Abstract
The effect of the active fraction of Elephantopus scaber L. (Asteraceae) (ES) on skin papillomas induced by 7,12-dimethylbenz(a)anthracene (DMBA) as an initiator and croton oil as promoter was studied in mice. The active fraction of E. scaber (100 mg/kg) on topical application delayed the onset of papilloma formation and reduced the mean number of papillomas and the mean weight of papillomas per mouse. The intraperitoneal administration of the active fraction of E. scaber also had a significant effect on subcutaneous injection of 20-methylcholanthrene (20-MCA)-induced soft tissue sarcomas in mice. It inhibited the incidence of sarcomas and reduced the tumor diameter compared to MCA-treated control animals. The subcutaneous administration of the active fraction of E. scaber significantly inhibited the growth of subcutaneously transplanted DLA and EAC solid tumors, delayed the onset of tumor formation, and increased the life span of tumor bearing mice. The present study thus indicates the tumor inhibitory activity of the active fraction of E. scaber against chemically induced tumors and its ability to inhibit the development of solid tumors.
Introduction
Elephantopus scaber L. (Asteraceae), in Sanskrit known as “Gojhiva (Anachuvadi)”, is an indigenous perennial herb commonly found in India. E. scaber exhibits anti-snake venom, analgesic, anti-inflammatory (CitationRuppelt et al., 1991), diuretic (CitationLaranja et al., 1992), antiviral (CitationLi et al., 2004), and antihepatotoxic (CitationLin et al., 1991; CitationRajesh & Latha, 2001) activities. The plant is also used in wound healing (CitationDixit & Pandey, 1984), liver diseases, hepatitis (CitationRao, 1981; CitationLin & Kan, 1990), skin diseases — mainly leucoderma — scabies, sexually transmitted diseases (CitationSahu, 1984; CitationDilara et al., 2000), and to prevent inflammation after birth (Burkill, 1966). Its antitumor properties have been reported (CitationGeetha et al., 2003; CitationGeetha, 2004).
In recent years in cancer chemoprevention studies, the identification of better antitumor-promoting agents has been highly desired, because they may have a wider applicability against the development of clinical cancers (CitationKelloff, 2000). Both epidemiological and animal studies have suggested that chemicals present in the diet, several herbs and plants with diversified pharmacological properties are useful agents for the prevention of human cancers (CitationBradlow et al., 1999). Herein, we report the presumptive role of the active fraction of E. scaber, in the inhibition of chemical carcinogenesis induced by DMBA/croton oil which induces papillomas, 20-methylcholanthrene which induces fibrosarcomas and the development of DLA and EAC solid tumors in mice.
Materials and methods
Preparation of the active fraction of Elephantopus scaber
Elephantopus scaber was collected from the herbal garden of the Institute and a voucher specimen of the plant (TBGT 25419 dated 22/08/2001) after authentication by Mathew Dan, the plant taxonomist, has been deposited in the Herbarium of the Institute. The shade-dried whole plant of E. scaber (900 g) was extracted with petroleum ether (2500 ml) and then with chloroform using a Soxhlet apparatus (16 h each). The crude chloroform extract (9.2 g) was then fractionated by column chromatography over silica gel (60-120 mesh, 300 g). The column was eluted with hexane and gradients of hexane ethyl acetate (EtOAc) mixtures. Seventy fractions of 100 mL each were collected and the fractions were pooled, according to similarities in TLC. Fractions 18 to 25 (2.8%, w/w, with respect to the dried plant material) eluted with 15% and 25% EtOAc in hexane, was referred to as the active fraction of E. scaber (ES). It was suspended in 10% DMSO, to required concentrations and used for the experiments.
Animals
Swiss albino mice (males) in the age group of 8-10 weeks, weighing 20-30 g were used for the experiments. The animals were housed under conventional laboratory conditions and fed with standard pellet diet (Lipton India, Mumbai, India) and boiled water ad libitum. All experiments involving animals were done, strictly adhering to the guidelines for animal experimentation and handling, issued by the Government of India.
Chemicals
7,12-Dimethylbenz(a)anthracene (DMBA), croton oil and 20-methyl cholanthrene (20-MCA) were obtained from Sigma (St. Louis, Missouri, USA) and dimethylsulphoxide were from SD Fine Chemicals, Mumbai, India.
Maintenance of Dalton’s Lymphoma Ascites (DLA) and Ehrlich Ascites Carcinoma (EAC)
DLA and EAC tumor cells were maintained as ascites by serial transplantation weekly in mice by i.p. injection of 1 × 106 cells per mouse. The tumor cells were aspirated from the tumor bearing mice aseptically and washed thrice in phosphate buffered saline, before transplantation.
