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Protective effect of p-coumaric acid against 1,2 dimethylhydrazine induced colonic preneoplastic lesions in experimental rats
Biomedicine & Pharmacotherapy 94 (2017) 577–588
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Protective effect of p-coumaric acid against 1,2 dimethylhydrazine induced colonic preneoplastic lesions in experimental rats Sharada H. Sharmaa , David Raj Chellappanb , Prabu Chinnaswamyb , Sangeetha Nagarajana,* a b
School of Chemical and Biotechnology, SASTRA University, Thirumalaisamudram, Thanjavur, 613401, Tamil Nadu, India Central Animal Facility, SASTRA University, Thirumalaisamudram, Thanjavur, 613401, Tamil Nadu, India
A R T I C L E I N F O
Article history: Received 15 May 2017 Received in revised form 27 July 2017 Accepted 27 July 2017 Keywords: Colon cancer Chemoprevention p-Coumaric acid Oxidative stress Gut microbial enzymes Preneoplastic lesions 1,2 di-methyl hydrazine
A B S T R A C T
Oxidative stress and gut microbial enzymes are intricately linked to the onset of colon carcinogenesis. Phytochemicals that modulate these two factors hold promise for the development of such agents as anticancer drugs. The present study evaluates the chemopreventive potential of p-coumaric acid (p-CA) – a phenolic acid in rats challenged with the colon specific procarcinogen DMH (1,2 di-methyl hydrazine). Rats were randomized into six groups (n = 7/group). Group 1 (control); Group 2 (p-CA 200 mg/kg b.w.); Group 3 (DMH 40 mg/kg b.w.); Groups 4 (DMH + p-CA 50 mg/kg b.w.) and Group 5 (DMH + p-CA 100 mg/ kg b.w.) and Group 6 (DMH + p-CA 200 mg/kg b.w.). After the experimental duration of 15 weeks’ rats were subjected to necropsy and tissues were collected for the histological and biochemical investigations. DMH induced colonic preneoplastic lesions viz., aberrant crypt foci (ACF), dysplastic ACF (DACF), mucin depleted foci (MDF) and beta catenin accumulated crypts (BCAC) were significantly suppressed by p-CA supplementation. Glucuronide conjugation of DMH in liver and its subsequent deconjugation mediated by microbes in the colon induced the formation of colonic preneoplastic lesions. p-CA inhibited these lesions and protected the rat colon against genotoxic insult by scavenging the free radicals via its strong antioxidant response and detoxification mechanism as measured by TBARS and enzymic antioxidants in control and experimental rats. Of the three tested doses, p-CA at a dose of 100 mg/kg body weight is found to exhibit a significant optimum effect compared to the other two doses 50 mg/kg body weight and 200 mg/kg body weight. © 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction Worldwide, colorectal cancer (CRC) remains the fourth most common cause of cancer related mortality with over 1.2 million new cases diagnosed each year along with >600,000 death per year [1]. In the United States in 2017, 135,430 individuals newly diagnosed with CRC and 50,260 deaths from the disease are projected [2]. According to the ICMR statistics, in India the annual incidence rates for colon cancer in men are 4.4 per 100,000 and in women are 3.9 per 100,000 [3]. This situation persists despite the fact that colon cancer is highly preventable by consumption of high fiber and low fat diet instead of high fat and low fiber diet. Chemoprevention involves the prevention or delaying the process of carcinogenesis through chronic administration of one or more
* Corresponding author. E-mail address: [email protected] (S. Nagarajan). http://dx.doi.org/10.1016/j.biopha.2017.07.146 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
chemical compounds and this chemoprevention concept is found to be the key in reducing the tumor burden and mortality [4]. Reactive oxygen species (ROS) plays a key role in microbiota-linked colon cancer via mechanisms that include bacterial-derived genotoxins, microbial-derived metabolism, the modulation of host defenses and inflammation pathways, oxidative stress induction, and anti-oxidative defense regulation [5]. Thus oxidative stress and gut microbiota are intricately linked to the onset of colon carcinogenesis. Phytochemicals that modulate these two factors holds promise for the development of such agents as anticancer drugs. p-Coumaric acid (p-CA), a phenolic acid of the hydroxycinnamic acid family, is found ubiquitously in free or bound form in mushroom, fruits (e.g. apples, pears, grapes, oranges, tomatoes and berries), vegetables (e.g. beans, potatoes and onions) and cereals (e.g. maize, oats and wheat) and is shown to possess anti-oxidant, anti-inflammatory, anti-ulcer, antiplatelet, anti-cancer and antimutagenic properties [6,7]. p-CA was reported to inhibit cell
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proliferation by presumably affecting different cell cycle phases in the human colonic cell line Caco-2 [8] and by inducing apoptosis through ROS-mitochondrial pathway in colon cancer cells like HT 29 and HCT 15 [9]. This is the first study attempted to explore the short term exposure of p-CA in rats challenged with DMH at a dose of 40 mg/ kg body weight for five weeks with a total experimental period of 15 weeks. This time period allows us to investigate the development of DMH induced preneoplastic lesions and the impact of p-CA on the same. 2. Materials and methods 2.1. Chemicals 1,2 dimethylhydrazine (DMH), p-Coumaric acid (p-CA), methylene blue and alcian blue were purchased from Sigma Chemical Co. (St Louis, MO, USA). b-catenin antibody and polymer HRP detection kit were purchased from BioGenex laboratories (San Ramon, CA, USA). All other chemicals and solvents used were of analytical grade and obtained from Hi-Media Laboratories (Mumbai, Maharashtra, India). 2.2. Animal care and diet Male albino Wistar rats (140–150 g) were procured from the Central Animal Facility, SASTRA University, Thanjavur, India. Animals were acclimatized to the laboratory conditions for 1 week and maintained under standard conditions. The animals were given standard rat feed (Altromin, Germany) and water ad libitum. All the animal experimental protocols were followed in accordance with the Indian National Law on animal care and use and approved by the Institutional Animal Ethics Committee of SASTRA University (CPSEA approval No.: 240/SASTRA/IAEC/RPP). 2.3. DMH and phytochemical dosage and administration DMH at a dose of 40 mg/kg body weight was dissolved in 1 mM EDTA, pH was adjusted to 6.5 with 1 mM NaOH and was given subcutaneously once a week for 5 weeks starting from the first
week of the experimental period. p-CA at a dose of 50 mg, 100 mg and 200 mg were suspended in 0.5% carboxymethylcellulose (CMC) and administered everyday via intragastric intubation for the total experimental period of 15 weeks. 2.4. Experimental design Rats were randomly divided into six groups (n = 7/per group). Group 1 (control group) was injected subcutaneously with normal saline once a week for five weeks and fed orally with 0.5% CMC for 15 weeks. Group 2 served as p-CA control (200 mg/kg b.w.) received oral administration of p-CA for 15 weeks. Group 3 served as DMH control (40 mg/kg b.w. s.c. once a week for 5 weeks). In addition to DMH, Group 4, Group 5 and Group 6 received oral administration of p-CA at a dose of 50 mg, 100 and 200 mg/kg b.w. respectively for 15 weeks. The total body weight was measured and recorded once per week. The experimental design is represented in Fig. 1 After the experimental period of 15 weeks the rats were sacrificed by CO2 asphyxiation and subjected to necropsy. 2.5. Macroscopic observations Colon was removed and flushed with ice cold PBS to remove debris. The colons were cut longitudinally and placed on clean tissue paper. The numbers of polyps were documented. 2.6. Determination of aberrant crypts The colonic tissues were flushed with ice cold PBS to remove debris and excised longitudinally. ACF was performed according to the method described by Bird et al. [10] Briefly the colon was cut in to proximal and distal. The sections were placed in between filter paper in a petri plate containing 10% neutral buffered formalin for 24 h at 4 C. The formalin fixed colon was then stained with 0.2% methylene blue for 20 min and then viewed under microscope (Model No. Leica ST4040). The same colon was further stained with 0.2% methylene blue for 20 min and then soaked in 70% methanol for destaining and then viewed under microscope (Model No. Leica ST4040) for DACF.
Fig. 1. Schematic representation of Experimental Design.
