Inhibition of Metastatic Lung Cancer in C57BL/6 Mice by Marine Mangrove Rhizophora apiculata

Cancer is a term used for disease in which abnormal cells proliferate without control and invade other tissues. Cancer disease is a leading cause of death worldwide, accounting for 7.6 million deaths in 2008 and could continue to rise 13.1 million deaths by year 2030 (WHO, 2008). The main reason for such high mortality from cancer is due to the highly invasive behavior of cancer cells, which usually results in metastasis (Khan and Mukhtar, 2010). Metastasis is a process by which cancer cells spread to other parts of the body through blood circulation and lymphatic system. A tumor formed by metastatic cancer cells is called as metastatic tumor (Klein, 2008). Many metastasis tumor develops at the first area of blood vessels that cancer cells arrive after leaving the primary tumor. After leaving the primary tumor, the lungs are one of the first site where metastatic cells are carried by the bloodstream (Hyoudou et al., 2004). The other common site for the cancer cells to metastasize includes brain, bones, liver, adrenal gland, lymph nodes, peritoneum, skin and other organs. Metastasis is an extremely complex process that remains to be a major problem in the management of cancer (Hunter et al., 2008). The metastatic process


Introduction
V Vinod Prabhu, C Guruvayoorappan* involves tumor cell invasion from the primary tumor, intravasation, arrest and extravasation of the circulatory system to form small tumors known as micrometastases that stimulate angiogenesis. Tumor cells break away from the primary tumor site and degrade proteins that make up the surrounding extracellular matrix (ECM) that separates tumor from neighboring tissues. Cancer cells degrades the protein, breach the ECM and metastasize to form secondary tumor at distant organs (Nguyen and Massagué, 2007). Metastatic cancer cells generally identical as cells of the primary cancer i.e. breast cancer that spreads to the lungs and forms a metastatic tumor is known as metastatic breast cancer, not lung cancer (Talmadge and Fidler, 2010). However, metastatic cancer cells and cells of the primary cancer usually have some molecular features in common such as the expression of certain proteins or the presence of specific chromosome changes. The competence of metastasize cells depends on the host's immune cells at the niche, blood circulation and capillary beds, but most of the cancer cells are been trapped by these barriers, but only few cancer cells prevail over these barriers and metastasize. Some time metastasized cancer cells can be dormant at distance niche for many years and could redevelop in later stage (Luzzi et al., 1998;Aragon Ching and Zujewski, 2007). More over there are many molecules involved during metastasis which includes adhesion molecules, proteases, cell mobility, ECM, growth factors, oncogenes, signal transductions and transcription factors.
Despite advancement in early cancer diagnosis and treatment included surgery, chemotherapy, radiotherapy and adjuvant therapies. Around 90% of cancer deaths are caused by metastasis that are resistant to conventional therapies (Gupta and Massague, 2006). Although there are several drugs that are used for cancer therapy, however there are no drugs available at present that blockade any single step in the metastatic process.
Natural products and their derivatives contribute more than 50% of all the drugs in clinical use of the world. Higher plants contribute not less than 25% of the total. Almost 60% of drugs approved for cancer treatment are of natural origin (Sithranga, 2010). Many experimental studies and clinical trial showed that many natural plants played an important role in blocking of lung metastasis from primary tumors (Schantz et al., 1987;Leyon and Kuttan, 2004;Leyon et al., 2005;Thejass and Kuttan, 2006).
Marine flora constitutes more than 90% of oceanic biomass that offer a great scope for discovery of new drugs. It is recognized that ocean contains a large number of natural products and novel chemical entities with unique biological activities that may be useful in finding the potential drugs with greater efficacy and specificity for the treatment of human diseases (Sithranga, 2010). The marine flora may contain novel compounds to withstand extreme variations in pressure, salinity, temperature and the chemicals produced are unique in diversity, structural, and functional features.
Mangroves have long been used in folk medicine to treat diseases and very few mangrove plants are reported for possible source of anticancer drugs, based on traditional knowledge and preliminary scientific work (Sithranga, 2010). Mangrove, Rhizophora apiculata (R.apiculata) family of Rhizophoraceae is halophytic used as folk medicine, based to on the fact that use of its root, leaf or stem extracts to a greater extent imparts an inhibitory effect on the growth of bacterial, viral and fungal pathogens (Premanathan et al., 1999;Antony et al., 2011). Polysaccharide extracted from the leaf of R.apiculata reported to inhibit HIV-1 or HIV-2 strains in various cell cultures (Premanathan et al., 1999). R.apiculata extract have a high content of flavanoids, tannins, catachin, anthroquinone, pyroligneous acid and syringol.
Our earlier report of phytochemical analysis of methanolic extract of R.apiculata by GC/MS and LC/ MS analysis shown the presence of pyrazole (alkaloid), ketone, thiazolidinediones and 4-pyrrolidinyl with a wide range of biological properties such as anti-tumor, antiinflammatory, immunostimulatory and chemoprotectant activities (Prabhu and Guruvayoorappan, 2012;2012a). Anti-metastasis activity of R.apiculata extract have not been reported elsewhere, to provide validity to the claims that R.apiculata extract has numerous polyphenol compounds with potential health benefits. In the present study we have evaluated the anti-metastatic activity of methanolic extract of R.apiculata in B16F-10 melanoma cell induced lung metastasis in C57BL/6 mice.

