BCL-2 and Bax Expression in Skin Flaps Treated with Finasteride or Azelaic Acid

Document Type: Research article

Authors

1 Department of pharmacognosy, School of Pharmacy, Shahid Beheshti University, M.C., Tehran, Iran. School of Pharmacy and Phytochemistry Research Centre, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

2 Faculty of Nutrition Sciences and Food Technology, Shahid Beheshti University of Medical Sciences and Health Services.

3 School of Pharmacy and Phytochemistry Research Centre, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

4 Physiology Research Center, Tehran University of Medical Sciences, Tehran, Iran.

5 Center for Research and Training in Skin Diseases and Leprosy, Tehran University of Medical Sciences, Tehran, Iran.

6 Faculty of Library and Information Science, Islamic Azad University, Tehran Science and Research Unit, Tehran, Iran.

7 Physiology Research Center, Tehran University of Medical Sciences, Tehran, Iran. Center for Research and Training in Skin Diseases and Leprosy, Tehran University of Medical Sciences, Tehran, Iran. Nano Vichar Pharmaceutical Ltd, Tehran, Iran.

Abstract

Despite all modern surgical techniques, skin flap that is considered as the main method in most reconstructive surgeries puts the skin tissue at danger of necrosis and apoptosis derived from ischemia. Therefore, finding a treatment for decreasing the apoptosis derived from flap ischemia will be useful in clinic.
In present study, we evaluated the effect of azelaic acid 20% and finasteride on expression of BCL-2 and bax proteins after the skin flap surgery. For this purpose, 21 rats were entered in three groups including control, azelaic acid 20% and finasteride, all experienced skin flap surgery and then flap tissue was assessed for determining the expression of proteins in 5 slices prepared from each rat that were graded between – to +++ scales.
Both azelaic acid and finasteride increased the expression of BCL-2 protein (p < 0.05) and decrease the expression of bax protein (p < 0.05).
These results suggested an antiapoptotic role for finasteride and azelaic acid in preserving the flap after the ischemia reperfusion insult.

Keywords

Main Subjects


Introduction

   Skin flap is considered as the main method in most reconstructive surgeries (1) and using it to provide suitable tissue coverage has an essential role in increasing patients’ life quality (2). Despite the rapid progresses in skin flap application in clinic, ischemia may restrict its utility (3). The phenomenon of ischemia-reperfusion (IR) has been postulated to be an important event in skin flap surgery that led to major complications and loss of tissue (4) but also involves in other chronic wounds such as venous stasis and diabetic foot ulcers (5-6). Therefore finding of treatments which reduce flap injury would be highly advantageous in reconstructive surgery.

   Oxidative stress derived from IR injury through production of reactive oxygen species (ROS) and oxidative damage causes DNA modification, lipid peroxidation and secretion of inflammatory cytokines (7-8). In addition apoptosis is induced by ischemic insults in skin tissue. (8)

    Plenty of proteins and number of genes regulate apoptosis, in which two pairs of proteins are more important that include Bcl2 and Bax. Bcl2 family of proteins are found in external surface of mitochondria and are divided to 3 groups: antiapoptotic proteins like Bcl2 and BclxL, proapoptotic proteins like Bax and BAD and the proteins with apoptotic activity like Bik (9-11). After a severe damage to the DNA of cells in a way that cannot be repaired, it will undergo apoptosis in which activation of p53 causes expression of Bax (Bcl2 associated x-protein). Bax protein are existing at external membrane of mitochondria with some other proteins make complex of Apoptosome,  that has key role in activating of caspases and induction of apoptosis. (12-13)

    Yet it has been shown that, various pharmacological agents including inducible nitric oxide synthsase (iNOS) (14), botulinum toxin A (15), Hemoglobin vesicles (16), Erythropoitin (17), University of Wisconsin solution (18) and Enalapril (19) protected against ischemia induced injury of skin flaps. In present study we evaluated the effects of two different drugs, finasteride and Azelaic acid on bcl2 and bax expression after skin ischemia reperfusion.

