Anti-Metastatic Effect of Boiled Garlic Extract on A 549 Lung Cancer Cells Line by FAK Mechanism

According to epidemiological and animal study, the daily intake of garlic have important role in decreasing of cancer prevalence and tumor metastasis. However, mechanism of anti-metastatic of garlic is not fully understood. In the present study, we examined the anti metastatic effect of a brief exposure to boiled garlic extract (BGE) on A549 Lung cancer cells. Cytotoxicity BGE on A549 lung cancer cells was tested using MTT Assay. Migration and invasion scratching wound assay A549 lung cancer cells was tested usingscratching wound assay and agarose dropt explants assay.The growth of A549 lung cancer cell was measured using Colony forming assay. To explore the molecular mechanism and effect of BGE on migration and invasion, western blotting was used to analyze the FAK/Paxillin pathway. We found that BGE inhibits the growth, migration and invasion of A549 lung cancer cells at 300 μg/mL. Interestingly, BGE inhibits migration and invasion of A549lung cancer cells by decreasing the total of FAK and P-Y397 expression at 6 hours after a brief exposure, but not influence of the paxillin expression. We think the present finding demonstrate a novel anti-metastatic effect of garlic and suggests a certain garlic-derived compounds, BGE may be potential agent for inhibiting migration and invasion through decreasing the total of FAK and P-Y397 expression in the cancer cell.


