Antitumor efficacy and amelioration of oxidative stress by Trichosanthes dioica root against Ehrlich ascites carcinoma in mice

Abstract

Context: Trichosanthes dioica Roxb. (Cucurbitaceae) is a dioecious climber, traditionally used in India for several medicinal purposes.

Objective: The present study assessed the hydroalcoholic extract of T. dioica root (TDA) for antitumor effect and antioxidant influence against Ehrlich ascites carcinoma (EAC) in Swiss albino mice.

Methods: Twenty four hours after intraperitoneal inoculation of tumor (EAC) cells in mice, TDA was administered at 5 and 10 mg/kg body weight daily for 9 consecutive days. On the 10th day, half of the mice were sacrificed for estimation of tumor proliferation, hematological, and liver antioxidant parameters viz. lipid peroxidation, reduced glutathione (GSH), glutathione S-transferase (GST), superoxide dismutase (SOD) and catalase (CAT); and the rest were kept alive for assessment of increase in life span. The antitumor effect of TDA was assessed by evaluating tumor weight, tumor volume, packed cell volume, viable and non-viable tumor cell counts, median survival time and percentage increase in life span of EAC bearing mice.

Results and discussion: TDA exhibited dose dependent and significant (p < 0.001) decrease in tumor weight, tumor volume, packed cell volume and viable cell count and extended the life span of EAC bearing hosts. Hematological profiles were significantly (p < 0.001) restored near to normal in TDA treated mice as compared to EAC control. TDA treatment significantly (p < 0.001) modulated the aforesaid liver antioxidant parameters as compared to EAC control.

Conclusion: The present study demonstrated that TDA possessed promising antitumor efficacy in mice, plausibly mediated by amelioration of oxidative stress by multiple mechanisms.

Keywords::

• Antioxidant
• lipid peroxidation
• glutathione
• glutathione-S-transferase

Introduction

Cancer can be defined as a rapid, abnormal, uncoordinated proliferation of aberrant cells in any tissue or organ of the body which may mass together to form a growth or tumor, or proliferate throughout the body indicating abnormal growth at other sites. If the process is not arrested, it may progress until it causes the death of the organism (Evans, 2002). Cancer is considered as one of the most fearsome causes of morbidity and mortality in all over the world. Although the disease has often been regarded principally as a problem of the developed world, more than half of all cancers occur in the developing countries (Stewart & Kleihues, 2003). Unfortunately, current available cancer chemotherapeutic agents insidiously affect the host cells especially bone marrow, epithelial tissues, reticulo-endothelial system and gonads (Mascarenhas, 1994). Most of the antineoplastic agents produce serious chronic or delayed toxicities that may be irreversible, particularly in heart, lungs and kidneys, thereby increasing morbidity (Nitha et al., 2005).

Plants have a long history of use in the treatment of cancer. The approach for minimizing unwanted toxicity is to employ newer natural products that may act by different and distinct mechanism(s) and/or precipitate less serious side effects. A number of plant or other natural product extracts have been studied for anticancer activity leading to the development of several clinically useful anticancer agents (da Rocha et al., 2001). Hence, the natural products now have been contemplated of exceptional value in the development of effective anticancer drugs with minimum host cell toxicity.

Trichosanthes dioica Roxb. (Cucurbitaceae), called pointed gourd in English, Potol in Bengali and Patola in Sanskrit, is a dioecious climber found wild throughout the plains of North and North-East India from Punjab to Assam and Tripura states of India. It is also cultivated in India for its fruits, a common culinary vegetable in India. In India, all parts of this plant have been traditionally used for various medicinal purposes. According to Ayurveda, the traditional system of Indian medicine, its root is a drastic purgative. The root has been traditionally used in India as purgative and as tonic, febrifuge, in treatment of jaundice, anasarca and ascites (Kirtikar & Basu, 1935; Anonymous, 1976; Nadkarni, 1976; Sharma et al., 2002). The leaves and tender shoots are also used medicinally and as culinary vegetable in West Bengal and Assam, called as Palta in Bengali.

