Enicostemma littorale prevents tumor formation in 7,12-dimethylbenz(a)anthracene-induced hamster buccal pouch carcinogenesis

Abstract

Oral cancer is one of the most common malignancies worldwide, and India has recorded the highest annual incidence of oral cancer in comparison with other countries. Altered lipid peroxidation and antioxidant status along with defect in detoxification cascade have been implicated in the pathogenesis of several cancers including oral cancer. The aim of this study was to investigate the chemopreventive potential of ethanolic extract of Enicostemma littorale leaves (ElELet) in 7,12-dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouch carcinogenesis. Oral tumor was developed in the buccal pouches of male golden Syrian hamsters by painting with 0.5% DMBA three times a week for 14 weeks. We observed 100% tumor formation with increase in tumor volume and tumor burden in the hamsters treated with DMBA alone. Imbalance in phase I (cytochrome P450 and cytochrome b₅) and phase II (glutathione reductase, glutathione-S-transferase, glutathione, and Deoxythymidine-diaphorase (DT)-diaphorase) detoxification agents and lipid peroxidation by-products (thiobarbituric acid reactive substances) and antioxidant (superoxide dismutase, catalase, glutathione peroxidase, and vitamins E and C) status was noticed in hamsters treated with DMBA alone. Oral administration of ElELet at a dose of 250 mg/kg body weight to hamsters treated with DMBA significantly prevented both precancerous and cancerous lesions in the oral cavity. ElELet modulated the status of phase I and II detoxification agents and antioxidants in favor of the suppression of oral carcinogenesis. This study thus suggests that E. littorale might have inhibited the oral carcinogenesis in DMBA-treated hamsters through its antioxidant potential. The present findings are also substantiated by histological studies during DMBA-induced oral carcinogenesis.

Keywords

Oral cancer DMBA detoxification enzymes lipid peroxidation antioxidants

Introduction

Oral cancer is one of the most life-threatening cancers worldwide and accounts for one-third of all malignancies in developing countries, especially in India, Sri Lanka, Pakistan, and Bangladesh. Recent epidemiological studies reported that oral cancer accounts for 40–50% of all cancers in India.¹ Oral cancer imposes significant health burden in Indian populations and is responsible for poor life quality of the patients. Tobacco and alcohol use are regarded as major risk factors of oral cancer in these regions. Despite medical advances in the treatment of oral cancer, late diagnosis and advanced tumor stages account for poor survival rate of oral cancer.² Oral cancer carries significant morbidity and mortality worldwide, and early diagnosis of this cancer would therefore improve the life quality and survival outcome of the patients.

Oral cancer proceeds through sequential pathological alterations, including leukoplakia, erythroplakia, and oral submucous fibrosis as well as chronic inflammation.³ 7,12-Dimethylbenz(a)anthracene (DMBA), the most preferred site and organ-specific procarcinogen, is widely employed to develop and induce oral tumors in the buccal pouch of golden Syrian hamsters. DMBA, on metabolic activation, is converted into its active carcinogenic metabolite, dihydrodiol epoxide, which mediates carcinogenesis through chronic inflammation, DNA damage, and overproduction of reactive oxygen species (ROS). DMBA-induced histopathological, biochemical, and molecular alterations closely resemble human oral carcinogenesis.^4,5

Oxidative stress has been implicated in the pathogenesis of several cancers, including oral cancer.⁶ Overproduction of ROS has been well documented in betel quid and tobacco chewers.⁷ It has been implicated in several disorders including cancer.

Chemoprevention is an innovative, feasible, and interesting approach to investigate the anticancer potential of medicinal plants and their active constituents. Extensive animal experimental studies and human phase I and phase II chemoprevention programs clearly pointed out that chemopreventive substances suppress or inhibit tumorigenesis through their antioxidant, anti-inflammatory, apoptotic, anticell proliferative, and antiangiogenic potential.^8
–10 Currently, there has been an increasing interest globally to identify anticancer compounds that are pharmacologically potent and have low or no side effects to use in the preventive medicine and in the food industry. Traditional herbal medicines form an important part of the health-care system in India. Ayurveda, supposed to be the oldest medical system in the world, provides potential leads to find active and therapeutically useful compounds from plants. Many naturally occurring substances were tested for anticancer activity in experimental animals, resulting in the present availability of some 30 effective anticancer drugs.¹¹