INTRODUCTION
polyclonal anti-FAK [pY ] antibody was purchased from Biosource International (Camarillo, CA, USA).Monoclonal The beneficial effect of garlic (Allium sativum) and their anti-GAPDH antibody (clone 6G5) was purchased from constituent include hypolipidaemic, hypoglycaemic, Biogenesis Ltd. (England, UK).All other reagents used were chelating agents, immune stimulants, antihypertensive, of the purest grade available.antimicrobial, and possibly anticancer effect.Consumption of fresh garlic may be linked to potential Preparation of Boiled Garlic extract cytotoxicity of intestinal cells, not boiled garlic.Therefore, Garlic cultivated as an environmental-friendly agricultural proteinaceous cytotoxic component of garlic has change product was purchased from local supermarket near their functional properties such assolubility, mobilityand Jember last July 2013.Fresh garlic cloves (250 g) were toxicity, during short boiling (1).Initial evidence for the homogenized in 500 ml ice-cold distilled water using anticancer effect of garlic was provided by kitchen blender for 30 sec.The homogenates were filtered epidemiological study, that high intake of garlic may be through four layers of medical gauze and the filtrate was associated with a protective effect against stomach and lyophilized.The yield of extract (23.5 gpowder) is stored at colorectal cancers.Preclinical animal studies have -20 °C until use.For experiments, one gram of the indicated that garlic is highly effective in affording lyophilized garlic powder was extracted with 10 ml PBS, by protection against cancer induced by a variety of chemical vigorous vortexing (30 sec for 5 times with intermittent carcinogens.For instance, Belman demonstrated that cooling on ice for 1 min) and centrifugation (10.000 ×g, 10 topical application of garlic and onion oil inhibited the min).Aliquot of the above fresh garlic extract was heated incidence of tumor promoted by phorbol-myristate in boiling water for 30 min, then centrifuged at 10.000 x g acetate (2).Cancer chemoprevention by garlic for 10 min and the supernatant was used as boiled garlic c o n s t i t u e n t s h a s b e e n o b s e r v e d a g a i n s t extract (BGE).Protein concentration of the supernatant benzo[a]pyreneinduced forestomach and pulmonary was determined using Bradford assay (8).cancer in mice, N-nitrosomethyl-benzylamine induced Cytotoxic Effect of Boiled Garlic Extract esophageal cancer in rats, azoxymethane-induced colon carcinogenesis in rats, and 2-amino-1-methyl-6-A549 lung cancer cells were seeded in 24-well plates at a 4 phenylimidazo [4,5-b] pyridine induced mammary density of 2 × 10 cells/well in 1 ml MEM containing 10% tumorigenesis in rats (2)(3)(4)(5).
FBS overnight at 37 °C.Non-adherent cells were removed by gentle washing.Then cells were treated for 24 hr with Elucidation of the mechanisms by which garlic may offer various concentrations of boiled garlic extract.To assess protection against cancer has been a passionate subject of cell viability, 100 μl of MTT solution (5 mg/ml) was added research for the past 20 year.There are summarizes of to each well and incubated for another 4 hr at 37 °C.After molecular targets of cancer chemoprevention by garlic, the removal of supernatant, the generated formazan including modulation of carcinogen activation, induction crystal was dissolved by adding 125 μl/well of DMSO and of phase 2 enzyme and modulation of anti-oxidative the absorbance was detected at 540 nm using ELISA reader enzymes, inhibition of post-translational modification of (Bio-Rad, USA).The percentage of cell viability was oncogenic Ras, inhibition of cell cycle progression, histone calculated as follows: modification, induction of apoptosis, as well as inhibition angiogenesis and metastasis.Focal adhesion kinase (FAK) is an important regulator of cell migration in which a function required for the invasion and metastasis of Scratching Wound Assay cancer cell (6).Because of its major role as kinase upstream of a number of signaling pathways involved in A549 lung cancer cells were seeded into six well culture the processes metastasis, manipulation of FAK is likely to plates and allowed to grow to approximately 90-95% be more effective than manipulation of downstream confluence.Similar sized wounds were introduced to targets (7).The adapter protein, paxillin, is important in monolayer cells using a sterile yellow pipette tip.Wounded scaffolding a large number of proteins to the focal monolayer cells were washed three times by PBS to adhesion kinase.Moreover, overexpression and/or remove cell debris and then cultured with or without BGE.increased activity of FAK are common in a wide variety of The speed of wound closure was monitored and human cancers, implicating a role for FAK in photographed every 8-12 hr using a phase-contrast carcinogenesis.Given the important role of FAK in a large microscope until complete wound closure was observed in number of processes involved in tumorigenesis, the untreated control (9).metastasis and survival signaling FAK should be regarded as a potential target in the development of anti-cancer Agarose Drop Migration Assay drugs.Therefore, selective inhibitors of FAK need to be 6 A549 lung cancer cells (10 cells) in 50 µl of free serum developed.Combination of these selective FAK inhibitors medium DMEM were mixed with 150 ul of 0.2% (w/v) with cytotoxic agents could be a very promising anti-SeaPlaque agarose (Cambrex Bio Science Rocklan, Inc., cancer therapy.In the present study, the precise ME, USA) in free serum medium DMEM.One or two mechanism whether non toxic dose of boiled garlic extract microliter size drops of cell suspension were gently could inhibit migration of A549 lung cancer cell line in vitro delivered onto the pre-cooled surface of 48-well plates.was analyzed.
The agarose was allowed to solidify for 10 min at 4 °C.Covered with 200 ul of complete medium (DMEM+FBS METHOD 20% + Ab 1%) with BGE 75, 150, and 300 µg/mL or vehicles Rabbit Polyclonal Anti-FAK Antibody were added, and centrifuge.It did incubate at 37 °C for 24 hr.Take a picture using phase contras and 100 x.Cells that Rabbit polyclonal anti-FAK antibody was purchased from migrated out of the rim of the drop were counted for Santa Cruz Biotechnology (Santa Cruz, CA,USA).Rabbit Various concentrations of BGE were treated A549 lung quantitative determination of migrating cells.The cancer cells for 24 hr.The cytotoxicity of BGE was distance of edge of the agarose drop to the leading edge of determined by MTT assay.Compared to that of controls, migrating cells was determined on 4 sides of each droplet.
the remaining cell viability was not significantly altered by Since test in quadruplicate, 16 reading were obtained boiled garlic extracts, even at a concentration as high as 5 from each measurement.The migration of cells out of the mg/mL (Fig. 1.).It was therefore clear that a 24 hr agarose drop produced a uniform, expanding corona of treatment of boiled garlic extracts, at the concentration the cells around the agarose drop.While the majority of ranging from 0 to 5 mg/mL, has no cytotoxicity to A549 the cells migrated out of agarose drop in the corona, few lung cancer cells, highly metastatic cancer cells.This cells migrated much farther out the major population of concentration range was then applied in all subsequent cells.The number of cells that migrated beyond the experiments.corona of cells was counted (9,10).

