Skip to main content

Main menu

  • Home
  • Current Issue
  • Archive
  • Info for
    • Authors
    • Subscribers
    • Advertisers
    • Editorial Board
  • Other Publications
    • In Vivo
    • Cancer Genomics & Proteomics
    • Cancer Diagnosis & Prognosis
  • More
    • IIAR
    • Conferences
    • 2008 Nobel Laureates
  • About Us
    • General Policy
    • Contact
  • Other Publications
    • Anticancer Research
    • In Vivo
    • Cancer Genomics & Proteomics

User menu

  • Register
  • Subscribe
  • My alerts
  • Log in
  • My Cart

Search

  • Advanced search
Anticancer Research
  • Other Publications
    • Anticancer Research
    • In Vivo
    • Cancer Genomics & Proteomics
  • Register
  • Subscribe
  • My alerts
  • Log in
  • My Cart
Anticancer Research

Advanced Search

  • Home
  • Current Issue
  • Archive
  • Info for
    • Authors
    • Subscribers
    • Advertisers
    • Editorial Board
  • Other Publications
    • In Vivo
    • Cancer Genomics & Proteomics
    • Cancer Diagnosis & Prognosis
  • More
    • IIAR
    • Conferences
    • 2008 Nobel Laureates
  • About Us
    • General Policy
    • Contact
  • Visit us on Facebook
  • Follow us on Linkedin
Research ArticleExperimental Studies

Effects of Artonin E on Migration and Invasion Capabilities of Human Lung Cancer Cells

KANOK PLAIBUA, VARISA PONGRAKHANANON, PREEDAKORN CHUNHACHA, BOONCHOO SRITULARAK and PITHI CHANVORACHOTE
Anticancer Research August 2013, 33 (8) 3079-3088;
KANOK PLAIBUA
1Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
VARISA PONGRAKHANANON
1Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand
3Cell-based Drug and Health Product Development Research Unit, Chulalongkorn University, Bangkok, Thailand
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
PREEDAKORN CHUNHACHA
1Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand
3Cell-based Drug and Health Product Development Research Unit, Chulalongkorn University, Bangkok, Thailand
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
BOONCHOO SRITULARAK
2Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
PITHI CHANVORACHOTE
1Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand
3Cell-based Drug and Health Product Development Research Unit, Chulalongkorn University, Bangkok, Thailand
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: pithi_chan@yahoo.com pithi.c@chula.ac.th
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Abstract

Background: Knowledge regarding substances that attenuate motility of cancer cells has gathered significant attention, as they benefit the development of novel anticancer strategies. The anti-migration and anti-invasion activities of artonin E, extracted from bark of Artocarpus gomezianus, were investigated in lung cancer cells in this study. Materials and Methods: Cytotoxicity and antiproliferative effects of artonin E were examined by 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Migration and invasion assays were performed on H460, H23, A549 and H292 human lung cancer cells. Cell morphology was determined by phalloidin-rhodamine staining. Motility-related proteins were investigated by western blotting. Results: Artonin E exhibited anti-migration and anti-invasion activities in H460 cells. Cell morphology revealed that treatment of the cells with non-toxic concentrations of artonin E resulted in a decrease of activated focal adhesion kinase (FAK), downstream protein kinase B (AKT) activation, and Cell division cycle-42 (CDC42), all of which were associated with the anti-motility effect of this compound. Artonin E inhibited invasion and migration of other lung cancer cells, namely H292, H23 and A549 cells. Conclusion: These results suggest that artonin E may be a promising candidate for anti-metastasis use.

  • Metastasis
  • migration
  • invasion
  • artonin E
  • lung cancer cells
  • H460
  • H23
  • A549
  • H292 cells

Metastatic lung cancer is the cause of more than 90% lung cancer-related deaths worldwide (1). Although the earliest stage of the disease presents as only pulmonary nodule without lymph node spread, some patients with disease at this stage will finally die from undetectable metastases (2). Cancer metastasis is a complex process of cell spreading which can be divided into several steps including migration, invasion, intravasation, survival in the circulation, extravasation, and metastatic colonization (3, 4). A growing body of evidence suggests that migration and invasion are crucial steps for successful metastasis (5); however, at present, there are no approved drugs that inhibit such cancer cell behavior.

Even though the molecular mechanisms, which cancer cells use for migration and invasion are not fully understood, based on previous research, they involve the ability of cancer cells to change their affinity for the extracellular matrix (ECM) and such alterations are due to modifications of various cellular signaling pathways including focal adhesion kinase FAK (6). Indeed, the activation of FAK through phosphorylation is important for FAK-induced focal adhesion turnover and cell movement (7). Activated FAK can transduce the signal through the phosphorylation of protein kinase B (AKT) resulting in cellular responses such as cell invasion and migration (6). Recently, the Rho family of small guanosine-5’-triphosphatases (GTPases), especially Cell division cycle-42 (CDC42), were shown to play an essential role in modulating actin re-organization and filopodia formation (8) and its overexpression was shown to be associated with enhanced migration and cancer aggressiveness (9, 10). The expression level of CDC42 was found to be up-regulated in many types of cancer (10, 11). In lung cancer, CDC42 was shown to be highly overexpressed in primary lung cancer cells (12). A previous study indicated that both curcumin-mediated CDC42 down-regulation and CDC42 knock-down attenuated cancer cell invasion in vivo (12).

