J Basic Clin Physiol Pharmacol 2015; 26(2): 185–200
Venkatachalam Karthikkumar, Gunasekaran Sivagami, Periyasamy Viswanathan and Namasivayam Nalini*
Rosmarinic acid inhibits DMH-induced cell proliferation in experimental rats Abstract Background: Colon cancer is one of the most common cancers in both men and women. The present study is an effort to unravel the anticarcinogenic effects of rosmarinic acid (RA) in 1,2-dimethylhydrazine (DMH)-induced rat colon carcinogenesis. Administration of DMH induces multiple tumors in the rat colon, which mimics human colon cancer. Methods: Male Wistar rats were divided into six groups and fed a high-fat diet. Group 1 served as control, group 2 rats were given RA [5 mg/kg body weight (b.w.)] orally every day for a total period of 30 weeks, and groups 3–6 were given weekly injections of DMH (20 mg/kg b.w. subcutaneous) once a week in the groin for the first 15 weeks. In addition to DMH, groups 4–6 received RA at a dose of 5 mg/kg b.w. during the initiation and postinitiation stages, and also throughout the entire study period. Colon tissues were examined histologically; further, the extent of oxidative stress was assessed by measuring lipid peroxidation and antioxidant levels in the colonic mucosa of rats. Results: Macroscopic and microscopic tumors were identified in all the groups that received DMH. The results revealed that supplementation with RA significantly inhibited the tumor formation and tumor multiplicity in DMH-treated rats. RA supplementation to DMH-administered rats significantly reduced the cell proliferation markers, namely, argyrophilic nucleolar organizing regions as well as proliferative cell nuclear antigen labeling index. In addition, RA supplementation reduces the expressions of tumor necrosis factor-α, interlukin-6, and cyclooxygenase-2, and modulates the expression of p65. *Corresponding author: Dr. Namasivayam Nalini, Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar-608002, Tamil Nadu, India, Phone: + 91-4144-239141, Fax: + 91-4144-238343, E-mail: [email protected] Venkatachalam Karthikkumar and Gunasekaran Sivagami: Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar, Tamil Nadu, India Periyasamy Viswanathan: Department of Pathology, Faculty of Medicine, Rajah Muthiah Medical College, Annamalai University, Annamalainagar, Tamil Nadu, India
Conclusions: The above findings clearly underline the chemopreventive efficacy of RA against DMH-induced colon carcinogenesis. Keywords: argyrophilic nucleolar organizing regions (AgNORs); chemoprevention; PCNA. DOI 10.1515/jbcpp-2014-0044 Received April 6, 2014; accepted July 14, 2014; previously published online September 11, 2014
Introduction Colorectal cancer is one of the most common forms of cancer worldwide, particularly among the Western population [1]. The incidence of colon cancer was formerly low in India and other Asian countries, but during the last decade a drastic increase in the colon cancer incidence has been observed in Asia [2]. Dietary habits play an important role in the development of colon cancer. In the olden days people used to consume natural substances with lots of medicinal properties. Massive economic growth and rapid urbanization influenced people to adopt a Western dietary style, which is composed of high fat, high protein, low carbohydrate, and low fiber, and this unbalanced diet is considered to be an important risk factor for the increased colon cancer incidence. Diet rich in fruits and vegetables is gaining prime importance to reduce the risk of colon cancer, as they contain various phytochemicals with diverse pharmacological properties [3]. 1,2-Dimethylhydrazine (DMH) is a potent procarcinogen for the induction of tumors in the colon and rectum of rats. The usefulness of this organospecific carcinogen, which induces selectively tumors in the colon, was confirmed by our previous studies [4, 5]. DMH is believed to form active intermediates, including azoxymethane and methylazoxymethanol (MAM), in the liver, which are subsequently transported into the colon through bile [6]. MAM is a long-lived metabolite found in the systemic circulation following DMH exposure. The enzymatic conversion of
