- Email: [email protected]
Chemopreventive effect of Cynodon dactylon (L.) Pers. extract against DMH-induced colon carcinogenesis in experimental animals
ARTICLE IN PRESS
Experimental and Toxicologic Pathology 62 (2010) 423–431 www.elsevier.de/etp
Chemopreventive effect of Cynodon dactylon (L.) Pers. extract against DMH-induced colon carcinogenesis in experimental animals Arul Albert-Baskar, Savarimuthu Ignacimuthu Division of Ethnopharmacology, Entomology Research Institute, Loyola College, Chennai – 600034, Tamil Nadu, India Received 3 February 2009; accepted 3 June 2009
Abstract The present study was aimed at evaluating the chemopreventive property of Cynodon dactylon. The antioxidant, antiproliferative and apoptotic potentials of the plant were investigated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, nitric oxide radical scavenging activity (NO) and MTT assay on four cancer cell lines (COLO 320 DM, MCH-7, AGS, A549) and a normal cell line (VERO). In vivo chemopreventive property of the plant extract was studied in DMH-induced colon carcinogenesis. The methanolic extract of C. dactylon was found to be antiproliferative and antioxidative at lower concentrations and induced apoptotic cell death in COLO 320 DM cells. Treatment with methanolic extract of C. dactylon increased the levels of antioxidant enzymes and reduced the number of dysplastic crypts in DMH-induced colon of albino rats. The present investigation revealed the anticancer potential of methanolic extract of C. dactylon in COLO 320 DM cells and experimentally induced colon carcinogenesis in rats. r 2009 Elsevier GmbH. All rights reserved. Keywords: Cynodon dactylon; 1,2-Dimethyl hydrazine; COLO 320 DM; DNA fragmentation; Chemopreventive property
Introduction Cancer is the second leading cause of death after cardiovascular diseases in India. Lung, breast, colon and stomach cancers are the four most common cancers worldwide (Bingham and Riboli, 2004). Medicinal plants are frequently used by traditional healers to treat a variety of ailments and symptoms including diabetes and cancer. According to the World Health Organization, over 80% of the world’s populations rely upon such traditional plant-based systems of medicine to provide them with primary healthcare (Calixto, 2005). Corresponding author. Tel.: +91 044 2817 8348; fax: +91 044 2817 4644. E-mail address: [email protected] (S. Ignacimuthu).
0940-2993/$ - see front matter r 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2009.06.003
Lipid peroxidation has gained more importance nowadays because of its involvement in pathogenesis of many diseases like cancer, diabetes and also aging (Halliwell et al., 1992). Free radicals or reactive oxygen species (ROS) are the main culprits in lipid peroxidation and cause destructive and irreversible damage to the components of a cell, such as lipids, proteins and DNA (Cheeseman and Slater, 1993; Lopaczyski and Zeisel, 2001). Although normal cells possess antioxidant defence systems against ROS, continuous accumulation of damage to the cells induces diseases such as cancer and aging (Mates and Sanchez-Jimenez, 2000). The continuous supply of antioxidant dose also plays a preventive role against diseases by removing the ROS in biological systems (Sgambato et al., 2001). Cancer cells, which are already irreversibly developed, obtain the
ARTICLE IN PRESS 424
A. Albert-Baskar, S. Ignacimuthu / Experimental and Toxicologic Pathology 62 (2010) 423–431
capability to escape apoptosis by various ways. The aim of anticancer agents is to trigger the apoptosis signaling system in these cancer cells whilst disturbing their proliferation (Bold et al., 1997). 1,2-Dimethyl hydrazine (DMH) is a colon specific carcinogen known to produce free radicals in circulation in experimental animal models (Arutiuian et al., 1997). Antioxidant enzymes are scavengers of free radicals and are modulated during carcinogenesis or after tumor formation. As a result of malignant state, alterations in the antioxidant enzymes occur; if they are required for the maintenance of the malignant state, then the recovery of decreased enzymes could help to reverse the malignancy (Sun et al., 2004). Cynodon dactylon (L.) Pers, root is used as cure for cancer in Indian Traditional medicine (Nadkarni, 1976).The present study was carried out to determine the anticancer potential of C. dactylon root extracts using in vitro models to minimize the use of experimental animals and the active extract was studied for chemopreventive potential using DMH-induced animal models.
Materials and methods Plant material and preparation of extract
and 25 mg of dried extract/ml solution. The reaction mixture contained 1000 ml of 0.25 mM DPPH with various concentrations of the extracts in 1 ml of ethanol. After 20 min incubation at room temperature the absorbance at l ¼ 517 nm was recorded using a Hitachi U-2000 spectrophotometer (Hitachi, Tokyo, Japan). The inhibition percentage (%) of radical scavenging activity was calculated using the following equation: Inhibition ð%Þ
A0 As 100 A0
where A0 is absorbance of the control and As is absorbance of the sample at 517 nm. From the inhibition (%), the amount of the samples (mg) reducing the absorbance by 50% was determined (IC50).
Nitric oxide scavenging assay In nitric oxide radical inhibition assay, Sodium nitroprusside in aqueous solution at physiological pH, spontaneously generates nitric oxide which interacts with oxygen to produce nitrite ions, which can be estimated by the use of Griess Illosvoy reaction (Garrat, 1964). In the present investigation, Griess Illosvoy reagent was modified using naphthylethylenediamine dihydrochloride (0.1% w/v) in place of 1-naphthylamine (5%). Scavengers of nitric oxide compete with oxygen leading to reduced production of nitric oxide (Marcocci et al., 1994). The IC50 value is the concentration of sample required to inhibit 50% of nitric oxide radical.
The medicinal plant (C. dactylon) was selected based on its use in traditional medicine. Roots of C. dactylon were collected washed with double distilled water twice and allowed to shade dry for about 10 days with free aeration. The plants were authenticated by Dr. Ayyanar (Plant taxonomist) at the Entomology Research Institute, Loyola College, Chennai. Voucher specimen (ERI 109) was preserved at the herbarium of Entomology Research Institute. Shade dried roots were powdered using an electric blender and the powder was used for extraction with three solvents sequentially using cold percolation method. 500 g of the root powder was soaked in hexane for 48 h, filtered and concentrated using a rotary evaporator at reduced pressure; to the residue ethyl acetate was poured and extracted as above. The residue was again used for methanol extraction. The crude extracts were dissolved in DMSO and used as a stock solution and filter sterilized (0.22 mm) before the experiment. The final working concentration of DMSO was less than 0.1%.
All the cell lines were purchased from National Center for Cell Science (NCCS, Pune) and subcultured using appropriate medium. COLO 320 DM (human colon adenocarcinoma cell line) was cultured in RPMI 1640. MCH-7 (human breast cancer cell), AGS (human stomach cancer cell line), A549 (human lung cancer cell line) and a normal cell line VERO (Monkey kidney cells) were cultured in DMEM. Both the media were supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco) and ampotericin B (Gibco). Cells were cultured as monolayers in 25 cm2 plastic tissue culture flasks at 37 1C under a humidified atmosphere of 5% CO2 in air.
DPPH free radical scavenging assay
Antiproliferative studies
The hydrogen-donating ability of solvent extracts was examined using the method of Blois (1958) in the presence of DPPH stable free radical. The extracts and positive control, Vitamin C, were diluted with ethanol to prepare sample solution equivalent to 400, 200, 100, 50
The survival of cells was determined by MTT assay using the method of Mosmann (1983). Briefly, the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide) (Sigma) solution was prepared freshly in phosphate-buffered saline (0.5 mg/ml)
Cell lines and culture medium
ARTICLE IN PRESS A. Albert-Baskar, S. Ignacimuthu / Experimental and Toxicologic Pathology 62 (2010) 423–431
(Gibco) just before use. Cells were seeded in 96 well plates (1 105 cells/well) and allowed for attachment for 6 h. The cells were treated with various concentrations of the extract (3.125, 6.25, 12.5, 25, 50, 100 and 200 mg/ml) for 24 h; then 100 ml of MTT dye was added to each well. The plates were incubated in a CO2 incubator for 4 h and optical density was determined by eluting the dye with DMSO and read using a 96 well plate reader at 560 nm.