Experimental protocol
Effect of ES on development of chemically induced skin papillomas
Inbred strains of male Swiss albino mice weighing 20-25 g were used for the experiment. Three days before the commencement of the experiment, the dorsal skin in the interscapular area of the mice was shaved with electric clippers and only those mice showing no hair regrowth were used for the study. Tumors were induced following initiation with 7, 12-dimethylbenz(a)anthracene (DMBA) and subsequent application of promoter-croton oil.
Treatments
The animals were divided into four groups of 15 animals per group. Group I control animals received topical application of 0.1 mL DMSO and group II animals received topical application of a single dose of ES, at a dose of 100 mg/kg body weight in 0.1 mL DMSO, 15 min prior to the application of DMBA (450 nmol in 0.1 mL acetone). Two weeks later, 0.1 mL croton oil (0.5% v/v in 100 μL acetone) was applied on the same site, twice weekly for 4 weeks. Group III control animals received topical application of 0.1 mL DMSO and group IV animals received topical application of ES at a dose of 100 mg/kg body weight in 0.1 mL DMSO, 15 min prior to the application of DMBA, and twice weekly, prior to the application of croton oil for four weeks.
During the 12 weeks of the study the mortality of the animals, an apparent sign of toxicity, was noted weekly. The skin lesions that persisted for two consecutive observations, more than 1 mm in diameter were considered to be papillomas. The incidence of skin papillomas, the number of mice with papilloma and the average number of papillomas formed per mouse were recorded over the entire period of the experiment. At the end of 12 weeks, both control and treated mice were sacrificed, the papillomas were surgically removed and the average weight of the papillomas was determined. The experiment was repeated once.
Effect of ES on development of chemically induced sarcomas
The mice were divided into three groups, with 15 animals per group. The hair was removed from the dorsal side of all animals and a single dose of 20-methylcholanthrene (200 μg/0.1 mL DMSO/mouse) was injected subcutaneously (s.c.) on the dorsal side to all group of animals. The group I animals that served as control were given intraperitoneal injection (i.p.) of 0.1 mLl DMSO twice weekly for eight weeks, group II and group III animals were given intraperitoneal injection of 100 and 200 mg/kg respectively of ES, twice weekly for eight weeks. The animals were observed for the onset of sarcoma as well as for their survival for 20 weeks. Tumor measurements were made using a caliper and tumor diameter (Td) was calculated using the formula as cited by CitationSalomi et al. (1991). The experiment was repeated once.
Effect of active fraction of E. scaber on the DLA solid tumor reduction in mice
The mice were divided into three groups of 9 mice per group. DLA tumor cells (1 × 106/0.5 ml) were subcutaneously injected on the right hind leg of the mice to form solid tumors as cited by CitationNair and Panikkar (1990) to all three groups. After 24 h, group I animals received 0.5 mL 10% DMSO, group II and group III animals received subcutaneous injection (in the same limb as the solid tumor) of 100 and 200 mg/kg of ES, respectively, untill the end of the experiment (30 days).
The measurement of tumor radii was made from day 10 of tumor induction and was repeated for a period of 30 days. The antitumor activity of the active fraction was assessed by comparing the tumor volume between the control and treated group, using the formula:
V = 4/3 π r12, r22
where r1 and r2 are the radii, along two directions. The experiment was repeated twice. The experiment was carried out using EAC tumor cells and data were recorded.
Effect of ES on the EAC solid tumor reduction in mice
The mice were divided into three groups of nine mice per group. EAC tumor cells (1 × 106/0.5 mL) were subcutaneously injected on the right hind leg of the mice to form solid tumors to all three groups of animals. After 24 h, group I animals received 0.5 mL 10% DMSO, group II and group III animals received subcutaneous injection (in the same limb as the solid tumor) of 100 and 200 mg/kg of ES, respectively, till the end of the experiment (30 days).
The measurement of tumor radii was made from day 10 of tumor induction and was repeated for a period of 30 days. The antitumor activity of the active fraction was assessed by comparing the tumor volume between the control and treated group. The experiment was repeated twice.
Statistical analysis
All data were expressed as the mean ± standard deviation (SD) and statistical analysis was performed by using the Student’s t-test (CitationBennet & Franklin, 1967). A p value less than 0.05 was taken as significant.