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186 Karthikkumar et al.: Rosmarinic acid inhibits uncontrolled proliferation MAM to a reactive aldehyde is considered to be rate limiting in DMH metabolism [7, 8]; that MAM breakdown may occur spontaneously or by enzyme-catalyzed reactions yields the alkylating methyldiazonium ion, capable of methylating cellular macromolecules. Carcinogenesis is a multistep process, beginning with initiation followed by promotion and progression [9]. Each step reflects the genetic alterations that drive the progressive transformation of normal cells into highly malignant derivatives that exhibit continuous disturbance of cellular and molecular signal cascades [10, 11]. Based on this theory, the biology of carcinogenesis is vital to understand the development of an effective prevention. With better understanding of possible ways to modulate carcinogenesis, preventive interventions during these stages became very attractive. Utilization of validated biomarkers can markedly facilitate the testing of potential chemopreventive agents [12]. The quantitative evaluation of argyrophilic nucleolar organizing regions (AgNORs) is the only method that permits information to be obtained on the rapidity of cell proliferation in routinely processed tissue samples. The AgNOR number has been proposed as a diagnostic parameter for the grade of malignancy. It has been suggested that the AgNOR number is significantly different in both benign and malignant tumors [1, 13]. With regard to the cancers of the alimentary tract, it has been reported that the AgNOR number can reflect the prognosis of colon cancer [14, 15]; it is higher in malignant lesions than in benign lesions of the stomach [16]. AgNORs can be visualized as black dots on reaction with silver nitrate. Fecal occult-blood screening may yield an additional benefit in detecting the risk of developing cancer: resulting from the detection and removal of premalignant adenomatous polyps. Fecal occult-blood test (FOBT), which detects blood in the stool, is a simple test used to diagnose disorders of the gut. Though FOBT is less sensitive for the presence of polyps, it is widely used to detect some, particularly large, polyps, which are more likely to bleed [17] and also to become cancerous [18]. Cyclooxygenase (COX)-2 is an inducible enzyme whose expression is upregulated by nuclear factor-κB (NF-κB)-mediated tumorigenesis. Enhanced COX-2 expression has been found in colon cancer tissues from subjects with clinically diagnosed colorectal cancer [19]. Most of the colon cancer drugs available share the property of inhibiting COX, a key enzyme in the conversion of arachidonic acid to prostaglandins. The NF-κB family of eukaryotic transcription factors influences a number of important cellular and organismal processes, including cellular growth control, apoptosis, immune and inflammatory responses, and cellular stress
responses [20]. Thus, inhibition of this chronic NF-κB activity can, in many cases, slow the growth of tumor cell lines or induce cell death. Tumor necrosis factor (TNF) is an important molecule in the cytokine network pathway, which regulates the expression of other cytokines such as interlukin-6 (IL-6). TNF-α induces phosphorylation of IκB, which triggers the rapid ubiquitination and subsequent degradation of IκB, resulting in the release and translocation of NF-κB to the nucleus where it binds to DNA sequences and induces a number of genes, including the antiapoptotic genes, thereby causing cell proliferation [21]. Natural products have a great therapeutic potential in a large number of ailments, including cancer and heart diseases. Compounds of plant origin have the ability to inhibit one or more stages of carcinogenesis and prevent or delay tumor development [22]. Rosmarinic acid (RA, α-o-caffeoyl-3,4-dihydroxyphenyl-lactic acid) is a naturally occurring hydroxylated phenolic compound found in Lamiaceae herbs, such as rosemary, sweet basil, and perilla [23–25]. RA has many pharmacological properties, which include antiinflammatory, antioxidant, antiangiogenic, and antitumor activities [26–29]. Based on the previous dose-dependent study, we have fixed RA at 5 mg/kg b.w., which has the preventive effect in a short-duration experiment [30]. Therefore, in the present long-term study, we have examined the colon cancer inhibitory effect of RA by evaluating its role in cell proliferation and tumor formation in the presence of the colon-specific carcinogen DMH. This study is also designed to gain an insight into the ability of RA to modulate oxidative stress by assessing the lipid peroxidation as well as the antioxidant status in carcinogen-exposed rats supplemented with RA.
Materials and methods Chemicals DMH, RA, hematoxylin, and eosin were purchased from the Sigma Chemical Co. (USA). All other chemicals used were of analytical grade and obtained from Hi-Media Laboratories (Mumbai, India). Antibodies for PCNA, p65 were purchased from Santa Cruz Biotechnology, USA. Primers for COX-2 was purchased from Sigma Chemical Co. (USA).