DNA fragmentation COLO 320 DM cells were seeded in 24 well plates and incubated at 37 1C in 5% CO2 until 100% confluence was attained. The cells were treated with three different concentrations (1000, 500 and 250 mg/ml) of C. dactylon methanol crude extracts and incubated for 24 h. The cells were harvested and centrifuged at 10000 rpm for 10 min. The cell pellet was suspended in 500 ml of TKM I buffer (10 mM Tris–HCl, 10 mM magnesium chloride, 10 mM potassium chloride, 2 mM EDTA, pH 7.6) vortexed vigorously and centrifuged at 10000 rpm for 10 min. The pellet was again suspended in 500 ml of TKM II (10 mM Tris–HCl, 10 mM Magnesium chloride, 10 mM potassium chloride, 2 mM EDTA, 0.4 M NaCl, pH 7.6). Again 40 ml of 10% SDS and 4 ml of RNAse A (10 mg/ml) were added and incubated at 55 1C for 10 min. Then 125 ml of 5 M NaCl was added, mixed and centrifuged at 10,000 rpm for 5 min. The supernatant was then transferred to a fresh tube and 1 th mixed with two volumes of absolute ethanol and 10 volume 3 M sodium acetate. The mixture was kept at 80 1C for 1 h and centrifuged at 12,000 rpm for 10 min. The pellet was washed in 70% ethanol and dried at 37 1C. The pellet was then suspended in 20 ml of TE buffer (pH 8.0) and electrophoresed in a 2% agarose gel.
Apoptosis COLO 320 DM (1 107) cells were treated with 6.25, 12.5, 25 and 50 mg/ml of extract for 24 h. At the end of the treatment cells were washed with phosphatebuffered saline (PBS) and resuspended in binding buffer (10 mM HEPES/NaOH Ph 7.4, 140 mM NaCl, 2.5 mM CaCl2). Aliquots of cells (100 ml) were incubated with 5 ml of Annexin-V FITC (fluorescein isothiocyanateconjugated), mixed and incubated for 15 min at room temperature in dark and stained with propidium iodide (5 mg/ml). The cells were then gently vortexed and 10,000 events were acquired and analyzed using Becton Dickinson FACS caliber. In brief, early apoptotic cells were defined as those cells exhibiting a fluorescein isothiocyanate-conjugated annexin-V-positive and propidium iodide-negative staining pattern, necrotic cells
425
exhibiting propidium iodide-positive and Annexin-V FITC negative.
DMH-induced colon carcinogenesis Animals and diet Experiments were carried out with 5 weeks old male albino Wistar rats, obtained from Central Animal House, Kings Institute, Chennai, Tamil Nadu. The animals were cared for in compliance with the principles and guidelines of Ethical Committee for Animal Care and institutional animal ethical committee in accordance with the Indian National Law on Animal Care and Use (Reg. No. 833/a/2004/CPCSEA). The animals were housed four per cage in polypropylene cages with a wire mesh top and a hygienic bed of husk in a specific pathogen-free animal room under controlled conditions of 12 h light/h dark cycle, with temperature of 2472 1C and relative humidity of 50710% till the end of the experimental period. The rats were held in quarantine for 1 week and had access to food and tap water ad libitum. Commercial pellet diet containing 4.2% fat (Hindustan Lever Ltd., Mumbai, India) was powdered and mixed with 15.8% peanut oil making a total of 20% fat in the diet (Table 1). This modified powdered pellet diet was fed to rats in all groups throughout the experimental period of 16 weeks. The total caloric intake of the rats in all the groups was adjusted to be the same. Carcinogen administration The experimental animals were divided into six groups. The animals in groups III–VI received subcutaneous injections of DMH at a dose of 20 mg/kg bwt once a week for the first four consecutive weeks. Prior to subcutaneous injection, DMH was dissolved in 1 mM EDTA, the pH adjusted to 6.5 with 1 mM NaOH to ensure the pH and stability of the chemical and was used immediately after preparation. Initial body weights of all animals in this study protocol were ensured to be between 80 and 120 g. The animal weights were recorded once a week throughout the experimental period and prior to sacrifice.
Table 1.
Composition of the diet.
Protein Fat Carbohydrates Fiber Minerals Vitamins
Commercial diet 84.2%
Peanut oil 15.8%
Total %
17.7 4.2 50.5 3.4 6.7 1.7
– 15.8 – – – –
17.7 20.0 50.5 3.4 6.7 1.7
ARTICLE IN PRESS 426
A. Albert-Baskar, S. Ignacimuthu / Experimental and Toxicologic Pathology 62 (2010) 423–431
Experimental design
Group 1
–
Group 2
–
Group 3
–
Group 4
–
Group 5
–
Group 6
–
Rats received modified pellet diet along with intragastric intubation of 0.1% CMC (1.0 ml), throughout the experimental period. Rats received modified pellet diet+100 mg/kg bwt methanolic extract of C. dactylon suspended in 0.1% CMC (1.0 ml), p.o. everyday throughout the experimental period. Rats were administered 20 mg/kg bwt DMH (carcinogen) s.c. once a week for four consecutive weeks and kept without any treatment for the next 12 weeks. The animals were treated as in group 3 along with methanolic extract of C. dactylon (25 mg/kg bwt, p.o.) throughout the entire experimental period of 16 weeks. The animals were treated as in group 3 along with methanolic extract of C. dactylon (50 mg/kg bwt, p.o.) throughout the entire experimental period of 16 weeks. The animals were treated as in group 3 along with methanolic extract of C. dactylon (100 mg/kg bwt, p.o.) throughout the entire experimental period of 16 weeks.
At the end of 16 weeks all the animals were sacrificed under anesthesia (i.p. administration of ketamine hydrochloride (30 mg/kg bwt), by cervical dislocation between 8 am and 10 am after an overnight fast. Blood samples were collected from all the experimental animals and plasma was separated and analyzed for antioxidant enzymes. Tumor and normal colon tissues were fixed in 10% formalin and were stained with hematoxylin and eosin for histopathological investigation. Total lipid peroxidation levels as evidenced by the formation of thiobarbituric acid reactive substances (TBARS) were estimated by the method of Yagi (1978) and reduced glutathione by the method of Ellman (1959). The activities of glutathione peroxidase (GPx) and glutathione S-transferase (GST) were estimated by the method of Rotruck et al. (1973) and Habig et al. (1974) respectively. Superoxide dismutase (SOD) was assayed by the method of Kakkar et al. (1984) and catalase (CAT) was assayed by the method of Sinha (1972). Serum protein was determined by the method of Lowry et al. (1951).
Statistical analysis Antioxidant and cytotoxic activities were performed in triplicate and the values were expressed as Mean7SD ANOVA was used to determine the significance level of the data obtained. All the biochemical analysis were expressed as means7SD. Statistical evaluation was done using one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (DMRT). The statistical significance was set at Po0.05. Analysis was performed using SPSS version 11.0 software (SPSS Inc., Chicago, IL, USA).
Results Several concentrations ranging from 3.125 to 200 mg/ml extracts of C. dactylon were tested for their antioxidant and antiproliferative activity using different in vitro models. The extracts exhibited antioxidant effects in a concentration dependant manner up to a given concentration (Table 2). Among the tested extracts methanolic extract exhibited strong antioxidant activity in the DPPH and the nitric oxide radical inhibition assays as evidenced by the low IC50 values (Table 2) of 63.03 and 98.89 mg/ml, respectively. Results of MTT assay revealed that all the extracts of C. dactylon exhibited antiproliferative effects towards the tested cell lines at various concentrations. Methanol extract showed promising effects with no toxicity to VERO cells in all tested concentrations (Fig. 1). To determine the mechanism of cytotoxicity either due to apoptosis or necrosis, DNA fragmentation and Annexin FITC/PI assays were carried out. The methanolic extract of C. dactylon exhibited fragmentation of DNA (Fig. 2) and proapoptotic activity in COLO 320 DM cells (Fig. 3). The chemopreventive potential of methanolic extract of C. dactylon was further validated in DMH-induced experimental carcinogenesis. Treatment with methanolic
Table 2.