Experimental

 

   This experiment received approval of ethical committee of the Center for Research and Training in Skin Disease and Leprosy, Tehran University of Medical Sciences. 21 Sprauge-Dawley male rats (200-250 grams) were chosen for random pattern cranial based skin flap elevation (19). The rats were divided to 3 equal number groups (7 rats in each) including one control and two treatment groups in which protein expression in their flap tissue were evaluated under receiving different pharmacologic agents for preconditioning the flaps. All along the study period the rats were situated in single cages supplied with ample water and food. Pre-operative preparations included shaving, administeration of artificial tears and scrubbing by isopropyl alcohol and povidone-iodine. Intraperitoneal administration of a mixture of ketamin and Xylazine (50 and 10 mg/kg respectively, Pake-Davis Pharmaceutical Co., Cambridge, UK) was performed for induction of general anesthesia.

   Dorsal surface of rat body was used for taking flap. Before surgery 1 mL of each pharmacologic agent under study was injected subcutaneously at spots 5.5, 6.5 and 7.5 cm distant from caudal margin of flap. For control group normal saline was merely used. In two experimental groups, the rats were injected azelaic acid (100 mg/flap, Merck) or finasteride (5 mg/kg, Reddy India).

   Flap elevation was performed by making two parallel caudal to cephalad incisions  extending from inferior angle of scapulae to superior border of pelvic bones with distance of 3 cm from each other on the mice backs, so 8×3 cm flaps were created after final incision connecting distal ends of two parallel cuts and leaving a 3 cm base connected to body for blood supply . After raising the flap and detaching it from underlying fascia, an impermeable plastic barrier similar in size to the flap (8×3 cm) was interpositioned between the flap and its corresponding bed (20).

   On the seventh post-operative day, tissue samples from flap area were prepared for measurement of apoptotic and anti apoptotic proteins in the tissue (21).

Immunohistochemical analysis

   Tissue samples were put in PBS 0.01 M and preserved in -70ºC and were studied by alkaline phosphatase immunohistochemistry. Five micron-thick sections were obtained by microtome and transferred onto adhesive slides. The sections were kept in the autoclave at 37 °C for 16 h and at 60 °C for 20 min. Then they were deparaffinized and dehydrated by immersion into xylene twice for 10 min and into alcohol twice for 2 min. Then, the specimens were incubated in 3% H2O2 for 5 min to inhibit activation of endogenous peroxidases. After reaction with primary antibody, reaction of secondary labeled antibodies (Rabbit polyclonal to Bcl2; phospho S87, ab73985, abcam and Rabbit polyclonal to Bax; ab69643, abcam) with alkaline phosphatase were applied for immunohistochemical analysis of bcl-2 and bax, respectively (dilution 1.100). In immunohistochemistry Bcl2 or Bax proteins take the dye and this shows the amount of protein expressed by cells quantitatively (22-23)

   Mayer's hematoxylin was used as counterstain and slides were examined by light microscopy. The results of the immunostaining were analyzed semiquantitatively. The percentage of positive keratinocytes (amount of staining) were recorded as follows: (–) no expression; (+/–) immunostaining in occasional cells (weak expression); (+) immunostaining in 1–25% of cells (mild expression); (++) immunostaining in 26–50% of cells (strong expression); and (+++) immunostaining in > 50% of cells (very strong expression) (24-25).

Statistical analysis

   The results of the study were statistically analyzed using SPSS 14 program (Windows, Microsoft, USA). Mann–Whitney's U-test was utilized for comparison of numerical data. A p-value of α ≤ 0.05 was considered significant.

 Results

   The results of immunohistochemical analysis of bcl-2 and bax are shown in Tables 1 and 2. In the control group expression of bcl2 was low and approximately in 71% of slides the level of expression of bcl2 was less than mild (+). However the expression of bax in the control group of skin flap after 7 days had been increased, while in more than 68.6% of slices the expression was more than weak grade. The results for azlaic acid and finasteride was converse, and the expression of bcl2 was increased in these groups to more than weak ( +/-) in approximately 51.5% and 60% of them, respectively (p < 0.05). On the other hand expression of bax protein was significantly decreased by treatment with azlaic acid and finasteride and reached less than mild grade in approximately 60% and  51.4% of them, respectively  (p < 0.05).

