups are shown in Figure 1.
Discussion In recent years, hyperglycaemia and a number of hyperglycaemia-related factors, including AGEs, have been linked to diabetic complications [12,13]. AGEs are formed by a complicated, non-enzymatic and irreversible process that links reducing sugar groups to proteins, lipids and nucleic acids. Evidence from studies using anti-AGE agents indicates that AGEs may play a role in the pathogenesis of diabetes complications [14]. RAGE, a member of immunoglobulin family of cell surface receptors, interacts with different ligands, especially AGEs [14]. The activation of RAGE engages critical signalling pathways linked to downstream responses, such as NF-kappaB pathway [15]. The involvement of NF-kappaB pathway in response to AGEs has been investigated in a macrophage cell line [16]. TNF-alpha and other proinflammatory cytokines, the potent angiogenic factor VEGF are regulated by NF-kappaB. During the inflammation period, fibrosis occurs in tissues through NF-kappaB pathway [6]. VEGF induces the proliferation of vascular endothelial cells in vitro via receptor-tyrosine-kinase [17]. Several tyrosine kinase inhibitors, such as lestaurtinib, sorafenib and sunitinib were identified as NF-kappaB antagonists [18]. These drugs have a broad spectrum of kinase inhibition and are shown to inhibit VEGF and tyrosine kinase [19]. Therefore, sunitinib plays a role in decreasing fibrosis and tissue injury. Sunitinib reduces the blood glucose levels both in diabetic and non-diabetic patients though the mechanism is unclear [20]. But various animal model studies evaluated the role of tyrosine kinases in the regulation of blood glucose levels. c-kit is a tyrosine kinase that is expressed in a variety of tissues including germ
Effect of sunitinib on diabetic ovary
DOI: 10.3109/09513590.2015.1005593
3
Table 1. The comparison of follicular degeneration, stromal degeneration, stromal fibrosis and NF-kappa B immune-expression scores of normal control group (Group 1), no-medication (water given) group (Group 2) and sunitinib-treated group (Group 3). Stromal degeneration score
Follicle degeneration score
Stromal fibrosis score
NF-kappaB immunoexpression percent (%)
0.18 ± 0.03 2.24 ± 0.21* 1.28 ± 0.18**
0.21 ± 0.10 2.65 ± 0.35* 1.43 ± 0.32**
0.28 ± 0.07 2.58 ± 0.54* 1.16 ± 0.15**
4.33 ± 1.15 38.14 ± 3.38* 11.23 ± 5.45**
Normal control (Group-1) Diabetic rat (water given) (Group-2) Sunitinib (Group-3)
Gynecol Endocrinol Downloaded from informahealthcare.com by University of Victoria on 02/26/15 For personal use only.
*p50.0001, Normal control group (Group-1) compared with diabetic rat (water given) group (Group-2). **p50.05, Sunitinib group (Group-3) compared with diabetic rat (water given) group (Group-2)
Figure 1. Hematoxylen & Eosine (HE) staining of sections from rat ovary (10/20 magnification) (a) Control group (Group-1), ovarian sections showed normal stroma (s), secondary follicle (sf), tertiary follicle (tf) (c) Water given diabetic rats (Group-2), ovarian sections showed fibrotic ovarian stroma (**), inflammation (*) (e) Decreased ovarian stromal fibrosis (**) and stromal inflammation (*) by sunitinib treatment in diabetic rats (Group-3). Immunohistochemical staining of sections from rat ovaries (100 magnification). (b) Normal expression of NF-kappaB in the control group (Group-1) ovary. (d): Marked expression of NF-kappaB in water given diabetic rats (Group-2) (f): Decreased expression of NF-kappaB in sunitinib treated diabetic rats (Group-3).
cells, hematopoietic progenitor cells, melanocytes and gastrointestinal pacemaker cells [21]. Women with diabetes have a delayed menarche age and higher risk of menstrual irregularities than non-diabetic women of similar age [22]. Nearly 35% of the women with diabetes have problems including amenorrhea, polymenorrhea, and oligomenorrhea throughout their reproductive years. This is approximately double the prevalence of menstrual disorders observed among women without diabetes [22]. Also, menopause age was significantly earlier in diabetic women when compared with nondiabetic women [23]. Several reports have suggested that early menopause in diabetic patients has an autoimmune etiology. Approximately 20–40% of women with premature ovarian failure also have autoimmune disorders [24,25]. Contrary to these reports, in our previous study, we showed that glucose toxicity occurs severely in ovary and this injury is originated by NF-kappaB way in diabetes
mellitus in rat models [7]. In this present study, we found similar results. Also, we showed the positive effects of sunitinib in ovarian injury, fibrosis and NF-kappaB immunoexpression. The effects of sunitinib in rat models gives hope to improved treatment of premature ovarian failure due to diabetes in humans. It is highly warranted to continue clinical investigations aiming the discovery of mechanisms and novel targets in diabetes mellitus-related premature ovarian failure.
