Chemopreventive Effect of Lantana camara Leaf Extract on 7,12-Dimethylbenz[a]anthracene-Induced Squamous Cell Carcinoma of Skin in Swiss Albino Mice

Abstract

The chemopreventive effect of Lantana camara. L. (Verbenaceae) methanol leaf extract (LCLE) on 7,12-dimethylbenz[a.]anthracene (DMBA)-induced skin cancer was investigated in Swiss albino mice. The skin lesions were induced by the twice-weekly topical application of DMBA (100 nmol/100 µL acetone) for 8 weeks on the shaved backs of mice. LCLE was administered at the maximal tolerated dose of 400 mg/kg twice a week, 1 week before DMBA application, and continued for 20 weeks thereafter. The results of the study revealed a significant decrease in incidence of skin papillomas in mice, slight weight gain in mice, and reduced death rate in comparison with DMBA alone at the end of 20 weeks. These results were supported by histopathologic studies. The skin section of LCLE-treated mice showed hyperplastic papillomatous lesions without the evidence of infiltration or cytological atypia. From the current study, it can be inferred that LCLE possesses potential chemopreventive activity in this experimental model of cancer.

Keywords:

• 7
• 12-Dimethylbenz[a.]anthracene
• Lantana camara
• squamous cell carcinoma

Introduction

The continuing magnitude and severity of the cancer problem make it imperative to develop a preventive approach to this disease. Chemoprevention represents a promising strategy that can slow, reverse, or completely halt the process of carcinogenesis (Hong & Sporn, 1997). The need of new agents with novel mechanisms of action to prevent cancer is perhaps the most urgent need in the entire field of chemoprevention (Holm et al., 1979). The mouse skin carcinogenesis model has become very useful in studying the genetic changes that are associated with the chemical initiation of papillomas and the transition to squamous cell carcinoma (Holden et al., 1997). All over the world, studies on plant materials have revealed their health promoting action including cancer prevention. Plants remain a large chemical library to be explored for new agents. Recently, plant-derived triterpenoids have attracted reasonable attention for their unique antineoplastic activity (Setzer & Setzer, 2003). Lantana (Lantana camara. L.; Verbenaceae), commonly known as red or wild sage, is one of the 10 most noxious weeds in the world (Sharma & Sharma, 1989). It has encroached upon a vast expanse of land area including pastures, orchards, tea gardens, forests, and agricultural lands in tropical and subtropical parts of the world and has imposed a great threat to grazing livestock and overall ecological balance (Ghisalberti, 2000). The plant foliage of lantana is very rich in pentacyclic triterpenoids (Sharma et al., 1991). All the parts of this plant have been used traditionally for several aliments throughout the world. The leaves are used as bechic, antitumoral, antibacterial, and antihypertensive (Ross, 1999). The methanol leaf extract of lantana showed in vitro. cytotoxicity against human lung carcinoma cell lines (A-549) and mouse melanoma (B16F₁₀) (Raghu et al., 2004). In the current work, the methanol extracts of leaves of lantana (LCLE) were examined for their antitumor activity on DMBA-induced skin cancer in Swiss albino mice. This model of carcinogenesis was used in order to test the efficacy of LCLE to prevent neoplastic development under in vivo. conditions. To our knowledge, this is the first in vivo. study on the effect of LCLE on DMBA-induced squamous cell carcinoma in Swiss albino mice.

Materials and Methods

The animal care and handling was done according to the guidelines set by the World Health Organization (WHO), Geneva, Switzerland, and the Indian National Science Academy (INSA), New Delhi, India.

Animals and chemicals

Female Swiss albino mice (6 weeks old) weighing 18–22 g were obtained from the Central Animal House of Panjab University, Chandigarh (India) and kept in the departmental animal rooms with controlled temperature of 23±5°C, 60±5% humidity and with a 12-h light/dark cycle. They were fed a basal diet and water. The mice were acclimatized for 1 week before experimentation. DMBA was purchased from Sigma-Aldrich (St. Louis, MO, USA). All other required chemicals were of highest purity and obtained from local suppliers.

Extraction of leaves of Lantana

The leaves of Lantana camara. were collected in September from Palampur (HP), India. The leaves were shade-dried and powdered. A voucher specimen was deposited in the herbarium at the University Institute of Pharmaceutical Sciences, Panjab University. For the preparation of extract, 1 L of methanol was added to Lantana. leaf powder (200 g) and kept for 24 h with intermittent shaking. The extract was separated by filtration through muslin cloth and the solvent was removed under reduced pressure. The residue R₁ was weighed and screened for their antitumor activity on DMBA-induced skin cancer in Swiss albino mice.

Experimental protocol

Skin cancer was induced according to the well established method of Azuine and Bhide (1992). Depilatory cream was used to remove hair from the backs of mice. The animals were left for 2 days and divided into three groups. Group I (n = 10) were treated with 100 µL vehicle (acetone). The acetone was topically applied on the depilated back of each mouse twice weekly for 8 weeks. The animals of group II (n = 15), were topically treated with DMBA (100 nmol/100 µL of acetone) on the depilated back of each mouse for 8 weeks. The animals of group III (n = 10) were orally treated with LCLE suspended in water and carboxymethyl cellulose (400 mg/kg body weight) and dosing was started 1 week before DMBA application and continued for 20 weeks thereafter.

