Essential Oil of Tridax procumbens L Induces Apoptosis and Suppresses Angiogenesis and Lung Metastasis of the B 16 F-10 Cell Line in C 57 BL / 6 Mice

Cancer is one of the main reasons for the cause of largest mortality worldwide. Many number of people die of lung cancer than colon, breast and prostate cancers (Zhi et al., 2007). Among cancer deaths, lung cancer is the one having top most mortality rate of 6 million per year Worldwide (Wenfeng et al., 2011). Among lung cancer types nonsmall-cell lung cancer accounts for 80% of deaths (Yang et al., 2008). In spite of having several chemotherapeutic and immunomodulating agents for treating cancer, there is still search for an ideal treatment that has minimal side effects and cost-effective. Most of the anticancer drugs available in the market like toxol and vinca alkaloids (vincristine, vinblastine) are obtained from medicinal plants (Shan et al., 1999). Evidences are there that these medicinal plant based drugs are believed to suppress the transformative,


Introduction
Cancer is one of the main reasons for the cause of largest mortality worldwide.Many number of people die of lung cancer than colon, breast and prostate cancers (Zhi et al., 2007).Among cancer deaths, lung cancer is the one having top most mortality rate of 6 million per year Worldwide (Wenfeng et al., 2011).Among lung cancer types nonsmall-cell lung cancer accounts for 80% of deaths (Yang et al., 2008).In spite of having several chemotherapeutic and immunomodulating agents for treating cancer, there is still search for an ideal treatment that has minimal side effects and cost-effective.Most of the anticancer drugs available in the market like toxol and vinca alkaloids (vincristine, vinblastine) are obtained from medicinal plants (Shan et al., 1999).Evidences are there that these medicinal plant based drugs are believed to suppress the transformative, activities may ultimately suppress the final steps of carcinogenesis such as angiogenesis and metastasis and also induce apoptosis (Bharat et al., 2006).According to cancer experts, metastasis is a serious aspect and is the common cause of cancer deaths.Most of the treatment for cancer involves the control of invasion and metastasis of tumors.Generally, angiogenesis is essential for the normal reproduction, development and organ repair.On the other Essential Oil of Tridax procumbens L Induces Apoptosis and Suppresses Angiogenesis and Lung Metastasis of the B16F-10 Cell Line in C57BL/6 Mice A Manjamalai 1 , MJ Mahesh Kumar 2 , VM Berlin Grace 1 * hand, angiogenesis is also important in a variety of tumor extreme activities are regulated by a variety of distinct pro-angiogenic and anti-angiogenic molecules.Several anti-angiogenic agents such as Angiostain, endostain and thrombspondins (endogenous proteins) have been proved both and studies for their anti-angiogenic Many anticancer drugs exert their cytotoxicity through 53 gene is an important protein in regulating DNA repair and triggering 53 act through many mechanisms for anticancer function, and plays a role in initiating apoptosis, genomic stability, 53 mutations are common in lung cancer and range from 33% in adenocarcinomas to 70% in small cell lung cancers (Oren et al., 1994).On the where it is responsible for chromatin condensation and DNA fragmentation.Caspase occupy a pivotal position in 53 does (Mraz et al., 2009).But the fact is that tumor cells are resistant to apoptosis.Hence, selective killing of tumor cells by way for treating cancer.Hence, the development of new anti-cancer drugs to promote apoptosis and suppress tumor directed angiogenesis is in need to overcome these problems.
plants often results in the discovery of new biologically history of use in the treatment of cancer (Mohammad et al., of such treatments should be viewed with some skepticism (Cragg et al., 1994).Hence, any discovery of anticancer compounds with considerable practical significance.Almost all terpenes have some biological activities in animals and play a meaningful role in human medicine.Hence, there is a growing interest in natural terpenoids in antiviral, cytotoxic, analgesic, anti-cancer, spermicidal and they are the dominant odorous compounds emitted vary depending on the composition of monoterpenes and extracts, essential oleoresins and oils of plants.Some reported for its cytotoxic activity on several cell lines like breast cancer and leukemic cell lines (Zhou et al., 2004).When these pinenes are the major constituents of analgesic activities (Erazo et al., 2006).Our study plant is an annual herb, native to tropical America and rapidly spread in many parts of tropical, plants extract has been reported by earlier studies that it is used for lowering blood pressure (Ikewuchi et al., 2010), blood glucose level (Bhagwat et al., 2008) and also its hepatoprotective activity in rats (Vilwanthan et al., 2005).All these studies and traditional use did not reveal any deleterious effect in rats and higher animals including man (Satish et al., 2012).Moreover, different solvent extracts from this plant as well as essential oil properties such as, anti-microbial (Mahato et al., 2005), et al., 2010), wound healing (Raina et al., 2008)  Extraction of essential oil from the study plant was done by hydro distillation method using clevenger-type were condensed on a cold surface using condenser attached to it.Essential oil gets separated based on the difference in density and immiscibility, is then collected and dried over anhydrous sodium sulphate and stored in a vial at low temperature until analysis.