Enicostemma littorale, a perennial herb, is commonly known as Mamajjakah in Sanskrit, Vellarugu in Tamil, and Indian gentian in English. It belongs to Gentianaceae family and is pungent and very bitter in taste. It is found throughout India and traditionally used in the treatment of malaria, diabetes, rheumatism, dropsy, swellings, itches, filariasis, and insect poisoning. The plant is locally (Tamil Nadu, India) used for inflammation, ulcer, and diabetes.¹² Preliminary phytochemical studies have revealed the presence of flavonoids, alkaloids, anthroquinones, tannins, sterols, swertiamarin, and glycosides.¹³ Reports also showed the presence of several minerals such as calcium, potassium, and iron.¹⁴ E. littorale is rich in several amino acids, such as alanine, serine, tryptophan, glutamic acid, phenylalanine, and ionone.¹⁵ Studies have reported its hypoglycemic, anti-inflammatory, antidiabetic, and hepatoprotective properties.^16
–18 It is also reported to have antitumor, antiarthritic, hypoglycemic, and antioxidant activities.¹⁹ E. littorale significantly improved the antioxidant defense mechanism in the diabetic rats.²⁰ Gopal et al.²¹ reported that E. littorale significantly reduced the lipid levels in rats bearing hepatoma. The anticancer activity of methanolic extract of the plant has been evaluated against Dalton’s ascitic lymphoma in Swiss albino mice.²² There were however no scientific studies on the chemopreventive potential of E.littorale leaves (ElELet) in DMBA-induced hamster buccal pouch carcinogenesis. The aim of this study is therefore to explore the chemopreventive potential of E. littorale by monitoring tumor incidence at regular intervals as well as by analyzing sequential histopathological alterations during DMBA-induced oral carcinogenesis.

Materials and methods

Chemicals

The carcinogen, 7,12-dimethylbenz(a)anthracene, was obtained from Sigma—Aldrich Chemical Pvt. Ltd (Banglore, Karnataka, India). All other chemicals used were of analytical grade and were purchased from Hi Media Laboratories (Mumbai, Maharashtra, India).

Animals

Forty male golden Syrian hamsters, 8 weeks old, weighing 80–120 g, were obtained from National Institute of Nutrition, Hyderabad, Telangana, India, and maintained in the Central Animal House, Rajah Muthiah Medical College and Hospital, Annamalai University, Annamalainagar, Tamil Nadu, India. The animals were housed in polypropylene cages and provided standard pellet diet and water ad libitum. The animals were maintained under controlled conditions of temperature and humidity with a 12-h light/12-h dark cycle.

Plant material

ElELet were collected from Cuddalore District of Tamil Nadu, India. The taxonomic identification of the plant was compared with the existing herbarium in the Department of Botany, Annamalai University.

Preparation of the plant extract

Five hundred grams of dried, finely powdered ElELet were soaked in 1500 ml of 95% ethanol overnight. The residue obtained after filtration was again resuspended in equal volume of 95% ethanol for 48 h and filtered again. The above two filtrates were mixed, and the solvents were evaporated in a rotavapor at 40–50% under reduced pressure. A semisolid material (9%) obtained was stored at 4° C until use. A known volume of the residual extract (250 mg/kg body weight (b.w.)) was suspended in distilled water and orally administered to the animals by gastric intubation using force feeding tube during the experimental period.

Dose-dependent study

The dose of the extract (250 mg/kg b.w.) was selected based on the dose-dependent study. E. littorale at a dose of 250 mg/kg b.w. has shown potent chemopreventive efficacy in hamsters treated with DMBA as compared to the rest of the doses (150 and 200 mg/kg b.w.). We have thus chosen the dose of 250 mg/kg b.w. for this study.

Experimental design

The institutional animal ethics committee (reg no: 160/1999/CPCSEA), Annamalai University, Annamalainagar, Tamil Nadu, India, approved the experimental design (proposaln: 889 dated May 29, 2012). A total of 40 hamsters were divided into 4 groups with 10 animals in each group. The group I hamsters served as control and were painted with liquid paraffin alone three times a week for 14 weeks on their left buccal pouches. Groups II and III hamsters were painted with 0.5% DMBA in liquid paraffin three times a week for 14 weeks on their left buccal pouches. Group II hamsters received no other treatment. Group III hamsters were orally administered with ethanolic extract of ElELet at a dose of 250 mg/kg b.w. on alternative days of DMBA painting. Group IV hamsters received oral administration of ElELet alone throughout the experimental period. The experiment was terminated at the end of 16th week. and all the animals were killed by cervical dislocation. Biochemical studies were conducted in the liver and buccal mucosa of control and experimental animals in each group. For histopathological studies, buccal mucosa tissues were fixed in 10% formalin and routinely processed and embedded with paraffin, 2–3 µm sections were cut in rotary microtome, and stained with hematoxylin and eosin.