Declaration of interest The authors report no declaration of interest.
References 1. Brownlee M. The pathobiology of diabetic complications: a unifying mechanism. Diabetes 2005;54:1615–25.
Gynecol Endocrinol Downloaded from informahealthcare.com by University of Victoria on 02/26/15 For personal use only.
4
O. Erbas et al.
2. Candido R, Forbes JM, Thomas MC, et al. A breaker of advanced glycation end products attenuates diabetes-induced myocardial structural changes. Circ Res 2003;92:785–92. 3. Wautier JL, Schmidt AM. Protein glycation: a firm link to endothelial cell dysfunction. Circ Res 2004;95:233–8. 4. Stitt AW, Moore JE, Sharkey JA, et al. Advanced glycation end products in vitreous: structural and functional implications for diabetic vitreopathy. Invest Ophthalmol Vis Sci 1998;39:2517–23. 5. Stitt AW, Li YM, Gardiner TA, et al. Advanced glycation end products (AGEs) co-localize with AGE receptors in the retinal vasculature of diabetic and of AGE-infused rats. Am J Pathol 1997; 150:523–31. 6. Kiriakidis S, Andreakos E, Monaco C, et al. VEGF expression in human macrophages is NF-kappaB-dependent: studies using adenoviruses expressing the endogenous NF-kappaB inhibitor IkappaBalpha and a kinase-defective form of the IkappaB kinase 2. J Cell Sci 2003;116:665–74. 7. Pala HG, Erbas O, Oltulu F, et al. Glucose injury in diabetic rat’s ovaries and effect of NF-kappaB way. Ege J Med 2013;52:32–6. 8. Patel TV, Morgan JA, Demetri GD, et al. A preeclampsia-like syndrome characterized by reversible hypertension and proteinuria induced by the multitargeted kinase inhibitors sunitinib and sorafenib. J Natl Cancer Inst 2008;100:282–4. 9. Launay-Vacher V, Derey G. Hypertension and proteinuria: a classeffect of antiangiogenic therapies. Anticancer Drugs 2009;20:81–2. 10. Tickenbrock L, Muller-Tidow C, Berdel WE, Serve H. Emerging Flt3 kinase inhibitors in the treatment of leukaemia. Exp Opin Emerg Drugs 2006;11:153–65. 11. Mori S, Cortes J, Kantarjian H, et al. Potential role of sorafenib in the treatment of acute myeloid leukemia. Leuk Lymphoma 2008;49: 2246–55. 12. Chen XF, Lin WD, Lu SL, et al. Mechanistic study of endogenous skin lesions in diabetic rats. Exp Dermatol 2010;19: 1088–95. 13. Yan SF, Berile GR, D’Agati V, et al. The biology of RAGE and its ligands: uncovering mechanisms at the heart of diabetes and its complications. Curr Diab Rep 2007;2:146–53.