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2.7. Homogenization of tissues
2.9. Biochemical investigations
Immediately after sacrifice, the colon tissues were washed with PBS. The tissues were chopped finely and homogenized with appropriate volume of ice-cold 0.1 M phosphate buffer using bead bug homogenizer (Z763713) at 4000 rpm for 90 s. The tubes were the centrifuged at 12000 rpm for 20 min at 4 C. The supernatant was collected and used for further biochemical analysis.
2.9.1. Lipid peroxidation assay Thiobarbituric acid reactive substances (TBARS) were analyzed by Ohkawa et al. method [11]. To 0.5 ml of tissue homogenate, 1.5 ml TBA (0.8%), 0.2 ml SDS (8.1%), 1.5 ml acetic acid was added. The mixture was made up to 4 ml using distilled water and heated in water bath at 95 C for 60 min. After cooling 1 ml distilled water and 5 ml n-butanol-pyridine mixture (15:1) was added and shaken vigorously. The solution was centrifuged at 4000 rpm for 5 min. The pink organic upper phase was read at 432 nm in UV–vis spectrophotometer (Thermo ScientificTM).
2.8. Histological investigations 2.8.1. Histopathology Liver, colon and kidney sections were fixed in 10% neutral buffered formalin. After 48 h of fixation, tissues were processed using automated tissue processing and embedding system (Leica TP 1020; Leica FG1150). Tissue sections of 3 mm thickness were cut using microtome (Leica RM 2125 RTS). Tissues were stained using Haematoxylin & Eosin stain in automated linear stainer (Model No. Leica ST4040) and were observed under microscope with in-built camera (Nikon Ci-DS-Fi2). 2.8.2. Immunohistochemistry 3 mm paraffin embedded sections were heated in hot air oven for 60 min at 60 C. The sections were then deparrafinized in xylene and rehydrated through graded alcohol. For antigen retrieval the sections were heated in pressure cooker for 10 min in trisodium citrate buffer (pH 6.0). The slides were then allowed to cool and rinsed with distilled water followed by tris buffered saline (TBS) (pH 7.6). Immunohistochemical staining was performed with polymer kit (Biogenex, San Ramon, CA). In order to block the nonspecific background, the slides were incubated with power block (Biogenex, San Ramon, CA) in a humid chamber for 10 min. The sections were then incubated at 4 C with pre-diluted primary antibody (Biogenex, San Ramon, CA) overnight. After this slides were rinsed in TBS and then covered with polymer-HRP reagent for 30 min in humid chamber and after rinsing with TBS thrice, slides were reacted with 3,30 -diaminobenzidene (Liquid DAB substrate pack, Biogenex, CA) for 10 min. The slides were counter stained with hematoxylin rinsed under tap water, dried and mounted. The slides were then observed under 40 magnification of microscope (Model No. Leica ST4040) and the images were analyzed using Image J software. 2.8.3. Mucin depleted foci Paraffin embedded sections were deparafinized and rehydrated through graded alcohol. The sections were then stained in Alcian blue for 30 min. The sections were washed in running tap water and rinsed in distilled water and kept in periodic acid solution for 30 min. After distilled water rinse the sections were placed in Schiff’s reagent for 60 min, followed by wash in running tap water and then counter stained with Mayer’s hematoxylin. The sections were viewed under microscope (Model No. Leica ST4040) and photographed.
2.9.2. Antioxidant enzymes assay Superoxide dismutase (SOD, EC.1.15.1.1) was analyzed by Kakkar et al. method [12]. Briefly, to 0.5 ml of tissue homogenate, 1 ml of H2O, 2.5 ml ethanol, 1.5 ml chloroform was added and centrifuged at 3000 rpm for 10 min at 4 C. The supernatant was collected. To an assay mixture of 1.2 ml sodium pyrophosphate buffer, 0.1 ml phenazine methosulfate, 0.3 ml nitroblue tetrazolium, 1.4 ml supernatant added 0.2 ml of NADH and incubated at 30 C for 90 s. The reaction was arrested by adding 1 ml glacial acetic acid and shaken vigorously with 2 ml butanol and allowed to stand for 10 min. The reaction mixture was centrifuged at 3000 rpm for 10 min. The upper pink organic layer was read at 520 nm in UV–vis spectrophotometer (Thermo ScientificTM). Catalase (CAT, EC.1.11.1.6) was analyzed by the method of Sinha et al. [13]. To a reaction mixture containing 0.9 ml, 0.01 M phosphate buffer (pH 7.0) and 0.1 ml tissue homogenate, 0.4 ml of H2O2 was added and incubated for 45 s. The reaction was terminated by addition of 2 ml dichromate acetic acid mixture (1:3). The tubes were incubated in boiling water bath for 10 min and cooled. The green color solution was read at 620 nm in UV–vis spectrophotometer (Thermo ScientificTM). Glutathione peroxidase (GPx, EC.1.11.1.9) was analyzed by Rotruck et al. method [14]. To a reaction mixture containing 0.2 ml Tris-HCL buffer (0.4 M), 0.2 ml EDTA (0.4 mM), 0.1 ml sodium azide (10 mM) and 0.5 ml tissue homogenate, 0.2 ml GSH, 0.1 ml H2O2 (1 mM) was added. The reaction mixture was mixed and incubated at 37 C for 10 min 0.5 ml of TCA was added and centrifuged. The supernatant was collected. To 0.1 ml of supernatant, 0.3 ml phosphate buffer (0.2 M) and 0.5 ml Ellman’s reagent was added. The yellow color solution was read at 412 nm in UV–vis spectrophotometer (Thermo ScientificTM). 2.9.3. Gut microbial enzymes b-glucuronidase was measured by the method of Freeman [15]. To 0.1 ml of homogenate, 0.4 ml of p-nitrophenyl-b-D-glucuronide (0.01 M), 0.2 ml EDTA (0.1 mM), 0.3 ml PBS (0.02 M) was added and incubated at 37 C for 30 min. The reaction was terminated by addition of 4 ml glycine buffer (pH 10.4). The reaction mixture was read at 540 nm in UV–vis spectrophotometer (Thermo ScientificTM). Values are expressed as micrograms of p-nitrophenol liberated/h/g protein of mucosal tissue. Mucinase activity was
Table 1 Effect of p-CA on body weight and polyp incidence. Groups
Control pCA (200 mg) DMH DMH + pCA (50 mg) DMH + pCA (100 mg) DMH + pCA (200 mg)
Body weight (g)
Poly Incidence
Initial
Final
Total no. of rats
No. of Poly bearing rats
Polyp incidence (%)
149.19 10.53 150.11 10.59 150.24 10.60 151.23 10.67 151.99 10.72 150.03 10.58
331.54 23.40a 353.63 24.96 a,b 366.50 25.86 a,b 347.17 24.50 a,b 350.31 24.72 a,b 361.07 25.48b
7 7 7 7 7 7
– – 7 5 4 3
100a 71.42 b 57.14c 42.85d
Data are expressed as mean SD. Data sharing a common superscript do not differ significantly at p < 0.05. (Duncan’s multiple range test).
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Fig. 2. (A)Topographic view of ACF in the unsectioned rat colon (20): Control and p-CA control rat showed normal crypts; DMH rat showed ACF (arrow) with >4 crypts; p-CA supplemented DMH rat showed ACF (arrow) with 1to 3 crypts. (B) Bar graph of ACF with different crypt multiplicity. Data are expressed as mean SD. Data sharing a common superscript do not differ significantly at p < 0.05. (Duncan’s multiple range tests).
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Fig. 3. Topographic view of DACF in the unsectioned rat colon (20): Control and p-CA control rat showed normal crypts; DMH rat showed DACF (arrow) with >4 crypts; p-CA supplemented DMH rat showed suppressed DACF in a dose dependent manner.
measured by method of Shiau and Chang method [16]. To 0.9 ml of homogenate, 0.1 ml of mucin was added and incubated at 37 C for 25 min. The reaction mixture was kept in boiling water bath for 3 min and 2 ml of TCA (3%), 3.5 ml of o-Toluidene reagent was added and heated in water bath for 10 min. The reaction mixture was read at 610 nm in UV–vis spectrophotometer (Thermo ScientificTM).
2.9.4. Serum transaminases Serum AST (EC 2.6.1.1) and ALT (EC 2.6.1.2) were measured using a diagnostic kit based on the method of Reitman and Frankel [17].
2.10. Statistical analysis Data are presented as means S.D. of seven rats per group. Data from polyp incidence, biochemical investigations, were analyzed using one way analysis of variance (ANOVA) and the group means were compared by Duncan’s Multiple Range Test (DMRT) using the SPSS (version 11.0). The results were considered statistically significant if the “p” < 0.05.