DPPH and NO scavenging property of C. dactylon.
Plant parts and extracts used
IC50 (lg/ml) DPPH
IC50 (lg/ml) NO
Cynodon dactylon (root) Hexane Ethyl acetate Methanol
874.15766.56 311.05723.69 63.0374.82
ND 450.08734.27 111.2278.47
Vitamin C
570.38
2571.90
Each value is mean7SD for 6 replication in each group. ND – Not Detectable.
ARTICLE IN PRESS A. Albert-Baskar, S. Ignacimuthu / Experimental and Toxicologic Pathology 62 (2010) 423–431
120
C. dactylon Hexane C. dactylon Ethyl acetate C. dactylon Methanol
d
100 IC50 (µg/ml)
427
80 c
60
ab
b a
40
b b
d a
20
a
b
a a a
0 VERO
AGS
MCF-7
A549
COLO 320 DM
Fig. 1. Antiproliferative effect of the crude extract of C. dactylon in three different cancer cell lines and a normal cell line. Each value is mean7SD for 6 replications in each group. Values not sharing a common superscript differ significantly at (a–d)Po0.05 (DMRT).
Fig. 2. Agarose gel pattern for the DNA from COLO 320 DM cells. Lane 1–100 mg/ml C. dactylon crude extract; lane 2-untreated cells; lane 3–1000–200 Kb marker.
extract of C. dactylon at three different concentrations (25, 50 and 100 mg/kg bwt) reduced the levels of lipid peroxides and increased the antioxidant enzymes in
circulation. Lipid peroxidation products and antioxidant enzyme levels determined in the plasma of experimental animals are shown in Tables 3 and 4. The levels of DC, LOOH and TBARS were significantly higher in DMH alone treated animals as compared to control rats (Po0.05). Treatment with three different doses of methanolic extract of C. dactylon in DMH treated rats, significantly (Po0.05) decreased the extent of lipid peroxidation when compared to the DMH alone treated rats (group 3). Among the three doses group 5 showed pronounced effect (Po0.01) when compared to the other doses in decreasing the levels of lipid peroxidation. At the end of 16 weeks, in DMH treated rats, the activities of SOD, CAT and GSH-dependent enzymes were significantly (Po0.05) decreased when compared to the control rats (group 1). Supplementation of methanolic extract of C. dactylon showed a significant (Po0.05) increase in the activities of free radical metabolizing enzymes in 100 mg/kg bwt treated animals. Tissue sections of Group 1 animals displayed normal colonic architecture with no signs of apparent abnormality (Fig. 4A). In the carcinogen treated Group 3, well differentiated signs of dysplasia were evident. Nuclei were enlarged; thickening of epithelium was seen; cells were hyper-chromatic and showed increased mitotic activity. Simultaneously, there was a loss in nuclear polarity (Fig. 4C). The tumor section of the colon from DMH treated rats (Fig. 5) also revealed poorly differentiated adenocarcinoma (Fig. 5). In the treatment group (Groups 4–6), histoarchitecture revealed no signs of dysplasia but indicated a little loss of nuclear polarity (Fig. 4D). The size and shape of the cells were uniform and the cells regained the near normal histoarchitecture (Fig. 4F). Occasionally, hyper-chromatic nucleus was evident. There were no signs of dysplasia or toxicity observed in Group 3 rats administered with methanolic extract of C. dactylon alone (Fig. 4B).
ARTICLE IN PRESS 428
A. Albert-Baskar, S. Ignacimuthu / Experimental and Toxicologic Pathology 62 (2010) 423–431
Fig. 3. Induction of apoptosis by methanolic extract of C. dactylon in COLO 320 DM cells. Cells in each quadrant reflect the percentage of cells.
Table 3.
Effect of methanolic extract of C. dactylon on circulatory lipid peroxidation status in rats subjected to DMH treatment.
Groups
TBARS (nmol/ml)
DC (mmol/ml)
LOOH (mmol/ml)
CON CON+SITO DMH DMH+25 mg/kg extract DMH+50 mg/kg extract DMH+100 mg/kg extract
1.8270.14e 1.8970.14e 4.1370.31a 3.2570.25b 2.9770.23c 2.5470.19d*
0.5970.04e 0.6470.05e 1.8870.14a 1.6470.12b 1.0470.08c 0.8270.06d*
0.2970.02e 0.3270.02e 0.8370.06a 0.7270.05b 0.5970.04c 0.5170.04d*
Data are presented as means7SD of 10 rats in each group. (a–e)Po0.05 values not sharing a common superscript letter are significantly different. *Po0.01, values are significantly different as compared to DMH alone treated group.
Table 4. Effect of methanolic extract of C. dactylon on antioxidant defence system in erythrocyte lysate of rats subjected to DMH treatment. Groups
z
CON CON+SITO DMH DMH+25 mg/kg extract DMH+50 mg/kg extract DMH+100 mg/kg extract
3.970.30a 4.070.30a 2.070.15e 2.170.16d 2.670.20c 3.570.27b*
SOD
y
CAT
2.270.17a 2.370.18a 1.170.08e 1.370.10d 1.670.12c 2.070.15b*
z
y
#
56.574.30a 57.074.34a 41.473.15c 44.373.37bc 48.57 3.69b 54.974.18a*
27.272.07a 28.872.19a 15.871.20c 17.471.33c 20.871.58b 26.772.03a*
3.370.25a 3.470.26a 1.570.11e 1.870.14d 2.170.16c 2.870.21b*
GR
GPx
GST
Data are presented as means7SD of 10 rats in each group. (a–e)Po0.05 values not sharing a common superscript letter are significantly different. *Po0.01, values are significantly different as compared to DMH alone treated group. z Enzyme required for 50% inhibition of NBT reduction/min/mg Hb. y m mol of H2O2 utilized/min/mg Hb. z mmol of NADPH oxidized/min/mg Hb. y mmol of GSH utilized/min/mg Hb. # mmol of CDNB-GSH conjugate formed/min/mg Hb.
Discussion Many commercially proven drugs used in modern medicine were initially used in crude form in traditional or folk healing practices, or for other purposes that suggested potentially useful biological activity. Most of the secondary plant compounds employed in modern medicine were first discovered through ethnobotanical
investigation (Gurib-Fakim, 2006). Natural antioxidants have a wide range of biochemical activities, including inhibition of ROS generation, direct or indirect scavenging of free radicals, and alteration of antioxidant potential (Finkel and Holbrook, 2000). Antioxidants have been used to inhibit apoptosis because apoptosis was initially thought to be mediated by oxidative stress (Hockenhery et al., 1993). Many
ARTICLE IN PRESS A. Albert-Baskar, S. Ignacimuthu / Experimental and Toxicologic Pathology 62 (2010) 423–431
429
Fig. 4. Histopathological findings of DMH-induced colon treated with three different concentrations of methanolic extract of C. dactylon. (A) Normal colon, (B) control animals treated with 100 mg/kg bwt methanolic extract of C. dactylon, (C) animals treated with DMH alone, (D) animals treated with DMH and treated with 25 mg/kg bwt methanolic extract of C. dactylon, (E) animals treated with DMH and treated with 50 mg/kg bwt methanolic extract of C. dactylon and (F) animals treated with DMH and treated with 100 mg/kg bwt methanolic extract of C. dactylon.