Gynecol Endocrinol, Early Online: 1–4
14. Peppa M, Stavroulakis P, Raptis SA. Advanced glycoxidation products and impaired diabetic wound healing. Wound Repair Regen 2009;17:461–72. 15. Bao W, Min D, Twigg SM, et al. Monocyte CD147 is induced by advanced glycation end products and high glucose concentration: possible role in diabetic complications. Am J Physiol Cell Physiol 2010;299:1212–19. 16. Zhang F, Banker G, Liu X, et al. The novel function of advanced glycation end products in regulation of MMP-9 production. J Surg Res 2011;171:871–6. 17. Swendeman S, Mendelson K, Weskamp G, et al. VEGF-A stimulates ADAM17-dependent shedding of VEGFR2 and crosstalk between VEGFR2 and ERK signaling. Circ Res 2008;101:916–18. 18. Miller SC, Huang R, Sakamuru S, et al. Identification of known drugs that act as inhibitors of NF-kappaB signaling and their mechanism of action. Biochem Pharmacol 2010;1;79:1272–80. 19. Mori S, Cortes J, Kantarjian H, et al. Potential role of sorafenib in the treatment of acute myeloid leukemia. Leuk Lymphoma 2008;49: 2246–55. 20. Agostino NM, Chinchilli VM, Lynch CJ, et al. Effect of the tyrosine kinase inhibitors (sunitinib, sorafenib, dasatinib, and imatinib) on blood glucose levels in diabetic and nondiabetic patients in general clinical practice. J Oncol Pharm Pract 2011;17:197–202. 21. Akin C, Metcalfe DD. The biology of Kit in disease and the application of pharmacogenetics. J Allergy Clin Immunol 2004;114: 13–19. 22. Yeshaya A, Orvieto R, Dicker D, et al. Menstrual characteristics of women suffering from insulin-dependent diabetes mellitus. Int J Fertil 1995;40:269–73. 23. Dorman JS, Steenkiste AR, Foley TP, et al. Familial Autoimmune and Diabetes (FAD) Study. Menopause in type 1 diabetic women: is it premature? Diabetes 2001;50:1857–62. 24. Kalantaridon SN, Davis S, Nelson L. Premature ovarian failure. Endocrinol Metab Clin North Am 1998;27:989–1006. 25. Hoek A, Schoemaker J, Drexhage HA. Premature ovarian failure and ovarian autoimmunity. Endocrinol Rev 1997;18:102–34.
ORIGINAL ARTICLE
Therapeutic effect of sunitinib on diabetes mellitus related ovarian injury: an experimental rat model study Oytun Erbas1, Halil Gursoy Pala2, Emel Ebru Pala3, Burcu Artunc Ulkumen2, Levent Akman4, Tulay Akman5, Fatih Oltulu6, Huseyin Aktug6, and Altug Yavasoglu6
Gynecol Endocrinol Downloaded from informahealthcare.com by University of Victoria on 02/26/15 For personal use only.
1
Physiology Department, Istanbul Bilim University School of Medicine, Istanbul, Turkey, 2Obstetrics and Gynecology-Perinatology Department, Celal Bayar University School of Medicine, Manisa, Turkey, 3Pathology Department, Tepecik Training and Research Hospital, Izmir, Turkey, 4Obstetrics and Gynecology Department, Ege University School of Medicine, Izmir, Turkey, 5Internal Diseases-Medical Oncology Department, Tepecik Training and Research Hospital, Izmir, Turkey, and 6Histology and Embryology Department, Ege University School of Medicine, Izmir, Turkey
Abstract
Keywords
The aim of our study is to investigate the effect of sunitinib on diabetes mellitus related-ovarian injury and fibrosis in rat models. An experimental diabetes mellitus model was created in 16 rats, and eight rats with normal blood glucose levels were included in control group (Group-1). The diabetic rats were divided into two groups:diabetic control group (water given) – Group-2 and sunitinib treatment group – Group-3. After four weeks, bilateral oophorectomy was performed and ovaries were examined histologically. The groups were compared by Student’s t-test, analysis of variance (ANOVA) and Mann–Whitney’s U-test. There was a significant increase in no-medication (water given) diabetic rat’s ovary (Group-2) in terms of follicular degeneration, stromal degeneration, stromal fibrosis and NF-kappaB immuneexpression compared with control group normal rats’ ovary (Group-1) (p50.0001). Stromal degeneration (p ¼ 0.04), stromal fibrosis (p ¼ 0.01), follicular degeneration (p ¼ 0.02), NF-kappaB immune-expression (p ¼ 0.001) significantly decreased in sunitinib-treated diabetic rat’s ovary (Group-3) when compared with no-medication (water given) diabetic rat’s ovary (Group-2) (p50.05). When we used sunitinib in the treatment of diabetic rats, ovarian injury, fibrosis and NF-kappaB immunoexpression decreased significantly. The effects of sunitinib in rat models give hope to the improved treatment of premature ovarian failure due to diabetes mellitus in humans.