Biochemical estimations

Determination of phase I and phase II detoxification agents

Liver and buccal mucosa tissues from hamsters were washed with ice-cold saline and homogenized with Teflon pestle and then used for biochemical estimations. The levels of cytochrome P450 and cytochrome b₅ in liver and buccal mucosa were determined according to the method described by Omura and Sato.²³ The activity of glutathione-S-transferase (GST) in the liver and the buccal mucosa was assayed by the method followed by Habig et al.²⁴ Glutathione reductase (GR) activity in the liver was assayed by the method of Carlberg and Mannervik.²⁵ Reduced glutathione (GSH) level was determined by the method described by Beutler and Kelley.²⁶ The activity of DT-diaphorase in the liver was estimated according to the method described by Ernster.²⁷ The oxidized glutathione (GSSG) level in the buccal mucosa was determined by the method followed by Tietze.²⁸

Estimation of lipid peroxidation by-products

Lipid peroxidation was estimated as evidenced by the formation of thiobarbituric acid reactive substances (TBARS). TBARS in plasma were assayed by the method described by Yagi.²⁹ Buccal mucosa lipid peroxidation was done using the method followed by Ohkawa et al.³⁰

Determination of enzymatic and nonenzymatic antioxidants

Superoxide dismutase (SOD) was assayed in plasma and buccal mucosa using the method of Kakkar et al.³¹ The activity of catalase (CAT) in plasma and the buccal mucosa was assayed by the method of Sinha.³² The activity of glutathione peroxidase (GPx) in plasma and the buccal mucosa was determined by the method described by Rotruck et al.³³ The reduced GSH levels in plasma and buccal mucosa were determined by the method of Beutler and Kelly.³⁴ The vitamin E level in plasma was determined colorimetrically using the method of Palan et al.³⁵ Buccal mucosa vitamin E was measured by the method of Desai.³⁶ The level of plasma vitamin C was determined by the method of Omaye et al.³⁷

Statistical analysis

The data are expressed as mean ± standard deviation. Statistical comparisons for biochemical parameters were performed by one-way analysis of variance (ANOVA), followed by Duncan’s multiple range tests (DMRT). The results were considered statistically significant if the p values were less than 0.05. After carrying out the ANOVA, the significance between the groups is compared with the help of DMRT. In this analysis, if the two groups receive a common superscript (e.g. a and a) they are statistically nonsignificant. If the two groups receive different superscript (e.g. a and b), they are statistically significant.

Results

Table 1 shows the tumor incidence, tumor volume, and tumor burden of control and experimental animals in each group. We observed 100% tumor formation with mean tumor volume (415.95 mm³) and tumor burden (1576.90 mm³) in hamsters treated with DMBA alone-painted hamsters (Figure 1). Oral administration of ethanolic extract of ElELet leaves at a dose of 250 mg/kg b.w. for 14 weeks significantly prevented the tumor incidence, tumor volume, and tumor burden in DMBA-painted hamsters. No tumors were observed in the control and ElELet alone-treated hamsters.

Table 1.

Incidence of oral neoplasm in control and experimental hamsters in each group.^a

Parameters	Group I	Group II	Group III	Group IV
Parameters	Control	DMBA alone	DMBA + ElELet	ElELet alone
Tumor incidence	–	100%	–	–
Total number of tumors/animal	–	38/10	–	–
Tumor volume (mm³)/animal	–	415.95 ± 20.6	–	–
Tumor burden (mm³)/animal	–	1576.9 ± 70.3	–	–
Dysplasia	–	Severe	Mild	–
Hyperplasia	–	Severe	Mild	–
Hyperkeratosis	–	Severe	Moderate	–
Oral squamous cell carcinoma	–	Well-differentiated squamous cell carcinoma	–	–

ElELet: Enicostemma littorale leaves; DMBA: 7,12-dimethylbenz(a)anthracene.

^a n = 10. Tumor volume was measured using the formula, v = 4/3 (D ₁/2) (D ₂/2) (D ₃/2), where D ₁, D ₂, and D ₃ are the three diameters (in cubic millimeter) of the tumor. Tumor burden was calculated by multiplying tumor volume and the number of tumors/animal.