Previous workers reported different phytochemical and pharmacological studies on T. dioica fruits and seeds in experimental animal models (Sharma & Pant, 1988a,b,c; Sharma et al., 1990; Kabir, 2000; Sultan et al., 2004; Sultan & Swamy, 2005; Sharmila et al., 2007; Ghaisas et al., 2008; Rai et al., 2008a,b). In our earlier studies, we reported anthelmintic effects of leaf and root, antibacterial and antimitotic activities of the root of T. dioica (Bhattacharya et al., 2009, 2010; Bhattacharya & Haldar, 2010a,b). As there are no experimental reports on antitumor activity on T. dioica root (TDA), we found it necessary to evaluate the hydroalcoholic extract of TDA for possible antitumor effect and influence on antioxidant status against Ehrlich ascites carcinoma (EAC) bearing Swiss albino mice.

Materials and methods

Plant material

The mature tuberous roots of T. dioica were collected during December 2008 from Majdia, Nadia district, West Bengal, India. The species was identified by Dr. M. S. Mondal, at the Central National Herbarium, Botanical Survey of India, Howrah, West Bengal, India, and a voucher specimen (SB-02) was deposited at Pharmacognosy Research Laboratory, Bengal School of Technology, Delhi Road, Hooghly 712102, India. Just after collection, the plant material was washed thoroughly with running tap water and shade dried at room temperature (24–26°C) and ground mechanically into a coarse powder.

Drugs and chemicals

Bovine serum albumin and 5-fluorouracil from Sigma Chemical Co., St. Louis, Mo; trichloroacetic acid and 1-dichloro-2,4-dinitrobenezene (CDNB) from Merck Ltd., Mumbai, India; thiobarbituric acid, nitroblue tetrazolium chloride from Loba Chemie, Mumbai, India; 5,5′-dithio bis-2-nitro benzoic acid, phenazonium methosulphate, nicotinamide adenine dinucleotide and reduced glutathione (GSH) from SISCO Research Laboratory, Mumbai, India. All the other reagents used were of analytical reagent grade obtained commercially. Doubled distilled water from all-glass still was employed throughout the study.

Preparation of extract

The powdered plant material (644 g) was macerated at room temperature (24–26°C) with 20% ethanol water (950 mL) for 4 days with occasional shaking, followed by re-maceration with the same solvent for 3 days. The macerates were combined, filtered and evaporated to dryness in vacuo (at 35°C and 0.8 MPa) in a Buchi evaporator, R-114. The dry extract (TDA, yield: 12.15%) was kept in a vacuum desiccator until use. Preliminary phytochemical analysis (Harborne, 1998) revealed the presence of reducing sugars, amino acids, triterpenoids and steroids in TDA. Presence of cucurbitacin aglycones in TDA was ascertained by thin layer chromatography on silica gel pre-coated high-performance thin layer chromatography (HPTLC) plates (Silica gel 60 F₂₅₄ Merck, Germany) detected with vanillin-phosphoric acid reagent (Wagner & Bladt, 1996). TDA was dispersed in distilled water as per required concentrations and sonicated for 10 min immediately prior to administration.

Animals

Adult male Swiss albino mice of about 2 months of age weighing 20 ± 2 g were obtained from Laboratory Animal Centre, Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India. The mice were grouped and housed in polyacrylic cages (38 × 23 × 10 cm) with not more than four animals per cage and maintained under standard laboratory conditions (temperature 25 ± 2°C with dark/light cycle 12/12 h). They were allowed free access to standard dry pellet diet (Hindustan Lever, Kolkata, India) and water ad libitum. The mice were acclimatized to laboratory conditions for 10 days before commencement of the experiment. All procedures described were reviewed and approved by the University Animal Ethical Committee, Jadavpur University (Reg. no. 367001/C/CPCSEA).

Preparation of tumor cells

The transplantable murine tumor cell line namely EAC cells were obtained from Chittaranjan National Cancer Institute (CNCI), Kolkata, India. The EAC cells were maintained in the ascitic form in vivo in Swiss mice by means of serial intraperitoneal transplantation of 2 × 10⁶ cells/mouse after every 10 days. Ascitic fluid was drawn out form EAC bearing mouse 8 days after transplantation. The freshly drawn fluid was diluted with ice-cold sterile isotonic saline and the tumor cell count was adjusted to 2 × 10⁷ cells/mL by sterile isotonic saline.