  

Table 1: Amount of immunostaining for bcl-2 protein in skin flap specimens in three different groups.       

Groups

 

Bcl2 expression

Total

 

-

+/-

+

++

+++

 

Control

12 (34.3%)

13 (37.1%)

8 (22.9%)

2 (5.7%)

0 (.0%)

35 (100.0%)

Azelaic acid *

7 (20.0%)

10 (28.6%)

12 (34.3%)

5 (14.3%)

1 (2.9%)

35 (100.0%)

Finasteride *

7 (20.0%)

7 (20.0%)

14 (40.0%)

6 (17.1%)

1 (2.9%)

35 (100.0%)

                    *p < 0.05 compared to the control group.

 

 

Table 2: Amount of immunostaining for bax protein in skin flap specimens in three different groups.

 Group

 

Bax expression

Total

-

+/-

+

++

+++

 

Control

3 (8.6%)

8 (22.9%)

11 (31.4%)

10 (28.6%)

3 (8.6%)

35 (100.0%)

Azelaic acid *

7 (20.0%)

14 (40.0%)

7 (20.0%)

7 (20.0%)

0 (.0%)

35 (100.0%)

Finasteride *

6 (17.1%)

12 (34.3%)

12 (34.3%)

4 (11.4%)

1 (2.9%)

35 (100.0%)

 

Discussion

   In present study treatment with azelaic acid and finasteride before skin flap procedure produced a significant and different profile of apoptotic protein expression after 7 days, in which the ratio of bcl2 to bax has been increased compared to control group.

    In our previous study we evaluated the effects of finasteride or azelaic acid 20% on skin flap viability and showed that each of these two treatments is able to significantly reduce the necrotic area of skin flap (26). In addition to the anti necrotic properties of azelaic acid and finasteride, results of present study confirmed the protective effects of azelaic acide and finasteride against I/R injury by changing protein expression in which led to the decreased apoptosis rate after skin flap procedure.  

    The effects of finasteride and azelaic acid on increasing of bcl2 and decreasing of bax expression in skin flaps in present work have some controversies with studies on the prostate cancer cells. The effect of finasteride on prostate tissue in patients undergoing radical prostatectomy showed decreased apoptotic factors caspase-7 and IGFBP-3 in cancer cells, while having little to no effect on caspase-3, insulin growth factor-1, bcl-2, p53 and p21 (27). Howevwer, in an In vitro model for prostate cancer, Finasteride caused the apoptosis and increased immunoreactivity for pro-apoptotic Bax whereas decreased antiapoptotic Bcl-2 and Bcl-xL expression (28). Treatment of rats with finasteride led to a slight decrease in Bax and observed.Significant reduction in Bcl-2 expression at the dose of 100 mg/kg body weight (29). In the case of azelaic acid, also there are studies that have showed converse results, for example it has been showed that the cardioprotective and antiapoptotic effects of 17Beta-estradiol were blocked by azelaic acid, as a thioredoxin (Trx) reductase inhibitor, because 17Beta-estradiol acts via activation of Trx reductase (30), that regulates the levels of intracellular ROS and modulates intracellular oxidative states, which may be important for cellular function, survival, and death (31).