Fig. 5. Morphological view of DMH-induced colon.
antioxidant substances have anticancer or anticarcinogenic properties (Johnson et al., 1994). Anticancer agents have been isolated from Andrographis paniculata, Phyllanthus amarus, Piper longum, Semecarpus anacardium, Withanica somnifera, Moringa oleifera, Aloe vera, Curcuma longa, Allium sativum and Tinospora cordifolia (Balachandran and Govindarajan, 2005). The methanolic extract of C. dactylon reduced the DPPH radical to the corresponding hydrazine by its
ability to donate hydrogen atom and acted as an antioxidant in a concentration dependant manner. The nitrite produced by the incubation of solutions of sodium nitroprusside in standard phosphate-buffered saline at 25 1C was reduced by the methanolic extract of C. dactylon root extract. This might be due to the antioxidant principles in the extract which competed with oxygen to react with nitric oxide and thus inhibited the generation of nitrite. The antiproliferative potential of the methanolic extract of C. dactylon studied by MTT assay is considered to be diagnostic of mitochondrial malfunction. Damage of this organelle by the extract could be involved in activation of the apoptotic pathway. The available semi synthetic anticancer drugs have more side effects and are cytotoxic to human beings since they are not cancer cell specific. Hence scientists are interested in finding a potent phytotherapeutic agent with noncytotoxic properties. Methanolic extract of C. dactylon exhibited antiproliferative activity towards all the cancer cells (COLO 320 DM, AGS, MCF-7 and A549) with a significant effect towards COLO 320 DM cells and was not toxic to VERO cells indicating it to be safe to normal cells. The mode of cell death caused by the extract had to be determined. COLO 320 DM cells treated with methanolic extract of C. dactylon were
ARTICLE IN PRESS 430
A. Albert-Baskar, S. Ignacimuthu / Experimental and Toxicologic Pathology 62 (2010) 423–431
subjected to DNA fragmentation and Annexin -V-FITC/PI assays, and both data indicated the mode of cell death to be by apoptosis. The ethanolic extract of C. dactylon had been reported to posses’ moderate antioxidant activities and was not cytotoxic towards PC12 cells up to 1 mg/ml (Auddy et al., 2003). Reducing oxidative stress may suppress the proliferation of tumor cells and enhance cancer cell apoptosis (Sun et al., 2004). A variety of cytotoxic/anticancer drugs has been reported to induce apoptosis of malignant cells in vitro (Muller et al., 1997). The chemopreventive potential of the methanolic extract of C. dactylon was further investigated in DMH-induced experimental colon carcinogenic model using albino rats. It was interesting to note that treatment of 100 mg/kg bwt of the extract exhibited significant protective protection to carcinogenesis by increasing the antioxidant enzymes and lowering the histopathological alterations caused by DMH administration. DMH is metabolized to a methyl free radical and generates hydroxyl radical or hydrogen peroxide in the presence of metal ions that may contribute to the initiation of lipid peroxidation (Dudeja and Brasitus, 1990). DC and LOOHs are primary products of lipid peroxidation used to quantify the oxidative damage in membrane lipids. Increased levels or accelerated generation of ROS and toxic degradation products of lipid peroxidation have been reported in circulation of DMHinduced animal models (Sengottuvelan et al., 2006). Removal of free radicals in biological systems is achieved through enzymic and non enzymic antioxidants, which act as principle defence systems against free radicals (Fang et al., 2002). In conclusion, the results of the present work show that the methanolic extract of C. dactylon activated the apoptotic pathway in mammalian colon adenocarcinoma cells and lowered the levels of lipid peroxides in circulation in DMH-induced animals treated with the extract. The ability of the extract to trigger and execute apoptosis in cancer cells is unclear but the MTT assay suggests a mitochondrial involvement. C. dactylon methanolic extract offers protection towards the damage caused by DMH in experimental animals indicating it to be a safe drug for colon cancer.
References Arutiuian AV, Prokopenko VM, Burmistrov SO, Oparina TI, Frolova EV, Zabezhinskii MA, et al. Free-radical processes in blood serum, liver and large bowel during 1,2-dimethylhydrazine-induced carcinogenesis in rats. Vopr Onkol 1997;43:618–22. Auddy B, Ferreira M, Blasina F, Lafon L, Arredondo F, Dajas F, et al. Screening of antioxidant activity of three Indian medicinal plants, traditionally used for the manage-
ment of neurodegenerative diseases. J Ethnopharmacol 2003;84:131–8. Balachandran P, Govindarajan R. Cancer – an ayurvedic perspective. Pharmacological Research 2005;51:19–30. Bingham S, Riboli E. Diet and cancer – the European prospective investigation into cancer and nutrition. Nature Rev Cancer 2004;4:206–15. Blois MS. Antioxidant determinations by the use of a stable free radical. Nature 1958;181:1199–200. Bold RJ, Termuhlen PM, McConkey DJ. Apoptosis, cancer and cancer therapy. Surg Oncol 1997;6:133–42. Calixto JB. Twenty five years of research on medicinal plants in Latin America: a personal review. J Ethnopharmacol 2005;100:131–4. Cheeseman KH, Slater TF. An introduction to free radical biochemistry. Br Med Bull 1993;49:481–93. Dudeja PK, Brasitus TA. 1,2-Dimethylhydrazine-induced alterations in lipid peroxidation in preneoplastic and neoplastic colonic tissues. Biochim Biophys Acta 1990; 1046:267–70. Ellman GL. Tissue sulphydryl groups. Arch Biochem Biophys 1959;82:70–7. Fang YZ, Yang S, Wu G. Free radicals, antioxidant, and nutrition. Nutrition 2002;18:872–9. Finkel T, Holbrook NJ. Oxidants, oxidative stress and the biology of ageing. Nature 2000;408:239–47. Garrat DC. The quantitative analysis of drugs, Vol. 3. Japan: Chapman & Hall; 1964. p. 456–8. Gurib-Fakim A. Review – medicinal plants: traditions of yesterday and drugs of tomorrow. Mol Aspect Med 2006;27:1–93. Habig WH, Pabst MJ, Jakoby WB. Glutathione S-transferase, the first enzymatic step in mercapturic acid formation. J Biol Chem 1974;249:7130–9. Halliwell B, Gutteridge JMC, Cross CE. Free radicals, antioxidants and human disease: where are we now? J Lab Clin Med 1992;119:598–620. Hockenhery DM, Oltavai ZN, Korsmeyer SJ. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 1993;75:241–51. Johnson IT, Williamson G, Musk SR. Anticarcinogenic factors in plant foods: a new class of nutrients. Nutr Rev 1994;7:175–204. Kakkar P, Das B, Viswanathan PN. A modified spectrophotometric assay of superoxide dismutase. Ind J Biochem Biophys 1984;21:130–2. Lopaczyski W, Zeisel SH. Antioxidant, programmed cell death, and cancer. Nutr Res 2001;21:295–307. Lowry OH, Roseborough NJ, Farr AL, Randll AJ. Protein measurement with Folin’s phenol reagent. J Biol Chem 1951;193:265–75. Marcocci P, Sckaki A, Albert GM. Antioxidant action of Ginkgo biloba extracts EGP761. Methods Enzymol 1994; 234:462–75. Mates JM, Sanchez-Jimenez FM. Role of reactive oxygen species in apoptosis: implications for cancer therapy. Int J Biochem Cell Biol 2000;32:157–70. Mosmann T. Rapid colorimetric assay for cellular growth and survival application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55–63.
ARTICLE IN PRESS A. Albert-Baskar, S. Ignacimuthu / Experimental and Toxicologic Pathology 62 (2010) 423–431
Muller I, Jenner A, Bruchelt G, Niethanmmer D, Halliwell B. Effect of concentration on the cytotoxic mechanism of doxorubicin-apoptosis and oxidative DNA damage. Biochem Biophys Res Commun 1997;230:254–7. Nadkarni KM. Indian materia medica. Bombay, India: Popular Prakashan; 1976. Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra WG. Biochemical roles as a component of glutathione peroxidase. Science 1973;179:588–90. Sengottuvelan M, Senthilkumar R, Nalini N. Modulatory influence of dietary resveratrol during different phases of 1,2-dimethylhydrazine induced mucosal lipid-peroxidation, antioxidant status and aberrant crypt foci development in
431
rat colon carcinogenesis. Biochim Biophys Acta 2006;1760: 1175–83. Sgambato A, Ardito R, Faraglia B, Boninsegna A, Wolf FI, Cittadini A. Resveratrol, a natural phenolic compound, inhibits cell proliferation and prevents oxidative DNA damage. Mutat Res 2001;496:171–80. Sinha AK. Colorimetric assay of catalase. Anal Biochem 1972; 47:389–94. Sun SY, Hail Jr. N, Lotan R. Apoptosis as a novel target for cancer chemoprevention. J Natl Cancer Inst 2004;96: 662–72. Yagi K. Lipid peroxides and human disease. Chem Physiol Lipids 1978;45:337–51.