Artonin E (Figure 1), an active flavonoid, obtained from the bark of Artocarpus gomezianus Wall. exTréc. (Moraceae), is known as “Ka-Noon-Pah” in Thailand (13). Artonin E was shown to exhibit promising growth-inhibition action against breast cancer cells (14). However, the effects of artonin E on cancer cell migration and invasion are unknown. In our view, the knowledge regarding such activities of the compound would benefit the development of novel anti-metastasis drugs, as well as strategies to overcome cancer.

Materials and Methods

Test compound. Artonin E, a pure compound in dry powder form, was obtained from Associate Professor Boonchoo Sritularak, Departments of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University (BKK, Thailand), and was dissolved in ethanol and RPMI-1640 to achieve the desired concentrations, containing less than 0.1% ethanol at final dilution.

Cell culture. Human lung cancer H460, H292, H23 and A549 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultivated in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 100 units/ml penicillin/streptomycin in a 5% CO2 environment at 37°C.

Chemicals. Hoechst 33342, propidium iodide (PI), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were obtained from Sigma Chemical, Inc. (St. Louis, MO, USA). Rabbit antibodies to CDC42, caveolin-1 (CAV1), pFAK (Tyr 397), FAK, pAKT (Ser 473), AKT, and β-actin were obtained from Cell Signaling Technology, Inc. (Danver, MA, USA).

Cytotoxicity and cell proliferation assay. To determine artonin E-mediated cytotoxicity and effects on cell proliferation, cell viability was determined by the MTT assay, as previously described (15), which measures cellular capacity to reduce MTT (yellow) to purple formazan crystal by mitochondrial dehydrogenase. Cells were seeded in a 96-well plate and allowed to attach for 12 h. Cells were treated with different concentrations of artonin E (0-50 μg/ml) and incubated for 24 h for cytotoxicity assay and 12, 24, 48 and 72 h for proliferation assay, and cells were incubated with 100 μl of 500 μg/ml MTT solution for 4 h at 37°C. Then, MTT solution was removed and 100 μl of 99.9% DMSO was added to dissolve the formazan crystal. The intensity of formazan product was measured at 570 nm using a microplate reader. All analyses were performed in at least three independent replicate cultures. The percentage of cell viability was calculated as follows: Embedded Image

Apoptosis and necrosis assay. Apoptotic and necrotic cell death were determined by Hoechst 33342 and PI co-staining as described elsewhere (15). Cells were seeded in 96-well plates and treated with different concentrations (0-0.5 μg/ml) of artonin E for 24 h, cells were incubated with 10 μM of the Hoechst 33342 and 5 μg/ml PI dye for 30 min at 37°C. The apoptotic cells with condensed chromatin and/or fragmented nuclei and PI-positive necrotic cells were visualized under a fluorescence microscope (Olympus IX51 with DP70; Olympus, Center Valley, PA, USA).

Figure 1.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 1.

Structure of artonin E.

Wound-healing assay. The wound-healing assay was determined as previously described (16). Briefly, a monolayer of cells was cultured in 96-well plates, and then a wound space was created by a 1-mm width tip. After rinsing with PBS, the cell monolayers were treated with different concentrations of artonin E (0-0.5 μg/ml) and allowed to migrate for 24, 48, and 72 h. Micrographs were taken under a phase-contrast microscope (×100; Olympus IX51 with DP70), and wound spaces were measured from 10 random fields of view using Olympus DP controller software. Quantitative analysis of cell migration was performed by using an average wound space from random fields of view, and the relative migration was calculated.

Invasion assay. An invasion assay was conducted using a 24-well Transwell unit with polyvinylidene difluoride (PVDF) filters (8 μm pore size) as previously described (16). Each well was pre-coated with 50 μl Matrigel (BD Biosciences, Bedford, MA, USA). The lower chamber of the unit contained with RPMI medium containing 10% FBS. Cells at the density of 3×105 cells/100 μl were incubated with artonin E at different concentrations (0-0.5 μg/ml) in RPMI containing 1% FBS, and then added to the upper chamber. After 24 h, the top medium and Matrigel were completely removed and the bottom side was fixed with 3.7% paraformaldehyde. After staining cells with Hoechst 33342, cells were visualized and scored under a fluorescence microscope (Olympus IX51 with DP70).