Acute toxicity study

The acute oral toxicity of TDA in male Swiss albino mice was studied as per OECD guideline 425 (Anon., 2008). The median lethal dose, i.e., LD₅₀ value of TDA was determined using the method of maximum likelihood.

Effect on normal peritoneal cells

The naive mice were divided into three groups (n = 6). The first group received TDA at the dose of 10 mg/kg body weight, p.o., once for a single day and the second group received the same treatment for two consecutive days. The untreated third group served as control. Peritoneal exudate cells were collected after 24-h treatment by repeated intraperitoneal wash with isotonic saline and counted by a hemocytometer in each of the treated groups and compared with those of the untreated group (Sur & Ganguly, 1994).

Experimental design

The animals were divided into five groups (n = 12). Except the first group, all groups received 0.1 ml of EAC cell suspension (2 × 10⁶ cells/mouse, i.p.). This was taken as day ‘0’. The first group served as normal saline control (received isotonic saline, 3 mL/kg body weight, i.p.). The second group served as EAC control. After 24 h of tumor inoculation the third and fourth group received TDA at the doses of 5 and 10 mg/kg body weight, p.o., respectively, and the fifth group received the reference drug 5-fluorouracil (20 mg/kg body weight, p.o.) for 9 consecutive days. 24 h after the last dose and after 18 h of fasting, blood was collected from six mice of each group, by cardiac puncture for the estimation of hematological parameters and then sacrificed by cervical dislocation for the study of antitumor and liver antioxidant parameters. The rest six mice of each group were kept alive with food and water ad libitum to assess the increase in the life span of the tumor bearing hosts. The effect of TDA on tumor proliferation and host’s survival time was assessed by observation of tumor volume, tumor weight, packed cell volume, viable and non-viable cell count, median survival time (MST) and percentage increase in lifespan (% ILS) (Haldar et al., 2010).

Body weight

The body weight of mice of each group was measured just before and 9 days after TDA treatment.

Tumor volume and packed cell volume

The mice were dissected and the ascitic fluid was collected form the peritoneal cavity. The volume of collected ascitic fluid was measured by taking it in a graduated centrifuge tube and the packed cell volume was determined by centrifuging at 1000 g for 5 min.

Tumor weight

The tumor weight was measured by weighing the mice before and after the collection of the ascetic fluid from the peritoneal cavity.

Tumor cell count

The ascitic fluid was taken in a white blood cell (WBC) pipette and diluted 100 times. Then one drop of the diluted suspension was placed on the Neubauer counting chamber and the numbers of cells in the 64 small squares were counted.

Viable and non-viable tumor cell count

The cells were then stained with trypan blue (0.4 % in isotonic saline) dye (Trypan blue dye exclusion assay). The cells that did not take up the dye were viable and those took the dye were non-viable. These viable and non-viable cells were counted.

Cell count = (No. of cells × dilution factor)/(area × thickness of liquid film).

MST and % ILS

The animals were observed for their mortality daily until their death or up to a maximum of 45 days. The mortality was monitored by recording MST and % ILS and as per the following formulae:

MST* = [First death + Last death]/2

*Time denoted by number of days.

% ILS = [(MST of TDA treated group / MST of EAC control group) − 1)] × 100

Determination of hematological parameters

Collected blood was used for the estimation of hemoglobin (Hb) content; red blood cell (RBC) count and WBC count (D’Armour et al., 1965; Wintrobe et al., 1961). Differential count of WBCs was carried out from Leishmen stained blood smears (Dacie & Lewis, 1958).

Determination of liver antioxidant parameters

Lipid peroxidation (TBARS)

The levels of thiobarbituric acid reactive substances (TBARS) in the liver tissue were measured as per reported method (Ohkawa et al., 1979). The levels of lipid peroxides (TBARS) were expressed as µmoles of malondialdehyde (MDA)/g of liver tissue.

