Tumor Biol. (2016) 37:5999–6006 DOI 10.1007/s13277-015-4469-9
ORIGINAL ARTICLE
Chronic unpredictable stress deteriorates the chemopreventive efficacy of pomegranate through oxidative stress pathway Shirin Hasan 1,2 & Nida Suhail 1,6 & Nayeem Bilal 1 & Ghulam Md. Ashraf 1,7 & Syed Kashif Zaidi 3 & Sultan AlNohair 4 & Naheed Banu 1,5
Received: 2 November 2015 / Accepted: 17 November 2015 / Published online: 23 November 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Chronic unpredictable stress (CUS) can influence the risk and progression of cancer through increased oxidative stress. Pomegranate is known to protect carcinogenesis through its antioxidative properties. This study is carried out to examine whether CUS affects the chemopreventive potential of pomegranate through oxidative stress pathway. Role of CUS on early stages of 7, 12 dimethyl benz(a) anthracene (DMBA) induced carcinogenesis, and its pre-exposure effect on chemopreventive efficacy of pomegranate juice (PJ) was examined in terms of in vivo antioxidant and biochemical parameters in Swiss albino rats. Rats were divided in various groups and were subjected to CUS paradigm, DMBA administration (65 mg/kg body weight, single dose), and PJ treatment. Exposure to stress (alone) and DMBA (alone) led to increased oxidative stress by significantly decreasing the antioxidant enzymes activities and altering the glutathione
(GSH), malondialdehyde (MDA), glutamate oxaloacetate transaminase (GOT), and glutamate pyruvate transaminase (GPT) levels. A significant increase in DNA damage demonstrated by comet assay was seen in the liver cells. Stress exposure to DMBA-treated rats further increased the oxidative stress and disturbed the biochemical parameters as compared to DMBA (alone)-treated rats. Chemoprevention with PJ in DMBA (alone)-treated rats restored the altered parameters. However, in the pre-stress DMBA-treated rats, the overall antioxidant potential of PJ was significantly diminished. Our results indicate that chronic stress not only increases the severity of carcinogenesis but also diminishes the anti-oxidative efficacy of PJ. In a broader perspective, special emphasis should be given to stress management and healthy diet during cancer chemoprevention. Keywords Carcinogenesis . Chronic unpredictable stress . DMBA . DNA damage . Oxidative stress . Pomegranate
* Naheed Banu
[email protected];
[email protected]
Introduction 1
Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh 202002, Uttar Pradesh, India
2
Present address: Department of Surgery, Loyola University Chicago, Maywood, IL, USA
3
Center of Excellence in Genomic Medicine Research, King Abdulaziz University, Jeddah, Saudi Arabia
4
Department of Family and Community Medicine, College of Medicine, Qassim University, Qassim, Kingdom of Saudi Arabia
5
Present address: College of Medical Rehabilitation, Qassim University, P.O. Box 2100, Buraydah 51451, Kingdom of Saudi Arabia
6
Department of Biochemistry, Faculty of Applied Medical Sciences, Northern Border University, Arar, Saudi Arabia
7
King Fahd Medical Research Center, King Abdulaziz University, P.O. Box 80216, Jeddah 21589, Saudi Arabia
Carcinogenesis is a multistage process; hence, chemoprevention has become an important approach to regulate cancer in addition to chemotherapy. Components present in fruits and vegetables like dietary fiber, phytochemicals, and micronutrients inhibit carcinogenesis by regulating cellular machineries of defense and apoptosis [1]. Pomegranate, famous for its medicinal properties is now being researched as a potential chemopreventive and anticancer agent. Pomegranate possesses strong antioxidant, anti-inflammatory, antiatherogenic, and anticarcinogenic properties [2, 3]. Pomegranate juice is rich in many active compounds which include the polyphenols such as flavonoids (anthocyanins like cyanidin-3-glucoside, cyanidin-3,5-diglucoside, delphinidin3-glucoside; catechins and other complex flavonoids) and hydrolyzable tannins (punicalin, pedunculagin, punicalagin, gallagic,