3. Results 3.1. Effect of p-CA on polyp incidence Table 1 shows the effect of p-CA on body weight and colonic polyp incidence in experimental rats. There was no significant difference in the initial and final body weight between different experimental groups other than the DMH administered rats supplemented with p-CA at a dose of 200 mg/kg b.w. which was shown to improve body weight. All animals survived throughout the study period of 15 weeks. Control and p-CA control rats showed 0% polyp incidence whereas DMH alone administered rats showed 100% polyp incidence. Supplementation of p-CA at different doses viz., 50 mg, 100 mg and 200 mg/kg b.w significantly reduced the polyp incidence to 71.42%, 57.14% and 42.85% respectively. p-CA exhibited dose dependent inhibitory effect in the colon of rats challenged with the procarcinogen DMH. 3.2. Effect of p-CA on preneoplastic lesions Supplementation of p-CA to DMH administered rats significantly suppressed the formation of pre neoplastic lesions. The size
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Fig. 4. Alcian blue staining for colonic mucin content (40): Control and p-CA control rat showed rich mucin content as evidenced by blue coloration; Lack of blue color in DMH treated rats showing absence of mucin; p-CA supplemented DMH rats showed restoration of blue color thereby mucin content. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
of ACF (Fig. 2A & B) and DACF (Fig. 3) are significantly reduced in a dose dependent manner in p-CA treated groups compared to DMH control. p-CA exhibited colonic mucosal protective effect by preventing mucin depletion induced by DMH (Fig. 4). Intense membrane and nuclear staining of b-catenin in the colonic crypts of DMH administered rats showed the onset of tumorigenesis and significant reduction in the b – catenin immunoreactivity in the colonic crypts of p-CA treated DMH administered rats (Fig. 5A and B) revealed the anti-tumorigenic potential of p-CA.
3.4. Effect of p-CA on gut microbial enzymes Table 3 depicts the activity of gut microbial enzymes viz.,
b-glucuronidase, and mucinase from control and experimental
rats. Compared to control rats, the colon of DMH alone administered rats showed significantly increased activity of these enzymes. Supplementation with p-CA at different doses (50 mg, 100 mg and 200 mg) significantly decreased their activity, and a significantly pronounced effect was observed in the rats supplemented 100 mg/kg b.w.
3.3. Effect of p-CA on oxidative stress
3.5. Effect of p-CA on organ toxicity
The effect of p-CA on the levels of oxidative stress is shown in Table 2. Compared to control rats, the colon of DMH alone administered rats showed increased levels of both TBARS and antioxidants viz., SOD, CAT and GPx which reflects the tissue homeostasis in the presence of DMH insult favoring cell survival. Decreased levels of TBARS and increased levels of SOD, CAT and GPx in the colon of DMH administered rats supplemented with pCA revealed that p-CA exhibited cytostatic effect in a dose dependent manner.
To ensure the protective effect of p-CA against DMH induced pathological alterations major organs viz., liver, kidney and the target organ colon tissue were subjected to H&E staining and the stained tissues were scored by a veterinary pathologist who is unaware of the experimental groups. Fig. 6A showed the H & E staining of the colon tissue of experimental rats. Control rats showed normal colonic crypt structures. DMH alone administered rats showed sessile adenomatous polyp with dysplasia and severe lymphoid hyperplasia. Moderate hyperplasia was observed in
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Fig. 5. (A) Immunohistochemical staining of b-catenin accumulated crypts (40): Control and p-CA control colonic crypts showing normal membrane staining; DMH alone showed nuclear and cytoplasmic accumulation of b-catenin; p-CA treated DMH rats showed treated and DMH + p-CA (50 mg/kg) treated colonic crypts showing dose dependent decrease in b-catenin accumulation. (B) The bar graph shows percentage difference in immunostaining between control and experimental rats as analyzed by Image J software. Data are expressed as mean SD. Data sharing a common superscript do not differ significantly at p < 0.05. (Duncan’s multiple range test).
DMH rats administered with p-CA at a dose of 50 mg/kg b.w; whereas p-CA at doses of 100 mg and 200 mg/kg b.w showed mild hyperplasia retaining almost near normal colonic architecture. Microscopical observation of colon tissue analyzing various parameters like severity of tissue damage, proportion of tissue affected, nature of tissue damage was used to grade
histopathological changes. The scored histopathology grade is shown in Fig. 6B. Lack of tissue damage in the liver and kidney reflects that every day supplementation of p-CA was well tolerated during the experimental period of 15 weeks (Fig. 7A, B). Assay of liver marker enzymes also validate the protective effect of p-CA in experimental rats (Fig. 7C).
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Table 2 Effect of p-CA on colonic oxidative stress markers. Groups
TBARS1
SOD#
CAT2
GPXy
Control pCA (200 mg) DMH DMH + pCA (50 mg) DMH + pCA (100 mg) DMH + pCA (200 mg)
0.70 0.05a 0.68 0.05 a 0.97 0.07b 0.84 0.06 c 0.78 0.05d 0.76 0.05d
4.42 0.31 a 4.58 0.32 a, 6.64 0.46 b 7.45 0.53 c 8.45 0.60d 8.84 0.62d
37.67 2.65 a 38.82 2.74 a 47.71 3.36 b 57.17 4.03 c 61.47 4.34 c,d 63.64 4.49 d
6.28 0.44 a 6.67 0.47 a 8.38 0.59 b 9.12 0.64 c 9.73 0.68c 11.12 0.78 d
Data are expressed as mean SD. 1 mmol/mg tissue. # enzyme required for 50% inhibition of NBT reduction. 2 mmol H2O2 utilized/min/mg protein. y mmol GSH utilized/min/mg protein. Data sharing a common superscript do not differ significantly at p < 0.05. (Duncan’s multiple range test).
Table 3 Effect of p-CA on colonic microbial enzymes. Groups
b – Glucuronidase mg of p-nitrophenol liberated/min/g protein
Mucinase mg of glucose liberated/min/mg protein
Control pCA (200 mg) DMH DMH + pCA (50 mg) DMH + pCA (100 mg) DMH + pCA (200 mg)
41.51 2.93a 40.61 2.87a 81.10 5.72b 78.41 5.53b 61.21 4.31c 58.34 4.11c
2.39 0.17a 2.33 0.16a 3.74 0.26b 3.58 0.25b 2.78 0.20c 2.58 0.18c
Data are expressed as mean SD. Data sharing a common superscript do not differ significantly at p < 0.05. (Duncan’s multiple range test).