Animal care and diet Male albino Wistar rats weighing 130–150 g were obtained from the Central Animal House, Rajah Muthiah Medical College and Hospital (RMMCH), Annamalai University (Tamil Nadu, India), and housed under standard environmental conditions (12 h light/dark cycle; 50% humidity; temperature 25 ± 2 °C). Commercial pellet diet containing
Brought to you by | Penn State - The Pennsylvania State University Authenticated Download Date | 5/26/15 12:07 AM
Karthikkumar et al.: Rosmarinic acid inhibits uncontrolled proliferation 187 4.2% fat (Hindustan Lever Ltd., Mumbai, India) was powdered and mixed with 15.8% peanut oil, making 20% total fat in the feed to prepare a high-fat, Western diet (modified pellet diet). The modified pellet diet and water were fed ad libitum to all the rats in the experiment. The rats were cared for in compliance with the principles and guidelines of the Ethical Committee for Animal Care of Annamalai University in accordance with the Indian National Law on Animal Care (Reg. No. 160/1999/CPCSEA/437). Food intake, water intake, and body weight (b.w.) changes were monitored regularly.
DMH and RA administration DMH was dissolved in 1.0 mM ethylenediaminetetraacetic acid just prior to use and the pH was adjusted to 6.5 with 1 mM NaOH to ensure the stability of the chemical. Animals received subcutaneous injections of DMH (20 mg/kg b.w.), once a week for 15 weeks [4]. RA was suspended in water just before treatment and was administered every day orally at a dose of 5 mg/kg b.w. for 30 weeks.
Treatment schedule Rats were randomly distributed into six groups of 12 rats each. Group 1 rats served as control, and group 2 received RA (5 mg/kg b.w.) every day throughout the experimental period. Groups 3–6 received DMH (20 mg/kg b.w. subcutaneous) once a week for the first 15 weeks, which represent the colon cancer-bearing rats. Group 4 rats were colon cancer-bearing rats treated with RA (as in group 2) for the first 15 weeks (initiation), group 5 rats were colon cancer-bearing rats administered with RA (as in group 2) starting 1 week after the cessation of DMH injections and continued till the end of the experimental period (postinitiation), and group 6 rats were colon cancer-bearing rats administered with RA (as in group 2) throughout the experimental period (entire period) of 30 weeks (Figure 1).
Histological observations The experiment was terminated at the end of 30 weeks; all the animals were euthanized under anesthesia. The colons were split open
longitudinally for macroscopical observation and gross tumors were counted. The liver and the colon segments were dissected, fixed immediately in 10% formalin, and paraffin embedded; 2–3 μm sections were cut in a rotary microtome, stained with hematoxylin and eosin, and viewed under a light microscope. Neoplastic lesions of colorectal mucosa were histopathologically classified into adenomas and adenocarcinomas. Adenomas are well-demarcated lumps of epithelial dysplasia, which can be classified into three major histological types: tubular, villous, and tubulo villous. Multiplicity of adenomas bears an increased risk of dysplasia and cancer. An adenoma is pedunculated when it possesses a stalk; sessile adenomas rise above the background mucosa without any stalk. Small (approx. 1 cm) adenomas are also known as “diminutive”. Further colorectal carcinoma can be graded into well-differentiated, moderately differentiated, and poorly differentiated lesions as described by Bosman [31].
AgNORs – Silver staining method AgNOR staining was carried out by the method of Nyska et al. [32]. Silver colloid solution was made up with 2% gelatin in 1% aqueous formic acid. This solution was mixed with twice its volume of 50% aqueous silver nitrate solution. Sections were exposed to the staining solution for 40 min at room temperature in the dark. The slides were washed in distilled water for 10 min, rehydrated through graded alcohol, cleared in xylene, and mounted using dibutyl phathalate xylene. The number of AgNORs per crypt cell nucleus was counted on one-step silver stained sections. The measurement was performed on 25 crypts per rat.
Proliferating cell nuclear antigen [PCNA and p65 (NFκB)] – immunohistochemsitry The paraffin-embedded sections (4 μm) were heated at 60 °C for 60 min, deparaffinized in xylene, and rehydrated through graded alcohol at room temperature. Tris-HCl buffer (0.05 M; pH 7.6) was used to prepare solutions and for washes between various steps. Sections were treated for 40 min at room temperature with 2% bovine serum albumin and incubated with primary antibodies against the
Figure 1 Experimental design.
