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Inhibition of pulmonary metastasis by Emilia sonchifolia (L.) DC: An in vivo experimental study George K Gilcy, Girija Kuttan∗

Q1

Department of Immunology, Amala Cancer Research Centre (Affiliated to the University of Calicut), Thrissur 680 555, Kerala State, India

a r t i c l e

i n f o

Article history: Received 28 March 2015 Revised 15 November 2015 Accepted 24 November 2015 Available online xxx Keywords: Emilia sonchifolia B16F10 melanoma Cytokines Vascular endothelial growth factor Matrix metalloproteinase Tissue inhibitor of matrix metalloproteinase

a b s t r a c t Background: Emilia sonchifolia (L.) DC is a widely distributed medicinal herb used mainly in the indigenous Ayurvedic system of medicine in India. This plant is one among the ten sacred plants of Kerala state in India, collectively known as Dasapushpam. Purpose: To assess the therapeutic efficacy of this well-known medicinal plant in a catastrophic complication like metastatic cancer progression. This study further aimed to scientifically validate the traditional medicinal use of this sacred plant. Study design: Highly metastatic B16F10 melanoma will spontaneously metastasize in C57BL/6 mice and is accepted as a useful murine model for the study on metastasis. Three different experimental modalities of prophylactic, simultaneous and after tumour development were used for data accumulation and analysis. Methods: Whole plant genuine extract of E. sonchifolia (25 mg/kg bodyweight) was administered intraperitoneally to C57BL/6 mice. Animals were sacrificed on 21st day after tumour induction and the lung tumour nodules were counted. Various lung and serum biochemical parameters along with major cytokine levels were recorded. Survival rate was monitored. Histopathology of the lung tissue and expression studies of the major genes involved in metastasis was also carried out. Results: E. sonchifolia significantly inhibited pulmonary tumour formation and increased the life span of animals. Lung collagen hydroxyproline, uronic acid, hexosamine, serum sialic acid, γ -glutamyl transpeptidase, vascular endothelial growth factor (VEGF), granulocyte monocyte colony-stimulating factor and other cytokine levels were significantly lowered in the treated group of animals. Histopathological analysis was also correlated with these findings. E. sonchifolia down regulated the expression of matrix metalloproteinases; extracellular signal-regulated kinases and VEGF at the same time up regulated the expression of tissue inhibitor of matrix metalloproteinases. Conclusion: Previous studies on E. sonchifolia proved its significant biological properties including antitumour, anti-inflammatory and antioxidant activities. Present report is so far the first study to demonstrate the anti-metastatic potential of this medicinal herb justifying its conventional use in the traditional medicine. © 2015 Published by Elsevier GmbH.

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Introduction

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One of the major causes of death in cancer patients is due to the ability of tumour cells to metastasize. Metastasis is a complex process that requires malignant cells to leave the primary tumour and proliferate at a distant site. The incidence of serious side

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Abbreviations: Erk, extracellular signal-regulated kinase; GGT, gamma glutamyl transpeptidase; GM-CSF, granulocyte monocyte colony-stimulating factor; IL-1β , interleukin-1β ; IL-6, interleukin-6; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of matrix metalloproteinase; TNF –α , tumour necrosis factor-α ; VEGF, vascular endothelial growth factor. ∗ Corresponding author. Tel.: +91 487 2307968; fax: +91 487 2307968. E-mail address: [email protected] (G. Kuttan).

effects limits the therapeutic application of cancer treatment using chemotherapeutic agents and ionizing radiations. Therefore, additional therapeutic approaches to eliminate these limitations must be established. Many traditional cancer therapies can improve key aspects of anti-cancer immunity by inducing tumour cell death in a way that is immunostimulatory or by modulating tumourinduced immunosuppression (Nowak et al. 2003). Agents that are able to block metastatic process of tumour cells have wide potential as anticancer agents therefore, it is essential to search for novel antimetastatic agents with minimum side effects. Hence there is an incitement to find out newer drugs with less toxic effects to prevent metastasis. Emilia sonchifolia (L.) DC (E. sonchifolia), the Lilac tassel flower belonging to the family Asteraceae with the local name of

http://dx.doi.org/10.1016/j.phymed.2015.11.017 0944-7113/© 2015 Published by Elsevier GmbH.