   It should be mention that prostatic hyperplasia demonstrates increased expression of the Bcl-2 protein, but no change in the expression of Bax, Bcl-x, and Bak  (32). Therefore interaction of finasteride or azelaic acid with this cells could be different from skin tissue after ischemia. Moreover, there are some findings that confirm the results of present study. Finasteride primarily was marketed for the treatment of benign prostatic hypertrophy (33), and this was derived from its’ inhibitory effects on 5-alpha-reductase activity (34). Also azelaic acid has showed a potent inhibitory effect on 5-alpha-reductase activity and this effect was detectable at concentrations as low as 0.2 mmol/L and was complete at 3 mmol/L (35). Enzyme 5-alpha-reductase is involved in catalyzing of testosterone to dihydrotestosterone (DHT) conversion (36). Inhibition of dihydrotestosterone (DHT) has been shown that increase the expression of iNOS in testis and epididymis of rats (37). In our previous study, administration of L-NAME prior to the finasteride and azelaic acid blocked its’ effects on reducing of flap necrotic area that suggested involvement of NO in this pathway. Therefore we suggested that finasteride and azelaic acid by inhibition of 5-alpha-reductase activity and decreasing of testosterone to dihydrotestosterone (DHT) conversion has eliminated the inhibitory effects of DHT on iNOS expression, and as a result this NO dependent pathway of IPC has been triggered. 

   Cytotoxic effects of nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) are considered to be one of the major causes of inflammatory diseases. On the other hand, protective effects of NO on toxic insults-induced cellular damage/apoptosis have been demonstrated recently. In the study by Yamaoka J et al., it has been demonstrated that NO from NO donor suppressed UVB-induced apoptosis of murine keratinocytes. In addition, NO significantly suppressed activities of caspase 3, caspase 8 and caspase 9 that had been upregulated by UVB radiation. NO also suppressed p53 expression that had been upregulated by UVB radiation and upregulated Bcl-2 expression that had been down-regulated by UVB radiation. These findings suggested that NO might suppress UVB-induced keratinocyte apoptosis by regulating apoptotic signaling cascades in p 53, Bcl-2, caspase3, caspase 8 and caspase 9 (38). In fact UV irradiation of human skin leads to inducible nitric-oxide synthase (iNOS) expression in keratinocytes and endothelial cells (ECs). In search of the molecular mechanism responsible for the protective effect, Suschek CV et al. found that protection from UVA-induced apoptosis is tightly correlated with NO-mediated increases in Bcl-2 expression and a concomitant inhibition of UVA-induced overexpression of Bax protein. NO either endogenously produced or exogenously applied , iNOS-derived NO, fully protects against UVA-induced cell damage and death via modulation of proteins of the Bcl-2 family (39). Therefore it is possible for azelaic acide and finasteride to modulate apoptotic proteins after ischemia reperfusion by regulating NO and related pathways.

   In conclusion, the results of present study suggested an antiapoptotic role for finasteride and azelaic acid in skin flap model by increasing of bcl-2 expression and decreasing of bax expression, however the precise mechanism and involved pathways remained to be determine.

 

(1)    Pavletic MM. Skin flaps in reconstructive surgery. Vet Clin. North Am. Small Anim. Pract. (1990) 20: 81-103.

(2)     Hold A, Kamolz L and Frey M. The need for flaps in burn surgery. Handchir. Mikrochir. Plast. Chir. (2009) 41: 343-7.

(3)    Kerrigan CL. Skin flap failure: pathophysiology. Plast. Reconstr. Surg. (1983) 72:766-77.

(4)    Delaney A, Diamantis S and Marks VJ. Complications of tissue ischemia in dermatologic surgery. Dermatol. Ther. (2011) 24 : 551-7.

(5)     Loots MA, Lamme EN, Zeegelaar J, Mekkes JR, Bos JD and Middelkoop E. Differences in cellular infiltrate and extracellular matrix of chronic diabetic and venous ulcers versus acute wounds. J. Invest. Derm (1998) 111: 850-7.

(6)     Angel M, Ramasastry SS, Swartz WM, Basford RE and Futrel JW. The causes of skin ulcerations associated with venous insufficiency: A unifying hypothesis. Plast. Reconstr. Surg. (1987) 79: 289-97.

(7)     Briganti S and Picardo M. Antioxidant activity, lipid peroxidation and skin diseases. What's new? J. Eur. Acad. Dermatol. Venereol. (2003) 17: 663-9.