Experimental and Toxicologic Pathology 62 (2010) 423–431 www.elsevier.de/etp
Chemopreventive effect of Cynodon dactylon (L.) Pers. extract against DMH-induced colon carcinogenesis in experimental animals Arul Albert-Baskar, Savarimuthu Ignacimuthu Division of Ethnopharmacology, Entomology Research Institute, Loyola College, Chennai – 600034, Tamil Nadu, India Received 3 February 2009; accepted 3 June 2009
Abstract The present study was aimed at evaluating the chemopreventive property of Cynodon dactylon. The antioxidant, antiproliferative and apoptotic potentials of the plant were investigated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, nitric oxide radical scavenging activity (NO) and MTT assay on four cancer cell lines (COLO 320 DM, MCH-7, AGS, A549) and a normal cell line (VERO). In vivo chemopreventive property of the plant extract was studied in DMH-induced colon carcinogenesis. The methanolic extract of C. dactylon was found to be antiproliferative and antioxidative at lower concentrations and induced apoptotic cell death in COLO 320 DM cells. Treatment with methanolic extract of C. dactylon increased the levels of antioxidant enzymes and reduced the number of dysplastic crypts in DMH-induced colon of albino rats. The present investigation revealed the anticancer potential of methanolic extract of C. dactylon in COLO 320 DM cells and experimentally induced colon carcinogenesis in rats. r 2009 Elsevier GmbH. All rights reserved. Keywords: Cynodon dactylon; 1,2-Dimethyl hydrazine; COLO 320 DM; DNA fragmentation; Chemopreventive property
Introduction Cancer is the second leading cause of death after cardiovascular diseases in India. Lung, breast, colon and stomach cancers are the four most common cancers worldwide (Bingham and Riboli, 2004). Medicinal plants are frequently used by traditional healers to treat a variety of ailments and symptoms including diabetes and cancer. According to the World Health Organization, over 80% of the world’s populations rely upon such traditional plant-based systems of medicine to provide them with primary healthcare (Calixto, 2005). Corresponding author. Tel.: +91 044 2817 8348; fax: +91 044 2817 4644. E-mail address: [email protected] (S. Ignacimuthu).
0940-2993/$ - see front matter r 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2009.06.003
Lipid peroxidation has gained more importance nowadays because of its involvement in pathogenesis of many diseases like cancer, diabetes and also aging (Halliwell et al., 1992). Free radicals or reactive oxygen species (ROS) are the main culprits in lipid peroxidation and cause destructive and irreversible damage to the components of a cell, such as lipids, proteins and DNA (Cheeseman and Slater, 1993; Lopaczyski and Zeisel, 2001). Although normal cells possess antioxidant defence systems against ROS, continuous accumulation of damage to the cells induces diseases such as cancer and aging (Mates and Sanchez-Jimenez, 2000). The continuous supply of antioxidant dose also plays a preventive role against diseases by removing the ROS in biological systems (Sgambato et al., 2001). Cancer cells, which are already irreversibly developed, obtain the
ARTICLE IN PRESS 424
A. Albert-Baskar, S. Ignacimuthu / Experimental and Toxicologic Pathology 62 (2010) 423–431
capability to escape apoptosis by various ways. The aim of anticancer agents is to trigger the apoptosis signaling system in these cancer cells whilst disturbing their proliferation (Bold et al., 1997). 1,2-Dimethyl hydrazine (DMH) is a colon specific carcinogen known to produce free radicals in circulation in experimental animal models (Arutiuian et al., 1997). Antioxidant enzymes are scavengers of free radicals and are modulated during carcinogenesis or after tumor formation. As a result of malignant state, alterations in the antioxidant enzymes occur; if they are required for the maintenance of the malignant state, then the recovery of decreased enzymes could help to reverse the malignancy (Sun et al., 2004). Cynodon dactylon (L.) Pers, root is used as cure for cancer in Indian Traditional medicine (Nadkarni, 1976).The present study was carried out to determine the anticancer potential of C. dactylon root extracts using in vitro models to minimize the use of experimental animals and the active extract was studied for chemopreventive potential using DMH-induced animal models.
Materials and methods Plant material and preparation of extract
and 25 mg of dried extract/ml solution. The reaction mixture contained 1000 ml of 0.25 mM DPPH with various concentrations of the extracts in 1 ml of ethanol. After 20 min incubation at room temperature the absorbance at l ¼ 517 nm was recorded using a Hitachi U-2000 spectrophotometer (Hitachi, Tokyo, Japan). The inhibition percentage (%) of radical scavenging activity was calculated using the following equation: Inhibition ð%Þ
A0 As 100 A0
where A0 is absorbance of the control and As is absorbance of the sample at 517 nm. From the inhibition (%), the amount of the samples (mg) reducing the absorbance by 50% was determined (IC50).
Nitric oxide scavenging assay In nitric oxide radical inhibition assay, Sodium nitroprusside in aqueous solution at physiological pH, spontaneously generates nitric oxide which interacts with oxygen to produce nitrite ions, which can be estimated by the use of Griess Illosvoy reaction (Garrat, 1964). In the present investigation, Griess Illosvoy reagent was modified using naphthylethylenediamine dihydrochloride (0.1% w/v) in place of 1-naphthylamine (5%). Scavengers of nitric oxide compete with oxygen leading to reduced production of nitric oxide (Marcocci et al., 1994). The IC50 value is the concentration of sample required to inhibit 50% of nitric oxide radical.
The medicinal plant (C. dactylon) was selected based on its use in traditional medicine. Roots of C. dactylon were collected washed with double distilled water twice and allowed to shade dry for about 10 days with free aeration. The plants were authenticated by Dr. Ayyanar (Plant taxonomist) at the Entomology Research Institute, Loyola College, Chennai. Voucher specimen (ERI 109) was preserved at the herbarium of Entomology Research Institute. Shade dried roots were powdered using an electric blender and the powder was used for extraction with three solvents sequentially using cold percolation method. 500 g of the root powder was soaked in hexane for 48 h, filtered and concentrated using a rotary evaporator at reduced pressure; to the residue ethyl acetate was poured and extracted as above. The residue was again used for methanol extraction. The crude extracts were dissolved in DMSO and used as a stock solution and filter sterilized (0.22 mm) before the experiment. The final working concentration of DMSO was less than 0.1%.
All the cell lines were purchased from National Center for Cell Science (NCCS, Pune) and subcultured using appropriate medium. COLO 320 DM (human colon adenocarcinoma cell line) was cultured in RPMI 1640. MCH-7 (human breast cancer cell), AGS (human stomach cancer cell line), A549 (human lung cancer cell line) and a normal cell line VERO (Monkey kidney cells) were cultured in DMEM. Both the media were supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco) and ampotericin B (Gibco). Cells were cultured as monolayers in 25 cm2 plastic tissue culture flasks at 37 1C under a humidified atmosphere of 5% CO2 in air.
DPPH free radical scavenging assay
Antiproliferative studies
The hydrogen-donating ability of solvent extracts was examined using the method of Blois (1958) in the presence of DPPH stable free radical. The extracts and positive control, Vitamin C, were diluted with ethanol to prepare sample solution equivalent to 400, 200, 100, 50
The survival of cells was determined by MTT assay using the method of Mosmann (1983). Briefly, the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide) (Sigma) solution was prepared freshly in phosphate-buffered saline (0.5 mg/ml)
Cell lines and culture medium
ARTICLE IN PRESS A. Albert-Baskar, S. Ignacimuthu / Experimental and Toxicologic Pathology 62 (2010) 423–431
(Gibco) just before use. Cells were seeded in 96 well plates (1 105 cells/well) and allowed for attachment for 6 h. The cells were treated with various concentrations of the extract (3.125, 6.25, 12.5, 25, 50, 100 and 200 mg/ml) for 24 h; then 100 ml of MTT dye was added to each well. The plates were incubated in a CO2 incubator for 4 h and optical density was determined by eluting the dye with DMSO and read using a 96 well plate reader at 560 nm.