Please cite this article as: G.K. Gilcy, G. Kuttan, Inhibition of pulmonary metastasis by Emilia sonchifolia (L.) DC: An in vivo experimental study, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2015.11.017

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Muyalchevian in Kerala state of India, is an edible plant used in the Ayurvedic system of medicine for the treatment of gastropathy, diarrhoea, ophthalmia, nyctalopia, cuts and wounds, intermittent fevers, pharyngodyma and asthma (Nair and Chopra 1996). This plant is used in the folklore medicine for treating tumour and inflammation (Shylesh et al. 2005). Previous studies conducted on this plant revealed its anti inflammatory (Nworu et al. 2012) and anti tumour (Shylesh and Padikkala 2000) properties. There are reports on E. sonchifolia that evince its protective effect on oxidative stress and modulation of selenite cataract (Lija et al. 2006) and antinociceptive effect (Couto et al. 2011). Most recently the immunomodulatory effect was reported from our laboratory (Gilcy and Kuttan 2015). Studies on the apoptotic activity of this plant on cancer cells (Lan et al. 2012) further proved its anticancer potential. The present study was designed to investigate the antimetastatic activity of E. sonchifolia, with already proven antitumour activity. Materials and methods Plant material

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The authenticated whole plants, including roots and areal parts of E. sonchifolia (L.) DC collected locally was obtained from Amala Ayurveda Pharmacy, Thrissur district, Kerala state, India in July 2011, and the voucher specimen is deposited at the herbarium of Amala Cancer Research Centre (Voucher No. 108/ACRC). The plant name has been checked with http://www.theplantlist.org and the original publication was the contribution to the botany of India by Robert Wight (International plant names index (IPNI) with id 203080-1). Standardized whole plant genuine extract of E. sonchifolia (L.) DC was used for the study in accordance to the European Medicines Agency (EMA) guidelines. The whole plants of E. sonchifolia including roots stem and leave were dried at 45 °C, then crushed and coarsely powdered. Approximately 100 g of the whole plant powder was extracted with 500 ml, 70% v/v methanol in a soxhlet apparatus for 24 h. After extraction the solvent was evaporated to dryness at 42 °C under reduced pressure using rotary evaporator. The yield of the dried whole plant genuine extract of E. sonchifolia was 17% (w/w) with a drug extract ratio of 100 g of the initial whole plant powder; 17 g of the whole plant genuine extract. The composition of the extract was analysed using Saturn 2200 (Varian, Inc., CA, USA) GC/MS system equipped with fused silica capillary column (30 m x 0.25 mm x 0.25 μm) coupled to a mass selective detector, operated in EI mode (70 eV). Helium was the carrier gas at flow rate of 1 ml/min. The injector and detector temperature were maintained at 250 °C and 300 °C respectively. The oven temperature was programmed from 100 to 150 °C at 4 °C/min and then held at 270 °C for 20 min. Sample volume 1 μl was injected with 1:20 split ratio. Scan interval was 0.5 s with mass range, m/z 40–600. Standardization of the genuine E. sonchifolia whole plant extract with unknown composition and the identification of the marker compounds (Gilcy and Kuttan 2015) was made by comparing retention indices and mass spectra with those in the literature, as well as by computerized matching of the acquired mass spectra with those stored in the NIST and Wiley mass spectral library and other published mass spectra.

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Animals, cell line and experimental protocol

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Healthy adult male C57BL/6 mice (6–8 weeks old) weighing 25–28 g were accommodated in individual ventilated cages fed with normal mice chow and water ad libitum. All the animal experiments were conducted in accordance with the internationally accepted principles for laboratory animal use and care and carried out with the prior approval of the Institutional Animal