4. Discussion p-Coumaric acid (p-CA), an ubiquitous plant phenolic acid, has been proven to render protection against N2a neruoblastoma cells [18], colon cancer cells like Caco-2 [8], HT-29 and HCT-15 [9]. The present study demonstrated the in vivo antiproliferative effect of pCA by evaluating colonic preneoplastic lesions as end points. Preneoplastic lesions represent the precursors of neoplasms as they harbor morphologically or functionally altered populations of cells and are used as end points in short term experiments [19]. ACF, DACF, MDF and BCAC are the most commonly used preneoplastic lesions in colon cancer chemoprevention studies. Though all the above mentioned abnormal crypts are categorized as pre-neoplastic lesions each one differs from the other in their morphology as well as in their tumor initiating potential. ACF are cryptic lesions which can be clearly distinguished from the normal crypts by their ability to uptake intense methylene blue dye and are characterized by increased size, thicker epithelial lining and increased peri-cryptic zone [20]. Appearance of ACF in rodents as well as in humans represents ACF as a surrogate endpoint biomarker in cancer prevention studies [21]. p-CA exhibited a dose dependent inhibitory effect on DMH driven ACF formation. Though ACF are considered as putative pre-neoplastic lesions for colon cancers and surrogate endpoint for colon cancer chemoprevention studies it was also found that not all the ACF had progressed to tumors suggesting a lack of correlation between ACF formation and tumor initiation [22]. This finding was based on the observation that large portion of ACF is hyperplastic and only a very small portion of ACF showed dysplasia, a hall mark of malignant potential [23]. So we analyzed the occurrence of DACF, MDF and BCAC as these lesions are closely related to APC mutations. MDF are originated from dysplastic crypts devoid of mucin and possess mutations in genes influencing colon carcinogenesis (APC and K-ras) and display Wnt signaling activation, MUC2 down regulation along with induction of inflammatory cascade suggesting MDF are precancerous [24–26]. Intense nuclear staining of b catenin in the colonic crypt cells of DMH alone administered rats provides a hint on the activation of Wnt
signaling pathway which might be suppressed by p-CA supplementation. In order to explore the potential mechanism of action of p-CA, it is necessary to investigate the mechanism of action of DMH on the onset of preneoplastic lesions. The main pathway involves the hepatic conversion of DMH to azomethane, azoxymethane and azoxymethanol which subsequently undergoes glucuronic acid conjugation and biliary excretion [27,28]. The detoxified methylazoxymethanol (MAM) glucuronide undergo bacterial hydrolysis regenerating MAM which then undergoes either a spontaneous or a tissue specific enzyme catalyzed breakdown to formaldehyde, hydroxyl ion and the active methylating species methyldiazonium (MD). Thus DMH exhibit mutagenic effect through methylation of guanine both at N-7 and O-6 position. The first step in DMH induced colon carcinogenesis appears to be alkylation of target organ DNA [29]. Thus, the carcinogenicity of DMH is attributed via gut microbial enzymes that conjugated MAM glucuronide and subsequent generation of free radicals. Hence we performed the gut microbial enzyme assays, lipid peroxidation and antioxidants in the colon tissue of control and experimental rats. Increased activity of b-glucuronidase in DMH rats confirmed the carcinogenicity of DMH via deconjugation of MAM glucuronide. Further increased mucinase activity correlates with the MDF i.e. the destruction of protective mucin layer of colonic epithelial cells. Supplementation of p-CA at different doses to DMH administered rats decreased the activity of these gut microbial enzymes which might be due to the ability of p-CA to promote the survival of beneficial organisms in the colon [30]. It has also been proposed that the high concentration of p-CA conjugates in the colon exerts various physiological actions like inhibition and promotion of some microorganisms and their metabolism and absorption of those metabolites by colon epithelial cells exerts biological activities to the host [31]. In cancer cells there is an enhanced oxidative stress that leads to overproduction of ROS which in turn contribute to pro-tumorigenic signaling pathways [32]. Increased levels of TBARS in the colon tissue of DMH alone administered rats could be explained by the generation of free radicals by DMH via microsomal oxidation reactions [33]. Intestinal epithelial cells are in constant exposure to
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Fig. 6. (A) Cross section of rat colon stained with hematoxylin and eosin (40): Colon of control rats showed normal architecture; Colon of DMH alone treated rats showed sessile adenoma with lymphoid hyperplasia; Colon of DMH +p-CA (50 mg/kg) treated rats showed moderate hyperplasia; Colon of DMH + p-CA (100 mg/kg & 200 mg/kg)
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Fig. 7. (A) Cross section of rat liver stained with hematoxylin and eosin (40): No significant pathological changes observed in control and experimental groups. (B) Cross section of rat kidney stained with hematoxylin and eosin (40): No significant pathological changes observed in control and experimental groups. (C) Serum alanine transaminase and aspartate transaminase activity showing no significant difference between the experimental groups. Data are expressed as mean SD. Data sharing a common superscript do not differ significantly at p < 0.05. (Duncan’s multiple range test).
oxygen radicals originating from diet or from microbial activity. It was reported that increased ROS production is accompanied with the phagocyte oxidative burst. In addition to these increased levels of ROS in the defective vascular system in the vicinity of the neoplastic lesions and intensified metabolic activity of neoplastic cells also translates into increased production of the superoxide anion radical [34,35]. Increased oxidative stress in the course of neoplastic process is also evidenced by the increased activity of antioxidants [36,37]. Thus increased levels of antioxidants in the
DMH group could be an outcome of cellular adaptation to oxidative stress in order to maintain redox homeostasis and survival advantage by transformed cells. Thus increased levels of antioxidants in the DMH group substantiate the maintenance of redox homeostasis and survival advantage by transformed cells. Decreased levels of TBARS in p-CA supplemented DMH administered rats showed that p-CA prevents the accumulation of free radicals responsible for lipid peroxidation. Our findings are in consistent with the previous report which
showing mild hyperplasia. (B) Histopathology grade expressed in mean SD (n = 4). Data sharing a common superscript do not differ significantly at p < 0.05(Duncan’s multiple range test).
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Fig. 8. Mechanism of DMH and its intervention by p-CA: DMH is converted to methyl azoxymethanol (MAM) in liver which gets conjugated with glucuronic acid to form MAM glucuronide that enters colon via extra hepatic circulation where it undergoes bacterial hydrolysis to form MAM. This MAM eventually releases formaldehyde, hydroxyl ion and active methylating species that methylates the DNA and causes mutations and uncontrolled cell proliferation. On the other hand, DMH also increased the levels of H2O2 that could increase ROS and antioxidants as an adaptive response to increased oxidative stress. p-CA improves the detoxification of DMH by improving the levels of beneficial gut microbes and by decreasing the levels of ROS thus exhibited a cytostatic effect on colon cells.
stated that p-CA protects against mutagenesis induced by H2O2 by scavenging hydroxyl radical [7]. Further induction of endogenous antioxidants viz., SOD, CAT and GPx in DMH administered rats treated with p-CA indicates the activation of antioxidant defense. The outcome of the present findings revealed that p-CA significantly inhibits precancerous lesions and reduced polyp incidence. In addition, preneoplastic lesions, histolopathological investigations and liver function enzyme assays validate the absence of adverse effect and antiproliferative potential of p-CA. 5. Conclusion The outcome of the present findings revealed that p-CA significantly inhibits precancerous lesions and reduces polyp incidence in a dose dependent manner. p-CA supplementation weakens the carcinogenicity of DMH via its antioxidant and prebiotic activity (Fig. 8). Of the three tested doses, p-CA at a dose of 100 mg/kg body weight is chosen for long term in vivo study as it exhibited the significant optimum effect compared to the other two doses 50 mg/kg body weight and 200 mg/kg body weight. Employing the dose by factor method [38] the human equivalent dose for the chosen optimum dose is found to be 15.2 mg/kg. Unlike most dietary phenolic compounds, orally consumed pcoumaric acid has reasonable bioavailability both in animals and man [39], reaching plasma levels of up to 5 mg/mL, with a Tmax between 60 and 180 min [40,41]. Its bioavailability to plasma is likely mediated by passive diffusion through the stomach wall as well as its affinity to the monocarboxylic acid transporter [40]. Consumption of fruits like apples, oranges, grapes, pears, tomatoes and berries; vegetables such as beans, potatoes and onions and cereals like maize, oats and wheat will ensure the intake of p-CA and there by dietary intervention of colon cancer.
Funding This work was supported by the Department of Science and Technology (SB/YS/LS-187/2014), New Delhi, India. Conflict of interest The authors declare that there are no conflicts of interest Acknowledgements The authors thank Dr. S. Swaminathan (Director, CeNTAB) and Dr. S. Panchapekesan (Co-oridinator, Central Animal Facility) of SASTRA University for their great support to perform the study. The authors would also like to thank Mr. Sundarkrishnan of DIMENSION Art, Mumbai, India for editing the manuscript. References [1] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, CA Cancer J. Clin. 66 (2016) (2016) 7–30. [2] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, CA Cancer J. Clin. 67 (2017) (2017) 7–30. [3] S. Gosavi, R.R. Mishra, V.P. Kumar, Study on the relation between colorectal cancer and gall bladder disease, J. Clin. Diagn. Res. 11 (2017) OC25–OC27. [4] L.W. Wattenberg, Chemoprevention of cancer, Cancer Res. 45 (1985) 1–8. [5] J. Gagnière, J. Raisch, J. Veziant, N. Barnich, R. Bonnet, E. Buc, M.A. Bringer, D. Pezet, M. Bonnet, Gut microbiota imbalance and colorectal cancer, World J. Gastroenterol. 22 (2016) 501–518. [6] S.J. Pragasam, V. Venkatesan, M. Rasool, Immunomodulatory and antiinflammatory effect of p-coumaric acid: a common dietary polyphenol on experimental inflammation in rats, Inflammation 36 (2013) 169–176. [7] L.R. Ferguson, I.F. Lima, A.E. Pearson, J. Ralph, P.J. Harris, Bacterial antimutagenesis by hydroxycinnamic acids from plant cell walls, Mutat. Res. 542 (2003) 49–58.