Plant collection
R.apiculata (Vernacular name -Surapunnai in Tamil), whole plant were collected from the Pichavaram mangrove forest located in Cuddalore District, Tamil Nadu on the southeast coast of India. Pichavaram is the second largest mangrove forest in the world.

Experimental animals
C57BL/6 mice (male 4-6-wk-old) weighing (20-25g) were kept in a pathogen free air controlled room maintained at 24°C with an ~50% relative humidity and 12-hr light/dark cycle, and provided ad libitum access to normal mice chow (Sai Feeds, Bangalore, India) and filtered water. All animal experiments were performed according to the rules and regulations of the Institutional Animal Ethics Committee of the Government of India.

Cell lines
B16F10 melanoma cells were obtained from the National Centre for Cell Sciences (Pune, India) and maintained in DMEM (Hi Media, Mumbai, India) containing 10% fetal bovine serum (FBS; Hi Media) and 1% antibiotic/antimycotic solution. All culture steps took place at 37°C in a 5% CO 2 /95% air incubator.

Chemicals
Gum acacia was purchased from Hi-Media, Formaldehyde solution was procured from Universal Laboratories Pvt. Ltd. (Hyderabad, India). All chemicals used were analytical or reagent grade.

Methanolic extract preparation
R.apiculata samples collected from the Pichavaram mangrove forest (Tamil Nadu) were taxonomically identified and authenticated at the Department of Botany at M.E.S. Kalladi College (Mannarkkad, India), and a voucher specimen was deposited (Accession Number: Rhiz-018). The plant material was dried at 45°C and then powdered. Ten gram of the material was stirred overnight in 70% methanol (100 ml), and then centrifuged at 10,000 rpm (10 min, 4°C). The resultant supernatant was collected and the methanol removed by evaporation (yield of final product=12% [w/w]).

Biochemical measurements
C57BL/6 mice were separated into two groups (9 Asian Pacific Journal of Cancer Prevention, Vol 14, 2013 1835 DOI:http://dx.doi.org/10.7314/APJCP.2013.14.3.1833 Rhizophora apiculata Inhibits Lung Metastasis in C57BL/6 Mice nos/group) for the experiment. All the animals in two groups were induced metastasis by injecting metastatic B16F10 melanoma cells (1x10 6 cells/animal) via lateral tail vein. Group I was kept as metastasis induced untreated control and received only vehicle. Group II were treated with R.apiculata extract (10 mg/kg b.wt. (i.p.) for ten consecutive days. The animals were euthanized and blood samples were collected from the animals on different day intervals (7 th 11 th and 21 st day) serum samples were isolated for biochemical measurements such as NO, GGT and sialic acid levels.