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Ethics Committee (IAEC) and were conducted strictly adhering to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) constituted by the Animal Welfare Division of Government of India (Sanction No. 149/1999/CPCSEA). B16F-10 melanoma cell line was obtained from the National Centre for Cell Sciences, India. Intraperitoneal route of drug administration is the commonly used route in small animals like mice because of the greater bioavailability, to reduce the chance of degradation by gastric juice and it offers a convenient alternative model to the intravenous administration of anticancer drugs in humans. Moreover the intraperitoneal administration of this immunomodulatory herbal extract (Gilcy and Kuttan 2015) with fast absorption to the vasculature has a direct effect on the cancer cells. The whole plant genuine E. sonchifolia methanolic extract in different concentrations (200, 100, 50, 25 mg/kg body wt.) were administrated intraperitoneally in mice for 14 days and observed for mortality, behavioural changes, and change in body weight. On 15th day, all the animals were sacrificed by cervical dislocation and selected organs such as liver, spleen, thymus, kidney, and lungs were dissected out and weights were recorded. Blood was collected by heart puncture; serum separated and was used for the analysis of hepatic and renal functions. Liver function markers, such as alkaline phosphatase (ALP), glutamate pyruvate transaminase (GPT), and kidney function markers such as creatinine and blood urea nitrogen were determined. For animal experiments 25 mg/kg of the whole plant genuine extract was resuspended in 1% gum acacia and administered intraperitoneally (ip) in 3 different modalities as follows: (I) Prophylactically with tumour induction: animals were treated with 10 consecutive doses prior to B16F10 tumour cell administration. (II) Simultaneously with tumour induction: given to animals simultaneously with B16F10 metastatic tumour cells for 10 consecutive days. (III) After tumour development: administration was done 7 days after B16F10 melanoma induction for 10 consecutive days.

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Apparatus and chemicals

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Thermal cycler (MJ Research, USA), Gel documentation system (Vilber Lourmat, France) and ELISA plate reader (ThermoLabsystems, USA) were used for the study. Hydroxyproline, glucuronic acid lactone and oligonucleotide primer sequences were purchased from Sigma Aldrich (Bangalore, India). N-Acetyl neuraminic acid was purchased from Sisco Research Laboratory (Mumbai, India). cDNA kit was purchased from Ambion Inc, Austin, Texas, USA. Specific quantitative sandwich ELISA kits for mouse IL-1β , IL-6, TNF-α and GMCSF were obtained from Pierce Biotechnology, Rock ford, IL, USA. ELISA kit for VEGF and TIMP was purchased from R&D Systems (Minneapolis, Minn., USA). All other chemicals used were of analytical reagent grade.

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Determination of antimetastatic activity of E. sonchifolia

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B16F10 melanoma cells (106 cells/animal) were injected through the lateral tail vein of C57BL/6 mice which serve as the metastatic tumour model.

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Lung tumour nodule formation and rate of survival

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C57BL/6 mice were divided into 4 groups (16 animals/ group). Pulmonary metastasis was induced to all the animals as described above. Group I animals were kept as untreated metastatic tumour

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bearing control. Treatment in Group II animals were done prophylactically, in group III animals simultaneously and in group IV animals after tumour development. On the 21st day after tumour challenge 8 animals from each group were sacrificed, blood was collected, lungs were excised and the surface lung tumour nodules were counted. For histopathological analysis lung tissues were fixed in 10% formalin, dehydrated in different concentrations of alcohol and embedded in paraffin wax. Sections (4 mm) were stained with eosin and hematoxylin. The remaining 8 animals in each group were observed for their survival. The mortality was observed and the percentage increase in life span (%ILS) was calculated using the formula %ILS = [(T – C)/C] × 100, where T (number of survival days of treated animals), C (number of survival days of control animals). The excised lungs were used to estimate following three parameters.

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Lung collagen hydroxyproline content

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The lungs were homogenized; protein precipitated with trichloroacetic acid (TCA), hydrolysed and hydrolysate evaporated to dryness and lung collagen hydroxyproline estimation was done by the chloramine-T method with hydroxyproline as standard (Bergman and Loxley 1970).