Cell morphological characteristics. Cells were washed with phosphate buffer saline (PBS) and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. Afterwards, cells were permeabilized by 0.1% Triton-X100 in PBS for 4 min, washed with PBS three times, and blocked with 0.2% bovine serum albumin (BSA) for 30 min as described elsewhere (17). After washing, cells were incubated with rhodamine-phalloidin in PBS for 30 min and rinsed three times. Images of stained cells with rhodamine-labeled phalloidin were taken under a fluorescence microscope (Olympus IX51 with DP70).

Western blot. Cells were seeded in 6 well-plates and treated with different concentrations of artonin E (0-0.5 μg/ml) for 24 and 72 h. After specific treatments, cell lysates were obtained by incubating the cells in ice-cold lysis buffer containing 20 mM Tris-HCl (pH 7.5), 0.5% Triton X-100, 150 mM sodium chloride, 10% glycerol, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 100 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN, USA) for 60 min on ice. Protein content was determined using the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA) and an equal amount of protein from each sample (60-80 μg) was heated at 95°C for 5 min with Laemmli loading buffer. The lysates were then loaded onto 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel for electrophoresis. After separation, proteins were transferred onto 0.45 μm nitrocellulose membranes (Bio-Rad). The transferred membranes were blocked for 1 h in 5% non-fat dry milk in TBST [25 mM Tris-HCl (pH 7.5), 125 mM NaCl, 0.05% Tween 20]. Membranes were washed twice with TBST for 7 min and incubated with the primary antibodies at 4°C for 10 h. Membranes were then washed three times with TBST for 7 min and incubated with horseradish peroxidase-coupled isotype-specific secondary antibodies for 2 h at room temperature. After washing again, the immune complexes were detected by enhancing with chemiluminescence substrate (Supersignal West Pico; Pierce, Rockford, IL, USA), and quantified using analyst/PC densitometry software (Bio-Rad) normalized to the level of β-actin protein.

Figure 2.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 2.

Effect of artonin E on cell viability of human lung cancer H460 cells. Cells were treated with different concentrations of artonin E (0-50 μg/ml) for 24 h. A: Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the concentration for 50% cell survival (IC50) was determined. B: Percentage of cell viability and cell apoptosis were analyzed by MTT assay and Hoechst 33342 staining assays, respectively. C: Morphology of apoptotic nuclei stained with Hoechst 33342 and propidium iodide (PI). D: Proliferation of H460 cells in response to artonin E 0.05-0.5 μg/ml for 12, 24, 48, and 72 h was investigated by the MTT assay. Values are means of triplicate samples±SD.

Statistical analysis. Mean data from at least three independent experiments were normalized to the result of the untreated control. Statistical differences between means were determined using an analysis of variance (ANOVA) and post hoc test at a significance level of p<0.05, and data are presented as the mean±SD.

Results

Cytotoxic effect of artonin E on human non-small cell lung cancer H460 cells. Although the anticancer activity of artonin E has been previously demonstrated in a breast cancer cell model (14), it is not clear whether such a compound causes cytotoxicity to lung cancer cells. The present study first-characterized the cytotoxic effect of artonin E on H460 human lung cancer cells by incubating the cells in the presence and absence of artonin E (0-50 μg/ml) for 24 h, and cell viability was analyzed by the MTT assay. Figure 2A shows that treatment with artonin E caused a dose-dependent decrease in cell survival and 50% inhibition (IC50) was observed in response to artonin E at the concentration of 31.93 μg/ml. Because the study aimed to investigate antimigration and anti-invasion activities of the compound, the concentrations of artonin E which cause neither toxic nor proliferative effects were further clarified. Results indicated that treatment with artonin E at concentrations of 0.05-0.5 μg/ml caused no significant effect on H460 cell viability (Figure 2B). The nuclear morphology study shown in Figure 2C supported the above finding that no apoptotic or necrotic cell death was detected in response to artonin E at these concentrations. In addition, artonin E at concentrations of 0.05-0.5 μg/ml did not significantly alter proliferation of the cells up to 72 h (Figure 2D).

Figure 3.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 3.

Effects of artonin E on H460 cell migration and invasion. For migration assay, wound-healing assay was performed. Wound space was created and the cells were treated with sub-toxic concentrations of artonin E for different times. For invasion assays, cells were seeded onto Matrigel-coated membrane and treated with sub-toxic concentrations of artonin E for 24 h. A: Wound space was visualized under a phase-contrast microscope at the indicated times. B: The relative cell migration was analyzed by comparison of the relative change in wound space of the treated groups over that of the untreated control. C, D: The invaded cells were stained with Hoechst 33342, visualized under fluorescence microscopy, and the relative cell invasion was determined. Values are means of triplicate samples±SD.; *p<0.05 versus untreated control.