Figure 2 shows the histopathological features of control and experimental hamsters. A myriad of histopathological changes (severe hyperkeratosis, hyperplasia, and dysplasia) and well-differentiated squamous cell carcinoma of the epithelium was observed in hamsters painted with DMBA alone. Mild-to-moderate preneoplastic lesions (hyperplasia, hyperkeratosis, and dysplasia) were noticed in DMBA + ElELet-treated hamsters. Hamsters administered with ethanolic extract of E. littorale showed well-defined and intact epithelium layers similar to that of control hamsters.

Figure 3 shows the status of phase I (cytochromes P450 and b₅) and phase II (GR, GST, GSH, and DT-diaphorase) detoxification agents in the liver of control and experimental hamsters in each group. The status of phase I detoxification agents was significantly increased, whereas phase II agents was decreased in the liver of DMBA-treated hamsters as compared to control hamsters. Oral administration of ElELet to DMBA-treated hamsters brought back the status of phase I and phase II detoxification agents to near-normal range. Oral administration of ElELet alone showed no significant difference in the status of phase I and II detoxification agents as compared to control hamsters.

Figure 4 shows the status of phase I (cytochromes P450 and b₅) and phase II (GST, GSH, and GSSG) detoxification agents in the buccal mucosa of control and experimental hamsters in each group. The status of phase I and phase II detoxification agents were significantly increased, whereas GSSG content was decreased in tumor-bearing hamsters as compared to control hamsters. Oral administration of ElELet to DMBA-treated hamsters brought back the status of phase I and II detoxification agents as compared to near-normal range. Oral administration of ElELet alone showed no significant difference in the status of phase I and II detoxification agents as compared to control hamsters.

Figure 5 shows the status of plasma TBARS and antioxidants (SOD, CAT, GPx, and GSH) in control and experimental hamsters in each group. The concentration of TBARS was increased, whereas enzymatic antioxidants activities were decreased in DMBA-painted hamsters as compared to control hamsters. Oral administration of ethanolic extract of ElELet leaves at a dose of 250 mg/kg b.w. restored the concentration of TBARS and antioxidants to near-normal range in DMBA-treated hamsters. Hamsters treated with E. littorale alone showed no significant difference in TBARS and antioxidants status as compared to control hamsters.

Figure 6 shows the status of buccal mucosa, TBARS, and antioxidants (SOD, CAT, GPx, GSH, and vitamin E) in control and experimental hamsters in each group. The status of TBARS, SOD, and CAT was decreased in the buccal mucosa, whereas the levels of GSH, GPx, and vitamin E were increased in DMBA-painted hamsters as compared to control hamsters. Oral administration of ethanolic extract of ElELet leaves at a dose of 250 mg/kg b.w. restored the concentration of TBARS to near-normal range in DMBA-treated hamsters. Hamsters treated with E. littorale alone showed no significant difference in TBARS and antioxidant status as compared to control hamsters.

Discussion

Oral cancer is the fifth most frequent cancer worldwide. Oral carcinogenesis is a multifocal disease, preceded by distinct premalignant lesions. DMBA-induced precancerous and cancerous lesions in the buccal mucosa of hamsters resemble the pathological alterations of human oral cancer. In this study, we investigated the chemopreventive potential of E. littorale during DMBA-induced oral carcinogenesis. The tumor incidence, tumor volume, and tumor burden in the hamster buccal pouch were determined. The buccal pouch from hamster treated with DMBA alone revealed severe keratosis, hyperplasia, dysplasia, and well-differentiated squamous cell carcinoma. Mild-to-moderate preneoplastic lesions, such as hyperplasia, keratosis, and dysplasia, were noticed in DMBA + ElELet-treated hamsters. Oral administration of E. littorale at a dose of 250 mg/kg b.w. to DMBA-treated hamsters significantly prevented the tumor incidence, tumor volume, and tumor burden. Our results suggest that E. littorale showed significant chemopreventive potential during DMBA-induced oral carcinogenesis.

While phase I detoxification agents (cytochromes P450 and b₅) are involved in the metabolic activation of carcinogens, phase II enzymes (GR, GST, GSH, GSSG, and DT-diaphorase) are involved in the excretion of carcinogenic metabolites in conjugation with reduced GSH. A fine balance thus exists between the status of phase I and phase II detoxification enzymes in the process of eliminating/excreting the carcinogenic metabolites. An imbalance in the above status, therefore, leads to neoplasia.^38,39 Increase in phase I detoxification agents accompanied by increase in phase II agents and reduction in GSSG. Phase II enzymes in the buccal mucosa of hamsters treated with DMBA alone revealed that oral carcinoma arises in the buccal mucosa due to imbalance in the status of phase I and II detoxification cascade. Repeated DMBA exposure to the buccal mucosa might be the reason for the increased activity of phase I and phase II detoxification agents. Increase in phase I enzymes in the liver, however, indicates the accumulation of carcinogenic metabolites of DMBA, which was not effectively excreted from the liver, as evidenced by decreased activities of phase II detoxification agents.