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Table 1 Primer sequences of genes used in the expression study. Genes

Primer sequence

Product size

MMP-2

Forward 5 -GAGTTGGCAGTGCAATACCT-3 Reverse 5 -GCCGTCCTTCTCAAAGTTGT-3

354bp

MMP-9 Erk-1 Erk-2 VEGF TIMP-1 TIMP-2 GAPDH

Forward 5 -AGTTTGGTGTCGCGGAGCAC-3 Reverse 5 -TACATGAGCGCTTCCGGCAC-3

Forward 5 -GCACGACCACACTGGCTTTC- 3 Reverse 5 -GATCAACTCCTTCAGCCGCTC-3 Forward 5 -ACAGGACCTCATGGA- GACGG-3 Reverse 5 -GATCTGCAACACGGGCA- AGG-3 Forward 5 -TGCTCACTTCCAGAAACACG-3 Reverse 5 -GGAAGGGTAAGCCACTCACA-3 Forward 5 -CTGGCATCCTCTTGTTGCTA-3 Reverse 5 -AGGGATCTCCAGGTGCACAA-3 Forward 5 -AGACGTAGTGATCAGGGCCA-3 Reverse 5 -GTACCACGCGCAAGAACCAT-3 Forward 5 -TGCTGG CGCTGAGTACGTCGT-3 Reverse 5 -GTGGAGGAGTGGGTGTCGCTG-3

327bp 512bp 216bp 350bp 414bp 525bp 557bp

Statistical analysis

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The data obtained were analysed using Graphpad InStat software and expressed as mean ± SD. Statistically significant differences between groups were calculated by the application of an analysis of variance (ANOVA) followed by Turkey’s multiple comparison test.

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Lung hexosamine content

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Results and discussion

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Lyophilized tissue samples were hydrolysed and evaporated to dryness. Hexosamine was estimated in the presence of Ehrlich’s reagent and glucosamine was used as standard (Elson and Morgan 1933).

Composition and toxicity profile of whole plant genuine extract of E. sonchifolia

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Lung uronic acid content

The principal constituents of the whole plant genuine extract were listed in Table 2. γ -humulene (47.6%), 2,6 dimethyl indolizine, (12.9%) and 3,7,11,15-tetramethyl-2-hexadecen-1-ol (phytol) (10.4%) were found to be the major compounds present. γ -humulene was reported to have apoptotic effects on human colorectal cancer cell HT29 (Lan et al. 2011). Indolizine and their derivatives were reported as highly active molecules having anticancer property and potent antioxidant activity (Vikas and Vipin 2014). 3,7,11,15tetramethyl-2-hexadecen-1-ol is a diterpene which has got antimicrobial, anticancer, anti inflammatory, antioxidant, antidiuretic and hypocholesterolemic properties (Mujeeb et al. 2014). Thus it can be hypothesized that the pharmacological properties of the plant may be either due to the combined action of these components or the activity of a specifically dominating active principle. The toxicity study revealed the no observed-adverse-effect level (NOAEL) of 50 mg/kg body wt. The doses of 25, 50 and100 mg/kg body wt., administered for 14 days did not produce any mortality, change in behaviour, body weight, relative organ weight, hepatic and renal functions when compared with normal untreated animals. Meanwhile, the administration of 100 mg/kg body wt. produced non-significant increase in the serum GPT and blood urea nitrogen when compared to normal animals as well as those animals treated with 50 mg/kg body wt. Hence an immunomodulatory study was conducted using three different concentrations (10, 25, 50 mg/kg body wt.) which are observed to be non-toxic. Since 25 and 50 mg/kg showed similar effects on the immune parameters and also the concentration of 10 mg/kg does not showed any significant effects, the data of minimum dose which is nontoxic but immunologically effective (25 mg/kg body wt.) as revealed from the immunomodulatory study was selected for further studies. The potent immunoregulatory activity of E. sonchifolia is evident from our recently published work (Gilcy and Kuttan 2015) on this medicinal herb, which belongs to Dasapushpam or a group of 10 sacred medicinal plants used in the Ayurvedic system of medicine.

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The lungs were digested with crude papain and hydrolyzed. The hydrolysate treated with sulphuric acid and uronic acid level was estimated in the presence of carbazole reagent with glucuronic acid lactone as standard (Bitter and Muir 1962).

