Anti-migration and Anti-invasion Abilities of Methanolic Leaves Extract of Clerodendrum Inerme on Lung Cancer Cells

Lung cancer is the most common cancer types in men and fourth cancer in women. It is the major cause of cancer-related death in the world. There are about 1,350,000 newly developed cases and 1,180,000 death cases of lung cancer, globally. Lung cancer is divided into two major types, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC).1 Non-small cell lung cancer (NSCLC) is the most common type that accounts for 80-90% of lung cancers, which is divided into 3 major pathologic subtypes including adenocarcinoma, squamous cell carcinoma and large cell carcinoma.2 About 30-40% of patients with advanced NSCLC will develop metastasis, which is the lung cancer cells migrate to and invade to the distant organs, such as bones, brain and contralateral lung.3 Metastasized lung cancer is difficult for treatment because it is highly resistant to radiation and conventional chemotherapeutic agents. It is the main cause of cancer-related death in lung cancer patients.4,5 Thus, the potential therapeutic agent for treating lung cancer metastasis is an urgent therapeutic need.


INTRODUCTION
Lung cancer is the most common cancer types in men and fourth cancer in women. It is the major cause of cancer-related death in the world. There are about 1,350,000 newly developed cases and 1,180,000 death cases of lung cancer, globally. Lung cancer is divided into two major types, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). 1 Non-small cell lung cancer (NSCLC) is the most common type that accounts for 80-90% of lung cancers, which is divided into 3 major pathologic subtypes including adenocarcinoma, squamous cell carcinoma and large cell carcinoma. 2 About 30-40% of patients with advanced NSCLC will develop metastasis, which is the lung cancer cells migrate to and invade to the distant organs, such as bones, brain and contralateral lung. 3 Metastasized lung cancer is difficult for treatment because it is highly resistant to radiation and conventional chemotherapeutic agents. It is the main cause of cancer-related death in lung cancer patients. 4,5 Thus, the potential therapeutic agent for treating lung cancer metastasis is an urgent therapeutic need.
Clerodendrum inerme is a medicinal plant in the family Verbenaceae. Its common names include glory bower, bag flower, and bleeding-heart. 6 It is widely distributed in South and South-east Asia, Australia and Pacific islands. 7 Different parts of C. inerme are used as a traditional medicine for the treatment of rheumatism, cough, scrofulous infection, venereal infection, skin diseases, beriberi, and tumors. [8][9][10] The leaves of C. inerme are used for treating fever, cough, skin rashes, chronic pyrexia and boils, and are used in conjunction with other plant leaves. 11 C. inerme exhibited many pharmacological effects including hepatoprotective, 12 antiinflammatory, 13 anti-bacterial, [14][15][16] anti-oxidant, 17 antidiabetic, 18 antiparasitic, 19 antiallergic, 20 and anticancer [21][22][23][24] activities. Recent studies showed that C. inerme leaves extract displays antiproliferation and anticancer activities on lung cancer cell line A549. 25,26 However, the anti-migration, anti-invasion and anti-adhesion activities of C. inerme leaves extract on cancer cells have not been reported. Therefore, the present study investigated the inhibitory effect of methanolic leaves extract of C. inerme on cancer migration, invasion and adhesion in A549 cells.

Collection of plant material and extract preparation
Province. Fresh leaves were washed under running tap water and then dried for 36 hours using a hot air oven at 60°C. Dried leaves were crushed and subjected to extraction using the maceration process in methanol for 7 days at temperature. The extracts were collected and concentrated at 40°C under reduced pressure using a rotary evaporator. The dried extracts were stored at 4°C until further use.

Cell culture
The human non-small cell lung cancer (NSCLC) cell lines, A549 was obtained from the American Type Culture Collection (Rockville, MD). A549 cells were cultured in DMEM supplemented with 10% FBS (Life Science, Little Chalfont, UK), 100 U/mL penicillin and 100 µg/mL streptomycin (Life Science, Little Chalfont, UK) at 37°C in a humidified atmosphere of 5% CO 2 .

Cell viability assays
An MTT assay was carried out to determine the cytotoxic effect of methanolic extract of C. inerme leaves on A549 cells. The cells were plated in 96-well plate at a density of 7x10 3 cells/well and treated with methanolic extract at different concentrations (0, 6.25, 12.5, 25, 50, 100, 200, and 400 µg/mL) for 24 hours. After incubation, 0.5 mg/mL of MTT solution was added to each well and the plates were further incubated for 2 hours at 37ºC. The supernatant was removed, and 100 µL of DMSO was added to solubilize the water-insoluble purple formazan crystals. The absorbance was measured using a Microplate spectrophotometer at 570 nm and cell viability percentage (%) was calculated relative to the control. IC 50 (inhibitory concentration 50%) value was calculated using the software GraphPad Prism 3.03 (GraphPad Software Inc., San Deigo, CA). Cell viability assay was performed with three independent experiments.