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188 Karthikkumar et al.: Rosmarinic acid inhibits uncontrolled proliferation PCNA or p65 protein (BioGenex Laboratories, Inc., San Ramon, CA, USA) for 1 h. Horseradish peroxidase activity was visualized after treatment with H2O2 and diaminobenzidine hydrochloride for 5 min using the labeled streptavidin-biotin method (BioGenex Kit, San Ramon, CA, USA). Immunoreactivities were regarded as positive if the apparent staining was detected in the nuclei. The PCNA labeling index was determined by calculating the ratio PCNA-positive nuclei/ total number of nuclei.
Lipid peroxidation and antioxidant enzyme levels The colonic mucosa was minced and homogenized with 3 vol (w/v) of the appropriate buffer using a Potter-Elvehjam homogenizer with a Teflon pestle and centrifuged at 12,000 g for 20 min at 4 °C. The supernatant was used for biochemical estimations. Lipid peroxidation was estimated by measuring the levels of the tissue lipid peroxidation byproduct thiobarbituric acid reactive substances (TBARS) by using the method of Jiang et al. [33]. Superoxide dismutase (SOD) and catalase (CAT) activities were assayed by the methods of Kakkar et al. and Sinha, respectively [34, 35]. Glutathione peroxidase (GPx) activity was assayed by the method of Carlberg and Mannervik and Rotruck et al. [36, 37].
PCR primer design Primers were designed using the primer express software (Eppendorf, USA). Primers specific for rat COX-2 (gene of interest) and internal standard GAPDH were used. COX-2 Forward primer: 5′-CCG CCA GCT TCA CTT GCC AC-3′ Reverse primer: 5′-CAG CAC AGC TCG GAA GAG CAT-3′ GADPH Forward primer: 5′-CTG CAC CAC CAA CTG CTT AGC C-3′ Reverse primer: 5′-ACA GCC TTG GCA GCA CCA GT-3′
Real-time PCR conditions and analysis PCR reactions were performed in 96-well plates with a Realplex detection system (Eppendorf) using one-step SYBR Green (QuantiFast SYBR Green RT-PCR Kit, QIAGEN, Germany). Reactions were done in 25 μL volumes containing 200 nM of each primer, 5 μL total RNA, and 12.5 μL 2 × SYBR Green Master Mix Reagent. Reactions were run using the manufacturer’s recommendations for first-step cycling, reverse transcription (50 °C for 10 min) and PCR initial step (95 °C for 5 min), and two-step cycling, 30 cycles of 95 °C for 15 s (denaturation) and 60 °C for 1 min (combined annealing/extension). Data were analyzed using the Realplex cycler (Eppendorf). Expression levels were determined as the number of amplification cycles needed to reach a fixed threshold in the exponential phase of the PCR reaction (Ct). The real-time assay results were quantified using the “threshold line” and the “cycle threshold”. The “threshold line” is the point at which a reaction reaches a fluorescent intensity above background. The “threshold line” is set in the exponential phase of the amplification for most accurate reading. The number
of cycles at which the samples reach this level is called the “cycle threshold”, Ct. Once the calculation for each gene was performed, the results were normalized to the GAPDH reference gene.
Determination of TNF-α and IL-6 The ELISA strip of each well was coated with the specific primary antibody against the specific cytokine protein. The test sample was allowed to react simultaneously with pairs of two antibodies, resulting in the cytokine being sandwiched between the solid phase and enzyme-linked antibodies. After incubation, the wells were washed to remove unbound-labeled antibodies. A horseradish peroxidase substrate, tetramethylbenzidine, was added and then arrested with the addition of stop solution changing the color to yellow. The concentration of the cytokine factors was directly proportional to the color intensity of the test sample. Absorbance was measured using an ELISA reader at 450 nm.