GC-MS analysis was performed in Indian Institute
IISR/28(13)09], using CARBOWAX capillary column and helium as carrier gas to identify the major compounds 250 o C and the oven temperature was programmed as 60 o C for 15 minutes, and then gradually increased to 280 o C for their mass spectra and retention indices.cage) were purchased from National Institute of Nutrition plastic cages and maintained at 12 hour light/12 hour dark cycle with free access to food and water.All the experiments involving animals were performed according to the standard protocol and guidelines after getting proper approval from Institutional Animal Ethical Committee.20-25 g were divided into 6 groups of 6 animals each.Each group of animals was given different doses of drug (essential oil) such as 50, 100, 200, 500, 1,000 and 2,000 DOI:http://dx.doi.org/10.7314/APJCP.2012.13.11.5887Tridax procumbens L Induces Apoptosis and Suppress Angiogenesis and Lung Metastasis of B16F-10 Cells supplemented with dextrose, penicillin, streptomycin and which were injected through tail vein with 1×10 6 /0.1 ml activity from small inoculums (Sheeja et al., 2010).Animals were divided into 4 groups of 6 mice in each group.Group 1 normal mice were left as such without any treatment.Group 2 cancer controls in which cancer cells were injected to develop cancer in mice and were left without any treatment.Group 3 vehicle controls injected with cancer cells and were treated with 0.1 ml of 50% of ethanol.Group 4 essential oil treated group injected with cancer cells and were treated with 0.1 ml brief experimental design is given below (6mice/group for 21 days); Group 1: Normal mice.Group 2: Cell line alone.Group 3: Cell line+vehicle treated.Group 4: Cell line+essential oil of treated nd ) day in all the experimental groups.group.
On the 22 nd excised, weighed, and placed in phosphate-buffered saline the surface of the lung was counted and the percentage inhibition of lung tumor nodule formation was calculated (Korangath et al., 2010).
Blood was collected from all mice by cardiac puncture nd day and the blood parameters such as hemoglobin (Hb) and white blood cells (WBC) were observed in all the groups (Mahesh et al., 2007).and examined under a microscope for histopathological changes of lung cancer (Gopal et al., 2010).2800 Woods Hollow Road, Madison, USA) following the rehydrated by sequentially graded ethanol washes (100%, 95%, 85%, 70%, and 50%) for 3 minutes each at room temperature.Again washed the samples by immersing K to each slide to cover the tissue section and incubated and cells to the staining reagents in subsequent steps.
immersing the slides in 4% methanol-free formaldehyde each slide and kept until a light brown background was microscope (Olympus BH-2).
Cell death was quantitated by counting 200 cells in the percentage of apoptotic cells based on morphological appearance.
P 53 53 and Caspase 3 was checked 20 minutes followed by permeabilization with 0.5% (v/v) the sections were incubated with primary antibody [mouse monoclonal antibodies to 53 and Caspase 3 (Santa Cruz Biotechnology, USA] for 1 hour and then incubated with were treated with CuSO4 (10 mM) in ammonium acetate buffer (50 mM CH 3 COONH 4 , pH 5.5) for 30 minutes.
6-diamidino-2-phenylindole) for 5 minutes and mounted tumor sections treated as above, but without primary antibody, served as negative control.After mounting the confocal microscope.Image analysis was done using 6 ) were injected intradermally on the shaven ventral skin of which group 1 receives no treatment left as mice injected with cancer cells alone, group 2 was injected with cancer cells and receives vehicle 0.1 ml of 50% ethanol and group 3 the drug treatment group was injected with cancer cells washed with phosphate buffered saline and the number of tumor directed capillaries was counted using a dissection formation was also calculated using the formula as capillaries of cancer control×100.
Data was statistically analyzed using one-way ANOVA as primary test followed by Dunnett's test one way ANOVA, using Graph pad InStat3.0 software (Guruvayoorappan et al., 2007).All the results were expressed as mean±S.D of 6 animals in each group and