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and ellagic acid esters of glucose) that account for 92 % of its antioxidant activity [4]. Pomegranate juice (PJ) was demonstrated to show better bioactivity compared to its purified components punicalagin and ellagic acid, thus highlighting the multifactorial effects of multiple compounds compared to single purified active ingredients [5]. A recent study has shown that chronic stress is a significant risk factor for higher rates of morbidity and mortality [6]. Increasing influence of psycho-socio-political, behavioral, and environmental factors in human lives has led to an enhanced susceptibility to stress related chronic diseases [7]. The major neural pathways activated by stressors are the hypothalamic-pituitaryadrenal (HPA) axis and the sympathetic nervous system that results in enhanced release of catecholamines and glucocorticoids [8]. Chronic exposures to these mediators of the stress response cause a substantial decline in endogenous antioxidant status and a rise in oxidative stress markers [9]. Various animal studies have shown that stress may alter tumor growth. However, results are conflicting as both exacerbation and attenuation of tumor development have been reported. Previous studies in our lab have shown the enhancement of 7, 12 dimethyl benz(a) anthracene (DMBA) induced oxidative stress and DNA damage on prior exposure to chronic unpredictable stress (CUS), thus exhibiting a promoter effect of stress on the process of carcinogenesis [10]. In view of the above findings, we studied the effect of CUS that utilizes both physical and psychological stressors, on the chemopreventive properties of pomegranate in Swiss albino rats. Antioxidant and biochemical parameters were studied in the circulation and liver. Efficacy of PJ in alleviating the deteriorated biochemical parameters was checked in three different conditions viz., CUS alone, DMBA alone, and CUS along with DMBA exposure.
Table 1
Materials and methods Chemicals DMBA, Histopaque 1077, HBSS, and RPMI 1640 were purchased from Sigma (St Louis, MO); reduced nicotinamide adenine dinucleotide phosphate (NADPH), reduced glutathione (GSH), glutathione oxidized (GSSG), 5,5-dithiobis(2nitrobenzoic acid) (DTNB), 1-chloro-2,4-dinitrobenzene (CDNB), Pyrogallol, and pNPP were purchased from SRL India. All other chemicals were of analytical grade. Commercial kits of SGOT, SGPT, and uric acid were purchased from Span Diagnostics Ltd. India. Animals Adult, female, Swiss albino rats, weighing 180±10 g obtained from Central Drug Research Institute, Lucknow, India, were used for the experiment. All experimental protocols adhered to the guidelines of the Animal Welfare Committee of the Aligarh Muslim University as per the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA, 714/02/a/CPCSEA) norms. The rats were housed under standard conditions of temperature and humidity and a 12-h light/dark cycle with free access to standard pellet diet (Ashirwad Industries, Chandigarh, India) and water (except during various treatments as mentioned in the protocol). Treatment of animals The experiment was set for a total of 5 months. After 1 week of acclimatization, the rats were divided into eight groups of 16 animals each as follows:
Chronic unpredictable stress procedure
Days
Stressor
Time
Days
Stressor
Time
Day 1
1 h restraint stress 30 min cold room (4 °C) 1 h shaking/crowding 5 min cold water swim 4 h wet bedding Lights on over night 3 h high density housing 5 min warm water swim 2 h restraint stress 30 min cold room (4 °C) 6 h isolation Lights on over night 5 min cold water swim 16 h food and water deprivation 2 h restraint stress 1 h shaking/crowding
08:00 a.m 11: 00 p.m 11:00 a.m 03:00 p.m 09:00 a.m 06:00 p.m 10:00 a.m 06:00 p.m 08:00 a.m 03:00 p.m 10:00 a.m 07:00 p.m 09:00 a.m 03:00 p.m 10:00 a.m 02:00 p.m
Day 9
4 h high density housing lights on over night 4 h wet bedding 16 h food and water deprivation 3 h restraint stress 2-h isolation 5 min cold water swim 1 h min shaking/crowing 10 min tail pinch in restrainer 1 h shaking/crowing 2 h restraint stress 3 h high density housing 24 h food and water deprivation
08:00 a.m 07:00 p.m 09:00 a.m 04:00 p.m 11:00 a.m 05:00 p.m 08:00 a.m 11:00 a.m 09:00 a.m 04:00 p.m 11:00 a.m 04:00 p.m 08:00 a.m
Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8
Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
Tumor Biol. (2016) 37:5999–6006 Control
a,b
9
*
CUS
8 MDA (nmol/mg protein)
Fig. 1 Changes in MDA levels on PJ supplementation in circulation and liver. Each value represents mean±SEM of eight animals. aP