Reduced glutathione and glutathione-S-transferase

The GSH level of liver tissue was determined as per reported method (Ellman, 1959) and expressed as µg/g of liver tissue. The enzymatic activity of glutathione S-transferase (GST) was measured by the reported method (Habig et al., 1974) and expressed as nanomole of CDNB-GSH conjugate formed/min/mg protein. Total protein of liver was estimated for this purpose as per reported method (Lowry et al., 1951).

Superoxide dismutase and catalase

The activity of superoxide dismutase (SOD) in liver tissue was assayed as per reported method and the SOD activity was expressed as unit of SOD/mg of liver tissue (Kakkar et al., 1984). Catalase (CAT) activity was assayed according to the reported method (Aebi, 1974). The specific activity of CAT was expressed in terms of µmol of hydrogen peroxide (H₂O₂) decomposed/min/mg of liver tissue.

Statistical analysis

All data are presented as the mean ± standard error of mean. The results were analyzed for statistical significance by one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test of significance. P values <0.05 (p ≤ 0.05) were considered as statistically significant.

Results

Acute toxicity

The oral LD₅₀ value of the hydroalcoholic extract of TDA in mice was 2800 mg/kg body weight.

Normal peritoneal cell count

The average peritoneal exudate cell count in naïve mice was found to be 5.41 ± 0.8 × 10⁶. Single treatment with TDA enhanced peritoneal cells to 8.73 ± 1.3 × 10⁶, while two consecutive treatments enhanced the count to 9.67 ± 0.8 × 10⁶. Both the enhancements were found to be significant (p < 0.001).

Body weight

The body weight of mice from EAC control group (after 9 days) was significantly (p < 0.001) increased when compared with normal control group. TDA significantly (p < 0.05) maintained the body weight toward normal in a dose related way as compared to EAC control animals (Table 1).

Table 1.  Influence of TDA on body weight of normal and EAC bearing mice.

Treatment	Initial body weight (g)	Final body weight (g)
Normal saline (3 mL/kg)	21.12 ± 0.78	21.17 ± 1.20
EAC control (2 × 10^6 cells/mouse)	21.27 ± 0.63	26.24 ± 0.74^§a
EAC + TDA (5 mg/kg)	20.65 ± 1.16	24.85 ± 0.58*^b
EAC + TDA (10 mg/kg)	21.54 ± 0.86	22.08 ± 1.12*^b
EAC + 5-FU (20 mg/kg)	20.89 ± 1.02	21.21 ± 0.91*^a

Data are expressed as mean ± SEM (n = 6); ^§final body weight of EAC control group vs. normal control group; *final body weight of all treated groups vs. EAC control group, ^ap < 0.001, ^bp < 0.05.

5-FU, 5-Fluorouracil.

Tumor proliferation and survival parameters

TDA at 5 and 10 mg/kg body weight reduced the tumor volume, tumor weight, packed cell volume and viable tumor cell count, significantly (p < 0.001) at a dose-dependent manner as compared to EAC control. Furthermore, TDA increased non-viable tumor cell counts and decreased viable tumor cell counts significantly (p < 0.001) as compared with the EAC control. In EAC control group, the MST was 19.42 ± 1.58 days, whereas in TDA treated groups these were 27.93 ± 1.35 (5 mg/kg) and 38.68 ± 1.12 (10 mg/kg) days, respectively. The reference drug 5-fluorouracil (20 mg/kg) showed MST 42.86 ± 1.64 days (Table 2).

Table 2.  Influence of TDA on tumor volume, tumor weight, packed cell volume, cell counts, median survival time (MST) and life span (% ILS) in EAC bearing mice.