Advanced glication end products, diabetes mellitus, NF-kappaB, premature ovarian failure, receptor of advanced glication end products, sunitinib
Introduction High levels of glucose causes tissue injury through five major mechanisms: (1) the polyol pathway, (2) the activation of protein kinase-C isoforms, (3) overactivity of the hexosamine pathway, (4) increased intracellular formation of advanced glycation end products (AGEs) and (5) increased expression of the receptor for AGEs and its activating ligands. Evidence of most studies indicate that all of these mechanisms are activated by mitochondrial overproduction of reactive oxygen species (ROS) [1]. AGEs are formed by the nonenzymatic reaction of glucose and other glycating components derived from glucose and fatty acid oxidation in arterial endothelial cells and heart with proteins [2,3]. In diabetes mellitus, AGEs increase in extracellular matrix [4,5]. Intracellular AGE precursors’ production can damage cells by three ways: (a) functional alterations,
Address for correspondence: Halil Gursoy Pala, Obstetrics and Gynecology-Perinatology Department, Celal Bayar University School of Medicine, Manisa 45210, Turkey. Tel: 902364652494, 905055252332. Fax: 902364652434. E-mail: [email protected]
History Received 26 March 2014 Revised 19 December 2014 Accepted 6 January 2015 Published online 23 February 2015
(b) abnormal interactions with other matrix components and integrins, (c) activation of the pleiotropic transcription factor nuclear factor (NF)-kappaB, by ROS. AGE receptors (RAGE) on cells, such as endothelial cells, vascular smooth muscle cells and macrophages, induce the production of ROS that leads to multiple pathological changes in gene expression. The activation of NF-kappaB pathway causes the production of vascular endothelial growth factor (VEGF) and inflammatory cytokines like TNF and IL-1 [6]. Like the other organ systems, glucose toxicity in ovary occurs through NF-kappaB pathway in diabetes mellitus [7]. Sunitinib is a receptor tyrosine kinase inhibitor in oral broad spectrum. It has been approved by the United States Food and Drug Administration (FDA) for the treatment of gastrointestinal stromal tumors and advanced stage renal cell carcinoma [8,9]. This drug was chosen for the investigation of adhesion prevention because it differs from other receptor tyrosine kinase inhibitors. It is more specific for VEGF receptors, although it also has an activity against platelet derived growth factor, stem cell factor receptor (C-kit), glial cell-line derived neutrophilic factor receptor (RET) and Fms-like tyrosine kinase-3 [10,11]. In this present work, our aim was to investigate the effect of sunitinib on diabetes mellitus related-ovarian injury and fibrosis in rat models.
2
O. Erbas et al.
Materials and methods Animals In this study, 24 female Sprague Dawley albino mature rats of 8 weeks, weighing 200–220 g were used. Animals were fed ad libitum and housed in pairs in steel cages having a temperaturecontrolled environment (22 ± 2 C) with 12-hour light/dark cycles. The experimental procedures were approved by the Committee for Animal Research of Gaziosmanpasa University. All animal studies are strictly conformed to the animal experiment guidelines of the Committee for Human Care.
Gynecol Endocrinol Downloaded from informahealthcare.com by University of Victoria on 02/26/15 For personal use only.
Experimental protocol Diabetes was induced by intraperitoneal (i.p.) injection of streptozocin (STZ) (Sigma-Aldrich, Inc.; Saint Louis, MO) (60 mg/kg in 0.9% NaCl, adjusted to a pH 4.0 with 0.2 M sodium citrate) for 16 rats. No drug was administered to the remaining rats whose blood glucose levels were under 120 mg/dl (n ¼ 8) (control group, Group-1). Diabetes was verified after 24 h by evaluating blood glucose levels with the use of glucose oxidase reagent strips (Boehringer–Mannheim, Indianapolis, IN). The rats with blood glucose levels 250 mg/dl and higher were included in this study as diabetic rat group (n ¼ 16). We waited 21 days for the development of diabetes-related microvascular complications. Then, 16 diabetic rats were randomly divided into 2 groups: Group-2 (diabetic control group, 8 rats) were given 4 ml/per day tap water by oral gavage and the rats in Group 3 (sunitinib group, 8 rats) were given 1 mg/kg/day of oral sunitinib for four weeks. Capsul containing 50 mg sunitinib (Sutent, Pfizer Inc, NYC, NY) was crushed and suspended in tap water to yield a concentration of 10 mg/ml. According to the weight of each rat, suspended drug solution was completed to 4 ml with tap water. The medications were given via orogastric tubes. Then, the animals were euthanized and blood samples were collected by cardiac puncture for biochemical analysis and bilateral oophorectomy was performed for histopathological examination. Histopathological examination of ovary Formalin-fixed/paraffin embedded ovary sections (4 mm) were stained with hematoxylen & eosine (HE). All sections were photographed with Olympus C-5050 digital camera mounted on Olympus BX51 microscope (Olympus, Tokyo, Japan). Follicular degeneration, stromal degeneration, and stromal fibrosis were scored by three histologists (FO, HA, AY) and one pathologist (EEP) from 