Chemopreventive potential of adrenergic blocker in behavioral stress accelerated prostate cancer development in rats

 

Prakash Nargatti¹*, Sudhir Patil², Sandip Patil^3,4, Nilofar Naikwade²

¹Department of Pharmacology, Annasaheb Dange College of Pharmacy, Ashta, Sangli, Maharashtra, India.

²Department of Pharmacology, Appasaheb Birnale College of Pharmacy, Sangli, Maharashtra, India.

³Department of Pharmacology, Dr. Shivajirao Kadam College of Pharmacy,

Kasbe Digraj, Sangli, Maharashtra, India

⁴Biocyte Institute of Research & Development, Sangli, Maharashtra, India

 

ABSTRACT:

Psychosocial stress increasingly recognized as an important health issue in development and progression of cancer. Prostate cancer patients have increased levels of stress and anxiety. Several studies suggest that environmental factors are important in prostate cancer development. Patients with prostate cancer reportedly show higher levels of anxiety compared with other cancer patients. Based on psychoneuroimmunology we report that stress promotes prostate cancer in rats through stress response system. The sympathetic nervous system potentially regulate tumour α and β-adrenergic signalling both via circulating adrenaline/nor-adrenaline and via local nor-adrenaline release from SNS nerve fibres. Activation of the sympathetic nervous system promotes cancer progression via β-adrenoreceptor–mediated activation of protein kinase A and exchange protein activated by adenylyl cyclase signalling pathways. Stimulation of α­ receptor leads to the activation of Ca⁺⁺ permeable no selective cationic channels and transient receptor potential (TRP) channel family, which are responsible for cell proliferation, which leads to prostate cancer progression. Certain adrenergic blockers can exert anticancer activity by blocking α and β adrenergic pathway. Thus current study examined association between prostate cancer progression and behavioural study. Our findings could be used to new clinical and treatment strategies in cancer therapy.

 

 

 

INTRODUCTION:

Clinical and epidemiological studies have identified that stress, chronic depression and lack of social support are considered as risk factors for cancer progression.^1-2 Psycho emotional stress activates hypothalamic-pituitary-adrenal axis (HPA) and sympathetic nervous system (SNS) that leads to release of glucocorticoids from adrenal gland, adrenaline and nor-adrenaline from adrenal medulla and sympathetic neurons.^3-4

 

According to Reiche E. 2004hypothalamus secretes corticotrophin - releasing factor (CRF) and arginine vasopressin, which activate the HPA axis, leading to release of adrenocorticotrophic hormone, enkephalins, and endorphins.

 

The activation of the sympathetic nervous system by CRF is mediated by direct innervation of the locus coeruleus in the brainstem, which leads to widespread release of nor epinephrine throughout the brain and peripheral tissues. Activation of the sympathetic nervous system also stimulates the release of CRF by hypothalamic parventricular nuclei. Thus, activation of one component of the system stimulates the other component.⁵

 

β adrenergic pathway:⁶

SNS activation regulates cancer-related molecular pathways by direct regulation of β-receptor bearing tumour cells and regulation of other β-receptor bearing cells present in the tumour microenvironment, such as macrophages and vascular cells.β₂ receptor are present on prostate gland cell. Both catecholamines bind to β₂ adrenergic receptor resulting in activation of adenylyl cyclase and subsequent conversion of ATP to cAMP. cAMP activates protein kinase A (PKA) and exchange protein activated by adenylyl cyclase (EPAC).

 

α adrenergic pathway:⁷

Among α₁-adrenergic receptor subtypes, α_1a is present in the fibro muscular stroma of the prostate. Its stimulation leads to the activation of phospholipase C (PLC), resulting in an increase in intracellular free Ca⁺⁺ via inositol triphosphate (IP₃) and diacylglycerol (DAG) production. Recent studies in vascular smooth muscles have shown that α₁-adrenergic receptor may also activate Ca⁺⁺-permeable nonselective cationic channels and that TRPC6 a member of the transient receptor potential (TRP) channel family.

 

Currently, carvedilol acts as adrenergic blocker as a non-selective β adrenergic blocker and selective α₁ blocking activity and it has strong antioxidant action.^8-9 In the present study, we selected Carvedilol to investigate; chemopreventive potential of adrenergic blocker using behavioral stress models (immobilization and cold-water swimming).^10-12

 

MATERIALS AND METHODS:

Materials:

PC-3 cell lines were obtained from National Center for Cell Sciences (NCCS) Pune, Carvedilol were obtained from Cipla, Monosodium glutamates were purchased from Sigma. All reagents and solvents used in the current investigation were of analytical grade.