Treatment	Tumor volume (mL)	Tumor weight (g)	Packed cell volume (mL)	Viable cell count (×10^6 cells /mL)	Non-viable cell count (×10^6 cells /mL)	MST (days)	Increase in life span (% ILS)
Normal saline (3 mL/kg)	—	—	—	—	—	All animals alive	All animals alive
EAC control (2 × 10^6 cells/mouse)	4.08 ± 0.35	4.63 ± 0.87	2.25 ± 0.18	8.03 ± 0.13	0.57 ± 0.04	19.42 ± 1.58	—
EAC + TDA (5 mg/kg)	2.35 ± 0.07*^a	2.56 ± 0.05*^a	0.77 ± 0.06*^a	3.11 ± 0.11*^a	1.15 ± 0.09*^a	27.93 ± 1.35*^a	43.82
EAC + TDA (10 mg/kg)	1.28 ± 0.27*^a	1.41 ± 0.26*^a	0.18 ± 0.21*^a	1.22 ± 1.07*^a	2.90 ± 0.03*^a	38.68 ± 1.12*^a	99.18*^a
EAC + 5-FU (20 mg/kg)	0.31 ± 0.06*^a	0.35 ± 0.13*^a	—	0.76 ± 0.07*^a	3.34 ± 0.05*^a	42.86 ± 1.64*^a	120.70*^a

Data are expressed as mean ± SEM (n = 6).

*all treated groups vs. EAC control group, ^ap < 0.001.

5-FU, 5-Fluorouracil.

Hematological parameters

Hematological parameters of tumor bearing mice were found to be significantly altered compared to those of normal saline control group. The leukocyte (WBC) count was found to be increased and RBC and Hb decreased in EAC control animals significantly (p < 0.001) when compared with the normal control group. Treatment with TDA at both test doses significantly (p < 0.001) increased the Hb content and RBC count toward the normal levels and brought down WBC count toward the normal counts when compared to EAC control group animals. In differential count of leucocytes, lymphocytes and monocytes were found to be decreased and the neutrophils were increased in EAC control group when compared with normal saline treated group. TDA treatment significantly (p < 0.001) brought them toward the normal counts (Table 3).

Table 3.  Influence of TDA on hematological parameters of normal and EAC bearing mice.

Treatment	Hb content (g/dL)	RBC (cells ×10^6/mm^3)	WBC (cells ×10^3/mm^3)	Differential count
				Monocytes (%)	Neutrophils (%)	Lymphocytes (%)
Normal saline (3 mL/kg)	12.33 ± 0.15	5.34 ± 0.31	3.86 ± 0.26	1.91 ± 0.06	20.78 ± 1.16	72.46 ± 1.88
EAC control (2 × 10^6 cells/mouse)	5.80 ± 0.46^§a	2.54 ± 0.17^§a	5.76 ± 0.72^§a	1.24 ± 0.05^§a	68.17 ± 0.98^§a	30.28 ± 1.57^§a
EAC + TDA (5 mg/kg)	8.37 ± 0.18*^a	3.26 ± 0.42*^b	4.58 ± 0.63*^b	1.52 ± 0.04*^b	37.72 ± 1.27*^a	57.27 ± 2.08*^a
EAC + TDA (10 mg/kg)	10.46 ± 0.23*^a	4.68 ± 0.15*^a	3.71 ± 0.12*^a	1.85 ± 0.06*^a	25.46 ± 1.15*^a	68.29 ± 1.96*^a
EAC + 5-FU (20 mg/kg)	11.85 ± 0.36*^a	5.22 ± 0.29*^a	3.82 ± 0.23*^a	1.88 ± 0.07*^a	22.20 ± 1.58*^a	70.75 ± 1.32*^a

Data are expressed as mean ± SEM (n = 6).

^§EAC control group vs. normal control group; *all treated groups vs. EAC control group, ^ap < 0.001, ^bp < 0.05.

5-FU, 5-Fluorouracil.

Liver antioxidant parameters

Lipid peroxidation (TBARS)

The levels of TBARS represented as MDA were significantly (p < 0.001) increased in EAC control animals when compared to normal control group. Treatment with TDA dose-dependently and significantly (p < 0.001) reduced the MDA level when compared with EAC control animals (Table 4).

Table 4.  Influence of TDA on liver antioxidant parameters of normal and EAC bearing mice.