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Protective effect of p-coumaric acid against 1,2 dimethylhydrazine induced colonic preneoplastic lesions in experimental rats Sharada H. Sharmaa , David Raj Chellappanb , Prabu Chinnaswamyb , Sangeetha Nagarajana,* a b
School of Chemical and Biotechnology, SASTRA University, Thirumalaisamudram, Thanjavur, 613401, Tamil Nadu, India Central Animal Facility, SASTRA University, Thirumalaisamudram, Thanjavur, 613401, Tamil Nadu, India
A R T I C L E I N F O
Article history: Received 15 May 2017 Received in revised form 27 July 2017 Accepted 27 July 2017 Keywords: Colon cancer Chemoprevention p-Coumaric acid Oxidative stress Gut microbial enzymes Preneoplastic lesions 1,2 di-methyl hydrazine
A B S T R A C T
Oxidative stress and gut microbial enzymes are intricately linked to the onset of colon carcinogenesis. Phytochemicals that modulate these two factors hold promise for the development of such agents as anticancer drugs. The present study evaluates the chemopreventive potential of p-coumaric acid (p-CA) – a phenolic acid in rats challenged with the colon specific procarcinogen DMH (1,2 di-methyl hydrazine). Rats were randomized into six groups (n = 7/group). Group 1 (control); Group 2 (p-CA 200 mg/kg b.w.); Group 3 (DMH 40 mg/kg b.w.); Groups 4 (DMH + p-CA 50 mg/kg b.w.) and Group 5 (DMH + p-CA 100 mg/ kg b.w.) and Group 6 (DMH + p-CA 200 mg/kg b.w.). After the experimental duration of 15 weeks’ rats were subjected to necropsy and tissues were collected for the histological and biochemical investigations. DMH induced colonic preneoplastic lesions viz., aberrant crypt foci (ACF), dysplastic ACF (DACF), mucin depleted foci (MDF) and beta catenin accumulated crypts (BCAC) were significantly suppressed by p-CA supplementation. Glucuronide conjugation of DMH in liver and its subsequent deconjugation mediated by microbes in the colon induced the formation of colonic preneoplastic lesions. p-CA inhibited these lesions and protected the rat colon against genotoxic insult by scavenging the free radicals via its strong antioxidant response and detoxification mechanism as measured by TBARS and enzymic antioxidants in control and experimental rats. Of the three tested doses, p-CA at a dose of 100 mg/kg body weight is found to exhibit a significant optimum effect compared to the other two doses 50 mg/kg body weight and 200 mg/kg body weight. © 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction Worldwide, colorectal cancer (CRC) remains the fourth most common cause of cancer related mortality with over 1.2 million new cases diagnosed each year along with >600,000 death per year [1]. In the United States in 2017, 135,430 individuals newly diagnosed with CRC and 50,260 deaths from the disease are projected [2]. According to the ICMR statistics, in India the annual incidence rates for colon cancer in men are 4.4 per 100,000 and in women are 3.9 per 100,000 [3]. This situation persists despite the fact that colon cancer is highly preventable by consumption of high fiber and low fat diet instead of high fat and low fiber diet. Chemoprevention involves the prevention or delaying the process of carcinogenesis through chronic administration of one or more
* Corresponding author. E-mail address: [email protected] (S. Nagarajan). http://dx.doi.org/10.1016/j.biopha.2017.07.146 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
chemical compounds and this chemoprevention concept is found to be the key in reducing the tumor burden and mortality [4]. Reactive oxygen species (ROS) plays a key role in microbiota-linked colon cancer via mechanisms that include bacterial-derived genotoxins, microbial-derived metabolism, the modulation of host defenses and inflammation pathways, oxidative stress induction, and anti-oxidative defense regulation [5]. Thus oxidative stress and gut microbiota are intricately linked to the onset of colon carcinogenesis. Phytochemicals that modulate these two factors holds promise for the development of such agents as anticancer drugs. p-Coumaric acid (p-CA), a phenolic acid of the hydroxycinnamic acid family, is found ubiquitously in free or bound form in mushroom, fruits (e.g. apples, pears, grapes, oranges, tomatoes and berries), vegetables (e.g. beans, potatoes and onions) and cereals (e.g. maize, oats and wheat) and is shown to possess anti-oxidant, anti-inflammatory, anti-ulcer, antiplatelet, anti-cancer and antimutagenic properties [6,7]. p-CA was reported to inhibit cell
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proliferation by presumably affecting different cell cycle phases in the human colonic cell line Caco-2 [8] and by inducing apoptosis through ROS-mitochondrial pathway in colon cancer cells like HT 29 and HCT 15 [9]. This is the first study attempted to explore the short term exposure of p-CA in rats challenged with DMH at a dose of 40 mg/ kg body weight for five weeks with a total experimental period of 15 weeks. This time period allows us to investigate the development of DMH induced preneoplastic lesions and the impact of p-CA on the same. 2. Materials and methods 2.1. Chemicals 1,2 dimethylhydrazine (DMH), p-Coumaric acid (p-CA), methylene blue and alcian blue were purchased from Sigma Chemical Co. (St Louis, MO, USA). b-catenin antibody and polymer HRP detection kit were purchased from BioGenex laboratories (San Ramon, CA, USA). All other chemicals and solvents used were of analytical grade and obtained from Hi-Media Laboratories (Mumbai, Maharashtra, India). 2.2. Animal care and diet Male albino Wistar rats (140–150 g) were procured from the Central Animal Facility, SASTRA University, Thanjavur, India. Animals were acclimatized to the laboratory conditions for 1 week and maintained under standard conditions. The animals were given standard rat feed (Altromin, Germany) and water ad libitum. All the animal experimental protocols were followed in accordance with the Indian National Law on animal care and use and approved by the Institutional Animal Ethics Committee of SASTRA University (CPSEA approval No.: 240/SASTRA/IAEC/RPP). 2.3. DMH and phytochemical dosage and administration DMH at a dose of 40 mg/kg body weight was dissolved in 1 mM EDTA, pH was adjusted to 6.5 with 1 mM NaOH and was given subcutaneously once a week for 5 weeks starting from the first
week of the experimental period. p-CA at a dose of 50 mg, 100 mg and 200 mg were suspended in 0.5% carboxymethylcellulose (CMC) and administered everyday via intragastric intubation for the total experimental period of 15 weeks. 2.4. Experimental design Rats were randomly divided into six groups (n = 7/per group). Group 1 (control group) was injected subcutaneously with normal saline once a week for five weeks and fed orally with 0.5% CMC for 15 weeks. Group 2 served as p-CA control (200 mg/kg b.w.) received oral administration of p-CA for 15 weeks. Group 3 served as DMH control (40 mg/kg b.w. s.c. once a week for 5 weeks). In addition to DMH, Group 4, Group 5 and Group 6 received oral administration of p-CA at a dose of 50 mg, 100 and 200 mg/kg b.w. respectively for 15 weeks. The total body weight was measured and recorded once per week. The experimental design is represented in Fig. 1 After the experimental period of 15 weeks the rats were sacrificed by CO2 asphyxiation and subjected to necropsy. 2.5. Macroscopic observations Colon was removed and flushed with ice cold PBS to remove debris. The colons were cut longitudinally and placed on clean tissue paper. The numbers of polyps were documented. 2.6. Determination of aberrant crypts The colonic tissues were flushed with ice cold PBS to remove debris and excised longitudinally. ACF was performed according to the method described by Bird et al. [10] Briefly the colon was cut in to proximal and distal. The sections were placed in between filter paper in a petri plate containing 10% neutral buffered formalin for 24 h at 4 C. The formalin fixed colon was then stained with 0.2% methylene blue for 20 min and then viewed under microscope (Model No. Leica ST4040). The same colon was further stained with 0.2% methylene blue for 20 min and then soaked in 70% methanol for destaining and then viewed under microscope (Model No. Leica ST4040) for DACF.
Fig. 1. Schematic representation of Experimental Design.
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2.7. Homogenization of tissues
2.9. Biochemical investigations
Immediately after sacrifice, the colon tissues were washed with PBS. The tissues were chopped finely and homogenized with appropriate volume of ice-cold 0.1 M phosphate buffer using bead bug homogenizer (Z763713) at 4000 rpm for 90 s. The tubes were the centrifuged at 12000 rpm for 20 min at 4 C. The supernatant was collected and used for further biochemical analysis.