Results
The results of treatment with single and repeated applications of ES on skin papilloma formation induced by DMBA/croton oil is depicted in . It is evident that by week 5, the occurrence of papilloma was noted in the control groups of animals. Thereafter, the mean number of papillomas increased in control groups which were found to be between 3.4 and 3.66. In the groups treated with single dose or repeated doses of ES, the occurrence of papilloma was noted only from week 8. The tumor responded better to the treatment using repeated doses of ES than the single dose treatment. The repeated dose of ES at a dose of 100 mg/kg significantly (p <0.001) reduced the mean number of papillomas (0.93) and the mean weight of papillomas (12.5 mg), while single dose treatment was less effective. All the treated and control mice survived the 12-week experimental period. The percentage of mice with papilloma for the groups treated with the single dose and repeated dose of ES (100 mg/kg) were 66.66% and 40% respectively, as compared to their respective DMBA control.
Table 1. Effect of ES on DMBA induced papillomas in mice.
ES had a significant effect on carcinogenesis induced by 20-MCA. It was noted that 46% of animals treated with methylcholanthrene developed sarcomas by week 10 and 90% of animals developed sarcomas by week 12. In the groups treated with ES (100 and 200 mg/kg), palpable tumors appeared in week 10 and only 41.6% and 41.1% of mice, respectively, developed tumors by week 12. The mean tumor diameter of control mice was found to be 2.8 cm whereas for animals treated with ES (100 mg/kg), it was found to be 1.1 cm (). ES at the 200 mg/kg dose level produced almost similar results to the 100 mg/kg. It was found that none of the control mice survived after 13 weeks but 50% of the animals treated with ES survived up to 20 weeks (till the end of the experiment).
Table 2. Effect of ES on 20-methylcholanthrene (MCA) induced sarcomas in mice.
The DLA solid tumor responded better to the treatment of ES than the EAC solid tumor. ES at a dose of 200 mg/kg significantly (p <0.001) inhibited the growth of subcutaneously transplanted DLA solid tumor and delayed the onset of tumor formation. The tumor volume on day 30 for the DLA solid tumor-treated group which received 100 and 200 mg/kg doses of ES was found to be 2.34 and 1.95 cc, respectively, as compared to the control group, and a significant increase in life span of 47.48% and 67.86% respectively was noted (). The administration of ES at doses of 100 and 200 mg/kg inhibited the growth of EAC solid tumor, and the tumor volume on day 30 was found to be 3.9 and 3.8 cc, respectively, as compared to the control group, and the increase in life span noted was 32.94% and 45.39%, respectively ().
Table 3. Effect of ES on the life span of DLA solid tumor bearing mice.
Table 4. Effect of ES on the life span of EAC solid tumor bearing mice.
Discussion
In the present study, the animals treated with ES could prolong the latent period of tumor occurrence, compared to control animals treated with DMBA/croton oil that had a shorter latency period. The two-stage technique of initiation and promotion on skin carcinogenesis induced by DMBA, has been shown to produce a high frequency of papillomas as a function of exposure to 12-O-tetradeconylphorbol-13-acetate (TPA), present in croton oil. The animals treated with a single dose of ES showed reduction of the mean number of papillomas and average weight of papillomas, but a more profound effect was noted with the groups treated with repeated doses of ES. There was no mortality of animals of any of the groups of animals during the 12-week experimental period, but the control animals became weak and died by about 14 weeks (data not shown). The groups treated with ES survived for a significantly longer period of time (21 weeks). In the present study, the ES in repeated doses produced a significant effect on DMBA-induced papillomas, and therefore its inhibitory action on tumor promotion cannot be ruled out. It is evident that a significant reduction in percentage of mice with papillomas was noted in groups treated with repeated doses of ES (40.1%), whereas control mice treated with DMBA/croton oil developed 86.66% of visible papillomas.
The intraperitoneal administration of ES inhibited the incidence of sarcomas and significantly reduced the tumor diameter, compared to control animals. This indicates the tumor inhibitory activity of E. scaber, against chemically induced tumors. The ES itself was antitumor-promoting (CitationGeetha et al., 2003; CitationGeetha, 2004), and thus inhibited the action of the promoter. These findings are relevant because of the exposure of human beings to environmental carcinogens in everyday life. The therapeutic efficacy of sobatum from Solanum trilobatum L. (Solanaceae) (CitationMohanan & Devi, 1997), red ginseng from Panax ginseng L. (Araliaceae) (CitationXiaoguang et al., 1998) and extract of Acanthus ilicifolius L. (Acanthaceae) (CitationBabu et al., 2002) against tumor progression and carcinogen-induced skin papillomas, supports our studies. Studies have also shown that curcumin present in the spice turmeric (CitationStoner & Mukhtar, 1995), Iscador from Viscum album L. (Loranthaceae) (CitationKuttan et al., 1996), fatty acids from Psoralea corylifolia L. (Papillionaceae) seeds (CitationLatha & Panikkar, 1999) and thymoquinone from Nigella sativa L. (Ranunculaceae) (CitationBadary & Gamal, 2001) afforded protection against 20-methylcholanthrene-induced sarcomas.