DNA fragmentation COLO 320 DM cells were seeded in 24 well plates and incubated at 37 1C in 5% CO2 until 100% confluence was attained. The cells were treated with three different concentrations (1000, 500 and 250 mg/ml) of C. dactylon methanol crude extracts and incubated for 24 h. The cells were harvested and centrifuged at 10000 rpm for 10 min. The cell pellet was suspended in 500 ml of TKM I buffer (10 mM Tris–HCl, 10 mM magnesium chloride, 10 mM potassium chloride, 2 mM EDTA, pH 7.6) vortexed vigorously and centrifuged at 10000 rpm for 10 min. The pellet was again suspended in 500 ml of TKM II (10 mM Tris–HCl, 10 mM Magnesium chloride, 10 mM potassium chloride, 2 mM EDTA, 0.4 M NaCl, pH 7.6). Again 40 ml of 10% SDS and 4 ml of RNAse A (10 mg/ml) were added and incubated at 55 1C for 10 min. Then 125 ml of 5 M NaCl was added, mixed and centrifuged at 10,000 rpm for 5 min. The supernatant was then transferred to a fresh tube and 1 th mixed with two volumes of absolute ethanol and 10 volume 3 M sodium acetate. The mixture was kept at 80 1C for 1 h and centrifuged at 12,000 rpm for 10 min. The pellet was washed in 70% ethanol and dried at 37 1C. The pellet was then suspended in 20 ml of TE buffer (pH 8.0) and electrophoresed in a 2% agarose gel.
Apoptosis COLO 320 DM (1 107) cells were treated with 6.25, 12.5, 25 and 50 mg/ml of extract for 24 h. At the end of the treatment cells were washed with phosphatebuffered saline (PBS) and resuspended in binding buffer (10 mM HEPES/NaOH Ph 7.4, 140 mM NaCl, 2.5 mM CaCl2). Aliquots of cells (100 ml) were incubated with 5 ml of Annexin-V FITC (fluorescein isothiocyanateconjugated), mixed and incubated for 15 min at room temperature in dark and stained with propidium iodide (5 mg/ml). The cells were then gently vortexed and 10,000 events were acquired and analyzed using Becton Dickinson FACS caliber. In brief, early apoptotic cells were defined as those cells exhibiting a fluorescein isothiocyanate-conjugated annexin-V-positive and propidium iodide-negative staining pattern, necrotic cells
425
exhibiting propidium iodide-positive and Annexin-V FITC negative.
DMH-induced colon carcinogenesis Animals and diet Experiments were carried out with 5 weeks old male albino Wistar rats, obtained from Central Animal House, Kings Institute, Chennai, Tamil Nadu. The animals were cared for in compliance with the principles and guidelines of Ethical Committee for Animal Care and institutional animal ethical committee in accordance with the Indian National Law on Animal Care and Use (Reg. No. 833/a/2004/CPCSEA). The animals were housed four per cage in polypropylene cages with a wire mesh top and a hygienic bed of husk in a specific pathogen-free animal room under controlled conditions of 12 h light/h dark cycle, with temperature of 2472 1C and relative humidity of 50710% till the end of the experimental period. The rats were held in quarantine for 1 week and had access to food and tap water ad libitum. Commercial pellet diet containing 4.2% fat (Hindustan Lever Ltd., Mumbai, India) was powdered and mixed with 15.8% peanut oil making a total of 20% fat in the diet (Table 1). This modified powdered pellet diet was fed to rats in all groups throughout the experimental period of 16 weeks. The total caloric intake of the rats in all the groups was adjusted to be the same. Carcinogen administration The experimental animals were divided into six groups. The animals in groups III–VI received subcutaneous injections of DMH at a dose of 20 mg/kg bwt once a week for the first four consecutive weeks. Prior to subcutaneous injection, DMH was dissolved in 1 mM EDTA, the pH adjusted to 6.5 with 1 mM NaOH to ensure the pH and stability of the chemical and was used immediately after preparation. Initial body weights of all animals in this study protocol were ensured to be between 80 and 120 g. The animal weights were recorded once a week throughout the experimental period and prior to sacrifice.
Table 1.
Composition of the diet.
Protein Fat Carbohydrates Fiber Minerals Vitamins
Commercial diet 84.2%
Peanut oil 15.8%
Total %
17.7 4.2 50.5 3.4 6.7 1.7
– 15.8 – – – –
17.7 20.0 50.5 3.4 6.7 1.7
ARTICLE IN PRESS 426
A. Albert-Baskar, S. Ignacimuthu / Experimental and Toxicologic Pathology 62 (2010) 423–431
Experimental design
Group 1
–
Group 2
–
Group 3
–
Group 4
–
Group 5
–
Group 6
–
Rats received modified pellet diet along with intragastric intubation of 0.1% CMC (1.0 ml), throughout the experimental period. Rats received modified pellet diet+100 mg/kg bwt methanolic extract of C. dactylon suspended in 0.1% CMC (1.0 ml), p.o. everyday throughout the experimental period. Rats were administered 20 mg/kg bwt DMH (carcinogen) s.c. once a week for four consecutive weeks and kept without any treatment for the next 12 weeks. The animals were treated as in group 3 along with methanolic extract of C. dactylon (25 mg/kg bwt, p.o.) throughout the entire experimental period of 16 weeks. The animals were treated as in group 3 along with methanolic extract of C. dactylon (50 mg/kg bwt, p.o.) throughout the entire experimental period of 16 weeks. The animals were treated as in group 3 along with methanolic extract of C. dactylon (100 mg/kg bwt, p.o.) throughout the entire experimental period of 16 weeks.
At the end of 16 weeks all the animals were sacrificed under anesthesia (i.p. administration of ketamine hydrochloride (30 mg/kg bwt), by cervical dislocation between 8 am and 10 am after an overnight fast. Blood samples were collected from all the experimental animals and plasma was separated and analyzed for antioxidant enzymes. Tumor and normal colon tissues were fixed in 10% formalin and were stained with hematoxylin and eosin for histopathological investigation. Total lipid peroxidation levels as evidenced by the formation of thiobarbituric acid reactive substances (TBARS) were estimated by the method of Yagi (1978) and reduced glutathione by the method of Ellman (1959). The activities of glutathione peroxidase (GPx) and glutathione S-transferase (GST) were estimated by the method of Rotruck et al. (1973) and Habig et al. (1974) respectively. Superoxide dismutase (SOD) was assayed by the method of Kakkar et al. (1984) and catalase (CAT) was assayed by the method of Sinha (1972). Serum protein was determined by the method of Lowry et al. (1951).
Statistical analysis Antioxidant and cytotoxic activities were performed in triplicate and the values were expressed as Mean7SD ANOVA was used to determine the significance level of the data obtained. All the biochemical analysis were expressed as means7SD. Statistical evaluation was done using one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (DMRT). The statistical significance was set at Po0.05. Analysis was performed using SPSS version 11.0 software (SPSS Inc., Chicago, IL, USA).
Results Several concentrations ranging from 3.125 to 200 mg/ml extracts of C. dactylon were tested for their antioxidant and antiproliferative activity using different in vitro models. The extracts exhibited antioxidant effects in a concentration dependant manner up to a given concentration (Table 2). Among the tested extracts methanolic extract exhibited strong antioxidant activity in the DPPH and the nitric oxide radical inhibition assays as evidenced by the low IC50 values (Table 2) of 63.03 and 98.89 mg/ml, respectively. Results of MTT assay revealed that all the extracts of C. dactylon exhibited antiproliferative effects towards the tested cell lines at various concentrations. Methanol extract showed promising effects with no toxicity to VERO cells in all tested concentrations (Fig. 1). To determine the mechanism of cytotoxicity either due to apoptosis or necrosis, DNA fragmentation and Annexin FITC/PI assays were carried out. The methanolic extract of C. dactylon exhibited fragmentation of DNA (Fig. 2) and proapoptotic activity in COLO 320 DM cells (Fig. 3). The chemopreventive potential of methanolic extract of C. dactylon was further validated in DMH-induced experimental carcinogenesis. Treatment with methanolic
Table 2.
DPPH and NO scavenging property of C. dactylon.