Determination of the effect of R.apiculata on serum NO level of metastatic tumor bearing animals
On day (7 th 11 th and 21 st day) serum NO level was estimated by the use of a Griess reaction. The reaction mixture containing sodium nitroprusside and serum was incubated at 25ºC for 150 min. After incubation, 1ml of the reaction mixture mixed with equal volume of Griess reagent and allowed to stand for 15 min at room temperature. The absorbance of pink colored chromophore formed was measured at 546 nm against the corresponding blank solutions and expressed in µm (Green et al., 1982).

Determination of the effect of R.apiculata on serum GGT level of metastatic tumor bearing animals
On day (7 th 11 th and 21 st day) serum GGT level was estimated by measuring the release of p-nitroaniline from gamma glutamyl p-nitroaniline in the presence of glycyl glycine. The GGT content was determined from the graph plotted using p-nitroaniline as the standard (Tate and Meister, 1974).

Determination of the effect of R.apiculata on serum sialic acid level of metastatic tumor bearing animals
C57BL/6 mice were separated into two groups (9 nos/group) for the experiment. All the animals in two groups were induced metastasis by injecting metastatic B16F10 melanoma cells (1x10 6 cells/animal) via lateral tail vein. Group I was kept as metastasis induced untreated control received only vehicle. Group II were treated with R.apiculata extract (i.p.) for ten consecutive days. Blood samples were collected by cardiac puncture on (Day 21) and serum was separated for the estimation of protein bound sialic acid level was determined by thiobarbituric acid assay (Skoza and Mohos, 1976;Bhavanandan et al., 1981). The serums were hydrolyzed using 0.2 N sulphuric acid. The hydrolysate was oxidized with periodic acid and incubated at 37 o C for 1 min. After terminating oxidation using sodium arsenate, 6% thiobarbituric acid was added. Sialic acid was estimated at 549 nm with reference to 532 nm after adding DMSO. Sialic acid content was determined from the standard graph plotted using n-acetyl neuraminic acid.
Determination of the effect of R.apiculata on lung collagen hydroxyproline, hexosamine, uronic acid and tumor nodules count of metastatic tumor bearing animals C57BL/6 mice were separated into two groups (9 nos/ group) for the experiment. All the animals in two groups were induced metastasis by injecting highly metastatic B16F10 melanoma cells (1x10 6 cells/animal) via lateral tail vein. Group I was kept as metastasis induced untreated control received only vehicle. Group II were treated with R.apiculata extract (i.p.) for ten consecutive days. Animals from each group were euthanized on 21 st days (final day of experiment) of tumor induction and the lungs were excised for counting tumor nodules and were used for the estimation of lung hydroxyproline (Bergman and Loxley, 1970), lung hexosamine (Elson and Morgan, 1933) and lung uronic acid (Bitter and Muir, 1962).

Determination of the effect R. apiculata on lung collagen hydroxyproline of metastatic tumor bearing animals
Lung collagen hydroxyproline was determined by the method of Bergman and Loxley (1970). The lungs were homogenized and protein precipitated with TCA were hydrolyzed for 24 hr at 110 0 C in sealed glass tubes using 6 N HCl. The HCl was evaporated and the remaining hydrolysate residue was allowed to dryness. The residue was dissolved in distilled water and assay by chloramines -T method. The absorbance was measured at 560nm. Standard graph was plotted using reagent hydroxyproline standard.

Determination of the effect R. apiculata on lung hexosamine of metastatic tumor bearing animals
The hexosamine content present in the lung tissue was estimated by the method of Elson and Morgan (1933). Lyophilized tissue samples were hydrolyzed with 2 N HCl at 100 0 C for 6 hr in sealed glass tubes. After hydrolysis, the HCl was evaporated and the remaining hydrolysate residue was allowed to dryness. The residue was dissolved in distilled water and treated with 2% acetyl acetone. The hexosamine level was determined in the presence of Ehrlich's reagent at 530 nm. Standard graph was plotted by using glucosamine standard.