The application of polycyclic hydrocarbons such as DMBA and methylcholanthrene to skin at appropriate doses results in tumor formation. It is known that they are metabolized by mixed function oxidase to reactive electrophiles, which react with nucleophilic centers in cellular macromolecules to initiate carcinogenesis (CitationDiGiovanni et al., 1978). Thus, DMBA is metabolized to its active form, and thus it is assumed that there is a lag period before its effects appear. As treatment with ES was started immediately before the application of DMBA, it is presumed that the drug might have prevented the transformation of DMBA to its reactive form or prevented some direct reaction with the reactive form of DMBA.
The mechanism(s) underlying the prevention of skin papillomas by ES is not clear. Since the biochemical alterations were not studied, it was not possible to comment on the changes leading to skin tumor promotion. Studies of CitationKelloff et al. (1999) and CitationHursting et al. (1999) have shown that many compounds both natural and synthetic, belonging to diverse structural and functional classes such as vitamins and minerals (vitamin A, E, selenium), phytochemicals (polyphenols, triterpenoidal glycosides, sesquiterpenes, allylic indole compounds, and cinnamic acids), and synthetic compounds such as retinoids have been identified to inhibit chemical carcinogenesis through various mechanisms. The antitumor activity exhibited by sesquiterpene lactones scabertopin, isoscabertopin, deoxyelephantopin and isodeoxyelephantopin isolated from Elephantopus scaber may be due, at least in part, to induction of apoptosis in vitro (CitationXu et al. 2006). Isodeoxyelephantopin inhibits NF-κB activation and NF-κB-regulated gene expression thereby explaining the ability of the compound to enhance apoptosis and inhibit invasion and osteoclastogenesis (CitationIchikawa et al., 2006). Studies of CitationZou et al. (2008) have shown that deoxyelephantopin isolated from Elephantopus carolinianus Willd. functions as a selective partial agonist against nuclear hormone receptor peroxisome proliferator-activated receptor-γ (PPARγ) and inhibits cancer cell proliferation and caused cell cycle arrest at G2/M phase. Studies of CitationLiang et al. (2008) have shown that sesquiterpene lactones, elescaberin, deoxyelephantopin and isodeoxyelephantopin exhibited significant inhibitory activities against human SMMC-7721 liver cancer cells in vitro. In view of this in the present study, we cannot rule out the possible participation or the synergistic action of the active principles of E. scaber such as deoxyelephantopin, isodeoxyelephantopin, scabertopin, isoscabertopin, molephantin, and molephantinin in the inhibition of chemical carcinogenesis.
In vivo experiments indicated that ES was found to be more sensitive against solid tumor development with DLA tumors than EAC tumors, indicating the specific nature of the drug towards the tumor cells. The present study also reveals that with DLA solid tumor, both the concentrations of ES (100 and 200 mg/kg) used, significantly reduced the tumor growth as indicated by reduction in tumor volume, but the reduction in EAC tumor volume was not as significant as that with DLA solid tumor, even at the highest concentration of ES (200 mg/kg) used in the present study. In the antitumor studies on subcutaneously transplanted solid tumor, therefore, there was preferential sensitivity of DLA tumor to ES, than EAC solid tumor. This could possibly be that simultaneous administration of ES after tumor transplantation resulted in reduction of tumor volume in both the tumor types, indicating that the earlier the drug administration, the greater the increase in the efficacy of the drug was noted. This property of solid tumor prevention is a useful index in testing the drug on higher animals, as per recognized protocol systems, since solid tumors are less sensitive to chemotherapeutic interventions.
There are reports that Iscador from Viscum album L. (Loranthaceae) reduced solid tumor produced by DLA and EAC ascitic tumor cells (CitationKuttan et al., 1990), saffron extract inhibited the growth of DLA and S-180 solid tumor (CitationNair et al., 1994) and sobatum from Solanum trilobatum L. (Solanaceae) (CitationMohanan & Devi, 1996) was more active against EAC-induced solid tumor than against DLA-induced solid tumor. The present study indicates that ES may have specific inhibitory effects on solid tumors. This is probably due to the difference in mechanism of tumor destruction, the difference in the ability of the drugs to penetrate cell membranes and various other metabolic processes within the cell.
In conclusion, our results provide evidence that ES has modulatory influence on the experimentally induced carcinogenesis and thus exhibits a chemopreventive action and also has the ability to inhibit solid tumors in mice.
Acknowledgements
The authors are thankful to the Kerala Forest and Wildlife Department and Dr. G.M. Nair, the Director of the Institute for facilities.
Declaration of interest
The authors are thankful to the Kerala Forest and Wildlife Department, Thiruvananthapuram for the financial grant.
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