Artonin E inhibits migration and invasion of H460 cells. To examine the effect of artonin E on migration of the cells, a wound healing assay was performed. Briefly, the confluent monolayer of H460 cells was scratched and cells were cultured with or without sub-toxic concentrations of artonin E (0.05-0.5 μg/ml) for 24, 48, and 72 h. Figures 3A and B show that the incubation of cells with artonin E at a concentration of 0.5 μg/ml significantly reduced the spreading of H460 cells to the wound area as early as 24 h, whereas artonin E at 0.05 μg/ml had no significant effect on cell migration in comparison to that of the untreated controls. In addition, artonin E at concentrations of 0.1 and 0.25 μg/ml significantly inhibited migration of H460 cells at 72 h. These results indicate that artonin E possesses the ability to inhibit cancer cell migration.

Figure 4.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 4.

Effect of artonin E on filopodia alteration. After treating with sub-toxic concentrations of artonin E for 24 h, cells were stained with phalloidin and filopodia were examined under fluorescent microscopy. Filopodia are indicated by arrow.

For invasion assays, the cancer cells were added to a 24-well transwell pre-coated with Matrigel and treated with different concentrations of artonin E. The invaded cells were determined as described in Materials and Methods. As shown in Figures 3C and D, artonin E at 0.25 and 0.5 μg/ml significantly inhibited cancer cell invasion through Matrigel matrix at 24 h.

Artonin E inhibits filopodia formation in lung cancer cells. During cell movement, membrane protrusions called filopodia were shown to be increased and the formation of filopodia was shown to be closely involved with cancer cell migration and invasion (18). Having shown that artonin E at sub-toxic concentrations significant inhibited lung cancer cell migration and invasion, we further tested whether the compound has an effect on filopodia formation. Cells were treated with 0-0.5 artonin E and phalloidin-labeled filopodia were detected under fluorescence microscopy. Figure 4 shows that in untreated controls, cells exhibited several membrane protrusions of filopodia. Interestingly, treatment with artonin E dramatically reduced directional stress fibers and filopodia in H460 cells in comparison to that of untreated cells. These data and the above findings suggest that artonin E has a negative impact on cell migration and invasion and this may, at least in part, involve the reduction of cellular filopodia.

Artonin E inhibits FAK signaling and suppresses CDC42 expression in H460 cells. In order to clarify the mechanism of artonin E in suppression of cancer cell motility, the expression level and activation status of proteins regulating cell motility, namely FAK and AKT, were investigated. Cells were seeded and incubated in the presence or absence of artonin E (0.05-0.5 μg/ml) and the expression of proteins was determined by western blotting. While artonin E had only a minimal effect on the level of total FAK, treatment of artonin E at concentrations of 0.1-0.5 μg/ml significantly reduced the level of phosphorylated FAK at Tyr 397 (activated FAK). It has been well-established that the phosphorylation of FAK at Tyr 397 activates its kinase activity that further triggers AKT signaling (6). We, thus, investigated the possible effect of artonin E on AKT activation. Figure 5 demonstrates that artonin E significantly reduced phosphorylation of AKT at Ser 473 in a concentration-dependent manner while it had no significant effect on total AKT expression. These findings suggested that artonin E inhibits cell migration via an FAK-AKT-dependent mechanism.

Figure 5.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 5.

Effect of artonin E on focal adhesion kinase (FAK), protein kinase B (AKT), Cell division cycle (CDC42), and caveolin-1(CAV1) proteins. A: Cells were seeded and treated with different concentrations of artonin E (0-0.5 μg/ml) for 24 and 72 h and then the expressions of pFAK (Tyr 397), FAK, pAKT (Ser 473), AKT, CDC42 and CAV1 proteins were determined by Western blotting. To confirm equal loading of samples, blots were re-probed with β-actin antibody. B: The immunoblot signals were quantified by densitometry and mean data from four independent experiments were presented. Values are means of samples±SD. (n=4); *p<0.05 versus untreated group.

The high expression level of CDC42, as well as CAV1 was shown to be tightly-associated with an increase in aggressiveness of cancer (9, 19). Since the CDC42 was shown to be up-regulated in primary lung cancer cells and such an increase of the protein was shown to be associated with high TNM stage and lymph node metastasis, it is interesting to investigate whether artonin E treatment could affect the cellular CDC42 level. We assessed the effect of artonin E on CDC42 level by western blot analysis and found that CDC42 protein expression was down-regulated in response to artonin treatment at the concentrations of 0.25 and 0.5 μg/ml in comparison to that of the untreated control. However, the expression of CAV1 protein was found to be only slightly affected in response to artonin E treatment. These results suggest that artonin E attenuates lung cancer cell migration and invasion through an FAK-AKT-dependent mechanism and via reduction of CDC42.