Plant parts and extracts used
IC50 (lg/ml) DPPH
IC50 (lg/ml) NO
Cynodon dactylon (root) Hexane Ethyl acetate Methanol
874.15766.56 311.05723.69 63.0374.82
ND 450.08734.27 111.2278.47
Vitamin C
570.38
2571.90
Each value is mean7SD for 6 replication in each group. ND – Not Detectable.
ARTICLE IN PRESS A. Albert-Baskar, S. Ignacimuthu / Experimental and Toxicologic Pathology 62 (2010) 423–431
120
C. dactylon Hexane C. dactylon Ethyl acetate C. dactylon Methanol
d
100 IC50 (µg/ml)
427
80 c
60
ab
b a
40
b b
d a
20
a
b
a a a
0 VERO
AGS
MCF-7
A549
COLO 320 DM
Fig. 1. Antiproliferative effect of the crude extract of C. dactylon in three different cancer cell lines and a normal cell line. Each value is mean7SD for 6 replications in each group. Values not sharing a common superscript differ significantly at (a–d)Po0.05 (DMRT).
Fig. 2. Agarose gel pattern for the DNA from COLO 320 DM cells. Lane 1–100 mg/ml C. dactylon crude extract; lane 2-untreated cells; lane 3–1000–200 Kb marker.
extract of C. dactylon at three different concentrations (25, 50 and 100 mg/kg bwt) reduced the levels of lipid peroxides and increased the antioxidant enzymes in
circulation. Lipid peroxidation products and antioxidant enzyme levels determined in the plasma of experimental animals are shown in Tables 3 and 4. The levels of DC, LOOH and TBARS were significantly higher in DMH alone treated animals as compared to control rats (Po0.05). Treatment with three different doses of methanolic extract of C. dactylon in DMH treated rats, significantly (Po0.05) decreased the extent of lipid peroxidation when compared to the DMH alone treated rats (group 3). Among the three doses group 5 showed pronounced effect (Po0.01) when compared to the other doses in decreasing the levels of lipid peroxidation. At the end of 16 weeks, in DMH treated rats, the activities of SOD, CAT and GSH-dependent enzymes were significantly (Po0.05) decreased when compared to the control rats (group 1). Supplementation of methanolic extract of C. dactylon showed a significant (Po0.05) increase in the activities of free radical metabolizing enzymes in 100 mg/kg bwt treated animals. Tissue sections of Group 1 animals displayed normal colonic architecture with no signs of apparent abnormality (Fig. 4A). In the carcinogen treated Group 3, well differentiated signs of dysplasia were evident. Nuclei were enlarged; thickening of epithelium was seen; cells were hyper-chromatic and showed increased mitotic activity. Simultaneously, there was a loss in nuclear polarity (Fig. 4C). The tumor section of the colon from DMH treated rats (Fig. 5) also revealed poorly differentiated adenocarcinoma (Fig. 5). In the treatment group (Groups 4–6), histoarchitecture revealed no signs of dysplasia but indicated a little loss of nuclear polarity (Fig. 4D). The size and shape of the cells were uniform and the cells regained the near normal histoarchitecture (Fig. 4F). Occasionally, hyper-chromatic nucleus was evident. There were no signs of dysplasia or toxicity observed in Group 3 rats administered with methanolic extract of C. dactylon alone (Fig. 4B).
ARTICLE IN PRESS 428
A. Albert-Baskar, S. Ignacimuthu / Experimental and Toxicologic Pathology 62 (2010) 423–431
Fig. 3. Induction of apoptosis by methanolic extract of C. dactylon in COLO 320 DM cells. Cells in each quadrant reflect the percentage of cells.
Table 3.
Effect of methanolic extract of C. dactylon on circulatory lipid peroxidation status in rats subjected to DMH treatment.
Groups
TBARS (nmol/ml)
DC (mmol/ml)
LOOH (mmol/ml)
CON CON+SITO DMH DMH+25 mg/kg extract DMH+50 mg/kg extract DMH+100 mg/kg extract
1.8270.14e 1.8970.14e 4.1370.31a 3.2570.25b 2.9770.23c 2.5470.19d*
0.5970.04e 0.6470.05e 1.8870.14a 1.6470.12b 1.0470.08c 0.8270.06d*
0.2970.02e 0.3270.02e 0.8370.06a 0.7270.05b 0.5970.04c 0.5170.04d*
Data are presented as means7SD of 10 rats in each group. (a–e)Po0.05 values not sharing a common superscript letter are significantly different. *Po0.01, values are significantly different as compared to DMH alone treated group.
Table 4. Effect of methanolic extract of C. dactylon on antioxidant defence system in erythrocyte lysate of rats subjected to DMH treatment. Groups
z
CON CON+SITO DMH DMH+25 mg/kg extract DMH+50 mg/kg extract DMH+100 mg/kg extract
3.970.30a 4.070.30a 2.070.15e 2.170.16d 2.670.20c 3.570.27b*
SOD
y
CAT
2.270.17a 2.370.18a 1.170.08e 1.370.10d 1.670.12c 2.070.15b*
z
y
#
56.574.30a 57.074.34a 41.473.15c 44.373.37bc 48.57 3.69b 54.974.18a*
27.272.07a 28.872.19a 15.871.20c 17.471.33c 20.871.58b 26.772.03a*
3.370.25a 3.470.26a 1.570.11e 1.870.14d 2.170.16c 2.870.21b*
GR
GPx
GST
Data are presented as means7SD of 10 rats in each group. (a–e)Po0.05 values not sharing a common superscript letter are significantly different. *Po0.01, values are significantly different as compared to DMH alone treated group. z Enzyme required for 50% inhibition of NBT reduction/min/mg Hb. y m mol of H2O2 utilized/min/mg Hb. z mmol of NADPH oxidized/min/mg Hb. y mmol of GSH utilized/min/mg Hb. # mmol of CDNB-GSH conjugate formed/min/mg Hb.
Discussion Many commercially proven drugs used in modern medicine were initially used in crude form in traditional or folk healing practices, or for other purposes that suggested potentially useful biological activity. Most of the secondary plant compounds employed in modern medicine were first discovered through ethnobotanical
investigation (Gurib-Fakim, 2006). Natural antioxidants have a wide range of biochemical activities, including inhibition of ROS generation, direct or indirect scavenging of free radicals, and alteration of antioxidant potential (Finkel and Holbrook, 2000). Antioxidants have been used to inhibit apoptosis because apoptosis was initially thought to be mediated by oxidative stress (Hockenhery et al., 1993). Many
ARTICLE IN PRESS A. Albert-Baskar, S. Ignacimuthu / Experimental and Toxicologic Pathology 62 (2010) 423–431
429
Fig. 4. Histopathological findings of DMH-induced colon treated with three different concentrations of methanolic extract of C. dactylon. (A) Normal colon, (B) control animals treated with 100 mg/kg bwt methanolic extract of C. dactylon, (C) animals treated with DMH alone, (D) animals treated with DMH and treated with 25 mg/kg bwt methanolic extract of C. dactylon, (E) animals treated with DMH and treated with 50 mg/kg bwt methanolic extract of C. dactylon and (F) animals treated with DMH and treated with 100 mg/kg bwt methanolic extract of C. dactylon.