(8)     Reid RR, Sull AC, Mogford JE, Roy N and Mustoe TA. A novel murine model of cyclical cutaneous ischemia-reperfusion injury. J. Surg. Res. (2004) 116: 172-80.

(9)     Hengartner MO. The biochemistry of apoptosis. Nature (2000) 407: 770-6.

(10)    Zamzami N, Brenner C, Marzo I, et al???: Subcellular and submitochondrial mode of action of Bcl2-like oncoproteins, Oncogene (1998) 16: 2265-82.

(11)    Cain K, Brown PG, Langlais C and Cohen GM. Caspase activation involves the formation of aposome, a large ( approximately 700 KD ) Caspase-activating complex. J. Biol. Chem. (1999) 274: 22686-22692.

(12)    Earnshaw WC, Martins LM and Kaufmann SH. Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Anu. Rev. Biochem. (1999) 383-424.

(13)     Thornberry NA and Lazebnik Y. Caspases: enemies within. Science (1998) 281: 1312-1316

(14)    Ozyazgan I, Ozköse M and Bakol G. Nitric oxide in flow-through venous flaps and effects of L-arginine and nitro-L-arginine methyl ester (L-NAME) on nitric oxide and flap survival in rabbits. Ann. Plast. Surg. (2007) 59: 550-7.

(15)    Kim YS, Roh TS, Lee WJ, Yoo WM and Tark KC. The effect of botulinum toxin A on skin flap survival in rats. Wound Repair. Regen. (2009) 17: 411-7.

(16)     Plock JA, Rafatmehr N, Sinovcic D, Schnider J, Sakai H, Tsuchida E, Banic A and Erni D. Hemoglobin vesicles improve wound healing and tissue survival in critically ischemic skin in mice. Am. J. Physiol. Heart Circ. Physiol. (2009) 297: H905-10.

(17)     Contaldo C, Elsherbiny A, Lindenblatt N, Plock JA, Trentz O, Giovanoli P, Menger MD and Wanner GA. Erythropoietin enhance oxygenation in critically perfused tissue through modulation of nitric oxide synthase. Shock (2009) 31: 599-606 17.

(18)     Eichhorn W, Blake FA, Pohlenz P, Gehrke G, Schmelzle R and Heiland M. Conditioning of myocutaneous flaps. J. Craniomaxillofac. Surg. (2009) 37: 196-200.

(19)    Pazoki-Toroudi H, Ajami M, Habibey R, Hajiaboli E and Firooz A. The Effect of enalapril on skin flap viability is independent of angiotensin II AT1 receptors. Ann. Plast. Surg. (2009) 62: 699–702.

(20)     De Carvalho EN, Ferreira LM, de Carvalho NA, et al????. Viability of a random pattern dorsal skin flap, in diabetic rats. Acta Cir. Bras. (2005) 20: 225-8.

(21)    Mc Farlane RM, De Young G and Henry RA. The design of a pedicle flap in the rat to study necrosis and its prevention. Plast. Reconstr. Surg. (1965) 35: 177-182.

(22)    Habibey R, Ajami M, Ebrahimi SA, Hesami A, Babakoohi S and Pazoki-Toroudi H. Nitric oxide and renal protection in morphine-dependent rats. Free Radic. Biol. Med. (2010) 49: 1109-18.

(23)    Ajami M, Eghtesadi S, Razaz JM, Kalantari N, Habibey R, Nilforoushzadeh MA, Zarrindast M and Pazoki-Toroudi H. Expression of Bcl-2 and Bax after hippocampal ischemia in DHA + EPA treated rats. Neurol. Sci. (2011) 32: 811-8.

(24)    Pazoki-Toroudi H, Ajami M, Babakoohi S, Khaki L, Habibey R, Akhiani M, Seirafi H and Firooz A. Effects of diphencyprone on expression of Bcl-2 protein in patients with alopecia areata. Immunopharmacol. Immunotoxicol. (2010) 32: 422-5.