2.9.1. Lipid peroxidation assay Thiobarbituric acid reactive substances (TBARS) were analyzed by Ohkawa et al. method [11]. To 0.5 ml of tissue homogenate, 1.5 ml TBA (0.8%), 0.2 ml SDS (8.1%), 1.5 ml acetic acid was added. The mixture was made up to 4 ml using distilled water and heated in water bath at 95 C for 60 min. After cooling 1 ml distilled water and 5 ml n-butanol-pyridine mixture (15:1) was added and shaken vigorously. The solution was centrifuged at 4000 rpm for 5 min. The pink organic upper phase was read at 432 nm in UV–vis spectrophotometer (Thermo ScientificTM).
2.8. Histological investigations 2.8.1. Histopathology Liver, colon and kidney sections were fixed in 10% neutral buffered formalin. After 48 h of fixation, tissues were processed using automated tissue processing and embedding system (Leica TP 1020; Leica FG1150). Tissue sections of 3 mm thickness were cut using microtome (Leica RM 2125 RTS). Tissues were stained using Haematoxylin & Eosin stain in automated linear stainer (Model No. Leica ST4040) and were observed under microscope with in-built camera (Nikon Ci-DS-Fi2). 2.8.2. Immunohistochemistry 3 mm paraffin embedded sections were heated in hot air oven for 60 min at 60 C. The sections were then deparrafinized in xylene and rehydrated through graded alcohol. For antigen retrieval the sections were heated in pressure cooker for 10 min in trisodium citrate buffer (pH 6.0). The slides were then allowed to cool and rinsed with distilled water followed by tris buffered saline (TBS) (pH 7.6). Immunohistochemical staining was performed with polymer kit (Biogenex, San Ramon, CA). In order to block the nonspecific background, the slides were incubated with power block (Biogenex, San Ramon, CA) in a humid chamber for 10 min. The sections were then incubated at 4 C with pre-diluted primary antibody (Biogenex, San Ramon, CA) overnight. After this slides were rinsed in TBS and then covered with polymer-HRP reagent for 30 min in humid chamber and after rinsing with TBS thrice, slides were reacted with 3,30 -diaminobenzidene (Liquid DAB substrate pack, Biogenex, CA) for 10 min. The slides were counter stained with hematoxylin rinsed under tap water, dried and mounted. The slides were then observed under 40 magnification of microscope (Model No. Leica ST4040) and the images were analyzed using Image J software. 2.8.3. Mucin depleted foci Paraffin embedded sections were deparafinized and rehydrated through graded alcohol. The sections were then stained in Alcian blue for 30 min. The sections were washed in running tap water and rinsed in distilled water and kept in periodic acid solution for 30 min. After distilled water rinse the sections were placed in Schiff’s reagent for 60 min, followed by wash in running tap water and then counter stained with Mayer’s hematoxylin. The sections were viewed under microscope (Model No. Leica ST4040) and photographed.
2.9.2. Antioxidant enzymes assay Superoxide dismutase (SOD, EC.1.15.1.1) was analyzed by Kakkar et al. method [12]. Briefly, to 0.5 ml of tissue homogenate, 1 ml of H2O, 2.5 ml ethanol, 1.5 ml chloroform was added and centrifuged at 3000 rpm for 10 min at 4 C. The supernatant was collected. To an assay mixture of 1.2 ml sodium pyrophosphate buffer, 0.1 ml phenazine methosulfate, 0.3 ml nitroblue tetrazolium, 1.4 ml supernatant added 0.2 ml of NADH and incubated at 30 C for 90 s. The reaction was arrested by adding 1 ml glacial acetic acid and shaken vigorously with 2 ml butanol and allowed to stand for 10 min. The reaction mixture was centrifuged at 3000 rpm for 10 min. The upper pink organic layer was read at 520 nm in UV–vis spectrophotometer (Thermo ScientificTM). Catalase (CAT, EC.1.11.1.6) was analyzed by the method of Sinha et al. [13]. To a reaction mixture containing 0.9 ml, 0.01 M phosphate buffer (pH 7.0) and 0.1 ml tissue homogenate, 0.4 ml of H2O2 was added and incubated for 45 s. The reaction was terminated by addition of 2 ml dichromate acetic acid mixture (1:3). The tubes were incubated in boiling water bath for 10 min and cooled. The green color solution was read at 620 nm in UV–vis spectrophotometer (Thermo ScientificTM). Glutathione peroxidase (GPx, EC.1.11.1.9) was analyzed by Rotruck et al. method [14]. To a reaction mixture containing 0.2 ml Tris-HCL buffer (0.4 M), 0.2 ml EDTA (0.4 mM), 0.1 ml sodium azide (10 mM) and 0.5 ml tissue homogenate, 0.2 ml GSH, 0.1 ml H2O2 (1 mM) was added. The reaction mixture was mixed and incubated at 37 C for 10 min 0.5 ml of TCA was added and centrifuged. The supernatant was collected. To 0.1 ml of supernatant, 0.3 ml phosphate buffer (0.2 M) and 0.5 ml Ellman’s reagent was added. The yellow color solution was read at 412 nm in UV–vis spectrophotometer (Thermo ScientificTM). 2.9.3. Gut microbial enzymes b-glucuronidase was measured by the method of Freeman [15]. To 0.1 ml of homogenate, 0.4 ml of p-nitrophenyl-b-D-glucuronide (0.01 M), 0.2 ml EDTA (0.1 mM), 0.3 ml PBS (0.02 M) was added and incubated at 37 C for 30 min. The reaction was terminated by addition of 4 ml glycine buffer (pH 10.4). The reaction mixture was read at 540 nm in UV–vis spectrophotometer (Thermo ScientificTM). Values are expressed as micrograms of p-nitrophenol liberated/h/g protein of mucosal tissue. Mucinase activity was
Table 1 Effect of p-CA on body weight and polyp incidence. Groups
Control pCA (200 mg) DMH DMH + pCA (50 mg) DMH + pCA (100 mg) DMH + pCA (200 mg)
Body weight (g)
Poly Incidence
Initial
Final
Total no. of rats
No. of Poly bearing rats
Polyp incidence (%)
149.19 10.53 150.11 10.59 150.24 10.60 151.23 10.67 151.99 10.72 150.03 10.58
331.54 23.40a 353.63 24.96 a,b 366.50 25.86 a,b 347.17 24.50 a,b 350.31 24.72 a,b 361.07 25.48b
7 7 7 7 7 7
– – 7 5 4 3
100a 71.42 b 57.14c 42.85d
Data are expressed as mean SD. Data sharing a common superscript do not differ significantly at p < 0.05. (Duncan’s multiple range test).
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Fig. 2. (A)Topographic view of ACF in the unsectioned rat colon (20): Control and p-CA control rat showed normal crypts; DMH rat showed ACF (arrow) with >4 crypts; p-CA supplemented DMH rat showed ACF (arrow) with 1to 3 crypts. (B) Bar graph of ACF with different crypt multiplicity. Data are expressed as mean SD. Data sharing a common superscript do not differ significantly at p < 0.05. (Duncan’s multiple range tests).
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Fig. 3. Topographic view of DACF in the unsectioned rat colon (20): Control and p-CA control rat showed normal crypts; DMH rat showed DACF (arrow) with >4 crypts; p-CA supplemented DMH rat showed suppressed DACF in a dose dependent manner.
measured by method of Shiau and Chang method [16]. To 0.9 ml of homogenate, 0.1 ml of mucin was added and incubated at 37 C for 25 min. The reaction mixture was kept in boiling water bath for 3 min and 2 ml of TCA (3%), 3.5 ml of o-Toluidene reagent was added and heated in water bath for 10 min. The reaction mixture was read at 610 nm in UV–vis spectrophotometer (Thermo ScientificTM).
2.9.4. Serum transaminases Serum AST (EC 2.6.1.1) and ALT (EC 2.6.1.2) were measured using a diagnostic kit based on the method of Reitman and Frankel [17].
2.10. Statistical analysis Data are presented as means S.D. of seven rats per group. Data from polyp incidence, biochemical investigations, were analyzed using one way analysis of variance (ANOVA) and the group means were compared by Duncan’s Multiple Range Test (DMRT) using the SPSS (version 11.0). The results were considered statistically significant if the “p” < 0.05.