 

Selection of Animals:

Male wistar rats weighing between 250-350gm are obtained from animal house of Appasaheb Birnale College of Pharmacy, Sangli for experimental purpose. The animals were maintained under controlled conditions of temperature (23± 2^oC), humidity (50± 5%) and 12 h light-dark cycles. All the animals were acclimatized or seven days before the study. The animals were randomized into experimental, normal and control groups, housed individually in sanitized polypropylene cages containing sterile paddy husk as bedding. They had free accessed to standard pellets as basal diet and water ad libitum. Animals were habituated to laboratory conditions for 48 hr prior to experimental protocol to minimize if any of non-specific stress. All studies conducted are approved by the Institutional Animal Ethical Committee (IAEC) of Appasaheb Birnale College of Pharmacy, Sangli, Maharashtra (Registered no. 843/AC/04/CPCSEA), India.

 

In-vitro cytotoxicity studies:

Brine Shrimp Lethality Assay¹³:

Brine shrimp lethality assay of carvedilol was performed to evaluate its cytotoxic activity according to method decribed by Mayer et al. Brine shrimp were hatched in a special chamber consisting of 2 liters of seawater along with 40mg of eggs. After 48 hrs the napulli are collected in test tube by capillary and used for bioassay. Drug solution of 10, 20, 30, 40, 50μg concentrations were prepared with artificial seawater. Then these napullis were treated with 0.5ml of different concentraions of drug. In control vial added 4.5ml of artificial seawater and 0.5ml artificial sea water with 0.2% DMSO water. After 24hrs survivors were counted, by using 3 x magnifying glasses or against lighted background, and the percent deaths and LC50 values were calculated by dose-response data which is transformed into a straight line by means of a trend line fit linear regression analysis (MS Excel version 7).

 

MTT assay¹⁴:

The MTT colorimetric assay was performed to evaluate the cytotoxicity of carvedilol. The monolayer cell (PC3 cell lines) culture was trypsinized and the cell count was adjusted to 3 lac cells/ ml using medium. Pre incubate cells at a concentration of 1× 10⁶ cells/ml in culture medium for 3 h at 37°C and 6.5% CO₂. The cells were seeded at a concentration of 5× 10⁴ cells/well in 100μl culture medium and incubated at 37ºC in 5% CO₂ incubator for 24 hrs. After 24 hours, when the monolayer formed, the supernatant was flicked off and added previously diluted with media of 100μl of different concentrations of test in microtitre plates and kept for incubation at 37ºC in 5 % CO₂ incubator for 72 hour and cells were periodically checked for granularity, shrinkage, swelling. After 72 hours, the sample solution in wells was flicked off and 10μl of MTT dye was added to each well. The plates were gently shaken and incubated for 4 hours at 37^o C in 5% CO₂ incubator. The supernatant was removed, 100μl of Isopropanol was added, and the plates were gently shaken to solubilize the formed formazan. The absorbance was measured using a micro plate reader at 590nm with a reference filter of 630nm.

 

In-vivo chemo preventive effect of adrenergic blocker:

The chemopreventive property of adrenergic blocker was determined using monosodium glutamate induced prostate cancer in rats.¹⁵

 

Experimental design:

Total 32 animals were randomly divided into the following 4 groups to evaluate chemopreventive potential of adrenergic blocker in behavioral stress accelerated prostate cancer development in rats.

 

Control I:

(n=8) Animals of this group received monosodium glutamate (MSG) 5mg/kg orally for 30 days.

 

Control II:

(n=8) Animals of this group received monosodium glutamate (MSG) 5mg/kg orally for 30 days. From 31^st to 40^th day animals are subjected to behavioral stress by immobilization and cold-water immersion.

 

Carvedilol 50 (C50):

(n=8) Animals of this group received monosodium glutamate (MSG) 5mg/kg orally for 30 days. From 31^st to 40^th day animals were receive 50mg/kg of carvedilol orally and after 1hr each animal were subjected to behavioral stress by immobilization and cold water immersion.

 

Carvedilol 100 (C100):

(n=8) Animals of this group received monosodium glutamate (MSG) 5mg/kg orally for 30 days. From 31^st to 40^th day animals were receive 100mg/kg of carvedilol orally and after 1hr each animal were subjected to behavioral stress by immobilization and cold water immersion.

 

Behavioral models:

Immobilization¹⁶:

Immobilization chamber used in this study was made up of acrylic having size (20cm long, 7cm diameter). Animal was kept immobilized in cylindrical acrylic tube with proper holes on it for breathing, that allows ample air but animals will be unable to move. The rat was totally restrained by packing the other end of tube with closer. Rat was exposed to chronic stress in the form of immobilization for 1 hour per day for a period of 10 days.