(25)    Koçak M, Bozdogan O, Erkek E, Atasoy P and Birol A. Examination of Bcl-2, Bcl-X and bax protein expression in psoriasis. Int. J. Dermatol.(2003) 42: 789-93.

(26)     Ajami M, Nilforoushzadeh M,  Babakoohi Sh, Habibey R, Banimostafa Arab F, Pazoki-Toroudi N, Rashighi-Firoozabadi M, Firooz A, Dowlati Y and Pazoki-Toroudi H. The effects of finasteride and azelaic acid on skin flap viability in rats. Skin Leishmaniasis (2010) 1: 12-17

(27)    Bass R, Perry B, Langenstroer P, Thrasher JB, Dennis KL, Tawfik O and Holzbeierlein J. Effects of short-term finasteride on apoptotic factors and androgen receptors in prostate cancer cells. J. Urol. (2009) 181: 615-20.

(28)     Golbano JM, Lóppez-Aparicio P, Recio MN and Pérez-Albarsanz MA. Finasteride induces apoptosis via Bcl-2, Bcl-xL, Bax and caspase-3 proteins in LNCaP human prostate cancer cell line. Int. J. Oncol. (2008) 32: 919-24.

(29)     Huynh H. Induction of apoptosis in rat ventral prostate by finasteride is associated with alteration in MAP kinase pathways and Bcl-2 related family of proteins. Int. J. Oncol.(2002) 20: 1297-303.

(30)     Satoh M, Matter CM, Ogita H, Takeshita K, Wang CY, Dorn GW and Liao JK. Inhibition of apoptosis-regulated signaling kinase-1 and prevention of congestive heart failure by estrogen. Circulation (2007) 115: 3197-204.

(31)     Nordberg J and Arner ES. Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radic. Biol. Med. (2001) 31: 1287–1312.

(32)      Cardillo M, Berchem G, Tarkington MA, Krajewski S, Krajewski M, Reed JC, Tehan T, Ortega L, Lage J and Gelmann EP. Resistance to apoptosis and up regulation of Bcl-2 in benign prostatic hyperplasia after androgen deprivation. J. Urol.(1997) 158: 212-6.

(33)     Matzkin H, Barak M and Braf Z. Effect of finasteride on free and total serum prostate-specific antigen in men with benign prostatic hyperplasia. Br. J. Urol. (1996) 78: 405-8

(34)     Tarter TH and Vaughan ED Jr. Inhibitors of 5alpha-reductase in the treatment of benign prostatic hyperplasia. Curr. Pharm. Des. (2006) 12: 775-83.

(35)     Stamatiadis D, Bulteau-Portois MC and Mowszowicz I. Inhibition of 5 alpha-reductase activity in human skin by zinc and azelaic acid. Br. J. Dermatol. (1988) 119: 627-32.

(36)     Bartsch G, Rittmaster RS and Klocker H. Dihydrotestosterone and the concept of 5alpha-reductase inhibition in human benign prostatic hyperplasia. World J. Urol. (2002) 19: 413-25.

(37)     Kolasa A, Marchlewicz M, Kurzawa R, Głąbowski W, Trybek G, Wenda-Różewicka L and Wiszniewska B. The expression of inducible nitric oxide synthase (iNOS) in the testis and epididymis of rats with a dihydrotestosterone (DHT) deficiency. Cell. Mol. Biol. Lett. (2009) 14: 511-27.

(38)     Yamaoka J, Kawana S and Miyachi Y. Nitric oxide inhibits ultraviolet B-induced murine keratinocyte apoptosis by regulating apoptotic signaling cascades. Free Radic. Res. (2004) 38: 943-50.

(39)      Suschek CV, Krischel V, Bruch-Gerharz D, Berendji D, Krutmann J, Krncke KD and Kolb-Bachofen V. Nitric oxide fully protects against UVA-induced apoptosis in tight correlation with Bcl-2 up-regulation. J. Biol. Chem. (1999) 274: 6130-7.