Determination of the effect of R. apiculata on lung uronic acid content of metastatic tumor bearing animals
The uronic acid content present in the lungs of metastatic tumor bearing animals was estimated by the carbazole reactions (Bitter and Muir, 1962). The lungs were digested with crude papain and were hydrolyzed at 100 0 C for 20 min in a sealed glass tubes. After hydrolysis, the hydrolysate was allowed to treat with sulfuric acid. The uronic acid level was determined by using carbazole reagent at 530 nm. Standard graph was plotted using by glucuronic acid lactone.

Determination of the effect of R. apiculata on the survival rate of metastatic tumor bearing animals
The remaining animals (n=3) of each group from the above experiment were observed for their survival rate. The mortality rate of each animal were observed and the percentage increase in life span (%ILS) was calculated using the formula %ILS=T-C/C×100. Where 'T' represents the number of survival days of treated animals and 'C' represents the number of survival days of control animals.

Histopathological examination
A portion of excised lung tissue were fixed in 10% formalin, cut into 5-µm thickness, stained using H&E (hematoxylin and eosin) and then examined for histopathological changes. The stained sections of lungs tissue samples were examined for large metastasis, hyperchromatic nucleus and pleomorphism.

Statistical analysis
The results are expressed as mean±SD. Statistical evaluation was performed using a one-way analysis of variance (ANOVA) followed by a Dunnett test using Graph Pad Instat (Version 3.0 for Windows 95; Graph Pad Software, San Diego, CA). p-values<0.05 were considered statistically significant.

Effect of R.apiculata on the serum NO and GGT level of metastatic tumor bearing animals
The effect of R.apiculata on serum NO and GGT level in metastases tumor bearing animals is shown in Table 1. The administration of methanolic extract of R.apiculata significantly (p<0.01) reduced the serum NO level (27.20±0.31µm) on 21 st day when compared with metastasis control (34.29±1.13µm) on the same day. The administration of R.apiculata extract also significantly (p<0.01) decrease the serum GGT level (44.30±1.4 nmol p-nitroaniline/ml) on 21st day when compared with metastasis control (81.44±4.0 nmol p-nitroaniline/ml) on the same day.

Effect of R.apiculata on serum sialic acid, lung hydroxyproline, hexoamine and uronic acid of metastases bearing animals
The effect of R.apiculata on serum sialic acid, lung hydroxyproline, hexoamine and uronic acid of metastases bearing animals is presented in The lung hexoamine level of R.apiculata extract treated animals were significantly (p<0.01) decreased (1.35±0.14 mg/100 mg tissue dry weight) when compared with metastatic control (3.17±0.17 mg/100 mg tissue dry weight). After the treatment of R.apiculata extract the lung uronic acid level significantly (p<0.01) reduced (93.20±2.30 µg/100 mg tissue wet weight) when compared with metastatic control animals (238.30±4.25 µg/100 mg tissue wet weight) on the same day.

Effect of R.apiculata on inhibition of lung metastatic nodule formation and on survival of animals
Effect of R.apiculata on inhibition of lung metastatic nodule formation and on survival of animals is shown in Table 3. B16F-10 melanoma cells which form lung tumor in experimental animals (Engbrin et al., 2002). The treatment with R.apiculata significantly (p<0.01) reduced (26.6±8.1) the number of countable colonies of pulmonary tumor nodules formation of B16F-10 melanoma cells in the lungs of the experimental mice when compared with metastasis control animals which shows massive growth of tumor nodules in lungs (45.2±9.0). The percentage of decrease in lung nodule formation of R.apiculata extract treated animals were (41.1%).
The survival rate of R.apiculata extract treated animals was increased when compared to metastatic control animals. The maximum survival rate were observed when R.apiculata extract administered (81±3.2) (107.6%) when    Table 3. The percentage increase in life span=T-C/ Cx100, where T and C are the number of days survived by the treated and control group of animals respectively. The increase in survival days and increase in life span were ILS%=107.3%.