0 to 3 according to the severity of injury, where 0 represented no pathologic findings, and 1, 2 and 3 represented pathologic findings of less than 33%, 33% to 66%, and more than 66% of the ovarian section, respectively. NF-kappaB immunoexpression For immunohistochemistry, sections were incubated with H2O2 (10%) for 30 min to eliminate endogenous peroxidase activity and blocked with 10% normal goat serum (Invitrogen) for 1 hour at room temperature. Subsequently, sections were incubated with primary antibodies (NF-kappaB, Bioss Inc.; dilution 1/100) for 24 h at 4 C. Antibody detection was performed with the Histostain-Plus Bulk kit (Bioss Inc, Woburn, MA) against rabbit IgG, and 3,30 diaminobenzidine (DAB) was used to visualise the final product. All sections were washed in PBS and photographed with an Olympus C-5050 digital camera attached to Olympus BX51 microscope. Brown cytoplasmic staining in oocyte was scored positive for NF-kappaB. The number of NF-kappaB positive cells was assessed systematically by scoring at least 100 ovarian stromal cells per
Gynecol Endocrinol, Early Online: 1–4
10 fields of tissue sections at 100 magnification by three histologists (FO, HA, AY) and one pathologist (EEP). Statistical analysis Data analyses were performed using SPSS version 15.0 for Windows (Chicago, IL). The groups of parametric variables were compared by Student’s t-test and analysis of variance (ANOVA). The groups of nonparametric variables were compared by the Mann–Whitney U-test. Results were given as mean ± standard error of mean (SEM). A value of p50.05 was accepted as statistically significant. p50.001 was accepted as statistically highly significant.
Results The comparison of follicular degeneration, stromal degeneration, stromal fibrosis and NF-kappaB immune-expression scores of normal control group (Group 1), no-medication (water given) group (Group 2) and sunitinib-medication group (Group 3) are summarized in Table 1. There was a significant increase in follicular degeneration, stromal degeneration, stromal fibrosis and NF-kappaB immuneexpression in no-medication (water given) diabetic rat’s ovary (Group-2) when compared with control group normal rats (Group-1) (p50.0001). Stromal degeneration (p ¼ 0.04), stromal fibrosis (p ¼ 0.01), follicular degeneration (p ¼ 0.02), NF-kappaB immune-expression (p ¼ 0.001) were significantly decreased in sunitinib-treated diabetic rat’s ovary (Group-3) when compared with no-medication (water given) diabetic rat’s ovary (Group-2). In diabetic rat’s ovary (Group-2), the morphological examination showed degeneration and stromal inflammation. Hematoxylen & Eosine and immunohistochemical staining results of these 3 groups are shown in Figure 1.
Discussion In recent years, hyperglycaemia and a number of hyperglycaemia-related factors, including AGEs, have been linked to diabetic complications [12,13]. AGEs are formed by a complicated, non-enzymatic and irreversible process that links reducing sugar groups to proteins, lipids and nucleic acids. Evidence from studies using anti-AGE agents indicates that AGEs may play a role in the pathogenesis of diabetes complications [14]. RAGE, a member of immunoglobulin family of cell surface receptors, interacts with different ligands, especially AGEs [14]. The activation of RAGE engages critical signalling pathways linked to downstream responses, such as NF-kappaB pathway [15]. The involvement of NF-kappaB pathway in response to AGEs has been investigated in a macrophage cell line [16]. TNF-alpha and other proinflammatory cytokines, the potent angiogenic factor VEGF are regulated by NF-kappaB. During the inflammation period, fibrosis occurs in tissues through NF-kappaB pathway [6]. VEGF induces the proliferation of vascular endothelial cells in vitro via receptor-tyrosine-kinase [17]. Several tyrosine kinase inhibitors, such as lestaurtinib, sorafenib and sunitinib were identified as NF-kappaB antagonists [18]. These drugs have a broad spectrum of kinase inhibition and are shown to inhibit VEGF and tyrosine kinase [19]. Therefore, sunitinib plays a role in decreasing fibrosis and tissue injury. Sunitinib reduces the blood glucose levels both in diabetic and non-diabetic patients though the mechanism is unclear [20]. But various animal model studies evaluated the role of tyrosine kinases in the regulation of blood glucose levels. c-kit is a tyrosine kinase that is expressed in a variety of tissues including germ
Effect of sunitinib on diabetic ovary
DOI: 10.3109/09513590.2015.1005593
3
Table 1. The comparison of follicular degeneration, stromal degeneration, stromal fibrosis and NF-kappa B immune-expression scores of normal control group (Group 1), no-medication (water given) group (Group 2) and sunitinib-treated group (Group 3). Stromal degeneration score
Follicle degeneration score
Stromal fibrosis score
NF-kappaB immunoexpression percent (%)
0.18 ± 0.03 2.24 ± 0.21* 1.28 ± 0.18**
0.21 ± 0.10 2.65 ± 0.35* 1.43 ± 0.32**
0.28 ± 0.07 2.58 ± 0.54* 1.16 ± 0.15**
4.33 ± 1.15 38.14 ± 3.38* 11.23 ± 5.45**
Normal control (Group-1) Diabetic rat (water given) (Group-2) Sunitinib (Group-3)
Gynecol Endocrinol Downloaded from informahealthcare.com by University of Victoria on 02/26/15 For personal use only.