Artonin E inhibits migration and invasion of other lung cancer cell lines. In order to confirm the negative regulatory effect of artonin E on lung cancer cell migration and invasion, human lung cancer cell lines H292, H23 and A549 were treated with the non-toxic concentrations of artonin E and subjected to migration as well as invasion assays. Our results, shown in Figure 6, revealed that artonin E at 0.25 and 0.5 μg/ml significantly inhibited the migratory behavior of H292, H23, and A549 cells in comparison to that of the respective untreated controls. Consistently, the invasion assay indicated that artonin E at such concentrations significantly suppressed the invasion of H292, H23, and A549 cells (Figure 7). These data strengthen the observations of the present study that artonin E has the ability to inhibit migration and invasion of lung cancer cells.

Discussion

Advanced and novel strategies for cancer therapies, including those inhibiting metastasis of cancer, have gathered increasing attention. Limited efficacy has been obtained from the available therapy, resulting in only 68% 5-year survival of patients with cancer in the United States (20), and the major cause of death found in such patients involves metastasis. As a hallmark of cancer metastasis, the ability of cancer cells to migrate away from the original tumor and to invade the blood or lymphatic circulation is considerably important (3). Herein we demonstrated, to our knowledge for the first time that artonin E, a plant-derived pure compound has a promising ability to inhibit lung cancer cell movement. Activation of cancer cell motility involves several mechanistic pathways including FAK (6), AKT (21), CDC42 (22), and CAV1 (19). Tumor progression and metastasis can be stimulated by FAK signaling pathways through the regulation of cell migration and invasion. Mutagenesis of FAK gene at the 397 site by the insertion technique eliminated the ability of FAK to activate motility (23). Likewise, many studies demonstrate that phosphorylation of FAK at Tyr 397 is necessary for FAK to promote cell migration (24-26). Invasion of malignant cells is also promoted by FAK signaling (27). Moreover, evidence has indicated that FAK triggers and activates the Phosphoinositide 3-kinase (PI3K)-AKT pathway (6). AKT activation was shown to be associated with cell migration and invasion by several means (21, 28). For instance, AKT regulates the stability of microtubules, which has an essential role in cell motility (29) and its function was shown to increase invasiveness of cells (21).

Figure 6.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 6.

Effects of artonin E on migration of H292, H23, and A549 cells. H292, H23, and A549 cells were subjected to migration assay and visualized under a phase-contrast microscope. The relative cell migration of H292, H23, and A549 cells are shown in A, B, and C, respectively. Values are means of triplicate samples±SD.; *p<0.05 versus untreated control.

Figure 7.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 7.

Effects of artonin E on invasion of H292, H23, and A549 cells. H292, H23, and A549 cells were added to Matrigel and treated with sub-toxic concentrations of artonin E for 24 h. The invaded cells were stained by Hoechst 33342 and visualized under fluorescence microscopy. Values are means of triplicate samples±SD.; *p<0.05 versus untreated control.

CDC42 protein, a member of Rho GTPase family, has been shown to regulate many cellular processes, including actin re-organization and cell polarity (30). CDC42 is also implicated in filopodia formation which leads to migration and invasion of cancer cells (31). Some evidence indicate that CDC42 was overexpressed in many types of human carcinoma, resulting in increased aggressiveness of the cancer (9, 11). Furthermore, the knock-down of CDC42 generally resulted in inhibition of cancer cell migration and invasion suggesting the significance impact of this protein on cell motility (32). In the present study, we found that treatment of the lung cancer H460 cells with artonin E resulted in the reduction of the cellular CDC42 levels (Figures 5A and B), along with the findings indicating that filopodia protrusions were reduced in the cells treated with artonin E (Figure 4). This substance may, at least in part, inhibit migration and invasion through CDC42 suppression. Even though we did not observe a significant effect of artonin E on the cellular CAV1 level, we and others have previously found the importance of CAV1 in regulating lung cancer cell migration and invasion (16, 33). Recently, CAV1 has been shown to play an important role in cancer metastasis (19). CAV1 was shown to mediate cancer cell migration and invasion in head and neck squamous cell carcinoma (34). Furthermore, overexpression of CAV1 was shown to enhance migration and invasion ability of H460 lung carcinoma cells, while its suppression using ShRNA-CAV1 had the opposite effect (16, 35).

In addition, we have provided evidence indicating that the inhibitory effect of artonin E on cancer cell motility can also be observed in other lung cancer cell models. Three human lung carcinoma cell lines, namely, H292, H23 and A549, were tested with non-toxic concentrations of artonin E and the results indicated that artonin E had similar activities in these cells as those found in H460 cells.

In summary, this investigation reveals that artonin E can inhibit migration and invasion of lung cancer cells via suppression of activated FAK, activated AKT, and CDC42; therefore, artonin E may be a promising agent for anti-metastasis therapy or used as adjuvant with standard therapies to improve survival of patients with cancer.

Acknowledgements

This work is supported by grants from the 90th Anniversary of Chulalongkorn University Fund and the Ratchadaphiseksompot Endowment Fund, Chulalongkorn University. The Authors would like to thank Mr. Krich Rajprasit for proofreading.