Treatment	MDA (µM/g of wet liver tissue)	GSH (µg/g of wet liver tissue)	GST (nM CDNB-GSH conjugate/min/mg of protein)	SOD (IU/mg of wet liver tissue)	CAT (µM of H2O2 decomposed/min/mg of wet liver tissue)
Normal saline (3 mL/kg)	167 ± 0.02	29.2 ± 0.23	16.4 ± 0.13	8.7 ± 0.28	38 ± 0.14
EAC control (2 × 10^6 cells/mouse)	494 ± 0.03^§a	8.2 ± 0.46^§a	7.3 ± 0.09^§a	3.4 ± 0.63^§a	8 ± 0.25^§a
EAC + TDA (5 mg/kg)	249 ± 0.06*^a	17.3 ± 0.19*^a	10.4 ± 0.21*^b	4.8 ± 0.44*^b	13 ± 0.17*^b
EAC + TDA (10 mg/kg)	182 ± 0.03*^a	25.8 ± 0.33*^a	15.2 ± 0.17*^a	6.6 ± 0.34*^a	27 ± 0.30*^a
EAC + 5-FU (20 mg/kg)	213 ± 0.01*^a	27.9 ± 0.12*^a	16.2 ± 0.10*^a	7.3 ± 0.59*^a	33 ± 0.15*^a

Data are expressed as mean ± SEM (n = 6).

^§EAC control group vs. normal control group; *all treated groups vs. EAC control group, ^ap < 0.001, ^bp < 0.05.

5-FU, 5-Fluorouracil.

Reduced GSH and GST

In the EAC control group, GSH content and GST activity were found to be significantly (p < 0.001) lowered from those of normal control animals. The level of GSH and GST activity were found to be significantly (p < 0.001) elevated toward normal by treatment with TDA as compared with EAC control group (Table 4).

SOD and CAT

There were significant (p < 0.001) reduction in SOD and CAT activities in EAC control group compared with normal saline group. Treatment with TDA significantly (p < 0.001) recovered their activities near to normal values when compared with EAC control animals (Table 4).

Discussion

The present work was aimed to study the antitumor activity of hydroalcoholic extract of TDA in EAC bearing mice. The results of this study revealed that TDA at the doses of 5 and 10 mg/kg demonstrated marked antitumor effect as evidenced by significant reduction in tumor volume, tumor weight, packed cell volume, viable tumor cell count and increase in normal peritoneal cell count, non-viable tumor cell count, MST and life span of tumor bearing hosts. TDA significantly restored the altered hematological parameters toward normal values. The TDA also significantly recuperated the hepatic antioxidant parameters, viz., lipid peroxidation, reduced GSH level, activities of GST, SOD and CAT in tumor bearing mice.

The EAC is a transplantable, poorly differentiated malignant tumor which appeared originally as a spontaneous murine mammary adenocarcinoma. It grows in both solid and ascitic forms. It is a rapidly growing carcinoma with very aggressive behavior and is able to grow in almost all strains of mice. In ascitic form, it has been used as a transplantable murine tumor model to investigate the antitumor effects of several natural and synthetic chemical substances (Chen & Watkins, 1970; Segura et al., 2000).

The effect of TDA treatment on the peritoneal exudates cell count of naïve mice is an indirect method of evaluating its inhibitory effect on tumor cell growth. Normally a mouse contains about 5 × 10⁶ peritoneal cells, 50% of which are macrophages (Sur & Ganguly, 1994). TDA treatment was found to enhance the peritoneal cell count. These results demonstrated the indirect inhibitory effect of TDA on EAC cells, which is probably mediated by the enhancement and activation of either macrophage or cytokine systems.

Intraperitoneal inoculation of EAC cells resulted in appearance of the ascitic fluid. The ascitic fluid is essential for tumor growth since it constitutes the direct nutritional source for tumor cells (Gupta et al., 2004a). So, a rapid increase in ascitic fluid would be a means to meet the nutritional requirements of growing tumor cells. This hypothesis was evident in the present study, since inoculation of EAC cells into mice caused significant increase in the body weight of EAC bearing mice. Such increase was due to accumulated ascitic fluid volume in the peritoneal cavity. TDA treatment dose dependently maintained the body weight near to normal in EAC treated mice.