*p50.0001, Normal control group (Group-1) compared with diabetic rat (water given) group (Group-2). **p50.05, Sunitinib group (Group-3) compared with diabetic rat (water given) group (Group-2)
Figure 1. Hematoxylen & Eosine (HE) staining of sections from rat ovary (10/20 magnification) (a) Control group (Group-1), ovarian sections showed normal stroma (s), secondary follicle (sf), tertiary follicle (tf) (c) Water given diabetic rats (Group-2), ovarian sections showed fibrotic ovarian stroma (**), inflammation (*) (e) Decreased ovarian stromal fibrosis (**) and stromal inflammation (*) by sunitinib treatment in diabetic rats (Group-3). Immunohistochemical staining of sections from rat ovaries (100 magnification). (b) Normal expression of NF-kappaB in the control group (Group-1) ovary. (d): Marked expression of NF-kappaB in water given diabetic rats (Group-2) (f): Decreased expression of NF-kappaB in sunitinib treated diabetic rats (Group-3).
cells, hematopoietic progenitor cells, melanocytes and gastrointestinal pacemaker cells [21]. Women with diabetes have a delayed menarche age and higher risk of menstrual irregularities than non-diabetic women of similar age [22]. Nearly 35% of the women with diabetes have problems including amenorrhea, polymenorrhea, and oligomenorrhea throughout their reproductive years. This is approximately double the prevalence of menstrual disorders observed among women without diabetes [22]. Also, menopause age was significantly earlier in diabetic women when compared with nondiabetic women [23]. Several reports have suggested that early menopause in diabetic patients has an autoimmune etiology. Approximately 20–40% of women with premature ovarian failure also have autoimmune disorders [24,25]. Contrary to these reports, in our previous study, we showed that glucose toxicity occurs severely in ovary and this injury is originated by NF-kappaB way in diabetes
mellitus in rat models [7]. In this present study, we found similar results. Also, we showed the positive effects of sunitinib in ovarian injury, fibrosis and NF-kappaB immunoexpression. The effects of sunitinib in rat models gives hope to improved treatment of premature ovarian failure due to diabetes in humans. It is highly warranted to continue clinical investigations aiming the discovery of mechanisms and novel targets in diabetes mellitus-related premature ovarian failure.
Declaration of interest The authors report no declaration of interest.
References 1. Brownlee M. The pathobiology of diabetic complications: a unifying mechanism. Diabetes 2005;54:1615–25.
Gynecol Endocrinol Downloaded from informahealthcare.com by University of Victoria on 02/26/15 For personal use only.