Statistical analysis All the values are expressed as means ± SD. Data were analyzed by one-way analysis of variance (ANOVA) and the significant difference among treatment groups was considered statistically significant at p
Venkatachalam Karthikkumar, Gunasekaran Sivagami, Periyasamy Viswanathan and Namasivayam Nalini*
Rosmarinic acid inhibits DMH-induced cell proliferation in experimental rats Abstract Background: Colon cancer is one of the most common cancers in both men and women. The present study is an effort to unravel the anticarcinogenic effects of rosmarinic acid (RA) in 1,2-dimethylhydrazine (DMH)-induced rat colon carcinogenesis. Administration of DMH induces multiple tumors in the rat colon, which mimics human colon cancer. Methods: Male Wistar rats were divided into six groups and fed a high-fat diet. Group 1 served as control, group 2 rats were given RA [5 mg/kg body weight (b.w.)] orally every day for a total period of 30 weeks, and groups 3–6 were given weekly injections of DMH (20 mg/kg b.w. subcutaneous) once a week in the groin for the first 15 weeks. In addition to DMH, groups 4–6 received RA at a dose of 5 mg/kg b.w. during the initiation and postinitiation stages, and also throughout the entire study period. Colon tissues were examined histologically; further, the extent of oxidative stress was assessed by measuring lipid peroxidation and antioxidant levels in the colonic mucosa of rats. Results: Macroscopic and microscopic tumors were identified in all the groups that received DMH. The results revealed that supplementation with RA significantly inhibited the tumor formation and tumor multiplicity in DMH-treated rats. RA supplementation to DMH-administered rats significantly reduced the cell proliferation markers, namely, argyrophilic nucleolar organizing regions as well as proliferative cell nuclear antigen labeling index. In addition, RA supplementation reduces the expressions of tumor necrosis factor-α, interlukin-6, and cyclooxygenase-2, and modulates the expression of p65. *Corresponding author: Dr. Namasivayam Nalini, Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar-608002, Tamil Nadu, India, Phone: + 91-4144-239141, Fax: + 91-4144-238343, E-mail: [email protected] Venkatachalam Karthikkumar and Gunasekaran Sivagami: Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar, Tamil Nadu, India Periyasamy Viswanathan: Department of Pathology, Faculty of Medicine, Rajah Muthiah Medical College, Annamalai University, Annamalainagar, Tamil Nadu, India
Conclusions: The above findings clearly underline the chemopreventive efficacy of RA against DMH-induced colon carcinogenesis. Keywords: argyrophilic nucleolar organizing regions (AgNORs); chemoprevention; PCNA. DOI 10.1515/jbcpp-2014-0044 Received April 6, 2014; accepted July 14, 2014; previously published online September 11, 2014
Introduction Colorectal cancer is one of the most common forms of cancer worldwide, particularly among the Western population [1]. The incidence of colon cancer was formerly low in India and other Asian countries, but during the last decade a drastic increase in the colon cancer incidence has been observed in Asia [2]. Dietary habits play an important role in the development of colon cancer. In the olden days people used to consume natural substances with lots of medicinal properties. Massive economic growth and rapid urbanization influenced people to adopt a Western dietary style, which is composed of high fat, high protein, low carbohydrate, and low fiber, and this unbalanced diet is considered to be an important risk factor for the increased colon cancer incidence. Diet rich in fruits and vegetables is gaining prime importance to reduce the risk of colon cancer, as they contain various phytochemicals with diverse pharmacological properties [3]. 1,2-Dimethylhydrazine (DMH) is a potent procarcinogen for the induction of tumors in the colon and rectum of rats. The usefulness of this organospecific carcinogen, which induces selectively tumors in the colon, was confirmed by our previous studies [4, 5]. DMH is believed to form active intermediates, including azoxymethane and methylazoxymethanol (MAM), in the liver, which are subsequently transported into the colon through bile [6]. MAM is a long-lived metabolite found in the systemic circulation following DMH exposure. The enzymatic conversion of
Brought to you by | Penn State - The Pennsylvania State University Authenticated Download Date | 5/26/15 12:07 AM