Cancer is a pathological state involving uncontrolled proliferation of tumor cells. The increase in ascitic volume was accompanied by an increase in total cell count. This is associated with an increase in peritoneal vascular permeability (Fastaia & Dumont, 1976). Treatment with TDA reduced intraperitoneal tumor burden, thereby reducing the tumor volume, tumor weight, packed cell volume and viable tumor cell count. The results of Trypan blue dye exclusion assay demonstrated that the viable cell count decreased with increased count of non-viable cells by TDA treatment. This implied that the antitumor action of TDA had a direct inhibitory relationship with the proliferation of tumor cells indicating loss of viability i.e. death of the TDA treated cells. These results could indicate either a direct cytotoxic effect of TDA on tumor cells or an indirect effect, which may involve macrophage activation and vascular permeability inhibition.

The reliable criteria for judging the value of any anticancer agent are the prolongation of life span of the tumor bearing host and decrease in leukocyte count of blood (Clarkson & Burchneal, 1965). It has been reported that ≥25 % increase in life span of EAC bearing animals is considered to be indicative of significant antitumor activity (Andreani et al., 1983). It can therefore be inferred that, TDA significantly enhanced the life span of EAC bearing mice which may be due to the prevention of tumor progression indicating its antitumorigenic potential.

Usually in cancer chemotherapy, the major problems that are being encountered are of myelosuppression and anemia (Price & Greenfield, 1958; Hogland, 1982). The anemia encountered in tumor bearing mice is mainly due to reduction in erythrocytes or Hb content, and this may occur either due to iron deficiency or due to hemolytic or myelopathic conditions (Gupta et al., 2004b). Results of present study indicated that TDA dose dependently and significantly raised the erythrocyte count and hemoglobin content when compared to those of EAC control mice. The WBC count was significantly reduced as compared with that of EAC control mice. These indicating parameters revealed that TDA exerted less toxic effect to the hemopoietic system and plausibly possessed selective cytotoxicity toward EAC and thereby it could maintain the normal hematological profile.

The redox state of the cell is known to regulate its growth behavior (Pahl & Baeuerle, 1994). The relationship between the endogenous antioxidant systems and growth of malignant cells is a feature observed in several studies. Neoplastic growth has been found to co-exist with an impairment in the endogenous antioxidant status (Oberley, 2002). Low activity of endogenous antioxidant system was found in cancer patients (Balasubramaniyan et al., 1994; Casado et al., 1995) and in experimental carcinoma cell lines (Yellin et al., 1994; Sharma et al., 1993). Several evidences suggest that EAC induces oxidative stress in mice (Baumgartner et al., 1978; Gupta et al., 2004a,b) Oxidative stress is caused by a relative overproduction of oxidative free radicals or reactive oxygen species (ROS). ROS results in lipid peroxidation and subsequently increases MDA and other TBARS levels which lead to degradation of cellular macromolecules (Yoshikawa et al., 1983). A marked increase in the concentration of TBARS in EAC control mice indicated enhanced lipid peroxidation leading to tissue injury and failure of the endogenous antioxidant defense mechanisms to prevent overproduction of ROS. MDA, the end product of lipid peroxidation, a biomarker of oxidative stress, was reported to be higher in carcinomatous tissue than in non-diseased organ (Yagi, 1987; Neilson et al., 1997). The present study revealed that TBARS levels measured as MDA in the EAC control liver tissues were higher than those in normal saline treated liver tissues. Treatment with TDA inhibited hepatic lipid peroxidation as revealed by reduction of MDA levels toward normal. This implied the inhibition in free radical (ROS) generation by TDA in tumor bearing mice. Decrease in lipid peroxidation by TDA in this study implies that its antitumor action may be mediated by preventing or reducing the genetic damage caused by oxidative free radicals.