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Serum sialic acid and γ -glutamyl transpeptidase levels

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Blood was collected by heart puncture, serum separated, and used to estimate serum sialic acid by thiobarbituric acid assay (Skoza and Mohos 1976) with N-acetyl neuraminic acid as standard. γ -glutamyl transpeptidase level in the serum was estimated by measuring the release of p-nitro aniline from γ -glutamyl pnitroanilide in the presence of an acceptor (glycylglycine) using a p-nitro aniline standard (Szasz 1976). Determination of serum VEGF, TIMP, IL-1β , TNF-α , IL-6, and GM-CSF levels Blood was collected from the caudal vein of all the experimental animals prior to B16F10 tumour induction and on 21st day after tumour challenge. Serum was separated and used for the estimation of VEGF, TIMP, IL-1β , TNF-α , IL-6, and GM-CSF using highly specific ELISA kits according to the manufacturer’s instructions.

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Expression of genes involved in metastasis

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Total RNA was isolated from the lungs, and cDNA was synthesized using Moloney murine leukaemia virus reverse transcriptase. Amplification was performed using specific primers of MMP2, MMP-9, Erk-1, Erk-2, TIMP-1, TIMP-2 and VEGF (Table 1). GAPDH was selected as a stable reference gene under the experimental conditions in this study.

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G.K. Gilcy, G. Kuttan / Phytomedicine xxx (2015) xxx–xxx Table 2 Chemical composition of the whole plant genuine extract of E. sonchifolia by gas chromatography mass spectrometry analysis (GCMS). Compound

Percentage

Compound

Percentage

2,6 dimethyl Indolizine Benzoic acid Benzene, 1,4-bis(1-methylethyl) 1, 3-Benzodioxole 4-tert-Butoxystyrene 2-Furancarboxaldehyde (2-methoxyethyl) benzene m-Aminophenylacetylene 2-Naphthalenol 2,6-Dimethoxyphenol 3-Hydroxy-4-methoxymandelate 2-(2-Propenyloxy)phenol 2(4H)-Benzofuranone

12.997 1.822 1.560 1.037 0.282 2.556 0.932 0.606 0.321 5.192 0.325 0.445 0.542

γ -humulene 4-[(1E)-3-Hydroxy-1-propen-1-yl]-2-methoxyphenol N-(4-Fluorophenyl) 2-Cyclohexen-1-one Cyclohexane, 1,1,3-trime 4-(3,3-Dimethyl-but-1-yne) Benzenepropanoic acid 2,7-Octanedione Palmitic anhydride 3,7,11,15-Tetramethyl-2-hexadecen-1-ol (phytol) 9,12- Octadecadienoic acid methyl ester Campesterol Cholesta-22,24-dien-5-ol

47.618 0.740 0.518 2.171 0.462 0.228 0.135 0.186 1.689 10.405 6.927 0.214 0.077

Table 3 Effect of E. sonchifolia on lung colonization of B16F10 melanoma cells and survival of animals. Treatment

Number of lung tumour nodules

Control E. sonchifolia (25 mg/kg body weight) Simultaneous Prophylactic Developed

250a

71.62 ± 19.30 195.87 ± 36.3 200 ± 30.96

Percentage inhibition of nodule formation

71.3∗∗∗ 22∗∗ 20∗∗

Percentage increase in survival (% ILS)

68∗∗∗ 31∗∗ 29∗∗

Note: The lungs were dissected out and observed for metastases on the 21st day after induction of B16F10 melanoma cells (106 cells) through the lateral tail vein. Values are mean ± S.D. a An arbitrary number of 250 is given for massive number of tumour nodules. ∗∗∗ P < 0.001, ∗∗ P < 0.01. Table 4 Effect of E. sonchifolia on the serum biochemical parameters of B16F10 melanoma bearing animals. Treatment

Sialic acid (μg/ml serum)

GGT (nmol p-nitroaniline/ml serum)

Normal Control E. sonchifolia (25 mg/kg body weight) Prophylactic Simultaneous Developed

21.86 ± 1.51 130.65 ± 6.54

24.45 ± 2.94 118.85 ± 9.98

113.1 ± 10.17∗∗ 62.66 ± 11.06∗∗∗ 112.16 ± 9.28∗∗

98.26 ± 12.39∗∗ 58.23 ± 9.39∗∗∗ 98.1 ± 8.01∗∗

Note: The serum was collected and assayed for different biochemical parameters on the 21st day after induction of B16F10 melanoma cells (106 cells) through the lateral tail vein. Values are mean ± S.D. ∗∗∗ P < 0.001, ∗∗ P < 0.01 significantly different from untreated control.