186 Karthikkumar et al.: Rosmarinic acid inhibits uncontrolled proliferation MAM to a reactive aldehyde is considered to be rate limiting in DMH metabolism [7, 8]; that MAM breakdown may occur spontaneously or by enzyme-catalyzed reactions yields the alkylating methyldiazonium ion, capable of methylating cellular macromolecules. Carcinogenesis is a multistep process, beginning with initiation followed by promotion and progression [9]. Each step reflects the genetic alterations that drive the progressive transformation of normal cells into highly malignant derivatives that exhibit continuous disturbance of cellular and molecular signal cascades [10, 11]. Based on this theory, the biology of carcinogenesis is vital to understand the development of an effective prevention. With better understanding of possible ways to modulate carcinogenesis, preventive interventions during these stages became very attractive. Utilization of validated biomarkers can markedly facilitate the testing of potential chemopreventive agents [12]. The quantitative evaluation of argyrophilic nucleolar organizing regions (AgNORs) is the only method that permits information to be obtained on the rapidity of cell proliferation in routinely processed tissue samples. The AgNOR number has been proposed as a diagnostic parameter for the grade of malignancy. It has been suggested that the AgNOR number is significantly different in both benign and malignant tumors [1, 13]. With regard to the cancers of the alimentary tract, it has been reported that the AgNOR number can reflect the prognosis of colon cancer [14, 15]; it is higher in malignant lesions than in benign lesions of the stomach [16]. AgNORs can be visualized as black dots on reaction with silver nitrate. Fecal occult-blood screening may yield an additional benefit in detecting the risk of developing cancer: resulting from the detection and removal of premalignant adenomatous polyps. Fecal occult-blood test (FOBT), which detects blood in the stool, is a simple test used to diagnose disorders of the gut. Though FOBT is less sensitive for the presence of polyps, it is widely used to detect some, particularly large, polyps, which are more likely to bleed [17] and also to become cancerous [18]. Cyclooxygenase (COX)-2 is an inducible enzyme whose expression is upregulated by nuclear factor-κB (NF-κB)-mediated tumorigenesis. Enhanced COX-2 expression has been found in colon cancer tissues from subjects with clinically diagnosed colorectal cancer [19]. Most of the colon cancer drugs available share the property of inhibiting COX, a key enzyme in the conversion of arachidonic acid to prostaglandins. The NF-κB family of eukaryotic transcription factors influences a number of important cellular and organismal processes, including cellular growth control, apoptosis, immune and inflammatory responses, and cellular stress
responses [20]. Thus, inhibition of this chronic NF-κB activity can, in many cases, slow the growth of tumor cell lines or induce cell death. Tumor necrosis factor (TNF) is an important molecule in the cytokine network pathway, which regulates the expression of other cytokines such as interlukin-6 (IL-6). TNF-α induces phosphorylation of IκB, which triggers the rapid ubiquitination and subsequent degradation of IκB, resulting in the release and translocation of NF-κB to the nucleus where it binds to DNA sequences and induces a number of genes, including the antiapoptotic genes, thereby causing cell proliferation [21]. Natural products have a great therapeutic potential in a large number of ailments, including cancer and heart diseases. Compounds of plant origin have the ability to inhibit one or more stages of carcinogenesis and prevent or delay tumor development [22]. Rosmarinic acid (RA, α-o-caffeoyl-3,4-dihydroxyphenyl-lactic acid) is a naturally occurring hydroxylated phenolic compound found in Lamiaceae herbs, such as rosemary, sweet basil, and perilla [23–25]. RA has many pharmacological properties, which include antiinflammatory, antioxidant, antiangiogenic, and antitumor activities [26–29]. Based on the previous dose-dependent study, we have fixed RA at 5 mg/kg b.w., which has the preventive effect in a short-duration experiment [30]. Therefore, in the present long-term study, we have examined the colon cancer inhibitory effect of RA by evaluating its role in cell proliferation and tumor formation in the presence of the colon-specific carcinogen DMH. This study is also designed to gain an insight into the ability of RA to modulate oxidative stress by assessing the lipid peroxidation as well as the antioxidant status in carcinogen-exposed rats supplemented with RA.
Materials and methods Chemicals DMH, RA, hematoxylin, and eosin were purchased from the Sigma Chemical Co. (USA). All other chemicals used were of analytical grade and obtained from Hi-Media Laboratories (Mumbai, India). Antibodies for PCNA, p65 were purchased from Santa Cruz Biotechnology, USA. Primers for COX-2 was purchased from Sigma Chemical Co. (USA).