Histopathology analysis
Histopathology analysis of lung metastatic tumor bearing animals is shown in Figure 1

Discussion
Metastasis is one of the hallmarks of malignant neoplasm or cancer which is the leading cause of death in many cancer patients (Nonaka et al., 1993;Lee et al., 2008). The degree of ability to spread varies between different types of tumors. In most cases, cancer patients with localized tumors have a better chance at survival than those with metastatic tumors. The metastatic capacity of a tumor cell is reliant on several factors. Tumor cells invade the tissues adjacent of primary tumor, increase their motility and migrated into the blood circulation (intravasate). Then the tumor cells elude the host defense mechanisms and adhere to a suitable niche. Consequently the tumor cells (extravasate) and invade into the secondary niche and evade apoptosis, regulates proliferation and angiogenesis (Chambers et al., 2001;Herzig and Christofori, 2002;Prabhu et al., 2012b). Among the metastasis cancers, the lung is the first organ to be encountered by the tumor cells making it a major site for tumor metastasis (Hyoudou et al., 2004). The survival rate for patients with metastatic melanoma is less than 10% (Bhatia et al., 2009). Tumor metastases are treated with surgery, radiotherapy, chemotherapy and hormone therapy or "multimodal therapy" (Wang et al., 2012). The metastatic treatment depends on, type of primary cancer, size and location of the metastases. Therefore, metastasis is a major target of cancer therapy and it is complicated to treat metastasis effectively with the available treatment with lesser survival rate (Bhatia et al., 2009). Many plants serve as novel drugs throughout the world. World Health Organization (WHO) reveals around 80% of the world population depends on plant derived medicine for their little of no adverse effects (WHO, 2008). Therefore in this study we have investigated the anti-metastatic potential of R.apiculata extract on B16F-10 metastatic lung cancer cell line in C57BL/6 mice.
Nitric oxide (NO) is a pleiotropic regulator with numerous biological processes, including vasodilatation, neurotransmission, macrophage-mediated immunity and immune defenses (Nathan, 1992). Concurrently NO also play an important role in tumor progression causing DNA damage, metastasis and angiogenesis (Shi et al., 1999;Fukumura and Kashiwagi, 2006;Prabhu and Guruvayoorappan, 2010). Increased NO generation in cancer cells has direct correlation with circulating tumor cells for their survival, high metastatic ability and angiogenesis by up-regulating vascular endothelial growth factor (VEGF) and VEGF-induced neovascularization (Edwards et al., 1996;Ambs et al., 1998). NO regulates some of the cellular adaptive responses to hypoxia which are associated with increased metastatic potential (Branco-Price et al., 2012). In the present study the R.apiculata extract significantly reduced the NO production level preventing high metastatic ability of tumor cells in experimental animals. The plant extract protective effect was further proved by showing reduction in lung tumor nodules (41.1%inhibition) compared to the metastatic control. This reduction in tumor nodules is correlated with an increase in the life span (107.6%) of the metastatic tumor bearing animals.
γ-glutamyl transpeptidase (GGT) is the only known enzyme that cleaves the g-glutamyl-cysteine peptide bond in GSH and other g-glutamyl compounds. GSH is synthesized intracellularly which provides energy to the tumor cells through gamma glutamyl cycle. GGT catalyzes glutathione (GSH) breakdown. An increase of GGT levels is a common finding in human tumors and as an important aspect of the tumor cell phenotype. In tumor cells, GGT over expression and its effects on GSH metabolism may affect several specific functions. It has been reported that variations in GGT expression and cellular levels of GSH is associated with modulation of metastatic properties of several tumors (Carretero et al., 1999). During tumor progression the serum GGT level increases due to cellular proliferation therefore serum GGT level can be used as marker for cellular proliferation (Pradeep and Kuttan, 2002). The administration of R.apiculata extract significantly reduced the serum GGT level in metastatic tumor bearing animals which is directly correlated with decrease in tumor cell proliferation and metastasis.