Fig. 5. Morphological view of DMH-induced colon.
antioxidant substances have anticancer or anticarcinogenic properties (Johnson et al., 1994). Anticancer agents have been isolated from Andrographis paniculata, Phyllanthus amarus, Piper longum, Semecarpus anacardium, Withanica somnifera, Moringa oleifera, Aloe vera, Curcuma longa, Allium sativum and Tinospora cordifolia (Balachandran and Govindarajan, 2005). The methanolic extract of C. dactylon reduced the DPPH radical to the corresponding hydrazine by its
ability to donate hydrogen atom and acted as an antioxidant in a concentration dependant manner. The nitrite produced by the incubation of solutions of sodium nitroprusside in standard phosphate-buffered saline at 25 1C was reduced by the methanolic extract of C. dactylon root extract. This might be due to the antioxidant principles in the extract which competed with oxygen to react with nitric oxide and thus inhibited the generation of nitrite. The antiproliferative potential of the methanolic extract of C. dactylon studied by MTT assay is considered to be diagnostic of mitochondrial malfunction. Damage of this organelle by the extract could be involved in activation of the apoptotic pathway. The available semi synthetic anticancer drugs have more side effects and are cytotoxic to human beings since they are not cancer cell specific. Hence scientists are interested in finding a potent phytotherapeutic agent with noncytotoxic properties. Methanolic extract of C. dactylon exhibited antiproliferative activity towards all the cancer cells (COLO 320 DM, AGS, MCF-7 and A549) with a significant effect towards COLO 320 DM cells and was not toxic to VERO cells indicating it to be safe to normal cells. The mode of cell death caused by the extract had to be determined. COLO 320 DM cells treated with methanolic extract of C. dactylon were
ARTICLE IN PRESS 430
A. Albert-Baskar, S. Ignacimuthu / Experimental and Toxicologic Pathology 62 (2010) 423–431
subjected to DNA fragmentation and Annexin -V-FITC/PI assays, and both data indicated the mode of cell death to be by apoptosis. The ethanolic extract of C. dactylon had been reported to posses’ moderate antioxidant activities and was not cytotoxic towards PC12 cells up to 1 mg/ml (Auddy et al., 2003). Reducing oxidative stress may suppress the proliferation of tumor cells and enhance cancer cell apoptosis (Sun et al., 2004). A variety of cytotoxic/anticancer drugs has been reported to induce apoptosis of malignant cells in vitro (Muller et al., 1997). The chemopreventive potential of the methanolic extract of C. dactylon was further investigated in DMH-induced experimental colon carcinogenic model using albino rats. It was interesting to note that treatment of 100 mg/kg bwt of the extract exhibited significant protective protection to carcinogenesis by increasing the antioxidant enzymes and lowering the histopathological alterations caused by DMH administration. DMH is metabolized to a methyl free radical and generates hydroxyl radical or hydrogen peroxide in the presence of metal ions that may contribute to the initiation of lipid peroxidation (Dudeja and Brasitus, 1990). DC and LOOHs are primary products of lipid peroxidation used to quantify the oxidative damage in membrane lipids. Increased levels or accelerated generation of ROS and toxic degradation products of lipid peroxidation have been reported in circulation of DMHinduced animal models (Sengottuvelan et al., 2006). Removal of free radicals in biological systems is achieved through enzymic and non enzymic antioxidants, which act as principle defence systems against free radicals (Fang et al., 2002). In conclusion, the results of the present work show that the methanolic extract of C. dactylon activated the apoptotic pathway in mammalian colon adenocarcinoma cells and lowered the levels of lipid peroxides in circulation in DMH-induced animals treated with the extract. The ability of the extract to trigger and execute apoptosis in cancer cells is unclear but the MTT assay suggests a mitochondrial involvement. C. dactylon methanolic extract offers protection towards the damage caused by DMH in experimental animals indicating it to be a safe drug for colon cancer.
References Arutiuian AV, Prokopenko VM, Burmistrov SO, Oparina TI, Frolova EV, Zabezhinskii MA, et al. Free-radical processes in blood serum, liver and large bowel during 1,2-dimethylhydrazine-induced carcinogenesis in rats. Vopr Onkol 1997;43:618–22. Auddy B, Ferreira M, Blasina F, Lafon L, Arredondo F, Dajas F, et al. Screening of antioxidant activity of three Indian medicinal plants, traditionally used for the manage-
ment of neurodegenerative diseases. J Ethnopharmacol 2003;84:131–8. Balachandran P, Govindarajan R. Cancer – an ayurvedic perspective. Pharmacological Research 2005;51:19–30. Bingham S, Riboli E. Diet and cancer – the European prospective investigation into cancer and nutrition. Nature Rev Cancer 2004;4:206–15. Blois MS. Antioxidant determinations by the use of a stable free radical. Nature 1958;181:1199–200. Bold RJ, Termuhlen PM, McConkey DJ. Apoptosis, cancer and cancer therapy. Surg Oncol 1997;6:133–42. Calixto JB. Twenty five years of research on medicinal plants in Latin America: a personal review. J Ethnopharmacol 2005;100:131–4. Cheeseman KH, Slater TF. An introduction to free radical biochemistry. Br Med Bull 1993;49:481–93. Dudeja PK, Brasitus TA. 1,2-Dimethylhydrazine-induced alterations in lipid peroxidation in preneoplastic and neoplastic colonic tissues. Biochim Biophys Acta 1990; 1046:267–70. Ellman GL. Tissue sulphydryl groups. Arch Biochem Biophys 1959;82:70–7. Fang YZ, Yang S, Wu G. Free radicals, antioxidant, and nutrition. Nutrition 2002;18:872–9. Finkel T, Holbrook NJ. Oxidants, oxidative stress and the biology of ageing. Nature 2000;408:239–47. Garrat DC. The quantitative analysis of drugs, Vol. 3. Japan: Chapman & Hall; 1964. p. 456–8. Gurib-Fakim A. Review – medicinal plants: traditions of yesterday and drugs of tomorrow. Mol Aspect Med 2006;27:1–93. Habig WH, Pabst MJ, Jakoby WB. Glutathione S-transferase, the first enzymatic step in mercapturic acid formation. J Biol Chem 1974;249:7130–9. Halliwell B, Gutteridge JMC, Cross CE. Free radicals, antioxidants and human disease: where are we now? J Lab Clin Med 1992;119:598–620. Hockenhery DM, Oltavai ZN, Korsmeyer SJ. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 1993;75:241–51. Johnson IT, Williamson G, Musk SR. Anticarcinogenic factors in plant foods: a new class of nutrients. Nutr Rev 1994;7:175–204. Kakkar P, Das B, Viswanathan PN. A modified spectrophotometric assay of superoxide dismutase. Ind J Biochem Biophys 1984;21:130–2. Lopaczyski W, Zeisel SH. Antioxidant, programmed cell death, and cancer. Nutr Res 2001;21:295–307. Lowry OH, Roseborough NJ, Farr AL, Randll AJ. Protein measurement with Folin’s phenol reagent. J Biol Chem 1951;193:265–75. Marcocci P, Sckaki A, Albert GM. Antioxidant action of Ginkgo biloba extracts EGP761. Methods Enzymol 1994; 234:462–75. Mates JM, Sanchez-Jimenez FM. Role of reactive oxygen species in apoptosis: implications for cancer therapy. Int J Biochem Cell Biol 2000;32:157–70. Mosmann T. Rapid colorimetric assay for cellular growth and survival application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55–63.
ARTICLE IN PRESS A. Albert-Baskar, S. Ignacimuthu / Experimental and Toxicologic Pathology 62 (2010) 423–431
Muller I, Jenner A, Bruchelt G, Niethanmmer D, Halliwell B. Effect of concentration on the cytotoxic mechanism of doxorubicin-apoptosis and oxidative DNA damage. Biochem Biophys Res Commun 1997;230:254–7. Nadkarni KM. Indian materia medica. Bombay, India: Popular Prakashan; 1976. Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra WG. Biochemical roles as a component of glutathione peroxidase. Science 1973;179:588–90. Sengottuvelan M, Senthilkumar R, Nalini N. Modulatory influence of dietary resveratrol during different phases of 1,2-dimethylhydrazine induced mucosal lipid-peroxidation, antioxidant status and aberrant crypt foci development in
431
rat colon carcinogenesis. Biochim Biophys Acta 2006;1760: 1175–83. Sgambato A, Ardito R, Faraglia B, Boninsegna A, Wolf FI, Cittadini A. Resveratrol, a natural phenolic compound, inhibits cell proliferation and prevents oxidative DNA damage. Mutat Res 2001;496:171–80. Sinha AK. Colorimetric assay of catalase. Anal Biochem 1972; 47:389–94. Sun SY, Hail Jr. N, Lotan R. Apoptosis as a novel target for cancer chemoprevention. J Natl Cancer Inst 2004;96: 662–72. Yagi K. Lipid peroxides and human disease. Chem Physiol Lipids 1978;45:337–51.