Animal care and diet Male albino Wistar rats weighing 130–150 g were obtained from the Central Animal House, Rajah Muthiah Medical College and Hospital (RMMCH), Annamalai University (Tamil Nadu, India), and housed under standard environmental conditions (12 h light/dark cycle; 50% humidity; temperature 25 ± 2 °C). Commercial pellet diet containing
Brought to you by | Penn State - The Pennsylvania State University Authenticated Download Date | 5/26/15 12:07 AM
Karthikkumar et al.: Rosmarinic acid inhibits uncontrolled proliferation 187 4.2% fat (Hindustan Lever Ltd., Mumbai, India) was powdered and mixed with 15.8% peanut oil, making 20% total fat in the feed to prepare a high-fat, Western diet (modified pellet diet). The modified pellet diet and water were fed ad libitum to all the rats in the experiment. The rats were cared for in compliance with the principles and guidelines of the Ethical Committee for Animal Care of Annamalai University in accordance with the Indian National Law on Animal Care (Reg. No. 160/1999/CPCSEA/437). Food intake, water intake, and body weight (b.w.) changes were monitored regularly.
DMH and RA administration DMH was dissolved in 1.0 mM ethylenediaminetetraacetic acid just prior to use and the pH was adjusted to 6.5 with 1 mM NaOH to ensure the stability of the chemical. Animals received subcutaneous injections of DMH (20 mg/kg b.w.), once a week for 15 weeks [4]. RA was suspended in water just before treatment and was administered every day orally at a dose of 5 mg/kg b.w. for 30 weeks.
Treatment schedule Rats were randomly distributed into six groups of 12 rats each. Group 1 rats served as control, and group 2 received RA (5 mg/kg b.w.) every day throughout the experimental period. Groups 3–6 received DMH (20 mg/kg b.w. subcutaneous) once a week for the first 15 weeks, which represent the colon cancer-bearing rats. Group 4 rats were colon cancer-bearing rats treated with RA (as in group 2) for the first 15 weeks (initiation), group 5 rats were colon cancer-bearing rats administered with RA (as in group 2) starting 1 week after the cessation of DMH injections and continued till the end of the experimental period (postinitiation), and group 6 rats were colon cancer-bearing rats administered with RA (as in group 2) throughout the experimental period (entire period) of 30 weeks (Figure 1).
Histological observations The experiment was terminated at the end of 30 weeks; all the animals were euthanized under anesthesia. The colons were split open
longitudinally for macroscopical observation and gross tumors were counted. The liver and the colon segments were dissected, fixed immediately in 10% formalin, and paraffin embedded; 2–3 μm sections were cut in a rotary microtome, stained with hematoxylin and eosin, and viewed under a light microscope. Neoplastic lesions of colorectal mucosa were histopathologically classified into adenomas and adenocarcinomas. Adenomas are well-demarcated lumps of epithelial dysplasia, which can be classified into three major histological types: tubular, villous, and tubulo villous. Multiplicity of adenomas bears an increased risk of dysplasia and cancer. An adenoma is pedunculated when it possesses a stalk; sessile adenomas rise above the background mucosa without any stalk. Small (approx. 1 cm) adenomas are also known as “diminutive”. Further colorectal carcinoma can be graded into well-differentiated, moderately differentiated, and poorly differentiated lesions as described by Bosman [31].
AgNORs – Silver staining method AgNOR staining was carried out by the method of Nyska et al. [32]. Silver colloid solution was made up with 2% gelatin in 1% aqueous formic acid. This solution was mixed with twice its volume of 50% aqueous silver nitrate solution. Sections were exposed to the staining solution for 40 min at room temperature in the dark. The slides were washed in distilled water for 10 min, rehydrated through graded alcohol, cleared in xylene, and mounted using dibutyl phathalate xylene. The number of AgNORs per crypt cell nucleus was counted on one-step silver stained sections. The measurement was performed on 25 crypts per rat.