Results
In the GC-MS analysis of the essential oil of we found that the oil contains 14 compounds Sabinene were found to be the major compounds with 96% similarity with Wiley and NBS library.studies which would be much lower than the IC 50 1, and lung weight of mice among the groups as shown in nd day it was found to be decreased (19.81±0.19)whereas, in essential relative lung weight of the normal mice was found to be (2.31±0.02)and it was found to be increased in the tumor induced group (3.22±0.06)whereas, in the treated group nodule formation in which a high % of inhibition (71.67%) was found in the mice treated with the essential oil of when compared with cancer induced group is 157.66±2.81 and that of in the treated group is 44.66±4.63.nd day.It was found to be (7325±17.60)in the normal mice and (7148±17.65) in the essential oil treated groups.On the other hand WBC was found to be high (7443±10.23)in the tumor control mice.Similarly, on the 22 nd day, the hemoglobin level also was found to be increased (13.43±0.17) in the essential oil treated group when compared with tumor control groups (11.25±0.21).
normal mice shows normal architecture of lungs with lungs of cancer control animals showed massive tumor of metastatic colonies of melanoma in the interstitium of leads to reduction in vital capacity of the lung.Multiple between and within lobules.Neoplastic tubular epithelial of lungs were occupied by multiple lobes of tubular tumor mass, metastatic foci and regeneration of alveolar epithelial-like morphology with well differentiated secretory glands was seen.
We have observed an increase in the number of apoptotic cells with mean apoptotic nuclei of 32.66±3.77 in treated mice which is significantly higher when compared with that of in the normal mice 5.33±1.51and Examination of the expression of 53 and Caspase expressions of 53 and Caspase 3 were found in the neoplastic epithelial cells of all the sections of cancer group when compared with the normal lung tissue.Expressions of 53 and Caspase 3 in the essential oil treated group were found to be increased when compared with   formation of tumor directed new blood vessels accounting for 39.47%.

Discussion
was found to have 14 compounds and out of which four compounds phellandrene (C10H16) and Sabinene (C10H16) were found to be the major compounds.All the major biological activities of each compound have been reported decades back by different researches.effect when they were used in breast cancer and studies showed that they are cytotoxic to human cancer cells but not on the healthy cells like the red blood cells.On the other hand, researchers have reported on the possible synergistic effect of these molecules with other monoterpenes, sesquiterpenes like Caryophyllene (Mercier et al., 2008).All these studies have proven the potential use of terpenes or a mixture of terpenes as the inducers of apoptosis in cancer cells.A number of medicinal values have been reported for both by leaf extract as well as by essential oil.
In the present study, we have evaluated the antimetastatic activity on lung cancer development of the essential oil of drug toxicity study it is clear that the drug even in its highest dosage did not show any lethal effect/abnormality on highly metastatic and form tumor cell colonies in the dependent cytotoxic effect for the essential oil of the procumbens showed a high cytotoxicity of cancer cell .studies for chemotherapeutic effect of essential oil were carried out by involving tumor nodule assay), Assay of tumor directed capillary vessel formation (anti-angiogenesis assay) and histopathological analysis.Modern therapies focus on the anti-angiogenic therapy as a   et al., 2007).Metastasis is one of the three hallmarks of cancer.Metastatic tumors are very common in the late stages of the metastases to occur are the lungs, liver, brain and the bones (Ait et al., 2007).A recent study showed that the essential oil of black seeds inhibits the metastasis in mouse essential oils effectively kill cancer cells while being non-toxic to normal cells (non neoplastic cells).Some of the most effective oils studied included sandalwood essential oil which inhibited growth by up to 90% of several different types of cancer cells (cervical, breast, skin and prostate) even at very small concentrations while having little or no harmful effect on normal cells.In our cell line, having a high metastatic potency to host in the of tumor nodule formation in the lungs by essential oil of may because of the major bioactive compounds present pinene which have been reported to inhibit the formation of new blood vessel and thereby arresting the metastasis (Guy et al., 2012).Metastasis is associated with loss of body weight and changes in haematological parameters (Richard et al., 2007).In our study we have observed a decrease in the body weight, increase in WBC and decrease in haemoglobin in cancer group, whereas they were almost normalized like normal group in essential oil treated group.
Apoptosis and necrosis are two typical types of cell death and has been recognized earlier as a key feature of normal animal development and it is a regulated progress that is under the control of several signaling pathways, such as caspase and mitochondrial pathways accumulating evidence has indicated that the dysregulation it has been suggested that the chemotherapeutic agents should have the ability to enhance apoptosis.Apoptosis is characterized by several biochemical criteria such as changes in mitochondrial membrane permeability, caspase signaling activation, internucleosomal DNA cleavage, and the release of intermembrane mitochondrial proteins underlying this phenomenon have been exclusively studied for a long time and it has become apparent that it that may be regulated by interactions with other cells (Konig et al., 1997).Caspase is an important apoptosis signaling molecule trigger a cascade of molecular interaction leading to apoptosis.Studies have shown that the extrinsic activation triggers the hallmark Caspase cascade characteristic of the apoptotic pathway, in which 53 is a mutated gene in all human tumors to about its half and is a transcription factor whose activity gives rise to a variety of cellular outcomes, most commonly cell cycle arrest and apoptosis, eliminating cancer-prone cells.Usually, 53 protein is present within a cell in minute 53 protects against cancer by regulating the cellular response to DNA damage, apoptosis, and oncogene activation (Chendil et al., 2004). 53has many mechanisms for anticancer function, and plays a role in apoptosis, genomic common method for detecting DNA fragmentation that results from apoptotic signaling cascades (Ramirezfor the evaluation of effect of the essential oil on apoptotic activity.Evaluation of the expression of 53 and Caspase level of positive apoptotic nuclei in the essential oil treated mice when compared with the cancer mice and this may be because of the activity of the major compounds present in the essential oil.Correspondingly, expression of 53 and caspase was found to be high in the treated cases than the normal and it clearly indicates that the essential has induced apoptosis.Many anti-cancer drugs currently on the market were developed from formation of mouse melanoma tumors and decreased invasion and metastasis of advanced tumors (Klein et al., apoptosis in human leukemia cells and in several human cancer cell lines such as human colon, neuroblastoma, and sarcoma cancer cell lines (Glucksmann et al., 1951).essential oil of warrant for further studies at the molecular level for detecting the detailed mechanism of the drug in preventing cancer.therapy targeting angiogenesis in cancer came to the herbs with anti-angiogenic and little toxicity properties have gained importance in the last decade.Non-toxic anti-angiogenic phytochemicals are useful in combating cancer by preventing the formation of new blood vessels including cancer.Extensive research over the last 50 years has indicated that Curcumin, Withanolide, Boswellic acid, Zerumbone and Resveratrol are plant derived major compounds from essential oil which both prevent and treat cancer by inhibiting angiogenesis and thereby preventing also have demonstrated that the extracts, natural pigments, anti-proliferative effects (Kitanaka et al., 1999).Our study has shown that the essential oil of tumor model with 39.47% inhibition on formation of tumor directed blood vessels.
In conclusion, based on the results obtained from our study and other related studies it can be concluded that the synergistic effects of essential oil of 10 injected mice makes them potentially valuable drug for cancer treatment.Essential oils have a long history of use proven to be safe for use.