At physiological concentrations, ROS play a vital role in protecting cells against infection, stimulating mitogenic response, and cell signaling.⁴⁰ However, the excessive generation of these ROS due to harmful chemicals and mutagen exposure resulted in several pathophysiological diseases including cancer. ROS has been implicated in all the three stages (initiation, promotion, and progression) of carcinogenesis.⁴¹ Overproduction of ROS in the body causes extensive DNA damage that could in turn contribute to neoplastic transformation. ROS-mediated DNA damage could cause structural modifications in DNA, activation of proto-oncogene, and inactivation of tumor suppressor genes, which eventually leads to neoplastic transformation.⁴² Cell membranes are much vulnerable to ROS-mediated oxidative damage, and ROS-induced lipid peroxidation has thus been implicated in the pathogenesis of carcinogenesis. Increased levels of circulatory TBARS have been well documented in oral cancer.⁴³ Antioxidants, the biological fighters against ROS, play a prominent role in the protection of cells and tissues against ROS-mediated oxidative damages.⁴⁴ Antioxidants act synergistically in stabilizing or deactivating ROS before they attack cells. Excessive production of ROS and compromised antioxidants has been well documented in several cancers, including oral carcinoma.⁴⁵ A poor or insufficient antioxidant potential was reported in both human and experimental oral carcinogenesis.⁴⁶ This could be partly due to their utilization by cancerous tissues or due to scavenging or neutralizing the harmful effects of ROS. Decreased TBARS levels accompanied by an increase in vitamins E and C content in tumor tissues of the DMBA-treated hamsters could lend credence to the above findings. Vitamin E, vitamin C, and GSH have synergistic role in scavenging free radicals as well as in maintaining their cellular levels. GSH plays a pivotal role in maintaining the cellular levels of vitamins C and E in an active form.⁴⁷ Vitamin C plays an important role in the regeneration of vitamin E.⁴⁸ Low levels of TBARS in the oral tumor tissues may either be due to abnormal cell proliferation or due to low content of polyunsaturated fatty acid, a substrate of lipid peroxidation. Mounting evidences pointed out lowered SOD and CAT activities in oral tumor tissues of human and golden Syrian hamsters.^49,50

GSH play diverse roles in various cellular processes in addition to its role as a nonenzymatic antioxidant. Extensive studies documented that GSH and GPx have regular effects on cell proliferation and are overexpressed in tumor tissues of several malignant cancers. Our results corroborate these observations.^51,52

Oral administration of E. littorale at a dose of 250 mg/kg b.w. to hamsters treated with DMBA significantly improved the status of lipid peroxidation and antioxidants both in circulation as well as in the tumor tissues. The plant E. littorale possesses multiple antioxidant components, including catechin, saponin, triterpenoids, and xanthones.⁵³ Daniel and Sabins⁵⁴ have isolated six phenolic compounds from E. littorale, which include vanillic acid, syringic acid, p-coumaric acid, and ferulic acid. Ghosal and coworkers⁵⁵ isolated seven flavanoids from E. littorale including apigenin and swertisin. Abirami et al.⁵⁶ reported that phenolic compounds of E. littorale are responsible for its antioxidant potential. Extensive in vitro studies documented the free radical scavenging potential of E. littorale using 2,2-diphenyl-1-picrylhydrazyl assay.⁵⁷ In vivo studies have also exhibited its antioxidant role.⁵⁸ Present results also revealed that ElELet have prominent and promising antioxidant potential, and these properties might have contributed to the suppression or delayed the formation of oral tumors during DMBA-induced hamster buccal pouch carcinogenesis. It is thus clearly evident that the antioxidant components present in the ElELet might have played a role in the recovery of redox condition in the hamsters treated with DMBA + ElELet.

To conclude, E. littorale significantly prevented or delayed the tumor formation in the buccal mucosa of hamsters treated with DMBA. The chemopreventive potential of E. littorale is substantiated with its modulating effect on the detoxification cascade and antioxidant potential during DMBA-induced oral carcinogenesis. Further studies are in progress to isolate and characterize the active constituents of E. littorale.

Footnotes

Conflict of interest

The authors declared no conflicts of interest.

Funding

Financial support from Indian Council of Medical Research (ICMR), New Delhi, India, to DR in the form of Senior Research Fellowship (SRF) is gratefully acknowledged.

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