3. Results 3.1. Effect of p-CA on polyp incidence Table 1 shows the effect of p-CA on body weight and colonic polyp incidence in experimental rats. There was no significant difference in the initial and final body weight between different experimental groups other than the DMH administered rats supplemented with p-CA at a dose of 200 mg/kg b.w. which was shown to improve body weight. All animals survived throughout the study period of 15 weeks. Control and p-CA control rats showed 0% polyp incidence whereas DMH alone administered rats showed 100% polyp incidence. Supplementation of p-CA at different doses viz., 50 mg, 100 mg and 200 mg/kg b.w significantly reduced the polyp incidence to 71.42%, 57.14% and 42.85% respectively. p-CA exhibited dose dependent inhibitory effect in the colon of rats challenged with the procarcinogen DMH. 3.2. Effect of p-CA on preneoplastic lesions Supplementation of p-CA to DMH administered rats significantly suppressed the formation of pre neoplastic lesions. The size
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Fig. 4. Alcian blue staining for colonic mucin content (40): Control and p-CA control rat showed rich mucin content as evidenced by blue coloration; Lack of blue color in DMH treated rats showing absence of mucin; p-CA supplemented DMH rats showed restoration of blue color thereby mucin content. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
of ACF (Fig. 2A & B) and DACF (Fig. 3) are significantly reduced in a dose dependent manner in p-CA treated groups compared to DMH control. p-CA exhibited colonic mucosal protective effect by preventing mucin depletion induced by DMH (Fig. 4). Intense membrane and nuclear staining of b-catenin in the colonic crypts of DMH administered rats showed the onset of tumorigenesis and significant reduction in the b – catenin immunoreactivity in the colonic crypts of p-CA treated DMH administered rats (Fig. 5A and B) revealed the anti-tumorigenic potential of p-CA.
3.4. Effect of p-CA on gut microbial enzymes Table 3 depicts the activity of gut microbial enzymes viz.,
b-glucuronidase, and mucinase from control and experimental
rats. Compared to control rats, the colon of DMH alone administered rats showed significantly increased activity of these enzymes. Supplementation with p-CA at different doses (50 mg, 100 mg and 200 mg) significantly decreased their activity, and a significantly pronounced effect was observed in the rats supplemented 100 mg/kg b.w.
3.3. Effect of p-CA on oxidative stress
3.5. Effect of p-CA on organ toxicity
The effect of p-CA on the levels of oxidative stress is shown in Table 2. Compared to control rats, the colon of DMH alone administered rats showed increased levels of both TBARS and antioxidants viz., SOD, CAT and GPx which reflects the tissue homeostasis in the presence of DMH insult favoring cell survival. Decreased levels of TBARS and increased levels of SOD, CAT and GPx in the colon of DMH administered rats supplemented with pCA revealed that p-CA exhibited cytostatic effect in a dose dependent manner.
To ensure the protective effect of p-CA against DMH induced pathological alterations major organs viz., liver, kidney and the target organ colon tissue were subjected to H&E staining and the stained tissues were scored by a veterinary pathologist who is unaware of the experimental groups. Fig. 6A showed the H & E staining of the colon tissue of experimental rats. Control rats showed normal colonic crypt structures. DMH alone administered rats showed sessile adenomatous polyp with dysplasia and severe lymphoid hyperplasia. Moderate hyperplasia was observed in
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Fig. 5. (A) Immunohistochemical staining of b-catenin accumulated crypts (40): Control and p-CA control colonic crypts showing normal membrane staining; DMH alone showed nuclear and cytoplasmic accumulation of b-catenin; p-CA treated DMH rats showed treated and DMH + p-CA (50 mg/kg) treated colonic crypts showing dose dependent decrease in b-catenin accumulation. (B) The bar graph shows percentage difference in immunostaining between control and experimental rats as analyzed by Image J software. Data are expressed as mean SD. Data sharing a common superscript do not differ significantly at p < 0.05. (Duncan’s multiple range test).
DMH rats administered with p-CA at a dose of 50 mg/kg b.w; whereas p-CA at doses of 100 mg and 200 mg/kg b.w showed mild hyperplasia retaining almost near normal colonic architecture. Microscopical observation of colon tissue analyzing various parameters like severity of tissue damage, proportion of tissue affected, nature of tissue damage was used to grade
histopathological changes. The scored histopathology grade is shown in Fig. 6B. Lack of tissue damage in the liver and kidney reflects that every day supplementation of p-CA was well tolerated during the experimental period of 15 weeks (Fig. 7A, B). Assay of liver marker enzymes also validate the protective effect of p-CA in experimental rats (Fig. 7C).
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Table 2 Effect of p-CA on colonic oxidative stress markers. Groups
TBARS1
SOD#
CAT2
GPXy
Control pCA (200 mg) DMH DMH + pCA (50 mg) DMH + pCA (100 mg) DMH + pCA (200 mg)
0.70 0.05a 0.68 0.05 a 0.97 0.07b 0.84 0.06 c 0.78 0.05d 0.76 0.05d
4.42 0.31 a 4.58 0.32 a, 6.64 0.46 b 7.45 0.53 c 8.45 0.60d 8.84 0.62d
37.67 2.65 a 38.82 2.74 a 47.71 3.36 b 57.17 4.03 c 61.47 4.34 c,d 63.64 4.49 d
6.28 0.44 a 6.67 0.47 a 8.38 0.59 b 9.12 0.64 c 9.73 0.68c 11.12 0.78 d
Data are expressed as mean SD. 1 mmol/mg tissue. # enzyme required for 50% inhibition of NBT reduction. 2 mmol H2O2 utilized/min/mg protein. y mmol GSH utilized/min/mg protein. Data sharing a common superscript do not differ significantly at p < 0.05. (Duncan’s multiple range test).
Table 3 Effect of p-CA on colonic microbial enzymes. Groups
b – Glucuronidase mg of p-nitrophenol liberated/min/g protein
Mucinase mg of glucose liberated/min/mg protein
Control pCA (200 mg) DMH DMH + pCA (50 mg) DMH + pCA (100 mg) DMH + pCA (200 mg)
41.51 2.93a 40.61 2.87a 81.10 5.72b 78.41 5.53b 61.21 4.31c 58.34 4.11c
2.39 0.17a 2.33 0.16a 3.74 0.26b 3.58 0.25b 2.78 0.20c 2.58 0.18c
Data are expressed as mean SD. Data sharing a common superscript do not differ significantly at p < 0.05. (Duncan’s multiple range test).