Wound healing assay
Wound-healing assay was performed to determine cell motility of A549. Briefly, the cells were seeded a density of 30x10 4 cells/mL in 6-well plates until 90% confluence. Then the cells were scratched with a sterile 200 µL pipette tip to generate the wound area. Cellular debris was washed with phosphate buffer saline, and cells were treated with non-toxic concentrations of methanolic extract (0, 200 and 400 µg/mL) for 24 hours. The wound closure was observed and photographed at 24 hours with an inverted microscope. The wound area was measured by NIH ImageJ software (NIH, Bethesda, MD). The experiments were repeated three times.

In vitro migration assay
Effect of methanolic extract of C. inerme leaves on cell migration was assayed by 24 well transwell chambers. Cells were seeded in 35 mm 3 dishes and treated with 0, 200 and 400 µg/mL for 24 hours. After incubation time, cells were harvested and seeded to the upper chamber of the transwell insert (polyethylene terephthalate (PET) filters with 8 µM pore size, corning life sciences) in serum-free medium. FBS medium as a chemoattractant was added to the lower chamber, and thereafter these transwell inserts were incubated for 24 hours at 37˚C in 5% (v/v) CO 2 . After incubation, the non-migratory cells were removed with a cotton swab and those on the lower surface of the membrane were fixed with methanol and stained with 0.5% crystal violet. Cells that migrate through the membrane were viewed and photographed under an inverted microscope. The percentage of the migratory cells for each treatment was calculated by NIH ImageJ software (NIH, Bethesda, MD).

Cell invasion assay
Effect of methanolic extract of C. inerme leaves on cancer cells to invade through membrane coated with 30 µg of Matrigel (Corning incorporated, Bedford, MA, USA) was measured by Modified Boyden chamber assay. 27 A549 cells were pretreated with 0, 200 and 400 µg/mL of the extracts for 24 hours. Cells in serum-free medium were seeded onto the upper chamber and medium containing 10% FBS was added into the lower chamber as a chemoattractant. The Boyden chamber was incubated for 24 hours at 37°C. At the end of incubation, the cells in the upper surface of the membrane were removed with a cotton swab. Cells that invade across the Matrigel to the lower surface of the membrane were fixed with methanol, stained with 0.5% crystal violet and washed with PBS. The invading cells on the lower surface of the membrane filter were viewed and photographed under an inverted microscope. The data are presented as the percentage of cell invasion from five random fields.

Cell adhesion assay
Effect of methanolic extract of C. inerme leaves on adhesion ability of A549 cells was determined by 3-(4,5-dimethyl 2-thiazolyl)-2,5diphenyltetrazolium bromide (MTT) assay. 28,29 Cells were pretreated with different concentrations of the extracts (0, 200 and 400 µg/mL) for 24 hours, then 1x10 4 cells/well were seeded into the Matrigel-coated 96well plate for 1 hour. After incubation time, the non-adherent cells were removed with PBS. The adherent cells were reacted with 0.5 mg/mL of MTT solution at 37°C for 2 hours. After that 100 µL of DMSO was added to each well to solubilize the water-insoluble purple formazan crystals. The absorbance was measured at 570 nm using a microplate reader. The percentage (%) of cell adhesion was calculated relative to the control.

Statistical analysis
All data were edited and entered for the statistical analyses using the SPSS 17.0 software package. Data were reported as mean±standard deviation (±SD) of three independent experiments. Statistical significance was analyzed by the one-way ANOVA test. The differences between treatment groups and the untreated group were considered statistically significant at p < 0.05.

Cytotoxic effect of methanolic leaves extract on C. inerme on A549 cells
The cytotoxicity of methanolic leaves extract of C. inerme on A549 cells was determined by MTT assay and the results are shown in Figure 1. There was no significant effect on cell viability after treatment with methanolic leaves extract of C.inerme (6.25-200 µg/mL) but the high concentration, the extract significantly inhibited cell growth of A549 cells. The 20% inhibitory concentration (IC 20 ) and 50% inhibitory concentration (IC 50 ) of the extracts were greater than 400 µg/mL. The non-cytotoxic and sub-cytotoxic concentrations (200 and 400 µg/mL showing >80% cell viability) of methanolic leaves extract of C. inerme were used for subsequent experiments.

The effect of methanolic leaves extract on C. inerme on cell migration
The anti-migration effects of the methanolic leaves extract of C. inerme were determined by using wound healing and transwell chamber assay. The cell monolayers were scratched with a micropipette tip to create the wound area. After treatment with non-toxic concentrations of C. inerme leaves extract for 24 hours, cells migrated to the wound area and the wound closure was analyzed. The result showed that the methanolic leaves extract of C. inerme could reduce migration of A549 cells to the wound area in a dose-dependent manner when compared with the untreated cells (Figure 2A and 2B). Treatment with methanolic leaves extract of C. inerme at 200 and 400 µg/mL inhibited 71.1±1.91% and 39.2±12.67% of cell migration, respectively. Furthermore, the extract also significantly suppressed cell migration as detected with the transwell chamber as shown in Figure 3A and 3B (**p<0.01). These results indicated that methanolic leaves extract of C. inerme significantly inhibited cell migration in A549 cells.