  • Received May 10, 2013.
  • Revision received May 22, 2013.
  • Accepted May 28, 2013.
  • Copyright© 2013 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved

References

  1. ↵
    1. Keshamouni V,
    2. Arenberg D,
    3. Kalemkerian G
    1. Ray MR,
    2. Jablons DM
    : Lung cancer metastasis novel biological mechanisms and impact on clinical practice. In: Hallmarks of Metastasis. Keshamouni V, Arenberg D, Kalemkerian G (eds.). New York, Springer, pp. 29-46, 2009.
  2. ↵
    1. Maslyar DJ,
    2. Jahan TM,
    3. Jablons DM
    : Mechanisms of and potential treatment strategies for metastatic disease in non-small cell lung cancer. Semin Thorac Cardiovasc Surg 16(1): 40-50, 2004
    OpenUrlPubMed
  3. ↵
    1. Hanahan D,
    2. Weinberg R
    : The hallmarks of cancer. Cell 100(1): 57-70, 2000
    OpenUrlCrossRefPubMed
  4. ↵
    1. Mina LA,
    2. Sledge GW
    : Rethinking the metastatic cascade as a therapeutic target. Nat Rev Clin Oncol 8(6): 325-332, 2011
    OpenUrlPubMed
  5. ↵
    1. Harlozinska A
    : Progress in molecular mechanisms of tumor metastasis and angiogenesis. Anticancer Res 25(5): 3327-3333, 2005
    OpenUrlAbstract/FREE Full Text
  6. ↵
    1. Bolos V,
    2. Gasent JM,
    3. Lopez-Tarruella S,
    4. Grande E
    : The dual kinase complex FAK-SRC as a promising therapeutic target in cancer. Onco Targets Ther 3: 83-97, 2010.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Vicente-Manzanares M,
    2. Choi CK,
    3. Horwitz AR
    : Integrins in cell migration-the actin connection. J Cell Sci 122(2): 199-206, 2009
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Raftopoulou M,
    2. Hall A
    : Cell migration: Rho GTPases lead the way. Dev Biol 265(1): 23-32, 2004
    OpenUrlCrossRefPubMed
  9. ↵
    1. Kamai T,
    2. Yamanishi T,
    3. Shirataki H,
    4. Takagi K,
    5. Asami H,
    6. Ito Y,
    7. Yoshida K
    : Overexpression of RhoA, Rac1, and Cdc42 GTPases is associated with progression in testicular cancer. Clin Cancer Res 10(14): 4799-4805, 2004
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Yoshioka K,
    2. Nakamori S,
    3. Itoh K
    : Overexpression of small GTP-binding protein RhoA promotes invasion of tumor cells. Cancer Res 59(8): 2004-2010, 1999
    OpenUrlAbstract/FREE Full Text
  11. ↵
    1. Jiang LC,
    2. Zhang Y,
    3. Qu XC
    : Effects of Cdc42 overexpression on the estrogen-enhanced multidrug resistance in breast cancer cells. Zhonghua Zhong Liu Za Zhi 33(7): 489-493, 2011 (in Chinese).
    OpenUrlPubMed
  12. ↵
    1. Chen QY,
    2. Jiao DM,
    3. Yao QH,
    4. Yan J,
    5. Song J,
    6. Chen FY,
    7. Lu GH,
    8. Zhou JY
    : Expression analysis of Cdc42 in lung cancer and modulation of its expression by curcumin in lung cancer cell lines. Int J Oncol 40(5): 1561-1568, 2012
    OpenUrlPubMed
  13. ↵
    1. Sritularak B,
    2. Tantituvanont A,
    3. Chanvorachote P,
    4. Meksawan K,
    5. Miyamoto T,
    6. Kohno Y,
    7. Likhitwitayawuid K
    : Flavonoids with free radical-scavenging activity and nitric oxide inhibitory effect from the stem bark of Artocarpus gomezianus. J Med Plants Res 4(5): 387-392, 2010
    OpenUrl
  14. ↵
    1. Shajarahtunnur J
    : Phytochemical and Bioactivities of Malaysian Artocarpus lowii king, a. Scortechinii king and a. Teysmanii miq. Doctoral dissertation, University Technology Malaysia, Malaysia, 2006.
  15. ↵
    1. Chanvorachote P,
    2. Pongrakhananon V
    : Ouabain down-regulates MCL-1 and sensitizes lung cancer cells to TRAIL-induced apoptosis. Am J Physiol Cell Physiol 304(3): 263-272, 2013
    OpenUrl
  16. ↵
    1. Luanpitpong S,
    2. Talbott SJ,
    3. Rojanasakul Y,
    4. Nimmannit U,
    5. Pongrakhananon V,
    6. Wang L,
    7. Chanvorachote P
    : Regulation of lung cancer cell migration and invasion by reactive oxygen species and caveolin-1. J Biol Chem 285(50): 38832-38840, 2010
    OpenUrlAbstract/FREE Full Text
  17. ↵
    1. Halim H,
    2. Chanvorachote P
    : Long-term hydrogen peroxide exposure potentiates anoikis resistance and anchorage-independent growth in lung carcinoma cells. Cell Biol Int 36(11): 1055-1066, 2012
    OpenUrlPubMed
  18. ↵
    1. Arjonen A,
    2. Kaukonen R,
    3. Ivaska J
    : Filopodia and adhesion in cancer cell motility. Cell Adh Migr 5(5): 421-430, 2011
    OpenUrlCrossRefPubMed
  19. ↵
    1. Sotgia F,
    2. Martinez-Outschoorn UE,
    3. Howell A,
    4. Pestell RG,
    5. Pavlides S,
    6. Lisanti MP
    : Caveolin-1 and cancer metabolism in the tumor microenvironment: markers, models, and mechanisms. Annu Rev Pathol 7: 423-467, 2012.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Merrill RM,