Morphologic observations of papillomas

The incidence of skin papillomas, average number of papillomas per mouse, body weight, and number of animals that survived the 20-week period were recorded. The body weight, number of deaths, and papillomas appearing on depilated skin were recorded at weekly intervals. Only those papillomas that persisted for 2 weeks or more have been taken into consideration for final evaluation of the data. The skin papillomas were excised and fixed in Zenker, routinely processed, and embedded in paraffin. Sections (7 µm thick) were stained with hematoxylin and eosin and examined under a light microscope to carry out histopathology.

Statistical analysis

The data were analyzed using Student's unpaired t.-test and χ² test; p < 0.05 was considered significant.

Results

The effect of LCLE on DMBA-induced incidence of skin cancer at weeks 1–20 is shown in Figure 1. The onset of papillomas was observed at week 5 (36.3%) in the DMBA-treated mice. There was gradual rise in the incidence of cancer that reached 100% during the 8th week. The incidence of DMBA-induced papillomas was delayed by 3 weeks in the LCLE group. An overall significant decrease in incidence of cancer (23.6% vs. 100%) was seen in the LCLE group at the end of week 20 compared with DMBA alone. There was a significant decrease in average number of papillomas per mouse (papilloma yield) in comparison with DMBA-treated mice after the end of study. The average number of papillomas in LCLE-treated animals was 1.3, whereas in DMBA-treated animals it was 5.5 at the termination of studies (Fig. 2). Application of acetone alone on the depilated backs of mice did not induce the papillomas throughout the experiment. The survival rate of mice decreased significantly in DBMA-treated mice compared with the vehicle-treated group. Survival of the LCLE-treated group was significantly higher (75%) in comparison with the DMBA-treated group (37.5%; Fig. 3). The average body weight of DMBA-treated mice did not differ from that of the acetone-treated mice throughout the study. However, there was a slight increase in the average body weight of LCLE-treated mice at the termination of the study (Fig. 4). The histopathologic examination of depilated backs of mice revealed normal skin and presence of subcutaneous tissue in acetone-treated mice. Twice weekly application of DMBA for 8 weeks on depilated mice induced well differentiated squamous cell carcinomas with formation of keratin pearls. There was marked infiltration of cancer cells in the underlying dermis. The skin section of LCLE-treated mice showed hyperplastic papillomatous lesions without evidence of infiltration or cytological atypia.

Figure 1 Effect of LCLE on DMBA-induced incidence of papillomas in mice, *p < 0.001 versus DMBA alone.

Figure 2 Effect of LCLE on average number of papillomas per mouse (papillomas yield) in contrast with DMBA-treated group, *p < 0.001 versus DMBA alone.

Figure 3 Effect of LCLE on survival of mice, *p < 0.01 versus DMBA alone.

Figure 4 Effect of LCLE on body weight of mice, *p < 0.01 versus DMBA alone.

Discussion

Carcinogenesis is a multistep process involving the sequential phases of initiation, promotion, and progression. Chemoprevention is one of the desirable strategies to reverse, arrest, or inhibit carcinogenesis (Sporn & Suh, 2000). The leaves of Lantana. have antitumor properties and they are used in the traditional system of medicine. Therefore, LCLE was evaluated for its chemopreventive activity against DMBA-induced skin cancer in mice. The mouse skin carcinogenesis model is a well-characterized model for studying the genetic and biological changes associated with the chemical initiation of lesions and their subsequent transition to squamous cell carcinoma. Administration of LCLE at the maximal tolerated dose (400 mg/kg, twice a week, 1 week before initiation followed by treatment for 20 weeks thereafter) has shown a significant delay in the onset and overall reduction of papillomas in mice, suggesting its chemopreventive activity. Pretreatment could be associated with the extract's ability to interfere with the initiation, which is a relatively rapid process, and the continuous treatment after DMBA with the promotion, which is a slow process. The LCLE also increased the survival of the animals. This effect could be associated with the low papilloma burden as a result of inhibitory effect of extract on carcinogenesis. Slight weight gain in LCLE-treated mice could be a result of recovery from the effect of DMBA or, alternatively, better papilloma control. DMBA is a potent polycyclic aromatic hydrocarbon capable of inducing skin papilloma when applied repeatedly on the mouse skin (Shukla et al., 1998). The primary bioactivation of DMBA occurs via formation of DMBA-3, 4-dihydrodiol. DMBA-diol-epoxide has been suggested to be the ultimate carcinogen responsible for inducing chronic inflammation, ROS production, and oxidative damage of DNA resulting in the transformation of normal cells to tumor cells (Slaga et al., 1979). When mouse skin is repeatedly exposed to DMBA, the Langerhans cells are depleted and local immunosuppression takes place (Qu et al., 1997). The mechanism by which LCLE inhibits the DMBA-induced skin carcinogenesis has not been clearly elucidated. However, it is hypothesized that LCLE reverses the depletion of skin Langerhans cells and local immunosupression. It is evident from the histopathologic studies that the severity of DMBA-induced malignancy was reduced markedly by LCLE administration. Based on these results, it is concluded that LCLE significantly reduced skin papillomas in this model of cancer and, therefore, Lantana. has potential for drug development. This strategy will help to utilize lantana as a source for drug development.

Acknowledgment

We thank ICMR, New Delhi, for providing funds to carry out this research work.