2
at 37ºC.When needed for experiments (or during routine passaging 0.03 [W/V] solution, and then washed in phosphate cell survival and proliferation.It depends on the cell type, cellular metabolism and incubation time with on the capacity of mitochondrial enzyme succinate dehydrogenase of viable cells to reduce the yellow 2-yl)-2, 5-diphenyl tetrazolium bromide] into a purple is a direct proportionality between the formazan produced and the number of viable cells.Different concentrations were treated with the cell line (1×10 6 formazan crystals and the absorbance was taken at 570 follows and expressed as percentage of viable cells, %Cell viability=[(O.D of control-O.D of test compound)/(O.D. of control)]x100.

Figure 1 .
Figure 1.In vitro Cytotoxicity Activity of Essential Oil Towards B16F-10 Cell Line by MTT Assay

53 and Caspase 3 Figure 4 .
Figure 4. Histopathological Analysis of Lung.A) Normal mice-[Shows normal architecture of lungs with bronchioles, 10 cell line (Cancer alone)-[Shows massive abnormal cell the interstitium of the lung, coarse granules of black melanin pigment are seen both inside the macrophages as well as outside in tumor mass and regeneration of alveolar passage with ciliated columnar epithelial cells and reduced metastatic foci in lungs showing luminal epithelial-like morphology with well differentiated secretory glands are seen and reduction in the coarse granules of black melanin pigment are also seen

Figure 3 .
Figure 3. A) Effect of Essential Oil of Tridax procumbens L, on Total Leukocyte Count (WBC).Data are expressed

Figure 6 .FigureFigure 8 .
Figure 6.Level of Apoptosis by TUNEL Assay.A) Normal lung section showing the minimal number of apoptotic

Table 1 . Effect of Essential Oil of Tridax procumbens L in the Inhibition of Lung Tumor Nodule Formation
* Figure 5. Levels of Apoptosis by TUNEL Assay.Data are

Table 2 . Effect of Essential oil of Tridax procumbens L on Tumor Directed Capillary Vessel Formation
*highly recognized strategy for cancer treatment (Eichhorn