The effect of methanolic leaves extract on C. inerme on A549 cell invasion
Matrigel-coated Boyden chamber assay was performed to investigate the anti-invasion of A549 by methanolic leaves extract on C. inerme. The invaded cells were stained by crystal violet and the percentage of invaded cells was analyzed by NIH ImageJ. The result demonstrated that methanolic leaves extract of C. inerme significantly suppressed A549 cell invasion compared with the untreated control (**p<0.01) as shown in Figure 4A and 4B. The percentage of invaded cells was 26.20±4.40% when treated with the extract at the concentration of 400 µg/mL. This result indicated that methanolic leaves extract of C. inerme significantly inhibited cell invasion in A549 cells.

Effect of methanolic leaves extract on C. inerme on cell adhesion
Effect of methanolic leaves extract on C. inerme on cell adhesion was performed by MTT assay. The result demonstrated that methanolic leaves extract of C. inerme could inhibit cell adhesion ability of A549 to Matrigel-coated wells at 29.25.0±4.17% after treatment with 400 µg/mL of the extract as shown in Figure 5. This result indicated that the methanolic extract of C. inerme leaves significantly inhibited the adhesion ability of lung cancer cells

DISCUSSION
Metastasis is the major problem for cancer treatment and is responsible for more than 90% of all cancer-related deaths. Cancer metastasis consists of multi-step and multifunctional biological events which include cell detachment, migration, invasion and adhesion. 30 At the earliest stage, cancer cells disseminate from the primary tumor, migrate and invade through the basement membrane, survive in the circulatory system, invade into a secondary site, then adhesion and     start to proliferate. 31,32 Disruption of any metastatic steps is a target for preventing the development of cancer metastases. Herein, we first demonstrated that the methanolic leaves extract on C. inerme inhibited lung cancer cell metastasis by suppressing cell growth, migration, and invasion and adhesion abilities of A549 cells. Before determination of cell migration, the cytotoxicity test was performed to determine a range of crude extract concentrations that provide more than 80% cell viability. We found that 6.25-200 µg/mL methanolic leaves extract of C. inerme showed no significant effect on cell viability but the high concentration, the extract significantly inhibited cell growth of A549 cells. The IC 20 and IC 50 were greater than 400 µg/mL. This value was higher than the previous studies that showed ethanol leaves extract and hydroalcoholic aerial parts C. inerme exhibited anti-proliferation and anti-cancer activities on lung cancer cell line with IC 50 values at 15.6 µg/ mL and 259.5 μg/mL, respectively. 25,26 To determine the anti-metastatic effects of the extract, the non-toxic concentrations at 200 and 400 µg/ mL and allowing over 80% cell survival were selected. Cell migration and invasion are important steps for the metastasis of cancer cells. The ability of cancer cells to migrate and invade other tissues finally lead to metastasis. In this study, we demonstrated methanolic leaves extract of C. inerme significantly suppressed cell migration and invasion of A549 cells in a dose-dependent manner when compared with the untreated cells (**p<0.001). Moreover, we found that the methanolic leaves extract of C. inerme could inhibit cell adhesion ability of A549 after treatment with 400 µg/mL of the extract as shown in Figure 5. These results indicated that the methanolic left extract of C. inerme inhibits cell metastasis of the A549 human lung without apparent cytotoxicity. Several phytochemical constituents have been isolated from C. inerme leaves such as alkaloids, flavonoids, terpenes, diterpenes, triterpene which possess many mechanisms of actions such as anti-cancer, anti-proliferation, anti-metastasis, anti-angiogenesis, anti-oxidation activities, cell cycle arrest and induction of apoptosis. 33 Shen and colleagues demonstrated that acacetin, a flavonoid, inhibits the invasion and migration of human prostate cancer DU145 cells via inactivation of the p38 MAPK signaling pathway. 34 Previous reports demonstrated that fatty acids extracted from C. volubile leaves could suppress tumor metastasis and invasion in human neuronal glioblastoma cells. 35 and breast cancer cells. 36 Although the mechanism underlying antimigration, anti-invasion and anti-adhesion effects are still unclear, the result demonstrated that C. inerme methanolic leaves extract could suppress cancer metastasis of lung cancer cell lines. Further study needs to focus on the mechanism involving anti-metastasis and the major active components both in vitro and in vivo for developing a novel chemotherapeutic agent in the future

CONCLUSION
In the present study, we first demonstrated that methanolic leaves extract of C. inerme inhibited cell migration, invasion and adhesion of A549 cells ( Figure 6). These findings indicated that methanol leave extracts of C. inerme may be used as an anti-metastatic agent for lung cancer. However, further biochemical researches are needed to isolate and identify the active compounds responsible for this pharmacological activity.