    2. Hunter BD
    : Conditional survival among cancer patients in the United States. Oncologist 15(8): 873-882, 2010
    OpenUrlAbstract/FREE Full Text
  21. ↵
    1. Kim D,
    2. Kim S,
    3. Koh H,
    4. Yoon SO,
    5. Chung AS,
    6. Cho KS,
    7. Chung J
    : Akt/PKB promotes cancer cell invasion via increased motility and metalloproteinase production. FASEB J 15(11): 1953-1962, 2001
    OpenUrlAbstract/FREE Full Text
  22. ↵
    1. Sinha S,
    2. Yang W
    : Cellular signaling for activation of Rho GTPase CDC42. Cell Signal 20(11): 1927-1934, 2008
    OpenUrlCrossRefPubMed
  23. ↵
    1. Cary LA,
    2. Chang JE,
    3. Guan JL
    : Stimulation of cell migration by overexpression of focal adhesion kinase and its association with Src and Fyn. J Cell Sci 109(7): 1787-1794, 1996
    OpenUrlAbstract/FREE Full Text
  24. ↵
    1. Niwa Y,
    2. Kanda H,
    3. Shikauchi Y,
    4. Saiura A,
    5. Matsubara K,
    6. Kitagawa T,
    7. Yamamoto J,
    8. Kubo T,
    9. Yoshikawa H
    : Methylation silencing of SOCS-3 promotes cell growth and migration by enhancing JAK/STAT and FAK signaling in human hepatocellular carcinoma. Oncogene 24(42): 6406-6417, 2005
    OpenUrlPubMed
    1. Sieg DJ,
    2. Hauck CR,
    3. Ilic D,
    4. Klingbeil CK,
    5. Schaefer E,
    6. Damsky CH,
    7. Schlaepfer DD
    : FAK integrates growth-factor and integrin signals to promote cell migration. Nat Cell Biol 2(5): 249-256, 2000
    OpenUrlCrossRefPubMed
  25. ↵
    1. Zhao J,
    2. Guan JL
    : Signal transduction by focal adhesion kinase in cancer. Cancer Metastasis Rev 28(1-2): 35-49, 2009.
    OpenUrlCrossRefPubMed
  26. ↵
    1. Shibata K,
    2. Kikkawa F,
    3. Nawa A,
    4. Thant AA,
    5. Naruse K,
    6. Mizutani S,
    7. Hamaguchi M
    : Both focal adhesion kinase and c-Ras are required for the enhanced matrix metalloproteinase 9 secretion by fibronectin in ovarian cancer cells. Cancer Res 58(5): 900-903, 1998
    OpenUrlAbstract/FREE Full Text
  27. ↵
    1. Grille SJ,
    2. Bellacosa A,
    3. Upson J,
    4. Klein-Szanto AJ,
    5. van Roy F,
    6. Lee-Kwon W,
    7. Donowitz M,
    8. Tsichlis PN,
    9. Larue L
    : The protein kinase Akt induces epithelial- mesenchymal transition and promotes enhanced motility and invasiveness of squamous cell carcinoma lines. Cancer Res 63(9): 2172-2178, 2003
    OpenUrlAbstract/FREE Full Text
  28. ↵
    1. Onishi K,
    2. Higuchi M,
    3. Asakura T,
    4. Masuyama N,
    5. Gotoh Y
    : The PI3K-AKT pathway promotes microtubule stabilization in migrating fibroblasts. Genes Cells 12(4): 535-546, 2007
    OpenUrlCrossRefPubMed
  29. ↵
    1. Schmitz AA,
    2. Govek EE,
    3. Böttner B,
    4. Van Aelst L
    : Rho GTPases: Signaling, migration, and invasion. Exp Cell Res 261(1): 1-12, 2000
    OpenUrlCrossRefPubMed
  30. ↵
    1. Machesky LM
    : Lamellipodia and filopodia in metastasis and invasion. FEBS Lett 582(14): 2102-2111, 2008
    OpenUrlCrossRefPubMed
  31. ↵
    1. Reymond N,
    2. Im JH,
    3. Garg R,
    4. Vega FM,
    5. Borda d'Agua B,
    6. Riou P,
    7. Cox S,
    8. Valderrama F,
    9. Muschel RJ,
    10. Ridley AJ
    : Cdc42 promotes transendothelial migration of cancer cells through β1 integrin. J Cell Biol 199(4): 653-668, 2012
    OpenUrlAbstract/FREE Full Text
  32. ↵
    1. Williams TM,
    2. Hassan GS,
    3. Li J,
    4. Cohen AW,
    5. Medina F,
    6. Frank PG,
    7. Pestell RG,
    8. Di Vizio D,
    9. Loda M,
    10. Lisanti MP
    : Caveolin-1 promotes tumor progression in an autochthonous mouse model of prostate cancer: Genetic ablation of Cav-1 delays advanced prostate tumor development in tramp mice. J Biol Chem 280(26): 25134-25145, 2005
    OpenUrlAbstract/FREE Full Text
  33. ↵
    1. Nohata N,
    2. Hanazawa T,
    3. Kikkawa N,
    4. Mutallip M,
    5. Fujimura L,
    6. Yoshino H,
    7. Kawakami K,
    8. Chiyomaru T,
    9. Enokida H,
    10. Nakagawa M,
    11. Okamoto Y,
    12. Seki N
    : Caveolin-1 mediates tumor cell migration and invasion and its regulation by miR-133a in head and neck squamous cell carcinoma. Int J Oncol 38(1): 209-217, 2011
    OpenUrlPubMed
  34. ↵
    1. Park J,
    2. Bae E,
    3. Lee C,
    4. Yoon SS,
    5. Chae YS,
    6. Ahn KS,
    7. Won NH
    : RNA interference-directed caveolin-1 knockdown sensitizes SN12CPM6 cells to doxorubicin-induced apoptosis and reduces lung metastasis. Tumour Biol 31(6): 643-650, 2010
    OpenUrlPubMed
PreviousNext
Back to top