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E. sonchifolia whole plant extract could enhance the immune parameters, trigger stem cell proliferation as well as differentiation, and heighten antibody responses in a well-regulated way along with an enhanced cytotoxic T lymphocyte (CTL) activity in tumourbearing animals which signifies the augmented involvement of a cell-mediated immune system to defend against tumour. E. sonchifolia provides a stimulated and optimized immune response and the results may be considered as a solid scientific evidence for its conventional and traditional medicinal uses by regulating the body’s defence system against pathological manifestations. In the present study the immunologically effective dosage of the same genuine whole plant extract was selected to find out its therapeutic effectiveness to prevent metastatic progression, the most dreadful condition in cancer and the results obtained were promising that complements the claimed immunoregulatory effect. Effect of E. sonchifolia on Lung tumour nodule formation, rate of survival and on the serum levels of sialic acid and γ -glutamyltranspeptidase (GGT) The effect of E. sonchifolia on the inhibition of pulmonary tumour nodule formation is shown in Table 3. There was a sig-

nificant inhibition in the number of lung tumour colonies in the metastatic tumour bearing mice along with an increase in the life span. Untreated control animals developed massive numbers of tumour nodules on their lungs and were assigned an arbitrary number of 250. The haematoxylin and eosin (H&E) stained sections of lung tissues were shown in Fig. 1. Compared to normal animals (Fig. 1A) the control animals with tumour (Fig. 1B) developed massive tumour growth and fibrosis that reduced the alveolar space, thereby, reducing the vital capacity of the lung. The same areas were characterized by necrosis around the alveolar passages and bronchioles. Reduction in tumour mass around the alveoli and pleura was observed especially in the simultaneous modality (Fig. 1C) in comparison to the other two modalities prophylactic (Fig. 1D) and after tumour development (Fig. 1E). Simultaneous administration significantly (P < 0.001) reduced the serum sialic acid and GGT levels in the treated animals compared to the control groups with metastatic tumour (Table 4). Elevated expression of sialoglycans in the circulation of tumour bearing animals correlate with tumour aggressiveness and ability to metastasize and invade surrounding tissues. Aberrant sialylation contribute to therapy resistance of cancer. Sialic acids promote tumourigenesis by facilitating escape from apoptosis, formation of

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Fig. 1. Histopathology of the lungs of metastatic tumour bearing animals (A) Normal control (B) Tumour control with nodules; E. sonchifolia treated (C) simultaneously with tumour induction (D) Prophylactically with tumour induction (E) after tumour development.

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metastasis, and resistance to therapy. Selective approaches interfering with sialic acid expression would therefore have great potential to counteract tumour growth and metastatic formation (Bull et al. 2014). Higher serum levels of GGT are associated with an increased cancer risk. GGT is dysregulated in malignant cells and this will produce reactive oxygen species resulting in more aggressive tumour formation (Fentiman 2012). Previous studies conducted on E. sonchifolia proved its antioxidant activity and this is in agreement with the free radical scavenging activity of this plant as evident from the reduced serum levels of GGT. Effect of E. sonchifolia on the Lung collagen hydroxyproline, hexosamine and uronic acid levels The lung collagen hydroxyproline content was drastically elevated in the control group compared with normal levels, indi-

cating fibrosis of lung tissue. This elevated level was significantly (P < 0.001) reduced in simultaneous treatment. Simultaneous administration significantly (P < 0.001) reduced high level of lung hexosamine content and similarly the uronic acid level compared to tumour bearing control group (Table 5). Hydroxy proline is post-translationally produced from proline and forms a major component of collagen the main cellular matrix protein. The degradation of collagen in the metastatic tumour tissues will result in an elevated level of hydroxyproline (Phang et al. 2008). The acidic and basic modifications of monosaccharides found in the extracellular matrix yield uronic acids (glucuronic acid) and amino sugars (hexosamines) and they form a vital part in many structural polysaccharides and glycosaminoglycans (GAG) found in the ECM. Hexosamine found in the ECM serves as ground substratum for collagen synthesis (West et al. 1985). Reduction in the enhanced level of these three parameters were well in