Proliferating cell nuclear antigen [PCNA and p65 (NFκB)] – immunohistochemsitry The paraffin-embedded sections (4 μm) were heated at 60 °C for 60 min, deparaffinized in xylene, and rehydrated through graded alcohol at room temperature. Tris-HCl buffer (0.05 M; pH 7.6) was used to prepare solutions and for washes between various steps. Sections were treated for 40 min at room temperature with 2% bovine serum albumin and incubated with primary antibodies against the
Figure 1 Experimental design.
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188 Karthikkumar et al.: Rosmarinic acid inhibits uncontrolled proliferation PCNA or p65 protein (BioGenex Laboratories, Inc., San Ramon, CA, USA) for 1 h. Horseradish peroxidase activity was visualized after treatment with H2O2 and diaminobenzidine hydrochloride for 5 min using the labeled streptavidin-biotin method (BioGenex Kit, San Ramon, CA, USA). Immunoreactivities were regarded as positive if the apparent staining was detected in the nuclei. The PCNA labeling index was determined by calculating the ratio PCNA-positive nuclei/ total number of nuclei.
Lipid peroxidation and antioxidant enzyme levels The colonic mucosa was minced and homogenized with 3 vol (w/v) of the appropriate buffer using a Potter-Elvehjam homogenizer with a Teflon pestle and centrifuged at 12,000 g for 20 min at 4 °C. The supernatant was used for biochemical estimations. Lipid peroxidation was estimated by measuring the levels of the tissue lipid peroxidation byproduct thiobarbituric acid reactive substances (TBARS) by using the method of Jiang et al. [33]. Superoxide dismutase (SOD) and catalase (CAT) activities were assayed by the methods of Kakkar et al. and Sinha, respectively [34, 35]. Glutathione peroxidase (GPx) activity was assayed by the method of Carlberg and Mannervik and Rotruck et al. [36, 37].
PCR primer design Primers were designed using the primer express software (Eppendorf, USA). Primers specific for rat COX-2 (gene of interest) and internal standard GAPDH were used. COX-2 Forward primer: 5′-CCG CCA GCT TCA CTT GCC AC-3′ Reverse primer: 5′-CAG CAC AGC TCG GAA GAG CAT-3′ GADPH Forward primer: 5′-CTG CAC CAC CAA CTG CTT AGC C-3′ Reverse primer: 5′-ACA GCC TTG GCA GCA CCA GT-3′
Real-time PCR conditions and analysis PCR reactions were performed in 96-well plates with a Realplex detection system (Eppendorf) using one-step SYBR Green (QuantiFast SYBR Green RT-PCR Kit, QIAGEN, Germany). Reactions were done in 25 μL volumes containing 200 nM of each primer, 5 μL total RNA, and 12.5 μL 2 × SYBR Green Master Mix Reagent. Reactions were run using the manufacturer’s recommendations for first-step cycling, reverse transcription (50 °C for 10 min) and PCR initial step (95 °C for 5 min), and two-step cycling, 30 cycles of 95 °C for 15 s (denaturation) and 60 °C for 1 min (combined annealing/extension). Data were analyzed using the Realplex cycler (Eppendorf). Expression levels were determined as the number of amplification cycles needed to reach a fixed threshold in the exponential phase of the PCR reaction (Ct). The real-time assay results were quantified using the “threshold line” and the “cycle threshold”. The “threshold line” is the point at which a reaction reaches a fluorescent intensity above background. The “threshold line” is set in the exponential phase of the amplification for most accurate reading. The number
of cycles at which the samples reach this level is called the “cycle threshold”, Ct. Once the calculation for each gene was performed, the results were normalized to the GAPDH reference gene.
Determination of TNF-α and IL-6 The ELISA strip of each well was coated with the specific primary antibody against the specific cytokine protein. The test sample was allowed to react simultaneously with pairs of two antibodies, resulting in the cytokine being sandwiched between the solid phase and enzyme-linked antibodies. After incubation, the wells were washed to remove unbound-labeled antibodies. A horseradish peroxidase substrate, tetramethylbenzidine, was added and then arrested with the addition of stop solution changing the color to yellow. The concentration of the cytokine factors was directly proportional to the color intensity of the test sample. Absorbance was measured using an ELISA reader at 450 nm.
Statistical analysis All the values are expressed as means ± SD. Data were analyzed by one-way analysis of variance (ANOVA) and the significant difference among treatment groups was considered statistically significant at p