Colony forming assay
The effects of BGE on colony formation on plates were measured as previously described (11,12).500 cells of A549 lung cancer cells in 2 ml of DMEM complete media were plated into each well of 6-well plates.The cells were cultured in the absence or presence of BGE concentrations of 150 and 300 µg/mL.After 7 days incubation, stop experiment and stain by crystal violet.Total number of colony was counted by ImageJ open software (National Institute of Health, Bethesda, Maryland, USA).

BGE Treatment and Western Blotting
Serum-starved A549 lung cancer cells were treated with orthovanadate, 1 mM PMSF, 1 µg/mL leupeptin, 5 µg/mL aprotinin and 2 µM pepstatin A) on ice.Lysates were scraped from the plates with a rubber policeman and the Boiled Garlic Extract Inhibits Cell Migration of A549 Lung supernatants were obtained after centrifugation at Cancer Cells 15.000 rpm for 10 min at 4 °C.Protein concentrations of In order to determine the inhibitory effect of boiled garlic the clarified cell lysates were determined using Bradford extracts on cell migration of A549 lung cancer cells, assay (8).The cell lysates were added with (5x) SDS sample analyses were carried out using two distinct cell migration buffer and heated at 95 °C for 5 min.The proteins were assays, such as scratch wound assay and agarose drop separated by 6-16% SDS-PAGE, and then transferred to migration assay.The results indicate that boiled garlic nitrocellulose membranes.Blocking was performed with extract inhibits the cell migration of A549 lung cancer cells TTBS buffer (10 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.1% in a dose dependent manner using scratch wound assay Tween 20) containing 5% skim milk powder.The (Fig. 2; Fig. 3); and agarose drop migration assay (Fig. 4; Fig. membranes were then incubated with primary antibody 5) for 4 hr at room temperature.Immunoblots were washed for 2 hr with changing TTBS buffer for every 20 min, and then incubated with horseradish peroxidase-linked secondary antibody for 30 min at room temperature.Immunoblots were washed for 1 hr with changing TTBS buffer for every 10 min, and developed with horseradish peroxidasedependent chemiluminescence (ECL) (Amersham Corp.).

Statistical Analysis
The values are represented as means standard deviation (S.D.).A paired Student's t-test was used to assess the significance of differences between two mean values or one-way analysis of variance (ANOVA).Results were considered to be statistically significant at P<0.05.

RESULT
Institute of Health, Bethesda, Maryland, USA).The results are expressed as mean ± SD (n=3).**P < 0.01 when compared to control.

Boiled Garlic Extract Inhibits Colony Forming of A549 Lung Cancer Cells
In order to determined inhibition effect of boiled garlic extracts on colony forming of A549 lung cancer cells, 500 cells of A549 lung cancer cells in 2 ml of DMEM complete media were pleted into each well of 6-well plates.The cells were cultured in the absence or presence of BGE concentrations of 150 and 300 µg/mL.After 7 days incubation, stop experiment and stain by crystal violet.

Note: Briefly, 500 cells of A549 lung cancer cells in 2 ml of DMEM
The results indicate that boiled garlic extract inhibits complete media were platted into each well of 6-well plates.The cells colony forming of A549 lung cancer cells at dose were cultured in the absence or presence of BGE concentrations of 150 dependent (Fig. 4).and 300 µg/mL.After 7 days incubation, stop experiment and stain by crystal violet.Total number of colony was counted by ImageJ open software (National Institute of Health, Bethesda, Maryland, USA).**P < 0,01 when compared to control.

Western Blotting Analysis
Activation of FAK on cancer cells related with increasing cell migration and proliferation (18).Paxillin is an adaptor protein down-regulation of FAK that related with cell motility.Therefore, the western blotting analysis was performed to see the effect boiled garlic extracts on FAK and its phosphorylation.The result was shown that boiled garlic extracts inhibited migration A549 lung cancer cell by   A problem on anti cancer agent, which it generally using toxic dose with some side effect.BGE was not containing toxic compound or decreasing beneficial compound.The bioactive compound such as selenium and copper content of rawgarlic is not altered by boiling (1,(15)(16)(17).Therefore, BGE can be used as nutriceutical compound for chemopreventive of cancer metastasis.In addition, the mechanism of migration and colony formation inhibitory effect of boiled garlic were confirmed by Western blotting analysis.Interestingly, BGE inhibits migration and colony forming of A549 lung cancer cells by decreasing the level of 397