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Fig. 2. Effect of E. sonchifolia on the serum cytokine levels (A) IL (interleukin)-1β , GM-CSF (granulocyte monocyte colony-stimulating factor), VEGF (vascular endothelial growth factor) (B) TNF (Tumour necrosis factor)-α , IL (interleukin) -6, TIMP (tissue inhibitor of matrix metalloproteinase). The serum was collected from the caudal vein on 21st day after induction of B16F10 melanoma cells (106 cells) and on the day before tumour induction (Pre). Values are mean ± S.D. a P < 0.001, b P < 0.01 significantly different from untreated control. Abbreviations: TC, tumour control; PR, prophylactic; SM, simultaneous; DV, developed.

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correlation with histopathological analysis of the lung tissue pointing towards the ability of this potent medicinal plant to combat with pulmonary metastasis. Effect of E. sonchifolia on the serum VEGF, TIMP, IL-1β , TNF-α , IL-6, and GM-CSF levels The production of IL-1β , GM-CSF, VEGF (Fig. 2A); TNF-α , IL-6 (Fig. 2B) were elevated in the serum of metastatic tumour bearing animals. This was significantly reduced (P < 0 0.001) by the simultaneous administration. The level of TIMP (Fig. 2B) was lowered in the tumour bearing animals and that was significantly (P < 0.001) increased by the extract treatment. Numerous studies have indicated that tumour cells exhibit an elevation in the constitutive production of proinflammatory cytokines such as TNF-α , IL-1β , IL-6, and GM-CSF. TNF-α can act as endogenous tumour promoter involved in the tumour progression

with pathways leading to activation of NF-κ B and AP-1 transcription complexes (Balkwill 2006). IL-1 expression increases the tumour invasiveness and metastasis by enhancing the expression of adhesion molecules on endothelial and malignant cells into the circulation and their dissemination to remote tissues. IL-6 aid tumour growth by inducing angiogenesis and inhibiting apoptosis and it plays an important role in cell migration, invasion and growth of malignancies. IL-6 activates different pathways most importantly stat 3 pathway that together contribute to its tumorigenic and antiapoptotic effects (Guo et al. 2012). Elevated serum levels of tumour promoting factor GM-CSF in cancer patients is considered as marker with high diagnostic sensitivity. GM-CSF will enhance invasive capacity of human lung cancer cells via increased expression of matrix degrading proteins. It is also involved in enhancing the expression of MMPs and this was reversible by GMCSF blocking (Gutschalk et al. 2013). Lowering of the serum levels of these cytokines indicate the potent effect of this plant on the

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Table 5 Effect of E. sonchifolia on the lung biochemical parameters of metastases bearing animals. Treatment

Hydroxyproline (μg/mg protein)

Uronic acid (μg/100 mg tissue wet wt.)

Hexosamine (mg/100 mg tissue dry wt.)

Normal Control E. sonchifolia (25 mg/kg body weight) Prophylactic Simultaneous Developed

0.95 ± 0.07 24.53 ± 3.13

35.65 ± 3.64 341.01 ± 38.54

0.44 ± 0.06 4.07 ± 0.69

19.93 ± 2.53∗∗ 11.08 ± 1.40∗∗∗ 20.37 ± 2.07∗∗

276.77 ± 38.76∗∗ 147.68 ± 30.33∗∗∗ 273.93 ± 33.23∗∗

2.95 ± 0.38∗∗ 2.02 ± 0.52∗∗∗ 2.90 ± 0.53∗∗

Note: The lungs were dissected out and assayed different biochemical parameters on the 21st day after induction of B16F-10 melanoma cells (106 cells) through the lateral tail vein. Values are mean ± S.D. ∗∗∗ P < 0.001, ∗∗ P < 0.01 significantly different from untreated control.