4. Discussion p-Coumaric acid (p-CA), an ubiquitous plant phenolic acid, has been proven to render protection against N2a neruoblastoma cells [18], colon cancer cells like Caco-2 [8], HT-29 and HCT-15 [9]. The present study demonstrated the in vivo antiproliferative effect of pCA by evaluating colonic preneoplastic lesions as end points. Preneoplastic lesions represent the precursors of neoplasms as they harbor morphologically or functionally altered populations of cells and are used as end points in short term experiments [19]. ACF, DACF, MDF and BCAC are the most commonly used preneoplastic lesions in colon cancer chemoprevention studies. Though all the above mentioned abnormal crypts are categorized as pre-neoplastic lesions each one differs from the other in their morphology as well as in their tumor initiating potential. ACF are cryptic lesions which can be clearly distinguished from the normal crypts by their ability to uptake intense methylene blue dye and are characterized by increased size, thicker epithelial lining and increased peri-cryptic zone [20]. Appearance of ACF in rodents as well as in humans represents ACF as a surrogate endpoint biomarker in cancer prevention studies [21]. p-CA exhibited a dose dependent inhibitory effect on DMH driven ACF formation. Though ACF are considered as putative pre-neoplastic lesions for colon cancers and surrogate endpoint for colon cancer chemoprevention studies it was also found that not all the ACF had progressed to tumors suggesting a lack of correlation between ACF formation and tumor initiation [22]. This finding was based on the observation that large portion of ACF is hyperplastic and only a very small portion of ACF showed dysplasia, a hall mark of malignant potential [23]. So we analyzed the occurrence of DACF, MDF and BCAC as these lesions are closely related to APC mutations. MDF are originated from dysplastic crypts devoid of mucin and possess mutations in genes influencing colon carcinogenesis (APC and K-ras) and display Wnt signaling activation, MUC2 down regulation along with induction of inflammatory cascade suggesting MDF are precancerous [24–26]. Intense nuclear staining of b catenin in the colonic crypt cells of DMH alone administered rats provides a hint on the activation of Wnt
signaling pathway which might be suppressed by p-CA supplementation. In order to explore the potential mechanism of action of p-CA, it is necessary to investigate the mechanism of action of DMH on the onset of preneoplastic lesions. The main pathway involves the hepatic conversion of DMH to azomethane, azoxymethane and azoxymethanol which subsequently undergoes glucuronic acid conjugation and biliary excretion [27,28]. The detoxified methylazoxymethanol (MAM) glucuronide undergo bacterial hydrolysis regenerating MAM which then undergoes either a spontaneous or a tissue specific enzyme catalyzed breakdown to formaldehyde, hydroxyl ion and the active methylating species methyldiazonium (MD). Thus DMH exhibit mutagenic effect through methylation of guanine both at N-7 and O-6 position. The first step in DMH induced colon carcinogenesis appears to be alkylation of target organ DNA [29]. Thus, the carcinogenicity of DMH is attributed via gut microbial enzymes that conjugated MAM glucuronide and subsequent generation of free radicals. Hence we performed the gut microbial enzyme assays, lipid peroxidation and antioxidants in the colon tissue of control and experimental rats. Increased activity of b-glucuronidase in DMH rats confirmed the carcinogenicity of DMH via deconjugation of MAM glucuronide. Further increased mucinase activity correlates with the MDF i.e. the destruction of protective mucin layer of colonic epithelial cells. Supplementation of p-CA at different doses to DMH administered rats decreased the activity of these gut microbial enzymes which might be due to the ability of p-CA to promote the survival of beneficial organisms in the colon [30]. It has also been proposed that the high concentration of p-CA conjugates in the colon exerts various physiological actions like inhibition and promotion of some microorganisms and their metabolism and absorption of those metabolites by colon epithelial cells exerts biological activities to the host [31]. In cancer cells there is an enhanced oxidative stress that leads to overproduction of ROS which in turn contribute to pro-tumorigenic signaling pathways [32]. Increased levels of TBARS in the colon tissue of DMH alone administered rats could be explained by the generation of free radicals by DMH via microsomal oxidation reactions [33]. Intestinal epithelial cells are in constant exposure to
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Fig. 6. (A) Cross section of rat colon stained with hematoxylin and eosin (40): Colon of control rats showed normal architecture; Colon of DMH alone treated rats showed sessile adenoma with lymphoid hyperplasia; Colon of DMH +p-CA (50 mg/kg) treated rats showed moderate hyperplasia; Colon of DMH + p-CA (100 mg/kg & 200 mg/kg)
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Fig. 7. (A) Cross section of rat liver stained with hematoxylin and eosin (40): No significant pathological changes observed in control and experimental groups. (B) Cross section of rat kidney stained with hematoxylin and eosin (40): No significant pathological changes observed in control and experimental groups. (C) Serum alanine transaminase and aspartate transaminase activity showing no significant difference between the experimental groups. Data are expressed as mean SD. Data sharing a common superscript do not differ significantly at p < 0.05. (Duncan’s multiple range test).
oxygen radicals originating from diet or from microbial activity. It was reported that increased ROS production is accompanied with the phagocyte oxidative burst. In addition to these increased levels of ROS in the defective vascular system in the vicinity of the neoplastic lesions and intensified metabolic activity of neoplastic cells also translates into increased production of the superoxide anion radical [34,35]. Increased oxidative stress in the course of neoplastic process is also evidenced by the increased activity of antioxidants [36,37]. Thus increased levels of antioxidants in the
DMH group could be an outcome of cellular adaptation to oxidative stress in order to maintain redox homeostasis and survival advantage by transformed cells. Thus increased levels of antioxidants in the DMH group substantiate the maintenance of redox homeostasis and survival advantage by transformed cells. Decreased levels of TBARS in p-CA supplemented DMH administered rats showed that p-CA prevents the accumulation of free radicals responsible for lipid peroxidation. Our findings are in consistent with the previous report which
showing mild hyperplasia. (B) Histopathology grade expressed in mean SD (n = 4). Data sharing a common superscript do not differ significantly at p < 0.05(Duncan’s multiple range test).
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Fig. 8. Mechanism of DMH and its intervention by p-CA: DMH is converted to methyl azoxymethanol (MAM) in liver which gets conjugated with glucuronic acid to form MAM glucuronide that enters colon via extra hepatic circulation where it undergoes bacterial hydrolysis to form MAM. This MAM eventually releases formaldehyde, hydroxyl ion and active methylating species that methylates the DNA and causes mutations and uncontrolled cell proliferation. On the other hand, DMH also increased the levels of H2O2 that could increase ROS and antioxidants as an adaptive response to increased oxidative stress. p-CA improves the detoxification of DMH by improving the levels of beneficial gut microbes and by decreasing the levels of ROS thus exhibited a cytostatic effect on colon cells.
stated that p-CA protects against mutagenesis induced by H2O2 by scavenging hydroxyl radical [7]. Further induction of endogenous antioxidants viz., SOD, CAT and GPx in DMH administered rats treated with p-CA indicates the activation of antioxidant defense. The outcome of the present findings revealed that p-CA significantly inhibits precancerous lesions and reduced polyp incidence. In addition, preneoplastic lesions, histolopathological investigations and liver function enzyme assays validate the absence of adverse effect and antiproliferative potential of p-CA. 5. Conclusion The outcome of the present findings revealed that p-CA significantly inhibits precancerous lesions and reduces polyp incidence in a dose dependent manner. p-CA supplementation weakens the carcinogenicity of DMH via its antioxidant and prebiotic activity (Fig. 8). Of the three tested doses, p-CA at a dose of 100 mg/kg body weight is chosen for long term in vivo study as it exhibited the significant optimum effect compared to the other two doses 50 mg/kg body weight and 200 mg/kg body weight. Employing the dose by factor method [38] the human equivalent dose for the chosen optimum dose is found to be 15.2 mg/kg. Unlike most dietary phenolic compounds, orally consumed pcoumaric acid has reasonable bioavailability both in animals and man [39], reaching plasma levels of up to 5 mg/mL, with a Tmax between 60 and 180 min [40,41]. Its bioavailability to plasma is likely mediated by passive diffusion through the stomach wall as well as its affinity to the monocarboxylic acid transporter [40]. Consumption of fruits like apples, oranges, grapes, pears, tomatoes and berries; vegetables such as beans, potatoes and onions and cereals like maize, oats and wheat will ensure the intake of p-CA and there by dietary intervention of colon cancer.
Funding This work was supported by the Department of Science and Technology (SB/YS/LS-187/2014), New Delhi, India. Conflict of interest The authors declare that there are no conflicts of interest Acknowledgements The authors thank Dr. S. Swaminathan (Director, CeNTAB) and Dr. S. Panchapekesan (Co-oridinator, Central Animal Facility) of SASTRA University for their great support to perform the study. The authors would also like to thank Mr. Sundarkrishnan of DIMENSION Art, Mumbai, India for editing the manuscript. References [1] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, CA Cancer J. Clin. 66 (2016) (2016) 7–30. [2] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, CA Cancer J. Clin. 67 (2017) (2017) 7–30. [3] S. Gosavi, R.R. Mishra, V.P. Kumar, Study on the relation between colorectal cancer and gall bladder disease, J. Clin. Diagn. Res. 11 (2017) OC25–OC27. [4] L.W. Wattenberg, Chemoprevention of cancer, Cancer Res. 45 (1985) 1–8. [5] J. Gagnière, J. Raisch, J. Veziant, N. Barnich, R. Bonnet, E. Buc, M.A. Bringer, D. Pezet, M. Bonnet, Gut microbiota imbalance and colorectal cancer, World J. Gastroenterol. 22 (2016) 501–518. [6] S.J. Pragasam, V. Venkatesan, M. Rasool, Immunomodulatory and antiinflammatory effect of p-coumaric acid: a common dietary polyphenol on experimental inflammation in rats, Inflammation 36 (2013) 169–176. [7] L.R. Ferguson, I.F. Lima, A.E. Pearson, J. Ralph, P.J. Harris, Bacterial antimutagenesis by hydroxycinnamic acids from plant cell walls, Mutat. Res. 542 (2003) 49–58.
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