Figure 7b. Western Blotting Analysis
FAK and its phosphorylation (P-Y ) from 3 hr after the treatment.Cell migration is a complex process that involves rapid change in dynamic of actin filaments, together with the formation and disassembly of cell adhesion site.FAK is crucial signaling component that is activated by numerous stimuli and function as biosensor DISCUSSIONS or integrator to control cell motility.FAK can influence the cytoskeleton structure of cell adhesion site and membrane Cancer metastasisis the leading cause of cancer deaths.It protrusion to regulate cell movement.Activation of FAK on is a multi-step process that a tumor cell migrates to distant cancer cells related with increasing cell migration and organ as new colony.There are several important event, proliferation (18).The adapter protein, paxillin, is including infiltration, invasion, migration, angiogenesis, important in scaffolding a large number of proteins to the escape from immune surveillance as well as forming new focal adhesion kinase (19-22).colony.In addition, cell migration plays an important role in diverse biological processes, such as development, the For decades, the fighting cancer has been an ongoing immune response, and cancer metastasis.The actin battle, and untill now, there is no exact way to treat this cytoskeleton is key component in cell migration.In which disease.Conventional treatments include surgery and tumor cell attachment to the extra-cellular matrix chemotherapy, nevertheless, these methods have several components, the gradation of the matrix by tumor celllimitation including physical pain, increased relapse and associated proteases, and tumor cell progression into the lower survival rate (2,6).One of the main reasons of cancer region where the matrixes were modified by proteolysis.
treatment is to eliminate cancer cells with minimal or no Evidence from several investigations suggests that some side effect; it is also favorable that it inhibits the metastatic components of the diet supplements, including garlic, process as well.Natural products have an important role in have shown anti-metastatic effect on cancer cells (13,14).
search for new drugs, even some of the most famous The mechanism of anti-metastatic of garlic extract was not widely used drugs are discovered from natural sources.fully understood yet and therefore, this study attempts to BGE that used in this study, was still crude extract.Its not elucidate the mechanism an anti metastatic effect of a clear which compound of BGE is responsible for the brief exposure to boiled garlic extract (BGE) on A549 Lung metastatic observed in the study.It can be one of the cancer cells line in vitro.
above or an unidentified one yet.To clarify this, further studies will be needed in the future.In the present study, the effects non-toxic concentrations of boiled garlic on cell migration of A549 lung cancer cells Taken together, the results suggested that the present were analyzed.Instead of using colony forming assay for finding demonstrates a novel anti-metastatic effect of analysis capacity of individual cancer cell forming new garlic and a certain garlic-derived compounds of BGE may colony, were carried out by two distinct cell migration be a potential agent for inhibiting cell migration and colony assays, using scratch wound assay and agarose drop forming by the modulation of FAK in cancer cells.migration assay.It was found that boiled garlic extract inhibited not only the cell migration, but also the colony CONFLICT OF INTEREST formation of A549 lung cancer cells at the concentration of 300 µg/mL.This result may be can answer some No competing financial interests exist.

Figure 1 .
Figure 1.Cytotoxic effect of boiled garlic extract on A549 lung

Figure 2 .
Figure 2. Statistical analysis of scratch wound assay Note: Statistical analysis was used image open software (National

Figure 5 .Figure 3 .
Figure 5. Agarose drop migration assay Note: A, Representative microscope photographic of cell migration out of

Figure 6 .
Figure 6.Statistical analysis of colony forming assay

Figure 4 .
Figure 4. Statistical analysis of agarose drop migration assay

Figure 5 .
Figure 5. Agarose drop migration assay Figure 7a.Western blotting analysis Note: A, Representative microscope photographic of cell migration out of agarose barrier ring; B, Schematic illustration of agarose drop migration assay using 12-well plates.
of BGE on protein expression of FAK and its phosphorylation (

Note:
B. Analysis results shown that BGE inhibits cell migration of A549 lung cancer cell by decreasing the level (Relative of optical density) of FAK 397 and its phosphorylation (P-Y ) from 3 hr after the treatment.