 

 

Figure No. 01: Rat exposed to immobilization stress

 

Cold water swimming^17-18:

In this method, the rat was placed in individually in tank of cold water (depth = 30cm, temperature = 5-10^oC) where they either swim or remain in an upright position keeping their heads above water level. The rat was kept for 30 minutes per day for 10 days to develop chronic stress.

 

Figure No. 02: Rat exposed to cold-water swimming

 

Parameters monitored:

The different parameters which are monitored during this study are tumour parameters like weight of prostate gland and size of prostate gland after washing with ethanol was recorded per 100g body weight¹⁹, haematological parameters, biochemical parameters like Prostatic acid phosphatase (PAP) and Total acid phosphatase (TAP), Estimation of tissue antioxidant biomarkers^20-26. The histopathological slides were prepared according to Godkar P. 2003.²⁷

 

Statistical analysis:

The data were statistically analyzed by Mean ± SD. The data were also analyzed by one way ANOVA followed by Dunnett’s t-test and values *p<0.05 were considered as significant and **p<0.01 were considered as more significant.

 

RESULTS AND DISCUSSION:

In –Vitro Cytotoxicity Studies:

Brine Shrimp Lethality Bioassay:

The test drug Carvedilol at concentrations of 10, 20, 30, 40, and 50μg showed 30, 50, 63.33, 93.33, 96.67% inhibition and it is found that there is dose dependent increase in % inhibition. The LC₅₀ of carvedilol was found to be 20μg. The Table No. 01 reflects effect of carvedilol on brine shrimp lethality bioassay.

 

MTT assay:

The test drug Carvedilol at concentrations of 10, 20, 30, 40, and 50μg showed 45.74, 54.45, 60.33, 80.65, 85.35 % inhibition and it is found that there is dose dependent increase in % inhibition. The Table no.02 reflects effect of Carvedilol in % inhibition against cell lines.

 

 

Table No. 01: Effect of Carvedilol on Brine shrimp lethality bioassay

Conc.(μg)	Total shrimp taken in each test tube	Shrimp Survived			Total no. of shrimp survived	% Inhibition	LC50 (μg)
		T1	T2	T3			20
10	10	6	7	8	21	30
20	10	6	4	5	15	50
30	10	3	5	3	11	63.33
40	10	2	0	0	2	93.33
50	10	1	0	0	1	96.67

 

 

 

Table No.02: % Growth inhibition of carvedilol

Sr. No.	Drug conc. (μg/ml)	% Inhibition
1	10	45.74
2	20	54.45
3	30	60.33
4	40	80.65
5	50	85.35

 

In-Vivo results:

Tumour Parameters:

Prostate Gland Size and Prostate Gland Weight:

The animals, which are treated with monosodium glutamate and exposed to different stress conditions (Control II group), showed significant increase in prostate gland weight and prostate gland size when compared to control I group indicating progression of prostate cancer progression. The drug treated group C50 and C100 group shows highly significant decrease in prostate gland weight and prostate gland size (0.3973± 0.0159gm and 1.20±0.017cm) and (0.372±0.0103gm and 1.25±0.020cm) as compared to control I and control II group. The effect of carvedilol on prostate gland size and prostate gland weight were summerized in Table no.03.

 

Table No. 03: Effect of carvedilol treatment on prostate gland size and prostate gland weight in male rats exposed behavioral stress accelerated prostate cancer

Groups	Prostate gland weight (gm)	Prostate gland size (cm)
Control I	0.5797 ± 0.0188	1.587 ± 0.020
Control II	0.7097 ± 0.0375	1.843 ± 0.0120
C 50	0.3937 ± 0.0159	1.207 ± 0.017
C 100	0.372 ± 0.0103	1.253± 0.020

 

Haematological Parameters:

Hb, RBC and WBC:

In hematological parameters, the control II group shown significant decrease in Hb (9.413±0.1225) and RBC (6.443±0.1357) level as compared to control I group, while group C50 and C100 showed increase in Hb and RBC (13.66±0.075 and 9.05±06807) and (11.67± 0.0622 and 8.620±0.0923) levels respectively.

 

The WBC level was observed to be increased in control II group (12.50±0.1365) while carvedilol treated group showed significant decrease in WBC level in both C50 (7.793 ± 0.0933) and C100 (8.273 ± 0.095) respectively. The effects of carvedilol on hematological parameters were summerized in Table no.04.