In this issue

Anticancer Research: 33 (8)
Anticancer Research
Vol. 33, Issue 8
August 2013
  • Table of Contents
  • Table of Contents (PDF)
  • Index by author
  • Back Matter (PDF)
  • Ed Board (PDF)
  • Front Matter (PDF)
Print
Download PDF
Article Alerts
Sign In to Email Alerts with your Email Address
Email Article

Thank you for your interest in spreading the word on Anticancer Research.

NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. We do not capture any email address.

Enter multiple addresses on separate lines or separate them with commas.
Effects of Artonin E on Migration and Invasion Capabilities of Human Lung Cancer Cells
(Your Name) has sent you a message from Anticancer Research
(Your Name) thought you would like to see the Anticancer Research web site.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
1 + 9 =
Solve this simple math problem and enter the result. E.g. for 1+3, enter 4.
Citation Tools
Effects of Artonin E on Migration and Invasion Capabilities of Human Lung Cancer Cells
KANOK PLAIBUA, VARISA PONGRAKHANANON, PREEDAKORN CHUNHACHA, BOONCHOO SRITULARAK, PITHI CHANVORACHOTE
Anticancer Research Aug 2013, 33 (8) 3079-3088;

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Reprints and Permissions
Share
Effects of Artonin E on Migration and Invasion Capabilities of Human Lung Cancer Cells
KANOK PLAIBUA, VARISA PONGRAKHANANON, PREEDAKORN CHUNHACHA, BOONCHOO SRITULARAK, PITHI CHANVORACHOTE
Anticancer Research Aug 2013, 33 (8) 3079-3088;
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Jump to section

  • Article
    • Abstract
    • Materials and Methods
    • Results
    • Discussion
    • Acknowledgements
    • References
  • Figures & Data
  • Info & Metrics
  • PDF

Related Articles

  • No related articles found.
  • PubMed
  • Google Scholar

Cited By...

  • No citing articles found.
  • Google Scholar

More in this TOC Section

  • 5-Azacytidine (5-aza) Induces p53-associated Cell Death Through Inhibition of DNA Methyltransferase Activity in Hep3B and HT-29 Cells
  • Prognostic Value of WNT1, NOTCH1, PDGFRβ, and CXCR4 in Oral Squamous Cell Carcinoma
  • Hypoxia-adapted Multiple Myeloma Stem Cells Resist γδ-T-Cell-mediated Killing by Modulating the Mevalonate Pathway
Show more Experimental Studies

Similar Articles

Keywords

  • Metastasis
  • migration
  • invasion
  • artonin E
  • lung cancer cells
  • H460
  • H23
  • A549
  • H292 cells
Anticancer Research

© 2023 Anticancer Research

Powered by HighWire