Sialic acid is a derivative of neuraminic acid occurs as a terminal component of carbohydrate chain of glycoproteins. Sialic acid at the terminal position is involved in cellular adhesion (components of many cell surface receptors) and have ability to cellular recognition sites during invading foreign cells including cancer cells (Schauer, 1985). Metastatic cancer cells often express a high density of sialic acid-rich glycoproteins (Fuster et al., 2005). Total serum sialic acid level has been recognized as a valuable non-specific monitor of tumor burden in melanomas. The increase in the sialic acid level on the surface of malignant cells creates a negative charge on cell membrane which creates electrostatic repulsion between cells that initiates the malignant cells to metastasize through blood circulation (Fuster et al., 2005). The amount of sialic acid on the surface of malignant cells is directly correlated with the metastatic ability of the tumor cells (Vedolva and Borovansky, 1994). The treatment of R.apiculata extract significantly decreased the serum sialic acid level in metastatic tumor bearing animals. The R.apiculata extract prevented the expression of sialic acid on the surface of malignant cells preventing negative charge and electrostatic repulsion thus inhibiting metastasis. The decreased in serum sialic acid level were directly correlated with the reduction in metastasis.
Hydroxyproline is a major component of the protein collagen. Hydroxyproline and proline play key roles for collagen stability (Nelson and Cox, 2005). In the lung, collagen is found associated with bronchi, with blood vessels, and with the alveolar interstitium; it plays an important role in the maintenance of lung structure and function. During lung metastasis there will be enormous accumulation of fibrosis in lungs resulting in substantial deposit of collagen (hydroxyproline) in the alveoli of lungs (Pradeep and Kuttan, 2002). Elevated level of collagen in the lung is associated with pulmonary fibrosis resulting in inability of the lung to function normal gas exchange (Voet and Voet, 1995). Therefore lung hydroxyproline level has been used as an indicator to determine collagen amount and lung fibrosis (Voet and Voet, 1995). In the present study the administration of R.apiculata extract significantly reduced the lung hydroxyproline level in metastatic tumor bearing animals. The R.apiculata extract inhibited excessive accumulation of collagen in lungs preventing pulmonary fibrosis during lung metastasis.
Hexosamine is the acidic and basic modification of monosaccharide yield uronic acids (glucoronic acid) and amino sugars (hexosamine). Hexosamine plays an important role in the production of N-acetyl neuraminic acid (sialic acid) which is present on the surface of malignant cells responsible for metastasis. Hexosamine, an integral part of many structural polysaccharides and glycosaminoglycans (GAG) found in the ground surface of ECM which promotes metastasis by opening the spaces for malignant cells to migrate. The excess hexosamine level is directly correlated with the active growth and proliferation of malignant cells (West et al., 1985). Therefore it is known marker and promoter of metastasis (Lipponen et al., 2001). In the study the administration of R.apiculata extract resulted in significant reduction in lung hexosamine level of metastatic tumor bearing animals. The R.apiculata extract significantly inhibited the excess synthesis of hexosamine by blocking the ECM thus preventing malignant cells metastasis and lung fibrosis (Pradeep and Kuttan, 2002).
Malignant cells yield uronic acid by the oxidation of the primary alcohol group of aldoses (monosaccharide) sugar derivatives. This leads to the formation of glucuronic acid lactone which is an essential form of uronic acid. In the present study the R.apiculata extract significantly decreased the lung uronic acid level in metastatic tumor bearing animals. The histopathological analysis of the lungs of R.apiculata treated animals also shows consonant results as above by reduction in the lung tumor nodules. Therefore, the above experimental evidences substantiate the anti-metastasis activity of the R.apiculata extract on experimentally induced lung metastasis in C57BL/6 mice.
In conclusion, this study is the first of its kind to report on the anti-metastatic activity of R.apiculata extract in metastatic tumor bearing animals. The inhibitory effect exhibited by R.apiculata extract could be attributed to the high content of pyrazole, 4-pyrrolidinyl, ketone derivatives and thiazolidinediones found in the methanolic extract (Prabhu and Guruvayoorappan, 2012). Therefore R.apiculata extract may be used as a therapeutic target to inhibit metastasis during tumor progression. Further investigations are required to trace out the exact mechanisms involved in the anti-metastatic property of R.apiculata.