4
O. Erbas et al.
2. Candido R, Forbes JM, Thomas MC, et al. A breaker of advanced glycation end products attenuates diabetes-induced myocardial structural changes. Circ Res 2003;92:785–92. 3. Wautier JL, Schmidt AM. Protein glycation: a firm link to endothelial cell dysfunction. Circ Res 2004;95:233–8. 4. Stitt AW, Moore JE, Sharkey JA, et al. Advanced glycation end products in vitreous: structural and functional implications for diabetic vitreopathy. Invest Ophthalmol Vis Sci 1998;39:2517–23. 5. Stitt AW, Li YM, Gardiner TA, et al. Advanced glycation end products (AGEs) co-localize with AGE receptors in the retinal vasculature of diabetic and of AGE-infused rats. Am J Pathol 1997; 150:523–31. 6. Kiriakidis S, Andreakos E, Monaco C, et al. VEGF expression in human macrophages is NF-kappaB-dependent: studies using adenoviruses expressing the endogenous NF-kappaB inhibitor IkappaBalpha and a kinase-defective form of the IkappaB kinase 2. J Cell Sci 2003;116:665–74. 7. Pala HG, Erbas O, Oltulu F, et al. Glucose injury in diabetic rat’s ovaries and effect of NF-kappaB way. Ege J Med 2013;52:32–6. 8. Patel TV, Morgan JA, Demetri GD, et al. A preeclampsia-like syndrome characterized by reversible hypertension and proteinuria induced by the multitargeted kinase inhibitors sunitinib and sorafenib. J Natl Cancer Inst 2008;100:282–4. 9. Launay-Vacher V, Derey G. Hypertension and proteinuria: a classeffect of antiangiogenic therapies. Anticancer Drugs 2009;20:81–2. 10. Tickenbrock L, Muller-Tidow C, Berdel WE, Serve H. Emerging Flt3 kinase inhibitors in the treatment of leukaemia. Exp Opin Emerg Drugs 2006;11:153–65. 11. Mori S, Cortes J, Kantarjian H, et al. Potential role of sorafenib in the treatment of acute myeloid leukemia. Leuk Lymphoma 2008;49: 2246–55. 12. Chen XF, Lin WD, Lu SL, et al. Mechanistic study of endogenous skin lesions in diabetic rats. Exp Dermatol 2010;19: 1088–95. 13. Yan SF, Berile GR, D’Agati V, et al. The biology of RAGE and its ligands: uncovering mechanisms at the heart of diabetes and its complications. Curr Diab Rep 2007;2:146–53.
Gynecol Endocrinol, Early Online: 1–4
14. Peppa M, Stavroulakis P, Raptis SA. Advanced glycoxidation products and impaired diabetic wound healing. Wound Repair Regen 2009;17:461–72. 15. Bao W, Min D, Twigg SM, et al. Monocyte CD147 is induced by advanced glycation end products and high glucose concentration: possible role in diabetic complications. Am J Physiol Cell Physiol 2010;299:1212–19. 16. Zhang F, Banker G, Liu X, et al. The novel function of advanced glycation end products in regulation of MMP-9 production. J Surg Res 2011;171:871–6. 17. Swendeman S, Mendelson K, Weskamp G, et al. VEGF-A stimulates ADAM17-dependent shedding of VEGFR2 and crosstalk between VEGFR2 and ERK signaling. Circ Res 2008;101:916–18. 18. Miller SC, Huang R, Sakamuru S, et al. Identification of known drugs that act as inhibitors of NF-kappaB signaling and their mechanism of action. Biochem Pharmacol 2010;1;79:1272–80. 19. Mori S, Cortes J, Kantarjian H, et al. Potential role of sorafenib in the treatment of acute myeloid leukemia. Leuk Lymphoma 2008;49: 2246–55. 20. Agostino NM, Chinchilli VM, Lynch CJ, et al. Effect of the tyrosine kinase inhibitors (sunitinib, sorafenib, dasatinib, and imatinib) on blood glucose levels in diabetic and nondiabetic patients in general clinical practice. J Oncol Pharm Pract 2011;17:197–202. 21. Akin C, Metcalfe DD. The biology of Kit in disease and the application of pharmacogenetics. J Allergy Clin Immunol 2004;114: 13–19. 22. Yeshaya A, Orvieto R, Dicker D, et al. Menstrual characteristics of women suffering from insulin-dependent diabetes mellitus. Int J Fertil 1995;40:269–73. 23. Dorman JS, Steenkiste AR, Foley TP, et al. Familial Autoimmune and Diabetes (FAD) Study. Menopause in type 1 diabetic women: is it premature? Diabetes 2001;50:1857–62. 24. Kalantaridon SN, Davis S, Nelson L. Premature ovarian failure. Endocrinol Metab Clin North Am 1998;27:989–1006. 25. Hoek A, Schoemaker J, Drexhage HA. Premature ovarian failure and ovarian autoimmunity. Endocrinol Rev 1997;18:102–34.