GSH, the most abundant tripeptide thiol, exists as GSH (reduced form) and GSSG (oxidized form) in cells and participates in diverse biological processes, including detoxification of xenobiotics. GSH, a potent inhibitor of the neoplastic process, plays an important role in the endogenous non-enzymatic antioxidant system. It is in reduced form (GSH) found in particularly high concentration in the liver and is known to have a key function in the protective process. Primarily it acts as reducing agent and detoxifies H₂O₂ in presence of an enzyme GSH peroxidase (Arias & Jakoby, 1976; Meister & Anderson, 1983). Besides its involvement in the detoxification process, GSH probably also plays an important role in lymphocyte function and depletion in GSH content were found to be associated with impaired immune response and increased risk of malignancy (Gmünder & Dröge, 1991). Lowered GSH content was found in human cancer cell lines (Yellin et al., 1994; Sharma et al., 1993). The depleted reduced GSH may be due to reduction in GSH synthesis or degradation of GSH by oxidative stress in EAC bearing animals. TDA treatment significantly elevated the reduced hepatic GSH levels toward normal in tumor bearing mice. The results exhibited that, the antitumor activity of TDA was accompanied with the enhancement of cellular non-enzymatic antioxidant defense by which TDA may have exerted its antitumor role.

GSTs are a family of multigene and multifunctional dimeric enzymes that catalyze the nucleophilic (conjugation) attack of the thiol moiety of the GSH on the electrophilic centre of various carcinogens, mutagens and other xenobiotic compounds (Devi et al., 2002). GSTs play an important role in initiating detoxification by catalyzing the conjugation of GSH to the eletrophilic foreign compounds for their elimination from the system (Mulder et al., 1995). Lower GST activity in human cancer tissues was reported (Yellin et al., 1994; Coursin et al., 1996). Our present study revealed decrease in GST activity in EAC control mice. TDA treatment significantly enhanced the diminished hepatic GST activity in EAC bearing mice. Augmented by TDA, GSH and GSTs plausibly played a critical role in prevention of tumor progression in EAC bearing mice.

The enzymatic antioxidant mechanisms are involved in the protection of tissues from oxidative stress by playing an important role in the elimination of free radicals (ROS). SOD and CAT are involved in the clearance of superoxide and H₂O₂ radicals (Oberley, 2002). It has been established that SOD activity is inhibited in cancer (Jiau-Jian & Larry, 1977; Oberley & Oberley, 1997). The lowering of both SOD and CAT activities as a result of tumor growth was reported (Casado et al., 1995; Marklund et al., 1982). The decrease in hepatic SOD and CAT activities was also reported in EAC bearing mice (Gupta et al., 2004b), and the similar findings were observed in our present investigation in EAC control mice. The administration of TDA at both doses significantly improved the SOD and CAT activities toward normal in a dose-dependent manner. Elevation of enzyme activities like GSTs, SOD and CAT, in TDA treated tumor bearing mice revealed the activation of enzymatic antioxidant defense mechanisms by which TDA resulted in amelioration of EAC-induced oxidative stress.

The presence of triterpenoids and cucurbitacin aglycones was affirmed in TDA by qualitative phytochemical analysis and HPTLC. Cucurbitacins are known to possess several biological activities including anticancer property (Miro, 2006). The presence of putative cucurbitacin aglycones could provide the chemical basis of its antitumor efficacy in mice. Recently, the authors have reported antimitotic potential of T. dioica root with an Allium test (Bhattacharya & Haldar, 2010b). This notable property may be responsible for its antitumor activity against tumor cell line in vivo.

In the present study, it was found that TDA treatment significantly reduced tumor proliferation, viability of tumor cells, normalized the hematological profiles, extending the survival time (lifespan) as compared with those of EAC control mice. Also TDA treatment resulted in significant amelioration of tumor-induced oxidative stress by more than one mechanisms involving modulation of lipid peroxidation, endogenous non-enzymatic (GSH) and enzymatic (GST, SOD, CAT) antioxidant and detoxification systems. Therefore, it can be concluded that the hydroalcoholic extract of TDA demonstrated remarkable antitumor efficacy against EAC in Swiss mice, mediated plausibly by virtue of its ameliorating oxidative stress by augmenting endogenous antioxidant defense mechanisms.

Acknowledgement

The authors are thankful to the authority of Jadavpur University, Kolkata 700032, India, for providing necessary facilities for the present study.

Declaration of interest

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.