metastasis. MMP-9 is critical for the formation of metastatic niche due to its ability to liberate VEGF and thereby support angiogenesis. MMP-2 and MMP-9 enhance tumour cell migration and contribute to the establishment of metastasis-prone sites at tumour distant organs (Kessenbrock et al. 2010). MMPs are produced in the Zymogen form and their activation and activities are regulated by TIMPs. The disruption of MMP-TIMP balance will result in tumour invasion and metastasis (Chirco et al. 2006). In the present study, E. sonchifolia treatment downregulated the expression of MMPs at the same time upregulated the expression and elevated the serum level of TIMPs, indicating its regulatory effect on the inhibition of MMP. When MMPs are lacking or TIMPs are overproduced, formation of new tumours decreases as evident from these results. Autocrine VEGF signalling crucial in highly aggressive cancers is mediated by VEGF RTKs (VEGF receptor tyrosine kinases) and NRPs (neuropilin) can promote growth, survival, migration and invasion of cancer cells most commonly by activating the P13K-AKT pathway. Erk signalling pathway is involved in the regulation of cell motility and its inhibition is expected to result in antimetastatic as well as antiangiogenic effects in tumour cells (Kohno and Pouyssegur 2006). In this study, the expression of ERK-1, ERK-2, and VEGF have been downregulated and as supporting evidence the serum VEGF level was also reduced by the treatment. These results show that E. sonchifolia could inhibit all the pathways that link the MMPs and VEGF to tumour survival, proliferation, and invasion.

Fig. 3. Effect of E. sonchifolia on the expression of MMP-2, MMP-9, Erk-1, Erk2, TIMP 1, TIMP2, VEGF and GAPDH: lane 1, metastasis bearing control; lane 2, E. sonchifolia treated Abbreviations: MMP, matrix metalloproteinase; Erk, extracellular signal-regulated kinase; TIMP, tissue inhibitor of matrix metalloproteinase; VEGF, vascular endothelial growth factor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

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inhibition of cytokine mediated signalling that play an important role in tumourigenesis. Effect of E. sonchifolia on MMP-2, MMP-9, ERK-1, ERK-2, TIMP 1,TIMP 2 and VEGF, expression in metastatic lungs The mRNA expression of prometastatic genes such as MMP-2, MMP-9, ERK-1, ERK-2 and VEGF was found to be upregulated in the lung tissues of metastatic-tumour control animals (Fig. 3). It was interesting to note that the expression of these genes were downregulated or inhibited in mice treated with simultaneous modality. mRNA expression of antimetastatic genes TIMP-1, and TIMP-2 were absent or down regulated in metastatic tumour bearing animals and the treatment could increase its expression. MMPs are a family of zinc dependent endopeptidases and MMP mediated ECM degradation leads to cancer cell invasion and

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Conclusion

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The above experimental evidences like significant reduction in lung colonization and extension in the life span, reduction in tissue and serum metastatic markers and gene level expression studies clearly underline the antimetastatic property of E. sonchifolia on experimentally induced metastasis in C57BL/6 mice. These results may be considered as a solid scientific evidence for its conventional and traditional medicinal uses. In addition, these properties of the plant may be due to the synergistic effect of various active components or due to specific activity of certain compounds that are predominantly present in the plant. These results were well in correlation with the claimed immunoregulatory activity of the medicinal herb and in the present study the apt modulation of the immune parameters like cytokine levels once again prove the role of the immune regulation in a drastic metastatic condition.

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Conflict of interest

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The authors declare that they have no conflict of interest.

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Acknowledgment

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The authors express gratitude to Council of Scientific and Industrial Research (CSIR), Government of India, for the senior research fellowship provided to Ms. Gilcy George K.

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Please cite this article as: G.K. Gilcy, G. Kuttan, Inhibition of pulmonary metastasis by Emilia sonchifolia (L.) DC: An in vivo experimental study, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2015.11.017

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Please cite this article as: G.K. Gilcy, G. Kuttan, Inhibition of pulmonary metastasis by Emilia sonchifolia (L.) DC: An in vivo experimental study, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2015.11.017

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