Table No. 04: Effect of carvedilol treatment on Hb, RBC, WBC in male rats exposed to behavioral stress accelerated prostate cancer

Groups	Hb (g/dl)	RBC (millions/mm3)	WBC (thousand/mm3)
Control I	10.65 ± 0.1193	7.243 ± 0.1891	10.46 ± 0.1069
Control II	9.413± 0.1225	6.443± 0.1357	12.50± 0.1365
C 50	13.66± 0.075	9.050± 0.06807	7.793± 0.0933
C 100	11.67 ± 0.0622	8.620 ± 0.0923	8.273± 0.095

 

Total acid phosphatase (TAP) and prostatic acid phosphatase (PAP):

The animals, which are treated with monosodium glutamate and exposed to stress (Control II group) showed significant increase in serum TAP and PAP level when compared with control I group. The group C50 shows decrease in serum TAP (33.82±1.948) and PAP (24.34±1.737) and group C100 shows decrease in serum TAP (41.04±0.772) and PAP (30.58±1.070) levels. The enhanced level of serum TAP and PAP were noted in prostate cancer and present test drug carvedilol shows decrease in TAP and PAP level. The effects of carvedilol on TAP and PAP were summerized in Table no.05

 

Table No. 05: Effect of carvedilol treatment on TAP and PAP level in male rats exposed to behavioral stress accelerated prostate cancer

Groups	TAP (IU/L)	PAP (IU/L)
Control I	52.15 ± 0.8063	39.97 ±1.050
Control II	67.03 ± 2.752	51.48 ± 1.880
C 50	33.82± 1.948	24.34± 1.737
C 100	41.04 ± 0.772	30.58 ± 1.070

 

Tissue antioxidant markers and lipid peroxidation of liver tissue homogenate:

In present study, there is marked decline in the levels of SOD (18.99±0.3831), CAT (8.886±0.574) and GSH (3.089±0.0327) in control II group, due to behavioral stress as well as oxidative stress with cancer. The drug treated group enhances the level of SOD, CAT and GSH, which will be helpful in delaying the progression of cancer. The C100 group shows more significant increase in SOD, CAT and GSH level as compared to C50 group. In addition, there is marked increase in the levels of MDA in control II group (5.003±0.0948) as compared to control I group. In C50 and C100 group, there is decrease in level of MDA (4.561±0.06590) and (3.123±0.0568) respectively. From above results, it is observed that drug carvedilol has antioxidant activity in dose dependant manner.⁸

 

 

Table No. 06: Effect of Carvedilol treatment on level of SOD, CAT, GSH and MDA in male rat exposed to behavioral stress accelerated prostate cancer

Groups	SOD (U/mg of protein)	CAT (U/mg of protein)	GSH (nmol/mg protein)	MDA (nmol/mg of protein)
Control I	29.12 ± 0.2391	15.71 ± 0.6236	3.246 ± 0.0581	4.141 ± 0.0338
Control II	18.99 ± 0.3831	8.886 ± 0.5741	3.089 ± 0.0327	5.003 ± 0.0948
C 50	26.98 ± 0.1633	24.71± 0.6600	3.824± 0.0648	4.561± 0.06590
C 100	38.48± 0.2679	26.31 ± 0.7014	4.951± 0.0879	3.123± 0.0568

 

 

Histopathological study:

The histological changes in the prostate cancer induced rats were prepared with haematoxylin and eosin staining of the paraffin sections in treatment groups.

 

The control I group shows moderate proliferation of glands with infolding of mucosal lining epithelium. The stroma appears odematous. This shows less prominent evidences of malignant cells where as control II group shows, marked proliferation of glands with complex mucosal infoldings, fibro muscular stroma shows odema fluid. The epithelial layer shows severe hyperplastic condition. It indicates that there is formation of malignancy.

 

The C50 group shows less hyperplastic condition and epithelium layer is around normal. This indicates that there is less evidence of malignancy as compared to control I and control II group. The C100 group animals showed mild proliferation with infoldings of mucosal lining epithelium. Stroma appears less odematous as compared to control I and control II group. The histopathological changes of all groups were showed in Fig no.03.

 

Figure No. 03: Histopathological changes

 

CONCLUSION:

From the present study, it is concluded that the drug adrenergic blocker (Carvedilol) shows a potential chemo preventive action. The chemo preventive action of carvedilol is due to reduction in prostate gland weight and prostate gland size, reduction in oxidative stress produced by monosodium glutamate followed by behavioral stress as carvedilol itself has antioxidant action. The histological studies further confirms that the adrenergic blocker (Carvedilol) inhibits cell proliferation as well as hyperplasia, dysplasia and it restores the architecture of epithelial layer near to normal which suggest that carvedilol has anti-tumour promotion. In cytotoxicity studies, the carvedilol showed significant cytotoxic potential in Brine shrimp lethality assay and MTT assay.

 

Therefore, the result of the present study, suggest that adrenergic blocker has potential chemopreventive effect against behavioral stress accelerated prostate cancer development in rats and may act as chemopreventive agent.

 

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Received on 17.03.2020            Modified on 19.05.2020

Accepted on 20.08.2020         © RJPT All right reserved